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Page 1: Immunity
Page 2: Immunity

Immunobiology

PRINCIPLES OF MEDICAL BIOLOGY A Multi-Volume Work, Volume 6 Editors: E. EDWARD BITTAR, Department of Physiology,

University of Wisconsin, Madison

NEVILLE BITTAR, Department of Medicine,

University of Wisconsin, Madison

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Principles of IVIeA Multi-Volume Work

Edited by E, Edward Bittar, Department of Physiology, University of Wisconsin, Madison and Neville Bittar, Department of Medicine University of Wisconsin, Madison

This work provides:

* A holistic treatment of the main medical disciplines. The basic sci­ences including most of the achievements in cell and molecular biology have been blended with pathology and clinical medicine. Thus, a special feature is that departmental barriers have been overcome.

* The subject matter covered in preclinical and clinical courses has been reduced by almost one-third without sacrificing any of the essentials of a sound medical education. This information base thus represents an integrated core curriculum.

* The movement towards reform in medical teaching calls for the adoption of an integrated core curriculum involving small-group teaching and the recognition of the student as an active learner.

* There are increasing indications that the traditional education sys­tem in which the teacher plays the role of expert and the student that of a passive learner is undergoing reform in many medical schools. The trend can only grow.

* Medical biology as the new profession has the power to simplify the problem of reductionism.

* Over 700 internationally acclaimed medical scientists, patholo­gists, clinical investigators, clinicians and bioethicists are participants in this undertaking.

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Immunobiology

Edited by E. EDWARD BITTAR Department of Physiology

University of Wisconsin

Madison, Wisconsin

NEVILLE BITTAR Department of Medicine

University of Wisconsin

Madison, Wisconsin

( ^ jAI PRESS INC.

Greenwich, Connecticut London, England

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Library of Congress Cataloging-in-Publication Data

Immunobiology / edited by E. Edward Bittar, Neville Bittar. p. cm.—(Principles of medical biology ; v. 6)

Includes index. ISBN 1-55938-811-0

1. Immunology. 2. Molecular immunology. I. Bittar, E. Edward. II. Bittar, Neville. III. Series.

[DNLM: 1. Immune System. 2. Immunity. QW 50413223 1996] QR181.I454 1996 616.07'9—dc20

DNLM/DLC 96-35160 for Library of Congress CIP

Copyright © 1996 by JAI PRESS INC.

55 Old Post Road, No. 2

Greenwich, Connecticut 06836

JAI PRESS LTD.

38 Tavistock Street

Covent Garden

London, England

All rights reserved. No part of this publication may be reproduced,

stored on a retrieval system, or transmitted in any form or by any

means, electronic, mechanical, photocopying, filming, recording, or

otherwise, without prior permission in writing from the publisher.

ISBN: 1-55938-811-0

Library of Congress Catalog No.: 96-35160

Manufactured in the United States of America

Page 8: Immunity

CONTENTS

List of Contributors ix

Preface

£ Edward Bittar and Neville Bittar xii i

Chapter 1

The Thymus in Immunity

J.FA.P. Miller 1

Chapter 2

The B-Cell in Immunity

David Tarlinton 21

Chapter 3

Cell-to-Cell Interactions in the Immune System

William A. Sewell and Ronald Penny 47

Chapter 4

Immunological Tolerance

J.FA.P. Miller 63

Chapter 5

The Generation of Diversity in the Immune

System

E.J. Steele and H.S. Rothenfluh 85

Chapter 6

The Antigen-Antibody Complex: Structure and

Recognition

P.M. Colman 107

vii

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viii CONTENTS

Chapter 7

The Major Histocompatibility Complex

Brian D.Tait 121

Chapter 8

B and T Cell Signaling at the Molecular Level

Tomas Mustelin and Paul Bum 137

Chapter 9

Cytokines in Immunology

Andrew J. Hapel and Shaun R. McColl 151

Chapter 10

Activation and Control of the Complement System

B.Paul Morgan 171

Chapter 11

Phagocytes in Immunity and Inflammation

Philip ISA. Murphy 197

Chapter 12

Anaphylaxis

Caiman Prussin and Michael Kaliner 231

Chapter 13

Autoimmunity and Autoimmune Disease

Sudershan K. Bhatia and Noel R. Rose 239

Chapter 14

Cell Death and the Immune System

R.M. Kluck and].W. Halliday 265

Chapter 15

Designer Antibodies

Andy Minn and Jose Quintans 281

Chapter 16

Psychoneuroimmunology

Ruth M. Benca 303

INDEX 315

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LIST OF CONTRIBUTORS

Ruth M, Benca

Sudershan K. Bhatia

Paul Bum

P.M. Colman

J.W. Halliday

Andrew J. Hapel

Michael Kaliner

R.M. Kluck

Department of Psychiatry

University of Wisconsin

Madison, Wisconsin

Department of Immunology and

Infectious Diseases

The John Hopkins University School of

Hygiene and Public Health

Baltimore, Maryland

Department of Biology

Hoffmann-La Roche Inc.

Nutley, Ne Jersey

CSIRO Division of Biomolecular

Engineering

Parkville, Victoria, Australia

Liver Unit

Queensland Institute for Medical

Research

Queensland, Australia

Experimental Haematology Group

John Curtin School of Medical Research

Australian National University

Canberra, Australian Capital

Territory, Australia

Institute for Asthma and Allergy

Washington, D.C.

Liver Unit

Queensland Institute for Medical

Research

Queensland, Australia

IX

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LIST OF CONTRIBUTORS

J.FA,P. Miller

Andy Minn

B. Paul Morgan

Philip M. Murphy

Tomas Mustelin

Ronald Penny

Caiman Prussin

Jose Quintans

Noel R. Rose

The Walter and Eliza Hall Institute of

Medical Research

Royal Melbourne Hospital

Melbourne, Victoria, Australia

Department of Pathology

The University of Chicago

Chicago, Illinois

Department of Medical Biochemistry

University of Wales College of Medicine

Heath Park, Cardiff, Wales

The Laboratory of Host Defenses

National Institute of Allergy and

Infectious Diseases

National Institutes of Health

Bethesda, Maryland

La Jolla Institute for Allergy and

Immunology

La Jolla, California

Centre for Immunology

St. Vincent's Hospital and University of

New South Wales

Sydney, New South Wales, Australia

National Institute of Allergy and

Infectious Diseases

National Institutes of Health

Bethesda, Maryland

Department of Pathology

The University of Chicago

Chicago, Illinois

Department of Immunology and

Infectious Diseases

The Johns Hopkins University School of

Hygiene and Public Health

Baltimore, Maryland

Page 12: Immunity

List of Contributors

H.S. Rothenfluh

William A. Sewell

E.I. Steele

David Tarlinton

Brian D. Tail

XI

Division of Immunology and Cell Biology

The John Curtin School of Medical

Research

Australian National University

Canberra, Australia

Centre for Immunology

St. Vincent's Hospital and University of

New South Wales

Sydney, New South Wales, Australia

Department of Biological Sciences

University of Wollongong

Wollongong, New South Wales, Australia

The Walter and Eliza Hall Institute of

Medial Research

Royal Melbourne Hospital

Melbourne, Victoria, Australia

Tissue Typing Laboratories

Royal Melbourne Hospital

Parkville, Melbourne, Australia

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As this volume demonstrates, immunobiology is a young science which is under-

going explosive growth. Judged by results, it is already an elaborate discipline

which cuts across every other area in biomedical research and even has its own

vocabulary (e.g., the effect). Rather than inculcate the habit of superficial

learning by having the student go through a maze of details, we have sought to

gather together sixteen essays that range from T-cells to psychoneuroimmunology.

This is in keeping with the growing understanding that the student is expected to

read and think far more for herselfhimself.

Next to nothing is known about innate immunity. However, recent evidence

suggests that collectins might bridge the gap between innate immunity and specific

clonal immune responses. Collectins are soluble effector proteins that include

serum mannose-binding protein, and lung surfactants A and D. They are considered

to be ante-antibodies.

Our most grateful thanks are due to the contributors who have made this volume

possible. They are also due to Ms. Lauren Manjoney and the production staff of

JAI Press for their skill and courtesy.

E. EDWARD BITTAR

NEVILLE BITTAR

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Chapter 1

The Thymus in Immunity

J.F.A.P. MILLER

Introduction 2

Historical Background 4

Antigen Recognition and the Major Histocompatibility Complex (MHC) 7

Peripheral T Cell Subsets 10 T Cell Migration 12

Recirculation of Naive T Cells 13

Tissue-Selective Homing of Activated and Memory T Cells 14 Intrathymic Events 15 The Thymus in Disease States 17 Summary 18 Recommended Readings 19

Principles of Medical Biology, Volume 6 Immunobiology, pages 1-20. Copyright © 1996 by JAI Press Inc.

All rights of reproduction in any form reserved.

ISBN: 1-55938-811-0

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2 J.F.A.P. MILLER

INTRODUCTION

The cells in the immune system responsible for specifically targeting and causing the removal of foreign material or antigen are known as lymphocytes. They circulate in blood and lymph and populate areas of the body known as lymphoid tissues which include the spleen, lymph nodes, thymus, tonsils, adenoids, and Peyer's patches, the last three being located along the alimentary tract.

The thymus in mammals is situated in the upper part of the thoracic cavity where it overlies the heart and some of the major blood vessels (Figure 1). It is unique among lymphoid tissues, both as regards structure and function. Relatively large in the infant, its maximum size is reached at the time of puberty, after which it regresses slowly, becoming reduced to little more than a vestigial structure in old age. It is divided into lobules each with a central part or medulla and a peripheral part or cortex (Figure 2). The main types of cells are the lymphocytes and the so-called stromal cells which include the cells of the epithelial framework and of the dendritic-macrophage lineages. T cell precursors (derived from fetal liver or later from bone marrow) enter from vessels at the cortico-meduUary junction and first associate with macrophages. Two or three days later they are found m the subcapsular cortex. They eventually give rise to more differentiated thymus lym­phocytes.

The cell composition of the thymus may be divided into three distinct layers. (1) In the outer cortex, beneath the capsule, is a layer of dividing primitive lymphocytes (lymphoblasts), which constitute 5 to 15% of the total thymic lymphocyte popula­tion. Some lymphoblasts interact with specialized epithelial cells, the "nurse cells," which promote their proliferation and differentiation to more mature smaller forms. (2) The newly derived lymphocytes migrate from the cortex towards the medulla. In the deep cortex are three major classes of cells: small lymphocytes, dendritic cortical epithelial cells and macrophages. The lymphocytes have a thin rim of cytoplasm, make up about 80 to 85% of the thymic lymphocyte population and are

Thyroid

Figure 1. The location of the thymus in the chest.

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The Thymus in Immunity

capsule

subcapsular blasts - c O .

cortical dendritic epithelial cell

medullary

epithelial

cells

MEDULLA

nurse cells

small cortical thymus lymphocytes

Q interdigitating O dendritic cells

medullary thymus lymphocytes

Figure 2. Structure of the thymus. Diagram to show cellular architecture of the

thymus (see text).

in intimate contact with the dendritic epithelial cells. These have long processes and are connected to one another by junctions known as desmosomes. They may be involved in selecting the T cell repertoire (see later and Figure 11). Interspersed among the network of dendritic epithelial cells are macrophages which engulf the many lymphocytes that have died or are destined to die. On the medullary side of the cortico-meduUary junction lie structures called Hassall's corpuscles which constitute the final graveyards for the massive numbers of dying lymphocytes. (3) The medulla contains medium sized thymic lymphocytes, macrophages, spatulate medullary epithelial cells and bone-marrow derived interdigitating dendritic cells. The latter are most conspicuous near the cortico-meduUary junction and are involved in negative selection of those lymphocytes which have the potential to inflict damage on the body's own tissues, the so-called self-reactive lymphocytes (see below). Some medullary mature T cells may be derived partly from the intrathymic maturation process and partly from extrathymic circulating T cells.

The proportion of lymphocytes undergoing cell division (mitosis) is much higher in the thymus than in any other lymphoid tissues throughout the life of the individual. Furthermore, thymus lymphocyte mitotic activity, unlike similar activ­ity elsewhere, is not dependent on antigenic stimulation but is preprogrammed and hence controlled intrinsically (from within the thymus).

During development, the thymus, unlike other lymphoid tissues, is a purely epithelial organ. Lymphocytes first appear in the epithelial network at about 10

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4 J.F.A.P. MILLER

weeks of gestation in the human and 12 days in the mouse. They are derived by differentiation of hemopoietic ancestral or stem cells which enter from the blood stream. It is only much later that lymphocytes make their appearance in other lymphoid organs. The thymus is thus often referred to as a primary or central lymphoid organ and the other lymphoid tissues as secondary or peripheral.

When animals are immunized by antigen, characteristic cellular changes occur in lymph nodes and spleen. For example, small lymphocytes enlarge to larger "blast" cells which stain with a particular RNA-staining dye (methyl green py-ronin). These undergo mitosis and antibody forming "plasma cells" accumulate in certain areas. None of these antigen-induced changes have ever been found in the intact thymus of immunized animals under normal conditions. These findings raised questions as to whether the thymus played any role in immunity.

HISTORICAL BACKGROUND

Prior to I960, the functions of the thymus and its lymphocytes were obscure. By contrast, the circulating small lymphocytes, as found in blood, lymph and lymphoid tissues, were proven to be immunologically competent by the work that Gowans and his collaborators performed in the late fifties and early sixties (Gowans, 1961). Yet although the thymus was known to be a lymphocyte-producing organ, immu-nologists did not consider it to have any immunological function. This may have been because some investigators, for example. Good and his collaborators (MacLean et al., 1957), concluded from experiments, in which the thymus was removed from adult rabbits, that they had obtained "evidence that the thymus gland does not participate in the control of the immune response." In the early sixties, Medawar (1963) even suggested that "we shall come to regard the presence of lymphocytes in the thymus as an evolutionary accident of no very great signifi­cance." What then was responsible for reversing the tide?

In the late fifties and early sixties, Miller, then working with a leukemogenic virus of mice, surgically removed the thymus (thymectomized) of newborn (neo­natal) mice to determine whether the virus, when introduced at birth, had first to multiply in thymus tissue. He found that neonatally thymectomized mice died prematurely from causes unrelated to leukemia induction and suggested "that the thymus at birth may be essential to life" (Miller, 1961a). Further experiments showed clearly that mice thymectomized at one day of age, but not later, were highly susceptible to infections, had a marked deficiency of lymphocytes in the circulation and in lymphoid tissues and were unable to reject skin grafts taken from incompatible mice of other strains (Miller, 196 lb). These results led to the hypothe­sis that "during embryogenesis the thymus would produce the originators of immunologically competent cells many of which would have migrated to other sites at about the time of birth. This would suggest that lymphocytes leaving the thymus are specially selected cells" (Miller, 1961b). In adult mice, thymectomy had for long been known not to have any untoward effects. Miller (1962a), however.

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The Thymus in Immunity 5

exposed adult thymectomized mice to total body irradiation which partially de­

stroyed the lymphoid system and was able to show that the recovery of lymphoid

and immune functions was thymus-dependent. Implanting thymus tissue into

neonatally thymectomized or adult thymectomized and irradiated mice allowed a

normal immune system to develop. When the thymus graft came from a foreign

strain, the neonatally thymectomized recipients failed to reject skin from mice of

the strain that had donated the thymus, although they could reject skin graft from

other incompatible strains. This led to the suggestion that "when one is inducing a

state of immunological tolerance in a newly born animal," for example by the

classical technique of injecting foreign bone marrow cells at birth (see Chapter 4),

"one is in effect performing a selective or immunological thymectomy" (Miller,

1962b). Thus, lymphocytes developing in the thymus in the presence of foreign

cells would be deleted, implying that the thymus should be the seat where tolerance

to the body's own tissues (self tolerance) is imposed. Some of these findings were

soon confirmed by groups working independently, notably those headed by

Waksman and by Good (Amason et al., 1962; Martinez et al., 1962).

In the late fifties and early sixties, only a single variety of lymphocyte was

believed to be involved in performing all types of immune responses in mammalian

species. In birds, however, it seemed that two distinct subsets of lymphocytes

performed those immune responses mediated by antibody (the "humoral" immune

responses) and those in which cells, but not antibody, were involved (the "cell-

mediated" immune responses). The latter include transplant rejection, delayed-

hypersensitivity reactions such as tuberculin sensitivity, and killing or "lysis" of

target cells. The finding of a division of labor among avian lymphocytes was first

reported by Szenberg and Warner (1962) using newly hatched chicks: surgical

removal of the bursa (an organ found only in birds and analogous to the thymus but

situated near the cloaca) soon after hatching was associated with defects in antibody

formation and early thymectomy with defects in cellular immune responses. Since

mice do not have a bursa and since neonatal thymectomy in that species prevented

both cellular and most humoral immune responses, it was widely believed that the

mammalian thymus fulfilled the fimctions of both the avian thymus and bursa. A

hint that two distinct lymphocyte subsets may indeed be involved in immune

responses in mice, however, came from the experiments of Claman and his

colleagues in 1966. They showed that irradiated mice receiving a mixed population

of marrow and thymus cells produced far more antibody than when given either

cell source alone. Having no genetic markers on their cells, they could not, however,

determine whether the antibody-forming cells were derived from the thymus or the

marrow. In independent investigations, (Miller and Mitchell, 1967,1968; Mitchell

and Miller, 1968) introduced genetically marked cells into neonatally thymecto­

mized or thymectomized irradiated hosts and established beyond doubt and for the

first time that antibody-forming cell precursors (subsequently known as B cells)

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6 J.F.A.P. MILLER

were derived from bone marrow, and that thymus-derived cells (now called T cells) were essential to help B cells to respond to antigen by producing antibody.

The existence of two distinct lymphocyte subsets, T and B cells, was not only confirmed but led to a re-investigation of numerous immunological phenomena including memory, tolerance, autoimmunity, and genetically determined unrespon­sive states. T cells were clearly responsible for the "cell-mediated" immunities, and T cells were themselves soon subdivided into subsets based on function, cell surface markers and secreted products or "lymphokines."

In 1957, prior to the discovery of T and B cells, Burnet postulated that lympho­cytes had predetermined reactivities. A cell with a receptor that best fitted a given antigenic determinant is selected by that antigen and activated to divide producing a clone of daughter cells, all with the same specificity (Figure 3). The antigen receptor on the membrane of these progeny cells would be identical in its binding site to the antibody eventually secreted by members of the clone. The theory has stood the test of time and for B cells, it was clear that the antigen recognition unit or receptor was an accurate sample of the antibody or immunoglobulin (Ig) which that cell would produce after successfiil antigenic stimulation. It was also found that a small proportion of naive B lymphocytes could specifically bind labeled antigen and that this binding could be blocked by antibody directed against the immunoglobulin receptor itself Yet T cells could never be shown to bind antigen

Clonal Selection

lymphocytes

antigen

lymphocyte-antigen

interaction

lymphocyte proliferation

and differentiation

clone

^^R ^ A ^ ^

antibody Jli\ k 1 i ' 'A^

Figure 3. Burnet's clonal selection theory. The antigen-specific receptor is unique on mature lymphocytes. A cell with a receptor into which a given antigenic determinant can be accommodated is selected by the antigen to divide and produce a clone of daughter cells, each with the same antigen specificity. In the case of B cells, as shown in this diagram, the membrane receptor is identical in its binding site to the antibody which members of the stimulated clone will eventually secrete.

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The Thymus in Immunity 7

and great controversy raged for many years over the nature of the antigen receptor

on T cells.

ANTIGEN RECOGNITION AND THE MAJOR HISTOCOMPATIBILITY COMPLEX (MHC)

Unlike B cells, T cells perceive, not naked antigen, but antigen presented on the

surface of other cells. Highly visible to T cells are molecules encoded by the major

histocompatibility complex (MHC), a series of genes which code for molecules on

the surface of a variety of cell types (see Chapter 7). They provoke violent rejection

reactions on the part of responding T cells and are perfect targets for killer or

cytotoxic T cells. Following virus infection and virus entry into cells, as first shown

by Zinkemagel and Doherty in 1974, T cells recognize not just the virus derived

antigenic determinants, but these in association with MHC-encoded molecules on

CLASS CLASS II

peptide groove peptide groove

^ 2 ^

Figure 4, Class I and class II MHC molecules. The class I molecule is composed of two polypeptide chains. The heavy chain has 3 external domains, a l , a2 and a3, a transmembrane portion (TM) and a cytoplasmic tail (CY). It is associated in its extracellular portion with the light chain, p2-microglobulin (p2m), a molecule not encoded by the MHC gene locus. The polymorphic regions of the class 1 heavy chain are those where the amino acid sequences of the polypeptide chain differ among unrelated individuals. They are situated in the a1 and a2 domains which form a groove that can accommodate peptide fragments derived from the processing of proteins synthesized within the cell (e.g., self proteins or virus-derived proteins in virus infected cells). The class II molecules are composed of two polypeptide chains, a and p, both encoded by the MHC gene locus. Each chain spans the membrane and hence has a transmembrane region, a cytoplasmic tail and an extracellular portion. Both the a and the p chains have two external domains, a1, a2, and p i , p2. The polymorphic regions lie in the a1 and pi portions which also form a groove into which can be accommo­dated peptides generally derived from proteins taken up by the cell from the external milieu (see also Figure 5). A separate class of antigens known as "superantigens'' (e.g., certain bacterial toxins) bind not to the groove of the class II molecules but to the external face of the domains and to the p chain of the TCR.

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8 J.F.A.P. MILLER

the cell surface. This phenomenon became known as MHC restriction and the MHC

molecules involved as restriction elements.

The MHC molecules that serve as targets of T cell responses occur in two major

forms, termed class I and II (Figure 4). The former are found on most tissue cells

and are composed of two noncovalently linked polypeptide chains—SL heavy one

(molecular weight 45 kD) spanning the cell membrane and having three extracel­

lular portions or domains (a l , a2 and a3), and a lighter chain termed p2-micro-

globulin. This does not span the membrane and is encoded by a gene distinct from

the MHC genes. The class II molecules consist of two noncovalently linked a (28

kD) and P (34 kD) chains, both encoded by the MHC and both having two

extracellular domains. The distribution of class II molecules is restricted mostly to

B cells, macrophages, and dendritic cells. Both class I and II molecules exhibit a

striking degree of structural variation or polymorphism within individuals of the

same species. The polymorphic regions of the molecules, where there are differ-

Endogenous pathway

r degradation

I proteins

peptides O

class I

\ \

endogenous synthesis

Exogenous

pathway

JL

r [o]*-(c

t class 11

O**—O exogenous antigen

endocytosis

Figure 5. Antigen-presenting cell (APC) and processing pathways. Professional ARC

present processed antigen in association with MHC class I and ii molecules. Two

pathways of antigen processing operate: they are referred to as endogenous and

exogenous. (1) Some proteins synthesized by the APC are chopped into fragments

(degraded into peptides) by cellular enzymes. Most newly synthesized class I mole­

cules are unstable unless peptide is associated with them. The binding of peptides to

MHC class I molecules occurs in an intracellular compartment known as the endoplas­

mic reticulum and the peptide-MHC complex can then be transported to the surface.

This particular route is known as the endogenous pathway. (2) External antigens taken

up by the APC ("endocytosis") are degraded In compartments known as endocytic

vesicles which fuse with other vesicles that contain class ii but not class I molecules.

This type of transport is referred to as the exogenous pathway.

Page 24: Immunity

The Thymus in Immunity 9

ences in amino acid sequences among unrelated individuals, are situated in the a l

and a2 domains of the class I molecules and the a l and pi domains of the class II

molecules. These domains form a groove or pocket capable of binding fragments

derived from the enzymatic degradation or processing of self or foreign components

(Bjorkman et al., 1987; Brown et al., 1993). Such fragments derived from protein

antigens are known as peptides and are made up of short sequences of amino acids

with a carboxyl end or "terminus" and an amino terminus. Cells which perform the

processing task and transport the peptide-MHC complex to their cell membrane

where T lymphocytes can examine them, are termed professional antigen-present­

ing cells (APC) (Figure 5).

The antigen-specific receptor on T cells (the "TCR") has specificity for both the

peptide and the external surface of the MHC molecule which accommodates the

peptide (Davis and Bjorkman, 1988). Most TCRs are composed of two disulfide-

linked polypeptide chains, a and p (Figure 6), although less common TCR use other

chains termed y and 5. Each chain has a constant amino acid sequence in its carboxyl

terminus (C) and a variable sequence in its amino terminus (V). Other molecules

intimately associated with the TCR are the CD3 complex composed of three

polypeptide chains (y, 5, and s) and the so-called q-q "homodimer" composed of a

pair of identical polypeptide chains. The CDS and q-q molecules are essential for

TCR

extracellular

membrane

cytoplasm

Figure 6. The antigen-specific T cell receptor (TCR) and associated CD3 and q-q

complexes. The antigen-specific TCR is composed of two disulfide-linked polypeptide

chains, a and |3. Each chain has a constant amino acid sequence in its carboxyl

terminus (C) and a variable sequence in its amino terminus (V). The CD3 complex,

composed of three polypeptide chains y, 6, and 8, and the homodimer q-q are

intimately associated with the TCR and are involved in TCR assembly and signal

transduction when the TCR has bound a peptide-MHC complex.

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10 J.F.A.P. MILLER

the assembly and transport to the cell surface of the TCR and play a role in transducing signals after occupation of the TCR by a peptide-MHC complex.

The TCR chains are encoded by several genes which rearrange during T cell development and contribute to the great diversity of specificities associated with TCRs (Davis and Bjorkman, 1989). This is described in detail in Chapter 5. Briefly, individuals inherit from their parents sets of "germline genes" which code for the combining site of antigen-specific receptors on both T and B cells. A variety of mechanisms then operate during T and B cell differentiation to rearrange and join together the germline elements and eventually give rise to the active gene which is a mosaic of these units. Hence, an enormous diversity can be generated and a great variety of antigen-specific receptors is made available to ensure that lymphocytes can recognize an infinite number of antigenic determinants.

PERIPHERAL T CELL SUBSETS

Although almost all T cells bear the Thy-1 marker, they are heterogeneous with respect to function and other cell surface markers. The two major subsets of T cells are termed CD4'" and CD8" T cells (Figure 7). The former are characterized by the presence on the membrane of the CD4 molecule and act as "helper" cells by assistmg B cells in producing certam types of antibody. The collaboration between T and B cells is described in Chapter 3, The CDS" T cells have CDS molecules on

Figure 7. The CD4 and CDS co-receptor molecules on T cells. The CD4 and CD8 molecules characterize mature peripheral T cells which recognize peptides m asso­ciation with MHC class II and class I molecules, respectively. The CD8 molecule has an affinity for specific sites on the a3 domain of the class I molecule and the CD4 for some sequences on the nonpolymorphic portion of the class 11 molecule.

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The Thymus in Immunity 11

their surface and, after direct contact with their target cells, act as killer or cytotoxic cells destroying foreign cells and cells infected by viruses. In some situations CDS" T cells require help from 004"" T cells for cytotoxic activity. The CD4 and CDS molecules are "coreceptors" as they act m concert with the TCR. The CD4 co-receptor has a binding site specific for a portion of the MHC class II molecule and the CDS co-receptor has one specific for a part of the a3 region of the class I molecule. Co-aggregation of CD4 or CDS molecules with the CD3-(;-c; complex and the TCR, once bound to its specific peptide-MHC complex, initiates a signaling cascade to "activate" the T cell, turning on its functional and lymphokine-secretion machinery (Janeway, 1992). T cell-derived lymphokines control the differentiation of a wide variety of cells of the hemopoietic and lymphoid systems and are active in initiating inflammatory responses such as delayed-type hypersensitivity.

CD4' T helper (Th) cells are themselves heterogeneous in terms of their lym-phokine release pattern. Although the naive CD4 cell can synthesize a variety of lymphokines immediately after activation, the way in which antigen is presented by different cells eventually restricts the secretion pattern. Thus Th cells can be divided into ThO (which can secrete various lymphokines) and into further differ­entiated forms known as Thl and Th2 cells (Mosmann and Coffman, 1989). Although both these cells can secrete the lymphokines IL-3, GM-CSF and TNF-a, they differ in their pattern of release of other lymphokines. Thus, Thl cells produce interferon-y and interleukin-2 while Th2 cells secrete interleukin-4, 5, 6, 10, and 13. Thl and Th2 can antagonize each other thus playing a role in immunoregulation (see also Chapters 4 and 9).

Under certain conditions, T cells can suppress the responsiveness of other lymphocytes. Whether such "suppressor T cells" exist as a distinct subset or reflect the production of inhibitory cytokines, such as TGF-(3 (A. Miller et al., 1992), has yet to be established.

T cells can also be subdivided according to their previous antigenic experience. Those which have not met antigen are termed "naive" or "virgin" cells and are characterized by the presence of distinct molecules on their surface. For example, they express the high molecular weight forms of the CD45 molecule (notably CD45RA), low levels of the molecule known as CD44 and high levels of L-selectin (also called MEL-14). Those T cells which have been stimulated by antigen are the progeny of naive T cells and are large "blasts" known as effector or activated T cells. They may become small "memory" cells whose existence may depend on continuous antigenic stimulation. Both activated and memory T cells exhibit on their cell membrane the CD45RO molecules, high levels of CD44, low levels of L-selectin and various adhesion molecules such as LFA-1 and CD2. All these molecules are involved in various T cell functions including intracellular signaling, adhesion to APC or to cells lining blood vessels ("endothelial cells") (Mackay, 1993; Sprent, 1993). Some of the molecular interactions occurring between specific T cells and professional APC are shown in Figure S.

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12 J.F.A.P. MILLER

APC or

target cell

Figure 8. Adhesion and co-stimulatory molecules involved during the interaction of T cells with TCR specific for an MHC-peptide complex presented by a professional APC. Cell surface molecules expressed on T cells may play a role in immune responses by functioning as receptors for cell surface molecules expressed on APC. The interaction of such molecules may strengthen the binding between the T cell and the APC and may be Involved in transmembrane signals initiated by TCR occupancy or independent of the TCR. The B7 molecule is characteristically expressed by profes­sional APCs and its binding to the T cell's CD28 molecule produces a powerful co-stimulator signal to ensure that the T cell becomes fully activated following the binding of its own TCR to the MHC-peptide complex presented by the APC.

As stated above, most T cells utilize TCR a and P genes but a smaller subset use

the genes y and 6. Some 76 cells exist in certain epithelial environments and, unlike

aP T cells, exhibit a highly restricted TCR specificity. They may thus express

invariant TCRs and perform totally different tasks. Much remains to be learned

about their functions.

T CELL MIGRATION

The total number of cells released from the thymus is small being of the order of 1

to 2 million per day in young mice. Output is maximal at an early age and declines

when thymic atrophy sets in, reaching very low levels in old age. The cells leaving

the thymus are typical CD4^ and CDS"*" T cells which may have to undergo a further

period of maturation during several days. The emigrants migrate non-randomly

along well defined routes, the actual pathway depending on whether the T cells are

naive or activated.

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The Thymus in Immunity

Recirculation of Naive T Cells

13

Naive small T cells recirculate from blood through lymphoid tissues and back to

blood directly or via the lymph. They have a long lifespan and do not divide unless

stimulated by antigen. Recirculation allows naive T cells to patrol the body and

home in on sites in lymph nodes and spleen which have trapped antigens and

invading micro-organisms.

Naive T cells have specialized receptors ("L-selectin") which allow them to bind

to distinct molecules on the surface of endothelial cells lining specialized venules

in the lymph nodes known as "post-capillary" or "high endothelial" venules (HEV)

(Figure 9). They then enter lymph nodes through a region known as the paracortex

which contains a network of specialized APCs including the so-called dendritic

cells. This area of the node is known as the T-cell dependent area and antigens

which provoke cellular immunity produce histological changes in this area, the

small T cells enlarging to large blasts which divide. Other areas of the lymph nodes,

including the follicles are known as the B-cell dependent areas (see Chapter 2).

After traversing the paracortex, the recirculating T cells enter the medulla of the

nodes and leave by efferent lymphatics which drain into other lymph nodes or end

up in the thoracic duct. This large vessel empties into a major blood vessel in the

neck.

The spleen is divided into red and white pulps (Figure 10), the former containing

many hemopoietic cells such as red blood cells, the latter being populated by

lymphocytes. The spleen does not have significant lymphatics and T cells enter via

the splenic artery which terminates in a loose network of vessels in the red pulp.

The T cells migrate to the area of the white pulp around the arterioles (the

"periarteriolar lymphocyte sheath" or PALS), a T-cell dependent area rich in APCs

cortex

medulla

afferent lymphatic

subcapsular sinus

primary follicle (B area)

medullary cord (B area)

medullary sinus

efferent lymphatic

Figure 9, Microanatomy of a lymph node (see text).

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14 I.F.A.P. MILLER

red pulp

marginal

sinus

central

arteriole

primary

follicle (B area)

marginal

zone (B area)

t

Figure 10. Microanatomy of a section of the spleen (see text).

including dendritic cells, and the B cells migrate to the follicles in the white pulp.

T cells leave the spleen by going to the red pulp and entering tributaries of the

splenic vein.

If recirculating T cells encounter antigen presented by APCs in the T-cell

dependent areas of lymph nodes or spleen, those T cells with TCR specific for the

antigen are sequestered from the circulating pool and activated to proliferate and

to produce effector cells which perform the cell-mediated immune responses

(Sprent et al., 1971).

Tissue-Selective Homing of Activated and Memory T Cells

T cells homing to the alimentary canal ("gut-tropic cells") are either activated

blasts or smaller CD45RO"^ memory-type cells. After antigen activation, T cells

downregulate the expression of L-selectin which was present on naive T cells and

instead express the a4(37 adhesion molecule ("integrin"). This allows them to bind

to specific molecules on endothelial cells found in gut mucosa and gut-associated

lymphoid tissues and thus to enter such tissues.

The skin represents a major entry point for microorganisms. T cells that home to

the skin are almost exclusively of the memory type and express the a4pi integrin.

This serves as an adhesion receptor for the molecule E-selectin which is found in

inflamed skin.

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The Thymus in Immunity 15

Memory-type T cells predominantly localize to inflamed sites. The inflammatory response can also affect lymph nodes and antigen stimulation induces the expres­sion of defined vascular adhesion molecules on the endothelial cells of HEV. This in turn allows a marked increase in the migration of memory-type T cells to the node.

The different migration behavior of naive and effector or memory T cells ensures that the immune system provides a most economical way of displaying its resources to fight against dangerous intruders.

INTRATHYMIC EVENTS

Most T cells arise in the thymus as a result of the programmed differentiation of incoming stem cells. There is little evidence for extra-thymic production of T cells. Since T cells are specialized to recognize antigenic determinants in association with self MHC molecules, the thymus must provide a repertoire of T cells by selecting those cells with TCR that have some degree of specificity for these MHC molecules. But because TCR specificities are randomly generated and there is extensive MHC polymorphism among individuals of a particular species, the specificities of T cells in the preselected pool of differentiating thymus lymphocytes must by chance be directed to all the MHC molecules expressed in the species. Most thymocytes will therefore lack the correct specificity and hence will be unsuitable. This presumably accounts for the vast numbers of thymocytes generated each day (about 10^ in mice), the massive rate of cell death and the small number exported to the periphery (about 10^ per day).

The earliest T cell precursor derived from stem cells entering the thymus is characterized by the surface expression of the molecule CD44 and very low levels of CD4. Soon after, these early cells lose the CD4 marker and transiently express, in addition to CD44, the CD25 molecule (which is a receptor for the interleukin, IL-2). At this stage, the cells are referred to as "double negative (DN) cells" because they lack expression of both CD4 and CDS which characterize mature T cells. The DN cells then lose CD44 expression and begin to rearrange and express the (3 chain genes of the TCR. This is followed by rearrangement of the TCR a locus, expression of low levels of the aP TCR on the surface and loss of CD25. A separate subset of DN cells rearrange the y and 8 locus of the TCR to express a y8 TCR.

The early thymocytes account for less than 3% of the cells in a mouse thymus. They proliferate extensively in the subcapsular cortical zone presumably as a result of interacting with cortical epithelial cells which produce a number of factors or "cytokines" influencing cell proliferation. The DN cells then migrate to the deeper cortex. A few can go out to the periphery without expressing the CD4 or CDS coreceptor molecules, but most give rise to cells that express both CD4 and CDS (termed double positive cells) and low to intermediate levels of the TCR. They are now subjected to stringent selection tests (Figure 11).

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16 J.F.A.P. MILLER

Random expression Positive selection Mature T of ap TCR genes by MHC molecules Negative selection cell pool

towa/S high a/S high a/S TCR expression TCR expression TCR expression

cor t ica l t dendritic ceHs

epi the l ium macr^hages

precursor low TCR a/? ^ (7)<

I medullary epithefium

J

N i f 4* j < no interaction — • T 4* J<

(^ no engagement > programmed cell death

Figure 11. Differentiation of thymus lymphocytes and repertoire selection. Pre-T cells entering the thymus from the circulation pass through a number of differentiation steps that lead either to death or to maturity. The diagram shows the stages at which the cells express the TCR, acquire or lose the coreceptors CD4 and CDS and are subjected to positive and negative selection forces. The timing of the selection processes depends on a variety of factors including the combined avidity of the TCR and coreceptors for the presented antigen and the intra-thymic localization of the selecting cells (see text).

Positive selection ensures that T cells expressing TCRs that have some degree of binding for polymorphic regions of the MHC molecules displayed on cortical epithelial cells are selected for survival. The binding is presumed to protect the cells from "programmed cell death" vv hich affects the bulk of the double positive thymocytes that express TCRs unable to bind to MHC molecules expressed intrathymically. Positive selection by MHC class I or II molecules involves concomitant engagement of either CDS or CD4, respectively, and dow^nregulation of the unengaged coreceptor. The single positive cells express high levels of TCR and are allowed to emigrate if they pass the negative selection test (see belovv ). Positive selection is thus a mechanism that allows the CD8" T cell to respond to peptides associated with the individual's own self MHC class I molecules, and the CD4' T cell to recognize peptides complexed with self class II molecules. It is therefore the basis of the phenomenon of MHC restriction, but it does not prevent the differentiation of cells with high affinity receptors for self peptides presented in association with MHC molecules. A negative selection mechanism is therefore required.

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The Thymus in Immunity 17

Developing T cells expressing TCRs able to bind strongly to peptide-MHC complexes presented by cells of the dendritic-macrophage lineages or by some medullary epithelial cells are deleted. This negative selection process ensures that T cells will not react to self antigens, at least to those existing in the thymus. It is thus essential for the induction of self-tolerance (Chapter 4). The timing and exact intrathymic site of this process depends on many factors among which are the accessibility of differentiating T cells to self-antigens, the combined avidity (bmd-ing strength) of the TCR and coreceptors for the self-MHC-peptide complexes and the intra-thymic location of the deleting cells.

If both positive and negative selection involve recognition by the TCR of the same self peptide-MHC complexes, how do all T cells avoid elimination? Several possibilities have been suggested to explain this paradox. For example, low affinity interactions of the TCR with self peptide MHC complexes may be sufficient to trigger positive selection but insufficient to induce deletion. Furthermore, T cells at different stages of maturation or in different intrathymic locations ("microenvi-ronments") may exhibit a difference in the structure of their TCR which does not affect antigen specificity but does influence the outcome of intracellular signaling. It is also clear that while high affinity interactions between the TCR and antigen leads to negative selection in the thymus, such interactions in fully mature T cells outside the thymus can lead to activation and a powerful immune response.

THE THYMUS IN DISEASE STATES

Congenital immune deficiency states arise when the thymus does not develop normally during gestation as, for example, in the diGeorge syndrome. Deletion or mutations affecting the gene coding for the enzyme, adenosine deaminase (ADA), are associated with severe immune deficiencies. ADA is an important enzyme in the purine catabolic pathway. If it is lacking, the normal catabolism of purines does not occur and this leads to the accumulation of metabolic products that are especially toxic to lymphocytes. Defective DNA repair and anomaly of the recom-binase enzymes essential for the rearrangement of genes which code for the T and B cell antigen-receptors, will also result in severe immunodeficiencies.

Malignancy of various types can arise in the thymus, either from the epithelium when they are termed thymomas, or from the lymphocytes as in acute lymphoblastic leukemia or lymphoma. The virus, HTLV-1, infects and causes malignant transfor­mation of mature T cells. It is the cause of some adult T cell leukemias.

The human retrovirus, HIV, is the cause of Acquired Immunodeficiency Syn­drome or AIDS. The CD4 molecule is the receptor for virus entry into cells and hence CD4" T cells are the principal targets for the virus. As CD4 is also present on monocytes, macrophages, dendritic cells and some brain cells (microglia), some of these cells also become infected and act as reservoirs for the virus. There follows a relentless loss of CD4' T cells and disruption of the architecture of lymph nodes

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18 J.F.A.P. MILLER

and of the thymus, itself. This leads to extreme susceptibility to infections with other micro-organisms.

Autoimmune diseases often arise as the result of the activation of T cells that have the potential to respond to autoantigens as described in Chapter 4.

SUMMARY

The thymus is a lymphoid organ which differs from other lymphoid tissues such as spleen and lymph nodes both with regards to structure and function. It is within the thymus that T lymphocytes differentiate from incoming stem cells, express genes which code for the specific antigen receptor (TCR), and are subjected to stringent selection procedures. These ensure that the mature T cell that emigrates from the thymus will not be able to react strongly to the body's own tissues and will recognize and be activated by foreign antigenic determinants only if these can associate with cell surface molecules encoded by the major histocompatibility complex (MHC) presented on the surface of so-called "professional" antigen-presenting cells (APC). Two distinct subsets of mature T cells exist as distinguished by the expression of the co-receptor molecules CD4 and CDS, the CDS'*" T cells able to perceive antigen in association with MHC class I molecules and the CD4" T cells recognizing antigen complexed with class II molecules. The migration of T cells follows well-defined routes, naive (non-activated) T cells recirculating as non-dividing cells from blood through certain "T-cell dependent" areas of the lymphoid tissues, to lymph and back to blood. Once successfully stimulated by antigen, naive T cells become effector or memory T cells, can secrete products known as lymphokmes, perform immunoregulatory and cytotoxic functions and express on their surface distinct molecules allowing them to migrate into nonlym-phoid tissues and into areas where inflammation has occurred. Immune deficiency diseases occur when the thymus or the T cells derived from it either fail to develop normally or are the targets of infection by various retroviruses, such as HIV which causes AIDS.

REFERENCES

Amason, B.G., Jankovic, B.D., & Waksman, B.H. (1962). Effect of thymectomy on "delayed"

hypersensitive reactions. Nature 194, 99-100.

Bjorkman, P.J., Saper, M.A., Samraoui, B., Bennett, W.S., Strominger, J.L., & Wiley, D.C. (1987).

Structure of the human class I histocompatibility antigen, HLA-2. Nature 329, 506-511.

Brown, J.H., Jardetzky, T.S., Gorga, J.C, Stem, L.J., Urban, R.G., Strominger, J.L., & Wiley, D.C.

(1993). Three-dimensional structure of the human class II histocompatibility antigen HLA-DRl.

Nature 364, 33-39.

Burnet, F.M. (1959). The Clonal Selection Theory of Acquired Immunity. Cambridge University Press,

New York.

Claman, H.N., Chaperon, E.A., & Triplett, R.F. (1966). Thymus-marrow cell combinations—synergism

in antibody production. Proc. Soc. Exp. Biol. Med. 122, 1167-1171.

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The Thymus in Immunity 19

Davis, M.M., & Bjorkman, P.J. (1988). T-cell antigen receptor genes and T-cell recognition. Nature

334, 395-402.

Gowans, J.L. (1961). The immunological activity of lymphocytes. In: Biological Activity of the

Leucocyte. Ciba Fdn. Study Group (Wolstenholme, G.E.W. and O'Connor, M., Eds.), pp. 32-44.

Churchill, London.

Janeway, C.A. (1992). The T cell receptor as a multicomponent signalling machine: CD4/CD8

co-receptors and CD45 in T cell activation. Ann. Rev. Immunol. 10, 645-674.

Mackay, C.R. (1993). Homing of naive, memory and effector lymphocytes. Curr. Opinion Immunol. 5,

423-427.

MacLean, L.D., Zak, S.J., Varco, R.L., & Good, R.A. (1957). The role of the thymus in antibody

production: An experimental study of the immune response in thymectomized rabbits. Transpl.

Bull. 41, 21-22.

Martinez, C, Kersey, J., Papermaster, B.W., & Good, R.A. (1962). Skin homograft survival in

thymectomized mice. Proc. Soc. Exp. Biol. Med. 109, 193-196.

Medawar, P.B. (1963). Discussion after Miller, J.F.A.P., & Osoba, D. The role of the thymus in the

origin of immunological competence. In: The Immunologically Competent Cell, Ciba Fdn. Study

Group (Wolstenholme, G.E.W. & Knight, J., Eds.), p. 7. Churchill, London.

Miller, A., Lider, O., Roberts, A.B., Spom, M.B., & Wiener, H.L. (1992). Suppressor T cells generated

by oral tolerization to myelin basic protein suppress both in vitro and in vivo immune responses

by the release of transforming growth factor p after antigen-specific triggering. Proc. Natl. Acad.

Sci. USA 89, 421-425.

Miller, J.F.A.P. (1961a). Analysis of the thymus influence in leukaemogenesis. Nature 191, 248-249.

Miller, J.F.A.P. (1961b). Immunological function of the thymus. Lancet 2, 74^749.

Miller, J.F.A.P. (1962a). Immunological significance of the thymus of the adult mouse. Nature 195,

1318-1319.

Miller, J.F.A.P. (1962b). Effect of neonatal thymectomy on the immunological responsiveness of the

mouse. Proc. Roy. Soc. London 156B, 410-428.

Miller, J.F.A.P., & Mitchell, G.F. (1967). The thymus and the precursors of antigen-reactive cells.

Nature 216, 659-663.

Miller, J.F.A.P., & Mitchell, G.F. (1968). Cell to cell interaction in the immune response. I. Hemolysin-

forming cells in neonatally thymectomized mice reconstituted with thymus or thoracic duct

lymphocytes. J. Exp. Med. 128, 801-820.

Mitchell, G.F., & Miller, J.F.A.P. (1968). Cell to cell interaction in the immune response. II. The source

of hemolysin-forming cells in irradiated mice given bone marrow and thymus or thoracic duct

lymphocytes. J. Exp. Med. 128, 821-837.

Mosmann, T.R., & Coffman, R.L. (1989). THl and TH2 cells: Different patterns of lymphokine

secretion lead to different functional properties. Ann. Rev. Immunol. 7, 145—173.

Sprent, J. (1993). Lifespans of naive, memory and effector lymphocytes. Curr. Opinion Immunol. 5,

433-438.

Sprent, J., Miller, J.F.A.P., & Mitchell, G.F. (1971). Antigen-induced selective recruitment of circulat­

ing lymphocytes. Cell. Immunol. 2, 171-181.

Szenberg, A., & Warner, N.L. (1962). Dissociation of immunological responsiveness in fowls with a

hormonally arrested development of lymphoid tissue. Nature 194, 146-147.

Zinkemagel, R.M., & Doherty, D.C. (1979). MHC-restricted cytotoxic T cells: Studies on the biological

role of polymorphic major transplantation antigens determining T cell restriction-specificity

function and responsiveness. Adv. Immunol. 27, 51-177.

RECOMMENDED READINGS

Boyd, R.L., & Hugo, P. (1991). Towards an integrated view of thymopoiesis. Immunol. Today 12, 71-79.

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20 J.F.A.P. MILLER

Kelso, A. (1989). Cytokines: Structure, function, and synthesis. Current Opinion Immunol. 2,215-225. Klem, J., & Nagy, Z.A. (1982). MHC restriction and Ir genes. Adv. Cancer Res. 37, 233-317. Miller, J.F.A.P. (1992). The Croonian Lecture. The key role of the thymus in the body's defence

strategies. Phil. Trans. Roy. Soc. 337B, 105-124.

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Chapter 2

The B-Cell in Immunity

DAVID TARLINTON

Introduction 22

B Cell Development in the Bone Marrow 23 B Cell Migration 30

B Cell Responses to Antigen 31

T Cell Dependent Responses 33 A Secondary Response 37

T Cell Independent Responses 38 Other Types of B Cells 39 B Cell Deficiencies 39

X-Linked Agammaglobulinemia (X-LA) 39 X-LA with Hyper-IgM 41 Selective Immunoglobulin Isotype Deficiencies 41

Common Variable Immunodeficiency (CVID) 42 B-Cell Development and Function Analyzed by Gene Targeting 42

Summary 44

Recommended Readings 45

Principles of Medical Biology, Volume 6 Immunobiology, pages 21-45. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-811-0

21

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22 DAVID TARLINTON

INTRODUCTION

The role of B cells in the immune system is to secrete antibody molecules in response to stimulation by antigen. B cells must recognize and respond to a potentially infinite number of foreign antigens while simultaneously ignoring self. The initial receptor diversity necessary to achieve this is generated through the immunoglobulin gene recombination process itself. This process, acting through the early stages of B cell development in the absence of antigen, generates what can be considered the primary B cell repertoire. This repertoire is then purged of reactivity to self and the remaining B cells are allowed to enter the peripheral lymphocyte pool where they are available to respond to exogenous antigen. When a B cell encounters its cognate antigen (that is, an antigenic determinant to which the immunoglobulin molecule binds with sufficiently high affinity) it will differ­entiate along one of three interrelated pathways. The most straightforward is for the responding B cell to terminally differentiate into a specialized antibody secret­ing cell called a plasma cell. Alternatively, the antigen specific B cell may enter a unique and transient structure called the germinal center wherein the affinity of the B cell's immunoglobulin molecule for antigen is selectively increased through the process of somatic mutation. The B cells of enhanced affinity which exit the germinal center appear to have two choices open to them. They may either become plasma cells and secrete their "improved" antibody as part of the primary humoral response or they may become memory B cells. As the name implies, memory B cells are a reservoir of cells expressing high affinity immunoglobulin molecules to previously encountered antigens. The purpose of such a population of cells is to provide a rapid and efficient response to an antigen upon subsequent re-exposure. Somewhat paradoxically, however, it appears that the persistence of memory B cells may itself depend on the persistence of antigen. The formation of antigen specific memory cells forms the basis of vaccination programs designed to elicit humoral responses. The main stages of B cell development and differentiation are shown in Figure 1. An additional feature of B cell development overlayed on the events outlined above is the phenomenon of isotype switching, whereby the constant region of an immunoglobulin molecule is exchanged. This exchange can lead to an array of different effector functions being associated with the same antigenic specificity.

The purpose of this chapter is to provide an overview of B cell function and, as such, to describe the events of B cell development both in the absence and presence of antigen. The stages of B cell development in the bone marrow are described and, where applicable, correlated with the molecular events of immunoglobulin gene rearrangement. Particular attention is paid to the phenotypic subsets and to the role of growth factors in early B cell development. The migration and population dynamics of newly formed B cells are described as are the selective processes which may operate to determine whether a particular B cell persists in the periphery or not. The differentiation of B cells induced by exposure to antigen is also described.

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The B'Cell in Immunity 23

Pro-B Pre-B Virgin B

0*0*0 self reactive

self reactive

unseiected (?)

Memory Bceii

o

t

Plasma cell

Figure / . A schematic representation of the life cycle of a B cell. B cells develop in

the absence of foreign antigens in the bone marrow where any self-reactive B cells

are deleted (indicated by the cross). Upon leaving the bone marrow, only a fraction

of the B cells is recruited into the recirculating B cell pool. This recruitment may be

the result of positive selection. B cells which react with self antigens in the periphery

are deleted, as are B cells which do not gain access to the peripheral pool. When a B

cell is exposed to antigen in the presence of T cell help, it either terminally differen­

tiates into an antibody secreting plasma cell or enters a germinal center. Within the

germinal center, the affinity of the antibody for antigen is improved by somatic

mutation and selection. Cells which exit the germinal center either become plasma

cells or long lived memory B cells.

especially the events associated with the generation of memory B cells. Finally,

consideration is given to the immunological basis of several B cell deficiencies.

B CELL DEVELOPMENT IN THE BONE MARROW

The purpose of B cell development is to produce a pool of circulating B cells which

is capable of recognizing and responding to foreign antigens in the appropriate

circumstances and which will not recognize and/or respond to self antigens.

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24 DAVID TARLINTON

Examination of the stages and events of B cell development in the bone marrow reveal how evolution has produced a system which achieves this goal.

B cells develop by the ordered differentiation of an uncommitted precursor cell. It is now widely accepted and well supported by experimentation that in higher animals all hematopoietic lineages (such as monocytes, lymphocytes, erythrocytes etc.) arise from a common precursor cell population whose members are called stem cells (Spangrude et al., 1988). These cells are thought to be self-renewing with each stem cell having the ability to give rise to progeny cells in any and possibly all hematopoietic lineages. As the progeny of a given stem cell differentiate, however, they become increasingly restricted in the cell lineages to which they can contribute (Figure 2). Evidence for this comes from the analysis of single bone marrow precursor cells cultured in vitro and allowed to differentiate by altering the culture conditions. By examining the mature cell types which develop in such clonal cultures, common precursor cells for different lineages can be inferred as can the activity of potential growth and differentiation factors. Similarly tagging experi­ments where stem cells are individually "labeled" with a genetic tag—for example, a retroviral insertion—^and then used to reconstitute the hematopoietic system of recipients such as lethally irradiated animals have also revealed relationships between lineages. The different lineages which develop in such a recipient can be examined for the genetic tag they contain. If T and B cells showed identity of a viral insertion site, for example, which was distinct from other lineages, this would indicate a common precursor cell for the T and B lineages. In such a way one can formulate relationships between the various hematopoietic lineages.

At one time there was thought to be a lymphoid stem cell which could replenish both the B and T lymphocyte populations but could not contribute to the myeloid lineage. Persistent searching for such a cell has so far been unsuccessful, leading most researchers to think that it probably does not exist. Conversely, there is a myeloid stem cell which cannot give rise to lymphocytes, but can reconstitute macrophages, monocytes, neutrophils, and so forth. The lineage relationships shown in Figure 2 should not be considered as absolute since there are still many things which are unknown or at the least unclear. For example, it has been shown that in the fetal liver of mice (in both human and murine development, the liver is a major organ of hematopoiesis in the fetus) a precursor cell exists which can give rise to both macrophages and B cells (Cumano et al., 1992), suggesting that the lineage relationships shown in Figure 2 may be something of a simplification. They do, however, provide a useful framework for discussing lymphocyte development.

Commitment to the B cell lineage is first apparent by the expression of surface markers known to be restricted to and expressed on all B cells. The subsequent stages of B cell development can be monitored by the successive acquisition and/or loss of various cell surface markers and growth factor receptors, both lineage specific and not. These changes are shown schematically in Figures 3 and 4. Surprisingly, since it represents the whole point of B cell development, the

Page 40: Immunity

The B'Cell in Immunity 25

STEM CELLS CX)MMITED PROGENITORS MATURE CELLS

T-Lymphocyt*

B-Lymphocyt* /Ptatma o«U

Erythrocyte

M*gakaryocyt«

Basophil /Mast call

Eosinophil

Nauuophll

Monocyte/ Macrophaga/ Kupffar call Langarhans call ate

Ostaodast

YOLK SAC FOETAL UVER BONE MARROW PERIPHERAL TISSUES BLOOD

Figure 2. All hematopoietic cells are derived from a common stem cell. A pluripotent stem cell is thought to give rise to lineage specific stem cells u/hich in turn give rise to the various terminally differentiated cell types v^hich appear in the blood. B cells although closely related to T cells probably do not have a common precursor. There are also reports consistent with B cells and macrophages being more closely related than is indicated in this figure (see text).

appearance of immunoglobulin molecules on the cell surface is quite a late event.

Although a number of nomenclatures exist for describing the stages of B cell

development, in the one used here the earliest B lineage cells are referred to as pro-B

cells which give rise to pre-B cells and subsequently immature or virgin B cells.

The immature B cell population is the first to express a fully assembled immuno­

globulin molecule on the cell surface. Immature B cells subsequently leave the bone

marrow and give rise to and maintain the recirculating B cell population. Further

distinctions between these various B cell subsets are described below.

The earliest identifiable B lineage compartment is that of the pro-B cell. In the

mouse this population is phenotypically defined by the presence of the pan-B cell

marker B220, low levels of the adhesion molecule leukosialin (CD43) and c-kit,

receptor for a growth factor called stem cell factor (SCF). These cells represent

around 3% of the nucleated cells in the marrow of a young adult (see for example

Hardy et al., 1991). In humans the corresponding population probably expresses

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26 DAVID TARLINTON

Growth factor receptor

Pro-B

IL3

IL7

c-kit

IGF-1

Pre-B

IL7

Virgin B

IL4

Mature B

IL4

Activated B

IL1

IL2

IL4

IL5

IL6

IL10

Plasma cell

IL6

Figure 3, By varying the receptors they express B cells respond to different lympho-kines at different stages In development. Early in development B cells tend to respond to lymphokines which promote proliferation such as IL7 and SCF. Later on, after activation, they become responsive to signals important for differentiation and anti­body secretion such as IL4, IL5, and IL6.

CD 19 (a pan B cell marker) and CD 10 (a neutral endopeptidase). Functionally, pro-B cells can be identified by their ability to proliferate in an undifferentiated state on stromal cells lines derived from either bone marrov^ or fetal liver. The stromal cells provide IL7 and express SCF on their surface, both of which are critical lymphokines in early B cell development. Lymphokine responsiveness changes w ith development as receptors are turned off and on (Figure 3). There are subdivisions within the pro-B cell compartment which can be resolved by the expression of other cell surface molecules. The earliest pro-B cell stage appears to have all immunoglobulin (Ig) loci in germline configuration, that is, gene rearrange­ment has yet to commence. As the cell progresses through the later pro-B cell stages Ig gene rearrangement starts at the heavy chain locus. By the end of the pro-B compartment, essentially every cell has reached at least the stage of joining a DH to a JH element on both IgH loci, while the four light chain loci (two kappa and two lambda) remain germline (Ehlich et al, 1993). The full details of Ig gene rearrangement are described in Chapter 5. While it is not entirely clear what signals are necessary to induce a pro-B cell to become a pre-B cell, it is probable that it involves the expression of a functional Ig heavy chain. This evidence comes from the analysis of mice carrying mutations which perturb B cell development. One such mutation is the so-called scid mutation (for severe combined immunodefi­ciency) which, as the name implies, resuhs in an absence of both B and T cells. Close analysis of B cell development in scid mice reveals that they have a numerically normal pro-B compartment but that all subsequent stages of B cell differentiation are missing. Since the scid mutation in mice blocks Ig gene rear-

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The B-Cell in Immunity 27

rangement, it suggests that a functionally rearranged IgH gene may be one of the

stimuli necessary to induce a pro-B cell to differentiate into a pre-B cell.

Pre-B cells are numerically the largest B cell subset in the bone marrow. They

differ phenotypically from their pro-B cell precursors in the mouse by, for example,

the loss of CD43 and c-kit expression and the onset of MHC class II expression. In

the human, pre-B cells lose surface expression of CD 10 and intracellular expression

of terminal deoxynucleotidyltransferase (TdT, an enzyme involved in Ig gene

rearrangement in adults). It appears that in the human MHC class II antigens, called

HLA-DR, are expressed from a very early point (see Figure 4). Pre-B cells

proliferate poorly in vitro on stromal cultures in the presence of IL7. In vivo,

however, the pre-B population shows extensive proliferation indicating that not all

the factors involved in B cell development have yet been defined. The pre-B cell

stage is when Ig light chain rearrangement commences and probably where IgH

gene rearrangement is completed. Pre-B cells develop into immature B cells upon

successful rearrangement and expression of one IgH locus and one of the four IgL

loci. It appears that at this point B cells lacking a productive Ig gene rearrangement

are lost. Although it is not known precisely how such cells die, a likely explanation

is that Ig-null cells fail to get a positive signal delivered through the Ig molecule

and therefore die from what amounts to suicide. It is currently postulated that the

s

> JO

m 30

DR

CD10

CD19

CD20

CD23

CD38

CD40

B7

Pro-B

O DtoJH

Pre-B

© VHtoDJH

VLtoJL

Virgin B

Q igM

Mature B

0 1 ^ IgM

IgD

Activated B

Q„ Memory B

0 IgGor

IgA

Plasma cell {

(§5 IgM, IgQ or IgA

secretion |

Figure 4. The cell surface proteins expressed by B cells change with development.

As a B cell matures it loses expression of some markers, such as CD10, and gains

others, such as CD23. These changes define developmental stages and often correlate

with changes occurring at the immunoglobulin gene locus with respect to gene

rearrangement or isotype switching. Plasma cells, which specialize in antibody

secretion, remove practically all cell surface proteins including the B lineage specific

markers. They are however easily identifiable by their shape and the presence of

immunoglobulin in the cytoplasm. A similar scheme can be drawn for mouse

development with some minor differences.

Page 43: Immunity

28 DAVID TARLINTON

positive signal is delivered to a pre-B cell through the surface expression of the Ig heavy chain in association with two proteins called collectively the pseudo light chain complex, and individually VpreB and 0:^5. Only those pre-B cells with a productive IgH gene rearrangement can express such a molecule and thus receive the growth stimulus necessary to remain viable and to initiate light chain gene rearrangement. Whatever the mechanism, cells with two nonproductive IgH gene rearrangements are efficiently removed from the B cell population since they are not seen beyond the pro-B cell stage and they do not appear to accumulate in the pre-B compartment.

Immature B cells represent the first time that IgM is expressed on the cell surface. This is a delicate time in B cell development since at this point any autoreactivity in the B cell repertoire will be exposed. The way in which the immune system deals with self-reactive cells is the study of tolerance and is discussed in greater detail in Chapter 4. While it is difficult (if not impossible) to follow the fate of self-reactive B cells which develop in a normal individual because of the heterogeneity of the population, a number of animal models have shed light on the fate of such cells. In these models, mice have been made transgenic for the genes necessary to produce an immunoglobulin molecule of a known specificity. That is, all the B cells developing in these mice will express identical immunoglobulin molecules with identical specificity. These Ig-transgenic mice can then be crossed with mice which express the antigen recognized by the transgene derived Ig. In such a way, every B cell which develops in the doubly positive progeny will be autoreactive, and the fate of individual B cells is now reflected in the fate of the population. Furthermore, the nature or context of the autoantigen can be altered. In two well characterized examples such pseudo-self antigens have been expressed as integral membrane proteins in either the bone marrow (Hartley et al., 1991) or the periphery, specifi­cally the liver (Russell et al., 1991). In both cases self-reactive B cell were killed either as soon as they were formed in the bone marrow or upon entry into the periphery. Finally, the effect of the self-antigen being soluble (i.e., a serum component) has been examined (Goodnow et al., 1988). In this case, the autoreac­tive B cells were not deleted but rather rendered incapable of responding to the antigen. This state is referred to as B cell anergy and in conjunction with clonal deletion (outlined above) constitutes the major form of purging the B cell repertoire of self-reactivity. While it should be borne in mind that these transgenic systems are model systems and are by necessity somewhat contrived, they adequately demonstrate the potential fate of self-reactive B cells.

Provided an immature B cell is not deleted because of its self-reactivity, it then leaves the bone marrow and enters the periphery. Whether the newly emigrant immature B cell gains entry to the recirculating B cell pool appears to depend on a number of factors, one of them being self-reactivity as outlined above. In summary, there appears to be a period in the development of a B cell in which stimulation by antigen leads to cell death rather than activation. The reasons why the same stimulus

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The B-Cell in Immunity 29

should have such different outcomes at different stages in B cell development are as yet unknown.

Approximately fifty million B cells leave the bone marrow of an adult mouse and enter the periphery every day, sufficient to replace the entire peripheral B cell pool in about five days (Osmond, 1986). Lifespan studies, however, indicate that this is not the case. In fact, in adult rats (these studies have been best performed in rodents) only around five percent of emigrant B cells gain access to the peripheral pool. This low level of replacement suggests that the majority of peripheral B cells are long lived, a fact that has recently been directly demonstrated in a number of experimental systems. Entry into the recirculating B cell population is also accom­panied with a further change in cell surface markers. The most obvious is the expression of IgD in addition to IgM on the cell surface (Figure 4). On a given B cell, both the IgM and IgD molecules have the same antigen specificity since these heavy chains are generated by the alternative splicing of a single variable region unit onto the two different constant regions. It was at one time maintained that since the onset of IgD expression correlated with the end of the period during which deletion of B cells could be induced by antigen, its expression was somehow related to this event. Recent analysis of mice unable to express IgD, however, suggests that this is either wrong or an oversimplification. Indeed, the analysis of these IgD""" mice has yet to reveal any clear-cut function for IgD. In addition to IgD, recircu­lating B cells differ from their precursors by expressing CD23 (the low affinity receptor for IgE) and higher levels of MHC class II. The recirculating B cell population is also uniformly positive for expression of CD40, a molecule which is critical for the activation of B cells by T cells. Prior to this stage, not all B lineage cells express CD40 (Figure 4).

In principle, access to the recirculating B cell pool may be by either of two processes: entry is either random or selective. While current experimental evidence is not conclusive, it tends to favor the selective recruitment of B cells into the recirculating pool. The most suggestive evidence comes from analyzing B cell populations for changes in Ig variable region gene usage. On two levels there is a shift in VH gene usage associated with becoming a long lived B cell. The best characterized example is that in which the VH gene families located closest the DH and JH elements are used preferentially in pre-B cells, while peripheral B cells utilize VH families according to their number of members (Malynn et al , 1990). The importance of this observation is that since pre-B cells do not express complete Ig on the surface, their repertoire is probably unselected. If entry into the peripheral pool were stochastic, then the peripheral repertoire would be expected to be the same as that of the pre-B cells. The fact that these two repertoires differ indicates selection on the basis of the expressed Ig. This selection could be either positive, negative or a mixture of both. That is, certain B cells could be either recruited because of some benefit their antigenic specificity endowed to the animal or, alternatively, immature B cells could be deleted because of self reactivity with the

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30 DAVID TARLINTON

few survivors becoming long lived B cells. The correct answer will probably be a mixture of both.

B CELL MIGRATION

In order to appreciate how B cells function in the immune system, it is necessary to understand a little of how they are distributed in the body and how they migrate from place to place. As has been detailed above, once generated in the bone marrow B cells then leave and enter the blood as immature B cells. These cells then enter the spleen at the periarteriolar lymphocytic sheath (PALS), an area rich in T cells and another specialized cell type called interdigitating dendritic cells (IDCs). One view of B cell maturation holds that the immature B cells are exposed to antigen at these locations and are thereby selected for entry into the peripheral pool (Gray and MacLennan, 1988). It certainly appears that there is a bifurcation in B cell circulation at this point, since beyond it only a small fraction of the newly produced B cells survive. This small fraction of the emigrant B cell population migrates from the PALS into adjacent B cell rich areas called follicles.

Follicles are organized structures in the secondary lymphoid organs with a reproducible cell distribution and a critical role to play in immune responses. The distribution of follicles in spleen and a typical follicle are shown in Figure 5. Within the follicle greater than 90% of the cells are B cells with the phenotype of recirculating B cells, namely IgM" IgD '. The remaining cells are CD4"*" T cells, a specialized kind of antigen presenting cell called a follicular dendritic cell (FDC) and macrophages. These various cell types cooperate in generating germinal centers in response to antigenic challenge. Surrounding the follicle is the marginal sinus and surrounding this the marginal zone. Recirculating B cells enter the secondary lymphoid organs via high endothelial venules in the marginal zone, and then migrate to the follicles. The B cells traverse the follicle and if they do not encounter their cognate antigen, they exit through the marginal sinus, which represents the efferent lymph, and repeat the whole process.

The marginal zone is typically considered to be part of the so-called white pulp in the spleen, meaning that it is part of the lymphocyte area. It does however form the junction between the red and white pulps and may be involved in the trafficking of lymphocytes between the two areas. B cells which reside in the marginal zone (as opposed to traversing it) have a phenotype which is distinct from that of the follicular B cells. MZ B cells express high levels of IgM, low levels of IgD and are negative for CD23, the inverse of follicular B cells. The exact role of the marginal zone B cells is not precisely known, but some experimental evidence suggests that these B cells may be involved in immune responses which are independent of T cell help and in transporting immune complexes into the follicles.

The basic organization of a follicle is quite well conserved in evolution. While present in essentially all secondary lymphoid organs it may have evolved from the organ in which the early events of B cell development occurred in more primitive

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The B'Cell in Immunity 31

> . ^ ^ .

^

%l D

Figure 5, Histological staining of B cell structures in secondary lymphoid organs. {A)

a transverse section of a mouse spleen, stained with an antibody specific for B cells.

Note how the B cells are organized Into horseshoe-shaped follicles, the center of

which Is the T-cell rich area. (B) a close up of one follicle. The lightly staining cluster

of cells Indicated by the arrow is a germinal center formed within the follicle. (Q cells

of a germinal center revealed more clearly with a specific stain and (D) a focus revealed

by an antl-immunoglobulln stain. Note the high level of staining which Indicates a

high level of immunoglobulin production by the B cells in the focus. (Courtesy of B.

Pulendran, The Walter & Eliza Hall Institute.)

animals. Indeed, in chickens and sheep the antigen independent phase of B cell development occurs in follicles structurally similar to those described above. In chickens these follicles are localized to an organ called the bursa of Fabricius (hence the B in B cell).

B CELL RESPONSES TO ANTIGEN

At this point the development of B cells has been chartered from stem cells up to recirculating B cells in the periphery. The various selective forces which operate on B cell development and which shape the repertoire have been described. What now needs to be considered is what happens when B cells encounter antigen in the periphery. That is, how do B cells participate in an immune response.

Page 47: Immunity

32 DAVID TARLINTON

3

s

GERMINAL CENTER

©

0

I immune complexes

O -*(©-"•"

• O — ' ( £ ) - . IgG

3 4

DAYS POST-IMMUNIZATION

Figure 6. The early stages of a primary T-cell dependent immune response. Antigen

complexes are localized on the surface of interdigitating dendritic cells (IDCs) outside

the follicle. B and T cells are stimulated by the antigen and begin the response. In the

extrafollicular pathway, B cells proliferate and differentiate into plasma cell in a focus.

In the intra-follicular pathway, B cells migrate into the follicle, proliferate and form a

germinal center. Immune complexes become localized on the follicular dendritic cells

(FDCs) and are important for B cell selection during affinity maturation.

When antigen is introduced into an animal it initiates a cascade of events that

eventually lead to its clearance and to the production of cells which will more

efficiently respond to the same antigen upon re-exposure. An outline of the early

events in an immune response is shown in Figure 6. Not all antigens, however, elicit

the same kind of response. Protein antigens generally elicit the participation of T

cells in the production and secretion of antigen specific immunoglobulin while

non-protein antigens do not. This basic distinction of T-dependent/T-independent

responses has several important ramifications. Since most antigens encountered are

T-dependent, these shall be considered first. The basic components of a T-depend-

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The B'Cell in Immunity 33

ent response are the production of antigen specific plasma cells, the germinal center reaction and the production of memory B cells.

T CELL DEPENDENT RESPONSES

The first thing to appreciate about immune responses is that they are actually quite difficult to initiate. The antigen has to be of a particular nature in order to elicit a response, it must either be given in the form of an aggregate or it must aggregate soon after introduction. The reason for this is that the cells of the immune system basically will not recognize soluble antigen. Indeed, antigen given in such a form is usually tolerogenic rather than immunogenic. Soluble antigen can be aggregated by immunoglobulin already circulating in the animal. Although this immunoglobu­lin will bind poorly to the antigen because of its low affinity, this can be compen­sated for by the increased avidity of binding that comes from being IgM. Secreted IgM is a pentameric molecule and therefore has ten antigen binding sites rather than the usual two. These multiple binding sites help to stabilize low affinity interactions.

While in general, immune complexes are removed from the system by phagocytic cells such as macrophages, a fraction is transported to secondary lymphoid organs where it acts to initiate a humoral immune response. This transportation is carried out by specialized dendritic cells in the periphery called Langerhan's cells. These cells migrate with antigen to the secondary lymphoid organs where they differen­tiate into extrafoUicular or interdigitating dendritic cells (IDCs). These dendritic cells are efficient in priming T-dependent responses because they express at high levels receptor molecules necessary for binding immune complexes (receptors for immunoglobulin Fc and complement component C3b), high levels of MHC class II antigens and they constitutively express T cell co-stimulatory molecules such as B7 (these are described in greater detail in Chapter 3). Both the class II and B7 molecules are important for priming helper T cells. As indicated earlier, the dendritic cells are visited both by recirculating B cells and newly generated virgin B cells and they exist in areas rich in T cells. If B cells from either of these sub-populations recognize the antigen on the surface of the dendritic cells in the presence of antigen primed T cells, they then follow one of two pathways; they either stay in the extrafoUicular PALS area and differentiate into an antibody producing plasma cell or they migrate to the follicle and initiate the formation of a germinal center (Figure 6).

The direct differentiation of antigen specific cells into plasma cells in the PALS forms the earliest B cell response to antigen. These antigen specific plasma cells are localized in a histologically identifiable structure called a focus, shown in Figure 5. The observation that each focus is adjacent to a germinal center has led to speculation about the relationship between the B cells in the focus and those in the germinal center. Are they, for example, clonally related or unrelated? Do B cells migrate from the germinal centers to the focus and there begin antibody secretion? While the answers to these questions are not yet known, they do provide for some

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34 DAVID TARLINTON

interesting speculations, some of which are outlined below. Foci persist for about one week, after which they dissipate. Although antibody production continues essentially unabated after this time, it presumably derives from plasma cells located in either the red pulp of the spleen or other organs entirely. Indeed, significant numbers of plasma cells are located in the bone marrow and the lamina propria, especially during a secondary response. An additional feature of B cell differentia­tion is also frequently visible in the plasma cells of the foci, namely immunoglobu­lin isotype switching.

Isotype switching is the name given to the process of genetic recombination whereby the constant region of an immunoglobulin molecule is replaced with another. All B cells start out life expressing IgM. They then co-express IgD, although this is not by isotype switching since it does not involve recombinatorial deletion of the intervening DNA. Upon stimulation by antigen, however, some fraction of the stimulated B cells will switch isotype at the heavy chain locus, transposing the VHDHJH rearrangement from upstream of IgM to being upstream of one of the other constant regions. For example, a cell may switch fi-om IgM to IgG, IgA, or IgE. The choice is not random, quite the opposite with the downstream isotype selected being determined by the stimulus the B cell receives. Certain T cell derived lymphokines act to dictate the isotype selected. Immune responses which elicit helper T cells of the so-called T^l subset, for example, are dominated by immunoglobulin of the IgG4 (human) or IgGl (mouse) isotype. The reason for this is that TH2 cells secrete the lymphokine IL4 which acts as a switch commitment factor to IgG4 and IgGl (and to IgE at higher concentrations). By itself IL4 cannot induce switching, but it does insure that if switching occurs, then it is directed to one isotype.

There is a certain symmetry to this system in that the nature of the antigen determines the nature of the response. That is, different antigens elicit TH subsets which produce different sets of lymphokines which, in turn and among other things, favor different immunoglobulin isotype switch outcomes. The different isotypes have different effector functions, and thus the response is tailored to the antigen. IgM and IgA, for example, are efficiently and specifically transported across epithelia and into the mucosa and one finds extensive switching to IgA in the gut associated lymphoid tissues (GALT). Although exactly how different antigens elicit TH cells secrefing different lymphokine profiles is currently unknown.

A small number of the B cells stimulated by antigen on the extrafollicular dendritic cells move into the follicle where they form a germinal center. Germinal centers derive from the extensive proliferation of these B cell clones and form distinct histological structures examples of which appear in Figure 5. The compo­sition of germinal centers is the same as that of the follicle in which they develop, namely B cells (90%), CD4'' TH cells (5%), follicular dendrific cells (2%) and macrophages (3%). These macrophages are unique in that they contain small dense bodies called Tingible bodies which represent the nuclei of dead lymphocytes that

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The B-Cell in Immunity 35

have been phagocytozed. The requirement for such a housekeeping cell in the germinal center will become apparent shortly. Germinal centers are usually first detectable about five days after the introduction of the antigen, although the day prior to their appearance rapidly dividing centroblasts can be seen in the primary follicle. Indeed, centroblasts have been estimated to have a doubling time of 6 hours! During the period seven to fourteen days post-immunization, the full architecture of the GC is resolved with the so-called light and dark zones becoming apparent. The dark zone is full of dividing centroblasts which are surface Ig negative while the light zone contains non-dividing centrocytes and is rich in follicular dendritic cells. The germinal center reaction can persist for several weeks with the average being four to five weeks. Finally, it appears that all of the cell types found in the germinal center are essential for its continued functioning. If the CD4" TH cells are removed by antibody treatment, for example, then the reaction immedi­ately stops.

The germinal center is not only a site of extensive and rapid B cell proliferation but also a site of cell death. Indeed, the whole reaction appears to be in a sort of equilibrium in that despite the proliferation, the germinal centers reach their maximum size at about day 14. What then happens to the majority of the cells being generated in the germinal center? While some presumably exit and become plasma cells or memory cells during the course of the reaction, it appears that most die. The reason for this extensive death is intimately linked with the processes of affinity maturation occurring in the germinal center.

Affinity maturation is the name given to the phenomenon whereby the average affinity of antibody for antigen increases during the course of a response. It is the result of two processes. One is the preferential growth of B cell clones whose Ig molecules have an intrinsically higher affinity for antigen. Such clones will be favored as the response continues and antigen becomes limiting. The other is the process of somatic mutation (or hypermutation) whereby higher affinity variants are created by deliberately introducing point mutations into the variable region gene segments of the immunoglobulin heavy and light chain genes. Although the mutational process is not random in that some nucleotide changes are more likely than others, and some positions are more likely to be mutated than others, it is not very selective. Thus, while some mutations will improve antigen binding, others will have either no effect or be deleterious. Some mutations may in fact destroy the ability of the immunoglobulin molecule either to bind antigen or even to be made at all. In order to function properly the system requires an efficient method of selecting B cells with improved affinity for antigen and deleting the remainder. This selection occurs in the germinal center.

A current model for the functioning of a germinal center is as follows. Extensive clonal expansion of a B cell occurs in the dark zone of the germinal center. These centroblasts have down regulated surface Ig expression and are actively undergoing somatic mutation. After each round of mutation, which may be after every round

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36 DAVID TARLINTON

of division, the cells migrate from the dark zone to the light zone. The light zone is rich in follicular dendritic cells which are covered in immune complexes and which express molecules important for B cell proliferation and differentiation. In the light zone, the centroblast has now become a centrocyte and again expresses surface Ig, presumably the most recently mutated form. In order to survive, this B cell has to gain access to the antigen on the surface of the FDC. This can only happen if the newly mutated surface Ig can displace the antibody already coating the antigen on the surface of the FDC. If the mutations improve affinity, then the B cell has a good chance of displacing lower affinity antibody. If the mutations are deleterious to antigen binding, then the B cell will not gain access to the antigen on the FDC and will rapidly die. These dead B cells are removed by the resident macrophages. Selected B cells on the other hand, probably re-enter the dark zone for further rounds of mutation and selection. There are two lines of evidence to support this model. First, in somatically mutated high affinity B cells, the mutations are not distributed randomly but are concentrated in regions of the genes which encode the antigen binding segments of the protein called complementarity determining regions (CDRs). Second, members of single B cell clones have been recovered which show hierarchical distributions of mutations (Jacob et al., 1991). Such a scheme is depicted in Figure 7. The simplest explanation for the concentration of mutations in the CDRs and the occurrence of mutational trees is for rounds of mutation to be punctuated by selection.

Not all somatic mutants re-enter the mutational process. Some apparently exit from the germinal center and become antibody secreting cells in the extrafollicular areas. Thus, as the response continues the average affinity of the antibody in circulation improves. The means by which the choice between continued mutation or antibody secretion is made are unknown. One possibility is that it depends on the availability of unbound antigen on the surface of the FDCs, the more free antigen the more likely that the cell will become a plasma cell. It has in fact been proposed that the germinal center reaction itself is terminated when there is no free antigen on the surface of the FDCs.

The final cell type to emerge from the germinal center is the memory B cell. This cell is isotype switched and its V genes are extensively mutated and selected. That is, the immunoglobulin expressed by this cell is close to the optimum possible for the antigen. It is not clear whether memory B cells are generated throughout the course of the germinal center reaction or only at the end when the reaction ceases. However it happens, the end result is a population of cells with high affinity for antigen. These cells then enter the circulation and persist for many months, even years. Recent experiments have indicated both that antigen is necessary for the persistence of memory B cells (Gray and Skarvall, 1988) and that memory B cells are not dividing (Schittek and Rajewsky, 1990). These are not necessarily contra­dictory statements, since they imply that memory B cells need to be regularly exposed to antigen but apparently not in a form which induces a response. It has

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The B-Cell in Immunity

DIVISION

37

e / \ / \

/ \ / \

Figure 7, A schematic representation of somatic mutation and selection in a single

B cell clone. As the daughter cells divide, their progeny incorporate different mutations

which may or may not be selected for. If they are, the cell continues to divide; if not,

the cell dies. The B cells at each generation possess the mutations of their antecedents

v^/hich aHou/s for the order in which the mutations were introduced to be determined.

been known for some time that immune complexes remain on the surface of FDCs and IDCs for many months if not years, and this may be the source of the continual antigenic exposure necessary to maintain the memory B cell population.

One final thing to say about T-dependent responses concerns the relationship between the cells of the foci and those of the germinal center. Two ideas have been proposed for the formation of these structures. In one the B cells which populate the GC are derived from a different "lineage" than those which enter the foci (Linton et al., 1988). That is, entry into the GC or the focus is intrinsic to the B cell and is determined before the exposure to antigen. The alternative view holds that the choice is made at the point of exposure to antigen, and that in fact progeny from the one B cell clone may populate both the GC and its adjacent focus (Jacob and Kelsoe, 1992). Once again, there is experimental evidence supporting both views.

A SECONDARY RESPONSE

When an animal is re-exposed to an antigen, immune complexes are again neces­sary for triggering the secondary or memory response. In this case there is often antigen specific immunoglobulin still circulating from the primary response. These

Page 53: Immunity

38 DAVID TARLINTON

complexes are again localized on extra-follicular dendritic cells in the secondary

lymphoid organs where they are exposed to circulating memory B cells. When

memory B cells encounter antigen, they rapidly begin to proliferate and differenti­

ate into antibody secreting plasma cells. These plasma cells migrate to the bone

marrow where they remain and continue to secrete immunoglobulin for a consid­

erable time. Since the memory B cell population is already isotype switched,

somatically mutated and affinity selected, the secondary response is both more

rapid and of higher initial affinity than the primary response. In fact, the peak

antibody titer is reached three to five days after secondary antigen exposure

compared to the two weeks required in the primary response. Whether or not

germinal centers arise as part of the secondary response is not completely clear.

Some investigators have found that they do, others that they do not. The difference

appears to reflect differences in immunization protocols, such as the length of time

between primary and secondary challenges.

T CELL INDEPENDENT RESPONSES

Some antigens do not require T cell participation in order to elicit an antibody

response. This is graphically demonstrated by the ability of congenitally athymic

nude mice (which lack all thymus derived lymphocytes) to respond, albeit weakly,

to certain antigens. The common feature of T-independent antigens is that they lack

protein epitopes and are therefore unable to be presented in a recognizable form to

T cells. Antigens such as polysaccharides and lipids are examples of T-independent

antigens. T-independent antigens are further divided into Type 1 and Type 2. Type

1 antigens have intrinsic polyclonal B cell activating properties, in that they are

themselves mitogenic. Lipopolysaccharide (LPS) is an example of a murine Type

1 antigen. Type 2 antigens are defined by the inability of mice bearing the X-linked

immunodeficiency (xid, see below) mutation to respond to them. An example of a

Type 2 antigen is the polysaccharide of pneumococcus.

There are a number of important differences between the response induced

by T-dependent and independent antigens. Foremost among them is that T-in­

dependent antigens do not result in memory B cells or affinity maturation

through V gene somatic mutation. Indeed, T-independent responses are gener­

ally composed of IgM antibodies, are of short duration and low affinity. Some

isotype switching may occur, typically to IgG3 and IgG2a in the mouse and

IgG2 in humans. The significance of T-independent antigens comes from the

fact that a number of pathogens elicit only this kind of response. Since these

responses do not produce memory B cells, these antigens are difficult to

vaccinate against. Finding a way of circumventing this is a major goal of current

immunological research.

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The B'Cell in Immunity 39

OTHER TYPES OF B CELLS

A number of recent studies have suggested that not all B cells in the periphery are the same. Sub-populations have been identified by their cell surface phenotype and, in some cases, by functional and developmental differences from the majority of B cells. One well characterized sub-population is that of the Ly-1 B cell. These B cells express the pan-T cell antigen CD5 (which, in the mouse, used to be called Ly-1, hence the name). Additionally, Ly-1 B cells express low levels of IgD, high levels of MHC class II and lack CD23. They are most frequent in the neonatal period and, in mice, are localized to peritoneal and pleural cavities. The equivalent B cell population expressing CD5 exists in humans, but it differs somewhat in its location, being absent from the peritoneum and mainly restricted to the blood. The develop­mental differences between Ly-1 B cells and follicular B cells are so numerous that a number of investigators have proposed that these B cells represent a different hematopoietic lineage. Needless to say, this is quite a contentious issue and the evidence is not yet conclusive.

At present the significance of the CD5+ B cell population is difficult to assess. These B cells have been implicated in the production of a number of autoantibodies in both humans and mice, and as being the population from which the vast majority of B chronic lymphocytic leukemias (B-CLLs) develop (greater than 90% of human B-CLL are CD5+). It is postulated that the inherent characteristics of CD5 B cells predispose them to becoming CLLs in that these cells are thought to be self-renew­ing IgM positive cells, thereby providing a greater opportunity for oncogenic transformation.

B CELL DEFICIENCIES

A number of immunodeficiencies involving different aspects of B cell immunology are known. These are listed in Figure 8, giving the human and mouse forms and the underlying genetic lesion where known. In fact, in the very recent past, the exact molecular nature of the defects in both X-linked agammaglobulinemia (XLA) and XLA with hyper-IgM were discovered. These findings represent the culmination of years of basic research in immunology and offer great hope of successful intervention in these diseases.

X-Linked Agammaglobulinemia (X-LA)

This condition, first described by Bruton in 1952, manifests as an almost complete absence of circulating immunoglobulin with peripheral B cell numbers also being profoundly decreased (less than 1% of normal). During early life affected individuals are protected by maternal IgG passed through the placenta, but as this declines, they become susceptible to recurrent infections, particularly of the respi­ratory tract. They can, however, be kept healthy by frequent infusions of gam-

Page 55: Immunity

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The B'Cell in Immunity 41

maglobulin. X-LA patients show a normal pro-B compartment but a severely reduced pre-B cell population. Some surface immunoglobulin positive B cells do, however, develop but they are of an immature phenotype and are mostly unable to mature further.

The X-LA mutation maps to the long arm of the X-chromosome. The gene responsible (now called btk for ruton's agammaglobulinemia tyrosine Ainase) was recently cloned and found to be a cytoplasmic tyrosine kinase (Vetrie et al , 1993). Such proteins are critical for signal transduction from surface receptors. It is thought that the mutation in /?rA: prevents appropriate signalling through the immunoglobu­lin molecule at all stages of B cell development. This almost completely blocks pre-B cell expansion and the development and differentiation of the immature B cells which do survive. Shortly after the cloning of btk, it was reported that mutation of the equivalent gene in the mouse was responsible for the xid phenotype described earlier (Rawlings et al., 1993; Thomas et al., 1993). The differences between human X-LA and murine xid are presumably due to the severity of the mutation; in mouse it is a missense mutation while in human it is a nonsense mutation.

X-LA with Hyper-IgM

This disease, also an X-linked recessive, is characterized by a severe reduction in all circulating Ig isotypes other than M which exists at somewhat elevated levels. Affected individuals are susceptible to severe bacterial infections. The molecular basis of this disease has also been elucidated and found to involve a molecule essential for the T cell mediated activation of B cells (Allen et al., 1993; Aruffo et al., 1993). The molecule found to be defective in this disease is the ligand for CD40 which is a membrane-bound protein called gp39 (Noelle et al., 1992) the gene for which also maps to the X chromosome. Engagement of CD40 is a powerful stimulant for B cells, inducing extensive proliferation and, in the presence of the lymphokines IL4, IL5, and ILIO, differentiation into antibody secreting cells of both IgM and downstream isotypes. Mutation of the gp39 gene effectively means that such individuals are bereft of T cell help. An additional feature of this condition is that many of the circulating IgM antibodies are directed against self antigens, particularly on red blood cells, leukocytes, platelets and some tissue antigens of the gastrointestinal tract.

Selective Immunoglobulin Isotype Deficiencies

The most common of these is selective IgA deficiency, affecting about 1 in 700 Caucasians. It is characterized by low levels of IgA (less than 0.5 mg/ml) and normal to slightly elevated levels of IgM and IgG. The consequences of IgA deficiency are variable, ranging from being asymptomatic (usually) to severe recurrent infections (rarely). The nature of the defect is currently unknown, al­though these patients appear to have normal surface IgA positive B cells and

Page 57: Immunity

42 DAVID TARLINTON

normally functioning T cells. The defect may be the result of a failure of the B cells to respond to lymphokines which enhance IgA production (IL5 and transforming growth factor-P, for example) or a failure of IgA B cells to differentiate into plasma cells.

Selective deficiencies of IgM and the IgGs have also been reported. The absence of IgM is associated with severe infections such as bacterial meningitis. In both these cases the B cells appear to be normal in that they express immunoglobulin on the surface, but they are blocked in the differentiative steps leading to a plasma cell. It is not known if the defects lie in the delivery or receipt of T cell help.

Common Variable Immunodeficiency (CVID)

The hypogammaglobulinemia associated with this condition usually manifests in early adult life. It appears to be autosomally inherited with both patients and relatives showing an increased incidence of autoimmune diseases such as rheuma­toid arthritis. CVID patients in general have peripheral B cells which do not function properly, although no single common defect has been observed. These patients are susceptible to bacterial infections.

B-Cell Development and Function Analyzed by Gene Targeting

The ability to generate mice carrying mutations in specific genes, introduced through homologous recombination in embryonic stem cells grown in vitro, has meant that several tenants of B cell development can be tested directly in animal models (reviewed in Pfeffer and Mak, 1994). Thus the importance of specific molecules can be determined by analyzing B-cell development and function in animals which lack that molecule. While in many instances such analyses have confirmed the role of the molecule in question, in some cases novel phenotypes have been discovered. Thus a number of genes not thought to have any involvement in B cell development have been revealed to be important by examination of knockout mice. A number of genes shown to be involved in B cell development and differentiation are listed in Table 1. Analysis of animals which are unable to rearrange their Ig heavy chain loci, such as the RAG deficient mice for example, has proven that a productive IgH gene rearrangement is absolutely required for the transition from the pro-B to the pre-B cell stage. Similarly, knockouts that prevent expression of the so-called pre-B receptor, such as deletion of the membrane exon of IgM and deletion of the gene encoding the pseudo light chain XS, also block B cell development at the pro-B to pre-B transition. A large number of factors which control transcription of the IgH locus also block B cell development at an early stage. The majority of these factors are associated with the activity of the transcrip­tion enhancer located in the intron between the JH elements and the constant region of IgM. It is also clear from the complex phenotypes of mice in which mature B cell function is effected, such as the lyn and IL2 receptor alpha chain knockouts

Page 58: Immunity

The B-Cell in Immunity 43

Table 1. Gene Knockouts with a B Cell Related Phenotype

Gene(s)

Ikaros^

pax-5* sox-4^, EBF,

E2a*

RAG-1,RAG-2, E^JH |iMT, X5, mb-1, B29

TdT

syk*, p50(csk)

IL7, IL7R

JAK3

JK, C K

IL2RY

Bcl-X*, Bel-2*

Bax

IL2Ra

lyn

BTK

HS-1

oct-2 , c-rel

MHC class II

CD21,CD35

CD28

C D 4 a CD40L

IL4

IL6

FcyRII

CD45 (B220)

IL5Ra

Fas (CD95)

Syl

Function

zinc finger protein

transcription factors

gene rearrangement

pre-BCR and BCR

expression

nucleotide addition

signal transduction

growth factors &

receptors

signal transduction

kappa light chain

cytokine receptor

cell survival factors

cell death factor

cytokine receptor

signal transduction

signal transduction

signal transduction

transcription factors

antigen presentation

complement receptors

T cell co-stimulator

T-B collaboration

switch commitment

factor

enhance Ig secretion

IgG receptor

phosphatase

cytokine receptor

cell death signal

IgGI switch region

Developmental Stage

of Effect

lymphocyte precursor

pro-B to pre-B

pro-B to pre-B

pre-B cell

pro-B cell

pro-B to pre-B

pro-B to pre-B

pre-B cell

pre-B to virgin B

early B lineage

virgin & mature B

mature B cells

mature B cell

mature B cell

mature B cell

mature B cell

mature B cell

mature B cell

mature B cell

mature B cells

mature B cell

mature B cells

mature B cell

mature B cells

B lineage

mature B cells

mature B cell

mature B cell

Phenotype

no B & T lineage cells

no mature B cells

no mature B cells

no mature B cells

no N region in Ig

rearrangements

no mature B cells

reduced B cells

no mature B cells

reduced B cells, all \gX

reduced B cells, hyper-

IgM

reduced B cells

B cell hyperplasia

B cell hyperplasia,

autoimmune

reduced B cells,

autoimmune

reduced B cell, Tl

responses

reduced TD & Tl

responses

reduced TD & Tl

responses

no TD responses

reduced TD, no GC

reduced TD, no GC

no TD responses

no IgE, reduced IgGI

reduced mucosal IgA

increased TD & Tl

responses

reduced BCR signaling

reduced IgM & lgG3

severe B cell

hyperplasia

reduced switching to

IgGI

Note: Symbols and abbreviations used in Table 1. TD, T-cell dependent; Tl, T-cell independent; BCR, B-cell receptor; # indicates an embryonic lethal, and * indicates a perinatal lethal mutation when in homozygous form.

Page 59: Immunity

44 DAVID TARLINTON

which develop autoimmune disease, that much is yet to be learned about the regulation of B cell function.

SUMMARY

B cells, the source of all antibody molecules in the immune system, are produced throughout life from stem cells in the bone marrow in an ordered process which maximizes diversity and deletes self-reactivity. In the periphery B cells are avail­able to respond to foreign antigen. For antigens which require T cell help, the B cell response not only produces antibody which helps in the clearance of the antigen, but also generates long lived memory cells which can respond to the same antigen years later. These memory B cells also have antibody molecules whose fit for the antigen has been improved during the response by the process of affinity maturation, carried out in a special structure called a germinal center. A number of immunodeficiencies have been defined in which specific stages of B cell develop­ment or function are affected. For some of these, the exact nature of the mutafion is now known, raising the possibility of gene therapy at some time in the future.

REFERENCES

Allen, R.C., Armitage, R.J., Conley, M.E., Rosenblatt, H., Jenkins, N.A., Copeland, N.G., Bedell, M.A.,

Edelhoff, S., Disteche, CM., Simoneaux, D.K., Fanslow, W.C, Belmont, J., & Spriggs, M.K.

(1993). CD40 ligand gene defects responsible for X-linked hyper-IgM syndrome. Science 259,

990-993.

Aruffo, A., Farrington, M., Hollenbaugh, D, Li, X., Milatovich, A., Nonoyama, S., Bajorath, J.,

Grosmarie, L.S., Stenkamp, R., Neubauer, M., Roberts, R.L., Noelle, R.J., Ledbetter, J.A., Franke,

U., & Ochs H.D. (1993). The CD40 ligand, gp39, is defective in activated T cells from patients

with X-linked hyper-IgM syndrome. Cell 72, 291-300.

Cumano A., Paige, C.J., Iscove, N.N., & Brady, G. (1992). Bipotential precursors of B cells and

macrophages in murine fetal liver. Nature 356, 612-615.

Ehlich, A., Schaal, S., Gu, H., Kitamura, D., Mueller, W., & Rajewsky, K. (1993). Immunoglobulin

heavy and light chain genes rearrange independently at early stages of B cell development. Cell

72, 695-704.

Goodnow, C.C, Crosbie, J., Adelstein, S., Lavoie, T.B., Smith-Gill, S.J., Brink, R.A., Pritchard-Briscoe,

H., Wotherspoon, J.S., Loblay, R.H., Raphael, K., Trent, R.J., & Basten, A. (1988). Altered

immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic

mice. Nature 334, 676-^82.

Gray, D., & Skarvall, H. (1988). B-cell memory is short lived in the absence of antigen. Nature 336,

70-73.

Hardy, R.R., Carmack, C.E., Shimon, S.A., Kemp, D.J., & Hayakawa, K. (1991). Resolution and

characterisation of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med.

173, 1213-1225.

Hartley, S.B., Crosbie, J., Brink, R., Kantor, A.B., Basten, A., & Goodnow, C.C. (1991). Elimination

from peripheral lymphoid tissues of self-reactive B lymphocytes recognizing membrane-bound

antigens. Nature 353, 765-769.

Jacob, J., Kelsoe, G., Rajewsky, K., & Weiss, U. (1991). Intraclonal generation of antibody mutants in

germinal centres. Nature 354, 389-392.

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The B-Cell in Immunity 45

Jacob, J., & Kelsoe, G. (1992). In situ studies of the primary immune response to (4-hydroxy-3-ni-

trophenyl)acetyl. II. A common clonal origin for periarteriolar lymphoid sheath-associated foci

and germinal centers. J. Exp. Med. 176, 679-687.

Linton, P.-J., Decker, D.J., & Klinman, N.R. (1988). Primary antibody forming cells and secondary B

cells are generated from separate precursor cell subpopulations. Cell 59, 1049-1059.

MacLennan, I.C.M., & Gray, D. (1988). Antigen-driven selection of virgin and memory B cells.

Immunol. Rev. 91,61-85.

Malynn, B.A., Yancopoulos, G.D., Barth, J.E., Bona, C.A., & Alt, F.W. (1990). Biased expression of

J^-proximal V^ genes occurs in the newly generated repertoire of neonatal and adult mice. J. Exp.

Med. 171,843-859.

Noelle, R.J., Roy, M., Shepherd, D.M., Stamenkovic, I., Ledbetter, J.A., & Aruffo, A. (1992). A 39-KDa

protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of

B cells Proc. Natl. Acad. Sci. USA. 89, 6550-6554.

Osmond, D.G. (1986). Population dynamics of bone marrow B lymphocytes. Immunol. Rev. 93,

103-124.

Pfeffer, U., & Mak, T.W. (1994). Lymphocyte ontogeny and activation in gene targeted mutant mice.

Ann. Rev. Immunol. 12, 367-411.

Rawlings, D.J., Saffran, D.C., Tsukada, S., Largaespada, D.A., Grimaldi, J.C, Cohen, L., Mohr, R.N.,

Bazan, J.F., Howard, M., Copeland, N., Jenkins, N.A., & Witte, O.N. (1993). Mutation of unique

region of Bruton's tyrosine kinase in immunodeficient mice. Science 261, 358-361.

Russell, D.M., Dembic, Z., Morahan, G., Miller, J.F.A.P., Burki, K., & Nemazee, D. (1991). Peripheral

deletion of self-reactive B cells. Nature 354, 308-311.

Schittek, B., & Rajewsky, K. (1990). Maintenance of B-cell memory by long-lived cells generated from

proliferating precursors. Nature 346, 749-751.

Spangrude, G.J., Heimfeld, S., & Weissman, I.L. (1988). Purification and characterisation of mouse

hematopoietic stem cells. Science 241, 58-62.

Thomas, J.D., Sideras, P., Smith, CLE., Vorechovsky, I., Chapman, V., & Paul, W.E. (1993).

Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes. Science

261,355-358.

Vetrie, D., Vorechovsky, I, Sideras, P., Holland, J., Davies, A., Flinter, F., Hammarstrom, L., Kinnon,

C, Levinsky, R., Bobrow, M., Smith, CLE., & Bentley, D.R. (1993). The gene involved in

X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases. Nature

361,226-233.

RECOMMENDED READINGS

Forster, I., & Rajewsky, K. (1990). The bulk of the peripheral B-cell pool in mice is stable and not

rapidly renewed from the bone marrow. Proc. Natl Acad. Sci. USA. 87, 4781-4784.

Gu, H., Tarlinton, D., Mueller, W., Rajewsky, K., & Forster, 1. (1991). Most peripheral B cells in mice

are ligand selected. J. Exp. Med. 173, 1357-1371.

Kipps, T.J. (1989). The CD5 B cell. Adv. Immunol. 47, 117-185.

Lalor, P.A., Nossal, G.J.V., Sanderson, R., & McHeyzer-Williams (1992). Functional and molecular

characterization of single, (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific, IgGl+ B cells from

antibody-secreting and memory B cell pathways in C57BL/6 immune response to NP. Eur. J.

Immunol. 22, 3001-3011.

Melchers, F., Rolink, A., Grawunder, U., Winkler, T.H., & Karasuyama, H. (1995). Positive and

negative selection events during lymphopoiesis. Curr. Op. Immunol. 7, 214-227.

Nahm, M.H ., Kroese, F.G.M., & Hoffmann, J.W. (1992). The evolution of immune memory and

germinal centres. Immunol. Today 13, 438-441.

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46 DAVID TARLINTON

Relink, A,, & Melchers, F. (1993). Generation and regeneration of cells of the B-lymphocyte lineage.

Curr. Opinion in Immunol. 5, 207-217. van Rooijen, N. (1990). Direct intrafollicular differentiation of memory B cells into plasma cells.

Immunol. Today 11, 154—157. Wabl, M., & Steinberg, C. (1996). Affinity maturation and class switching. Curr. Op. Immunol. 8,

89-92.

Page 62: Immunity

chapter 3

Cell-to-Cell Interactions in the

Immune System

WILLIAM A. SEWELL and RONALD PENNY

Molecular Basis of Cell-to-Cell Interaction 48 Cell Surface Molecules 48 Cytokines 50

Antigen Presentation to Lymphocytes 51 Introduction 51 Antigen Presentation to B Cells 52 Antigen Presentation to T Cells 52

Effector Mechanisms of T Cells 55 Interaction Between T and B Cells 55

Interaction Between T Cells and Macrophages 58 Thl and Th2 Cells 58 Cytotoxic Cells 59

Interaction between Leukocytes and Endothelium 60

Principles of Medical Biology, Volume 6 Immunobiology, pages 47-62. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-811-0

47

Page 63: Immunity

48 WILLIAM A. SEVVELL and RONALD PENNY

Summary 59

Recommended Readings 60

MOLECULAR BASIS OF CELL-TO-CELL INTERACTION

Cell Surface Molecules

Cells participate in cell-to-cell adhesion when regions of the extracellular do­mains of surface molecules on different cells bind to each other. Cell surface molecules may also act as receptors for soluble factors (such as antibodies, complement components, or cytokines), they may bind to the extracellular matrix, or they may act as receptors for viruses. Any of these events may be followed by functional changes in the cell, which may be mediated by the intracellular domains of the cell surface molecules.

When stable adhesion between cells of two different lineages takes place, several different types of molecules on each cell may be involved. In most cases, one adhesion molecule on one of the cells binds to one molecule on the other cell, and the two molecules are different. The overall stability of cell-to-cell adhesion is greater when the number of different types of bonds increases. Cells may only make appropriate responses when several different pairs of adhesion molecules have bound. Thus, when T cells recognize antigen, the specificity of the T cell is determined by the T cell antigen receptor, but its adhesion to antigen is weak, and functional changes in the T cell depend on the achievement of stable cell-to-cell adhesion by several pairs of adhesion molecules.

The three most widely recognized classes of cell adhesion molecules are illus­trated in Table 1. The immunoglobulin superfamily, as its name implies, consists of molecules with one or more domains which resemble those of immunoglobulins. Most of the members of this superfamily exist principally as molecules bound to the cell surface, with immunoglobulin domains in their extracellular regions (Williams and Barclay, 1988).

Each domain consists of about 100 amino acids, and forms a compact beta-pleated sheet structure sometimes known as a "barrel." The surfaces of the domains are able to bind to other structures. Members of this superfamily are found on many cell types and are especially prominent in cells of the immune and nervous systems. Immunoglobulin and the Ti chains of the T cell receptor contain variable domains, which are different in each different B and T cell respectively. All other immuno­globulin domains are the same on all cells that express them. Some of these molecules have been found in very primitive organisms. It has therefore been suggested that in evolution, the first function of the immunoglobulin superfamily was in cell-to-cell adhesion, and that the development of antigen receptor molecules (immunoglobulin and Ti) was a later event. In many cases, immunoglobulin

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Cell-tO'Cell Interactions 49

Table I. Cell Adhesion Superfamilies in the Immune System

Structure

Superfamily Extracellular/lntracellular Examples Discussed in this Chapter

Immunoglobulin

Integrin

-Q

EGF

Selectin ^)-iH-ii lectin L^-^n

Ti, MHC (membrane proximal domains

only), p2-microglobulin, CD2, CD3,

CD4, CDS, CD28, CD50, CD54, CD58,

CD80, B7-2, CD102, CD106

CD11 a/CDI 8 (LFA-1); CD49d/CD29

(VLA-4)

L-selectIn, E-selectin, P-selectin

Notes: For the immunoglobulin superfamily, Ti, MHC Class II and CDS exist as dimers i.e., two separate structures are bound together on the cell surface. For immunoglobulins, n relates to the number of domains in the one chain; n = 1 for p2-microglobulin, CD3, CDS, and CD28; n = 2 for Ti, CD2, CD58, CD80, B7-2 and CD102; n = 4 for CD4; n = 5 for CD50 and CD54; n = 7 for CD106. For the selectins, n = 2 for L-selectin, n = 6 for E-selectin and n=9 for P-selectin. The lectin-like domain is capable of binding carbohydrate; the EGF domain is similar to the cytokine epidermal growth factor; SCR stands for short consensus repeat, a domain found in many proteins that regulate the complement pathway.

superfamily molecules bind to other members of the superfamily. Alternatively, some members of the immunoglobulin superfamily bind to another major class of surface molecules, the integrins.

The integrins are a large class of cell surface molecules, present on leukocytes and on other cells (Hynes, 1992). Some integrins bind to extracellular matrix proteins. Other integrins bind to other cell surface molecules, especially to members of the immunoglobulin superfamily. Integrins consist of two non-covalently asso­ciated chains, alpha and beta, and their adhesiveness can be rapidly altered. For example, after T cells are activated, they become more sticky as a result of increased adhesiveness of their Leukocyte Function Antigen-1 (LFA-1) (CD 11 a/CD 18) molecules. Leukocyte egress from the bloodstream is regulated by integrin sticki­ness, as described later.

The initial contact between leukocytes and endothelium depends on the third major class of binding proteins, the selectins. These are molecules in which the N-terminal region, furthest from the cell, is a lectin like domain, capable of binding carbohydrate. In some cases, the carbohydrate ligands are sulphated (Bevilacqua and Nelson, 1993). The latter moieties may be carried by a variety of surface proteins, and their expression is determined by the presence within the cell of the appropriate transferase enzyme, which attaches the carbohydrate to the protein. Selectins may be expressed on circulating blood cells and on endothelium.

Page 65: Immunity

50 WILLIAM A. SEWELL and RONALD PENNY

Cytokines

Cytokines are secreted proteins with predominantly local actions. They have a number of similarities with hormones. For example, they are produced by special­ized cells in response to particular kinds of stimuli, and they bind with high affinity to specific receptors on cells to elicit characteristic responses. Unlike hormones, many cytokines are extremely difficult to detect in the bloodstream, as they are produced in very small quantities and their action is mainly on nearby cells, "paracrine," or on the same cell that made the cytokine, "autocrine." This is particularly true of cytokines produced by T cells, which mainly act locally. By contrast, some of the cytokines produced by macrophages, such as tumor necrosis factor (TNF), interleukin-1 (IL-1), and IL-6 are more readily detectable in the bloodstream and can act in an endocrine fashion. Some of the principal cells that make cytokines, such as T cells and macrophages, are mobile, and cytokines are only released after the cells have moved to sites where they are required. Therefore, only very small quantities of cytokines are often secreted, as they need to be present at a concentration of only 10" M or less in a very small volume of extracellular fluid. Because of their low abundance, cytokines have been difficult to study. It is only in recent times, with the advent of molecular cloning and other tools of modern biology, that cytokines have been well characterized.

Many cytokines have polypeptide backbones of around 100 to 150 amino acids. Cytokines are usually glycosylated, although this is not essential for function. Their monomeric molecular weights are often about 15,000 including glycosylation. Cytokines may exist in monomeric, dimeric or trimeric form. In any immune reaction, many different cytokines may be present, some of which may have overlapping functions. Also, many cells have receptors for several different cyto­kines. The concept of the cytokine network has been proposed, whereby the response of a cell depends on the balance between the many different cytokines acting on it.

Because cytokines are potent effector molecules, it is not surprising that there are many ways in which their production and effects are controlled. There is often strict regulation of the abundance of their mRNA. Their genes are transcribed only in activated and not in resting cells, and their mRNAs often have short half-lives. The activity of cytokines may also be controlled after they have been secreted. In the case of IL-1, a naturally occurring receptor antagonist, IL-lra, has been described. Soluble forms of the receptors for several cytokines exist; these bind cytokines but, because they are not attached to cells, are unable to deliver signals. Thus, production of soluble receptors causes cytokines to be "mopped up" and is a mechanism for inhibition of cytokine activity. In each of the following sections, the role of cytokines in cell-to-cell interaction will be described. Important cytokines in the immune system are listed in Table 2. Further information is available in Nicola (1994).

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Cell-to-Cell Interactions 51

Table 2, Summary of Cytokines in the Immune System

Name

IL-1

IL-2 IL-3 IL-4

IL-5 IL-6

IL-7 IL-8 IL-9 IL-10 IL-11 IL-12

IL-13 IL-14 IL-15

IFN-a, p

IFN-Y

TNF-a and

TNF-p G-CSF M-CSF GM-CSF

SCF

Major Source(s)

Macrophages

T-cells T-cells, mast cells T-cells, mast cells

T-cells, mast cells Macrophages

Bone marrow stromal cells Macrophages T-cells T-cells, macrophages Bone marrow stromal cells Macrophages, dendritic cells

T-cells T-cells Many cell types

various cells T-cells

-P TNF-a-macrophages

TNF-6-T-cells T-cells, macrophages Macrophages Macrophages Macrophages, T-cells

Bone marrow stromal cells

Major Act ion (s) within the Immune System

Mediates local and systemic inflammatory

response, fever T and B-cell proliferation Growth of immature hemopoietic cells B-cell growth and switch to IgE, inhibition

of cytokine production by macrophages Growth and survival of eosinophils Acute phase protein synthesis, B cell

growth B- and T-cell development Chemotaxis of neutrophils Growth of T-cells and hemopoietic cells B-cell growth, inhibition of macrophages Growth of hemopoietic cells Stimulation of T- and natural killer cell

function and IFN-y production Similar to IL-4 B-cell proliferation Similar to IL-2

Antiviral, antiproliferative macrophage activation, antiviral

Both mediate local and systemic

inflammatory response, fever, cachexia Many effects; in general, anti-inflammatory Growth of neutrophil precursors Growth of macrophage precursors Growth of macrophage and neutrophil

precursors Growth of hemopoietic stem cells, mast

cells

Notes: Abbreviations: IL, interleukin; IFN, interferon; TNF, tumor neurosis factor; TGF, transforming growth factor; CSF, colony stimulating factor; SCF, stem cell factor. Only major sources and major actions within the immune system are shown. The three CSFs are also made by endothelial cells and fibroblasts. There is significant overlap between the actions of the TNFs, IL-I and IL-6.

ANTIGEN PRESENTATION TO LYMPHOCYTES

Introduction

T and B lymphocytes respond to antigen by the process of clonal selection. Each

new lymphocyte contains a unique type of antigen receptor on its surface, as a result

of rearrangements in the genes encoding the variable domains of their antigen

receptor molecules, i.e. surface immunoglobulin in B cells and Ti in T cells (see

Chapter 5). Any new antigen will bind firmly to antigen receptors on only a very

few lymphocytes, which are "selected" to proliferate, and give rise to a "clone," in

which all cells have the same type of antigen receptor, and their products curtail

the infective process. Hence the phrase "clonal selection" was coined. Some of

these T and B cells are long-lived and are known as memory cells. If exposure to

the same microorganism takes place on a second or subsequent occasion, there is

Page 67: Immunity

52 WILLIAM A. SEWELL and RONALD PENNY

usually no clinical disease, because the numerous memory cells promptly inactivate the microorganism.

Antigen Presentation to B Cells

Surface immunoglobulin on B cells is capable of binding free extracellular antigen. B cell epitopes (the parts of an antigen bound by immunoglobulin) are typically on the surface of antigen molecules, and antigen does not need to be metabolized by the host for B cells to recognize it. Some B cell protein epitopes consist principally of linear peptides, but many epitopes are "conformational," comprised of distant regions of the primary sequence that are adjacent in the mature, folded protein. Furthermore, B cells can recognize not only proteins but also several other types of organic chemicals, including carbohydrates, DNA, and many drugs. In secondary lymphoid tissues, follicular dendritic cells (FDC) form a network in the B cell rich areas, or follicles. FDC have receptors for complement and for the Fc portion of antibody, and are therefore able to bind antigen when it is part of an antigen-antibody complex (Tew et al., 1990). Antigen may be held by FDC for very long periods and this may contribute to the phenomenon of immunological memory.

Antigen Presentation to T Cells

The Ti chains of the T cell antigen receptor do not respond to free antigen; they only detect antigen which is bound by MHC (Major Histocompatibility Complex) molecules on the surface of other cells. The normal function of MHC molecules is to present an antigen to T cells. There are two major types of MHC molecules— Class I and Class II, features of which are summarized in Table 3. The genes

Table 3, Summary of MHC Class I and Class II Molecules

MHC chains in antigen-binding

Associated molecule on cell sur

Expression

Source of antigen

Size of peptide*

Compartment where peptide

binds to MHC

Type of T cell bound

Type of T cell response

Major role of T cell response

site

•face

Class 1

1 chain only

P2-microglobulin

Most nucleated cells

endogenous

9

endoplasmic reticulum

CD8

Cytotoxic

Antiviral

Class II

2 chains, a and P

None

Dendritic cells

B cells

Monocytes/macrophages

exogenous

13-17

endosomal

CD4

Help to antibody

formation; macrophage

activation

Co-ordination of immune

response

Note: * Length in amino acids

Page 68: Immunity

Cell'to-Cell Interactions 53

encoding both types are located on the short arm of human chromosome 6. The extracellular domains of MHC molecules contain an antigen-binding site, which consists of two roughly parallel alpha helices on a beta-pleated sheet. Antigen, in the form of a linear peptide, is bound in the space between the helices. The membrane proximal regions, between the antigen-binding site and the cell mem­brane, consist of immunoglobulin-like domains.

In Class I molecules, a single polypeptide chain comprises the antigen binding site. This chain is non-covalently bound to another immunoglobulin superfamily member, p2-microglobulin. Class I MHC molecules are very widely expressed, and can be found on almost all types of nucleated cells, though in the absence of interferon their expression may be difficult to detect on some cells (Germain and MarguHes, 1993).

In the case of Class II molecules, two separate polypeptide chains, alpha and beta, together make up the antigen binding site. Cells expressing MHC Class II molecules are sometimes collectively referred to as "antigen presenting cells," but this term is often employed loosely, and in this chapter, the names of specific cell lineages will be used. MHC Class II, which is much less widely expressed than Class I, is most abundant on dendritic cells. Their major function is to present antigen to T cells; they are separate from the follicular dendritic cells described above (Stein-man, 1991). Dendritic cells develop in the bone marrow, migrate through the bloodstream, and lodge in the tissues as Langerhans cells in the skin, or as mucosal dendritic cells in mucosal sites. After they bind antigen, they travel through afferent lymphatics to lymph nodes, where they lodge in the T cell areas and are known as dendritic or interdigitating cells. These cells then make contact with many different T cells which pass through these regions. Dendritic cells are very effective at activating T cells and, unlike other types of Class II positive cells, can stimulate primary T cells, i.e. those which have not previously encountered antigen. Dendritic cells are also found in the thymus, where they are involved in T cell development (see Chapter 14.)

The other cell types that express Class II MHC abundantly are B cells and macrophages. Both can present antigens to T cells, but are more effective at stimulating memory T cells, i.e., those that have previously been exposed to antigen. B cells are efficient at presenting antigen that has been captured and internalized after binding to their surface immunoglobulin. Because of the high affinity of surface immunoglobulin for antigen, this is an effective system for presentation of antigen when it is present in low amounts. Macrophages circulate in the bloodstream as monocytes and can enter the tissues at sites of inflammation. They can present antigen derived from organisms that have been phagocytosed.

There are two different intracellular pathways of antigen presentation (Germain and Margulies, 1993). Class II MHC deals with antigen that has been derived from outside the cell and internalized, where it enters an endosomal compartment and is degraded by proteolytic digestion. Some of the resulting peptides, typically of

Page 69: Immunity

54 WILLIAM A. SEWELL and RONALD PENNY

around thirteen to seventeen amino acids, bind firmly to the antigen binding cleft

of the MHC molecules and the combination of peptide and MHC is then expressed

on the cell surface. In the case of Class I, the peptides that bind to MHC are produced

from proteins synthesized within the cell and are known as endogenous. These

peptides may be derived from proteins of intracellular microorganisms such as

viruses, and are mostly 9 amino acids long. Binding of peptide to MHC takes place

in the endoplasmic reticulum, and the MHC molecule, with peptide bound to it, is

then transported to the cell surface.

The structure on the T cell that binds to the combination of peptide and MHC is

known as the T cell receptor complex. This consists of two parts, the Ti chains and

the CD3 complex. The Ti chains are structurally related to immunoglobulin and

their variable domains are different in each newly formed T cell (see Chapter 5).

The Ti chains bind to the combination of antigenic peptide and MHC molecules.

The CD3 component is identical on all T cells. It is not expressed on other types of

cells and is therefore the most widely used surface marker to identify and enumerate

T cells in diagnostic immunology laboratories.

Two other molecules on the T cell surface, CD4 and CDS, aid in stabilizing the

interaction between the MHC/peptide and the Ti chains. They bind to Class II and

Class I MHC molecules respectively. MHC Class I molecules therefore present

antigen to CDS T cells. As this class of T cells is predominantly involved in

cytotoxic responses, this interaction enables cytotoxic T cells to recognize and

destroy virally infected cells that present antigen to them. Because Class I MHC is

so widely expressed, almost any cell that is infected by a virus can be destroyed by

cytotoxic T cells. By contrast, CD4 cells respond to antigen presented by highly

specialized MHC Class II positive cells and have a more general role in co­

ordinating immune responses.

For optimal stimulation of T cells, other cell surface molecules are also involved.

This process is sometimes known as co-stimulation. A scheme of the interaction

between a CD4 T cell and a cell presenting the antigen is shown in Figure 1. The

most important co-stimulatory molecule is CD2S, which is present on the T cell

surface, and binds to B7-1 (CDSO) or B7-2 on dendritic cells, B cells and macro­

phages (Bluestone, 1995). CD2S, B7-1 and B7-2 belong to the immunoglobulin

superfamily. For a full T cell response, it is necessary for CD2S to be engaged.

Other molecules contribute to adhesion between T cells and cells presenting

antigen (Springer, 1990). These include the immunoglobulin superfamily pair CD2

and CD5S (LFA-3). CD2 is on T cells and binds to CD5S which is expressed on a

number of other types of cell. Another important pair of adhesive molecules is

LFA-1, an integrin, and CD54 (intercellular adhesion molecule-1, ICAM-1), a

member of the immunoglobulin superfamily. Both of these can be either on the T

cell or on the cell presenting the antigen. Other members of the ICAM family,

ICAM-2 (CD 100) and ICAM-3 (CD50) can also bind to LFA-1.

Page 70: Immunity

Cell-to-Cell Interactions 55

Dendritic cell/ B cell/ Macrophage

plasma \ ^ ^ membrane

extra­cellular region

plasma membrane/^

Tcell

MHC Class

a, p

CD80 CD58 CD54 (B7/BB1) (LFA-3) (ICAM-1)

CD2

c o n a/18 (LFA-1)

Figure 1, Antigen presentation to CD4+ T cells. Several of the important cell adhesion

events are shown. The MHC Class II positive cell is shown at the top of the figure, and

the T cell is underneath. The antigen-binding site on the MHC Class II molecule is

visualized end-on as is the linear peptide antigen, marked by #. The variable (V) and

constant (C) domains of the Ti chains are shown. Each circle represents an immuno-

globulin-like domain. In this figure, CD54 is on the cell presenting the antigen and

CD11 a/18 is on the T cell; adhesion can also occur between CD54 on the T cell and

CD1 la/18 on the other cell.

Once stable interaction between a T cell and a cell presenting antigen takes place, the T cell becomes activated and its total amount of protein and RNA increases markedly over the next 12-24 hours. Activated cells secrete the cytokine IL-2 and express IL-2 receptors on their surface (Smith, 1988). After IL-2 binds to IL-2 receptors, the cell enters the S phase of the cell cycle, that is, synthesizes DNA, and clonal expansion commences. This process is sometimes known as autocrine proliferation, because IL-2 is secreted by, and acts on, the same cells. The activated T cells are able to interact with other types of cells.

EFFECTOR MECHANISMS OF T CELLS

Interaction Between T and B Cells

In most antibody responses, T and B cells collaborate. B cells present antigen to T cells, and T cells respond by providing "help" to the process of antibody formation. T cells and B cells may collaborate in response to different epitopes on

Page 71: Immunity

56 WILLIAM A. SEWELL and RONALD PENNY

the same antigens. T cells need not recognize the same part of the molecule bound by the B cell. As described above, T cells will only recognize linear peptides, whereas B cells mainly recognize epitopes on the surfaces of proteins and other types of organic molecules. Provided the molecular complex recognized by the B cell contains protein, it is possible for the B cell to receive T cell help to produce antibodies to a variety of different types of structure in the antigen. An example is the response to tetanus toxoid, where the antibodies neutralize tetanus toxin by binding to its carbohydrate, whereas T cell help depends on the protein components of the toxoid.

In the absence of T cells, a response can be elicited to certain types of antigens. These are typically large polymers with multiple repeating subunits, such as polysaccharide antigens of bacterial capsules. These antigens are known as T-independent antigens. The responses are often limited to the IgM class of antibody, and the induction of memory is relatively poor.

T cell help contributes to the activation and proliferation of B cells, to their antibody secretion and to their differentiation into plasma cells, and is essential for class switching and affinity maturation. Class switching can take place within the activated clone so that IgG, IgA, or IgE classes of antibody may be made in addition to the IgM which is most prominent in the earliest responses. Class switching is achieved by the use of different parts of the heavy chain immunoglobulin gene, so that different constant regions are expressed. Within a clone, the variable domains are not substituted, so that the antibodies, although of different classes, all bind to the same epitope. However, more subtle changes do take place in the variable domains, leading to secretion of high affinity antibody, a process known as affinity maturation, which is discussed in Chapter 14.

Activated T cells help B cells in two ways: (1) by secretion of several cytokines (Seder and Paul, 1994); (2) by expression of a surface molecule on T cells, the CD40 ligand (CD40L) (Clark and Ledbetter, 1994). The molecular basis of T cell help to B cell antibody formation has only been elucidated over the last several years, and it is possible that other, as yet unidentified, cytokines and cell surface molecules will be found to play an important role.

In the development of antibody responses, T cells and B cells are closely apposed, and cytokine secretion has been observed predominantly from the pole of the T cell closest to the B cell. Thus, only minute amounts of cytokines need to be secreted into the small intercellular space between the lymphocytes to reach a concentration sufficient to activate the cytokine receptors on the B cell surface. In addition, CD40L binds to CD40 on the B cell surface (Figure 2). All these factors act together on the B cell, along with signals derived from the binding of antigen by surface antibody on the B cell.

The cytokines include IL-2, IL-4, IL-6, IL-10, IL-13, IL-14, interferon-gamma (IFN-y) and transforming growth factor-beta (TGF-P). Different cytokines can

Page 72: Immunity

Cell-tO'Cell Interactions 57

surface immuno­globulin

A native antigen

BCELL

L. processing and presentation of antigen

proliferation antibody formation class switching

TCELL

MHC Class I TcR

X^I

CD40 CD40L

cyto­kines

activation

antibody secretion

Figure 2. Collaboration between B and T cells. Antigen, shaded, enters B cells after

binding to surface immunoglobulin, is processed into small peptide fragments, and

bound to MHC Class II. The combination of MHC and peptide is then expressed on

the cell surface and activates the T cell by engagement of the T cell receptor (TcR).

Other cell surface molecules involved in this process are shown in Fig. 1. The activated

T cell expresses CD40L on its surface and secretes several cytokines (see text for

details). The B cell then proliferates, secretes antibody and undergoes class switching.

produce similar effects, and the same cytokine can produce different effects. No single cytokine is either necessary or sufficient for B cell activation and prolifera­tion (Paul, 1991). Cytokines alone do not cause proliferation in resting B cells; the cytokines must be accompanied by CD40L. IL-2, IL-4, IL-6, IL-10, IL-13, and IL-14 all play a role in the stimulation of B cell proliferation (Mosmann and Moore, 1991; Nicola, 1994). This gives rise to clonal expansion of antigen- specific B cells. Cytokines and CD40L not only increase B cell numbers but also promote B cell differentiation. In the presence of CD40L and cytokines, some cells in the activated B cell clone sv itch the class of antibody they make to IgG, IgA or IgE. Class switching can be modulated by the type of cytokines secreted by T cells. Several of the cytokines listed above can contribute to class switching to IgG. IL-4 and IL-13 are particularly important in the induction of IgE antibody, and switching to IgE can be inhibited by IFN-y. IL-10 and TGF-p are important in the induction of IgA antibody secretion.

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58 WILLIAM A. SEWELL and RONALD PENNY

Interaction Between T Cells and Macrophages

Macrophages have a major role in controlling the spread of various microorgan­isms, including intracellular bacteria such as mycobacteria, and a large number of fungi and protozoa. Macrophages are most effective in controlling such organisms when they have been activated by T cells, principally of the CD4 subset. Activated macrophages are larger, more effective in phagocytosis and lysis of microorgan­isms, and they secrete a large number of factors. This component of the immune system is traditionally measured by delayed hypersensitivity reactions to the local deposition of antigen. Similar reactions take place in responses to many microor­ganisms, in transplantation rejection and in certain autoimmune diseases. There are several T cell cytokines that have an important role in such reactions, the most important of which is IFN-y (Farrar and Schreiber, 1993). It stimulates the micro­bicidal activity of macrophages on intracellular and extracellular microorganisms, enhances the secretion of complement proteins, and promotes the production of other cytokines such as TNF-a. In addition, it promotes antigen presentation, as described above. Other T cell cytokines which stimulate macrophage function include lymphotoxin (also known as TNF-(3) and granulocyte macrophage-colony stimulating factor (GM-CSF). The importance of CD4 T cells and macrophages is demonstrated in conditions where deficits occur in both compartments, as seen in HIV infection, when infection with a wide range of bacteria, fungi and protozoa takes place.

Thl and Th2 Cells

It has been of great interest whether the T cells that help antibody formation are the same as those that activate macrophages. As described above, it is now clear that different sets of cytokines are involved in mediating these two types of effector functions. A number of stable T cell clones have been prepared experimentally that secrete one but not the other group of cytokines. T helper 1 (Thl) clones, but not Th2 clones, make IL-2, IFN-y, and TNF-P, and elicit delayed hypersensitivity reactions. By contrast, Th2 but not Thl clones make IL-4, IL-5, IL-6, IL-10, and IL-13 and are more effective than Thl in helping antibody formation. These patterns were first described in the mouse, but have subsequently been reported in human T cell clones. These patterns have been best described in Leishmania

infections, where Th 1 responses are associated with healing and Th2 with progres­sive disease. Many T cell clones do not conform to the Thl and Th2 models described above. However, this model has enabled immune responses to be classified as either Type 1, associated with the cytokines made by Thl clones, or Type 2, associated with Th2 cytokines. Type 1 responses are found in protective reactions to mycobacteria and in a number of auto-immune diseases. Type 2 responses are present in infestation with helminthic parasites and in allergic reactions (Mossman and Sad, 1996).

Page 74: Immunity

Cell'tO'Cell Interactions 59

Target cell

plasma \ ^ membrane

extra­cellular region

plasma membrane

CDS Tcell

CD58 CD54 MHCCIassI (LFA-3) (ICAM-1)

CD2

CDl la / IS (LFA-I)

Figure 3. Target recognition by CDS T cells. Several of the important cell-cell

adhesion events are shown. As in Fig. 1, the antigen-binding site and the peptide

antigen are shown end-on, with the antigen indicated by #. P2m stands for P2-'T^'cro-

globulin. The variable (V) and constant (C) domains of Ti are shown.

Cytotoxic Cells

Some T cells, mainly of the CDS phenotype, are cytotoxic to other cells of the

body. This is thought to be a mechanism whereby virally infected cells can be

eliminated before they can produce and release large amounts of infective virus.

Cytotoxic T cells are also involved in the rejection of transplants and in the lysis

of certain types of tumor cells. As described above, CDS T cells recognize antigen

presented by Class I MHC (Germain and Margulies, 1993). This system thereby

enables cells to alert T cells to the fact that they are infected by a virus, even before

intact viral particles are made, or complete viral proteins are embedded in the cell

surface. In addition to interaction between the T cell receptor, MHC Class I, antigen,

and CDS, other cell surface molecules stabilize the interaction between the T cell

and its target. These include binding between CD2 on the T cell and CDS8 on the

target, and interaction between LFA-1 and CD54 (Figure 3). After cytotoxic T cells

recognize antigen, they lyse their targets using mechanisms common to another

type of cytotoxic cell, natural killer (NK) cells, which are defined in the blood as

large granular lymphocytes. The mechanism of cytotoxicity and target cell death

is described in detail in Chapter 14.

Page 75: Immunity

60 WILLIAM A. SEWELL and RONALD PENNY

Cytotoxic T cells can secrete cytokines, which contribute to immune responses. A prominent cytotoxic T cell product is IFN-y, which has antiviral activity.

INTERACTION BETWEEN LEUKOCYTES AND ENDOTHELIUM

Leukocytes circulate in the bloodstream and leave at well defined locations. T and B lymphocytes have complex recirculation pathways. They travel in the blood­stream and can enter lymphoid organs through high endothelial venules (HEV). Resting T lymphocytes leave vascular endothelium by binding to HEV in lymphoid organs. Resting T cells express high levels of L- selectin, which binds to sulphated carbohydrates on HEV. Lymphoid organs are major sites where lymphocytes may make contact with antigen. Lymphocytes leave lymph nodes by efferent lymphatics and return to the bloodstream by the thoracic duct. This large flow of lymphocytes through the lymphoid organs enables antigen to encounter large numbers of lymphocytes, so that the tiny proportion of lymphocytes with antigen receptors that recognize individual antigens will have the chance to make contact and be activated. Although T and B cells are involved in recirculation processes, T cells are more active, moving more rapidly through lymphoid tissue.

At inflammatory sites, lymphocytes and other leukocytes are able to bind to endothelium and enter the tissues. Among T cells, memory cells, which have previously contacted antigen, are more effective than naive cells in this process. Compared with naive cells, they have lower surface levels of L-selectin but higher amounts of several adhesion molecules in the immunoglobulin and integrin super-families. Expression of E-selectin by activated endothelium is an important factor in the egress of neutrophils, monocytes and some memory T lymphocytes into sites of inflammation (Bevilacqua and Nelson, 1993).

Interaction between leukocytes and endothelium first depends on contact be­tween selectins and their ligands, causing cells to roll along the endothelium. This is followed by the secretion of factors, such as the cytokine IL-8 and other chemotactic factors, by the endothelium. These act on receptors on leukocytes to increase the stickiness of their integrins, leading to the arrest of the leukocyte (Figure 4) (Butcher and Picker, 1996). These integrins, such as LFA-1 and VLA-4 (CD49d/CD29), may bind to members of the immunoglobulin superfamily, namely to ICAM-1, -2, or -3, and to VCAM-1 (CD 106), respectively. Once the leukocyte has stopped, it is then able to migrate between endothelial cells and reach an inflammatory focus. This complex system, involving at least three recognition steps (Figure 4) and different ligand-receptor pairs at each step, enables particular types of leukocytes to home to specific locations, even though some of the processes employ structures that are widely expressed. For example, although L-selectin is present on the surface of many types of leukocytes, it is only lymphocytes that enter secondary lymphoid tissue through HEV.

Page 76: Immunity

Cell'to-Cell Interactions 61

I II III

LEUKOCYTE LEUKOCYTE LEUKOCYTE

ENDOTHELIUM ENDOTHELIUM ENDOTHELIUM

kfiy

II

r L-selectin

/

Carbohydrate

ligand

# soluble

factor

1

ij i integrin

e.g. LFA-1

rPO

f\ integrin

receptor

e.g. ICAM-1

Figure 4. Interaction between leukocytes and endothelium. The three-step model is

illustrated. In the first step, adhesion is initiated by binding between selectins and their

carbohydrate ligands. In the figure, L-selectin on the leukocyte is shown. In the case

of E-selectin and P-selectin, the selectin is on the endothelium. In the second step, the

endothelium secretes a cytokine or other soluble factor, which acts on receptors on

the leukocyte. In the third step, the adhesiveness of integrins on the leukocyte Is rapidly

up-regulated, enabling firm binding to ligands on the endothelium. The conforma­

tional change shown in the integrin is highly schematic.

SUMMARY

The development of full immune responses requires the interaction between

different types of cells. This is achieved by molecules on the surface of cells making

contact with each other, and by the secretion of cytokines which convey information

from one cell to another. Many of these interactions take place in secondary

lymphoid tissue, i.e., lymph nodes, spleen, tonsils, Peyer's patches and other

mucosal-associated lymphoid tissues. In these sites, T cells, B cells, antigen

presenting cells and antigen may be all found together.

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62 WILLIAM A. SEWELL and RONALD PENNY

Cell-to-cell interaction is important in the presentation of antigen to B and T cells. In particular, T cells only respond to antigen when it is presented bound to MHC molecules on the surface of other cells. In this process, several other molecules on the T cell surface bind to surface molecules on the cell presenting the antigen. T cell responses are principally mediated by the secretion of cytokines, and are often directed at the very cell that presented antigen to the T cell. T cells greatly augment the capacity of B cells to produce antibody and the ability of macrophages to lyse microorganisms. Cytotoxic lymphocytes recognize adjacent virally infected cells by contact between cell surface molecules, and lyse such cells by the secretion of toxic granules. Egress of leukocytes from the bloodstream is regulated by contact between cell surface molecules and by cytokines.

REFERENCES

Bevilacqua, M.P., & Nelson, R.M. (1993). Selectins. J. Clin. Inv. 91, 379-387.

Bluestone, J.A. (1995). New perspectives of CD28-B7-mediated T cell costimulation. Immunity 2,

555-559.

Butcher, E.C., & Picker, L.J. (1996). Lymphocyte homing and homeostasis. Science 272, 60-66.

Clark, E.A., & Ledbetter, J.A. (1994). How T and B cells talk to each other. Nature 367, 425-428.

Farrar, M.A., & Schreiber, R.D. (1993). The molecular cell biology of interferon-y and its receptor.

Ann. Rev. Immuol. 11,571-611.

Germain, R.N., & Margulies, D.H. (1993). The biochemistry and cell biology of antigen processing and

presentation. Ann. Rev. Immunol. 11, 403-450.

Hynes, R.O. (1992). Integrins: Versatility, modulation, and signaling in cell adhesion. Cell 69, 11-25.

Mosmann, T.R., & Moore, K.W. (1991). The role of IL-10 in cross-regulation of Th 1 and Th2 responses.

Immunol. Today 12, A49-A53.

Mosmann, T.R., & Sad, S. (1996). The expanding universe of T-cell subsets: Thl, Th2 and more.

Immunol. Today 17, 138-146.

Nicola, N.A. (Ed.) (1994). Guidebook to cytokines and their receptors, pp. 1—261. Sambrook and Tooze

Publication at Oxford University Press, Oxford.

Paul, W.E. (1991). Interleukin-4: A prototypic immunoregulatory lymphokine. Blood 77, 1859-1870.

Seder, R.A., & Paul, W.E. (1994). Acquisition of lymphokine-producing phenotype by CD4+ T cells.

Ann. Rev. Immunol. 12, 635-673.

Smith, K.A. (1992). Interleukin-2. Curr. Opin. Immunol. 4, 271-276.

Springer, T.A. (1990). Adhesion receptors of the immune system. Nature 346, 425-434.

Steinman, R.M. (1991). The dendritic cell system and its role in immunogenicity. Ann. Rev. Immunol.

9,271-296.

Tew, J.G., Kosco, M.H., Burton, G.F., & Szakal, A.K. (1990). Follicular dendritic cells as accessory

cells. Immunol. Rev. 117, 185-211.

Williams, A.F., & Barclay, A.N. (1988). The immunoglobulin superfamily—Domains for cell surface

recognition. Ann. Rev. Immunol. 6, 381—405.

RECOMMENDED READINGS

Germain, R.N. (1994). MHC-dependent antigen processing and peptide presentation: providing ligands

for T lymphocyte activation. Cell 76, 287-299.

Paul, W.E., & Seder, R.A. (1994). Lymphocyte response and cytokines. Cell 76, 241-251.

Springer, T.A. (1990). Adhesion receptors of the immune system. Nature 346,425-434.

Page 78: Immunity

Chapter 4

Immunological Tolerance

J.F.A.P. MILLER

Introduction 64 Historical Background 64

Possible Fate of Self-Reacting Lymphocytes 68 Self-Tolerance in T Cells 70

Intra-thymic Clonal Deletion 70

Post-thymic Tolerance 72

Self-Tolerance in B Cells 74 Artificially Induced Tolerance 77

In the Living Animal {In Vivo) 11

In Tissue Culture {In Vitro) 80

Autoimmunity and the Breakdown of Immunological Self Tolerance 81 Potential Therapeutic Applications 82 Summary 82 Recommended Readings 84

Principles of Medical Biology, Volume 6 Immunobiology, pages 63-84. Copyright © 1996 by JAI Press Inc.

All rights of reproduction in any form reserved. ISBN: 1-55938-811-0

63

Page 79: Immunity

64 J.F.A.P. MILLER

INTRODUCTION

The immune system provides us with a powerful weapon against infections, eliminating invading micro-organisms and killing infected cells. Yet, under normal conditions, it does not attack the body's own healthy cells. How, then, does it distinguish what is self from what is foreign or nonself, and how does it tolerate self?

Immune cells (lymphocytes) randomly generate a great diversity of antigen-specific receptors (Chapters 1,2, and 5). Therefore, some cells will be self-reactive and have the potential to cause damage. To prevent self-reactivity, they should be eliminated in some way, either by being functionally inactivated or physically deleted. This state of unresponsiveness induced by exposure to self antigens is known as immunological tolerance. It is antigen specific and can be mimicked experimentally by antigen exposure under a variety of conditions, as will be described in this chapter.

The inability to respond to self is not preprogrammed in the individual's gene pool (the germline) but is acquired during development. Consider, for example, the immune response to skin in laboratory animals of highly inbred strains in which individuals in any given strain are identical to one another in their genetic make-up. Mice of incompatible strains A and B can respond immunologically against each other's skin by reciprocally rejecting a skin graft. However, although the first generation offspring (F1 hybrid) of matings between A and B (which express the antigens of both parents) will have inherited the potential to react against the A or B skin, they cannot reject such skin. Yet the ability to reject this skin reappears in some of the F2 progeny of matings between the Fl hybrids. The discrimination between what is self and what is nonself is therefore not encoded in the germline but must be learned.

What constitutes self? From an immunological viewpoint, "self must encom­pass all antigenic determinants or "epitopes" encoded in the individual's DNA. Other epitopes must be considered as nonself Yet there is nothing in the structure of a molecule which would allow a given individual to distinguish self from nonself Attributes other than the structural characteristics of an epitope must therefore also be sensed. Among these are the stage in development when lymphocytes are first confronted with antigen, the site of encounter, the nature of the cells presenting the epitopes, and the production by these cells of so-called "co-stimulatory" molecules which play a role in influencing lymphocyte responsiveness. These attributes are discussed below.

HISTORICAL BACKGROUND

At the turn of the century, Paul Ehrlich (1900) realized that some mechanism must operate to prevent autoantibody formation. He coined the term "horror auto-toxicus", implying the need for a "regulating contrivance" to stop the production

Page 80: Immunity

Immunological Tolerance 65

of such antibodies. In 1938, Traub experimentally induced antigen-specific toler­ance by injecting a particular virus (LCMV) into fetal mice in the uterus of their mothers. The injected mice maintained the viral infection throughout life, but unlike uninoculated mice, they did not produce neutralizing antibodies following virus challenge in adult life. That cells carrying different antigens could develop and co-exist within the same host, was reported by Owen in 1945. He described an "experiment of nature" occurring naturally in non-identical twin cattle with fused placentae (Figure 1). The developing fetuses exchanged hemopoietic cells via the shared placental blood vessels and each animal carried throughout life the erythro­cyte markers of both calves. They exhibited life-long tolerance to the otherwise foreign cells in that they could not mount an antibody response to each other's erythrocyte antigens. This observation led Burnet and Fenner to postulate in 1949 that "body cells carry 'self-marker' components which allow recognition of their 'self character", and that antigen encounter in early life was the critical factor determining responsiveness and hence recognition of nonself epitopes. The hy­pothesis seemed logical, since the immune system is usually confronted with most self components before birth and only later with nonself antigens. The classical experiments of Billingham, Brent and Medawar performed in 1953 thoroughly vindicated this hypothesis (Figure 2). They induced specific immunological toler­ance to foreign skin grafts (allografts) in mice by injecting foreign ("allogeneic") cells in fetal life or in the immediate neonatal period. By contrast, injection of such

reciprocal skin

grafts

accepted

Figure 1, Natural tolerance in nonidentical bovine twins fused at the placenta.

Placental fusion leads to an exchange of blood cells in fetal life. Among these cells

are stem cells or ancestral cells capable of self renewal and differentiation to more

mature cells. After birth, the separated twins were shown by Owen to be permanently

tolerant of each other's tissue type. They were chimeric, i.e., cells, which normally

would be incompatible, co-existed in each twin and skin grafts from either twin failed

to be rejected.

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66 J.F.A.P. MILLER

Donors Recipients

' bone V ' ^ "•

marrow

cells

skin

Figure 2. Artificial induction of specific immunological tolerance. Billingham, Brent

and Medawar were the first to establish immunological tolerance experimentally. Fetal

or newborn mice of a given strain A were inoculated with hemopoietic cells obtained

from donor mice of an incompatible strain B. The recipients were later grafted with

skin from donors of strain B and failed to reject such skin, although they could reject

skin from donors of a third incompatible strain C in the normal way.

cells first in adult mice generally induced an acceleration of the immune response to a subsequent skin graft from the same donor.

The phenomenon of immunological tolerance could easily be interpreted within the framework of Burnet's 1957 clonal selection theory (see Figure 3 in Chapter 1). This states that an immunocompetent cell bearing a given antigen-specific receptor is selected by the antigen and then activated to divide. As a result, it produces a "clone" of daughter cells, all with the same specificity. Antigens encountered after birth would activate specific clones to proliferate and produce antibody, whereas antigen encountered before birth would induce the deletion of these clones, which Burnet termed "forbidden clones." Implicit in this theory is the requirement for prenatal generation of the entire immune repertoire. This of course is not the case, since lymphocyte differentiation continues during postnatal life. The key factor in determining the nature of the response, whether tolerance or immunity, cannot therefore be the developmental stage of the individual but rather the state of maturity of the lymphocyte at the time it encounters antigen. This was suggested by Lederberg in 1959, in a modification of the clonal selection theory: contact of immature lymphocytes with antigen would lead to deletion or functional inactiva-tion, whereas contact with mature cells would result in immunity.

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Immunological Tolerance 67

The 1960s witnessed key discoveries which established the immunological competence of the small lymphocyte, the crucial role of the thymus in the devel­opment of the immune system, and the existence of two interacting subsets of lymphocytes, T and B cells (see Chapters 1 and 2). This set the stage for a thorough investigation of the cellular mechanisms involved in the induction of tolerance or immunity. Both T and B cells were shown to be independent potential targets of tolerance induction, immature cells generally being more susceptible. Since most of the cells of the immune system in early life have yet to reach maturity, the individual is highly susceptible to tolerance induction at this stage.

In 1970, Bretscher and Cohn proposed that the distinction between immunity and tolerance depended on whether the immunocompetent lymphocyte received a "second" or co-stimulator signal in addition to the epitope presented to it. Lafferty and his group (1983) provided much evidence in transplantation systems, support­ing the view that specialized "professional" antigen-presenting cells (APC) were required for the activation of T cells, primarily because these APC were fully equipped to deliver the second signal. For example, pancreatic islet tissue could be transplanted into incompatible hosts without rejection provided passenger leuko­cytes (APC capable of delivering the second signal) were removed by culturing this tissue under special conditions. Survival of the tissue deprived of APC was attributed to its inability to deliver the "second signal" for initiating an immune response. If antigen in combination with the second signal was later supplied by injecting leukocytes expressing the graft antigen, then the islet tissue was rejected.

Until recently, only artificially induced tolerance had been investigated. This was done by injecting antigens or foreign cells into an animal and following the fate of responding T or B cells under a variety of circumstances. Such an experimental system could not reveal to what extent unresponsiveness resulting from the inocu­lation of such foreign material mimicked self tolerance or was an epiphenomenon reflecting the induction of some immunoregulatory feedback loop inhibiting an incipient response. Only recently has the introduction of transgenic technology (Palmiter and Brinster, 1986) made it possible to investigate natural tolerance to self constituents. This technique allows a known gene to be introduced into mice of defined genetic background (Figure 3). Provided the gene becomes integrated appropriately, its effects upon the development of the immune system can be studied. If the introduced gene is linked to a tissue-specific promoter, its expression can be directed to particular cell types: for example, a fissue compatibility (histo­compatibility) gene linked to the promoter for insulin will be expressed in the insulin-producing cells of the pancreas. The encoded product of the introduced "transgene" is treated by the body essentially as an authentic self antigen and its effects can be studied in vivo in the absence of the injury and inflammation that accompany the grafting of foreign cells or tissues. Furthermore, the parent strain and the transgenic strain provide ideal congenic partners, i.e. individuals identical in their genetic make-up except for the introduced transgene. They can therefore

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68 J.F.A.P. MILLER

recently inseminated

female mouse

• one-cell embryo

( ^ DNA micro-injected

into male pronucleus

injected embryos transferred to

oviduct of pseudo-pregnant

female

^^—\ ^ 3 ^ \ tails of offspring tested by

^ ^ " ^ ^ ^ ^ ^ " ^ ^ j i 'Southern' technique - « = ^ - — = ^ f o r incorporation of injected DNA

Figure 3, Transgenic technology. One-cell embryos are obtained from a recently

inseminated pregnant female mouse and the DNA corresponding to a known gene is

micro-injected into the pronucleus. The inoculated eggs are inserted into the oviduct

of a pseudo-pregnant female (one mated with a vasectomized male). Small sections

of tails from offspring are tested for transgene incorporation by Southern blotting, i.e.

by determining the presence or absence of the inoculated DNA with a specific probe.

Mice that have integrated the gene are known as transgenic mice.

be used for control experiments and lymphocyte transfer studies. Transgenic models dealing with the problem of immune tolerance have used two independent strategies: the introduction of genes encoding either defined antigens or rearranged antigen-specific receptors (either antibodies or T cell receptors).

POSSIBLE FATE OF SELF-REACTING LYMPHOCYTES

As stated above, immunological tolerance demands that self-reactive lymphocytes should be either inactivated or deleted. Experiments have established five possible ways in which self-reactive lymphocytes may fail to respond to self antigens:

1. Clonal ignorance: the self antigen may not, under normal circumstances, be perceived by self-reactive lymphocytes, either because it is not situated in the pathway of circulating cells, or because it is present on cells which cannot activate lymphocytes.

2. Clonal deletion: the self-reactive cells are physically deleted from the repertoire at some stage during their maturation.

3. Clonal abortion: the further differentiation of the immature self-reactive cell is prevented.

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Immunological Tolerance 69

4. Clonal anergy: the intrinsic mechanism of response is downregulated in the mature self-reactive cell.

5. Suppression: activity of the mature self-reactive cell is continuously inhib­ited by its interaction with other cells, such as cells producing inhibitory lymphokines or cells reacting against the antigen-receptor itself

Which of these fates awaits the self-reactive lymphocyte depends on numerous factors, among which are: (1) the stage of maturity of the cell being silenced, (2) the affinity of its receptor for the autoantigen, (3) the nature of this antigen, (4) its concentration, (5) its tissue distribution, (6) its pattern of expression, and (7) the availability of co-stimulatory signals. In general, in the absence of co-stimulation, antigen is more likely to signal a cell negatively, switching off its ability to respond (Figure 4). More work must, however, be done to define precisely the exact conditions required for inducing anergy, aborfion or deletion, and the biochemical and molecular mechanisms which lead to those results.

peptide

no costimulation costimulation

Figure 4. Co-stimulation. Both antigen and a second signal are required for T cells to be activated. The first signal is transmitted through the antigen-specific T cell receptor (TCR) provided this is specific for a peptide (a protein degradation product) accommodated in the groove of an MHC molecule on the surface of antigen-present­ing cells (APC). This signal alone fails to activate the T cell and may even prevent its capacity to respond or render it tolerant, if it is not accompanied by the second or co-stimulatory signal. Other molecules on the surface of activated "professionar' APC, such as the B7 molecule shown here, bind to complementary T cell molecules, such as CD28, and transmit a powerful co-stimulatory signal leading to T cell activation.

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70 J.F.A.P. MILLER

SELF-TOLERANCE IN T CELLS

The thymus is responsible for the development of T lymphocytes, whether cyto­toxic T cells (those equipped to destroy foreign or infected cells) or helper T cells (those able to assist other lymphocytes in performing their functions). In the thymus, T cells develop from precursors which have not undergone rearrangement of their T cell receptor (TCR) genes. Once these genes are rearranged, the T cells express TCR which enable them to perceive antigen degradation products or peptides which fit in the groove of the MHC molecules encoded by the body's own major histocompatibility complex (MHC) (Chapters 1 and 7). It seems therefore reasonable that it is within the thymus that self-reactive T cells are prevented from exerting their function.

Intra-thymic Clonal Deletion

As described in Chapter 1, the high proliferative rate of thymocytes is paralleled by a massive rate of cell death, the vast majority of the double positive, CD4"^CD8' , cells dying within the thymus. Among the factors which account for this are aberrant TCR gene rearrangement, negative selection and lack of positive selection. Developing T cells are positively selected for survival only if they can express TCR that allow them to bind with a certain degree of strength (avidity) to MHC molecules encountered on thymic cortical epithelial cells. The binding presumably protects the cells from programmed cell death and the process of positive selection ensures that the mature T cell will be able to recognize antigen (peptides) that are accom­modated in the binding cleft of self MHC molecules, and so will be self-MHC restricted. This selection will not, however, prevent the differentiation of T cells bearing TCR of high binding strength for self peptides and MHC molecules. Some form of negative selection must therefore operate to silence such self-reactive cells. This generally occurs by the physical deletion of clones of T cells specifically reactive to self-antigens presented intra-thymically as shown experimentally in the late 1980s. For example, in mice transgenic for rearranged TCR directed to the antigen, H-Y, which is expressed only on cells in male mice, H-Y autospecific T cells were deleted in male though not in female mice (von Boehmer, 1990, Figure 5).

Exactly where and when negative selection occurs in thymocyte development depends on a variety of factors, including the accessibility of developing T cells to self-antigen, the combined avidity of the TCR and accessory molecules, CDS or CD4, for the self MHC-self peptide complex, and the identity of the deleting cells. Elimination of self-reactive cells is usually performed by thymic dendritic cells or macrophages which are rich in class I and II molecules and situated predominantly at the cortico-medullary junction (Chapter 1). Other cells involved in deletion may be specialized intra-thymic "veto" cells bearing self epitopes. On being perceived by self-reactive T cells, these veto cells would impart a negative signal killing the

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Immunological Tolerance 71

No T cell deletion. Most H-Y-specific T cells

Most CD8 T cells kill deleted in thymus.

H-Y* target cells. Peripheral T cells show

downregulation of

TCR and CD8.

Figure 5. Intra-thymic negative selection demonstrated in transgenic mice. Mice

were made transgenic for a TCR specific for the male histocompatibility antigen, H-Y,

seen in association with a particular MHC class I molecule. Most peripheral T cells in

female mice of that MHC genotype expressed anti-H-Y TCR and a large proportion of

CDS'*" T cells reacted in response to H-Y in vitro. In male mice of the same genotype,

H-Y specific T cells were deleted in the thymus and most peripheral T cells either

lacked the CD8 co-receptor or expressed low CDS levels.

self-reactive T cell clone (Miller, 1980) (Figure 6). Some of the medullary epithelial

cells may also impose negative selection. Hence, although most self epitopes can

be ubiquitous, those occurring within the thymus are presented to maturing T cells

in a way that leads to the deletion of Burnet's "forbidden clones".

Figure 6. The veto effect. Veto signals may be generated by specialized veto cells (a special type of CDB" T cell), provided the TCR of the T cell (the cell to be vetoed) has bound peptide-MHC complexes presented by the veto cell, and the veto celTs own accessory molecule CDS binds to its usual site on the a3 domain of the class I MHC molecule of the T cell. This may constitute one mechanism ensuring self tolerance.

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72 J.F.A.P. MILLER

Post-thymic Tolerance

Self antigens expressed only outside the thymus may not provoke an immune response if they elude the immune system. This may be the case if they are sequestered in privileged sites away from the circulating routes of T cells, or exposed on certain cell types which do not express MHC molecules and hence cannot present peptides derived from those antigens to T cells. It may also be the case if the auto-antigens occur in amounts too low to be detected by T cells, or if the avidity of the combined TCR and accessory molecules is not high enough for T cells to make effective contact with the autoantigen-presenting cells. Under these conditions, as shown by Zinkemagel et al. (1991), Miller and Heath (1993) and Heath et al. (1995), the T cells ignore the existence of the autoantigens but the resulting lack of T cell activation is not equivalent to tolerance induction since presentation of the autoantigen by professional AFC would immunize.

Evasion of the immune system by extra-thymic autoantigens may be one way in which the hazards of self-reactivity and the occurrence of autoimmune responses may be avoided. Yet it is a precarious one, given that molecules may be released from dying cells and hence processed and presented by professional antigen-

A/b A/b-specifIc TCR

T ceil response

to b-bearing cells

double transgenic

Figure 7, Transgenic models of peripheral Tcel l deletion and energy. Mice of a given

haplotype (e.g., A) were made transgenic for a foreign class I molecule (e.g., b)

expressed, under the influence of a promoter specific for an extra-thymic tissue. They

were then mated to mice transgenic for a TCR directed to ^ (T), such that more than

90% of the T cells were anti-^ and traceable by an antibody specific for that particular

TCR. Double transgenic offspring obtained from these matings were found not to

respond to b-bearing cells in contrast to normal mice. Investigations showed that the

transgenic T cells either ignored the extra-thymic tissue bearing b molecules or had

been rendered anergic and unable to respond. Anergy was associated with downregu-

lation of the TCR and accessory molecules. In some cases the transgenic T cells were

actually deleted following encounter with b in the periphery. The outcome depended

on the tissue expressing the transgene b and the extent of expression.

Page 88: Immunity

Immunological Tolerance 73

presenting cells, such as macrophages, cells which have co-stimulatory function and which are well equipped to activate T cells. As autoimmunity is not the rule, fail-safe mechanisms exist to ensure tolerance induction to extra-thymic autoan-tigens in peripheral T cells. Both peripheral T cell deletion and anergy have been demonstrated under certain experimental conditions using transgenic mice (Figure 7). Evidence is also mounting for the existence of some type of T cell-dependent suppression of potentially autoaggressive T cells. The idea of suppressor T cells was first suggested in 1974 by Gershon, but the failure to isolate a distinct subset of suppressor T cells led many to question their existence. Nevertheless, one well documented way in which T cells may suppress immune response is by the inhibitory effects of cytokines. The release of TGF-p by T cells after some forms of antigen stimulation is one example (A. Miller et al., 1992). Furthermore, the evidence obtained by Mosmann and Coffman (1989) for two types of helper T cells, Thl and Th2 with a distinct antagonistic lymphokine profile (Chapters 1 and 9, Figure 8), strongly suggests that T-cell dependent immunoregulation of lympho­cyte responses is a reality that needs further exploration at both the cellular and molecular levels.

IFN-7 ^ 0

MHC Q peptide "^ ^ complex

Figure 8, Reciprocal regulation of helper T cell subsets. Two subsets of helper T cells,

T h l , and Th2, exist. Their lymphokine products exert an influence on their differen­

tiation and subsequent effector function. Thus, for example, through its production of

IL-10, the Th2 cells may render Thl cells anergic by interfering with the co-stimulator

function of antigen-presenting cells. IL-4 stimulates Th2 development and also inhibits

Thl effector function. This results in a strong enhancement of Th2 activity at the

expense of Th1 cells. On the other hand, interferon-y (IFN-y), produced by Th1 cells

inhibits the proliferation of Th2 cells but not Thl cells. + indicates stimulation, -

indicates inhibition.

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74 J.F.A.P. MILLER

SELF-TOLERANCE IN B CELLS

The production of high affinity antibodies of the IgG class is T-cell dependent (Chapters 1 and 3). Such antibodies are more Hkely to cause tissue damage than low affinity or IgM antibodies. For this reason, and since the threshold of tolerance for T cells is lower than for B cells, the simplest explanation for lack of self-reac­tivity in the B cell repertoire is absence of T cell help resulting from T cell tolerance to self antigens. Nevertheless circumstances do exist in which B cells may become self-reactive unless subjected to tolerance induction. For example, exposure to antigens derived from micro-organisms, expressing both foreign T-cell epitopes and B-cell epitopes cross-reacting with self antigens, could result in a vigorous antibody response (Figure 9). Furthermore, in contrast to the TCR, the Ig receptor on mature, antigenically-stimulated, B cells can undergo hypermutation and may acquire anti-self reactivity. Tolerance must thus be imposed on B cells, both during their development and following antigenic stimulation in secondary lymphoid tissues.

The fate of self-reactive B cells has been determined in transgenic mice, in experiments by Goodnow, Basten and colleagues (1988) and by Nemazee and Burki (1989) (Figure 10 and 11). In these models, tolerization by self-antigens was shown to lead to one of several end results:

1. Clonal deletion. Encounter with cell membrane-associated self-antigens able to cross-link Ig receptors on B cells, led to their elimination from secondary lymphoid tissues (Figure 10). This type of tolerance occurred with self-antigens, irrespective of whether they were expressed on cells located within the bone marrow or elsewhere. In both situations the bone marrow contained residual self-reactive B cells, suggesting that immature B cells were less readily deleted than immature T cells during the early stages of differentiation.

Figure 9. The response of B cells to self or foreign antigens. (1) B cells with Ig receptors

specific for an epitope of a foreign antigen X can engulf the antigen and process it to

produce determinants which associate with MHC class II molecules on the B cell

surface. T helper (Th) cells with TCR specific for other epitopes of that antigen,

perceived as peptides in association with the class II molecules, wil l then be able to

interact with the B cell to activate it. The B cell, receiving T cell help (in the form of

interleukins and various molecular interactions) wil l then produce antibodies against

the foreign antigen X (see Chapter 3). (2) If the specific Th cells are not available, either

because of a genetic gap in the T cell repertoire (Chapter 9), or because of deletion

resulting from self-tolerance achieved in the thymus, any self-reactive B cells wi l l not

be able to produce auto-antibodies. (Continued.)

Page 90: Immunity

Immunological Tolerance 75

1. foreign antigen X

surface Ig

anti-X antibodies

, self antigen

no anti-self antibodies

antigen S —foreign T cell epitope

—self B cell epitope

No T cell help

Anti-self T cell

deleted through

self tolerance

anti-self antibodies

Figure 9, (Continued.) (3) These can, however, be produced if a self-reactive B cell

can receive help from an anti-foreign helper T cell In response to antigens made up

of various determinants, including some which are foreign T cell epitopes and others

which are self B cell epitopes. Similarly, the response to a foreign antigen could in

some cases lead to antibodies that cross-react with self-constituents. There is therefore

a need to purge the B cell repertoire of self-reactive B cells.

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76 J.F.A.P. MILLER

2. Clonal anergy. Self-reactive B cells, exposed to monomeric, soluble, antigen above a critical concentration threshold, were not deleted from secondary lymphoid tissues, where they occurred in normal numbers, but became anergic (Figure 11). This was associated with downregulation of the membrane IgM receptor, and a maturation arrest of the self-reactive B cells.

No evidence for the activity of suppressor T cells or of anti-idiotypic B cells was found in these transgenic models.

The fact that matuie B cells can be rendered tolerant to self-antigens that exist in either soluble or cell membrane-associated forms, provides a mechanism for preventing the escape of B cells acquiring auto-reactivity through antigen-driven hypermutation.

transgenic for X transgenic for Ig anti-X

anti-X

B ceU

response

non transgenic

normal

X-transgenic anti-X Ig transgenic double transgenic

no anti-X

activity

anti-X antibody

present

anti-X B cells

deleted from

secondary

lymphoid tissue

Figure 10. Clonal deletion of peripheral B cells responsive to integral membrane protein antigens. Mice were made transgenic for a foreign MHC class I molecule, "\",

expressed, under the influence of the metallothionein promoter which directs expres­sion to tissues such as the liver. Other mice were made transgenic for Ig genes coding for antibodies to this class I molecule, and mated to the former transgenic mice to obtain double transgenic offspring. In these, B cells with specificities for the foreign class I molecule and exported from the marrow were partially deleted in the spleen and completely in the lymph nodes. Tolerance to membrane bound self antigens can thus be induced in B cells by clonal deletion.

Page 92: Immunity

Immunological Tolerance 77

transgenic for S

X

transgenic for Ig anti-S

e

non transgenic S-transgenic anti-S Ig transgenic double transgenic

normal tolerant of S anti-S normal tolerant of S most B cells S-specific

B cell express anti-S B cells not

response Ig transgene deleted but

anergic with

down regulated

surface IgM

Figure 11. Clonal Anergy in peripheral B cells responsive to soluble protein antigens.

Double transgenic mice were produced by mating two different sets of transgenic

parents. One carried a gene for an otherwise foreign antigen produced by liver cells

and expressed largely in soluble form ("S'O. This resulted in both T and B cell tolerance.

A second transgenic line carried the rearranged heavy and light chain genes encoding

a high-affinity antibody to the soluble antigen and distinguishable from endogenous

Ig by means of a genetic marker on the heavy chain (an "allotype"). The double

transgenic offspring were profoundly tolerant to the soluble antigen producing no

antibody. Nevertheless, B cells with specificities for the soluble antigen had not been

deleted but had downregulated their surface IgM, not their IgD, receptors and were

therefore anergic.

ARTIFICIALLY INDUCED TOLERANCE

In the Living Animal (In Vivo)

Tolerance can be induced artificially in vivo by various means which may be summarized as follows:

/ . Establishment of Chimerism (Co-existence of Cells from Genetically

Different Individuals)

Tolerance to foreign cells can be established following inoculation of allogeneic cells into neonatal hosts, or into adult hosts after immunosuppressive regimes such as total body irradiation, drugs (e.g., cyclosporin A), or anti-lymphocytic antibodies (e.g., anti-lymphocyte globulin, anti-CD4 antibodies, etc.). For tolerance to be long lasting, some of the allogeneic cells should persist in their host, i.e. a certain degree

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78 J.F.A.P. MILLER

of cellular chimerism must be achieved. This is best obtained if the inoculum contains cells capable of self-renewal (e.g., bone marrow). If mature allogeneic T cells are present in the injected cell population, they may react against the histo­compatibility antigens of their host and induce a severe and often fatal disease known as graft-versus-host disease.

2. Antibodies to T Cell Accessory Molecules

The work of Waldmann and colleagues (1989) indicated that tolerance of transplanted tissues could also be achieved in adult animals by T-cell depleting or non-depleting monoclonal antibodies directed to the T cell accessory molecules (or co-receptors), CD4 and CDS. In this case, tolerance of skin allografts could be obtained even in the absence of chimerism.

3. Soluble Antigens

Early work by Dresser and Mitchison (1968) showed that tolerance could be induced in neonatal and adult animals by soluble protein antigens given in deag-gregated form. T and B cells differ in their susceptibility to tolerization by these antigens. Thus, for example, tolerance is achieved in spleen and thymus T cells with very low antigen doses and within a few hours. Tolerance of spleen B cells requires a much longer time and a much higher antigen dose. Levels of antigen which have produced B cell tolerance in neonates are of the order of 100-fold less than those in adults.

4. Oral Administration

Antigens administered orally have been able to induce tolerance through a mechanism that may involve an immunoregulatory or suppressor cell network.

5. Targeting Antigen to Naive B Cells

In some experimental situations, antigen presented by naive B cells induced antigen-specific T cell tolerance. This may be because naive B cells lack co-stimu­lator functions and yet can efficiently process antigen and present it to T cells. Accordingly, T cell tolerance may be achieved by targeting the antigen to resting B cells simply by coupling it to a monoclonal antibody directed against the IgD molecule on the B cell surface.

6. Clonal Exhaustion

Tolerance in T lymphocytes and to a lesser extent in B cells can be the outcome of clonal exhaustion occurring at the end of a powerful immune response. A very strong or a repeated antigenic challenge may stimulate all the antigen-responding

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Immunological Tolerance 79

cells to differentiate into short-lived end cells, leaving no cells able to respond to a subsequent challenge with antigen.

7. Antagonist Peptides

Some peptides that fit in the MHC pocket fail to stimulate T cells with specific TCR. This may be because there is a low affinity interaction which is sufficient to occupy the TCR sites but unable to stimulate a critical number of TCR to trigger the response. Alternatively, the antagonist may fail to induce a necessary confor­mational change in the TCR and other coreceptors. Whatever the case may be the antagonist peptide will block the response of T cells to the corresponding agonist peptide, a variant form which normally could stimulate the cells. Specific T cell tolerance is thus achieved by antagonist peptides.

8. T'Cell Independent Antigens

These antigens are generally slowly metabolized in vivo and thus tend to produce long-lasting B cell tolerance, if a large enough antigen dose is given. A very high concentration of these antigens can even blockade the immunoglobulin binding sites of fully differentiated antibody-forming cells and prevent them from secreting antibody.

9. Generation of Anti-idiotypic Responses

The antibody-combining site may act as an antigen (termed "idiotype") and stimulate the formation of "anti-idiotypic antibodies." These antibodies can block the responsiveness of the B cell by cross-linking its surface immunoglobulin molecules. Since, in some animals, most of the antibodies produced in response to some antigens carry a particular idiotype, suppression of this idiotype by anti-idio­typic antibody can alter the response. Tolerance will thus be partial since it will affect only those B cells carrying the idiotype.

/ 0. Veto and Suppression

In some experimental systems, tolerance appears to have been induced and to be transferable by veto cells (Miller, 1980) (Figure 6) which can reside extra-thymi-cally, or by "suppressor T cells" which may exert their effects through the release of lympho-kines such as TGF-P (Miller et al., 1992).

/ / . Immune Deviation

Although this is not strictly tolerance in terms of the functional or physical inactivation of cells with specific antigen receptors, the use of certain cytokines may allow manipulation of selective immune responses so that those that predomi­nate are not damaging. For example, Thl-type cells are most active in cell-mediated

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80 I.F.A.P. MILLER

immune responses which destroy tissue grafts. Although both Thl and Th2 cells secrete the lymphokines IL-3, GM-CSF and TNF-a, they differ in their production of other lymphokines. Thus, Thl cells secrete IL-2, y-interferon and TNF-P, and Th2 cells produce IL-4, IL-5, IL-6, IL-10 and IL-13 (Figure 8). IL-10 can suppress the activities of Thl cells by an effect on antigen-presenting cells and may thereby diminish the damage caused by Thl cells (see also Chapter 9).

Persistence of antigen is essential to maintain the tolerance state in vivo. Once the antigen concentration drops below a certain threshold level, tolerance breaks and responsiveness returns. When tolerance occurs as a result of clonal deletion or permanent anergy, recovery is related to the time required to generate new lympho­cytes from their precursors. It can be prevented by thymectomy.

In Tissue Culture {In Vitro)

B cells can readily be made tolerant in vitro as clearly demonstrated by Nossal and colleagues (Nossal, 1983). Tolerance can be achieved by antigens which cross-link the Ig receptor, but which do not possess intrinsic mitogenic capacity. To be tolerized in this way, mature B cells require high antigen concentration and immature B cells low concentrations. Monoclonal anti-IgM antibodies can be used to mimic antigen cross-linking of the B cell Ig receptors. High concentrations result in clonal abortion of pre-B cells, preventing their further differentiation to surface membrane IgM-bearing B cells. On the other hand, lower concentrations allow the pre-B cells to develop into morphologically normal B cells, with normal numbers of Ig receptors, but they become profoundly anergic. Thus, at the critical time of acquisition of Ig receptors for antigen, namely, at the pre-B to B cell transition, both B cell ftinction and B cell numbers can be modulated via their surface Ig receptors.

Whether antigen induces B cell abortion or anergy has been worked out by Nossal and his collaborators (1983). The outcome appears to depend on the degree of Ig receptor occupancy and cross-linking. Contributing factors include antigen valency and concentration, Ig receptor affinity for the binding epitope and state of maturity of the B cell. At one end of the spectrum is clonal abortion: this tends to occur in less mature cells and in those possessing a higher affinity epitope-binding receptor or those encountering antigens of higher valency and concentration (Table 1). At the other end of the spectrum, no effect will occur at low concentrations and affinities. Clonal anergy is induced when conditions exist between these two extremes. A second window of susceptibility to tolerance induction occurs tran­siently during the generation of B cell memory. Such a tolerance-susceptible stage may ensure that newly derived memory B cells, that have acquired self reactivity as a result of accumulated somatic mutations, would be purged from the repertoire.

T cells can also be tolerized outside the body. For example. Lamb, Feldmann and colleagues (1983) used an influenza-specific T cell clone and incubated its cells for a few hours at 37°C with a high concentration of influenza peptide. When this was performed in the absence of cells that can present antigen to T cells and activate

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Immunological Tolerance 81

Table 1, Sensitivity of B Cells, at Various Stages of Development, to Tolerance Induction

Stage of B Cell

Development

Pre-B — B cell

Bcel l

Antibody-forming cell

Memory B cell

Very Strong

abortion

deletion

blockade

abortion

Strength of

Strong

abortion

anergy

no effect

abortion

Tolerogenic Signal

Intermediate

anergy

no effect

no effect

anergy

Weak

no effect

no effect

no effect

no effect

them (i.e., cells possessing co-stimulator activity), the T cells remained alive but were anergic. The outcome could be influenced by various lymphokines: thus, IL-2, but not interferon y or IL-1, inhibited tolerance induction, and addition of IL-2 reversed established tolerance. These and other experimental results have suggested that altered regulation of the IL-2 pathw^ay may be an intracellular lesion that characterizes the induction of tolerance in T cells.

AUTOIMMUNITY AND THE BREAKDOWN OF IMMUNOLOGICAL SELF TOLERANCE

As stated above, T cells exist that are potentially autoreactive but that normally ignore the presence of autoantigens, either because these are located in some "immunologically privileged" or T cell-shielded site, or because they are exposed on cells unable to activate T cells through the lack of co-stimulator molecules. In such cases, autoimmunity may be induced by a process known as "molecular mimicry." A foreign antigen, e.g., one derived from an invading micro-organism, may by chance possess epitopes which cross-react with self epitopes. The foreign antigen will of course be processed by "professional" antigen-presenting cells— those which do possess the necessary co-stimulator activities—^and specific T cells will therefore be activated to respond. Activated T cells, unlike naive T cells, can traverse endothelial barriers and enter various nonlymphoid tissues. The T cells responding to the foreign antigen will therefore be fully equipped for aggression against the cells bearing the cross-reactive self epitopes. In this scenario, autoim­munity does not result from the breakdown of self tolerance but from its circum­vention through the display by professional APC of antigenic determinants to which the individual was never tolerant.

Since many diverse tolerance-inducing mechanisms exist (see above), it is likely that there are multiple ways in which tolerance can break down. For example, flaws in thymic negative selection may occur in individuals with certain MHC genotypes. Some MHC molecules may not negatively select self-reactive T cells or may not positively select immunoregulatory T cells involved in preventing the activation of autoreactive T cells. Other MHC molecules may negatively select T cells with

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82 J.F.A.P. MILLER

receptors specific for epitopes of micro-organisms and failure to eliminate such infection may be associated with tissue damage and processing of autoantigen by professional APC. Factors other than MHC genotype may also determine whether autoimmunity occurs. The fact that clonal anergy is reversible in the presence of IL-2 suggests that whatever causes disturbances in cytokine production may in some cases predispose to autoimmunity. Likewise, defects in the regulation of Thl and Th2 cells may be associated with the predominance of certain types of immune responses and apparent loss of tolerance. The pathogenesis of autoimmune disease is further discussed in Chapter 13.

POTENTIAL THERAPEUTIC APPLICATIONS

It is of course essential to learn how to induce tolerance to a foreign tissue graft, or to control the undesirable immune responses which occur in hypersensitivity reactions or in autoimmune conditions. The various ways of establishing artificial tolerance in adult animals as stated above are being examined for their potential clinical applications. Some success has been achieved with transplants associated with chimerism and performed under the umbrella of immunosuppressive agents. Treatment with monoclonal non-depleting anti-CD4 and anti-CD8 antibodies has also been used successfully in patients. The possibility that tolerance can be induced in patients by oral feeding the target antigen and by peptide antagonists is being examined.

It is also important to learn how to activate ignorant T cells or to break peripheral tolerance to a particular self component to enable the body to mount an active immune response which could limit the growth of tumors that may express their own unique tumor-specific genes as a membrane-bound antigen which is ignored by T cells.

The finding that non-depleting CD4 antibodies can induce transplant tolerance has obvious clinical implications.

SUMMARY

Several mechanisms exist to prevent lymphocytes from reacting against self antigens. As T cells develop in the thymus and express antigen-specific receptors, those with high affinity to self components existing within the thymus are deleted. Low affinity self-reactive T cells and T cells with receptors against antigens not represented intra-thymically will mature and join the peripheral T cell pool. They may either ignore self antigens expressed by tissues unable to activate T cells through lack of the appropriate co-stimulator signals, or they may under certain conditions be deleted or rendered anergic and unable to respond. Likewise B cells which express surface immunoglobulin receptors with high binding affinity to self membrane-bound antigens will be deleted after leaving their birth place in the bone marrow, while those with potential to respond to soluble self proteins will be

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Immunological Tolerance 83

rendered anergic by downregulating their surface IgM receptor. Tolerance can be induced artificially by various regimens which may eventually be used clinically to prevent rejection of foreign transplants and to manage autoimmune diseases. Autoimmunity may occur under certain conditions, e.g. if T cells that ignore autoantigens are stimulated professional antigen presenting cells that can deliver a powerful co-stimulator signal and happen to present an antigen, derived from invading microorganisms, that cross reacts with a self antigen.

REFERENCES

Billingham, R.E., Brent, L., & Medawar, P.B. (1953). Actively acquired tolerance of foreign cells.

Nature 172, 603-606.

Bretscher, P.A., & Cohn, M. (1970). A theory of self-nonself discrimination: Paralysis and induction

involve the recognition of one and two determinants on an antigen, respectively. Science 169,

1042-1049.

Burnet, F.M. (1959). The Clonal Selection Theory of Acquired Immunity. Cambridge University Press.

Burnet, P.M., & Fenner, F. (1949). The Production of Antibodies. Macmillan, London.

Dresser, D.W., & Mitchison, N.A. (1968). The mechanism of immunological paralysis. Adv. Immunol.

8, 12^181.

Ehrlich, P. (1900). On immunity with special reference to cell life. Proc. Roy. Soc. 66B, 424-448.

Gershon, R.K. (1974). T-cell control of antibody production. Contemp. Topics Immunobiol. 3, 1^0.

Goodnow, C.C, Crosbie, J., Adelstein, S., Lavoie, T.B., Smith-Gill, S.J., Brink, R.A., Pritchard-Briscoe,

H., Wotherspoon, J.S., Loblay, R.H., Raphael, K., Trent, R.J., & Basten, A. (1988). Altered

immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic

mice. Nature 334, 676-682.

Heath, W.R., Karamalis, F., Donoghue, J., & Miller, J.F.A.P. (1995). Autoimmunity caused by ignorant

CD8+ T cells is transient and depends on avidity. J. Immunol. 25, 2339-2349.

Lafferty, K.J., Prowse, S.J., & Simeonovic, C.J. (1983). Immunobiology of tissue transplantation: A

return to the passenger leukocyte concept. Ann. Rev. Immunol. 1, 143-173.

Lamb, J.R., Skidmore, B.J., Green, N., Chiller, J.M., & Feldmann, M. (1983). Induction of tolerance in

influenza virus-immune T lymphocyte clones with syngeneic peptides of influenza hemagglutinin.

J. Exp. Med. 157, 1434-1447.

Lederberg, J. (1959). Genes and antibodies. Science 129, 1649-1653.

Miller, A., Lider, O., Roberts, A.B., Spom, M.B., & Wiener, H.L. (1992). Suppressor T cells generated

by oral tolerization to myelin basic protein suppress both in vitro and in vivo immune responses

by the release of transforming growth factor p after antigen-specific triggering. Proc. Natl. Acad.

Sci. USA 89, 421-425.

Miller, J.F.A.P., & Heath, W.R. (1993). Self-ignorance in the peripheral T cell pool. Immunol. Rev.

133, 131-150.

Miller, R.G. (1980). An immunological suppressor cell inactivating cytotoxic-T lymphocyte precursor

cells recognizing it. Nature 287, 544-546.

Mosmann, T.R., & Coffman, R.L. (1989). TH1 and TH2 cells: different patterns of lymphokine secretion

lead to different functional properties. Ann. Rev. Immunol. 7, 145—173.

Nemazee, D.A., & Burki, K. (1989). Clonal deletion of B lymphocytes in a transgenic mouse bearing

anti-MHC class I antibody genes. Nature 337, 562-566.

Nossal, G.J.V. (1983). Cellular mechanisms of immunologic tolerance. Ann. Rev. Immunol. 1, 33-62.

Owen, R.D. (1945). Immunogenetic consequences of vascular anastomoses between bovine twins.

Science 102,400-401.

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84 J.F.A.P. MILLER

Palmiter, R.D., & Brinster, R.L. (1986). Germ line transformation of mice. Ann. Rev. Genet. 20, 465-499.

Traub, E. (1938). Factors influencing the persistence of choriomeningitis virus in the blood of mice after clinical recovery. J. Exp. Med. 68, 229-250.

von Boehmer, H. (1990). Developmental biology of T cells in T cell-receptor transgenic mice. Ann.

Rev. Immunol. 8, 531-556. Waldmann, H., Cobbold, S.P., & Qin, S. (1989). Tolerance induction using CD4 and CD8 monoclonal

antibodies. Progr. Immunol. 7, 147-155. Zinkemagel, R.M., Pircher, H.P., Ohashi, P., Oehen, S., Odermatt, B., Mak, T., Amheiter, H., Burkit,

K., & Hengartner, H. (1991). T and B cell tolerance and responses to viral antigens in transgenic

mice: Implications for the pathogenesis of autoimmune versus immunopathological disease.

Immunol. Rev. 122, 133-171.

RECOMMENDED READINGS

Basten, A. (1989). Self tolerance: the key to autoimmunity. Proc. Roy. Soc. Lond. 238B, 1-23.

Goodnow, C. (1992). Transgenic mice and analysis of B-cell tolerance. Ann. Rev. Immunol. 10,

489-518.

Miller, J.F.A.P., & Morahan, G. (1992). Peripheral T cell tolerance. Ann. Rev. Immunol. 10, 51-69.

Moller, G. (Ed.) (1993). Peripheral T-cell immunological tolerance. Immunol. Rev. vol. 133.

Nossal, G.J.V. (1992). Immunity versus tolerance: the cell biology of positive and negative signalling

of B lymphocytes. Adv. Mol. Cell Biol. 6, 56-77.

Rose, N.R., & Mackay, I.R., (Eds.) (1985). The Autoimmune Diseases. Academic Press, London.

Waldmann, H., & Cobbold, S. (1993). The use of monoclonal antibodies to achieve immunological

tolerance. Immunol. Today 7, 247-251.

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Chapter 5

The Generation of Diversity in the

Immune System

E.J. STEELE and H.S. ROTHENFLUH

Introduction 86

Clonal Selection—Strategy of B and T Cell Responses 87

The Generation of Diversity in the Immune System 92

Functional and Structural Isotypes of Immunoglobulins 92 The Germline Repertoire 94 The Generation of the Somatic Repertoire 95

"Directed Mutagenesis"—Evolution of the Immune System? 102 Summary 103 Recommended Readings 106

Principles of Medical Biology, Volume 6 Immunobiology, pages 85-106. Copyright © 1996 by JAI Press Inc.

All rights of reproduction in any form reserved. ISBN: 1-55938-811-0

85

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86 E.J. STEELE and H.S. ROTHENFLUH

INTRODUCTION

The primary purpose of the evolutionarily advanced immune system as represented

in contemporary mammals (humans and mice) is to protect the individual against

disease. At the same time the individual must not mount specific autoimmune

responses. These two evolutionary forces have shaped a molecular and cellular

strategy so that each individual can respond to antigens presented on unexpected

invaders (bacteria, viruses), yet at the same time, prevent immune responses against

self antigens. Here we will outline the mechanisms which generate the antigen

^ L 1

{ —

— C D R l -

— FR2—

- C D R 2 -

— FR3—

"^CDRS

FR4v

> V H

(continued)

Figure 1, Simplified diagrams of the antigen receptors. The amino terminal (NH2)

and carboxyl terminal (COOH) ends of each chain are indicated. (A) Generalized

structure of an Ig molecule. Each Ig molecule is composed of two identical heavy (H)

chains and two identical light (L) chains each of which contains an antigen binding

variable region (V) at the amino end and a constant region (C) at the carboxyl end.

The positions of the three antigen contact sites - the complementarity determining

regions (CDR) - and the four framework regions (FR) are indicated in the magnification.

The two H chains and each H-L light chain pair are held together by disulfide bonds

(indicated by a straight line connecting the chains). (B) Generalized structure of an ap

T cell antigen receptor (TcR) complex. The variable (V) and the constant (C) regions

of each chain are indicated. All TcRs (both aP and yd) are noncovalently associated

with either an a-d-y-E-C^-C, CD3 complex or with an a-5-y-e-^-r| CD3 complex. The

TcR complex also contains a CD4 molecule on T helper cells (Th) or a CD8 molecule

on cytotoxic T cells (Tc). The dark areas of the V regions indicate the positions of the

CDRs, whilst the light areas indicate the positions of the FRs. (Adapted from Sinha, A.

A. et al., 1990, and from Coleman et al., 1992.)

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The Generation of Diversity in the Immune System 87

B

Figure 1. (Continued.)

recognition repertoire. The issue of the mechanism of 'self tolerance' will not be considered in this chapter although the process will be touched on at various points where it is necessary to show how this 'force' has shaped the diversification of antigen-specific receptor genes.

The immune system has developed mechanisms for generating a virtually limit­less variety of different antigen receptor specificities as the antigenic universe of both self and foreign epitopes is immense. It is a complex molecular universe consisting of numerous conformational and linear epitopes presented by many different biologically important polymers (proteins, polysaccharides, nucleic acids) and their degradation products (peptides, monosaccharides to oligosaccharides, oligonucleotides). The size of the repertoire is estimated to be in the range of 10^ to greater than 10 ^ (Berek and Milstein, 1988). Antigen combining sites of B cell and T cell receptors (Igs and TcRs, Fig. 1.) must therefore be specific and restricted to short linear or conformational oligopolymers (recognizing from a few to about ten residues) in order to achieve the fine specificity necessary to assist in the self vs non-self discrimination between the inner and outer antigenic universes.

CLONAL SELECTION—STRATEGY OF B AND T CELL RESPONSES

The enormous diversity of antigen recognition is reflected at the cellular level in the number of antigen specific T and B lymphocytes circulating in each individual lymphatic system. Each specific B cell and most T cells express one specific surface

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88 E.J. STEELE and H.S. ROTHENFLUH

receptor, immunoglobulin and T cell receptor, respectively. Until very recently it was thought that all lymphocytes express only one receptor. However, it has since been shown that a significant number of T cells can express two TcRs of different specificities (Padovan et al., 1993).

The antigen combining site in each type of receptor consists of an interaction of two polypeptide chains creating an epitope binding pocket composed of a heavy (H) and light (L) chain pair for Igs, and ap or y6 heterodimer for TcRs (Figure 1). The walls of the binding pocket are lined with amino acids which are thought to make direct contact with the antigenic epitopes and constitute the complementar­ity-determining regions (CDRs) of the variable (V) region. The spacial interactions of the CDRs of each variable region are determined by the framework regions (FR). Thus, the V regions of both receptors are involved in direct contact with the epitope, whilst the constant regions are involved in the various effector functions.

It is now generally agreed that whilst Igs and TcRs bind different types of antigenic epitope, the antigen binding site structure in the amino terminal portions of the polypeptide chains are very similar (Figure 1). Indeed, in the V regions of both types of receptor molecule, the CDRs correspond to the hypervariable do­mains, while the FRs correspond with the conserved domains. This indicates similar selective forces driving the evolution of the genes encoding antigen-specific receptor molecules in general (Steele et al., 1993 and below).

As will be discussed in more detail below, Igs and TcRs molecules are not encoded as a single gene, but as separate genetic elements which need to be brought together in a certain sequence to form a functional receptor. The programmed assembly of these genetic elements also involves stochastic processes which ensure that millions of potential specific cells are generated continuously from the bone marrow. These cells differentiate further in peripheral lymphoid tissues giving rise to mature antigen-responsive T and B lymphocytes. However, due to the random nature of some of the steps involved many lymphocytes will produce non-func­tional or self-specific receptors, and as a result, each step of lymphocyte differen­tiation also involves various mechanisms to eliminate such cells. For example, fewer than 10% of the cells undergoing differentiation in the thymus actually leave this organ to enter the periphery as mature, antigen-responsive T cells.

In the late 1950s Sir MacFarlane Burnet, adding to the earlier work of Niels Jeme and David Talmage, formulated the Clonal Selection Theory to explain the specific adaptive properties of acquired immunity (Burnet, 1959). The idea at the heart of Burnet's theory was that each B cell expressed and secreted only one specific antibody i.e. each specific cell was potentially selectable (and therefore potentially able to be deleted by the self tolerance mechanism) by a specific antigenic epitope leading to clonal growth of that specific cell line. The organism could therefore biologically amplify in a few days the antigen specific cell (or cells) and manufac­ture at high rate those specific antibody molecules required by the host, ensuring that high concentrations flood the lymphatics and blood plasma systems. In this

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The Generation of Diversity in the Immune System 89

way, they help clear the body of the infective agent via direct neutralization and indirect complement-mediated reactions (target cell lysis, opsonization and phago­cytosis, chemotaxis and white cell infiltration to the depot of infection).

Unlike antibodies, TcRs cannot recognize either conformational or linear epi­topes on intact, soluble high molecular weight polymers. TcRs only recognize breakdown products of proteins (peptides) in the conformational context of cell surface major histocompatibility complex (MHC) antigens (Davis and Chien, 1993) which are displayed on antigen presenting cells (APC). Several different types of white blood cells have been shown to have antigen processing and presenting functions: Macrophages (Unanue, 1984), Dendritic cells (Steinman et al., 1983), B cells (Lanzavecchia, 1985) and, although only inefficiently, T cells (Lanzavecchia et al., 1988). Thus, whilst there can be some molecular cross reactivity or even 'molecular mimicry' in the universe of epitopes which T and B cells recognize, it is clear they 'look at' different types of protein epitope.

In addition, different subsets of T cells can only recognize peptide in association with a certain class of surface MHC molecule. Helper T cells (Th) and cytotoxic T cells (Tc) recognize the conformational complex of small peptides respectively contained in the peptide binding clefts of MHC Class II and Class I antigen presenting structures displayed on the surface APCs and target cells (Figure 2).

It is necessary to be ftilly aware of these differences because they are important in understanding the different immune strategies employed by B and T cells. In order to be effective, antibodies must neutralize soluble toxins or viruses before

! Voces;

Figure 2. Presentation of a processed antigen to T cells. Helper T cells (Th) only recognize antigen when it is presented with a MHC class II molecule, whilst cytotoxic T cells (Tc) cells can only recognize antigen when it is associated with a MHC class I molecule.

Page 105: Immunity

90 E.J. STEELE and H.S. ROTHENFLUH

sig negative Bcl-2 negative Non Ig secretor

;^W 7hr generation time '^/ Hypermutator on?

sIg positive ^ Bcl-2 negative ^^i. No division

Apoptosis

\ LOW \ \ AFFINITY

COMBINING SITES

{continued)

Follicular Dendritic Cell (FDC)

Figure 3, (A) Somatic hypermutation followed by antigenic selection. B centroblasts

undergo a period of rapid proliferation inside the germinal center. It is thought that

somatic hypermutation of Ig V regions takes place during this phase. Following this

stage the cells commence expression of mutated surface antibody (sIg) to become B

centrocytes. If the antibody has increased Ag binding ability due to somatic mutations,

then the B centrocyte wil l bind to one of the complete Ag molecules displayed on the

surface of a FDC. The act of binding the Ag possibly coupled with other signals induces

the expression of the bcl-2 gene product. This protein has been shown to prevent

apoptosis, hence any B centrocyte with a non-functional sIg or one that has lost or

reduced its Ag binding ability wi l l continue to undergo apoptosis. (Adapted from

Steele, E.J. et al., 1993). (B) Germinal center formation and progression during an

immune response. At day 1 of the immune response small numbers of lymphoblasts

(as few as 2-3) enter the primary follicles where they proliferate. As cell numbers

increase with time, a secondary follicle or germinal center forms within the original

primary follicle. It is thought that the proliferating cells also undergo somatic hyper­

mutation. Following its proliferative phase, the mutated cells are then selected by

antigen displayed on the surface of FDCs. Between days 3-5 of the immune response

macrophages start to appear in the germinal center, presumably to remove the cell

debris caused by apoptosis. At this time plasma B cells begin to form the medullary

cords which also increase in size over time. Successfully mutated B cells eventually

leave the germinal center and become detectable in the peripheral circulatory system

after day 5. As the numbers of these cells in circulation increases, the overall affinity

of the serum antibodies for the immunizing antigen increases—this is known as affinity

maturation. (Adapted from Hood et al., 1984.)

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77ie Generation of Diversity in the Immune System 91

B Lymphoblast

Medulla Paracortical area (T cells)

Primary lymphoid follicle

Day 2 of immune response

Medullary cords of plasma cells

Day 3-5 of immune response

Day 5-10 of immune response

Figures. (Continued.)

they bind or enter their target cells. This means that Igs need to be of relatively high

binding power (affinity) so that toxins and viruses can be neutralized at very low

concentrations. This is the key strategy positively selected for in the evolution of

the antibody system (Langman and Cohn, 1987). In fact, there has been further

evolutionary improvement in higher vertebrates: during a secondary or hyperim­

mune response, the memory B cells arising from the first encounter with the

pathogen now produce and secrete antibodies with binding affinities several orders

of magnitude higher than those made during the primary response. This phenome­

non is called affinity maturation and is now known to be a direct consequence of

the process of somatic hypermutation of the IgV region genes (see Figure 3) coupled

to antigen mediated selection of high affinity cells emerging from the mutation

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92 E.J. STEELE and H.S. ROTHENFLUH

process. These post-antigenic differentiation events occur in specialized post-anti-genic structures in lymphoid tissue called germinal centers (Figure 3).

The evolutionary significance of higher affinity antibodies produced as a conse­quence of a secondary infection (or booster vaccination) is that viruses or toxin molecules can be quickly bound and disposed of when they are initially present at low concentrations at the portal of entry in a wound or at a focus of infection at mucosal surfaces. This is less likely to occur if the antibodies are of low affinity. Moreover, it is obvious that the 'protective' antibody must bind native or unde-graded antigenic epitopes on the toxin or virus (unlike Th or Tc cells that bind peptides in the MHC+Peptide complex derived from the normally inaccessible portions of a globular protein antigen).

At this stage in the development of our knowledge clonally distributed antigen specific TcRs are not thought to undergo the phenomenon of affinity maturation nor indeed somatic mutation of their V region genes (Eisen, 1986; Steele et al., 1993). However, T cells interact with and recognize MHC+Peptide complexes at cell-cell interfaces and due to multipoint interactions the thermodynamics of the binding affinities in this situation is very different from that of soluble Ab-Ag interactions (Blanden et al., 1987). It is still not clear if a form of specificity maturation in T cells can ensue following a secondary or hyperimmune challenge (Steele et al., 1993).

Th cells are the orchestrators of a successful specific immune response. They activate antigen specific B cells which then either become antibody secreting plasma cells or undergo the process of somatic hypermutation (see below) to become memory cells and they control the level of activity of the other T cell subsets. Th cells are also a target cell that is infected by the human immunodefi­ciency viruses (HIV), hence, these viruses have overcome the specific immune response by attacking the key players in the system. The strategy of the Tc cell is to locate and kill (early in infection) those small numbers of cells containing intracellular parasites (most often viruses but also certain bacterial pathogens such as L. monocytogenes, T. pallidum, M. leprae). It is now established that Tc effectors are directly responsible for clearing the body of infected target cells (Kees and Blanden, 1976). Indeed, a recent dramatic demonstration of the protective power of Tc cells during intracellular pathogenic infections has been provided by the lack of clearance of L. monocytogenes infection in mice genetically engineered to lack the "killing channel" proteins necessary for cytotoxic T cells to kill their infected targets (Kagi et al., 1993).

THE GENERATION OF DIVERSITY IN THE IMMUNE SYSTEM

Functional and Structural Isotypes of Immunoglobulins

This chapter is primarily concerned with the generation of the diversity in the antigen recognition variable region genes. However, it is necessary to be aware that

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The Generation of Diversity in ttie Immune System 93

Igs are also functionally diverse. This effector diversity is specified by amino acid sequences in the constant region domains of the Ig molecule. The different Ig classes, or serologically defined isotypes of Igs are respectively IgM, IgD, IgG (+subclasses l,2a,2b,3), IgE and IgA. This classification is based on serologically defined amino acid sequence differences in the heavy chain constant region domains: |i chains (in IgM), 6 chains (in IgD), y chains (in IgG), 8 chains (in IgE) and a chains (in IgA).

The structural diversity is associated with differences in effector function. In a prototypical antibody response IgM antibodies (highly effective at complement

Functional Ig ^ or Ig§ rearrangement:

P L VDJ E Sj C| C5 S- C^ Syi Cyi SY2b C^b

-D-jH [ ] -H ,^ / /K=>- [~ l - / / - |M- / / - - Hh

P L VDJ E S^ Syi C i

-<^{EJ

Looping out of intervening sequence

SY2b ^Y2b

Excision of DNA loop

Functional Igyj rearrangement:

^ ^ ^^\ ^ M ^yl ^ 1 ^72b ^72b

Figure 4. Heavy chain isotype switch of a functional Ig^ or Igs to the Igyi isotype (the

IgM and IgD C region gene both utilize the same switch region - S^). The initial step

involves the pairing up or the switch regions involved, in this case S^ and Syi, and

hence, the looping out of the intervening sequence. The circular DNA is excised and

the remaining portions of the two switch regions are joined to form a hybrid switch

region (8^/5) and a new functional rearrangement: IgGi. Note, the C region genes are

composed of varying numbers of introns and exons which are not shown in this figure.

P = promoter, E = enhancer, L = leader sequence. (Adapted from Coleman et al.,

1992.)

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94 E.J. STEELE and H.S. ROTHENFLUH

fixing and promoting phagocytosis of opsonized pathogens) are produced first, followed by the other classes: IgG for systemic (or serum) antibody responses, and secretory IgA for responses induced at mucosal surfaces (lungs, gastrointestinal tract, genitourinary tract). This is achieved at the molecular (DNA) level by an isotype switching mechanism, whereby a rearranged variable region gene initially linked to the \x chain coding exons is physically switched (translocated) at the DNA level, and positioned in front of the next set of heavy chain exons (Figure 4, and see Honjo et al., 1989a). Specialized DNA sequences in the 'switch region' are thought to direct the switching enzyme system to efficiently and accurately remove (delete by looping out) the intervening DNA in a given cell line between the rearranged variable gene (a so-called -V[D]J-) and the new constant region exons.

Additionally, there are alternative splice sites for the secreted and membrane bound forms of each isotype. The exception is IgD which is co-expressed with IgM; it is strictly membrane bound and the function of the receptor is still not clear. It has been thought to play a role in regulatory and tolerance processes. Recent work in genetically engineered mice deficient in IgD suggests that IgD may augment efficient recruitment of B cells into the "affinity maturation" pathway during germinal center formation (Figure 3) in the early phase of a T cell-dependent primary response (Roes and Rajewsky, 1993).

The Germline Repertoire

Over the past 20 years the structure of the germline DNA encoding the genes for the assembly of Igs and TcRs has been elucidated (Tonegawa, 1983; Wilson et al., 1988; Honjo et al., 1989b; Davis and Bjorkman, 1988). There is also no doubt that when the current Human Genome Project (HUGO) is completed early next century the entire DNA sequences of most of the prototypical variable region gene segments for Igs and TcRs will be known.

The diagram in Figure 5 summarizes the structure of the genetic elements encoding mouse Ig heavy chains on chromosomes 12 (Rathbun et al., 1989). In humans and mice the genes encoding heavy and light chains of Igs are dispersed,

Ig heavy chain:

LVj LV2 LV„ D>,2 J1.4 C^

40-H-^ W^#^(^^-|HI H h ^ ° ^5' C ' Sl' C72b' CY2a C , C , n>300 " ^

10-20kb

Figure 5, Germline organization of the IgH chain genetic elements in the mouse. L = Leader sequence which encodes the signal peptide which directs secretion of the Ig protein to the cell surface or into the medium, kb = kilobases. Note: The C, introns /exons are not shown in this diagram (Adapted from Honjo, 1983).

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The Generation of Diversity in the Immune System 95

Table 1, Chromosomal Location of the Ig Heavy and Light Chain Gene Loci in the Murine and

Human Genome

Human Murine Chain type Chromosome Chromosome

_ _ _

K 2 6

?t 22 16

located at loci on different chromosomes (Table 1). A similar organization applies for the Va and Vp loci for TcRs (Davis and Bjorkman, 1988; Wilson et al , 1988).

The tandemly arranged repertoire of germline V segments can be very large. In some strains of inbred mice the immunoglobulin VH gene complex can number 1000 V genes (Rathbun et al., 1989). Each V segment is centrally located within a much larger element of 10-20 kb in size (e.g., see Bothwell, 1984). These V gene containing elements are duplicated many times at each chromosomal locus. The major biological problem is to understand how such a large tandem repertoire of homologous yet distinctly very different DNA sequences has evolved in the face of genetic instabilities such as unequal crossing over during meiosis and random genetic drift.

The Generation of the Somatic Repertoire

Rearrangement

During lymphocyte development the germline encoded V segments (which are not transcribed nor translated into protein) are rearranged in the sequence shown in Figure 6 for mouse Ig heavy chains. The V regions of Ig heavy chains and TcR P chains are composed of three rearranged genetic elements: a variable gene (V), diversity genes (D) and a joining gene (J), whilst the V regions of the other chains consist of a V gene rearranged to a J gene. The programmed DNA rearrangement occurs in the absence of stimulation by extrinsic antigens. Heptamer and nonamer recognition sequences in the 3' flanking regions of V segments and in the 3'5' flanking regions of D and J elements guide a system of recombinase enzymes which promote V[D]J rearrangement (Rathbun et al., 1989; Alt et al., 1992, Fig. 7). Thus, a given V segment will rearrange to D or J elements to position the rearranged V[D]J several kb upstream of the constant region exons. A fully assembled V gene can then be transcribed and expressed as a protein receptor as shown in the figure. A similar process occurs at the chromosomal locus encoding the other complemen­tary polypeptide chain (in this case the L chain). The two polypeptide chains are then assembled in the cytoplasmic vesicles as complete Ig molecules for anchoring in the cell surface membrane or secretion.

Page 111: Immunity

96

L Vi LV LV.

^W-^Hh^HMf-w-i

E.J. STEELE and H.S. ROTHENFLUH

^ mh

Germline DNA

D-Jjoin precedes V-DJjoin

L VDJ C i

Rearranged DNA Introns are spliced out

L VDJ C^

c a p - | | III hAAAAA

Processed mRNA

Figure 6, Schematic diagram of the rearrangement process. Initially a D gene is joined to a J gene. Following this a V gene is joined to the Dj rearrangement to form the complete rearranged DNA which is then transcribed. During processing of the pre-mRNA in the nucleus, the introns are removed, thus producing a mature mRNA molecule, cap = cap structure at 5' end of processed RNA. AAAAA = poly-A tail added at 3' end of processed RNA. (Adapted from Tonegawa, S. 1983).

In any given lymphocyte, with the exception of dual TcR T cell subsets, a process of allelic exclusion ensures that only one of the alleles in the diploid cell supplies a productively rearranged gene. Thus, in a maturing B cell, H or L loci on both chromosomes can be rearranged and a complex intracellular regulatory process (as yet unknown) ensures that the cell expresses only one allelic product in the fully assembled Ig. In this way one mature B or T cell expresses only one antigen receptor specificity enabling clonal selection and proliferation following antigen stimula­tion.

All genetic combinations of V, J and/or D are thought to be possible (Manser et al., 1984). Simple calculations show that the potential repertoire of fully assembled receptors is enormous (Berek and Milstein, 1988). Thus, it is apparent that a germline repertoire of 500 VH segments, 20 D elements and 4 JH elements will produce 40,000 heavy chains, and 500 VL segments rearranging at random to 5 Jk elements will generate 2,500 light chains. If all possible combinatorial VH/VL polypeptide pairs are realized, this gives 10^ different antibodies. This repertoire has been built up during phylogenetic evolution. The potential germline encoded antibody repertoire is therefore very large and this, by itself, should be sufficient armory to protect the individual against unexpected diseases.

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The Generation of Diversity in ttie immune System 97

Table 2, Comparison of Germline and Somatic Diversification Mechanisms in Ig Heavy and k Light Chains and in TCR a and p Chains

No. of V genes

No. of D genes

No. of J genes

D genes in ail reading frames

CDRs present in germline V

genes

N region addition

Somatic hypermutation

Immunoglobulins

H

>300

>12

4

Rare

+

+ +

K

200-250

0

4

-+

-+

a

100

0

50

-+

+ ?

TcR

P 25-50

2

12

Common

+

+ ?

Source: Adapted from Steele, E.j. et al., 1993.

The situation is not as simple as it first appears, as many of the VH/VL [ and Va/Vp ] combinations are likely to be non-viable (Cohn, 1968). Data on this point for TcRs is not yet available but it is known that preferential association of polypeptide chains appears to be a feature of Ig assembly from H and L polypeptide chains (called the Mannik Phenomenon, see Cohn, 1968).

Junctional Diversity

Additional diversity can be created during the rearrangement process. During Ig heavy chain and TcR (3 chain rearrangement, two D genes can be rearranged giving rise to a V-D-D-J variable region which may be fully functional (see Table 2). Such a rearrangement was recently found in an antibody heavy chain isolated from a systemic lupus erythematosus patient (Davidson et al., 1990).

When the various genetic elements are lined up for joining via the hep-tamer/nonamer recognition sequences, an exonuclease may remove nucleotides from the ends of the genetic elements involved to create a novel and unique join (Figure 7). Many Ig H chain and TcR a and (3 chain V regions have been found to possess varying numbers of non-templated nucleotides at the joins i.e., nucleotides not coded for by the genetic elements being rearranged. The additional sequences are known as N regions and they are inserted by the enzyme terminal deoxynu-cleotidyl transferase (Desiderio et al., 1984, Figure 7). N region additions are not evident in fetal and early neonatal Igs but are prominent features of rearranged V[D] J genes in adults (Feeney, 1990). In many V regions there is evidence of both removal of nucleotides from the joins and N region additions. No such deletions or N regions have yet been reported for antibody light chains; however, they too are capable of producing extensive junctional diversity during rearrangement. During

Page 113: Immunity

98 E.I. STEELE and H.S. ROTHENFLUH

Exonuclease

A. I D I l7l l9l

I J I inw D-J ends held together joining enzyme. Signal

by a putative sequences are joined

m TZA

An exonuclease removes nucleotidyltransferase

bases. Terminal deoxy-adds bases.

"GTGAACCG i -CACTTGGC L

N-Region

Figure 7. Nucleotide removal and N region addition during the D-J rearrangement

of an IgH chain. The heptamer (7)/nonamer (9) signal sequences are involved in lining

up the genetic elements involved. The same mechanism is also involved in the V-DJ

rearrangement step which follov^s the D-J rearrangement. Often the ends of the genetic

elements involved are removed by the exonuclease, indeed it is not unusual for the

D gene to be almost completely removed and replaced by a N region. (Adapted from

A l t , F. W., and Baltimore, D., 1982).

Ig L chain rearrangement the joining sites of the V and J elements involved can vary from one rearrangement to the next, thus resulting in many unique rearrange­ments even v^hen the same two genetic elements are involved.

However, the immune system pays a heavy price for being able to generate so much junctional diversity: many if not most of these nucleotide deletions, N regions and imprecise joins will result in a non-functional protein due to loss of the reading frame. All the cells with non-functional receptors must be deleted from the repertoire, and hence, the high rate of lymphocyte precursor production in the bone marrow is coupled to a high rate of cell death.

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The Generation of Diversity in the Immune System 99

Secondary Rearrangements

A supplementary somatic process has been identified termed a "secondary rearrangement." Here the V segment of a rearranged V[D]J is replaced by a DNA recombination event which inserts a different upstream V segment (Reth et al., 1986). Heptamer recognition sequences (palindromes) embedded within the 3' ends of V segments and possibly related to heptamers immediately 5' and 3' of D and J elements are thought to direct the specificity of the secondary recombinase en­zymes. The significance of variable region replacement is still not clear. Recent developments on the mechanism of self tolerance induction in B cells suggest it may be the genetic mechanism underlying the process called "receptor editing." If a developing B cell encounters a self epitope, the self reactive cell has a 'second chance' to change the specificity of its Ig receptor by V gene replacement (reviewed in Nossal, 1993). T cells developing in the thymus may also employ a similar V replacement strategy (Petrie et al., 1993).

o •a

-1000 1000 2000 3000 Nucleotides from cap site

4000

P c L VDJHI JH2JH3 JH4

Figure 8. Distribution of somatic mutations around a putative V-D-JHI rearrange­ment. The distribution of somatic mutations is asymmetric and the upstream boundary for the somatic hypermutation process lies around the promoter (P)/transcription start site (c) region. The downstream boundary seems to lie around the enhancer region (E). (Adapted from Rothenfluh, H. S. et al., 1993).

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100 E.J. STEELE and H.S. ROTHENFLUH

Somatic Hypermutation of Rearranged V(D)J Genes

Additional somatic diversity can be generated as a consequence of antigen-stimu­lated somatic hypermutation, a process that introduces nucleotide changes (mainly point mutations) into the DNA sequence within and adjacent to rearranged V genes. Antigenic stimulation is obligatory to activate the process and it is entirely depend­ent on T cell help. T-independent antigens, such as lipopolysaccharide, do not activate the mutator. Whilst the detailed molecular mechanism is still unknown, many molecular and cellular features of the process are now clear (reviewed in Steele, 1991).

In mice it is established that somatic point mutations are introduced mainly into the transcribed region of the genomic DNA of rearranged V[D]J regions (Steele et al., 1992; Rothenfluh et al, 1993). A comparison of many somatically mutated genes shows an asymmetrical distribution of mutations around rearranged V genes with a single major mode centered on the V[D]J region and a positively skewed tail into the non-translated J-C intron (Figure 8). A subset of mature B cells once specifically activated in a T-dependent manner migrate to primary lymphoid follicles to form a germinal center (Figure 3). In these sites the selected 'founder' B cell mutates its productively rearranged V[D] J genes at a very high rate, estimated to be 10' to 10"" per base pair per replication event (mutation rates in other eukaryotic genes are 10" to 10'^'). The founder B cell undergoes clonal growth and rapid point mutation targeted to V[D]J within the germinal center (Jacob et al., 1991; MacLennan, 1991). A stringent Ag mediated selection process ensures the emergence of higher affinity mutants only (Figure 3).

Ag-Ab complexes consisting of Abs of lower affinity produced in the initial phase of the primary response present selecting epitopes from the surface of interdigitating follicular dendritic cells (FDCs). Mutated B cell progeny ('centro­cytes') which no longer bind Ag (or bind at a much lower affinity) do not receive an activating cross-linking signal and 'commit suicide' via a process called apop-tosis, or programmed cell death. However, those progeny with mutant Ig receptors of higher affinity compete to dislodge the lower affinity antibodies in the Ag-Ab complex, and are 'selected' to survive as long lived memory cells. It can be seen that we have a process of rapid antigen-mediated Darwinian positive selection or 'evolution in microcosm' (Cunningham, 1977), supplying the host organism with high affinity protective antibodies specific for native conformational epitopes.

Thus, the somatic hypermutation and selection process within a germinal center basically results in the fine tuning of the specificity repertoire during an immune response (Goverman et al., 1986). It only contributes to the diversification process with respect to cross reactive specificities. However, the target area of mutation extends into the flanking regions, particularly into the 3' J-C intron Figure 8 and the resultant concentration of mutational differences to the CDRs or hypervariable domains (Wu and Kabat, 1970) are clearly the result of antigen binding selection for functional antibodies (Weigert et al., 1970) i.e., amino acid replacement

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The Generation of Diversity in ttie Immune System 101

L V j LV2 LV„ Di.„ J i ^ Cj,

-ii«r[HZ:i''-l»-^HHHF^^^H}{}ff-^HlHlHllB-i

L VDJ Cfi

Productive VDJ rearrangement in a mature B cell

Somatic hypermutation in germinal center

1 ^ Antigenic stimulation

of rearranged V region

L VDJ Cil

DNA

L VDJ C i

cap-||»**»|« AAAAA mature mRNA

Further antigenic germinal

selection within center

-IH

cap-H

m m i: CDRl CDR2 CDR3

iA m AAAAA

Figure 9. The sequence of events resulting in the selection for somatic mutations in the CDRs (Wu-Kabat structures). When a mature B cell expressing a functionally rearranged Ag specific Ig comes in contact with the Ag it may become an unmutated plasma B cell or it may undergo the somatic hypermutation pathv^ay. In the latter case, the Ag stimulated B cell will enter a germinal center in the spleen or in a lymph node. Mutations in the cartoon (represented by the concentrations of black dots) are introduced into the VDJ and its flanking regions. The mutation mechanism does not extend into the constant region exons. During antigenic selection within the germinal center, B cells bearing surface Ig with mutated CDRs but conserved FRs are selected for providing the mutated Ag binding site has a higher affinity for Ag. Thus B cells leaving germinal centers have accumulated changes in their CDRs but few if any in their FRs. CDR3 is crosshatched to simply indicate that most of its diversity results from junctional diversity rather than somatic mutations, cap = cap structure at 5' end of processed RNA. AAAAA = poly-A tail added at 3' end of processed RNA. Note, in the DNA molecule of the lower diagram only the first exon of C^ is shown.

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102 E.J. STEELE and H.S. ROTHENFLUH

mutations in the CDRs and conserved (silent) nucleotide changes in the FR regions. The sequence of events resulting in the emergence of somatically fashioned 'Wu-Kabat' structures within V[D]Js (Steele et al., 1993) is shown in Fig. 9.

It should be noted at this point that of the three CDRs in a rearranged variable region the CDR3 is almost solely a somatic construction in that it spans the V-J join in IgL chains or the V-D and D-J joins in IgH chains. Thus, most of the diversity found in CDR3 is due to imprecise joining, nucleotide deletions and/or N region addition rather than somatic hypermutation. The position of the other two CDRs, CDRl and CDR2 are abeady embedded in the germline encoded V segment (below).

The somatic mutator is clearly regulated in both space and time. This is necessary given the potential to generate lethal genetic errors in somatic cells. The possibility that autoimmune B cells may be generated cannot be excluded in this schema and such a danger exists for all hyperimmune memory B cell responses. However, this would be limited by the relative lack of T cell help as mature post-thymic T cells themselves are unlikely to undergo antigen driven somatic mutation of their TcRs (Steele et al., 1993) i.e., autoreactive Th cells would be clonally deleted in the thymus.

We will now turn to a new concept that is emerging from work in our laboratory on how the germline V gene repertoire has evolved (Rothenfluh and Steele, 1993a).

"DIRECTED MUTAGENESIS"—EVOLUTION OF THE IMMUNE SYSTEM?

A moments' reflection on the facts of somatic hypermutation shows that the mammalian immune system has evolved a tightly regulated process of "directed mutation" (Steele, 1989). It is also clear that the process depends very much on positive Darwinian selection but now occurring within a somatic cell population of a multicellular organism. Indeed, it was this aspect which proved decisive in the acceptance of Burnet's Clonal Selection Theory over its more instructionalist forerunners (Steele, 1991b). Rapid somatic mutation and antigen-binding selection results in a brisk affinity maturation of the memory antibody response. It is important to re-emphasize that this is not old fashioned 'directed mutation' in the sense of the environment instructing genetic change directly on the DNA.

A key evolutionary question can therefore be posed: Does this acquired and clearly beneficial somatic adaptation die with the individual or can acquired somatic mutations in V genes be inherited through the germline? (Steele, 1979; Rothenfluh and Steele, 1993a,b). Current research in our laboratory on DNA sequences of a large number of germline V segments and their non-transcribed and non-translated 5' flanking regions (from the genomic DNA of inbred mice) lead us to deduce that the germline V genes bear all the hallmarks of powerful somatic selection pressure namely, positive Darwinian selection occurring first in the soma coupled to physical transfer and integration of these somatically fashioned V genes into homologous target sequences in the germline DNA (Rothenfluh et al., 1995).

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The Generation of Diversity in the Immune System 103

SUMMARY

The mammalian immune system has evolved sophisticated germline and somatic strategies for the generation of an immense repertoire of antigen-specific lympho­cytes. The key evolutionary selective forces have been the need to protect the individual against 'unexpected' infections and to avoid autoimmune disease. The germline is composed of a large tandem array of V segments located upstream of joining elements (D,J) which are themselves proximal 5' to the constant region coding exons at Ig and TcR genetic loci. During lymphocyte development V genes rearrange to produce complete V[D]J variable regions which are transcribed, translated and the protein chains assembled into functional antigen-specific Ig and TcR receptors. Such receptors are clonally distributed such that any mature B cell or most T cells express only one antigen-specific receptor on their surface mem­brane.

Combinatorial DNA recombination of the germline encoded elements (V-to-[D]-to-J) together with combinatorial association and assembly of complete polypeptide chains can by itself generate a potentially very large recognition repertoire (>10' ), although many of these 'random' combinations may not be functional receptors. Additional somatic diversification processes include V to [D] J junctional diversity, nucleotide deletions, N region additions and 'secondary rearrangements' that can lead to receptor replacement and therefore a complete change of clonal specificity. Finally, there is the tightly regulated antigen-driven process of somatic hypermuta-tion of rearranged IgV genes. It is confined to a subset of mature B cells during differentiation to memory cells in specialized post-antigenic lymphoid structures called germinal centers. Memory B cells arising from germinal centers express and secrete mutated high affinity antibodies. In this way the specificity of the antibodies is fine tuned during an immune response. It is not known whether a similar process occurs in T cells but if it does it would have to occur during T cell development in the thymus to ensure clonal deletion of autoreactive cells. Positive Darwinian selection drives the development and evolution of both the germline and somatic variable gene repertoires. Indeed, there is emerging evidence from the structure and pattern of germline V gene sequences that acquired somatic mutations in V[D]J genes may be inherited in the germline DNA.

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Reth, M., Gehrmann, P., Petrac, E., & Weise, P. (1986). A novel VH to VHDJH joining mechanism in

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RECOMMENDED READINGS

Ada, G.L., & Nossal, G. (1987). The clonal selection theory. Sci. Amer. 255, 62-69.

Moller, G. (Ed.) (1987). The role of somatic mutation in the generation of lymphocyte diversity.

Immunol. Rev. Vol. 96, Munksgaard, Copenhagen. Moller, G. (Ed.) (1992). Germinal centers in the immune response. Immunol. Rev. Vol. 126.

Munksgaard, Copenhagen. Tonegawa, S. (1985). The molecules of the immune system. Sci. Amer. 253, 104-113.

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Chapter 6

The Antigen-Antibody Complex: STRUCTURE AND RECOGNITION

P.M. COLMAN

Introduction 107

Antibody Structure 108

Antigen Structure 113

Antibody-Antigen-Complexes 116 Summary 119

Recommended Readings 120

INTRODUCTION

Biological processes generally are controlled by interactions between molecules. Mostly these interactions are the result of evolutionary refinement and optimization of the interacting molecular species. Specific immune responses to an antigen call

Principles of Medical Biology, Volume 6 Immunobiology, pages 107-120. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-811-0

107

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108 P.M.COLMAN

for an interaction between immune receptors and that antigen, but in this case the antigen may not necessarily have been encountered during either the evolutionary history or the lifetime of the animal. Survival of the animal depends on a system for generating an ensemble of diverse molecules, one or more of which is capable of attachment to a particular antigen. The amino acid sequence variability among antibodies which results from gene segment rearrangement and somatic mutation is a major source of diversity of antibody specificities. The purpose of this chapter is to examine the structural basis of antigen binding by antibody, and antibody variability is a large part of the story. However, beyond that there are strictly conserved structural features of antibodies whose role in binding of antigen is quite fundamental and we shall discuss also those special architectural features of antibody molecules which suit them well for their task.

ANTIBODY STRUCTURE

We consider here immunoglobulin type yl (IgGl) although the general principles extend to all types. IgG is a four chain structure, a dimer of heavy (H) and light (L) chains which are covalently joined by disulfide bonds. These polypeptides are arranged into a -Y- shaped structure (Figure 1). Each arm of the -Y- contains one

Figure 1. Quaternaryarrangementof immunoglobulin domains in an IgGl molecule. Variable (V) and constant (C) domains have the structure shown in Figure 2.

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The Antigen-Antibody Complex 109

light chain and the amino terminal half of the heavy chain. The C-terminal halves

of the two heavy chains constitute the stem of the molecule. Proteolysis of many

antibodies results in three fragments being produced, two Fab (fragment antigen

binding) being the arms and one Fc (fragment crystalline) being the stem.

Heavy and light chains are composed of multiple copies of a single structural

domain. This domain of circa 100 amino acids is the building block of many

molecules of the immune system and indeed of some non-immune system mole­

cules. Heavy chains contain four domains (two in the Fab and two in the Fc) and

light chains contain two domains. In both heavy and light chains, the N-terminal

domains are highly variable across antibodies of different specificity, whereas the

remaining domains display conserved amino acid sequences amongst different

molecules. To reflect this pattern of variable and constant chemical structure, the

domains on the two polypeptides are referred to as VH, CHI, ^m ^^^ ^HS, and VL

and CL, reading in each case from the N-terminus (Figure 1).

The basic domain structure is illustrated in Figure 2. It is a p-sheet sandwich

composed of seven P-strands labelled A through G. Strands ABED form one sheet

and CFG the other. The two sheets are covalently connected through cysteine

residues on strands B and F. The strand orientations in the two sheets are almost

parallel to each other and in this respect the domain structure is typical of many

other P-sheet structures in proteins which display a similar 'aligned' packing of

CDR1

CDR 2

Figure 2. Immunoglobulin domain structure. Constant domains comprise seven

strands, A through G. Variable domains have two additional strands C and C\

Complementarity determining regions of variable domains are labeled CDR1 through

CDR3.

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110 P.M.COLMAN

p-sheets. Variable and constant domains are distinguished from each other struc­turally by the inclusion in variable domains of two additional P-strands between strands C and D. These are labelled C and C Two aspects of this elaboration of the seven-strand structure are important for the special function of variable do­mains. The C'C" loop forms part of the antigen binding site and the extended P-sheet C'CFG plays a crucial role in determining the pairing of VH and VL domains (see below).

Sequence variation between antibody molecules is not uniformly distributed throughout the V^ and VH domains. Rathermore, in each case, it is concentrated in three places in loops between the p-strands. These hypervariable loops, BC, C'C" and FG, are associated directly with the specificity of a particular antibody and are referred to as the three Complementarity Determining Regions or CDRs. They are all located at the same end of the domain structure. The three CDRs themselves are not equally variable. Most variation occurs in the third CDR of VH ( C D R H 3 ) followed by CDR L3 and, as illustrated below, these two CDRs are centrally located in the antigen binding site. CDR H3 is also most variable with respect to length and to the conformation of its polypeptide backbone.

Immunoglobulin variable domains have a segmented gene structure. VL domains are encoded by a V gene and a J gene which correspond respectively to strands A through F (and including most of CDR L3) and strand G. VH domains have an additional level of complexity in their gene structure. The V gene in that case does not include CDR H3. That loop is encoded by a separate element known as D (for diversity), and thus explains the supravariability of CDR H3 compared with the other five CDRs. The J gene structure for VH domains is similar to that for VL domains, i.e., it encodes strand G of the p-sandwich structure. In man, heavy chain variable domains derive from combinatorial association of one each of 500 V genes, 15 D genes and 4 J genes, giving rise to some 30,000 possible VH domains. For light chains, there are 200 V genes and 4 J genes, and 800 possible VL domains. Different pairwise association of VL and VH domains potentially produces over 20 million different antigen binding sites. Other aspects of the recombination process, such as variability in the joining sites of the genes, further expands the repertoire of different structures.

The organization of the domains into the -Y- shaped molecule is illustrated in Figure 1. Domains associate laterally along the length of the molecule through extensive noncovalent interfaces. CHI and CL domains form a dimer through association of the ABED face of each domain. CH3 domains dimerize in a very similar manner. In both cases the P-strand orientation of one of the ABED faces in the interface is approximately 90° to that on the partner ABED face. Like the aligned packing described for the domain structure itself, orthogonal packing of P-sheets is also a very common structural motif in protein molecules. Association of CH2 domains is unusual because of the role of carbohydrate in covering the ABED face there.

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The Antigen-Antibody Complex 111

In contrast to constant domains, VL and VH domains dimerize through their C'CFG faces, and they do so in a way which is quite unusual compared to other known protein structures. The packing of the two C'CFG P-sheets conforms to neither the ahgned nor orthogonal classes. Rather, the strand orientations in this case are inclined at approximately 60° to each other (Figure 3). The basis for this unusual interaction is two-fold. First, there are some very characteristic and conserved amino acid sequences in the C and G strands which introduce bulges into the regular p structure of these strands and give the C'CFG face a strong curvature (Figure 3). Secondly, conserved amino acids projecting outwards from the C'CFG face contribute to the complementary surfaces of the VL and VH domains at this interface. These two features of variable domain structure are both special to and conserved in variable domain structures. The VL-VH association brings into close spatial proximity the six CDRs of the two domains (Figure 3), CDRs HI, H2, LI and L2 being peripheral and CDRs H3 and L3 central. This arrangement is important in view of the fact that H3 and L3 are more variable than the other four CDRs. Antigens do not always interact with all six CDRs but in all cases studied so far CDRs H3 and L3 are part of the interaction. Together, the six CDRs form the entire surface of the extremities of the Fab arms. It is generally very common to find the 'active sites' of protein molecules located either at the subunit boundaries of oligomeric proteins or at structural domain interfaces.

There is an approximate two fold symmetry relationship between the VL and VH domains within the heterodimer and Figure 3 is a view down this pseudo symmetry axis. In that figure, close to the viewer, the CDRs are seen to participate in interactions across the dimer interface. Most of the CDR contacts in this interface involve H3 and L3. The variability in CDR sequences and structures from molecule to molecule result in small perturbations of the geometry of pairing of VL and VH domains between different antibodies. It appears that any given antibody molecule has a well defined and preferred pairing geometry for the variable domains, but variation among molecules can be as large as 15°, i.e., after alignment of, say, the VH domain of two different antibodies, their VL domains may differ in alignment by up to 15°. Thus, despite the conserved structures in this interface (to the rear of the view in Figure 3), the variable CDR structures modulate the interacfion. The VL-VH interface structure can also be modified during encounter with an antigen (see below).

Fv fragments are VL-VH domain pairs which can sometimes be generated by proteolytic digestion of antibodies. They are now readily producible by genetically engineering bacteria to over-express VL and VH genes. Fv can be made this way either as a two chain structure which forms a heterodimer VL-VH pair or as a single chain entity in which the two domains on a single polypeptide are joined by an appropriate linker. Linkers of 15 amino acids suffice to allow the C terminus of one V domain to be joined to the N terminus of the other without distorting the Fv quaternary structure. The three dimensional structures of Fv fragments, either of

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112 P.M. COLMAN

Figure 3, V L - V H pairing to form the antigen binding surface. This view is down the

approximate two fold symmetry axis relating V H (above) and VL (below) with the six

CDRs towards the reader. The C',C,F, and G strands VL V H on the two domains are

shaded. In each case strand C is farthest from the reader and strand G is closest.

the two chain or single chain type, have shown that VL-VH pairing is largely

unaffected by the removal of constant domains. This result was anticipated during

the 1970s when studies with Bence-Jones proteins (dimers of light chains) showed

that the presence of the CL domains did not influence the way in which VL domains

dimerized. It is possible that some CDR sequences are not very well accommodated

at the VL-VH interface and in those cases the presence of constant domains may be

important in maintaining the structure of the antigen binding site by stabilizing the

interaction between the heavy and light chains.

The linkages between domains along the length of the polypeptide chains are less

extensive than the lateral contacts described above and are of varying flexibility.

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The Antigen-Antibody Complex 113

The VL-CL and VH-CHI links are loose and allow an elbow movement within the Fab arm of the molecule. The CHI-CH2 link is through a cysteine rich peptide known as the hinge which allows large movements of the Fab arms with respect both to each other and to the Fc stem. The CH2-CH3 link is less flexible. The antigen binding site is distant from and loosely held to the sites of effector functions in the Fc region. Such a structure is consistent with the idea that these functions are not fired by a specific conformational trigger but rather by aggregation of bivalent antibody by multivalent antigen.

Amino acid sequences of T cell receptors suggest features common to Fab fragments of antibodies, including the special architecture at the interface of the variable domains of the a and p chains. However, no direct three dimensional structures are yet available either of the receptor or of its complex with MHC antigen and peptide.

ANTIGEN STRUCTURE

The definition of the antigen binding site of an antibody can be done with some precision by a study of antibody structure and the comparison of molecules with different specificities. In contrast, the definition of an antibody binding site on an antigen is not only difficult a/^r/on but also has meaning only in the context of a specific antibody molecule. Antigenic molecules can be proteins, polysaccharides, nucleic acids (or the oligomeric units of any of these), or naturally occurring or man made organic compounds (usually conjugated with larger carrier molecules). Most of the attention here will be on proteins and peptides because (i) more is known in these cases about the structures of complexes, (ii) there is an extensive database of protein-protein interactions against which to compare antibody-antigen complexes, and (iii) protein antigens are generally important in the development of protective immunity to pathogens.

We consider here one example of a well-characterized antigen which addresses some of the practical issues. What are the physico-chemical determinants of antibody binding to antigen? What determines cross-reactivity of anti-sera to two antigens? What minimal changes to antigen can abolish binding by a monoclonal antibody and conversely what antigenic changes can be tolerated by a monoclonal antibody?

Influenza viruses undergo continuous antigenic variation. The selection pressure of antibodies to strains of the virus currently and previously infecting man ensures a survival advantage to virus variants to which these antibodies cannot bind The virus has two different glycoproteins displayed on its envelope, a neuraminidase and a hemagglutinin, and both of these are subject to two types of antigenic variation. On the one hand, single amino acid sequence changes in the antigens accumulate continuously and can lead to a variant capable of reinfecting an individual. On the other hand, there occur occasional and sudden dramatic changes in antigenic structure caused by reassortment of the segmented viral genome and

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114 P.M.COLMAN

resulting in a new neuraminidase or hemagglutinin molecule with only 50% amino acid sequence identity to the antigens of previously circulating strains. Such events characterize new subtypes of the antigens, defined experimentally by the lack of cross-reactivity between antisera to antigens of different subtype. Cross- reacting anti-sera characterize variants within a subtype, where amino acid sequence simi­larities are usually higher than about 80%.

Studies of the three dimensional structure of neuraminidase of different subtypes show, as expected, identical three dimensional structures at the level of the fold of the polypeptide chain. However, a comparison of the surface structures of two different neuraminidase subtypes (Figure 4) reveals that conserved structures are dispersed around the surface and do not segregate into any single, large patch. It is known (see below) that antibodies need to attach themselves to a large surface on the antigen (-700A^ or more and involving 15 or more amino acids) in order to bind effectively and the absence of such large conserved surface patches between antigens of different subtypes is the likely cause of the failure of antisera to cross-react. The largest conserved surface structural feature across neuraminidase subtypes is the enzyme active site, about 600A^ in area (Figure 4).

Monoclonal antibodies have been used in many virus systems to suppress wild-type virus growth and to select variants which are able to grow, presumably because of the failure of the antibody to bind to them. These variants typically differ by a single amino acid from wild-type and three dimensional structure analysis of such variants of both influenza virus hemagglutinin and neuraminidase show that the structural consequences of these amino acid substitutions are usually very localized to the site of mutation. Furthermore, sometimes the single change in amino acid which abolishes the antibody binding is a change which is considered structurally conservative, e.g., alanine to valine, or asparagine to aspartic acid (or vice versa). Superficially, these observations sit uncomfortably with the require­ment for large surfaces of interaction. Why should one amino acid sequence change out of 15 or more in the binding site to antibody make such a large difference?

One answer is that in some cases it does not. Examples are known in which the substitution of one amino acid within the interface by another, even of dissimilar physico-chemical properties, has only a small effect on the binding affinity and only a local structural effect on the antibody-antigen interface. In other cases, the contribution to the total binding energy by a single amino acid can be sufficiently large that its substitution by another residue effectively abolishes binding. Note that this does not imply a particularly strong contribution for any one amino acid in the interaction. An antibody-antigen complex with a dissociation constant Kd~10^^M will be severely compromised by the loss of a single tight hydrogen bond (4kcal/mole) which will raise K^ by a factor of nearly 1000. Thus, a change as subtle as a single serine to alanine, and the concomitant loss of the hydroxyl group, can produce a variant antigen to which a monoclonal antibody no longer binds. It is not possible to formulate rules about the effects of general or specific amino acid

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116 P.M. COLMAN

sequence changes in an antigen on the binding of a particular antibody. In some cases, subtle changes can have large effects and in other cases more substantial changes have little effect. A particular outcome depends on the structural context of the mutated amino acid within the antibody-antigen interface.

ANTIBODY-ANTIGEN-COMPLEXES

X-ray crystallography of antibody-antigen complexes has allowed a direct exami­nation of the type and extent of chemical interactions which form. In several cases, it has also enabled a comparison of the bound and free forms of the antibody. The diverse nature of antigen and the capacity of antibody to accommodate it suggests that conclusions from a handful of structures will not paint a complete picture.

Antibody-antigen interfaces are extensive (Figure 5). The lower limit of buried surface area between two interacting protein molecules in a biological complex stands at around 1200A^, i.e., ~600A^ from each partner. Buried surface areas in antibody-protein interfaces are typically somewhat higher than this, but the figure may be a little less for antibody-peptide interactions. The physico-chemical nature of the interaction can include hydrophobic interactions, hydrogen bonding and ion pair formation. In the handful of protein antigen-antibody complexes studied in three dimensions at this time, there are between ten and fifteen hydrogen bonds, and up to three ion pairs within the interfaces. There is an unusually high proportion of aromatic residues within the CDRs of the VL and VH domains. In comparison, aliphatic amino acids contribute less to the binding site. This may relate to the fact that antigen binding to antibody will reduce the conformational entropy of side chains within the binding site by virtue of restricting rotational freedom around side chain bonds. Aromatic amino acids can contribute large buried surface areas to the interface with minimal unfavorable entropic consequences.

Not only do the surfaces display a measure of chemical complementarity, but their shapes are also complementary (Figure 5). This means that water molecules are generally excluded from the buried interior of the interface. This exclusion is not absolute and, in addition, water molecules can bridge the antibody and antigen around the solvent exposed perimeter of the interface. The antibody binding surface of protein antigens is usually comprised of several discontinuous peptide segments (2-5 in currently available structures). The antibody does not always engage all six of its CDRs in forming the complex. In cases studied so far, CDRs H3 and L3 are always participating in binding antigen, and at least two other CDRs also contribute. Sometimes an amino acid outside of the CDRs, but structurally adjacent to them, forms part of the contact surface with the antigen.

Anti-peptide antibody complexes with peptide have been observed to bind peptide either in extended or folded conformations. Buried surface areas are somewhat less than the 1200A^ lower limit of protein-protein interfaces and the number of amino acid residues of the peptides seen to interact specifically with the antibody is about half the usual 15 or so seen in protein antigen-antibody com-

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The Antigen-Antibody Complex 117

Figures, Antibody-antigen interfaces are large surfaces of complementary shape and

chemistry. Here the influenza virus neuraminidase is shown on the left with the

variable domains of an antibody on the right. The right hand view has separated the

antigen and antibody by 8A to illustrate the shape complementarity of the interfaces.

In this example, antigen and antibody both bury 900 A^ of their surface in the interface.

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118 P.M.COLMAN

plexes. In the case of antibody-peptide interactions, the binding site on the antibody is not so much the surface of the six CDRs as a groove running between the V^ and VH domains. For low molecular weight haptens, the binding site is usually a pocket near the center of the CDR surface. The interactions between antibodies and small antigens are more reminiscent of enzyme-substrate interactions than of complexes between macromolecules. In regard to the chemical and physical complementarity of the interfaces there is little to distinguish an antibody-antigen complex from other known protein-protein or protein-ligand complexes.

During secondary immune responses, somatic hypermutation introduces addi­tional diversity into the antibody repertoire, and from these somatic variants antibodies with higher affinity to the antigen are selectively produced. The sites of amino acid sequence changes in the antibody introduced in this way are sometimes within the CDRs and sometimes within framework regions of the variable domains. In the case of changes in the CDRs it is likely that an antibody-antigen interface residue has been altered. It is also likely that the cause of the increased affinity is a local improvement in physical and/or chemical fit around the site of the mutated residue, rather than any gross rearrangement of the antibody on the antigen. Where the somatic changes are not in CDRs it may be plausible to argue an indirect effect on affinity for antigen. One example of an indirect effect on CDR structure is in VH domains, where the size of a residue in the DE comer of the domain influences the conformation of CDR H2. Similar 'knock-on' effects have been observed in the structures of dimers of VL domains where sequence changes in CDR L3 affect the structure of CDR L2.

Binding studies reveal that the affinity of Fv fragments for antigen may be several fold weaker than of the parent Fab fi-agment. Fv fragments have been crystallized in complex with antigen, and studies of these structures show that they bind to antigen in the same way as the Fab fragment from which they derive. Genetically engineered Fv fragments can now be used to probe many aspects of antibody-an­tigen binding and recognition, including the contributions of individual amino acids to the binding energy.

In protein-ligand complexes generally it is observed that sometimes the comple­mentarity of the partners pre-exists quite precisely prior to engagement. In other cases, conformational changes in the protein are required to achieve complemen­tarity. These conformational changes frequently involve movements of side chains on the surface of the protein and sometimes also include changes in the conforma­tion of the polypeptide backbone. Both types of conformational change have been seen to occur in the CDRs of antibodies as a consequence of antigen binding. However, there is an additional and unique dimension to the capacity of antibody to adapt its shape to an antigen. In some cases it has been observed that the VL-VH

interface is perturbed by binding antigen. Thus, in addition to local structural effects within the CDRs, the three VH CDRs are able to be moved en bloc with respect to the three VL CDRs. This movement in some cases is as much as a few A, i.e,

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The Antigen-Antibody Complex 119

approaching the distance between adjacent amino acids on a polypeptide chain (3.8A). Since the purpose of such a rearrangement of the VL-VH interface is to bind antigen, it is not surprising that the magnitude and direction of the movement is different in different complexes, and in some cases may be very small or zero. The special design features of the VL-VH interface described earlier appear to allow it to function as an adaptor whose purpose is to improve the binding of poorly fitting structures.

When ligands bind to proteins at subunit or domain interfaces, it is commonly observed that some rearrangement of the subunit or domain structures occurs. Such quaternary structure changes are a special feature of allosteric proteins where the altered arrangement of subunits usually affects the affinity of the protein for ligand binding at remote sites. Hemoglobin is the best studied structural example of this phenomenon. There is no evidence to suggest that the antibody adaptor is function­ing in this way. Unlike allosteric proteins where a single ligand induces a specific quaternary structure change, antibodies utilize the adaptor to maximize interaction with an antigen, resulting in quaternary structure changes which are antigen and antibody dependent.

As important as this adaptor may be for binding particular antigen, it should not, and apparently does not, seriously compromise the specificity of the immune response. However, there are some situations in which one antibody is able to bind two quite different antigens. Antiidiotypy is an example of this. If antibody 1 (Abl) is raised against antigen (Ag) and Ab2 against the idiotype of Abl, it might be expected that Ab2 would resemble Ag, since both are bound by Abl. Such resemblances are rare although some are known. They require not only that Ab 1 is rigid, at least in the sense that its structure is identical in complex both with Ag and Ab2, but also that all of the amino acids of Ab 1 in the interface with Ag or Ab2 are behaving in the same way, e.g. an asparagine residue should in both cases donate hydrogen bonds to the interface. More common is the observation that Ag and Ab2 are structurally unrelated. Structural differences of Ab 1 in the two complexes is one possible explanation for this, and different physico-chemical utilization of particular amino acids of Abl in the two complexes is another.

SUMMARY

The antigen binding site of antibodies is a chemically and structurally variable surface of amino acids located at the extremities of the arms of the -Y- shaped molecule. Six hypervariable loops (Complementarily Determining Regions) con­tribute to this surface and determine the specificity of the antibody. Three of these CDRs are on the VH domain and three are on the V^ domain, and their relative positions in space are determined by the interactions at the VL-VH dimer interface. That interface has unusual architectural features when compared with the database of protein structures.

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120 P.M. COLMAN

Antibodies behave like other protein molecules when they associate with a ligand. They may change their structure, at the level either of the peptide backbone or side chain conformation, to achieve improved fit with the antigen. In addition, the VL and VH domains themselves may undergo some rearrangement across the VL-VH interface upon binding an antigen. The VL-VH interface plays the role of an adaptor, permitting movement of VL CDRS relative to VH CDRS SO that shape complementarity of the entire CDR surface to the antigen can be optimized. The diversity of CDR sequences, coupled with the ability to adopt a number of related conformations, results in a formidable armada of antibody specificities.

RECOMMENDED READINGS

Colman, P.M. (1988). Structure of antibody-antigen complexes: Implications for immune recognition. Adv. Immunol. 43, 99-132.

Colman, P.M. (1989). Neuraminidase enzyme and antigen. In: The Influenza Viruses (Krug, R.M., Ed.)

pp. 175-218. Plenum N.Y. Colman, P.M. (1991). Antigen-antigen receptor interactions. Curr. Opin. Struct. Biol. 1, 232-236. Davies D.R., & Chacko, S. (1993). Antibody Structure. Ace. Chem. Res., 26, 421^27. Herron, J.N., He, X.M., Ballard, D.W., Blier, P.R., Pace, P.E., Bothwell, A.L.M., Voss, E.W., Jr., &

Edmundson, A.B. (1991). An autoantibody to single-stranded DNA: Comparison of the three-dimensional structures of the unliganded Fab and a deoxynucleotide-Fab complex. Proteins:

Structure, Function, and Genetics 11, 159-175. Stanfield, R.L., Takimoto-Kamimura, M., Rini, J.M., Profy, A.T., & Wilson, I.A. (1993). Major

antigen-induced domain rearrangements in an antibody. Structure 1, 83-93. Tulip, W.R., Varghese, J.N., Baker, A.T., van Donkelaar, A., Laver, W.G., Webster, R.G., & Colman,

P.M. (1991). Refmed atomic structures of N9 subtype influenza virus neuraminidase and escape

mutants. J. Mol. Biol. 221, 487-497. Tulip, W.R., Varghese, J.N., Laver, W.G., Webster, R.G, & Colman, P.M. (1992). Refmed crystal

structure of the influenza virus neuraminidase-NC41 Fab complex. J. Mol. Biol. 227, 122-148. Wharton, S.A., Weis, W., Skehel, J.J., & Wiley, D.C. (1989). Structure, function and antigenicity of the

haemagglutinin of influenza virus. In: The Influenza Viruses (Krug, R.M., Ed.), pp. 153-174. Plenum, New York.

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Chapter 7

The Major Histocompatibility Complex

BRIAN D. TAIT

Introduction 122

Basic Genetic Structure of the MHC 122 Class 1 Region 122 Class 11 Region 124

Class III Region 125

Protein Structure of MHC Molecules 126 Class I Molecules 126

Class II Molecules 127 MHC Polymorphism and Nomenclature 128

Class I 128 Class II 129

Techniques for Detecting MHC Polymorphism 129 Serology 129 Molecular Techniques 129

Function of Class I and Class II Molecules 130 HLA in Transplantation 130

Matching 131

Principles of Medical Biology, Volume 6

Immunobiology, pages 121-136.

Copyright © 1996 by JAI Press Inc.

All rights of reproduction in any form reserved. ISBN: 1-55938-811-0

121

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122 BRIAN D.TAIT

Sensitization 131

HLA and Disease Associations 132 Molecular Mimicry 132 Restriction of Antigen Presentation 132

Class III Association 133 Summary 133

INTRODUCTION

The major histocompatibility complex (MHC) is found in all vertebrate species and its fundamental biological function is the recognition of self from non-self As such, the MHC plays a pivotal role as a regulator of immune function. This role is achieved by a variety of molecules that are the products of a gene cassette with varying but coordinated functions. In the human, the MHC gene cluster is found on the short arm of human chromosome 6.

The majority of the genes which comprise the human MHC are polymorphic, the degree of polymorphism varying between loci. The clinical relevance of MHC polymorphism is threefold. First, the study of MHC polymorphism and its effect on the functioning of the various products of the MHC gives insights into how these molecules operate at the molecular level in normal immune responses. Secondly, many human diseases, particularly those of autoimmune nature, show associations with particular MHC alleles; the study of these associations can assist in the understanding of these disease processes and the role MHC products play. Thirdly, some of the MHC gene products are powerful stimulators of alloresponsiveness in the clinical transplant situation. This alloresponsiveness takes the form of both antibodies and cytotoxic T cells directed at polymorphic regions of certain MHC molecules. These various facets of the MHC will be discussed in this chapter.

BASIC GENETIC STRUCTURE OF THE MHC

The MHC consists of 4,000 Kb of DNA on the short arm of human chromosome 6 and is divided into three classes of genes based on structure and function (Figure 1). The genes within the MHC are mherited in a co-dominant fashion, i.e., alleles on both chromosomes code for a protein product. The combination of MHC genes on one chromosome is termed a haplotype.

Class I Region

HLA (originally termed histocompatibility locus -A) -A,B,C are three highly polymorphic genes within the class I region. The HLA-A gene was the first gene discovered in the human MHC, one allele of which was shown to code for the

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specificity MACl described by Dausset in 1958 (Dausset, 1958). Three additional expressed class I genes termed HLA-E,F,G have recently been found (Geraghty et al., 1987;Kolleretal., 1988; Geraghty etal., 1990). Five alleles have been described for HLA-E, (Ohya et al., 1990) but little information is available on the polymor­phism of HLA-F,G. It does appear, however, that these newly discovered class I genes are not as polymorphic as the classical HLA-ABC genes. There is evidence of numerous other genes within the class I region but the majority such as HLA-H appear to be pseudogenes (i.e., they have no expressed protein product). The class I gene products have widespread distribution being expressed on most cell types.

The technique used for defining the polymorphism of the products of the class I genes over the last thirty years has been a serologically based technique termed the microlymphocytotoxicity assay (Terasaki and McClelland, 1964). Recently, with the sequencing of the majority of class I alleles and the use of techniques such as one dimensional isoelectric focusing (lEF) (Yang, 1989a), it has become evident that not all class I molecule sequence differences can be detected serologically (Yang, 1989b).

Class II Region

The class II region consists of four subregions termed DR, DQ, DO/DN and DP containing both expressed and non-expressed genes.

DR Sub-region

The DR sub region consists of one expressed DRA gene and four expressed DRB

genes; DRBl, DRB3, DRB4, and DRB5. In addition, there are a number of non-expressed pseudogenes—Z)/?^2, DRB6, DRB 7, DRB8, DRB9, The number and combination of DRB genes varies on different haplotypes.

The DRB] gene is highly polymorphic; limited polymorphism is seen with DRA,

DRB3, DRB5, and DRB4. Many of the DR specificities were first defined by serology, but as with class I, the polymorphism is far more extensive than is detected by this technique.

DQ Sub-region

The DQ subregion contains four genes; DQAI, DQA2, DQBl, and DQB2. The DQAl and DQBl genes are polymorphic, while DQA2 and DQB2 are non-expressed genes.

DP Sub-region

The structure of the DP subregion is similar to DQ, i.e., DPAl and DPBJ are expressed polymorphic genes, while DPA2 and DPB2 are non-expressed genes.

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The Major Histocompatibility Complex 125

The DP product is not expressed to the same level on the cell surface as DR and

DQ and as a result there has been little serological definition of DP historically

definition of polymorphism has relied on a cellular technique termed the primed

lymphocyte test (PLT) (Shaw et al, 1980).

DO/DN Sub-region

The DO/DN genes are newly discovered and less well characterized than DR,

DQ or DP. The degree of polymorphism of these genes has not been adequately

studied and the protein products have not been identified.

The class II gene products have limited cell expression being expressed mainly

on B cells, macrophages and dendritic cells (antigen presenting cells). (See section

on fimction of class I and class II molecules.)

The reader is referred to recent Nomenclature Committee reports (Bodmer et al ,

1995) listing alleles and serological epitopes currently defined at each class I and

class II locus.

Class III Region

Unlike the class I and class II region genes which code for cell bound molecules,

the class III region genes code for a variety of circulating molecules which also

serve immunoregulatory functions. These molecules include complement compo­

nents C2, C4, and properdin factor (Bf). Also included in the class III region are

genes for tumor necrosis factor A and B and heat shock protein 70. There are a large

number of genes within the class III region whose fiinction is not known. These

include the Bat (B Associated Transcript) series of genes, so called because the

first Bat gene was discovered near the HLA-B locus. Of special interest are the

21 hydroxylase genes. Only one of these genes 21-OHB, is functional and the

other, 21-OHA, is non-expressed (White et al., 1986). The 21-OHB gene codes

for cytochrome P450, an enzyme involved in the mineralocorticoid/glucocorti-

coid biochemical pathway. The presence of these duplicated genes in the class

III region is intriguing, since the functional 270//gene does not appear to have

an immunoregulatory function. In some haplotypes unequal crossing over

during meiosis has resulted in the absence of a functional 210HB gene. In

individuals where this has occurred on both haplotypes, 21 OH deficiency

occurs. This deficiency is expressed in utero in its severest form as salt wasting

and masculinizafion of the female fetus. HLA typing of amniotic fluid cells is

a useful predictor of an affected fetus in a family where a previous child is

affected.

The reader is referred to papers dealing with specific class III genes in order to

obtain a more in depth description of their individual structure and function.

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126 BRIAN D. TAIT

PROTEIN STRUCTURE OF MHC MOLECULES

Class I Molecules

The relationship of class I genes with the protein product is shown in Figure 2a. The gene exons code for specific regions of the protein product (the class I molecules), the introns having been spliced out during the production of messenger RNA (mRNA). The first exon comprises the signal sequence, which initiates transcription of the gene. The second, third and fourth exons code for the alpha 1, alpha 2, and alpha 3 extracellular domains of the class 1 molecule, respectively. The fifth, sixth, and seventh exons code for the transmembrane and cytoplasmic portion, which anchors the molecule in the cell. The class I molecule is non-covalently associated on the cell membrane with beta-2-microglobulin which is coded for by a gene on chromosome 15.

{continued)

Figure 2, A. The relationship of the class I gene exon/intron structure with the

expressed protein molecule. Note the class I molecule is associated with beta-2-

microglobulin which is coded for by a non-MHC gene. (Reproduced with permission

from Tait B.D. (1990). Life Science 2,30. Adapted from Kaufman et al. Cell 36:1,1984.)

B. The relationship of the class II gene exon/intron structure with the expressed protein

molecule. The class II molecule is a dimeric structure consisting of an alpha chain

(coded for by a class II A gene) and a beta chain (coded for by a class II B gene).

(Reproduced with permission from Tait, B.D. (1990). Life Science 2,30. Adapted from

Kaufman et al. Cell 36:1, 1984.)

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The Major Histocompatibility Complex Ml

The majority of class I polymorphism is located in the alpha 1 and alpha 2 domains (exons 2,3). Crystallography data (Bjorkman et al., 1987) has revealed that the first and second domains of the class I molecule combine to form a peptide binding groove or "Bjorkman's groove" (Figure 3). This groove consists of a base of beta pleated sheets bordered by alpha helices into which foreign and self peptide can bind. (See section on Function of HLA class I and class II molecules.)

The level of expression of class I molecules is influenced by cytokines such as interferon gamma (IFN gamma) and tumor necrosis factor (TNF) which can upregulate expression under certain conditions such as viral infection or rejection of foreign tissue.

Class II Molecules

The gene organization and corresponding protein domains of the class II mole­cules are shown in Figure 2b. The class II molecules are presented on the cell surface as a dimeric structure, consisting of the product of an A gene and B gene (i.e., alpha chain and beta chain). The basic relationship between the gene exons and the domain structure of the molecule is similar to that found in the class I molecule with one important difference; the majority of class II polymorphism occur only in the second exon which codes either for the alpha 1 or beta 1 domain, with the exception of the DR A gene which has limited polymorphism (2 alleles) (see

Figure 2, (Continued.)

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128 BRIAN D. TAIT

Figure 3, The structure of the class I and class II molecules as derived from crystal­

lography data. In the class I molecule the first and second domains combine to form

a peptide binding groove or 'Bjorkman's groove/ This groove consists of a base of

beta pleated sheets bordered by alpha helices into v^hich foreign and self peptide bind.

In class II the groove is formed in a similar manner by the alpha I and beta I domains

of the tv^o protein chains.

structure of class II region). The essential difference therefore between DR and other class II molecules is that for DQ and DP both alpha 1 and beta 1 domains are polymorphic. The evolutionary significance of this difference is not understood.

The structure of the class II molecules is similar to that of class I but in the case of class II the alpha 1 and beta 1 domains comprise the peptide groove (Figure 3). The class II molecules unlike class I are not associated with beta 2 microglobulin on the cell surface.

MHC POLYMORPHISM AND NOMENCLATURE

Class I

The class I nomenclature allows for the description of alleles and serological specificities (i.e., epitopes detected by antibody). Alleles are designated by a prefix which indicates the locus, i.e., A,B,C followed by an asterisk which signifies an

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The Major Histocompatibility Complex 129

allele. The first two numbers refer to the serological specificity, e.g., 2,24 and so on and the last two numbers refer to the allele. For example, there are three alleles which code for the A33 serological specificity—^A*3301, A*3302 and A*3303. These three alleles while differing in sequence share a common motif which when translated into the protein product represents the A3 3 epitope detected by antisera of this specificity. The reader is referred to Amett and Parham (1995) for a complete listing of class I allele sequences.

Class I amino acid substitutions are not randomly distributed, the majority being located either in the base of the peptide binding groove or in the alpha helices. Amino acid variations in the base of the groove are thought to influence peptide binding into the groove. Variations in the alpha helices can influence both peptide binding, and interaction of the peptide MHC complex with the T cell receptor. (See section on function of class I and class II molecules.)

Class II

The nomenclature system used for naming class II alleles is identical to that used for class I, with the exception that the first four digits are used for identifying the locus involved, i.e., DRBl refers to the Bl locus of the DR subregion. There is extensive polymorphism observed for most of the class II loci with a greater degree of polymorphism observed in the B genes than the A genes. In the DRB1 molecule there are three hypervariable regions centered around positions 10, 30 and 70. Positions 10 and 30 are in the base of the groove while position 70 is located in the alpha helix. The position of the variable amino acids differ with the different class II isotypes. The reader is referred to Marsh and Bodmer (1995b) for a full listing of class II sequences.

TECHNIQUES FOR DETECTING MHC POLYMORPHISM

Serology

Serology is the "cornerstone" technique which has been used to define the polymorphism of both class I and class II molecules. The original microlympho-cytotoxicity assay was first described by Terasaki and McClelland (1964) and little change has occurred in the performance of this assay over the subsequent period. The test is performed in wells of a microtiter tray (Terasaki Tray) using lympho­cytes as the target cell and a battery of antisera or monoclonal antibodies with specificity for both allele specific and public or shared epitopes.

Molecular Techniques

The demand for more discriminating methods of typing, particularly for class II alleles has led to the development of molecular techniques which examine DNA rather than protein polymorphism. The most common method in use is termed the

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130 BRIAN D.TAIT

Polymerase Chain Reaction—Sequence Specific Oligonucleotide technique (PCR-

SSO). This method involves the amplification of a specific segment of DNA in the

polymerase chain reaction, followed by probing with short length oligoprobes

which are designed to detect sequence differences between alleles. The reader is

referred to Erlich et al. (1989) for a complete description of the PCR reaction and

to Kimura and Sasazuki (1992) for a description of SSO typing.

FUNCTION OF CLASS I AND CLASS II MOLECULES

Foreign and self peptides are recognized by the immune system in the context of

MHC class I and class II molecules. Class II molecules bind exogenous peptides

in their groove for presentation to CD4 positive or helper T cells. The peptides are

derived from protein molecules which are phagocytosed into the cell interior and

bound to class II molecules in the endosome. In contrast, class I molecules bind

peptides derived from endogenous antigen which can be foreign (replicating

micro-organisms) or self (breakdown products of normal cell metabolism). The

peptide class I complex is recognized by CDS positive or cytotoxic T cells.

Presentation of foreign peptide in the context of class II molecules therefore initiates

a proliferative T helper cell response, leading via T-B and T-T cell collaboration

to antibody and cytotoxic T cell responses. The helper T cell response is therefore

class II restricted. Since CDS positive cells recognize peptide in the context of class

I molecules, cytotoxic responses are therefore class I restricted.

Class I and class II molecules are synthesized in the endoplasmic reticulum (ER).

In order for endogenous peptides to bind to class I molecules in the ER there is a

requirement for proteolysis of the intact protein molecules and subsequent transport

across the ER membrane. Recently with the aid of deletion mutant cells four genes

have been identified within the class II region which are responsible for both the

proteolytic process and transport of the resultant peptide. Two genes {LMP2,

LMP7) which are homologous in sequence and structure to the proteosome genes

appear to be responsible for the proteolytic process which reduces complex protein

molecules to short length peptides (Spies et al., 1990; Powis et al., 1992). Two

further genes (Tap 1 and Tap 2) are responsible for the transport of the peptide

fragments across the endoplasmic reticular membrane (Glynne et al., 1991; Kelly

et al., 1991). The location of these genes is shown in Figure 1.

HLA IN TRANSPLANTATION

The HLA system influences transplant survival in two ways. 1 Firstly, matching

donor and recipients for both class I and class II specificities results in improvement

of graft survival, and secondly, antibodies in the recipient with specificity for HLA

molecules expressed by the donor can result in early rejection of the graft.

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The Major Histocompatibility Complex 131

Matching

Solid Organs

The effect of HLA matching in renal transplant survival is reflected in the improved results obtained with one and two HLA haplotype matched living related donors when compared with transplants from cadaveric donors with varying degrees of mismatches (Opelz, 1992).

The Collaborative Transplant Study (CTS) centered in Heidelberg, Germany and organized by Professor G. Opelz collects data from a large number of transplant centers worldwide and analyses graft survival according to numerous parameters. The effect of HLA matching on renal transplant survival is clearly demonstrable in this large data set with an approximately 20% difference in graft survival at I year between the best and the worst HLA matched transplants (Opelz et al., 1992).

Analyses of the effect of HLA matching on renal graft survival to date have relied on serological data. With the advent of molecular typing it is now possible to examine the effect of allele matching in addition to epitope matching. This data is expected to emerge over the next 2-3 years as sufficient numbers of transplant pairs for meaningful analysis are DNA typed.

Although there is less data on cardiac transplantation, the effect of HLA-B,DR serological matching is clearly seen in the CTS study (Opelz et al., 1992). The effect of HLA matching on liver transplants survival is less obvious than that seen in renal and cardiac transplantation. Recent results from the CTS study (Opelz et al., 1993) indicate that the effect of HLA matching is dependent on the original disease of the recipient.

Bone Marrow Transplantation

In bone marrow transplantation immunocompetent cells from the donor are transplanted into an immunodeficient host. Cells in the bone marrow inoculum are able to recognize and mount an immune response against allo-antigenic differences on the recipient cells. The clinical sequelae of this "reverse rejection" is termed graft versus host disease (GVHD). In its most severe form it can result in damage to several tissues and organs of the recipient, leading to death. Although some GVHD is clearly due to non MHC differences, the frequency and severity of acute GVHD can be reduced by accurate HLA matching. The development of molecular matching (e.g., PCR-SSO) therefore has allowed high resolution allele matching which is essential in matched unrelated bone marrow transplantation.

Sensitization

The presence of circulating antibodies in the recipient directed at HLA-class I specificities of the donor can lead to hyperacute rejection of both renal and cardiac transplants. It is essential therefore in order to detect class I antibody to perform a

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132 BRIAN D.TAIT

pre transplant crossmatch by testing the recipients serum against donor T lympho­cytes. In contrast, the liver has a unique capacity to withstand antibody mediated rejection via absorption of antibody by non-parenchymal cells. High titers of HLA antibody can however result in rejection of liver allografts (Ratner et al., 1993).

HLA AND DISEASE ASSOCIATIONS

Many human diseases, particularly autoimmune diseases, show associations with certain HLA alleles or haplotypes. Included in these diseases are arthropathies (rheumatoid arthritis), neurological diseases (multiple sclerosis) and organ specific diseases such as chronic active hepatitis and insulin dependent diabetes mellitus (Tiwari and Terasaki, 1985). With several exceptions, the most notable being the strong association of B27 with ankylosing spondylitis, uveitis and reactive arthritis, the majority of HLA and disease associations appear strongest with class II alleles.

It is beyond the scope of this chapter to detail all the HLA and disease associations described to date but rather make general statements concerning possible mecha­nisms to explain these associations.

Molecular Mimicry

Certain HLA associations may be explained by a shared amino acid sequence between a disease causing organism and a class I or class II molecule. When this occurs an individual may respond inadequately to a foreign organism, resulting in chronic infection and ensuing disease pathogenesis. Conversely, an autoimmune response to a self protein may occur as a result of a shared amino acid sequence with a foreign organism.

The B27 association with AS and the reactive arthropathies may reflect such a mechanism. B27 shares amino acid sequences with several microorganisms and injection of rabbits with B27 positive lymphocytes produces antibodies that react with Klebsiella and enterobacter antigens (Ebringer et al., 1980).

Restriction of Antigen Presentation

Presentation to the T cell receptor of either self or foreign peptide on class II molecules could explain some of the class II associations with disease. Certain peptides have been shown to preferentially bind to particular class II allelic products (O'Sullivan et al., 1990). In the case of, for example, viral infection, failure to bind peptide may result in lack of immune response and persistence of viral infection with subsequent disease pathogenesis. An HLA association with an autoimmune response may also reflect preferential binding of a self peptide to a particular class II molecule.

In addition to class II molecules consideration must be given to the role of class I molecules as targets of autoimmune and viral specific cytotoxic T cells. Varying levels of class I restricted cytotoxicity and cell damage may reflect different degrees

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The Major Histocompatibility Complex 133

of peptide binding to class I molecules. Other factors such as allelic variation in the

degree of class I upregulation as a result of cytokine stimulation could also play a

role.

Class III Association

Class III allelic polymorphism may be responsible for susceptibility in some

diseases, e.g., Systemic Lupus Erythematosus (SLE). HLA haplotypes associated

with this disease appear to contain a null C4 allele, suggesting complement

deficiency may play a role (Schneider et al., 1991). In addition, the class III region

contains many genes whose functions have not been elucidated and which may

influence susceptibility in other diseases.

In summary, there may be several MHC genetic loci acting alone or in concert

to confer susceptibility to a range of human diseases. The reader is referred to

additional references listed at the end of this chapter which cover in greater detail

the genetic associations and molecular mechanisms underlying these diseases.

SUMMARY

The major histocompatibility complex (MHC) consists of a cluster of genes on the

short arm of human chromosome six which serves an immunoregulatory function

and is critical in the recognition of self from non-self There are three gene regions

in the MHC. The class I genes (HLA-A,B,C) code for molecules which are

expressed on most tissue cells and function as presentation molecules for endo­

genous self and foreign peptides to CDS cytotoxic T cells. The class II gene products

(HLA-DR,DQ,DP) are expressed on a limited range of cell types termed antigen

presenting cells, and present exogenous peptide to CD4 helper T cells. Located in

between the class I and class II genes are the class III genes which code for soluble

mediators of immune function such as complement components, heat shock pro­

teins and tumor necrosis factor.

The HLA class I and class II genes are highly polymorphic, and as such, are

powerful stimulators of alloresponsiveness, representing the major human trans­

plantation system. Matching for class I and class II molecules is critical therefore

in improving graft survival in many forms of transplantation.

Many human diseases particularly those of autoimmune nature show associations

with particular class I and class II alleles. Although the exact mechanisms for these

associations are not fully understood, the concepts of molecular mimicry (i.e.,

shared epitopes between microorganisms and MHC molecules) and preferential

binding of certain viral or auto-peptides to class I and II molecules are popular

concepts. There is also evidence that polymorphic genes within the class III region

may also explain some MHC and disease associations.

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134 BRIAN D.TAIT

REFERENCES

Amett, K.L., & Parham, P. (1995a). HLA class I nucleotide sequences. Tissue Antigens 45, 217-257.

Bodmer, J.G., Marsh, S.G.E., Albert, E.D., Bodmer, W.F., Bontrop, R.E., Charron, D., Dupont, B.,

Erlich, H.A., Mach, B., Mayr, W.R., Parham, P., Sasazuki, T., Schreuder, G.M.Th., Strominger,

J.L., Svejgaard, A., & Terasaki, P.I. (1995). Nomenclature for factors of the HLA system, 1995.

Human Immunology 43, 149-164.

Bjorkman, P.J., Saper, M.A., Samraoui, B., Bennett, W.S., Strominger, J.L., & Wiley, D.C. (1987).

Structure of the human class I histocompatibility antigen HLA-A2. Nature 329, 506-512.

Dausset, J. (1958). Iso-leuco-anticorps. Acta Haematol. 20, 156-166.

Ebringer, A., Avakian, H., Cowling, P., Welsh, J., Wooley, P., & Panayi, G. (1980). Ankylosing

spondylitis, HLA B27 and Klebsiella. Cross reactivity studies with rabbit anti-lymphocyte sera

and human tissue typing sera. Ann. Rheum. Dis. 39, 194.

Erlich, H.A. (Ed.) (1989). PCR Technology. Principles and application of DNA amplification. Stockton

Press, New York.

Geraghty, D.E., Koller, B.H., & Orr, H.T. (1987). A human major histocompatibility complex class I

gene that encodes a protein. Proc. Natl. Acad. Sci. USA 84, 9145-9149.

Geraghty, D.E., Wei, X., Orr, H.T., & Koller, B.H. (1990). Human leukocyte antigen F (HLA-F)—An

expressed HLA gene composed of a class I coding sequence linked to a novel transcribed repetitive

element. J. Exp. Med. 171,1-18.

Glynne, R., Powis, S.H., Beck, S., Kelly, A., Kerr, L-A., & Trowsdale, J. (1991). A proteosome—related

gene between the two ABC transporter loci in the class II region of the human MHC. Nature 353,

357-360.

Kelly, A., Powis, S.H., Glynne, R., Radley, E., Beck S., & Trowsdale, J. (1991). Second proteosome—

related gene in the human MHC class II region. Nature 353, 667-668.

Kimura, A., & Sasazuki, T. (1992). Eleventh International Histocompatibility Workshop reference

protocol for the HLA-DNA typing technique. In: HLA 1991. Proceedings of the Eleventh

International Histocompatibility Workshop and Conference, Vol. 1 (Tsuji, K., Aizawa, M.,

Sasazuki, T., Eds.), pp. 397-419. Oxford University Press.

Koller, B.H., Geraghty, D.E., Shimizu, Y., DeMars, R., & Orr, H.T. (1988). A novel HLA class I gene

expressed in resting T lymphocytes. J. Immunol. 141, 897—904.

Marsh, S.G.E., & Bodmer, J.G. (1995). HLA class II nucleotide sequences 1995. Human Immunology

45, 258-280.

Ohya, K-I., Kondo, K., & Mizuno, S. (1990). Polymorphism in the human class I MHC locus in Japanese.

Immunogenetics 32, 205-209.

Opelz, G. (1992). Collaborative Transplant Study—10 year report. Trans. Proc. 24, 2342-2355.

Opelz, G. (1993). Collaborative Transplant Study. Newsletter 1, 93. Ruprecht-Karls Universitat,

Heidelberg, Germany.

O'Sullivan, D., Sidney, J., Appella, E., Walker, L., Phillips, L., Colon, S.M., Miles, C, Chesnut, R.W.,

& Sette, A. (1990). Characterization of the specificity of peptide binding to four DR haplotypes.

J. Immunol. 145, 1799-1808.

Powis, S.H., Mockridge, I., Kelly, A., Kerr, L-A., Glynne, R., Gileadi, U., Beck, S., & Trowsdale, J.

(1992). Polymorphism in a second ABC transporter gene located within the class II region of the

human major histocompatibility complex. Proc. Natl. Acad. Sci. USA 89, 1463—1467.

Ramer, L.E., Phelan, D., &. Brunt, E.M. (1993). Probable antibody mediated failure of two sequential

ABO compatible hepatic allografts in a single recipient. Transplantation 55, 814—819.

Schneider, P.M., Hartung, K., Seuchter, S.A., Albert, E., Baur, M.P., Coldewey, R., Kalden, J.R.,

Lakomek, H.J., Peter, H.H., Ritmer, C, Schendel, D., & Deicher, H.R. (1991). Association of

MHC class I, II and III genes with systemic lupus erythematosus—^Results of a German collabo­

rative study. In: HLA Vol. 2 (Tsuji, K., Aizawa, M., Sasazuki, T., Eds.), pp. 525-528. Oxford

University Press, Oxford.

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The Major Histocompatibility Complex 135

Shaw, S., Johnson, A., & Shearer, G. (1980). Evidence for a new segregant series of B cell antigens that

are encoded in the HLA-D region and that stimulate secondary allogeneic proliferation and

cytotoxic responses. J. Exp. Med. 152, 565-580.

Spies, T., Bresnahan, M., Barham, S., Arnold, D., Blanck, G., Mellins, E., Pious, D., & DeMars, R.

(1990). A gene in the human major histocompatibility complex class II region controlling the class

I antigen presentation pathway. Nature 348, 744-747.

Terasaki, P.I., & McClelland, J.D. (1964). Microdroplet assay of human serum cytotoxins. Nature 204,

99^-1000.

Tiwari, J.L., & Terasaki, P.I. (1985). HLA and Disease Associations. Springer-Verlag, New York.

White, P.C, New, M.I., & Dupont, B. (1986). Structure of human steroid 21-hydroxylase genes. Proc.

Natl. Acad. Sci. USA 83, 5111-5115,

Yang, S.Y. (1989a). A standardized method for detection of HLA-A and HLA-B alleles by one

dimensional isoelectric focusing (lEF) gel electrophoresis. In: Immunology of HLA (Dupont, B.,

Ed.), Vol. I, pp. 332-335. Springer-Verlag, New York.

Yang, S.Y. (1989b). Nomenclature for HLA-A and HLA-B alleles detected by one dimensional

isoelectric focusing (ID-IEF) gel electrophoresis. In: Immunology of HLA (Dupont, B., Ed.), Vol.

I, pp. 54-57. Springer-Verlag, New York.

FURTHER READINGS

General Genetic Structure

Campbell, R.D., & Trowsdale, J. (1993). Map of the human MHC. Immunol. Today 14, 349-352.

Tait, B.D. (1990). MHC-From serology to sequence. Today's Life Science January 1990, 30-40.

Tsuji, K., Aizawa, M., & Sasazuki, T., (Eds.) (1992). HLA 1991. Proceedings of the Eleventh

International Histocompatibility Workshop and Conference. Oxford University Press, Oxford.

Class III

Campbell, R.D., Carroll, M.C., & Porter, R.R. (1986). The molecular genetics of components of

complement. In: Advances in Immunology Vol. 38, pp. 203-244. Academic Press Inc., Orlando.

Carroll, M.C., Katzman, P., Alicot, E.M., Koller, B.H., Geraghty, D.E., Orr, H.T., Strominger, J.L., &

Spies, T. (1987). Linkage map of the human major histocompatibility complex including the

tumour necrosis factor genes. Proc. Natl. Acad. Sci. USA 84, 8535-8539.

Higashi, Y., Yoshioka, H., Yamane, M., Gotoh, O., & Fujii-Kuriyama, Y. (1986). Complete nucleotide

sequence of two steroid 21-hydroxylase genes tandemly arranged in human chromosome: A

pseudogene and a genuine gene. Proc. Nad. Acad. Sci. USA 83, 2841-2845.

Milner, CM., & Campbell, R.D. (1991). Polymorphic analysis of the three MHC linked HSP70 genes.

In: HLA 1991 Vol. 2 (Tsuji, K., Aizawa, M., & Sasazuki, T., Eds.), pp. 157-161. Oxford University

Press.

Spies, T., Blanck, G., Bresnahan, M., Sands, J., & Strominger, J.L. (1989). A new cluster of genes within

the human major histocompatibility complex. Science 243, 214-217.

Speiser, P.W., New, M.I., & White, P.C. (1988). Molecular genetic analysis of non classical steroid

21 hydroxylase deficiency associated with HLA-B 14, DRl. New Eng. J. Med. 319, 19-23.

Techniques

Tsuji, K., Aizawa, M., & Sasazuki, T. (Eds.) (1992). HLA1991. Proceedings of the Eleventh Interna­

tional Histocompatibility Workshop and Conference. Oxford University Press, Oxford.

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136 BRIAN D.TAIT

Function of Class I and Class II Molecules

Neefles, J.J., & Momburg, F. (1993). Cell biology of antigen presentation. Current Opinions in Immunology 5, 27-34.

HLA and Transplantation

Opelz, G. (1992). Collaborative transplant study—10 year report. Transplant Proceedings 24, 2342-2355.

Tait, B.D. (1993). Seeking the perfect match: HLA and transplantation. Medical J. Australia 159, 696-700.

Terasaki, P.I. (Ed.). Series entided "Clinical Transplants" 1986-1995. UCLA Tissue Typing Laborato­ries.

HLA and Disease

Tiwari, J.L., & Terasaki, P.I. (1985). HLA and Disease Associations. Springer-Verlag, New York.

Tsuji, K., Aizawa, M., & Sasazuki, T. (Eds). (1992). HLA 1991. Proceedings of the Eleventh

Histocompatibility Workshop and Conference. Oxford University Press, Oxford.

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Chapter 8

B and T Cell Signaling at the

Molecular Level

TOMAS MUSTELIN and PAUL BURN

Introduction 138

The BCR and TCR Receptors 138

Inositol Phospholipid Hydrolysis and Calcium Mobilization 140

Tyrosine Phosphorylation 141 The Src Family of Nonreceptor PTKs 143

Lck: A Co-receptor Associated PTK 143 Fyn\ A Src Family PTK Associated with the TCR Complex 143 Src Family PTKs in B cells: Blk, Lyn, and Fyn 144

The Syk Family of Nonreceptor PTKs 144 Other PTKS in Lymphocytes 144 Regulation of Src Family PTKs 144

CD45 PTPase and Csk PTK: Regulators of Src Family Kinases 146 Toward the Nucleus 147

Principles of Medical Biology, Volume 6 Immunobiology, pages 137—150. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved.

ISBN: 1-55938-811-0

137

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138 TOMAS MUSTELIN and PAUL BURN

Summary 149

Recommended Readings 149

INTRODUCTION

The major function of mature B and T lymphocytes is to recognize a foreign antigen and respond to it by inducing a complex activation process. This ultimately leads to recruitment of other cell types and initiation of a complete immune response. On the molecular level, recognition of antigen is mainly mediated by unique antigen receptors, namely the B cell antigen receptor complex (BCR) on B lymphocytes and the T cell antigen receptor complex (TCR) on T cells. Binding of properly presented antigen to these receptors initiates an ordered cascade of biochemical changes and generates signals that are transmitted sequentially from the cell surface to the nucleus, causing activation of previously silent genes in a highly co-ordinated manner. This finally results in the transformation of the resting cells to metabo-lically active and often proliferating lymphoblasts, actively secreting immuno­globulins or immunoregulatory lymphokines or displaying targeted cytotoxicity. This chapter will present a critical overview of the most important recent advances in our understanding of how signals from the BCR and TCR are transduced. Additional details can be obtained from the recommended literature at the end of this chapter.

THE BCR AND TCR RECEPTORS

The BCR complex consists of a transmembrane immunoglobulin D or M molecule, having two heavy chains and two light chains, and at least two invariant glycopro­teins, BCR-a (MB-1) and BCR-p (B29) (Figure 1). Recognition of antigen is mediated by the variable portion of the Ig chains and leads to oligomerization of BCRs and concomitantly signal transduction into the cell. While the Ig heavy chains have a very short cytoplasmic tail (the light chains are solely extracellular), the BCR-a and BCR-P polypeptides have long intracellular tails and are thought to be mainly responsible for signal transmission.

The TCR complex is structurally and functionally related to the BCR complex. On most mature T cells the TCR consists of two highly polymorphic disulfide-linked proteins (a and p chains), similar to immunoglobulins in their overall primary structure and gene organization, and CD3, a complex of four (or five) invariant polypeptides termed CD3 y, 6, 8, C„ and r|, (r| is a splice-variant of Q. These are 16-28-kD transmembrane proteins noncovalently associated with the clonotypic aP-chains. About 80-90% ofC, is found as a disulfide-linked homodi-mer: the rest forms a heterodimer with the r| subunit. As in the case of the BCR complex, the antigen-recognizing aP-TCR polypeptides each contain a single

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B and T Cell Signaling at the Molecular Level 139

TCR

CDS B29 MB-1

Figure 1, Structure of the TCR and BCR. The antigen (Ag) binding sites are shaded. LC, light chain; HC, heavy chain of immunoglobulin.

membrane spanning region, but very short (« 12 amino acids) cytoplasmic domains, while the other subunits, notably C,, have large cytoplasmic tails that participate in signal generation.

Recent studies have also begun to unravel the structures in the receptor complexes that couple to signal transduction components. A conserved tyrosine-containing amino acid motif is found in the small subunits of both the TCR and BCR complexes. The precise role of these motifs is not clear yet, but there is evidence that their presence is required for signal transduction and that they may bind other signaling proteins involved in propagation of the signal.

The expression of either CDS or CD4 glycoproteins on the surface of T lympho­cytes correlates with their ability to recognize antigen in the context of class I and II major histocompatibility complex (MHC) molecules, respectively (Figure 2).

-ABC

Figure 2. Participation of the CD4 and CD8 co-receptors during recognition of an antigenic peptide (Ag) on class II (HLA-D) or class I (HLA-ABC) MHC molecules.

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140 TOMAS MUSTELIN and PAUL BURN

CD4" cells are usually of a helper/inducer phenotype, while CDS'*" cells predomi­nantly exhibit cytotoxic or suppressor properties. CD8/CD4 play an important accessory role in T cell activation by binding to MHC class I or class II molecules. They stabilize the interaction of the latter with the TCR/CD3 complex and serve an active role in transmembrane signalling during T cell activation. Furthermore, recent studies indicated that CD4/CD8 have an important function in the thymic selection of the T cell repertoire.

INOSriOL PHOSPHOLIPID HYDROLYSIS AND CALCIUM MOBILIZATION

Some 10 years ago it was found that antigen receptor-mediated activation of B and T cells is associated with rapid hydrolysis of inositol phospholipids, mainly phosphatidylinositol-4,5-bisphosphate, by phospholipase C (PLC). This response is also induced by a variety of other receptors in many different cell types, and leads to the production of two second messengers: (i) diacylglycerol, which activates the serine/threonine-specific protein kinase C (PKC), and (ii) inositol-1,4,5-trisphos-phate, which leads to the mobilization of Ca " from intracellular stores (Figure 3). This also provided a mechanism for the increase in the intracellular Ca "" concen­tration ([Ca " ]i) observed in activated lymphocytes.

WY^:^ IR

Figure 3. Receptor-induced activation of phospholipase C (PLC) leads to hydrolysis of phosphatidyl-inositol-4,5-bisphosphate (PIP2), producing inositol-l,4,5-triphos-phate (IP3), which liberates Ca " from intracellular stores; and diacylglycerol (DC), which activates protein kinase C (PKC). Activated PKC, in turn, phosphorylates a number of cellular proteins on serine and threonine residues.

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B and T Cell Signaling at the Molecular Level 141

The fact that increased inositol phospholipid hydrolysis was detected as early as 20 seconds after receptor triggering and [Ca " ]i started to rise within 30-60 seconds made researchers conclude that these events were among, if not the earliest, biochemical events induced by BCR or TCR triggering. This notion was further supported by the finding that a combination of Ca^'^-ionophores, raising [Csi^^]i

similarly to receptor triggering, and phorbol esters, which bind to and directly activate PKC, leads to several events characteristic of lymphocyte activation such as lymphokine secretion, morphological changes and proliferation. Today we know that many other biochemical events, notably tyrosine phosphorylation, take place before or in parallel with the activation of PLC, and that phorbol esters not only mimic the diacylglycerol-induced activation of PKC, but also affects many other important enzymes. Nevertheless, PKC and [Ca " ]i seem to be an important part of signal transduction in B and T lymphocytes.

TYROSINE PHOSPHORYLATION

While the activation of PLC by BCR and TCR stimulation has been known for more than a decade, the involvement of tyrosine phosphorylation as an important mechanism of lymphocyte signal transduction has only recently been recognized. The first polypeptide found to be tyrosine phosphorylated upon receptor-triggering (reported in 1986) was the C^-chmn of the TCR/CDS complex itself It took several years until other substrates were found. Initial studies addressed the question how PLC was activated and whether tyrosine phosphorylation might be involved. Experiments using genistein, a specific inhibitor of protein tyrosine kinases (PTKs),

Table 7, Tyrosine Phosphorylated Proteins in Activated Lymphocytes

Protein

TCR/CD3 (;

TCR/CD3 8

p42"^^^ p44"'^'^

She

CDS

Zap

Syk

Raf

PI3-K

Vav

VCP

rasGAP

PLCY2

PLCyl

CD45

Mr (kD)

16-21

23

42/44

46/52

70

70

72

72-75

85+110

95

100

120

145

148

180-240

Lymphocyte^

f T

T, B

T

T

T

T, B

T

T, B

T, B

T

T, B

B

T

T

Induced by

TCR, CD2, CD4

TCR

TCR, BCR, Lck in vitro

TCR

TCR, in vitro

TCR

TCR, BCR

IL2 receptor

TCR, BCR, Lck in vitro

TCR, BCR, Lck in vitro

TCR

TCR ?, BCR, Lck in vitro

BCR

TCR, Lck in vitro

TCR,PTPase inhibitors, Csk in vitro

Notes: ^ This list is not intended to be complete.

Abbreviations used: J, T cells; B, B cells.

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142 TOMAS MUSTELIN and PAUL BURN

indicated that PLC activation occurs as a result of tyrosine phosphorylation. This observation has later been confirmed by several groups and it is now well docu­mented that direct tyrosine phosphorylation of the yl and Y2 isoforms of PLC (PLCyl and PLCy2) is the event that results in the activation of these enzymes. The list of known tyrosine phosphorylated polypeptides (Table 1) now also includes many other proteins with known enzymatic activities or assumed functions, such as lipid kinases, protein kinases, nucleotide exchange factors, GTPase activating proteins (GAPs) and cytoskeletal components. To the extent known, the targets for rapid tyrosine phosphorylation are identical or similar, not only in various subsets of lymphocytes but also in other cell types. PTK inhibitors which prevent phospho­rylation of all these substrates, also block all examined early and late parameters of cell activation, including increased phosphatidylinositol turnover, and increase in [Ca "* ]i, IL-2 receptor expression, cytotoxicity, blast transformation and DNA synthesis.

Unlike many growth factor receptors, the TCR and BCR receptors do not have intrinsic PTK activity themselves. Thus, the induction of tyrosine phosphorylation during activation of lymphocytes implies that these receptors can either recruit cellular PTKs or suppress one or more phosphotyrosine phosphatases (PTPases). Current understanding favors the first possibility but does not rule out the latter. A large number of recent reports indicate that two families of nonreceptor PTKs, the Src (Table 2) and Syk families, are involved in signal transduction in lymphocytes. In T cells, the two Src family kinases, Lck (p56'' ) and Fyn (p59 '") and the Syk family PTK Zap (ZAP-70; pWP) all play active roles in TCR-induced signal transduction. Mice lacking a functional lck or fyn gene display severely deficient capability to respond to TCR-stimulation. In addition, Lck, but not Fyn, seems to be crucial for T cell maturation in the thymus. During T cell activation, both kinases are activated and at least Lck binds and presumably phosphorylates PLCyl. Less

Table 2, Expression of Src Family Tyrosine Kinases in Leukocytes

Gene

c-src

c-yes 1

fyn

c-fgr

lyn

lck

hck

bik

Mr (kD)

60

62 (59)

59

55/53^

56/53^

56 (58, 60,

59/56^

55

62,.,

Expression in Leukocytes^

thrombocytes, low or absent in others

ubiquitous, thrombocytes, T

ubiquitous, thrombocytes, T, B, NK

NK, B, G

B, G, Mo, thrombocytes

.f T, NK

G, M

B

Notes: ^ Abbreviations used: T, T cells; B, B cells; NK, natural killer cells; G, granulocytes; Mo, monocytes.

Two Mr forms due to alternative mRNA splicing. ^ Multiple Mr forms due to posttranslational modification.

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B and T Cell Signaling at the Molecular Level 143

information is available regarding the importance of the various PTKs present in

B lymphocytes, but the Src family PTKs (Table 2), Blk (p55 ' ), Lyn (p56'y") and

Fyn, and Syk (p72^^ ) are all activated by BCR triggering and co-immunoprecipitate

with the BCR.

THE Src FAMILY OF NONRECEPTOR PTKs

Lck: A Co-receptor Associated PTK

Much of our current understanding of the function and regulation of Src family

PTKs stems from the breakthroughs in the work with Lck over the past 4—5 years.

This PTK, which is expressed at high levels only in T lymphocytes, is located at

the inner surface of the plasma membrane, largely due to the covalent attachment

of myristic acid to its NH2-terminus (a feature of all Src family PTKs). In addition,

Lck is non-covalently associated with the cytoplasmic domain of the CD4 or CDS

glycoproteins and with the P-subunit of the interleukin-2 receptor.

The high stoichiometry of Lck association with the CD4 or CDS co-receptors

suggests a direct role of Lck in T cell activation. Moreover, a mutant T cell line that

was deficient in T cell activation due to lack of CD4 could be rescued by wild-type

CD4, but not by mutant forms of CD4 that were unable to associate with Lck. A

genetic approach provided additional evidence for the involvement of Lck in T cell

signaling. Mutant cell lines were selected that upon TCR triggering showed no

increase in [Ca " ]i. One of them was analyzed in detail and was found to express a

non-functional form of Lck. This defect could be restored by transfecting the

/cA:-deficient cell line with wild-type lck. Lck also plays an essential role in thymic

development. Studies using lck knock-out mice revealed that thymocyte develop­

ment was blocked at a very early stage of maturation. Thus, Lck is clearly a key

factor in both thymocyte development and signal transduction in mature T lympho­

cytes.

Fyn: A Src Family PTK Associated with the TCR Complex

The simple model of Lck being the important kinase was complicated by the

finding that another Src family PTK, Fyn, is directly associated with the TCR

complex. Further support for an important role for Fyn came from observations that

thymocytes from mice expressing ajyn transgene were more readily triggered by

TCR stimulation and produced higher levels of IL-2 than controls. Conversely, T

cells from mutant mice lacking Fyn displayed diminished responses. Genetic

studies using « knock-out mice yielded surprising results: thymocyte develop­

ment inj^n-negative mice was essentially normal, and T cell signaling was affected

only at specific stages of maturation.

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144 TOMAS MUSTELIN and PAUL BURN

Src Family PTKs in B cells: Bik, Lyn, and Fyn

In B lymphocytes, triggering of the BCR has been shown to activate the Src family PTKs Blk, Lyn and (to a lesser degree) Fyn. Furthermore, at least Lyn and Fyn co-immunoprecipitate with the BCR, suggesting a direct or indirect physical association. It is not yet known if any of these Src family PTKs is more important than the others in signal transmission from the BCR and whether these different Src family members interact with distinct sets of substrate proteins.

THE SYK FAMILY OF NONRECEPTOR PTKs

The Syk family PTKs are "newcomers" in the field of lymphocyte signal transduc­tion. Although Syk was purified from thymus in 1986, it was not until 1991 that this kinase was found to become tyrosine phosphorylated and activated by BCR triggering. In T cells, another member of this family. Zap, is similarly phosphory­lated upon receptor stimulation, and associates with the C, chain of the TCR/CDS complex in a tyrosine phosphorylation-dependent manner. Syk co-immunoprecipi-tates with the BCR, but this association seems to be independent of activation and tyrosine phosphorylation. Whether this difference between Syk and Zap is only apparent, or reflects a true difference in their biology, remains uncertain.

OTHER PTKs IN LYMPHOCYTES

Lymphocytes also express several other types of nonreceptor PTKs, as well as some receptor-type PTKs. The former class includes kinases of the abl-, tec/itk/atk-Jak-,

c-fes-, Jak- and c^/r-families, while the latter includes the receptor for insulin. In addition, novel PTKs are discovered at an increasing rate, suggesting that between 1,000 and 3,000 kinases exist in the mammalian genome. If half of these are PTKs and an estimated 10% of these are expressed in lymphocytes, each lymphoid cell may contain some 50-150 different PTKs. It is puzzling that so many different PTKs are required in a single cell type. Apparently, these PTKs have specialized functions, are differentially regulated, have different substrates, and regulate each other in complex networks or cascades. Obviously, elucidation of these pathways will require a lot more of hard work.

REGULATION OF SRC FAMILY PTKs

The function of Src family PTKs in antigen receptor signaling, as well as in other aspects of lymphocyte physiology, is tightly regulated at all available levels: transcription of the genes, translation and processing of the resulting RNA, post-translational modifications and protein-protein interactions (see Fig. 4). Best char­acterized are the post-translational mechanisms, including reversible phosphorylation of tyrosine and serine residues, myristylation at Gly-2, association

Page 160: Immunity

B and T Cell Signaling at the Molecular Level 145

myristylation site

\

unique domain

auto- negative phosphorylation regulatory

site site

B — ( s H 3 ) 4 ^ ^ ^ H ^"^

£ domain J src -family PTKs

syk -family PTKs

T auto-

phosphorylation site(s)

Figure 4, Schematic representation of Src and Syk family protein tyrosine kinases.

with transmembrane receptors and cytoskeletal elements and complex formation

with regulatory proteins or substrates. Different Src family PTKs seem to be

regulated very similarly, but may associate with different transmembrane or regu­

latory proteins. These associations are largely dictated by the unique N-terminal

80-100 amino acids of Src family PTKs, and two independently folded hemispheri­

cal domains termed SH2 and SH3 (for ^rc //omology). SH3 domains (« 50 amino

acids) bind proline-rich motifs on other proteins and may functionally link the PTKs

to G-protein signaling pathways or the cytoskeleton. SH2 domains, on the other

hand, comprise « 100 amino acids and were originally identified on PTKs such as

c-Src (p60"-''"), Fps (p98^P') and Abl (pHS^*'^ One or two SH2 domains are also

found in many other proteins known to be involved in signal transduction, e.g.,

PLCyl, PLCy2, GAP, phosphatidylinositol 3-kinase (PI 3-K), Grb-2, She (p52 ' ' )

and in the PTKs of the Syk family. Distinct SH2 domains, which display a 3O--60%

sequence homology, were shown to bind different phosphotyrosine-containing

peptide motifs, thereby inducing specific protein-protein interactions. Together,

SH2 and SH3 domains are also important for the regulation of the catalytic activity

of Src kinases. The enzymatic activity of Src family kinases depends on the

phosphorylation state of two tyrosine residues (Figure 4). The major site of in vitro

autophosphorylation (Tyr-394 for Lck and Tyr-417 for the hematopoietic form of

Fyn) is normally phosphorylated to low stoichiometry in vivo, but a high level

phosphorylation at this site correlates with situations in which Lck and Fyn are

activated. In contrast, a more carboxy-terminal site (Tyr-505 for Lck and Tyr-528

for Fyn) has a negative regulatory function, i.e. phosphorylation correlates with

suppression of enzyme activity. This is thought to take place via an intramolecular

folding mechanism in which the phosphorylated C-terminus loops back onto its

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146 TOMAS MUSTELIN and PAUL BURN

own SH2 domain, resulting in sterical inactivation of the catalytic domain. Corre­

spondingly, mutation of either the conserved carboxy-terminal tyrosine residue to

phenylalanine (an amino acid that is similar to tyrosine but cannot be phosphory-

lated) or mutations within the SH2 domain, both, in turn, lead to a constitutively

increased PTK activity of the enzyme and lead to cellular transformation when

overexpressed in NIH3T3 fibroblast cells. The effects of active Src family PTKs

in these cells are not, however, directly applicable to lymphoid cells. Thus, although

transfection of Tyr-505 to Phe-505 mutated Ick into a CD4~8" T cell line did not

alter its level of basal tyrosine phosphorylation, it augmented TCR-induced tyro­

sine phosphorylation and interleukin-2 production. This indicates that the level of

tyrosine phosphorylation in T cells does not simply reflect the catalytic activity of

PTKs, but that other mechanisms, such as PTPases and substrate accessibility, play

major roles.

CD45 PTPase and Csk PTK: Regulators of Src Family Kinases

The enzymes acting on the conserved C-terminal negative regulatory tyrosine residues of Src family PTKs seem to include the CD45 PTPase and the Csk (p50^^ ) PTK. The latter is a cytosolic nonreceptor PTK expressed in all cell types examined. Structurally, Csk resembles the Src family kinases in having SH3, SH2 and kinase domains, but differs in lacking an N-terminal membrane attachment motif and the two tyrosine residues corresponding to the autophosphorylation site and the C-ter­minal negative regulatory site typical of Src family kinases. Csk seems to be highly specific for the C-terminus of Src family PTKs.

CD45 is a large (180-220 kD) transmembrane receptor-like PTPase with a highly glycosylated and variable extracellular portion and an invariable cytoplasmic part consisting of two PTPase domains in tandem. CD45 appears to be essential for TCR-induced T cell activation, as indicated by the findings that CD45-negative T cell mutants failed to respond to TCR-triggering with proliferation, early tyrosine phosphorylation or inositol phospholipid hydrolysis. The response to antigen was regained in a revertant CD45" clone. Although some controversy remains regarding the preference for specific Src family members, data from several laboratories agree with the notion that at least Lck and Fyn are physiologically relevant substrates for CD45 in vivo. Dephosphorylation of these kinases at their C-terminal negative regulatory site by CD45 seems to correlate with responsiveness of the T cells to TCR stimulation, suggesting that the role of CD45 is, at least partly, to keep Src family PTKs sufficiently active to participate in signal transmission. Findings with CD45-negative B lymphocytes suggest that B cell-specific kinases are similarly regulated by CD45. It should also be noted that other PTPases seem to perform the same type of task. For example, expression of the CD45-related receptor-like PTPase, PTPa, in fibroblasts activated the transforming potential of c-Src by dephosphorylating its negative regulatory Tyr-527.

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B and T Cell Signaling at the Molecular Level

TOWARD THE NUCLEUS

147

Even if many details are elusive, it is clear that triggering of lymphoid antigen

receptors causes an intracellular protein tyrosine phosphorylation signal which

leads to a change in the transcription of genes and eventually results in cell

activation. Much of the current research focuses on the targets of tyrosine phospho­

rylation (Table 1). Obviously, these substrate proteins must be responsible for

transmission of the signal from the membrane receptors through the cytoplasm to

the nucleus (Figure 5). Some of these immediate down-stream effector proteins are

already known and their regulation and function is under investigation.

t\ ,. Ca++

V

Figures. The TCR-induced tyrosine phosphorylation cascade. For abbreviations see text.

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148 TOMAS MUSTELIN and PAUL BURN

T cell activation is not only accompanied by hydrolysis of inositol phospholipids, but also by enhanced formation of inositol phospholipids phosphorylated at the 3-position of the inositol ring. These lipids are poor substrates for PLC and their physiological function remains unknown. Their production, however, seems to correlate with cell proliferation as well as malignant transformation in many cell systems. The enzyme producing 3-phosphorylated inositol lipids, PI 3-K, is a heterodimer consisting of a regulatory 85 kD (p85a) and a catalytic 110 kD (pi 10) subunit. It has been found that both subunits of PI 3-K become tyrosine phospho­rylated during T cell activation and that direct phosphorylation of PI 3-K by Lck can activate the lipid kinase. Furthermore, PI 3-K activation was deficient in a Lck-negative cell line, suggesting that it may be a direct substrate for Lck.

The GTP binding protein Ras (p2 T ) has been known as an oncogene for several years. It can be viewed as a molecular switch with an "off state, in which it is bound to GDP, and an "on" state, in which it is bound to GTP. Stimulation of the TCR results in replacement of GDP by GTP and thereby activation of Ras proteins. However, it remains unclear by what molecular mechanism the TCR activates Ras and how the signal is transmitted to the next components of the signaling machin­ery. Very recent work revealed that at least two pathways link TCR signaling to Ras activation (Figure 5).

One of the earliest events upon triggering of the TCR/CD3 complex is tyrosine phosphorylation on the C^ chain. As a result. She, an adaptor protein containing an SH2 domain but no obvious catalytic domain, binds to phosphorylated C^ chain and becomes in turn phosphorylated on tyrosine. Another adaptor protein, Grb2, binds via its SH2 domain to tyrosine phosphorylated She and via its two SH3 domains to a second protein named Sos. Sos was shown to possess a guanine nucleotide exchange activity that activates Ras by replacing GDP by GTP. Thus, activation of Ras is most likely due to physical recruitment of Sos to the plasma membrane, where it can interact with Ras, which is anchored in the membrane, too.

As an alternative pathway of Ras activation, another nucleotide exchange factor, Vav, is phosphorylated on tyrosine very rapidly after activation of T or B cells. In the case of Vav, phosphorylation of the exchange factor stimulates its activity and thereby switches on Ras proteins. It remains to be determined how these two pathways operate in T cells to activate Ras, and whether alternate means of Ras activation may occur under different conditions of T cell stimulation.

From Ras the signal is passed on to c-Raf (p72^"'' , a serine/threonine-protein kinase. This kinase can, in turn, activate a series of enzymes termed the mitogen activated protein (MAP) kinases, and thereby initiates a cascade of phosphorylation events which culminates in the phosphorylation of nuclear proteins and transcrip­tion factors.

In contrast to this highly complex pathway, a more direct mode of activating transcription factors has recently been discovered. Several receptors, e.g., epider­mal growth factor (EGF) receptor, interferon receptors, and various interleukin

Page 164: Immunity

B and T Cell Signaling at the Molecular Level 149

receptors, respond to ligand binding by directly activating latent cytoplasmic transcription factors. This is mediated by tyrosine phosphorylation of these tran­scription factors, either directly through receptors with intrinsic PTK activity or via yet unidentified C3^osolic PTKs. In response to their phosphorylation the transcrip­tion factors are translocated into the nucleus where they bind to specific DNA sequences, resulting in transcription of distinct sets of genes. This mechanism has so far not been described for the BCR and TCR, but it will be most challenging to elucidate the relevance of such a pathway for B-cell and T-cell activation.

SUMMARY

The BCR and TCR mediate specific recognition of antigen by lymphocytes. They are multisubunit cell surface proteins that in contrast to some growth factor receptors lack intrinsic tyrosine kinase activity. However, their smaller subunits contain distinct motifs that can interact with cytoplasmic protein tyrosine kinases. Additionally, the T cell co-receptors CD4 and CDS are complexed with the Src family kinase, Lck. Stimulation of BCR and TCR results in tyrosine phosphoryla­tion and activation of a subset of cellular proteins. Three pathways of intracellular signal transduction have so far been found to play a role in lymphocyte activation: (i) PLC Y activation, leading to elevated concentration of intracellular free Ca^^ and to activation of PKC, (ii) phosphorylation of inositol phospholipids at position 3 of the inositol ring, and (iii) the Ras pathway that results in a cascade of ser­ine/threonine phosphorylations. Together, these events result in changes in cellular structure and metabolism, in transcription of a distinct set of genes, and, eventually, in cell activation.

ACKNOWLEDGMENTS

We thank Drs. K. E. Amrein and T. Jascur for critical reading and comments on this

manuscript.

RECOMMENDED READINGS

Altman, A., Coggeshall, M., & Mustelin, T. (1990). Molecular events mediating T cell activation. Adv.

Immunol. 48, 277-360.

Egan, S.E., & Weinberg, R.A. (1993). The pathway to signal achievement. Nature 365, 781-783.

Feig, L.A. (1993). The many roads that lead to Ras. Science 260, 767-768.

Klausner, R.D., & Samelson, L.E. (1991). T cell antigen receptor activation pathways: The tyrosine

kinase connection. Cell 64, 875-878.

Montminy, M. (1993). Trying on a new pair of SH2s. Science 261, 1694-1695.

Mustelin, T. (1994). Src family of tyrosine kinases in leukocytes. Molecular biology intelligence unit.

RG Landes Co, Austin, Texas.

Mustelin, T., & Bum, P. (1993). Regulation of Src family tyrosine kinases in lymphocytes. Trends

Biochem. Sci. 18,215-220.

Page 165: Immunity

150 TOMAS MUSTELIN and PAUL BURN

Penninger, J.M., Wallace, V.A., Kishishara, K., & Mak, T.W. (1993). The role of p56''^ and p59^ " tyrosine kinases and CD45 protein tyrosine phosphatase in T-cell development and clonal selection. Immunol. Rev. 135, 183-214.

Perlmutter, R.M., Levin, S.D., Appleby, M.W., Anderson, S.J., & Alberola-Ila, J. (1993). Regulation

of lymphocyte function by protein phosphorylation. Ann. Rev. Immunol. 11, 451-499. Reth, M. (1995). The B-cell antigen receptor complex and co-receptors. Immunol. Today 16, 310-313. Robey, E., & Allison, J.P. (1995). T-cell activation: integration of signals from the antigen receptor and

costimulatory molecules. Immunol. Today 16, 306-310. Rudd, C.E., Janssen, O., Cai, Y.-C, da Silva, A.J., Raab, M., & Prasad, K.V.S. (1994). Two-step

TCR(7CD3-CD4 and CD28 signaling in T cells: SH2/SH3 domains, protein-tyrosine and lipid

kinases. Immunol. Today 15, 225—234. Thomas, M.L. (1989). The leukocyte common antigen family. Ann. Rev. Immunol. 7, 339-369. Weiss, A., & Littman, D.R. (1994). Signal transduction by lymphocyte antigen receptors. Cell 76,

263-274. Zenner, G., zur Hausen, J.D., Bum, P., & Mustelin, T. (1995). Towards unraveling the complexity of

T cell signal transduction. BioEssays 17, 967-975.

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Chapter 9

Cytokines in Immunology

ANDREW J. HAPEL and SHAUN R. McCOLL

Introduction 151

Cytokine Families and Their Cellular Sources 152

Lymphokines 154 Monokines 159 Chemokines 161

Receptors and Signaling Pathways 163

Clinical Applications 164

INTRODUCTION

Immunity is due to a family of cells called lymphocytes. Lymphocytes are highly differentiated end cells derived from progenitors found in adult bone marrow. Lymphocytes comprise two major cell types, 'T' cells that mature in the environ-

Principles of Medical Biology, Volume 6 Immunobiology, pages 151-169. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-811-0

151

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152 ANDREW J. HAPEL and SHAUN R. McCOLL

ment of the thymus, and antibody-producing 'B' cells that mature in the periphery (B is derived from the Bursa of Fabricius, the site of B cell production in birds. The organ does not exist in mammals). Hematopoiesis and lymphopoiesis are virtually inseparable components of the same system that throughout life replaces the cells of blood. The cells of blood provide an interactive system that cooperates to provide immunity to antigenic challenge, whether this be in the form of microorganisms, toxins or tumors. Lymphocytes rely on other blood cells (dendritic cells and macrophages) to present antigen to them so that immunity can be generated, and once activated by antigen, T lymphocytes in particular, direct blood cells to achieve their final function through the production of cytokines. Mature blood cells may be involved in the production of a particular class of immunoglobulin (B cells), the phagocytosis and destruction of microorganisms (macrophages and neutrophils), or release of mediators (mast cells and eosinophils).

The different cells of the immune and blood systems are able to interact because they are mobile, and because they are able to produce chemoattractants that orchestrate the accumulation of appropriate cell types to a focal point where the interactions leading to immune induction or effector function can occur. In addition, the different cell types involved in immune induction and effector function produce a range of soluble polypeptide molecules that are involved variously in chemoat-traction, cell activation, co-stimulation, and cell maturation. These cytokines comprise the lymphokine, colony-stimulating factor, chemokine, and monokine families. Various other names such as "interleukin" are subsumed within these groupings. The purpose of this chapter is to provide a listing and brief description of the sources and properties of the soluble mediators that function in immunity, and to provide a conceptual framework for their function. Finally we provide a brief overview of present and possible future clinical applications.

CYTOKINE FAMILIES AND THEIR CELLULAR SOURCES

Cytokines are made by virtually every cell of the body. The cytokines that regulate aspects of immunity are derived primarily from blood cells, and the reticulo-endo-thelial system. They include cytokines produced at sites of tissue damage to attract and activate granulocytes and monocytes, cytokines produced by granulocytes and monocytes to attract lymphocytes (the chemokines), cytokines produced by lym­phocytes and macrophages that act on myeloid cells and their progenitors (the colony-stimulating factors [CSFs]), and cytokines that are produced by lympho­cytes and monocytes that act predominantly on lymphocytes to regulate the immune response (lymphokines and interleukins [ILs]). In addition factors such as tumor necrosis factor a (TNF a) and lymphotoxin (TNF P), transforming growth factors (TGF) and other molecules can play a role in modulating immunity (see Table 1). The nomenclature of the cytokines and their families is a hostage to history, for many of them were named and classified based on the cells that were first found to produce them or on the basis of the cells on which they acted. As some cytokines

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Cytokines in Immunology 153

Table h

Cytokine

IL-1a, IL- ip

IL-2

IL-3

IL-4

IL-5

IL-6

IL-7

C-X-C

chemokines

C-C

chemokines

IL-9

IL-10

IL-11

IL-12

IL-13

C-CSF

M-CSF

GM-CSF

TNF-a

TNF-p

IFN-a

IFN-p

IFN-Y

Chromosome

2q 13-21

4q 26-28

5q 23-31

5q31

5q31

7p 15-21

8q 12-13

4q 13-21 (MIP-2)

17q 11.2-12 (MCP-1)

17q 11-21 (MIP-1)

5q 22-35 ?

19q 13.3-13.4

IL-12a,3p 11.2-12.2

IL-12b,5q 31-33

5q

17q 11.2-21

1p 13-21

5q 23-31

6p

6p

9p

9p21

12q24.1

Source

MACROPHAGES,

LYMPHOCYTES,

FIBROBLASTS

T CELLS T CELLS, MAST CELLS

T CELLS, MAST CELLS,

BONE MARROW

STROMAL CELLS

T CELLS, MAST CELLS

MACROPHAGES, MAST

CELLS, T CELLS,

FIBROBLASTS

BONE MARROW

STROMAL CELLS

MONOCYTES, T CELLS,

NEUTROPHILS,

ENDOTHELIAL CELL,

FIBROBLASTS

AS ABOVE

?

T CELLS

T CELLS

B CELLS AND

MACROPHAGES

T CELLS

MACROPHAGES,

FIBROBLASTS,

ENDOTHELIAL CELLS

FIBROBLASTS

T CELLS,

MACROPHAGES,

ENDOTHELIUM,

FIBROBLASTS

MACROPHAGES,

LYMPHOCYTES

T CELLS,

MACROPHAGES

MOST LEUKOCYTES

LEUKOCYTES,

FIBROBLASTS

T CELLS

Target

monocytes, endothelial

cells, fibroblasts, T

cells, keratinocytes

T cells

myeloid progenitor cells

mast cells

eosinophils

B cells

T cells

mainly neutrophils

monocytes, select T

cells, B cells,

basophils, eosinophils ?

macrophages

B cells

B cells, macrophages,

NK cells

macrophages

neutrophils, stem cells

monocytes,

macrophages, stem

cells, B cells

monocytes.

macrophages.

neutrophils, stem cells

monocytes, neutrophils.

endothelial cells,

fibroblasts, T cells,

keratinocytes

monocytes, endothelial

cells, fibroblasts, T

cells, keratinocytes

monocytes, neutrophils,

stem cells,

endothelial cells,

fibroblasts

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154 ANDREW J. HAPEL and SHAUN R. McCOLL

were identified repeatedly in different biological systems there is necessarily considerable redundancy in their nomenclature.

Lymphokines

Lymphokines are cytokines that are made by lymphocytes, most of which also act on lymphocytes. The primary source of lymphokines is antigen-triggered immune T cells. To understand the central role of T cell-derived lymphokines in regulation of the immune response it is important to realize that T cells themselves are subject to regulation by cytokines throughout their development and during their functional life span. Interleukins are cytokines that are used as communication signals between leukocytes. Several comprehensive reviews exist describing their properties (see Kishimoto, 1992).

Lymphokines in T Cell Responses

Thymus-derived lymphocytes fall into several categories defined by properties such as immune status (naive or memory), function (cytotoxic-suppressor or helper-inducer), or cytokine production (T helper 0, 1, and 2). Naive and memory T cells are most readily distinguished by antibodies directed against different isoforms of the cell surface molecule CD45. These isoforms differ only in the extracellular portion of the molecule, which is encoded by exons A, B and C. None of these exons is expressed in human memory T cells. They are instead recognized by anti-CD45RO antibody. Transition from the CD45RA to the CD45RO pheno-type accompanies differentiation of naive to memory T cells. A primary response to antigen occurs when naive T cells first encounter antigen presented by macro­phages or dendritic cells. Naive T cells respond to antigenic stimulation by the production of IL-2, followed by later production of IL-4 as the naive cells undergo a transition to become memory cells (Akbar et al., 1991).

Both CD4 and CDS T cells can synthesize lymphokines. The memory CD4 T cell is primarily responsible for synthesis of IL-3, IL-4, IL-6 and IFN-y. Addition of IL-4 enhances the proliferation of naive T cells stimulated in vitro through the cell surface molecules CD2 and CD3 using antibodies against these cell surface markers. Similar effects have been observed when naive T cells are stimulated with PHA, suggesting that memory T cells are required to amplify the response of naive T cells. Production of IL-4 may be triggered in memory cells because the challenge antigen is cross reactive with environmental antigens that provide chronic stimu­lation of the immune response. In mice the picture is complicated by the subdivision of T cells into Thl and Th2 subsets where Thl cells synthesize IL-2 and IFN-y while Th2 cells synthesise IL-4 and IL-5. Human memory T cells do not always appear to fit this pattern. However, a large panel of human T cell clones specific for bacterial PPD and Toxocara canis have been isolated and these can be classified as Th 1 or Th2 based on the profile of cytokines that they produce. In a similar vein.

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filaria-induced eosinophilia is associated with significantly more IL-5 producing T cells in infected patients (Mossman and Moore, 1991, Scott and Kaufman, 1991).

Naive T cells produce considerably more IL-2 than do memory cells but prolif­erate poorly in response to it, while memory cells proliferate readily in IL-2. Antibody to the IL-2 receptor has been used to inhibit proliferation of memory T cells. On the other hand, production of IL-6 and IL-4 by memory T cells seems essential to an optimum response by naive T cells to IL-2 and antigen, so to some extent the two populations can be seen as having a symbiotic relationship.

As is the case with CD4 T cells, the CDS or cytotoxic-suppressor T cell population can be divided into subsets based on the cytokines that they produce. The most convincing evidence for the existence of discrete subsets of CDS T cells in the human comes from studies using T cell clones isolated from patients infected with Mycobacterium leprae. T cells taken from healed skin lesions of patients with tuberculoid leprosy were cytotoxic, secreted IFN-y but not IL-4, and were restricted to recognition of antigen in association with class I MHC. T cells taken from patients with lepromatous leprosy on the other hand, suppressed the killing of M. leprae by M. leprae-specific CD4 T cell clones, secreted IL-4, IFN-y, IL-5, and IL-10 but not IL-6 , and were restricted to recognizing antigen in association with class II MHC.

lymphokines in B Cell Responses

T cell cytokines are required for the phenomenon of B cell help. IL-4 selectively induces immunoglobulin gene switching to the 8 and yl loci, and suppresses production of IgM, IgG3, IgG2b, and IgG2a while IFN-y inhibits IL-4-induced switching and induces IgG2a. Switching to the 8 locus enhances IgE production and this, combined with the effects of IL-3 and IL-4 on mast cell production, and IL-5 on eosinophil production, provide an environment where the numbers of cells that bind IgE, or synthesize IgE, are enhanced simultaneously (Bergstedt-Lindqvist etal., 1988; Kelso, 19S9).

IL-2 and IL-5 do not directly affect Ig class switching but IL-5 acts synergistically with IL-4 to enhance IgE production by human B cells, and both cytokines enhance IgGl secretion by anti-Ig-activated B cells. IgA production, induced by IL-5, is enhanced by IL-4. There appear to be differences in the mechanism of action of IL-4 on one hand, and IL-5 and IL-6 on the other. IL-4 actively promotes Ig class switching rather than expanding a population of cells that have already switched, whereas IL-5 and IL-6 enhance IgA production by expanding a population of cells that has already switched (Beagley et al., 19S9; Murray et al , 1987; Pene et al., 19S8a,b; Purkeson et al., 1988; Schoenbeck et al., 1989).

The molecular events involved in isotype switching are complex. IL-4 induces transcription of non-productive germ line mRNA from Cnyl and s genes prior to DNA rearrangement while transcription of germline y3 and y2b is suppressed. IFN-y on the other hand inhibits the induction of Cnyl by IL-4. The overall effect

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156 ANDREW J. HAPEL and SHAUN R. McCOLL

of IL-4 may be to cause exposure of selected CH genes to a recombinase that results

in rearrangement (Berton et al., 1989; Esser and Radruch, 1989; Rothman et al.,

1988; 1989; Stavnezer et al, 1988).

One clear message from the preceeding discussion is that there is reciprocity

between IL-4 and IFN-y in their effects on B cell differentiation. This may be

significant when the overall outcome of an immune response is directed against

helminths where production of IgGl and IgE, in association with mast cells

(IL-4-mediated effects) is essential, or when the immune response must instead

deal with viral infections where IgG2a production and enhancement of FcyR on

monocytes, NK cells and neutrophils (IFN-y mediated events) are important.

B cells have been shown to promote the preferential growth of T cells which

secrete cytokines of the Th2 phenotype, possibly because they produce IL-12 and

IFN-y. Macrophages, on the other hand, are more likely to favor induction of Thl

cells. The role of mast cells, NK cells and other hemopoietic elements can be

deduced from what is known about the cytokines that they secrete and the circum­

stances under which this occurs (Wodnar-Filipowicz, 1989).

IL-12 markedly inhibits IL-4-stimulated production of IgE, but has no effect on

production of IgG, IgM or IgA induced by pokeweed mitogen. Part of this effect

is indirectly mediated by the induced release of IFN-y. IL-12 does not however

inhibit IgE responses by B cells that have already switched to membrane expression

of IgE.

IL-12 acts on hemopoietic stem cells synergistically to produce colonies in the

presence of stem cell factor, and may be a product of the central macrophage of

hemopoietic islets.

IL-13 is a product of activated T cells, which, like IL-4, regulates human

monocyte and B cell functions. IL-4 and IL-13 may have arisen as a result of gene

duplication. They share a common signal transducing subunit of their receptor with

IL-2, but do not cross-compete for each others receptors (Zurawski and deVries,

1994).

IL-13 is produced by both CD4 and CD8 T cell clones, and in the case of CD4+

cells, those with properties of Thl, Th2, and ThO subsets are all capable of making

this cytokine in response to antigen-specific stimuli. IL-13 is produced within two

hours of antigenic stimulation of T cell clones and its production persists for at least

72 hours. These kinetics are very different from those for the production of IL-4

which is produced only transiently 24 hours after T cell stimulation.

Human B cells respond to IL-4 and IL-13 by increasing their expression of CD23,

MHC class II antigen, sIgM, CD71, and CD72. Similarly, both cytokines induce

proliferation of B cells. IL-13 also acts as a switch factor inducing production of

IgE. IgE switching is preceded by induction of germline 8 mRNA synthesis. IL-13

does not act synergistically with IL-4 which is more potent in all effects attributed

to IL-13 and most probably acts through a similar signaling pathway. There has

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been speculation that IL-13 is involved in generation of the symptoms of allergy, partly because of its very rapid induction.

Lymphokines in Cell-mediated Immunity

Cell-mediated immunity is that part of the immune response that is mediated by cells rather than antibody. It is particularly relevant to inflammation and to genera­tion of cytotoxic T cells. One of the primary manifestations of a cell-mediated immune response is the attraction and activation of monocytes and neutrophils to a focus of infection or to a tumor. This attraction of inflammatory cells is orches­trated primarily by chemokines (discussed below). The principal effector molecules involved in phagocyte activation include the T cell-derived cytokines GM-CSF, IL-3, TNF-a, IFN-y, and IL-4. The combination of cytokines that is present can influence differentiation and proliferation to a limited extent. IL-3 is primarily a mitogenic cytokine, as is GM-CSF, while TNF-a, IFN-y, and IL-4 tend to act in synergy to cause differentiation. Differentiated macrophages and neutrophils have limited if any proliferative capacity and tend to be activated by GM-CSF. Activa­tion includes enhanced expression of a number of cell surface markers and of various functional capabilities as outlined below.

The reciprocity seen between IL-4 and IFN-y in their effects on Ig synthesis extends to other cellular functions in immunity. For instance, IL-4 inhibits the LPS and IFN-y induce secretion of TNF-a, IL-1 and PGE2 by human monocytes. IL-4 and IFN-y together modulate expression of class II MHC antigen expression on, production of complement components by, and cytotoxicity of monocytes. As a general principle most cytokines act synergistically depending on the target cell and their availability.

GM-CSF production by T cells is induced by antigenic challenge. GM-CSF induces proliferation of myeloid progenitor cells to produce neutrophils, eosino­phils and monocytes, and activates mature cells. GM-CSF is also released from endothelial cells, mesenchymal cells and fibroblasts following stimulation with IL-1 (a macrophage product) or TNF-a (a macrophage and T cell product). Since one action of GM-CSF is to activate cytokine release by mature macrophages (eg, TNF-a, IL-1, and chemokines), there is considerable scope for amplification of local GM-CSF production by recruitment of many cell types in the locality of an inflammatory process or immune focus.

GM-CSF enhances phagocytosis and intracellular killing of microorganisms by neutrophils and macrophages, possibly by enhancing superoxide production. Neu­trophil and macrophage adherence is enhanced by GM-CSF, as are expression of a large number of proteins including cytokines (chemokines and other cytokines including IL-l, TNFa, GM-CSF), enzymes, FcR and production of u-PA, a regulator of monocyte migration. GM-CSF therefore most likely plays an important role in immunity to intracellular microorganisms by indirectly attracting appropri­ate effector cells and their progenitors into foci of infection and enhancing matu-

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158 ANDREW J. HAPEL and SHAUN R. McCOLL

ration and function of these cells. In addition, because GM-CSF acts to amplify numbers of cells that can ingest and eliminate antigen, this cytokine may play an indirect role in regulating the power and duration of an immune response (McColl et al., 1992, Beaulieu et al , 1992, Pouliot et al., 1994a,b).

lnterleukin-3 is primarily a T cell product but is also made in significant amounts by basophils stimulated with antigen-antibody complexes (Wodnar-Filipowicz, 1989). IL-3 causes proliferation of progenitor cells of a wide range of hemopoietic lineages and has been described as a multi-CSF. Human IL-3 activates macro­phages with increased integrin and MHC class II antigen expression. IL-3 also induces the production of the proinflammatory cytokines IL-1, IL-6 and TNF-a from human monocytes. The cell surface antigen CD40 is induced in monocytes and production of uPA is increased in macrophages. IL-3 can also cause the release of histamine from basophils. Thus IL-3 most likely acts to enhance certain aspects of antigen-handling by macrophages, and in concert with IL-4 and IL-5 activates the effector cells responsible for rejection of helminths. IL-3 also has pronounced synergistic activity with M-CSF, causing rapid expansion of the monocyte/neutro­phil progenitor pool through its ability to negate the differentiative effects of M-CSF. Since IL-3 is made by all classes of helper T cell it would appear that the ability of this cytokine to rapidly induce proliferation of a variety of cell types, and particularly its synergistic effect with M-CSF (which tends to be ubiquitously expressed in all tissues), are important effectors of some aspect of immune regula­tion. Whether this is because of increased antigen-presenting activity of the induced populations, or because of an increased ability to remove antigen from the system (positive or negative regulation) is not clear (Morris et al., 1990).

IL-5 seems to be the most selective of the cytokines produced by T cells in that it has a specific and unduplicated role in inducing eosinophil production and survival. IL-5 also affects basophil production, and activates both mature basophils and eosinophils. Eosinophil activity in ADCC, phagocytosis, and superoxide production is enhanced. Expression of the integrin molecules CDl lb and VLA-4 is enhanced in IL-5-treated eosinophils. Ig-A and IgG induced degranulation of eosinophils is enhanced by IL-5 as is the capacity to migrate. The effects of IL-5 tend to be eosinophil-specific in the human and IL-5 can be viewed as a cytokine with particular importance in resistance and immunity to parasites (Sanderson, 1990).

Interleukin 12 is produced primarily by macrophages but also by B cells. Toxoplasma gondii. Staphylococcus aureus Cowan I strain, and Listeria monocy­togenes all induce production of IL-12 but no clear physiologic stimulus for its production has yet been described although IL-10 has been implicated. IL-12 enhances the cytolytic activity of a number of cell types including T cells, macro­phages, NK and LAK cells. In addition, the proliferation of activated NK cells and T cells is increased, possibly because IL-12 induces increased expression of the IL-2 receptor. Expression on NK cells of receptors for IL-4, TNF-a and IL-12 is

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enhanced. Production of IFN-y is enhanced. IL-12 stimulates the induction of Thl cells and is a synergistic factor in the growth of hematopoietic stem cells.

There is considerable evidence that IL-12 is involved in regulating the induction of Thl and Th2 cells. IL-12 particularly appears to induce development of Thl from naive T cells. IL-4 on the other hand seems to preferentially induce Th2 cells. Production of IL-12 may be regulated in macrophages by IL-10 which inhibits development of Thl cells through an indirect effect on macrophages. Human CD4 T cell lines produced from atopic individuals exposed in vitro to Dermatophagoides pteronysius group I antigen are usually of the Th2-like subclass of helper cells in that they produce IL-4 but little or no IFN-y. When IL-12 is added to the induction system the T cells generated produce cytokines characteristic of the Thl or ThO subclass. PPD-stimulated T cells on the other hand more closely resemble Thl cells, producing IFN-y but little or no IL-4. When T cells are generated in vitro using PPD in the presence of neutralizing antibodies against IL-12 the ThO phenotype of helper cell predominates (Brunda 1994).

IL-13 does not act on T cells, but has pronounced effects on NK cells. IL-1 and IL-13 have synergistic effects in inducing IFN-y in NK cells. In contrast, IL-4 inhibits IFN-y production induced by IL-2.

Monokines

Mononuclear phagocytes, including the peripheral blood monocyte, the macro­phage and the dendritic cell are major sources of cytokines in the immune system. The cytokines derived from these cell types have traditionally been referred to as monokines, however, this term is no longer an adequate description in light of the discovery that cell types other than mononuclear phagocytes produce these factors. The range of cytokines produced by mononuclear phagocytes is broad and includes interleukins, chemokines as well as CSFs, interferons, TNF-a, and peptide growth factors.

Mononuclear phagocytes produce interleukins-1 a, -Ip and -6, TNF-a and sev­eral CSFs. All four of these cytokines are produced by mononuclear phagocytes activated by a variety of agonists, including IL-1 a and P and TNF-a. However, as mentioned above, they are also produced by other cells, including B and T cell subsets, endothelial cells, keratinocytes and fibroblasts. The first four cytokines, are multi-functional cytokines. They collectively exert a broad range of biological effects in many different systems, not just the immune system. IL-1 was the first "interleukin" to be discovered and named. What was originally defined as IL-1, was eventually found to consist of two closely-related polypeptides now known as IL-1 a, and IL-1 p. These two cytokines are the products of separate genes. A similar situation exists for TNF-a and TNF-P (Akira et al., 1990, Dinarello, 1991; Fiers, 1991).

IL-1 a and P are critical mediators of the immune response. They enhance B and T cell proliferation, thereby increasing humoral immunity. More important perhaps

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160 ANDREW J. HAPEL and SHAUN R. McCOLL

is their ability to stimulate the afferent limb of the immune response via their ability

to increase the level of gene expression in target cells. For instance, the level of

cytokine and adhesion molecule gene expression in mononuclear phagocytes,

endothelial cells, keratinocytes and fibroblasts is controlled by IL-la and P, as well

as TNF-a. These effects of IL-1 a, IL-1 p and TNF-a, have led to their being referred

to as proinflammatory cytokines (Dinarello, 1991; Fiers, 1991).

IL-6 stimulates differentiation of B cells and induces antibody production. IL-6

also stimulates acute-phase protein release by hepatocytes and growth of T cells,

hybridomas and melanomas. In addition, this cytokine, in conjunction with IL-3

and IL-1, stimulates growth of myeloid precursors (Akira et al.; 1990, Gauldie et

al., 1987; Ikebuchi et al., 1987).

TNF-a as the name implies, was originally characterized as a cytokine possessing

the ability to induce necrosis in tumors. TNF-a was independently discovered as

the major mediator of cachexia. However, it is now realized that this cytokine plays

important roles in host defense against infections and also exerts important effects

on a variety of different cells of the immune system. The cellular targets of TNF-a

include T cells, in which IL-2 receptor expression and production of IFN-y are

regulated, B cells, in which proliferation and production of immunoglobulin are

regulated, macrophages, and neutrophils, in which chemokine and monokine

production are regulated, and endothelial cells and fibroblasts in which adhesion

molecule expression and cytokine production are regulated. In addition, TNF-a,

like GM-CSF is capable of directly activating and priming mature phagocytes such

as neutrophils and monocytes for enhanced functional responses in vitro and may

therefore play critical amplification roles in the inflammatory response (Akira et

al., 1990, Fiers, 1991; DiPersio et al., 1988; McColl et al., 1990; McColl et al,

1991a).

Interferons a and pi are also produced by activated mononuclear phagocytes,

although IFN-a is the major IFN produced by mononuclear leukocytes. These

cytokines have potent antiviral effects, and inhibit proliferation of a wide range of

cell types (Nathan, 1992).

In addition to producing the cytokines mentioned above, mononuclear phago­

cytes also release a range of peptide growth factors including fibroblast growth

factor (FGF), platelet-derived growth factor (PDGF), epidermal growth factor

(EGF) and transforming growth factor (TGF) (see Massague, 1990; Martin et al.,

1992; Westermark and Heldin, 1993; HoUenberg, 1994 for reviews on these growth

factors).

Finally, while this section has focussed on what are termed monokines, note

should be taken that granulocytes, particularly neutrophils, can also be a

significant source of most of the cytokines mentioned above (McColl and

Showell, 1994).

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Cytokines in Immunology 161

Chemokines

Recruitment of specific leukocyte populations to sites of inflammation and infection is a feature of both normal and pathological immune responses. The recent discovery of the chemokine gene superfamily, whose members exhibit chemotactic specificity for both common and distinct sets of leukocytes has provided consider­able insight into the mechanism(s) by which specific leukocyte attraction may be regulated. The members of the chemokine superfamily are related by predicted primary structural similarities and by the conservation of a four-cysteine motif The superfamily has two branches, classified according to the position of the first two cysteines in the conserved motifs. The "C-X-C" branch, which includes PF4, interleukin 8 (IL-8), melanocyte growth-stimulatory activity (MGSA or gro-a), ENA-78, IP-10 and Nap-2 is characterized by the separation of the first two cytsteines in the primary structure by an intervening amino acid, whereas in the "C-C" branch, the two cysteines are directly adjacent. Members of the C-C branch include monocyte chemotactic proteins (MCP)-1, -2 and -3, RANTES, macrophage inflammatory proteins (MlP)-la and -ip and 1-309 (Stoeckle and Barker, 1990; Schall, 1991; Oppenheim et al., 1991; Miller and Krangel, 1992).

The various members of the chemokine gene superfamily are also functionally related by their in vitro ability to stimulate the recruitment (chemotaxis) of both overlapping and specific subsets of leukocytes. They are thus likely to play an important role in allergy, inflammation, host resistance to infection as well as in various autoimmune diseases where selective recruitment of leukocyte subsets is involved. While the biological function for which they have been named is chemotaxis, like most chemotaxins (eg., C5a and leukotriene B4), the chemokines also activate other important leukocyte functions including degranulation, adhesion and cytoskeletal rearrangement. In addition, they have also been identified as stem cell growth inhibitors in vitro. The signal transduction pathways involved in these responses are discussed in more detail below.

In general, members of the C-X-C chemokine subfamily, with the exception of IP-10, which is also a chemoattractant for CD4+ve and CD29+ve T lymphocytes (Taub et al., 1993a) are chemotactic and activating factors for neutrophils but not for mononuclear leukocytes, whereas the C-C chemokines are chemotactic and activating factors for various mononuclear cells and granulocytes other than neutrophils. MCP-1, 2, 3 and 1-309 are chemotactic for monocytes, whereas RANTES, MlP-la and MIP-ip, while also being chemotactic for monocytes, specifically mediate the recruitment of different subpopulations of T cells (Schall et al., 1990; Taub et al., 1993a,b; Schall et al., 1993). RANTES is a chemoattractant for memory T cells (CD4+ve, CD45RO+ve), whereas MIP-1 p recruits naive T cells (CD4+ve). MIP-1 a, on the other hand, mediates the chemotaxis of naive (CD4+ve) and cytotoxic (CD8+ve) T cells as well as B cells. Recent studies have also shown that MCP-1, -3, RANTES and MIP-la induce to varying extents, mediator release

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162 ANDREW J. HAPEL and SHAUN R. McCOLL

and chemotaxis of basophils and eosinophils and are thus likely to be involved in

allergic inflammation (Baggiolini and Dahinden, 1994).

Various studies have been carried out to assess the effect of chemokines in vivo.

Tumor cells engineered to express MCP-1 or IP-10 fail to form tumors in vivo when

injected into mice (Rollins and Sunday, 1991, Luster and Leder, 1993). This effect

is associated with an influx of monocytes to the tumor site. The effect of injections

of purified or recombinant groa, IL-8,1-309, MlP-la and -ip, and MCP-1 into

sites on the skin (dermal or footpad) of mice, rats and rabbits has also been

examined. In most cases, the leukocyte infiltrate detected in vivo, matched that

observed in vitro. However, this was not the case for 1-309, or MIP-1 a or -1P which

all led to neutrophil influx in vivo, but do not attract these cells in vitro. It has also

been reported that IL-8 is chemotactic for basophils in vitro, however, this was not

confirmed when in vivo experiments were performed. Because of these discrepan­

cies between in vivo and in vitro effects of different chemokines, the question of

whether specific chemokines actually control the recruitment of the same leukocyte

subsets in vivo, as suggested by in vitro studies, remains to be adequately addressed

(Miller and Krangel, 1992).

Consistent with their specificity with respect to biological targets, the chemokine

genes exhibit a high degree of cell-specific expression. Chemokines are produced

by every cell of the body thus far examined, including myeloid and lymphoid cells,

endothelial cells and fibroblasts. However, while these genes share several common

structural features, they are not all regulated in the same way, or indeed, expressed

in the same cell types. For instance, in a functional T cell-line, mRNA for both

MIP-la and P is rapidly expressed in response to antigen stimulation, whereas that

of RANTES is decreased and MCP-1 mRNA is not expressed at all (Schall 1991).

MCP-1 and IL-8 are expressed in monocyte/macrophages, fibroblasts and endothe­

lial cells (Oppenheim et al., 1991, Schall, 1991). However, IL-8, MIP-la and P are

expressed in neutrophils whereas MCP-1 and RANTES are not (Bazzoni et al.,

1992; McColl and Showell, 1994; Hachicha et al., 1994). On the other hand IL-8,

RANTES, MCP-1 and MCP-2 are expressed in synovial fibroblasts, whereas

MIP-la, MIP-ip and 1-309 have not yet been detected in these cells (Hachicha et

al., 1993, Rathanaswami et al., 1993a,b; Rathanaswami et al., 1994).

The major physiological regulators of chemokine gene expression appear to be

the monokines TNF-a and IL-1 (both IL-la and IL-ip). Synthesis of select

chemokines, including MCP-1, MCP-2 and IP-10, occurs in response to lymphok-

ines such as IFN-y and IL-4 (Schall, 1991), and in addition, these lymphokines can

influence the level of synthesis of chemokines induced by TNF-a and IL-ip

(Hachicha et al., 1993; Rathanaswami et al., 1993b; Rathanaswami et al., 1994).

These latter observations further illustrate the complexity of the immune cytokine

network in man.

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Cytokines in Immunology 163

RECEPTORS AND SIGNALING PATHWAYS

Cytokines provide their signals to cells through receptors on the cell surface. These receptors may be simple, comprising a single transmembrane molecule as is the case for the two alternative receptors for TNF-a, or complex, comprising a variety of molecules some conferring specificity for ligand and others providing a link to the machinery of the cell by specialized transducing elements, as is the case for the IL-2 receptor.

The receptor for IL-2 is probably the best understood of the cytokine receptors that have a role to play in immunity. It is made up of three chains, a, P, and y. The dissociation constant for IL-2 binding is 10"' M for the aPy trimer. The y chain alone does not bind IL-2 but the combination of an a and a P chain, or any heterodimer will bind IL-2, though with lower affinity than the trimer. The P molecule is critical for signal transduction, interacting with the rc-family of protein tyrosine kinases including p56lck, and p59fyn, ultimately resulting in activation of c-fos and c-jun, possibly through activation ofpllras. The g subunit is essential for internalization of IL-2 by the cell (Taniguchi and Minami, 1993).

GM-CSF, IL-3 and IL-5 receptors share a common P chain that is involved in signal transduction, but utilize different a chains that confer specificity for the ligand. Different portions of the intracellular part of the receptor are linked to different intracellular signaling pathways. Protein kinase C, PI3 kinase, vav. She,

Grb, and SOS are all phosphorylated as the result of phophotyrosine kinase activity, leading to regulation of the MAPK cascade at the level of Ras. Jak2 is activated, as well as Gia and phophodiesterase. Pertussis toxin-sensitive G proteins and tyrosine kinases are involved in the signal transduction pathways utilized by GM-CSF in mature phagocytes (McCoU et al., 1989, McColl et al., 1991b). Migration of Gi to the nucleus, seems to enhance the rate of cell division induced by MAPK, induction of c-fos and c-jun, and expression of c-myb and c-myc. By depressing the levels of intracellular cAMP these cytokines regulate protein kinase A. In the absence of cAMP, PKA cannot inhibit Raf-l, the target of Ras in the MAPK cascade, thus cell division is preferred to differentiation (Foxwell et al., 1992, Kishimoto et al., 1994; Miyajima et al., 1993; Townsend et al., 1993).

There is no available data that adequately addresses the question of why the outcome of cell signaling by the three cytokines is so different. IL-5 induces eosinophil differentiation, IL-3 basophil differentiation, and GM-CSF macro­phage/neutrophil differentiation. The signals that confer this sort of specificity on the three cytokines remain undefined.

In some cases cytokines may bind to soluble forms of receptor that can result either in cellular activation by the complex, or in inhibition of the cytokine by competition of soluble receptors with those bound on the cell surface.

Chemokines tend to use the same intracellular signaling pathways as other well-characterized chemotactic factors such as the oligopeptide fMet-Leu-Phe and the lipid mediator leukotriene B4 (Gerard and Gerard, 1994). This has been shown

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164 ANDREW J. HAPEL and SHAUN R. McCOLL

for IL-8, NAP-2, groa in neutrophils, and MCP-1 in monocytes (Peveri et al., 1988, Moser et al., 1989; Walz et al., 1989; Sozzani et al, 1991). Two IL-8 receptors, one binding IL-8 only, and another to which IL-8, groa, Nap-2, and most other C-X-C chemokines also bind, have been cloned (Holmes et al., 1991, Murphy and Tiffany, 1991). To date, three C-C chemokine receptors have been isolated. One receptor, referred to as JOSHl, binds MlP-la, RANTES, MIP-lp and MCP-1, although signal transduction (assessed by the mobilization of intracellular calcium in trans-fected cells expressing the receptor) is observed only in response to RANTES or MlP-la (Neote et al., 1993). Two cDNAs encoding functional MCP-1 receptors with alternatively-spliced carboxyl terminals have recently been cloned (Charo et al., 1994). Transfection studies have shown that these receptors bind MCP-1 with high affinity. MCP-1, but none of the other chemokines tested, including MlP-la, MIP-ip, RANTES or IL-8, elicits calcium mobilization in cells transfected with the receptor cDNAs. All of the functional chemokine receptors cloned thus far conform to the seven membrane-spanning structure that is characteristic of the G protein receptor superfamily. Accordingly, it has been shown that the cellular actions of several chemokines are inhibited by prior treatment of the cells with pertussis toxin, an inhibitor of Gia2. They appear to activate several phospholipase signaling pathways including phospholipase C which leads to the release of inositol trisphosphate and the subsequent release of calcium from intracellular stores and influx of calcium from the extracellular space. In addition, phospholipase A2, phospholipase D and various tyrosine kinases have all been implicated in leukocyte activation by chemokines (McDonald et al., 1993; Kelvin et al., 1993; Rollet et al., 1994; L'Heureux et al., 1994).

Of considerable recent interest is the identification of a promiscuous chemokine-binding receptor on the surface of erythrocytes in humans. This receptor has now been cloned and identified as the Duffy antigen which is used by the malarial pams'itQ,Plasmodium vivax to gain access to erythrocytes. RANTES, IL-8, MCP-1, MIP-ip, but not MlP-la all bind to this receptor, but fail to induce any detectable signal. This latter observation has led to the theory that this receptor may act as a sink for chemokines in whole blood thereby attenuating inflammation (Horuk, 1994).

CLINICAL APPLICATIONS

While there is enormous potential to use cytokines or their inhibitors in vivo to modulate the immune response, at the time of writing, few cytokines have yet been through extensive clinical trials. Of these only IL-2, IFN-y and the colony stimu­lating factors G- and GM-CSF and IL-3 have effects that are related to improved immune function. For the most part IL-2 has been used in an attempt to augment anti-tumor immunity, sometimes in conjunction with administration of activated NK cells or T cells. The benefits have been unspectacular. Toxicity has proven

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Cytokines in Immunology 165

difficult to overcome and little if any benefit has been achieved in terms of enhanced

immune function.

On the other hand it may be possible to use IL-2 as a potent accessory to

immunization regimes where live recombinant vaccines are used to deliver anti­

gens. One system currently under development is the use of vaccinia virus con­

structs that contain the IL-2 (or other cytokine) gene along with the gene for an

immunizing antigen. The live virus containing the antigen and cytokine genes

infects cells of the recipient and expresses the genes causing production of antigen

and cytokine in large amounts locally. When such vaccines are used, enhanced

responses to the target antigen can be achieved, and by incorporating other cytokine

genes into the immunizing virus it may be possible to influence the class of

immunity, that is, the isotype of antibody that is induced.

Of the CSFs, G-CSF has specific effects in enhancement of neutrophil numbers

in patients who have received chemotherapy and bone marrow replacement. This

has been useful in combatting infections of various sorts. GM-CSF is less specific

than G-CSF and induces all types of granulocyte. It is a potent activator of

monocytes and macrophages as well. In addition GM-CSF induces production of

other cytokines such as tumor necrosis factor and IL-l. A further difference

between G and GM-CSF is that the latter is less effective at inducing neutrophil

migration. M-CSF has been shown in very limited clinical trials to cause an increase

in the number and activation state of monocytes and macrophages, to enhance

antibody-dependent cellular cytotoxicity and to enhance phagocytosis and intracel­

lular killing.

While there has been great success in reducing myelotoxicity and mucositis seen

in aggressive chemotherapy there is no evidence that the use of any cytokines

actually results in an increase in patient survival. This presently unfortunate

situation may change with more effective delivery protocols, and more appropriate

targets for therapy. While it may be unrealistic to expect cytokines to be effective

in prolonging life expectancy in crisis situations such as following aggressive

chemotherapy, it is entirely reasonable to expect a cytokine to be of some use as an

immune enhancer in a recombinant vaccine (Lloyd and Johnston, 1993).

Research into the possibility of inhibiting the biological activity of specific

chemokines is currently very intense. The major effort at this stage centers around

design of specific and potent receptor antagonists. Receptor mutation studies are

also being conducted in order to facilitate peptide design for antagonism and since

the chemokines are small, they can be synthesized relatively efficiently, and thus

amino acid substitutions are relatively easy to make. For the most part, this type of

work has been conducted on IL-8 and its receptor. While no antagonists have

appeared as yet, in vivo studies using antibodies inhibiting biological activity

suggest that targeting of specific chemokines may pay dividends in treating immune

disorders. One major problem that will have to be overcome, however, is the

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166 ANDREW J. HAPEL and SHAUN R. McCOLL

binding of several chemokines to the same receptor. This promiscuity makes the

design of chemokine receptor antagonists particularly challenging.

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Chapter 10

Activation and Control of the

Complement System

B. PAUL MORGAN

Introduction and Basic Concepts 172

Activation of Complement 175

The Classical Activation Pathway 175

The Alternative Activation Pathway 180 Amplification on Activator Surfaces 180 Cleavage of C5 181

Control of the Complement System 182 Control in the Classical Pathway 182

Control in the Alternative Pathway 184

Control in the Membrane Attack Pathway 185 Physiological Roles of Complement 185

Bacterial Killing by Complement 186 Immune Complex Solubilization by Complement 186 Cell Activation by Complement 187

Principles of Medical Biology, Volume 6 Immunobiology, pages 171-196. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-811-0

171

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172 B. PAUL MORGAN

Complement and the Immune Response

Pathology of Complement

Complement Deficiencies

Complement in Autoimmune Diseases

Evasion of Complement Killing by Microorganisms

Iatrogenic Activation of Complement

Control of Complement Activation In Vivo

Summary

Recommended Readings

187

188

188

189

190

190

191

191

196

INTRODUCTION AND BASIC CONCEPTS

Complement was discovered just over a century ago as a heat-labile component of blood plasma which had bactericidal properties and was capable of lysing foreign erythrocytes (Buchner, 1889). The multi-component nature of the system was also recognized at the turn of the century (Ferrata, 1907), but it was not until the 1960s that the components began to be isolated and characterized (Mayer, 1984), begin­ning an exciting period during which the intricacies of this fascinating system have

CLASSICAL PATHWAY

C4-l->-C4b

C5b . ,

MEMBRANE ATTACK PATHWAY

ALTERNATIVE PATHWAY

C3bBb(P)

- C5b —!>• C5b6 _VC5b67 — i ^ C5b678

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1/ C5b678{9)n

(MAC)

Figure 1, The complement system. The three pathways of the system and their

constitutive proteins are shown. Thick arrows represent enzymatic cleavages; dotted

lines represent cooperative events.

Page 188: Immunity

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Page 190: Immunity

Activation and Control of ttie Complement System 175

emerged. Despite this long history, new discoveries continue to be made and this superficially simple system continues to surprise.

In this chapter I will provide a concise but up-to-date summary of the complement system, focussing on the activation of the system, its regulation, its biologically active products and its involvement in disease. Complement plays a central role in innate immune defense. The primary purpose of complement is to kill invading microorganisms but it is also important in the mediation of inflammatory reactions and the clearance of immune complexes. Complement, via interactions with other components of the immune system, also plays a role in the development of immunity.

The complement system consists of a group of eleven soluble plasma proteins which interact with one another in two distinct enzymatic activation cascades (the classical and alternative pathways) and in the non-enzymatic assembly of a cy­tolytic complex (the membrane attack pathway) (Figure 1). Control of these enzymatic cascades is essential to prevent the rapid consumption of complement in response to trivial stimuli and is provided by ten or more plasma and membrane-bound inhibitory proteins which act at multiple stages of the system to regulate activation (Table 1).

In the first section of this chapter I will describe the activation of the system, the components of the activation and membrane attack pathways and their interactions. In the second section I will discuss the control of the pathway and the regulatory proteins involved. In the third section I will outline the physiological importance of complement, its role in host defence and the biological activities of the products of activation. In the final section I will focus on pathological processes involving complement.

ACTIVATION OF COMPLEMENT

The Classical Activation Pathway

Activation of CI

The classical pathway is so called because it was the first activation pathway to be recognized. The pathway is initiated by the binding of CI to an activator (Hughes-Jones, 1986; Loos, 1988). Antibody bound to antigen is the best known activator of the classical pathway but many other substances can also bind CI and initiate the classical pathway and these non-antibody activators may be of consid­erable physiological and pathological significance (Taylor, 1993). CI is a large heterooligomeric complex (molecular weight approx. 800 kD) consisting of a single molecule of Clq and two molecules each of Clr and Cls (Ziccardi, 1983; Arlaud et al., 1990). These components are held together noncovalently in a Ca^^-dependent complex (Figure 2). Clq is itself composed of six copies of each of three distinct polypeptide chains (A, B, and C). One copy each of the A, B and

Page 191: Immunity

176 B. PAUL MORGAN

Bound C1

Figure 2. Structure and activation of C I . The fluid-phase CI complex, consisting of

one molecule of C1 q and two molecules each of CI r and C1 s, binds via at least two

of its six 'heads' to immunoglobulin. (1) binding causes conformational changes in

C l q , resulting in activation; (2) this causes conformational changes and activation of

the first CI r molecule which (3) cleaves and activates the second CI r which in turn

(4) cleaves and activates CI s. Reproduced from Morgan (1990). Complement: Clinical

aspects and relevance to disease. Academic Press, London, with permission.

C chains are wound around each other in a triple helix containing an extended, collagen-like tail and a large globular head (Reid and Porter, 1981). In the intact Clq molecule the collagen tails of the six triple helices are tightly associated along the amino terminal half but then diverge to form six connecting strands, each bearing a globular head. This complicated molecule is encoded by three closely linked genes present on chromosome Iq in the order A-C-B (Sellar et al., 1991).

Clq binds via its globular heads to the Fc portion of IgG. Activation requires the binding of multiple heads of a C1 q molecule by aggregates of IgG. This multivalent interaction greatly increases the strength of binding and triggers conformational changes within Clq which induce activation. IgM is a multivalent molecule and can thus activate Clq efficiently and without the need for aggregate formation. Among the human IgG subclasses the ranking of efficiency for activation of CI is IgG3 > IgGl > IgG2; IgG4 does not activate CI.

Clr and C Is are homologous, single chain molecules of molecular weight 80 kD which, in the presence of Ca " , form an elongated Clr2Cls2 complex which sits

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Activation and Control of the Complement System 177

between the globular heads of Clq (Figure 2) (Reid, 1986; Sim and Reid, 1991). They are encoded by closely linked genes on chromosome 12. Conformational changes within Clq upon binding antibody allow the auto-activation of the proen­zyme Clr, a process which involves cleavage at a single site within the molecule, thereby revealing the active site. Clr then activates Cls in the complex, again by cleaving at a single site in the molecule (Sim and Reid, 1991).

Of the non-antibody activators of CI, mannose binding protein (MBP) is of particular importance. MBP is a high molecular weight serum lectin made up of many copies of a single 32 kD chain (Taylor et al., 1989). Its role in vivo is to bind mannose and N-acetyl glucosamine residues in bacterial cell walls. Once bound, MBP can develop the capacity to activate the classical pathway of complement either by binding Clr and Cls to form a MBP-Clr2-Cls2 complex or by binding a novel protein termed MBP-associated serine protease or MASP (Matsuhita and Fujita, 1992). MBP thus provides a rapid, antibody-independent means of activat­ing the classical pathway on bacteria and deficiency is associated with defective bacterial opsonization and repeated infections (Turner, 1991). Other lectins have also been shown to activate complement, leading to the proposal that they represent a distinct complement activation pathway (Holmskov et al., 1994).

Binding and Cleavage of C4

The next component in the classical pathway is C4, a large plasma protein (200 kD) which consists of three disulfide-bonded chains (a, (3 and y) (Shreiber and Muller-Eberhard, 1974; Janatova and Tack, 1981). C4 is synthesized as a single-chain precursor which is processed to give the three-chain molecule prior to secretion. The gene for C4 is in the class III region of the major histocompatibility complex (MHC) on the short arm of chromosome 6. In fact, C4 is encoded by two closely linked genes which give rise to the two isotypic variants, C4A and C4B (Campbell et al., 1990). These variants differ by only six amino acids but this small difference causes significant changes in function, C4A binding preferentially to amino groups after cleavage and C4B to hydroxyl groups (see below). C4B is also more efficient in propagating continued activation of complement.

C1 s cleaves fluid-phase C4 at a single site near the amino-terminus of the a chain, releasing a small fragment, C4a and in the process exposing a reactive thiolester group in the a chain of the large fragment, C4b. Exposure of this reactive group bestows upon C4b the capacity to bind to membranes or other surfaces (Law et al., 1980; Law and Dodds, 1990). The thiolester group forms covalent amide or ester bonds with exposed amino or hydroxyl groups respectively on the activating surface (Figure 3). The thiolester is extremely short-lived due to its susceptibility to inactivation by hydrolysis. As a consequence, C4b binding to surfaces is a very inefficient process, most of the C4b formed decaying in the fluid phase and C4b binding being restricted to the immediate vicinity of the activating CI complex.

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178 B.PAUL MORGAN

^ C3 con

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Gly Cys ^ G l n Thr 988 \ /991

Gly Glu 989 990

Figure 3. Activation and decay of C3. A. Activation of C3 involves a single cleavage

in the a-chain mediated by the C3-convertase of the classical or alternative pathway.

A small fragment, C3a, is released and a thiolester group is exposed in the large

fragment C3b. Decay of C3b is mediated by factor I which, in the presence of an

appropriate cofactor (factor H, CR1 or MCP), cuts at 2 sites in the a'-chain, releasing

a 2 kD fragment, C3f, and yielding iC3b (i = inactive). A further cleavage by factor I

releases C3c, leaving the fragment C3dg (35 kD) attached to the activator surface.

Finally, serum proteases cleave a small fragment (C3g, 5 kD) from C3dg. B. Detail of

the thiolester group formed by residues 988-992 in the C3d region of the a-chain of

C3. A similar thiolester moiety is also found in the structurally related proteins C4 and

a2-macroglobulin.

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Activation and Control of the Complement System 179

Binding and Activation of C2

The next component of the classical pathway is C2, a single chain plasma protein of molecular weight 102 kD which, like C4, is encoded by a gene in the class III region of the MHC on chromosome 6. The gene for C2 is closely linked to that for factor B, its functional homologue in the altemative pathway (see below). Membrane-bound C4b expresses a binding site which, in the presence of Mg^^ ions, can bind C2 near its amino terminus and present it for cleavage by an adjacent CI complex to yield a 30 kD amino-terminal fragment, C2b, and a 70 kD carboxy-terminal fragment, C2a (Nagasawa and Stroud, 1977). The C2b fragment may be released or remain loosely attached to C4a but is not required for enzymatic activity. The C2a fragment binds to C4b through a newly exposed site near its new amino terminus to form the C4b2a complex, the next enzyme in the classical pathway.

Binding and Cleavage of C3

The penultimate component of the classical pathway, C3, is the most abundant of the complement components (1-2 mg/ml in serum). It is a large (185 kD) molecule composed of two chains (a, 110 kD and P, 75 kD) held together by disulfide bonds (Lambris, 1988). Like C4, with which it shares many structural features, C3 is synthesized as a single chain precursor molecule and cleaved intracellularly prior to secretion. The gene for C3 is on chromosome 19.

C2a in the C4b2a complex cleaves C3 in the fluid phase at a single site in the a chain, releasing a small fragment from the amino-terminus (C3a, 9 kD) and exposing in the large fragment, C3b, a thioester group and binding sites for several complement receptors and regulatory proteins (see below). The labile thioester group confers upon C3b the capacity to bind covalently to the activating surface as described above for C4b (Figure 3). Binding is inefficient and the bulk of the C3b formed decays in the fluid phase to form C3bi ('i' = inactive). Only C3b bound to the activating surface in close proximity to the C4b2a complex takes any further part in activation.

Binding and Cleavage of C5

The final component of the classical pathway, and also the first component of the membrane attack pathway, is C5. C5 is a two-chain plasma protein of 190 kD molecular weight, encoded on chromosome 9, which is structurally related to C3 and C4. Like these molecules it is synthesized as a single-chain precursor and cleaved prior to secretion, however, C5 does not contain a thioester group and thus cannot itself bind covalently to surfaces (Law and Dodds, 1990). C3b attached to the membrane binds C5 and presents it for cleavage by C2a in an adjacent C4b2a complex. The C4b2a complex can only cleave C5 bound to C3b and hence the C5 cleaving enzyme (C5 convertase) of the classical pathway is termed C4b2a3b. Cleavage occurs at a single site in the a chain of C5, releasing a small amino-

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180 B.PAUL MORGAN

terminal fragment, C5a (approx. 10 kD), and exposing on the larger fragment, C5b, a labile hydrophobic surface binding site and a site for binding C6, neither of which are expressed by uncleaved C5 (Hughes-Jones, 1986; Loos, 1988).

The Alternative Activation Pathway

Initiation of the Alternative Pathway

The alternative pathway provides a rapid, antibody independent route for com­plement activation and amplification on invading microorganisms and other for­eign surfaces. C3 is the key component but two other proteins, unique to the alternative pathway, are also required. Factor B is a single-chain 93 kD plasma protein, structurally and functionally homologous with C2 which, once activated, can cleave C3 at a site identical to that used by the C4b2a enzyme. C2 and factor B have 40% protein sequence identity and are encoded by closely linked genes in the MHC on chromosome 6. Activation of factor B is accomplished by factor D, a 26 kD serine protease enzyme present in plasma in its active form, which cleaves factor B at a single site, releasing a 30 kD fragment, Ba, and exposing a serine protease domain on the large (60 kD) fragment, Bb (Gotze, 1986). Factor D can only cleave factor B bound to C3b. Initiation of the alternative pathway on a surface therefore requires that C3b be deposited in a conformation which allows factor B to bind and be cleaved by factor D. How then is the initiating C3b formed?

In many circumstances some degree of classical pathway activation may have occurred, depositing sufficient C3b to trigger the alternative pathway. Neverthe­less, it appears that the alternative pathway can be initiated independent of the classical pathway. In biological fluids C3 is continuously hydrolysed at a slow rate to form a metastable C3 (H2O) molecule which, in the presence of Mg " ions, can bind factor B in solution and render it susceptible to cleavage by factor D. Bb in the fluid phase C3 convertase thus formed (C3(H20)Bb) cleaves C3, releasing C3a and exposing the thioester group in C3b which can then bind to adjacent surfaces (Lachmann and Hughes-Jones, 1984; Law and Dodds, 1990). As a result of this "tickover" phenomenon C3b is continuously deposited in small amounts on all cells in the body.

Amplification on Activator Surfaces

Deposition of C3b on host cells does not result in continued activation because the surface features do not favor a binding of factor B (non-activator surfaces) and bound C3b decays, a process catalyzed by fluid-phase and membrane inhibitors (see below). In contrast, the surface features of many microorganisms and foreign cells favor amplification (activator surfaces). C3b binds factor B (Mg^'^-dependent) and presents it for cleavage by fluid-phase factor D, thus forming the alternative pathway C3 convertase C3bBb which cleaves more C3. Activating surfaces thus rapidly become coated with C3b molecules, each one of which can itself recruit

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Activation and Control of the Complement System 181

factor B to form more convertases and further amplify activation. The precise nature of the surface features which determine whether activation will occur are still not clear; however, surface carbohydrates, particularly sialic acid, appear to be impor­tant (Pangbum and Muller-Eberhard, 1984; Pangburn, 1986).

Cleavage of C5

As in the classical pathway, bound C3b acts as a receptor for C5, enabling the cleavage of C5 by Bb in an adjacent C3bBb complex. The site of cleavage is identical to that caused by C2a in the classical pathway convertase (C4b2a3b). C5a is released and labile membrane- and C6-binding sites are exposed on C5b as described above for the classical pathway.

The Membrane Attack Pathway

Cleavage of C5 by the C5 convertase of either the classical or alternative pathways is the final enzymatic step in the complement cascade. The membrane attack pathway involves the non-covalent association of C5b with the four terminal complement components, all of which are hydrophilic plasma proteins, to form an amphipathic membrane-inserted complex. While still attached to C3b in the con­vertase C5b binds C6, a large (120 kD) single chain plasma protein. Binding of C6 stabilizes the membrane binding site in C5b and exposes a site for C7, a 110 kD single chain plasma protein which is homologous to C6 (see below). Attachment of C7 causes conformational changes in the complex which result in its release from the convertase to the fluid phase and the transient expression of a hydrophobic membrane binding site. If the C5b67 complex encounters a membrane during the brief lifetime of this site, it binds tightly. However, the complex does not penetrate deeply into the membrane and does not disturb the integrity of the lipid bilayer. In addition to the natural decay of the membrane binding site, attachment of C5b67 is further limited by the tendency of the complex to aggregate and by the presence of multiple fluid-phase inhibitors (see below). Deposition of C5b67 is thus limited to the target cell. A small proportion of complexes may attach to the membranes of closely apposed host cells and cause damage or lysis, so-called innocent bystander lysis (Podack and Muller-Eberhard, 1978).

The penultimate component of the membrane attack pathway, C8, is a complex molecule made up of three chains, a, p and y (molecular weights 65 kD, 65 kD and 22 kD respectively), encoded by separate genes, a and p closely linked on chromosome 1, y on chromosome 9. The a and p chains are homologous with each other and with C6 and C7 whereas C8y displays no homology. The a and y chains are covalently linked whereas the P chain is non-covalently associated in the complex. The P chain in C8 binds C7 in the C5b67 complex and the resulting complex, C5b-8, becomes more deeply buried in the membrane and forms small pores, causing the cell to become slightly leaky.

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182 B.PAUL MORGAN

The final component in the pathway, C9, is a single chain plasma protein (molecular weight 69 kD) which is homologous with C6, C7, C8a, and C8(3. The first C9 molecule to enter the C5b-8 complex binds to C8a and undergoes a major conformational change from a globular, hydrophilic form to an elongated, amphi-pathic form which traverses the membrane and exacerbates membrane leakiness. Unfolding of C9 also exposes binding sites which enable additional C9 molecules to bind, unfold and insert in the membrane. The pore thus grows with the recruit­ment of additional C9 molecules, individual complexes (membrane attack com­plexes, MACs) containing anything up to 18 C9 molecules. MACs containing multiple C9 molecules can be visualized in the electron microscope as ring-like structures enclosing a 10 nm pore. Evidence that the pore is formed mainly from C9 molecules is provided by the observation that C9 incubated in vitro in the presence of Zn " ions forms identical ring structures (Podack, 1988; Esser, 1990). The exact mechanism by which the MAC causes lysis is still the subject of debate between those who state that the MAC ring surrounds a rigid, transmembrane pore and those who consider that the MAC induces areas of lipid perturbation (leaky patches) in the membrane (Bhakdi and Tranum-Jensen, 1991; Esser, 1991). What­ever the exact mechanism, the MAC forms functional pores in cell membranes through which ions and small molecules pass, bringing about osmotic lysis of the cell. This strategy of pore formation as a means of killing targets is widely used in nature, notably by bacterial toxins and by mammalian lymphocytes (Peitsch and Tschopp, 1991). The MAC component proteins C6, C7, C8a, C8p and C9 are all highly homologous and have clearly arisen from a common ancestor. C6 and CI

are closely linked on chromosome 5 with C9 more distant on the same chromosome (Rogne et al., 1991). C8a and C8p are closely linked on chromosome I. These duplication events have probably arisen to increase the efficiency of targeting and of cytolysis by the MAC.

CONTROL OF THE COMPLEMENT SYSTEM

As a consequence of the proteolytic cascade nature of the complement system, small initiating events could result in catastrophic activation of the system. In order to prevent this each part of the complement system is tightly controlled. This control is mediated in part by the inherent instability of the activation pathway enzymes but is also provided by a number of fluid-phase and membrane-associated inhibitors (Figure 4).

Control in the Classical Pathway

The first step of the classical pathway is regulated by CI-inhibitor (Clinh), a 90 kD plasma protein, a member of the serine protease inhibitor (Serpin) family, which binds to CI and prevents its activation. Clinh also binds activated CI, physically removing Clr and Cls and thus causing disruption of the multimolecular CI

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Activation and Control of the Complement System

Alternative pathway

fH

Ifl)

Membrane attack

pathway

S protein

clusterin

C8

183

Classical pathway

Ct/^----.cT

fH

C4bp

Ifl)

^C4b2aY

< \

C4b2a3b

C5b-9

Figure 4, Regulation of the complement system. The complement system is tightly

controlled by a number of fluid-phase and membrane-associated proteins acting either

to inhibit the enzymes of the activation pathways (activated C I , C3 convertases, C5

convertases) or to restrict assembly of the MAC. Membrane-bound regulators are

boxed, fluid-phase inhibitors are bracketed. CI inh, C1 inhibitor; C4bp, C4b binding

protein; DAF, decay accelerating factor; MCP, membrane cofactor protein; CR l ,

complement receptor 1; HRF, homologous restriction factor.

complex (Davis, 1988). CI inh is the only serum protease inhibitor capable of inhibiting activated C1. Control of the later steps of the classical pathw^ay primarily involves Factor I, a two-chain (55 kD and 42 kD) plasma serine protease, together with a number of fluid-phase and membrane proteins which act as cofactors for factor I and/or accelerate the dissociation of the classical pathway C3- and C5-con-vertases (C4b2a and C4b2a3b). From the fluid-phase two proteins are involved, C4b-binding protein (C4bp), a large, multimeric plasma protein (6 or 7 a chains, each of approx. 75 kD, and zero or 1 (3 chain) and Factor H, a single-chain 150 kD plasma protein. C4bp binds C4b in the C4b2a complex and accelerates the disso­ciation (decay) of the enzyme into its component molecules (Gigli et al., 1979: Reid et al., 1986). It also acts as a cofactor for cleavage of C4b in the complex by factor I, a process which inactivates the enzyme complex (see below). The (3 chain confers upon C4bp the capacity to bind Protein S, a cofactor involved in the coagulation system (Hessing, 1991). Factor H binds C3b in the C4b2a3b complex and causes inactivation of the enzyme complex both by accelerating dissociation and by acting as a cofactor for factor I cleavage of C3b (Vik et al., 1990).

On the membrane, at least three proteins help regulate the classical pathway convertases. Decay accelerating factor (DAF) is a 65 kD single-chain protein

Page 199: Immunity

184 B.PAUL MORGAN

tethered to the membrane by a glycosyl phosphatidylinositol (GPI) anchor. DAF

binds to and causes dissociation of the enzyme complexes C4b2a and C4b2a3b on

the cell membrane. Membrane cofactor protein (MCP) is a 60 kD transmembrane

protein which, like DAF, binds to the C4b2a and C4b2a3b enzyme complexes but,

instead of causing dissociation, acts as a cofactor for the cleavage of C4b and C3b

in the complexes by factor I, thus inactivating the enzyme. Complement receptor

1 (CRl) is a large (approx. 200 kD) transmembrane protein which inactivates the

C4b2a and C4b2a3b enzymes both by causing dissociation (like DAF) and by

acting as a cofactor for cleavage by factor I (like MCP). DAF, MCP, CRl, factor

H and C4bp all share important structural features and all are tightly linked in a

region known as the Regulators of Complement Activation (RCA) cluster on the

short arm of chromosome 1. These important molecules are thoroughly described

in several recent reviews (Reid et al., 1986; Hourcade et al., 1989; Liszewski et al.,

1991; Morgan and Meri, 1994).

Factor I, in the presence of an appropriate cofactor (C4bp, factor H, MCP or

CR1), can enzymatically cleave both C4b and C3b and hence inactivate the classical

pathway convertases. The cleavage events are similar. In the case of C3b, Factor I

sequentially cleaves at two sites in the a-chain, releasing a small fragment (C3f, 2

kD) and yielding iC3b ('i' = inactive). A third factor I cleavage in the a chain

releases a large fragment (C3c, 140 kD), leaving the C3dg fragment (40 kD)

attached to the membrane. Factor H, C4bp, MCP and CRl all act as cofactors for

the first two cleavages (factor H only for C3b and C4bp only for C4b) but only CR 1

has good cofactor activity for the third factor I cleavage. Further cleavage of C3dg

by serum proteases may occur to yield the cell-bound fragment C3d (Figure 3).

Control in the Alternative Pathway

Control in the alternative pathway involves many of the same membrane and

fluid-phase regulatory proteins described above for the classical pathway. Factor I

again assumes a pivotal role. From the fluid phase, factor H binds C3b in the C3bBb

and C3bBbC3b complexes and inactivates the enzyme both by causing dissociation

and by acting as a cofactor for factor I-mediated cleavage of C3b. On the membrane,

control is mediated by DAF, MCP and CRl. DAF and CRl cause dissociation of

Bb from the C3bBb and C3bBbC3b complexes, and MCP and CRl act as cofactors

for factor I-mediated cleavage of C3b in these complexes. A unique feature of the

alternative pathway is the existence of a protein which stabilizes the C3 and C5

cleaving enzymes. Properdin (P) is a large, multimeric (2,4 or more subunits each

of 56 kD) plasma protein which binds to C3b in the complexes and inhibits the

spontaneous and accelerated (Factor H, DAF, CRl) decay (Smith et al., 1984;

Pangbum, 1989).

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Activation and Control of the Complement System 185

Control in the Membrane Attack Pathway

Like the activation pathways, the membrane attack pathway is tightly regulated

by fluid phase and membrane inhibitors. The fluid-phase C5b-7 complex is intrin­

sically unstable, its hydrophobic membrane binding site being susceptible to

inactivation by hydrolysis. Binding of C5b-7 to membranes is further restricted by

several serum proteins. The most studied of these is S protein (vitronectin), an 80

kD single-chain protein, a single molecule of which binds tightly to the C5b-7

complex and prevents its binding to membranes. C8 and C9 bind but the resultant

SC5b-9 complex is inactive (Podack et al., 1984). The fluid-phase C5b-7 complex

can also be inactivated by binding to a recently described serum protein, clusterin,

which consists of two disulfide-bonded 40 kD chains (Jenne and Tschopp, 1992).

Clusterin is a muki-fiinctional protein which is a serum lipoprotein, is present in

high concentration in seminal plasma and also appears to be a marker for cell death.

C5b-7 is also inactivated by binding other lipoproteins and, perhaps most important

of all, by binding C8 in the fluid phase. With the exception of C8, all these

fluid-phase inhibitors have other functions and it is likely that their MAC inhibiting

activity is a rather non-specific by-product of their affinity for hydrophobic mole­

cules. Their importance in vivo as complement inhibitors is unproven. On the

membrane, two inhibitors have been described, homologous restriction factor

(HRF), a 70 kD GPI-anchored protein the significance of which is unproven

(Zalman, 1992), and CD59 antigen (CD59), a 20 kD GPI-anchored molecule

which is the major MAC inhibitor on most cells. CD59 binds to C8 in the C5b-8

complex and blocks incorporation of C9, thereby preventing formation of the

lytic MAC (Lachmann, 1991; Davies and Lachmann, 1993). CD59 is encoded

on chromosome 11, and shows no structural similarities with any of the other

complement inhibitors.

PHYSIOLOGICAL ROLES OF COMPLEMENT

The physiological importance of complement is most dramatically demonstrated

in individuals deficient in components of the system (see below). The main

problems encountered by such individuals are recurrent and severe bacterial

infections and immune complex disease, vividly demonstrating that the principal

roles of complement are to mediate killing of invading bacteria and to solubilize

immune complexes. Complement also plays an important physiological role as an

initiator or enhancer of inflammation at sites of infection or injury and in recent

years it has become evident that complement also makes an important contribution

to the induction of antibody responses. These roles are all mediated by the products

of complement activation and are described below.

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186 B.PAUL MORGAN

Bacterial Killing by Complement

Most bacteria, with the important exception of the Gram negative organisms belonging to the genus Neisseria, are resistant to the lytic effects of the MAC, a consequence of their thick cell walls and capsules. Nevertheless, complement contributes to bacterial killing by attracting phagocytic cells to the site of infection and coating the bacteria in readiness for phagocytosis. The surfaces of most bacteria activate complement via the alternative pathway and rapidly become coated with complement fragments, particularly C3b and its breakdown product iC3b (Taylor, 1983). This process, known as opsonization, enables phagocytic cells to recognize bacteria by binding to fragments of C3 through specific receptors, complement receptors 1 and 3 (CRl and CR3) on the phagocyte membrane. The bacterium can then be efficiently internalized, killed and digested within the cell (Shreiber, 1984; Ross, 1986). Opsonization can also be achieved by bacterium-bound antibody interacting with Fc receptors on phagocytes but this process requires preexisting antibacterial antibody.

Complement activation at the site of infection also attracts phagocytic cells and activates them for efficient killing. These activities are mediated by the small fragments released from C3, C4, and C5 during the activation process. These fragments, C4a, C3a, and C5a, are all very similar in structure and have similar biological effects although their potencies differ, C5a being the most active, C3a of intermediate activity and C4a the least active. All bind to receptors on mast cells and cause degranulation with the consequent release of vasoactive amines which mediate vasodilatation and increased vascular permeability (anaphylaxis). Re­moval of the carboxy-terminal arginine residue by a plasma carboxypeptidase N (anaphylatoxin inactivator) completely abrogates anaphylactic activity of these fragments, thus restricting their activity to the site of activation (Hugh, 1981). C5a and, to a lesser extent, C3a also attract phagocytic cells to the site of activation (chemotaxis). This activity is mediated through specific receptors on phagocytes. Binding of C5a stimulates cell adhesion to and migration through vessel walls and the concentration gradient of C5a guides the cells towards the inflammatory site. In addition, C5a also causes activation of phagocytic cells, priming them for bacterial killing. Importantly, C5a retains some chemotactic activity even after loss of the carboxy-terminal arginine (C5adesArg) and is thus able to signal chemotaxis at some distance from the activation site (Till, 1986). It has recently been demon­strated that cells other than phagocytes, including hepatocytes and brain cells, express receptors for C5a and perhaps also C3a (Haviland et al., 1995; Gasque et al., 1995). The physiological relevance of these findings have yet to be elucidated.

Immune Complex Solubilization by Complement

Immune complexes consist of aggregates of antibody and antigen which form in the plasma under physiological and pathological conditions. The complexes effi-

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ciently activate the classical pathway of complement and hence become coated with complement fragments. Coating with complement serves two main purposes; it masks antigenic sites, limiting further growth of the immune complex, and it provides a ligand for immune complexes to attach to cells bearing complement receptors (opsonization). These events are essential for efficient immune complex solubilization and clearance. Erythrocytes are a central component of the immune complex clearance system. CRl on the erythrocyte surface binds C3b on the immune complex, effectively removing it from the circulation. Erythrocyte-bound complexes are then carried to the liver and spleen where transfer to tissue phago­cytes occurs with subsequent digestion (Pascual and Schifferli, 1992). Individuals with deficiencies of early components of the classical pathway, particularly those with C1 deficiency, are unable to solubilize immune complexes efficiently and as a consequence have severe immune complex disease.

Cell Activation by Complement

Several of the products of complement cause priming of phagocytic cells for more efficient bactericidal activity at the site of inflammation. C5a binds specific receptors on the phagocyte surface and enhances the capacity of the cell to mount an oxidase response and to produce enzymes. The MAC, which forms pores in membranes and can thus bring about lysis of some target cells, is also important as a cell activator in vivo. Metabolically active nucleated cells at the inflammatory site are resistant to lysis by the MAC and are activated to release proinflammatory molecules, thus enhancing the inflammatory response. These important nonlytic effects of the MAC are discussed in several recent reviews (Morgan, 1989; 1992).

Complement and the Immune Response

Several of the complement components and fragments generated during comple­ment activation influence the cellular immune response. Most work has focussed on C3 and its fragments (Pepys, 1974). C3 and the fragments C3b and iC3b have been reported to enhance the mitogen-induced proliferative response of B and T lymphocytes, probably acting through complement receptors (CRl, CR2, and CR3) on phagocytic cells and lymphocytes (Kinoshita, 1993). A novel mechanism for the potentiation of immune responses by fragments of C3 has recently been revealed by Fearon and co-workers (Fearon and Carter 1995). C3 fragments bound to antigen act as a bridge between B cell membrane immunoglobulin and a signaling complex containing CD 19, CR2 (to which C3 fragments bind) and TAPA-1. The resulting close association of membrane immunoglobulin with this complex greatly en­hances the B cell response to antigen.

In vivo, fragments of C3 play an important role in the trapping of antigen-con­taining immune complexes in lymph nodes. Follicular dendritic cells express complement receptors which bind C3 fragments on the complexes. Antigen in the

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trapped complexes can then be presented to B cells. Individuals deficient in C3 trap antigen very poorly and as a consequence do not mount a good antibody response (Klaus and Humphrey, 1986; Erdei et al., 1991). The anaphylatoxin C3a has been shown to inhibit lymphocyte activation induced by mitogen or antigen. These effects are probably mediated through C3a receptors on T lymphocytes. C3adesArg (C3a which has lost its carboxy-terminal arginine) has no inhibitory effect. Other fragments of C3 have also been demonstrated to influence the cellular immune system in vitro although the physiological relevance of these findings is unclear.

C5a indirectly enhances antibody responses by stimulating cytokine release from macrophages. Importantly, C5adesArg, which is much more abundant and stable than C5a in vivo, retains this activity. As a consequence, the stimulatory effect of C5a and C5adesArg is likely to outweigh the inhibitory effect of C3a and the net result is that complement activation causes enhanced humoral immune responses (Weiler, 1987).

PATHOLOGY OF COMPLEMENT

Complement Deficiencies

Deficiencies of almost every complement protein and regulator have been described and more detailed accounts of the various complement deficiencies can be found in several recent reviews (Morgan and Walport, 1991; Colten and Rosen, 1992;Figueroaetal., 1993).

Deficiencies of components of the classical pathway (C1, C4, or C2) are particu­larly associated with an increased susceptibility to immune complex disease, a consequence of the failure of immune complex solubilizafion. The role of comple­ment in immune complex solubilization is detailed on page 186. The frequency and severity of disease is greatest with Clq deficiency, closely followed by total C4 deficiency, each giving rise to a severe immune complex disease which closely resembles systemic lupus erythematosus (SLE). Subtotal deficiency of C4 is common, due to the extremely high frequency of null alleles at both the C4A and C4B loci, but total C4 deficiency is very rare. Deficiency of C2 is the commonest homozygous complement deficiency in Caucasoids but causes much less severe disease because deposition of the early components, C1 and C4, provides some solubilization of immune complexes.

C3 is an essential component of both activation pathways and is vital for efficient opsonization of bacteria. Deficiency thus causes a marked propensity to bacterial infections. Factors I and H are essential for fluid phase regulation of complement activation and deficiency of either causes uncontrolled activation with a consequent secondary deficiency of C3 which is often severe and predisposes to bacterial infections.

Deficiencies of components of the alternative pathway are rare and do not predispose to immune complex disease or pyogenic infections. A few individuals

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deficient in factor D have been described all of whom have presented with recurrent Neisseria infections, usually meningococcal meningitis. Properdin deficiency is the commonest disorder of the alternative pathway. It is inherited in an X-linked manner and also predisposes to infection with Neisseria.

Deficiencies of terminal pathway components (C5, C6, C7, C8, or C9) also cause susceptibility to infection with Neisseria. Deficiency of C6 is the second most common complement deficiency among Caucasians and is frequently associated with meningococcal meningitis and systemic infection with the meningococcus. C9 deficiency, rare in Caucasians, is by far the most frequent complement defi­ciency in Japan with an incidence approaching 1 in 1000.

CI inhibitor (Clinh) is the sole regulator of activated CI. Partial (heterozygous) deficiency of Clinh is relatively common and is associated with the disease hereditary angioedema (HAE) (Kerr & Yeung-Laiwah, 1987; Davis, 1988). HAE is thus inherited in an autosomal dominant manner. Minor trauma initiates comple­ment activation in skin or mucous membranes and the low tissue levels of Clinh are quickly exhausted. Complement activation (and other enzymatic events usually inhibited by Clinh) then proceeds in an uncontrolled manner, resulting in tissue inflammation. The individual presents with recurrent episodes of edema in skin and mucous membranes. Although most episodes of edema are of little clinical conse­quence, edema of the gut wall may present with acute abdominal symptoms, and laryngeal or tracheal edema can cause acute airways obstruction.

Deficiencies of cell membrane regulators or receptors may also cause disease. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hemolytic disorder caused by an absence of the GPI-anchored complement regulatory molecules DAF and CD59 on an expanding clone of blood cells. Affected erythrocytes and platelets become susceptible to damage by complement and hence hemolysis and thrombotic episodes occur (Rosse, 1990). The molecular basis of this fascinating disorder has recently been revealed. Mutations in the gene for one of the key enzymes, termed PIG-A, in the GPI anchor biosynthesis pathway have been found in every case so far examined. All cells in the affected clone are deficient in all GPI-linked molecules (Takahashi et al., 1993). Deficiency of erythrocyte CRl has been reported in association with systemic lupus erythematosus (SLE) but this associa­tion is probably a consequence rather than cause of the disease.

Complement in Autoimmune Diseases

The complement system contributes to tissue damage in a large number of autoimmune diseases. Autoantibodies and immune complexes in the affected organs activate complement and exacerbate inflammation. In many autoimmune diseases, from the organ specific (e.g., autoimmune thyroid disease) to the dissemi­nated (e.g., systemic lupus erythematosus), complement deposition can be detected in the affected tissues and products of complement activation are found in the plasma (Laurell, 1986; Rauterberg, 1986; Whaley, 1989). In all these autoimmune

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diseases, complement is just one of several factors which contribute to pathogene­sis. Nevertheless, modulation of complement activation may be of therapeutic benefit in these conditions (see below).

Evasion of Complement Killing by Microorganisms

Many microorganisms have developed mechanisms for avoiding complement mediated damage and a few have even taken advantage of complement activation to enhance their infectivity (Fishelson, 1994). Complement is activated on the surfaces of most microorganisms, with deposition of fragments of C3 and other components. This process of opsonization, by rendering the microorganisms more susceptible to uptake by phagocytes, is among the most important roles of the complement system. However, some microorganisms have subverted this process to their own advantage (Cooper, 1991; Frank, 1992). Organisms which infect cells must find a way of entering the cell and it is here that complement activation may act to enhance infectivity. The strategy is dramatically illustrated by the human retroviruses HIV and HTLV-1. HIV activates complement efficiently through the classical pathway but is resistant to lysis (Ebenblicher et al., 1991). The virus thus becomes coated with complement fragments which can bind complement receptors CRl and CR2 on cells and facilitate uptake into the cell. In this way, complement activation enhances uptake of HIV into CD4-positive T cells (the main receptor being CD4 itself) and may also enable the virus to infect CD4-negative cells, including monocytes, macrophages and B lymphocytes (Haeffner-Cavaillon et al., 1992). Other viruses directly utilize host complement receptors or regulators as receptors to gain entry into the cell. The Epstein-Barr virus binds to CR2 (McClure, 1992). Recently it has been reported that MCP is the receptor for the measles virus (Naniche et al., 1993) and the group A streptococcus (Okada et al., 1995), and that DAP is a receptor for some strains of Echo virus (Bergelson et al., 1994). Many pathogenic microorganisms are highly resistant to complement killing and this complement resistance correlates closely with virulence. It has recently been shown that in organisms as diverse as vaccinia viruses and schistosome worms, comple­ment resistance is achieved by the expression of complement inhibitor molecules which mimic the effects of the human complement inhibitors (Parizade et al., 1991; Isaacs et al., 1992). In many cases it is clear that the microorganism has acquired its complement regulators from the human host—a particularly spectacular piece of molecular piracy.

Iatrogenic Activation of Complement

Many materials used in the tubing and membrane components of extrocorporeal circuits are potentially complement activating and exposure of blood to these materials in renal hemodialysis, cardiopulmonary bypass or plasmapheresis is hazardous (Mollnes et al., 1991). In hemodialysis blood is in contact with a large

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surface area of dialysis membrane, commonly made of cuprophane, a complex polysaccharide material which activates complement via the alternative pathway. Release of active fragments, particularly C3a and C5a, causes activation of neutro­phils and monocytes which aggregate and become deposited in the lung. Release of toxic molecules from these cells causes the lung damage which is a common complication of long-term dialysis.

Activation of complement may also occur in transplanted organs. When a poorly matched organ is used for transplant it is rejected within minutes due to complement activation in the graft, the phenomenon of hyperacute rejection. Complement-mediated hyperacute rejection is an almost universal outcome of cross-species transplants and is thus the major barrier to the use of animal organs for human recipients (xenotransplantation) (White, 1992). Attempts are now underway to produce transgenic animals (pigs) expressing human complement regulatory mole­cules in the hope that the organs from these animals will be less complement activating, enabling them to be used for human recipients.

Control of Complement Activation In Vivo

Despite extensive research, safe, non-toxic pharmacological agents capable of efficiently inhibiting complement activation have not been found. Those agents which do inhibit complement efficiently in vivo, for example CVF, are toxic and not suitable for therapy. Many substances, including antiinflammatory agents, cause some inhibition of complement but are of low potency. A promising new approach to the inhibition of complement activation in vivo involves the use of molecular biological techniques to produce soluble, recombinant forms of the natural regulatory molecules (Fearon, 1992). Soluble recombinant CRl has been shown to inhibit complement activation and pathology in several animal models of human diseases and has recently begun clinical trials in man. The use of recombi­nant forms of CRl and other complement regulatory molecules in therapy is likely to be an area of considerable interest and importance in the next few years.

SUMMARY

The complement system consists of a group of plasma proteins which interact with one another in a sequential manner to enhance inflammation and to help kill bacteria and other foreign organisms. The two distinct activation pathways, one triggered through antibody-antigen complexes (the classical pathway), the other triggered by foreign surfaces (the alternative pathway), are both proteolytic cascades with considerable potential for the amplification of complement activation. The activa­tion pathways converge on a common membrane attack pathway during which the component proteins complex together and insert as a pore into cell membranes, thus bringing about cell lysis. During activation several biologically active fragments are produced from the complement proteins which are important in defence against

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infection. The system is tightly controlled by a battery of regulatory molecules in plasma and on cell membranes which act to minimize damage to self cells.

Overactivation of the complement system can exacerbate tissue damage in many diseases, including autoimmune disease and infection. Inherited deficiencies of components of the complement system graphically reveal the biological importance of complement. Although the precise consequences depend on the component involved, most individuals with complement deficiencies are prone to bacterial infections and to immune complex diseases.

The recent development of specific complement inhibitors opens the possibility of preventing activation in the numerous diseases in which complement contributes to tissue injury.

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Takahashi, M., Takeda, J., Hirose, S. et al. (1993). Deficient biosynthesis of N-acetylglucosaminyl-

phosphatidylinositol, the first intermediate of glycosyl phosphatidylinositol anchor biosynthesis,

in cell lines established from patients with paroxysmal nocturnal hemoglobinuria. J. Exp. Med.

177,517-521.

Taylor, M.E., Brickell, P.M., Craig, K.K., Summerfield, J.A. (1989). Structure and evolutionary origin

of the gene encoding a human serum mannose-binding protein. Biochem. J. 262, 763—767.

Taylor, P.W. (1983). Bactericidal and bacteriolytic activity of serum against gram negative bacteria.

Microbiol. Rev. 47, 46-76.

Taylor, P.W. (1993). Non-immunoglobulin activators of the complement system. In: Activators and

Inhibitors of Complement (Sim, R.B., Ed.), pp. 37-68. Kluwer, Amsterdam.

Till, G.O. (1986). Chemotactic factors. In: The Complement System (Rother, K., & Till, G.O., Eds.),

pp. 354—367. Springer, Berlin.

Turner, M.W. (1991). Deficiency of mannan binding protein—^a new complement deficiency syndrome.

Clin. Exp. Immunol. 86(suppl.l), 53-56.

Vik, D.P., Nunoz-Canoves, P., Chaplin, D.D., & Tack, B.F. (1990). Factor H. Curr. Topics Microbiol.

Immunol. 153, 147-162.

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196 B.PAUL MORGAN

Weiler, J.M. (1987). Complement and the immune response. In: Complement in Health and Disease

(Whaley, K., Ed.), pp. 289-316. MTP Press, Lancaster.

Whaley, K. (1989). Measurement of complement activation in clinical practice. Complement Inflamm.

6,96-103.

White, D.J.G. (1992). Transplantation of organs between species. Int. Arch. Allergy Appl. Immunol.

98, 1-5.

Zalman, L.S. (1992). Homologous restriction factor. In: Current Topics in Microbiology and Immunol­

ogy Vol. 178. "Membrane defenses against attack by complement and perforins" (Parker, C.J.,

Ed.), pp. 87-101. Springer-Verlag, Berlin.

Ziccardi, R.J. (1983). The first component ofhuman complement (CI): Activation and control. Springer

Semin. Immunopathol. 6, 213-231.

RECOMMENDED READINGS

Morgan, B.P. (1990). Complement: Clinical aspects and relevance to disease. Academic Press, London.

A monograph describing the complement system and focussing on the role of complement in

disease. Law, S.K., & Reid, K.B.M. (1989). Complement in Focus. IRL Press, London. An excellent, concise

summary of structural and functional aspects of the complement system (second edition may now

be available). Rother, K., & Till, G.O. (1988). The Complement System. Springer-Verlag, Berlin. A multi-author text

which provides an extremely detailed and complete review of all aspects of the complement system.

Immunology Today (\99\). 12 (9), 291-342. A complete issue of this readable journal covering several

important aspects of the complement system. Springer Seminars in Immunopathology (1994). 15 (4), 303—431. An issue dealing with various topical

issues relating to the biology of the complement system.

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Chapter 11

Phagocytes in Immunity and

Inflammation

PHILIP M. MURPHY

Introduction: General Role of Phagocytes in Homeostasis 198 Historical Background 198

Phagocytes and Infection 199 Phagocytes and Immunity 201 Phagocytes and Inflammation 202

Phagocyte Development 203 Phagocytes Differentiate from Totipotent Stem Cells in the Bone Marrow 203 Mature Phagocytes 205 Monocytes and Macrophages 209

Specialized Functions of Phagocytes 211 Cell Migration 211 Phagocytosis/Opsonization 217 Degranulation 219 Production of Antimicrobial Oxidants 221

Principles of Medical Biology, Volume 6 Immunobiology, pages 197-229. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-811-0

197

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198 PHILIP M. MURPHY

Production of Pro-Inflammatory Mediators 224 Immunoregulation 225

Summary 225

Recommended Readings 228

INTRODUCTION: GENERAL ROLE OF PHAGOCYTES IN HOMEOSTASIS

Historical Background

The study of phagocytes in the 19* century linked the phenomena of infection, inflammation and immunity, which had had quite separate histories. Prior to that time, epidemics of plague, pox, typhus, typhoid, and other scourges were usually attributed to pariahs, foreigners, or the wrath of God: contagion was visible, germs were not. Inflammation (from the Latin for "on fire") was defined by Celsus in the first century A. D. as a pathologic state characterized by redness, warmth, swelling and pain, a definition that has remained clinically useful to the present day. Inflammation was regarded correctly as a consequence of injury, but was not yet appreciated for its role in fighting infection. Immunity (from the Latin for "exemp­tion") was the word given to describe resistance to contagion, although there was no understanding of its root causes. It was commonly recognized long before Jenner, for example, that milkmaids enjoyed unusually fair skin, because they were spared the scourges of the pox that afflicted nearly everyone else. We now know that exposure to cowpox, a viral disease of cattle that causes limited infections in humans, can protect an individual, as it did the milkmaids, from acquiring smallpox. It was only after the development of scientific methodology that such observations could be transformed through hypothesis, experiment and invention into theories of disease, enabling the intellectual histories of infection, inflammation and immu­nity to emerge from superstition, to become fully and mechanistically linked.

The invention of the microscope by Leeuwenhoek in Holland, the development of aniline dyes in the nineteenth century by the new German chemical industry, and the invention of in vitro culture techniques by Koch and others, followed by their application to the study of pathologic specimens, led to the development of the germ theory of disease and the birth of the experimental science of microbiology, without any initial connection of infection to immunity or inflammation. The crown jewels of microbiology are Koch's postulates for determining the microbial etiol­ogy of specific diseases. Jenner, who knew nothing about germs, and later Pasteur, who shared in their discovery, subjected the phenomenon of immunity to the scrutiny of experimental test. Remarkably, the development of vaccination as a safe treatment modality, and the establishment of immunity as a scientific concept were both outcomes of the same series of experimental inoculations (Pasteur, 1880;

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Phagocytes in Immunity and Inflammation 199

Oppenheim and Potter, 1981). Jenner's simple experiment ultimately led to perhaps the greatest practical achievement of medical science: the eradication of smallpox as a threat to human health. Pasteur went further scientifically by showing that infection with microorganisms is causally linked to the development of immunity. However, the host factors responsible for the phenomenon remained elusive.

"White corpuscles" were first discovered in the blood by William Hewson in the 18* century (Robinson and Mangalik, 1975). In 1824, Dutrochet in France, and later Julius Cohnheim in Germany, accurately described the microscopic response of an animal to local experimental irritation (Dutrochet, 1824; Cohnheim, 1889). Adherence of leukocytes to blood vessels, and movement of both fluid and cells to the extravascular space led to local accumulation of leukocytes. Thus, leukocyte-blood vessel interactions and leukocyte accumulation in the tissue spaces were proposed as the cellular bases of inflammation. The leukocytes were thought to respond to injury, but were not yet linked to host defense or immunity. In fact, leukocytes were thought by many to actually facilitate the dissemination of germs throughout the body. The next great intellectual contribution was that of Elie Metchnikoff, a Russian zoologist, who connected his experiments on the engulf-ment of rose thorns by the wandering phagocytes of transparent starfish larvae to Pasteur's observations on microbial pathogenesis in vertebrates. Metchnikoff s seminal proposal was that the relationship between phagocytes and microorganisms was not conspiratorial but was instead adversarial, and could explain immunity (Metchnikoff, 1905). Thus, Metchnikoff s study of phagocytes produced the theory of cell-mediated immunity that articulated the links among infection, inflammation and immunity.

The work of Von Behring, Ehrlich and many others extended and corrected Metchnikoff s initial theory, showing that immunity can be the consequence of both cellular and humoral factors (Silverstein, 1979). Phagocytes are now known to lack the memory and target specificity characteristic of immunity, these being the province of lymphocytes. With this intellectual foundation firmly in place, the last 100 years have been spent in reductionist pursuit of the detailed molecular and cellular connections that exist among infection, inflammation and immunity. From the nineteenth century focus on microbes as the targets of the immune system, it is now known that non-self targets also include the cells and proteins of genetically distinct individuals (leading to serum sickness, tissue transplant rejection, and blood transfusion reactions), as well as inappropriate targets of the same individual (leading to autoimmune phenomena). Phagocytes may participate in the pathogene­sis of all of these phenomena. See Table 1.

Phagocytes and Infection

In order to accomplish its host defense mission, the phagocyte must quickly learn that a site of infection exists, then crawl to the site, bind to the offending cells, and finally engulf and digest them while ignoring nornial host structures. This process

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200 PHILIP M. MURPHY

Table 1, Phagocytes and Human Disease

Inflammation: diseases where the pathogenesis may be partly due to the presence of

phagocytes and their products

Rheumatoid arthritis

Gout

Neutrophilic dermatosis

Myocardial infarction

Inflammatory bowel disease

Asthma

Emphysema

Vasculitis

Glomerulonephritis

Adult respiratory distress syndrome

Dialysis-related leukoaggregation

Malignant neoplasms at sites of chronic inflammation

Impaired immunity: disorders of phagocytes associated with increased susceptibility to

infection with pyogenic microorganisms

I. Disorders of phagocyte number

A. Decreased production

-Acquired

iatrogenic (chemotherapy, radiotherapy, drugs)

nutritional

infections

myelophthisis

drug-induced

idiopathic

-Congenital

Cyclic neutropenia

Kostmann's syndrome

Reticular dysgenesis

Chronic idiopathic neutropenia

B. Increased destruction

autoimmune neutropenia (e.g., Felty's syndrome, drug-induced)

II. Disorders of phagocyte function

-Acquired

Association with numerous primary metabolic, neoplastic, and immunologic

disorders (e.g., uremia, hypergammaglobulinemia and diabetes mellitus)

Drug-induced

Thermal injury

-Congenital

Leukocyte adhesion deficiency-1

Leukocyte adhesion deficiency-2

Neutrophil-specific granule deficiency

Myeloperoxidase deficiency

Chronic granulomatous disease

Chediak-Higashi syndrome

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Phagocytes in Immunity and Inflammation 201

is not random but instead involves a highly regulated communication network and specific targeting events. Extracellular signaling molecules known as chemoattrac-

tants are generated at the target site by the microbe, as well as by the host in response to the microbe. As the chemoattractant diffuses out from the target site, phagocytes detect it by means of specific cell surface receptors. The spatial and temporal interaction of the receptors with the chemoattractant conveys information to the cell regarding the location of the target. In response to this information, the cell extensively remodels its cytoskeleton, and biases its otherwise random movements in the direction of higher concentrations of the chemoattractant, thereby moving towards the target. This is known as chemotaxis. Phagocytes leave the bloodstream by crawling between vascular endothelial cells, in a complex process known as diapedesis. The phagocyte recognizes and binds to the target most efficiently when it has first been decorated with molecular tags such as specific antibodies or generic complement fragments, for which it possesses additional sets of receptors. The tagging process is known as opsonization (from the Greek for "to prepare food"). The phagocyte then engulfs the bound target forming a cytoplasmic phagosome.

Phagocytosis in turn triggers the delivery of antimicrobial proteins and digestive enzymes to the phagosome by fusion with cytoplasmic granules, and the production of an array of germ-killing oxidants by activation of a latent NADPH oxidase.

Phagocytes and Immunity

Immunity can be operationally defined as the phenomenon whereby sublethal natural infection with a pathogenic microorganism, or exposure to its products, induces changes in the host that protect it from disease upon subsequent challenge with the same or a related organism. Vaccination is the intentional exploitation by man of this natural phenomenon. While the immune system can respond ferociously to non-microbial antigens, such as allergens or mismatched tissues, it is clear that these are evolutionary or iatrogenic sideshows to the main immunological event: defense of the host against microbial intruders.

The hallmarks of the immune system— fine target specificity, memory, and the ability to distinguish self from non-self—^are poorly-developed properties of phagocytes, but are highly-developed properties of lymphocytes. Nevertheless, the phagocytes, particularly macrophages, can distinguish normal cells from senescent cells, neoplastic cells and foreign cells. Specialized phagocytic receptors for the generic Fc portion of specific antibody molecules (FcRs), and for complement-de­rived opsonins, are critical determinants of target recognition by phagocytes (Ravetch and Kinet, 1991; Wright, 1992).

Since they recognize many different targets indirectly by these generic adaptors, phagocytes can be regarded as performing a non-specific effector function on the efferent limb of the immune system. Nevertheless, phagocytes also serve as critical components on the afferent limb of the immune system by preparing complex microbial protein antigens for specific recognition by T lymphocytes, a process

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202 PHILIP M. MURPHY

known as antigen presentation (Unanue and Allen, 1987). The engraftment in higher species of specific immune functions onto the more ancient scavenger functions of phagocytes over evolutionary time has produced a system of host defense and repair with greater flexibility and specificity, newly capable of identi­fying cryptic invaders such as viruses, and certain bacteria and protozoa that replicate inside of host cells, often inside of phagocytes themselves.

Phagocytes and Inflammation

Like livers and hearts, a healthy immune system usually functions imperceptibly: a silent war is waged and won against microbial challengers. When an encounter of the immune system with a microbe is clinically apparent, the condition is termed inflammation. From the perspective of antimicrobial host defense, inflammation can be regarded as an imperfection of the immune system, resulting from either an inefficient or overzealous reaction to the microbe. Inflammation occurs, in part, because many of the same oxidants, antimicrobial proteins, and degradative en­zymes that damage microbes inside the protected environment of the phagosome, can also damage blood vessels, extracellular matrix and epithelial cells when they are released into the extracellular space by cellular degranulation or cytolysis. In fact, phagocytes and their products can fan the flames of an inflammatory reaction long after the microbial arson has itself been consumed. Poststreptococcal

glomerulonephritis is a classic clinical example in which neutrophils contribute to the acute loss of kidney function when immune complexes become trapped in the subendothelial spaces of renal glomeruli, after the streptococcus has been eradi­cated from the pharynx or other local site.

In addition to microbes and immunological insults, chemical and physical irritants can also trigger an inflammatory response. In these cases, inflammation can be regarded as a normal response of the immune system to acute tissue injury. Sunburn, blunt trauma, and cold-induced asthma are examples that differ dramati­cally in their clinical characteristics, and involve distinct effector components of the immune system. Natural inflammatory reactions may be acute and transient, or chronic and persistent; they may be systemic or localized, depending on the nature of the inciting irritant and individual host factors.

Acute inflammation typically involves rapid changes in vascular smooth muscle tension and vascular permeability, as well as neutrophil accumulation and activa­tion, all of which are induced by soluble inflammatory mediators. The complement system is a particularly important source of inflammatory mediators, since it can be activated by direct contact with immune complexes or target particles. A detailed description of the complement system can be found in Chapter 10 of this volume. Complement activation gives rise to partial cleavage products of specific comple­ment proteins that acquire new functional properties, such as opsonization (C3b, C3bi), phagocyte chemoattraction (C5a), cytolysis (C5b-9, the membrane attack complex), and modulation of vascular permeability and vascular smooth muscle

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Phagocytes in Immunity and Inflammation 203

tension (C3a, C5a, the complement-derived anaphylatoxins). Acute inflammatory lesions can also occur without local accumulation of phagocytes, highlighting the importance of inflammatory mediators and vascular changes in inflammation (Ognibene et al., 1986).

Chronic inflammation involves delayed and persistent tissue infiltration by macrophages and/or lymphocytes. Fibroblast and capillary proliferation and in­creased production of extracellular matrix proteins are also found. The granuloma

is a classic histopathological lesion of chronic inflammation composed of macro­phages and fibroblasts that often forms when macrophages cannot degrade an ingested particle.

Intradermal antigenic challenges of sensitized subjects in vivo have been impor­tant in linking specific effector components of the immune system with specific types of inflammatory pathology and specific classes of antigens. By the turn of the century the Arthus reaction (IgG-immune complexes and neutrophils), imme­

diate type hypersensitivity reactions (IgE and basophils), and delayed type hy­

persensitivity reactions (T lymphocytes) had been established as important experimental models of immunologically-mediated inflammatory reactions (Co­hen etal., 1979).

PHAGOCYTE DEVELOPMENT

Phagocytes Differentiate from Totipotent Stem Cells in the Bone Marrow

The development of phagocytes can be divided into mitotic and non-mitotic stages, lineage-committed and non-committed stages, and bone marrow, blood and tissue stages. Extensive overlap exists for these classifications (Figure 1). Thus, the mitotic stage occurs in two distinct operationally defined compartments of the bone marrow: the stem cell compartment, in which lineage decisions have not yet been made, and the progenitor cell compartment, in which the lineage of individual cells is defined. Furthermore, progenitor cells and possibly even stem cells can also be found in the peripheral blood.

All of the formed elements of the blood, namely erythrocytes, leukocytes and platelets, are thought to arise from the proliferation and differentiation of single totipotent stem cells in the bone marrow. The first evidence for this idea came from experiments in which lethally irradiated mice were rescued by transfusion with very small numbers of syngeneic undifferentiated bone marrow-derived cells (Span-grude et al., 1988). Reconstitution has been achieved with as few as thirty donor-derived cells. Given experimental inefficiencies, these experiments have been interpreted to mean that a single totipotent stem cell can fully reconstitute all cell lineages of the blood. Since it is impossible to completely ablate the recipient marrow without killing the animal by damaging other less radiosensitive organs, it has so far not been possible to rigorously define the very earliest totipotent stem cell. Nor has it been established whether the earliest stem cells are capable of

Page 219: Immunity

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Phagocytes in Immunity and Inflammation 205

self-renewal, or instead are selected for differentiation by stochastic processes from a cohort of identical totipotent cells that becomes depleted over time, much like gonadal oocytes. Bone marrow stromal cells contribute to a hematopoietic induc­

tive microenvironment that may also play a role in lineage commitment. Cyclic neutropenia is an autosomal recessive condition in which the number of

cells in all of the blood cell lineages decreases in a periodic manner owing to a transient maturation arrest (Wright et al., 1981). The period of each cycle is -21 days. Circulating neutrophil numbers are the most seriously affected because of their short half-life, and can be reduced to zero. Thus, the clinical features of the disease—periodic infections—^are determined by the degree of neutropenia. Trans­plantation studies in a natural animal model of cyclic neutropenia, the gray collie dog, have indicated that the defect is cellular and not humoral and that it probably lies in an early hematopoietic progenitor cell, possibly the totipotent stem cell. The rarity of the human disease and the lack of fortuitous cytogenetic abnormalities has impeded progress in defining the molecular genetics.

While the stem cell compartment is defined by in vivo reconstitution experiments in lethally irradiated animals, lineage-specific progenitor cells are defined by panels of monoclonal antibodies for specific cell surface markers and by the ability to give rise to colonies of mature, morphologically identifiable blood cell lineages in vitro.

The progenitor cells are designated colony forming unit-X (CFU-X), where X designates the lineage that arises. Three major cell lineage commitments occur early on: the erythroid lineage which leads to the production of red blood cells, the lymphoid lineage which leads to the production of T and B lymphocytes, and the myeloid lineage which leads to the production of granulocytes (neutrophils, eosi­nophils and basophils), monocytes and megakaryocytes. The molecular basis of lineage commitment is poorly understood, but appears to involve the expression of receptors for lineage-specific peptide growth factors that act at specific stages of differentiation (Golde and Gasson, 1988; Dexter et al., 1990). These include the colony-stimulating factors (CSF) for the myeloid series, Granulocyte-CSF (GCSF), Macrophage-CSF (MCSF), Granulocyte/Macrophage-CSF (GMCSF), and inter-leukin 3 (IL-3; granulocyte, macrophage, erythroid and mast cell colony-sfimulat-ing factor). GMCSF and GCSF are multifunctional molecules that also potentiate certain functional properties of mature granulocytes. GMCSF and GCSF have important pharmacological properties and are clinically useful in restoring the neutrophil count to safe levels after cytoreductive chemotherapy (Groopman et al., 1989).

Mature Phagocytes

The myeloid differentiation pathway is the most kinetically active, largely due to the rapid turnover of neutrophils, the most numerous cells of the myeloid lineage (Table 2). Two-thirds of bone marrow precursor cells can be shown to bear markers characterisfic of the myeloid lineage (Robinson and Mangalik, 1975).

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206 PHILIP M . M U R P H Y

Table 2. Differential Features of Phagocytes

Feature

Specific growth factor

Life-span

Mitotic

Nucleus

~% of blood leukocytes

Staining property

Inflammation

Microbial target

Surface marker

Granule marker

Selective activator

Phagocytosis

Degranulation

Antigen presentation

Oxidant production

ADCC

Lipid mediator

Cytokine production

Monocyte/

Macrophage

M-CSF^

months-years

yes

reniform

; 15%

neutral

chronic

bacteria, fungi

CD14

non-specific

esterase

MCP-1

++++

+ + + + LTB4

++++

Phagocyte

Neutrophil

G-CSF

days

no

multilobed

45%

neutral

acute

bacteria, fungi

— lactoferrin

IL-8

+++ + -+ + LTB4

+

Granulocyte

Eosinophil

IL-5

months

no

bilobed 5%

acidic

acute/chronic

helminths

FCgRII

MBP, ECP,

EDN, CLC, EPO

IL-5

+ +-I-+

-+ + LTC4 ?

Basophil^

IL-4

?

no

cleft

0.5%

basic

allergic ?

Fc,RI

histamine

allergen

-++++

---LTC4 ?

Notes: ^ Basophils are not phagocytic, but arise from a proximal progenitor cell, and have features in common with other granulocytes.

Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; CSF, colony-stimulating factor; M, macrophage; G, granulocyte; IL, interleukin;CD, cluster designation; LT, leukotriene;MCP, monocyte chemoattractant protein; MBP, major basic protein; ECP, eosinophil cationic protein; EDN, eosinophil-derived neurotoxin; CLC, Charcot-Leyden crystal protein; EPO, eosinophil peroxidase.

Neutrophils, eosinophils and basophils are terminally differentiated granulocytes that can be distinguished by morphology and function. All have multi-lobed nuclei and conspicuous granules. Differences in granule content and in specialized surface receptors account in large part for the distinct functional properties of these cells.

Neutrophils

The neutrophil has a high capacity for motility, phagocytosis and microbial killing. Sixty percent of nucleated cells in the bone marrow belong to the neutrophil series (Robinson and Mangalik, 1975). The mitotically active precursors of the neutrophil are the myeloblast (2%), promyelocyte (5%), and myelocyte (12%). The mitotic phase of neutrophil development lasts -7.5 days in the marrow. The post-mitotic phase lasts 6.5 days and includes the metamyelocyte and band forms (22%), and finally mature neutrophils (20%). Approximately 10'^ mature neutro­phils are produced by the marrow each day. They do not return to the marrow. The

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Phagocytes in Immunity and Inflammation 207

marrow contains a large reserve of neutrophils that can be rapidly mobilized by pro-inflammatory stimuli. The blood pool is divided equally between a pool of circulating cells and a pool of cells that are adherent to endothelial cells in the microvasculature, commonly known as the marginated pool. Neutrophils in the marginated pool can be induced to demarginate by stress, exercise, glucocorticoids or epinephrine.

In contrast, bacterial lipopolysaccharide, a potent pro-inflammatory component of the outer membrane of Gram negative bacteria that is also known as endotoxin, increases margination, transiently decreasing the total circulating neutrophil con­centration. Later an increase in circulating neutrophils is observed in response to stimulation by endotoxin of neutrophil release from the marrow reserves. Gluco­corticoids have a similar effect on the marrow reserves, but they further increase the circulating pool by decreasing both the marginated pool and diapedesis into the tissues. Neutrophil kinetics during challenges with bacterial endotoxin are similar to those that are observed during septic shock. After circulating for ~8 hours in the peripheral blood, neutrophils enter the tissue pool where they survive for 1 to 2 days. They are then cleared from the tissues in the spleen, gut and other sites.

Abnormalities in the circulating concentration of neutrophils are common. Neutrophilia is a normal response to infection. Neutropenia is most commonly an acquired condition, especially as a consequence of cytoreductive chemotherapy or drug toxicity. Regardless of etiology, the risk of infection is particularly great when the circulating concentration of neutrophils is sustained below 500 cells/|Lil. In the few reported cases of inherited neutropenia, deficiencies of other blood cell types are also present. Kostmannn's syndrome is a particularly interesting autosomal recessive condition characterized by severe neutropenia. The bone marrow has normal cellularity, but has few or no myelocytes. Patients with Kostmann's syndrome typically die from overwhelming bacterial infections by 6 months of age (Kostmann, 1975).

Eosinophils

Eosinophils have a characteristic bilobed nucleus and possess granules that stain red with the acid aniline dye eosin. Eosinophil and neutrophil precursors can be distinguished as early as the promyelocyte stage. Eosinophils represent only - 5 % of circulating leukocytes. Unlike neutrophils, they can live for weeks in the tissues. They are especially numerous at mucosal surfaces, such as the gut and respiratory tract. Eosinophil kinetics differ significantly from those of neutrophils in three respects: (1) unlike neutrophils, eosinophils can be found in the thoracic duct lymph and in lymph nodes, (2) unlike neutrophils, glucocorticoids, epinephrine and stress all decrease the circulating concentrations of eosinophils, and (3) unlike neutro­phils, increased production of eosinophils is regulated in large part by the T cell cytokine IL-5. IL-3, IL-5, and GMCSF have important effects on both eosinophil development and functional activation. Of these three cytokines, only IL-5 is

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208 PHILIP M. MURPHY

specific in its actions for eosinophils (Weller, 1991). The plasma membrane, the sensing surface of the cell, contains many of the same chemoattractant and phago­cytic receptors in eosinophils and neutrophils, although the relative abundance of individual receptors may differ substantially.

The eosinophil appears to be an important effector element in host defense against helminths (Butterworth, 1984; Weller, 1991). Natural helminth infections are associated with eosinophilia. Moreover, eosinophils are able to kill helminths in

vitro, and rodents experimentally depleted of eosinophils are highly susceptible to helminth infections in vivo. High plasma levels of IgE are also characteristic of helminth infections. Although this finding is not fully understood, specific anti-parasite IgE may serve as a physiologically important opsonin able to bind helminths to special IgE receptors on the surface of eosinophils (Capron et al., 1984).

Blood eosinophilia and eosinophilic tissue infiltrates are also frequently found in allergic inflammation, and certain forms of malignancy. The Charcot-Leyden crystal is a hallmark of eosinophil-related disease. It is composed of a lysophos-pholipase from eosinophil granule and plasma membranes, and is most commonly found in sputum from patients with allergic asthma (Weller et al., 1980). Eosino­phils and their products have been strongly linked to airway hyperreactivity, and increased mucus secretion in allergic asthma (Durham et al., 1989).

Basophils

Unlike other leukocytes of the myeloid lineage, basophils lack FCyRs and are unable to efficiently phagocytize particles. Basophils have a segmented nucleus and large cytoplasmic granules rich in histamine that stain purplish-red with blue aniline dyes, a property known as metachromasia. They have many structural and functional similarities with tissue mast cells (Metcalfe et al., 1992). Both cell types develop from hematopoietic stem cells, and both participate in immediate type hypersensitivity reactions. With the exception of the stomach and the central nervous system, blood basophils and tissue mast cells account for all of the histamine that is found in the blood and tissue. Basophils express high affinity receptors for IgE (FCgRI) that differ from the low affinity FCgR on eosinophils (FCgRII). FCgRI mediates basophil activation when it is cross-linked by IgE mole­cules bound to allergens (Ravetch and Kinet, 1991). Allergens are typically proteinaceous; polysaccharide antigens are poor allergens. After activation by allergen, basophils release preformed histamine from granules and newly synthe­size a variety of inflammatory mediators such as leukotrienes, prostaglandins, and platelet-activating factor. Only a few FCgRs need to be cross-linked to obtain maximal amounts of histamine release.

Basophils represent a small fraction (-0.5%) of circulating leukocytes yet play a paradoxically large role in human disease, owing to the potent vasoactive properties of histamine. Clinical examples of diseases in which basophils play an

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Phagocytes in Immunity and Inflammation 209

important role in pathogenesis include asthma, urticaria, anaphylaxis, and rhinitis. While their involvement in the pathogenesis of allergic inflammation is clearcut, the restorative and defensive roles of basophils have not yet been clearly identified. Studies with rodents have implicated basophils in host defense against ectoparasites such as ticks, although convincing data are lacking in humans (Brown et al., 1982). Allergy may be a disadvantageous remnant of the immune system that originally evolved to control parasites that may no longer confront the host because of extinction, or an altered host tropism. Thus, the basophil acts as an endogenous pathogen in allergic subjects.

Monocytes and Macrophages

The promonocyte is the first morphologically identifiable cell of the mononuclear phagocyte system. Monocytes circulate in the blood, then enter and take up residence in the tissues where they differentiate into macrophages. Unlike granu­locytes, mature cells of the mononuclear phagocyte system are capable of cell division, long life, and further differentiation in the tissues where they acquire generic, as well as tissue-specific functions (Adams and Hamilton, 1992). During differentiation, the cell changes dramatically: it more than doubles in size, increases its content of mitochondria, granules, and granule enzymes, and may acquire increased phagocytic and microbicidal capacity. Tissue macrophages (also known as histiocytes) may be adherent or free. Examples of adherent macrophages are Kupffer cells of the liver, osteoclasts, Langerhans cells of the skin, pulmonary alveolar macrophages, microglial cells of the brain, splenic macrophages, and multinucleated giant cells in granulomata. Examples of free macrophages are the fluid phase macrophages found in the peritoneal, pleural, and synovial spaces. There are structural and functional differences among macrophages from different sites.

Together with the neutrophil, monocytes and macrophages are the major effector cells for killing bacteria and fungi. However, certain bacteria, such as the myco­bacteria and Listeria monocytogenes, and certain protozoa, such as Leishmania spp.

and Toxoplasma gondii, have evolved mechanisms for eluding the killing mecha­nisms of macrophages, and are in fact obligate intracellular pathogens of resting macrophages (Densen and Mandell, 1980; Cohn, 1986) (Figure 2). The killing capacity of the macrophage for these organisms can be increased dramatically by stimulation of the cell with interferon y, which markedly increases the ability of the cell to produce anti-microbial oxidants that can lethally damage the cell wall and other microbial structures (Nathan, 1987). Macrophages also kill tumor cells in

vitro, although the physiologic importance of this function has yet to be convinc­ingly demonstrated in vivo. Macrophages are also involved in the clearance of senescent cells and in detoxification of endogenous and exogenous wastes. They accumulate at sites of devitalized tissue (necrotaxis), and are involved in tissue remodeling and wound healing. In addition to their killing, scavenging and repara-

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Page 226: Immunity

Phagocytes in Immunity and Inflammation 211

tive actions, macrophages are the source of a wide range of soluble mediators that regulate the function of endothelial cells, lymphocytes and other phagocytes (Nathan, 1987). Macrophages or their products also regulate hematopoiesis, anti­gen presentation, coagulation, and lipid and iron metabolism (Adams and Hamil­ton, 1992). The diversity of macrophage functions is reflected in the complexity of its sensing surface, the plasma membrane, which deploys receptors for a phenome­nal number of extracellular signalling molecules, including neurotransmitters, hormones, chemoattractants, immunoglobulins, adhesion molecules, carbohy­drates, opiates, and cytokines.

SPECIALIZED FUNCTIONS OF PHAGOCYTES

In the blood, phagocyte gene transcription, protein synthesis and oxygen consump­tion are all at very low levels: the cell circulates essentially as a quiescent sphere. Anti-microbial proteins and enzymes that were synthesized during maturation in the bone marrow are in a latent state in intracellular granules. The phagocyte's trigger is cocked; a chemoattractant or opsonized microbe pulls it. Nevertheless, unlike a gun that is fired, phagocyte activation is not a monotonous, uniform process, but instead is a complex web of metabolic changes that may occur in parallel or in series, that may vary in magnitude, and that can be dissociated from one another depending upon the identity and concentration of the stimulus. In general, however, phagocyte activation involves dramatic changes to cell mem­branes: granules fuse; pseudopods, phagosomes, and phagolysosomes are formed; and the plasma membrane becomes stickier. The cell becomes highly elongated in shape and highly polarized. Associated biochemical changes are equally dramatic with new production and release of oxidants and autacoids, mobilization of cal­cium, cytoskeletal remodeling, increased oxygen consumption, increased glucose metabolism, and increased lipid turnover.

The cell biology of mature phagocytes has largely involved the study of their idiosyncratic functions, namely cell migration, phagocytosis, anti-microbial killing (degranulation and oxidant production), autocrine and paracrine regulation of the inflammatory response, and antigen presentation.

Cell Migration

The study of cell motility in vitro has shown that the cell may undergo random displacements whose rate may be increased by extracellular stimuli, a process known as chemokinesis. If the stimulated displacement has a directional bias, it is then referred to as chemotaxis. When the extracellular stimulus is not soluble, but is instead a molecule bound to a surface, directed movement is referred to as haptotaxis. The relative contribution of random movement, chemokinesis, chemo­taxis, and haptotaxis to the migration of cells in multicellular organisms in vivo is unknown. Directed movement of cells is the basis for physiologic processes as

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212 PHILIP M. MURPHY

diverse as nutrient acquisition by bacteria, stalk formation by slime molds, yeast

mating, oocyte fertilization by spermatocytes, embryonal pattern formation, wound

healing, tumor metastasis and pus formation.

Prokaryotic cells and spermatocytes are propelled through an aqueous environ­

ment by extracellular appendages (cilia or flagella). In contrast, non-spermatocyte

eukaryotic cells do not swim but instead crawl along surfaces by concerted

movement of the plasma membrane and reorganization of the cytoskeleton (Stossel,

1993). The environment that is traversed, the type of molecular motor and the

sensitivity of the cell to chemoattractants all greatly influence the nature and

velocity of cell movement. The rapid, ameboid movements of phagocytes in

response to chemoattractants enable them to serve as the first line of cell-mediated

host defense against infection.

The molecular basis of phagocyte activation has been the subject of intense study

over the past 10 years. Greater than twenty different phagocyte chemoattractants

Table 3. Phagocyte Chemoatt ractants

Chemoattractants and Secretagogues

N-formyl peptides

C5a

Leukotriene B4

Platelet-activating factor

Thromboxane B2

Histamine

Pure Chemoattractants

Substance P

Transforming growth factor p

lnterleukin-5

Chemokines (chemoattractant and cytokine activityf

lnterleukin-8 (neutrophil-activating peptide 1)

Neutrophil-activating peptide 2

GROa

GROp

GROy

Epithelial-derived neutrophil activator-78

Y interferon-inducible protein-10

Macrophage inflammatory protein-1a

Macrophage inflammatory protein-ip

RANTES

Monocyte chemoattractant protein-1

Monocyte chemoattractant protein-2

Monocyte chemoattractant protein-3

Note: ^ Chemokines are structurally and functionally related

70-78 amino acid peptides encoded by distinct genes on

human chromosomes 4 and 17. A detailed description of

the sources, cel l targets, receptors and actions of

chemoattractants can be found in Oppenheimetal . , 1991

and Murphy, 1994.

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Phagocytes in Immunity and Inflammation 213

have been purified (Table 3). Known phagocyte chemoattractants are mostly peptides or lipids. While different types of phagocytes respond to many of the same chemoattractants (e.g. C5a), cell-type specific chemoattractants are also known (e.g., histamine and IL-5 for eosinophils, and IL-8 for neutrophils) (Weller, 1991; Oppenheim et al., 1991). Many but not all of these molecules also activate other phagocyte functions such as degranulation (secretion) and oxidant production.

With few exceptions (IL-5, transforming growth factor pi), phagocyte chemoat­tractants bind to structurally related receptors with seven predicted plasma mem­brane-spanning helices, that are functionally coupled to heterotrimeric GTP-binding proteins (G proteins) (Figure 3). Molecular cloning studies have shown that the G protein-coupled chemoattractant receptors are related to G protein-coupled neurotransmitter, hormone, and odorant receptors, and to the

Extracellular Milieu

Figure 3, Model for activation of phagocytes by chemoattractants. An extracellular

ligand, IL-8 for example, binds to a G protein-coupled receptor with seven transmem­

brane helices, causing activation of the coupled G protein. Py subunits activate

membrane phospholipases resulting in lipid remodeling, second messenger produc­

tion, and mobilization of calcium stores from calciosomes. These events presumably

activate other intracellular signalling molecules (black box) leading to the activation

of specialized phagocyte functions. See text for details. C, C-terminus; N, N-terminus;

1-7, proposed transmembrane helices of the chemoattractant receptor; - , negative

charge of the N-terminal segment of the IL-8 receptor; ++, IL-8 is a basic polypeptide;

PIP2, phosphatidylinositol bisphosphate; IP3, inositol-1,4,5-trisphosphate; IP3R, IP3

receptor; DAG, diacylglycerol; PLC, phospholipase C; PLD, phospholipase D; PC,

phosphatidylcholine; PA, phosphatidic acid; PPH, phosphatidate phosphohydrolase;

PKC, protein kinase C.

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214 PHILIP M. MURPHY

opsins that mediate light reception. In some cases, a single receptor can specifically

bind several different chemoattractants, and in other cases one chemoattractant can

bind to several different receptors (Murphy, 1994). While specialized roles have

been identified for certain chemoattractants, functional redundancy is a more

prominent feature of the chemoattractants, perhaps serving to secure the whole

system against inactivating mutations in any single component.

Surprisingly, functional homologues of the IL-8 and the MIP-la/RANTES

chemoattractant receptors have been found in Herpesvirus saimiri, a. transforming

T lymphotropic primate virus, and human cytomegalovirus, respectively. More­

over, the Duffy red cell antigen that mediates invasion of red cells by the malaria

parasite Plasmodium vivax, is also an IL-8 and RANTES binding protein. Thus,

the chemoattractant receptors may have been turned to the host's disadvantage by

these pathogens through molecular mimicry (reviewed in Murphy, 1994).

The activated chemoattractant receptor catalyzes the exchange of GDP for GTP

by the G protein, resulting in G protein activation. Many distinct G protein subtypes

have been characterized. However, chemoattractant receptors probably are coupled

to only a few of these. G^ appears to play a particularly prominent role. Upon

activation, the Py subunit of the G protein dissociates from the a subunit. Py

activates a phosphatidylinositol-specific phospholipase C, probably the P2 isoform,

which catalyzes the hydrolysis of phosphatidylinositol bisphosphate, forming

inositol-1, 4, 5-trisphosphate (IP3) and diacylglycerol (DAG). These molecules

serve as intracellular second messengers: IP3 stimulates the release of stored

calcium from structures known as calciosomes resulting in a rapid elevation of

intracellular Ca " concentration; DAG activates protein kinase C, which may

regulate critical downstream signalling molecules by phosphorylation. A delayed

but more abundant source of DAG is membrane phosphatidylcholine which is

hydrolyzed by a phospholipase D. Early PLC- and late PLD-activation events have

been associated with the migratory and cytotoxic responses of phagocytes, respec­

tively (reviewed in Snyderman and Ewing, 1992).

Phagocytes are incredibly sensitive to chemoattractant gradients: they are capa­

ble of sensing a gradient of less than 1% across the cell diameter (Zigmond, 1978).

Consequently, the cell sends out a thin veil of membrane known as Sipseudopodium

(or lamellipodium), which is highly ruffled. Cytoplasm then streams forward into

this process bringing with it cytoplasmic granules which may fuse with the leading

edge of the plasma membrane, thereby releasing proteolytic enzymes into the

extracellular milieu. The formerly spherical cell thus becomes highly polarized;

granules segregate towards the leading edge, whereas the nucleus is at the trailing

edge or uropod (Snyderman and Goetzl, 1981; Stossel, 1993) (Figure 4). Paul

Ehrlich first suggested that the release of granule contents during diapedesis could

facilitate the movement of the phagocyte into the tissue (Ehrlich, 1900). It is now

known that the released enzymes include elastase and coUagenase, which can

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Phagocytes in Immunity and Inflammation 215

degrade extracellular matrix proteins that form barriers to phagocyte movement (Janoff, 1985; Weiss, 1989; Henson et al , 1992).

As first detailed by Julius Cohnheim in the nineteenth century, transendothelial migration of leukocytes (diapedesis) occurs in the post-capillary venules via specific leukocyte-endothelial cell contacts (Cohnheim, 1889) (Figure 4). Leuko­cytes in the marginated pool first make weak, reversible contacts that mediate rolling of the cell along inflamed endothelium. IL-1, bacterial endotoxin, and tumor necrosis factor are examples of stimuli that can promote the rolling phenomenon by activating endothelial cells. Dramatic advances in the molecular and cell biology of leukocyte adhesion have occurred in the last ten years, pioneered by the work of Springer, Weissman, Lasky, and their colleagues. Rolling is now known to be mediated by adhesive molecules known as selectins that are expressed on the plasma membrane of both leukocytes and activated endothelial cells, and that bind to specific sialylated, fucosylated carbohydrate counter-receptors expressed on the target cell surface (Lasky and Rosen, 1992). At least one of the natural ligands appears to be the sialyl Lewis^ blood group antigen. The selectins form a protein family with N-terminal lectin-like domains, complement domains and epidermal growth factor-like domains. E-selectin is expressed only on endothelial cells and requires de novo synthesis after activation. In contrast, P-selectin, which is also produced by platelets, is pre-synthesized and stored. L-selectin, is produced by leukocytes.

Phaqocyte-Endothelium Interaction Stage

Intravascular Space

Activator Adhesins Disease

Rolling IL-1,TNF,lps Selectins-CHO LAD-2 Cou nter-receptors

Firm Adhesion Chemoattractant lntegrins-ICAi\/ls l-AD-1

Transendothelial Chemoattractant PECAM Migration

Degranulation Chemoattractant none

LAD-1, LAD-2

SGD

Extravascular Space

Figure 4. Model for the movement of phagocytes from the blood to the tissues. Ips, bacteria! lipopolysaccharlde; TNF, tumor necrosis factor; IL, interleukin; CHO, car­bohydrate; ICAM, Intercellular adhesion molecule; PECAM, platelet-endothellal cell adhesion molecule; LAD, leukocyte adhesion deficiency; SGD, specific granule deficiency.

Page 231: Immunity

216 PHILIP M. MURPHY

Selectin-mediated binding may be necessary for diapedesis, but it is not suffi­

cient. A second stimulus directed at the leukocyte appears to be required to stimulate

increased expression and functional activation of a second class of adhesion

molecules known as the leukocyte integrins, a subset of the integrin superfamily of

adhesion molecules (Springer, 1990). The leukocyte integrins are Mac-1, pi50,95

and LFA-1 which have distinct expression patterns on different leukocytes and

mediate a broad array of adhesive functions, such as homotypic (leukocyte to

leukocyte) and heterotypic (leukocyte to endothelium) aggregation, spreading on

surfaces, and binding to opsonized particles. Mac-1 and LFA-1 bind tightly to

endothelial cell counter-receptors known as intercellular adhesion molecules 1 and

2 (ICAM-1 and ICAM-2). The best candidates for the second stimulus for leuko-

cyte-endothelial cell binding by integrins are chemoattractants, such as platelet-ac­

tivating factor. After integrins become activated and bind to the endothelial

counter-receptors, the spherical leukocytes stop rolling, rapidly flatten and begin

to crawl between adjacent endothelial cells to arrive in the subendothelial space.

The three leukocyte integrins are membrane heterodimers that share a common

P subunit (CD 18) that is non-covalently associated with related but distinct a

subunits (CDl la, b, and c) (Kishimoto and Anderson, 1992). Their endothelial cell

counter-receptors, the ICAMs, are members of the immunoglobulin superfamily of

proteins. Two experiments of nature, the human diseases designated leukocyte

adhesion deficiency 1 and 2 (LAD-1 and LAD-2), have highlighted the importance

of the integrins and the selectins, respectively, in the normal function of the immune

system.

Patients with LAD-1 have a mutation in the gene for the shared (3 subunit of

Mac-1, pi50,95 and LFA-1 integrins that is transmitted as an autosomal recessive

trait on chromosome 21. As a consequence, leukocytes from patients with LAD-1

fail to express functional heterodimeric leukocyte integrins (Springer et al., 1984;

Anderson and Springer, 1987). Predictably, LAD-1 leukocytes fail to adhere to

endothelial cells in vitro and in vivo, and therefore have a defect in transendothelial

migration and fail to accumulate at sites of infection and inflammation. Moreover,

phagocytes from patients with LAD-1 do not recognize C3bi-coated particles. The

clinical expression of the disease is recurrent, life-threatening bacterial and fungal

infections in children, often presenting in the neonatal period. Blood from unin­

fected patients with LAD-1 exhibits a sustained and marked leukocytosis, due to

marked reduction in the size of the marginated pool. Leukocyte concentrations in

the blood can elevate even ftarther during infectious episodes. Because of the

inability of leukocytes to cross endothelial barriers, infected sites have delayed or

insufficient inflammation (infection without pus), which often leads to life-threat­

ening delays in diagnosis. There may be a history in patients with LAD-1 of

impaired wound healing, often manifested as delayed separation of the umbilical

cord after birth. In a reciprocal clinical condition, sera from patients with homozy-

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Phagocytes in Immunity and Inflammation 217

gous deficiency of C3 lack C3-dependent opsonic activity. As in LAD-1, C3 deficiency also results in recurrent infections with pyogenic micro-organisms.

Only two patients have been described with LAD-2, both of whom were Arab children living in Israel who presented with severe bacterial infections. There was no clear history of parental consanguinity. Leukocytes from the patients expressed normal numbers of leukocyte integrins but failed to adhere properly to surfaces in

vitro and in vivo. Both patients had deletions on chromosome 19, mental retardation and the rare Bombay blood group phenotype. The molecular defect appears to be in a fucosyl transferase responsible for the construction of the sialyl Lewis^ counter-receptor for the endothelial cell selectins. This enzyme is also required for formation of the Bombay blood group. Although it is theoretically possible to cure the adhesion defect by feeding the patients fucose, the new antigens thus formed may be recognized as foreign by the patients' immune system (Etzioni et al., 1993).

The identification of the molecular basis of leukocyte adhesive phenomena has opened the door to the possibility of developing specific anti-adhesive treatments for acute and chronic inflammatory diseases, and for vascular occlusive phenom­ena, such as myocardial infarction, where phagocytes and their products may participate in pathogenesis.

Phagocytosis/Opsonization

Phagocytosis involves the recognition, binding and internalization of particles (Stossel, 1974; Wright, 1992). While most cells can phagocytize particles to a limited extent, neutrophils and macrophages possess a high capacity for phagocy­tosis because of the expression of specialized receptors. Classical opsonic attach­ment occurs via receptors for antibody and the complement-derived opsonins C3b and C3bi (Ravetch and Kinet, 1991; Wright, 1992). Classical opsonization is particularly important for the phagocytosis of encapsulated bacteria such as Strep­

tococcus pneumoniae. Two other types of attachment of phagocytes to microbes have been delineated: non-opsonic attachment of the integrins CR3, pi 50,95, and LFA-1 to bacterial lipopolysaccharide; and non-classical opsonic attachment via receptors for the plasma-derived opsonins mannose binding protein (MBP) and lipopolysaccharide-binding protein (LBP) (Wright, 1992) (Figure 5). Fewer bind­ing sites are required for attachment of particles to phagocytes than for their ingestion. Complement receptors may be most responsible for binding, whereas FCyRs may be most responsible for internalization (Mantovani, 1975).

The cell biology of phagocytosis can be conceptualized as localized cell motility, involving localized cytoskeletal remodeling. As the particle binds to and deforms the plasma membrane, oriented microfilaments surround the particle, and cyto­plasm streams forward entraining granules that may fuse with the nascent phagosome. Formation of the phagosome requires energy, but can occur under anaerobic conditions. To form the phagosome, the plasma membrane fuses to itself, resulting in an inside-out membrane configuration.

Page 233: Immunity

218 PHILIP M. MURPHY

p150,95 CR3

Non-opsonic Attachment Classical

Opsonization

Non-classical Opsonization

\P\h^^®©^^(B

Figure 5. Molecular apparatus for microbial attachment to phagocytes. Hair-like projections from the microbe represent bacterial llpopolysaccharlde. Ag, antigen; C3b, C3bi are complement-derived opsonins; LBP, lipopolysaccharide-binding pro­tein; MBP, mannose binding protein; Man R, mannose receptor; CR, complement receptor; FcR, immunoglobulin Fc receptor (multiple subtypes for IgG and IgE); LFA, leukocyte function associated. Note that CR3 can bind directly to llpopolysaccharlde or to C3bi.

Three antigenically distinct Fcy receptors have been characterized on phagocytes. FCYRI is a high affinity receptor (Kd~10-^M) for IgG v^hereas FCyRll and FCYRIII

are low affinity receptors for IgG (K^-KT^M). Fc^Rs I and II are expressed on macrophages, whereas Fc^Rs II and III are expressed on neutrophils. Unexpected complexity has been identified in this system by molecular cloning of multiple distinct genes encoding additional Fc^R subtypes (Ravetch and Kinet, 1991). C3b binds to complement receptor type I (CRl), whereas C3bi binds to complement receptor type 3 (CR3). CR3 is identical to the Mac-1 integrin which as we have already seen is able to serve an alternative adhesive function, binding to ICAM-1 during transendothelial migration (Kishimoto and Anderson, 1992). Thus CR3 mediates both diapedesis and phagocytosis, illustrating at the molecular level the relationship between cell migration and phagocytosis. Adhesive and phagocytic ligands appear to bind to distinct sites on CR3.

The phagocytic receptors explain the ability of phagocytes to target micro­organisms, but less well their ability to distinguish normal from senescent, damaged and neoplastic host cells. Targetting may occur by additional adhesion systems that

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Phagocytes in Immunity and Inflammation 219

involve the recognition of new carbohydrate structures or oxidized proteins on the surface of abnormal host cells by molecules such as the macrophage scavenger receptors or the mannose receptor (Stahl, 1990). Neutrophils are more phagocytic than monocytes, although the converse is true when the particles or cells are particularly large or numerous.

Degranulation

One of the features that distinguishes phagocytes from lymphocytes is the abundance of granules packed into the cytoplasm. The granules contain the micro­bicidal weaponry of the cell that acts in the confines of the phagolysosome, but that also has the potential to damage macromolecules present on adjacent host cells (Gallin, 1984; Henson et al, 1992). In 1900, Paul Ehrlich proposed that neutrophil granules may contribute to inflammation by secreting their contents into the extracellular space. This hypothesis was examined by the work of Weissmann and his coworkers at New York University 70 years later, and has been extensively supported from the biochemical characterization of granule components (Table 4). To convey the destructive potential of their granules, Weissmann and others have referred to phagocytes as "secretory organs of inflammation" (Weissmann et al., 1973). Today the products of phagocyte granules have been implicated in the pathology of gout, rheumatoid arthritis, vasculitis, inflammatory bowel disease, a wide variety of dermatopathic disorders, periodontitis, myocardial infarction, asthma, emphysema, and the adult respiratory distress syndrome, among other human diseases (Malech and Gallin, 1987) (Table 1). In addition, granule proteins catalyze the production of anti-microbial oxidants that, in vitro, have been impli-

Table 4. Microbicidal and Degradative Proteins Contained in Neutrophil and Eosinophil Granules^

Granule

Neutrophil

Specific

Azurophilic

Tertiary

Eosinophil

Specific

Microbicidal Proteins

Oxidant Production

flavocytochrome 6553 (O2)

apolactoferrin (OH-)

; myeloperoxidase (HOC1)

eo-peroxidase (OH-)

flavocytochrome bsss (O2)

Cationic Proteins

defensins

bactericidal/permeability-

increasing protein (BPI)

eo-derived neurotoxin

eo-cationic protein major

basic protein

_ Degradative Enzymes

lysozyme

collagenase

acid hydrolases

lysozyme

elastase

neutral proteases

gelatinase

acid hydrolases

Note: ^ Specific granules also contain chemoattractant receptors and integrins that are brought to the plasma membrane upon cell activation.

Page 235: Immunity

220 PHILIP M. MURPHY

cated in DNA damage and cellular transformation (Weitzman et al., 1985). This could explain the high incidence of carcinoma associated with chronic inflamma­tion in vivo, as occurs in patients with chronic hepatitis B, ulcerative colitis, and scars from old tuberculous lung lesions.

In addition to modifying the inflammatory site, degranulation adds new mem­brane and fresh supplies of chemoattractant receptors and phagocytic/adhesion receptors from storage pools in the granule to the leading or sensing edge of the cell, thereby recharging the sensing and effector pathways for phagocyte migration (Wright and Gallin, 1978; Henson et al., 1992). Exocytosis of granule contents may also be important for killing of helminths by eosinophils (Weller, 1991) (Figure 2). Finally, detonation of basophils during allergic reactions depends on rapid and massive fusion of granule membrane to plasma membrane (Metcalfe et al., 1992).

Granule fusion appears to be regulated in part by calcium, calcium-binding proteins such as the synexins, GTP, low molecular weight GTP-binding proteins, and cytoskeletal remodeling by proteins such as gelsolin. Shortening of actin filaments by gelsolin may enhance the cytoplasmic motility of granules, permitting them to approach and fuse with other membrane-delimited compartments (Henson etal., 1992;Stossel, 1993).

Subcellular fractionation techniques pioneered by deDuve, Palade and their collaborators at Rockefeller University in the 1950s and 1960s were applied to phagocytes, and led to rapid advances in understanding of the structure and biochemical composition of phagocytic cell granules. It is now known that neutro­phils possess at least three types of granules (Baggiolini et al., 1969; Henson et al., 1992). The primary, or azurophil, granules appear during the promyelocyte stage of neutrophil maturation before the other granules are detectable. Azurophil gran­ules are lysosomes that contain a variety of degradative enzymes such as neutral proteases, acid hydrolases and several highly cationic antimicrobial proteins such as the defensins and bactericidal/permeability-increasing protein. Myeloperoxidase is the most abundant primary granule enzyme, and is responsible for the green color of pus. Myeloperoxidase catalyzes the generation of hypochlorous acid, a potent antimicrobial oxidant, from hydrogen peroxide and chloride after activation of the respiratory burst oxidase (Klebanoff, 1992).

A distinct and more abundant granule appears later in neutrophil development and contains a set of proteins that in most cases are unique to neutrophils. It has therefore been named the secondary or neutrophil-specific granule (Gallin, 1984). Specific granules contain apolactoferrin, collagenase, histaminase, chemotactic and adhesion receptors, and the flavocytochrome component of the NADPH oxidase (see below). Both primary and secondary granules contain lysozyme which breaks down bacterial cell wall proteoglycan. A third granule type, designated the tertiary granule, contains gelatinase.

Membrane fusion events are under separate regulatory control for primary, secondary and tertiary granules. Thus, release of tertiary granule contents occurs

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Phagocytes in Immunity and Inflammation 221

with minor perturbation of the cell membrane. In vitro and in vivo studies suggest that primary granules may preferentially fuse with phagosomes and play an intracellular, lysosomal, degradative role, whereas the contents of specific granules are more readily released into the extracellular milieu (Wright and Gallin, 1978; Henson et al., 1992). Specific granule products may amplify the inflammatory reaction by inducing the production of monocyte chemoattractants, thereby setting up a second, monocyte-dependent wave of inflammation (Gallin, 1984). In addi­tion, specific granule proteases may degrade extracellular matrix proteins to facili­tate movement of the cell through the tissues. While many details regarding the structure, ftmction and regulation of neutrophil granules remain unknown, their importance in homeostasis is most clearly established by the congenital disorder known as Neutrophil-Specific Granule Deficiency (SGD).

SGD is a rare disorder of uncertain inheritance that is associated with recurrent bacterial infections. Leukocytes from affected patients are severely deficient in chemotaxis, adherence, and microbicidal functions when tested in vitro. Specific granule morphology is abnormal and biochemical markers of specific granules, such as lactoferrin, are absent or severely deficient. Defects in the production of azurophil and gelatinase-containing granule proteins, as well as in the production of eosinophil-specific granule proteins have also been reported in a patient with SGD (Rosenberg and Gallin, 1993). These findings plus the observation that lactoferrin is present in normal amounts in nasal secretions from patients with SGD, suggest that the underlying defect is in a phagocyte-specific regulatory factor controlling biosynthesis of the deficient proteins (Gallin et al., 1982; Gallin, 1985; Lomaxetal., 1989).

The contents of eosinophil-specific granules are quite idiosyncratic (Weller, 1991). Lysosomal hydrolases and an NADPH oxidase are present in this granule, whereas in neutrophils they are separated into primary and secondary granules, respectively (Table 4). Like neutrophils, eosinophils also contain a peroxidase and highly basic anti-microbial proteins, although they are distinct from the neutrophil examples. The dense core of the eosinophil-specific granule is composed of crystals of major basic protein (MBP). The core is surrounded by a matrix composed of eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), eosino­phil peroxidase (EPO), and several other lysosomal enzymes. The most potent antihelminthic products of eosinophils are MBP, ECP and EPO. MBP and ECP may amplify inflammatory responses by inducing histamine release from basophils and mast cells. Basophil granules are distinct from both eosinophils and neutro­phils. They contain histamine and sulfated proteoglycans of uncertain function (Metcalfe et al., 1992).

Production of Antimicrobial Oxidants

When phagocytes engulf particles, they dramatically increase their consumption of oxygen, a phenomenon known as the respiratory burst. This process leads to the

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222 PHILIP M. MURPHY

accumulation in the phagolysosome of a series of oxidants (e.g., superoxide anion, hydrogen peroxide, hypochlorous acid, hydroxyl radical) that chemically attack macromolecules of the ingested microbe (Babior, 1978) (Figure 6). Oxidant pro­duction also causes an increase in phagolysosomal pH to levels at which lysosomal enzymes are active, leading to degradation of the ingested microbe (Segal and Abo, 1993).

Specific Granule

NADP

NADPH Oxidase Assembly

^f^f^

NADPH

t fHexose Monophos-'l I Dhate Shunt J

Azurophil Granule

Figure 6. Oxidative killing mechanisms of phagocytes. Upon formation of the

phagosome, a latent NADPH oxidase is activated by assembly of the indicated

cytosolic and membrane components, and superoxide anion is produced, leading to

the generation of a series of antimicrobial oxidants. The phagosome membrane is

derived from the plasma membrane. Lactoferrin (LF) and myeloperoxidase (MPO) are

delivered to the phagosome by fusion with specific and azurophil granules, respec­

tively. The phagosomal membrane flavocytochrome component of the oxidase (gp91,

p22) Is derived from both the plasma membrane and the specific granule membrane.

See text for details and Table 4 for non-oxidatlve killing pathways. SOD, superoxide

dismutase.

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Phagocytes in Immunity and Inflammation 223

The importance of oxygen and its toxic metabolites in the efficient killing of micro-organisms by phagocytes is most clearly revealed by Chronic Granuloma­tous Disease (CGD), a rare inherited human disorder of the phagocyte respiratory burst that is associated with recurrent life-threatening infections and high mortality rates in childhood. The phagocytes of patients with CGD ingest organisms normally but lack the ability to produce oxidants, and thus fail to efficiently kill certain bacteria and fiingi. Since micro-organisms themselves can produce hydrogen peroxide, phagocytes from patients with CGD can take advantage of this source to kill the organism by generating more toxic oxidants such as hypochlorous acid. Organisms that possess the enzyme catalase, however, metabolize H2O2 to O2 and H2O, thereby depriving the phagocyte of the H2O2 that they themselves produce. Thus, patients with CGD typically become infected with catalase positive organ­isms such as Staphylococcus aureus. The hallmark pathologic lesion of CGD, the granuloma, resembles granulomata formed during infection of macrophages by intracellular pathogens such as mycobacteria, which possess mechanisms for eluding the normal antimicrobial weaponry of the cell. Granulomatous inflamma­tion persists even after the infectious process has resolved, suggesting the impor­tance of oxidants in the resolution of the inflammatory response.

The molecular details of oxidant production are now partly understood from combined genetic and biochemical studies of CGD (Rotrosen, 1992; Rotrosen, 1993). Upon cell activation by a chemoattractant or by phagocytosis, a multisubunit NADPH oxidase is assembled from three distinct cytosolic regulatory proteins, known as p47P' ° , p67P^ ^ and the low molecular weight GTP-binding protein rac-1, and a heterodimeric flavocytochrome located in the membrane of specific granules and plasma membranes. The flavocytochrome comprises the catalytic moiety of the oxidase enzyme. The fully assembled oxidase is an intramolecular electron transporter that transfers electrons from NADPH to flavin adenine dinucleotide (FAD), then to heme, and finally to molecular oxygen, thereby producing super­oxide anion. NADPH is formed during the respiratory burst by Ci glucose meta­bolism via the hexose monophosphate shunt. Superoxide anion is rapidly converted by superoxide dismutase to hydrogen peroxide, a potent anti-microbial oxidant. Myeloperoxidase, which can be delivered to the phagosome by fusion with azuro­phil granules, then catalyzes the formation of HOCl from hydrogen peroxide and chloride. The microbicidal potency of HOCl (most commonly known as the active antiseptic ingredient in household bleach) is - 50 times greater than that of hydrogen peroxide. Lactoferrin, delivered to the phagosome from specific granules, may be the most important iron source for the Fenton reaction in which hydroxyl radical is formed from hydrogen peroxide in the presence of reducing agents such as superoxide (Ambruso and Johnston, 1981).

The most common genetic mutations (--65%) found in patients with CGD occur in the gene for the large subunit of the flavocytochrome (gp9 P^ ' ) which is located on the X chromosome. Thus inheritance of CGD in these families displays a classic

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224 PHILIP M. MURPHY

X-linked Mendelian pattern, affecting -50% of males. The next most common mutations (--30%) are in the gene for p47P' ^ . These families display autosomal recessive inheritance. Mutations in the autosomal genes for p67P^ ^ and the small subunit of the flavocytochrome are extremely rare accounting for only a few % of all cases of CGD (Clark et al., 1989). No mutations have thus far been reported for rac-1. All four disease-related genes have been cloned, paving the way for the development of gene therapy.

While its importance in human phagocytes is not yet clear, the ability of murine macrophages to produce nitric oxide by the action of the enzyme nitric oxide synthase on L-arginine is a second oxidant-generating system of phagocytes that may be important for killing certain intracellular parasites, viruses, and tumor cells by macrophages (Liew and Cox, 1991).

Although the phagocyte needs to produce oxidants to kill microbes, it also needs to control the level of oxidant production so as not to cause unnecessary damage to host structures. Oxidants can damage extracellular matrix proteins directly or by activating latent proteases such as collagenase, elastase and gelatinase (Weiss, 1989). The NADPH oxidase appears to be a highly regulated enzyme, lacking constitutive activity, and turned off with time after activation. Moreover, the phagocyte possesses several important mechanisms for reducing oxidant concen­trations. These include catalase, superoxide dismutase, iron-free transferrin, and the glutathione redox system. NADPH, in addition to being the source of electrons for the production of toxic oxygen species, is also the reducing agent for production of reduced glutathione which in turn can detoxify hydrogen peroxide.

Production of Pro-Inflammatory Mediators

Phagocytes, especially macrophages, are important sources of cytokines, chemoattractants, and vasoactive mediators (Nathan, 1987). These substances have profound effects on humoral, cellular and vascular components of the inflammatory response. Local concentrations of mediators can be regulated at multiple levels, including gene transcription, latent enzyme activation and release of presynthesized products. IL-1, IL-6, and tumor necrosis factor are just three of the best charac­terized examples of multi-functional cytokines that participate in the pathogenesis of many aspects of inflammation from fever to septic shock.

Activation of phagocytes also induces the synthesis and release of a variety of 20-carbon fatty acid derivatives of arachidonic acid, that upon cell activation is itself cleaved from membrane phospholipids by the action of phospholipase A2 (Samuelsson et al., 1987). Arachidonic acid is a substrate for the aspirin-inhibitable enzyme cyclo-oxygenase which catalyzes the production of cyclic endoperoxides. These can be further metabolized to produce either a series of prostaglandins or thromboxanes. Prostaglandin E2 is a particularly potent vasodilator that contributes to the edema and erythema present in inflammatory sites. Thromboxane B2, in contrast, is vasoinactive, but possesses chemotactic activity for phagocytes.

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Alternatively, arachidonic acid can be oxygenated by the enzyme 5-lipoxygenase

to begin the metabolic pathway for the production of leukotrienes and lipoxins.

Leukotriene B4 is the principal chemotactic leukotriene produced by activated

neutrophils. Eosinophils produce primarily the sulfidopeptide leukotrienes C4, D4

and E4 from LTA4 and glutathione (Weller et al, 1983; Samuelsson et al., 1987;

Weller, 1991). In contrast to LTB4, these substances are not leukotactic but instead

are myotropic: they cause bronchoconstriction, increased mucus production and

increased vascular permeability of post-capillary venules during late phase allergic

reactions. For this reason they are also known collectively as the slow reacting

substance of anaphylaxis. The lipid known as platelet-activating factor (PAF) is

produced by activated phagocytes by replacing the 2-acyl group in 1-alkyl-2-acyl-

glycerol phosphocholine, a membrane phospholipid, with acetate. PAF is a potent

platelet secretagogue, a spasmogen for smooth muscle cells, and a chemoattractant

and activating factor for phagocytes. Thus, activation of phagocytes leads to the

production of peptide and lipid autocoids which amplify the signal that initiated the

inflammatory reaction.

Immunoregulation

Macrophages are unique among phagocytes in their ability to support a specific

immune response by presenting complex protein antigens to T lymphocytes

(Unanue and Allen, 1987). They do this by first engulfing antigens such as immune

complexes or bacteria, and degrading them in the phagolysosome. Small antigenic

fragments ~9 amino acids in length known as epitopes, then bind to a specific

groove of class II major histocompatibility (MHC) molecules (Figure 2). The

MHC-peptide complex is then transported to the cell surface where T lymphocytes

bearing specific T cell receptors bind to the peptide and become activated. A

detailed account of macrophages as antigen-presenting cells can be found in the

earlier chapter of this volume.

SUMMARY

Phagocytes represent the last vestige in higher species of the ancient cellular function for which they are named. Human genetic diseases indicate that phagocy­tosis and oxidant production are critical to the defense of the host against pathogenic bacteria and fungi, which undoubtedly served to select and maintain these functions over evolutionary time. Phagocytes may also be deleterious to the host, contributing to acute and chronic inflammation. Many chronic inflammatory conditions occur in adulthood after reproduction has occurred, and therefore do not impose selective pressure upon the germ line. Moreover, mechanisms for regulating phagocyte functions would by necessity evolve more slowly than the micro-organisms they are designed to control. Thus, inflammation may be an intrinsic property of a system

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226 PHILIP M. MURPHY

that only crudely responds over evolutionary time to hypermutable microbial targets, while awaiting establishment of a finely tuned, specific immune response.

ACKNOWLEDGMENTS

I thank Sunil Ahuja, John Gallin, Helene Rosenberg, Fei Li, and Robert Sokolic for helpful comments about the manuscript.

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Chapter 12

Anaphylaxis

CALMAN PRUSSIN and MICHAEL KALINER

Introduction 232

Classification of Causes of Anapliylaxis 233

IgE Mediated Anaphylaxis 233 Complement Mediated Anaphylaxis 233

Non-immunologically Mediated Mast Cell Reactions 234 Clinical Findings 235

Pathogenesis 235 Differential Diagnosis 236 Treatment 237

Summary 237 Recommended Readings 238

Principles of Medical Biology, Volume 6 Immunobiology, pages 231-238. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved.

ISBN: 1-55938-811-0

231

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INTRODUCTION

Anaphylaxis represents the most impressive and potentially life threatening mani­festation of immediate (IgE mediated) hypersensitivity. This syndrome can affect almost any organ system, although reactions involving the pulmonary, circulatory, cutaneous and gastrointestinal systems are most common. Reactions can vary from mild urticaria (hives) to anaphylactic shock and death.

Portier and Richet used the term anaphylaxis to describe the fatal reaction induced by the introduction of minute amounts of antigen into dogs that had been previously sensitized to that antigen. Attempting to induce protective immunity to sea anenome toxin by prior injection, they unexpectedly observed that some of the dogs had an immediate fatal reaction when reimmunized. The dramatic and unexpected fatal response was the opposite (ana=Greek: backwards) of the expected protection (phylax=Greek: guard). Richet was subsequently awarded the Nobel prize in medicine and physiology in 1913 for his work in the field.

Anaphylaxis is the clinical syndrome elicited in a hypersensitive individual on subsequent exposure to the sensitizing antigen. The necessary components of the anaphylactic response are: (1) a sensitizing antigen, usually administered parenter-ally, (2) an IgE class antibody response resulting in systemic sensitization of mast cells and basophils, (3) reintroduction of the sensitizing antigen, usually systemi-cally, (4) mast cell degranulation with mediator release, generation, or both, and (5) production of several pathologic responses by the mast cell derived mediators manifested as anaphylaxis.

Mast cell activation can be induced by a number of disparate stimuli. In classic IgE mediated anaphylaxis, activation is induced via cross linking of mast cell high affinity IgE receptors (FcSRI) by antigen. Upon activation, a number of both preformed and newly synthesized mediators with potent biological activity are released into the surrounding tissue.

Mast cells are rich in granules containing a large number of preformed mediators, in particular histamine. Upon activation, the contents of these granules are exocy-tosed into the surrounding tissue with subsequent release of mediators. Activation causes the de novo synthesis and release of a number of active lipid mediators derived from arachidonic acid, including prostaglandin D2, Leukotriene C4 and platelet activating factor. These lipid mediators are all synthesized after activation. Recent reports demonstrate that mast cells are sources of both preformed and newly synthesized cytokines, such as tissue necrosis factor-a and interleukin-4. The significance of these mediators in anaphylaxis remains to be determined.

Because the mediators that are released or generated by mast cells cause anaphy­laxis, any event associated with mast cell activation may produce the same clinical picture. Anaphylaxis usually refers to IgE mediated, antigen stimulated mast cell activation. Whereas anaphylactoid reactions denote other non-IgE mediated re­sponses where immunologic recognition of antigen does not occur; for example.

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those produced by chemical agents capable of directly causing mast cell degranu-lation (e.g., radiocontrast agents, opiates).

CLASSIFICATION OF CAUSES OF ANAPHYLAXIS

IgE Mediated Anaphylaxis

IgE mediated anaphylaxis has been implicated in untoward reactions elicited by drugs, insect stings, chemicals, foods, preservatives, and environmental factors (Table 1). An agent capable of causing IgE antibody formation must be either a whole antigen or a hapten. For example, horse serum, formerly used as a source of antitoxin, contains many antigenic proteins. Subsequent reexposure to horse serum predictably caused anaphylaxis. Horse antisera are now rarely used therapeutically, an exception being antivenom therapy for snakebite. More common sources of antigens include allergenic extracts, insulin, Hymenoptera venom (e.g., bee sting), foods, latex rubber, seminal plasma, and chymopapain.

Haptens are molecules that are too small to elicit an immune response by themselves; however, haptens may bind to endogenous serum proteins, such as albumin, and become antigenic. The most important haptens are penicillins and related antibiotics. Most instances of IgE mediated anaphylaxis are due to the administration of penicillin drugs.

Complement Mediated Anaphylaxis

Anaphylactic reactions have been observed after the administration of whole blood or its products, including serum, plasma, fractionated serum and immuno-

Table h Causes of Anaphylaxis/Anaphylactoid Reactions

IgE Mediated Reactions Antibiotics and other drugs Foreign Proteins (horse serum, chymopapain) Foods Allergen Immunotherapy Hymenoptera Stings

Latex Rubber (gloves, medical supplies) Seminal Plasma

Complement Mediated Reactions Blood, blood products

Nonimmunologic Mast Cell Activators Radiocontrast Media Opiates

Modulators of Arachidonic Acid Metabolism

Nonsteroidal anti-inflammatory agents Exercise Induced Anaphylaxis Idiopathic Anaphylaxis Catamenial Anaphylaxis

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234 CALMAN PRUSSIN and MICHAEL KALINER

globulins. One of the mechanisms responsible for these reactions is the formation of immune complexes, resulting in the activation of the complement cascade. Many active species are generated by complement; the anaphylotoxins C3a, C4a, and C5a are capable of causing mast cell activation and consequent systemic reactions. In addition, anaphylotoxins may directly induce vascular permeability and contract smooth muscles. This complement mediated anaphylaxis is thus immunologically mediated, though not through IgE.

Typical reactions are seen in blood transfusions in which the recipient has developed antibodies (IgG or IgM) to a component of the donor's blood, usually either cell surface antigens or serum proteins. An example of this is seen in ABO blood incompatibility reactions, in which the recipient has antibodies (typically IgG and IgM) to the donor ABO red cell surface antigens.

Non-immunologically Mediated Mast Cell Reactions

Direct mast cell activators can cause a syndrome clinically indistinguishable from anaphylaxis through release of mast cell granules. To differentiate these reactions from immunologically mediated (IgE, IgG/complement) reactions, they are com­monly referred to as anaphylactoid reactions. Although the steps leading up to mast cell activation differ from classic anaphylaxis, release of mast cell mediators yields a syndrome that is clinically indistinguishable. Contrary to the pathogenesis of IgE mediated anaphylaxis, no priming is required, since the reaction is due to direct mast cell activation.

The clinically most important agents include iodinated radiocontrast media, narcotics and neuromuscular blocking agents. Mild reactions are seen in approxi­mately 5% of individuals receiving radiocontrast media. Severe systemic reactions occur in 1 in 1000 exposures. The pathogenesis of radiocontrast reactions is still uncertain. Contrast media is hypertonic and may cause direct mast cell degranula-tion via this mechanism; in addition, activation of the complement and coagulation systems with subsequent mediator release may play a role. Newer low osmolality non-ionic radiocontrast media have a significantly lower rate of reactions.

Approximately 5-10% of asthmatic individuals react to nonsteroidal antiinflam­matory drugs (NSAID), such as aspirin and indomethacin, with rhinorrhea, bron-chospasm and rarely, vascular collapse. Typically these patients have the "aspirin triad"—^aspirin sensitivity, chronic sinusitis with nasal polyps and asthma. The exact mechanism underlying NSAID sensitivity is not yet known. The cause of this disorder appears at least in part to be due to a modulation of arachidonic acid metabolism by shifting from the cyclo-oxygenase to the lipoxygenase enzyme pathways. The consequences of this are: (1) a reduction in the production of prostaglandins and thromboxanes, and (2) enhanced production of leukotrienes and HETEs (hydroxy-eicosatetraenoic acids).

These patients react to all NSAID's despite the diversities of their chemical structures. This is taken as ftirther evidence that the syndrome is not antibody

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Anaphylaxis 235

mediated via immunologic recognition of the drug molecule, but rather through their common pharmacologic effects.

Exercise induced anaphylaxis is a disorder in which individuals experience anaphylaxis with exercise. The pathogenesis of this unusual disorder is not clear. The syndrome is sometimes linked to ingestion of particular foods.

A syndrome termed idiopathic anaphylaxis has been described in a group of patients who experience anaphylaxis due to no recognized cause. These patients present with classical anaphylaxis, yet despite extensive workup, no cause is found. The diagnosis is based on clinical signs and symptoms of anaphylaxis and the exclusion of known causes of anaphylaxis. The diagnosis of anaphylaxis can be confirmed in difficult diagnostic situations by performing a plasma tryptase deter­mination (see differential diagnosis, below).

A rare cause of repeated episodes of anaphylaxis without a readily identifiable etiology is catamenial anaphylaxis, a syndrome of hypersensitivity to endogenous progesterone secretion. Some, but not all, patients with catamenial anaphylaxis exhibit a cyclic pattern of attacks that intensifies during the luteal phase of the menstrual cycle. These patients have positive skin tests to medroxyprogestrone, experience systemic reactions to infusions of lutenizing hormone releasing hor­mone (LHRH), and respond favorably to ovarian suppression with LHRH agonists or oophorectomy.

Clinical Findings

The primary organ systems involved in anaphylaxis are the cutaneous, gastroin­testinal, respiratory and cardiovascular systems. Characteristically, patients de­scribe an immediate sense of impending doom coincident with flushing, tachycardia and often pruritus. If left untreated, the initial signs and symptoms may rapidly evolve to urticaria, angioedema, rhinorrhea, bronchorrhea, asthma, laryn­geal edema, abdominal cramps, cardiac arrhythmias, faintness, syncope, shock, and death. The organ systems involved in these responses contain the largest numbers of mast cells and thus are the most affected by mast cell activation.

Urticaria is due to mast cell degranulation in the epidermis, with a consequent increase in vascular permeability and localized edema, causing the characteristic raised skin or wheal. Histamine is the mediator responsible for the associated pruritus. Angioedema has a similar pathogenesis; however, the location of mast cell degranulation is in the dermis or subcutaneous tissue. As such, the swelling is not as discrete and there is less pruritus, due to a relative lack of sensory nerve endings in this anatomic location.

Pathogenesis

When mast cells degranulate, preformed and rapidly generated mediators are released into the connective tissue. Although many of these mediators induce

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236 CALMAN PRUSSIN and MICHAEL KALINER

Table 2, Processes Invo lved in the Symptoms of Anaphy lax is

Pathologic Process Sign or Symptom Putative Mediator

Vascular permeability

Vasodilation

Urticaria, angioedema,

laryngeal edema

Flushing, headache

Smooth muscle contraction Wheezing (asthma),

gastrointestinal cramps

diarrhea

Tachycardia Palpitations

Reduced peripheral vascular Faintness, syncope,

resistance hypotension

Histamine (Hi), leukotrienes,

prostaglandins

Histamine (Hi and H2),

leukotrienes, prostaglandins

Histamine (Hi), leukotrienes,

prostaglandins

Histamine (Hi) and

leukotrienes

Histamine (Hi and H2)

dramatic effects, few other than histamine are capable of entering the circulation in an active state. As summarized in Table 2, most of the changes occurring in anaphylaxis can be attributed to histamine (acting through Hi and H2 receptors), prostaglandins and leukotrienes.

The consequences of mast cell mediator release include increased vascular permeability, contraction of smooth muscle and vasodilatation. Increased vascular permeability is due to the formation of intercellular gaps between endothelial cells in postcapillary venules. The increased vascular permeability causes tissue edema leading to urticaria (if the reaction is limited to the epidermis); angioedema (if the reaction is in the dermis or subcutaneous tissue); laryngeal edema; nasal congestion; and gastrointestinal swelling, with abdominal bloating and cramps. Contraction of smooth muscle leads to bronchospasm and asthma, as well as abdominal cramping and diarrhea. Vasodilation leads to flushing, reduced peripheral vascular resistance, hypotension and syncope. Cardiac histamine receptor stimulation leads to tachy­cardia and possible arrhythmias.

Differential Diagnosis

The diagnosis of anaphylaxis is usually based upon history and presenting signs compatible with a diagnosis of anaphylaxis. Patients typically note the onset within minutes following exposure to a provocative stimulus. Anaphylaxis is easily confused with a vasovagal reaction; distinction of the two is critically important as the prognosis and therapy of the two are quite different. Patients with vasovagal reactions typically present with pallor, diaphoresis and bradycardia; in contrast, flushing, urticaria, angioedema, pruritus, wheezing and tachycardia are typical of anaphylaxis.

If laryngeal edema is the presenting problem, hereditary angioedema must be considered. Hereditary angioedema is not associated with flushing, asthma or urticaria. Systemic mastocytosis is characterized by a generalized overabundance

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Anaphylaxis 237

of mast cells. These mast cells may degranulate, producing local effects or rarely causing systemic effects identical to anaphylaxis. Suspicion of the diagnosis should be raised with the recognition of the classic reddish-brown maculopapular lesions that urticate on trauma (Darier's sign), the history of flushing attacks, evidence of bone involvement, gastrointestinal pain and peptic ulcers. Increased urinary hista­mine and PGD2 metabolites are also seen. Bone marrow biopsy is usually diagnos­tic.

In difficult diagnostic situations (e.g. critically ill or surgical patients), many of the signs of anaphylaxis may be obscured. In situations where the diagnosis of anaphylaxis itself is in doubt, measurement of plasma tryptase may confirm the diagnosis. Tryptase is an enzyme found in mast cell granules and is released into the circulation following systemic mast cell degranulation. Whereas the circulating half life of histamine is on the order of minutes, tryptase remains in the circulation long enough to be measured 3 to 4 hours later.

Treatment

Anaphylaxis is an acute medical emergency requiring immediate attention. If possible, the source of antigen should be removed. Subcutaneous epinephrine is the mainstay of therapy. This drug maintains the blood pressure, antagonizes many of the mediators of anaphylaxis and reduces the subsequent release of mediators through its action on mast cells and basophils. Often both Hi and H2 antihistamines are given. Blood pressure should be maintained with fluid and pressors as needed. Corticosteroids have no immediate effect but should be administered to prevent prolonged or recurrent anaphylaxis.

Treatment of anaphylaxis has been complicated by the increased use of beta-adrenergic blocking agents in many patients. In the presence of beta-blockers, anaphylactic reactions may be more severe, prolonged and refractory to treatment.

SUMMARY

Anaphylaxis is a major medical emergency, the pathogenesis of which is remark­ably well understood. The signs and symptoms of anaphylaxis are due to mast cell activation with subsequent release of potent mediator substances. Activation is typically brought about by antigen binding to IgE on the surface of the mast cell. Anaphylactoid reactions differ in being caused by direct mast cell activators, which are not recognized by IgE. Histamine, prostaglandins and leukotrienes are the most significant mediators in the pathogenesis of anaphylaxis.

Anaphylaxis is usually associated with an exposure to a causative substance such as medications, foreign proteins, foods, insect stings and blood products. Diagnosis is based largely on clinical findings, which include flushing, tachycardia, urticaria, angioedema, asthma, laryngeal edema, cardiac arrhythmias, shock and death. Treatment requires prompt diagnosis and institution of appropriate therapy.

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238 CALMAN PRUSSIN and MICHAEL KALINER

RECOMMENDED READINGS

Atkinson, T.P., & Kaliner, M.A. (1992). Anaphylaxis. Med. Clin. North Amer. 76, 841-855. Bochner, B.S., & Lichtenstein, L.M. (1991). Anaphylaxis. New Engl. J. Med. 324, 1785-1790. Marquardt, D.L., & Wasserman, S.I. (1993). Anaphylaxis. In: Allergy. Principles and Practice. (Mid-

dleton, E., Ed.), pp. 1525-1536. Mosby, St Louis, MO.

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Chapter 13

Autoimmunity and Autoimmune Disease

SUDERSHAN K. BHATIA and NOEL R. ROSE

Introduction 240

Autoimmunity 240

Self and Non-Self 240

Mechanisms of Positive Selection 241

Negative Selection and Self-Tolerance 242 Autoimmune Disease 244

Autoimmune Thyroid Disease 245 Incidence 245 Histopathological Changes 245 Autoantigens 246 Animal Models 247 Multifactorial Etiology 247

Thyroglobulin 254 Summary 255

Principles of Medical Biology, Volume 6 Immunobiology, pages 239-263. Copyright © 1996 by JAI Press Inc.

All rights of reproduction in any form reserved.

ISBN: 1-55938-811-0

239

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240 SUDERSHAN K. BHATIA and NOEL R. ROSE

INTRODUCTION

A cardinal property of the immune system is its ability to distinguish self from non-self The fundamental mechanisms involved in self-recognition are gradually coming to light. Clearly, more than one mechanism is responsible for this biologi­cally important phenomenon. Yet, there are many instances where self/non-self discrimination breaks down and autoimmune disease results. In fact, it has been the study of these exceptions to the rule that has taught us most about the essential mechanisms required to maintain normal unresponsiveness to self

In this chapter, we discuss the mechanisms known to underlie self/non-self discrimination. Then we describe in detail one of the best characterized examples of an autoimmune disease, autoimmune thyroid disease.

AUTOIMMUNITY

A basic function of the immune system is to discriminate between self and non-self. Immunological unresponsiveness to self-antigens is termed self-tolerance. The importance of tolerance to self-antigens was first recognized at the advent of this century by Paul Ehrlich (see Himmelweit, 1901). "Horror autotoxicus" was the term that he used to imply that an immune response to self-antigens would be harmful. A major breakthrough in our current understanding of autoimmunity occurred in 1956, with the discovery of the induction of autoimmune thyroiditis in rabbits by immunization with rabbit thyroid extracts (Rose and Witebsky, 1956). At the same time, autoantibodies to thyroglobulin (Tg) were found in the serum of patients with Hashimoto's thyroiditis (Roitt et al., 1956; Witebsky et al., 1957).

These events marked the beginning of our recognition of autoimmune disease as a major cause of human disease. In the latter (Witebsky et al., 1957), criteria, known as Witebsky's postulates, for the recognition of human autoimmune diseases were formulated. Based on these criteria, an autoimmune disease is characterized by: (i) identification of circulating or cell-bound antibodies in patients with the disease, (ii) demonstration of the specific antigen against which the immune response is directed, (iii) induction of antibody against this antigen in experimental animals, and (iv) development of pathological changes similar to the disease in question in the appropriate tissues of the experimental animal. As the recognition of self is an integral component of the immune response, the changing concepts of autoimmune disease over the years have enhanced our understanding of the immune response itself

SELF AND NON-SELF

The mechanisms by which self/non-self distinction is achieved are now beginning to be understood. The cells of the immune system (the B cells and the T cells) are rendered tolerant to molecules of the host organism by three processes: one that

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Autoimmunity and Autoimmune Disease 241

results in elimination of the cells that would otherwise produce an anti-self response (clonal deletion) (Burnet, 1959); another that results in their inactivation (clonal anergy) (Nossal, 1983), and a regulatory mechanism of active suppression of autoreactive cells (clonal balance) (Rose et al., 1981).

The major responsibility for distinguishing self from non-self lies with the T-cell receptor (TCR) (Sha et al, 1988). T cells recognize antigen exclusively in associa­tion with self-MHC^ (Bevan and Fink, 1978). This is not an inherent property of the developing T cells, but is "learned" during thymocyte maturation (Lo and Sprent, 1986). The "self refers to the MHC haplotype expressed on the thymic epithelium in which the thymocytes matured. When the thymic progenitor cells arrive from the bone marrow, they express no TCR. The TCR is a heterodimeric surface protein composed of an a and a P chain. Rearrangement of variable (V) diversity (D) and joining (J) gene segments at the P locus allows for cytoplasmic expression of the p chain. This event is followed by V-J rearrangement at the a locus, culminating in ap TCR expression on the cell surface of immature thymo­cytes (Fowlkes and Pardoll, 1989). Random rearrangement of TCR gene segments and combinatorial association of the two chains give rise to a large T-cell repertoire of at least 10^ different specificities. This randomly generated T-cell repertoire is shaped by two processes that occur in the thymus, positive and negative selection (Kruisbeek et al., 1992). Positive selection is responsible for generating a T-cell repertoire that has the ability to recognize antigenic peptides in association with self-MHC molecules. The other process is called negative selection, and assures that tolerance for self-antigens is achieved. During negative selection, potentially autoreactive T cells are actually deleted from the T-cell repertoire (Kappler et al., 1987) or are clonally inactivated (Ramsdell and Fowlkes, 1990).

MECHANISMS OF POSITIVE SELECTION

The use of naturally occurring mouse mutants as well as genetically manipulated animals in the form of gene-deficient or transgenic mice has clarified many of the questions raised by earlier studies on positive selection in chimeric mice (von Boehmer, 1990). Immature T cells do not express the TCR or the auxiliary surface molecules, CD4 orCD8.'^ As described previously, lymphopoietic T-cell precursors first rearrange and express the TCR P gene and only later the TCR a gene (Raulet et al., 1985). TCR p can be expressed on the surface of immature, but not mature, T-cell lines in disulfide linkage to a developmentally regulated protein, named gp33 (Groettrup et al., 1993). The biological significance of this early P expression was confirmed by introduction of TCR p transgene into various rearrangement-deficient mice (von Boehmer, 1990). The results indicated that expression of TCR P favors the development of very immature CD4-, CD8-, TCR negative thymocytes into intermediate CD4+CD8+ thymocytes. The maturation due to this positive selection by TCR P chain involves signaling through a CD3 surface molecule and requires the cytoplasmic /c^-related tyrosine kinase (Levelt et al., 1993). Once the signal is

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242 SUDERSHAN K. BHATIA and NOEL R. ROSE

delivered, TCR a rearrangement and expression are promoted, while the expression gp33 is terminated (Kishi et al, 1991; Goettrup et al., 1993). Therefore, further TCR P rearrangement is suppressed in the maturing cells, ensuring expression of only one TCR p allele.

Further maturation and positive selection of the CD4+CD8+ thymocytes into functionally mature CD4+CD8- or CD4-CD8+ precursors of helper and cytotoxic T cells, respectively, are mediated by the ap TCR. The intrathymic ligands that must bind to the ap TCR to promote development are the polymorphic MHC molecules that are also involved in transport and presentation of antigenic peptides (von Boehmer, 1990). Experiments have shown that the specificity of the ap TCR for either class I or class II MHC molecules determines whether the rescued cells will be of the CD4-CD8+ cytotoxic or CD4+CD8- helper precursor phenotype (Scott et al., 1989). In accord with these conclusions, it was shown that class I MHC and class II MHC-deficient mice lack mature CD4-CD8+ and CD4+8- T cells, respectively (Cosgrove et al., 1991). Positively selected cells eventually leave the thymus and patrol the body as long-lived resting lymphocytes (von Boehmer and Hafen, 1993) that can only be activated by foreign peptides presented by self-MHC molecules.

Similar to the role of TCR in positive selection of T cells in the thymus, the immunoglobulin (Ig) receptor has been shown to operate for positive selection of B cells in the germinal centers of spleen (von Boehmer, 1994). As a result of antigen binding and T-cell help, proliferating B cells undergo extensive somatic hypermu-tation of the variable regions of the Ig heavy and light chain genes (Jacob et al., 1991). Mutants that no longer bind the antigen presented on the follicular dendritic cells die (Weiss et al., 1992). A few cells, namely those with mutated Ig-receptor genes coding for a receptor that binds most efficiently to antigen, are rescued from programmed cell death (apoptosis) through the binding of their surface Ig receptor to antigen and binding of the CD40 molecule by the gp39 ligand (CD40 molecules are present on the surface of B cells, while their gp39 ligand is present on the surface of activated T cells) (Liu et al., 1989; Noelle et al, 1992). As a result, some of the rescued cells become long-lived memory cells (Schittek and Rajewsky, 1990) that, upon encounter with the same antigen, will produce high-affinity antibodies. The differences seen between the processes involved in T-cell and B-cell selection are that the starting point in B-cell selection is a mature rather than an immature cell and that the ligand that induces positive selection represents foreign rather than self protein (von Boehmer, 1994).

NEGATIVE SELECTION AND SELF-TOLERANCE

A central tenet of the immune system is self-tolerance, or lack of immune respon­siveness to self-components. A random germ line repertoire, positively selected to recognize antigen plus self-MHC, will certainly include autoreactive receptors. Negative selection describes the process whereby a lymphocyte/antigen interaction

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Autoimmunity and Autoimmune Disease 243

results in the death of autoreactive lymphocytes. Elimination of a self-reactive

lymphocyte is termed clonal deletion. However, lymphocytes can be functionally

silenced by antigen without being killed, a phenomenon termed clonal anergy

(Nossal and Pike, 1980).

Clonal deletion as a major mechanism of T-cell tolerance was first directly

demonstrated by the absence of self-reactive clones in mice expressing the corre­

sponding superantigen. For example, Vpi7a+ T cells, which frequently recognize

MHC antigen I-E, were found to be virtually absent in the periphery of I-E

expressing mouse strains (Kappler et al, 1987). Examination of TCR expression

in the thymus of these animals showed that clonal deletion occurred during

thymocyte development. Whereas there was significant expression of the autore­

active receptors in the immature thymocyte pool, expression was almost completely

absent in the mature, medullary population of thymocytes. Clonal deletion was

subsequently shown in other cases of Vp-dominated recognition of superantigens.

For example, T cells that express Vp8.1, Vp6, and Vp9 are eliminated in mice

expressing the superantigen/MHC combination with which they can react, Mis-la

(the minor lymphocyte-stimulating antigen-1) plus the appropriate class II alleles

(Kappler et al., 1988). Correspondingly, Vp3-expressing T cells are absent from

Mls-2a- or Mls-3a-expressing mice (Pullen et al., 1988). In addition to Vpi7a-ex-

pressing cells, Vp5- and vp 11-expressing cells are deleted in most I-E-expressing

strains of mice (Kappler et al., 1987). Clonal deletion in the thymus has also been

induced experimentally in vivo. Injection from birth of Mis-la (MacDonald et al.,

1988) or staphylococcal enterotoxin B (White et al., 1989) resulted in the efficient

elimination of the mature thymocytes expressing Vps reactive with the antigens.

Experiments demonstrated that the expression of different self-antigens causes

deletion of thymocytes at different stages of development (Pircher et al., 1989). It

is suggested that the timing of deletion is determined by the affinity of the TCR for

antigen. Interaction of receptors with an antigen for which they have a high affinity

causes earlier thymocyte deletion than interaction of receptors with a low-affinity

antigen (Kappler et al, 1988). The affinity hypothesis proposes that whereas

thymocytes with both high- and low-affinity receptors are positively selected on

self-MHC, only the high-affinity clones are subsequently deleted (Sprent and

Webb, 1987). In other words, the affinity theory suggests that very weak engage­

ment is positively selective, whereas stronger engagement is lethal.

The question arises as to how tolerance to self-antigens that are not constitutively

expressed in the thymus is accomplished. According to one view, circulating

self-antigens may be captured by the thymic epithelial cells and effectively pre­

sented, in complex with their cell surface MHC, to the developing T cells in the

thymus, subsequently resulting in negative selection (Blackman et al., 1990).

Another view is that nondeletional mechanisms of peripheral tolerance, such as

clonal anergy or suppression, may operate (Kruisbeek et al., 1992).

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244 SUDERSHAN K. BHATIA and NOEL R. ROSE

Immunological self-tolerance comprises a summation of all of the regulatory influences that prevent autoimmunity. It is not fully clear why some self-antigens induce tolerance by one mechanism or the other. Nevertheless, some potentially autoimmune T cells slip through the traps of selection to escape and give rise, at a later date, to autoimmune disease.

The importance of the thymus in inducing tolerance to self-antigens is strength­ened by the evidence that neonatally thymectomized mice develop a variety of tissue-specific autoimmune diseases later in life (Kojima and Prehn, 1981). Neo­natal thymectomy (NTx) probably interferes with clonal deletion and permits self-reactive T cells to escape. It may also eliminate a population of regulatory or suppressor T cells. Although factors controlling these autoimmune diseases are poorly understood, defects in clonal selection and clonal deletion have been assigned to be the major causes of (NTx) autoimmune diseases (Smith et al., 1989).

Tolerance induction in B cells was documented long ago (Nossal and Pike, 1975; Metcalf and Klinman, 1977). Immature B cells in the bone marrow were considered especially sensitive to tolerance induction by one nondeletional mechanism— clonal anergy (Nossal and Pike, 1980). Mature B cells could also be rendered tolerant if they encounter antigen in the absence of T-cell help or other co-stimu­latory influences, but only at much higher antigen concentrations (Pike et al., 1981). In recent years, with the help of transgenic mouse technology, it has been revealed that all three mechanisms of tolerance induction (deletion, anergy and clonal balance) may be involved in B-cell tolerance (reviewed in von Boehmer, 1994).

During development, some lymphocytes with self-reactivity ignore a self-antigen because of low affinity, poor accessibility, lack of suitable presentation, or absence of appropriate help. Such lymphocytes may induce strong autoimmune responses following changed operational circumstances (Nossal, 1994).

AUTOIMMUNE DISEASE

Autoimmune disease results from an attack on the host's own tissues by elements of the immune system. Examples of diseases with an autoimmune component range from multi-system disorders, such as systemic lupus erythematosus or rheumatoid arthritis, to localized diseases, such as multiple sclerosis, Type I diabetes, and thyroiditis (Basten, 1989). Many different pathways can lead to the development of autoimmune disease. The central event is the proliferation of high-affinity self-reacfive T cells by a variety of inifiators. Molecular mimicry of self-antigens with bacterial, viral or other external antigens may be one such triggering event (Penhale and Young, 1988; Oldstone, 1989). Alterafion of self-antigens by chemi­cals, heavy metals or other toxic environmental substances may also potentially contribute to induction of an autoimmune response (Weigle and High, 1967). Aberrant expression of class II MHC has been proposed to play a role in the development of autoimmune diseases (Bottazzo et al., 1983).

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Autoimmunity and Autoimmune Disease 245

Once the T cell has been stimulated, it can induce autoimmune disease through a variety of effector mechanisms. It may produce lymphokines that directly injure surrounding cells (Sinha et al., 1990); cytotoxic cells directly produce tissue destruction (Sarvetnick et al., 1988); T-cell and B-cell interaction may activate self-reactive B cells to generate autoantibodies (Mamula et al., 1992), and the autoantibodies may directly affect target organs (Stanley et al., 1984); or antibody may cooperate with complement or "killer" cells to produce tissue injury (Mamula etal., 1994).

The immunological recognition of an antigen by T-helper cells is directed to a limited number of sites on the antigen molecule. All antigenic proteins have stretches of amino acids which, in peptide form, are able to fit a common peptide-binding site on the class II molecule recognized by the T-cell receptor (Buus et al., 1986). The identification of these antigenic determinants of autoantigens could enable the development of effective therapies to block the antigen-specific genera­tion of the autoimmune response.

AUTOIMMUNE THYROID DISEASE

Autoimmune thyroid disease (AITD) is a prototype of an autoimmune disease, where the thyroid gland is targeted by the immune system. AITD clinically presents in different forms; the two most commonly encountered are Hashimoto's thyroiditis (HT), usually causing hypothyroidism, and Graves' disease (GD) in the form of hyperthyroidism. The recognition of HT as a classical example of an autoimmune disorder is credited to the independent discoveries of experimental thyroiditis and anti-Tg autoantibodies by Rose and Witebsky (1956) and Roitt et al. (1956). The concept that GD can also be included in the group of thyroid autoimmune disorders stems from the discovery by Adams and Purves (1956) of the long-acting thyroid stimulator (L ATS) and the subsequent demonstration that L ATS is a thyroid-stimu­lating autoantibody responsible for the hyperthyroidism of GD.

Incidence

HT occurs more frequently in females than in males, with a ratio of about 4:1. The peak incidence is between 30 to 50 years of age (Bigazzi and Rose, 1985). A juvenile variant occurs in children and adolescents. The prevalence of thyroiditis in the general population varies between 0.5% to 1.2%. Epidemiological studies have demonstrated that the incidence of HT is increasing, especially in younger age groups (Safran et al., 1987).

Histopathological Changes

The thyroid glands of patients with AITD are usually enlarged. In HT, the thyroid gland shows diffuse mononuclear cell infiltration, consisting mainly of lympho­cytes. These cells may aggregate to form lymphoid follicles with germinal centers.

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246 SUDERSHAN K. BHATIA and NOEL R. ROSE

Plasma cells and macrophages are also present. The glands may show parenchymal

damage, hyperplasia of thyroid epithelial cells and variable degrees of fibrosis. In

GD, the thyroid is diffusely enlarged owing to parenchymal hypertrophy and

hyperplasia. Lymphocytic and plasma cell infiltration often occur.

The lymphocytic infiltrates are composed of approximately equal proportions of

T cells and B cells (Totterman, 1978). The percentages of CD4+ and CD8+ cells,

as reported by various studies, vary considerably (Del Prete et al., 1986; Bagnasco

et al, 1987; Margolick et al., 1988; Cohen and Weetman, 1988), but CD8+

lymphocytes seem to predominate in HT (Mariotti and Pinchera, 1990). Cytotoxic

T cells have been cultured from lymphocytic infiltrates, and their phenotypes have

been mainly CD4-CD8+.

Aberrant expression of class IIMHC by thyroid cells is an observation by some

investigators (Davies et al., 1988). MHC class II antigens are not detectable on the

surface of normal thyroid epithelial cells; however, HLA-DR and -DQ molecules

have been found on thyroid epithelial cells from patients with AITD (Most et al.,

1986). This led to the hypothesis that such epithelial cells could become APCs,

capable of stimulating autoreactive T cells by presentation of endogenous autoan-

tigens (Bottazzo et al., 1983; Weetman et al., 1986). However, the idea that this

phenomenon could be the initiating event in AITD seems unlikely, as the phenome­

non is dependent on local T-cell infiltration (Cohen et al., 1988). Nevertheless, class

Il-expressing thyrocytes may play a role in perpetuating the autoimmune response

(Weetman, 1992).

Autoantigens

Three different autoantigens, namely, thyroglobulin (Tg), thyroid peroxidase

(TPO) or microsomal antigen and thyroid-stimulating hormone receptor (TSHR)

have been recognized as playing a role in the development of AITD. Autoantibodies

against these antigens have been detected in patients suffering from HT and GD.

Until recently, Tg was the only antigen known to induce experimental autoimmune

thyroid disease in animal models. TPO was recently reported to have induced

experimental murine thyroiditis (Kotani et al., 1990).

Among other antigens to which autoantibodies have been detected in thyroiditis

patients are the second colloid antigen (Bigazzi, 1990); autoimmune thyroid disease

related autoantigen (ATRA-1), recognized by about 27% of Hashimoto's sera

(Hirayu et al., 1987); and a 70 kD antigen (Chan et al., 1989). The significance of

these antigens is unknown. Antibodies against T4 and T3 have also been detected,

although infrequently, in patients. They may represent a subset of anti-Tg antibod­

ies, specifically recognizing a short Tg sequence containing iodothyronine (Pearce

et al., 1981). There may be other, as yet unknown, cell surface antigens involved

in AITD.

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Autoimmunity and Autoimmune Disease 247

Animal Models

Several animal models of experimentally induced thyroiditis (EAT) or sponta­

neously induced thyroiditis (SAT) have been described (Kuppers et al., 1988). EAT

may be induced by injection of homologous or autologous Tg in many animal

species, including rabbit, guinea pig, mouse, rat, dog, monkey, chicken, and cat. T

cells play a central role in the mechanisms responsible for EAT, as the disease can

be induced by adoptive transfer of T cells (Felix-Davies and Waksman, 1961;

Twarog and Rose, 1970; Romball and Weigle, 1987).

SAT has been described in chickens, rats, and dogs. The OS chicken is the best

studied model of human HT, being characterized by spontaneous appearance of

high titers of antibodies to Tg. The lymphocytic infiltration of the thyroid gland

and the subsequent destruction of thyroid follicles presents similarities to the human

disease. Another model of spontaneous thyroiditis was introduced by Penhale et al.

(1973); it involves neonatal thymectomy and irradiation of Wistar rats. Recently,

thyroiditis has been reported to develop in NOD mice (Bernard et al., 1992). It

might prove to be a good model to study human AITD as a large collection of

murine immunological reagents is available.

So far, no counterpart of GD in experimental animals has been established.

Multifactorial Etiology

Evolving knowledge of the human immune system and the results of studies on

thyroid autoimmunity suggest that the cause of autoimmune disease is multifacto­

rial. The development of autoimmune disease involves genetic and environmental

influences that amplify self-reactivity. The complex interplay between these mul­

tiple factors may shift the balance and contribute to the pathogenesis of AITD. The

OS chicken is one of the best established animal models for a spontaneously

occurring autoimmune disease. Much of our knowledge of a multifactorial etiology

of AITD comes from this model of SAT. The various factors that seem to be

involved in the autoimmune process in the OS chicken include: (i) immune

dysregulation, including severe mononuclear cell infiltration, induction of anti-Tg

autoantibodies, hyper-responsiveness to ConA stimulation, decreased suppression,

(ii) genetically determined susceptibility of the target organ (thyroid gland) such

that there is a lower threshold of thyroid epithelial cells for MHC expression via

gamma IFN, which may perhaps take over the role of antigen presentation from the

initially involved macrophages and endothelial cells, (iii) hormonal influences—

presence of high amounts of corticosteroid which, in turn, leads to a heightened

immune responsiveness in the OS chicken, and (iv) environmental factors, includ­

ing endogenous virus (ev 22) and dietary iodine (Wick et al, 1986). Similar

mechanisms may be operative in HT.

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248 SUDERSHAN K. BHATIA and NOEL R. ROSE

Immunological Response

Humoral and eel I-mediated immunity. Antibodies to Tg are present in 80-90% of patients with HT, and 50-60% of patients with GD (Amino et al., 1976). B cells secreting anti-Tg antibodies have been demonstrated in the peripheral blood and among the intra-thyroidal lymphocytes of patients with AITD (McLachlan, 1983; litaka et al., 1988). Since there are relatively few circulating Tg-specific antibody-secreting B cells, it has been suggested that antibody formation occurs mainly in the thyroid, where antibody-secreting cells are more easily demonstrated (McLachlan et al., 1979; Weetman et al., 1982). Marked dissociation has been observed between the extent of histological thyroiditis and the levels of autoanti­bodies. The natural experiment of transplacental antibody passage from mother to the fetus suggests that antibody alone cannot destroy the thyroid gland (Parker et al., 1961). Autoantibodies to Tg in combination with mononuclear cells can produce antibody-dependent cell-mediated cytotoxic activity (Calder et al., 1974) or may cause injury by forming immune complexes and activating the complement system (Kalderon et al., 1977). In animal model studies of OS chickens, autoanti­bodies have been shown to increase the severity of mild thyroiditis but, by themselves, do not have the ability to induce disease (Jaroszewski et al., 1978).

Isotype restriction of Tg autoantibodies has also been reported. One study states that anti-Tg antibodies in HT patients are primarily IgG4 and IgGl (Thompson et al., 1983). Another study found that, although IgG4 is over-represented, it accounts for around only 30% of the Tg antibody contained in total IgG (Weetman et al., 1988). Recently, Kuppers et al. (1993) have reported that all four IgG subclasses were used in the response to human Tg, although the pattern of usage varied between AITDs. Patients with HT showed a higher proportion of IgG2 antibody than did GD patients. The predominance of a particular subclass of autoantibodies may have diagnostic significance as a predictor of disease. There is speculation that Tg autoantibodies of different IgG subclasses interact with different epitopes on Tg (Fukuma et al., 1989).

Animal experiments have demonstrated that autoimmune thyroiditis can be induced by transfer of Tg-specific T cells into naive syngeneic animals (Felix-Davies and Waksman, 1963; Twarog and Rose, 1970; Romball and Weigle, 1987), thus providing evidence for a T-cell-mediated immune mechanism as a cause for the development of autoimmune thyroid disease. A specific T-cell reaction to Tg has generally been found in AITD patients (Weetman and McGregor, 1984), although optimal antigen concentration appears to be variable. Peripheral and intra-thyroidal T cells proliferate in vitro when cultured with autologous DR+ thyroid cells (Del Prete et al., 1987). The precise characterization of antigenic determinants recognized by the T cells needs to be investigated. Many studies have examined T-cell subsets in thyroid tissues of patients with active autoimmune thyroid disease. Increased CD8 (cytotoxic or suppressor) T cells and increased CD4 helper T cells have been reported in GD patients (Margolick et al., 1984). Increased

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proportions of activated cytotoxic/suppressor T cells, expressing HLA-DR antigen, have been found in HT patients (Canonica et al, 1984).

Role of cytokines. Cytokines produced by T cells, macrophages and other cell lines regulate the function and activity of T cells. There is evidence of the production of gamma interferon (IFN-y) and alpha tumor necrosis factor (TNF-a) in intrathyroidal lymphocyte infiltrates (Del Prete et al., 1989). In situ production of IFN-y in the thyroid gland of AITD patients has ak D been demonstrated (Margolick et al., 1986). The increased production of IFN-y has been shown to induce HLA-DR antigen expression on the surface of human thyroid cells. The aberrant expression of class II MHC antigens on thyroid epithelial cells has been hypothesized to be an early event of thyroid autoimmunity (Hanafusa et al., 1983).

Production of IL-1 and IL-6 by the thyroid cells has also been demonstrated (Zheng et al., 1991). This phenomenon could be particularly relevant, as a DR+ thyroid cell producing IL-1 and IL-6 could be considered a fully functioning antigen-presenting cell, able and sufficient to trigger autoimmune reactions. IL-6 is one of the most important cytokines affecting B-cell development. The produc­tion of IL-6 is enhanced by TNF and IL-1 (Weetman et al., 1990). As thyroid cells are exposed to these cytokines in thyroiditis, it is likely that the enhanced IL-6 production within the diseased gland, in turn, stimulates plasma cell formation, leading to localized autoantibody formation. In addition, both IL-1 and IL-6 activate T cells; their localized production by thyrocytes may further promote the development of thyroiditis by stimulating cell-mediated effector mechanisms.

Recent reports demonstrate that patients receiving cytokines as therapeutic agents seem unusually prone to develop thyroiditis. The immunological effects produced by IL-2 and GM-CSF therapy sometimes result in the common develop­ment of autoimmune thyroiditis (Burman et al., 1986; Von Leissum et al., 1989; Hoekman et al., 1991).

Suppressor cell defect A partial antigen-specific defect in suppressor T lym­phocytes as the basis for AITD has been suggested by Volpe (1991). Self-tolerance to Tg is hypothesized to be maintained by an immunoregulatory balance between helper and suppressor T cells (Volpe, 1988). Volpe suggests that an HLA-related gene abnormality in antigen presentation to T lymphocytes may result in inadequate activation of antigen-specific suppressor T cells. Environmental factors adversely affecting overall suppressor T-cell function may serve to precipitate AITD. The consequent activation of specific helper T cells leads to cytokine and thyroid antibody production, and the combined effects of cytokines, thyroid antibodies and cellular components on thyrocytes complete the pathological picture. Recently, Volpe's group has demonstrated that peripheral blood mononuclear cells (PBMC) from HT and GD patients, when stimulated in culture with Tg, have markedly higher proliferative responses when they are CD8 depleted and CD4 enriched (Akasu et al., 1993). This finding suggests that suppressor CD8 cells play a role in

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preventing CD4 cells from proliferating due to Tg-stimulation. Other investigators do not agree on the role of antigen-specific T-suppressor cells (Weetman, 1989). Some have even questioned the very existence of suppressor cells (MoUer, 1988).

Genetic Predisposition

HT and GD frequently occur in the same family and several cases of twins with either of the two conditions have been reported (Hall et al., 1972; Volpe et al., 1973; Doniach, 1975). Thyroid antibodies are found in approximately 50% of first-degree relatives of patients with AITD (Farid et al., 1977). Using more sensitive assays, segregation analysis of families with thyroiditis suggested that the inheritance of thyroid autoantibodies is a Mendelian dominant trait in women, with reduced penetrance in men (Philips et al., 1990).

HL A genes are supposed to control immune responsiveness to a variety of foreign and self-antigens. In many autoimmune conditions, associations have been reported with certain MHC alleles. An association of HLA-DR5 with HT was reported (Farid et al., 1981). In one small study from Newfoundland, HT was associated with DR4 (Thompson and Farid, 1985). Another study in patients from Toronto and Denmark reported an association with DR5 (Vargas et al., 1988), while from Hungary DR3-association was reported (Stenzsky et al., 1987). The DNA-restriction frag­ment length polymorphism (RFLP) method of DR typing, which is less prone to error, has shown that DR3 was more frequent in English patients with HT (Tandon et al., 1991). An RFLP study from the USA found no HLA-DR associations with HT (Mangllabruks et al., 1991).

Transracial comparisons of HLA association with HT are inconsistent. HLA-B17 and HLA-Bw46 have been reported in blacks and orientals, respectively (Hawkins et al., 1987). In Japanese, B16 is increased and DR2 is decreased (Nakao et al., 1978; Sakurami et al., 1982). In a more recent report from Japan, an association with HLA-DRw53 was noted (Honda et al., 1989). In contrast, a significantly increased prevalence of HLA-B8 and -DR3 has been reported in Caucasians with GD (Farid, 1981). Thus, HLA associations are either weak or inconsistent between populations.

As described previously, the T-cell receptor (TCR) is a heterodimer, composed of two chains, a and p. Another heterodimer, 76, also exists but its ftinctional significance in autoimmune disease is not clear. Restricted heterogeneity of the gene for the variable regions of the a-chain of the human TCR has also been reported (Davies et al, 1991). Restriction of the T-cell repertoire at the site of autoimmune attack would imply a role for a selected subset of T cells in the pathology of the disease. Such restriction would have therapeutic implications: therapy with monoclonal antibodies to specific TCR, peptide fragments of the TCR or with the T cells themselves might be possible. However, recent reports by other investigators have failed to show evidence of limited variability of antigen receptors on intrathyroidal T cells (Mcintosh et al., 1993).

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Animal studies also lend support to the role of genetic control of susceptibility. Variation in the susceptibility of various strains of mice to induction of experimen­tal autoimmune thyroiditis (EAT) indicates the influence of genetic factors. The response was found to be predictable on the basis of their MHC (H-2) genotype (Vladutiu and Rose, 1971). A number of other genes, such as the IgG heavy chain allotypes, may also be involved (Kuppers, 1988). Recently, Gleason et al. (1990) investigated the binding specificities and nucleotide sequences of murine mono­clonal anti-Tg antibodies. They report no evidence for preferential use of any V-region family or gene segment, suggesting that the antigenic epitopes on mouse thyroglobulin (mTg) elicit a very diverse autoantibody response that is derived from a large number of V-region gene segments. A recent study in humans (Chazenbalk et al., 1993), using another thyroid antigen—^TPO, demonstrated that the putative germ line genes used by the TPO human autoantibodies involved only five different heavy and light (H and L) chain combinations. In addition, the study found that the same H and L combinations were used by mukiple patients. Further analysis revealed that the F(ab) proteins expressed by these genes define two major, closely associated domains in an immunodominant region on TPO. These domains contain the binding sites of 80% of IgG-class TPO autoantibodies in the sera of patients with autoimmune thyroid disease, thus indicating that there is a restriction in H and L chain usage in relation to the interaction with specific antigenic domains in human, organ-specific autoimmune disease.

Genetic defects, such as haplotypes associated with hyper-responsiveness of the immune system (evidenced by high Tg autoantibody titers and severe SAT) observed in OS chickens, also contribute to our understanding of the role of genetic loci for the altered reactivity of the immune system and the susceptibility of the target organ for autoimmune effector mechanisms (Wick et al., 1986).

Endocrine Abnormality

A hypothesis presented by Wilkin (1989) argues that the primary lesion of autoimmune disease is in the target organ. According to this theory, a heightened immune response is a physiological reaction to the excessive production and turn-over of the antigens from the primary lesion. Hence, autoimmune thyroiditis is a result of the excessive production of Tg by the thyroid gland; on the other hand, there are some reports that raising the serum Tg levels can downregulate autoim­mune thyroiditis in mice (Lewis et al., 1987).

Structural abnormality of Tg has been suggested to be a possible factor in the induction of autoimmune disease, but no link so far of any heterogeneity at the molecular level has been observed in AITD. A mutation within exon 10 of the Tg gene has been demonstrated in patients with non-toxic goiter, leading to less efficient production of thyroid hormone (Corral et al., 1993).

Hormones also appear to play an important role in the regulation of the autoim­mune process. A strong female:male predominance for autoimmune thyroid disease

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suggested an influence by sex hormones. The sex hormones have a variety of effects on the immune system. Investigations showed that estrogens exacerbate experi­mental autoimmune thyroiditis (EAT), whereas testosterone ameliorates it (Okayasu et al., 1981; Weetman, 1991). Prolactin may have a role because asymp­tomatic autoimmune thyroiditis is more common in hyperprolactinemic disorders (Ferrara et al., 1983). Altered levels of these hormones and other changes which accompany pregnancy presumably account for the remission seen in the last trimester, and the subsequent exacerbation of subclinical thyroiditis and rise in autoantibody levels which constitute the entity of post-partum thyroiditis (Lazarus and Othman, 1991). Similarly, after onset of puberty, the prevalence of thyroid autoimmunity rose among females (Burek et al., 1982).

Environmental Factors

Infectious agents. Immune reaction to an environmental antigen could lead to antibodies that cross-react with thyroglobulin or other thyroid antigens. Molecular mimicry between bacterial and viral antigenic determinants and thyroid autoan-tigens could lead to the induction of an immune response against the target organ and to the development of autoimmune disease. A high frequency of antibodies directed to human thyroid cell cytoplasm has been observed in sera of patients with Yersinia enterocolitica infection (Lidman et al., 1976). Conversely, a high fre­quency of antibodies to Y. enterocolitica has been reported in patients with various thyroid diseases (Safron, 1987). TSH-specific binding sites sharing several prop­erties with the human TSH receptor have been found in Y. enterocolitica and to a lesser extent in other bacteria (Weiss et al., 1983). A similar phenomenon has also been observed in patients with infections due to leishmania and mycoplasma (Safron, 1987).

Virus infections of thyroid cells have been frequently suggested as potential triggers of AITD through induction of structural alterations of autoantigens or enhancement of antigen presentation to the immune system (Joasoo et al., 1975), but evidence in favor of this hypothesis has been difficult to obtain. Epstein-Barr virus infection has been implicated in the development of autoimmune thyroiditis in three patients (Coyle et al., 1989). A recent report of nucleotide and amino acid homology between human thyrotropin receptor and the HIV-1 nef protein suggests an avenue through which a shared response against an HIV-1 related retrovirus could play a role in the pathogenesis of GD (Burch et al., 1991).

Role of iodine. Various reports on the role of increased dietary iodine con­sumption in humans suggest that a significant relationship exists between high iodine intake and increased prevalence of autoimmune thyroid disease (Gaitan et al , 1985; Tajiri et al., 1986; Roti et al, 1987; Koutras et al., 1987). The exact mechanism of this relationship is not well understood. Weetman et al. (1983) have demonstrated that iodine enhances IgG synthesis by human peripheral blood

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lymphocytes in vitro. Studies of animal models provide ample evidence for the

pathogenic role of iodine in AITD. A significant increase in lymphocytic thyroiditis

in young BBAV rats was observed with increased iodine ingestion (Braverman,

1989). Similar observations have been made in the spontaneous thyroiditis model

in OS chickens (Bagchi et al., 1985). Experimental evidence suggests that incor­

poration of dietary iodine into thyroglobulin increases its immunogenicity (Sundick

etal., 1987).

Current information suggests that iodine is a major contributing environmental

factor in the pathogenesis of the multifactorial etiology of AITD. More detailed

investigations are needed to establish a correlation and to understand the immu­

nological effects of iodine.

Other Factors

Stress. The role of stress in triggering and modulating autoimmune mecha­

nisms responsible for GD has been envisaged (Safran et al., 1987). Physical and

psychological stressful events have been associated with the onset of GD (McKen-

zie and Zakarja, 1989). It is hypothesized that stress could aggravate the putative

defect of both nonspecific and thyroid-specific T-suppressor lymphocytes (Volpe,

1986). It may also induce changes in levels of cytokines (Abraham, 1991).

Drugs. Serum anti-thyroid antibodies and overt clinical AITD have been re­

ported to be increased in patients receiving lithium therapy (Safran et al., 1987).

The in vitro activity of lithium on some immune parameters is consistent with

augmentation of helper T-cell activity and macrophage phagocytosis (Safran et al.,

1987). Animal experiments demonstrate that lithium may increase anti-Tg antibod­

ies in rat EAT but does not induce thyroid antibodies in normal nonimmunized

animals (Hassman et al., 1985).

Treatment with amiodarone (an iodine-containing drug) has been reported to

affect thyroid function, inducing either hyper- or hypo-thyroidism (Martino et al.,

1984). Thyroid autoantibodies developed in several patients treated with amio­

darone, but disappeared after withdrawal of the drug (Monteiro et al., 1986).

Amiodarone has also been shown to affect T-cell function, increasing the number

of both helper and suppressor/cytotoxic T cells (Rabinowe et al., 1986). The cause

of this abnormality, whether due to amiodarone itself or to its iodine content,

remains to be elucidated.

Toxins. Several environmental toxins have been proposed as potential inducers

of AITD. They include polybromated biphenyls, phenols, thiocyanate, hy-

droxypyridines, substituted dihydroxybenzenes, and resorcinol derivatives (Safran

et al., 1987). Carcinogens, such as methylcholanthrene and dimethylbenzan-

thracene, have also been shown to induce EAT in certain genetically susceptible

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strains of rats (Reuber, 1970). Similar factors in humans genetically predisposed to the development of AITD could be the initiating factors of the disease.

THYROGLOBULIN

Human Tg is a huge molecule of 660,000 molecular weight. It is a homodimer, with each chain containing 2748 amino acids. The nucleotide structure of the mRNA is composed of 8448 base pairs. The gene sequence of Tg is comprised of over 300 kb, with 37 exons (Malthiery et al., 1989). The only known function of this protein is serving as a prohormone for thyroxine, the hormone which regulates the vital metabolic functions of the body. Of 110 tyrosine residues in each chain, only four have been shown so far to be involved in the formation of the hormone. The rest of the structure of this huge molecule is not known to serve any other function, except for conserving iodine for efficient production of the hormone.

Some structural homology of Tg has been seen with the acetylcholinesterase molecule (Schumacher et al., 1986), and the alternatively expressed domain of the invariant chain of the la molecule associated with the MHC system (Koch et al., 1987). Recently, McLachlan and Rapoport (1989) showed evidence of structural homology of a T-cell epitope between human TPO and human Tg, and possibly certain bacterial proteins. They suggest that this common T-cell epitope may play a role in the pathogenesis of AITD.

From a pathological aspect, Tg is the major antigen involved in autoimmune thyroid disease. In order to understand the process of antigen recognition for production of autoantibodies and to determine the immunodominant regions of Tg involved in T-cell activation, it is essential to characterize the B-cell and T-cell epitopes of Tg. The results of such an investigation can aid in the understanding of the immunological process of the autoimmune disease. The knowledge so obtained can further be extended for designing preventive measures and immunotherapeutic approaches for autoimmune thyroid disease. Overall, it can form a basis for the study of other, more crippling, autoimmune diseases.

Investigations reported to date on the characterization of B-cell epitopes of Tg initially suggested 40 antigenic sites (Heidelberger, 1938) on the basis of recogni­tion by xenogeneic antibodies. Analysis performed by competitive binding between autoantibodies and monoclonal anti-Tg antibodies suggests 4 to 6 major antigenic determinants of Tg (Nye et al., 1980). Results also suggest different determinants for pathological and natural autoantibodies (Piechaczyk et al., 1987; Bresler et al., 1990). Species cross-reactivity has also been utilized to demonstrate multiple epitopes of Tg (Chan et al, 1987). Some experiments suggest that autoantibodies recognize only the conformational epitopes (Kondo and Kondo, 1984). Recent reports, using recombinant peptides of Tg, have shown that Tg autoantibodies are highly heterogeneous (Henry et al., 1990). There is some evidence of certain epitopes being more frequently involved in autoimmune reactions.

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Recently, the identification of three different T-cell epitopes of Tg have been reported by three different investigators. All of these epitopes are in different regions of Tg. A 40 amino acid synthetic peptide in the 1672—1711 residue region of human Tg was shown to be recognized by a human Tg-specific cytotoxic hybridoma (Texier et al., 1992). Another group of investigators (Hutchings et al., 1992) report a 12 mer synthetic peptide containing a thyroxine at position 2553 in Tg to be recognized by two clonotypically distinct murine Tg autoreactive T-cell hybridomas. This peptide could not directly induce thyroiditis in susceptible mice, but adoptive transfer of in vivo primed lymph node cells boosted in vitro with peptide could transfer thyroiditis in naive recipients. A third group of investigators (Chronopoulou and Carayanniotis, 1992) has reported a 17 mer peptide correspond­ing to amino acids 2495 to 2511 of the human Tg, with one residue difference at position 2510. This peptide has been shown to induce mononuclear cell infiltration of the thyroid gland in susceptible mice.

SUMMARY

The mechanisms for achieving self/non-self distinction include clonal deletion, clonal anergy, and clonal balance. Clonal deletion results in the elimination of self-reactive T cells during their maturation in the thymus. T cells reactive with self-antigens not represented in the thymus, and probably some low-affmity T cells, escape clonal deletion and populate peripheral lymphoid tissues and blood. These self-reactive T cells are normally held in check by clonal anergy or clonal balance. Anergy occurs when a T cell receives its antigen-specific signal in the absence of required co-stimulatory signals. Clonal balance depends upon the ratio of stimula­tory and inhibitory signals delivered to the T cells. Self-tolerance of B cells probably depends upon similar mechanisms, but is less effective. Autoantibodies, therefore, are more common than self-reactive T cells.

Autoimmune disease results from an attack on the host's own tissues by self-re­active T cells or antibodies. Autoimmune thyroid disease is a well-characterized paradigm of disease caused by autoimmunity. Graves' disease results from an over-active thyroid gland produced by autoantibodies directed to the receptor for the thyroid-stimulating hormone. Hashimoto's thyroiditis is associated with thy­roid damage produced by self-reactive T cells directed to thyroglobulin or, possibly, thyroperoxidase. The disease can be reproduced in experimental animals by immu­nization with autologous thyroglobulin, providing firm evidence that the thyroiditis is caused by autoimmunization.

The etiology of autoimmune disease is multifactorial and includes both genetic and environmental factors. Several genes are involved, affecting both immune regulation and vulnerability of the target organ. Among the environmental factors are infectious agents, drugs, diet, and toxins. Hormones and even emotional status also play a role. Both T cells and antibodies are involved in the pathogenesis and the demonstration of autoantibodies is useful in diagnosis. Future treatment and