The Complement System

The Complement System

060525

沈 弘 德

台北榮總教研部

Innate and adaptive immunity

ADCC

CytokinesAPCsDCs

Abbas AK & Lichtman AH. Cellular and Molecular Immunology 5th ed. 2003

Types of adaptive immunity

(CD4+) (CD8+)

Abbas AK & Lichtman AH. Cellular and Molecular Immunology 5th ed. 2003

The differentiation and functions of TH1 and TH2 subsets of CD4+ helper T lymphocytes

IL-12 IL-4

Abbas AK & Lichtman AH. Cellular and Molecular Immunology 5th ed. 2003

Opsonization

- Deposition of opsonins on an antigen, therebypromoting a stable adhesive contact with anappropriate phagocytic cell.

Opsonin

- A substance (e.g., an antibody or C3b) thatpromotes the phagocytosis of antigens bybinding to them.

Schematic representation of the roles of C3b and antibody in opsonization.

Kuby J et al., Immunology 2003

Complement

• 1890 Jules Bordet, Paul Ehrlich

• Bacteriolytic activity requires two different substances.

• Heat-labile

• Augments the opsonization and killing of bacteria byantibodies (the major effector of the humoral branchof the immune system).

• Evolved as part of the innate immune system.

• The Functions of Complement

• The Complement Components

• Complement Activation

• Regulation of the Complement System

• Biological Consequences of Complement Activation

• Complement Deficiencies

The multiple activities of the complement system.

Kuby J et al., Immunology 2003

The complement cascade

The complement components• Synthesized mainly by liver hepatocytes (blood monocytes,

tissue macrophages, epithelial cells of GI &GU tracts).

• Most circulate in the serum in functionally inactive formsas proenzymes (zymogens).

• Designated by numbers, by letter symbols, or by trivial names.

• Peptide fragments formed by activation of a component- the larger fragments: bind to the target near the site of activation- the smaller fragments: local inflammation

• Complexes with enzymatic activity are designated by a bar over the number or symbol.

The classical pathway

Overview of the complement activation pathways.

Kuby J et al., Immunology 2003

Structure of C1

The C1q molecule is composed of 18polypeptide chains that associate toform six collagen-like triple helicalarms, the tips of which bind to exposed C1q-binding sites in the CH2 domain of the Ab molecule.

Abbas AK & Lichtman AH. Cellular and Molecular Immunology 5th ed. 2003

(b)

Structure of the C1 macromolecular complex

Kuby J et al., Immunology 2003

C1q molecule

C1 binding to the Fc portions of IgM and IgG

The formation of an Ag-Ab complexinduces conformational changes in the Fc portion of the pentameric IgMmolecule that expose at least three binding sites for the C1q component of the complement system.

Each C1 molecule must bind by its C1q globular heads to at least two Fc sites for a stable C1-Ab interactionto occur.

Abbas AK & Lichtman AH. Cellular and Molecular Immunology 5th ed. 2003

(a)

(b)

Kuby J et al., Immunology 2003

Model of pentamericIgM in planar form

Model of pentamericIgM in staple form

(c) (d)

Electron micrographs of IgM antiflagellum antibody bound to flagella, showing the planar form (c) and stape form (d)

Kuby J et al., Immunology 2003

Schematic diagram of intermediates in the classical pathway of complement activation (1).

C1q binds antigen-bound antibody. C1r activates auto-catalyticallyand activates the second C1r; both activate C1s.

Kuby J et al., Immunology 2003

Schematic diagram of intermediates in the classical pathway of complement activation (2).

C1s cleaves C4 and C2. Cleaving C4 exposes the binding site for C2. C4 binds the surface near C1 and C2 binds C4, forming C3 convertase.

Kuby J et al., Immunology 2003

Schematic diagram of intermediates in the classical pathway of complement activation (3).

C3 convertase hydrolyzes many C3 molecules. Some combine with C3 convertase to form C5 convertase.

*A single C3 convertase molecule can generate over 200 molecules of C3b.

C3 convertase

Kuby J et al., Immunology 2003

Schematic representation of the roles of C3b and antibody in opsonization.

Kuby J et al., Immunology 2003

Internal thioester bonds of C3 molecules

Cleavage of C4 exposes a highly reactive thioester bond on the C4b molecule that allows it to bind covalently to molecules in the immediate vicinity of its site of activation.

C3 contains anunstable thioesterbond.

C3b undergoes hydrolysis by the time it hasdiffused 40 nm away from the convertases.

Abbas AK & Lichtman AH. Cellular and Molecular Immunology 5th ed. 2003

_____

Hydrolysis of C3 by C3 convertase C4b2a

The membranes of most mammalian cells have high levels of sialic acid,which contributes to the rapid inactivation of bound C3b molecules on host cells;consequently this binding rarely leads to further reactions on the host cell membrane.

(a labile internalthioester bond)

Kuby J et al., Immunology 2003

Overview of the complement activation pathways.

Kuby J et al., Immunology 2003

The alternative pathway

• does not depend on antibody for its activation

• being initiated in most cases by cell-surfaceconstituents that are foreign to the host

1.C3 hydrolyzes spontaneously, C3b fragment attaches to foreign surface.

2.Factor B binds C3a, exposes site acted on by Factor D. Cleavage generates C3bBb, which has C3 convertase activity.

3.Binding of properdin stabilizes convertase.

4.Convertase generates C3b; some binds to C3 convertase activating C5’ convertase. C5b binds to antigenic surface.

Schematic diagram of intermediates in the formation of bound C5b by the alternative pathway of complement activation

b

b

*More than 2 x 106 molecules of C3b can be deposited on anantigenic surface in less than 5 minutes.

Kuby J et al., Immunology 2003

Schematic representation of the roles of C3b and antibody in opsonization.

Kuby J et al., Immunology 2003

Overview of the complement activation pathways.

Kuby J et al., Immunology 2003

The mannose-binding lectin pathway

• does not depend on antibody for its activation

• originates with host proteins (MBL) binding microbial surfaces

The mannose-binding lectin (MBL) pathway

- MBL, an acute phase protein, binds to mannose residues, and tocertain other sugars on many pathogens.

- MBL, like C1q, is a two- to six-headed molecule that forms a complex with two protease zymogens (MASP-1 and MASP-2).

- When the MBL complex binds to a pathogen surface, MASP-2 is activated to cleave C4 and C2.

- A C3 convertase is formed from C2a bound to C4b.

- The MBL pathway is of importance in innate host defensemechanisms in early childhood.

Mannose-binding lectin forms a complex with serine proteases that resembles the complement C1 complex.

*MBL is an acute phase proteinproduced in inflammatory responses.

The acute-phase response produces molecules that bind pathogens but not host cells.

On vertebrate cells, these mannose residues are covered by other sugar groups, especially by sialic acid while avoiding complement activation on host cell surfaces.

The three complement pathways

converge

at the membrane-attack complex

Overview of the complement activation pathways.

Kuby J et al., Immunology 2003

Schematic diagram of intermediates in the classical pathway of complement activation (4).

The C3b component of C5 convertase binds C5, permitting C4b2a to cleave C5.

The production of C5b initiates the assembly of the terminalcomplement components.

The C5b component becomesinactive within 2 minutes unlessC6 binds to it and stabilizesits activity.

4b 2a 3b

Kuby J et al., Immunology 2003

Schematic diagram of intermediates in the classical pathway of complement activation (5).

C5b binds C6, initiating the formation of the membrane-attack complex.

The MAC complex forms a large channel through the membrane of the target cell,enabling ions and small molecules to diffuse freely across the membrane.

C9: a perforin-like molecule

Kuby J et al., Immunology 2003

Late steps of complement activation and formation of the MAC (membrane attack complex)

(10-17 molecules)

Abbas AK & Lichtman AH. Cellular and Molecular Immunology 5th ed. 2003

(a) (b)poly-C9 complex

Kuby J et al., Immunology 2003

Complement-induced lesions on themembrane of a red blood cell

The main components and effector actions of complement

Antibody-mediated mechanisms for combating infection by extracellular bacteria

The critical function of the complementsystem in convertinga humoral antibodyresponse into aneffective defensemechanism.

Kuby J et al., Immunology 2003

The multiple activities of the complement system.

Kuby J et al., Immunology 2003

Biological consequences of

complement activation

Kuby J et al., Immunology 2003

(a) (b)

Schematic representation of the roles

of C3b and antibody in opsonization.

Opsonins: C3b, C4b, iC3bCR1: 5,000/resting phagocytes

50,000/activated cells

Kuby J et al., Immunology 2003

Electron micrograph of EB virus coated with antibody and C3b and bound to theFc and C3b receptor (CR1) on a B lymphocyte

Clearance of circulating immune complexes

Defects in complement activation↓

Failure to clear circulating immune complexes↓

Deposition in blood vessel walls & tissues↓ (eg. kidney)

Activate leukocytes by Fc receptor-dependent pathways & produce local inflammation

and tissue injury

Erythrocytes account for about 90% of the CR1 in the blood (~ 5 x 102/RBC).

Erythrocytes play an important role in binding C3b-coated immune complexes and carring these complexes to the liver and spleen.

Kuby J et al., Immunology 2003

Scanning electron micrographs of E. coli showing (a) intact cells and (b, c) cells killed by complement-mediated lysis.

(a) (b) (c)

Kuby J et al., Immunology 2003

Microbial evasion of complement-mediated damage

Kuby J et al., Immunology 2003

The complement system neutralizes viral infectivity

• formation of larger viral aggregates- reduce the net number of infectious viral particles

• a coating of Ab and/or complement to the surfaceof a viral particle

- blocking attachment to susceptible host cells- facilitate binding of the viral particle to cells possessing Fc or CR1

- lysing most enveloped viruses

(a) (b) (c)

Electron micrographs of negatively stained preparations of EB virus

Control withoutantibody

Antibody-coated

particles

Particles coated withantibody and complement

Kuby J et al., Immunology 2003

Regulation of the complement system

Regulation of the complement system

• Inclusion of highly labile components thatundergo spontaneous inactivation if they are notstabilized by reaction with other components.

• A series of regulatory proteins(regulators of complement activation [RCA]gene cluster - chromosome 1 in humans).

DAF

DAF

Kuby J et al., Immunology 2003

Regulation of the complement system by regulatory proteins (1)

1. C1 inhibitor (C1Inh) binds C1r2s2, causing dissociation from C1q.

2. Association of C4b and C2a is blocked by binding C4b-binding protein (C4bBP),complement receptor type I, or membrane cofactor protein (MCP).

3. Inhibitor-bound C4b is cleaved by Factor 1.

4. In alternative pathway, CR1, MCP, or Factor H prevent binding of C3b and Factor B.

5. Inhibitor-bound C3b is cleaved by Factor 1.

(serine protease inhibitor)

(serine protease)

Kuby J et al., Immunology 2003

Inactivation of bound C4b and C3b by regulatory proteins of the complement system

Kuby J et al., Immunology 2003

Regulation of the complement system by regulatory proteins (2)

C3 convertases are dissociated by C4bBP, CR1, Factor H, and decay-accelerating Factor (DAF or CD55).

C2a C4b CR1

Bb C3bKuby J et al., Immunology 2003

Regulation of the complement system by regulatory proteins (3)

1. S protein prevents insertion of C5b67 MAC component into the membrane.

2.Homologous restriction factor (HRF) and membrane inhibitor of reactive lysis (MIRL or CD59) bind C5b678, preventing assembly of poly-C9 and blocking formation of MAC.

Kuby J et al., Immunology 2003

DAF

DAF

Kuby J et al., Immunology 2003

Complement-binding receptors

• Many of the biological activities of the complement systemdepend on the binding of complement fragments to complement receptors, which are expressed by various cells.

*iC3b (incomplete C3b) designates breakdown products of C3b

Kuby J et al., Immunology 2003

Role of complement in B cell activation

B-cell coreceptor

Abbas AK & Lichtman AH. Cellular and Molecular Immunology 5th ed. 2003

104 molecules of mIgM had to be engaged byantigen for B-cell activation to occur when theco-receptor was not involved.

When CD19/CD2/CD81 co-receptor wascrosslinked to the BCR, only 102 moleculesof mIgM had to be engaged for B-cell activation.

NK cells use a variety of receptors to identify target cells to be killed

Doan et al. Concise Medical Immunology 2005

The complement system in disease

Complement deficiencies

• immune-complex diseases (genetic deficiencies)

• recurrent infection

• C3 deficiencies: - with the most severe clinical manifestations

• hereditary angioedema:- deficiency of C1Inh- localized edema of the tissue

• paroxymal nocturnal hemoglobinuria (PNH)- defect in cell-surface DAF and MIRL

• studies using knock-out mice

Paroxysmal nocturnal hemoglobinuria (PNH)- A defect in regulation of complement lysis

• The defect lies in a posttranslational modification of thepeptide anchor (glycolipid GPI anchor) that binds DAF andMIRL to the cell membrane.

• The defect identified in PNH lies early in the path to formationof a GPI anchor and residues in the pig-a gene.

* X-linked pig-a gene (phosphatidylinositol glycan complementation class A gene)

The complement system in disease (1)

A. Complement deficiencies

1. genetic deficiencies in classical pathway components(C1q, C1r, C4, C2 and C3)

2. deficiencies in components of the alternative pathway(properdin, factor D, C3)

3. deficiencies in the terminal complement components(C5, C6, C7, C8, C9, Neisseria bacteria)

4. deficiencies in complement regulatory proteins(abnormal complement activation)

5. deficiencies in complement receptors(CR3 & CR4 – inadequate adherence of neutrophilsto endothelium at tissue sites of infection)

The complement system in disease (2)

B. Pathologic effects of a normal complement system

- The immune complexes produced in autoimmune diseasesmay bind to vascular endothelium and kidney glomeruliand activate complement (MAC generation).

- It initiates the acute inflammatory responses that destroy thevessel walls or glomeruli and lead to thrombosis, ischemicdamage to tissues, and scarring.

- Some of the late complement proteins may activateprothrombinases in the circulation that initiate thrombosis.

Mechanisms postulated to account for the survival of the fetus as an allograft in the mother

Complement inhibitor

- Trophoblast and decidua may also be relatively resistant to complement-mediated damage becausethey express high levels of a C3 and C4 inhibitor called Crry.

- Crry may block maternal alloantibody-mediated damagethrough the classical pathway of complement activation.

- Crry-deficient embryos die before birth and show evidenceof complement activation on trophoblast cells.

Summary

1. The complement system comprises a group of serum proteins, many of which exist in inactive forms.

2. Complement activation occurs by the classical, alternative, orlectin pathways, each of which is initiated differently.

3. The three pathways converge in a common sequence ofevents that leads to generation of a molecular complex that causes cell lysis.

4. The classical pathway is initiated by antibody binding to acell target; reactions of IgM and certain IgG subclassesactivate this pathway.

5. Activation of the alternative and lectin pathways is antibody-independent. These pathways are initiated by reaction of complement proteins with surface molecules of microorganisms.

6. In addition to its key role in cell lysis, the complement systemmediates opsonization of bacteria, activation of inflammation, and clearance of immune complexes.

7. Interactions of complement proteins and protein fragmentswith receptors on cells of the immune system control bothinnate and acquired immune responses.

8. Because of its ability to damage the host organism, the complement system requires complex passive and active regulatory mechanisms.

9. Clinical consequences of inherited complement deficiencies range from increases in susceptibility toinfection to tissue damage caused by immunecomplexes.