Immunology Overview

Immunology Overview

W. Robert Fleischmann, Ph.D.

Department of Urologic Surgery

University of Minnesota Medical School

[email protected]

(612) 626-5034

Objectives

• Provide an overview of immunological principles

• Provide a framework for future lectures

• Introduce immunological terminology

Immune System Function• Normal Immune System:

– Protects against non-self– Defends against microbes, foreign antigens, and tumors– Results in a state of well-being most of the time

• Deficient Immune System:– Cannot protect against non-self– Develop infections and tumors– Results in immunodeficiency diseases

• Hyperactive Immune System:– Over-reacts to stimulus– Can be fatal, e.g. bee sting– Results in allergic and asthmatic diseases

• Blind Immune System:– Cannot distinguish self from non-self– Results in autoimmune diseases

A Conundrum• We live in a environment rife with

microorganisms that seek to grow on or in us. If the microorganisms succeed, we will become ill and might die.

• Because of mutations that arise, we are constantly producing precancerous and cancerous cells. If the cancerous cells grow, we will die.

• Yet, most of us are in a state of well-being most of the time.

• How is this possible? We have a well developed immune system that protects us from microorganisms and cancerous cells.

What Kinds of Microorganisms Seek to Infect Us?

• Viruses

• Bacteria

• Fungi

• Protozoan parasites

• Helminth parasites

Why Don’t Most Microorganisms Cause

Infections?• The skin provides a protective physical

barrier to microorganisms.• Mucous secretions help to protect the nose,

lungs, and other orifices by entrapment.• Saliva (lysozyme) helps to protect the mouth.• However, some breaks in these defenses

inevitably occur and we become infected.

Why Don’t We Die When Infected by Microorganisms?

• Is it because of man-made interventions?– Vaccinations

• Can prevent infections• Available for the past several hundred years

– Antivirals• Very few antivirals are available

– Antibiotics• Can cure established infections or prevent infections• Available for only the past 75 years or so

Why Don’t We Die When Infected by Microorganisms? • Is it because of Innate Immunity?

– Innate immunity stops most infections before they can cause symptoms.

– Innate immunity reduces the number of invading organisms. So, the dose of microorganisms needed to cause symptomatic infection may be hundreds or thousands of microorganisms.

– Still, some microorganisms do evade our innate immunity.

Why Don’t We Die When Infected by Microorganisms? • Is it because of Adaptive Immunity (aka

Acquired Immunity)?– Adaptive immunity does not develop

immediately.– Adaptive immunity primarily protects us

from re-exposure.– When an infection overwhelms innate

immunity and persists for a long enough period of time, adaptive immunity can provide protection (Ex., Mycobacterium tuberculosis).

Why Don’t We Die When Infected by Microorganisms? • Actually, it is a combination of the

following.– Innate Immunity– Adaptive Immunity– Man-made interventions

Types of Immunity

• Innate Immunity– Pre-existing defenses that are non-specific– Pre-existing defenses that do not change

with repeated exposure

• Adaptive Immunity– Reactive defenses that are specific– Reactive defenses that have memory

Introduction to Innate Immunity

Functions That Are Activated When Innate Immunity Is

Stimulated

Three Major Features/Functions of Activated Innate Immunity

• Complement Activation

• Inflammation

• Cell Activation – Cytokine and lymphokine production– Phagocytosis or other killing

How Is Innate Immunity Stimulated?

Stimulation of Innate Immunity

• It is the recognition of patterns that are present in or on microorganisms and not in our cells that stimulates or activates innate immunity.– Because innate immunity relies on pattern

recognition, it is non-specific and does not generate immunologic memory

– Pattern recognition stimulates complement activation and activation of non-specific cell-mediated immunity

Factors of Innate Immunity

Barrier defenses Skin, mucous secretions

Inflammation Low pH, low O2

Phagocytes and other cytotoxic cells

Macrophages, PMNs, NK cells

Soluble mediators Complement, IFNs, ILs, TNFs, chemokines (leukotrienes, prostaglandins

Antimicrobial peptides -defensin, -defensin, cathelicidins

Acute Phase Response proteins C-reactive protein, mannose-binding lectin

What Kind of Patterns Are Recognized by Innate

Immunity?

Molecules Recognized by Pattern Recognition

• Polyanions• Lipoproteins• Lipoteichoic acid• Lipoarabinomannan• Other mannose-

containing compounds

• Lipopolysaccharides• Formyl peptides• Muramyl peptides• Peptidoglycans• Phosphorylcholine

Effects of Pattern Recognition• Non-cellular effects of pattern recognition

– Bind to C-reactive protein– Mannose-binding lectins bind to mannose-

containing molecules, triggering complement– LPS triggers complement activation by the

alternate pathway

• Cellular effects of pattern recognition – Phagocytosis by macrophages and PMNs– Production of reactive oxygen and reactive

nitrogen molecules– Killing by NK cells

Non-Cellular (Soluble) Pattern Recognition by Innate Immunity

Soluble mediators Complement, IFNs, ILs, TNFs, chemokines (leukotrienes, prostaglandins

Antimicrobial peptides -defensin, -defensin, cathelicidins

Acute Phase Response proteins C-reactive protein, mannose-binding lectin

Antimicrobial Peptides:Defensins are cationic proteins 29-35 aa in length produced by

neutrophils, epithelial cells of kidney and pancreas, and by paneth cells in the gut. They kill S. aureus, S. pneumoniae, E. coli, P. aeruginosa, and H. influenzae. They disrupt microbial membrane, block DNA, RNA, protein synthesis.

Cathelicidin, a single protein, has chemotactic activity for neutrophils, monocytes, mast cells, and T cells; degranulates mast cells; and, promotes wound healing.

Acute-Phase Response Proteins: C-reactive proteins bind to polysaccharide on S. pneumoniae

and to phosphoryl choline on many microbial surfaces and act as opsonins. High levels of C-reactive protein are associated with higher risk of coronary heart disease.

Mannose-binding lectins recognize mannose-containing

patterns on microbes but not on host cells. They direct complement to attack the microbes to which they bind.

Cellular (Cell-Associated) Pattern Recognition by Innate Immunity

• Toll-Like Receptors (TLRs)– 11 TLRs have been identified– Responsible for recognition

and binding to patterns present in/on viruses, bacteria, parasites, and fungi

– Each TLR recognizes a distinct repetoire of highly conserved molecules on the different pathogens

– Extracellular domain has leucine-rich repeats (LRRs)

– Intracellular domain has three conserved sequences

Ligands of Different TLRs

TLR Signalling• Different TLRs are

found on different cells. Most are found on macrophages, PMNs, and B cells.

• TLRs can serve as heterodimers (TLRs 1, 2, and 6), homodimers (TLR4 and TLR5?), or monomers (TLRs 3, 7, 8, and 9).

• Some TLRs are surface proteins, others are internal proteins.

• Ligand binding to TLR activates phosphorylation of second messengers, activating NFB and turning on transcription.

Cellular Responses to TLR Signaling

• Activation of the transcription factor NFB causes– Expression of pro-inflammatory genes

• Production of prostaglandins and leukotrienes• Production of interleukins and other cytokines

– Increased phagocytosis and synthesis of reactive oxygen and nitrogen molecules in macrophages and neutrophils

– Increased efficiency of antigen presentation

Complement Activation

Three Pathways of Complement Activation

• Classical pathway (adaptive immunity)– C1q binds to Ag:Ab complex cleaves C4 and and C2 to form C4b2a

(C3 convertase) and C4b2b (C5 convertase) to initiate a cleavage cascade C1qr2s2 - C4 - C2 - C3 - C5. C3b also cleaves C5 to C5b.

– C5b binds to a membrane and initiates formation of the attack complex (C6, C7, C8, C9).

– C9 binds to C6, C7, C8 to form a pore in the membrane.

• Lectin pathway (innate immunity)– Mannose binding protein bound to bacterial carbohydrates (lectins)

mimics C1q, binds and activates serum proteases, and activates C4 cleavage. This initiates the rest of the cascade.

• Alternate pathway (innate immunity)– C3 is spontaneously cleaved or cleaved to C3b by a serum protease

activated by bacteria. Normally this C3b would turn over.– C3b binds to bacterial cell walls (Gram + and Gram - [LPS]), yeast

cell walls, and viral envelopes and is stabilized by this binding.– Bound C3b, in turn, binds to Factors B and D and properdin to

become activated as C3 convertase and cleaves more C3 to C3b which then cleaves C5 to C5b, initiating the rest of the cascade.

Complement Pores Versus Perforin Pores

Important Products of Complement Activation

• C3 cleaved to C3a and C3b

• C4 cleaved to C4a and C4b

• C5 cleaved to C5a and C5b

Activities of C3a, C4a, And C5a

• Chemotactic factors that increase directional migration of PMNs and macrophages

• Activating factors that cause PMNs and macrophages to degranulate– Release of digestive enzymes (cauterization)

– Release of adhesion molecules

• Activating factors that cause respiratory burst in PMNs and macrophages

• Anaphylactic factors that cause mast cells and basophils to degranulate releasing large quantities of histamine (vascular collapse and shock)

• Potency: C5a >>> C3a >>> C4a

Activity of C3b and C4b

• C3 and C4 are cleaved to highly reactive C3b and C4b, respectively.

• C3b and C4b are deposited on any surface with an exposed amine or hydroxyl, such as a bacterium.– Act as opsonins– Act in a feedback loop to continue cleaving C3

• C3b and C4b can be down-regulated– When C3b and C4b bind to body cells they are inactivated

by membrane bound decay-accelerating factor (DAF).– Soluble factors Factor H, Factor 1, and anaphylatoxin

inactivator can block or inactivate C3b and C4b.

Activity of C5b

• Binds to microorganisms or host body cells• Acts as a focal point for the deposition of C6-C9

– C9 is the critical part of the membrane attack complex that punches a hole in the cell wall or cell membrane killing bacteria.

• C5b can be down-regulated– Soluble S protein can bind to soluble C5b and prevent its

binding to a cell membrane. – Body cells have protectin (CD59) and homogolous

restriction factor (HRF) on their surface that bind to C8, preventing C8 binding of C9 and preventing formation of the membrane attack complex.

Genetic Deficiencies Occur For Each Complement Component And Regulatory Factor

• Deficiencies in C1q, C1r, C1s, C4, and C2– Predispose individuals to systemic lupus erythematosus,

glomerulonephritis, and vasculitis, due to a lack of C3b generation and resulting in a lack of clearance of immune complexes

– Increased incidence of Streptococcus and Staphylococcus infections because of reduced opsonization

• Deficiencies in C3 are most severe, with increased immune-complex disease and recurrent bacterial infections.

• Deficiencies in C5 and the membrane attack complex lead to recurrent Neisseria infections.

• Deficiency in C1 inhibitor (C1Inh) cause hereditary angioedema, a disease with trauma-induced or spontaneous edema. Airway obstruction can be fatal.

Inflammation

Hallmarks of Inflammation

• Influx of fluid (edema)

• Increased temperature (hyperthermia)

• Decreased oxygenation (local hypoxia)

• Influx of white blood cells (extravasation)

Triggers of Inflammation• Complement C5a stimulation of basophil and mast

cell degranulation and activation– Histamine = increased vascular permeability– Prostaglandin E2 = vasodilation, increased vascular

permeability– Leukotriene D2 = neutrophil chemotaxis, increased vascular

permeability– Leukotriene D4 = increased vascular permeability

• Macrophages– TNF = can cause fever; stimulates expression of E-selectin– IL-1 = endogenous pyrogen; stimulates expression of E-

selectin– IL-8 = chemotaxis

• NK cells – IFN- = activation of phagocytic cells and NK cells

Extravasation of PMNs

Cell Activation

Cells of the Innate Immune System

• Polymorphonuclear Neutrophils (PMNs)

• Eosinophils

• Basophils and Mast cells

• Macrophages/Dendritic Cells/Interdigitating Cells

• Natural Killer cells (NK cells)

Origin and Distribution of Cells of the Immune System

Polymorphonuclear Leukocytes

• Called PMNs or polymorphonuclear leukocytes for their tri-lobed nuclei

• Also know as Polys, neutrophils or granulocytes

• Contain granules that do not stain with either acidic or basic stains

• Mediators of innate immunity

• Short-lived cells

PMN Functions• Primary phagocytic cells in the blood• First cells to migrate to a site of inflammation or

infection– Effector cells of acute inflammation or infection

• Phagocytize bacteria, viruses, and antigens• Phagosome fuses with granules forming a

phagolysosome– Contain enzymes that digest bacteria– Contain myeloperoxidases that make reactive oxygen

species and reactive nitrogen species

• Have receptors for Fc portion of antibodies, so can also kill by antibody-dependent cellular cytotoxicity

Eosinophils

• Another subset of polymorphonuclear leukocytes

• Have a bi-lobed nucleus

• Contain eosinophilic granules

Eosinophil Functions

• Eosinophilic granules contain agents that are anti-parasitic

• Release histaminase and aryl sulphatase that inactivate histamine and leukotrienes to reduce the inflammatory response and reduce PMN recruitment

• Activated by complement C5a and C3a to degranulate

• Mediators (with Th1 cells and basophils) of the delayed reaction of the allergic response

Basophils

• Multilobed, polymorphonuclear leukocytes

• Express Fc receptors for IgE and, thus have IgE on their surface

• Contain an abundance of basophilic granules

Basophil and Mast Cell Functions

• Basophils – Found at low levels in the blood– Mediators of the delayed reaction of the allergic response

• Mast cells – Mononuclear cells with analogous function to basophils found in

tissues (skin, lungs)– Also contain basophilic granules; IgE on their surface– Mediators of the immediate reaction of the allergic response

• Both release proinflammatory cytokines– Release preformed histamine when IgE on surface is cross-linked

by antigen– Synthesize and release prostaglandins and leukotrienes

• Activated by complement C5a and C3a to degranulate

Monocytes• Differentiate to form

macrophages in the peripheral tissues where they are the first line of defense against microbial invasion– Kupfer cells in liver– Microglial cells in brain– Bronchial alveolar

macrophages in lung

• Contain a horse-shoe shaped nucleus and cytoplasmic granules

• Long-lived cells

Macrophage Functions• Late migrators to sites of inflammation (effector cells

of chronic inflammation)• Major producers of cytokines and lymphokines

– IFN-: antiviral properties– IL-1, IL-6, and TNF-: mediators of fever– CXCL8: chemotactic factor recruits PMNs, basophils, and T

cells– IL-12: activation of NK cells and CD4 Th1 helper T cells

• Prodigious phagocytic cells: professional phagocytic cells (the “big boys”) that can be highly activated by IFNs to phagocytize and kill, using reactive oxygen and nitrogen species.

• Also present antigen to the adaptive immune system

Natural Killer Cells

• Lymphocytes that participate in innate immunity

• Also known as large granular lymphocytes

• Contain acentric and slightly irregular nucleus, with granules visible in the cytoplasm

Natural Killer Cell Functions• NK cells recognize

damaged cells by their deficiency in MHC antigens (HLA in humans)– Virus-infected cells– Tumor cells

• Exposure to IFNs (especially IFN-) highly activates NK cell killing function (20-100X)

• IL-12 and TNF- activate NK cells to secrete cytokines, principally IFN-

Deficiencies of Innate Immunity• Congenital neutropenia

– Lack of GM-CSF – Frequent bacterial infections

• Glucose-6-phosphate dehydrogenase deficiency (G6PD) – Unable to produce NADPH by pentose phosphate pathway, buildup of reduced

glutathione– RBC denaturation and hemolysis

• Chronic granulomatous disease– Inability to produce hydrogen peroxide and hypochlorous acid– Inability to kill phagocytosed bacteria

• Leukocyte adhesion deficiency (LAD)– Lack of integrin subunit, the common chain– Inability to recruit innate immune cells to site of inflammation– Increased susceptibility to bacterial, fungal, and viral infections.

• Complement defects– Increased susceptibility to bacterial infections– Reduced ability to remove immunocomplexes

• Chediak-Higashi Syndrome– Defect in gene LYST (CHS1), a lysosomal trafficking gene that affects lysosomes

and melanosomes– Increased susceptibility to bacterial infections.

Major Virus Diseases• SARS Coronavirus• West Nile encephalitis Flavivirus• Yellow fever Flavivirus• Hepatitis B Hepadnavirus• Chickenpox Herpesvirus• Mononucleosis Herpesvirus• Influenza Orthomyxovirus• Measles Paramyxovirus• Mumps Paramyxovirus• Poliomyelitis Picornavirus• Jaundice Picornavirus• Smallpox Poxvirus• AIDS Retrovirus• Rabies Rhabdovirus• Common Cold Rhinovirus• Diarrhea Rotavirus• Rubella Togavirus

Major Bacterial Diseases

• Trachoma Chlamydia trachomatis• Bacillary dysentery Shigella flexneri• Food poisoning Salmonella enteritidis,

typhimurium• Plague Yersinia pestis• Tularemia Pasteurella tulaensis• Typhoid fever Salmonella typhi• Gonorrhea Neisseria gonorrhoeae• Meningococcal meningitis Neisseria meningitidis• Meningitis, pneumonia Haemophilus influenzae• Legionnaire’s disease Legionella pneumophila• Whooping cough Bordetella pertussis• Cholera Vibrio cholerae• Anthrax Bacillus anthracis• Diphtheria Corynebacterium diphtheriae• Tetanus Clostridium tetani• Gastroenteritis Clostridium difficile• Boils, wound infection Staphylococcus aureus• Pneumonia, scarlet fever Streptococcus pneumoniae• Tonsilitis Streptococcus pyogenes• Leprosy Mycobacterium leprae• Tuberculosis Mycobacterium tuberculosis• Respiratory disease Mycoplasma pneumoniae• Typhus Richettsia prowazeckii• Lyme disease Borrelia burgdorferi• Syphillis Treponema pallidum

Fungal, Protozoan, Helminth Diseases

• Fungal– Aspergillosis Aspergillus species– Athlete’s foot Tinea pedis– Candidiasis, thrush Candida albicans– Pneumonia Pneumocystis jirovecii

• Protozoan parasites– Leishmaniasis Leishmania major– Malaria Plasmodium falciparum– Toxoplasmosis Toxoplasma gondii– Trypanosomiasis Trypanosoma brucei

• Helminth parasites– Common roundworm Ascaris lumbricoides– Schistosomiasis Schistosoma mansoni