The Complement System

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Innate and adaptive immunity

Cytokines APCs DCs ADCC

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, thereby promoting a stable adhesive contact with an appropriate phagocytic cell. Opsonin - A substance (e.g., an antibody or C3b) that promotes the phagocytosis of antigens by binding 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 by antibodies (the major effector of the humoral branch of 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 forms as 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 18 polypeptide chains that associate to form six collagen-like triple helical arms, 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

Structure of the C1 macromolecular complex

C1q molecule

(b)

Kuby J et al., Immunology 2003

C1 binding to the Fc portions of IgM and IgG

The formation of an Ag-Ab complex induces conformational changes in the Fc portion of the pentameric IgM molecule 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 interaction to occur.
Abbas AK & Lichtman AH. Cellular and Molecular Immunology 5th ed. 2003

(a)

Model of pentameric IgM in planar form

(b)

Model of pentameric IgM in staple form

Kuby J et al., Immunology 2003

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

(c)

(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-catalytically and 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.

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

C3 contains an unstable thioester bond.

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.

C3b undergoes hydrolysis by the time it has diffused 40 nm away from the convertases.
Abbas AK & Lichtman AH. Cellular and Molecular Immunology 5th ed. 2003

_____

Hydrolysis of C3 by C3 convertase C4b2a

(a labile internal thioester bond)

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.
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-surface constituents that are foreign to the host

Schematic diagram of intermediates in the formation of bound C5b by the alternative pathway of complement activation
1.C3 hydrolyzes spontaneously, C3b fragment attaches to foreign surface. b 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.
*More than 2 x 106 molecules of C3b can be deposited on an antigenic 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 to certain 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 defense mechanisms in early childhood.

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

*MBL is an acute phase protein produced 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 terminal complement components.
The C5b component becomes inactive within 2 minutes unless C6 binds to it and stabilizes its 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)

poly-C9 complex

(b) Complement-induced lesions on the membrane of a red blood cell

Kuby J et al., Immunology 2003

The main components and effector actions of complement

Antibody-mediated mechanisms for combating infection by extracellular bacteria

The critical function of the complement system in converting a humoral antibody response into an effective defense mechanism.

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

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

(a)

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

(b)

Kuby J et al., Immunology 2003

Opsonins: C3b, C4b, iC3b

CR1: 5,000/resting phagocytes 50,000/activated cells

Clearance of circulating immune complexes

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.

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

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 surface of 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

Electron micrographs of negatively stained preparations of EB virus

(a) (b) (c)

Control without antibody

Antibodycoated particles

Particles coated with antibody and complement

Kuby J et al., Immunology 2003

Regulation of the complement system

Regulation of the complement system

Inclusion of highly labile components that undergo spontaneous inactivation if they are not stabilized 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).

(serine protease inhibitor)

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

(serine protease)

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.

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 decayaccelerating Factor (DAF or CD55).

C2a C4b

CR1

Bb C3b
Kuby 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 system depend 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

104 molecules of mIgM had to be engaged by antigen for B-cell activation to occur when the co-receptor was not involved.

B-cell coreceptor

When CD19/CD2/CD81 co-receptor was crosslinked to the BCR, only 102 molecules of mIgM had to be engaged for B-cell activation.

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

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 the peptide anchor (glycolipid GPI anchor) that binds DAF and MIRL to the cell membrane. The defect identified in PNH lies early in the path to formation of 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 neutrophils to 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 diseases may bind to vascular endothelium and kidney glomeruli and activate complement (MAC generation). - It initiates the acute inflammatory responses that destroy the vessel walls or glomeruli and lead to thrombosis, ischemic damage to tissues, and scarring. - Some of the late complement proteins may activate prothrombinases 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 because they express high levels of a C3 and C4 inhibitor called Crry. - Crry may block maternal alloantibody-mediated damage through the classical pathway of complement activation. - Crry-deficient embryos die before birth and show evidence of 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, or lectin pathways, each of which is initiated differently. 3. The three pathways converge in a common sequence of events that leads to generation of a molecular complex that causes cell lysis. 4. The classical pathway is initiated by antibody binding to a cell target; reactions of IgM and certain IgG subclasses activate this pathway.

5. Activation of the alternative and lectin pathways is antibodyindependent. 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 system mediates opsonization of bacteria, activation of inflammation, and clearance of immune complexes. 7. Interactions of complement proteins and protein fragments with receptors on cells of the immune system control both innate 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 to infection to tissue damage caused by immune complexes.