Complement system

Complement System

The complement system includes serum and membrane-bound proteins that functions in both natural and acquired defense system. These proteins are highly regulated and interact via a series of proteolytic cascades. The term ‘complement’ refers to the ability of these proteins, to complement (augment) the effects of other components of the immune system (e.g. antibody). Complement has several main effects: 1. Lysis of cells (e.g. bacteria, tumor cells and enveloped virus directly). 2. Production of peptide fragments that participate in inflammation and attract phagocytes. 3. Opsonization of organisms and immune complexes for clearance by phagocytosis and enhancement of antibody-mediated immune responses. Further more the complement goes to work, as soon as an invading microbe is detected; the system makes up an effective host immune defense long before specific host defenses are mobilized.

COMPLEMENT ACTIVATION The complement system works as a cascade. A cascade is a set of reactions that amplify some effects, i.e. more products are formed in the second reaction than the first, still more in the third and so on. Of the proteins, so far identified in the complement system, 13 participate in the cascade itself, seven

7

activate or inhibit reactions in the cascade. The components of the classical pathway are numbered from C1 to C9 and the reaction sequence is C1-C4-C2-C3-C5-C6-C7C8-C9. Up to C5, activation involves proteolytic cleavage, liberating smaller fragments from C2 through C5 except for C2, where for historical reasons the larger fragment that remains bound to the complex is termed C2a, the smaller fragments are by the letter ‘a’ (e.g. C4a) and the larger fragments by letter ‘b’ (e.g. C5b). Activation of the complement system can be initiated, either by antigenantibody complexes or by a variety of nonimmunological molecules. Sequential activation of complement components occurs via three main pathways.

Classical Pathway Only, immunoglobulin M (IgM) and immunoglobulin G (IgG) (IgG1, IgG2, IgG3 not IgG4) activate or fix complement via the classical pathway. The formation of an antigenantibody complex induces conformational changes in the Fc portion of the IgM molecule that expose a binding site for the C1 component of the complement system. C1 in serum is a macromolecular complex consisting of C1q and two molecules of each of C1r and C1s, held together in a complex (C1qr2, s2) stabilized by Ca2+ (Fig. 7.1). One molecule of IgM or two molecules of IgG can initiate the process. C1q binding in the presence

78 Textbook of Immunology of Escherichia coli, Salmonella of low virulence), viruses (parainfluenza virus, human immunodeficiency virus) and even apoptotic cells interact with C1q directly, causing C1 activation and there by classical pathway.

Alternative Pathway (Properdin Pathway)

Fig. 7.1: C1 macromolecular complex consisting of C1q and two molecules of each C1r and C1s (1qr2s2)

of calcium ions, leads to sequential activation of C1r and C1s. C1s cleaves C4 and C2 to form C4bC2a, the latter is an active C3 convertase, which cleaves C3 molecule into two fragments as C3a and C3b. C3a is an anaphylatoxin. C3b forms complex with C4bC2a producing a new enzyme called C5 convertase (C4bC2aC3b). C5 convertase cleaves C5 to form C5a and C5b. C5a is an anaphylatoxin and chemotactic factor. The C5b component is extremely labile and becomes inactive within 2 minutes, unless C6 binds to it and stabilizes its activity. Attachment of C5b to the bacterial membrane initiates formation of the membrane attack complex (MAC) and lysis of the cell (Fig. 7.2). The attachment of C5b leads to the addition of components C6, C7 and C8. C8 provides a strong anchor into the membrane and facilitates the subsequent addition of multiple C9 molecules to form a pore in the membrane. Loss of membrane integrity results in the unregulated flow of electrolytes and causes lysis and death of cell. Non-immunological classical pathway activators: Certain bacteria (e.g. some strains

The alternative pathway does not involve immune complex. Many unrelated substances such as bacterial endotoxin, IgA and IgD antibodies, cobra venom factor, nephritic factor (protein present in the serum of glomerulonephritis) initiate alternative pathway. This pathway does not involve C1, C2 or C4. C3 is cleaved so that C3 convertase is generated via the action of factors B, D and properdin. The alternative C3 convertase (C3bBb) generates more C3b. The additional C3b binds to the C3 convertase to form C3bBbC3b, which is the C5 convertase of the alternative pathway that generates C5b, leading to production of MAC described earlier (Fig. 7.3).

Mannan-binding Lectin Pathway Lectins are proteins that bind to specific carbohydrate. Mannose-binding lectin (MBL) or mannan-binding lectin is an acute phase protein, which binds sugar residues like mannose, found on microbial surface (e.g. Listeria species, Salmonella species, Candida albicans). MBL level can rise rapidly in response to infection, inflammation and other forms of stress. MBL once bound to appropriate mannose containing residues, can interact with MBL-activated serum protease (MASP). Activation of MASP leads to subsequent activation of components C2, C4 and C3 (Fig. 7.4).

Regulation of the Complement System Following the activation of the complement, its components and split products are capable of attacking host cells, as well as foreign

Complement System 79

Fig. 7.2: Complement pathway

80 Textbook of Immunology Regulation After Assembly of Convertases

Fig. 7.3: Terminal or membrane attack complex (MAC) of complement. The MAC forms pore on the surface of microbes causing lysis.

cells and microorganisms. Elaborate regulatory mechanisms have been evolved, to direct activity on designated targets rather than the host cells. First of all, the components formed in the complement pathway are highly labile substances that stabilized by reaction with other components. Secondly, a series of regulatory proteins can inactivate various complement components disallowing over activation and tissue damage. The activation of complement system is regulated at different stages.

Regulation Before Assembly of Convertase Activity 1. C1 inhibitor (C1 INH) binds to C1r2s2, causing dissociation from C1q. 2. Association of C4b and C2a is blocked by binding C4b-binding protein (C4b BP), complement receptor type1 (CR1) or membrane cofactor protein (MCP). 3. Inhibitor-bound 4b is cleaved by factor I to form bound 4d and soluble 4c. 4. In alternate pathway, CR1, MCP or factor H prevent binding of C3b and factor B. 5. Inhibitor-bound C3b is cleaved by factor I to form iC3b and soluble C3f fragment. Further cleavage of iC3b by factor I releases C3c and leaves C3dg bound to the membrane.

1. C3 convertases are dissociated by C4b BP, CR1, factor H and decay-accelerating factor (DAF). 2. Factor ‘P’ (properdin) protects C3b and stabilizes this C3 convertases of the alternative pathway. 3. Anaphylatoxin inactivator is an alpha globulin that enzymatically degrades C3a, C4a and C5a, which are anaphylatoxins released during ‘C’ cascade.

Regulation at Assembly of Membrane Attack Complex 1. ‘S’ protein prevents insertion of C5b678 MAC components into the membrane. 2. Homologous restriction factor (HRF) and membrane inhibitor of reactive lysis (MIRL or CD59) binds C81 preventing assembly of poly C9 and blocking formation of MAC.

Biological Consequences of Complement Activation Complement plays an important role in humoral immunity by amplifying the response and converting into an effective defense mechanism to destroy invading pathogens. The MAC mediates cell lysis, while the other complement components or split products take part in inflammation, opsonization of antigen, viral neutralization and clearance of immune complexes. Many biological activities of the components are the outcome of the interactions of complement fragments with their receptors expressed on various cells. Some of the complement receptors also help in regulating complement activity by binding, biologically active complement components and degrading them into inactive form. The complement receptors and their primary ligands, which include various complement components and their

Complement System 81

Fig. 7.4: Mannose-binding lectin (MBL) pathway of complement activation. MBL bound to mannose containing residues (–CHO) interact with MBL-activated serine proteases (MASP). Activation of MASP leads to activation of component C2, C4 and C3.

proteolytic products are listed (Table 7.1). The major biological effects of the complement (Figs 7.5A and B) are: 1. Opsonization: Cells, immune complexes are easily phagocytosed much more efficiently in the presence of C3b receptors in most of the cells. 2. Chemotaxis: C3a and C5a stimulate the movement of neutrophils. 3. Inflammation (C3a, C4a and C5a). 4. Anaphylatoxins: C3a, C4a and C5a can stimulate the degranulation of mast cells, releasing mediators.

5. Phagocytic function is carried out by C3b and C5b. 6. Increased capillary permeability: C2 kinins are vasoactive amines, which increases capillary permeability. 7. Virus neutralization: May require participation of ‘C’ for neutralization of herpes virus by IgM antibody. 8. Bacteriolysis or cytolysis: Insertion (C5b 6789, MAC) into the cell surface leads to killing or lysis of erythrocytes, bacteria and tumor cells. 9. Immune adherence: ‘C’ bound to immune complex adhere to erythrocytes or to non-primate platelets. The immune adherence (C3 and C4) contributes to defense against pathogenic microorganisms, since such adherent particles are rapidly phagocytosed. Besides the biological effects stated above, ‘C’ participate in the cytotoxic (type II) and immune complex (type III) hypersensitivity reactions and play a major role in the pathogenesis of autoimmune hemolytic anemia, paroxysmal nocturnal hemoglobinuria and hereditary angioneurotic edema. Endotoxin is an effective activator of alternate ‘C’ pathway. In endotoxic shock, there is massive platelet lysis and release of large amount of platelet factor leads to disseminated intravascular coagulation (DIC) and thrombocytopenia.

Defects in the Complement System Deficiency in the complement system affects both innate and acquired immunity. A number of gene defects involving complement components or their regulatory proteins lead to susceptibility to infections or risk to autoimmune disorders. Homozygous deficiency in any of the earlier components (C1q, C1r, C1s, CH, C2) exhibit similar symptoms, notably marked increase in immune complex disorders, such

82 Textbook of Immunology

Figs 7.5A and B: Major biological effects of the complement. A. Classical and alternative pathways of the complement cascade. Although the two pathways are initiated in different ways, they combine to activate the complement system; B. Activation of the classical complement pathway. In this cascade, each complement protein activates the next one in the pathway. The action of C3b is critical for opsonization and along with C5b for formation of membrane attack complexes. C4a, C3a and C5a also are important to inflammation and phagocyte chemotaxis.

Complement System 83

Table 7.1 Complement binding receptors Receptor

Major ligands

Activity

Cellular distribution

CR1 (CD35)

C3b, C4b

Blocks formation of C3 convertase, binds immune complex to cell

B cells, T cells, macrophages, monocytes, neutrophils, erythrocytes, etc.

CR2 (CD21)

C3d, C3dg, iC3b

Part of B cell coreceptor, binds Epstein-Barr virus

B cells, some T cells and follicular dendritic cells

CR3 (CD11b/18)

iC3b

Bind immune complexes enhancing their phagocytosis, binds cell adhesion molecules on neutrophils

Monocytes, macrophages, neutrophils, natural killer (NK) cells, etc.

C3a/C4a receptor

C3a, C4a

Initiate degranulation of mast cells and basophils

Mast cells, basophils, granulocytes

C5a receptor

C5a

Induces degranulation of mast cells and basophils

Mast cells, basophils, monocytes, macrophages, platelets, etc.

CR4 (CD11c/18)

as systemic lupus erythematosus (SLE), glomerulonephritis and vasculitis. In addition, the individuals with such complement deficiency are more prone to suffer from repeated pyogenic infections by bacteria such as staphylococci and streptococci. Deficiency of factor D and properdin leads to increased susceptibility to infection, specially by Neisseria, because of impaired alternative pathway.

level of C1 inactivation of classical pathway. As a result uncontrolled inflammatory episodes occur involving vascular system, gastrointestinal (GI) tract and respiratory tract. Deficiency in DAF or CD59 allow accumulation of complement complexes including the MAC on host cell membrane causing cell injury.

C3 deficiency results in serious problems with recurrent infections and with immune complex-mediated diseases because of the central position of C3 in all three of the complement activation pathways.

Complements are synthesized by liver, spleen and phagocytic cells.

Deficiency in the complement components (C5, C6, C7, C8 and C9) involved in MAC, develop recurrent meningococcal and gonococcal infections. Individuals, deficient of MAC components, rarely suffer from immune complex diseases. This suggests that they produce enough C3b to clear immune complex by opsonization.

1. What is complement? Explain in detail about complement pathway. 2. Discuss the complement-mediated serological tests. 3. Discuss the biological activities of complements.

Deficiency in regulatory proteins causes serious disorders. The most common is hereditary angioneurotic edema with reduced

Synthesis of Complement

STUDY QUESTIONS Essay Questions

Short Notes 1. Complement. 2. Complement deficiency diseases. 3. Complement receptors.

84 Textbook of Immunology 4. Mannan-binding lectin pathway of complement activation.

SUGGESTED READING 1. Black JG. Microbiology. Principles and Applications, 3rd edition. USA: Prentice Hall College div; 1996. 2. Coligan JE, Kruisbeek AM, Margulies DH, et al. Current protocols in immunology. New York: Wiley; 1997. 3. Daniel P Stites. Basic and Clinical Immunology, 8th edition. USA: Lange (Medical Book); 2007. 4. Goldsby RA, Kindt Thomas J, Osborn Barbara A Kuby. Immunology, 6th edition. New York: WH Freeman and Company; 2007.

5. Greenwood D, Slack R. Medical Microbiology, 15th edition; 1997. 6. Jawetz. Melnick and Adelberg’s Medical Microbiology, 25th edition. USA: McGraw Hill Lange Basic Science; 2010. 7. Male David, Brostoff Jonathan, Roth David B, et al. Immunology, 7th edition. Mosby: Elsevier; 2006. 8. Thao Doan, Roger Melvold, Susan Viselli, et al. Lippincott’s illustrated reviews: Immunology, 1st Indian print. Baltimore, USA: Lippincott Williams and Wilkins; 2008. 9. Tortora, Funke, Case. Microbiology an Introduction, 6th edition; 1998.