The complement system in immunology
- 1. • SUBMITTED TO: • Dr. BINDU BATTAN • ASSISTANT PROFESSOR • (DEPARTMMENT OF BIOTECHNOLOGY) • IMMUNOLOGY • SUBMITTED BY: • SHASHI (1) • TEENA(5) • MEHEK(9) • CHITRANGNA(15) • KIRTI(16) • (MSc. FINAL YEAR STUDENTS)
- 2. COMPLEMENT SYSTEM • Major effector of the humoral branch of immune system . •Research on complement began in 1890 when Jules Border at Institute Pasteur in Paris showed that sheep antiserum to the bacterium Vibrio cholerae caused lysis of the bacteria and that heating the antiserum destroyed it's activity. •Paul Ehrlich coined term 'complement' defining it as"activity of blood serum that completes the action of antibody". Activation of complement cascade may be initiated by bye several proteins that circulate in normal serum.These molecules, termed accused face protein acute phase protein, dresses pattern recognition capacity and undergo changes in concentration during inflammation.
- 3. Functions of the complement • Lysis of cells ,bacteria and viruses • Opsonization, which promotes phagocytosis of particulate antigen • binding to specific complement receptors on cells of the immune system, triggering specific cell functions, inflammation, and secretion of immunoregulatory molecules • Immune clearance, which removes immune complexes from the circulation and deposits them in the spleen and liver
- 5. Components of complement • Soluble proteins and glycoproteins synthesized mainly by liver hepatocytes • Constituted 5% of the serum global in fraction in functionally inactive form as proenzyme or zymogens. • Complement components are designated by numerals (C1-C8),by letter symbols (eg factor D) or by trivial names(eg homologous restriction factor). Peptide fragment formed by activation of component are denoted by small letters. • the larger fragments bind to the target near the site of activation and the smaller fragment diffuse from the side and can initiate localised inflammatory response is binding to the specific receptors. • The complement fragments interact with one another to form functional complexes, those complexes that have enzyme attack activity are designated by bar over the number of symbol(eg C42a, C3Bb)
- 6. Complement activation • The early steps culminating in the formation of C5b can occur by classical pathway, the alternative pathway or the lectin pathway. • the final steps that lead to the formation of the membrane attack complex or MAC are identical in all three pathways.
- 7. Classical Pathway • The classical pathway of complement activation is considered part of adaptive immune response since it begins with the formation of antigen-antibody complexes. • These complexes may be soluble, or they may be formed when an antibody binds to antigenic determinants, or epitopes, situated on viral, fungal, parasitic, or bacterial cell membranes. • Only complexes formed by IgM or certain subclasses of IgG antibodies are capable of activating the classical complement pathway. • The initial stage of activation involves the complement components C1, C2, C3 and C4, which are present in plasma as zymogens.
- 8. • The formation of an antigen-anribody complex induces conformational changes in the nonantigen-binding (Fc) portion of the antibody molecule. This conformational change exposes a binding site for the C1 component of complement. • In serum, C1 exists as a macromolecular complex complex consisting of one molecule of C1q and two molecules each of serine proteases, C1r and C1s, held together in a Ca+² stabilized complex. • The C1q molecule itself is composed of 18 polypeptide chains that associate to form six collagen like triple helical arms, the tips of which bind the CH² domain of antigen bound antibody molecule. • Binding of C1q to the CH²domains of Fc regions of the antigen complexed antibody molecule induces a conformational change in one of the C1r molecules that converts it to an avtive serine protease enzyme.This C1r molecule then cleaves and activate its partner C1r molecule.
- 10. • The two C1r proteases then cleave and activate the two C1s molecules.C1s has two substrates, C4 and C2.C4 is activated when C1s hydrolyzes a small fragment (C4a) from the amino terminus of one of its chains. • The C4b fragment attaches covalently to the target membrane surface in the vicinity of C1, & then binds C2.On binding C4b, C2 becomes susceptible to cleavage by the neighboring C1s enzyme, and the smaller C2b fragment diffuses away, leaving behind an enzymatically active C4b2a complex. C4b2a complex is called C3 convertase. • The membrane bound C3 convertase enzyme, C4b2a,now hydrolyzes C3, generating two unequal fragments; the small anaphylatoxin C3a and the pivotal fragment C3b.
- 12. • C3b acts in three important and different way to protect the host. ① Similar to that of C4b, C3b binds covalently to microbial surfaces, providing a molecular "tag" & thereby allowing phagocytic cells with C3b receptors to engulf the tagged microbes.This process is called Opsonization. ② C3b molecules can attach to the Fc portion of antibodies participating in soluble antigen-antibody complexes. These C3b - tagged immune complexes are bound by C3b receptors on phagocytes or red blood cells, & are either phagocytosed, or conveyed to the liver where they are destroyed. ③ Some molecules of C3b bind the membrane - localized C4b2a enzyme to form the trimolecular, membrane bound, C5 convertase complex C4b2a3b.The C3b component of this complex binds C5 , & the complex then cleaves C5 into two fragments: C5b and C5a. • This trio of tasks accomplished by C3b molecule places it right at the centre of complement attack pathways.
- 15. LECTIN PATHWAY • Also called as MBL pathway. • The pathway is independent of antibodies for its activation like classical pathway • For initiation of complement activation process this pathway uses lectin proteins that recognise specific carbohydrate components primarily found on microbial surface as it’s receptor molecules.
- 16. MBL-mannose binding lectin, the first lectin demonstrated to be capable of Initiating complement activation. MBL are found in several microorganisms such as Salmonella, Listeria and Niesser of bacteria. Cryptococcus neoformans and Candida albicans strains. MBL also recognise structure in addition to mannose, including N- acetylglucosamine, D-glucose and L-fucose.
- 19. LECTINPATHWAY • After MBL binds to the surface of a cell or a pathogen, MBL associated Serine protease, MASP- 1 and MASP-2, binds to MBL. • The active complex formed by this association causes cleavage and activation of C4 and C2 . • The MASP-1 and MASP-2 proteins have structural similarity to C1r and C1s and mimic their activities . • This means of activating the C2-C4 components to form a C3 andC5 convertase without need for specific antibody binding represents an important innate defence mechanism comparable to the alternative pathway , but utilising the elements of the classical pathway except for C1 protein.
- 21. TheAlternativePathway • The alternative pathway generates bound C5b, the same product that the classical pathway generates, but it does so without the need for antigen-antibody complexes for initiation. • Because no antibody is required, the alternative pathway is a component of the innate immunesystem. • This major pathway of complement activation involves four serum proteins: C3,factor B,factor D,andproperdin. • The alternative pathway is initiated in most cases by cell-surface constituents that are foreign to the host . For example, both gram- negative and gram-positive bacteria have cell-wall constituents that canactivate the alternativepathway.
- 22. • In the classicalpathway,C3is rapidly cleaved to C3aand C3bby the enzymatic activity of the C3convertase. In the alternative pathway, serum C3,which contains an unstable thioester bond, is subject to slow spontaneous hydrolysisto yield C3aand C3b. • The C3b component can bind to foreign surface antigens (such as those on bacterial cells or viral particles) or even to the host’s own cells. • 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 cellmembrane. • Because many foreign antigenic surfaces (e.g., bacterial cell walls, yeast cell walls, and certain viral envelopes) have only low levels of sialic acid, C3b bound to these surfaces remains active for alonger time.
- 30. Regulation of complement system
- 31. Regulato -ry Proteins 1. Factor H • Regulate alternative pathway. • Reduce amount of C5 convertase available . • With both cofactor activity for the factor (-mediated C3b cleavage, and decay accelerating activity. 2. Cl inhibitor • Important regulator of classic pathway . • A serine protease inhibitor (serpin). • It acts by forming a complex with the C1 proteases, C1r2s2, causing them to dissociate from C1q and preventing further activation of C4 or C2 . • C1INH inhibits both C3b and the serine protease MASP2. • It is the only plasma protease capable of inhibiting the initiation of both the classical and lectin complement pathways.
- 32. Regulat -ory Proteins 3. Factor I • Cleaves cell-bound or fluid phase C3b and C4b into inactivate fragments C3b and C4b . 4. Decay accelerating factor (DAF) • Glycoprotein on surface of human cells . • Accelerate the decay of the C4b2a on the surface of host cells, including decay accelerating factor, or DAF (CD55), CR1, and C4BP (C4 binding protein) . • These decay accelerating factors cooperate to accelerate the breakdown of the C4b2a complex into its separate components. • Th enzymatically active C2a diffuses away, and the residual membrane-bound C4b is degraded by factor I. • Acts on both classical and alternative pathway.
- 33. Regulat ory Proteins 6. C4b-binding protein (C4BP) • Inhibits the action of C4b in classical pathway • Splits C4 convertase and is a cofactor for factor I 7. Complement Receptor 1 (CR-1) • Co-factor for factor I, together with CD46 8. Protectin (CD59) and Vitronectin (S protein) • Inhibits formation of MAC by binding C5b678 • Present on "self" cells to prevent complement from damaging them
- 34. Regulat ory Protein 10.Carboxypeptidases Can Inactivate the Anaphylatoxins C3a and C5a • Anaphylatoxin activity is regulated by cleavage of the C-terminal arginine residues from both C3a and C5a by serum carboxypeptidases, resulting in rapid inactivation of the anaphylatoxin activity . • The specific enzymes that mediate the control of anaphylatoxin concentrations are carboxypeptidases N, B, and R. • These enzymes remove arginine residues from the carboxyl termini of C3a and C5a to form the so-called des-Arg (“without Arginine”), inactive forms of the molecules.
- 36. Consequences Of Complement System 1. Cell lysis by MAC • MAC damages cell membrane by making pores or channels in it and allowing the free passage of various ions and water in to the cell. This ultimately leads to cell death. Bacteria, enveloped viruses, irreversibly damaged cells, cancerous cells etc. are killed by this mechanism commonly referred to as complement mediated cell lysis. • Gram positive bacteria, which are protected by their thick peptidoglycan layer, bacteria with a capsule or slime layer around their cell wall, or non-enveloped viruses are less susceptible to lysis.
- 37. 2. Inflammatory Response • C3a, C4a and C5a are called anaphylatoxins. They bind to receptors on mast cells and induce its degranulation leading to release of histamine and other pharmacologically active mediators of inflammation. • They cause vasoconstriction and increase in vascular permeability. Along with the C5b67, they also induce the migration of neutrophils and monocytes to the site of complement activation. • This leads to an inflammatory response which is a local protective response.
- 38. 3. Opsonization • C3b and C4b act as major opsonin that coat the immune complexes and particulate antigen. • Phagocytic cells express receptors (CR1, CR3 and CR4) for complement components (C3b, C4b etc.) and are able to bind the complement coated antigen and enhance its phagocytosis. • C5a augments this process by enhancing the CR1 expression on phagocytes by 10 folds.
- 39. 4. Viral Neutralization • Complement plays a very important role in viral neutralization in many ways: • Most viral particles bind to their serum antibody and form particulate immune complex which stimulates the classical pathway. Many viruses are also capable of activating the alternative and lectin pathway in the absence of antibody. So, the MAC is able to destroy the viruses. • C3b helps in the formation of viral aggregates by acting as opsonin and thus decrease the net number of infective viral particles. This effect is enhanced in the presence of serum antibody. • Complement products also coat the viral particles. This coating neutralizes the viral infectivity by blocking its attachment to target cell and enhancing its phagocytosis by macrophages through complement. • Complement cytolyze most enveloped viruses causing fragmentation and disintegration.
- 40. 5. Solubilization of Immune Complex • C3b plays an important role in removing immune complex from the blood. its binding to complexes facilitates their binding to CR1 on RBCs. • CR1 are present in higher number is granulocytes than the RBCs, but because of much larger number of RBCs than granulocytes in blood, RBCs account for the 90% of the total CR1 in blood. • Immune complexes bound to the RBCs are taken to liver and spleen where they are phagocyted after separation from RBCs.