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complement.pptx
- 1. Complement Pathways Dr. ArunKumar Parthasarathy Ph.D
- 2. COMPLEMENT 2 ▰ Represents agroup of proteins normally found in serum in inactive form, but when activated they augment the immune responses. ▰ Complements constitute about 5% of normal serum proteins. ▰ Their level does not increase following either infection or vaccination.
- 3. General properties 3 ▰ Bindto Fc region of antibody ▰ Role of antigen ▰ Species nonspecific ▰ Heat labile ▰ Cytolytic activity
- 4. Complement Components 4 ▰ Complementsystem comprises of about 30 serum proteins grouped into complement components, the properdin system and the regulatory proteins. ▰ Complement components are named by numerals. There are nine components; C1 to C9. C1 has three subunits- C1q, C1r and C1s. ▰ Properdin system and the regulatory proteins are named by letter symbols, e.g., factor-B
- 5. Synthesis 5 ▰ Liver isthe major site of synthesis of complement proteins. ▰ Minor sites include blood monocytes, tissue macrophages, and epithelial cells of GIT and genitourinary tract.
- 6. Complement Activation 6 ▰ Allthe complement proteins are synthesized in inactive form (e.g. zymogens) and are activated by proteolysis. ▰ Complements have two unequal fragments (large and small fragment). ▰ The larger fragments are usually designated as ‘b’ (e.g. C3b) and the smaller fragments are designated as ‘a’ (e.g. C3a). ▰ An exception is C2a which is larger fragment. ▰ During proteolysis, the smaller fragment is removed exposing the active site of the larger fragment. ▰ The larger fragment participates in the cascade reaction of complement pathway and the smaller fragment diffuses away to mediate other functions.
- 7. 7 ▰ Cascade reaction-Fragments of complements interact in a definite sequential manner with a cascade like effect, which leads to formation of complex. ▰ Such complex having enzymatic activity is designated by putting a bar over the number or symbol (e.g. C 3bBb).
- 8. COMPLEMENT PATHWAYS 8 ▰ Classicalpathway- Antibody dependent pathway. Pathway is triggered by the antigen antibody complex formation. ▰ Alternative pathway- Antibody independent pathway, triggered by the antigen directly. ▰ Lectin pathway - recently described pathway. It resembles classical pathway but it is antibody independent.
- 9. Stages of complementactivation 9 ▰ There are four main stages in the activation of any of the complement pathways. Initiation of the pathway Formation of C3 convertase Formation of C5 convertase Formation of membrane attack complex (MAC) All the three pathways differ from each other in their initiation till formation of C3 convertase. Then, the remaining stages are identical in all the pathways
- 10. Classical Pathway 10 ▰ Antibodydependent ▰ Not all antibodies can bind to complements of classical pathway. ▰ Decreasing order of ability of antibodies to fix complement is- IgM (most potent) > IgG3> IgG 1> IgG2. ▰ The other classes of antibodies do not fix complements. ▰ CH2 domain on IgG, CH4 on IgM participate in complement binding. ▰ The classical pathway begins with activation of C1 and binding to antigen- antibody complex.
- 11. Initiation 11 ▰ The firststep is the binding of C1 to the antigen- antibody complex. ▰ The first binding portion of C1 is C1q, which reacts with the Fc portion of IgM or IgG bound to antigen. ▰ C1q is a hexamer having six globular heads each acting as a combining site. ▰ Effective activation of classical pathway begins only when C1q is attached to the Fc portion of antibody by at least two of its globular binding sites. ▰ C1q binding in the presence of calcium ions, in turn activates sequentially C1r followed by C1s.
- 12. Formation of C3Convertase 12 ▰ Activated C1s acts as an esterase (C1s esterase), which can cleave C4 to produce C4a (an anaphylatoxin), and C4b which binds to C1 and participates further in complement cascade. C14b in the presence of magnesium ions cleaves C2 into C2a, which remains linked to complement complex, and C2b (has kinin like activity), which is released outside. C14b2a is referred to as C3 convertase of the classical pathway.
- 13. Formation of C5Convertase 13 ▰ C3 convertase hydrolyses many C3 molecules into two fragments: C3a (an anaphylatoxin) C3b which remains attached to C14b2a to form C14b2a3b complex which acts as C5 convertase of classical pathway.
- 14. Formation of MembraneAttack Complex 14 ▰ Begins with C5 convertase cleaving C5 into C5a (an anaphylatoxin, released into the medium) and C5b, which continues with the cascade. C5b is extremely labile, gets stabilized by binding soon with C6 and C7 to form C5b67 followed by addition of C8. Hydrophobic regions on C7 and C8 help in penetration into the target cell membrane.
- 15. 15 This insertedmembrane complex (C5b678) has a catalytic property to bind to C9 molecule and then it polymerizes the C9 into a tubular channel of 10 nm diameter. Penetration of C9 - channels or pores on the target cell membrane Each tubular channel - hydrophobic outside, hydrophilic inside - free passage of ions and water into the cell - cellular swelling - lysis. C5b6789 destroys the target cell by attacking the cell membrane – MAC. Process of cytolysis is referred to as complement-mediated cytotoxicity.
- 16. Formation of MembraneAttack Complex (Cont..) 16
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- 18. Alternative Pathway 18 ▰ Independentof antibody; hence is considered as a part of innate immunity. ▰ Four stages. ▰ Differs from the classical pathway in first two stages. ▰ Three complement components C1, C4 and C2 are not involved. Requires three other complement proteins present in serum named factor B, factor D and properdin.
- 19. Initiation 19 Antigens from pathogenNon microbial initiators Endotoxin or LPS (lipopolysaccharide) from Gram negative bacteria Human antibodies in complexes- IgA, IgD Teichoic acid from Gram positive bacteria Tumor cells Fungal cells- Yeast cells Cobra venom factor Heterologous RBCs from mouse, rabbit and chicken Parasites like Trypanosomes Anion polymer like dextran sulphate Virus infected cells Pure carbohydrates like agar, inulin
- 20. 20 ▰ First complementcomponent to be involved in alternative pathway is free C3 in the serum. ▰ C3 hydrolyzes spontaneously, to generate C3a which diffuses out and C3b fragment which attaches to foreign cell surface antigen. ▰ Factor B binds to C3b coated foreign cells. ▰ Factor D - acts on factor B, and cleaves it into Ba (diffuses out) and Bb (remains attached). ▰ C3bBb - C3 convertase. ▰ C3bBb has a very short half-life of 5 minutes. ▰ Stabilized by properdin (half-life is increased to 30 minutes). Formation of C3 Convertase Formation of C5 convertase and formation of membrane attack complex - identical to that of classical pathway.
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- 22. Lectin Pathway 22 ▰ Complementpathway of innate immunity -works independent of antibody. ▰ Mediated through lectin proteins of the host that interact with mannose residues present on microbial surface. ▰ Lectin pathway involves all complement components used for classical pathways except C1. ▰ Instead of C1, host lectin protein called mannose binding lectins mediate the first ‘initiation’ stage.
- 23. Initiation 23 ▰ Activation -Mannose carbohydrate residues of glycoproteins present on microbial surfaces. ▰ Mannsoe binding lectins (MBL) bind to mannose residues on microbial surface. ▰ MBL is an acute phase reactant protein, similar to C1q in structure. ▰ After binding of MBL to microbial surface, another host protein called MASP (MBL associated serine protease) gets complexed with MBL. ▰ MASP is similar or C1r and C1s and mimics their functions. ▰ MBL-MASP complex cleaves C4 which in turn splits C2. ▰ MBL/MASP-C4b2a acts as C3 convertase.
- 24. Essentials of MedicalMicrobiology by Apurba S Sastry © Jaypee Brothers Medical Publishers 24
- 25. Differences between thethree complement pathways 25 Features Classical pathway Alternative pathway Lectin pathway Activator (initiator) Antigen antibody complex Endotoxin IgA, IgD, Cobra venom, Nephritic factor Carbohydrate residue of bacterial cell wall (mannose binding protein) that binds to host lectin antigen. First complement activated C1 C3b C4 C3 convertase C14b2a C3bBb MBL/MASP-C4b2a C5 convertase (C3 convertase + 3b) C14b2a3b C3bBb3b MBL/MASP-C4b2a3b Complement level in the serum All C1-C9: Low C1,C4,C2- Normal Others- Low C1- Normal Others- Low Immunity Acquired Innate Innate
- 26. EFFECTOR FUNCTIONS OF COMPLEMENT 26 ▰MAC and other complement by-products produced during the activation augment the immune response in many ways. Target cell lysis by MAC Inflammatory response Opsonization Removing the immune complexes from blood- Viral neutralization
- 27. Target cell lysisby MAC 27 ▰ MAC makes pores or channels in the target cell membrane. ▰ Allows the free passage of various ions and water into the cell leading to cell swelling, lysis and death. ▰ E.g. Bacteria, enveloped viruses, damaged cells, tumor cells, etc
- 28. Inflammatory response 28 ▰ C3a,C4a and C5a - Anaphylatoxins. ▰ Bind to surface receptors of mast cells and induce their degranulation leading to release of histamine and other inflammatory mediators. ▰ Cause vasoconstriction, and increased vascular permeability.
- 29. Opsonization 29 ▰ C3b andC4b - major opsonins - coat the immune complexes and particulate antigens. ▰ Phagocytic cells express complement receptors (CR1, CR3 and CR4) for complement components (C3b, C4b). ▰ Bind to complement coated antigens and enhance phagocytosis. ▰ C5a - enhances the CR1 expression on phagocytes by 10 folds.
- 30. Removing the immunecomplexes from blood 30 ▰ C3b - important role. ▰ C3b bound immune complexes - Recognized by complement receptor CR1 present on RBCs. ▰ Immune complexes bound to RBCs are taken to liver and spleen where they are phagocytosed after being separated from the RBCs.
- 31. Viral neutralization 31 ▰ Complementscoated on virus surfaces neutralize the viral infectivity by blocking their attachment sites. ▰ C3b mediated opsonization of viral particles ▰ Lysis of the enveloped viruses by: Activation of classical pathway (most viruses) Alternative or lectin pathways (viruses like Epstein Barr virus, rubella etc)
- 32. COMPLEMENT RECEPTORS 32 ▰ Playan important role in mediating the activities of complement products as well as in regulating their activities. ▰ There are many complement receptors (CR1 to CR5) - distributed on various cell types and bind to specific ligands to mediate specific function. ▰ Example - CR2 is present on B cells and is involved in humoral immune response - also acts as receptor for Epstein-Barr virus.
- 33. EVASION OF COMPLEMENTSYSTEM BY MICROORGANISMS 33 Mechanisms Examples Shown by Gram negative bacteria Long polysaccharide side chain of bacteria can prevent MAC insertion Escherichia coli Salmonella Non covalent interactions between bacterial cell wall components can prevent MAC insertion Neisseria gonorrhoeae Elastases destroy C3a & C5a Pseudomonas Shown by Gram positive bacteria Thick peptidoglycan cell wall prevents MAC insertion Staphylococcus Streptococcus Bacterial capsule forms a physical barrier between C3b and CR1 interaction Streptococcus pneumoniae
- 34. 34 Mechanisms Examples Shown byother microbes Proteins mimicking complement regulatory proteins Vaccinia virus, Herpes simplex virus, Epstein-Barr virus, Trypanosoma cruzi, Candida albicans
- 35. REGULATION OF COMPLEMENTPATHWAYS 35 ▰ Antigen non-specific. ▰ Capable of attacking microorganisms as well as host cells. ▰ Several regulatory mechanisms have evolved to restrict complement activity only to the designated target cells. ▰ There are a series of regulatory proteins, which inactivate various complement components at different stages. Examples: ▰ C1 inhibitor (or C1 esterase inhibitor): soluble glycoprotein, inhibits the action of C1q by splitting C1qrs into C1rs and C1q - whole classical pathway is inhibited. ▰ DAF (Decay accelerating factor):CD55 molecule present on cell membrane, accelerates dissociation of C3 convertase - inhibiting all three pathways.
- 36. COMPLEMENT DEFICIENCIES 36 Complement protein deficiencies Pathway(s)involved Disease/pathology C1, C2, C3, C4 C1, C2,C4-Classical pathway C3- Common deficiency SLE, glomerulonephritis & pyogenic infections Properdin, Factor D Alternative pathway Neisseria and pyogenic infection Membrane attack complex (C5- C9) Common deficiency Disseminated Neisseria infection C1 esterase inhibitor Overactive classical pathway Hereditary angioneurotic edema DAF (Decay accelerating factor) & CD59 Deregulated C3 convertase Increased RBC lysis PNH (Paroxysmal nocturnal hemoglobinurea)
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