Complement System PDF

THE COMPLEMENT SYSTEM https://www.youtube.com/watch?v=uKzeBikWbvs https://www.youtube.com/watch?v=9Y0q_z2o-_c https://www.youtube.com/watch?v=YGN1eEsD1uU Prof. Asit Ranjan Ghosh/ Sr. Prof./ SBST 8/30/2024 Prof....

THE COMPLEMENT SYSTEM https://www.youtube.com/watch?v=uKzeBikWbvs https://www.youtube.com/watch?v=9Y0q_z2o-_c https://www.youtube.com/watch?v=YGN1eEsD1uU Prof. Asit Ranjan Ghosh/ Sr. Prof./ SBST 8/30/2024 Prof. ASIT RANJAN GHOSH/ 1 SBST/VIT  Why study the complement system? Innate & Adaptive Immunity Infection Inflammation Cell lysis Immune complex disease Autoimmune disease 8/30/2024 Prof. ASIT RANJAN GHOSH/ 2 SBST/VIT Definition: Complement consists of about 30 proteins , produced by liver, present in plasma and on cell surfaces that interact with each other to produce biologically active inflammatory mediators that promote cell and tissue injury, cell lysis. They are pro-enzyme (convertases) [ ZYMOGENS]. Nomenclature: a. the first component of complement is named C1 (etc.) [C1-C9] other components are designated by capital letters and names: Factor B, Properidin, CD 55, CD 59 b. when cleaved: fragments of complement components are designated by small letters (e.g. C3a and C3b); C2, C3 & C4, C5 get fragmeneted 8/30/2024 Prof. ASIT RANJAN GHOSH/ 3 SBST/VIT  C3a C3 C3b Factor B Ba + Bb Factor H Plasma = serum + clotting factor + COMPLEMENT Factor I COMPLEMENT: serum & plasma 8/30/2024 Prof. ASIT RANJAN GHOSH/ 4 SBST/VIT  Complement System Activation Regulation Amplification Biologic Function 8/30/2024 Prof. ASIT RANJAN GHOSH/ 5 SBST/VIT Summary of Complement Pathways 3 pathways for activation: 1. classical: most specific (antibody dependent activation, binds C1) [Adaptive Immunity] 2. lectin binding: some specificity (mannose binding protein (MASP1 & MASP2, binds C4) [Innate Immunity] 3. alternative: most primitive (non-specific, auto-activation of C3) [Innate Immunity] 8/30/2024 Prof. ASIT RANJAN GHOSH/ 6 SBST/VIT THE CLASSICAL PATHWAY OF ACTIVATION Activation via antigen-antibody complexes or by aggregated immunoglobulins: 1. IgG (mainly IgG1, IgG2, and IgG3) and IgM are most efficient in reacting with complement. 2. Classically, an antigen-antibody complex is designated by EA, where E is the antigen and A is the antibody. EA might represent immune complexes or antibody-coated bacteria, tumor cells, or lymphocytes. Complement components bind to EA in an orderly sequence to form a macromolecular complex, EAC 1,4,2,3,5,6,7,8,9. The classical pathway involves the following components and steps: 1. C1: When IgG or IgM reacts with antigen, the Fc fragment of the immunoglobulin provides a C1 binding site. C1, also called the recognition unit, contains three polypeptides-C1q, C1r and C1s-that are held together by calcium ions. With the removal of the calcium, C1 breaks down into its three subunits. a. C1q, the portion of the molecule that attaches first to immunoglobulin and initiates complement activation, has six binding sites. Because of its multivalency it can cross-link multiple immunoglobulin molecules. b. C1q binding leads to activation of C1r proenzymes. c. Activated C1r cleaves the proenzyme C1s. The latter acquires esterase activity and is referred to as C1 esterase (C1s). Calcium ions are essential for activation of C1. 8/30/2024 Prof. ASIT RANJAN GHOSH/ 7 SBST/VIT The classical pathway involves the following components and steps (cont’d): 2. C4: C1s mediates cleavage of native C4, the next component in the complement cascade, into C4a and C4b. One molecule of C1s can cleave several C4 molecules, thus serving as one of the sites in the amplification process. a. C4a, one of three anaphylatoxins, is released into the fluid phase. b. C4b can bind to cell membranes, but rather inefficiently. 3. C2: C1s in the presence of C4b cleaves the next component, C2, into C2a and C2b. a. C2a remains linked to the cell-bound C4b, thus forming the bimolecular complex C4b2a. Magnesium ions are required for formation of the C4b2a complex. C4b2a has enzymatic activity and is referred to a classical pathway C3 convertase. b. C2b is released into the fluid phase. 4. C3: The substrate for C3 convertase is C3. Circulating C3 binds to the C4b2a complex and is cleaved into two fragments, C3a and C3b. a. C3a is an anaphylatoxin and remains unbound. b. C3b is bound to the membrane in the vicinity of the C4b2a complex (C3 convertase), forming a trimolecular complex, C4b2a3b, which has enzymatic activity. The C4b2a3b complex is also referred to as C5 convertase, which acts on C5, the first complement component of the membrane attack pathway. 8/30/2024 Prof. ASIT RANJAN GHOSH/ 8 SBST/VIT  Classical Complement Pathway C1qrs C2 C4 C3 antibody C5 C4b C4a Bacteria C1= C1qr2s2 -> C1q +r2 +s2 Complement can be activated through three pathways: classical, lectin, and alternative. The classical pathway is activated when C1q binds to antibody attached to antigen, activating C1r and C1s, which cleave C4 and C2 8/30/2024 Prof. ASIT RANJAN GHOSH/ 9 SBST/VIT  Classical Complement Pathway C1qrs C2 C4 C3 antibody C5 C4b C2b C4a C2a Bacteria 8/30/2024 Prof. ASIT RANJAN GHOSH/ 10 SBST/VIT  Classical Complement Pathway C1qrs C2 C4 C3 antibody C5 C4b C2b C3b C4a C2a Bacteria C3a 8/30/2024 Prof. ASIT RANJAN GHOSH/ 11 SBST/VIT  Classical Complement Pathway C1qrs C2 C4 C3 antibody C5 C4b C2b C3b C4a C5b C2a Bacteria C3a C5a Animation complete 8/30/2024 Prof. ASIT RANJAN GHOSH/ 12 SBST/VIT  Classical Complement Pathway C1qrs antibody C4b C6 C2b C3b C7 C5b C8 Bacteria C6 C9 C7 C8 C9 C9 C9 C9 C9 C9 Animation complete 8/30/2024 Prof. ASIT RANJAN GHOSH/ 13 SBST/VIT  MAC PORES Source: undetermined Source: undetermined 10nm=100 Angstrom MAC diameter (approx.) Source: www.wikimedia.org 8/30/2024 Prof. ASIT RANJAN GHOSH/ 14 SBST/VIT  Lectin Binding Complement Pathway MBP C2 C4 C3 C5 C4b C4a Bacteria 8/30/2024 Prof. ASIT RANJAN GHOSH/ 15 SBST/VIT  Lectin Binding Complement Pathway MBP C2 C4 C3 C5 C4b C2b C4a C2a Bacteria C1r2 +s2 = MASP1 + MASP2 MBL=~ C1 (q1r2s2) MASP= Mannose Associated Serine Proteases; MASP1 & MASP2 8/30/2024 Prof. ASIT RANJAN GHOSH/ 16 SBST/VIT  Lectin Binding Complement Pathway MBP C2 C4 C3 C5 C4b C2b C3b C4a C2a Bacteria C3a 8/30/2024 Prof. ASIT RANJAN GHOSH/ 17 SBST/VIT  Lectin Binding Complement Pathway MBP C2 C4 C3 C5 C4b C2b C3b C4a C5b C2a Bacteria C3a C5a Animation complete 8/30/2024 Prof. ASIT RANJAN GHOSH/ 18 SBST/VIT  Lectin Binding Complement Pathway MBP C4b C6 C2b C3b C7 C5b C8 Bacteria C6 C9 C7 C8 C9 C9 C9 C9 C9 C9 Animation complete 8/30/2024 Prof. ASIT RANJAN GHOSH/ 19 SBST/VIT  MAC PORES Source: undetermined Source: undetermined 10nm=100 Angstrom MAC diameter (approx.) Source: www.wikimedia.org 8/30/2024 Prof. ASIT RANJAN GHOSH/ 20 SBST/VIT  THE ALTERNATIVE PATHWAY OF ACTIVATION The alternative pathway, also referred to as the properdin pathway, is considered to be a primitive defense system, a bypass mechanism that does not require C1, C4, and C2 interaction. The alternative pathway involves the following components and steps: 1. C3: The initial recognition event necessary for alternative pathway activation is the presence of C3, specifically C3b, which is probably continuously generated in small amounts in the circulation. a. Factor B. C3b interacts with factor B, also called the C3 proactivator, to form C3b, B, which is a magnesium ion-dependent complex. b. Factor D. The complex C3b, B is susceptible to enzymatic cleavage by factor D, also called C3 proactivator convertase, into two fragments, Ba and Bb. The Ba fragment is released. An active site is exposed in the Bb fragment, which remains bound to C3b, forming th C3b, Bb complex, also called amplification C3 convertase. When stabilized by the binding of properdin (P), which slows the dissociation of Bb, the C3b, Bb complex becomes a C3 convertase that cleaves C3 and generates more C3b. C3b then fixes to the activator surface so that more factor B binding sites are exposed, and the amplification loop amplifies the initial recognition event. 8/30/2024 Prof. ASIT RANJAN GHOSH/ 21 SBST/VIT As more C3b is generated, the complex expands (C3bn’, Bb; where n > 1) and becomes a C5 convertase capable of cleaving C5 into C5a and C5b and initiating the membrane attack pathway. Cobra venom factor (CVF) contained in cobra venom has the interesting property of activating the alternative complement pathway. Like C3b, it complexes with Bb to form CVF, Bb which is resistant to factors H and I and capable of continuously activating the C3 -to-C3b conversion leading to complement depletion. Factor H is a regulator of complement activation that blocks the formation of C3 convertase and is a cofactor for cleavage of C3b by factor I. Deficiencies of factor H result in unregulated elaboration of C3b on activation of the alternative pathway. 8/30/2024 Prof. ASIT RANJAN GHOSH/ 22 SBST/VIT  Alternative Complement Pathway C3 B C5 C3b C3a Bacteria 8/30/2024 Prof. ASIT RANJAN GHOSH/ 23 SBST/VIT  Alternative Complement Pathway C3 B C5 C3b P= properdin Bb C3a Bacteria 8/30/2024 Prof. ASIT RANJAN GHOSH/ SBST/VIT 24  Alternative Complement Pathway C3 B C5 C3b Bb C5b C3a Bacteria C5a Animation complete 8/30/2024 Prof. ASIT RANJAN GHOSH/ 25 SBST/VIT  Alternative Complement Pathway C6 C3b C7 Bb C5b C8 C6 C9 C7 Bacteria C8 C9 C9 C9 C9 C9 C9 C9 MAC Animation complete 8/30/2024 Prof. ASIT RANJAN GHOSH/ 26 SBST/VIT  MAC PORES Source: undetermined Source: undetermined 10nm=100 Angstrom MAC diameter (approx.) Source: www.wikimedia.org 8/30/2024 Prof. ASIT RANJAN GHOSH/ 27 SBST/VIT V. Amplification: C3 convertase: binds and cleaves multiple C3 molecules on surface to form C3b +C3a - classical: C4b2b = MBL pathway - alternative: C3bBb 8/30/2024 Prof. ASIT RANJAN GHOSH/ 28 SBST/VIT THE (COMMON) MEMBRANE ATTACK PATHWAY [Terminal Pathway] The pathway of membrane attack, also called the common pathway, is marked by the convergence of the classical, MBL and alternative pathways at the point of C5 activation. Activation of the membrane attack complex is initiated by C5 convertase (i.e., C4b2a3b in the classical pathway, MBL pathway and C3bn’Bb in the alternative pathway). This is the only component in the attack complex that has enzymatic activity, with cleavage occurring only once. All other components bind spontaneously. The membrane attack complex involves the following components and steps. 1. C5 is cleaved by C5 convertase into a smaller C5a fragment and a larger C5b fragment. a. C5a, an anaphylatoxin, is released into the surrounding fluid medium. b. C5b is the first component of the membrane attack complex. It is the receptor for the C6 and C7 components. 2. C6 and C7: Unstable C5b binds to C6, forming a stable C5b67 complex that is bound to the target cell membrane. 3. C8 attaches to the membrane-bound C5b67 complex and membrane leakage begins. Cell lysis can occur by the C5b678 complex in the absence of C9. 4. C9 attachment to the C5b678 complex serves a primary function of greatly accelerating cytolysis by the production of circular lesions in the membrane. 5, Cell lysis. The C5b6789 complex induces the formation of hollow cylinders (tubules) about 15 nm long and 8-12 nm in diameter in the lipid bilayer of the cell membrane, allowing passage of electrolytes and water across the membrane and leading ultimately to osmotic lysis of the cell. 8/30/2024 Prof. ASIT RANJAN GHOSH/ 29 SBST/VIT  SUMMARY OF COMPLEMENT ACTIVATION Classical Alternative Lectin-Binding Pathway Pathway Pathway C1q MBP C3 [C4b2b] [C3bBbP] P= properdin C3 Convertase C3a C3b C3b, C3bi anaphylatoxins (opsonlzation) C5a C5b C5b-C 9 (membrane attack complex) Terminal Pathway 8/30/2024 Prof. ASIT RANJAN GHOSH/ 30 Cell Injury SBST/VIT VI. Biologic Function: The complement system has four major function: 1. lysis of infectious organisms (all 3 pathways), 2. activation of inflammation (all 3 pathways, a-subunits) 3. opsonization (C3b = Opsonin) and (All 3) 4. immune clearance(classical, phagocytosis). 8/30/2024 Prof. ASIT RANJAN GHOSH/ 31 SBST/VIT  anaphylatoxins: C3a and C5a – smooth muscle contraction – mast cell degranulation mediator release (histamine, leukotrienes) – vascular changes: dilation, increased permeability (edema) – C5a also leukocyte adhesion and chemotaxis (recruitment) opsonization: C3b (opsonin), C3bi, C3d: (binding to complement receptors (CR) and enhanced phagocytosis by neutrophils and macrophages) clearance of circulating immune complexes membrane attack complex (MAC): C5b-C9 (cell lysis) 8/30/2024 Prof. ASIT RANJAN GHOSH/ 32 SBST/VIT BIOLOGICAL CONSEQUENCES OF COMPLEMENT ACTIVATION During complement activation, several materials with important biological activities are generated. C3 and C5 and their cleavage products appear to be the most important complement components in terms of biological function. 1. C3a and C5a are referred to as anaphylatoxins. a. They cause the release of vasoactive amines (e.g., histamine) from mast cells and basophils in a manner that stimulates mediator release by IgE. b. Mediator release causes smooth muscle contraction and increases vascular permeability, effects which can be counteracted by antihistamines and anaphylatoxin inactivator. c. C5a is much more active than C3a on a molar basis and, in addition to anaphylatoxin activity, has a wider range of biological activity, including the following: - Chemotactic factor - Neutropenia - Oxidative burst, and of degranulation of neutrophils - Production of leukotrienes 8/30/2024 Prof. ASIT RANJAN GHOSH/ 33 SBST/VIT  2. C3b generation and coating on target cells through complement receptors (CR1) on monocytes, neutrophils and B cells is perhaps the major biological function of complement. C3b also plays an important role in opsonization. C3b-coated cells also tend to aggregate (immune adherence), a process that also may promote phagocytosis. 3. C3e provokes a release of neutrophils from bone marrow causing prompt leukocytosis. 4. C3d, another cleavage product of C3b, can interact with receptors on lymphocytes. C3 nephritic factor (C3NeF) found in the circulation of patients with mesangiocapillary glomerulonephritis. It acts as an antibody against the C3bn Bb complex and leads to a marked hypocomplementemia. 8/30/2024 Prof. ASIT RANJAN GHOSH/ 34 SBST/VIT C4 and its cleavage products have certain important biological functions: 1. The binding of C1 and C4 by a virus-antibody complex can neutralize virus activity. 2. C4a, have anaphylatoxin activity, causing the release of histamine from mast cells and basophils. 3. C4b receptor sites exist on several cell types, suggesting a role for C4b in opsonization as seen with C3b. 8/30/2024 Prof. ASIT RANJAN GHOSH/ 35 SBST/VIT Ba and Bb. Generated exclusively by the alternative pathway have important biological functions. 1. Ba is chemotactic for neutrophils. 2. Bb activates macrophages and causes them to adhere to and spread on surfaces. Immunodeficiencies result from a lack of complement and faulty complement activation. 8/30/2024 Prof. ASIT RANJAN GHOSH/ 36 SBST/VIT  VII. Regulation: Activation of the complement components is associated with potent biological functions that, if left unchecked, would exhaust the complement system. Uncontrolled activation of the complement system is prevented by several serum proteins that bind to (inhibit) or enzymatically attack (inactivate) complement components. Inhibit activation: classical pathway – C1 inhibitor (C1INA): plasma protein spontaneous decay (hydrolysis) of C3 convertases: inhibit C3 convertase: (all pathways) – Plasma proteins: Factor I – Cell membrane proteins: - decay accelerating factor (DAF) (CD55): - membrane co-factor protein (MCP) (CD46): 8/30/2024 Prof. ASIT RANJAN GHOSH/ 37 SBST/VIT VII. Regulation Inactivate anaphylatoxins: cleave C3a and C5a – serum carboxypeptidase N (SCPN): Inhibit MAC: – Protectin (CD59): cell associated protein Calcium ions are essential for activation of C1. C2. C4: C1s mediates cleavage of native C4, the next component in the complement cascade, into C4a and C4b. One molecule of C1s can cleave several C4 molecules, thus serving as one of the sites in the amplification process. 8/30/2024 Prof. ASIT RANJAN GHOSH/ 38 SBST/VIT  Kouser L. Front. Immunol., 23 April 2013 | https://doi.org/10.3389/fimmu.2013.00093 8/30/2024 Prof. ASIT RANJAN GHOSH/ 39 SBST/VIT  SUMMARY OF COMPLEMENT ACTIVATION & REGULATION Classical Pathway Lectin-Binding Alternative Pathway Pathway C1q MBP C3 DAF : Decay accelerating factor Hydrolysis CD55 C1INA [C4b2b] [C3bBbP] DAF-cell C3 Convertase MCP: membrane cofactor protein Factor I CD46 SCPN : serum carboxypeptidase N C3a C3b MCP-cell SCPN C5a C5b C5b-C 9 (membrane attack complex) Protectin-cell CD59 8/30/2024 Prof. ASIT RANJAN GHOSH/ 40 Cell Injury SBST/VIT 8/30/2024 Prof. ASIT RANJAN GHOSH/ 41 SBST/VIT 8/30/2024 Prof. ASIT RANJAN GHOSH/ 42 SBST/VIT 8/30/2024 Prof. ASIT RANJAN GHOSH/ 43 SBST/VIT  Complement receptor (CR) A CR is a membrane-bound receptor belonging to the complement system. CRs bind effector protein fragments that are produced in response to antigen- antibody complexes or damage-associated molecules. CR activation contributes to the regulation of inflammation, leukocyte extravasation, and phagocytosis; it also contributes to the adaptive immune response. Different CRs can participate in either the classical complement pathway, the alternative complement pathway, or both. White blood cells, particularly monocytes and macrophages, express complement receptors on their surface. All four complement receptors can bind to fragments of C3 or C4 coated on pathogen surface, but the receptors trigger different downstream activities. Complement receptor (CR) 1, 3, and 4 function as opsonins which stimulate phagocytosis, whereas CR2 is expressed only on B cells as a co-receptor. Red blood cells (RBCs) also express CR1, which enables RBCs to carry complement-bound antigen-antibody complexes to the liver and spleen for degradation. 8/30/2024 Prof. ASIT RANJAN GHOSH/ 44 SBST/VIT 8/30/2024 Prof. ASIT RANJAN GHOSH/ 45 SBST/VIT  VIII. Complement Deficiencies: early components: auto-immune disease (if immune complex is not eliminated, then Autoimmune disorder!) middle and late components: pyogenic bacterial and Nisseria infections most common congenital deficiency: C2 C1INA deficiency: hereditary angioedema Inhibit activation of classical pathway DAF deficiency: paroxysmal nocturnal hemoglobinuria Inhibit C3 convertase 8/30/2024 Prof. ASIT RANJAN GHOSH/ 46 SBST/VIT  systemic lupus erythematosus 8/30/2024 Prof. ASIT RANJAN GHOSH/ 47 SBST/VIT 8/30/2024 Prof. ASIT RANJAN GHOSH/ 48 SBST/VIT Complement Inactivation: Denaturation of heat labile Complement proteins by Heating at 56o C x 30 minutes 8/30/2024 Prof. ASIT RANJAN GHOSH/ 49 SBST/VIT  C1 8/30/2024 Prof. ASIT RANJAN GHOSH/ 50 SBST/VIT Antibody present Antibody absent 8/30/2024 Prof. ASIT RANJAN GHOSH/ 51 SBST/VIT