COMPLEMENT

History History In the late 19 th century, Hans Ernst August Buchner found that blood serum contained a “factor” or “principle” capable of killing bacteria. In the late 19 th century, Hans Ernst August Buchner found that blood serum contained a “factor” or “principle” capable of killing bacteria.

Founders Paul Ehrlich and Jules Bordet Founders Paul Ehrlich and Jules Bordet

Complement : History Discovered in 1894 by Bordet It represents lytic activity of fresh serum Its lytic activity is destroyed when heated at 56 0 C for 30 min

Complement : Introduction Complement – A series of serum and membrane expressed proteins involved in the effector role of the immune response to pathogens Complement – A series of serum and membrane expressed proteins involved in the effector role of the immune response to pathogens Made up of approximately 30 circulating and membrane-bound proteins. Made up of approximately 30 circulating and membrane-bound proteins. Synthesized in the liver and by cells involved in the inflammatory response. Synthesized in the liver and by cells involved in the inflammatory response.

COMPLEMENT Complements are the heat labile factors present in the normal serum which are activated characteristically by Ag- Ab interaction and subsequently mediate a number of biologically significant consequences Complements are the heat labile factors present in the normal serum which are activated characteristically by Ag- Ab interaction and subsequently mediate a number of biologically significant consequences Biological effector mechanism (Triggered Enzyme Cascade) includes Coagulation, Fibrinolytic and Kinin systems Biological effector mechanism (Triggered Enzyme Cascade) includes Coagulation, Fibrinolytic and Kinin systems General Properties :- General Properties :- 1. Non specific serological reagent 2. 5% of normal serum proteins 3. Heat labile, Inactivated at 56 0 C for 30 min heating 4. Binds to Fc portion of Ig bound to Ag 5. Only IgM, IgG 3,1,2 fix the complement

Complement Functions Complement has a central role in inflammation as it causes chemotaxis of phagocytes, opsonization and lysis of pathogens and clearence of immune complexes.

(a)Schematic representation of the roles of C3b and antibody in opsonization (b) Electron micrograph of Epstein-Barr virus coated with antibody and C3b and bound to the Fc and C3b receptor (CR1) on a B lymphocyte

Clearance of circulating immune complexes by reaction with receptors for complement products on erythrocytes and removal of these complexes by receptors on macrophages in the liver and spleen. Because erythrocytes have fewer receptors than macrophages, the latter can strip the complexes from the erythrocytes as they pass through the liver or spleen. Deficiency in this process can lead to renal damage due to accumulation of immune complexes.

COMPLEMENT COMPONENTS Around 20 serum proteins as complement components, properdin system and control proteins Around 20 serum proteins as complement components, properdin system and control proteins Complement components : 9 fractions C1 to C9 Complement components : 9 fractions C1 to C9 Fraction C1 is a Ca dependent complex which on chelation with EDTA yields 3 protein subunits – C1q,r,s Fraction C1 is a Ca dependent complex which on chelation with EDTA yields 3 protein subunits – C1q,r,s Immune cytolysis – Erythrocyte lysis by Ab – EAC complex Immune cytolysis – Erythrocyte lysis by Ab – EAC complex EAC 14235, 4 comes between 1 and 2 EAC 14235, 4 comes between 1 and 2 When C component acquires enzymatic or other biological activity it is indicated by a bar over the number When C component acquires enzymatic or other biological activity it is indicated by a bar over the number Fragments cleaved from the complement cascade are indicated by small letters – C3a, C3b Fragments cleaved from the complement cascade are indicated by small letters – C3a, C3b Inactivated forms of complement - iC3b Inactivated forms of complement - iC3b

COMPLEMENT ACTIVATION Complement cascade is a series of reactions in which preceeding components act as enzymes on succeeding components, cleave them into dissimilar fragments Complement cascade is a series of reactions in which preceeding components act as enzymes on succeeding components, cleave them into dissimilar fragments Large fragments join the cascade, smaller ones are biologically active and mediate : Large fragments join the cascade, smaller ones are biologically active and mediate : - Inflammation & Increase vascular permeability - Induce smooth muscle contraction - Chemotaxis of leucocytes - Promote virus neutralisation & Detoxify endotoxins - Release of histamine from mast cells Two parallel and independent mechanisms Two parallel and independent mechanisms 1. Classical complement pathway 2. Alternative or Properdin pathway

Pathways of complement activation CLASSICAL PATHWAY ALTERNATIVE PATHWAY activation of C5 LYTIC ATTACK PATHWAY antibody dependent LECTIN PATHWAY antibody independent Activation of C3 and generation of C5 convertase

The Classical Pathway Antigen-Antibody complexes are main activators of this pathway. Antigen-Antibody complexes are main activators of this pathway. Activated by the formation of soluble Ag-Ab complexes or binding of Ab (IgM or IgG) to Ag on a target cell. Activated by the formation of soluble Ag-Ab complexes or binding of Ab (IgM or IgG) to Ag on a target cell. C-reactive protein binds to the surface of many bacteria and are also activators. C-reactive protein binds to the surface of many bacteria and are also activators.

Activation Effectiveness IgM is more effective at activating complement than IgG IgM is more effective at activating complement than IgG C1q binds to the CH2 domain of Ig and requires at least two adjacent Fc regions C1q binds to the CH2 domain of Ig and requires at least two adjacent Fc regions Activation of the Thiol-Ester bond and covalent attachment to antigen Activation of the Thiol-Ester bond and covalent attachment to antigen

C1q binds to IgM and IgG molecules Binding

Fraction C1 is a Ca dependent complex which on chelation with EDTA yields 3 protein subunits – C1q,r

CLASSICAL PATHWAY - summary Involved in Specific active immunity, starts with activation of C1 and culminate in immune cytolysis Involved in Specific active immunity, starts with activation of C1 and culminate in immune cytolysis EA EAC1 EA EAC1 C1q,r,s Ca 2+ C4 EAC14b C4a C2 Mg 2+ EAC 14b2a C2b CYTOLYSIS EAC 14b2a3b C3 C5,6,7 C3a EAC 14b2a3b5b67 C5a EAC 14b2a3b5B6789 C8,9

Components of the Classical Pathway C4 C2 C3 C1 complex Ca ++ C1r C1s C1q C1s is an enzyme and cleaves C4 and C2

Ca ++ C1r C1s C1q C4 C4a b Classical Pathway Generation of C3-convertase Cleavage of C4 by C1s produces C4a and C4b

Classical Pathway Generation of C3-convertase C4b Mg ++ C4a Ca ++ C1r C1s C1q C2 C2b a C2 a C4b binds C2 and C4b2 is cleaved by C1s. C2b is released but C2a remains bound to C4b on the surface. C4b2a is C3 Convertase

Classical Pathway Generation of C5-convertase C4b Mg ++ C4a Ca ++ C1r C1s C1q C2b C2 a C3 C3a b ________ C4b2a3b is C5 convertase; it leads into the Membrane Attack Pathway (Lytic pathway)

Generation of C5 convertase leads to the activation of the Lytic pathway

Components of the lytic pathway C6 C9C9 C8 C7 C5

Lytic pathway C5-activation C3b C2 a C4b C5 b C5a

Lytic pathway assembly of the lytic complex C5 b C6 C7 C5b first binds C6 and then C7 from the plasma. Membrane bound C5b67 recruits C8 and C9 to form the Membrane Attack Complex (MAC)

Lytic pathway: insertion of lytic complex into cell membrane C5 b C6 C7 C8 C9C9 C9C9 C9C9 C9C9 C9C9 C9C9 C9C9 C9C9 C9C9

ALTERNATE PATHWAY - summary The activation of C3 without prior participation of C142 The activation of C3 without prior participation of C142 Pillemer (1954) – Properdin system a system in nonspecific antimicrobial defence Pillemer (1954) – Properdin system a system in nonspecific antimicrobial defence C3b in circulation Free C3b inactivated by Factor H & I Stabilsed with Activator, Zymosan C3bFactor B + C3b, B + Factor D BaC3b, Bb + Properdin C3b, Bb, P + C3Cascade

Components of the alternative pathway C3 fB fD P

Spontaneous C3 activation C3 H2OH2O B D Generation of C3 convertase Factor B activate F -D which then cut F-B releasing Ba, while Bb becomes an active protease C3Bb complex has a very short half life b C3 C3a b

B D b C3b If spontaneously-generated C3b is not degraded C3-activation the amplification loop C3 C3a b

B D BbBb C3b C3 b C3-activation the amplification loop C3b C3a b

BbBb C3b BbBb BbBb C3a C3-activation the amplification loop C3b

Control of spontaneous C3 activation via DAF C3b DAF prevents the binding of factor B to C3b B Autologous cell membrane DAF CR1

Control of spontaneous C3 activation via DAF DAF dislodges C3b-bound factor Bb B bb C3b Autologous cell membrane DAF CR1 B b

Autologous cell membrane C3b B b H I iC3b Control of spontaneous C3 activation via CR1 B b I iC3b DAF CR1 DAF CR1

Degradation of spontaneously produced C3b C3b iC3b II C3dg C3c

C3b stabilization and C5 activation C3b C3b finds an activator (protector) membrane C3 C3a b B D b P This is stable C5 convertase of the alternative pathway

C3b regulation on self and activator surfaces C3b

C5-convertase of the two pathways C3b BbBb C5-convertase of the Alternative Pathway C4b C2a C3b C5-convertase of the Classical and lectin Pathways

Components of mannose-binding lectin pathway C4 MBL C2 MASP1 MASP2 Pathogen

Mannose-binding lectin pathway C4 MBL C4b C4a C4b C2 C2b C2a _____ C4b2a is C3 convertase; it will lead to the generation of C5 convertase MASP1 MASP2 Binding to lectins cause autocatalytic activation of MASPs which then cleave C4 & C2

REGULATION OF C - ACTIVATION Inhibitors & Inactivators Inhibitors & Inactivators Inhibitors :- Inhibitors :- 1. C1 esterase :- Heat labile alpha neuramino glycoprotein inhibits other esterases found in blood- plasmin, kininogen & Hageman factor. It checks the autocatalytic prolongation 2. S protein (vitronectin):- Binds to C567 and modulates cytolytic action of the membrane attack complex Inactivators :- Inactivators :- 1. Factor I :- Serum beta globulin cause homeostatic control of C3 activation in alternate pathway 2. Factor H :- Acts with factor I modulating C3 activation 3. Anaphylatoxin inactivator :- Alpha globulin which degrades C3a, C4a and C5a 4. C4 binding protein :- Controls cell bound C4b

Regulation of complement system by regulatory proteins (black)

BIOLOGICAL EFFECTS Mediates Immunological membrane damage (Cytolysis) Mediates Immunological membrane damage (Cytolysis) Amplifies inflammatory response : C2 kinins Amplifies inflammatory response : C2 kinins In Hypersensitivity reactions (Type II & Type III) In Hypersensitivity reactions (Type II & Type III) Pathogenesis of Autoimmune disesases : Haemolytic anaemia, Paroxysmal nocturnal hemoglobinuria, Hereditory angioneurotic edema Pathogenesis of Autoimmune disesases : Haemolytic anaemia, Paroxysmal nocturnal hemoglobinuria, Hereditory angioneurotic edema Antiviral activity & Endotoxic shock Antiviral activity & Endotoxic shock Promotes phagocytosis : CR receptors, CR 1,2,3,4 & C1q Promotes phagocytosis : CR receptors, CR 1,2,3,4 & C1q Immune adherence : C3 & C4 Immune adherence : C3 & C4 Interacts with coagulation, fibrinolytic and kininogenic systems of blood Interacts with coagulation, fibrinolytic and kininogenic systems of blood Conglutinaion : Bovin serum protein, Conglutinin (K) reacts with bound C3, immuno conglutinin : Abs with K like activity Conglutinaion : Bovin serum protein, Conglutinin (K) reacts with bound C3, immuno conglutinin : Abs with K like activity

Biological effects of C5a

Biological Activities of Classical Pathway Components Component Biological Activity C2b Prokinin; cleaved by plasmin to yield kinin, which results in edema C3a Anaphylotoxin; can activate basophils and mast cells to degranulate resulting in increased vascular permeability and contraction of smooth muscle cells, which may lead to anaphylaxis C3bOpsonin Activation of phagocytic cells C4aAnaphylotoxin C4bOpsonin

ProductBiological EffectsRegulation Biological properties of C- activation products C2b (prokinin) edemaC1-INH C3a (anaphylatoxin) mast cell degranulation; enhanced vascular permeability; anaphylaxis carboxy- peptidase- B (C3-INA)

ProductBiological EffectsRegulation Biological properties of C- activation products as C3, but less potent (C3-INA) C4a (anaphylatoxin) opsonization; phagocytosis C4b (opsonin) C4-BP, factor I C3b (opsonin) opsonization; phagocyte activation factors H & I

ProductBiological EffectsRegulation Biological properties of C- activation products anaphylactic as C3, but much more potent; attracts & activates PMN causes neutrophil aggregation, stimulation of oxidative metabolism and leukotriene release C5a (chemotactic factor) carboxy- peptidase-B (C3-INA) C5b67protein-Schemotaxis, attaches to other membranes

Complement Deficiencies and Disease Classical Pathway Pathway Component DiseaseMechanism C1INH C1INHHereditaryAngioedema Overproduction of C2b (prokinin) C1, C2, C4 C1, C2, C4Predisposition to SLE Opsonization of immune complexes help keep them soluble, deficiency results in increased precipitation in tissues and inflammation

C1-inhibitor deficiency: hereditary angioedema

Complement Deficiencies and Disease : Lectin Pathway Pathway Component DiseaseMechanism MBL MBL Susceptibility to bacterial infections in infants or immunosuppressed Inability to initiate lectin pathway

Complement Deficiencies and Disease Alternative Pathway Pathway/ComponentDiseaseMechanism Factors B or D Susceptibility to pyogenic (pus- forming) bacterial infections Lack of sufficient opsonization of bacteria C3 Susceptibility to bacterial infections Lack of opsonization and inability to utilize the membrane attack pathway C5, C6, C7 C8, or C9 Susceptibility to Gram-negative infections Inability to attack the outer membrane of Gram- negative bacteria

Complement Deficiencies and Disease Alternative Pathway cont… Pathway Component DiseaseMechanism Properdin (X-linked) Susceptibility meningococcal meningitis Lack of opsonization of bacteria Factors H or I C3 deficiency and susceptibility to bacterial infections Uncontrolled activation of C3 via alternative pathway resulting in depletion of C3

Opsonization and phagocytosis

C 1 I nh C1qrs breakdown C1r C1s C1q C1r C1s