Complement

Complement: History Discovered in 1894 by Bordet It was used to refer to a heat-labile serum component Its lytic activity destroyed when heated at 56°C for 30 min However it is now known that complement contributes to many other functions Complement refers, historically, to fresh serum capable of lysing antibody (Ab)-coated cells. This activity is destroyed (inactivated) by heating serum at 56EC for 30 minutes. The lytic activity of complement is decreased in certain diseases, e.g. SLE, serum sickness, chronic infections, complement deficiencies, etc.

Complement Functions Host benefit: Host detriment: opsonization to enhance phagocytosis phagocytes attraction and activation lysis of bacteria and infected cells regulation of antibody responses clearance of immune complexes clearance of apoptotic cells Host detriment: Inflammation, anaphylaxis

Proteins of the complement system Complement comprises over 20 different serum proteins These proteins are produced by a variety of cells: Hepatocytes, macrophages, gut epithelial cells Some components can bind to Immunoglobulins While others are proenzymes that when activated can cleave and activate other components

Proteins of the complement system (nomenclature) C1(q r s), C2, C3, C4, C5, C6, C7, C8, C9 factors B, D, H and I, properdin (P) mannose binding lectin (MBL), MBL associated serine proteases (MASP-1 MASP-2) C1 inhibitor (C1-INH, serpin), C4-binding protein (C4-BP), decay accelerating factor (DAF), Complement receptor 1 (CR1), protein-S (vitronectin)

Definitions C-activation: alteration of C proteins such that they interact with the next component C-fixation: utilization of C by Ag-Ab complexes Hemolytic units (CH50): dilution of serum which lyses 50% of a standardized suspension of Ab-coated R.B.Cs C-inactivation: denaturation (usually by heat) of an early C-component resulting in loss of hemolytic activity Convertase/esterase: altered C-protein which acts as a proteolytic enzyme for another C-component

Activation product of complement proteins (nomenclature) Activated component are usually over-lined: e.g. C1qrs When enzymatically cleaved, the larger moiety, binds to the activation complex or membrane and the smaller peptide is released into the microenvironment Letter “b” is usually added to the larger, membrane-binding, peptide and “a” to the smaller peptide (e.g., C3b/C3a, C4b/C4a, C5b/C5a) EXCEPTION: C2 (the larger, membrane-binding moiety is C2a; the smaller one is C2b)

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

Components of the Classical Pathway C1r C1s C1q C4 C2 C3 Chelating agents dismantle the C1 complex and are anti-complementary. Heat destroys the C2 component. Sample for C measurement should be drawn in a green-top vial (no EDTA), must be kept cold and tested as soon as possible. C1 complex

Classical Pathway Generation of C3-convertase b Ca++ C1r C1s C1q C4

Classical Pathway Generation of C3-convertase C2b a C4a Ca++ C1r C1s C1q _____ C4b2a is C3 convertase Mg++ C4b

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

Biological Activities of Classical Pathway Components 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 C3b Opsonin Activation of phagocytic cells C4a Anaphylaotoxin C4b

Control of Classical Pathway Components Regulation All C1-inhibitor (C1-INH); dissociates C1r and C1s from C1q C3a C3a-inactivator (C3a-INA; Carboxypeptidase B) C3b Factors H and I; Factor H facilitates the degradation of C3b by Factor I C4a C3a-INH C4b C4 binding protein (C4-BP) and Factor I; C4-BP facilitates degradation of C4b by Factor I; C4-BP also prevents the association of C2a with C4b thus blocking formation of C3 convertase

Components of mannose-binding (lectin) pathway Mannose Binding Lectin (MBL), which binds to bacterial surface with mannose-containing polysaccharides MBL associated serine proteases (MASP-1 MASP-2)

Components of mannose-binding lectin pathway MASP2 C2 MASP1 MBL

Mannose-binding lectin pathway _____ C4b2a is C3 convertase; it will lead to the generation of C5 convertase C4b C4a C2b C2a C4b C4 C2a C2 MASP2 MASP1 MBL

Components of the alternative pathway factors B & D properdin (P)

Control and regulation of the alternative pathway Factor I & H DAF Complement receptor 1

the result is lysis of these organisms The alternative pathway can be activated by: Many gram-negative bacteria: Neisseria meningitidis and N. gonorrhoea Some gram-positive bacteria Certain viruses and parasites Aggregated immunoglobulina(particularly IgA) the result is lysis of these organisms

Activation of the alternative pathway Spontaneous hydrolysis of C3 to produce C3i C3i cleaves Factor B into Bb The C3iBb complex acts as C3 convertase(has very short half life) Once C3b is formed Factor B binds to it and becomes susceptible to cleavage by Factor D C3bBb is a more stable C3 convertase which continues to generate more C3b(amplfication loop)

Components of the alternative pathway D C3 B P

Degradation of spontaneously produced C3b iC3b C3dg C3c I

C3b stabilization and C5 activation When C3b finds the appropriate surface it binds to factor B, which is cleaved by Factor D to produce C3 convertase(C3bBb) (which is more stable) C3 convertase(C3bBb) is further stabilized by Poperdin

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

C3b regulation on self and activator surfaces

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

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

Components of the lytic pathway 9

Lytic pathway C5-convertase (of the Classical and lectin Pathways or the Alternative Pathway) cleaves C5 into C5a & C5b C5b associates with C6 &C7 and insrets into the cell membrane Then C8 binds, followed by several molecules of C9 C9 molecules form a pore in the membrane C5bC6C7C8C9 is MAC

Biological effects of C5a

Biological properties of C-activation products Biological Effects Regulation C2b (prokinin) edema C1-INH C3a (anaphylatoxin) mast cell degranulation; enhanced vascular permeability; anaphylaxis carboxy-peptidase- B (C3-INA)

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

Biological properties of C-activation products Biological Effects Regulation 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) C5b67 protein-S chemotaxis, attaches to other membranes

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

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

Complement Deficiencies and Disease Alternative Pathway Pathway/Component Disease Mechanism 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 Disease Mechanism 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