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THE MULTIPLE ROLES OF COMPLEMENT Dr Andrew Guirguis Haematology Registrar The Alfred Hospital Scientific Meeting – 22 nd May, 2008.

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Presentation on theme: "THE MULTIPLE ROLES OF COMPLEMENT Dr Andrew Guirguis Haematology Registrar The Alfred Hospital Scientific Meeting – 22 nd May, 2008."— Presentation transcript:

1 THE MULTIPLE ROLES OF COMPLEMENT Dr Andrew Guirguis Haematology Registrar The Alfred Hospital Scientific Meeting – 22 nd May, 2008

2 History of complement Ehrlich – role of ‘complementing’ antibodies in killing of bacteria. Ehrlich – role of ‘complementing’ antibodies in killing of bacteria – Bordet 1895 – Bordet Subsequent discovery of components Subsequent discovery of components Current knowledge:- Current knowledge:- –> 30 proteins in plasma + on cell surfaces –~ 15% of globulin fraction of proteins

3 Nomenclature Nomenclature C1 – C1q, C1r, C1s C1 – C1q, C1r, C1s C4, C2, C3, C5, C6, C7, C8, C9 C4, C2, C3, C5, C6, C7, C8, C9 Many referred to as ‘zymogens’ Many referred to as ‘zymogens’ ‘a’ and ‘b’ – added in to denote cleavage products. ‘a’ and ‘b’ – added in to denote cleavage products. ‘b’ – larger fragment ‘b’ – larger fragment Alternative pathway proteins:- ‘Factors’ or identified by single letters Alternative pathway proteins:- ‘Factors’ or identified by single letters Complement receptors:- named according to ligand (eg C6 receptor) or using CD system. Complement receptors:- named according to ligand (eg C6 receptor) or using CD system.

4 The basics! ‘Innate immune system’ ‘Innate immune system’ Cascade Cascade C3 – most important component C3 – most important component Activation:- innate or adaptive systems Activation:- innate or adaptive systems –Classical:- adaptive immune system – immune complexes bind to C1q –Alternative:- innate – chance binding of C3b to microorganism surface. Distinction of self from non-self! Distinction of self from non-self! Deficiencies:- increased susceptibility to recurrent infections (pyogenic bacteria) OR illnesses a/w production of autoantibodies + immune complexes. Deficiencies:- increased susceptibility to recurrent infections (pyogenic bacteria) OR illnesses a/w production of autoantibodies + immune complexes.

5 Main roles Defends against pyogenic bacterial infections Defends against pyogenic bacterial infections Bridges both the innate and adaptive immunity systems Bridges both the innate and adaptive immunity systems Assists in disposing of immune complexes etc Assists in disposing of immune complexes etc

6 Role in Inflammation 1. Opsonization:- C3b is important! 2. Chemotaxis:- complement fragments diffuse from target – stimulating cellular movement and activation. 3. Target cell lysis:- ‘membrane attack complex hydrophobic ‘plug’ inserted into lipid membrane bilayer

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8 Activation Pathways:- Pathways:- 1. Classical 2. Lectin 3. Alternative Common end point: formation of C3 convertase – cleaves to C3a and C3b Common end point: formation of C3 convertase – cleaves to C3a and C3b –Classical + Lectin pathways – C4b2a –Alternative pathway – C3bBb Ultimately:- converted into C5 convertase – by further addition of C3b. Production of MAC. Ultimately:- converted into C5 convertase – by further addition of C3b. Production of MAC.

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11 1. Classical pathway ‘Antibody’ directed ‘Antibody’ directed Begins with C1 Begins with C1 C1 C1 –Pentamolecule – C1q fragment (6 domains) + 2 x C1r + 2 x C1s –Antibody binds to two or more of the six domains (binds either IgG or IgM molecules) –C1 complex undergoes conformational change –‘Autocatalysis’ of C1r –C1s activation

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13 2. Lectin pathway Antibody independent Antibody independent –C1q – calcium-dependent lectin (collectin) –Other members:- mannan-binding lectin (MBL), conglutinin and lung surfactant A + D. –MBL – may bind mannose grps on bacterial surface – then interacts with associated Serine Proteases – MASP1 and 2 (homologous to C1r and C1s). –Antibody independent activation of classical pathway

14 Downstream effects C1 – cleaves C4 – forming activated C4b C1 – cleaves C4 – forming activated C4b –Two isotypes exist  C4A – binding amine grps (usually on proteins)  C4B – hydroxyl grps on CHO C4b – allows binding of C2. Acted on by C1s to release C2b. C4b – allows binding of C2. Acted on by C1s to release C2b. C4b + C2a = classical pathway convertase (C3) C4b + C2a = classical pathway convertase (C3) By definition:- C3 convertase – breaks up C3 to C3a and C3b (focus of further complement activation) By definition:- C3 convertase – breaks up C3 to C3a and C3b (focus of further complement activation)

15 What about regulation? C1 inhibitor – serine proteinase inhibitor (aka serprin) – binds and inactivates C1r and C1s C1 inhibitor – serine proteinase inhibitor (aka serprin) – binds and inactivates C1r and C1s Inhibition of formation of C3 convertase enzyme- C4b2a (by ongoing catabolization of C4b by Factor I and C4 binding protein) Inhibition of formation of C3 convertase enzyme- C4b2a (by ongoing catabolization of C4b by Factor I and C4 binding protein) Other complement control factors – inhibit complement binding to host cell surfaces Other complement control factors – inhibit complement binding to host cell surfaces –DAF: (Decay accelerating factor) – CD55 –CR1 –MCP: Membrane co-factor protein –Inhibit binding of C2 to C4b; promote ‘decay acceleration’ of C2a from C4b. Assist in catabolism of C4b by Factor I

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17 Alternative pathway Spontaneous activation – C3 is susceptible to spontaneous hydrolysis by water Spontaneous activation – C3 is susceptible to spontaneous hydrolysis by water ‘Tick over activation’ – to form C3i ‘Tick over activation’ – to form C3i C3i – acts as binding site for Factor B (cleaved by Factor D – to form Ba) C3i – acts as binding site for Factor B (cleaved by Factor D – to form Ba) C3iBb – alternative pathway C3 convertase C3iBb – alternative pathway C3 convertase Most C3b generated becomes inactivated in water. If it comes into contact with non-self – initiates amplification loop of alternative pathway. Most C3b generated becomes inactivated in water. If it comes into contact with non-self – initiates amplification loop of alternative pathway.

18 Regulation… it’s always about rules!!! Factor H and I Factor H and I DAF + CR1 – accelerate dissociation of C3bBb DAF + CR1 – accelerate dissociation of C3bBb ‘How C3b reacts is governed by the surface to which it attaches’ – protected vs non- protected

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20 Initiators of complement activation pathways Classical Classical –Immune complexes –Apoptotic cells –Viruses + GN bacteria –CRP bound to ligand Lectin Lectin –Mannose groups – terminal ends of microbes Alternative Alternative –Bacteria, fungi, viruses, tumour cells etc

21 Membrane attack complex Requires enzymatic cleavage of C5 Requires enzymatic cleavage of C5 Sequential binding of C6, C7 (hydrophobic status), C8, C9 (up to 14 monomers) Sequential binding of C6, C7 (hydrophobic status), C8, C9 (up to 14 monomers) Formation of lytic ‘plug’ – majority of damage caused by C9 Formation of lytic ‘plug’ – majority of damage caused by C9 C9 – analogous to perforin (used by T lymphocytes) C9 – analogous to perforin (used by T lymphocytes) C5b67 – can be inactivated by numerous means (S protein – vitronectin etc) C5b67 – can be inactivated by numerous means (S protein – vitronectin etc) RBC immunity: poorly lysed by homologous complement RBC immunity: poorly lysed by homologous complement –CD59: glycophospholipid foot. Inhibits insertion + unfolding of C9 into membranes.

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23 Clinically speaking… CH50 / THC (total haemolytic complement):- requires all nine components of classical pathway to give normal value – used to screen for deficiency of classical pathway. CH50 / THC (total haemolytic complement):- requires all nine components of classical pathway to give normal value – used to screen for deficiency of classical pathway. –If very low - ? Homozygous deficiency of classical pathway component –Less dramatic reduction during inflammatory process AH50: alternative pathway measure AH50: alternative pathway measure C3/4:- helpful as activity markers in those with SLE C3/4:- helpful as activity markers in those with SLE Anaphylatoxins:- C5a / C3a – if increased – complement activation Anaphylatoxins:- C5a / C3a – if increased – complement activation ? M’ment of split products ? M’ment of split products

24 Elevated complement levels = inflammatory response (i.e acute phase reaction) – esp C3 / C4 / B Elevated complement levels = inflammatory response (i.e acute phase reaction) – esp C3 / C4 / B Reduced levels: often a/w disease involving immune complexes / autoantibodies. May be useful for Dx + Mx of certain diseases (eg SLE, Sjogren’s, vasculitis etc) Reduced levels: often a/w disease involving immune complexes / autoantibodies. May be useful for Dx + Mx of certain diseases (eg SLE, Sjogren’s, vasculitis etc) –Low C4 / C3 + N FB – classical pathway activation –Low FB + C3 + N C4 – alternative p’way activation –C4 + FB – low = both p’ways activated

25 Clinical implications 1. Complement deficiencies 2. Glomerulonephritis 3. C1 inhibitor deficiency 4. SLE 5. PNH 6. Sepsis 7. APLS

26 1. Complement deficiency:- Increased susceptibility to pyogenic infections Contributing factors Contributing factors –Deficient opsonisation –Deficiency compromising lytic activity –Deficient manose-binding lectin pathway 1. Pyogenic infection:-  Site of defect:- antibody production, complement proteins of classical pathway, phagocyte function  Usually bacteria is opsonised with Ab – complement is then activated, phagocytosis occurs and intracellular killing  Key player:- C3b 2. Impaired lysis  MAC component deficiency – a/w Neisserial disease*  Risk of meningococcal disease ~ 0.5% / yr (RR 5000 cf normal population) 3. Deficient lectin  Deficiency occurs due to 1 of 3 point mutations – a/w reduced levels.  Associated with higher risk of infection in children – whilst losing passive immunity  ? Protective against mycobacterial infections

27 2. Glomerulonephritis… Key of C3b regulation:- whether Factor B or H binds to C3b If C3 regulation is defective:- often a/w GN. If C3 regulation is defective:- often a/w GN. –Due to C3 nephritic factor – increases stability of C3 convertase enzymes – association with membranoproliferative GN OR –Reduced function of Factor H or I  ? Associations with HUS (+/- low level of C3)

28 3. C1 INHIBITOR DEFICIENCY Autosomal dominant – inadequate production of physiologically adequate C1 inhibitor Autosomal dominant – inadequate production of physiologically adequate C1 inhibitor –Type 1:- 85% - reduced transcription of abnormal allele. Reduced levels of C1 inhibitor –Type 2:- point mutation in C1 inhibitor gene – altered activity (So levels may be normal or high – as not consumed) –Autoantibodies against C1 inhibitor Inhibits – C1r and C1s, activated FXI and XII Inhibits – C1r and C1s, activated FXI and XII Consumed by plasmin – trigger for angioedema attacks. Consumed by plasmin – trigger for angioedema attacks. Rx: C1 inhibitor infusion. Rx: C1 inhibitor infusion.

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30 4. Complement deficiency + SLE Inverse correlation with position of deficient protein in activation sequence of the classical pathway Inverse correlation with position of deficient protein in activation sequence of the classical pathway –Homozygous def of C1q, C1r and C1s + C4 – strongly a/w SLE (93%, 57%, 75%) –Cf. def of C2 – 10% prevalence. Protective role exists for those in whom activation of classical pathway up to C4 cleavage occurs. Protective role exists for those in whom activation of classical pathway up to C4 cleavage occurs.

31 5. PNH:- Acquired stem cell disorder Acquired stem cell disorder Deficiency of PIG-A (somatic mutation) – required for synthesis of glycosyl-PI phospholipid. Deficiency of PIG-A (somatic mutation) – required for synthesis of glycosyl-PI phospholipid. Important for anchorage of proteins to cell membranes Important for anchorage of proteins to cell membranes In PNH – lack of GPI-linked proteins (including complement- regulating surface proteins) - eg DAF (i.e CD55) which regulates formation of C3 convertase and CD 59 – restricts formation of MAC. In PNH – lack of GPI-linked proteins (including complement- regulating surface proteins) - eg DAF (i.e CD55) which regulates formation of C3 convertase and CD 59 – restricts formation of MAC. –Deficiency on RBCs:- does not allow protection against terminal complement Clinically: chronic haemolysis, fatigue, pain, thrombotic events. median age – early 30s; median survival as low as yrs. Clinically: chronic haemolysis, fatigue, pain, thrombotic events. median age – early 30s; median survival as low as yrs.

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33 Smooth muscle dystonia - ? 2’ to NO depletion during chronic haemolysis

34 Who to screen? Hb’uria Hb’uria Coombs –ve haemolytic anaemia Coombs –ve haemolytic anaemia Those with AA or MDS (annual screen) Those with AA or MDS (annual screen) Haemolytic anaemia Haemolytic anaemia VT without explanation (including unusual sites – eg mesenteric, portal, cerebral etc) VT without explanation (including unusual sites – eg mesenteric, portal, cerebral etc) Unexplained arterial thrombosis Unexplained arterial thrombosis Episodic dysphagia or abdo pain Episodic dysphagia or abdo pain Parker et al, 2005

35 Dx:- Flow cytometry: gold standard (peripheral blood). Granulocytes provide best estimate of PNH clone size. Flow cytometry: gold standard (peripheral blood). Granulocytes provide best estimate of PNH clone size.

36 Role of Soliris (eculizumab) Other Rx:- -Supportive transfusions - Haematinic supplementation - Anticoagulation (for those with Hx of thrombosis or for prophylaxis) -Therefore multiple benefits - Risks??

37 6. Complement system + sepsis C5a – anaphylatoxin – strong chemoattractant. C5a – anaphylatoxin – strong chemoattractant. Sepsis – excessive early production of C5a – upregulated proinflammatory response. Sepsis – excessive early production of C5a – upregulated proinflammatory response. ? Role for blockade of C5a with antibodies – shown to improve survival of septic mice. ? Role for blockade of C5a with antibodies – shown to improve survival of septic mice. ? Use in IHD to assist cardiac reperfusion ? Use in IHD to assist cardiac reperfusion

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39 7. Complement + APLS Nature medicine 2004:- – –Previous mouse models – shown that complement activation plays an important role in pregnancy + fetal growth restriction – –Likely induced by activation thru aPL antibodies (classical pathway) – –Anticoagulation alone – insufficient in completely averting miscarriage – –Heparin use - ? Additional role via inhibition of complement.

40 APLS Mouse model used:- – –Pregnant mice injected with aPL antibodies – –Rx:- heparin (UFH or LMWH) – reduced frequency of fetal resorption to that of healthy controls. – –To rule out mere ‘anticoagulant effect’ – use of fondaparinux or hirudin – both do not directly affect the complement systems.

41 In vivo:- – –Focus on C3 and degradation products – increased levels seen with aPL-IgG injection. – –Abolished by UFH or LMWH, but not by fondaparinux or hirudin. Separate study:- use of Crry-Ig (complement receptor 1- related gene / protein y)(exogenous inhibitor of C3 activation) OR C3 deficient mice – similar results. – –Associated with fewer resorptions and less antibody-mediated growth retardation Activation of complement – associated with thrombophilic state

42 A role for ‘complement’ary medicine??


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