THE MULTIPLE ROLES THE MULTIPLE ROLES OF COMPLEMENTOF COMPLEMENT

Dr Andrew GuirguisDr Andrew GuirguisHaematology RegistrarHaematology RegistrarThe Alfred HospitalThe Alfred HospitalScientific Meeting – 22Scientific Meeting – 22ndnd May, May, 20082008

History of complementHistory of complement

Ehrlich – role of ‘complementing’ Ehrlich – role of ‘complementing’ antibodies in killing of bacteria.antibodies in killing of bacteria.

1895 – Bordet1895 – Bordet Subsequent discovery of componentsSubsequent discovery of components Current knowledge:-Current knowledge:-

– > 30 proteins in plasma + on cell > 30 proteins in plasma + on cell surfacessurfaces

– ~ 15% of globulin fraction of proteins~ 15% of globulin fraction of proteins

NomenclatureNomenclature

C1 – C1q, C1r, C1sC1 – C1q, C1r, C1s C4, C2, C3, C5, C6, C7, C8, C9C4, 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 a’ and ‘b’ – added in to denote cleavage

products. products. ‘‘b’ – larger fragmentb’ – larger fragment Alternative pathway proteins:-Alternative pathway proteins:- ‘Factors’ or ‘Factors’ or

identified by single lettersidentified by single letters Complement receptors:-Complement receptors:- named according to named according to

ligand (eg C6 receptor) or using CD system.ligand (eg C6 receptor) or using CD system.

The basics!The basics! ‘‘Innate immune system’Innate immune system’ Cascade Cascade C3 – most important componentC3 – most important component Activation:- innate or adaptive systemsActivation:- innate or adaptive systems

– Classical:- adaptive immune system – immune Classical:- adaptive immune system – immune complexes bind to C1qcomplexes bind to C1q

– Alternative:- innate – chance binding of C3b to Alternative:- innate – chance binding of C3b to microorganism surface.microorganism surface.

Distinction of self from non-self!Distinction of self from non-self! Deficiencies:-Deficiencies:- increased susceptibility to recurrent increased susceptibility to recurrent

infections (pyogenic bacteria) OR illnesses a/w infections (pyogenic bacteria) OR illnesses a/w production of autoantibodies + immune production of autoantibodies + immune complexes.complexes.

Main rolesMain roles

Defends against pyogenic bacterial Defends against pyogenic bacterial infectionsinfections

Bridges both the innate and adaptive Bridges both the innate and adaptive immunity systemsimmunity systems

Assists in disposing of immune Assists in disposing of immune complexes etccomplexes etc

Role in InflammationRole in Inflammation1.1. Opsonization:-Opsonization:- C3b C3b

is important!is important!2.2. Chemotaxis:-Chemotaxis:-

complement complement fragments diffuse fragments diffuse from target – from target – stimulating cellular stimulating cellular movement and movement and activation.activation.

3.3. Target cell Target cell lysis:-lysis:-‘membrane ‘membrane attack complex attack complex hydrophobic ‘plug’ hydrophobic ‘plug’ inserted into lipid inserted into lipid membrane bilayer membrane bilayer

ActivationActivation

Pathways:-Pathways:-1. Classical1. Classical2. Lectin2. Lectin3. Alternative3. Alternative

Common end point:Common end point: formation of C3 formation of C3 convertase – cleaves to C3a and C3bconvertase – cleaves to C3a and C3b– Classical + Lectin pathways – C4b2aClassical + Lectin pathways – C4b2a– Alternative pathway – C3bBbAlternative pathway – C3bBb

Ultimately:-Ultimately:- converted into C5 convertase converted into C5 convertase – by further addition of C3b. Production of – by further addition of C3b. Production of MAC.MAC.

1. Classical pathway1. Classical pathway

‘‘Antibody’ directedAntibody’ directed Begins with C1Begins with C1 C1C1

– Pentamolecule – C1q fragment (6 domains) + 2 Pentamolecule – C1q fragment (6 domains) + 2 x C1r + 2 x C1sx C1r + 2 x C1s

– Antibody binds to two or more of the six Antibody binds to two or more of the six domains (binds either IgG or IgM molecules)domains (binds either IgG or IgM molecules)

– C1 complex undergoes conformational changeC1 complex undergoes conformational change– ‘‘Autocatalysis’ of C1rAutocatalysis’ of C1r– C1s activationC1s activation

2. Lectin pathway2. Lectin pathway

Antibody independentAntibody independent– C1q – calcium-dependent lectin (collectin)C1q – calcium-dependent lectin (collectin)– Other members:- mannan-binding lectin Other members:- mannan-binding lectin

(MBL), conglutinin and lung surfactant A + (MBL), conglutinin and lung surfactant A + D.D.

– MBL – may bind mannose grps on MBL – may bind mannose grps on bacterial surface – then interacts with bacterial surface – then interacts with associated associated SSerine erine PProteases – roteases – MASP1 and MASP1 and 22 (homologous to C1r and C1s). (homologous to C1r and C1s).

– Antibody independent activation of Antibody independent activation of classical pathwayclassical pathway

Downstream effectsDownstream effects

C1 – cleaves C4 – forming activated C4bC1 – cleaves C4 – forming activated C4b– Two isotypes exist Two isotypes exist

C4A – binding amine grps (usually on proteins)C4A – binding amine grps (usually on proteins) C4B – hydroxyl grps on CHOC4B – hydroxyl grps on CHO

C4b – allows binding of C2. Acted on by C4b – allows binding of C2. Acted on by C1s to release C2b.C1s to release C2b.

C4b + C2a = C4b + C2a = classical pathway convertaseclassical pathway convertase (C3)(C3)

By definition:- C3 convertase – breaks up By definition:- C3 convertase – breaks up C3 to C3a and C3b (focus of further C3 to C3a and C3b (focus of further complement activation)complement activation)

What about regulation?What about regulation? C1 inhibitorC1 inhibitor – – serserine ine prproteinase oteinase ininhibitor (aka hibitor (aka

serprin) – binds and inactivates C1r and C1sserprin) – binds and inactivates C1r and C1s Inhibition of formation of C3 convertase Inhibition of formation of C3 convertase

enzyme-enzyme- C4b2a (by ongoing catabolization C4b2a (by ongoing catabolization of C4b by Factor I and C4 binding protein)of C4b by Factor I and C4 binding protein)

Other complement control factorsOther complement control factors – inhibit – inhibit complement binding to host cell surfacescomplement binding to host cell surfaces– DAF:DAF: (Decay accelerating factor) – CD55 (Decay accelerating factor) – CD55– CR1CR1– MCP:MCP: Membrane co-factor protein Membrane co-factor protein– Inhibit binding of C2 to C4b; promote ‘decay Inhibit binding of C2 to C4b; promote ‘decay

acceleration’ of C2a from C4b. Assist in acceleration’ of C2a from C4b. Assist in catabolism of C4b by Factor Icatabolism of C4b by Factor I

Alternative pathwayAlternative pathway Spontaneous activation – C3 is susceptible Spontaneous activation – C3 is susceptible

to spontaneous hydrolysis by waterto spontaneous hydrolysis by water ‘‘Tick over activation’ – to form C3iTick over activation’ – to form C3i C3i – acts as binding site for Factor B C3i – acts as binding site for Factor B

(cleaved by Factor D – to form Ba)(cleaved by Factor D – to form Ba) C3iBb – alternative pathway C3 convertaseC3iBb – alternative pathway C3 convertase Most C3b generated becomes inactivated Most C3b generated becomes inactivated

in water. If it comes into contact with non-in water. If it comes into contact with non-self – initiates amplification loop of self – initiates amplification loop of alternative pathway.alternative pathway.

Regulation… it’s always about Regulation… it’s always about rules!!!rules!!! Factor H and IFactor H and I DAF + CR1 – accelerate dissociation DAF + CR1 – accelerate dissociation

of C3bBbof C3bBb

‘‘How C3b reacts is governed by the How C3b reacts is governed by the surface to which it attaches’ – surface to which it attaches’ –

protected vs non-protectedprotected vs non-protected

Initiators of complement Initiators of complement activation pathwaysactivation pathways ClassicalClassical

– Immune complexesImmune complexes– Apoptotic cellsApoptotic cells– Viruses + GN bacteriaViruses + GN bacteria– CRP bound to ligand CRP bound to ligand

LectinLectin– Mannose groups – terminal ends of Mannose groups – terminal ends of

microbesmicrobes AlternativeAlternative

– Bacteria, fungi, viruses, tumour cells etcBacteria, fungi, viruses, tumour cells etc

Membrane attack complexMembrane attack complex Requires enzymatic cleavage of C5Requires enzymatic cleavage of C5 Sequential binding of C6, C7 (hydrophobic Sequential binding of C6, C7 (hydrophobic

status), C8, C9 (up to 14 monomers)status), C8, C9 (up to 14 monomers) Formation of lytic ‘plug’ – majority of damage Formation of lytic ‘plug’ – majority of damage

caused by C9caused by C9 C9 – analogous to perforin (used by T C9 – analogous to perforin (used by T

lymphocytes)lymphocytes) C5b67 – can be inactivated by numerous means C5b67 – can be inactivated by numerous means

(S protein – vitronectin etc)(S protein – vitronectin etc) RBC immunity:RBC immunity: poorly lysed by homologous poorly lysed by homologous

complementcomplement– CD59:CD59: glycophospholipid foot. Inhibits insertion + glycophospholipid foot. Inhibits insertion +

unfolding of C9 into membranes.unfolding of C9 into membranes.

Clinically speaking…Clinically speaking… CH50 / THC (total haemolytic complement):CH50 / THC (total haemolytic complement):- -

requires all nine components of classical requires all nine components of classical pathway to give normal value – used to screen pathway to give normal value – used to screen for deficiency of classical pathway.for deficiency of classical pathway.– If very low - ? Homozygous deficiency of classical If very low - ? Homozygous deficiency of classical

pathway componentpathway component– Less dramatic reduction during inflammatory Less dramatic reduction during inflammatory

processprocess AH50:AH50: alternative pathway measure alternative pathway measure C3/4:- helpful as activity markers in those with C3/4:- helpful as activity markers in those with

SLESLE Anaphylatoxins:- C5a / C3a – if increased – Anaphylatoxins:- C5a / C3a – if increased –

complement activationcomplement activation ? M’ment of split products? M’ment of split products

Elevated complement levelsElevated complement levels = inflammatory = inflammatory response (i.e acute phase reaction) – esp C3 response (i.e acute phase reaction) – esp C3 / C4 / B/ C4 / B

Reduced levels:Reduced levels: often a/w disease involving often a/w disease involving immune complexes / autoantibodies. May immune complexes / autoantibodies. May be useful for Dx + Mx of certain diseases be useful for Dx + Mx of certain diseases (eg SLE, Sjogren’s, vasculitis etc)(eg SLE, Sjogren’s, vasculitis etc)– Low C4 / C3 + N FB – classical pathway Low C4 / C3 + N FB – classical pathway

activationactivation– Low FB + C3 + N C4 – alternative p’way Low FB + C3 + N C4 – alternative p’way

activationactivation– C4 + FB – low = both p’ways activatedC4 + FB – low = both p’ways activated

Clinical implicationsClinical implications

1.1. Complement deficienciesComplement deficiencies

2.2. GlomerulonephritisGlomerulonephritis

3.3. C1 inhibitor deficiencyC1 inhibitor deficiency

4.4. SLESLE

5.5. PNHPNH

6.6. SepsisSepsis

7.7. APLSAPLS

1. Complement deficiency:-1. Complement deficiency:-Increased susceptibility to pyogenic Increased susceptibility to pyogenic

infectionsinfections Contributing factorsContributing factors

– Deficient opsonisationDeficient opsonisation– Deficiency compromising lytic activityDeficiency compromising lytic activity– Deficient manose-binding lectin pathwayDeficient manose-binding lectin pathway

1.1. Pyogenic infection:-Pyogenic infection:- Site of defect:- antibody production, complement proteins of classical Site of defect:- antibody production, complement proteins of classical

pathway, phagocyte functionpathway, phagocyte function Usually bacteria is opsonised with Ab – complement is then activated, Usually bacteria is opsonised with Ab – complement is then activated,

phagocytosis occurs and intracellular killingphagocytosis occurs and intracellular killing Key player:-Key player:- C3b C3b

2.2. Impaired lysisImpaired lysis MAC component deficiency – a/w Neisserial disease*MAC component deficiency – a/w Neisserial disease* Risk of meningococcal disease ~ 0.5% / yr (RR 5000 cf normal Risk of meningococcal disease ~ 0.5% / yr (RR 5000 cf normal

population)population)

3.3. Deficient lectinDeficient lectin Deficiency occurs due to 1 of 3 point mutations – a/w reduced levels.Deficiency occurs due to 1 of 3 point mutations – a/w reduced levels. Associated with higher risk of infection in children – whilst losing Associated with higher risk of infection in children – whilst losing

passive immunitypassive immunity ? Protective against mycobacterial infections? Protective against mycobacterial infections

2. Glomerulonephritis…2. Glomerulonephritis…

Key of C3b regulation:- Key of C3b regulation:- whether Factor B whether Factor B or H binds to C3bor H binds to C3b

If C3 regulation is defective:-If C3 regulation is defective:- often a/w often a/w GN.GN.– Due to C3 nephritic factor – increases Due to C3 nephritic factor – increases

stability of C3 convertase enzymes – stability of C3 convertase enzymes – association with association with membranoproliferative GNmembranoproliferative GN OROR

– Reduced function of Factor H or IReduced function of Factor H or I ? Associations with HUS (+/- low level of C3)? Associations with HUS (+/- low level of C3)

3. C1 INHIBITOR DEFICIENCY3. C1 INHIBITOR DEFICIENCY Autosomal dominant – inadequate Autosomal dominant – inadequate

production of physiologically adequate C1 production of physiologically adequate C1 inhibitorinhibitor– Type 1:-Type 1:- 85% - reduced transcription of 85% - reduced transcription of

abnormal allele. Reduced levels of C1 inhibitorabnormal allele. Reduced levels of C1 inhibitor– Type 2:- Type 2:- point mutation in C1 inhibitor gene – point mutation in C1 inhibitor gene –

altered activity (altered activity (So levels may be normal or So levels may be normal or high – as not consumed)high – as not consumed)

– Autoantibodies against C1 inhibitorAutoantibodies against C1 inhibitor Inhibits – C1r and C1s, activated FXI and XIIInhibits – C1r and C1s, activated FXI and XII Consumed by plasmin – trigger for Consumed by plasmin – trigger for

angioedema attacks.angioedema attacks. Rx: Rx: C1 inhibitor infusion.C1 inhibitor infusion.

4. Complement deficiency + 4. Complement deficiency + SLESLE Inverse correlation with position of Inverse correlation with position of

deficient protein in activation deficient protein in activation sequence of the classical pathwaysequence of the classical pathway– Homozygous def of C1q, C1r and C1s + Homozygous def of C1q, C1r and C1s +

C4 – strongly a/w SLE (93%, 57%, 75%)C4 – strongly a/w SLE (93%, 57%, 75%)– Cf. def of C2 – 10% prevalence.Cf. def of C2 – 10% prevalence.

Protective role exists for those in Protective role exists for those in whom activation of classical pathway whom activation of classical pathway up to C4 cleavage occurs. up to C4 cleavage occurs.

5. PNH:-5. PNH:- Acquired stem cell disorderAcquired stem cell disorder Deficiency of PIG-A (somatic mutation) – required for Deficiency of PIG-A (somatic mutation) – required for

synthesis of glycosyl-PI phospholipid.synthesis of glycosyl-PI phospholipid. Important for anchorage of proteins to cell Important for anchorage of proteins to cell

membranesmembranes In PNH – lack of GPI-linked proteins (including In PNH – lack of GPI-linked proteins (including

complement-regulating surface proteins) - eg DAF (i.e complement-regulating surface proteins) - eg DAF (i.e CD55) which regulates formation of C3 convertase CD55) which regulates formation of C3 convertase andand CD 59 – restricts formation of MAC.CD 59 – restricts formation of MAC.– Deficiency on RBCs:- Deficiency on RBCs:- does not allow protection against does not allow protection against

terminal complementterminal complement Clinically: Clinically: chronic haemolysis, fatigue, pain, chronic haemolysis, fatigue, pain,

thrombotic events. median age – early 30s; median thrombotic events. median age – early 30s; median survival as low as 10-15 yrs.survival as low as 10-15 yrs.

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

Who to screen?Who to screen?

Hb’uria Hb’uria Coombs –ve haemolytic anaemiaCoombs –ve haemolytic anaemia Those with AA or MDS (annual screen)Those with AA or MDS (annual screen) Haemolytic anaemiaHaemolytic anaemia VT without explanation (including VT without explanation (including

unusual sites – eg mesenteric, portal, unusual sites – eg mesenteric, portal, cerebral etc)cerebral etc)

Unexplained arterial thrombosisUnexplained arterial thrombosis Episodic dysphagia or abdo pain Episodic dysphagia or abdo pain

Parker et al, 2005

Dx:-Dx:- Flow cytometry: Flow cytometry: gold standard (peripheral gold standard (peripheral

blood). Granulocytes provide best estimate of blood). Granulocytes provide best estimate of PNH clone size.PNH clone size.

Role of Soliris (eculizumab)Role of Soliris (eculizumab)

Other Rx:--Supportive transfusions- Haematinic supplementation- Anticoagulation (for those with Hx of thrombosis or for prophylaxis)

-Therefore multiple benefits- Risks??

6. Complement system + 6. Complement system + sepsissepsis C5a – anaphylatoxin – strong C5a – anaphylatoxin – strong

chemoattractant.chemoattractant. Sepsis – excessive early production of Sepsis – excessive early production of

C5a – upregulated proinflammatory C5a – upregulated proinflammatory response.response.

? Role for blockade of C5a with ? Role for blockade of C5a with antibodies – shown to improve survival antibodies – shown to improve survival of septic mice.of septic mice.

? Use in IHD to assist cardiac reperfusion? Use in IHD to assist cardiac reperfusion

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.

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.

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

A role for ‘complement’ary A role for ‘complement’ary medicine?? medicine??