THE MULTIPLE ROLES OF COMPLEMENT

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THE MULTIPLE ROLES OF COMPLEMENT

• Dr Andrew Guirguis
• Haematology Registrar
• The Alfred Hospital
• Scientific Meeting 22nd May, 2008

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History of complement

• Ehrlich role of complementing antibodies in
  killing of bacteria.
• 1895 Bordet
• Subsequent discovery of components
• Current knowledge-
• gt 30 proteins in plasma on cell surfaces
• 15 of globulin fraction of proteins

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Nomenclature

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

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The basics!

• Innate immune system
• Cascade
• C3 most important component
• 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!
• Deficiencies- increased susceptibility to
  recurrent infections (pyogenic bacteria) OR
  illnesses a/w production of autoantibodies
  immune complexes.

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Main roles

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

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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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Activation

• Pathways-
• 1. Classical
• 2. Lectin
• 3. Alternative
• 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.

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1. Classical pathway

• Antibody directed
• Begins with 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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2. Lectin pathway

• 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

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Downstream effects

• 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 C2a classical pathway convertase (C3)
• By definition- C3 convertase breaks up C3 to
  C3a and C3b (focus of further complement
  activation)

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What about regulation?

• 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)
• 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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Alternative pathway

• Spontaneous activation C3 is susceptible to
  spontaneous hydrolysis by water
• Tick over activation to form C3i
• C3i acts as binding site for Factor B (cleaved
  by Factor D to form Ba)
• 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.

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Regulation its always about rules!!!

• Factor H and I
• 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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Initiators of complement activation pathways

• Classical
• Immune complexes
• Apoptotic cells
• Viruses GN bacteria
• CRP bound to ligand
• Lectin
• Mannose groups terminal ends of microbes
• Alternative
• Bacteria, fungi, viruses, tumour cells etc

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Membrane attack complex

• Requires enzymatic cleavage of C5
• Sequential binding of C6, C7 (hydrophobic
  status), C8, C9 (up to 14 monomers)
• Formation of lytic plug majority of damage
  caused by C9
• C9 analogous to perforin (used by T
  lymphocytes)
• C5b67 can be inactivated by numerous means (S
  protein vitronectin etc)
• RBC immunity poorly lysed by homologous
  complement
• CD59 glycophospholipid foot. Inhibits insertion
  unfolding of C9 into membranes.

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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.
• If very low - ? Homozygous deficiency of
  classical pathway component
• Less dramatic reduction during inflammatory
  process
• AH50 alternative pathway measure
• C3/4- helpful as activity markers in those with
  SLE
• Anaphylatoxins- C5a / C3a if increased
  complement activation
• ? Mment of split products

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• 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,
  Sjogrens, vasculitis etc)
• Low C4 / C3 N FB classical pathway activation
• Low FB C3 N C4 alternative pway activation
• C4 FB low both pways activated

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Clinical implications

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

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1. Complement deficiency-Increased
susceptibility to pyogenic infections

• Contributing factors
• Deficient opsonisation
• Deficiency compromising lytic activity
• Deficient manose-binding lectin pathway
• 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
• Impaired lysis
• MAC component deficiency a/w Neisserial
  disease
• Risk of meningococcal disease 0.5 / yr (RR
  5000 cf normal population)
• 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

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2. Glomerulonephritis

• Key of C3b regulation- whether Factor B or H
  binds to C3b
• 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)

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3. C1 INHIBITOR DEFICIENCY

• 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
• Consumed by plasmin trigger for angioedema
  attacks.
• Rx C1 inhibitor infusion.

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4. Complement deficiency SLE

• 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.

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5. PNH-

• Acquired stem cell disorder
• Deficiency of PIG-A (somatic mutation) required
  for synthesis of glycosyl-PI phospholipid.
• 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.
• 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 10-15 yrs.

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

• Hburia
• Coombs ve haemolytic anaemia
• Those with AA or MDS (annual screen)
• Haemolytic anaemia
• VT without explanation (including unusual sites
  eg mesenteric, portal, cerebral etc)
• Unexplained arterial thrombosis
• Episodic dysphagia or abdo pain

Parker et al, 2005
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Dx-

• Flow cytometry gold standard (peripheral blood).
  Granulocytes provide best estimate of PNH clone
  size.

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Role of Soliris (eculizumab)

• Other Rx-
• Supportive transfusions
• Haematinic supplementation
• Anticoagulation (for those with Hx of thrombosis
  or for prophylaxis)
• Therefore multiple benefits
• Risks??

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6. Complement system sepsis

• C5a anaphylatoxin strong chemoattractant.
• Sepsis excessive early production of C5a
  upregulated proinflammatory response.
• ? Role for blockade of C5a with antibodies
  shown to improve survival of septic mice.
• ? Use in IHD to assist cardiac reperfusion

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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.

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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.

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• 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

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A role for complementary medicine??