Download THE ROLE OF COMPLEMENT

THE MULTIPLE ROLES
OF COMPLEMENT
Dr Andrew Guirguis
Haematology Registrar
The Alfred Hospital
Scientific Meeting – 22nd May, 2008
History of complement
Ehrlich – role of ‘complementing’
antibodies in killing of bacteria.
 1895 – Bordet
 Subsequent discovery of components
 Current knowledge:
– > 30 proteins in plasma + on cell surfaces
– ~ 15% of globulin fraction of proteins
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.
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.
Main roles
Defends against pyogenic bacterial
infections
 Bridges both the innate and adaptive
immunity systems
 Assists in disposing of immune complexes
etc

Role in Inflammation
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
1.
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.
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
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
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)
What about regulation?
C1 inhibitor – serine proteinase inhibitor (aka
serprin) – binds and inactivates C1r and C1s
 Inhibition of formation of C3 convertase enzymeC4b2a (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
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.
Regulation… it’s 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 nonprotected
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
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.
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
 ? M’ment of split products



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)

– Low C4 / C3 + N FB – classical pathway activation
– Low FB + C3 + N C4 – alternative p’way activation
– C4 + FB – low = both p’ways activated
Clinical implications
1.
2.
3.
4.
5.
6.
7.
Complement deficiencies
Glomerulonephritis
C1 inhibitor deficiency
SLE
PNH
Sepsis
APLS
1. Complement deficiency:Increased susceptibility to pyogenic
infections

Contributing factors
1.
Pyogenic infection:-
– Deficient opsonisation
– Deficiency compromising lytic activity
– Deficient manose-binding lectin pathway

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


MAC component deficiency – a/w Neisserial disease*
Risk of meningococcal disease ~ 0.5% / yr (RR 5000 cf normal population)


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


2.
3.
Impaired lysis
Deficient lectin

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)
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.

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.
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 complementregulating 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 1015 yrs.
Smooth muscle dystonia - ? 2’ to NO depletion during chronic haemolysis
Who to screen?
Hb’uria
 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
Dx:
Flow cytometry: gold standard (peripheral blood).
Granulocytes provide best estimate of PNH clone size.
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 + 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

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 1related 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
medicine??