COMPLEMENT SYSTEM

RAKESH SHARDA
Department of Veterinary Microbiology
NDVSU College of Veterinary Science & A.H.,
MHOW
 Complement: History

Discovered in 1894 by
Bordet
It represents lytic activity
of fresh serum

Its lytic activity destroyed

when heated at 56°C
for 30 min
 Complement

 A series of molecules present in serum which interact

with the bacterial cell membranes and with each other so
that, at completion, they punch a hole in the bacterial
membrane.
 It is composed of a series of reactions which act as a
cascade with the product of one active on the next in the
chain.
 The products amplify the reactions, increase permeability
of blood vessels and also act to attract cells to the site of
the reaction. The products make the target more
attractive to phagocytosis by immune cells
(“opsonization”).
 30+ components – 20 are soluble, rest attached to
membranes

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 Complement

 It can be activated by interaction of one of the

initial components with bacterial cell surface
substances (“Lectin pathway”), bacterial
products (the “Alternative pathway”), or by
antibodies that have bound to antigens (the
“Classical pathway”).
 There is a “recognition unit”, made up of some
of the initial components.
 The final product is the “membrane attack
complex”.

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 Complement functions

• Host benefit:
– opsonization to enhance phagocytosis
– phagocyte 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
 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.c

• 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
 Proteins of the complement
system (nomenclature)

• C1(qrs), 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)
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 in 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), EXCEPT C2 (the larger, membrane-
binding moiety is C2a; the smaller one is C2b)
 Pathways of complement
activation
CLASSICAL LECTIN ALTERNATIVE
PATHWAY PATHWAY PATHWAY

antibody antibody
dependent independent

Activation of C3 and
generation of C5 convertase

activation
of C5

LYTIC ATTACK
PATHWAY
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 Components of the Classical
Pathway

C3 C4

C1 complex

C1 a multi-subunit protein containing three different proteins, C1q,

C1r and C1s, binds to the Fc region of IgG and IgM antibody
molecules that have interacted with antigen.
 Classical Pathway
Generation of C3-convertase
 The binding of C1 to antibody is via C1q and C1q
must cross link at least two antibody molecules
before it is firmly fixed.
 The binding of C1q results in the activation of C1r
which in turn activates C1s. The result is the
formation of an activated “C1qrs”, which is an
enzyme that cleaves C4 into two fragments C4a and
C4b.
 The C4b fragment binds to the membrane and the C4a
fragment is released into the microenvironment.
 Activated “C1qrs” also cleaves C2 into C2a and C2b.
 C2a binds to the membrane in association with C4b
and C2b is released into the microenvironment. The
resulting C4bC2a complex is a C3 convertase, which
cleaves C3 into C3a and C3b.

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 Classical Pathway
Generation of C5-convertase
 C3b binds to the membrane in
association with C4b and C2a
 C3a is released into the
microenvironment.
 The resulting C4bC2aC3b is a C5
convertase.
 The generation of C5 convertase is the
end of the classical pathway.

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 Biological Activities of Classical
Pathway Components
Component 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 Opsonin
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 Control of Classical Pathway
Components
Component 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
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Components of mannose-binding lectin
pathway

MBL MASP1
 Mannose-binding lectin pathway

 Initiated by the binding of mannose binding lectin

(MBL) to bacterial surfaces with mannose-
containing polysaccharides
 Binding of MBL to a pathogen results in the
association of two serine proteases, MASP-1 and
MASP-2 (MBL-associated serine proteases).
 Formation of the MBL/MASP-1/MASP-2 tri-
molecular complex results in the activation of the
MASPs and subsequent cleavage of C4 into C4a
and C4b.
 The C4b fragment binds to the membrane and the
C4a fragment is released into the
microenvironment.

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 Mannose-binding lectin pathway

 Activated MASPs also cleave C2 into C2a and

C2b.
 C2a binds to the membrane in association with
C4b and C2b is released into the
microenvironment.
 The resulting C4bC2a complex is a C3
convertase, which cleaves C3 into C3a and C3b.
 C3b binds to the membrane in association with
C4b and C2a and C3a is released into the
microenvironment.
 The resulting C4bC2aC3b is a C5 convertase.
 The generation of C5 convertase is the end of the
lectin pathway.

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 Components of the
alternative pathway

C3
 Spontaneous C3 activation

 In serum there is low level spontaneous

hydrolysis of C3 to produce C3i.
 Factor B binds to C3i and becomes
susceptible to Factor D, which cleaves
Factor B into Bb.
 The C3iBb complex acts as a
C3 convertase and cleaves C3 into C3a
and C3b.
 C3iBb complex has a very short half life

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 C3-activation
the amplification loop
 Once C3b is formed, Factor B will
bind to it and becomes susceptible
to cleavage by Factor D.
 The resulting C3bBb complex is an
alternative C3 convertase that will
continue to generate more C3b, thus
amplifying C3b production.

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 C5-convertase of the two
pathways

C5-convertase of the C5-convertase of the

Classical and lectin Alternative Pathway
Pathways

C3b C3b C3b

C4b
 Lytic pathway

Generation of C5 convertase
leads to the activation of the

Lytic pathway
Components of the lytic pathway

C7
C6

C
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 Biological properties of C-activation
products

Product Biological Effects Regulation

C2b
edema C1-INH
(prokinin)

C3a mast cell degranulation; carboxy-

(anaphylatoxin) enhanced vascular peptidase- B
permeability; (C3-INA)
anaphylaxis
 Biological properties of C-activation
products

Product Biological Effects Regulation

C3b opsonization; factors H & I
(opsonin) phagocyte activation

C4a as C3, but less (C3-INA)

(anaphylatoxin) potent

C4b opsonization; C4-BP,

(opsonin) phagocytosis factor I
 Biological properties of C-activation
products

Product Biological Effects Regulation

C5a anaphylactic as C3, but carboxy-

(chemotactic much more potent; peptidase-B
factor) attracts & activates PMN (C3-INA)
causes neutrophil
aggregation, stimulation
of oxidative metabolism
and leukotriene release

C5b67 chemotaxis, attaches protein-S

to other membranes
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COMPLEMENT FIXATION TEST

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 COMPLEMENT FIXATION TEST

The activation of the classical complement pathway by

antibody bound to antigen results in the generation of
membrane attack complexes that can disrupt the
membranes. If the antibody is bound to red cells, these
are ruptured and haemolyis occurs. This phenomenon
can be used to measure serum antibody levels in a test
called the complement fixation test. Complement fixation
tests are most useful as an aid in the diagnosis of acute
or recent viral infection, because they primarily detect
IgM class of antibody. The test entails the use of viral
antigens, guinea pig complement and an indicator system
of “sensitized” sheep RBCs, ie.e sheep RBCs + anti-
sheep RBC antibody (haemolysin).
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COMPLEMENT FIXATION TEST

 The complement fixation assay

indicator system uses sheep red
blood cells (SRBC) and anti-SRBC
antibody (haemolysin).

 If the ANTIBODY SPECIFIC FOR

THE ANTIGEN IN THE ASSAY IS
PRESENT in the patient's serum,
then complement is completely
consumed in the reaction and
there is none left to bind to the
SRBC/anti-SRBC complexes.

 A Test Positive For Ab =

NO HEMOLYSIS

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COMPLEMENT FIXATION TEST

 If there is NO ANTIBODY
PRESENT in the patient's serum
the antigen is not bound, and the
complement reagent does not
have immune complexes with
which to react.
 Complement is still present in the
indicator reaction and binds
strongly to the SRBC/anti-SRBC
complexes. This causes the
SRBCs to burst in a process
called hemolysis.
 A Test Negative For Ab =
LOTS OF HEMOLYSIS
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 Complement Fixation
Serum with Serum without
antibodies antibodies
Day 1

Antigen binds Unbound

to antibodies antigen

Complement
Unbound
binds to Ag/Ab
complement
complex
Hemolysin sensitized Hemolysin sensitized
red blood cells red blood cells
Day 2

serve as an indicator serve as an indicator

No lysis Lysis
Positive Negative
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Complement Fixation

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