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Complement
Hazem M. Abu-Eisha
Ph.D , MD
Assistant Professor of Immunology
Pathology Department
King Saud University
King Khaled University Hospital
E mail : [email protected]
Outlines
1. General considerations
2. Complement Activation Pathways
3. Consequences of Complement Activation ( Functions )
4. Regulations of Complement Activity
5. Complement Deficiency
6. Measurement of complement activity
Story of Complement started in 1890s , in
Institute Pasteur in Paris
when Jules Bordet
found that sheep antiserum to bacterium Vibrio
Cholerae cause lysis of this bacteria
Heating of
this antiserum
destroy its
bacteriolytic activity & this ability was restored by
adding fresh serum that doesn't contain Abs
against this bacterium
Explanation of this bacteriolytic
activity is :
1.
Sheep antiserum contain substances { Specific
antibacterial Abs } survive heating process
2.
Heat sensitive component responsible for the
lytic activity
General
Considerations

A complex group of normally found inactive plasma
proteins that interact with each other in a cascading fashion
to protect against infectious agents

Historically,
the term complement (C) was
used to refer to a heat labile serum component that was
able to lyse bacteria
 Proteins of complement are mainly synthesized by liver hepatocytes.
Also , it can be formed by monocytes & macrophages
 Complement components are designated by numbers ( C1 - C9 ) or by
letters ( Factor B )
 Many components are proenzymes ( i.e. : Inactive until proteolytic
cleavage occurs )
 Activated components of complement are over-lined ( e.g. C1qrs )

When complement component was enzymatically cleaved , it is
divided into two fragments
(a&b)
The letter “a” refers to the smaller fragment which initiates localized
inflammatory response and the letter “b” refers to the larger fragment
which binds to the target ( e.g. C3a and C3b )
The exception is for C2 in which the larger fragment is given letter
“a” and the smaller fragment is given the letter “b”
Complement
proteins
* C1 ( qrs ) , C2 , C3, C4 , C5 , C6 , C7 , C8 & C9
* Factors B , D , H , I & Properdin ( P )
* Mannose - Binding Lectin (MBL ) , Mannose- associated serine proteases ( MASP
-1 & MASP - 2 )
* C1 inhibitor ( C1INH ) , C4b - binding protein ( C4 - BP ) & Decay accelerating
factor ( DAF )
* Complement receptors ( CR1, CR2, CR3 & CR4 )
Serine Proteases ARE :
Proteolytic enzymes that cleave the next components in the cascade
[ C1r, C1s , C2b & Factors D & B ]
1. Classical Pathway
•
Activation of classical pathway is
antibody - dependent
•
IgM & IgG ( IgG3 , IgG1 & IgG2 )
are the most important two
isotypes
in
activating
this
pathway
• Sequence :
1.
2.
3.
4.
C1 is firstly activated & cleaved (
C1q , C1r & C1s )
C1s activates C2 & C4
C2a & C4b activate C3 ( C3
convertase )
Finally , C4b2a3b is formed ( C5
convertase )
2. Alternative Pathway

Antibody- independent pathway
 It is activated when C3 binds to the
surface of a microorganism
 C3b cleaves factors B & D with release
of Ba & Bb fractions
 Together with C3b , factor Bb forms
C3bBb complex
( C3 convertase )
 Factor P ( Properdin ) stabilizes C3bBb
complex as it binds with it
 C3bBb binds other C3b fragments
producing C3bBb3b (
C5 convertase )
3. Lectin Pathway
•
Lectin pathway is antibody - independent
•
It is closely similar to classical pathway
•
Lectins are proteins that can bind carbohydrate ( CHO ) and normally found
in serum
•
The pathway is activated by binding of Mannose - Binding Lectin ( MBL ) to
mannose residues on glycoprotein or CHO on the surface of microorganisms
forming a complex ( MBL - serine protease 1 & 2 ) greatly similar to C1
•
MBL- associated serine protease ( MASP ) acts on C4 & C2 to generate C3
convertase
Membrane Attack Complex (MAC / C5b-9)
•
C5
convertase
from
the
classical
(C4b2a3b), lectin (C4b2a3b) or alternative
(C3bBb3b) pathways cleaves C5 into C5a
and C5b
•
C5b rapidly associates with C6 and C7
and inserts into the membrane. The C5b67
complex is referred to as the Membrane
Attack Complex (MAC)
•
Subsequently C8 binds, followed by
binding of several molecules of C9
•
The C9 molecules form a pore in the
membrane
through
which
the
contents leaks out and lysis occurs
cellular
Functions of Complement
Biological Consequences of
Complement Activation
o
Lysis of bacterial cells & viruses
o Antigen opsonization that facilitate phagocytosis
o Viral neutralization
o Initiation of immune responses as : Inflammation
o
Solubilization & clearance of immune complexes
Complement has a central role in inflammation causing chemotaxis of phagocytes, activation of mast
cells and phagocytes, opsonization and lysis of pathogens, and clearance of immune complexes.
1. Cell Lysis
Cells susceptible to complement mediated - lysis are :
1. Viruses
2. Gram negative bacteria
But some gram negative bacteria
& most of gram positive
bacteria are generally resistant to complement mediated lysis
WHAT ARE THE EVADING MECHANISMS ?
1.
Long polysaccharide side chain in the cell wall that prevents insertion of
MAC into the bacterial membrane
2.
Capsulated bacteria prevents interaction between C3b deposited in the
membrane & CR1 on phagocytic cells
3.
Some bacteria have elastase which inactivates C3a & C5a
2. Antigen
Opsonization
•
C3b is the major & potent
opsonin of complement system
Activation of C3
Coating
of C3b on immune complexes &
different
antigens
Phagocytic cells ( Neutrophils ,
monocytes
&
macrophages
)
express complement receptors (
CR1 , CR3 & CR4 ) can bind C3b
that will enhance phagocytosis
3. Viral Neutralization
Mechanisms of Viral neutralization :
1.
Binding of Ab & or complement to the viral particles
forms a thick protein coat which
neutralizes viral infectivity by blocking attachment to the host cells ( C4 )
2.
Binding of viral particles to cells possessing Fc or CR1 ( Phagocytic cells ) thus
enhancing phagocytosis & intracellular destruction of ingested viral particle
3.
Lysing enveloped viruses that leads to fragmentation of the envelope & disruption of the
nucleocapsid
4. Inflammatory Response
•
Smaller fragments resulting from complement cleavage , C3a,
C4a & C5a called ANAPHYLATOXINS which can bind to
receptors on basophiles & mast cells
degranulations with
release of pharmacologically active mediators :
1. Smooth muscle contraction
2. Increased vascular permeability
•
So complement activation
influx of fluids that carries
antibody & phagocytic cells to the site of antigen entry
•
C3a, C5a & C5b67 are the most important chemotactic factors with
C5a is the most potent in mediating this process
5. Solubilization of
Immune Complexes
•
•
This function is evident in patients with SLE
Complement deficiency ( C4 ) leads to SLE as
it interfere with effective solubilization & clearance of
immune complexes which in turn leads to their
persistence
TISSUE DAMAGE
( Type III hypersensitivity reaction )
•
RBCs express CR1. Coating
the immune complexes with C3b
helps in binding to CR1 on
RBCs.
•
These immune complexes are
carried to liver & spleen where
they are separated from RBCs to
be phagocytosed & prevented
from their deposition in tissues
Are you tired like this ?
Have a rest !!!!!!!
Regulation of the
Complement System
•
Complement
can
spontaneously through
pathway
•
It must be controlled by regulatory
proteins to prevent complement mediated
damage of healthy autologous cells
be
the
activated
alternative
•
C3 is unstable compound & without inhibition ,
spontaneous break down occur with production of very
reactive C3b fragment
•
C3b can react to two common chemical functional
groups ; Amino & Hydroxyl groups
•
Unless C3b is neutralized by water ; many organisms
contain these two functional groups where C3b attach to
the pathogen and is not broken down
Regulatory Mechanisms
1.
Serum proteins enzymatically attack
complement components
so inactivate
them
2.
Serum proteins bind
complement component
3.
to
&
inhibit
Regulatory proteins in cell membranes
Function
Component
Regulatory Component
C1
C1- Inh ( 2 )
Dissociates C1r and C1s from C1q
C3a
C3a inactivator ( 1 )
Inactivates C3a
C3b
Factors H ( 2 ) and I ( 1 )
Factor
H
facilitates
the
degradation of C3b by Factor I
MAC
CD59 ( MAC inhibitor ) ( 3 )
Prevents formation of MAC
C4b
C4b - binding protein (C4b-BP)
( 2 ) and Factor I ( 1 )
C4b-BP facilitates degradation of
C4b by Factor I.
C4b-BP also prevents association
of C2a with C4b thus blocking the
formation of C3 convertase
C3 convertase Decay accelerating factor
( DAF ) ( 3 )
C5b67
S protein ( Vitronectin ) ( 3 )
Accelerates decay & prevent
assembly of C3 convertase
Binds soluble C5b67 & prevent its
insertion into cell membrane
Complement Deficiency
•
Deficiency of one of the regulatory components can lead to a
significant disease
•
Example :
Deficiency of C1 inhibitor ( C1Inh )
Hereditary Angioedema
There is activation of Classical Pathway
It may be fatal if not treated & controlled ; as if it occurs in Larynx
that end with fatal swelling & oedema which can obstruct the
airway
Deficiency or dysfunction of CD59 can
leads to
erythrocytes
Increased susceptibility of
( RBCs )
autologous complement
required )
to lysis
in a diseases called
The upper diagram
( Low levels of
that is much lower than normally
Nocturnal Haemoglobinuria
•
(1):
Paroxysmal
( PNH )
Assembly of MAC in absence of
the regulator CD59. C9 binds C5b-8, with
further recruitment of
C9
molecules, which in turn forms MAC
•
In the lower diagram ( 2 ) : CD59 binds the C5b-8 complex
and prevents insertion of C9, which is essential for the initiation of MAC
pore formation.
11
2
• C3
is essential for all complement
pathway
( Classic , Alternative & Lectin )
• Patients
with C3 deficiency usually
suffer from severe bacterial infections,
reflecting the central role of C3 in
activating C5
that ends with MAC
formation
Measurement of
Complement Activity
•
Complement Fixation Test ( CFT )
depends on formation of Ag/Ab complex that
based on consumption of complement
•
CFT can be used to identify one of them if the
other is known ( Usually AB )
•
Mainly used in viral infections
First step :
o
Ag & Ab are mixed
o
Known amount of complement is added
o
If Ag/Ab complexes are
complement will be fixed
o
If Ag/Ab complexes are
will be fixed
not formed
formed
, no
, complement
Second step :
o Add an indicator system
{ Sensitized red blood cells }
Indicator system is used where Standard amount of
RBCs that have been pre-coated with anti-erythrocyte
antibodies is added
RESULT
o
If Ag/Ab complexes are not formed ,
complement will be fixed by the indicator system ,
All RBCs
will be lysed
& the test is
NEGATIVE
o
If Ag/Ab complexes are formed , complement
will be fixed by these complexes & some of the
complement will be consumed by these
complexes so : Not all RBCs of the indicator
system will be lysed & the test is POSITIVE
Simply, measuring the amount RBCs lysis by
measuring the release of haemoglobin into
the medium
: Indirectly measure the
amount of AG / AB complexes in the tested
sample
Complement Haemolytic
Assay ( CH50 )
o
Functional evaluation of Classical pathway with
assessment of MAC
o
CH50 measures complement required to obtain
50% haemolysis of sheep RBCs under standard
conditions
o
Haemolysis
is
measured
by
amount
haemoglobin released from lysed RBCs
of
1.
Classical
pathway
activity
is
measured
using
antibody-sensitized
sheep
erythrocytes in a buffer containing both Ca2+ and Mg2+ ions
2.
Standards and samples diluted in the specific buffer are incubated with the
relevant target cell and the amount of hemolysis measured
3.
The results are converted mathematically to standardized hemolytic units (CH50)
Measurement of Complement
Components
Measurement of Complement components
especially : C3 & C4
 ELISA
 Single radioimmunodiffusion
 Nephlerometry
Mainly in Immunodeficiency diseases &
autoimmune disorders ( SLE )
TEXT BOOK FOR
ADVANCED READING
Kuby
Immunology
4th edition
Richard A. Goldsby , Thomas J.
Kindt and
Barbara A. Osborne
ISBN
0 - 7167 - 3331 - 5
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