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COMPLEMENT SYSTEM

Historically, the term complement (C) was used to refer to a heat-labile serum component
that was able to lyse bacteria (activity is destroyed (inactivated) by heating serum at 56
degrees C for 30 minutes). However, complement is now known to contribute to host
defenses in other ways as well. Complement can opsonize bacteria for enhanced
phagocytosis; it can recruit and activate various cells including polymorphonuclear cells
(PMNs) and macrophages; it can participate in regulation of antibody responses and it can
aid in the clearance of immune complexes and apoptotic cells. Complement can also have
detrimental effects for the host; it contributes to inflammation and tissue damage and it can
trigger anaphylaxis.

Complement comprises over 20 different serum proteins (see Table 1) that are produced by
a variety of cells including, hepatocytes, macrophages and gut epithelial cells. Some
complement proteins bind to immunoglobulins or to membrane components of cells. Others
are proenzymes that, when activated, cleave one or more other complement proteins. Upon
cleavage some of the complement proteins yield fragments that activate cells, increase
vascular permeability or opsonize bacteria.

Table 1. Proteins of the Complement system

Classical Pathway Lectin Alternative Lytic
Pathway Pathway Pathway
Activation Proteins: Mannan
binding protein
C1qrs, C2, C3, C4 (MBP), C3, Factors B C5, C6, C7,
mannan- & D*, C8, C9
asociated Properdin (P)
serine protease
(MASP, MASP2)
Control Proteins:

C1-INH, C4-BP
Protein S
Factors I* & H,
decay
accelerating
factor (DAF),
Complement
receptor
1(CR1), etc.
Components underlined acquire enzymatic activity when
[Link] marked with an asterisk have enzymatic activity
in their native form.

PATHWAYS OF COMPLEMENT ACTIVATION

Complement activation can be divided into four pathways (figure 1): the classical pathway,
the lectin pathway, the alternative pathway and the membrane attack (or lytic) pathway.
Both classical and alternative pathways lead to the activation of C5 convertase and result in
the production of C5b which is essential for the activation of the membrane attack pathway.

CLASSICAL PATHWAY
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C1 activation
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. C1
binding does not occur to antibodies that have not complexed with antigen and binding
requires calcium and magnesium ions. (N.B. In some cases C1 can bind to aggregated
immunoglobulin [e.g. aggregated IgG] or to certain pathogen surfaces in the absence of
antibody). 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.

C4 and C2 activation (generation of C3 convertase)

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.

C3 activation (generation of C5 convertase)

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 classical pathway.

Several of the products of the classical pathway have potent biological activities that
contribute to host defenses. Some of these products may also have detrimental effects if
produced in an unregulated manner. Table 2 summarizes the biological activities of
classical pathway components.

Table 2. Biological Activity of classical pathway

products
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; promotes phagocytosis by binding
to complement receptors

Activation of phagocytic cells

C4a Anaphylotoxin (weaker than C3a)
C4b Opsonin; promotes phagocytosis by binding
to complement receptors

The importance of C1-INH in regulating the classical pathway is demonstrated by the result
of a deficiency in this inhibitor. C1-INH deficiencies are associated with the development
of hereditary angioedema.

LECTIN PATHWAY
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The lectin pathway is very similar to the classical pathway. It is initiated by the binding of
mannose-binding lectin (MBL) to bacterial surfaces with mannose-containing
polysaccharides (mannans). Binding of MBL to a pathogen results in the association of two
serine proteases, MASP-1 and MASP-2 (MBL-associated serine proteases). MASP-1 and MASP-
2 are similar to C1r and C1s, respectively and MBL is similar to C1q. 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. 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.

The biological activities and the regulatory proteins of the lectin pathway are the same as
those of the classical pathway.

ALTERNATIVE PATHWAY
The alternative pathway begins with the activation of C3 and requires Factors B and D and
Mg++ cation, all present in normal serum.

1. Amplification loop of C3b formation

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. Once C3b is formed,
Factor B will bind to it and becomes susceptible to cleavage by Factor D. The resulting
C3bBb complex is a C3 convertase that will continue to generate more C3b, thus amplifying
C3b production. If this process continues unchecked, the result would be the consumption of
all C3 in the serum. Thus, the spontaneous production of C3b is tightly controlled by the
Decay accelerating factor

2. Control of the amplification loop

As spontaneously produced C3b binds to autologous host membranes, it interacts with DAF
(decay accelerating factor), which blocks the association of Factor B with C3b thereby
preventing the formation of additional C3 convertase. In addition, DAF accelerates the
dissociation of Bb from C3b in C3 convertase that has already formed, thereby stopping the
production of additional C3b. Some cells possess complement receptor 1 (CR1). Binding of
C3b to CR1 facilitates the enzymatic degradation of C3b by Factor I. In addition, binding of
C3 convertase (C3bBb) to CR1 also dissociates Bb from the complex. Thus, in cells
possessing complement receptors, CR1 also plays a role in controlling the amplification loop.
Finally, Factor H can bind to C3b bound to a cell or in the in the fluid phase and facilitate the
enzymatic degradation of C3b by Factor I. Thus, the amplification loop is controlled by either
blocking the formation of C3 convertase, dissociating C3 convertase, or by enzymatically
digesting C3b. The importance of controlling this amplification loop is illustrated in patients
with genetic deficiencies of Factor H or I. These patients have a C3 deficiency and increased
susceptibility to certain infections.

3. Stabilization of C convertase by activator (protector)

When bound to an appropriate activator of the alternative pathway, C3b will bind
Factor B, which is enzymatically cleaved by Factor D to produce C3 convertase (C3bBb).
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However, C3b is resistant to degradation by Factor I and the C3 convertase is not rapidly
degraded, since it is stabilized by the activator surface. The complex is further stabilized by
properdin binding to C3bBb. Activators of the alternate pathway are components on the
surface of pathogens and include: LPS of Gram-negative bacteria and the cell walls of some
bacteria and yeasts. Thus, when C3b binds to an activator surface, the C3 convertase
formed will be stable and continue to generate additional C3a and C3b by cleavage of C3.

4. Generation of C5 convertase

Some of the C3b generated by the stabilized C3 convertase on the activator surface
associates with the C3bBb complex to form a C3bBbC3b complex. This is the C5 convertase
of the alternative pathway. The generation of C5 convertase is the end of the alternative
pathway. The alternative pathway can be activated by many Gram-negative (most
significantly, Neisseria meningitidis and N. gonorrhoea), some Gram-positive bacteria and
certain viruses and parasites, and results in the lysis of these organisms. Thus, the
alternative pathway of C activation provides another means of protection against certain
pathogens before an antibody response is mounted. A deficiency of C3 results in an
increased susceptibility to these organisms. The alternate pathway may be the more
primitive pathway and the classical and lectin pathways probably developed from it.

Remember that the alternative pathway provides a means of non-specific resistance against
infection without the participation of antibodies and hence provides a first line of defense
against a number of infectious agents.

Many gram negative and some gram positive bacteria, certain viruses, parasites,
heterologous red cells, aggregated immunoglobulins (particularly, IgA) and some other
proteins (e.g. proteases, clotting pathway products) can activate the alternative pathway.
One protein, cobra venom factor (CVF), has been extensively studied for its ability to
activate this pathway.

MEMBRANE ATTACK (LYTIC) PATHWAY

C5 convertase from the classical (C4b2a3b), lectin (C4b2a3b) or alternative (C3bBb3b)

pathway cleaves C5 into C5a and C5b. C5a remains in the fluid phase and the C5b rapidly
associates with C6 and C7 and inserts into the membrane. Subsequently C8 binds, followed
by several molecules of C9. The C9 molecules form a pore in the membrane through which
the cellular contents leak and lysis occurs. Lysis is not an enzymatic process; it is thought to
be due to physical damage to the membrane. The complex consisting of C5bC6C7C8C9 is
referred to as the membrane attack complex (MAC).

C5a generated in the lytic pathway has several potent biological activities. It is the most
potent anaphylotoxin. In addition, it is a chemotactic factor for neutrophils and stimulates
the respiratory burst in them and it stimulates inflammatory cytokine production by
macrophages. Its activities are controlled by inactivation by carboxypeptidase B (C3-INA).

Some of the C5b67 complex formed can dissociate from the membrane and enter the fluid
phase. If this were to occur it could then bind to other nearby cells and lead to their lysis.
The damage to bystander cells is prevented by Protein S (vitronectin). Protein S binds to
soluble C5b67 and prevents its binding to other cells.
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BIOLOGICALLY ACTIVE PRODUCTS OF COMPLEMENT ACTIVATION

Activation of complement results in the production of several biologically active molecules

which contribute to resistance, anaphylaxis and inflammation.

Kinin production
C2b generated during the classical pathway of C activation is a prokinin which becomes
biologically active following enzymatic alteration by plasmin. Excess C2b production is
prevented by limiting C2 activation by C1 inhibitor (C1-INH) also known as serpin which
displaces C1rs from the C1qrs complex). A genetic deficiency of C1-INH results in an
overproduction of C2b and is the cause of hereditary angioneurotic edema. This condition
can be treated with Danazol which promotes C1-INH production or with ε-amino caproic acid
which decreases plasmin activity.

Anaphylotoxins
C4a, C3a and C5a (in increasing order of activity) are all anaphylotoxins which cause
basophil/mast cell degranulation and smooth muscle contraction. Undesirable effects of
these peptides are controlled by carboxypeptidase B (C3a-INA).

Chemotactic Factors
C5a and MAC (C5b67) are both chemotactic. C5a is also a potent activator of neutrophils,
basophils and macrophages and causes induction of adhesion molecules on vascular
endothelial cells.

Opsonins
C3b and C4b in the surface of microorganisms attach to C-receptor (CR1) on phagocytic cells
and promote phagocytosis.

Other Biologically active products of C activation

Degradation products of C3 (iC3b, C3d and C3e) also bind to different cells by distinct
receptors and modulate their functions.

In summary, the complement system takes part in both specific and non-specific resistance
and generates a number of products of biological and pathophysiological significance

There are known genetic deficiencies of most individual C complement components, but C3
deficiency is most serious and fatal. Complement deficiencies also occur in immune complex
diseases (e.g., SLE) and acute and chronic bacterial, viral and parasitic infections.
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Table 4. Activities of Complement Activation Products and their

Control Factors
Fragment Activity Effect Control
Factor (s)
C2a Prokinin, accumulation of Edema C1-INH
fluids
C3a Basophil and mast cells Anaphylaxis C3a-INA
degranulation; enhanced
vascular permeability,
smooth muscle contraction
C3b Opsonin, phagocyte Phagocytosi Factors H
activation s and I
C4a Basophil and mast cells Anaphylaxis C3a-INA
degranulation; enhanced
vascular permeability, (least
smooth muscle contraction potent)

C4b Opsonin Phagocytosi C4-BP and

s Factor I
C5a Basophil and mast cells Anaphylaxis C3a-INA
degranulation; enhanced
vascular permeability, (most
smooth muscle contraction potent)
Chemotaxis, stimulation of Inflammatio
respiratory burst, n
activation of phagocytes,
stimulation of
inflammatory cytokines
C5bC6C7 Chemotaxis Inflammatio Protein S
n (vitronectin)
Attaches to other Tissue
membranes damage
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Table 5. Complement deficiencies and disease

Pathway/
Disease Mechanism
Component
Classical Pathway
Hereditary Overproduction of C2b
C1INH
angioedema (prokinin)
Opsonization of immune
complexes help keep them
Predisposition to
C1, C2, C4 soluble, deficiency results in
SLE
increased precipitation in
tissues and inflammation
Lectin Pathway
Susceptibility to
bacterial
infections in Inability to initiate the lectin
MBL
infants or pathway
immunosuppresse
d
Alternative
Pathway
Susceptibility to
pyogenic (pus- Lack of sufficient opsonization
Factors B or D
forming) bacterial of bacteria
infections
Susceptibility to Lack of opsonization and
C3 bacterial inability to utilize the
infections membrane attack pathway
Susceptibility to Inability to attack the outer
C5, C6, C7 C8, and
Gram-negative membrane of Gram-negative
C9
infections bacteria
Susceptibility
Lack of opsonization of
Properdin (X-linked) meningococcal
bacteria
meningitis
C3 deficiency and
Uncontrolled activation of C3
susceptibility to
Factors H or I via alternative pathway
bacterial
resulting in depletion of C3
infections