The Complement System

Complement
• 1890s Jules Bordet
• Sheep antiserum could lyse Vibrio cholera
• Heated antiserum could not lyse bacteria
• Added fresh serum (with no cholera antibodies) to heated serum
• Lysing ability restored!!!!
• Bordet correctly reasoned that bacteriolytic activity requires two
different substances:
• first, the specific antibacterial antibodies, which survive the heating
process,
• and a second, heat-sensitive component responsible for the lytic activity.
• Complement = activity in serum that completes the action of antibodies.
The Complement System

Overview
Sometimes the interaction of antibodies with antigen is useful by itself. For
example, coating a virus or bacterium thus preventing it from binding to -
and invading - a host cell (e.g., antipolio antibodies); binding to a toxin
molecule (e.g., diphtheria or tetanus toxin) thus keeping the toxin from
entering a cell where it does its dirty work.
But most of the time, the binding of antibodies to antigen erforms no useful
function until and unless it can activate an effector mechanism. The
complement system serves several effector roles. So, the complement
system provides the actual protection from the response while the
interaction of antibodies and antigen provides the specificity of the
response.
Put another way, antibodies "finger" the target, complement destroys it.
Complement includes more than 30 soluble and cell-bound proteins. The
biological activities of this system affect both innate and acquired immunity

After initial activation, the various complement components interact, in a highly

regulated cascade, to carry out a number of basic functions including:

1. Lysis of cells, bacteria, and viruses

2.Opsonization, which promotes phagocytosis of particulate antigens

3. Binding to specific complement receptors on cells of the immune system,

triggering specific cell functions, inflammation, and secretion of
immunoregulatory molecules

4. Immune clearance, which removes immune complexes from the circulation

and deposits them in the spleen and liver
 The Complement Components

The proteins and glycoproteins that compose the complement system are
synthesized mainly by liver hepatocytes, also blood monocytes, tissue
macrophages, and epithelial cells of the gastrointestinal and genitourinary
tracts.

These components constitute 5% of the serum globulin fraction.

Most circulate in the serum in functionally inactive forms as proenzymes, or

zymogens, which are inactive until proteolytic cleavage, which removes an
inhibitory fragment and exposes the active site.

The complement-reaction sequence starts with an enzyme cascade.

Complement components are designated by numerals (C1–C9), by letter
symbols (e.g., factor D), or by trivial names (e.g., homologous restriction
factor).

Peptide fragments formed by activation of a component are denoted by

small letters. In most cases, the smaller fragment resulting from cleavage of
a component is designated “a” and the larger fragment designated “b” (e.g.,
C3a, C3b; note that C2 is an exception: C2a is the larger cleavage
fragment).

The larger fragments bind to the target near the site of activation, and the
smaller fragments diffuse from the site and can initiate localized
inflammatory responses by binding to specific receptors.

The complement fragments interact with one another to form functional

complexes. Those complexes that have enzymatic activity are designated by
a bar over the number or symbol (e.g., C4b2a, C3bBb).
 Initiation of the complement cascade

The early steps, culminating in formation ofC5b, can occur

by the classical pathway, the alternative pathway, or the
lectin pathway. The final steps that lead to a membrane
attack are the same in all pathways.

• Classical pathway- Induced by immune complexes

• Alternative Pathway - Antibody Independent
• Lectin Pathway- Mannose binding lectin binds surface
glycoproteins, concentration increases during inflammation
Complement activation by the classical pathway commonly begins with
the formation of soluble antigen-antibody complexes or with the binding
of antibody to antigen on a suitable target, such as a bacterial cell.

IgM and certain subclasses of IgG (human IgG1, IgG2, and IgG3) can
activate the classical complement pathway.

The initial stage of activation involves C1, C2, C3, and C4, which are
present in plasma in functionally inactive forms.

The formation of an antigen-antibody complex induces conformational

changes in the Fc portion of the IgM molecule that expose a binding site
for the C1 component of the complement system.

C1 in serum is a macromolecular complex consisting of C1q and two

molecules each of C1r and C1s, held together in a complex (C1qr2s2)
stabilized by Ca2 ions.
The C1q molecule is composed of 18 polypeptide chains that associate to form
six collagen-like triple helical arms, the tips of which bind to exposed C1q-
binding sites in the CH2 domain of the antibody molecule. Each C1r and C1s
monomer contains a catalytic domain and an interaction domain; the latter
facilitates interaction with C1q or with each other.
 Each C1 molecule must bind by its C1q globular heads to at
least two Fc sites for a stable C1-antibody interaction to occur.

 When pentameric IgM is bound to antigen on a target surface it

assumes the so-called “staple” configuration, in which at least
three binding sites for C1q are exposed. Circulating IgM,
however, exists as a planar configuration and therefore cannot
activate the complement cascade.

 An IgG molecule, on the other hand, contains only a single C1q-

binding site in the CH2 domain of the Fc, so that firm C1q
binding is achieved only when two IgG molecules are within
30–40 nm of each other on a target surface or in a complex,
providing two attachment sites for C1q.

 This difference accounts for the observation that a single

molecule of IgM bound to a RBcs can activate the classical
complement pathway while some 1000 molecules of IgG are
required to assure that two IgG molecules are close enough to
each other on the cell surface to initiate C1q binding.
Binding of C1q to Fc binding sites induces a conformational change
inC1r that converts C1r to an active serine protease enzyme,C1r, which
then cleaves C1s to a similar active enzyme, C1s.

C1s has two substrates, C4 and C2. The C4 component is a

glycoprotein containing three polypeptide chains ,, and .

C4 is activated when C1s hydrolyzes a small fragment (C4a) from the

amino terminus of the chain, exposing a binding site on the larger
fragment (C4b).
C4a=anaphylatoxin
Some of the
C3b has other
functions like
coats immune
complexes
and
particulate
antigens,
functioning as
an opsonin
C3b can bind to cell membranes or bind to particulate
antigens or immune complexes.
The C5b component is extremely
labile and becomes inactive
within 2 minutes - C6 binds &
stabilizes its activity.

Up to this point, all the

complement reactions take place
on the hydrophilic surface of
membranes or on immune
complexes in the fluid phase.

As C5b6 binds to C7, the

resulting complex undergoes a
hydrophilic-amphiphilic
structural transition that exposes
hydrophobic regions, which
serve as binding sites for
membrane phospholipids
enabling the C5b67 complex to
insert into the phospholipid
bilayer.
 MAC
The C5b component is extremely labile and becomes inactive within 2
minutes - C6 binds & stabilizes its activity.

Up to this point, all the complement reactions take place on the hydrophilic
surface of membranes or on immune complexes in the fluid phase.

As C5b6 binds to C7, the resulting complex undergoes a hydrophilic-

amphiphilic structural transition that exposes hydrophobic regions, which
serve as binding sites for membrane phospholipids enabling the C5b67
complex to insert into the phospholipid bilayer

If, however, the reaction occurs on an immune complex or other noncellular

activating surface, then complex is released. Released C5b67 complexes can
insert into the membrane of nearby cells and mediate “innocent-bystander”
lysis. Regulator proteins normally prevent this from occurring.
 Binding of C8 to membrane-bound C5b67 induces a
conformational
 change in C8, so that it exposes a hydrophobic region, which
interacts with the plasma membrane.

 The C5b678 complex creates a small pore, 10 Å in diameter;

formation of this pore can lead to lysis of red blood cells but not of
nucleated cells. The final step in formation of the MAC is the
binding and polymerization of C9, a perforin- like molecule, to the
C5b678 complex. As many as 10–17 molecules of C9 can be
bound and polymerized by a single C5b678 complex. During
polymerization, the C9 molecules undergo a hydrophilic-
amphiphilic transition, so that they too can insert into the
membrane.

 The completed MAC, which has a tubular form and functional pore
size of 70–100 Å, consists of a C5b678 complex surrounded by a
poly-C9 complex. Since ions and small molecules can diffuse
freely through the central channel of the MAC, the cell cannot
maintain its osmotic stability and is killed by an influx of water and
loss of electrolytes.
Alternative pathway

•The alternative pathway generates bound C5b, the

same product that the classical pathway generates,
but it does so without the need for antigen-antibody
complexes for initiation.

•So it is a component of the innate immune system.

•Involves four serum proteins: C3, factor B, factor D,

and properdin.

•The alternative pathway is initiated in most cases by

cell-surface constituents that are foreign to the host
(Table).
 Hydrolysis of C3 initiates the alternative pathway
• Spontaneous hydrolysis of plasma C3
• C3b is produced at a significant rate by spontaneous cleavage (C3)
through spontaneous hydrolysis of the thioester in the C3 to form
C3(H2O), allowing binding factor B. factor D plasma protease cleave
factor B to form C3(H2O)Bb a fluid C3 convertase, and can cleave C3 to
C3a and C3b.
• The C3b component can bind to foreign surface antigens or even to the
host’s own cells. The membranes of most mammalian cells have high
levels of sialic acid, which contributes to the rapid inactivation of bound
C3b molecules on host cells; However many foreign antigenic surfaces
have only low levels of sialic acid, C3b bound to these surfaces remains
active for a longer time
Contains a labile thioester bond

C3b can bind to cell surfaces

Inactivated on host cells by
surface sialic acids
 The mannan binding lectin pathway

• The MB-lectin pathway uses a protein very similar to C1q

to trigger the complement cascade
• It does not depend on antibody for its activation.
• However, the mechanism is more like that of the classical
pathway, because after initiation, it proceeds, through the
action of C4 and C2, to produce a C5 convertase
• MB-lectin binds specifically to mannose residues on
pathogens surfaces
• It is present at low conc. in normal plasma and during
acute phase reaction its production increase by liver
• MB-lectin forms a complex
with two protease : MBL
associated serine protease;
MASP-1 and MASP-2
• Closely homologous to C1r
and C1s and activated to
cleave C4 and C2
• form a C5 convertase
without need for specific
antibody- innate defense
mechanism
 Regulation of the Complement System
Because many elements of the complement system are capable of
attacking host cells as well as foreign cells and microorganisms – thus
elaborate regulatory mechanisms has eloved.

A general mechanism of regulation in all complement pathways is the

inclusion of highly labile components that undergo spontaneous
inactivation if they are not stabilized by reaction with other components.

In addition, a series of regulatory proteins can inactivate various

complement components (Table 7-2).
The reaction catalyzed by the C3 convertase enzymes in all the pathways
is the major amplification step in complement activation, generating
hundreds of molecules of C3b.

The C3b generated by these enzymes has the potential to bind to nearby
cells, mediating damage to the healthy cells by causing their opsonization
by phagocytic cells bearing C3b receptors or by induction of the
membrane attack complex.

Damage to normal host cells is prevented as C3b undergoes spontaneous

hydrolysis by the time it has diffused 40 nm away from the C4b2a or
C3bBbconvertase enzymes, so that it can no longer bind to its target site.

The potential destruction of healthy host cells by C3b is further limited by

a family of related proteins that regulate C3 convertase activity. These
regulatory proteins all contain repeating amino acid sequences (or motifs)
of about 60 residues, termed short consensus repeats (SCRs). All these
proteins are encoded at a single location on chromosome 1 in humans,
known as the regulators of complement activation (RCA) gene cluster.
In the classical and lectin pathways, three structurally
distinct RCA proteins act similarly to prevent assembly of
C3 convertase. These regulatory proteins include soluble
C4b-binding protein (C4bBP) and two membrane- bound
protein , complement receptor type 1 (CR1) and membrane
cofactor protein (MCP). Each of these regulatory proteins
binds to C4b and prevents its association with C2a. Once
C4bBP, CR1, or MCP is bound to C4b, another regulatory
protein, factor I, cleaves the C4b into bound C4d and
soluble C4c. In the alternative pathway CR1,MCP, or a
regulatory component called factor H binds to C3b and
prevents its association with factor B
However, HRF and MIRL inhibition occurs only if the complement
components
are from the same species as the target cells. For this reason, MIRL and HRF
are said to display homologous restriction, for which the latter was named.
Biological Consequences of Complement Activation

Complement serves as an important mediator of the humoral response.

The MAC mediates cell lysis,

while other complement components or split products participate in the

inflammatory response,
opsonization of antigen,
viral neutralization, and
clearance of immune complexes
Many of the biological activities of the complement system depend on the
binding of complement fragments to complement receptors, which are
expressed by various cells. Also these receptors play an important role in
regulating complement activity by binding biologically active
complement components and degrading them into inactive products.
The Membrane-Attack Complex Can Lyse Cells

The membrane-attack complex formed by complement activation can

lyse gram-negative bacteria, parasites, viruses, erythrocytes, and
nucleated cells. Most enveloped viruses like herpesviruses,
orthomyxoviruses, paramyxoviruses, and retroviruses are susceptible to
complement mediated lysis. The viral envelope is largely derived from
the plasma membrane of infected host cells and is therefore susceptible to
pore formation by the membrane attack complex.

The complement system is generally quite effective in lysing gram-

negative bacteria. However, some gram-negative bacteria and most
gram-positive bacteria have mechanisms for evading complement-
mediated damage (Table 7.5 ).
 Lysis of nucleated cells requires formation of multiple membrane attack
complexes, whereas a single MAC can lyse a red blood cell. Many
nucleated cells, including the majority of cancer cells, can endocytose
the MAC.

 If the complex is removed soon enough, the cell can repair any
membrane damage and restore its osmotic stability.

 Thus complement-mediated lysis by antibodies specific for tumor-cell

antigens,which offers a potential weapon against cancer, may be
rendered ineffective by endocytosis of the MAC.
Cleavage Products of Complement Components Mediate
Inflammation

The smaller fragments resulting from complement cleavage, C3a, C4a, and
C5a, called anaphylatoxins, bind to receptors on mast cells and blood
basophils and induce degranulation, with release of histamine and other
pharmacologically active mediators. The anaphylatoxins also induce
smooth-muscle contraction and increased vascular permeability.
Activation of the complement system thus results in influxes of fluid that
carries antibody and phagocytic cells to the site of antigen entry. The
activities of these highly reactive anaphylatoxins are regulated by a serum
protease called carboxypeptidase N, which cleaves an Arg residue from
the C terminus of the molecules, yielding so-called des-Arg forms. The
des-Arg forms of C3a and C4a are completely inactive while that of C5a
retains about 10% of its chemotactic activity and 1% of its ability to cause
smooth
muscle contraction. C3a, C5a, and C5b67 can each induce monocytes and
neutrophils to adhere to vascular endothelial cells, extravasate through the
endothelial lining of the capillary, and migrate toward the site of
complement activation in the tissues. C5a is most potent in mediating these
processes, effective in picomolar quantities.
C3b and C4b Binding Facilitates Opsonization

C3b is the major opsonin of the complement system, although C4b

and iC3b also have opsonizing activity. The amplification that occurs
with C3 activation results in a coating of C3b on immune complexes
and particulate antigens. Phagocytic cells, as well as some other cells,
express complement receptors (CR1, CR3, and CR4) that bind C3b,
C4b, or iC3b (see Table 13-4). Antigen coated with C3b binds to cells
bearing CR1. If the cell is a phagocyte (e.g., a neutrophil, monocyte,
or macrophage), phagocytosis will be enhanced. Activation of
phagocytic cells by various agents, including C5a anaphylatoxin, has
been shown to increase the number of CR1s from 5000 on resting
phagocytes to 50,000 on activated cells, greatly facilitating their
phagocytosis of C3b-coated antigen. Furthermore C3b acts as an
adjuvant when coupled with protein antigens. C3b targets the antigen
directly to the phagocyte, enhancing the initiation of antigen
processing and accelerating specific antibody production.
The Complement System Also Neutralizes Viral Infectivity

For most viruses, the binding of serum antibody to the repeating

subunits of the viral structural proteins creates particulate immune
complexes ideally suited for complement activation by the classical
pathway.

Some viruses (e.g., retroviruses, Epstein-Barr virus, Newcastle disease

virus, and rubella virus) can activate the alternative, lectin, or even the
classical pathway in the absence of antibody.

The complement system mediates viral neutralization by a number of

mechanisms.

Some degree of neutralization is achieved through the formation of

larger viral aggregates, simply because these aggregates reduce the net
number of infectious viral particles.
 Although antibody plays a role in the formation of viral aggregates, in
vitro studies show that the C3b component facilitates aggregate
formation in the presence of as little as two molecules of antibody per
virion. For example, polyoma virus coated with antibody is neutralized
when serum containing activated C3 is added. The binding of antibody
and/or complement to the surface of a viral particle creates a thick
protein coating. This coating neutralizes viral infectivity by blocking
attachment to susceptible host cells.

 The deposits of antibody and complement on viral particles also

facilitate binding of the viral particle to cells possessing Fc or type 1
complement receptors (CR1). In the case of phagocytic cells, such
binding can be followed by phagocytosis and intracellular destruction
of the ingested viral particle.

 Finally, complement is effective in lysing most, if not all, enveloped

viruses, resulting in fragmentation of the envelope and disintegration of
the nucleocapsid.
 The Complement System Clears Immune Complexes
from Circulation
The importance of the complement system in clearing immune
complexes is seen in patients with the autoimmune disease systemic
lupus erythematosus (SLE).

These individuals produce large quantities of immune complexes and

suffer tissue damage as a result of complement-mediated lysis.

In SLE patients, deficiencies in C1,C2, and C4 each contribute to

reduced levels of C3b on immune complexes and hence inhibit their
clearance. The lower levels of CR1 expressed on the erythrocytes of
SLE patients also may interfere with the properbinding and clearance of
immune complexes.
 The complement deficiencies are thought to interfere with
effective solubilization and clearance of immune complexes; as a
result, these complexes persist, leading to tissue damage by the
very system whose deficiency was to blame.

 The coating of soluble immune complexes with C3b is thought

to facilitate their binding to CR1 on erythrocytes. Although red
blood cells express lower levels of CR1 than granulocytes do ,
there are about 103 red blood cells for every white blood cell;
therefore, erythrocytes account for about 90% of the CR1 in the
blood.

 For this reason, erythrocytes play an important role in binding

C3b-coated immune complexes and carrying these complexes to
the liver and spleen. In these organs, immune complexes are
stripped from the red blood cells and are phagocytosed, thereby
preventing their deposition in tissues.
 Complement Deficiencies
 Genetic deficiencies have been described for each of the
complement components. Homozygous deficiencies in any of the
early components of the classical pathway (C1q, C1r, C1s, C4, and
C2) exhibit similar symptoms, notably a marked increase in
immune-complex diseases such as systemic lupus erythematosus,
glomerulonephritis, and vasculitis.

 In addition, individuals with such complement deficiencies may

suffer from recurrent infections by pyogenic (pusforming) bacteria
such as streptococci and staphylococci as the early complement
components ordinarily prevent recurrent infection by mediating a
localized inflammatory response and opsonizing the bacteria.

 Deficiencies in factor D and properdin—early components of the

alternative pathway—appear to be associated with Neisseria
infections but not with immune-complex disease.
 Patients with C3 deficiencies have the most severe clinical
manifestations, reflecting the central role of C3 in activation of C5 and
formation of the MAC. The first patient identified with a C3 deficiency
was a child suffering from frequent severe bacterial infections and may
have immunecomplex diseases.

 Individuals with homozygous deficiencies in the components involved

in the MAC develop recurrent meningococcal and gonococcal
infections caused by Neisseria species. In normal individuals, these
gram-negative bacteria are generally susceptible to complement-
mediated lysis or are cleared by the opsonizing activity of C3b.MAC-
deficient individuals rarely have immune-complex disease, which
suggests that they produce enough C3b to clear immune complexes.

 Interestingly, a deficiency in C9 results in no clinical symptoms,

suggesting that the entire MAC is not always necessary for
complement-mediated lysis.
 Congenital deficiencies of complement regulatory proteins have also
been reported. The C1 inhibitor (C1Inh) regulates activation of the
classical pathway by preventing excessive C4 and C2 activation by C1.
Deficiency of C1Inh gives rise to a condition called hereditary
angioedema, which manifests clinically as localized edema of the
tissue, oftenfollowing trauma, but sometimes with no known cause.
THE END