Lecture-7

Complement System

The complement system, also known as complement cascade, is a part of

the immune system that enhances (complements) the ability of antibodies and phagocytic cells
to clear microbes and damaged cells from an organism, promote inflammation, and attack the
pathogen's cell membrane. It is part of the innate immune system, which is not adaptable and
does not change during an individual's lifetime. The complement system can, however, be
recruited and brought into action by antibodies generated by the adaptive immune system.

The complement system consists of a number of small proteins that are

synthesized by the liver, tissue macrophages, blood monocytes, and epithelial cells of
the genitourinary system and gastrointestinal tract and circulate in the blood as
inactive precursors. When stimulated by one of several triggers, proteases in the system cleave
specific proteins to release cytokines and initiate an amplifying cascade of further cleavages.
The end result of this complement activation or complement fixation cascade is stimulation
of phagocytes to clear foreign and damaged material, inflammation to attract additional
phagocytes, and activation of the cell-killing membrane attack complex. About 50 proteins and
protein fragments make up the complement system, including serum proteins, and cell
membrane receptors. They account for about 10% of the globulin fraction of blood serum.[2]

Pathways of complement activation (Complement fixation)

There are three biochemical pathways of activation of complement system:

1. The classical complement pathway,

2. The alternative complement pathway
3. The lectin pathway.[3]

The alternative pathway accounts for the majority of terminal pathway activation and so
therapeutic efforts in disease have revolved around its inhibition.
History:
In 1888, George Nuttall while working with anthrax bacilli, found that sheep blood serum had
mild killing activity against the bacterium. The killing activity disappeared when he heated the
blood. In 1891, Hans Ernst August Buchner, observing the same property of blood in his
experiments, named the killing property "alexin", which means "to ward off" in Greek. By
1894, several laboratories had demonstrated that serum from guinea pigs that had recovered
from cholera killed the cholera bacterium in vitro. Heating the serum destroyed its killing activity.
Nevertheless, the heat-inactivated serum, when injected into guinea pigs exposed to the cholera
bacteria, maintained its ability to protect the animals from illness. Jules Bordet, a
young Belgian scientist in Paris at the Pasteur Institute, concluded that this principle has two
components, one that maintained a "sensitizing" effect after being heated and one (alexin)
whose toxic effect was lost after being heated. The heat-stable component was responsible for
immunity against specific microorganisms, whereas the heat-sensitive component was
responsible for the non-specific antimicrobial activity conferred by all normal sera.
In 1899, Paul Ehrlich renamed the heat-sensitive component "complement”. He introduced
the term "complement" as part of his Side Chain Theory of the immune system. According to
this theory, the immune system consists of cells that have specific receptors on their surface to
recognize antigens. Upon immunization with an antigen, more of these receptors are formed,
and they are then shed from the cells to circulate in the blood. Those receptors, which we now
call "antibodies", were called by Ehrlich "amboceptors" to emphasize their bifunctional binding
capacity: They recognise and bind to a specific antigen, but they also recognize and bind to the
heat-labile antimicrobial component of fresh serum. Ehrlich, therefore, named this heat-labile
component "complement", because it is something in the blood that "complements" the cells of
the immune system. Ehrlich believed that each antigen-specific amboceptor has its own specific
complement, whereas Bordet believed that there is only one type of complement. In the early
20th century, this controversy was resolved when it became understood that complement can
act in combination with specific antibodies, or on its own in a non-specific way.

Functions of complement:
Complement triggers the following immune functions:

1. Membrane attack – by activation of the cell-killing membrane attack complex leading to

rupture of cell wall of bacteria. (Classical Complement Pathway)
2. Phagocytosis – stimulation of phagocytes to clear foreign and damaged material
by opsonizing antigens. C3b has most important opsonizing activity. (Alternative
Complement Pathway)
3. Inflammation –by attracting macrophages and neutrophils (Phagocytes). (Lectin
pathway)

➢ Most of the proteins and glycoproteins that constitute the complement system are
synthesized by hepatocytes.
➢ Significant amounts are also produced by tissue macrophages, blood monocytes,
and epithelial cells of the genitourinary system and gastrointestinal tract.
➢ All the three pathways of activation generate homologous variants of
the protease C3-convertase.
➢ The classical complement pathway typically requires antigen-antibody
complexes for activation (specific immune response).
➢ The alternative pathway can be activated by spontaneous complement component
3 (C3) hydrolysis, foreign material, pathogens, or damaged cells.
➢ The mannose-binding lectin pathway can be activated by C3 hydrolysis or antigens
without the presence of antibodies (non-specific immune response).
➢ In all three pathways, C3-convertase cleaves and activates component C3,
creating C3a and C3b, and causes a cascade of further cleavage and activation
events.
 ➢ C3b binds to the surface of pathogens, leading to greater internalization
by phagocytic cells by opsonization.
➢ In the alternative pathway, C3b binds to Factor B.
➢ Factor D releases Factor Ba from Factor B bound to C3b.
➢ The complex of C3bBb is a protease which cleaves C5 into C5b and C5a.
➢ C5 convertase is also formed by the classical pathway when C3b binds C4b and
C2b.
➢ C5a is an important chemotactic protein, helping recruit inflammatory cells.
➢ C3a is the precursor of an important cytokine (adipokine) named ASP and is
usually rapidly cleaved by carboxypeptidase B.
➢ Both C3a and C5a have anaphylotoxin activity, directly triggering
degranulation of mast cells as well as increasing vascular permeability
and smooth muscle contraction.
➢ C5b initiates the membrane attack pathway, which results in the membrane attack
complex (MAC), consisting of C5b, C6, C7, C8, and polymeric C9.
➢ MAC is the cytolytic end product of the complement cascade; it forms a
transmembrane channel, which causes osmotic lysis of the target cell.
➢ Kupffer cells and other macrophage cell types help clear complement-coated
pathogens.
Classical pathway
The classical pathway is triggered by activation of the C1-complex. The C1-
complex is composed of 1 molecule of C1q, 2 molecules of C1r and 2 molecules of C1s,
or C1qr2s2. This occurs when C1q binds to IgM or IgG complexed with antigens. A single
pentameric IgM can initiate the pathway, while several (ideally six) IgG mulecules are needed.
This also occurs when C1q binds directly to the surface of the pathogen. Such binding leads to
conformational changes in the C1q molecule, which leads to the activation of
two C1r molecules. C1r is a serine protease. They then cleave C1s (another serine protease).
The C1r2s2 component now splits C4 and then C2, producing C4a, C4b, C2a, and C2b. C4b and
C2a bind to form the classical pathway C3-convertase (C4b2a complex), C4b2a complex, or C3
convertase, catalyzes the cleavage of C3 into C3a and . C3b later joins with C4b2b to make
C5 convertase (C4b2a3b complex).
Alternative pathway
The alternative pathway is continuously activated at a low level as a result of
spontaneous C3 hydrolysis but C3b that is generated from C3 by a C3 convertase enzyme
complex in the fluid phase is rapidly inactivated by factor H and factor I, The surface-bound C3b
binds with factor B to form C3bB. This complex in the presence of factor D will be cleaved into
Ba and Bb. Bb will remain associated with C3b to form C3bBb, which is the C3 convertase in
alternative pathway.
The C3bBb complex is stabilized by binding oligomers of factor P (properdin). The stabilized C3
convertase, C3bBbP, then acts enzymatically to cleave much more C3, some of which becomes
covalently attached to the same surface as C3b. This newly bound C3b recruits more B, D and
P activity and greatly amplifies the complement activation. When complement is activated on a
cell surface, the activation is limited by endogenous complement regulatory proteins, which
include CD35, CD46, CD55 and CD59, depending on the cell.
Pathogens, in general, don't have complement regulatory proteins (there are many exceptions,
which reflect adaptation of microbial pathogens to vertebrate immune defenses). Thus, the
alternative complement pathway is able to distinguish self from non-self on the basis of the
surface expression of complement regulatory proteins. Host cells don't accumulate cell surface
C3b (and the proteolytic fragment of C3b called iC3b) because this is prevented by the
complement regulatory proteins, while foreign cells, pathogens and abnormal surfaces may be
heavily decorated with C3b and iC3b. Accordingly, the alternative complement pathway is one
element of innate immunity.
Once the alternative C3 convertase enzyme is formed on a pathogen or cell surface, it may bind
covalently another C3b, to form C3bBbC3bP, the C5 convertase. This enzyme then cleaves C5
to C5a, a potent anaphylatoxin, and C5b. The C5b then recruits and assembles C6, C7, C8 and
multiple C9 molecules to assemble the membrane attack complex. This creates a hole or pore
in the membrane that can kill or damage the pathogen or cell.[1]
Lectin pathway
The lectin pathway is homologous to the classical pathway, but with the opsonin, mannose-
binding lectin (MBL), and ficolins, instead of C1q. This pathway is activated by binding of MBL to
mannose residues on the pathogen surface, which activates the MBL-associated serine
proteases, MASP-1, and MASP-2 (very similar to C1r and C1s, respectively), which can then
split C4 into C4a and C4b and C2 into C2a and C2b. C4b and C2b then bind together to form
the classical C3-convertase, as in the classical pathway. Ficolins are homologous to MBL and
function via MASP in a similar way. Several single-nucleotide polymorphisms have been
described in M-ficolin in humans, with effect on ligand-binding ability and serum levels.
Historically, the larger fragment of C2 was named C2a, but it is now referred to as C2b. [17] In
invertebrates without an adaptive immune system, ficolins are expanded and their binding
specificities diversified to compensate for the lack of pathogen-specific recognition molecules.
.
Activation of complement (summary)
Activation
Classic Alternative Lectin
pathway

Ag–Ab spontaneous hydrolysis of MBL-Mannose

Activator
Complex C3 Complex

C3-convertase C4b2b C3bBb C4b2b

C5-convertase C4b2b3b C3bBbC3b C4b2b3b

MAC development C5b+C6+C7+C8+C9

Regulation of complement activity

The complement system has the potential to be extremely damaging to host tissues, meaning
its activation must be tightly regulated. The complement system is regulated by complement
control proteins, which are present at blood plasma and host cell membrane.[34] Some
complement control proteins are present on the membranes of self-cells preventing them from
being targeted by complement. One example is CD59, also known as protectin, which inhibits
C9 polymerization during the formation of the membrane attack complex. The classical pathway
is inhibited by C1-inhibitor, which binds to C1 to prevent its activation.
C3-convertase can be inhibited by decay accelerating factor (DAF), which is bound to
erythrocyte plasma membranes via a GPI anchor.
Complement and Diseases
Complement deficiency
It is thought that the complement system might play a role in many diseases with an immune
component, such as Barraquer–Simons syndrome, asthma, lupus
erythematosus, glomerulonephritis, various forms of arthritis, autoimmune heart
disease, multiple sclerosis, inflammatory bowel disease, paroxysmal nocturnal
hemoglobinuria, atypical hemolytic uremic syndrome and ischemia-reperfusion injuries,[36][37] and
rejection of transplanted organs.[38]
The complement system is also becoming increasingly implicated in diseases of the central
nervous system such as Alzheimer's disease and other neurodegenerative conditions such as
spinal cord injuries.
Deficiencies of the terminal pathway predispose to both autoimmune
disease and infections (particularly Neisseria meningitidis, due to the role that the membrane
attack complex ("MAC") plays in attacking Gram-negative bacteria).[42]
Infections with N. meningitidis and N. gonorrhoeae are the only conditions known to be
associated with deficiencies in the MAC components of complement.[43] 40–50% of those with
MAC deficiencies experience recurrent infections with N. meningitidis.
Deficiencies in complement regulators
Mutations in the genes of complement regulators, especially factor H, have been associated
with atypical hemolytic uremic syndrome, and C3 glomerulopathy. Both of these disorders are
currently thought to be due to complement overactivation either on the surface of host cells or in
plasma, with the molecular location of genetic variation in complement proteins providing clues
into the underlying disease processes. Moreover, several single nucleotide polymorphisms and
mutations in the complement factor H gene (the most common of which results in the protein
change p.Y402H) have been associated with the common eye disease age-related macular
degeneration Polymorphisms of complement component 3, complement factor B,
and complement factor I, as well as deletion of complement factor H-related 3 and complement
factor H-related 1, also affect a person's risk of developing age-related macular degeneration.
Mutations in the C1 inhibitor gene can cause hereditary angioedema, a genetic condition
resulting from reduced regulation of bradykinin by C1-INH.]
Paroxysmal nocturnal hemoglobinuria is caused by complement breakdown of RBCs due to an
inability to make GPI. Thus the RBCs are not protected by GPI anchored proteins such as DAF.
Diagnostic tools
Diagnostic tools to measure complement activity include the total complement activity test.
The presence or absence of complement fixation upon a challenge can indicate whether
particular antigens or antibodies are present in the blood. This is the principle of
the complement fixation test.