The complement system

Complement is defined as “the activity of blood serum that completes action of antibody”.

The term complement refers to a set of 50-plus serum proteins and glycoproteins that
cooperates with both the innate and the adaptive immune systems to eliminate
pathogens, dying cells, and immune complexes from the body.

The term was coined by Paul Ehrlich

The Nobel Prize in Physiology or Medicine in the year 1908

was awarded to Paul Ehrlich and Ilya Ilyich Mechnikov “in recognition of
their work on immunity.“

1
 Basic functions of activated complement components

After activation various complement components they 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 particular antigen.
3. Binding to specific complement receptors on cells of the immune system triggering
specific cell functions, infalmmation, and secretion of immunoregulatory molecules.
4. Immune clearance, which removes complexes from the circulation and deposits them
in the spleen and liver.
2
1. Complement system consists of soluble proteins and glycoproteins.

2. Most complement components are synthesized in the liver by hepatocytes.

3. Some are also produced by blood monocytes, tissue macrophages, fibroblasts, and
epithelial cells of the gastrointestinal and genitourinary tracts.

4. Complement components constitute approximately 15% of the globulin protein

fraction in plasma.

5. Several of the regulatory components of the system exist on cell membranes. So, the
term complement now embraces proteins and glycoproteins distributed between the
blood plasma and cell membranes.

6. Most circulate in the serum in functionally inactive form as proenzymes or zymogens.

7. Proteolytic cleavage removes an inhibitory fragment and exposes the active site of the
molecule.

8. The complement reaction sequence starts with an enzyme cascade.

3
1. Complement factors are designated by numerals (C1-C9)

2. By letter symbols (Factor D)

3. Or by trivial names (homologous restriction factor)

4. Peptide fragments formed by activation of components are denoted by small

letters.

5. Smaller fragments designated as “a” and larger as “b” (C3a, C3b).

6. C2 is an exception where C2a is larger in size than C2b.

7. The larger fragments bind to the target while smaller fragments diffuse from
the site and cause localized inflammatory reactions by binding to the
specific receptors.

8. The complement fragments interact with one another to form one functional
complex.

9. The complexes those have enzymatic activity are designated with a bar over
4
the number or symbol (C4b2a, C3bBb).
Complement components can be classified into seven functional categories
(1) The complement pathways
are initiated by proteins that
bind to pathogens, either
directly or via an antibody
or other pathogen-specific
protein. After a
conformational change,
(2) enzymatic mediators activate
other enzymes that generate
the central proteins of the
complement cascade, the C3
and C5 convertases, which
cleave C3 and C5, releasing
active components that
mediate all functions of
complement, including
(3) opsonization,

(4) inflammation
(5) generation of the membrane attack complex (MAC). Effector complement proteins can label an
antibody-antigen complex for phagocytosis (opsonins), initiate inflammation (anaphylatoxins), or
bind to a pathogen and nucleate the formation of the MAC. Often, these effectors act through
(6) complement receptors on phagocytic cells, granulocytes, or erythrocytes.
(7) Regulatory proteins limit the effects of complement by promoting their degradation or preventing
their binding to host cells. 5
1. Initiator complement components initiate their respective complement reactions by binding to
particular soluble or membrane-bound molecules. Once activated by their ligand, they undergo
conformational alterations resulting in changes in their biological activity.

2. Enzymatic mediators. Several complement components are proteolytic enzymes that cleave and
activate the next member of a complement reaction sequence. Proteins that are inactive until
cleaved by proteases are called zymogens. Some complement proteases become active by binding
to other macromolecules and undergoing a conformational change; others are zymogens
themselves, inactive until cleaved by another “upstream” protease. The two enzyme complexes
that cleave the complement components C3 and C5 are called the C3 and C5 convertases,
respectively, and occupy places of central importance in complement biology. The sequence of
proteins in a complement pathway from the initiator protein to the biological effector is referred to
as a “complement cascade.”

3. Phagocytosis-enhancing components, or opsonins. On activation of the complement cascade,

several complement proteins are cleaved into two fragments, each of which then takes on a
particular role. For C3 and C4, the larger fragments, C3b and C4b, serve as opsonins, binding
covalently to microbial cells and serving as ligands for phagocytic cells with receptors for C3b or
C4b.

4. Inflammatory mediators. Some small complement fragments act as inflammatory mediators.

These fragments bind to receptors on the endothelial cells lining small blood vessels and induce an
increase in capillary diameter, thus enhancing blood flow to the affected area. They also attract
other cells to the site of tissue damage. Since these effects can be harmful (even lethal) in excess,
these fragments are called anaphylatoxins, derived from the Greek phrase meaning “against
protection.” C3a and C5a are examples of anaphylatoxins. 6
5. Membrane attack proteins. Proteins of the membrane attack complex (MAC) insert into the cell
membranes of invading microorganisms and punch holes that result in lysis of the pathogen. The
MAC has been extensively imaged by electron microscopy. The complex itself forms a ring-
shaped multimer of complement proteins with a central hole through which cytoplasmic
contents can escape. MACs can also form on infected host cells, although the complement system
must first overcome the regulatory mechanisms designed to protect host cells from complement
attack.

6. Complement receptor proteins. Receptor molecules on cell surfaces bind complement proteins
and signal specific cell functions. For example, some complement receptors such as CR1 bind to
complement components such as C3b that have opsonized pathogens, triggering phagocytosis of
the C3b-bound pathogen. Binding of the anaphylatoxin complement component C5a to C5a
receptors (C5aRs) on neutrophils stimulates neutrophil degranulation and inflammation.

7. Regulatory complement components. Host cells are protected from unintended

complementmediated damage by the presence of regulatory proteins. These regulatory proteins
include factor I, which degrades C3b, and CD59 (protectin), which inhibits the formation of the
MAC on host cells.

7
Three major pathways of complement activation.

8
Fig. Generation of C3 and C5 convertases by the three major pathways of complement
activation.

The classical pathway is initiated when C1q binds to antigen-antibody complexes. The antigen is
shown here in dark red and the initiating antibody in green. The C1r enzymatic component of C1
(shown in blue) is then activated and cleaves C1s, which in turn cleaves C4 to C4a and C4b. C4b
attaches to the membrane and binds C2, which is then cleaved by C1s to form C2a and C2b. (C2b is
then acted on further to become an inflammatory mediator.) C2a remains attached to C4b, forming the
classical pathway C3 convertase (C4b2a).

In the lectin pathway, mannose-binding lectin (MBL, green) binds specifically to conserved
carbohydrate arrays on pathogens, activating the MBL-associated serine proteases (MASPs, blue). The
MASPs cleave C2 and C4, generating the C3 convertase as in the classical pathway.

In the alternative pathway, C3 undergoes spontaneous hydrolysis to C3(H O), which binds serum
factor B. On binding to C3(H O), B is cleaved by serum factor D, and the resultant C3(H O)Bb
complex forms a fluid-phase C3 convertase. Some C3b, released after C3 cleavage by this complex,
binds to microbial surfaces. There, it binds factor B, which is cleaved by factor D, forming the cell-
bound alternative pathway C3 convertase, C3bBb. This complex is stabilized by properdin.

There is a second set of convertase enzymes generated in the early stages of complement
activation.
The C5 convertases are formed by the addition of a C3b component to each of the two C3
convertases. C5 convertases cleave C5 into C5a, an inflammatory mediator, or anaphylatoxin, and
C5b, which is the initiating factor of the membrane attack complex.
9
10
The Classical pathway is initiated by antibody binding to antigens

The classical pathway of complement activation is considered as part of the adaptive immune
response since it begins with the formation of antigen-antibody complexes.

These complexes may be soluble, or they may be formed when an antibody binds to antigenic
determinants, or epitopes, situated on viral, fungal, parasitic, or bacterial cell membranes.

Soluble antibody antigen complexes are often referred to as immune complexes. Only complexes
formed by antigens with antibodies of the IgM class or IgG1, IgG2 and IgG3 subclasses of IgG
antibodies are capable of activating the classical complement pathway.

The initial activation involves interaction of these antibody-antigen complexes with the complement
components C1, C2, and C4, normally found in the plasma as inactive precursors or zymogens. The
formation of an antigen-antibody complex induces conformational changes in the nonantigen-binding
(Fc) portion of the antibody molecule.

This conformational change exposes a binding site on the antibody for the C1 component of
complement. In serum, C1 exists as a macromolecular complex consisting of one molecule of C1q
and two molecules each of the serine proteases C1r and C1s, held together in a Ca2+-stabilized
complex (C1qr2 s2 ). The C1q molecule itself is composed of 18 polypeptide chains that associate to
form six collagen-like triple helical arms, the tips of which bind the CH2 domain of the antigen-
bound antibody molecule.

11
In serum, C1 exists as a macromolecular complex consisting of one molecule of C1q and two
molecules each of the serine proteases C1r and C1s, held together in a Ca2+-stabilized complex
(C1qr2 s2 ). The C1q molecule itself is composed of 18 polypeptide chains that associate to form six
collagen-like triple helical arms, the tips of which bind the CH2 domain of the antigen-bound
antibody molecule.

Structure of the C1 macromolecular complex. C1q interacts with two molecules each of C1r and C1s
to create the C1 complex. The C1q molecule consists of 18 polypeptide chains in six collagen-like
triple helices.
12
Antigenic determinants are shown in dark red, initiating components (antibodies and C1q) are shown in
green, active enzymes are shown in blue, and anaphylatoxins in bright red.
13
1. Binding of C1q to the C 2 domains of the Fc regions of the antigen-complexed antibody
molecule induces a conformational change in one of the C1r molecules. This conformational
change in the C1r molecule converts it to an active serine protease enzyme that then cleaves and
activates its partner C1r molecule. The two C1r proteases then cleave and activate the two C1s
molecules.

2. Activated C1s has two substrates, C4 and C2. C4 is activated when C1s hydrolyzes a small
fragment (C4a) from the amino terminus of one of its chains.

3. The C4b fragment attaches covalently to the target membrane surface in the vicinity of C1, and
then binds C2. C4b binding to the membrane occurs when an unstable, internal thioester on C4b
is exposed on C4 cleavage and reacts with hydroxyl or amino groups of proteins or
carbohydrates on the cell membrane.

4. This reaction must occur quickly, before the unstable thioester is further hydrolyzed and can no
longer make a covalent bond with the cell surface .

5. Indeed, approximately 90% of C4b is hydrolyzed before it can bind the cell surface. C4b is also
capable of forming covalent bonds with the constant regions of antibody molecules involved in
antigenantibody complexes.

14
Binding of C4b to the microbial membrane surface occurs through a thioester bond via an exposed
amino or hydroxyl group.
(a) Both C3b and C4b contain highly reactive thioester bonds, which are subject to nucleophilic attack
by hydroxyl or amino groups on cell membrane proteins and carbohydrates.
(b) Breakage of the thioester bond leads to the formation of covalent bonds between the membrane
macromolecules and the complement components.
(c) If this covalent bond formation does not occur quickly after generation of the C3b and C4b
fragments, the thioester bond will be hydrolyzed as shown.

15
6. On binding C4b at the membrane surface or on an immune complex, C2 becomes susceptible to
cleavage by the neighboring C1s enzyme. A smaller C2b fragment diffuses away, leaving behind
an enzymatically active C4b2a complex. In this complex, C2a is the enzymatically active
fragment, but it is active only when bound by C4b. This C4b2a complex, is the C3 convertase
that converts C3 into its enzymatically active form.

7. The membrane-bound or immune complex–bound C3 convertase enzyme, C4b2a, now

hydrolyzes C3, generating two unequal fragments: the small anaphylatoxin C3a, and the
pivotally important fragment C3b. A single C3 convertase molecule can generate over 200
molecules of C3b, resulting in tremendous amplification at this step of the classical pathway.

8. The generation of C3b is an essential precursor to many of the subsequent reactions of the
complement system. Deficiencies of complement components that act prior to C3 cleavage leave
the host extremely vulnerable to both infectious and autoimmune diseases, whereas deficiencies
of components later in the pathway are generally of lesser consequence.

9. In particular, patients with deficiencies in C3 itself are unusually susceptible to infections with
both gram-positive and gram-negative bacteria. This is because C3b acts in three important and
different ways to protect the host:

16
In a manner very similar to that of C4b, C3b binds covalently to microbial surfaces, providing
a molecular “tag” that allows phagocytic cells with C3b receptors to engulf the tagged
microbes. This process is called opsonization.

C3b, like C4b, can attach to the Fc portions of antibodies participating in soluble
antigenantibody complexes. These C3b-tagged immune complexes are bound by C3b
receptors on phagocytes or red blood cells, and are either phagocytosed, or conveyed to the
liver where they are destroyed.

Some molecules of C3b bind the membrane-localized C4b2a enzyme to form the trimolecular,
membrane-bound, C5 convertase complex C4b2a3b .

The C3b component of this complex binds C5, and the complex then cleaves C5 into the two
fragments: C5b and C5a. C4b2a3b is therefore the C5 convertase of the classical pathway.

The trio of tasks accomplished by the C3b molecule places it right at the center of complement
attack pathways. C3b is thus a central component in all three complement activation
pathways.

17
The Lectin Pathway
This pathway is initiated when soluble proteins recognize microbial antigens . The lectin pathway of
complement activation, like the classical pathway, proceeds through the activation of a C3 convertase
composed of C4b and C2a.
This pathway uses lectins—proteins that recognize particular carbohydrate components—as its
specific receptor molecules .
Because it does not rely on antibodies from the adaptive immune system, the lectin pathway is
considered to be an arm of innate, rather than adaptive, immunity.

Initiation of the lectin pathway relies on lectin

receptor recognition of microbial cell surface
carbohydrates.
Lectin receptors, such as MBL (Mannose-binding lectin),
bind microbial cell surface carbohydrates. They bind the
(MBL-associated serine proteases) MASP family serine
proteases, which cleave C2 and C4 to mediate formation
of a lectin-pathway C3 convertase.

18
Mannose-binding lectin (MBL), the first lectin demonstrated to be capable of initiating complement
activation, binds close-knit arrays of mannose (sugar) residues that are found on the surfaces of
microbes such as Salmonella, Listeria, and Neisseria bacteria; Cryptococcus neoformans and
Candida albicans fungi; and even on the membranes of some viruses such as HIV1 and respiratory
syncytial virus (RSV).

Further characterization of MBL demonstrated that it also recognizes N-acetylglucosamine, Dglucose,

and L-fucose polymers on microbial surfaces. All these sugars, including mannose, present their
associated hydroxyl groups in defined three-dimensional arrays and thus MBL is acting as a classic
pattern recognition receptor .

Individuals with low levels of MBL suffer from repeated, serious bacterial infections.

MBL is constitutively expressed by the liver and belongs to the subclass of lectins known as
collectins.

Other lectin receptors have been recognized as initiators of the lectin pathway of complement
activation. These include collectin-10 and collectin-11 as well as several members of the ficolin
family: ficolin-1, ficolin-2, and ficolin-3.

These proteins share a common “stalk” consisting of a collagen-like triple helix, coupled to a
carbohydrate recognition structure. The nature of the recognition structure defines the lectin as
belonging to the collectin or the ficolin family.

19
In the blood, MBL is associated with MBL-associated serine proteases, or MASP proteins.

Three MASP proteins—MASP-1, MASP-2, and MASP-3—have been identified. MASP-2 is the most active
protein.

MASP-2 is structurally related to the serine protease C1s, and when MBL binds to a microbial surface,
associated MASP-2 molecules cleave both C2 and C4, giving rise to the C4b2a which is known as C3
convertase . This is similar to the C3 convertase of the classical pathway.

From this point on, the lectin pathway uses all the same downstream components as the classical pathway.

20
The alternative pathway is initiated in three distinct ways

Initiation of the alternative pathway of complement activation, like the lectin pathway, is
independent of antibody-antigen interactions. Therefore, this pathway is also considered to be
part of the innate immune system.

However, unlike the lectin pathway, the alternative pathway uses a different set of C3 and C5
convertases .

The alternative pathway C3 convertase, C3bBb, is made up of one molecule of the C3b protein
fragment and one molecular fragment unique to the alternative pathway, Bb.

A second C3b is then added to make the alternative pathway C5 convertase, C3bBbC3b.

Alternative pathway can be initiated in three distinct ways.

The first mode of initiation to be discovered, the “tickover” pathway, uses the four serum
components C3, factor B, factor D, and properdin (or factor P) .

Two additional modes of activation for the alternative pathway have also been identified: one is
initiated by properdin, and the other by proteases such as thrombin and kallikrein.

21
The Alternative (Tick over (slow speed or smooth) Pathway

The term tick over refers to the fact that C3 is constantly being made and spontaneously inactivated
and is thus considered to be “ticking over.”

The alternative tick over pathway is initiated when C3, which is at high concentrations in serum,
undergoes spontaneous hydrolysis at its internal thioester bond, yielding the molecule known as
hydrolyzedC3, C3(H2O).

The conformation of C3(H 2O) is different from that of the C3 parent protein.

C3(H 2O) accounts for approximately 0.5% of plasma C3. In the presence of serum Mg , C3(H 2O)
binds another serum protein, factor B .

When bound to C3(H 2O), factor B becomes susceptible to cleavage by a serum protease, factor D.

Factor D cleaves factor B, releasing a smaller Ba subunit that diffuses away, leaving a catalytically
active Bb subunit bound to C3(H2 O).

The C3(H2 O)Bb complex is referred to as the “fluid-phase C3 convertase” because it remains in the
blood plasma and is not bound to any cells.

In the plasma, the fluid-phase convertase cleaves many molecules of C3 into C3a and C3b .
The C3(H2 O)Bb complex is not very stable in a healthy host and it is rapidly degraded, hence the term
“tick over” for this pathway.
22
Initiation of the alternative tickover pathway of
complement.

(a) Spontaneous hydrolysis of soluble C3 to C3(H O)

allows the altered conformation of C3(H O) to bind
factor B, rendering it susceptible to cleavage by factor
D. The resulting complex C3(H O)Bb forms a fluid-
phase convertase capable of cleaving C3 to C3a and
C3b.

(b) Some of the C3b molecules formed by the

fluidphase convertase bind to cell membranes. C3b,
like C3(H O), binds factor B in such a way as to make
B susceptible to factor D– mediated cleavage.

(c) The membrane-bound C3bBb is stabilized by

properdin (factor P), which binds the C3bBb
complex on the membrane. Addition of a second C3b
molecule to the C3bBb complex forms the C5
convertase, which is also stabilized by properdin.

23
However, if there is an infection present, some of the newly formed C3b molecules bind nearby
microbial surfaces via their thioester linkages .

In the presence of a microbial infection, factor B binds the newly attached C3b molecules on the
microbial cell surface , and becomes susceptible to cleavage by factor D, with the generation of C3bBb
complexes.

These C3bBb complexes are now located on the microbial membrane surface. Like the C4b2a
complexes of the classical pathway, the cell-bound C3bBb complexes have C3 convertase activity, and
this complex now takes over from the fluid-phase C3(H O)Bb as the predominant C3 convertase.

24
To be clear, there are two C3 convertases in the alternative tickover pathway:
a fluid-phase C3(H O)Bb, which initiates the pathway, and
a membrane-bound C3bBb C3 convertase that amplifies it and results in microbial destruction.
The cell-bound alternative pathway C3 convertase is unstable until it is bound by properdin (otherwise
known as factor P), a serum protein. Once stabilized by properdin, these cell-associated, C3bBb C3
convertase complexes rapidly generate large numbers of C3b molecules on the microbial surface. Many of
these then bind factor B, which is cleaved in turn by factor D, thus facilitating the cleavage of yet more
molecules of C3 and amplifying the rate of C3b generation. This amplification pathway is rapid; once the
alternative pathway has been initiated, more than 2 × 10 molecules of C3b can be deposited on a microbial
surface in less than 5 minutes.

The alternative C5 convertase complex has the

composition C3bBbC3b, and like the alternative C3
convertase, it is also stabilized by binding to properdin.

Like the classical and lectin pathway C5 convertase,

C3bBbC3b cleaves C5, which goes on to form the MAC
(membrane attack complex).

25
The Alternative Properdin-Activated Pathway
In addition to stabilizing the ongoing activity of the alternative pathway, properdin may also serve to
initiate it. In vitro experiments demonstrated that if properdin molecules were attached to an artificial
surface and allowed to interact with purified complement components in the presence of Mg , the
immobilized properdin bound C3b and factor B . This bound factor B proved to be susceptible to
cleavage by factor D, and the resultant C3bPBb complex acted as an effective C3 convertase,
Thus, it seemed that properdin could initiate activation of the alternative pathway on an artificial
substrate.

Initiation of the alternative pathway by specific, noncovalent binding of properdin to the

target membrane. Properdin (factor P) binds to components of microbial membranes, and
stabilizes the binding of C3bBb complexes of the alternative complement pathway. The difference
between this and the tickover pathway is that properdin binds first and initiates complement
deposition on the membrane.

26
The Alternative Protease-Activated Pathway

The biochemical pathway that leads to complement activation is similar in concept to the blood
coagulation pathway. Both use protease cleavage and conformational alterations of key proteins to
modify enzyme activities, as well as amplification of various steps of the pathways by feed-forward
loops.

Several decades ago, it was shown that protein factors involved in blood clotting, such as thrombin,
could cleave the complement components C3 and C5 in vitro, with the release of the active
anaphylatoxins C3a and C5a. Since these cleavage reactions required relatively high thrombin
concentrations, they were at first thought not to be physiologically meaningful.

More recently, however, it has been demonstrated in a mouse disease model that initiation of the
coagulation cascade may result in the cleavage of physiologically relevant amounts of C3 and C5
to produce C3a and C5a.

Interestingly, when blood platelets are activated during a clotting reaction, they release high
concentrations of ATP and Ca2+ along with serine/threonine kinases.

These enzymes act to phosphorylate extracellular proteins, including C3b.

Phosphorylated C3b is less susceptible to proteolytic degradation than its unphosphorylated form, and
thus, by this route, activation of the clotting cascade enhances all of the complement pathways.

27
Summary of alternative pathway
The alternative tickover pathway is initiated when C3(H O) binds to factor B, which then
becomes susceptible to cleavage by factor D into Ba and Bb. The Bb fragment continues to
bind to the hydrolyzed C3(H O) and together they form a fluid-phase C3 convertase. Some of
the C3b generated by this convertase adheres to microbial surfaces; there it binds factor B,
which again, in the presence of factor D, is cleaved, resulting in the formation of the
membrane-bound C3 convertase, C3bBb. This complex is stabilized by properdin.

The alternative pathway may also be activated by the initial binding of properdin to a bacterial
surface.

Generation of the anaphylatoxin C5a can also be effected by thrombin cleavage of C5, linking
the coagulation and complement cascades.

The end result of the initiating sequence of the alternative pathway is the generation of
enzymes that cleave C3 into C3a and C3b, and C5 into C5a and C5b.

28
29
Three major pathways of complement activation.

30
The Three Complement Pathways Converge at the Formation of C5 Convertase and Generation
of the membrane attack complex) MAC
All three initiation pathways culminate in the formation of C5 convertase. For the classical and lectin
pathways, C5 convertase has the composition C4b2a3b; for the alternative pathway, C5 convertase has
the formulation C3bBbC3b. However, the end result of all types of C5 convertase activity is the same:
cleavage of the C5 molecule into two fragments, C5a and C5b.

The large C5b fragment is generated on the surface of the target cell or immune complex and provides
a binding site for the subsequent components of the membrane attack complex (MAC). However, the
C5b component is extremely labile and is not covalently bound to the membrane, as are C3b and C4b.
Therefore, it is rapidly inactivated unless it is stabilized by the binding of C6.

When C5b binds to the serum protein C6, the resulting complex interacts reversibly with the cell
membrane via both ionic and hydrophobic bonds. Binding of C7 to C5bC6 induces a conformational
change in C7 that exposes hydrophobic regions on its surface capable of inserting into the interior of
the microbial membrane .

The insertion of C7 into the cell membrane is the triggering event for the formation of the membrane
attack complex, which will ultimately cause cell death. If, however, C6 and C7 binding occurs on an
antigen-antibody (immune) complex or other noncellular surface, then the hydrophobic binding sites
will be unable to anchor the complex and it is released. Sometimes these complexes bind to C8 or even
to C9 before being released.

Released membrane attack complexes can potentially insert into the membrane of nearby cells and
mediate “innocent bystander” lysis. However, under physiologic conditions, such lysis is usually
minimized by regulatory proteins; the “orphan” complexes are bound by the regulatory protein S (also
called vitronectin) and then destroyed. 31
Formation of the membrane attack complex (MAC).
(a) Formation of the MAC, showing the addition of C6, C7, C8, and C9 components to the C5b
component.
(b) Photomicrograph of poly-C9 complex formed by in vitro polymerization of C9 and complement-
induced lesions on the membrane of a red blood cell. These lesions result from formation of
membrane attack complexes.
(c) Relative locations of the members of the membrane attack complex: C5b, C6, C7, C8, and C9 32
C8 is made up of two peptide chains: C8β and C8αγ. Binding of C8β to the C5b67 complex
induces a conformational change in the C8αγ dimer such that the hydrophobic domain of
C8αγ can insert into the interior of the phospholipid membrane.

The C5b678 complex is capable of creating a small membrane pore, 10 Å in diameter. The
final step in the formation of the MAC is the binding and polymerization of C9 to the
C5b678 complex. As many as 10 to 19 molecules of C9 can be bound and polymerized by a
single C5b678 complex.

During polymerization, the C9 molecules undergo a conformational transition, so that they,

too, can insert into the membrane.

The completed MAC, which has a tubular form and functional pore diameter of 70 to 100 Å,
consists of a C5b678 complex surrounded by a poly-C9 complex .

Loss of plasma membrane integrity leads irreversibly to cell death.

33
 Complement activation pathways

Tick over pathway

Thrombin
Protease pathway

Properdin pathway
34
Initiating and amplifying proteins of the classical and lectin-mediated complement pathways

35
Cont.
Initiating and amplifying proteins of the classical and lectin-mediated complement pathways

36
Initiating and amplifying proteins of the alternative complement pathways

Cont.
37
Initiating and amplifying proteins of the alternative complement pathways

38
The proteins of the complement membrane attack complex (MAC)

39
The three main classes of complement activity in the service of host defense

Cont.
40
The three main classes of complement activity in the service of host defense

41
42
Receptors that bind complement components and their breakdown products

Cont.43
Receptors that bind complement components and their breakdown products

44
The breakdown products of C3b—iC3b, C3d, and C3dg—are each bound specifically by the
complement receptor CD21 (CR2), which is expressed on B cells in noncovalent association
with the B-cell receptor.

Since C3b can form covalent bonds with antigens, and these bonds are not affected by
breakdown of C3b to iC3b, C3d, and C3dg, the close association of CD21 with the B-cell
receptor enables the B cell to simultaneously bind antigen via both the B-cell receptor and CD21
.
This has the effect of reducing the antigen concentration necessary for B-cell activation by
approximately 100-fold. Deficiencies in CD21 have been identified in patients suffering from
autoimmune diseases such as systemic lupus erythematosus.

Coligation of antigen to B cells via the B-cell receptor

and the B-cell coreceptor.

The B-cell coreceptor is a complex of three cell membrane

molecules: CD21 (CR2), CD81 (TAPA-1), and CD19.
Antigen that has been covalently bound to fragments of the
C3 complement component is bound by both the
immunoglobulin BCR and the CD21 complement receptor,
thus significantly increasing the avidity of the cell receptors
for the antigen and allowing lower concentrations of antigen
to trigger B-cell activation. The CD19 component is
important in B-cell signaling by antigen.

45
 Release of proinflammatory mediators

Binding of the anaphylatoxins C3a and C5a to the G protein–coupled receptors C3aR and C5aR. C3aR and C5aR are
G protein–coupled receptors. Binding of the anaphylatoxins to these receptors stimulates the release of
proinflammatory mediators from macrophages, neutrophils, basophils, eosinophils, and mast cells. 46
Opsonization of microbial cells by complement components and antibodies.

Opsonization with antibody and complement also provides

critical protection against viral infection. Antibody and
complement can create a thick protein coat around a virus that
neutralizes viral infectivity by preventing the virus from binding
to receptors on the host cell.

They then promote phagocytosis by activated macrophages via

the Fc and complement receptors, followed by intracellular
destruction of the digested particle.

Phagocytosis is mediated by many different complement

receptors on the surface of macrophages and neutrophils,
including CR1, SIGN-R1, and C1qRp.

Phagocytes, using their Fc receptors, also bind to antigens

opsonized by antibody binding.

47
Disposal of Apoptotic Cells and Bodies
Apoptotic cells express the phospholipid phosphatidylserine on the exterior surface of their plasma
membranes. In healthy cells, this phospholipid is normally restricted to the cytoplasmic side of the
membrane, and the change in its location as the cell enters apoptosis serves to signal the immune
system that the cell is dying. Exposed phosphatidylserine is then bound by the serum protein,
annexin A5, which in turn is recognized by C1q. Nuclear fragmentation, DNA cleavage, and the
expression of DNA on the cell surface are also hallmarks of apoptosis, and recent work has
demonstrated that C1q binds specifically to DNA as well as to glycoproteins and phospholipids
exposed on the surfaces of dying cells and apoptotic fragments . Once apoptosis begins, the dying
cell is broken down into membrane-bound vesicles termed apoptotic bodies, which also express
phosphatidylserine and/or DNA on their exterior membrane surfaces

C1q colocalizes with annexin A5 on the surface

of apoptotic cells. This figure demonstrates that
both C1q and annexin A5 are deposited on the
surface of human HeLa cells treated with lethal
doses of ultraviolet light. The three pictures on the
left show the locations of cells, stained purple to
highlight nuclear material; some of these cells can
also be seen in the merged images on the far right.
The cells were also stained green for annexin A5
(which binds to exposed phosphatidylserine
residues) and red for C1q before receiving lethal
doses of ultraviolet light. The second and third
rows show similar staining 2 hours and 24 hours
following treatment, respectively. At 2 hours, C1q
and annexin A5 binding are clearly visible. At 20
hours, nuclear fragmentation and nuclear blebbing,
hallmarks of apoptotic cell death, can be seen.
Scale bars represent 20 μm.
48
 Disposal of Immune Complexes

The coating of soluble immune complexes with C3b

facilitates their binding by CR1 on erythrocytes.

Although red blood cells express lower levels of CR1 (100–

1000 molecules per cell, depending on the age of the cell and
the genetic constitution of the host) than do granulocytes (5
× 104 per cell), there are about 1000 erythrocytes for every
white blood cell, and therefore erythrocytes account for
about 90% of the CR1 in blood.

Erythrocytes also therefore play an important role in clearing

C3b-coated immune complexes by conveying them to the
liver and spleen, where the immune complexes are stripped
from the red blood cells and phagocytosed

Clearance of circulating immune complexes by binding to erythrocyte complement receptors and

subsequent stripping from these receptors by macrophage complement receptors in the liver and spleen.
Because erythrocytes have fewer CR1 receptors than macrophages, the latter can strip the complexes from
the erythrocytes as they pass through the liver or spleen.

Deficiency in this process can lead to renal damage due to accumulation of immune complexes. 49
50
Complement Regulators

C1-inhibitor (C1INH)
functions as a regulator to
prevent excessive activation
of both classical and lectin
pathways

Other regulators
Complement receptor 1 (CR1),
C4 binding protein (C4BP),
decay accelerating factor (DAF),
membrane cofactor protein (MCP),
and factor I (FI)
factor H (FH)

51
 The Regulation of Complement Activity
All biological systems with the
potential to damage the host
are subject to rigorous
regulatory mechanisms.

Such a mechanism is essential

to destroy the appropriate
pathogen but not the healthy
tissue.

By this activity damage to

healthy host tissues is
minimized.

52
The various stages at which complement activity is subject to regulation is shown in the figure
The terminal complement pathway. The C5 convertase cleaves an inert molecule of C5 into a potent anaphylatoxin,
C5a, and a bioactive fragment C5b. C5b interacts with C6, C7, C8, and multiple copies of C9 to form the membrane
attack complex C5b-9 (MAC). C5b-8 inserts into the membrane and C9 polymerize to form a pore inducing Ca flux
and pathogen lysis. Host cells are protected from lysis by expression of CD59, which prevents the insertion and by
clusterin and vitronectin, which bind to C8 and prevents insertion in the membrane. If MAC is bound to the
membrane, host cells can internalize it or remove it by ectocytose.
53
Complement is only fully activated in cases of pathogen infection. During an infection, complement
leads to inflammation, opsonization, phagocytosis, and destruction of the pathogen and ultimately
results in activation of the adaptive immune response.

In a healthy individual, the Alternate Pathway is permanently active at low levels to

survey for presence of pathogens.
Healthy host cells are protected against complement attack and are resistant to
persistent low-grade activation.
The three pathways are activated on the surface of apoptotic cells, which are constantly
generated within the body during normal cellular homeostasis

54
 In the plasma, during normal physiological
conditions, the dominant active complement
pathway is the Alternative pathway

The rate of hydrolysis of C3 to C3(H2 O)

can be accelerated by interactions between
C3 and a number of biological and artificial
interfaces,
including gas bubbles,
biomaterial surfaces, and
lipid surfaces and complexes

Complement activation in physiological conditions.

(A) The alternative pathway is permanently active due to spontaneous transformation of bio-
inactive molecule C3 to bioactive C3(H2 O). This allows generation of C3b, which is rapidly
inactivated by FH and FI in fluid phase or is covalently bound to the surface and then inactivated
on host cells.
55
(B) Classical and lectin pathway recognition molecules bind to apoptotic cells and together with C3b
from the alternative pathway induce a low level of complement activation. Apoptotic cells are not
lysed, but rapidly cleared by phagocytes in an immunologically silent manner. Host cells and plasma
contain multiple regulatory proteins to assure that complement activation will be limited in
physiological conditions
56
Complement during infection with a pathogen.
The permanent activity of the alternative pathway allows it to immediately identify pathogens that are not specifically
protected against complement. Danger-associated molecular patterns on its surface of pathogens are recognized by
C1q, MBL, and ficolins allowing classical and lectin pathway activation, C3 convertase, C4b2a generation, and C3
cleavage. Opsonization due to covalent binding of C3b to the target is accelerated by the amplification loop of the
complement pathways. The effector functions resulting from this complement activation are: pathogen lysis by C5b-9
membrane attack complex, opsonization and phagocytosis of the pathogen, activation of host immune and non-
immune cells by complement anaphylatoxins, inflammation, stimulation of an adaptive immune response, and
antibody generation. Secreted antibodies will bind to the pathogen and create immune complexes that will be
recognized by C1q and will activate the classical pathway. Altogether these mechanisms contribute to pathogen 57
elimination