Complement System

The complement system is the major effector of the humoral branch of the immune system.
Research on complement began in the 1890s, when Jules Bordet at the Institut Pasteur in Paris showed that
sheep antiserum to the bacterium Vibrio cholera caused lysis of the bacteria and that heating the antiserum
destroyed its bacteriolytic activity. Surprisingly, the ability to lyse the bacteria was restored to the heated
serum by adding fresh serum that contained no antibodies directed against the bacterium and was unable to
kill the bacterium by itself.
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. Paul Ehrlich in Berlin independently carried out similar experiments and
coined the term complement, defining it as “the activity of blood serum that completes the action of
antibody.” In ensuing years, researchers discovered that the action of complement was the result of
interactions of a large and complex group of proteins.

The Functions of Complement

Research on complement now 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:
 Lysis of cells, bacteria, and viruses
 Opsonization, which promotes phagocytosis of particulate antigens
 Binding to specific complement receptors on cells of the immune system, triggering specific cell
functions, inflammation, and secretion of immunoregulatory molecules
 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, although significant amounts are also produced by blood monocytes, tissue macrophages, and
epithelial cells of the gastrointestinal and genitourinary tracts. 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). 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).

Complement Activation
The early steps, culminating in formation of C5b, 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.
The Classical Pathway Begins with Antigen-Antibody Binding
Complement activation by the classical pathway commonly begins with the formation of soluble antigen-
antibody complexes (immune 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 and IgG molecule that expose a binding site for the C1 component of the complement
system. Each C1 molecule must bind to at least two Fc sites for a stable C1-antibody interaction to
occur.
 Binding of C1 to Fc binding sites induces a conformational change that converts C1 to an active
serine protease enzyme that has two substrates, C4 and C2.
 The C4 component is activated when C1 hydrolyzes a small fragment (C4a) from the amino
terminus, exposing a binding site on the larger fragment (C4b).
 The C2 is cleaved by the C1; the smaller fragment (C2b) diffuses away and larger C2a binds with C4b
 The resulting C4b2a complex is called C3 convertase, referring to its role in converting the C3 into an
active form.
 Hydrolysis of a short fragment (C3a) from by the C3 convertase generates C3b.A single C3 convertase
molecule can generate over 200 molecules of C3b, resulting in tremendous amplification at this step
of the sequence.
 Some of the C3b binds to C4b2a to form a trimolecular complex C4b2a3b, called C5 convertase and
cleave C5 into C5a, which diffuses away, and C5b, which attaches to C6 and initiates formation of the
membrane attack complex.
 Some of the C3b generated by C3 convertase activity does not associate with C4b2a; instead it diffuses
away and then coats immune complexes and particulate antigens, functioning as an opsonin. C3b
may also bind directly to cell membranes.
The Alternative Pathway Is Antibody-Independent
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. Because no antibody is required, the
alternative pathway is a component of the innate immune system. This major pathway of complement
activation 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 For example,
both gram-negative and gram-
positive bacteria have cell-wall
constituents that can activate
the alternative pathway.
 In the alternative pathway,
serum C3, which contains an
unstable thioester bond, is
subject to slow spontaneous
hydrolysis to yield C3a and C3b.
The C3b component can bind to
foreign surface antigens (such
as those on bacterial cells or
viral particles).
 The C3b present on the surface of the foreign cells can bind another serum protein called factor B.
 Binding to C3b exposes a site on factor B that serves as the substrate for an enzymatically active
serum protein called factor D.
 Factor D cleaves the C3b-bound factor B, releasing a small fragment (Ba) that diffuses away and
generating C3bBb.
 The C3bBb complex has C3 convertase activity and thus is analogous to the C4b2a complex in the
classical pathway.
 properdin binds to C3bBb complex, stabilizing it
  The C3bBb can activate unhydrolyzed C3 to generate more C3b autocatalytically. As a result, the
initial steps are repeated and amplified, so that more molecules of C3b can be deposited on an
antigenic surface in.
 The C3 convertase generates the C3bBb3b complex, which exhibits C5 convertase activity, analogous
to the C4b2a3b complex in the classical pathway.
 The nonenzymatic C3b component binds C5, and the Bb component subsequently hydrolyzes the
bound C5 to generate C5a and C5b; the latter binds to the antigenic surface
The Lectin Pathway Originates With Host Proteins Binding Microbial Surfaces
Lectins are proteins that recognize and bind to specific carbohydratetargets.
 The lectin pathway is activated by the binding of mannose- binding lectin (MBL) to mannose
residues on glycoproteins or carbohydrates on the surface of microorganisms including certain
Salmonella, Listeria, and Neisseria strains, as well as Cryptococcus neoformans and Candida
albicans.
 MBL is an acute phase protein produced in inflammatory responses. Its function in the complement
pathway is similar to that of C1.
 After MBL binds to the surface of a cell or pathogen, MBL-associated serine proteases,MASP-1 and
MASP-2, bind to MBL.
 The active complex formed by this association causes cleavage and activation of C4 and C2.
 The MASP-1 and -2 proteins have structural similarity to C1 and mimic their activities.
 This means of activating the C2–C4 components to form a C5 convertase without need for specific
antibody binding represents an important innate defense mechanism comparable to the alternative
pathway, but utilizing the elements of the classical pathway except for the C1 proteins.
The Three Complement Pathways Converge at the Membrane-Attack Complex
The terminal sequence of complement activation involves C5b, C6, C7, C8, and C9, which interact
sequentially to form a macromolecular structure called the membrane-attack complex (MAC). This complex
forms a large channel through the membrane of the target cell, enabling ions and small molecules to diffuse
freely across the membrane.
 The end result of activating the classical, alternative, or lectin pathways is production of an active C5
convertase. This enzyme cleaves C5 This generates the small C5a fragment, which diffuses away, and
the large C5b fragment, which binds to the surface of the target cell and provides a binding site for
the subsequent components of the membrane-attack complex.
 The C5b component is extremely labile and becomes inactive within 2 minutes unless C6 binds to it
and stabilizes its activity.
 As C5b6 binds to C7, the resulting complex can bind to membrane phospholipids. hydrophobic
binding sites enable the C5b67 complex to insert into the phospholipid bilayer.
 Binding of C8 to membrane-bound C5b67 induces a conformational change in C8, so that it too
undergoes a hydrophilic-amphiphilic structural transition, exposing 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 as10–17 molecules of C9 can be bound and polymerized
by a single C5b678 complex.
 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.

Biological Consequences of Complement Activation

The Membrane-Attack Complex Can Lyse a Broad Spectrum of Cells
The membrane-attack complex formed by complement activation can lyse gram-negative bacteria, parasites,
viruses, erythrocytes, and nucleated cells. Among the pathogenic viruses susceptible to lysis by complement-
mediated lysis are herpesviruses, Orthomyxo viruses, paramyxoviruses, and retroviruses. The complement
system is generally quite effective in lysing gram-negative bacteria Gram-positive bacteria are generally
resistant to complement- mediated lysis because the thick peptidoglycan layer in their cell wall prevents
insertion of the MAC into the inner membrane.
Cleavage Products of Complement Components Mediate Inflammation
various peptides generated during formation of the MAC play a decisive role in the development of an
effective inflammatory response. 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. can 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.
C3b and C4b Binding Facilitates Opsonization
C3b is the major opsonin of the complement system, although C4b and C3b 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. 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.
Recent studies indicate that complement fragment 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
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.
The binding of antibody and/or complement to the surface of a viral particle creates a thick protein coating
that can be visualized by electron microscopy (Figure 13-13). 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).
The Complement System Clears Immune Complexes from Circulation
The coating of soluble immune complexes with C3b is thought to facilitate their binding to CR1 on
erythrocytes. 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