MLS 117: IMMUNOLOGY/ SEROLOGY (Complement System)

IMMUNOLOGY: Unit 7-The Complement System

Objectives:
• Name and compare the three complement activation pathways.
• Describe the mechanisms and consequences of complement activation.
• Explain the biological functions of the complement system.
• Name and describe alterations in complement levels.
• Briefly describe the assessment of complement levels.

Soluble Mediators of the Immune System

• The activation of complement is focused on the surface of invading microorganisms, with limited complement deposited on
normal cells and tissues.
• If the mechanisms that regulate this delicate balance malfunction, the complement system may cause injury to cells,tissues, and
organs, such as destruction of the kidneys in systemic lupus erythematosus or hemolytic anemias.

COMPLEMENT SYSTEM
Complement is a complex series of more than 30 soluble and cell-bound proteins that interact in a very specific way to
enhance host defense mechanisms against foreign cells.
Originally recognized in the 1890s as a heat-labile substance present in normal non immune serum, complement was so
coined by Paul Ehrlich because it complements the action of antibody in destroying microorganisms.
Jules Bordet was awarded the Nobel Prize in 1919 for his role in elucidating the nature of complement.

 While complement promotes opsonization and lysis of foreign cells and immune complexes, chronic activation (occurs in
autoimmune diseases) can lead to inflammation and tissue damage.
 Most plasma complement proteins are synthesized in the liver, with the exception of C1 components, which are mainly produced
by intestinal epithelial cells, and factor D, which is made in adipose tissue.
Other cells, such as monocytes and macrophages, are additional sources of early complement components, including C1,
C2, C3, and C4.
 Most of these proteins are inactive precursors, or zymogens, which are converted to active enzymes in a very precise order.

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The complement system can be activated in three different

ways:
• The first is the classical pathway, which involves nine
proteins that are triggered by antigen–antibody combination.
Pillemer and colleagues later discovered an antibodyindependent
pathway in the 1950s, and this plays a major role as a natural
defense system.
• The second pathway, the alternative pathway, was
originally called the properdin system, because the protein
properdin was thought to initiate this pathway. Now, however, it is
known that properdin’s major function is to stabilize a key enzyme
complex formed along the pathway
• The third pathway, the lectin pathway, is another
antibodyindependent means of activating complement proteins. Its
major constituent, mannose- (or mannan-) binding lectin (MBL),
adheres to mannose found mainly in the cell walls or outer coating
of bacteria, viruses, yeast, and protozoa

 The complement system plays a major part in the inflammatory

response directed against foreign antigens.
 Complement fragments acting as opsonins, for which specific
receptors are present on phagocytic cells, enhance metabolism
and clear immune complexes.
 Complement components are also able to
 increase vascular permeability,
 recruit monocytes and neutrophils to the area of antigen
concentration, and
 trigger secretion of immunoregulatory molecules that
amplify the immune response.
 Any deficiencies in the complement system can result in an
increased susceptibility to infection or in the accumulation of
immune complexes with possible autoimmune manifestations.

The Classical Pathway

The classical pathway or cascade is the main antibody directed
mechanism for triggering complement activation. Those classes that can
include IgM, IgG1, IgG2, and IgG3.

 IgM is the most efficient, because it has multiple binding sites; thus, it
takes only one molecule attached to two adjacent antigenic
determinants to initiate the cascade.
 Two IgG molecules must attach to antigen within 30 to 40 nm of each
other before complement can bind, and it may take at least 1000 IgG
molecules to ensure that two are close enough to initiate such binding.
 Within the IgG group, IgG3 is the most effective, followed by IgG1 and
then IgG2. (IG-, 3-1-2)

In addition to antibody, there are a few substances that can bind

complement directly to initiate the classical cascade. These include C-reactive
protein, several viruses, mycoplasmas, some protozoa, and certain gram-
negative bacteria such as Escherichia coli.
Complement activation can be divided into three main stages, each
of which is dependent on the grouping of certain reactants as a unit.
 The first stage involves C1, which is known as the recognition unit. Once
C1 is fixed, the next components activated are C4, C2, and C3, known
collectively as the activation unit.

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 C5 through C9 comprise the membrane attack complex, and it is this last unit that completes lysis of the foreign particle.

The Recognition Unit

The first complement component to bind is C1,
• It consists of three subunits: C1q, C1r, and C1s, which are stabilized by calcium. (C1 q-r-s)
 C1q unit is the part that binds to antibody molecules, the
 C1r and C1s subunits generate enzyme activity to begin the cascade.

 C1q “recognizes” the fragment crystallizable (FC) region of two adjacent antibody
molecules, and at least two of the globular heads of C1q must be bound to initiate the
classical pathway. This binding occurs at the CH2 region for IgG and at the CH3 region
for IgM.
 C1r and C1s are both serine protease proenzymes or zymogens. As binding of C1q
occurs, both are converted into active enzymes.
 Autoactivation of C1r results from a conformational change that takes place as C1q is
bound.
 Once activated, C1r cleaves a thioester bond on C1s, which activates it. Activated C1r is
extremely specific, because its only known substrate is C1s.
 C1s has a limited specificity, with its only substrates being C4 and C2. Once C1s is activated, the recognition stage ends.

The Activation Unit

Phase two, the formation of the activation unit, results in the production of an
enzyme known as C5 convertase.

 C1s cleaves C4 to split off a 77-amino acid fragment called C4a. In the process, it
opens a thioester-containing active site on the remaining part, C4b.
 C4b must bind to protein or carbohydrate within a few seconds, or it will react with
water molecules to form iC4b, which is rapidly degraded. Thus, C4b binds mainly to
antigen in clusters that are within a 40 nm radius of C1.

 C2 is the next component to be activated.

 The C2 gene is closely associated with the gene for factor B (alternative pathway) on
chromosome 6 in the major histocompatability complex, and each serves a similar
purpose in its particular pathway.
 When combined with C4b in the presence of magnesium ions, C2 is cleaved by C1s to
form C2a and C2b.
 Binding of C2 to C4b can occur in the fluid phase, but C4b attached to antigen is much
more efficient in accepting C2. This serves to keep the reaction localized.

 C4b + C2a is known as C3 convertase.

 This is written as C4b2a to indicate that the complex is an active enzyme. If binding
does occur, C3 is cleaved into two parts, C3a and C3b.

 C3 is the major constituent of the complement system. It serves as the pivotal point
for all three pathways.
 The cleavage of C3 to C3b represents the most significant step in the entire process
of complement activation.
 In addition to being required for the formation of the membrane attack complex, C3b
also serves as a powerful opsonin. Macrophages have specific receptors for it and this
contributes greatly to the process of phagocytosis.
 If C3b is bound within 40 nm of the C4b2a, then this creates a new enzyme known as C5 convertase.
 The cleaving of C5 with deposition of C5b at another site on the cell membrane constitutes the beginning of the membrane
attack complex (MAC).

The Membrane Attack Complex

 C5 convertase, consisting of C4b2b3b, splits off a 74-amino-acid piece known as C5a, and C5b attaches to the cell membrane,
forming the beginning of the MAC.
 The splitting of C5 and the cleavage of C3 represents the most significant biological consequences of the complement system.
However, C5b is extremely labile, and it is rapidly inactivated unless binding to C6 occurs.
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 Subsequent binding involves C6, C7, C8,and C9.

 Membrane damage is caused by at least two different mechanisms: channel

formation and the binding of phospholipids (causes a reordering and
reorientation of molecules that results in leaky patches).
 When complement proteins are bound, membrane phospholipids rearrange
themselves into domains surrounding the C5b6789 complex, and the integrity of
the membrane is destroyed. Ions then are able to pass freely out of the cell.
 The membrane attack unit begins when C6 binds to C5b, thereby stabilizing it.
 C7 binds next, this allows for insertion of the C7 part of the C5b67 complex into the
membrane of the target cell.
 Next, C8 binds to C7 and exposes a hydrophobic region that interacts with the cell
membrane to form a small hole in the membrane as C8 is inserted into the lipid
bilayer.
 Lysis can be observed at this stage, but the addition of C9 accelerates the process.
 Binding of C8 causes a loss of potassium from the cell, which is followed by leakage
of amino acids and ribonucleotides.
 The pore formed by the binding of C5b678 is small, capable of lysing erythrocytes
but not nucleated cells.
 For lysis of nucleated cells to be achieved, C9 must bind to the complex.
 C9 polymerizes only when bound, and it is believed that the C5–C8 complex acts as a
catalyst to enhance the rate of reaction.
 One such unit can lyse erythrocytes, but lysis of nucleated cells is a multihit
phenomenon.
 Destruction of target cells actually occurs through an influx of water and a
corresponding loss of electrolytes.

The Alternative Pathway

Pathogens can be destroyed in the absence of antibody by means of the
alternative pathway, which acts as part of innate or natural immunity.
• First described by Pillemer and his associates in the early 1950s, the pathway
was originally named for the protein properdin.
• Now it is known that properdin does not initiate this pathway but rather
stabilizes the C3 convertase formed from activation of other factors. In addition
to properdin, the serum proteins that are unique to this pathway include factor
B and factor D.

 Triggering substances for the alternative pathway include

Bacterial cell walls, especially those containing lipopolysaccharide
Fungal cell walls
Yeast
Viruses; virally infected cells
Tumor cell lines
Parasites, especially Trypanosomes.
 All of these can serve as sites for binding the complex C3bBb, one of the end
products of this pathway.

 The conversion of C3 is the first step in this pathway.

 Once activated, it can bind to factor B. Binding of C3b (sometimes called iC3)
to B causes a conformational change that makes B more susceptible to
cleavage by serine proteases. Thus, only bound factor B can be cleaved by
factor D.
 The role of factor B is thus analogous to that of C2 in the classical pathway,
because it forms an integral part of a C3 convertase.

 Factor D is a plasma protein that goes through a conformational change when

it binds to factor B. It is a serine protease, and its only substrate is bound
factor B. When bound to factor B, the catalytic site on factor D opens,
making it an active enzyme.
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 It cleaves factor B into two pieces: Ba and Bb.

 Bb remains attached to C3b, forming the initial C3 convertase of the alternative pathway.
 As the alternative pathway convertase, C3bBb is then capable of cleaving additional C3 into C3a and C3b.
 Some C3b attaches to cellular surfaces and acts as a binding site for more factor B. This results in an amplification loop that feeds
C3b into the classical and alternative pathways.
 All C3 present in plasma would be rapidly converted by this method were it not for the fact that the enzyme C3bBb is extremely
unstable unless properdin binds to the complex.
 C3bBb can also cleave C5, but it is much more efficient at cleaving C3. If, however, some of the C3b produced remains bound to
the C3 convertase, the enzyme is altered to form C3bBb3bP, which has a high affinity for C5 and exhibits C5 convertase activity.
 C5 is cleaved to produce C5b, the first part of the membrane attack unit. From this point on, both the alternative and classical
pathways are identical.

The Lectin Pathway

The lectin pathway represents another means of activating complement without antibody being present.
• This pathway provides an additional link between the innate and acquired
immune response, because it involves nonspecific recognition of carbohydrates
that are common constituents of microbial cell walls and that are distinct from
those found on human cell surfaces.
 One key LECTIN, called mannose-binding or mannan binding lectin (MBL),
binds to mannose or related sugars in a calcium- dependent manner to
initiate this pathway.
 MBL is considered an acute phase protein, because it is produced in the liver
and is normally present in the serum but increases during an initial
inflammatory response.
 It plays an important role as a defense mechanism in infancy, during the
interval between the loss of maternal antibody and the acquisition of a full-
fledged antibody response to pathogens.

 The structure of MBL is similar to that of C1q, and it is associated with three
MBL-serine proteases (MASPs):
 MASP-1,
 MASP-2, and
 MASP-3.
 Once MBL binds to a cellular surface, MASP-2, which is homologous to C1s,
autoactivates.
*MASP-2 thus takes the active role in cleaving C4 and C2.
 Once C4 and C2 are cleaved, the rest of the pathway is identical to the
classical pathway.

System Controls
 Activation of complement could cause tissue damage and have devastating systemic effects if it was allowed to proceed
uncontrolled.
 To ensure that infectious agents and not self-antigens are destroyed and that the reaction remains localized, several plasma
proteins act as system regulators. In fact, approximately one-half of the complement components serve as controls for critical
steps in the activation process.
 Because activation of C3 is the pivotal step in all pathways, the majority of the control proteins are aimed at halting
accumulation of C3b.

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Biological Manifestations of Complement Activation

Activation of complement is a very effective means of  The last major effect of complement-derived peptides is
amplifying the inflammatory response to destroy and clear OPSONIZATION.
foreign antigens.  C4b, C3b, and iC3b, which accumulate on cell
membranes as complement activation proceeds,
 Complement proteins also serve as a means of linking innate bind to specific receptors on erythrocytes,
and natural immunity. neutrophils, monocytes, and macrophages.
 They act as opsonins to facilitate destruction by  This facilitates phagocytosis and clearance of foreign
phagocytic cells, and they play a major role in uptake substances, and it is one of the key functions of the
and presentation of antigens so a specific immune complement system.
response can occur.
 They also facilitate B-cell activation and may, in fact, be
necessary for maintaining immunological memory.

 Effector molecules generated earlier in the cascade play a

major role in all these areas. Such molecules can be
classified into three main categories:
 anaphylatoxins
 chemotaxins
 opsonins.
An anaphylatoxin is a small peptide that causes
increased vascular permeability, contraction of smooth
muscle, and release of histamine from basophils and mast
cells.

 Proteins that play such a part are C3a, C4a, and C5a.

 C3a, C4a, and C5a attach to specific receptors on

neutrophils, basophils, mast cells, eosinophils, smooth
muscle cells, and vascular endothelium.
C3a and C4a attach to the C3a receptor (C3aR)
C5a attaches to the C5a receptor (C5aR).

 When binding occurs on basophils and mast cells,

histamine is released, thereby increasing vascular
permeability and causing contraction of smooth muscles.

 C5a causes neutrophils to release hydrolytic enzymes,

oxygen radicals, and prostaglandins, aiding in destruction
of foreign antigens.
 C5a also serves as a chemotaxin for neutrophils, basophils,
eosinophils, mast cells, monocytes, and dendritic cells.
 Binding of C5a to monocytes causes them to undergo an
oxidative burst that includes increased production of
hydrolytic enzymes, neutrophil chemotactic factor,
platelet activating factors, interleukin-1, and toxic
oxygen metabolites.
 This may produce fever and an increase in acute phase
reactants, both of which are characteristic of an
inflammatory response.

 As a means of localizing and controlling the effects of C3a,

C4a, and C5a, these substances are rapidly inactivated by
an enzyme in the plasma called carboxypeptidase N.