Complement System • C4b binds mainly to antigen in clusters that are within a 40-nm radius of

C1.
INTRODUCTION: • C2 is the next component to be activated.
• The C2 gene is closely associated with the gene for Factor B (alternative
• Complement is a complex series of more than 30 proteins that play a pathway) on chromosome 6 in the major histocompatibility complex
major part in amplifying the inflammatory response to destroy and (MHC)
clear foreign antigens. • When combined with C4b in the presence of magnesium ions, C2 is
cleaved by C1s to form C2a (which has a molecular weight of 70,000)
and C2b (which has a molecular weight of 34,000)
• Complement activation is also proinflammatory in its ability to increase
• The combination of C4b and C2a is known as C3 convertase. C3, the
vascular permeability, recruit monocytes and neutrophils to the area of major and central constituent of the complement system, is present in
antigen concentration, and trigger secretion of immunoregulatory the plasma at a concentration of 1 mg/mL to 1.5 mg/mL
molecules that amplify the immune response. • The molecule has a molecular weight of 190,000 and consists of two
• - Complement has important “housekeeping” roles as well. polypeptide chains, alpha (α) and beta (β). The α chain contains a highly
• Complement activation needs to be carefully regulated. reactive thioester group.
• C3b is estimated to have a halflife of 60 microseconds if not bound to
antigen
Pathways of the Complement System • The cleavage of C3 represents a second and major amplification process
because about 200 molecules are split for every molecule of C4b2a.
• If C3b is bound within 40 nm of the C4b2a, this creates a new enzyme
• The first pathway described, the classical pathway, involves nine
proteins that are triggered primarily by antigen–antibody combination. known as C5 convertase.
• This second pathway, the alternative pathway, was originally called the • The cleaving of C5 with deposition of C5b at another site on the cell
properdin system because the protein properdin was thought to be the membrane constitutes the beginning of the MAC
main initiator of this pathway.
• The third pathway, likely the most ancient of the three, is the lectin The Membrane Attack Complex (MAC)
pathway, another antibody-independent means of activating
complement proteins. • C5 consists of two polypeptide chains, α and β, which are linked by
• Most plasma complement proteins are synthesized in the liver with the disulfide bonds to form a molecule with a molecular weight of about
exception of C1 components. 190,000.
• C5 convertase, consisting of C4b2b3b, splits off a 74-amino acid piece
The Classical Pathway
known as C5a that is released into circulation, whereas C5b attaches to
the cell membrane, forming the beginning of the MAC.
• The classical pathway, the first activation cascade described, is the main
antibody-directed mechanism for triggering complement activation. • C5b is extremely labile and rapidly inactivated unless binding to C6
• The immunoglobulin classes that can activate the classical pathway occurs.
include IgM, IgG1, IgG2, and IgG3, but not IgG4, IgA, or IgE. • C6 and C7 each have molecular weights of approximately 110,000 and
• Two IgG molecules must attach to antigen within 30 to 40 nm of each have similar physical and chemical properties.
other before complement can bind; it may take at least 1,000 IgG • C8 is made up of three dissimilar chains joined by disulfide bonds and
molecules to ensure that there are two close enough to initiate such has a total molecular weight of about 150,000
binding.
• C9 is a single polypeptide chain with a molecular weight of 70,000
• In addition to antibodies, a few substances can bind complement
directly to initiate the classical cascade. • The Membrane Attack Complex (MAC)
• Complement activation can be divided into three main stages, each of • The carboxyterminal end is hydrophobic, whereas the amino-terminal
which is dependent on the grouping of certain reactants as a unit. end is hydrophilic. The hydrophobic part serves to anchor the MAC
• The first stage involves the recognition unit, which in the case of the within the target membrane.
classical pathway is C1. • The complex of C5b-C6-C7-C8 and C9 is known as C5b-9 or MAC.
• Once C1 is fixed, the next components activated are C4, C2, and C3, • When formed, the MAC presents a pore of 70 to 100Å that allows ions
known collectively as the activation unit of the classical pathway (and
to pass in and out of the membrane.
the lectin pathway)
• C5 through C9 comprise the membrane attack complex (MAC); • The presence of C9 greatly speeds this lysis.

The Recognition Unit The Lectin Pathway

• Instead of activation through antibody binding, the lectin pathway is

• The first complement component of the classical pathway to bind is C1,
a molecular complex of 740,000 d. activated by recognition of surface moieties that are found on
• The complex is made up of one C1q subunit and two each of the C1r pathogens.
and C1s subunits. • Although this pathway is the most recently described of the three
• C1q has a molecular weight of 410,000 and is composed of six strands activation pathways of complement, it is probably the most ancient.
that form six globular heads with a collagen-like tail portion. • The lectin pathway molecules are structurally similar to those of the
• C1q “recognizes” the fragment crystallizable (Fc) region of two adjacent classical; the classical and lectin pathways even share the components
antibody molecules, but at least two of the globular heads of C1q must
C4 and C2.
be bound to initiate the classical pathway.
• C1r and C1s are serine protease proenzymes, also called zymogens. • The role C1q serves in the classical pathway is filled by three classes of
• Autoactivation of C1r results from a conformational change that takes recognition molecules in the lectin pathway: lectins, ficolins, and CL-K1.
place as C1q is bound. • The Lectin Pathway
• Once activated, C1r cleaves a thioester bond on C1s which, in turn, • One key lectin, called mannose-binding, or mannan-binding, lectin
activates it. (MBL), binds to mannose or related sugars in a calcium-dependent
• C1s has a limited specificity, with its only substrates being C4 and C2. manner to initiate this pathway.
• Once C1s is activated the recognition stage ends.
• MBL is considered an acute phase protein because it is produced in the
The Activation Unit liver and is normally present in the serum but increases during an initial
inflammatory response.
• C4 is the second most abundant complement protein, with a serum • The enzymatic role played by C1r and C1s in the classical pathway is
concentration of approximately 600 μg/mL. played in the lectin pathway by serine proteases called MBL-associated
• C1s cleaves C4 to split off a 77-amino acid fragment called C4a. In the serine proteases (MASPs).
process, it opens a thioester containing active site on the remaining
part, C4b
• The lectin pathway plays an important role as a defense mechanism in • There are specific receptors on certain cells that also exert a
infancy, during the interval between the loss of maternal antibody and controlling influence on the activation process.
the acquisition of a full-fledged antibody response to pathogens. • Because activation of C3 is the pivotal step in all pathways, the
majority of the control proteins are aimed at halting accumulation
The Alternative Pathway of C3b.

• First described by Pillemer and his associates in the early 1950s, the Regulation of the Classical and Lectin Pathways
alternative pathway was originally named for the protein properdin, a
constituent of normal serum with a concentration of approximately 5 to • C1 inhibitor (C1-INH) inhibits activation at the first stages of both
15 μg/mL. the classical and lectin pathways.
• Properdin has been confirmed to bind and initiate activation, the • Its main role is to inactivate C1 by binding to the active sites of C1r
primary function of properdin is to stabilize the C3 convertase formed and C1s.
from activation of other factors. • C1-INH also inactivates MASP-2 binding to the MBL-MASP
• Triggering substances for the alternative pathway include bacterial complex, thus halting the lectin pathway.
cell walls, especially those containing lipopolysaccharide, fungal cell • Further formation of C3 convertase in the classical and lectin
walls, yeast, viruses, virally infected cells, tumor cell lines, and some pathways is inhibited by four main regulators: soluble C4-binding
parasites, especially trypanosomes. protein (C4BP) and three cell-bound receptors, complement
• Native C3 is not stable in plasma. Water is able to hydrolyze a receptor type 1 (CR1), membrane cofactor protein (MCP), and
thioester bond, thus spontaneously activating a small number of these decayaccelerating factor (DAF) Pathways
molecules. • C4BP is abundant in the plasma and has a molecular weight of
• The C3b binds to Factor B, which has a molecular weight of 93,000 about 520,000.
and is fairly abundant in the serum, at a level of 200 μg/ mL. • If C4BP attaches to cell-bound C4b, it can dissociate it from C4b2a
• Once bound to C3b, Factor B can be cleaved by Factor D. complexes, causing the cessation of the classical pathway.
• Factor D is a plasma protein that goes through a conformational • CR1, also known as CD35, is a large polymorphic glycoprotein with
change when it binds to Factor B. a molecular weight between 165,000 and 280,000.
• It cleaves Factor B into two pieces: Ba (with a molecular weight of • It binds C3b and C4b but has the greatest affinity for C3b. Once
33,000) and Bb (with a molecular weight of approximately 60,000) bound to CR1, both C4b and C3b can then be degraded by Factor
• Bb remains attached to C3b, forming the initial C3 convertase of the I.
alternative pathway. • A main function of CR1 is as a receptor on platelets and red blood
• As the alternative pathway convertase, C3bBb is then capable of cells (RBCs), which helps to mediate transport of C3bcoated
cleaving additional C3 into C3a and C3b. immune complexes to the liver and spleen.
• Binding of properdin increases the half-life of C3bBb from 90 seconds • MCP, or CD46, has a molecular weight between 50,000 and
to several minutes. In this manner, optimal rates of alternative pathway 70,000 and is found on virtually all epithelial and endothelial cells
activation are achieved. except erythrocytes.
• C3bBb can also cleave C5, but it is much more efficient at cleaving C3. • DAF or CD55, a 70,000 d membrane glycoprotein, is the third
• However some of the C3b produced remains bound to the C3 main receptor and has a wide tissue distribution.
convertase, the enzyme is altered to form C3bBb3bP, which has a high • DAF is capable of dissociating both classical and alternative
affinity for C5 and exhibits C5 convertase activity. pathway C3 convertases.
• The carboxy-terminal portion of DAF is covalently attached to a
glycophospholipid anchor that is inserted into the outer layer of
the membrane lipid bilayer.
• The presence of DAF on host cells protects them from bystander
lysis.

Regulation of the Alternative Pathway

• The principal soluble regulator of the alternative pathway is
Factor H, which has a molecular weight of 160,000. It acts by
binding to C3b, preventing the binding of Factor B.
• Factor H also accelerates the dissociation of the C3bBb complex
on cell surfaces.
• Additionally, Factor H acts as a cofactor that allows Factor I to
break down C3b.
• When Factor I binds, a conformational change takes place that
allows it to cleave C3b.
• iC3b is further broken down to C3c and C3dg by Factor I in
conjunction with another cofactor: the CR1 receptor.

Regulation of Terminal Components

• S protein is a soluble control protein that acts at a deeper level of

complement activation. Also known as vitronectin, S protein
interacts with the C5b-7 complex as it forms in the fluid phase and
prevents it from binding to cell membranes.
• A receptor, known by various terms, including membrane
System System Controls
inhibitor of reactive lysis (MIRL) or CD59, also acts to block
• Activation of complement could cause tissue damage and have formation of the MAC.
devastating systemic effects if it were allowed to proceed
uncontrolled.
Complement Receptors and Their Biological Roles
 • A second receptor, CR2 (or CD21), is found mainly on B Major Pathway Components
lymphocytes and follicular dendritic cells. • Hereditary deficiency of any complement protein, with the
• CR2 plays an important role as part of the B-cell co-receptor for exception of C9, usually manifests itself in increased susceptibility
antigen. to infection and delayed clearance of immune complexes.
• Another receptor, CR3 (CD11b/CD18), found on monocytes, • A lack of C2, the most common deficiency, is found in 1 in 20,000
macrophages, neutrophils, and natural killer (NK) cells, specifically individuals. Recent evidence indicates that atherosclerosis may be
binds particles opsonized with iC3b, a C3b degradation product. related to a C2 deficiency.
• The CR3 receptor plays a key role in mediating phagocytosis of • A second deficiency that occurs with some frequency is that of
particles coated with these complement fragments. MBL. Deficiencies and polymorphisms in MBL occur in about 30%
• Patients whose white blood cells (WBCs) lack these receptors fail of the population.
to exhibit functions such as chemotaxis, surface adherence, and • Low MBL has also been associated with the risk of some cancers,
aggregation. infection during chemotherapy, and certain autoimmune
• The CR4 (CD11c/CD18) receptor is very similar to CR3 in that it disorders such as systemic lupus erythematosus (SLE), but these
also binds iC3b fragments in a calcium-dependent fashion. connections are not yet well defined.
• CR4 proteins are found on neutrophils, monocytes, tissue • The most serious deficiency is that of C3 because it is the key
macrophages, activated T cells, dendritic cells, NK cells, and mediator in all pathways. C3 deficiencies are, however, extremely
activated B cells. rare.
• Collectin receptors, bind the collagen portion of C1q and generally • Individuals with a C3 deficiency are prone to developing severe,
enhance the binding of C1q to FC receptors. Interacting only with recurrent life-threatening infections with encapsulated bacteria
bound C1q, the receptors appear to increase phagocytic cells’ such as Streptococcus pneumoniae and may also be subject to
uptake of immune complexes opsonized with C1q. immune complex disease.
• It appears that a deficiency of any of the terminal components of
Biological Manifestations of Complement Activation the complement cascade (C5–C8) causes increased susceptibility
• Activation of complement is a very effective means of amplifying to systemic Neisseria infections, including meningococcal
the inflammatory response to destroy and clear foreign antigens. meningitis and disseminated gonorrheal disease.
• Complement proteins also serve as a means of linking innate and
adaptive immunity Regulatory Factor Component
• Recent work demonstrates that complement is necessary for • A prime example of a disease caused by a missing or defective
maintaining immunologic memory. regulatory component is paroxysmal nocturnal hemoglobinuria
• Effector molecules generated earlier in the cascade play a major (PNH). Individuals with this disease have RBCs that are deficient in
role in all these areas. Such molecules can be classified into three DAF.
main categories: anaphylatoxins, chemotaxins, and opsonins • These individuals appear to have a deficiency in the
• An anaphylatoxin is a small peptide that causes increased vascular glycophospholipid anchor of the DAF molecule that prevents its
permeability, contraction of smooth muscle, and release of insertion into the cell membrane.
histamine from basophils and mast cells. Proteins that play such a • Some studies indicate that a DAF deficiency is associated with a
part are C3a and C5a. lack of CD59 (MIRL) and both are implicated in PNH.
• C3a and C5a attach to specific receptors on neutrophils, basophils, • CD59 prevents insertion of C9 into the cell membrane by binding
mast cells, eosinophils, smooth muscle cells, and vascular to the C5b-8 complex, inhibiting formation of transmembrane
endothelium. channels
• C5a causes neutrophils to release hydrolytic enzymes, oxygen • Another complement deficiency disorder that has recently
radicals, and prostaglandins, which aid in the destruction of received considerable attention is hereditary angioedema (HAE).
foreign antigens • This disease is caused by a deficiency or lack of C1-INH, which
• C5a also serves as a chemotaxin for neutrophils, basophils, occurs with a population frequency of 2 in 10,000.
eosinophils, mast cells, monocytes, and dendritic cells. • C1-INH is a serpin (serine protease inhibitor) that controls many of
• Binding of C5a to monocytes causes them to undergo an oxidative the serine proteases on contact. The lack of C1-INH results in
burst that includes increased production of hydrolytic enzymes, localized swelling that can be either subcutaneous or found within
neutrophil chemotactic factor, plateletactivating factors, the bowel or upperrespiratory tract.
interleukin-1 (IL-1), and toxic oxygen metabolites. • HAE is separated into two types, type I and type II. Type I is
• C3a and C5a are rapidly inactivated by an enzyme in the plasma characterized by a decrease in the C1-INH protein; type II has
called carboxypeptidase N to localize and control their effects. normal levels of C1-INH, but the function is decreased.
• The last major effect of complement-derived peptides is • In addition to the hereditary forms of the disorder, there are
opsonization. C4b, C3b, iC3b, and C3dg, which accumulate on cell acquired forms that result from either consumption of C1-INH or
membranes as complement activation proceeds, bind to specific from autoantibodies blocking the function of C1-INH.
receptors on erythrocytes, neutrophils, monocytes, and • Hemolytic uremic syndrome (HUS) is the most common cause of
macrophages renal failure in children and is characterized by hemolytic anemia,
low platelet count, and acute renal failure.
Complement and Disease States • The atypical form of HUS (aHUS) occurs because of complement
Although complement acts as a powerful weapon to combat infection by dysregulation caused by genetic polymorphisms.
amplifying phagocytosis, in some cases it can actually contribute to tissue • Complement has also been implicated in C3 glomerulopathies
damage or death. Complement can be harmful if (C3G), which are diseases involving the glomeruli of the kidneys.
• It is activated systemically on a large scale as in gram negative • Analysis of these patients has shown that 71% to 100% of these
septicemia patients have mutation in a complement protein, specifically C3,
• It is activated by tissue necrosis such as myocardial infarction Factor B, Factor H, or Factor I.
• Lysis of red blood cells occurs • Other patients have an acquired autoantibody. These
autoantibodies are known as C3 nephritic factors (C3NeF).
• A C3NeF is an antibody that binds the C3-convertase from the
alternative pathway, C3bBb, holding it together and making it
Complement Deficiencies impervious to the normal control mechanisms.
 • C3G caused by C3NeF is clinically indistinguishable from the • Decreased levels of complement components or activity may be
hereditary form of the disorder. caused by decreased production, consumption, or in vitro
• An investigation of a possible complement deficiency can be consumption.
complicated by depletion of complement components because of • Specimen handling is extremely important. Blood should be
consumption through activation collected in a clot tube with no serum separator. The tube should
be spun down and the serum should be frozen or placed on dry
Laboratory Detection of Complement Abnormalities ice if it is not tested within 1 to 2 hours.
• If a complement deficiency is suspected, it is possible to narrow
Immunologic Assays of Individual Components down the possible candidate components with a CH50 and an
• The methods most frequently used to measure individual AH50 assay.
components include radial immunodiffusion (RID) and • Once a CH50 and AH50 hemolytic assay have been performed, it
nephelometry. is appropriate to move to testing the levels or function of the
• Nephelometry measures concentration according to the amount components as directed by the relative results of the CH50 and
of light scattered by a solution containing a reagent antibody and AH50.
a measured patient sample. • For C8, it is necessary to measure function because it is a
• Components for which there are standardized reagents include threesubunit protein. The loss of one of the three subunits would
C1q, C4, C3, C5, Factor B, Factor H, and Factor I. RID uses agarose not put the level out of normal range, but it would render the
gel into which specific antibody is incorporated. remaining two subunits nonfunctional.
• None of the assays for individual components are able to • C2 type II deficiency results from a genetic mutation in C2 that
distinguish whether the molecules are functionally active. Thus, renders the protein nonfunctional but does not decrease the level
although the preceding techniques give quantitative results and of expression, which is also true for C1-INH in type II HAE.
are relatively easy to perform, test results must be interpreted • A typical screening test for complement abnormalities usually
carefully includes determination of the following: C3 and C4, as well as
hemolytic content.
Assays for the Classical Pathway • Testing for products of complement activation such as C3a, C4a,
• The hemolytic titration (CH50) assay is most commonly used for C5a, and Ba (as well as breakdown products including iC3b and
this purpose. This assay measures the amount of patient serum C4d) can also be performed as a means of monitoring
required to lyse 50% of a standardized concentration of antibody- inflammatory processes such as rheumatoid arthritis and SLE.
sensitized sheep erythrocytes.
• The titer is expressed in CH50 units, which is the reciprocal of the
dilution that is able to lyse 50% of the sensitized cells.
• An additional CH50 test has also been developed based on lysis of
liposomes that release an enzyme when lysed. This lysis can be
read on an analyzer and is more accurate than traditional CH50
testing.
• Lytic activity can also be measured by radial hemolysis in agarose
plates. Rabbit RBCs that have been sensitized with antibody are
implanted in agarose and patient serum is added to wells
punched in the gel.
• Solid-phase IgM attached to the walls of microtiter plates is used
to initiate complement activation. Anti-human antibody to C9
conjugated to alkaline phosphatase is the indicator of
complement activation.
• The same method can detect split products that result from
complement activation. These products include C4a, C4d, C3a,
iC3b, C5a, and the soluble form of the MAC sC5b-9, all of which
are generated only if complement activation has occurred.
• An AH50 can be performed in the same manner as the CH50,
except magnesium chloride and ethylene glycol tetraacetic acid
(EGTA) are added to the buffer and calcium is left out.
• Rabbit RBCs are used as the indicator because they provide an
ideal surface for alternative pathway activation.
• An additional means of testing for alternative pathway function is
via ELISA. One such test can detect C3bBbP or C3bP complexes in
very small quantities.
• One test system has been developed that can determine the
activity of all three pathways. Strips used for the classical pathway
are coated with IgM, strips for the alternative pathway are coated
with lipopolysaccharide, and strips for the MBL pathway are
coated with mannose.

Interpretation of Laboratory Finding