Complement System

Ashley Frazer-Able, PhD,

7
and Christine Dorresteyn Stevens, EdD, MT(ASCP)

LEARNING OUTCOMES CHAPTER OUTLINE

After finishing this chapter, you should be able to: PATHWAYS OF THE COMPLEMENT
SYSTEM
1. Describe the roles of the complement system.
The Classical Pathway
2. Differentiate between the classical, alternative, and lectin pathways
and indicate proteins and activators involved in each. The Lectin Pathway
3. Discuss the formation of the three principal units of the classical The Alternative Pathway
pathway: recognition, activation, and membrane attack units. SYSTEM CONTROLS
4. Describe how initiation of the lectin pathway occurs. Regulation of the Classical and Lectin
5. Explain how C3 plays a key role in all pathways. Pathways
6. Describe regulators of the complement system and their role in the Regulation of the Alternative Pathway
complement system. Regulation of Terminal Components
7. Discuss the complement-related kidney disorders and applicable COMPLEMENT RECEPTORS AND THEIR
complement testing. BIOLOGICAL ROLES
8. Relate biological manifestations of complement activation to BIOLOGICAL MANIFESTATIONS
generation of specific complement products. OF COMPLEMENT ACTIVATION
9. Describe the deficiencies of complement components and the diseases COMPLEMENT AND DISEASE STATES
they cause. COMPLEMENT DEFICIENCIES
10. Differentiate tests for functional activity of complement from Major Pathway Components
measurement of individual complement components.
Regulatory Factor Components
11. Analyze laboratory findings and indicate disease implications in
LABORATORY DETECTION
relation to complement abnormalities.
OF COMPLEMENT ABNORMALITIES
Immunologic Assays of Individual
Components
Assays for the Classical Pathway
Alternative Pathway Assays
Interpretation of Laboratory Findings
SUMMARY
CASE STUDIES
REVIEW QUESTIONS

You can go to DavisPlus at davisplus.fadavis.com keyword Stevens for the

laboratory exercises that accompany this text.
91
92 SECTION 1 Nature of the Immune System

KEY TERMS
Activation unit Classical pathway Hemolytic uremic syndrome Membrane cofactor protein
Alternative pathway Complement receptor type (HUS) (MCP)
Anaphylatoxin 1 (CR1) Hereditary angioedema (HAE) Paroxysmal nocturnal
Decay-accelerating factor Immune adherence hemoglobinuria (PNH)
Bystander lysis
(DAF) Lectin pathway Properdin
C1 inhibitor (C1-INH)
Factor H Mannose-binding lectin Recognition unit
C3 glomerulopathies
(C3G) Factor I (MBL) S protein
C4-binding protein Hemolytic titration (CH50) Membrane attack complex
(C4BP) assay (MAC)

As described in Chapter 3, complement is a complex series of cell walls or outer coating of bacteria, viruses, yeast, and protozoa.
more than 30 proteins that play a major part in amplifying the Although each of these pathways will be considered separately,
inflammatory response to destroy and clear foreign antigens. activation seldom involves only one pathway.
These soluble and cell-bound proteins interact in a very spe- Most plasma complement proteins are synthesized in the
cific way and have powerful abilities. They can lyse foreign liver with the exception of C1 components; these are mainly
cells, opsonize and tag the invaders for clearance, and direct produced by intestinal epithelial cells and Factor D, which is
the adaptive immune system to the site of infection.1 Comple- made in adipose tissue.1,6 Other cells, such as monocytes and
ment activation is also proinflammatory in its ability to increase macrophages, are additional sources of early complement com-
vascular permeability, recruit monocytes and neutrophils to the ponents, including C1, C2, C3, and C4.6,7 Most of these pro-
area of antigen concentration, and trigger secretion of im- teins are inactive precursors, or zymogens, which are converted
munoregulatory molecules that amplify the immune response.2 to active enzymes in a very precise order. Table 7–1 lists the
In their proinflammatory role, complement proteins serve as characteristics of the main complement proteins.
an important link between innate and adaptive immunity.
Complement has important “housekeeping” roles as well. The Classical Pathway
Complement recognizes cellular debris such as apoptotic cells
and immune complexes, tagging them for removal by innate The classical pathway, the first activation cascade described, is
immune cells.3 Because of its potential for far reaching effects, the main antibody-directed mechanism for triggering comple-
complement activation needs to be carefully regulated. Chronic ment activation. However, not all immunoglobulins are able
activation can lead to inflammation and tissue damage to the to activate this pathway. The immunoglobulin classes that can
host. Any deficiencies to the complement system can result in activate the classical pathway include IgM, IgG1, IgG2, and
an increased susceptibility to infection or the accumulation of IgG3, but not IgG4, IgA, or IgE. IgM is the most efficient of
immune complexes resulting in possible autoimmune disor- the activating immunoglobulins because it has multiple bind-
ders.4 However, numerous proteins act as controls or regulators ing sites; thus, it takes only one molecule attached to two
of the system. These controls, as well as the major proteins adjacent antigenic determinants to initiate the cascade. Two
involved in activation, will be discussed in detail. IgG molecules must attach to antigen within 30 to 40 nm of
each other before complement can bind; it may take at least
1,000 IgG molecules to ensure that there are two close enough
Pathways of the Complement System to initiate such binding.1,8 Some epitopes, notably the Rh group,

The complement system can be activated in three different ways.

The first pathway described, the classical pathway, involves Connections
nine proteins that are triggered primarily by antigen–antibody
combination. Pillemer and colleagues discovered an antibody- Jules Bordet
independent pathway in the 1950s that plays a major role as a Jules Bordet was awarded the Nobel Prize in 1919 for his role in
natural defense system.5 This second pathway, the alternative elucidating the nature of complement. Complement was origi-
pathway, was originally called the properdin system because the nally recognized in the 1890s; Paul Ehrlich coined the term com-
protein properdin was thought to be the main initiator of this plement because the substance complements the action of
antibody in destroying microorganisms.1 Present as a substance
pathway. Now it is known that properdin’s major function is to
in normal nonimmune serum, complement is part of the innate
stabilize a key enzyme complex formed along the pathway and immune system. Complement is considered an acute phase
that the other forms of activation are more prominent. The third reactant because levels rise during an infection. It also has a
pathway, likely the most ancient of the three, is the lectin path- unique property of being easily inactivated (i.e., heat-labile) by
way, another antibody-independent means of activating comple- heating serum to 56°C for 30 minutes. Refer to Chapter 3 for a
ment proteins. Its prototypic constituent, mannose- (or mannan-) further description of the innate immune system.
binding lectin (MBL), adheres to mannose found mainly in the
Chapter 7 Complement System 93

Table 7–1 Proteins of the Complement System

SERUM PROTEIN MOLECULAR WEIGHT (KD) CONCENTRATION (µG/ML) FUNCTION
Classical Pathway
C1q 410 150 Binds to Fc region of IgM and IgG
C1r 85 50 Activates C1s
C1s 85 50 Cleaves C4 and C2
C4 205 300–600 Part of C3 convertase (C4b)
C2 102 25 Binds to C4b—forms C3 convertase
C3 190 1,200 Key intermediate in all pathways
C5 190 80 Initiates membrane attack complex
C6 110 45 Binds to C5b in MAC
C7 100 90 Binds to C5bC6 in MAC
C8 150 55 Starts pore formation on membrane
C9 70 60 Polymerizes to cause cell lysis
Alternative Pathway
Factor B 93 200 Binds to C3b to form C3 convertase
Factor D 24 2 Cleaves Factor B
Properdin 55 15–25 Stabilizes C3bBb–C3 convertase
MBL Pathway
MBL 200–600 0.0002–10 Binds to mannose
MASP-1 93 1.5–12 Unknown
MASP-2 76 Unknown Cleaves C4 and C2
Fc = Fragment crystallizable; Ig = immunoglobulin; MAC = membrane attack complex; MBL = mannose-binding lectin; MASP = MBL-associated serine protease.

are too far apart on the cell for this to occur; therefore, they three subunits—C1q, C1r, and C1s—which require the pres-
are unable to fix complement. Within the IgG group, IgG3 is ence of calcium to maintain structure.1,8 The complex is made
the most effective, followed by IgG1 and then IgG2.8 up of one C1q subunit and two each of the C1r and C1s sub-
In addition to antibodies, a few substances can bind com- units (Fig. 7–2). Although the C1q unit is the part that binds
plement directly to initiate the classical cascade. These include to antibody molecules, the C1r and C1s subunits generate
C-reactive protein (CRP), several viruses, mycoplasmas, some enzyme activity to begin the cascade.
protozoa, and certain gram-negative bacteria such as Escherichia C1q has a molecular weight of 410,000 and is composed
coli.8 However, most infectious agents can directly activate only of six strands that form six globular heads with a collagen-like
the alternative or lectin pathways. tail portion. This structure has been likened to a bouquet of
Complement activation can be divided into three main tulips with six blossoms extending outward (see Fig. 7–2). As
stages, each of which is dependent on the grouping of certain long as calcium is present in the serum, C1r and C1s remain
reactants as a unit. The first stage involves the recognition associated with C1q.
unit, which in the case of the classical pathway is C1. Once C1q “recognizes” the fragment crystallizable (Fc) region of
C1 is fixed, the next components activated are C4, C2, and C3, two adjacent antibody molecules, but at least two of the glob-
known collectively as the activation unit of the classical path- ular heads of C1q must be bound to initiate the classical path-
way (and the lectin pathway). C5 through C9 comprise the way. C1r and C1s are serine protease proenzymes, also called
membrane attack complex (MAC); this last unit completes zymogens. As binding of C1q occurs, both are converted into
the lysis of foreign particles. Each of these is discussed in detail active enzymes. Autoactivation of C1r results from a confor-
in the following sections. Figure 7–1 depicts a simplified mational change that takes place as C1q is bound. Once acti-
scheme of the entire pathway. vated, C1r cleaves a thioester bond on C1s which, in turn,
activates it. Activated C1r is extremely specific because its only
The Recognition Unit known substrate is C1s. Likewise, C1s has a limited specificity,
The first complement component of the classical pathway to with its only substrates being C4 and C2. Once C1s is activated
bind is C1, a molecular complex of 740,000 d. It consists of the recognition stage ends.
94 SECTION 1 Nature of the Immune System

Recognition
C1qrs
C4
C2
C4a Inactive C1qrs
complex
C2b

C4b2a
Activation
C3

C3a

C4b2a3b
Activated C1s
C5 C1q binds Fc

C5a

C5b C1r
Membrane
attack complex C6
C7
C8
C9

C5b6789 FIGURE 7–2 Structure of C1qrs. When two or more globular heads
of C1q attach to bound immunoglobulin molecules, the collagen-
FIGURE 7–1 The classical complement cascade. C1qrs is the recog- like stalks change their configuration. The resulting shape change
nition unit that binds to the FC portion of two antibody molecules. causes C1r to become a serine protease, which cleaves a small frag-
C1s is activated and cleaves C4 and C2 to form C4b2a, also known as ment of C1s, uncovering the C1s protease, whose only targets are
C3 convertase. C3 convertase cleaves C3 to form C4b2a3b, known as C4 and C2.
C5 convertase. The combination of C4b2a3b is the activation unit.
C5 convertase cleaves C5. C5b attracts C6, C7, C8, and C9, which
bind together, forming the membrane attack complex (MAC).
histocompatibility complex (MHC). Each serves a similar
C9 polymerizes to cause lysis of the target cell.
purpose in its particular pathway.8
When combined with C4b in the presence of magnesium
ions, C2 is cleaved by C1s to form C2a (which has a molecular
The Activation Unit weight of 70,000) and C2b (which has a molecular weight of
Phase two, the formation of the activation unit, begins when 34,000) (see Fig. 7–3A). This is the only case for the designa-
C1s cleaves C4 and ends with the production of the enzyme tion “a” to be given to the cleavage piece with enzyme activity,
C5 convertase (Fig. 7–3). C4 is the second most abundant though there are discussions to make the nomenclature of C2
complement protein, with a serum concentration of approx- match that of the other components of complement with the
imately 600 µg/mL.8 C1s cleaves C4 to split off a 77-amino “a” fragment being the smaller fragment. The short life of these
acid fragment called C4a. In the process, it opens a thioester- reactive species serves as a mechanism of control, keeping the
containing active site on the remaining part, C4b. C4b must reaction localized.
bind to protein or carbohydrate within a few seconds or it The combination of C4b and C2a is known as C3 conver-
will react with water molecules to form iC4b, which is rap- tase (see Fig. 7–3B). This is written as C4b2a to indicate that
idly degraded. Thus, C4b binds mainly to antigen in clusters the complex is an active enzyme. This complex is not very
that are within a 40-nm radius of C1. This represents the stable. The half-life is estimated to be between 15 seconds and
first amplification step in the cascade because for every C1 3 minutes, so C3 must be bound quickly. If binding does occur,
attached approximately 30 molecules of C4 are split and C3 is cleaved into two parts, C3a and C3b.
attached.1 C3, the major and central constituent of the complement
C2 is the next component to be activated. Complement pro- system, is present in the plasma at a concentration of 1 mg/mL
teins were named as they were isolated before the sequence of to 1.5 mg/mL.8 It serves as the pivotal point for all three path-
activation was known—hence the irregularity in the number- ways. The cleavage of C3 to C3b represents the most signifi-
ing system. The C2 gene is closely associated with the gene for cant step in the entire process of complement activation.9 The
Factor B (alternative pathway) on chromosome 6 in the major molecule has a molecular weight of 190,000 and consists of
Chapter 7 Complement System 95

C4b
two polypeptide chains, alpha (α) and beta (β). The α chain
contains a highly reactive thioester group. When C3a is
C2 removed by cleavage of a single bond in the α chain, the
C4a thioester is exposed; the remaining piece, C3b, is then capable
of binding to hydroxyl groups on carbohydrates and proteins
in the immediate vicinity.2,5,9 C3b is estimated to have a half-
life of 60 microseconds if not bound to antigen. Therefore,
C2b only a small percentage of cleaved C3 molecules bind to anti-
C4
gen; most are hydrolyzed by water molecules and decay in the
fluid phase.8,10
The cleavage of C3 represents a second and major amplifi-
cation process because about 200 molecules are split for every
molecule of C4b2a.10 In addition to being required for the for-
mation of the MAC, C3b also serves as a powerful opsonin.
Macrophages have specific receptors for it (discussed later in
C4b2a the chapter) and make a major contribution to the process of
(C3 convertase) phagocytosis. A large number of molecules are needed for this
A
to occur; hence, the need for amplification.
If C3b is bound within 40 nm of the C4b2a, this creates a
new enzyme known as C5 convertase. Figure 7–3C depicts
C3a this last step in the formation of the activation unit. The cleav-
C3
ing of C5 with deposition of C5b at another site on the cell
membrane constitutes the beginning of the MAC.

~200x
The Membrane Attack Complex (MAC)
C3b C5 consists of two polypeptide chains, α and β, which are linked
by disulfide bonds to form a molecule with a molecular weight
of about 190,000. C5 convertase, consisting of C4b2b3b, splits
off a 74-amino acid piece known as C5a that is released into cir-
C3 Convertase culation, whereas C5b attaches to the cell membrane, forming
the beginning of the MAC. The splitting of C5 and the cleavage
B of C3 represent the most significant biological consequences of
the complement system as explained in the section on biological
C5a
C5 manifestations of complement activation. However, C5b is
extremely labile and rapidly inactivated unless binding to C6
occurs.1
Once C6 is bound to C5b, subsequent binding involves
C7, C8, and C9. None of these proteins has enzymatic activ-
C5b ity; they are all present in much smaller amounts in serum
than the preceding components. C6 and C7 each have mo-
lecular weights of approximately 110,000 and have similar
physical and chemical properties. C8 is made up of three dis-
C4b2a3b
(C5 convertase)
similar chains joined by disulfide bonds and has a total mo-
lecular weight of about 150,000.6 C9 is a single polypeptide
C chain with a molecular weight of 70,000. The carboxy-
FIGURE 7–3 Formation of the activation unit. (A) Activated C1qrs terminal end is hydrophobic, whereas the amino-terminal
cleaves C4 and C2, with the larger pieces, C4b and C2a, binding to end is hydrophilic. The hydrophobic part serves to anchor
the target cell surface and forming the enzyme C3 convertase. the MAC within the target membrane. Formation of the mem-
(B) Each C3 convertase cleaves ~200 C3 molecules into C3a and C3b. brane attack unit is pictured in Figure 7–4. The complex
C3b is a powerful opsonin that binds to the target in many places. of C5b-C6-C7-C8 and C9 is known as C5b-9 or MAC. If the
(C) Some C3b associates with C4b2a, forming C4b2a3b, also complex is soluble in circulation, it is known as sC5b-9.
known as the C5 convertase. This convertase cleaves C5 into Measurement of the level of sC5b-9 is an indicator of the
the anaphylatoxin C5a and C5b, which binds to the target cell.
amount of terminal pathway activation that is occurring.
When formed, the MAC presents a pore of 70 to 100Å that
allows ions to pass in and out of the membrane.1,10 Destruc-
tion of target cells actually occurs through an influx of water
and a corresponding loss of electrolytes. The presence of C9
96 SECTION 1 Nature of the Immune System

acute phase protein because it is produced in the liver and is

normally present in the serum but increases during an initial
C8 C9 inflammatory response.13 The enzymatic role played by C1r
and C1s in the classical pathway is played in the lectin pathway
by serine proteases called MBL-associated serine proteases
(MASPs). There are currently three MASPs identified, labeled
C7 MASP-1, MASP-2, and MASP-3. The lectin pathway plays an
important role as a defense mechanism in infancy, during the
interval between the loss of maternal antibody and the acqui-
C6 sition of a full-fledged antibody response to pathogens.15

MAC
C5b The Alternative Pathway
First described by Pillemer and his associates in the early 1950s,
the alternative pathway was originally named for the protein
properdin, a constituent of normal serum with a concentration
of approximately 5 to 15 µg/mL.5 Although the alternative path-
way can be activated on its own, it appears that it functions
mainly as an amplification loop for activation started from the
FIGURE 7–4 Formation of the membrane attack unit. C5b binds classical or lectin pathways. Although properdin has been con-
to the target cell, whereas C6 and C7 attach to it. C8 binds to these firmed to bind and initiate activation, the primary function of
associated molecules and begins (along with C7) to penetrate the properdin is to stabilize the C3 convertase formed from activation
cell membrane. Multiple C9 molecules bind to C5b678 and polymer- of other factors. In addition to properdin, the serum proteins Fac-
ize to form a transmembrane channel, the membrane attack tor B and Factor D are unique to this pathway. C3 is a key com-
complex, which causes lysis of the cell. ponent of this pathway as well as the two other pathways. The
alternative pathway is summarized in Figure 7–5.
Triggering substances for the alternative pathway include
greatly speeds this lysis. However, sufficient perturbation of bacterial cell walls, especially those containing lipopolysaccha-
the membrane can occur in the absence of C9 so that defi- ride, fungal cell walls, yeast, viruses, virally infected cells,
ciencies in C9 appear largely benign. tumor cell lines, and some parasites, especially trypanosomes.1
All of these can serve as sites for binding the complex C3bBb,
one of the end products of this pathway. The conversion of C3
The Lectin Pathway is the first step in this pathway.
The lectin pathway represents another means of activating Native C3 is not stable in plasma. Water is able to hydrolyze
complement. Instead of activation through antibody binding, a thioester bond, thus spontaneously activating a small number
the lectin pathway is activated by recognition of surface moi- of these molecules.16 C3b, sometimes called iC3, is formed by
eties that are found on pathogens.11 This pathway provides an this spontaneous hydrolysis, from activation, or from the clas-
additional link between the innate and acquired immune re- sical or lectin pathways. It acts as the seed of activation of the
sponse because it involves nonspecific recognition of carbohy- alternative pathway. The C3b binds to Factor B, which has a
drates that are common constituents of microbial cell walls and molecular weight of 93,000 and is fairly abundant in the serum,
that are distinct from those found on human cell surfaces.12,13 at a level of 200 µg/ mL.8,17 Once bound to C3b, Factor B
Although this pathway is the most recently described of the can be cleaved by Factor D. The role of Factor B is thus anal-
three activation pathways of complement, it is probably the ogous to that of C2 in the classical pathway because it forms
most ancient. The lectin pathway molecules are structurally an integral part of a C3 convertase.
similar to those of the classical; the classical and lectin path- Factor D is a plasma protein that goes through a conforma-
ways even share the components C4 and C2. Once C4 and C2 tional change when it binds to Factor B.17,18 It is a serine pro-
are cleaved, the rest of the pathway is identical to the classical tease with a molecular weight of 24,000; its only substrate is
pathway. The role C1q serves in the classical pathway is filled bound Factor B. The concentration of Factor D in the plasma
by three classes of recognition molecules in the lectin pathway: is the lowest of all the complement proteins, approximately
lectins, ficolins, and CL-K1.11 The structure of all three classes 2 µg/mL.10 It cleaves Factor B into two pieces: Ba (with a molec-
of recognition molecules is similar to that of C1q because they ular weight of 33,000) and Bb (with a molecular weight of ap-
are all classed as collectins. One key lectin, called mannose- proximately 60,000). Bb remains attached to C3b, forming the
binding, or mannan-binding, lectin (MBL), binds to mannose initial C3 convertase of the alternative pathway. Bb is rapidly
or related sugars in a calcium-dependent manner to initiate inactivated unless it becomes bound to a site on one of the trig-
this pathway.14 These sugars are found in glycoproteins or car- gering cellular antigens.
bohydrates of a wide variety of microorganisms such as bacte- As the alternative pathway convertase, C3bBb is then capable
ria, yeasts, viruses, and some parasites. MBL is considered an of cleaving additional C3 into C3a and C3b. Some C3b attaches
Chapter 7 Complement System 97

C3a
1 H2O 1. C3 is hydrolyzed by water to produce C3b, which binds
Factor B and together they attach to target cell surface.
C3b B
C3 C3b B 2. B is cleaved by Factor D into the fragments Ba and Bb.
Bb combines with C3b to form C3bBb, an enzyme with
C3 convertase activity.

3. More C3 is cleaved, forming more C3bBb. This enzyme

is stabilized by properdin, and it continues to cleave
additional C3.
2 D Ba 4. If a molecule of C3 remains attached to the C3bBbP
enzyme, the convertase now has the capability to
C3b cleave C5. The C5 convertase thus consists of
C3bBbP3b. After C5 is cleaved, the pathway is identical
Bb to the classical pathway.

C3 C3a
3

C3b Bb P C3b

C5 C5a
4 C5b

C3b Bb P C3b

FIGURE 7–5 The alternative pathway.

to cellular surfaces and acts as a binding site for more Factor B, self-antigens are destroyed and that the reaction remains
resulting in an amplification loop; activation initiated by the clas- localized, several plasma proteins act as system regulators.
sical or lectin pathways is amplified to levels of biological con- In addition, there are specific receptors on certain cells that
sequence. All C3 present in plasma would be rapidly converted also exert a controlling influence on the activation process.
by this method were it not for the fact that the enzyme C3bBb In fact, approximately one-half of the complement compo-
is extremely unstable unless properdin binds to the complex. nents serve as controls for critical steps in the activation
Binding of properdin increases the half-life of C3bBb from process. Because activation of C3 is the pivotal step in all
90 seconds to several minutes.8,17 In this manner, optimal rates pathways, the majority of the control proteins are aimed at
of alternative pathway activation are achieved.14 halting accumulation of C3b. However, there are controls at
C3bBb can also cleave C5, but it is much more efficient at all crucial steps along the way. Regulators will be discussed
cleaving C3.19 If, however, some of the C3b produced remains according to their order of appearance in each of the three
bound to the C3 convertase, the enzyme is altered to form pathways. A brief summary of these is found in Table 7–2.
C3bBb3bP, which has a high affinity for C5 and exhibits C5
convertase activity.16,19 C5 is cleaved to produce C5b, the first
part of the membrane attack unit. From this point on the alter- Regulation of the Classical
native, lectin, and classical pathways are identical. Figure 7–6 and Lectin Pathways
shows the convergence of all three pathways. C1 inhibitor (C1-INH) inhibits activation at the first stages of
both the classical and lectin pathways. Its main role is to inacti-
System Controls vate C1 by binding to the active sites of C1r and C1s. Clr and
Cls become instantly and irreversibly dissociated from C1q.6,10
Activation of complement could cause tissue damage and C1q remains bound to antibody, but all enzymatic activity ceases.
have devastating systemic effects if it were allowed to pro- C1-INH10 also inactivates MASP-2 binding to the MBL-MASP
ceed uncontrolled. To ensure that infectious agents and not complex, thus halting the lectin pathway.12,18 C1-INH is a
98 SECTION 1 Nature of the Immune System

Classical Pathway Lectin Pathway Alternative Pathway

C1qrs
Teichoic acid
MBL
Zymosan
MASP-1 LPS

MASP-2
MASP-3

C3b ! B

Mannose
Factor D

C4

C2 C3

C4b C2a C3b Bb

C3 convertase
C3 convertase

Properdin stabilizes

C2a

C4b C3b C5 C3b Bb P C3b

C5 convertase
C5 convertase

C5a

C7

C5b
C6 C8

Membrane attack complex

FIGURE 7–6 Convergence of the classical, alternative, and lectin pathways. The binding of C1qrs to two antibody molecules activates
the classical pathway, whereas the alternative pathway is started by hydrolysis of C3. The lectin pathway is triggered by binding of MBP to
mannose on bacterial cell walls. MASP-1, MASP-2, and MASP-3 bind to form an activated C1-like complex. MASP-2 cleaves C2 and C4 and
proceeds like the classical pathway. Factor B and Factor D operate in the alternative pathway. Although C3 convertase is formed differently
in each pathway, C3 is a key component in each one. The C5 convertase in the alternative pathway consists of C3bBb3bP. In the classical
and lectin pathways, C5 convertase is made up of C4b2a3b. After C5 is cleaved, the pathway is common to all.
Chapter 7 Complement System 99

Table 7–2 Plasma Complement Regulators

SERUM PROTEIN MOLECULAR WEIGHT (KD) CONCENTRATION (MG/ML) FUNCTION
C1 inhibitor 105 240 Dissociates C1r and C1s from C1q
(C1-INH)
Factor I 88 35 Cleaves C3b and C4b
Factor H 150 300–450 Cofactor with I to inactivate C3b;
prevents binding of B to C3b
C4-binding protein 520 250 Acts as a cofactor with I to
(C4BP) inactivate C4b
S protein 84 500 Prevents attachment of the C5b67
(vitronectin) complex to cell membranes

glycoprotein with a molecular weight of 105,000. Like most fibroblasts, and on numerous types of epithelial cells.6–8 DAF
of the other complement proteins, it is mainly synthesized in is capable of dissociating both classical and alternative path-
the liver; however, monocytes also may be involved to some way C3 convertases. It can bind to both C3b and C4b in a
extent in its manufacture. manner similar to CR1.4 It does not prevent initial binding
Further formation of C3 convertase in the classical and of either C2 or Factor B to the cell but can rapidly dissociate
lectin pathways is inhibited by four main regulators: soluble both from their binding sites, thus preventing the assembly
C4-binding protein (C4BP) and three cell-bound receptors, of an active C3 convertase.
complement receptor type 1 (CR1), membrane cofactor The carboxy-terminal portion of DAF is covalently attached
protein (MCP), and decay-accelerating factor (DAF).1,18 All to a glycophospholipid anchor that is inserted into the outer
of these act in concert with Factor I, a serine protease that in- layer of the membrane lipid bilayer. This arrangement allows
activates C3b and C4b when bound to one of these regulators. DAF mobility within the membrane so it can reach C3 conver-
C4BP is abundant in the plasma and has a molecular weight tase sites that are not immediately adjacent to it (Fig. 7–7).2
of about 520,000. It is capable of combining with either fluid- The presence of DAF on host cells protects them from by-
phase or bound C4b; therefore, C4b cannot bind to C2 and is stander lysis. It is one of the main mechanisms used in dis-
made available for degradation by Factor I. If C4BP attaches to crimination of self from nonself because foreign cells do not
cell-bound C4b, it can dissociate it from C4b2a complexes, possess this substance. However, it does not permanently mod-
causing the cessation of the classical pathway. ify C3b or C4b; they are capable of re-forming elsewhere as
CR1, also known as CD35, is a large polymorphic glycopro- active convertases.
tein with a molecular weight between 165,000 and 280,000.7
It is found mainly on peripheral blood cells, including neu-
trophils, monocytes, macrophages, erythrocytes, eosinophils,
Regulation of the Alternative Pathway
B lymphocytes, some T lymphocytes, and follicular dendritic The principal soluble regulator of the alternative pathway is
cells.3 It binds C3b and C4b but has the greatest affinity for Factor H, which has a molecular weight of 160,000.18 It acts
C3b.6,20 Once bound to CR1, both C4b and C3b can then be by binding to C3b, preventing the binding of Factor B. C3b in
degraded by Factor I. the fluid phase has a hundredfold greater affinity for Factor H
A main function of CR1 is as a receptor on platelets and red than for Factor B, but on cell surfaces C3b preferentially binds
blood cells (RBCs), which helps to mediate transport of C3b- to Factor B. Factor H also accelerates the dissociation of the
coated immune complexes to the liver and spleen.7,20 It is there C3bBb complex on cell surfaces. When Factor H binds to
that fixed tissue macrophages strip the immune complexes C3bBb, Bb becomes displaced. In this manner, C3 convertase
from the RBCs, process the complexes, and return the RBCs activity is curtailed in plasma and on cell surfaces.
intact to circulation. The ability of cells to bind complement- Additionally, Factor H acts as a cofactor that allows Factor I
coated particles is referred to as immune adherence. to break down C3b. It appears that only those molecules with
MCP, or CD46, has a molecular weight between 50,000 and tightly bound Factor H acquire high-affinity binding sites for
70,000 and is found on virtually all epithelial and endothelial Factor I.21 When Factor I binds, a conformational change takes
cells except erythrocytes.7 MCP is the most efficient cofactor place that allows it to cleave C3b.21 On cellular surfaces, C3b
for Factor I-mediated cleavage of C3b. It can serve as a cofactor is cleaved into C3f, which is released into the plasma, and iC3b,
for cleavage of C4b, but it is not as effective as C4BP. MCP also which remains attached but is no longer an active enzyme.
helps to control the alternative pathway because binding of iC3b is further broken down to C3c and C3dg by Factor I in con-
Factor B to C3b is inhibited. junction with another cofactor: the CR1 receptor (Fig. 7–8).10
DAF or CD55, a 70,000 d membrane glycoprotein, is the With this key role in complement regulation, it should not be
third main receptor and has a wide tissue distribution. It surprising that Factor H has recently been shown to play a role
is found on peripheral blood cells, on endothelial cells and in a variety of disorders (discussed in the text that follows).
100 SECTION 1 Nature of the Immune System

DAF DAF

C4b C2a C4b

Classical
pathway

A
DAF DAF

C3b C3b

Bb

Alternative
pathway

B
FIGURE 7–7 A. In the classical pathway, DAF dissociates C2a from C4b. B. Inhibitory effects of DAF. In the alternative pathway, when C3b binds
to cell surfaces that have DAF present, DAF helps dissociate Bb from binding to C3b.

Regulation of Terminal Components include degradation products of C3b, such as C3dg, C3d, and
iC3b. In addition, the Epstein-Barr virus gains entry to B cells
S protein is a soluble control protein that acts at a deeper level by binding to this receptor. CR2 is present only on mature
of complement activation. Also known as vitronectin, S protein B cells and is lost when conversion to plasma cells occurs. CR2
interacts with the C5b-7 complex as it forms in the fluid phase plays an important role as part of the B-cell co-receptor for
and prevents it from binding to cell membranes.8 Binding of antigen. Acting in concert with CD19, it binds complement-
C8 and C9 still proceeds, but polymerization of C9 does not coated antigen and cross-links it to membrane immunoglobu-
occur; therefore, the complex is unable to insert itself into the lin to activate B cells. In this manner, immune complexes are
cell membrane or to produce lysis.4 more effective at enhancing B-cell differentiation and produc-
A receptor, known by various terms, including membrane tion of memory cells than is antigen by itself.2,22
inhibitor of reactive lysis (MIRL) or CD59, also acts to block Another receptor, CR3 (CD11b/CD18), found on mono-
formation of the MAC. MIRL is widely distributed on the cell cytes, macrophages, neutrophils, and natural killer (NK)
membranes of all circulating blood cells, including RBCs, and cells, specifically binds particles opsonized with iC3b, a C3b
on endothelial, epithelial, and many other types of cells.8,10 degradation product.7 It does this in a calcium-dependent
Table 7–3 lists the receptors and indicates the types of cells manner. The CR3 receptor plays a key role in mediating
on which they are found. phagocytosis of particles coated with these complement frag-
ments (Fig. 7–9). These proteins trigger surface adhesion and
increased activity of phagocytic cells.2 Patients whose white
Complement Receptors blood cells (WBCs) lack these receptors fail to exhibit func-
and Their Biological Roles tions such as chemotaxis, surface adherence, and aggregation.
Deficiencies in phagocytosis are also noted. These individuals
Some complement receptors found on host cells amplify and have an impaired capacity to bind iC3b-coated particles and
enhance the immune response by augmenting phagocytosis are subject to recurrent infections.
and stimulating accessory cells rather than acting as regulators The CR4 (CD11c/CD18) receptor is very similar to CR3 in
(see Table 7–3). CR1 has been discussed in the previous sec- that it also binds iC3b fragments in a calcium-dependent fash-
tion. A second receptor, CR2 (or CD21), is found mainly on ion. CR4 proteins are found on neutrophils, monocytes, tissue
B lymphocytes and follicular dendritic cells.22 Ligands for CR2 macrophages, activated T cells, dendritic cells, NK cells, and
Chapter 7 Complement System 101

1 1. Factor H (FH) competes with factor B (B) for binding

C3 FH
Blocks to spontaneously (hydrolytically) activated C3b.

B 2. Factor H dissociates any C3bBb complexes that form

C3a C3b
on self-cell surfaces.

3. Factor H is a cofactor with factor I (FI), enabling

cleavage of C3b. The resuting C3bi loses enzymatic
activity, but is still an opsonin.
2
4. CR1 is a cofactor with FI, enabling cleavage of
FH
Displaces C3bi. The resulting C3dg is an opsonin and a
cofactor in B cell stimulation.
C3b Bb

Self-cell surface

Enables cleavage
3 FI
FH

C3bi C3f

4 Enables
cleavage
FI
CR1

C3dg C3c

FIGURE 7–8 Complement controls. CR1 receptor acts as a cofactor in the inactivation of C3b. Factor I cleaves C3b to form C3dg and C3c.
C3dg is not an effective opsonin and is not capable of further participation in the complement cascade.

Table 7–3 Receptors on Cell Membranes for Complement Components

RECEPTOR LIGAND CELL TYPE FUNCTION
CR1 (CD35) C3b, iC3b, C4b RBCs, neutrophils, monocytes, Cofactor for Factor I;
macrophages, eosinophils, B and T cells, mediates transport of
follicular dendritic cells immune complexes
CR2 (CD21) C3dg, C3d, iC3b B cells, follicular dendritic cells, epithelial B-cell co-receptor for
cells antigen with CD19
CR3 (CD11b/CD18) iC3b, C3d, C3b Monocytes, macrophages, neutrophils, NK Adhesion and increased
cells activity of phagocytic
cells
CR4 (CD11c/CD18) iC3b, C3b Monocytes, macrophages, neutrophils, NK Adhesion and increased
cells, activated T and B cells, dendritic activity of phagocytic
cells cells

Continued
102 SECTION 1 Nature of the Immune System

Table 7–3 Receptors on Cell Membranes for Complement Components—cont’d

RECEPTOR LIGAND CELL TYPE FUNCTION
DAF (CD55) C3b, C4b RBCs, neutrophils, platelets, monocytes, Dissociates C2b or Bb
endothelial cells, fibroblasts, T cells, from binding sites, thus
B cells, epithelial cells preventing formation of C3
convertase
MIRL (CD59) C8 RBCs, neutrophils, platelets, monocytes, Prevents insertion of C9
endothelial cells, epithelial cells into cell membrane
MCP (CD46) C3b, C4b Neutrophils, monocytes, macrophages, Cofactor for Factor I
platelets, T cells, B cells, endothelial cells cleavage of C3b and C4b

RBC = red blood cell; NK = natural killer.

MAC. Complement proteins also serve as a means of linking

innate and adaptive immunity. They act as opsonins to facil-
itate recognition and subsequent destruction by phagocytic
cells; in addition, they play a major role in the uptake and
Antigen
presentation of antigens so a specific immune response can
occur. They also facilitate B-cell activation. Recent work
demonstrates that complement is necessary for maintaining
immunologic memory.24 Effector molecules generated earlier
in the cascade play a major role in all these areas. Such mol-
FcR ecules can be classified into three main categories: anaphy-
CR3 CRP-R latoxins, chemotaxins, and opsonins.
An anaphylatoxin is a small peptide that causes increased
vascular permeability, contraction of smooth muscle, and re-
lease of histamine from basophils and mast cells. Proteins that
FIGURE 7–9 Role of C3b in opsonization. C3b, antibody, and play such a part are C3a and C5a. Both of these have molecular
C-reactive protein are all important opsonins that coat antigens and weights between 9000 and 11,000 and are formed as cleavage
accelerate phagocytosis. Receptors for C3b (CR3), antibody (FcR), products from larger complement components. Of these mol-
and C-reactive protein (CRP-R) on the phagocytic cell membrane ecules, C5a is the most potent; it is at least 200 times more
bind the opsonins and pull the membrane around to envelop the
powerful than C3a.25
antigen.
C3a and C5a attach to specific receptors on neutrophils, ba-
sophils, mast cells, eosinophils, smooth muscle cells, and vas-
activated B cells.2,7 Neutrophils and monocytes, however, pos- cular endothelium.24,25 C3a attaches to the C3a receptor (C3aR),
sess smaller amounts of CR4 than of CR3. Their function ap- whereas C5a attaches to the C5a receptor (C5aR). When binding
pears to be similar to that of CR3 and they may assist occurs on basophils and mast cells, histamine is released, in-
neutrophil adhesion to the endothelium during inflammation. creasing vascular permeability and causing contraction of
Receptors specific for C1q are found on neutrophils, mono- smooth muscles. C5a causes neutrophils to release hydrolytic
cytes, macrophages, B cells, platelets, and endothelial cells.7,23 enzymes, oxygen radicals, and prostaglandins, which aid in the
These receptors, known as collectin receptors, bind the collagen destruction of foreign antigens.25
portion of C1q and generally enhance the binding of C1q to FC C5a also serves as a chemotaxin for neutrophils, basophils,
receptors. Interacting only with bound C1q, the receptors appear eosinophils, mast cells, monocytes, and dendritic cells. In this
to increase phagocytic cells’ uptake of immune complexes op- manner, these cells are directed to the source of antigen concen-
sonized with C1q. On neutrophils, they may also act to enhance tration. Because of increased vascular permeability, neutrophils
the respiratory burst triggered by IgG binding to FC receptors. migrate from blood vessels to the tissues and tend to aggregate.
Binding of C5a to monocytes causes them to undergo
an oxidative burst that includes increased production of
Biological Manifestations hydrolytic enzymes, neutrophil chemotactic factor, platelet-
of Complement Activation activating factors, interleukin-1 (IL-1), and toxic oxygen
metabolites.13,19 IL-1 is a protein that enhances T-cell acti-
Activation of complement is a very effective means of ampli- vation. The activation may produce fever and lead to an in-
fying the inflammatory response to destroy and clear foreign crease in acute-phase reactants, both of which are characteristic
antigens. The cycle does not always have to proceed to lysis of an inflammatory response.
for this to be accomplished; hence, some of the initiating C3a and C5a are rapidly inactivated by an enzyme in the
proteins are much more plentiful than proteins that form the plasma called carboxypeptidase N to localize and control their
Chapter 7 Complement System 103

effects. C3a is cleaved in seconds, whereas conversion of C5a B locus is nearby, C2-deficient persons are often reported to have
occurs more slowly. decreases in Factor B also. Other types of complement deficien-
The last major effect of complement-derived peptides is cies are less common.
opsonization. C4b, C3b, iC3b, and C3dg, which accumulate A second deficiency that occurs with some frequency is that
on cell membranes as complement activation proceeds, bind of MBL. Deficiencies and polymorphisms in MBL occur in about
to specific receptors on erythrocytes, neutrophils, monocytes, 30% of the population. The health consequences of such varia-
and macrophages as previously discussed. This binding facili- tion in MBL levels remain unclear, however. Lack of MBL has
tates phagocytosis and clearance of foreign substances or been associated with pneumonia, sepsis, and meningococcal dis-
cellular debris, which is one of the key functions of the com- ease in infants.13,15,31,32 Low MBL has also been associated with
plement system. In addition, attachment of C3 products to an the risk of some cancers, infection during chemotherapy, and
antigen has been found to enhance the B-cell response. certain autoimmune disorders such as systemic lupus erythe-
matosus (SLE), but these connections are not yet well defined.33
The most serious deficiency is that of C3 because it is the
Complement and Disease States key mediator in all pathways. C3 deficiencies are, however, ex-
tremely rare.28 Individuals with a C3 deficiency are prone to
Although complement acts as a powerful weapon to combat
developing severe, recurrent life-threatening infections with en-
infection by amplifying phagocytosis, in some cases it can ac-
capsulated bacteria such as Streptococcus pneumoniae and may
tually contribute to tissue damage or death. Complement can
also be subject to immune complex diseases.16 Such complexes
be harmful if
can lodge in the kidney and result in glomerulonephritis.26,34
• It is activated systemically on a large scale as in gram- It appears that a deficiency of any of the terminal components
negative septicemia of the complement cascade (C5–C8) causes increased suscepti-
• It is activated by tissue necrosis such as myocardial bility to systemic Neisseria infections, including meningococcal
infarction meningitis and disseminated gonorrheal disease.10,28 Table 7–4
• Lysis of red blood cells occurs lists the complement components and the disease states associ-
In the case of septicemia caused by a gram-negative organism, ated with the absence of each individual factor.
large quantities of C3a and C5a are generated, leading to neu-
trophil aggregation and clotting. Damage to the tiny pulmonary
capillaries and interstitial pulmonary edema may result.2
Table 7–4 Deficiencies of Complement
Tissue injury following obstruction of the blood supply,
Components
such as occurs in a myocardial infarction or heart attack, can
cause complement activation and deposition of MACs on cell DEFICIENT
surfaces. Receptors for C3a and C5a have been found in coro- COMPONENT ASSOCIATED DISEASE
nary plaques, indicating that complement components may in- C1 (q, r, or s) Lupuslike syndrome; recurrent infections
crease the damage to heart tissue.25,26 C2 Lupuslike syndrome; recurrent
Lysis may be another end result of complement activation. infections; atherosclerosis
Hemolytic diseases such as cold autoimmune hemolytic anemia
are characterized by the presence of an autoantibody that binds C3 Severe recurrent infections;
at low temperatures. When these cells warm up, complement glomerulonephritis
fixation results in lysis. (See Chapter 15 for a more complete C4 Lupuslike syndrome
discussion of complement-mediated autoimmune diseases.)
C5–C8 Neisseria infections
C9 No known disease association
Complement Deficiencies Hereditary angioedema
C1-INH

Major Pathway Components DAF Paroxysmal nocturnal hemoglobinuria

Although excess activation of the complement system can result MIRL Paroxysmal nocturnal hemoglobinuria
in disease states, lack of individual components also has a dele- Factor H Recurrent pyogenic infections
terious effect. Hereditary deficiency of any complement protein, or Factor I
with the exception of C9, usually manifests itself in increased
MBL Pneumococcal diseases, sepsis,
susceptibility to infection and delayed clearance of immune Neisseria infections
complexes. Most of these conditions are inherited on an auto-
somal recessive gene and are quite rare, occurring in 0.03% of Properdin Neisseria infections
the general population.26 A lack of C2, the most common defi- MASP-2 Pneumococcal diseases
ciency, is found in 1 in 20,000 individuals.4,27–29 Recent evidence
indicates that atherosclerosis may be related to a C2 deficiency.28 C1-INH = C1 inhibitor; DAF = decay-accelerating factor; MASP-2 =
mannose-associated serine protease; MBL = mannose-binding lectin;
C2-deficient individuals may also be more prone to recurrent
MIRL = membrane inhibitor of reactive lysis.
streptococcal and staphylococcal infections.30 Because the Factor
104 SECTION 1 Nature of the Immune System

Regulatory Factor Components connected to deficiencies, polymorphisms, or autoantibodies

involving one or multiple complement components. Key
A prime example of a disease caused by a missing or defective among these is a set of rare kidney disorders associated with
regulatory component is paroxysmal nocturnal hemoglo- complement. Hemolytic uremic syndrome (HUS) is the
binuria (PNH). Individuals with this disease have RBCs that most common cause of renal failure in children and is char-
are deficient in DAF. Hence, the RBCs are subject to lysis by acterized by hemolytic anemia, low platelet count, and acute
means of the bystander effect once the complement system renal failure.36 The primary cause of HUS is a Shiga toxin re-
has been triggered. These individuals appear to have a defi- lated to infection that is associated with acute diarrhea. The
ciency in the glycophospholipid anchor of the DAF molecule atypical form of HUS (aHUS) occurs because of complement
that prevents its insertion into the cell membrane.10,22 When dysregulation caused by genetic polymorphisms. The atypical
C3b is deposited on erythrocytes through activation of either HUS has a less acute onset and may not be associated with di-
pathway, the result is complement-mediated intravascular arrhea; otherwise, the clinical presentation is similar. The ge-
and extravascular hemolysis, resulting in a chronic hemolytic netic mutations associated with aHUS include those of Factor
anemia. H, MCP, Factor I, Factor B, and thrombomodulin, as well as
Some studies indicate that a DAF deficiency is associated autoantibodies to Factor H and Factor I.36
with a lack of CD59 (MIRL) and both are implicated in PNH.32 Complement has also been implicated in C3 glomeru-
CD59 has the same glycophospholipid anchor found in DAF; lopathies (C3G), which are diseases involving the glomeruli
therefore, the gene deficiency affects both molecules. As men- of the kidneys. Recently several forms of rare glomerulonephri-
tioned previously, CD59 prevents insertion of C9 into the cell tis (GN) were reclassified based on immunofluorescence and
membrane by binding to the C5b-8 complex, inhibiting for- the presence or absence of C3 and immunoglobulins. For the
mation of transmembrane channels.25 Both DAF and CD59 are C3 glomerulopathies, it is only C3 that is found in the deposits
important in protecting RBCs against bystander lysis. on the kidney. Analysis of these patients has shown that 71%
Another complement deficiency disorder that has recently to 100% of these patients have mutation in a complement pro-
received considerable attention is hereditary angioedema tein, specifically C3, Factor B, Factor H, or Factor I. Other pa-
(HAE). HAE involves recurrent attacks of swelling that affect tients have an acquired autoantibody. These autoantibodies are
the extremities, the skin, the gastrointestinal tract, and other known as C3 nephritic factors (C3NeF).
mucosal surfaces. This disease is caused by a deficiency or lack A C3NeF is an antibody that binds the C3-convertase from
of C1-INH, which occurs with a population frequency of 2 in the alternative pathway, C3bBb, holding it together and making
10,000.32 Although C1-INH was named for its role in control- it impervious to the normal control mechanisms. In this way,
ling complement, it is the function of C1-INH in controlling a C3NeF leads to uncontrolled cleavage of C3 with concomi-
the contact pathway of the coagulation system that is critical tant uncontrolled deposition of C3 on the kidneys. C3G caused
in this disease. C1-INH is a serpin (serine protease inhibitor) by C3NeF is clinically indistinguishable from the hereditary
that controls many of the serine proteases on contact. The lack form of the disorder.37 It is only with laboratory measurement
of C1-INH results in localized swelling that can be either sub- of the presence or absence of a C3NeF that the nature of the
cutaneous or found within the bowel or upper-respiratory disorder can be differentiated. In addition, an investigation of
tract.35 Normally, this spontaneously subsides in 48 to 72 hours, a possible complement deficiency can be complicated by de-
but if the edema occurs in the area of the oropharynx, life- pletion of complement components because of consumption
threatening upper-airway obstruction may develop.26 These through activation. For both the autoantibodies and activation-
attacks can be quite debilitating even when they are not life related consumption, laboratory testing is the key way to tell
threatening, so there is a need for proper diagnosis for these the acquired forms from the hereditary forms of complement
patients. disorders.
HAE is separated into two types, type I and type II. Type I
is characterized by a decrease in the C1-INH protein; type II
has normal levels of C1-INH, but the function is decreased. Laboratory Detection
The genetic cause of either type is an autosomal dominant gene of Complement Abnormalities
that codes for either a dysfunctional or an inactive protein. In
addition to the hereditary forms of the disorder, there are ac- Determining the levels of complement components can be use-
quired forms that result from either consumption of C1-INH ful in diagnosing disease. Hereditary deficiencies can be iden-
or from autoantibodies blocking the function of C1-INH. To tified and much can be learned about inflammatory or
differentiate the acquired and hereditary forms, measurement autoimmune states by following the consumption of comple-
of C1q can be helpful; C1q will be low in the acquired forms, ment proteins. Techniques to determine complement abnor-
but not in the hereditary forms. Measurement of C4 can also malities generally fall into two categories: (1) measurement of
be a very helpful screen for HAE, particularly during an attack, components as antigens in serum and (2) measurement of
because patients who do not exhibit a drop in C4 at that time functional activity.8 Many assays that are unavailable in routine
are rare.4,35 clinical laboratories are available in specialized laboratories.
In addition to these well-described diseases of comple- Some of the more common assays will be discussed in the text
ment, there are a growing number of disorders that are being that follows.
Chapter 7 Complement System 105

Immunologic Assays of Individual Assays for the Classical Pathway

Components Assays that measure lysis, the end point of complement activa-
The methods most frequently used to measure individual com- tion, are functional tests that are frequently run in conjunction
ponents include radial immunodiffusion (RID) and nephelom- with testing of individual components. The hemolytic titration
etry.26,34 C3 and C4 levels are routinely measured in most (CH50) assay is most commonly used for this purpose.26,32 This
clinical laboratories by nephelometry or by turbidity measure- assay measures the amount of patient serum required to lyse
ments that are often automated. Both of these types of meas- 50% of a standardized concentration of antibody-sensitized
urements and the methods used for the other components rely sheep erythrocytes. Because all proteins from C1 to C9 are nec-
on the precipitation of antigen (the complement component essary for this to occur, absence of any one component will result
being measured) and antibody. Nephelometry measures con- in an abnormal CH50, essentially reducing this number to zero.
centration according to the amount of light scattered by a solu- The titer is expressed in CH50 units, which is the reciprocal
tion containing a reagent antibody and a measured patient of the dilution that is able to lyse 50% of the sensitized
sample (refer to Chapter 10 for more details on nephelometry). cells.32,38 The 50% point is used because this is when the
Generally, the more antigen–antibody complexes that are pres- change in lytic activity per unit change in complement is at
ent, the more a beam of light will scatter as it passes through maximum (Fig. 7–11). Most laboratories need to establish
the solution. Such systems have a high degree of accuracy; re- their own normal values.
sults are available quickly and processing is easy because of the An additional CH50 test has also been developed based on
use of automation. However, they involve expensive equipment. lysis of liposomes that release an enzyme when lysed. This lysis
Components for which there are standardized reagents in- can be read on an analyzer and is more accurate than tradi-
clude C1q, C4, C3, C5, Factor B, Factor H, and Factor I. RID tional CH50 testing.25 However, lytic assays in general are com-
uses agarose gel into which specific antibody is incorporated. plicated to perform and lack sensitivity. Individual laboratories
Serum serves as the antigen and is placed in wells that are cut in must establish their own normal values. When the results are
the gel. Diffusion of the antigen from the well occurs in a circular abnormal, the reasons cannot be determined. Such procedures
pattern (Fig. 7–10). The radius of the resulting circle can be re- are useful in establishing functional activity or lack thereof.
lated to antigen concentration (see Chapter 10 for further details Additional testing for individual components should be
on RID). This is a sensitive technique when performed correctly, performed to follow up on any abnormality.
but at least 24 hours are needed before test results are available. Lytic activity can also be measured by radial hemolysis in
None of the assays for individual components are able to agarose plates. Rabbit RBCs that have been sensitized with an-
distinguish whether the molecules are functionally active. tibody are implanted in agarose and patient serum is added to
Thus, although the preceding techniques give quantitative re- wells punched in the gel. Lysis appears as a clear zone around
sults and are relatively easy to perform, test results must be in- each well; if complement standards are run, the size of the zone
terpreted carefully. Nephelometry and RID are both sensitive can be related to complement concentration.26,32,39 Solid-phase
tests.32 Enzyme-linked immunosorbent assay (ELISA) methods IgM attached to the walls of microtiter plates is used to initiate
are available for the measurement of the inhibitor C1-INH. complement activation. Anti-human antibody to C9 conju-
gated to alkaline phosphatase is the indicator of complement
activation. When a substrate is added, if any C9 is present and
the antibody conjugate has attached, a color change will be ev-
Unloaded gel ident. (Refer to Chapter 11 for a complete discussion of the
Calculations principle of ELISA techniques.) This type of testing, which is
Measure blue
precipitation ring very sensitive, is probably the best screen for complement ab-
normalities.25 The same method can detect split products that
Internal result from complement activation. These products include
diameter C4a, C4d, C3a, iC3b, C5a, and the soluble form of the MAC
sC5b-9, all of which are generated only if complement activa-
tion has occurred.
Gel after precipitating and staining

Outer
Alternative Pathway Assays
diameter
Alternative pathway activation can be measured by several
different means. An AH50 can be performed in the same
manner as the CH50, except magnesium chloride and ethyl-
ene glycol tetraacetic acid (EGTA) are added to the buffer and
calcium is left out.32 This buffer chelates calcium, which
Patient with low level blocks classical pathway activation. Rabbit RBCs are used as
FIGURE 7–10 Radioimmunodiffusion for measurement of comple- the indicator because they provide an ideal surface for alter-
ment component C5. native pathway activation.
106 SECTION 1 Nature of the Immune System

pathway are coated with mannose. Such testing is easy to per-

form and is not dependent upon the use of animal erythro-
cytes, which may be hard to obtain. Deficiencies can be
detected using the combined test results.

Interpretation of Laboratory Findings

Decreased levels of complement components or activity may
be caused by decreased production, consumption, or in vitro
consumption. The third condition must be ruled out before
either of the other two is considered. Specimen handling is
extremely important. Blood should be collected in a clot tube
with no serum separator.39 The tube should be spun down and
Antibody-coated
the serum should be frozen or placed on dry ice if it is not
red blood cells tested within 1 to 2 hours. If a specimen has been inadequately
refrigerated, been subjected to multiple freeze–thaws, or been
Patient serum Complement activation in prolonged storage, the results may be invalid and the test
A Red blood cell lysis needs to be repeated with a fresh specimen. Control serum
Serum to be tested is diluted serially and added to sensitized should also be included with each batch of test sera.
sheep red blood cells. The tubes are incubated at 37°C and then If a complement deficiency is suspected, it is possible to
centrifuged to pellet the unlysed cells.
narrow down the possible candidate components with a CH50
The CH50 is defined as the reciprocal of the dilution that causes and an AH50 assay. If the CH50 is low but the AH50 is normal,
lysis of 50% of the cells used in the assay. the components unique to the classical pathway should be in-
In the example shown, the CH50 would be about 80 U/mL.
vestigated. If the CH50 is normal but the AH50 is low, the al-
ternative pathway components need to be investigated. If both
the CH50 and AH50 are low, suspicion should be on the com-
100% ponents of the terminal pathway because that pathway is
shared by both the classical and alternative pathways. Although
this analysis will be true for most patients, it is also possible, if
there is sufficient activation of complement through any one
pathway, that the activation could consume enough compo-
Percent lysis

nents to lower the function of the other pathways. As previ-

ously stated, complement activation is rarely limited to just one
pathway. Such consumption can result from loss of a control
protein, presence of an autoantibody, an ongoing infection, or
other activation circumstances.
Once a CH50 and AH50 hemolytic assay have been per-
0% formed, it is appropriate to move to testing the levels or func-
1/320 1/160 1/80 1/40 1/20 tion of the components as directed by the relative results of the
B Serial dilutions CH50 and AH50. For many of the components, an antigen
FIGURE 7–11 CH50 testing. (A) Antibody-coated RBCs are added to level is sufficient to determine where the deficiency lies; how-
patient serum. This activates the complement in the sample and the ever, there are a few instances in which measurement of the
RBCs are lysed. Although only C1qrs is shown for simplicity, this test function would be more informative. For C8, it is necessary to
requires C1 through C9 to be present in order for lysis to occur. The measure function because it is a three-subunit protein. The loss
degree of lysis indicates the functional capacity of the complete of one of the three subunits would not put the level out of nor-
classical pathway. (B) CH50 methodology. mal range, but it would render the remaining two subunits
nonfunctional. C2 type II deficiency results from a genetic mu-
tation in C2 that renders the protein nonfunctional but does
An additional means of testing for alternative pathway func- not decrease the level of expression, which is also true for
tion is via ELISA. One such test can detect C3bBbP or C3bP C1-INH in type II HAE.
complexes in very small quantities. Microtiter wells are typi- A typical screening test for complement abnormalities usu-
cally coated with bacterial polysaccharide to trigger activation ally includes determination of the following: C3 and C4, as
of the alternative pathway. well as hemolytic content. Testing for products of complement
One test system has been developed that can determine the activation such as C3a, C4a, C5a, and Ba (as well as breakdown
activity of all three pathways.32,39 Strips used for the classical products including iC3b and C4d) can also be performed as a
pathway are coated with IgM, strips for the alternative pathway means of monitoring inflammatory processes such as rheuma-
are coated with lipopolysaccharide, and strips for the MBL toid arthritis and SLE. Table 7–5 presents some of the possible
Chapter 7 Complement System 107

Table 7–5 Diagnosis of Complement activation unit consisting of C2, C4, and C3; and the
Abnormalities MAC, consisting of C5, C6, C7, C8, and C9.
IMPAIRED • The lectin pathway is activated by carbohydrates pres-
FUNCTION OR CLASSICAL LECTIN ALTERNATIVE ent in microbial cell walls and serves as an important
DEFICIENCY PATHWAY PATHWAY PATHWAY link between the innate and adaptive immune re-
C1q, C1r, C1s Low Normal Normal sponses. Molecules distinct to the lectin pathway include
C4, C2 Low Low Normal
mannose-binding lectin (MBL), MASP-1, MASP-2, and
MASP-3.
MBL, MASP2 Normal Low Normal • The alternative pathway is triggered by bacterial and fun-
B, D, P Normal Normal Low gal cell walls, yeast, viruses, tumor cells, and certain par-
asites. Factors unique to the alternative pathway include
C3, C5, C6, Low Low Low Factor B, Factor D, and properdin.
C7, C8, C9
• The MAC is common to all three pathways.
C1-INH Low Low Low • Plasma protein regulators of the complement system
Factor H and I Low Low Low
play an extremely important role because if uncon-
trolled, complement activation could have devastating
Improperly Low Low Low systemic effects.
handled • Soluble regulators include C1 inhibitor (C1-INH),
sera C4-binding protein (C4BP), Factor H, Factor I, and
Adapted from Seelen MA, et al. An enzyme-linked immunosorbent assay- S protein.
based method for functional analysis of the three pathways of the comple- • Examples of cell-bound regulators are complement recep-
ment system. In: Detrick B, Hamilton RG, and Folds JD, eds. Manual of tor type 1 (CR1), membrane cofactor protein (MCP), and
Molecular and Clinical Laboratory Immunology. 7th ed. Washington, DC: decay-accelerating factor (DAF).
ASM Press; 2006:124. • Specific complement receptors found on host cells am-
plify the immune response by enhancing phagocytosis
screening results from ELISA testing and correlates these with and stimulating other accessory cells. Some of these
deficiencies of individual factors. An understanding of these receptors include CR1, CR2, CR3, CR4, and collectin
patterns may be helpful in differentiating hereditary deficien- receptors.
cies from activational states that consume available comple- • Effector molecules generated during complement activa-
ment components. Additional testing would be necessary, tion play a major role in recognition and presentation of
however, to actually pinpoint hereditary deficiencies. antigens, activation of B cells, and maintenance of im-
munologic memory. They are classified as anaphylatoxins,
chemotaxins, and opsonins.
SUMMARY • Anaphylatoxins increase vascular permeability, whereas
chemotaxins attract phagocytic cells to a specific area
• The complement system is a series of more than 30 soluble and opsonins coat damaged or foreign cells to enhance
and cell-bound proteins that interact with both the innate phagocytosis.
and adaptive immune systems to enhance host defenses • Deficiencies of complement components can place
against infection. an individual at risk for certain infections. Missing or
• Activities of complement include lysis of foreign or dam- deficient regulators are the cause of diseases such as
aged cells, opsonization, increase in vascular permeability, paroxysmal nocturnal hemoglobinuria and hereditary
and attraction of monocytes and macrophages to areas angioedema.
where needed. • Laboratory assays for individual complement components
• The classical complement pathway is triggered by IgG or include radial immunodiffusion and nephelometry.
IgM binding to the surface of pathogens. Nine major pro- • The hemolytic titration or CH50 assay is a measure of
teins are involved in this pathway. lysis, the end point of complement activation in the clas-
• Three distinct units are involved in the classical pathway. sical pathway. The AH50 assay is a similar test for mea-
They are the recognition unit consisting of C1qrs; the suring the activity of the alternative pathway.
108 SECTION 1 Nature of the Immune System

CASE STUDIES
1. A 3-year-old child has a history of serious infections and hands. She stated that she has had these symptoms on
is currently hospitalized with meningitis. The doctor sus- several previous occasions. After ruling out appendicitis,
pects that he may have a complement deficiency and or- the physician ordered a battery of tests, including some
ders testing. The following results are obtained: decreased for abnormalities of complement components. The
CH50, decreased AH50, and normal C4 and C3 levels. following results were obtained: red and white blood
cell count normal, total serum protein normal, CH50
Questions
decreased, alternative pathway function normal, C3 level
a. What do the results indicate about the possible normal, and C4 and C2 levels decreased.
pathway(s) affected?
b. Which component(s) are likely to be lacking? Questions
c. What sort of additional follow-up would be a. What symptoms led physicians to consider a possible
recommended? complement abnormality?
2. A 25-year-old female appeared at the local hospital’s b. What are possible reasons for a decrease in both C4
emergency department with symptoms of abdominal pain and C2?
as well as severe vomiting and swelling of the legs and c. What other testing would confirm your suspicions?

REVIEW QUESTIONS
1. The classical complement pathway is activated by 6. Which of the following describes the role of properdin
a. most viruses. in the alternative pathway?
b. antigen–antibody complexes. a. Stabilization of C3/C5 convertase
c. fungal cell walls. b. Conversion of B to Bb
d. mannose in bacterial cell walls. c. Inhibition of C3 convertase formation
d. Binding and cleavage of Factor B
2. Which of the following is characteristic of complement
components? 7. Which best characterizes the membrane attack
a. Normally present in serum complex (MAC)?
b. Mainly synthesized by B cells a. Each pathway uses different factors to form it.
c. Present as active enzymes b. C5 through C9 are not added in any particular
d. Heat stable order.
c. One MAC unit is sufficient to lyse any type
3. All of the following are true of the recognition unit except of cell.
a. it consists of C1q, C1r, and C1s. d. C9 polymerizes to form the transmembrane
b. the subunits require calcium for binding together. channel.
c. binding occurs at the FC region of antibody
molecules. 8. All of the following represent functions of the
d. C1q becomes an active esterase. complement system except
a. decreased clearance of antigen–antibody
4. Which of the following is referred to as C3 convertase? complexes.
a. C1qrs b. lysis of foreign cells.
b. C3bD c. increase in vascular permeability.
c. C3bBb d. migration of neutrophils to the tissues.
d. C4b5a
9. Which of the following is true of the amplification
5. Mannose-binding protein in the lectin pathway is loop in complement activation?
most similar to which classical pathway component? a. It is only found in the alternative pathway.
a. C3 b. The membrane attack unit is amplified.
b. C1rs c. C3b is the product that is increased.
c. C1q d. Increasing amounts of C1qrs are produced.
d. C4
Chapter 7 Complement System 109

10. Factor H acts by competing with which of the 16. A decreased CH50 level and a normal AH50 level
following for the same binding site? indicate which deficiency?
a. Factor B a. Decrease in components in the lectin pathway only
b. Factor D b. Decrease in components in the alternative pathway
c. C3B only
d. Factor I c. Decrease in components of both classical and
alternative pathways
11. A lack of CR1 receptors on RBCs would result in d. Decrease in components of the classical pathway
which of the following? only
a. Decreased binding of C3b to RBCs
b. Decreased clearance of immune complexes by the 17. Which best describes the role of an anaphylatoxin?
spleen a. Coats cells to increase phagocytosis
c. Decreased breakdown of C1qrs b. Attracts WBCs to the area of antigen concentration
d. Decreased binding of Factor H c. Increases production of interleukin-1
d. Increases permeability of blood vessels
12. Which best describes the role of CR2 on cell
membranes? 18. Which best describes the role of Factor H?
a. Binds C1qrs to inactivate it a. Acts with DAF to break down C3b
b. Acts as co-receptor on B cells for antigen b. Prevents binding of Factor B to C3b
c. Increases clearance of immune complexes c. Binds to the C5C6C7 complex
d. Binds particles opsonized with C3b d. Binds to C1q to shut down the classical pathway

13. Which of the following best characterizes hemolytic 19. A lack of C1-INH might result in which of the
uremic syndrome? following conditions?
a. It is a rare cause of renal failure in children. a. Paroxysmal nocturnal hemoglobinuria
b. It can be associated with deficiencies in Factor H. b. Hemolytic uremic syndrome
c. The major cause is lack of DAF on RBCs. c. Hereditary angioedema
d. It is associated with antibody-to-C3 convertase. d. Increased bacterial infections

14. The CH50 test measures which of the following? 20. Which would be most effective in measuring an
a. Patient serum required to lyse 50% of sensitized individual complement component?
sheep RBCs a. CH50 assay
b. Functioning of both the classical and alternative b. Radial immunodiffusion
pathways c. AH50 assay
c. Genetic deficiencies of any of the complement d. Lytic assay with liposome
components
d. Functioning of the lectin pathway only

15. Which of the following would be most effective in

preventing bystander lysis of RBCs?
a. C1-INH
b. Factor B
c. DAF
d. Factor H