Clinical utility of complement assessment.

CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Sept. 1995, p. 509–517 1071-412X/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 2, No. 5

MINIREVIEW Clinical Utility of Complement Assessment ALAA E. E. AHMED*

AND

JAMES B. PETER

Specialty Laboratories, Inc., Santa Monica, California 90404-3900 ciencies, as well as mutations that produce functionally inactive complement proteins, in which deficiencies cannot be defined by quantitation alone (57). These assays are modifications of the CH50 assay in which EAs are incubated with dilute serum containing an excess of complement proteins except for the particular component under investigation. Hemolytic assays also are used to evaluate fluid-phase regulatory proteins such as factors I and H and the C1 inhibitor (C1Inh) (85). Another simple method of evaluating C1Inh function (used in the diagnosis of hereditary and acquired angioedema) is based on activation of the C1 macromolecule by immune complexes or aggregated immunoglobulin G (IgG); this is followed by detection of C1r by radial immunodiffusion in agar containing antibodies to C1r (76). Because C1Inh binding masks the relevant epitopes on the C1r molecules, a normal precipitin ring after immune complex activation suggests inherited or acquired C1Inh deficiency or dysfunction. It is possible to differentiate between genetic type I (C1Inh deficiency) and genetic type II (C1Inh is present but lacks normal functional activity) hereditary angioedema and between acquired type I (functional protein at low concentration because of hypercatabolism by an autoantibody or malignancy) and acquired type II angioedema (normal concentration of protein with low-level function because of autoantibodies); concentrations of C1q, C1r, and C1s are characteristically low in individuals with the acquired forms but are normal in those with hereditary angioedemas (60). Similar enzyme immunoassays (EIAs) for the evaluation of complement activity in serum have also been described. Aggregated human IgG and IgM (classical pathway) or lipopolysaccharides (alternative pathway) are coated onto the solid phase to serve as complement activators. Following incubation with the serum, deposition of C3c, C9, or properdin (P) on the activator is detected by EIA (26, 66). In another approach, serum is incubated with a classical or alternative pathway activator, e.g., immune complexes or inulin, respectively. The multimolecular complexes formed in the fluid phase, i.e., C1rC1s-C1Inh (classical pathway), C3bBbP (alternative pathway), or SC5b-9 (terminal sequence), can be detected by specific antibodies by EIA techniques (6, 7). C4 and C3 nephritic factors (NeFs), autoantibodies to classical and alternative pathway convertases, are present in patients with a number of diseases, including systemic lupus erythematosus (SLE) and membranoproliferative glomerulonephritis (64). Crossed immunoelectrophoresis was initially described for the detection of C3NeF by measuring the fluid-phase conversion of C3. A more sensitive and specific hemolytic assay uses EAs carrying one of the C3 convertases (C4b2a or C3bBbP) incubated with heat-inactivated patient serum for the detection for C4NeF or C3NeF, respectively. The NeF antibodies stabilize the C3 convertases, making them inaccessible for inactivation by factor I and either factor H or C4 binding protein (C4BP). After the addition of guinea pig serum as a

INTRODUCTION Via its multiple functional activities, the complement system plays a major role in host defense against infection and in inflammatory processes. The system consists of more than 25 self-assembling proteins divided into three groups (classical pathway, alternative pathway, and terminal sequence; Table 1), as well as a series of receptors and regulatory proteins. These components act in series and in parallel to achieve several important functions (52, 84, 86). The sequence of complement activation has been reviewed extensively elsewhere (57, 86) and is summarized in Fig. 1. LABORATORY ASSESSMENT OF COMPLEMENT ACTIVATION IN VIVO Complement activation in vivo results in degradation of complement components, with the resultant expression, formation, and release of neoantigens, multimolecular complexes, and split products (Table 2) into the fluid phase and their deposition at the sites of complement activation in tissue. Currently, two types of assays are used to evaluate the activation status of the complement system: functional and immunochemical assays. Functional assays. Current complement functional assays permit evaluation of the total functional integrity of the classical, alternative, and terminal sequence pathways, as well as of individual complement components (59, 85). The 50% hemolytic complement (CH50) assay evaluates classical pathway and terminal sequence activities by determining the capacity of serum to lyse antibody-sensitized sheep erythrocytes (EAs) in the presence of Ca21 and Mg21; Ca21 is essential for the interaction of the subcomponents of the C1 macromolecule, while Mg21 is required for the binding of C4b and C2a (59, 85). Similarly, the functional activity of the terminal sequence can be measured directly with the terminal sequence hemolytic assay (TH50) by using EAs carrying the classical pathway C3 convertase, C4b2a; the patient’s serum serves as a source of complement components C3 to C9 (85). The functional activity of the alternative pathway is assessed with the alternative sequence hemolytic assay (AH50) by evaluating complement-mediated lysis of rabbit erythrocytes in Mg21EGTA [ethylene glycol-bis(b-aminoethyl ether)-N,N,N9,N9tetraacetic acid] buffer (85). EGTA chelates the Ca21 required for assembly of the C1 macromolecule, thereby inhibiting the activity of the classical pathway (85), and Mg21 is necessary for the interaction of C3b and factor B (85). Hemolytic functional assays for individual complement components are useful for defining homozygous and to some extent heterozygous defi* Corresponding author. Mailing address: Specialty Laboratories, Inc., 2211 Michigan Ave., Santa Monica, CA 90404-3900. Phone: (310) 828-6543. Fax: (310) 828-6634. 509

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CLIN. DIAGN. LAB. IMMUNOL. TABLE 1. Components of the classical, alternative, and terminal complement pathways

Pathway and component

Concn in serum (mg/dl)

Classical C1q C1r C1s C4

8.0–12.0 2.0–6.5 1.8–8.5 10–40

C2

1.6–3.6

Alternative Factor B

Activation product

C1r C1s C4a C4b C2a C2b

Terminal C3

0.1–0.4 2.5–4.0 60–100

C5

5.5–11.0

C6 C7 C8 C9

2.8–6.0 2.7–7.4 4.9–10.0 3.3–9.0

a

Binds to immune complexes Activates C1s Activates C4 and C4 Anaphylatoxin Binds to activator Binds C4b Kinin forming

12–28 Ba Bb

Factor D Properdin

Activity

C3a C3b iC3b C3c C3dg C3e C5a C5b

Chemotactic factor Part of C3bBb Cleaves factor B Stabilizes C3bBb Anaphylatoxin Part of C5 convertase ligand for CR1 Ligand for CR3 Ligand for CR2 Leukocytosis Anaphylatoxin Part of MACa Part of MAC Part of MAC Part of MAC Part of MAC

No. of genes

Chromosomal location

3 1 1 2 (4A, 4B)

1p (A, B chain) 12p 12p 6p

1

6p

1

6p

1 1

Not determined Xp

1

19q

1

9q

1 1 3 (A, B, G) 1

5 5 1p (A, B), 9q (G) 5

MAC, membrane attack complex.

source of terminal components, the degree of lysis is measured; the degree of lysis generally correlates with the titers of the autoantibodies (64). Complement immunochemical assays. Numerous immunochemical assays, including radial immunodiffusion, rocket immunoelectrophoresis, nephelometry (the method of choice, as assessed by accuracy and precision), turbidimetry, and EIA, are currently available to quantitate native complement proteins. Nephelometric reagents for C3b, C3c, C4b, and C4c, as well as native C3 and C4, are available commercially. However,

the concentrations of native complement proteins such as C3 and C4 in serum are unreliable indicators of complement activation in vivo (see below). New assays that use specific polyclonal and monoclonal antibodies for the detection and quantitation of neoantigens expressed on multimolecular complexes and on complement fragments (split products) promise to be of much greater value for the assessment of complement activation in vivo. These include EIAs suitable for the quantitation of C1r-C1s-C1Inh, C4bC4BP, C3bBbP, SC5b-9, C3dg, iC3b, C4d, Bb, and the anaphylatoxins C3a and C5a (8, 30, 33, 38, 39, 41, 48–50, 55, 56, 61, 63, 90, 91). Flow cytometric methods are available to measure the cellular expression of and to define the deficiencies of complement receptors and membrane regulatory proteins (41). LABORATORY DETECTION AND MONITORING OF HYPOCOMPLEMENTEMIA

FIG. 1. Classical and alternative complement pathways. Activation of either pathway results in the formation of C3 convertases, which in turn activate the terminal sequence. Ag, antigen; Ab, antibody; MAC, membrane attack complex.

Although deficiencies of most of the complement components and regulatory proteins are well characterized (Table 3) (18, 81), inherited homozygous deficiencies (primary hypocomplementemia) are rarely encountered (18, 81). The more common forms of hypocomplementemia typically result from complement consumption as a consequence of existing disease (secondary hypocomplementemia), e.g., immune complex diseases which activate complement through the classical pathway or bacterial infections which activate complement primarily via the alternative pathway (21, 82). Detection of complement consumption is essential for following the progression of diseases or predicting the extent of tissue damage caused by complement activation (21). Detection of primary hypocomplementemia. The combined use of the CH50 and AH50 hemolytic functional assays permits

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TABLE 2. Products of complement activation Product and component

Property

Multimolecular complexes C1r-C1s-C1Inh ......................................................................................................................................Inactivation product of C1 C3bBbP ..................................................................................................................................................Alternative pathway fluid-phase C5 convertase SC5b-9....................................................................................................................................................Soluble membrane attack complex Split products C4a .........................................................................................................................................................Anaphylatoxin, split product of C4 C4d .........................................................................................................................................................Inactivated C4b produced by factor I and C4BP Ba............................................................................................................................................................Fluid-phase activation product of factor B Bb ...........................................................................................................................................................Part of alternative pathway C5 convertase C3a .........................................................................................................................................................Anaphylatoxin, split product of C3 iC3b ........................................................................................................................................................Inactivated C3b produced by factors I and H C3c..........................................................................................................................................................Split product of C3b or iC3b C3dg .......................................................................................................................................................Split product of C3b or iC3b C5a .........................................................................................................................................................Anaphylatoxin, split product of C5 Components expressing neoantigens C4b .........................................................................................................................................................Activation product of C4 produced by C1s C3a, C3a(des Arg)................................................................................................................................Anaphylatoxin, split product of C3 iC3b ........................................................................................................................................................Inactivated C3b produced by factors I and H C5a .........................................................................................................................................................Anaphylatoxin, split product of C5 C9 ...........................................................................................................................................................Final component of membrane attack complex

screening for inherited complement deficiencies in the classical, alternative, and terminal sequence components and then specific evaluation of the defective pathway (Fig. 2), providing a low-cost indication of the functional integrity of the whole complement system (85). Such evaluations are commonly indicated in patients with the major recurrent infectious disease associations listed in Table 3. Homozygous-deficient individuals show no lysis, whereas heterozygous-deficient individuals show low levels of lysis (18, 81). No lysis or a low level of lysis in the CH50 assay but normal results in the AH50 assay indicates a deficiency in a component specific to the classical com-

plement pathway, i.e., C1, C4, and C2. On the other hand, a low level of lysis or no lysis in the AH50 assay but normal results in the CH50 assay suggests a deficiency in an alternative pathway-specific component, usually properdin or factor H or I. No lysis or a low level of lysis in both hemolytic assays suggests a deficiency in either C3 or one of the terminal pathway components, C5 to C9. C9 deficiency is better diagnosed by measuring the C9 antigen concentration rather than by the functional hemolytic assay, since some lysis does occur in the absence of C9. However, it should be noted that both the CH50 and AH50 functional assays detect only massive activation of

TABLE 3. Complement deficiencies and associated clinical patterns Disease association

Pathway or product and complement component

Major

Minor

Classical pathway C1q, C1r, C1s, C4 C2

Immune complex diseases (SLE-like) Immune complex diseases (SLE-like)

Alternative pathway Factor D Properdin

Recurrent upper respiratory tract infection (Neisseria spp.) Recurrent infection

Terminal pathway C3, C5, C6, C7, C8, C9

Recurrent infection

Immune complex disease (SLE-like)

Fluid-phase regulatory proteins Factor I Factor H C1Inh Protein S

Recurrent Recurrent Recurrent Recurrent

Immune complex disease (SLE-like) Hemolytic uremic syndrome Immune complex disease (SLE-like) Tissue necrosis

Membrane-phase regulatory proteins DAF, CD59, and C8BP

Paroxysmal nocturnal hemoglobinuria

Complement receptors CR2, CR3, CR4 CR1

Severe immunodeficiency SLE

pyogenic infection pyogenic infection subcutaneous swelling and mucosal edema thrombosis

Recurrent infection, including meningitis Recurrent infection, including meningitis, also in healthy individuals

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FIG. 2. Laboratory diagnosis of complement deficiency. The CH50, AH50, and TH50 assays are used to differentiate between classical, alternative, and terminal sequence complement component deficiencies. Rat serum containing EDTA is added to the reaction mixtures of each assay as a source of the terminal complement components C3 through C9.

complement resulting in depletion of complement components (85), and determination of the pathway involved requires further assessment. Detection of secondary hypocomplementemia. Concentrations of C4 and factor B in serum or plasma commonly are used as measures of turnover for the classical and alternative pathways, respectively; assessment of the C3 concentration can reflect the degree of activation of both pathways. This approach has a number of limitations, including the wide reference ranges of C4 and C3 concentrations and the fact that both C4 and C3 are acute-phase reactants (68). Indeed, even CH50 and AH50 activities can increase with inflammation (86). In addition, measurements of C4 concentrations are complicated by the high frequency of heterozygous C4 deficiency in the normal population (null alleles) (14, 74) and by the different hemolytic activities of the alleles of the C4 allotypes C4A and C4B (35). Isolated measurement of C4 therefore can be misleading. Because C3 is normally present in serum at relatively high concentrations (16), C3 is also an insensitive marker for complement activation; significant decreases in C3 concentration occur only after massive complement activation (86, 87). Decreased CH50 activity and low C4 or C3 concentrations also can result from decreased synthesis of complement components, increased consumption, or inadequate regulation (53). In the last case, uncontrolled complement activation causes depletion of C1, C4, and C2 and decreased CH50 in the case of C1Inh deficiency (60), depletion of C3 and factor B in the case of factor I or factor H deficiency (86), or depletion of C3 and C4 in the case of factor I or C4BP deficiency (86). Because most complement components are acute-phase reactants, ongoing inflammation can easily mask an increase in complement consumption (35). If analyses are restricted to CH50 and C4 and C3 concentration determinations, it can be difficult to detect, much less discriminate among, classical pathway activation, heterozygous C4 deficiency, or a regulatory protein deficiency with resultant complement consumption (53). Thus, the need for additional, more sensitive and incisive assays for complement activation is apparent. Assays for classical pathway activation. The formation of stable C1r-C1s-C1Inh complexes is evidence of classical pathway-mediated activation of complement in vivo. Both in vivo and in vitro studies demonstrate that elevated concentrations of C1r-C1s-C1Inh correlate with both C2 and C3 cleavage, as reflected by a decrease in C2 hemolytic activity, the generation of C3a, and low concentrations of circulating C1q, C4, and C3

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(7, 9). In a solid-phase assay developed for the specific detection of the C1r-C1s-C1Inh complexes, anti-C1s polyclonal antibodies are coated on the solid phase to capture the complexes which are subsequently detected by C1Inh-specific antibodies (33). Unfortunately, because native C1s competes with the C1s of C1r-C1s-C1Inh complexes for binding to the solid phase, the analytical sensitivity of this assay is expected to be suboptimal. Monoclonal antibodies that react exclusively with the activation-dependent neoepitope(s) on C1s are needed to improve this assay. C4a, an anaphylatoxin like C3a, is inactivated in vivo to C4a(des Arg) by serum carboxypeptidase N (46). Although there is good correlation between the generation of C3a and C4a during complement activation (7), C4a concentrations should probably not be used when there is verifiable or even suspected renal disease, because C4a is apparently cleared by the kidneys (1). A monoclonal antibody-based EIA for C4d, another C4 cleavage fragment, is now available (48), but its use cannot yet be recommended because C4 is highly polymorphic and the basis of the polymorphism resides within the C4d fragment; it is unclear whether the epitope recognized by the C4d-specific monoclonal antibody is present on all C4 allotypes (74). A promising new assay for C4b that uses a monoclonal antibody which recognizes a neoantigen expressed on C4b merits rigorous clinical evaluation (89). However, measurement of C4 and its fragments as a measure of complement activation by the classical pathway is complicated by the C4 null alleles (74). C1r-C1s-C1Inh appears to be a more reliable marker for classical pathway activation than C4d or C4b (9). Assays for alternative pathway activation. Assays for the measurement of alternative pathway activity use monoclonal antibodies which recognize neoantigens on the factor B split products Ba and Bb (50). Because Ba is cleared by the kidneys (50, 86), only measurement of Bb is likely to be uncomplicated in its clinical interpretation and, hence, worthy of further study. The alternative pathway C3 convertase C3bBbP can also be measured by EIA. The complex is trapped on the solid phase by properdin-specific antibodies; bound C3bBbP is detected by C3-specific antibodies. Under physiological conditions, properdin will not bind fluid-phase C3b in the absence of bound Bb (9). Because the half-life (t1/2) of the C3bBbP complex is only 30 to 45 min at 308C in vitro, it is likely to have an even shorter t1/2 at 378C in vivo. Thus, although the detection of the C3bBbP complex, i.e., the stabilized C3 convertase of the alternative pathway, in plasma may indicate ongoing complement activation in vivo at the time of venous sampling (8), its short t1/2 does not bode well for clinical utility in monitoring chronic diseases. Assays for terminal pathway activation. Several methods have been developed to measure the split products of C3. The anaphylatoxin C3a, a split product of C3 generated during complement activation via the classical or alternative pathways, is inactivated rapidly by the removal of its N-terminal arginine by serum carboxypeptidase N, forming C3a(des Arg) (46). A radioimmunoassay for C3a and/or C3a(des Arg) which includes a precipitation step to separate C3 and its larger fragments from C3a has been available for some time (37). An EIA for the direct quantitation of C3a(des Arg) in plasma uses a monoclonal antibody that reacts exclusively with C3a(des Arg) but not with C3a or native C3 (12). Accurate measurement of C3a(des Arg) is made difficult by the generation in vitro of C3a when blood samples are collected in EDTA tubes. Addition of the synthetic protease inhibitor FUT-175 (nafamostat mesilate [Futhan]) to EDTA in the collection tube minimizes spurious in vitro complement activation (69, 83). With proper precautions, measurement of plasma C3a(des Arg) is

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very promising for the accurate assessment of complement activation in patients with different disorders (1, 2, 10, 88). In contrast, the clinical utility of assays for other C3 activation products such as iC3b, C3d, C3dg, or C3c by using monoclonal antibodies against neoantigens remains to be defined (62, 65). Like C3a, C5a can be used as an indicator of terminal sequence activation if the same precautions are taken to avoid in vitro activation. C5a is rapidly (t1/2, ,20 min) cleared from the circulation by C5a receptors on neutrophils even before inactivation by carboxypeptidase N (46). Thus, C3a measurement has advantages over C5a measurement in assessing terminal pathway activation. Assays for activity of terminal complement sequence. Increased concentrations of SC5b-9 (the soluble complex of protein S and C5b-9) in serum reflect complement activation through either the classical or the alternative pathway (49). Both radioimmunoassay and EIA techniques with polyclonal or monoclonal antibodies raised against neoepitopes on the C9 moiety of the C5b-9 complex have been described (38, 49, 56, 90). Since the complexes are not generated spontaneously in vitro, are stable in vitro, and have long t1/2s in vivo, their detection offers clear advantages for assessing complement activation in body fluids (56). However, the levels of protein S in serum may affect the rate of SC5b-9 formation under inflammatory conditions. Effects of impaired renal function on levels of complement components in plasma. The kidneys play a major role in the clearance and catabolism of immunologically relevant low-molecular-weight plasma proteins including b2-microglobulin, C4a, factor D, and Ba (84). These proteins are filtered by the glomeruli and are catabolized in the proximal tubular epithelium. Decreased glomerular filtration in patients with chronic renal failure results in greatly elevated concentrations of these proteins in plasma (1). Thus, elevated C4a, factor D, or Ba concentrations may be attributed falsely to enhanced complement activation if the effect of altered renal function on the clearance of complement split products is not considered. Critical study of such obfuscating effects must take place early in the evaluation of any new marker for complement activation.

COMPLEMENT ASSESSMENT While the CH50 and AH50 assays are useful in screening for primary hypocomplementemia, changes in the concentrations of individual complement components in plasma because of synthesis and clearance or consumption make these traditional hemolytic functional assays insensitive monitors of complement activation and, hence, of disease activity; additional tests are needed for incisive evaluation (65). Traditionally, C3 and/or C4 concentrations are used to monitor the activity of immune complex-mediated disease (17, 36, 42, 58, 67, 70). While both C3 and C4 concentrations can be measured by nephelometry with high degrees of accuracy and precision, the wide normal ranges as well as the steady-state concentrations of plasma C3 and C4 concentrations are influenced by several factors. These assays are therefore of limited value in the assessment of complement activation in vitro and in vivo (53). The need for sensitive markers of complement activation in vivo has led to the introduction of EIA methods that can be used to detect and quantitate complement activation products in plasma (Table 2). These new assays provide potential tools for measuring the extent of complement activation, for discriminating between complement deficiency versus activation or consumption, and for detecting activation or consumption

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in the presence of inflammation, which increases the steadystate levels of complement proteins (62, 65). Initial assessment. In patients suspected of having primary hypocomplementemia, especially children, with appropriate findings, e.g., SLE or recurrent pyogenic infections (Table 3), assessment of complement function by the CH50 and AH50 assays is recommended because both assays are useful as screening tests for genetically determined complement deficiency (Fig. 2). If one or both of the screening tests show no lysis, use of a combination of immunochemical and functional assays is required to determine which component is deficient or inactive (Table 2). To minimize in vitro complement activation, blood specimens for all functional assays (except the C1 functional assay) must be drawn into EDTA tubes. Plasma must be separated within 1 h and must be stored at 2708C until it is assayed. Such precautions are minimal and mandatory because it is difficult to differentiate between in vivo and in vitro activation. For some assays, e.g., C3a, the addition of proteolytic enzyme inhibitors, e.g., FUT-175, is also necessary. Again, it should be noted that both the CH50 and AH50 assays detect only massive activation or genetically determined defects of complement which result in the depletion of complement components (85). Therefore, even though rigorous evidence of their clinical utility from prospective studies is lacking, assays for SC5b-9 and/or C3a concentrations are usually recommended for patients with secondary hypocomplementemia, since high concentrations indicate that complement activation has occurred, regardless of which pathway is involved (7). Determination of the affected pathway requires further assessment, which is best determined at present by measuring the concentrations of C4d for classical pathway activation (87) and those of Bb for alternative pathway activation (62). Serial assessment. Serial assessment of complement is commonly used to monitor disease activity or to predict a flare-up. Currently, most laboratories perform CH50, C3, and C4 measurements. However, the information obtained is often unpredictable, misleading, and insensitive (53). If the patient is known to suffer from a disease that involves classical pathway activation (e.g., SLE or rheumatoid arthritis), assessment of C1r-C1s-CInh, C4b, or C4d is recommended. For diseases which activate the alternative pathway (e.g., bacterial infection), measurement of C3bBbP or Bb is the assay of choice for serial assessment of alternative pathway activation. Despite the lack of proven predictive value for most of the new assays mentioned above, serial measurement of any of these parameters theoretically should parallel disease activity; prospective studies are needed. The introduction of the activation index is promising for the evaluation of some diseases (42, 58, 67). These indices are the ratio of the split product concentration (determined by EIA or radioimmunoassay) to the total component concentration (determined by nephelometry). The activation index is 0 or near 0 if low-level or no activation has occurred. Significant increases in the ratio of split product concentration to total component concentration, i.e., an elevated activation index, indicate the degree of activation independent of the basal level of the native component. Components of different activation indices include Bb:total factor B for the alternative pathway, C4d:total C4 for the classical pathway, and iC3b:total C3 or C3a:total C3 ratios for the terminal sequence (58). COMPLEMENT AND DISEASES Careful consideration of the history and clinical presentation is mandatory before initiating complement assessment. If, for

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example, a patient presents with a disease likely to involve classical pathway activation, then one of the aforementioned assays for classical pathway evaluation should be considered. The following section summarizes briefly the use of new assays for complement activation products in the diagnosis and tracking of a number of clinical situations. SLE. C3d, which has the longest in vivo t1/2 of all C3 split products (16), has been used to measure classical and alternative pathway turnover in 79 patients with SLE (67). Reported to reflect complement activation better than measurement of C3, C4, and CH50 (67), C3d is claimed to be a marker in predicting the activity of SLE (67). Indeed, the C3d:total C3 activation index is said to discriminate between patients with different disease severities better than the C3d concentration alone (67). On the other hand, increased concentrations of the iC3b neoantigen, which makes up the whole activated C3 (iC3b, C3dg, C3c), correlated with disease activity in patients with SLE in one study (58) but not in another one (17). The concentrations of the factor B split products Ba and Bb in plasma are reported to be better markers of disease activity in patients with SLE than either C3 or C4 concentrations (4, 50). In a 15-month prospective study of 86 patients with SLE, Ba concentrations closely paralleled disease activity (14). Although evaluation of C4d concentration may be more sensitive, Ba or SC5b-9 concentrations are reportedly more accurate indicators of disease activity than conventional CH50, C3, and C4 evaluations (14). That no effect of renal failure on Ba concentrations was noted presumably reflects the lack of severe renal disease in the patients in that study (14). During pregnancy, the ratio of CH50 to Ba concentration is higher in patients with preeclampsia than in those with active SLE (3, 78). A decline in the C3 or C4 concentration or CH50, accompanied by elevations of C4d, Ba, and SC5b-9 concentrations, appears to differentiate a lupus flare from non-SLE disease of pregnancy (4, 78). Rheumatoid arthritis and other rheumatic diseases. Consistent with classical pathway activation, the concentrations of C1r-C1s-C1Inh, G3bBbP, and SC5b-9 in plasma are significantly elevated in patients with SLE and rheumatoid arthritis (9, 40, 77, 78). There is a relationship between concentrations in plasma and synovial fluid, suggesting that these complexes diffuse between the synovial cavity and the intravascular compartment or that the degree of systemic activation is similar to that occurring within the joint (9). In patients with juvenile rheumatoid arthritis, the concentrations of both C4d and Bb in plasma correlate strongly with disease activity manifested by polyarticular and pauciarticular manifestations or an elevated erythrocyte sedimentation rate (40). Unfortunately, that study used heparinized plasma, which may affect C4d and Bb levels; there is no correlation with concentrations of immune complexes. In patients with leukocytoclastic cutaneous vasculitis, the concentrations of C3, factor B, CH50, C1q, C4, and C2 in plasma are generally within the reference ranges (23). In contrast, elevated C3dg and SC5b-9 concentrations are significantly correlated with the severity of vasculitis (23). In patients with Kawasaki syndrome, C3d, C4d, Bb, and SC5b-9 concentrations are elevated, strongly suggestive of classical pathway activation (47). In patients with juvenile dermatomyositis, C3 and C5b-9 complexes are deposited in the arterioles and capillaries of affected muscle (45). Clinically active disease associated with complement activation, as measured by C3d and C4 concentrations, has been reported in a small number of patients with juvenile dermatomyositis (71). Renal injury. Thirty-six percent of patients with immune complex-mediated renal injury (Henoch-Scho ¨nlein nephritis,


IgA nephropathy, membranoproliferative glomerulonephritis type I, SLE, some cases of microscopic polyarteritis, IgM glomerulonephritis, and minimal change glomerulonephritis) have elevated concentrations of C4a (1). Seventy-three percent of patients with renal diseases not related to immune complex pathology (polycystic kidney, diabetic nephropathy, hydronephropathy) have elevated C4a concentrations; 33% have elevated C3a concentrations (1, 2). Impaired clearance by the kidneys could account for the high concentrations of C4a in patients with nonimmune renal diseases. Certainly, evaluation of the C3a concentration provides a more reliable index of complement activation in vivo than evaluation of the C4a concentration in patients with impaired renal function (1, 2). Uncomplicated clinical interpretation of C3a and C4a assays in patients with renal disorders is, however, not promising. Elevated C3dg and SC5b-9 concentrations are detected in the urine of 43% of patients with membranous nephropathy; increased C3dg concentrations are detected in the plasma of 45% of patients with IgA nephropathy (20). No information about plasma C3dg and SC5b-9 levels is available. These observations may reflect different sites of complement activation in the two groups, i.e., a subepithelial activation site in patients with membranous nephropathy and a mesangial activation site in patients with IgA nephropathy. This may suggest that urinary C3dg and SC5b-9 may indicate ongoing active glomerular damage and might prove to be important determinants for the introduction and monitoring of therapy. Demyelinating disorders of the central and peripheral nervous systems. The C9 neoantigen is detected in affected tissues in patients with experimental allergic encephalomyelitis, experimental autoimmune neuritis, multiple sclerosis, and GuillainBarre´ syndrome lesions (19). Complement activation is evidenced by detection of C9 consumption in the cerebrospinal fluid of patients with multiple sclerosis (19). Elevated concentrations of SC5b-9 in plasma and cerebrospinal fluid and increased concentrations of C3a and C5a in the cerebrospinal fluid of patients with multiple sclerosis and Guillain-Barre´ syndrome are also reported (19, 34). No studies of predictive values for disease activity or for therapeutic monitoring are available. Allergy. Complement activation is certainly not characteristic of allergic states and is not useful for the diagnosis. However, there is evidence that complement contributes to the inflammatory reaction usually found in allergic states. Not unexpectedly, most studies show no evidence of complement activation, as assessed by the conventional evaluation of native complement components, e.g., C3 or C4 concentration or CH50. In contrast, in one study of patients with bronchial asthma, increased concentrations of multimolecular complexes of complement activation (C1r-C1s-C1Inh, C3bBbP, and SC5b-9) revealed elevated activity of the classical, alternative, and terminal sequence pathways (43). Nevertheless, no correlation was found between the concentration of complement activation products and the forced expiratory volume in 1 s, a measure of the severity of asthma (43); clinical utility is not promising. Diabetes. The concentrations of C4, C3, and SC5b-9 in plasma are not significantly different in patients with insulindependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM) compared with those in healthy individuals. On the other hand, SC5b-9 concentrations correlate with urine albumin excretion and the plasma von Willebrand factor, circulating immune complex, and anti-heparan sulfate cross-reactive single-stranded DNA antibody concentrations in patients with IDDM (79). Why the anaphylatoxin C4a, but not C3a, should be significantly higher in

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patients with adult-onset IDDM than in patients with juvenileonset IDDM and NIDDM and healthy individuals is unclear (11). Complement activation is not correlated with the presence of circulating antoantibodies (11). NIDDM is a major risk factor for coronary artery disease. Patients with ischemic heart disease show higher concentrations of C3d in their plasma, suggesting that complement may be involved in the development of macrovascular disease in patients with NIDDM (25). Cardiovascular disease. As in cardiovascular disease with diabetes mellitus, recent studies suggest that complement activation is involved in human atherosclerosis (73). C5b-9 complexes, as well as C3, iC3b, C3dg, and C3c fragments, are detected in human atherosclerotic lesions, indicative of complement activation, formation of C3dg and C5b-9 by C3 and C5 convertases, and deposition of C5b-9 at the site of injury (72). However, no data on the concentrations of complement activation products in the plasma of patients with cardiovascular disease in the absence of diabetes mellitus are available. Biocompatibility and transplantation. An inflammatory reaction may result following different forms of extracorporeal circulation, such as cardiopulmonary bypass (CPB), hemodialysis, and apheresis. Activation of complement, a consistent phenomenon during CPB surgery, may have adverse effects both directly (affecting the heart and pulmonary circulation and inducing hemolysis) and indirectly (activating granulocytes and monocytes with subsequent release of inflammatory mediators). The degree of complement activation is considered an index of biocompatibility (13). The clinical relevance of this index is supported by studies which demonstrate a significant correlation between the degree of complement activation and organ dysfunction after CPB or adverse symptoms during hemodialysis (29, 32, 54). Although evaluation of the C3a concentration alone has been recommended as a test of biocompatibility during CPB (13), SC5b-9 is likely to be a better marker of complement activation during CPB or related stresses (80), because the production of C3a but not that of SC5b-9 in vitro is a major problem unless stringent precautions are taken. Therefore, the biocompatibility during CPB (or during dialysis or apheresis) (54) is probably best measured as the concentration of SC5b-9 together with the concentrations of C3 activation fragments (C3a or iC3b). The complement activation assays now available could also be used in prospective, multivariable studies to assess the effects of new materials used in dialysis membranes on complement concentrations in vivo and in vitro and to determine which pathways are involved. It is widely accepted that complement activation during hyperacute rejection is mediated either by antibodies to the endothelium of the transplanted organ (classical pathway activation) or by the direct interaction of complement with the donor endothelium (alternative pathway activation) (22). In patients with acute renal allograft rejection, the concentrations of C1rC1s-C1Inh, C3bBbP, and SC5b-9 in plasma are elevated (44). C1r-C1s-C1Inh concentrations reportedly increase 4 to 7 days before clinical symptoms become obvious and prior to an appreciable elevation in creatinine or C3a levels. Measurement of the C1r-C1s-C1Inh concentration may be useful for the early recognition of patients at risk (44). Neonatal diseases. Neonates have a relative deficiency of most complement proteins compared with the concentrations in adults (5, 24). Whether low native complement protein concentrations contribute to susceptibility to infection in neonates is not known. The increase in C3a levels within the first few hours of life might reflect exposure to gram-negative and gram-positive bacteria. The increased plasma C3a level is paralleled by the increased formation of C3bBbP complexes (93).

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Thus, complement activation products might provide much earlier indicators of severe neonatal infection than C-reactive protein, the acute-phase reactant which, by comparison, appears within 6 to 8 h after infection (77, 93). ARDS. The acute respiratory distress syndrome (ARDS), a well-known cause of respiratory failure in adults, occurs in patients with a variety of diseases. Although easily diagnosed in adults, ARDS is not as easily recognized in children. In newborns, ARDS may be indistinguishable clinically from hyaline membrane disease. In adults with ARDS, elevated concentrations of C3a and SC5b-9 in plasma are predictive of a poor prognosis (92). Unlike the generalized inflammatory activation in ARDS, there is no significant complement activation (C3a, C3dg, C1r-C1s-C1Inh, or C3bBbP) in patients with hyaline membrane disease (15). Therefore, if confirmed in prospective studies, C3a concentrations may be helpful in the differential diagnosis of ARDS in infants. Septic shock syndrome. Activation of the alternative pathway commonly is implicated in patients with septic shock syndrome caused by gram-negative bacteremia (51). Elevated C3a and classical pathway C4a concentrations are strongly associated with mortality in patients with septic shock syndrome (31). A recent study of 12 patients with septic shock syndrome demonstrated that SC5b-9 and Bb concentrations, as well as the Bb:factor B activation index, were significantly increased in 3 of the 12 patients evaluated (93). Furthermore, although no increase in iC3b and C4d concentrations was noted, the iC3b:C3 activation index was higher in 17% of patients with septic shock syndrome compared with that in healthy individuals (75). Further studies with a larger patient population are needed to confirm these observations. Thermal injury. Complement activity is responsible for increasing vascular, smooth-muscle contraction, anaphylatoxininduced mast cell histamine secretion, and release of oxygen radicals which initiate the inflammatory response in patients with thermal injury (28). Increased concentrations of C3a, C4a, and C5a are usually found in the plasma of patients with thermal injury (27), but the clinical utility of these markers still must be evaluated. Alternative pathway components (factor B, properdin) have been shown to be depleted by 1 h postburn in proportion to the size of the burn (28). The significance of this, however, awaits clinical clarification. CONCLUSIONS The recent introduction of new techniques for assessing complement activation reflects both the disappointment with and consequent decline in the use of traditional methods based on the measurement of intact components. Only in unusual clinical circumstances are the functional assays for CH50 and AH50 useful as screening tests for complement deficiencies; identification and confirmation of a specific, genetically determined deficiency requires additional immunochemical and functional quantitation of individual complement components. Indeed, the only direct approach to the evaluation of complement activation is the detection of complement activation or catabolism products: either split products, multimolecular complexes, or neoantigens. Some of the markers for complement activation have severe limitations under specific pathologic conditions, whereas others offer only limited or unreliable information. As yet, none of the available assays has a well-defined clinical utility for evaluating complement activation in any disease. The prognostic value of serial assessment of complement activation products offers the possibility of identifying subsets of patients with smoldering disease who might benefit from early and more

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aggressive therapeutic intervention. Examination of body fluids other than plasma, e.g., pleural fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, and urine, for the presence of complement products may provide useful diagnostic and pathogenetic information, but the available data on complement products in body fluids other than plasma are even more meager than those for complement products in plasma. The focus over the last few decades has been to document the role of complement in the pathogenesis of inflammatory disorders. Relatively little attention has been given to the development of new assays for clinical evaluation of complement or to the clinical utility of either the established or the new assays. For effective clinical monitoring of complement activation, some necessary, albeit perhaps not sufficient, characteristics include the following: stability of the component, complex, or fragment in vitro, relatively long t1/2 in vivo, not an acute-phase reactant, not influenced by the renal function status, should be specific for one pathway, and should have a high clinical utility and predictive value. Definitive evaluation of the clinical utility of the assays for complement activation as tools for the routine monitoring of disease activity will require well-focused prospective simultaneous studies of multiple complement analytes by clinicians and immunology laboratories. Several promising, analytically reliable assays are already available for this research.


15. 16.

17. 18. 19. 20.

21. 22. 23.

24. 25.

ACKNOWLEDGMENTS We thank Nancy Campman for expert work on the schematic figures and Tsippi Rudy and Mindy Shaffer for assistance with the tables and typing of the manuscript. We thank and acknowledge Sharon Hunt Gerardo for editorial assistance and K. Whaley for reviewing the manuscript.

26.

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