AUTOANTIBODIES TO C1q

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AUTOANTIBODIES TO C1q MARK H. WENER, MD University of Washington, Seattle, WA 98195, USA

HISTORICAL NOTES AUTOANTIGEN AUTOANTIBODY CLINICAL UTILITY CONCLUSIONS TAKE-HOME MESSAGES REFERENCES

ABSTRACT Autoantibodies to C1q bind to epitopes on the collagen-like region (CLR) of the C1q molecule. These autoantibodies are frequently present in the serum of patients with systemic lupus erythematosus (SLE), particularly with active lupus nephritis. Antibodies to C1q serve as prognostic and disease activity markers in SLE, and are particularly associated with proliferative forms of lupus nephritis. Anti-C1q may play a pathogenic role in augmenting immune complex glomerulonephritis by leading to aggregation of immune complexes that have bound C1q. Anti-C1q antibodies are also characteristic of hypocomplementemic urticarial vasculitis syndrome (HUVS). Anti-C1q is often detected using C1q as the antigen in a high salt buffer, but definitive proof that binding to C1q is caused by autoantibodies rather than immune complexes requires the use of purified CLR of C1q as antigen. In routine clinical practice, measurement of antiC1q by solid-phase assays with either intact C1q or CLR is useful in assessing disease activity and prognosis in SLE patients and in support of a diagnosis of HUVS.

HISTORICAL NOTES In the early 1970s, Agnello et al. found that monomeric IgG that bound C1q was present in systemic lupus erythematosus (SLE) sera. Monomeric IgG which precipitated C1q was later found to be characteristic of the hypocomplementemic urticarial vasculitis syndrome (HUVS). In the late 1970s and early 1980s, several groups observed that the sera of patients with SLE contained IgG that was indistinguishable in size from monomeric IgG and that bound to solid-phase C1q in tests for presumptive circulating immune complexes. The presence of monomeric C1q-binding IgG was associated with proliferative forms of lupus nephritis. Autoantibodies, 2/e Copyright © 2007, Elsevier, B.V. All rights reserved.

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In a series of papers in the mid-1980s, Uwatoko et al. established that monomeric IgG from SLE sera bound to solid-phase but not fluid-phase C1q, that the binding was to the collagen-like region (CLR) rather than to the globular heads of C1q, and that the binding was due to antibodies to C1q. This autoantibody binding persisted in 1 M NaCl, whereas the binding of aggregated IgG (as a surrogate for immune complexes) was inhibited by 1 M NaCl. Uwatoko and Mannik [1] demonstrated conclusively that sera from selected SLE patients contained antibodies to the collagen-like region of C1q.

AUTOANTIGEN Definition C1q is a cationic pI = 93, 410–460 kDa glycoprotein that binds to the Fc portions of immunoglobulins in immune complexes to initiate complement activation via the classical pathway. In high resolution electron micrographs, the C1q molecule has a shape likened to a bouquet of six tulips, with the “stems” consisting largely of N-terminal collagen-like regions with repeating amino acid sequence (gly-x-y), and the “blossoms” consisting of six C-terminal globular protein domains. C1q consists of a total of eighteen 22–29 kDa polypeptide chains, with six copies each of the A, B, and C polypeptides, assembled as six disulfide linked A–B dimers and three disulfide linked C–C dimers [2]. The structurally similar A, B, and C peptides are encoded on the short arm of human chromosome 1.

Biological Function C1q serves as a recognition and regulatory protein for the complement cascade. The CLR of C1q is the scaffold for C1s and C1r, which together with C1q comprise the calcium-dependent C1 complex. Complement activation by immune complexes via the C1-dependent classical pathway is initiated by the binding of multiple Fc regions of IgG or IgM to the globular region of C1q, initiating a steric change in the CLR of C1q, and activation of the C1r and C1s esterases. Immune complexes or aggregated immunoglobulins are not unique in binding to C1q, since DNA, C-reactive protein, prion and amyloid proteins, and other substances also bind. C1q is related to the collectin family of proteins, which includes mannan binding protein, lung surfactant proteins A and D, and bovine conglutinin, each of which contains collagen-like domains adjacent to lectin binding domains. C1q participates in clearance of apoptotic material. Inherited C1q deficiency causes SLE, possibly because absence of C1q leads to failure of complement-mediated clearance of immunogenic apoptotic bodies. C1q also binds directly to a number of cellular receptors that are present on lymphocytes, fibroblasts, and other cell surfaces and intracellular membranes such as mitochondrial membranes. Binding of C1q to cell receptors may induce phagocytosis, chemotaxis, and pro-coagulant activity, although the physiological role of C1q in these processes is unclear.

Origin/Source C1q is isolated from blood. In the circulation, C1q exists primarily in the form of C1, a calcium-dependent complex with one molecule of C1q and two molecules of

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each of the C1 esterases C1r and C1s. The usual concentration of C1q in human serum is 60–200 mg/l.

Methods of Purification Several effective approaches exist for the isolation of C1q. General strategies include differential salt solubility, ion exchange chromatography followed by size-exclusion gel chromatography, or affinity isolation based on binding to IgG or DNA. Purified C1q is also available from several commercial sources. Purity of the antigen should be verified, since contamination with IgG and other plasma constituents is common. Removal of IgG can be achieved by passage over an anti-IgG or a staphylococcal protein A or protein G affinity column. Purification of C1q can also be improved by affinity chromatography on concanavalin A-agarose, with elution of C1q using alpha-methylglucopyranoside. CLR preparation: Enzymatic digestion of C1q allows preparation of its two major functional regions. The globular region (MW ∼39 kDa) can be prepared by digesting the CLR using collagenase. The CLR (MW ∼176 kDa) remains after enzymatic removal of the globular head of C1q by limited pepsin digestion (Figure 87.1), followed by gel chromatography. Under reducing conditions on SDS-PAGE analysis, the CLR polypeptides are approximately 16 and 14 kDa. The absence of binding of aggregated IgG and loss of hemolytic complement activity verify that the globular heads of C1q are destroyed. The epitopes recognized by autoantibodies to CLR of C1q are not well defined. According to most investigators, anti-C1q from patients with HUVS or SLE are not detected by Western blot, and the epitopes are probably conformational.

Immune Complex

Globular Heads

Collagen-like Region

Anti-C1q

FIGURE 87.1 Schematic drawing of the structure of C1q and antiC1q antibodies. The Fab 2 antigenbinding portion of antiC1q binds to epitopes in the collagen-like region (CLR) of C1q. In contrast, the globular heads of C1q are the binding sites for the Fc portions of IgG in immune complexes, as shown in the figure. The globular heads of C1q can be removed by limited pepsin digestion, as indicated by the dotted line, leaving the CLR of C1q.

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AUTOANTIBODY Definition Antibodies to C1q can be defined as immunoglobulins that bind to C1q via the antigen-binding region of the immunoglobulin molecules. Immunoglobulins, immunoglobulin aggregates, and immune complexes that bind to C1q via the Fc region of the immunoglobulin molecule should not be considered autoantibodies to C1q. Because the function of C1q involves binding to immune complexes and aggregates via their Fc portion, this distinction is important. Autoantibodies to C1q do not bind to C1q in solution; therefore, assays for anti-C1q must employ solid-phase or surface-bound antigen.

Pathogenic Role The association of anti-CLR with active lupus nephritis, especially proliferative forms of lupus nephritis associated with subendothelial immune deposits, suggested the possibility that anti-CLR is pathogenic. Anti-CLR is also associated with subendothelial deposits among patients with membranoproliferative glomerulonephritis (MPGN), and anti-CLR is most frequently found in patients with type I MPGN, which is characterized by the presence of these deposits. Anti-C1q contributes to the formation and/or persistence of subendothelial immune deposits by promoting aggregation of different C1q-containing immune complexes in the renal glomerular basement membrane [3]. Aggregation of C1q-bound complexes is enhanced by anti-CLR, leading to larger, longer-lasting, and possibly more pathogenic immune deposits. Anti-C1q antibodies from SLE patients probably do not influence complement activation directly, either in vitro or in vivo. IgG anti-CLR in both HUVS and SLE sera has been reported to be predominantly the IgG2 isotype, although all patients also have IgG1, IgG3, and/or IgG4 antibodies [4]. In contrast, IgG anti-CLR from patients with MPGN is predominantly of the IgG3 subclass. In other studies, isolated anti-CLR from SLE patients shows the same subclass distribution as normal IgG IgG1 > IgG2 > IgG3 > IgG4. The predominant class of anti-C1q in patients with rheumatoid vasculitis is IgA. Although anti-C1q is directed to the collagen-like region of C1q, no crossreactivity and no correlation between antibodies to human type II collagen and anti-CLR are found in patients with SLE or RA. Patients with HUVS may have severe pulmonary disease, but anti-CLR-containing sera from patients with HUVS does not react with the pulmonary surfactant proteins A or D, which are collectins with collagen-like regions, or with type IV collagen. It has been suggested that anti-C1q/anti-CLR could arise as an immunogen caused by the neoantigen expressed on C1q when C1q binds to immune complexes. The linkage of anti-C1q with a variety of immune complex–associated diseases makes that possibility attractive. Murine monoclonal antibodies have been developed which preferentially recognize C1q neoantigens that arise after C1q binds to immune complexes.

Genetics Antibodies to C1q are more frequent among lupus patients with the FcRIIa polymorphism, in which the amino acid in position 131 of the FcRIIa molecule

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contains an arginine, leading to decreased binding to IgG2 subclass molecules [5]. This polymorphism is also associated with higher risk of lupus nephritis and with decreased clearance of IgG2 subclass antibodies.

Methods of Detection ELISA technology is typically used to detect autoantibodies to the CLR of C1q. CLR (1–10 ug/ml concentration) is used to coat the solid phase. Sufficient concentrations of CLR are required to assure detectable binding of anti-CLR, since binding avidity characteristics differ in different sera, and tend to be lower in SLE than HUVS sera. After coating, plates are blocked, and then incubated with serum (typically diluted 1:50 to 1:200) in phosphate buffered saline (0.15 M NaCl) with Tween-20 to minimize non-specific adsorption of IgG. Appropriate dilutions of enzyme-linked  Fab 2 fragments of antibodies to the specific human immunoglobulins (IgG, IgA, or IgM) to be tested are added, incubated, then allowed to react with substrate for color development and recording of absorbance. A known positive serum is used at various dilutions to generate a calibration curve. When performing the assay, positive controls should include at least one positive serum, and negative controls should include normal serum as well as aggregated IgG as a surrogate immune complex. Investigators should consider testing diluted sera in wells that are blocked, but not coated with antigen, to detect non-specific binding to wells. An alternative approach toward assay of anti-C1q antibodies has been used in many studies. This approach is based on the finding that the binding of aggregated IgG (as a surrogate immune complex) to the globular head of C1q is totally eliminated in the presence of 1.0 M NaCl, whereas anti-CLR binding to C1q is retained with that salt concentration. Intact C1q is the target antigen on the solid phase, as used in the C1q solid-phase assay for immune complexes, but 1.0 M NaCl is substituted for 0.15 M NaCl during serum incubations and washes. The high-salt C1q-binding modification is sufficient to exclude the binding of aggregated IgG to C1q, but high salt concentration does not eliminate all binding of immune complexes to C1q [6]. Since high salt does not eliminate the binding to C1q of all IgG-containing immune complexes, the high-salt technique is not equivalent to the detection of anti-C1q using the isolated CLR of C1q, a technique that eliminates the potential binding of immune complexes. The high-salt method of detecting antiC1q is less rigorous and less definitive than using CLR as a target antigen. In the review of disease associations below, we have designated results using the 1.0 M NaCl modification as “anti-C1q,” and results using CLR as “anti-CLR.” Anti-C1q production has also been studied by the ELISPOT technique. This technique allows assay of antibody production at the single cell level, and holds promise for study of the short-term kinetics of anti-C1q production in response to therapy.

CLINICAL UTILITY Disease Associations Systemic lupus erythematosus: Several investigators have demonstrated the association of anti-C1q and anti-CLR with SLE (reviewed in [7]). Among SLE patients, the frequency of IgG anti-C1q in different series ranges from 17 to 46%, depending on the methods used and patient selection (Table 87.1). In contrast, the prevalence

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TABLE 87.1 Selected Studies of Prevalence of AntiC1q in Sera from Patients with SLE Method

Patient selection

Prevalence

Comments

Reference

Binding to C1q in 1.0 M NaCl

Positive tests for solid-phase C1q binding

15 of 15

Suggested that C1q-binding IgG was not an immune complex

Uwatoko et al. 1984

Binding of monomeric IgG to C1q

Patients with renal biopsies

Class IV: 11 of 14 (78.6%) Class III or IV+V: 6 of 11 (54.5%)

Binding of IgG to C1q was associated with proliferative glomerulonephritis

Wener et al. 1987

IgG binding to CLR

Nephrology and rheumatology patients with high prevalence of nephritis

31/68 (46%)

IgG binding to CLR

Unselected patients

7/20 (35%)

IgG antiCLR correlated highly r = 094 with C1q solid phase immune complex assay

Menzel et al. 1991

IgG binding to CLR

University hospital and clinics

48/174 (28%)

Some patients with mild or inactive disease

Wisnieski and Jones 1992

Binding of IgG or IgA to C1q in 1.0 M NaCl

University Rheumatology and Nephrology Clinics

IgG 30/88 (34%) IgA 0/88 (0%)

With nephritis 83% positive, without nephritis 21% positive p = 00001

Siegert et al. 1991 and 1992

Binding of IgG or IgA to C1q in 1.0 M NaCl

University Rheumatology and private clinics

IgG: 57/169 (34%) IgA: 8/169 (5%)

Younger patients more likely to have antiCLR

Siegert et al. 1993

Binding of IgG or IgA to C1q in 1.0 M NaCl

University center

IgG 10/60 (17%) IgA 6/60 (10%)

No correlation with antibodies to human type II collagen

Cook et al. 1994

Binding of IgG to C1q in 1.0 M NaCl

University center

50% (24/48)

100% (14/14) with active nephritis had antiC1q, including 3 in which antiC1q antedated LN

Trendelenburg 1999

Binding of IgG to C1q in 1.0 M NaCl

Hospital Nephrology Division

–

Correlated with lupus nephritis flares (sensitivity 87%, specificity 92%)

Moroni 2001

Binding of IgG to C1q in 1.0 M NaCl

Lupus referral center

49% (74/151)

Present in 74% with active lupus nephritis, 53% with inactive LN, and 32% without LN. Higher levels with active LN

Marto 2005 [10]

Wener et al. 1989

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of IgA antibodies to C1q is low in patients with SLE. Anti-C1q antibodies have generally been found with increased frequency among patients with lupus nephritis, compared to patients with non-renal lupus, with a frequency as high as 74% in patients with active lupus nephritis. Higher titers of anti-C1q are present in patients with active nephritis than in patients with inactive nephritis or without nephritis, and presence of anti-C1q in patients without active nephritis predicts renal flares. Elevated serum titers of anti-C1q tended to be associated with proliferative forms of lupus glomerulonephritis and subendothelial deposits of immune complexes, although anti-C1q has been found with multiple histologic types of lupus nephritis in some studies. It has been suggested that severe lupus nephritis does not occur in the absence of anti-C1q, particularly if anti-C1q and anti-dsDNA are both absent. Levels of anti-C1q are only weakly correlated with levels of anti-dsDNA, but tend to be associated with hypocomplementemia. Hypocomplementemic urticarial vasculitis syndrome: Antibodies to CLR are closely associated with the HUVS, a relatively rare condition that shares features, and may coexist, with SLE. Virtually all patients with active HUVS or SLE with coexisting HUVS have IgG anti-CLR in their serum, and the presence of anti-CLR may be considered a diagnostic component of HUVS [8]. Most patients with active HUVS have substantial elevations of anti-CLR, although in occasional patients the elevations may be only moderate. Patients with HUVS tend to have the highest levels of IgG anti-CLR of any diagnostic group. Rheumatoid arthritis (RA) and other rheumatic diseases: Patients with other rheumatic diseases may also have serum anti-C1q. Anti-C1q is found in few patients with uncomplicated RA, and most of those patients have low titers. In contrast, 77% of patients with rheumatoid vasculitis and Felty’s syndrome have serum antiC1q [9]. Interestingly, IgG anti-C1q was the dominant form of anti-C1q among patients with Felty’s syndrome (IgG in 76% of patients, IgA in 29%), whereas IgA anti-C1q predominated among patients with rheumatoid vasculitis (IgG in 32%, IgA in 68% of patients). Among sera of patients with primary Sjogren’s’ syndrome, 13% contain IgG anti-CLR. Other associations of anti-C1q with autoimmune rheumatic diseases are listed in Table 87.2. Renal diseases: Autoantibodies to C1q have been found in serum from a high proportion of patients with membranoproliferative glomerulonephritis (MPGN). Sera from patients with MPGN type I, with only subendothelial immune deposits in glomerular basement membranes, have a high prevalence of anti-C1q. Among patients with MPGN type II (with subendothelial and subepithelial electron dense deposits) or MPGN type III (with only subepithelial electron dense deposits), less than half of the patients have anti-CLR. Serial levels of anti-CLR do not parallel the disease course in MPGN patients. Patients with other renal diseases may also have anti-C1q (Table 87.2). Normal individuals and others: Neither IgG nor IgA anti-C1q is specific for any single diagnosis (see Table 87.2). Furthermore, normal individuals may have anti-C1q. As is true for many other autoantibodies, increased levels of anti-C1q are found in normal older individuals, and occasionally in younger normal subjects. Whereas only about 5% of randomly selected individuals in the 40–69 age ranges have IgG anti-C1q above the usual upper limit of a blood bank donor reference population, 18% of septuagenarians have elevated levels. Furthermore, anti-C1q in younger individuals with elevated levels are almost always minimally elevated above the upper limit of normal, but levels in elderly normal individuals may be elevated

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TABLE 87.2 Clinical Associations of AntiC1q Antibodies Established associations Systemic lupus erythematosus (SLE) Hypocomplementemic urticarial vasculitis syndrome (HUVS) Possible associations Autoimmune rheumatic diseases Rheumatoid arthritis (RA) Felty’s syndrome Rheumatoid vasculitis Sjögren’s syndrome (SS) Mixed connective tissue disease Polyarteritis nodosa Mixed cryoglobulinemia Renal disorders Focal glomerulosclerosis Membranoproliferative glomerulonephritis (MPGN) Anti-glomerular basement membrane nephritis Myasthenia gravis HIV infection Normal aging

substantially above the upper limit of normal. Non-specific binding may cause some false- positive results.

CONCLUSIONS IgG autoantibodies to the CLR of C1q are markers for HUVS. IgG anti-CLR are also frequent in patients with SLE, where they are associated with proliferative forms of glomerulonephritis. In SLE patients, anti-CLR, analogous to measurement of anti-dsDNA, is a useful marker for the progression of renal disease and for assessing disease activity. Anti-CLR is also present frequently in patients with membranoproliferative glomerulonephritis, especially type I. IgA anti-C1q is frequent in patients with rheumatoid vasculitis, but its diagnostic role in that condition remains unclear. C1q antibodies cause aggregation of C1q-containing immune complexes in the subendothelial portion of the glomerular basement membrane, and thus they augment the development of proliferative forms of glomerulonephritis.

TAKE-HOME MESSAGES • Autoantibodies to C1q are directed to the collagen-like region of C1q, and should be differentiated from immune complexes that bind the globular heads of the C1q molecule. • Anti-C1q is found in hypocomplementemic urticarial vasculitis, systemic lupus erythematosus, and membranoproliferative glomerulonephritis. • Anti-C1q is associated with proliferative forms of lupus nephritis, and absence of anti-C1q predicts the absence of severe proliferative lupus nephritis. • Anti-C1q serves as a prognostic and disease activity marker for proliferative lupus nephritis. • Anti-C1q is found enriched in the glomerular basement of kidneys from patients with lupus nephritis, and probably contributes to the pathogenesis.

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