C1q solid-phase radioimmunoassay: evidence for detection of antibody directed against the collagen-like region of C1q in sera from patients with systemic lupus erythematosus.

In earlier studies we showed that the C1q-binding IgG in the sera from patients with systemic lupus erythematosus (SLE) tested by C1q solid-phase radioimmunoassay is cofractionated with monomeric IgG on gel filtration and mostly binds to C1q via the F(ab')2 region. In this study, we found that C1q, even when stripped of its immune complex-binding globular regions by pepsin digestion, retained a substantial part of its ability to bind IgG from SLE sera, suggesting that the collagen-like region of C1q is involved in binding to the SLE IgG. Heat-inactivation of C1q also failed to abolish its ability to bind IgG from SLE sera. In contrast, the binding of C1q to heat-aggregated IgG was completely abrogated by these treatments. In addition, the reaction of heat-aggregated IgG with the solid-phase C1q was markedly dependent on ionic strength whereas the binding of IgG from SLE sera with the solid-phase C1q persisted at high concentrations of salt. These findings suggest that the Clq-binding IgG in SLE sera is, at least in part, antibody directed against the collagen-like region of C1q.     

Detection of C1q-bearing immune complexes by a monoclonal anti-C1q ELISA system

A monoclonal antibody directed against the collagenous portion of human C1q was used to detect C1q-bearing immune complexes in patients with rheumatic disorders. Sera of patients with rheumatoid arthritis, systemic lupus erythematosus (SLE), osteoarthritis, as well as normal human sera (NHS) used as controls were tested in an ELISA system. C1q-bearing immune complexes were bound to a solid-phase monoclonal anti-C1q antibody, and detected with F(ab')2 antibodies to human IgG. Heat-aggregated human IgG was adjusted to the same concentration as the WHO standard for immune complexes and used for the standard curve in NHS. The mean value in NHS was 19.5 micrograms/ml equivalents of aggregated IgG. Using 2 SD over the mean as the upper limit for normal values, samples greater than 43 micrograms/ml were considered positive. Patients with osteoarthritis were negative; high levels of C1q-bearing immune complexes were detected in patients with rheumatoid arthritis (up to 800 micrograms/ml equivalents of aggregated IgG). With our assay C1q-bearing immune complexes were detected with high frequency (81%) in the sera of patients with rheumatoid arthritis, while a C1q solid-phase binding assay (C1q SPBA) revealed positive results only in 67% of rheumatoid arthritis sera. Compared to NHS, CH50 titers and C1q values of sera from patients with rheumatoid arthritis were frequently high. In contrast, the sera of SLE patients with low CH50 titers and low C1q levels had IgG immune complexes which could be detected only in the C1q-SPBA. C1q-bearing immune complexes were not detectable in the sera of patients with SLE. Since C1q triggers activation of the classical C pathway, this assay with monoclonal anti-C1q antibody appears to be useful for detecting immune complexes in rheumatoid arthritis patients with normal or elevated CH50 and C1q values, especially in the early stage of the disease.     

Detection of autoantibodies against the globular domain of human C1q in the sera of systemic lupus erythematosus patients

The anti-C1q antibodies present in systemic lupus erythematosus (SLE) patients' sera are associated with renal involvement and the titer of these autoantibodies correlates with the clinical activity of the disease. It has previously been shown that anti-C1q antibodies bind neo-epitopes within the collagen region of human C1q. Evidence that these polyclonal autoantibodies recognize epitopes within the globular domain (gC1q) of the molecule has not been documented. In this study, we screened, using ELISA, a number of sera from SLE patients for the presence of anti-gC1q autoantibodies using recombinant globular head regions of individual A (ghA), B (ghB) and C (ghC) chains of human C1q. The recombinant proteins were used as test antigens to determine the levels of autoantibodies directed against ghA, ghB and ghC. SLE sera, containing high levels of anti-C1q antibodies, showed differentially increased binding towards ghA and ghB, which suggested that the gC1q domain can also be target of anti-C1q antibodies generated in SLE patients. Such antibodies can have severe pathophysiological consequences since these are likely to further impair the ability of C1q to clear immune complexes.     

Autoantibodies against mannose-binding lectin in systemic lupus erythematosus

In systemic lupus erythematosus (SLE), autoantibodies directed against complement components of the classical pathway, especially against C1q, are associated with severe disease and are of prognostic value for flares of lupus nephritis. Mannose-binding lectin (MBL), the recognition unit of the MBL pathway of complement activation, has structural similarities to C1q. Deficiencies of MBL have been shown to predispose to the development of SLE and to influence the course of the disease. We hypothesized that the presence of autoantibodies to MBL, analogous to autoantibodies to C1q in patients with SLE, may contribute to disease development. The occurrence of anti-MBL autoantibodies was assessed by enzyme-linked immunosorbent assay (ELISA) of 68 serum samples from 20 patients with SLE and in serum from 70 healthy controls. Levels of antibodies directed against MBL were significantly higher in patients with SLE compared to healthy subjects. No significant difference was found between patients with active disease compared to those with inactive disease. While the occurrence of anti-C1q autoantibodies was associated with renal involvement, no such relationship was found for anti-MBL autoantibodies. A significant correlation was found between anti-MBL and anti-C1q antibody levels. The level of anti-MBL antibodies was negatively correlated with MBL-complex activity of circulating MBL. Anti-MBL autoantibodies were of the immunoglobulin G (IgG) isotype and the binding site of IgG anti-MBL was located in the F(ab')2 portion. We conclude that anti-MBL are present in sera from SLE patients and influence the functional activity of MBL.     

Anti-fibronectin autoantibodies in systemic lupus erythematosus, rheumatoid polyarthritis, and various viral or bacterial infectious diseases

A micro-enzyme linked immunosorbent assay (ELISA) aimed at detecting anti-fibronectin (anti-Fn) antibodies has been developed and standardized. Fifty sera from systemic lupus erythematosus (SLE), 50 from rheumatoid arthritis (RA) patients, as well as 200 sera from patients with bacterial or viral infections were assayed for the presence of anti-Fn autoantibodies. The IgG fractions of three representative positive sera (1 SLE, 1 RA and 1 streptococcal endocarditis) were digested with pepsin and the resulting F(ab')2 fragment assayed in the test. The presence of the anti-Fn activity in these fragments as well as lack of correlation in individual sera between the level of anti-Fn (as determined by ELISA) and that of Ig or immune complexes, suggest that our anti-Fn autoantibodies are indeed detected in our assay. The meaning of these antibodies, which were also found with bacterial and viral infections is discussed within the frame of the fibronectin biological properties.     

Anti-endothelial cell antibodies in patients with rheumatoid arthritis complicated by vasculitis.

IgG antibodies reactive with human umbilical vein endothelial cells were found in 19 out of 28 patients with rheumatoid vasculitis (RV), in four out of 24 patients with rheumatoid arthritis (RA), in seven out of 10 patients with systemic lupus erythematosus (SLE), but not in healthy donors. In four patients with RV who were followed longitudinally, regression of vasculitic episodes coincided with decreasing titres of anti-endothelial antibodies (AEA). Binding activity to endothelial cells was observed in intact IgG and F(ab')2 fragments of IgG. AEA activity was unrelated to antibodies against nuclear, blood group or major histocompatibility complex antigens and did not involve immune complexes. AEA activity was not specific for endothelial cells since the AEA-positive sera and the IgG fractions prepared from these sera also reacted with fibroblasts. Adsorption of positive sera and corresponding IgG fractions with endothelial cells decreased the IgG binding reactivity on both fibroblasts and endothelial cells. These findings show that RV patients have IgG-AEA, and suggest that these antibodies may play a role in the pathogenesis of the disease.     

Detection of circulating immune complexes with polyethylene glycol precipitation F(ab')2 anti-C3 ELISA

A polyethylene glycol (PEG) precipitation F(ab')2 anti-C3 ELISA for the detection of complement-fixing IgG circulating immune complexes (CIC) is described. For this assay, test sera were treated with 3.5% PEG and then measured with F(ab')2 anti-C3 ELISA. The lower detection limit was 4 micrograms/ml of heat aggregated human IgG (HAHG). Intra-assay coefficient of variation (CV) was 4.9-8.3%. High levels of CIC are found in the sera of patients with systemic lupus erythematosus (SLE), hepatitis B and stomach cancer.     

C1q solid-phase radioimmunoassay: binding properties of solid-phase C1q and evidence that C1q-binding IgG complexes in systemic lupus erythematosus are not bound to endogenous C1q

The binding properties of C1q solid-phase radioimmunoassay (C1q SPRIA) were examined, using heat-aggregated IgG (HAG) as the model of immune complexes (IC). The free, liquid-phase C1q, which was added to the C1q-coated tubes prior to the addition of HAG, had little inhibitory effect on binding of HAG to the solid-phase C1q, suggesting that the solid-phase C1q has a higher affinity for HAG than the liquid-phase C1q. On the other hand, more than 60% inhibition was seen when HAG was preincubated with the liquid-phase C1q. These binding properties of HAG to the solid-phase C1q in the presence of the liquid-phase C1q were not essentially altered by the heat inactivation or the addition of EDTA, suggesting that these pretreatments are not essential in C1q SPRIA. Next, in similar kinds of experiments, the binding properties of C1q-binding IgG complexes in SLE sera were investigated. In contrast to HAG, the binding capacity of IgG complexes in SLE sera to the solid-phase C1q was not inhibited by the preincubation with excess liquid-phase C1q. These findings suggest that C1q-binding IgG complexes in SLE sera detected by C1q SPRIA may not be bound to endogenous C1q in the circulation.     

Parallelism of serum anti-F(ab')2 and anti-cationic IgG reactivities in patients with systemic lupus erythematosus

Cationic anti-DNA antibodies may be related to glomerular injury in murine lupus nephritis or in patients with systemic lupus erythematosus (SLE). Therefore, anti-cationic antibodies in SLE could include antibodies with regulatory function on such pathogenic cationic molecules. Since anti-F(ab')2 antibodies may be involved in the idiotype control of anti-DNA antibodies in some patients with inactive SLE, the present study was aimed to determine if SLE patients with significant serum levels of anti-F(ab')2 produce antibodies reacting with cationic IgG molecules. Three SLE sera with high titers of anti-F(ab')2 antibodies were individually adsorbed by sequential affinity chromatography on three Sepharose columns coupling normal IgG from Cohn Fraction II, pooled cationic IgG myeloma paraproteins displaying idiotypic anti-DNA markers (F4 and 8.12), and F(ab')2 fragment from allogeneic IgG, respectively. Eluates obtained from cationic IgG adsorption showed predominant anti-F(ab')2 reactivity. A similar profile was also detected in a serum from a normal control donor with high levels of anti-F(ab')2. Biotinylation of anti-cationic eluates showed that such antibodies were significantly more reactive with cationic than anionic or neutral IgG, confirming their apparent affinity for positively charged antigens on IgG molecules. Since anti-cationic absorptions were able to remove the anti-F(ab')2 activities in the SLE sera studied, it is possible that anti-cationic antibodies could function as immunoregulatory antibodies in the idiotypic control of some SLE autoreactive phenomena, including glomerular anti-DNA deposition.     

Quantitative immunoassay for IgA class circulating immune complexes using solid phase Facb fragment of anti-C3

A quantitative assay of IgA class circulating immune complexes (IgA-CIC) by a solid phase anti-C3 enzyme immunoassay (anti-C3 EIA) is described. A stable and reproducible standard for determination of IgA-CIC was prepared successfully by chemical binding of complement C3 to human serum IgA. Two of 27 sera from patients with systemic lupus erythematosus (SLE), however, contained high concentrations of IgA class anti-F(ab')2 antibodies that caused false positive results when the F(ab')2 of anti-C3 was used for EIA. Solid phase Facb of anti-C3 was found to eliminate the false positive results caused by IgA class anti-F(ab')2 and IgA class rheumatoid factor. Good reproducibility and recovery were observed with this Facb anti-C3 EIA using the IgA-C3, a stable standard material, and so this method should be useful clinically in elucidating the role of IgA-CIC.     

IgG naturally occurring antibodies stabilize and promote the generation of the alternative complement pathway C3 convertase

Normal human IgG contains naturally occurring anti-C3 antibodies (anti-C3 NAbs) that have been proposed to regulate complement amplification. Here, we report a novel procedure for anti-C3 NAb purification. Pooled human IgG was fractionated on a DEAE column prior to affinity chromatography on IgG and then on C3. Anti-C3 NAbs co-purified with anti-F(ab')2 NAbs. In a refined protocol, IgG fractions were absorbed on Fc, F(ab')2, and C3, which allowed to isolate the directly accessible NAbs and to remove IgG hinge-region-specific NAbs. Since a substantial fraction of total anti-C3 NAbs in whole IgG pre-existed as complexes, IgG that did not bind to the three affinity columns was treated with urea and the affinity chromatography repeated to collect the dissociated NAbs. The urea-accessible anti-F(ab')2 NAbs were rather pure but anti-C3 NAbs yet contained substantial amounts of anti-F(ab')2 NAbs. Anti-C3 NAbs showed up to 400-fold and anti-F(ab')2 NAbs, up to 30-fold enrichment as compared to pooled normal human IgG. Anti-C3 NAb preparations exhibited nephritic factor activity that was up to 60 times stronger than that of total IgG from a patient with membranoproliferative glomerulonephritis type 2. In addition, anti-C3 NAbs promoted C3 convertase generation, when added to the convertase precursor or during convertase assembly, suggesting a non-nephritic-factor mechanism. Factors H and I reduced the overall level of activity but had no influence on the NAb dose-response curve meaning that NAbs did not interfere with factor H binding. Convertase promoting activity during assembly correlated with the content of anti-C3 NAbs in NAb complexes. In conclusion, anti-C3 NAbs associated with framework-specific anti-idiotypic NAbs stabilize C3 convertase and promote its generation but their activity is compensated for in whole IgG.     

Antibodies against C1q in anti-glomerular basement membrane nephritis.

The prevalence of antibodies against the collagen-like region of the subcomponent of the first component of complement, C1q, was investigated in 11 patients with anti-glomerular basement membrane (GBM) nephritis. Anti-C1q antibodies (anti-C1qAb) were detected in seven patients. IgG anti-C1qAb were found in four and IgA anti-C1qAb in five patients. During follow up of the patients a relationship was observed between the levels of IgG anti-C1qAb and the levels of anti-GBM antibodies (anti-GBMAb). Gelfiltration experiments indicated that both IgG anti-C1qAb as well as IgG anti-GBMAb were monomeric and that binding also occurred with the F(ab')2 fragments of the antibodies. Although anti-C1qAb and anti-GBMAb are both directed against a collagen-like structure, it was demonstrated by means of inhibition experiments that anti-C1qAb and anti-GBMAb are directed against different antigenic sites. Comparison of patients with anti-GBM nephritis with and without anti-C1qAb revealed that there were no differences in disease activity or disease severity. Therefore, the results of this study suggest that anti-C1qAb do not play a direct pathogenetic role in anti-GBM nephritis.     

Current situation and problems in the estimation of circulating immune complexes]

Some methods employing murine monoclonal antibodies have been developed for the estimation of circulating immune complexes (ICs). In the assays using monoclonal antibodies to C1q and C3d, ICs attached by reaction of C1q or C3d with the corresponding antibodies are detected by enzyme-labelled anti-IgG antibody. The murine monoclonal rheumatoid factor (RF) of IgG class is employed in the assay for detection of ICs. ICs reacted with the RF on the solid phase are further detected by the reaction with the second anti-IgG antibody labelled with the enzyme. The anti-C1q antibody in the sera as well as ICs produces positive reactions in the solid phase C1q assay, the assays using monoclonal antibodies are recommended for use in the detection of circulating ICs. In the pretreatment of serum samples, heating at 56 degrees C induces aggregation of IgG to produce a positive reaction by these sensitive assays, and the addition of EDTA-Na2 increases free C1q detached from C1 to induce increased binding to IgG. Reactions of aggregated IgG with RF and C1q in the fluid phase inhibit the following binding of monoclonal RF and anti-C1q antibody on the solid phase. Sera of patients with SLE were examined for CH50, anti-DNA antibody and ICs. The levels of ICs determined by the anti-C1q and C3d antibody assay did not correlate with other parameters. Positivity of ICs was unexpectedly lower in SLE sera. To evaluate the significance of the estimation of ICs, more data must be analyzed by these methods.     

Isolation of human anti-idiotypes broadly cross reactive with anti-dsDNA antibodies from patients with Systemic lupus erythematosus

Antibodies to double stranded (ds)DNA play a central role in clinical diagnosis and disease expression in Systemic lupus erythematosus (SLE). This paper describes the isolation of anti-idiotype reagents (anti/antidsDNA) from four SLE sera and the demonstration of broad and quantitatively similar cross reactivity to both polyclonal and monoclonal anti-dsDNA antibodies isolated from SLE patients. Seven affinity-purified polyclonal and three monoclonal human anti-dsDNA preparations reacted preferentially with anti-idiotype F(ab')(2) coated plates compared to normal immunoglobulin (Ig)G F(ab')(2) coated plates in ELISA. In contrast, autoantibodies of other specificities (anti-Ro/SSA, anti-La/SSB, and anti-U(1)RNP) reacted equally with anti/anti-dsDNA F(ab')(2) and normal IgG F(ab')(2) coated plates. Such anti-idiotypic antibodies could play a significant role in the regulation of anti-dsDNA antibody levels in SLE.     

Human Autoantibodies Against C1q: Lack of Cross-Reactivity with the Collectins Mannan-Binding Protein, Lung Surfactant Protein A and Bovine Conglutinin

The collectins, a group of humoral C-type lectins, have globular and collagen-like regions and share structural features with the complement protein C1q. The question was asked if autoantibodies to the collagen-like region of C1q (anti-C1qCLR) might cross-react with collectins, such as mannan-binding protein (MBP), lung surfactant protein A (SP-A) and bovine conglutinin (BK). Anti-C1qCLR antibodies of the systemic lupus erythematosus (SLE) type and anti-C1qCLR antibodies of the hypocomplementemic urticarial vasculitis syndrome (HUVS) type were investigated. Cross-absorption and elution experiments combined with antibody detection by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis gave no evidence of cross-reactive anti-C1qCLR antibodies. However, one serum with HUVS type anti-C1qCLR antibodies contained anti-MBP antibodies that were cross-reactive with SP-A. Judging from results of ELISA inhibition experiments and immunoblot analysis, four SLE sera contained antibodies to native BK, while two sera with HUVS type anti-C1qCLR antibodies contained antibodies to epitopes of denatured BK. This might imply that autoimmunity to collagen-like structures is not restricted to C1qCLR in HUVS and HUVS/SLE overlap syndromes.     

Immune deposition of C1q and anti-C1q antibodies in the kidney is dependent on the presence of glomerular IgG

Anti-C1q autoantibodies are found frequently in patients with Systemic Lupus Erythematosus (SLE) and several studies indicate that these autoantibodies are associated with renal involvement. We have shown earlier that administration of anti-C1q antibodies to normal BALB/c mice results in the deposition of these antibodies and C1q in the kidney. In the present study we have investigated which factors are essential for this C1q-anti-C1q deposition. Injection of anti-C1q antibodies in C57BL/6 mice results in deposition of both C1q and anti-C1q in glomeruli, while administration of equal concentrations of anti-C1q to immunoglobulin deficient Rag2-/- mice did not result in deposition of anti-C1q antibodies. Analysis of renal sections of naive Rag2-/- mice revealed absence of mouse IgG and C1q in the glomeruli, while circulating C1q was within normal levels. Reconstitution of Rag2-/- mice with IgG, either by injection with purified mouse IgG or by splenocyte transfer, resulted in restored localization of mouse IgG together with C1q in the kidney. Subsequent injection of anti-C1q antibodies in these IgG reconstituted mice resulted in clear deposition of C1q together with anti-C1q in the kidneys comparable to that found in C57BL/6 mice receiving anti-C1q. We propose that the continuous presence of serum derived non-immune IgG in the glomerulus serves as a target for low affinity interactions with C1q, which then can serve as antigen for anti-C1q antibodies. Therefore we hypothesize that high and fluctuating levels of IgG as observed in patients with SLE may contribute to flares of renal inflammation in those patients with anti-C1q autoantibodies.     

Evaluation of anti-C1q capture assay for detecting circulating immune complexes and comparison with polyethylene glycol-immunoglobulin G, C1q-binding, and Raji cell methods.

An anti-C1q capture method kit (C1q-immunoglobulin G [IgG]) (Ortho Diagnostics, Inc., Raritan, N.J.) for measuring circulating immune complexes (CIC) was evaluated. The kit showed poor diagnostic sensitivity (P less than 0.005) for identifying CIC in patients with systemic lupus, rheumatoid arthritis, and bacterial endocarditis, as compared with polyethylene glycol-IgG and Raji cell tests (12, 24, and 24 positive, respectively, of 31 patients). Of the patients who were positive with the C1q-IgG test, 25% showed discrepancies when their results were compared with the polyethylene glycol-IgG and C1q-binding test results. Gel filtration chromatography of two of these discrepant sera showed the only peak of C1q-IgG activity to be associated with monomeric IgG (molecular weight, less than 200,000). We concluded that the kit method may be measuring substances other than CIC in some sera, because molecules of C1q attached to IgG should exhibit a molecular weight of greater than 500,000.     

Correlation of C3d fixing circulating immune complexes with disease activity and clinical parameters in patients with systemic lupus erythematosus

Using anti-C3d as a solid phase reagent, C3d fixing circulating immune complexes (CIC) were detected in sera from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis, membranous nephropathy and IgA nephropathy. Particularly, sera from SLE showed the highest CIC levels and highest incidence of positivity among these diseases. In the 51 serum samples from 48 patients with SLE we studied, the CIC detected by the anti-C3d assay correlated well (P less than 0.01) with the CIC detected by the solid phase C1q assay, but not with those detected by the conglutinin assay. In addition, the CIC detected by the anti-C3d assay correlated more significantly (P less than 0.001) with disease activity, as well as some clinical parameters (serum anti-dsDNA antibodies, CH50 and C3 levels) than CIC detected by the other two assays of SLE sera. The anti-C3d binding materials were found to be of intermediate (8-19S) and small (7S) sizes in a small number of SLE sera which we analysed.     

Immune complexes in Hodgkin's disease: isolation, immunochemical and physico-chemical analysis.

Immune complexes (IC) present in sera from patients with Hodgkin's disease (HD) were isolated using three different affinity columns: C1q-degalan, anti-C1q sepharose and conglutinin (K)-degalan. The isolated IC were analysed by immunoprecipitation, SDS-PAGE and sucrose density gradients and compared with IC similarly isolated from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and in vitro prepared BSA-anti-BSA complexes. Isolated material from each disease, and BSA-anti-BSA complexes contained proteins compatible with true immune complexes--IgM, IgG, C1q and C3 breakdown components. Albumin, fibronectin and CRP, whose affinity for IgM, C1q and C3 are known, were co-isolated along with IC material. The size of isolated IC in HD ranged from 8-40S on sucrose density gradients. Despite the operational difference in detecting and isolating HD complexes via the C1q ligand (C1q-degalan or anti-C1q column) and C3bi (K-degalan), material purified by both methods showed remarkable similarity on SDS-PAGE and immunoprecipitation analysis. Although IC isolated from different diseases showed disparate banding patterns on SDS-PAGE this was attributed to a variation in the relative concentrations of constituent proteins--IgM, IgG and C3 breakdown products. IgM, IgG and C3 bind loosely, and non-specifically, to macromolecular aggregates formed around immune complexes. Using the anti-C1q column, most of this material could be eluted using 0.02M EDTA. Least protein, yet the most specific for antigen and antibody was eluted at pH 3.0.     

Identification of HLA-linked antigens by pregnancy-associated non-cytotoxic alloantisera

Non-cytotoxic sera obtained from post-partum primiparous and multiparous women were examined by a rosette inhibition technique for the presence of antibodies mediating blockade of human B lymphocyte Fc receptors. Selective activity was demonstrated against a panel of normal human B lymphocytes and lymphocytes from patients with chronic lymphocytic leukaemia (CLL). A pattern of specific activity was found in sera and in their IgG fractions, which was not accounted for by antibodies directed to known HLA-A, -B or -DR antigens. Several sera were identified with selective activity in this assay. As the results of testing sera in a direct binding assay correlated with those of the EA inhibition assay, and since EA inhibitory activity occurred in F(ab')2 fractions of sera, it is possible that these non-cytotoxic antibodies bind directly to B cell surface antigens. Sera may therefore have been identified which possess antibodies to hitherto undefined HLA antigens.