Glomerular deposition of C1q and anti-C1q antibodies in mice following injection of antimouse C1q antibodies

Anti-C1q autoantibodies are present in the serum of patients with different autoimmune diseases such as systemic lupus erythematosus (SLE). The occurrence of these autoantibodies correlates with renal involvement. In the present study we examined whether injection of rabbit antimouse C1q antibodies in mice leads to deposition in kidneys. Injection of healthy mice with a single dose of rabbit IgG antimouse C1q antibodies resulted in deposition of both C1q and IgG anti-C1q in glomeruli. The pattern of deposition observed in the glomeruli of mice injected with antimouse C1q antibodies both at 24 h and 2 weeks was both glomerular basement membrane (GBM)-associated and mesangial. Injection of control IgG did not have a detectable effect on circulating C1q levels, and no deposition of either C1q or rabbit IgG was seen at 24 h. The deposition of rabbit antimouse C1q and C1q in glomeruli resulted in complement activation, as assessed by C3 deposition, and influx of leucocytes associated with albuminuria in some, but not all mice. In none of the control mice was albuminuria observed. This report is the first to show that anti-C1q antibodies deposit in the healthy glomerulus together with autologous C1q. This deposition is stable for at least 2 weeks, causes complement activation, leucocyte influx and can lead to mild albuminuria.     

Correlation between the renal C1q deposition and serum anti-C1q antibody: a potential role of anti-C1q antibody in lupus nephritis

The anti-C1q antibody has been shown to be associated with lupus patients with renal involvement. We conducted a study to determine the relationship between the serum anti-C1q titer and the renal deposition of C1q. The serum anti-C1q was measured in 26 healthy controls and 47 systemic lupus erythematosus (SLE) patients who were divided into 2 groups as non-nephritis and nephritis SLE. We analyzed the relationship between the anti-C1q titers and SLE, renal C1q staining and the WHO classification for lupus nephritis. The result revealed that the serum anti-C1q was present in 50.8% of the SLE patients, that its levels in those with renal involvement were significantly higher than in the normal control group (61.540 +/- 87.720 U/ml vs 15.750 +/- 2.530 U/ml, p = 0.005). Besides, the serum anti-C1q levels were higher in the patients with lupus nephritis with C1q deposition in the kidney tissue (66.038 +/- 91.141 U/ml vs 16.652 +/- 3.097 U/ml, p < 0.01). There seems to be evidence supporting that the autoantibody anti-C1q might play a pathogenic role in lupus nephritis.     

A method to differentiate between anti-C1q antibodies and C1q-binding immune complexes using collagenase-digested solid phase C1q.

A method for the detection of circulating immune complexes in the presence of autoantibodies to C1q is described. Solid phase C1q-digestion with bacterial collagenase results in the elimination of the collagen-like region of C1q. Binding of model immune complexes to this modified solid phase C1q is practically unaltered, while reactivity of anti-C1q antibodies is abolished by this procedure. In conjunction with an ELISA using the collagen-like region of C1q as antigen this modified C1q solid phase assay may be used to determine immune complexes and anti-C1q antibodies in the sera of patients with autoimmune rheumatic diseases.     

IgG4 antibodies from patients with asymptomatic bancroftian filariasis inhibit the binding of IgG1 and IgG2 to C1q in a Fc-Fc-dependent mechanism

A striking feature of lymphatic filariasis (LF) is the clinical heterogeneity among exposed individuals. While endemic normals (EN) remain free of infection despite constant exposure to the infective larvae, a small group of patients, generally microfilaria free (Mf-) develops severe pathology (CP) such as lymphedema or hydrocele. Another group of infected individuals remains asymptomatic while expressing large amounts of microfilariae (Mf+). This Mf+ group is characterized by an immune-suppressed profile with high levels of anti-inflammatory cytokines and elevated IgG4. This particular immunoglobulin is unable to activate the complement. The complement system plays a critical role in both innate and adaptive immunity. However, its importance and regulation during LF is not fully understood. Using affinity chromatography and solid-phase-enzyme-immunoassays, we investigated the ability of antibody isotypes from LF clinical groups to bind C1q, the first element of the complement’s classical pathway. The results indicate that while C1q is similarly expressed in all LF clinical groups, IgG1–2 in the plasma from Mf+ individuals presented significantly lower affinity to C1q compared to EN, Mf−, and CP. In addition, selective depletion of IgG4 significantly enhanced the affinity of IgG1–2 to C1q in Mf+ individuals. Strikingly, no effect was seen on the ability of IgG3 to bind C1q in the same conditions. More interestingly, papain-generated IgG4-Fc-portions interacted with Fc portions of IgG1–2 as revealed by far-western blot analysis. These data suggest that while being unable to bind C1q, IgG4 inhibits the first steps of the complement classical pathway by IgG1 or IgG2 via Fc-Fc interactions.

     

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.     

Platelet interactions with C1q in whole blood and in the presence of immune complexes or aggregated IgG.

Previous studies identified specific receptors for C1q on human blood platelets in purified systems using monomeric C1q. To assess the physiologic potential of platelet C1q receptors, C1q binding was evaluated in whole blood and in the presence of immune complexes or aggregated IgG. Blood was obtained from healthy volunteers and collected directly into EDTA (1 vol 100mM EDTA:9 vol whole blood) and purified, 125I-labeled C1q or 125I-C1q associated with albumin-anti-albumin immune complexes. Samples were incubated at 22 or 37 degrees C for 60 min, and total cell bound C1q and platelet associated C1q were quantified. Platelet-bound, monomeric C1q or immune complex-associated C1q represented 40-50% of total peripheral blood cell-associated C1q. C1q binding was unaffected by the incubation temperature, but the preincubation of 125I-C1q with immune complexes enhanced binding two- to threefold. This binding was partially inhibited by preincubating platelets with either the collagen-like amino-terminal fragments of C1q (c-C1q) or a monoclonal antibody Fab fragment recognizing platelet Fc receptors. A more complete inhibition was achieved if platelets were preincubated with both agents. Similar observations were made using washed platelets and 125I-C1q associated with aggregated IgG. The role of C1q and platelet C1q receptors in enhancing aggregated-IgG binding to platelets was further supported by experiments demonstrating increased 125I-aggregated IgG binding to platelets not only after preincubation of 125I-aggregated IgG with C1q but also following platelet preincubation with C1q. These data suggest that C1q receptors may participate in the localization and presentation of C1q-associated immune complexes on the platelet surface and demonstrate that platelets contribute significantly to the C1q binding activity of peripheral blood.     

抗 C1q 抗体及补体 C3、C4 在神经精神性狼疮诊断中的应用价值

目的:探讨抗C1q 抗体及补体C3、C4 在神经精神性狼疮诊断中的应用价值。方法:对22 例狼疮脑病及66例SLE 患者进行横断面研究。抗C1q 抗体采用ELISA 方法,C3 及C4 采用免疫比浊法检测。分析补体与狼疮脑病的临床表现相关性,采用Logistic 回归分析狼疮脑病的危险因素。结果:NPSLE 患者抗C1q 抗体水平明显高于非NPSLE 患者,补体C4明显低于非NPSLE 患者,采用ROC 曲线分析抗C1q 抗体诊断NPSLE 敏感性及特异性分别为63.6%、66.7%。单因素分析后发现抗C1q 抗体、抗核糖体P 蛋白抗体、抗核小体抗体与神经精神性狼疮相关,但多因素Logistic 回归分析并未发现其与狼疮脑病发生有关。补体C4 降低与SLE 患者脑血管意外发生有关。结论:抗C1q 抗体在狼疮脑病诊断中具有一定的诊断价值,且补体C4 可能参与狼疮脑病脑血管意外的发生。     

Carbodiimide crosslinking of human C1q and rabbit IgG

The water soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to covalently link carboxyl groups on rabbit IgG to lysyl groups on complement protein C1q. The interaction between C1q and IgG was disrupted by varying the pH, modifying essential residues in the IgG binding site of C1q and by reducing the interchain disulfides of IgG. Under each of these conditions the correlation found between binding and crosslinking indicated a strong requirement for the proteins to bind normally in order for crosslinking to occur. SDS-PAGE analysis of the crosslinked material showed a 210 kDa band consistent with one IgG crosslinked to two disulfide linked C1q chains. Blotting and autoradiography showed the crosslinking involved the A and/or B and C chains of C1q. The lysines flanking the intrachain half cystines are proposed as the likely candidates for crosslinking to IgG, thus delineating the immunoglobulin binding site of C1q.     

The IgG detected in the C1q solid-phase immune-complex assay is not always of immune-complex nature

The properties of the solid-phase C1q immune-complex assay as well as the nature of the IgG detected by this assay in patients' sera were investigated. Aggregated IgG was used as a model for immune complexes. Aggregated IgG bound to solid-phase C1q was detected by 125I-anti-IgG. Fluid-phase C1q (either in normal human serum or purified) neither inhibited the binding of aggregated IgG to solid-phase C1q nor dissociated bound aggregated IgG from the solid-phase C1q. Therefore, we concluded that the solid-phase C1q has a higher affinity for aggregated IgG than the fluid-phase C1q, probably because of the polymerization of the solid-phase C1q. To get more insight into the nature of the IgG detected by the C1q solid-phase assay in patients' sera, we investigated whether C4 and/or C3 were present on it. With the use of 125I-anti-C4 and 125I-anti-C3 instead of 125I-anti-IgG, C4 and C3, respectively, were easily detected on the aggregated IgG that had bound to the solid-phase C1q. The lower limit of detection of these assays was 30 micrograms aggregated IgG/ml of normal human serum. Sera of patients suffering from rheumatoid arthritis and systemic lupus erythematosus were tested with these assays and, despite positive results with 125I-anti-IgG, no positive results were obtained with either 125I-anti-C4 or 125I-anti-C3. So, on the IgG detected by the C1q solid-phase assay in patients' sera, neither C4 nor C3 are present. Furthermore, in five of the six sera tested, this IgG sedimented as monomeric IgG. Therefore, it seems unjustified to refer to this IgG as circulating immune complexes.     

Disulfide bridge formation between C1q and IgG in vitro

The globular heads of C1q are known to possess free-SH groups. Here we show that these groups, which are concealed in the native molecule, are exposed by interaction of C1q with dialysis membrane. During iodination, I+ and I2 oxidize these sulfhydryls to produce disulfide-linked C1q aggregates. Approximately 15% of C1q bound to immunoglobulin aggregates is resistant to high conductivity elution and reducing agent is required to release it. These data show that dialysis, adsorption to Ig and iodination of C1q result in structural and functional changes in the molecule, and suggest a mechanism by which these changes occur. Disulfide bridging between C1q and IgG in vitro suggests that this may be a normal physiological function of C1q for which the free cysteines of human, mouse and guinea pig C1q have been conserved.     

Anti-C1q antibodies in hepatitis C virus infection

Autoantibodies against C1q have been described in many immune-complex diseases including hypocomplementaemic urticarial vasculitis and systemic lupus erythematosus (SLE). No study has focused on the role of anti-C1q antibodies in hepatitis C virus (HCV) infection. The aim of this study was (i) to evaluate the prevalence of anti-C1q antibodies in HCV infection; and (ii) to analyse the association of anti-C1q antibodies with clinical and biological features of HCV-mixed cryoglobulinaemia (MC) vasculitis. We searched for anti-C1q antibodies using an enzyme-linked immunosorbent assay (ELISA) test in 111 HCV patients (75 had cryoglobulin and 23 systemic vasculitis), 60 SLE patients and 109 blood donors. Anti-C1q antibodies were detected in 26% of HCV patients compared to 10% of healthy donors (P < 0.01), and 38% in patients with SLE. Although there was a higher prevalence of anti-C1q antibodies among HCV patients with type III cryoglobulin (50%, P < 0.01), the overall prevalence of anti-C1q antibodies was similar in HCV patients being cryoglobulin-positive or cryoglobulin-negative (26%versus 25%, P = 0.98). A significant association was found between anti-C1q antibodies and low C4 fraction of complement (P < 0.05). No association was found between anti-C1q antibodies and HCV genotype, severity of liver disease or with specific clinical signs of HCV-MC vasculitis. This study shows an increased prevalence of anti-C1q antibodies in HCV-infected patients. Anti-C1q antibodies were associated with low C4 levels. No association was found between anti-C1q antibodies and HCV-MC vasculitis, nor between anti-C1q antibodies and cryoglobulinaemia.     

C1q rs292001 polymorphism and C1q antibodies in juvenile lupus and their relation to lupus nephritis

C1q deficiency is related strongly to systemic lupus erythematosus (SLE), but very few and inconsistent studies explored the single nucleotide polymorphisms of the C1q gene in relation to juvenile SLE (jSLE) and lupus nephritis (LN). The objective of this study was to analyse whether C1q rs 292001 polymorphism is associated with SLE and disease phenotype, especially nephritis, and to investigate the relation between this polymorphism and clinical data, treatment outcome, serum level of C1q protein and antibodies. Typing of C1q rs292001 polymorphism using restriction fragment length polymorphism and measuring serum levels of C1q protein and antibodies by enzyme-linked immunosorbent assay (ELISA) were performed for 130 children with SLE and 208 healthy controls. The A allele of C1q rs292001 was associated with jSLE and LN (P = 0·005 and 0·013, respectively) and the AA genotype was associated with jSLE (P = 0·036). Low serum levels of C1q protein were found in jSLE and LN (P < 0·001 and 0·009, respectively), and these levels were increased after treatment in patients with LN (P = 0·009) and active renal disease (P = 0·027). Higher titres of C1q antibodies were found in patients with LN (P = 0·015) and correlated negatively with C1q protein level (P < 0·001) and patient age (P = 0·04). The A allele and AA genotype of C1q rs292001 can be considered a susceptibility risk factor and the GG genotype could be considered protective for jSLE and LN in the studied cohort of Egyptian children. Decreased serum levels of C1q protein and increased titres of C1q antibodies may be involved in the pathogenesis of jSLE, especially LN.     

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.     

Enhancing effect of C1q on IgG monoclonal antibody binding to hapten

We have previously demonstrated that IgG antibody binding to microfilariae of Dirofilaria immitis increased in the presence of purified C1q. The present study was designed to examine the mechanism of the C1q effect using a system with an antihapten monoclonal antibody (MoAb) and a hapten as an antigen. Microtiter plates were coated with 4-hydroxy-3-nitrophenyl-acetyl (NP)-bovine serum albumin (BSA), and mouse anti-NP MoAb (IgG) was added in the presence of C1q. The amount of IgG which bound to NP-BSA increased with the addition of C1q (p less than 0.01) when the antibody had both specificity to the antigen and ability to fix C1q. The C1q effect, examined using two anti-NP MoAbs with different affinities, was more apparent with the low-affinity antibody (LAMoAb) than with the high-affinity (HAMoAb; percent enhancement of IgG binding was 19 vs. 12%). The C1q effect on LAMoAb binding was doubled when a small amount of HAMoAb was incubated with LAMoAb. The C1q effect on IgG binding might be operative in the early phase of infection, where a small amount of high-affinity antibody and a relatively large amount of low-affinity antibody are produced in the host.     

Detection of IgG Aggregates or Immune Complexes Using Solid-Phase C1q and Protein A-Rich Staphylococcus aureus as an Indicator System

A radioimmunoassay for detection of Clq-binding IgG aggregates and antigen-IgG antibody complexes is described The assay makes use of solid-phase Clq and ³²p-labelled protein A-rich Stphylococcusaureus as an indicator system. Both 19S and heavier IgG aggregates that fixed Clq were detected The sensitivity of the assay permitted detection of heavy (19–25S) IgG aggregates at a concentration of 8 μg/ml or less. The results indicated that detection of IgG in this assay is dependent on the degree of IgG polymerization and the molar ratio between the solid-phase Clq and the IgG polymers. Albumin-anti-albumin complexes, preformed at equilibrium with antibody to antigen molar ratios of 2:1 to 3:1 and at antigen concentrations of 25 to 40 μ g/ml. were also detectable using the described radioimmunoassay     

Pathogenic role of anti-C1q autoantibodies in the development of lupus nephritis--a hypothesis

A substantial proportion of patients with systemic lupus erythematosus (SLE) develop renal inflammatory disease, so-called lupus nephritis (LN). LN is a severe complication of SLE which is strongly associated with the presence of autoantibodies against C1q, the first component of the complement system, and other self-antigens (i.e. against DNA and nucleosomes) as well. In this review, the authors focus on anti-C1q autoantibodies and interpret the available data in order to explain how LN may develop and how anti-C1q autoantibodies contribute to its pathogenesis.     

The effect of VH residues 6 and 23 on IgG3 cryoprecipitation and glomerular deposition

MRL/lpr mice spontaneously develop a lupus-like autoimmune syndrome characterized by immunopathologic manifestations such as necrotizing vasculitis of the skin and glomerulonephritis. A feature of this autoimmune syndrome is the production of extremely large amounts of monoclonal IgG3 cryoglobulins. The structural basis of IgG3 cryoprecipitation is not well understood. Although the IgG3 isotype is necessary for cyroprecipitation, not all IgG3 antibodies cryoprecipitate. It has been postulated that electrostatic charge may be influential in cryoprecipitation. To investigate this problem, the V_H and V_L sequences of a panel of IgG3 cryoglobulins and non-cryoglobulins were compared, with particular attention to charged amino acid differences. At V_H residues 6 and 23 the cryoglobulins were more positively charged than their non-cryoglobulin counterparts. To analyze further the effect of charge on cryoprecipitation, the sequence of an IgG3 monoclonal cryoprecipitating rheumatoid factor was modified by site-directed mutagenesis. The more positive residues at V_H 6 and 23 present in some of the cryoglobulin antibodies were mutated to the more negative residues found in the non-cryoglobulins. The results show that V_H residue 6 affects cryoprecipitation while residue 23 does not. When injected into normal BALB/c mice, the unmutated antibody produced glomerular immune deposits and focal glomerulonephritis, whereas loss of cryoprecipitability by mutating residue 6 completely abrogated glomerular immune deposition and glomerular injury. In contrast, the mutation at residue 23 which retains cryoprecipitability reduced glomerular immune deposition and prevented glomerular injury.     

C1 inhibitor removes the entire C1qr2s2 complex from anti-C1Q monoclonal antibodies with low binding affinities.

Evidence is presented for a new C1 Inhibitor (C1 INH) function. C1 INH was capable of dislodging the entire C1qr2s2 complex from C1-activating substances that bound weakly to the globular heads of C1q. Two different mouse IgG1 monoclonal antibodies with different affinities for C1q globular heads were compared for their complement-activating properties in the presence of normal human serum. As expected the higher affinity monoclonal antibody (Qu) was more effective in binding C1q and causing C1-mediated C4b deposition. Unexpectedly, time responses of C1 (C1q) binding to immobilized 3C7 reached a peak then gradually decreased. However, C1q remained constantly bound to immobilized Qu. These results indicated that after C1 activation in human serum, the entire C1 complex (including C1q) was dislodged from 3C7, but not from immobilized Qu. The addition of purified C1 INH to purified C1, which had bound to immobilized 3C7, resulted in removal of C1 (C1q). Removal of the entire C1qr2s2 did not occur when C1 INH preparations were first neutralized by the addition of purified activated C1s. In summary, it is suggested that C1 INH plays a prominent role in dislodging the entire C1qr2s2 from immunoglobulin preparations which have a low binding affinity for the globular heads of C1q.