Anti-C1q autoantibodies specific against the globular domain of the C1qB-chain from patient with lupus nephritis inhibit C1q binding to IgG and CRP

Lupus nephritis is one of the most severe manifestations of systemic lupus erythematosus. Higher titers of serum anti-C1q autoantibodies correlate with disease activity in patients with lupus nephritis. Anti-C1q autoantibodies have been shown to bind neo-epitopes within the collagen region of human C1q. In a preliminary study, we recently reported that the anti-C1q autoantibodies could also recognize epitopes within the globular domain (gC1q) of the C1q molecule. Here, 38 sera from patients with renal biopsy-proven lupus nephritis were screened 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. We isolated anti-gC1q autoantibodies from three selected patients. Human C1q was pre-incubated with increasing concentrations of the isolated anti-ghA, anti-ghB or anti-ghC autoantibodies and its binding to different C1q target molecules such as IgG and CRP was then evaluated. Anti-ghB, but not anti-ghA and anti-ghC autoantibodies, markedly inhibited C1q interaction with IgG as well as CRP. These results appear to suggest that the anti-ghB autoantibodies may partially induce acquired functional C1q deficiency and thus may interfere with the biological function of C1q.     

Analysis of IgG subclasses and IgE antibodies across the clinical spectrum of bancroftian filariasis in an endemic area

Analysis of immune response in individuals with different clinical manifestations living in filaria endemic area will be of interest to understand the immunological events associated with the disease development in filaria infected endemic population. The levels of four IgG subclasses and IgE antibodies against Brugia malayi microfilarial excretory-secretory (Bm mf ES) antigen as well as circulating filarial antigen level were evaluated in 84 individuals belonging to different groups in an endemic area for bancroftian filariasis. Microfilaraemics showed significantly elevated levels of IgG4 and IgG3 antibodies compared to endemic normals (P < 0.02). As many as 70% of this group were positive for IgG4 & IgG3 antibodies. While Acute filarial cases had pronounced IgG1 antibodies(P < 0.001), the Grade I chronic cases showed higher levels of IgG3 and IgG4 antibodies (P < 0.02), Occult filarial cases had higher levels of IgG4 and IgG3 (P < 0.02) and also of IgG4 antibodies (P < 0.001). IgE antibodies were found to be elevated in microfilaraemics as well as other clinical filarial groups. Circulating filarial antigen was detected in 95% of microfilaraemics, 60% of acute cases, 75% to 90% of different grades of chronic filarial cases, 100% of occult cases and none of the endemic normals.     

Bullous pemphigoid IgG2 and IgG4 antibodies have a capability to inhibit a transient increase of intracellular Ca2+ induced by bullous pemphigoid IgG1 antibody.

To elucidate the role of Bullous pemphigoid (BP) IgG subclasses in the transmembrane signal transduction of keratinocytes, we examined whether BP IgG2 or IgG4 inhibits the intracellular Ca2+ concentration ([Ca2+]i) increase induced by BP IgG1. IgG2 from one of five BP patients inhibited the increase of [Ca2+]i in human squamous cell carcinoma cell line; DJM-1 cells induced by BP IgG1. IgG4 from two of four BP patients inhibited the increase of [Ca2+]i induced by BP IgG1. In addition, a two fold quantity of IgG2 inhibited the increase of [Ca2+]i induced by BP IgG1. A two fold quantity of IgG4 from two of five BP patients inhibited the increase of [Ca2+]i induced by BP IgG1. These results indicate that BP IgG2 and IgG4 have a capability to inhibit the increase of [Ca2+]i induced by BP IgG1.     

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.     

Identification of circulating parasite antigen in patients with bancroftian filariasis.

Because many cases of lymphatic filariasis cannot be diagnosed either clinically or by immunodiagnostic test based on antibody detection, recent efforts have been more directed towards developing methods for detecting parasite antigen in the blood or urine. Using a solid phase (Sepharose 4B) two-site immunoradiometric assay (IRMA) employing hyperimmune rabbit antifilarial antisera, we have previously shown (Hamilton et al., 1984) that essentially all cases of patent (ie. microfilaremic) infection in patients with bancroftian filariasis can be detected by this semi-quantitative assay as well as some individuals with amicrofilaremic (i.e., 'cryptic') infection. The present communication reports the results of studies that identify a prominent circulating antigen detected by this IRMA in sera from patients with microfilaremia. The antigen was eluted from Sepharose-bound rabbit polyclonal antiserum that had been reacted with known antigen positive sera. It was run in SDS-PAGE, blotted to nitrocellulose paper and identified autoradiographically using 125I-labelled rabbit antifilarial antiserum. Its high molecular weight (approximately 200 kD), stability to acid and boiling, and sensitivity to pronase and periodate suggest its being a glycoprotein. Isolation of this antigen will permit the development of specific reagents (such as monoclonal antibodies) which should enhance both the sensitivity and utility of the currently available antigen detection systems.     

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.     

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.     

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.     

Lack of IgG4 antibody response to carbohydrate antigens in patients with lymphatic filariasis.

It has been suggested that humans are genetically restricted from making IgG4 antibody responses to carbohydrate antigens. To test this hypothesis we examined sera from 35 patients with bancroftian filariasis (an infection known to induce very high levels of IgG4 antibodies to the parasite and known to be associated with repeated streptococcal infections) as well as from 15 normal individuals for their IgG and IgG subclass responses to streptococcal protein [streptolysin-O (SO), deoxyribonuclease B (DB)] and carbohydrate [group A carbohydrate (GAC)] antigens. Levels of IgG antibodies to all three antigens were found to be significantly higher in the filariasis patients compared to normals (P less than 0.01), and the subclass composition of these antibodies proved heterogenous. Although responses to all three antigens included IgG1, IgG2 and IgG3 antibodies and although IgG4 responses to the proteins SO and DB were significantly higher in the filariasis patients than in normals (P less than 0.001), more importantly there were no detectable anti-GAC IgG4 antibodies in either study group. These observations, coupled with our earlier finding of the absence of IgG4 responses to phosphocholine (PC) in patients with lymphatic filariasis, suggest that even the chronic antigenic stimulation of filarial helminth infection, which leads to very prominent IgG4 responses to protein antigens, cannot overcome the genetic restriction in humans for making IgG4 antibodies to carbohydrate antigens, whether of parasite or non-parasite origin.     

C1q binding to antibody-coated cells: Predictions from a simple multivalent binding model

We have derived a simple mathematical model describing the multivalent binding of Clq to IgG antibody-coated target cells. The model contains the assumptions that the Clq is hexavalent for IgG, that the cell-bound antibody is mobile in the plane of the plasma membrane, and that the six Clq subunits bind sequentially to the cell-surface antibody. We have calculated how Clq would bind at equilibrium to cells coated with different amounts of antibody, and how free monomeric IgG would inhibit this interaction. The model predicts that the degree of binding of Clq to the cells will be strongly enhanced by increasing the cell surface antibody density, but that even small amounts of cell-bound antibody will render the target cells capable of binding Clq.Scatchard plots of Clq binding invariably show negative curvature due to a fall in the density of free antibody sites as the cell-bound antibody becomes saturated with Clq. Over a wide range of cell-bound antibody densities little of the cell-associated Clq is bound pentavalently or hexavalently. While Clq binding is easily inhibitable by free IgG at low levels of cell-bound antibody, this inhibition is progressively overcome by increasing the density of antibody on the cell. This reflects the inherent property of multivalent ligands such as Clq to seek out regions of high site density.     

IgM and IgG antibodies to hepatitis C virus in patients with mixed cryoglobulinaemia

To assess the relationship between hepatitis C virus (HCV) infection and essential mixed cryoglobulinaemia (EMC), sera from 23 patients with EMC were tested for IgG and IgM antibodies to HCV antigens and for HCV RNA. Quantitative HCV antibody studies were performed on scrum and purified cryoglobulin fractions. HCV antibodies of both IgG and IgM class were found in 22 (96%) patients. Ten of these were also HCV-RNA positives. Higher litres of anti-HCV IgM were present in the 11 patients with evidence of liver damage. Anti-HCV IgG antibodies were shown to be concentrated in the IgG fraction of cryoglobulins in all eight patients studied. These results strongly suggest a role for HCV in the pathogenesis of EMC.     

C1q binding to chimeric monoclonal IgG3 antibodies consisting of mouse variable regions and human constant regions with shortened hinge containing 15 to 47 amino acids

We have constructed four different deletion mutants of a chimeric mouse-human IgG3 anti-(4-hydroxy-3-nitrophenyl)acetyl/(5-iodo-4 hydroxy-3 nitrophenyl) acetyl (NP/NIP) antibody lacking one or more of the four exons coding for the hinge region. The mutant variants all retained intact hinge region epitopes since they all reacted with IgG3 hinge-specific antibodies. Surprisingly, all the deletion mutants bound C1q equally well or even better than the wild type. Thus the high C1q binding activity of IgG3 compared to IgG1 is apparently not due to the total length of the IgG3 hinge, which is 62 amino acids, nor is it due to the length of the upper hinge which is the stretch from the end of CH1 to the first inter-heavy chain disulfide bond.     

Ultrastructural demonstration of specific IgG and IgE antibodies binding to Aspergillus fumigatus from patients with aspergillosis.

Aspergillus-induced diseases usually demonstrate elevated circulating antibodies belonging to different isotypes. The antigens currently used to detect antibodies are crude culture filtrate and mycelial extracts of A. fumigatus (Af). Most Af-associated diseases result from the inhalation of the spores of the organisms present in the environment. However, it is not known whether specific circulating antibodies directed only against spore or mycelia of Af exist in the sera of patients with Af-induced diseases. With colloidal gold we have investigated thin sections of spores and hyphae of Af for their reactivity with Af-specific IgG and IgE antibodies. The results indicate that both spores and hyphae reacted identically with IgG and IgE antibodies from patients. None of the sera from normal control subjects reacted in this system, although low levels of antibodies were detected in the sera by ELISA. Sera from both patients with allergic bronchopulmonary aspergillosis or aspergilloma reacted with cell envelope antigens, whereas sera from patients with invasive aspergillosis also bound to cell sap. This method therefore demonstrates localization of antigens binding to different isotypes in the sera from different clinical forms of aspergillosis and may be useful in purifying specific antigens for immunodiagnosis.     

C1q binding substances in pemphigus and bullous pemphigoid. Detection with a [131I] C1q binding assay.

A modification of the [125I]C1q binding assay was developed to allow the estimation of C1q binding activity (C1q BA) in pemphigus and bullous pemphigoid sera. The modifications include lower final concentration of PEG 6000 (1-5%) which permitted the use of sera that had been stored at -20 degrees C for extended periods of time; use of 131I instead of 125I and an [131I] C1q concentration of 5 microng/ml rather than 1 microng/ml. EDTA was used at a final concentration of 0-13 M to obviate the need for heat inactivation of sera. Sera from seventy-one patients with pemphigus and from 142 patients with bullous pemphigoid were tested for C1q BA. Of these 40% of the pemphigus and 20% of the bullous pemphigoid patients showed elevated C1q BA. A relationship between elevated C1q BA in serum and active disease was noted. Sequential samples from forty patients with pemphigus and thirty-seven patients with bullous pemphigoid demonstrated two different types of relationship between serum antibody titres to cutaneous antigens and C1a BA. In some patients serum antibody titres and C1q BA increased and decreased simultaneously; in others, increase of C1q BA followed increase of antibody titre and coincided with its decrease. The latter relationship supports the hypothesis that C1q BA may represent at least in part antigen-antibody complexes containing cutaneous antigens.     

C1q binding activity in the sera of patients with chronic lung diseases.

Sera from patients with chronic lung diseases were tested for the presence of immune complexes (ICs) by the 125I-C1q-binding assay. Contrary to earlier reports, modification of the test system by addition of heparin decreased rather than increased the ability of the test to discriminate between control and pathological sera. Using the unmodified system, elevated C1q-binding activity (C1qBA) was found in patients with asthma (18%), chronic bronchitis (18%), sarcoidosis (18%), fibrosing alveolitis (50%), bronchogenic carcinoma (52%) and bronchiectasis (67%). Studies with the reducing agent 2-mercaptoethanol (2-ME) suggested a role for IgM rheumatoid factor (RF) and/or IgG-containing complexes in the C1q-reactive material of sera from patients with bronchiectasis and bronchogenic carcinoma. In the latter two groups, C1qBA was found to correlate with serum levels of IgG and IgA but not with C3 and C4. A weak condition between levels of C-reactive protein (CRP) and C1qBA was found in the bronchogenic carcinoma group. Carcinoembryonic antigen (CEA) levels were elevated in all groups studied but no correlation with C1qBA was demonstrated, suggesting that CEA and CEA-ICs, if present, do not have an influence on the C1qBA of such sera. The results indicate that elevated serum C1qBA is a concomitant of both chronic inflammatory and neoplastic diseases of the lung but the extent of any similarity in the non-immunoglobulin components of the immune complexes in the respective conditions remains unknown.     

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.     

Similar specificity of IgG1 and IgG2a antibodies in individual mice.

In this study the previous finding of similar variable regions in individual IgG1 and IgG2a antibody populations is extended by the demonstration of similar fine specificity of IgG1 and IgG2a combining sites. Antibody populations from individual mice directed against oligo-D-alanine determinants were analyzed in their cross-reactivity towards 5 heterologous dipeptides. This was done by mixing antibody and hapten followed by determination of free antibodies in a kinetic red cells sensitization assay. The comparison of hyperimmune sera from 10 mice showed that genetically identical mice can differ significantly in their cross-reaction pattern. Within each serum the cross-reaction pattern was determined for IgG1 and IgG2a. With a few exceptions the same individual pattern was found in both IgG1 and IgG2a antibody populations. This was taken as evidence that the combining sites of IgG1 and IgG2a anti-oligo-D-alanine antibody populations in an individual mouse are similar.     

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.