Circulating immune complexes in IgA deficiency

Circulating immune complexes (IC) were demonstrated in patients with serum IgA deficiency. Sixteen of thirty-one IgA deficient patients had serum IC detected by solid phase C1q radioimmunoassay for IgG class complexes. The presence of cryoglobulins (thirteen out of thirty-one patients) and increased polyethylene glycol precipitation (ten out of thirty patients) provided additional evidence for the presence of IC. Fourteen patients were asymptomatic but seven had clinical evidence of disease which could have been IC mediated: two with glomerulonephritis, three with polyarthritis, one with vasculitis and one with thyroiditis. Serum IC remained detectable in multiple samples over several months but this correlated poorly with the presence or absence of disease. Serum antibody to IgA was detected in fifteen out of thirty-one patients. There was no direct relationship between the presence of IC and the level of serum anti-IgA antibody; however, this antibody was shown to be present in the IC isolate in eight patients. It is proposed that a considerable portion of the IC load in IgA deficiency results from defective antigen exclusion at the level of the mucosa.     

Circulating IgA immune complexes in aids

Circulating immune complexes were isolated from the serum of patients with AIDS, as well as patients with other acute and chronic viral diseases. Analysis of these immune complexes by methods of flow cytometry and by radioimmune (Raji cell) assay revealed a prevalence of IgA complexes in the serum of AIDS patients and a prevalence of IgG complexes in the serum of patients with other viral diseases. Raji cells bind immune complexes via Fc and complement (C,) receptors and may detect IgA immune complexes more efficiently than a Clq assay since IgA has no affinity for Clq.     

Analysis of circulating IgA and detection of immune complexes in primary IgA nephropathy.

The sera of 31 patients with primary IgA nephropathy were investigated for IgA containing immune complexes by Raji cell-binding IgA radioimmunoassay and conglutinin-binding IgA radioimmunoassay. Positive results, without correlation with IgA serum levels, were found in 68% of the patients using the first assay, in 39% of the patients with the second assay. Positive sera were analysed by gel chromatography. Conglutinin-binding IgA eluted in two peaks, a minor one of 400,000-800,000 daltons mol. wt and a major one corresponding to monomeric IgA. No increase of secretory IgA and of polymeric IgA was detectable. IgA immune complexes were likewise found in the sera of patients with systemic lupus (five of 12), rheumatoid arthritis (four of 12), subacute bacterial endocarditis (four of 12) and HB virus hepatitis (four of 16). However, the high prevalence on these sera of IgG and IgM immune complexes detected by polyethylene glycol precipitation, solid phase Clq binding assay contrasted strongly with their absence in IgA nephropathy. In addition, the presence of abnormal amounts of conglutinin reactive IgA correlated with the recurrence of IgA deposits after renal transplantation (20 patients studied). Conglutinin reactive IgA could contribute to the glomerular deposition of IgA and subsequently play a significant role in the pathogenesis of IgA nephropathy.     

IgA1 and IgA2 immune complexes in primary IgA nephropathy and Henoch-Sch?nlein nephritis.

The distribution of IgA subclasses in IgA immune complexes (IgA IC) in sera of patients with primary IgA glomerulonephritis and Henoch-Schönlein purpura nephritis was analysed. High levels of IgA IC containing both IgA1 and IgA2 subclasses were present in correlation with the phases of clinical activity. In these nephropathies the finding of IgA subclass distribution in IgA IC similar to that found in secretions may add further support to the hypothesis that IgA IC are of mucosal origin, albeit a primary derangement of the humoral immune system in these patients cannot be disregarded.     

The specific detection of IgA in immune complexes

An assay to detect IgA in circulating immune complexes (IC) using low avidity goat IgM antibody against human polyclonal IgA is described. The binding of this antibody to IgA coupled to Sepharose 6B is inhibited by IgA-containing IC. The specificity and sensitivity of this anti-IgA inhibition assay (a-IgA-InhA), was evaluated with aggregated purified immunoglobulins, sera of patients with Henoch-Schönlein purpura and normal human sera. Aggregated immunoglobulins of the IgA class, but not monomeric IgA, were reactive. Sucrose density ultracentrifugation showed that large IgA constituents (> 19S) were found only in the sera of patients with Henoch-Schönlen purpura. Both these sera and normal human serum contained smaller IgA components (between 7S and 19S), probably small polymers of IgA, which were reactive in this assay and interfered with detection of IgA-containing IC. Redissolved precipitates obtained from normal serum with polyethylene glycol showed reduced reactivity in the test, whereas the inhibitory activity of IgA-containing IC in sera of patients with Henoch-Schönlein purpura was retained in the precipitates. Precipitation of sera with polyethylene glycol allowed detection of smaller quantities of IgA-containing IC in patients with Henoch-Schölein purpura.     

Lack of complement activation by human IgA immune complexes.

Complement activation may play an important role in renal injury associated with glomerular deposition of IgA immune complexes. The ability of naturally occurring human IgA immune complexes (IgA-IC) and covalently cross-linked human IgA oligomers (X-IgA) to activate complement were examined in vitro and in vivo. Large-sized IgA-IC were isolated from a patient's serum by affinity purification (Jacalin-Sepharose) and gel chromatography. Stable X-IgA were prepared by chemical cross-linking with a heterobifunctional reagent, N-succinimdyl 3-(2-pyridyl-dithio) propionate (SPDP). Treatment of fresh normal human serum with large amounts of either IgA-IC or X-IgA failed to activate C3. The C3 consumption was measured immunochemically by the decrease of the B antigen on the native C3 and by the generation of iC3b. Addition of these complexes to serum did not result in cleavage of factor B. Administration of human IgA-IC or X-IgA to mice, killed after 6 h, resulted in glomerular deposition of IgA. Despite the presence of intense glomerular IgA deposits no C3 was detected. Collectively, these findings suggest that neither soluble nor renal localized human IgA complexes activate complement.     

Characterization of circulating idiotypes containing immune complexes and their presence in the glomerular mesangium in patients with IgA nephropathy.

The possible pathogenic role for idiotype-anti-idiotype interactions in kidney diseases has recently been suggested. Since patients with IgA nephropathy often present antibodies against alimentary antigens, like bovine serum albumin (BSA), we isolated an idiotypic antibody with BSA specificity from one of these patients. By means of a specific anti-idiotypic antibody raised in rabbits, we have studied the participation of these idiotypes in circulating and renal deposited immune complexes (IC) in patients with IgA nephropathy. On indirect immunofluorescence, the presence of cross-reactive idiotypes was detected in the glomeruli of 12 out of 42 (28%) patients with IgA nephropathy, but in none of 15 membranous or mesangiocapillary nephritis examined. The staining was located within mesangial and paramesangial areas, with a similar, but less intensive, pattern distribution than IgA. Previous adsorption of rabbit anti-idiotype antibodies on an idiotype-Sepharose column completely abolished that staining. A close relationship was found between the presence of cross-reactive idiotypes on mesangial immunoglobulins and the existence of increased levels of serum idiotypes and idiotype-containing IC. Serum analytical ultracentrifugation showed that circulating IC containing idiotypes have chiefly a large (greater than 19 S) and intermediate (13 S-19 S) size, while those containing anti-BSA antibodies were only between 7 S-13 S fractions, or absent. Our results suggest that in patients with IgA nephropathy, shared idiotypes participate in the formation of circulating and renal deposited IC. It is possible that the apposition of free anti-idiotype to idiotype already bound to glomeruli, and vice versa, could contribute to increasing the amount and size of mesangial immune deposits, and, therefore, facilitate or perpetuate tissue injury.     

Suppressive effect of IgA soluble immune complexes on neutrophil chemotaxis.

The effect of IgA and IgG soluble immune complexes (SIC) on neutrophil chemotaxis was investigated. Six types of SIC were prepared from kappa-type IgA and IgG myeloma proteins: IgA-anti-kappa chain antibody SIC in IgA excess, IgA-anti-kappa chain antibody in SIC in anti-kappa chain antibody excess, IgA-anti-alpha chain antibody SIC in IgA excess, IgG-anti-kappa chain antibody SIC in IgG excess, IgG-anti-kappa chain antibody SIC in anti-kappa chain antibody excess, and IgG-anti-gamma chain antibody SIC in IgG excess. Three types of IgA SIC had a marked suppressive effect on neutrophil chemotaxis, while IgG SIC, free IgA, free anti-kappa chain antibody, and free anti-gamma chain antibody showed no inhibitory activity. This suppressive effect on neutrophil chemotaxis caused specifically by polymerized IgA was a cell-directed one and was expressed in a concentration dependent manner.     

Experimental cholestasis promotes the deposition of glomerular IgA immune complexes.

Previous experimental and clinical studies support a role for the hepatobiliary system in the clearance of oligomeric IgA from serum, and alterations of this system have been associated with the deposition of IgA in the renal mesangium. The present studies in mice address the question of whether the mesangial deposition of IgA following cholestasis includes immune complexes. While bile duct ligation resulted in mesangial IgA deposition within several days in approximately 75% of animals, whether deliberately orally immunized, nonimmunized, or given injected immune complexes, mice that underwent sham operations had IgA deposits only if orally immunized. Moreover, mice that had been orally immunized or given injected immune complexes and whose bile ducts had been ligated contained deposits of specific IgA antibody and antigen. In the ligated mice some of the IgA was secretory IgA, as demonstrated by the presence of secretory component. Thus, bile duct ligation promotes the deposition of circulating IgA immune complexes, presumably by decreasing their clearance from serum, and gives rise to secretory IgA in the glomerular mesangium. The secretory immune system probably plays a role in the pathogenesis of idiopathic and cirrhosis-related human IgA nephropathy.     

Antibodies to polyclonal IgA,IgA1, and IgA2 and isotype-specific immune complexes in IgA nephropathy

The concentrations of serum IgG and IgM antibodies to polyclonal IgA (IgA_p), IgA₁, and IgA₂ were determined by enzyme immunoassay in 31 patients with IgA nephropathy and 30 healthy controls. Patients with IgA nephropathy had significantly raised concentrations of serum IgA compared to controls (Mann-WhitneyU test,P=0.001) and increased concentrations of conglutinin-binding IgA immune complexes (P=0.024). No differences in the median concentrations of IgG and IgM anti-IgA antibodies were found between the patients and the controls. In serum samples from healthy controls there was a significant positive correlation between IgM anti-IgA_p and IgA immune complex concentrations (P=0.05), which contrasted with the finding of an inverse correlation between IgM anti-IgA_p and IgA immune complex concentrations in patients with IgA nephropathy (P<0.05). In addition, the concentrations of conglutinin binding IgM immune complexes in serum were found to correlate with the concentration of IgM anti-IgA_p (0.010<P<0.025), IgM anti-IgA₁, and IgM anti-IgA₂ (P«0.005 for both) in patients with IgA nephropathy but not in controls. IgM anti-IgA antibodies may be important in augmenting the clearance of IgA immune complexes from the serum of patients with IgA nephropathy.     

Composition of IgA immune complexes precipitated with polyethylene glycol. A model for isolation and analysis of immune complexes

Covalently cross-linked large and intermediate-sized IgA oligomers, prepared with IgA anti-dinitrophenyl (DNP) and bis-DNP-pimelic acid ester, were used to examine the ability of different concentrations (3.5%, 5% and 7%, w/v) of polyethylene glycol (PEG) to precipitate IgA immune complexes (IgA-IC). The size of the IgA-IC precipitated with PEG was determined by gradient polyacrylamide gel electrophoresis and quantitative autoradiography. The standard concentration of 3.5% PEG precipitated only a minor fraction (20%) of the IgA-IC. In contrast, 5% and 7% PEG precipitated 45% and 79% of the complexes, respectively. To test the influence of the antigen on the PEG assay. IgA-IC prepared with IgA anti-DNP and DNP conjugates of either bovine serum albumin or Ficoll were also used. Approximately 38% of these IgA-IC were precipitated with 3.5%, PEG. By comparison, the concentration of 5% and 7% PEG precipitated 60% and 76% of the IgA-IC, respectively. Distilled water rather than the standard borate-buffered saline was shown to be the optimal solvent for resolubilization of the PEG precipitates. Serum samples from 22 IgA nephropathy patients and 12 normal donors were tested with 3.5%, 5% and 7% PEG. Only the 7% PEG assay showed a significant difference between patients and controls (P less than 0.001) in the IgA levels of precipitates. Thus, the use of 7% PEG is recommended for the detection, isolation and analysis of large- and intermediate-sized IgA-IC.     

Glomerular immune deposits in experimental IgA nephropathy. A continuum of circulating and in situ formed immune complexes.

Studies were undertaken to elucidate the primary pathogenetic mechanisms responsible for immunoglobulin (Ig) A immune complexes formation and glomerular deposition in vivo. Monomeric (mIgA) and polymeric IgA (pIgA) anti-dinitrophenyl (DNP) were purified from MOPC 315 myeloma. A DNP-conjugated Ficoll was used as an antigen. For simulation of natural conditions of in vivo immune complex formation, 131I-DNP-Ficoll and 125I-IgA were administered through the intravenous and intraperitoneal routes, respectively. The kinetics half-life (t1/2) of the antigen (2.9 hours) and either the pIgA (7.2 hours) or mIgA (6.3 hours) in the experimental groups was not significantly different from the control. Glomerular IgA immune deposits were detectable only in mice that received pIgA and DNP-Ficoll. Plasma samples analyzed by gradient polyacrylamide gel electrophoresis revealed formation of large- and intermediate-sized pIgA complexes in circulation prior to glomerular deposition. Although mIgA failed to interact with such complexes in the circulation, it did bind to the pIgA immune deposits in the glomerulus. These results indicate that glomerular IgA immune deposits evolve from the localization of preformed circulating pIgA complexes that eventuates an in situ mIgA-mediated complex formation.     

IgA and IgG immune complexes increase human macrophage C3 biosynthesis.

We have studied the effect of IgA- and IgG-containing immune complexes on the production of complement proteins C3, factor B and C2 by human monocyte-derived macrophages, using biosynthetic labelling, immunoprecipitation, sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) and autoradiography. There was a consistent increase in C3 production and secretion with both IgA and IgG immune complexes. This increase appeared after a 24-hr incubation period of the macrophages in the presence of immune complexes. No change in the biosynthesis of factor B and C2 proteins was observed in these experiments. Concomitant with the enhanced C3 biosynthesis, the immune complexes caused an increase in macrophage tumour necrosis factor (TNF) production; 310 + 24 U/ml/5 x 10(5) cells and 430 + 51 U/ml/5 x 10(5) cells for IgA and IgG immune complexes, respectively, versus 12 + 8 U/ml/5 x 10(5) cells in the control cells. The presence of prednisolone (2 x 10(-5) M) or dexamethasone (1 x 10(-7) M) inhibited the immune complex-induced TNF production, but had no effect on C3-increased synthesis, suggesting that the effect of immune complexes was not mediated by endogenous TNF production. These findings may be relevant to the local inflammatory response in IgA immune complex-mediated diseases, including IgA nephropathy.     

Glomerular deposition of immune complexes prepared with monomeric or polymeric IgA.

Clearance kinetics and renal deposition of soluble IgA immune complexes (IgA IC) were examined to determine the nephritogenic potential of IgA molecular form in mediating experimental IgA nephropathy. The immune complexes were prepared by mixing purified radiolabelled monomeric (mIgA) or polymeric (pIgA) IgA anti-dinitrophenyl (DNP), derived from MOPC-315 myeloma, with DNP conjugated Ficoll (DNP8-Ficoll). Clearance of IgAIC from the circulation was curve fitted by two exponential components. The first component was similar for both mIgA IC and pIgA IC. The second component was slightly more rapid for mIgA IC than for pIgA IC. Immunofluorescence studies, however, showed that only pIgA IC deposited in the kidneys. Analysis of IgA IC by gradient polyacrylamide gel electrophoresis indicated that mIgA formed only small latticed complexes. The critical role of IgA IC lattice size in renal deposition was confirmed by demonstrating that large latticed mIgA IC, prepared by covalent cross-linking mIgA with a specific affinity labelling antigen, deposited in the kidneys in a pattern similar to pIgA IC. Our results suggest that the monovalency of mIgA is responsible for its inability to form a large latticed nephritogenic complexes.     

Characterization of immune complexes containing cytomegalovirus-specific IgM antibodies following a kidney graft.

In order to demonstrate the viral specificity of IgM-containing immune complexes (IgM-CIC) detected by a C1q assay in renal allograft recipients developing a CMV infection, a technique is described allowing: 1) the dissociation of IgM-CIC by action of an acid buffer, and 2) the characterization of the viral specificity of IgM antibodies released by this treatment. This step was performed by ELISA and Western Blot. When technique was applied to the follow-up of a renal allograft recipient developing a recurrent CMV infection within 2 months post-graft, it was found that the IgM-CIC detected on the day of the graft were not CMV-specific, whereas the IgM-CIC detected during the second month after transplantation contained CMV-specific IgM antibodies. These CMV-specific IgM-CIC were detected as early as the urinary viral excretion. It was shown by Western Blot analysis that these IgM antibodies reacted with a 45-47 kDa viral polypeptide which is a viral target for specific humoral response at the early phase of CMV infection.     

The elimination of circulating complexes containing polymeric IgA by excretion in the bile.

Radiolabelled human dimeric IgA injected intravenously into rats behaved like oligomeric rat IgA in that 40% of the injected dose was recovered in the bile within 6 h. Monomeric IgA was not transported into bile. In addition, dimeric IgA was able to carry with it macromolecular material in the form of a complex that also included rat SC. This was demonstrated in rats which had received an intravenous injection of specifically purified radiolabelled rabbit antibody tao the idiotype of a human IgA myeloma: when a later injection of unlabelled IgA dimer with the corresponding idiotype was given up to 18% of the rabbit antibody when the monomeric form of the same IgA was injected. This mechanism for clearing complexes containing polymeric IgA is mediated by hepatocytes and is distinct from the phagocyte-mediated mechanisms which clear conventional complexes.     

Detection and characterization of circulating and glomerular immune complexes in experimental IgA nephropathy.

The different models of experimental IgA nephropathy described so far have provided insight into pathogenesis; however, the evidence for a role of IgA immune complexes (IC) has only been gained in passive systems. In an active model of IgA nephropathy, induced in mice by repeated injections of dextran, some of the mechanisms that could explain the formation of glomerular IgA deposits are studied in this report. Serum total IgA and anti-dextran IgA antibody levels increased significantly over the period of immunization. Only 13-30% of mice had total and/or specific IgA IC, determined by Raji cell and PEG assay in ELISA. Analytical ultracentrifugation showed that IgA IC were of small (7-13 S) or intermediate (13-17 S) size. There was a close correlation between total serum IgA levels and the presence of IC-containing IgA anti-dextran antibodies, with the existence of IgA in the mesangium. The percentage of animals (n = 76) with IgA mesangial deposits increased over the immunization period (88% at 10 weeks). Forty-three per cent of mice had polymeric IgA in the mesangium; by contrast, only 12% had dextran deposits. On the whole, these data suggest that in the dextran-induced IgA nephropathy, the glomerular IgA could be the result of circulating IgA complexes and/or IgA polymers deposition.     

Microbial IgA protease removes IgA immune complexes from mouse glomeruli in vivo: potential therapy for IgA nephropathy

The hallmark of IgA nephropathy (IgAN), the most common form of glomerulonephritis, is the presence of mesangial deposits containing IgA, specifically the IgA1 subclass, as the most prominent component. The deposited IgA is considered to be part of an immune complex. The family of enzymes known as bacterial IgA proteases exhibits substrate specificity that is essentially limited to the hinge region of IgA1. Here we demonstrate the ability of systemically administered IgA protease to remove glomerular IgA immune complexes, both the antigen and antibody components, in a passive mouse model of IgAN. Thus, IgA protease may have potential as a therapeutic agent for human IgAN.     

Detection of monomeric and polymeric IgA containing immune complexes in serum and kidney from patients with alcoholic liver disease

The purpose of this study was to characterize circulating IgA and the IgA deposited in the glomeruli of patients with alcoholic liver disease. In the 6 patients studied there was an increased proportion in monomeric IgA (3.5 fold) and IgA between 9-13S (8.94 fold), 13-17S (4.49 fold) and 17-21S (1.63 fold) fractions on 5-40% sucrose density gradient ultracentrifugation at physiological pH. All fraction between 9.12S decreased at acid pH, however a 3.28 fold increase in fractions where polymeric IgA is expected to appear. IgG eluted at acid pH from autopsy kidney was studied by the same procedures. At pH 7.4 about 55% of that IgA have a molecular weight comprised between 9-12S, decreasing to around 25% at acid pH. The existence of true polymeric IgA in serum and kidney was based on the capacity of high molecular weight IgA to bind human secretory component. The amount of immune complexes with monomeric IgA were higher than those with polymeric IgA in serum as well as in kidney. However, the percentage of heavy IgA (probably polymeric IgA) in kidney were, in each patient, higher than those observed in serum. Our results show the presence of high amounts of monomeric and polymeric IgA, both partially as immune complexes, in serum and kidneys of patients with alcoholic liver disease and IgA glomerulonephritis. Furthermore, our data suggest a role for human liver in the clearance of serum IgA such as has been demonstrated in the some animal species, especially in rats.     

IgA-containing immune complexes after challenge with food antigens in patients with IgA nephropathy.

The possibility that patients with IgA nephropathy (IgAN) might have abnormal IgA immune responses to immunogens commonly encountered at mucosal surfaces, resulting in the formation of circulating immune complexes (CIC), was examined. Since it is generally held that such increased IgA responses are characterized by detectable aberrancies in handling of IgA-containing CIC, IgAN patients and controls were given a large volume of bovine milk (after dietary deprivation of bovine antigens) and immune complex levels were measured over a period of 12 h. An assay based on binding of CIC containing C3 to solid-phase anti-C3 and subsequent development with isotype-specific antibody revealed no differences in responses of patients and controls with respect to IgG- and IgM-containing CIC. Although IgAN patients tended to have higher levels of IgA-containing CIC, there were no differences in response patterns when IgA CIC levels after ingestion of the milk stimulus were related to baseline levels. Polymorphonuclear leucocytes (PMNC), which bear surface receptors for IgA, were isolated from some subjects at the same times as the samples for CIC levels and examined by two-colour immunofluorescence for the coincident presence of IgA and milk antigens. In contrast to the data obtained in the CIC assays, these experiments revealed the simultaneous presence of IgA and two of three milk proteins in PMNC of IgAN patients but not controls. Follow-up experiments designed to assess more quantitatively the coincidental presence of IgA and milk antigens indicated no significant differences between patients and controls. However, milk proteins seemed to be more commonly associated with IgA in PMNC of IgAN patients, suggesting the presence of non-complement-fixing IgA/antigen CIC after mucosal challenge of some IgAN patients.