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.     

Presence of shared idiotypes in serum and immune complexes in patients with IgA nephropathy

Patients with IgA nephropathy often present a large array of antibodies against diet antigens and this disease can be experimentally induced by alimentary antigens. In this report, we have described the isolation from a patient with IgA nephropathy of antibovine serum albumin (BSA)-antibody idiotypes that are specifically recognized by auto-and heteroantiidiotypic antibodies. The fact that antigen (BSA) but not monomeric or aggregated human IgG inhibited the binding of antiidiotypic antibodies to the idiotypes, suggested that the idiotypic determinants are in or near the antigen binding site and that it is not a rheumatoid factor. By means of the heteroantiidiotypic antibodies raised in rabbits we observed the presence of increased levels of shared idiotypes in serum and/or immune complexes (IC) of 48 out of 70 (68.5%) genetically unrelated patients with IgA nephropathy. The close correlation (P less than 0.005) between the presence of IgA-IC, measured by Raji cell assay, and the existence of high levels of serum idiotypes, suggest that a portion of circulating IC could consist of idiotype-antiidiotype. A strong concordance between the presence and levels of idiotypes and the clinical activity, as defined by the existence of haematuria, was also noted. The discrepancies and absence of correlation observed in our study among the levels of anti-BSA antibodies of different classes and serum levels of idiotypes, circulating IC and haematuria could suggest that the antibodies reacting with the heterologous antiidiotypic antibodies could be directed to other more pathogenic antigens than dietary antigens. All together, our results suggest that IgA nephropathy might belong to the group of diseases that occur in susceptible individuals with a limited potential in the immunological response repertoire.     

Detection of bovine serum albumin in the circulating IgA immune complexes of patients with IgA nephropathy

Alimentary antigenic challenge has been postulated to have a role in the genesis of IgA circulating immune complexes (CIC), resulting in mesangial IgA disease. In this study, we examined the relationship between bovine serum albumin (BSA) and IgA CIC in patients with IgA nephropathy. Of the 47 patients studied, elevated IgA CIC levels were found in 32% by the F(ab')2 anti-C3 and Raji cell enzyme immunoassays (EIA). Elevated IgA anti-BSA antibody levels were found in 9 patients, and there was a positive correlation between these levels and IgA CIC as measured in the Raji cell EIA (R = 0.60, P less than 0.001). In 4 patients with elevation of both IgA CIC and IgA anti-BSA antibody levels, solubilization experiments were done to demonstrate the presence of BSA antigen in the IgA CIC. Using the Raji cell EIA, the IgA CIC levels decreased significantly after preincubating the sera with serial concentrations of excess BSA. No corresponding effect was seen with human serum albumin used as control. Hence, BSA may be the antigenic stimulus in the formation of IgA CIC in selected patients with IgA nephropathy. The pathogenic capacity of these IgA-BSA CIC remains to be determined.     

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.     

Detection of IgA class circulating immune complexes bound to anti-C3d antibody in patients with IgA nephropathy

IgA class circulating immune complexes (CIC) were detected by solid-phase fluorescent enzyme immunoassay of F(ab')2 anti-C3d antibody in the serum of 52 patients with IgA nephropathy. Conglutinin (Kg) binding IgA class CIC were also measured, and results by these assays were compared. Kg binding IgA class CIC and anti-C3d binding IgA class CIC were detected in 27% and 44%, respectively, of the patients with IgA nephropathy. Either or both of the two were found in 65% of the patients. There was no significant correlation between IgA class CIC detected by these methods and serum IgA. Although all samples with a very high level of anti-C3d binding IgA class CIC did not also have a very high level of Kg binding IgA class CIC, there was a slight quantitative correlation between the 2 assays. Ultracentrifugation analysis showed that anti-C3d binding IgA class CIC were of various sizes between polymeric (21 S) and monomeric IgA (7 S), whereas Kg binding IgA class CIC were mostly monomeric IgA (8 S) with a minor component of heavy fractions (14 S). Both IgA class CIC fixed iC3b and IgA class CIC fixed C3d are present in IgA nephropathy. These observations suggest that the different types of complement bound to IgA class CIC have different roles in IgA nephropathy.     

Studies on glomerular immune solubilization by complement in patients with IgA nephropathy

A study of the solubilization of glomerular immune deposits by serum or complement in patients with IgA nephropathy is described. Renal biopsy specimens were obtained from 15 patients with IgA nephropathy. These specimens were incubated with fresh and heated sera from healthy adults or with lyophilized complement components, i.e., C3 and C4, at 37 degrees C for one hour in plastic tubes. The sections were then stained with fluorescein isothiocyanate (FITC)-labelled anti-human IgA antisera and examined by fluorescence microscopy. Normal sera showed a marked capacity to solubilize the glomerular immune deposits characteristic of IgA nephropathy. The solubilization capacity was reduced after inactivation and absorption of sera with anti-human C3 antiserum. Lyophilized C3 or C4 did not show any ability to solubilize such deposits. It was concluded that the solubilization of glomerular immune deposits may require whole active (fresh) components of complement related to the alternative pathway.     

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-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.     

Evaluation of the presence of circulating immune complexes and their relationship to glomerular IgG deposits in streptozotocin-induced diabetic rats

Circulating immune complexes (CIC) have been postulated to contribute to the development of secondary complications in diabetes mellitus. In this study, CIC were measured in control rats and both insulin deficient and insulin treated streptozotocin-induced diabetic rats. CIC were more prevalent in both groups of diabetic rats as determined by the fluid and solid phase Clq binding assays. By 42 days after induction of diabetes, 80% of insulin deficient and 50% of insulin treated rats had detectable CIC by either/or both assays. As determined by direct immunofluorescence, there was progressive accumulation of rat IgG in the glomerular mesangium. The presence of CIC paralleled the glomerular deposition of IgG. The relationship of circulating insulin levels to the clearance of CIC and the glomerular deposition of IgG is discussed.     

Characterization of circulating Yersinia-specific immune complexes in patients with yersiniosis

The size of immune complexes (ICs) containing Yersinia enterocolitica antigens was studied by size exclusion high-pressure liquid chromatography and sucrose density gradient ultracentrifugation in sera of patients with recent yersiniosis. The ICs detected were relatively small, i.e., of equal size to or slightly larger than the corresponding anti-Yersinia antibodies. The size of the ICs was equal in the patients with Yersinia-triggered reactive arthritis and in those recovering without complications. No changes were observed during a follow-up. The equal size of ICs in the patients with and without arthritis also suggests that antigens and antibodies involved are similar in both patient groups. Taken together with our earlier findings indicating occurrence of high concentrations of Yersinia IgM ICs in the arthritic patients, the present results suggest that Yersinia--IgM ICs have a role in the pathogenesis of Yersinia-triggered reactive arthritis.     

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.     

Immune complexes in IgA nephropathy: presence of antibodies against diet antigens and delayed clearance of specific polymeric IgA immune complexes

Several features suggest that IgA nephropathy is an immune complex (IC)-mediated disease. The source of antigen(s) is unknown but the predominant involvement of IgA suggest that it is associated in some way with the gut or respiratory tract. Taking into account the specific hepatobiliary transport by polymeric IgA of circulating antigens entering through the mucosal surfaces we examined the possible involvement of antibodies against food antigens in the circulating IC and the existence of a defect in their blood clearance in patients with IgA nephropathy. A rise in multimeric IgA-IC (Raji assay) occurred in three of seven control subjects with a peak at 2-4 h after food ingestion. The amount of multimeric IgA-IC present at fasting in four out of six patients, diminished 2-4 h after food challenge, reaching a new peak around 6 h. At fasting, three out of six patients had IC containing antibodies against diet antigens (e.g. ovalbumin). These IC paralleled, both in patients and controls, the levels of multimeric IgA-IC. In patients small multimeric IgA-IC predominated at fasting and 24 h after food ingestion, while larger IC were detected at 2-4 h of food challenge. The specific polymeric IgA-IC showed in controls a maximal peak with similar distribution to that of multimeric IgA-IC, but with a quicker disappearance from the circulation. By contrast, polymeric IgA-IC remained elevated 24 h after food ingestion in most patients. These results suggest that antibodies against common antigens are within circulating IC and that a defect in the hepatic clearance of circulating polymeric IgA-IC exists in patients with 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.     

Effect of IgA deposits on the glomerular mesangium in Berger's disease

In mesangial IgA glomerulonephritis (Berger's disease), the immunoproteins appeared to gain access from the capillary lumen to the mesangium via endothelial fenestrae or via channels between the endothelial cells. The deposits are transported into the deeper mesangium by a process of inhibition or diffusion, with the matrix acting as the head. There are no true channels or grooves in the mesangial matrix for the transport of the immunoproteins. The contractility of the glomerular myoid fibrils may account for the movement of deposits to the hilus for possible removal. There was partial dissolution of the deposits in the mesangial matrix accompanied by loosening of the matrix. No evidence was found for any significant intracellular phagocytosis and digestion. The mesangial deposits directly or indirectly stimulated the cellular hypertrophy and hyperplasia and increased deposition of mesangial matrix. This was accompanied by formation of collagen fibrils within the thickened matrix and led to atrophy of the mesangial cells and sclerosis of the glomeruli.     

Peripheral glomerular capillary wall lesions in IgA nephropathy and their implications

Peripheral glomerular capillary walls were studied in 26 cases of IgA nephropathy by means of the transmission electron microscope. Ultrastructural abnormalities were identified in 11 cases (42%). Abnormalities of the glomerular basement membrane (GBM) were the most frequent change which consisted of localized thinning, lamination, irregular thickening, disruption, membranolysis and aneurysmal dilatation of the GBM. Subendothelial electron dense deposits were seen. Necrosis and detachment of the podocytes from the GBM were also encountered. The changes were correlated with the clinical findings at the time of diagnosis which showed a significant correlation of these peripheral glomerular capillary wall lesions with proteinuria. With light microscopy, crescents were significantly more frequently seen in the cases showing the ultrastructural capillary wall abnormalities than those without. This observation suggested that local peripheral glomerular capillary wall damage was an important factor in the pathogenesis of the extracapillary lesions in IgA nephropathy.     

Detection of circulating immune complexes in patients with glomerulonephritis.

143 patients were evaluated clinically on the basis of the renal biopsy. Three methods for detecting circulating immune complexes (CIC) were employed: complement consumption test, inhibition of erythrocyte antibody IgG-EA rosette forming test and optical density of 4% PEG precipitated sera. CIC were present in the sera of all patients with acute poststreptococcal glomerulonephritis (2 weeks after streptococcal infection of the throat). In a group of patients with chronic glomerulonephritis the highest values in positive results were observed in lupus nephritis, chronic proliferative glomerulonephritis and chronic submicroscopic glomerulonephritis. These results were compared with levels of total hemolytic complement, C3, C4 components and serum immunoglobulins (IgA, IgG, IgM).     

Abnormalities of the IgA immune system in members of unrelated pedigrees from patients with IgA nephropathy.

In the last few years many investigators have reported the recurrence of primary IgA nephropathy (IgAN) or the presence of persistent microhaematuria and/or proteinuria in family members of patients with IgAN. Our study was undertaken to investigate the relevance of abnormalities in the regulation of the IgA and IgM immune system in microhaematuric and asymptomatic family members of IgAN patients. Fifty-four out of 120 members of nine unrelated pedigrees were examined by urinalysis; polymeric IgA (pIgA), IgA rheumatoid factor (IgARF), IgA1-IgG immune complexes (IgA 1-IgG IC) and IgA 1-IgM IC, and other immunoglobulins were measured in serum samples. Moreover, we studied the production of immunoglobulins, pIgA and IgARF by peripheral blood mononuclear cells (PBMC) in basal conditions and after pokeweed mitogen (PWM) stimulation. Our data demonstrate that persistent microhaematuria was present in 24% of relatives. High serum levels of IgA, mainly pIgA and IgARF, IgA 1-IgG IC and IgA 1-IgM IC occurred in 66% of relatives. Abnormal spontaneous production of IgA by PBMC and after PWM stimulation was present in 64% of family members. Interestingly, high serum levels of IgM and abnormal production of this immunoglobulin by PBMC were observed in relatives. However, the immunological abnormalities did not correlate in any way with the presence of urinary abnormalities such as microhaematuria, which was most likely determined by an underlying glomerular alteration.     

Computer imaging analysis of glomerular extracellular components in patients with IgA nephropathy

Computer imaging analysis was used for quantitative evaluation of the extents, amounts and distributions of glomerular extracellular components, such as the 7S and NC-1 domains of type IV collagen, laminin (LN), fibronectin (FN) and IgA, in glomeruli from patients with IgA nephropathy. Renal biopsy specimens from 13 patients with IgA nephropathy were incubated with mouse monoclonal antibodies against the FN or non-collagenous (NC-1) domain of type IV collagen or polyclonal antiserum against the LN or 7S domain of human type IV collagen, and then stained with appropriate dilutions of FITC-labeled anti-mouse Ig antisera. Marked staining of the 7S or NC-1 domain of type IV collagen, LN or FN was detected in the glomerular capillary walls and/or mesangial areas in patients with IgA nephropathy. In particular, a prominent increase of FN was observed in the subendothelial regions of glomerular capillary walls, i.e. mesangial interposition, in the moderate or advanced stage of IgA nephropathy. Therefore, computer imaging analysis was shown to be useful for the quantitative determination of such components distributed in glomeruli from patients with IgA nephropathy.     

A quantitative analysis of the mesangium in children with IgA nephropathy: sequential study

Quantitative analysis of the mesangial matrix and cells was performed on serial renal biopsies from 41 children with IgA nephropathy. In the repeat renal biopsy, nine patients showed a significant increase of mesangial matrix, 29 showed no change and in three there was a significant decrease. Eight of the nine patients (89 per cent) with a matrix increase had persistent proteinuria at the second biopsy, whereas only 14 of the 32 (44 per cent) without a matrix increase had persistent proteinuria (P less than 0.05). Although the mesangial matrix increased in patients with persistent proteinuria, there was no decrease in patients with clinical remission. In contrast to the mesangial matrix, mesangial cells significantly decreased in 23 patients, did not change in 16, and significantly increased in only two in the second biopsy. These findings suggest that mesangial matrix increase is usually an irreversible change and that persistent proteinuria is associated with matrix increase with worsening in glomerular morphology and clinical outcome. This study indicates the importance of serial renal biopsy in children with IgA nephropathy with persistent proteinuria.