Alice C. Allen

Galactosylation of N- and O-linked carbohydrate moieties of IgA1 and IgG in IgA nephropathy.

The mechanism of IgA deposition in the kidneys in IgA nephropathy is unknown, Mesangial IgA is of the IgA I subclass, and since no consistent antigenic target for the IgA I has been described, we have investigated the glycosylation of the molecule, as a potential non‐immunological abnormality which may contribute to its deposition. IgA 1 is rich in carbohydrate, carrying N‐linked moieties in common with IgG, but also O‐linked sugars, which are rare in serum proteins, and not expressed by IgG or lgA2, Lectin binding assays were designed to examine the expression of terminal galactose on the N‐linked carbohydrate chains of purified serum IgG and IgAI, and the O‐linked sugars of IgAI and C1 inhibitor (one of the very few other serum proteins with O‐linked glycosylation). No evidence was found for abnormalities of N‐linked glycosylation of either isotype in IgA nephropathy compared with matched controls. However, in IgA nephropathy, reduced terminal galactosylation of the hinge region O‐linked moieties was demonstrated; this was not seen in C1 inhibitor, which showed normal or increased galactosylation of the O‐linked sugars. This abnormality of IgA1 has considerable implications for the pathogenesis of IgA nephropathy, since the O‐linked sugars lie in an important functional location within the IgA1 molecule, close to the ligand of Fc receptors. Changes in the carbohydrates in this site may therefore affect interactions with receptors and extracellular proteins, leading to anomalous handling of the IgA1 protein in this condition, including failure of normal clearance mechanisms, and mesangial deposition.

Increased dimeric IgA producing B cells in the bone marrow in IgA nephropathy determined by in situ hybridisation for J chain mRNA.

AIM: To investigate the possible role of the systemic IgA immune system in the pathogenesis of IgA nephropathy METHODS: J chain mRNA expression in the IgA cells of the bone marrow was studied. Bone marrow trephine biopsy specimens from seven patients with IgA nephropathy and seven matched controls were examined by (1) non-isotopic in situ hybridisation (ISH) and (2) combined immunofluorescence and non-isotopic ISH to identify the plasma cell type. Serum polymeric IgA was also determined using standard high pressure liquid chromatography and sandwich enzyme linked immunosorbent assay. RESULTS: Non-isotopic ISH revealed a similar number of J chain mRNA positive cells/unit length in biopsy specimens from patients (16.5 +/- 2.7 cells/mm) and controls (17.7 +/- 2.4 cells/mm). Combined immunofluorescence and ISH revealed a greater proportion of J chain mRNA positive IgA cells in patients (7.6 +/- 1.45%) compared with controls (3 +/- 0.8%). Serum polymeric IgA was similar in both patients (91 +/- 22 mg/l) and controls (77 +/- 24 mg/l). CONCLUSION: These data suggest that excess production of dimeric IgA occurs in the bone marrow in IgA nephropathy.

Antineutrophil cytoplasm antibodies (ANCA) of IgA isotype in adult Henoch-Schönlein purpura.

ANCA are associated with certain forms of systemic vasculitis, and have been reported previously to be of the IgG and IgM isotype. We examined ihc possible association between IgA ANCA and the IgA‐related diseases Henoch‐Schönlein purpura (HSP) and IgA nephropathy (IgAN). IgA and IgG ANCA were detected by isotype‐specific solid‐phase assays with a crude neutrophil extract, and their presence was confirmed by antigen‐specific fluid‐phase competitive inhibition tests and by indirect immunofluorescence. The possible interference by IgA rheumatoid factor was excluded. IgA ANCA were detected in sera from 11/I4 HSP patients (79%). from 1/30 IgAN patients (3%), from 1/40 patients with vasculitides classically associated with IgG ANCA (2‐5%), and in none or 60 sera from healthy blood donors. IgG ANCA were present with IgA ANCA in three patients with HSP. Only one HSP serum had anti‐myeloperoxidase (MPO) activity by both IgA and IgG isotype‐specific ELISA, and none was positive for proteinase 3 (PR3). Western blot analysis performed with neutrophil extract showed that the four strongest IgA ANCA‐positive HSP sera reacted with a 51 ‐kD protein; Western blot performed on cellular fractions showed that this protein is primarily membrane‐associated, and different from fibronectin. Our study suggests that adult HSP is closely associated with circulating IgA ANCA. which may be directed against a different autoantigen than that recognized by IgG ANCA.

Exaggerated systemic antibody response to mucosal Helicobacter pylori infection in IgA nephropathy.

Numerous studies in the literature report aberrant immune responsiveness in immunoglobulin A (IgA) nephropathy. However, all these studies investigate immune responses invoked by an artificially engineered antigen challenge. For the first time in IgA nephropathy, we report the systemic humoral responses generated as part of an active mucosal immune response against a common environmental pathogen, Helicobacter pylori (Hp). We studied 22 patients with IgA nephropathy and 9 controls without renal disease who were shown to be infected with Hp, using a 13C-urea breath test. Hp antigen-specific enzyme-linked immunosorbent assays were established to measure the anti-Hp IgA, IgG, and IgA and IgG subclass antibody levels. In addition, anti-Hp responses in the monomeric and polymeric (pIgA) fractions of serum IgA were measured after separation by gel filtration high-performance liquid chromatography. IgA nephropathy was associated with both a greater rate of IgA anti-Hp seropositivity (P < 0.05) and a more pronounced IgA anti-Hp antibody response (P < 0.01). In almost all cases, IgA anti-Hp was IgA1, and more than 90% was polymeric. There was no difference in the frequency of IgG anti-Hp seropositivity, but patients produced a much greater IgG anti-Hp response (P < 0.01). In addition, the IgG subclass responses were markedly different, with IgG1 predominant in controls and IgG2 and IgG3 the major subclasses produced in IgA nephropathy. We have shown an exaggerated systemic antibody response to mucosal infection caused by Hp in patients with IgA nephropathy, predominantly consisting of pIgA1, IgG2, and IgG3. This suggests that in IgA nephropathy, not only is pIgA1 production poorly controlled, but regulation of IgG isotype switching in response to mucosal pathogens is also deranged.

IgA myeloma presenting as Henoch-Schönlein purpura with nephritis

IgA nephropathy (IgAN) and Henoch-Schönlein purpura (HSP) are both characterized by IgA-mediated tissue injury, including mesangial proliferative glomerulonephritis. Abnormalities of IgA1 glycosylation are described in IgA nephropathy and HSP nephritis. IgA-antineutrophil cytoplasmic antibodies (ANCA) have been inconsistently described in the serum of patients with HSP. In IgA myeloma, the paraprotein-mediated renal lesion is typically cast nephropathy; IgAN or HSP have only rarely been reported in myeloma even when an IgA paraprotein is circulating in large concentrations. We report the case of a 50-year-old man with IgA myeloma who presented with HSP including nephritis and rapidly progressive renal failure. His IgA1 had altered O-glycosylation in the pattern seen in IgAN and also contained an IgA-ANCA. This case adds further weight to the evidence that IgA1 O-glycosylation abnormalities predispose to mesangial IgA deposition and also that IgA-ANCA may have a pathogenic role in the development of HSP.

Increased dimeric IgA-producing B cells in tonsils in IgA nephropathy determined by in situ hybridization for J chain mRNA

The origin of mesangial IgA deposits in IgA nephropathy (IgAN) remains obscure. A significant proportion of deposited immunoglobulin is dimeric (J chain‐positive). Previous studies of J chain expression within lymphoid tissue in IgAN have utilized antibodies which other investigators have found to be non‐specific. To address this problem, we have developed an in situ hybridization (ISH) method for the detection of J chain mRNA within IgA plasma cells. Tonsils from 12 patients with IgAN and 12 controls were studied using (i) non‐isotopic ISH for J chain mRNA, and (ii) combined immunofluorescence (IF) and fluorescent ISH. J chain mRNA‐positive cells were identified in germinal centres, and within the subepithelial and interfollicular zones. A greater number of J chain mRNA‐positive cells were found in the germinal centres of patients (mean 57.7±4.6 cells/105μm2) compared with controls (mean 36.9±3.5 cells/105μm2) (P < 0.001). Combined IF and fluorescent ISH showed a greater proportion of J chain mRNA‐positive interfollicular IgA cells in patient tonsils (3.2±3.4%) compared with controls (21±2.3%; P < 0.02). These results indicate a shift towards dimeric IgA production in the tonsils in IgAN. In addition, the finding of excess numbers of J chain‐positive IgA‐negative cells within germinal centres suggests that an abnormality may be present at the B cell differentiation stage before IgA switching. These results further highlight immune abnormalities within the tonsil as a central feature of abnormal polymeric IgA biology in this common form of glomerulonephritis.

Increased and prolonged production of specific polymeric IgA after systemic immunization with tetanus toxoid in IgA nephropathy

IgA nephropathy (IgAN) is a chronic form of glomerulonephritis which is characterized by THE deposition in the glomerular mesangium of polymeric IgA (pIgA), the source of which is unknown. In order to investigate the production of pIgA in IgAN, patients were immunized systemically with tetanus toxoid (TT). Two weeks after immunization patients and controls responded to TT with an IgA response of similar magnitude. HPLC separation of sera showed that patients with IgAN produce significantly more plgA anti‐TT than controls (7·7 versus 2·88 arbitrary units; P 0·04). At this time, 33% of serum IgA anti‐TT produced by patients with IgAN was polymeric, compared with 21% produced by controls (P < 0·02). Monomeric IgA (mIgA) anti‐TT levels were similar in both groups. Four weeks after immunization the proportion of pIgA anti‐TT in controls and patients was significantly reduced from the 2 week level (from 21% to 0%, P < 0·02 for controls: and from 33% to 8%, P 0·001, for patients). Only four out of 12 controls had any detectable pIgA anti‐TT at this time compared with nine out of 10 patients with IgAN (P<0·05), and IgAN patients produced proportionally more pIgA anti‐TT than did controls (median 8%, interquartile ranges (IQR) 4–10%versus 0% IQR 0–3%; P < 0·01). HPLC analysis under acid conditions did not alter the pattern of pIgA and mIgA anti‐TT, suggesting that the high molecular weight IgA fraction was not due to complexes. These data indicate that circulating pIgA results (at least in part) from a systemic response to antigen, which may be exaggerated in IgAN.

Increased Immunoglobulin A and Immunoglobulin A1 Cells in Bone Marrow Trephine Biopsy Specimens in Immunoglobulin A Nephropathy

The origin of mesangial immunoglobulin A (IgA) in IgA nephropathy remains unknown. To investigate potential abnormalities within the bone marrow in this condition, bone marrow trephine biopsy specimens from seven patients and matched controls were studied using two-color immunofluorescence. In addition, serum levels of IgA and IgA1 were determined by radial immunodiffusion. Serum levels of IgA and IgA1 were higher in patients than in controls (4.53 +/- 1.38 g/L v 2.56 +/- 1 g/L, P < 0.01 and 3.68 +/- 1.11 g/L v 1.92 +/- 0.7 g/L, P < 0.005, respectively). In addition, patient trephine biopsy specimens contained an increased percentage of IgA plasma cells (61.6% +/- 4.4%) compared with controls (47.3% +/- 2.5%) (P < 0.02). The proportion of IgA plasma cells bearing subclass IgA1 was also greater in the patient biopsy specimens (91.6% +/- 1.9%) compared with controls (81.4% +/- 2.7%) (P < 0.01). In patients a positive correlation between the percentage of marrow IgA plasma cells and serum IgA levels was found (r = 0.94, P < 0.002). However, our studies failed to demonstrate a similar correlation between serum IgA1 levels and IgA1 marrow cells. These findings support the hypothesis that mesangial IgA may derive from the bone marrow.

Henoch-Schönlein purpura with immunoglobulin a nephropathy and abnormalities of immunoglobulin a in a Wiskott-Aldrich syndrome carrier

Abnormalities of immunoglobulin A1 (IgA1) glycosylation have been described in patients with IgA nephropathy (IgAN), whether primitive or secondary to Henoch-Schönlein purpura. The Wiskott-Aldrich syndrome, an X-linked recessive disorder, is associated with abnormalities of IgA. Renal involvement with mesangial IgA deposition identical to that found in IgAN has been reported during this affection. We report the case of a female carrier of the Wiskott-Aldrich syndrome presenting with Henoch-Schönlein purpura and abnormalities of IgA glycosylation, as previously reported in patients with IgAN. The galactosylation abnormalities of IgA could be linked to the patients status as carrier of the Wiskott-Aldrich syndrome and could contribute to the pathogenesis of IgAN.

Expression of T cell receptor variable region families by bone marrow γδ T cells in patients with IgA nephropathy

IgA nephropathy (IgAN) is characterized by mesangial deposition of polymeric IgA (pIgA). Abnormalities of the IgA system include reduced mucosal and increased bone marrow (BM) pIgA production. γδ T cells are regulators of mucosal IgA production and oral tolerance. We have described previously a deficiency of γδ T cells expressing Vγ3 and Vδ3 from the duodenal mucosa in IgAN. Since pIgA production is displaced to the BM, we have now studied BM γδ T cells in IgAN.