Characterization of an acidic nuclear protein recognized by autoantibodies in sera from patients with IgA nephropathy.

A nuclear antigen is recognized by autoantibodies in the sera of some patients with IgA nephropathy. Using these autoantibodies as a reagent, this antigen was purified 77.2-fold by ammonium sulfate fractionation, DEAE chromatography and Sepharose 6BCL gel filtration. The antigenicity of this antigen was sensitive to trypsin but resistant to RNase and DNase, suggesting that the antigenic determinant resided in protein and not nucleic acids. This antigen was inactivated at 56 degrees C for 3 h. Isoelectrophoretic focussing showed that the pI was below 4. The immunoblotting (Western transfer) assay showed a single polypeptide (69,000 Daltons) which proved to be a reactive antigen.     

Detection of polymeric IgA in glomeruli from patients with IgA nephropathy.

A study on the detection of polymeric IgA in glomeruli from renal biopsy specimens in patients with IgA nephropathy is described. Renal biopsy specimens were obtained from patients with IgA nephropathy. These specimens were stained with FITC-labelled anti-human J chain antisera and then examined with a fluorescent microscope. The J chain was observed in the glomerular mesangium by immunofluorescent staining. In parallel studies, renal biopsy specimens were treated with citrate buffer (pH 3.2) and the 'eluate' was neutralized by sodium hydroxide. The eluate was labelled with iodine-125, and the radiolabelled 'eluate' was fractionated by sucrose density-gradient ultracentrifugation. Polymerized IgA in the 'eluate' obtained from patients with IgA nephropathy was found to sediment predominantly as 9S to 11S using a sucrose density gradient analysis. Polymeric IgA in the fractions of the density gradient analysis was determined by anti-human IgA and anti-human J chain antisera. It was demonstrated that IgA and J chain were eluted from the glomeruli in some patients with IgA nephropathy. It is concluded that IgA deposited in the glomeruli is composed of dimers and/or larger polymers of circulating IgA in some patients with IgA nephropathy.     

T-cell dysfunctions in IgA nephropathy: specific abnormalities in the regulation of IgA synthesis

This work was undertaken to determine the cellular abnormalities that could explain the high levels of serum IgA frequently found in patients with IgA nephropathy. Seventeen control subjects and twenty-seven patients who had received no therapy were studied. After in vitro pokeweed mitogen (PWM) stimulation, significantly higher amounts of IgA were produced by peripheral blood mononuclear cells (PBM) of patients when compared with those of the control group (560 +/- 97 vs 231 +/- 57 ng/ml, P less than 0.0025). No differences were observed in the synthesis of IgG and IgM. Twenty out of twenty-seven patients presented an increase in the percentages of OKT4+ cells (mean + 2 SD), in relation to the control group, with normal or elevated percentages of OKT8+ cells. The OKT4+/OKT8+ cell ratio was elevated in 12 out of 27 patients. All patients presented some abnormality in the generation of IgA-specific suppressor cells at variable doses of concanavalin A (Con A) on in vitro PWM-stimulated culture of PBM. In both assays low doses of Con A (2.5 micrograms/ml) induced a certain suppression of IgA synthesis in patients that was not observed in the majority of the control group. At these doses some patients also showed an enhancement in the synthesis of IgG and IgM. On the contrary, higher doses of Con A (50 micrograms/ml) produced significantly less IgA suppression than the controls. Normal IgA-suppression values were found at 10 micrograms/ml of Con A. T cells obtained from patients were significantly more efficient than T cells from controls in providing IgA-helper activity for normal allogeneic enriched B cells (P less than 0.025) in PWM-stimulated cocultures. These results show that patients with IgA nephropathy present, after mitogen stimulation in vitro, a specifically increased production of IgA as well as an augmentation in the activity of IgA-helper T cell and a deregulation on IgA-suppressor T-cell function. According to these data, it is suggested that the alteration observed in helper T cells might precede that of suppressor T cells. These immunoregulatory abnormalities might contribute to the pathogenesis of the disease.     

Increase of IgA specific helper T alpha cells in patients with IgA nephropathy.

Patients with IgA nephropathy show an emergence of IgA dominant circulating immune complexes (CIC) as well as increased levels of serum IgA and/or IgA bearing peripheral blood lymphocytes. In order to elucidate immunological aberrations responsible for the increased IgA synthesis in such patients, quantitative and qualitative analysis was performed on T alpha cells which have been recently identified as possessing IgA specific helper activity on human B cells. Three different methods were employed to quantitate T alpha cells. These methods included a rosette formation of T cells with either bovine red cells coated with the IgA fraction of anti-bovine red cell antiserum or those coated with TNP and anti-TNP IgA antibody, and an analysis of T cells combined with fluorescein conjugated human IgA myeloma protein. T alpha cells were sorted by a fluorescence activated cell sorter and co-cultured with a B cell rich fraction to evaluate whether there is a qualitative difference in IgA specific helper activity between patients and healthy adults. T alpha cells were significantly increased in patients with IgA nephropathy while there were no significant changes in patients with chronic proliferative glomerulonephritis without mesangial deposition of IgA. There was no qualitative difference in IgA specific helper activity of T alpha cells between patients and healthy adults. It is suggested that increased levels of T alpha cells in patients with IgA nephropathy may be responsible for increased synthesis of IgA in such patients.     

Inhibition of neutrophil migration by serum IgA from patients with IgA nephropathy.

Previously we showed that patients with IgA nephropathy present high serum levels of polymeric IgA and that in vitro polyclonal stimulation of their peripheral blood lymphocytes results in the synthesis of a large amount of true polymeric IgA. The aim of this study was to determine if the serum of patients with IgA nephropathy was capable of suppressing the directional migration of human normal polymorphonuclear cells (PMN), as do the polymeric fractions of IgA myeloma. Incubation of controls' PMN with fresh or heat-inactivated patients' plasma impaired the casein-induced directional migration significantly more than incubation with controls' plasma. This inhibitory effect was closely linked to polymeric IgA fractions and to a lesser extent with monomeric IgA immune complexes. The removal of IgA by immunoadsorption from patients' plasma completely abolished the migration suppression observed on controls' PMN. These results suggest that the high serum levels of polymeric IgA observed in patients with IgA nephropathy, by inhibiting directional migration and phagocytosis of PMN, and probably monocytes, could facilitate the persistent circulation and renal deposition of immune complexes.     

Effect of T-helper cytokine environment on specificity of T-cell responses to mycobacterial 65,000 MW heat-shock protein.

The purpose of this work was to determine if the fine specificity of T cells differed between mice immunized with an antigen in a T helper 1 (Th1) cytokine-dominated environment as compared with a T helper 2 (Th2) cytokine-dominated environment. It was found that splenic T cells from mice immunized with mycobacterial heat-shock protein (hsp 65) and interleukin-12 (IL-12) produced less interleukin-4 (IL-4) and more interferon-gamma (IFN-gamma) in response to stimulation with hsp 65 in vitro than did T cells from mice immunized with hsp 65 alone. The T-cell proliferative response to hsp 65 did not differ between the two groups of mice, although the responses were higher than those of T cells from non-immunized mice. Strikingly, T cells from mice given hsp 65 and IL-12 gave significantly higher responses to six peptides (corresponding to the sequence of hsp 65) to which T cells from mice immunized with hsp 65 alone did not respond. It is considered that different epitopes are presented to T cells (possibly owing to changes in antigen processing) if the environment is shifted, by IL-12, from Th2 towards Th1 cytokines.     

Binding of IgA to erythrocytes from patients with IgA nephropathy

Primate erythrocytes (RBCs) may be involved in the transport and processing of C3b-containing immune complexes (IC). Compared to RBCs from healthy controls, increased amounts of IgA were detectable on RBCs from 7 of 17 patients with IgA nephropathy (IgA NP). There was no difference in the amount of IgG or IgM. The highest amount of RBC-bound IgA corresponded to 6 ng IgA/10(8) cells. The mechanisms involved in the binding of IgA to RBCs were investigated in vitro. Isolated IgA1 or IgA2 did not bind to RCBs from a patient with IgA deficiency. In contrast, incubation of RBCs with a polyethyleneglycol (PEG) precipitate of serum from a patient with IgA NP which contained IgA-IC resulted in IgA1 binding. However, this binding was not inhibited by monoclonal anti-CR1 or by an excess of IgG or IgM. Factor I did not cause release of IgA from RBCs from patients with IgA NP. Heat-aggregated IgA1 also bound to RBCs and this binding was not affected by the presence of complement. We conclude that minute amounts of IgA-IC are bound to RBCs by a complement- and Fc receptor-independent mechanism. The quantity of IgA-IC associated with RBC is so small that it is unlikely to represent an important in vivo route of IgA-IC transport or processing.     

IgA polyspecific autoantibodies in IgA nephropathy.

The specificity of circulating and kidney-bound IgA during IgA nephropathy is still a matter of discussion. In the present study, high levels of IgA antibodies directed against a panel of self and non-self antigens were found in the serum from patients with IgA nephropathy and were eluted from four out of the seven kidney biopsies studied. After immunoadsorption of pooled selected serum samples on TNP and actin-coated columns, polyspecific IgA antibodies were eluted. This supports the hypothesis that IgA-bearing B cells clones most probably producing polyspecific antibodies are a major feature of human IgA nephropathy. These findings also suggest that it may be hazardous to draw conclusions from the finding of apparently monospecific IgA antibodies in this condition.     

Increased concentration of serum IgA antibody to pneumococcal polysaccharides in patients with IgA nephropathy.

It has been postulated that IgA nephropathy (IgAGN) is caused by deposition within the glomerular mesangium of IgA polymers and IgA containing immune complexes which are overproduced in response to antigens presented at mucosal surfaces. To test this, the concentrations of specific antibodies to capsular polysaccharides from pneumococci, which are common commensal and/or pathogenic bacteria in the respiratory tract, have been measured. Sera from 35 patients with IgAGN, six with systemic lupus erythematosus (SLE), eight with membranous glomerulonephritis (MGN) and six with Goodpasture's syndrome (GPS), and from 20 controls (C) were assayed. The concentrations of IgG and IgA antibodies specific for each of five pneumococcal polysaccharides (serotypes 2, 7F, 9N, 14 and 23F) were determined by ELISA. The results from the SLE, MGN and GPS patients were pooled and used as a control group of patients with forms of nephritis other than IgAGN (patient controls, PC). Groups were compared using the Wilcoxon test or the Chi square test. There were no significant differences in the concentrations of IgG antibody to any of the serotypes between the IgAGN and normals, but the PC sera had significantly lower concentrations than either the IgAGN or normals. By contrast, there were no differences between the PC and C in the proportion with detectable IgA antibody to four of the serotypes, but this was significantly increased in IgAGN. There was insufficient IgA antibody to serotype 2 to detect in the assay system used. It is concluded that IgAGN patients have greater concentrations of IgA antibodies, but not IgG, to these pneumococcal polysaccharides, compared with normal controls or patients with other forms of nephritis.     

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.     

Clearance kinetics and fate of macromolecular IgA in patients with IgA nephropathy

IgA glomerulonephritis is associated with macromolecules of polymeric IgA in the circulation and mesangial deposits. An impairment in the reticulophagocytic function of patients with IgA nephropathy has been postulated as the potential cause for persistence of IgA immune complexes in the circulation and their eventual glomerular deposition. Since the fate and removal mechanisms of circulating macromolecular IgA are unknown in humans, we examined the blood clearance and organ uptake of purified IgA polymers and macromolecules in patients with IgA nephropathy and normal controls. The IgA macromolecules were prepared by covalent cross-linking of purified human polymeric IgA with a heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate. After intravenous injection, large IgA molecules were removed rapidly from the circulation of patients (t1/2 = 3.8 +/- 1.0 minutes) and controls (t1/2 = 4.9 +/- 1.5 minutes). Dynamic gamma camera scintigraphy revealed the liver as the major organ that mediated the removal of the macromolecular IgA with no significant difference in the rate of hepatic uptake for patients (t1/2 = 3.4 +/- 0.6 minutes) and controls (t1/2 = 3.3 +/- 0.9 minutes). No significant amount of radioactivity could be detected in the lungs, kidneys, and spleen. The small polymers had a slower and similar clearance rates for patients (t1/2 = 29.3 +/- 7.9 h) and controls (t1/2 = 29.0 +/- 8.6 h). These findings have general significance in showing the liver as a major organ for removal of macromolecular IgA. In addition, the results have specific importance in showing that patients with IgA nephropathy do not suffer from an IgA removal dysfunction.     

Immunofluorescent studies on S-protein in glomeruli from patients with IgA nephropathy.

Detection of S-protein, which is a regulatory component of the membrane attack complex (MAC), and of C9 in glomeruli by immunofluorescence in 11 of 15 patients with IgA nephropathy is described. The study showed that glomerular injuries such as glomerular adhesion to Bowman's capsule and crescent formation were more marked in glomeruli with S-protein and/or C9 in patients with IgA nephropathy. S-protein co-existed with C9 in glomeruli from such patients. It is suggested that the deposition of S-protein might reflect certain types of histopathologic injuries in glomeruli from patients with IgA nephropathy. It is concluded that activation of terminal components of complement may be one of the exacerbating factors in patients with IgA nephropathy.     

Increase of IgA-specific switch T cells in patients with IgA nephropathy.

Enumeration and functional analysis of CD4+ T cells with receptors for the Fc portion of IgA (i.e. T alpha 4 cells) in the peripheral blood of patients with IgA nephropathy, their relatives and age-matched controls were performed to elucidate polyclonal activation of IgA production in this disease. Enumeration of T alpha 4 cells was performed by a fluorescence activated cell sorter, and functional analysis was carried out by separation of T alpha 4 cells, and IgM-, IgA- and IgG-bearing lymphocytes using panning methods followed by cultures of these cells for 7 days with pokeweed mitogen. There was a significant increase in the amount of peripheral blood T alpha 4 cells in patients with IgA nephropathy and their relatives. T alpha 4 cells specifically enhanced the switch of IgM-bearing cells to IgA-bearing cells, and this switch activity was inhibited by addition of human myeloma IgA. It is suggested that T alpha 4 cells may be responsible for polyclonal activation of IgA production in IgA nephropathy.     

Defective immunoglobulin A (IgA) glycosylation and IgA deposits in patients with IgA nephropathy

Defective glycosylation and immune complex (IC) formation may be of primary importance in immunoglobulin A nephropathy (IgAN) pathogenesis. The aim of this study was to determine whether defective IgA1 glycosylation might support renal deposition of IgA and disease activity. IgA was isolated from the serum of 44 IgAN patients and 46 controls and glycosylation analysed by ELISA using glycan‐specific lectins. IgA was measured by immunodiffusion and immune complexes by ELISA. IgA subclasses in IC deposits in kidney glomeruli were identified by immunohistochemical methods. A significant increase in N‐acetylgalactosamine (GalNAc) in terminal position (p = 0.02) observed in some of the IgAN patients, became more pronounced when sialic acid was removed from IgA1, indicating enhanced expression of α‐2,6‐sialyltransferase in patients compared with controls (p < 0.0001). Patients with defective galactosylation had lower serum IgA than other IgAN patients (p = 0.003). IgAN patients with both IgA1 and IgA2 glomerular deposits (21.7%) had increased GalNAc in terminal position (p = 0.003). Taken together, our results show that increased IgA glycosylation in IgAN associates with low levels of IgA, concomitant IgA1 and IgA2 glomerular deposits and poor clinical outcome.     

Antigenic epitopes of MAP2694 homologous to T-cell receptor gamma-chain are highly recognized in multiple sclerosis Sardinian patients

Mycobacterium avium ss. paratuberculosis (MAP) is an intracellular pathogen recently associated with multiple sclerosis (MS). Aiming to identify immunodominant epitopes belonging to MS related protein MAP2694 (UniProt accession no. Q73WG6), we investigated the binding activity of selected peptides against MS Sardinian sera. An overlapping 9-mers peptide library was synthesized spanning the entire aminoacidic sequence of the protein. Peripheral blood was collected from 47 MS patients and 42 sex and age matched healthy volunteers and subjected to enzyme-linked immunosorbent assay (ELISA) in order to investigate the reaction against the linear peptides generated. Two out of 58 synthetic 9-mers were strongly recognized by MS patients’ antibodies compared to controls. A competitive inhibition assay demonstrated that these two epitopes are immunodominant antibody targets within MAP2694 protein, as sera pre-adsorbed with these peptides were able to significantly block the antibody reaction to the MAP2694 protein, even if at a lesser extent than MAP2694 protein itself.     

Three T-cell epitopes within the C-terminal 265 amino acids of the matrix protein pp65 of human cytomegalovirus recognized by human lymphocytes

Although a T-cell response in human cytomegalovirus (HCMV)-immune individuals exists against the most abundantly expressed protein pp65 of the virus matrix, less is known about the determinants that evoke this response. The aim of the study was to identify regions within HCMV pp65 (ppUL83) that contain sequences for the cellular immune response by the use of three recombinant overlapping β-galactosidase pp65 fusion proteins (C74, C35, and C47), covering the C-terminal 265 amino acids of the entire pp65 sequence. Two T-cell epitope determinants were recognized by human lymphocytes of healthy, HCMV-seropositive, human leukocyte antigen (HLA)-typed individuals. One T-cell determinant (amino acids [aa] 303–388) was localized in the mid-region of the entire pp65 sequence and a second T-cell determinant (aa 477–561) within the C-terminal region. By fine mapping with synthetic hexadecamer peptides three T-cell epitopes were identified within these two regions: P10-I (aa 361–376) in the mid-region, P3-II (aa) 485–499), and P6-II (aa 509–524) in the C-terminal region. Inhibition studies with monoclonal antibodies to HLA class I or class II revealed a class I restricted response to peptides P10-I or P6-II, respectively. P10-I responders shared the HLA-DR11 allele and P6-II responders the -DR3 allele. Therefore, these T-cell epitopes of HCMV pp65 might be presented in association with particular HLA class II alleles. J. Med. Virol. 52:68–76, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Decrease of IgA-specific suppressor T cell activity in patients with IgA nephropathy.

The activity of IgA-specific suppressor T cells was lower in eight patients with IgA nephropathy than in six patients with chronic proliferative glomerulonephritis without glomerular deposition of IgA, two patients with acute glomerulonephritis, or five healthy adult controls. It was determined by the quantitation of immunoglobulins produced from pokeweed mitogen-stimulated B cells cultured with the T cell supernatant (TCS) obtained from concanavalin A-stimulated T cells. Results from a study on an identical twin sister with IgA nephropathy suggested that the decreased activity of IgA-specific suppressor T cells might not be a cause but a result of increased IgA-bearing lymphocytes and serum IgA in patients with IgA nephropathy.