Glomerular and serum immunoglobulin G subclasses in IgA nephropathy

The distribution of human IgG subclasses among mesangial glomerular deposits of 11 patients with IgA nephropathy (IgA-N) was examined by indirect immunofluorescence with subclass-specific mouse monoclonal antibodies (mAb). A subclass restriction was observed with mesangial deposits containing almost exclusively IgG1 (81% of the studied biopsies) and IgG3 (64%). IgG2 was present in only 1 out of the 11 cases studied and IgG4 was never found to be present, although seven different anti-IgG4 mAb were used. In addition, serum levels of total IgA and IgG, as well as serum IgG subclass levels, were measured in 27 patients with IgA nephropathy by an indirect competitive immunoenzymatic assay using mAb. It was noted in IgA-N patients, but not in normal individuals, that there was significant positive correlation between total IgA and IgG serum levels which was entirely due to a positive correlation between total serum IgA and IgG2 levels. This study provides no explanation for the subclass restrictions observed but suggests that (i) the presence of IgA-IgG1-IgG3 in mesangial deposits may be secondary to an antigenic stimulation, possibly viral, and (ii) the positive correlation between IgA and IgG2 serum levels may result from an increased T helper function.     

Profiles of immunoregulatory cytokine production in vitro in patients with IgA nephropathy and their kindred.

We hypothesized that the altered immunoglobulin synthesis and/or lymphocyte function apparent in patients with IgA nephropathy (IgAN) is due to a primary defect in lymphokine regulation. In addition, we reasoned that such changes in lymphokine production might be, at least partially, genetically determined. To assess the extent of lymphocyte abnormalities, we investigated the profile of cytokine production from peripheral blood mononuclear cells (PBMC) in 34 IgAN patients and 44 of their first degree relatives, 10 of whom had persistent microhaematuria. Compared with healthy volunteers (n = 34), PBMC from patients showed increased IL-2 production both spontaneously or after phytohaemagglutinin (PHA) (20 micrograms/ml) stimulation, whereas IL-4 and interferon-gamma (IFN-gamma) production were significantly higher only after stimulation. Microhaematuric relatives had a similar pattern of cytokine production, whereas non-microhaematuric relatives showed no significant difference versus normals. The altered pattern of cytokine production appeared to be quite specific to IgAN patients and their microhaematuric relatives, because patients with other forms of primary glomerulonephritis (n = 17) did not differ from normal individuals. Patients and relatives that hyperproduced IL-4 were also hyperproducers of IL-2. No such congruence was seen in any other group or with any other pairing of cytokines. We propose that a subpopulation of IgAN patients bear lymphocytes intrinsically hyperresponsive. Among those individuals such hyperresponsiveness may be causally related to the pathogenesis and/or character of IgAN.     

Serum levels of IgA1 and IgA2 in children and in patients with IgA deficiency

Serum levels of IgA1 and IgA2 were measured by solid phase radioimmunoassay in samples from 110 children between 3 months and 10 years of age. Both IgA1 and IgA2 were detectable in all samples, and both IgA1 and IgA2 increased with increasing age. The percent of total serum IgA that was IgA2 did not change with age and was the same in samples from children (15.05 +/- 10.2%) as in samples from adults (15.86 +/- 7.98%). The proportion of serum IgA that was IgA2 was much less variable within sibships than within the group at large (P less than 0.005). In the 16 patients with IgA deficiency, the proportion of serum IgA that was IgA1 or IgA2 was highly variable. IgA2 constituted more than 50% of the IgA in 5 patients and less than 7% of the IgA in an additional 5 patients. These findings suggest that regulation of serum concentrations of IgA1 and IgA2 is complex and influenced by genetic factors and probably other unidentified factors.     

In vivo alteration of antibody production in patients with IgA nephropathy

Augmentation of IgA production has been postulated for the development of IgA nephropathy. An influenza HA vaccine was administered to healthy adults and patients with IgA nephropathy to elucidate if there was any in vivo alteration of antibody production in response to antigenic stimulation in these patients. The vaccine was administered s.c. in a dose of 350 CCA units at an interval of 4 weeks. IgG, IgA and IgM class antibodies to influenza HA antigens and three classes of rheumatoid factors (RF) were determined using a solid phase fluorescence immunoassay. The titres of IgG class antibodies to influenza HA antigens did not change significantly in either group after the vaccination. No significant differences were observed in the titres of IgG antibodies between the two groups. IgA antibodies were significantly increased only in patients in the 4th week and continued to the 8th week. The titres of IgA antibodies were always higher in patients than in controls during the study period. IgM antibodies were significantly increased stepwise in both groups to an equal degree. IgG and IgA RF were always higher in patients than in controls. IgM RF were significantly increased and higher than in controls in the 8th week in patients. It is concluded that patients with IgA nephropathy might be high responders for IgA antibody production, and that polyclonal activation might be associated with increased IgA production following in vivo antigenic stimulation in these patients.     

Increased serum levels of IgA1-IgG immune complexes and anti-F(ab')2 antibodies in patients with primary IgA nephropathy

A solid-phase ELISA was used to detect IgA1 immune complexes (IgA1 ICs) containing IgG and IgM in 38 serum samples from 30 patients with primary IgA nephropathy (IgAN) and 14 subjects with non-IgA chronic glomerulonephritis. A jackfruit lectin, jacalin, was used as the substrate for the selective binding of human IgA1 ICs in serum PEG precipitate (7%). The presence of IgG, A and M antibodies against the F(ab')2 region of IgG was also investigated by the solid-phase ELISA. Six patients were studied during remission and relapse (fever, upper respiratory tract infection and macroheamaturia). The results showed significant increases in serum levels of IgA1 ICs (P less than 0.001) in 39.4% of the IgAN patients, IgA1-IgG ICs (P less than 0.001) in 68.4%, and IgA1-IgM ICs (P less than 0.002) in 10.5% of the patients. A significant increase in IgA1-IgG ICs was observed during relapse (P less than 0.02). Significantly high values of IgG (P less than 0.003) and IgA (P less than 0.001) antibodies directed at the F(ab')2 region of IgG were found. A significant increase in anti F(ab')2 antibodies (class IgA and IgM) was seen in the acute phase of the disease. The data suggest that an increased production of IgA1 ICs occurs in IgAN patients; ICs are mainly IgA1-IgG ICs during relapse. The presence of high serum levels of IgG and IgA antibodies against the F(ab')2 region of IgG indicates that in addition to the multiple anomalies of IgA regulation described in IgAN patients there may be further aberrances.     

Subpopulations of T alpha cells in patients with IgA nephropathy: correlation between T alpha 4 cells and in vitro IgA production

T cells play important roles in the regulation of the immune system and are divided into subpopulations by various kinds of markers on the membrane surface. T cells with Fc-receptors for IgA are termed T alpha cells, and the properties of this cell population have been revealed in recent years. T alpha cells are increased in patients with IgA nephropathy and possess IgA specific helper activity. T alpha cells consist of two subpopulations, T alpha cells with OKT4 antigen (T alpha 4 cells) and with OKT8 antigen (T alpha 8 cells). To investigate the immunological aberrations in patients with IgA nephropathy, we detected immunoglobulin produced by peripheral blood lymphocytes and enumerated the numbers of T alpha cells (including both T alpha 4 and T alpha 8 cells). The numbers of T alpha 4 cells (but not T alpha 8 cells) and in vitro IgA production were increased in patients with IgA nephropathy (IgA nephropathy, mean 1.9%. Control, mean 0.8%. P = 0.0075). In addition, the numbers of T alpha 4 cells and the amount of IgA in the supernatant of lymphocyte cultures were positively correlated in these patients (P = 0.025. r = 0.3836). From the results in the present study, it was suggested that T alpha 4 cells might be related to immunological aberrations, such as an increase in IgA seen in patients with IgA nephropathy.     

Serum IgE levels in patients with selective IgA deficiency.

In this study serum IgE levels were measured by a double-antibody radioimmunoassay in 31 patients with serum IgA concentration less than 0.01 mg/ml who were followed in the arthritis and allergy clinics. On a group basis there was no significant difference in mean serum IgE levels between the IgA deficient patients and normal subjects of the same age. However, in the absence of atopic disease, IgA deficient patients had significantly lower serum IgE levels. When atopy was associated with IgA deficiency IgE levels were the same as in the normal subjects but significantly lower than those of atopic non-IgA deficient patients. IgE levels in those with recurrent respiratory tract infection were not different. Adults with anti-IgA antibodies had significantly lower IgE values. IgE levels in patients with RA, JRA or SLE were not significantly different. Selective IgA deficient patients may have a relative deficiency of serum IgE depending on the comparison group.     

Decreased cervicovaginal production of both IgA1 and IgA2 subclasses in women with AIDS

Paired sera and cervicovaginal secretions from 35 HIV-1-infected women representing different CDC stages of HIV infection were evaluated for total IgA, IgA1 and IgA2, for IgA, IgA1 and IgA2 to gp160, and for albumin. Age-matched healthy women (n= 45) served as controls. The secretion rates of total IgA, IgA1 and IgA2 were evaluated by calculating their relative coefficients of excretion by reference to albumin. In HIV-infected women, total IgA1 and IgA2 in sera and in cervicovaginal secretions increased proportionately as early as stages II + III and more markedly at stage IV. By contrast, the secretion rates of total IgA, IgA1 and IgA2 were markedly reduced in AIDS women, the IgA2 secretion rate decreasing significantly as early as stages II +III. This apparent discrepancy was probably the result of increased transudation of serum-borne immunoglobulins into the vaginal cavity, since albumin levels in cervicovaginal secretions increased significantly according to the stages of disease. HIV-reactive IgA antibodies in serum, as in cervicovaginal secretions, were principally found within the IgA 1 subclass. In women at stage IV, a high local production of IgA1 to gp160 occurred in spite of the impairment of cervicovaginal IgA synthesis, probably because of marked genital HIV replication at advanced stages.     

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.     

The bone marrow as production site of the IgA deposited in the kidneys of patients with IgA nephropathy

Patients with primary IgA nephropathy have increased plasma levels of polymeric IgA1 and deposits of IgA1 in their kidneys. The origin of this material is unknown. The production of IgA and its subclasses was investigated in the bone marrow of 14 patients and 19 controls using two colour immunofluorescence and tissue culture. Patients had an increase in the percentage of plasma cells containing IgA (45.8 +/- 7.2 mean +/- s.d.) compared to controls (40.1 +/- 10.5) (P = 0.08). IgA plasma cells containing subclass IgA1 were significantly (P less than 0.01) increased in patients (89.9 +/- 2.7%) compared to controls (84.1 +/- 6.7%). Correspondingly IgA plasma cells containing subclass IgA2 were significantly decreased (P less than 0.01) in patients (7.4 +/- 3.0%) compared to controls (13.5 +/- 5.9%). Production of IgA in bone marrow culture in patients was increased (1,684 +/- 1,151 ng/culture) compared to controls (1,087 +/- 937), but this difference was not significant (P = 0.2). However, in patients the IgA1 subclass contributed significantly (P less than 0.01) more to the IgA synthesis in culture (ratio of IgA1 over IgA: 0.96 +/- 0.02) than in controls (ratio 0.90 +/- 0.06). These findings suggest that the bone marrow may well be the site of long-term overproduction of the IgA1 found in the circulation and mesangial deposits in IgA nephropathy.     

Serum IgA preferentially binds to cationic polypeptides in IgA nephropathy.

The observation of negatively charged IgA in the mesangium of patients with primary IgA nephropathy (IgA-GN) prompted us to study the charge of serum IgA in IgA-GN, Henoch Schönlein purpura (HSP), alcoholic liver cirrhosis (ALC), membranous nephropathy (MGN) and systemic lupus erythematosus (SLE). Since no abnormal distribution of IgA isoelectric points was detected by isoelectric focusing studies, we developed a sensitive charge-dependent assay using plates coated with either cationized BSA (cBSA) or poly-L-lysine. In 15 IgA-GN sera, the amount of IgA reacting specifically with cBSA (cBSA-IgA) was almost linearly correlated with the poly-L-lysine-binding IgA (r = 0.97, P = 0.0006), suggesting that both assays detect charge-dependent interactions and thus probably measure anionic IgA. Significantly high serum levels of cBSA-IgA were observed in 56% of IgA-GN patients and in 40% of ALC patients. In contrast, normal serum levels of cBSA-IgA were detected in HSP, MGN and SLE. Both, the mono- or polymeric IgA bound to cBSA in a patient''s serum studied. Contrasting with the presence of anionic IgA, no increase of cBSA-IgG was observed in IgA-GN. IgA rheumatoid factor (IgA-RF) assay showed high levels in IgA-GN (39%) and in ALC (25%). IgA-RF levels did not correlate with the amount of cBSA-IgA. When 18 patients with IgA-GN were tested after kidney transplantation, increased levels of cBSA-IgA and/or IgA-RF were found to be associated with the recurrence of mesangial IgA deposits in the graft. This suggests that both negatively charged IgA and IgA-RF may play a role in the recurrence of mesangial IgA deposits.     

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.     

Increased IL-10 production by stimulated whole blood cultures in primary IgA nephropathy

Most patients with primary IgA nephropathy (IgAN) have a significantly higher memory repertoire of IgA1-producing B lymphocytes in their bone marrow together with high plasma levels of IgA1. The connection between the mucosal immune system and the bone marrow compartment is probably based on traffic of either antigen-presenting cells (APC) or antigen-specific lymphocytes. Cytokines play an important role in the proliferation and differentiation of lymphoid cells. In order to mimic the in vivo situation as much as possible, we assessed cytokine production profiles ex vivo in 23 IgAN patients and matched controls, using lipopolysaccharide (LPS)- or phytohaemagglutinin (PHA)-stimulated whole blood (WB) cultures. Interferon-gamma (IFN-γ), IL-2, IL-6, IL-10 and tumour necrosis factor-alpha (TNF-α) production in culture supernatants were determined by cytokine-specific ELISAs. Compared with controls, PHA-stimulated cultures resulted in significantly higher IL-10 (P < 0.001), IL-2 (P < 0.005) and IFN-γ (P < 0.001) levels in IgAN patients, but no significant differences in TNF-α or IL-6 levels were found. In LPS-stimulated cultures, the only significant difference (P < 0.001) between the two groups was the increased IL-10 production in IgAN patients. The enhanced cytokine production in stimulated WB cultures suggests altered monocyte-related T cell responses in patients with IgAN. Increased IL-10 production may eventually result in an increased number of IgA-producing B lymphocytes in the bone marrow. In addition, high levels of endogenous IL-10 may down-regulate the effector functions of monocytes, or possibly APC in general, and consequently the IgA response at the mucosal level.     

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.     

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.     

Serum ceruloplasmin and copper levels in patients with primary brain tumors

Summary Serum copper and ceruloplasmin levels are known to increase in several malignancies such as osteosarcomas, some gastrointestinal tumors, and lung cancer. In this study serum copper and ceruloplasmin levels in 40 patients with primary brain tumors were studied. Both parameters were increased in sera of patients with tumors in comparison with healthy subjects or patients with non-tumorous neurological diseases. It is concluded that copper and ceruloplasmin represent a good complement to some other nonspecific parameters in evaluating the activity of malignancy and the therapeutic results.     

Altered production of IgE and IgA induced by IL-4 in peripheral blood mononuclear cells from patients with IgA nephropathy.

In order to elucidate the factors responsible for altered immunoglobulin production in patients with IgA nephropathy (IgAN), the in vitro effects of IL-4 and interferon-gamma (IFN-gamma) on the synthesis of IgE and IgA by peripheral blood mononuclear cells (PBMC) were studied. Spontaneous IgE and IgA synthesis by PBMC was significantly increased in patients with IgA nephropathy compared with controls. The maximum amounts of IgA and IgE synthesis by PBMC after stimulation with IL-4 were almost the same both in patients with IgAN and in controls. The enhancement rate of IL-4-induced IgE and IgA synthesis was significantly lower in IgAN than in the controls, suggesting in vivo preactivation of PBMC in IgAN patients. IFN-gamma suppressed IgA and IgE synthesis by PBMC from IgAN patients as well as controls. However, the suppressive effect on IgE synthesis was less prominent in patients with IgAN. These results suggested that altered IL-4 action might be involved in the development of IgA nephropathy.