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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-gamma), IL-2, IL-6, IL-10 and tumour necrosis factor-alpha (TNF-alpha) 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-gamma (P<0.001) levels in IgAN patients, but no significant differences in TNF-alpha 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.

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BACKGROUND: In IgA nephropathy (IgAN), the abnormalities in the IgA immune system are apparently restricted to the IgA1 subclass in the systemic compartment, as evidenced by the antigen-specific responses to recall antigens. Since precursors of IgA producing B cells in human peripheral blood belong predominantly to the mucosal compartment, we took the opportunity to assess the capacity of circulating B cells in peripheral blood (PBMC) of 20 IgAN patients and matched controls to produce IgA, IgA1, and IgA2. METHODS: Supernatants from T cell- (immobilized anti-CD3) and B cell-specific (CD40 ligation) activated cultures were assessed for immunoglobulin isotypes by ELISA. In addition, we compared the sensitivity of T and B cells to various cytokines (IL-2, IL-10, TGF-beta) in both culture systems. RESULTS: In contrast to significantly higher plasma IgA1 levels (P < 0.01), no significant differences in salivary IgA1 (P = 0.73) and IgA2 (P = 0.96) levels or ratios (P = 0.91) were found. In the absence of exogenous cytokines, none of the different culture systems led to significant differences in IgA or IgA subclass synthesis by PBMCs of patients and controls. However, in IgAN patients, the addition of IL-2 did not enhance the production of the IgA subclasses as was found in controls. Furthermore IL-10 led to significantly (P < 0.05) lower IgA1 and IgA2 synthesis in patients than in controls. TGF-beta induced suppression of all isotypes in patients and controls. None of the different conditions resulted in a selectively enhanced production of any one of the IgA subclasses. When both IL-10 and TGF-beta were added to the cultures, IgM was the predominant immunoglobulin synthesized both in patients and controls with a significantly (P < 0.05) lower synthesis of IgM, IgG, IgA1, and IgA2 in patients. CONCLUSION: These in vitro data suggest that PBMCs from patients contain more mature and further differentiated B cells. However, there was no selective IgA or IgA1 dysregulation of circulating B cells in IgAN. These results do not confirm the widely believed paradigm that patients with IgAN are primary hyperresponders.

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Apoptosis of glomerular cells (GMC) has been observed in the early phase as well as the resolution phase of Thy-1 nephritis. Recently, we and others reported that IgG2a (ER4G) and IgG1 (OX7) monoclonal mouse anti-Thy-1 antibodies (anti-Thy-1 MoAb) are able to induce apoptosis of rat GMC in vivo. The purpose of this study was to investigate whether cross-linking of Thy-1 would influence the degree of apoptosis in cultured rat GMC using monomeric and dimeric IgA anti-Thy-1 MoAb. IgA anti-Thy-1 MoAb (ER4A) was generated by class switching of the IgG producing ER4 (ER4G) hybridoma. The ER4A clone spontaneously produces monomeric (m-ER4A) and dimeric IgA anti-Thy-1 MoAb *di-ER4A). Unaltered epitope specificity of ER4A was confirmed by blocking experiments of the binding of fluorescence labeled ER4G to cultured rat GMC with unlabeled ER4A on FACS. For the experiments of apoptosis, quiescent rat GMC were incubated for eight hours with medium alone or with medium in the presence of 10 micrograms/ml of m-ER4A, di-ER4A or control IgA MoAb of corresponding sizes. Apoptosis was assessed by morphological studies, agarose gel electrophoresis and quantitative FACS analyses using terminal deoxynucleotidyl transferase (TDT) method and the annexin V method. The TDT method detects specific-DNA nicking in apoptosis. The annexin V method detects early membrane changes during apoptosis. In morphological studies, cells incubated with m-ER4A and di-ER4A showed typical apoptotic features such as nuclear condensation and fragmentation. DNA isolated from the cells incubated with di-ER4A was cleaved into a distinctive ladder pattern compatible with apoptosis. In contrast, both medium alone and control IgA MoAb did not reveal detectable changes in morphological studies and agarose gel electrophoresis. In quantitative analyses by FACS using the TDT method and the annexin method, both m-ER4A and di-ER4A induced significantly higher percentages of apoptosis in rat GMC as compared to the controls. Furthermore, di-ER4A was considerably more efficient than m-ER4A in inducing apoptosis possibly through additional cross-linking of Thy-1 on the cell surface. This notion was confirmed by experiments, in which the addition of goat anti-mouse kappa antibodies enhanced apoptosis of rat GMC pre-sensitized with m-ER4A. Taken together, our results indicate that apoptosis of rat GMC by anti-Thy-1 antibodies is enhanced by cross-linking of Thy-1 on the cell surface. These studies are of importance for our understanding of mechanisms that may play a role in glomerular diseases.

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The presence of immunoglobulin G (IgG) in the mesangial area in kidneys of patients with different forms of glomerulonephritis suggests a role for IgG in the inflammatory process. This study investigates whether IgG is able to bind to cultured human mesangial cells (MC) in vitro. Incubation of MC with 125I-aggregated IgG(125I-AIgG), as a model for immune complexes (IC), at 4 degrees C resulted in a time- and dose-dependent binding of 125I-AIgG to MC. The binding of 125I-AIgG to MC was inhibited by excess AIgG or Fc-fragments and not by F(ab')2-fragments or human serum albumin (HSA). Scatchard analysis revealed the presence of 2.8.10(6) receptors/cell with an affinity of 9.7.10(7) M-1. Incubation of MC with 125I-C1q resulted in a time- and dose-dependent binding of 125I-C1q to MC. The binding of 125I-C1q was inhibited by excess C1q or C1q talls and not by HSA. Scatchard analysis revealed the presence of 3.2.10(7) binding sites/cell with an affinity of 1.4.10(7) M-1. Immunoprecipitation of 125I-labeled MC membrane proteins with C1q or monoclonal antibodies directed against human C1q-R revealed a single 66 to 68 kd band under reducing conditions. Fluorescence-activated cell-sorter analysis revealed an average of 60.1% +/- 5.4% of the cells positive with a mean channel of fluorescence of 592. A cooperative effect between C1q-R and Fc gamma-R in the binding of 125I-AIgG to MC, was assessed by incubation of 125I-AIgG in the presence of increasing concentrations of C1q, C1q talls, or delta C1q. Only intact C1q showed a 6- to 11-fold enhancement in binding of 125I-AIgG to MC. These studies demonstrate the occurrence of C1q-R and Fc gamma-R on MC and indicate that binding of IC is enhanced after interaction of IC with C1q.

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Anti-Thy-1 nephritis is a model of mesangial proliferative glomerulonephritis. It has been suggested that apoptosis, which is a counteracting regulatory mechanism against undesired cell proliferation, is involved in sequential histological changes in this model. In the present study, we investigated whether IgG2a anti-Thy-1 monoclonal antibody (ER4) or its F(ab')2 fragments are able to induce apoptosis of rat glomerular mesangial cells (GMC) in vitro. After co-culture with ER4 or its F(ab')2 fragments, apoptosis was assessed by morphological studies with Hoechst 33258 stain and FITC-annexin V. The latter detects the dislocation of negatively charged phospholipid, phosphatidylserine, from the inner to the outer leaflet of the membrane during apoptosis. This is a sensitive method for the detection of apoptosis. Under fluorescent microscopy, distinct nuclear condensation and positive reactivity with FITC-annexin V were observed in cells co-cultured with ER4 or its F(ab')2 fragments. The results obtained by FACS analysis with annexin V showed a direct correlation with the detection of apoptosis with the terminal deoxynucleotidyl transferase reaction (TDT). Up to 19% and 23% of rat GMC, which were co-cultured for 24 hours with 1 microgram/ml (0.5 microgram/l x 10(5) cells) of ER4 or its F(ab')2 fragments, were labeled by TDT, respectively. With annexin V, up to 34% and 31% cells displaying apoptosis were seen. The degree of apoptosis as measured by the annexin V method was dependent on the concentration of ER4 and time of incubation in the presence of ER4. Finally, apoptosis was confirmed by gel electrophoresis of DNA isolated from the cells co-cultured with each monoclonal antibody (MAb). DNA extracts from cells co-cultured with ER4 or its F(ab')2 fragments demonstrated typical internucleosomal DNA fragmentation. Medium alone, controls of anti-human C3bi receptor MAb (IB4) and anti-rat MHC class I MAb (OX18) showed neither nuclear changes nor significant labeling of the cells with the TDT reaction or with the annexin V. Taken together, these results demonstrate for the first time that anti-Thy-1 MAb is able to induce apoptosis of rat GMC in vitro. The Thy-1 antigen on rat GMC, therefore, seems to function as one of the molecules regulating cell death and thereby may determine the degree of mesangial alteration in Thy-1 nephritis.

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Depositions of IgA in the renal glomerular mesangial area are a hallmark of IgA nephropathy, and are thought to be crucial for the onset of inflammation processes in IgA nephropathy. In this report we show that human mesangial cells (MC) in vitro bind IgA and that binding of IgA enhances the production of IL-6 by MC. Furthermore we show that the size of IgA is crucial in its capability to enhance IL-6 production. Monomeric IgA does not affect basic IL-6 production, whereas dimeric and polymeric IgA enhance IL-6 production up to 3- to 9-fold respectively. Additional studies demonstrate that enhanced IL-6 production by MC is not accompanied by increased proliferation of human mesangial cells, a finding which is distinct from that found with rat mesangial cells. Taken together, these fmdings suggest that deposition of dimeric and polymeric IgA in the mesangial area of human kidneys in IgA nephropathy may amplify local inflammation.

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The mesangium plays a crucial role in processes of inflammation in the kidney. Since deposits of IgA in the mesangium in patients with IgA nephropathy suggest a role for IgA in the inflammatory process, we investigated whether IgA is able to bind to cultured rat mesangial cells (MC) in vitro and induce activation of MC. As a source of IgA, monomeric (mIgA), dimeric (dIgA) and polymeric IgA-alpha-DNP (pIgA) rat monoclonal antibodies were used. FACS analysis indicated binding of dIgA and pIgA to MC while only a small percentage of the cells exhibited binding of mIgA. Additional experiments employing radiolabeled IgA revealed a time- and dose-dependent binding of 125I-dIgA and 125I-pIgA with 6 x 10(6) binding sites for dIgA with an affinity of 5.5 x 10(6) M-1 and 7.2 X 10(6) binding sites/cell for pIgA with an affinity of 1.2 x 10(6) M-1. As compared to 125I-dIgA and 125I-pIgA, little binding of 125I-mIgA to MC occurred; the binding of dIgA and pIgA was not influenced by excess cold BSA, IgG or asialofetuin. Since some studies have suggested that fibronectin might interact with IgA, the binding of IgA to MC in the presence or absence of fibronectin or the RGD fragment was also analyzed. However no influence of fibronectin or the RGD fragment on binding of dIgA and pIgA to MC was observed. As a measure for activation of MC by IgA, the production of IL-6 by MC was analyzed. Dimeric IgA and pIgA both induced a dose-dependent increase of IL-6 production by MC.(ABSTRACT TRUNCATED AT 250 WORDS)

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Previous reports have shown production of complement components C4, C2 and factor B by renal tissue. We have shown recently that human proximal tubular epithelial cells (PTEC) synthesize C3 in vitro, and that IL-2 enhances this production. In the present study we demonstrate that human mesangial cells (MC) in culture produce factor H and that supernatants of activated peripheral blood mononuclear cells (T cell growth factor (TCGF)) induce C3 production and enhance factor H synthesis in both a time- and dose-dependent manner. To investigate whether certain defined cytokines from TCGF were responsible for the observed effect, we tested various cytokines for their effect on complement production by MC. It is shown that IL-1 induces C3 synthesis whereas factor H production is up-regulated by IFN-gamma, in both a dose- and time-dependent manner. Antibody blocking experiments revealed that C3 synthesis induced by both TCGF and IL-1 could be blocked with antibodies specific for IL-1, and also that TCGF and IFN-gamma enhanced factor H synthesis could both be blocked with antibodies specific for IFN-gamma. Cycloheximide was able to inhibit C3 and factor H production, suggesting de novo synthesis of the proteins. mRNA-polymerase chain reaction (PCR) analysis revealed mRNA encoding for C3 after stimulation with TCGF and IL-1. Factor H genes are constitutively expressed in cultured mesangial cells and its expression is up-regulated by TCGF and IFN-gamma. Northern blot analysis with specific probes for C3 and factor H revealed bands which support the results obtained by PCR analysis.

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Previous reports have shown the presence of C1Q-R on monocytes, macrophages, polymorphonuclear cells, fibroblasts, platelets, lymphocytes, and endothelial cells. The present study demonstrates a functional C1Q-R on rat renal mesangial cells (MC). Incubation of MC with increasing concentrations of [125I]C1Q resulted in a dose-dependent binding of [125I]C1Q to MC; the binding of [125I]C1Q was inhibitable by excess unlabeled C1Q or C1Q stalks whereas BSA and C1Q globular heads had no effect. Scatchard analysis of the data revealed the presence of 6.2 x 10(7) binding sites/cell with an affinity of 4.9 x 10(6) M-1 for C1Q. Immunoprecipitation of 125I-labeled MC membrane proteins with C1Q or mAb directed against human C1Q-R revealed a single 66- to 68-kDa band under reducing conditions. We have shown previously that soluble stable aggregates of IgG bind to rat MC in a dose-dependent fashion. In addition the presence of a receptor for IgG has been described on rat MC. In order to find out whether there is a cooperative effect between C1Q and AlgG in binding of [125I]AlgG to MC, we incubated [125I]AlgG in the presence of increasing concentrations of C1Q, and showed a 5- to 15-fold enhancement of binding of [125I]AlgG to MC. Neither heat-inactivated C1Q nor C1Q stalks were able to enhance the binding of [125I]AlgG to MC. Enhanced binding by C1Q was only observed when aggregated IgG was used; the binding of monomeric IgG to MC was not affected by C1Q. These studies indicate that there is a cooperative effect between Fc gamma R and C1Q-R on MC in the recognition of immune complexes.

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Primary rat mesangial cells (MC) were cultured in RPMI-1640 containing 5% fetal calf serum (FCS). The cells produced interleukin-6 (IL-6) in vitro depending on the concentration of FCS in the medium. Binding of soluble aggregates of IgG (AIgG) to MC was visualized with AIgG coupled with aminomethyl coumarin acetic acid (AMCA). There was a dose-dependent binding of 125I-AIgG to MC at 4 degrees C. Scatchard analysis revealed binding of AIgG containing 20 to 24 molecules per aggregate, with an affinity of 2.2 x 10(8) M-1 and a total average number of 2.7 x 10(5) sites per cell. The binding of AIgG or immune complexes to MC resulted in enhanced production of IL-6 by MC in culture. This enhanced production of IL-6 was dependent on the concentration of AIgG. To our surprise, preparations of monomeric IgG (mIgG) also enhanced the production of IL-6 by MC, but to a lower extent when compared to levels induced by AIgG. Very little amounts of aggregated F(ab')2 fragments [AF(ab')2] bound to MC and consequently no significant enhancement of IL-6 release by AF(ab')2 was seen, suggesting that IL-6 production is an Fc receptor-mediated phenomenon. The production of IL-6 by MC is inhibitable by cycloheximide, thus indicating de novo synthesis. Northern blot analysis revealed a threefold increase in mRNA for IL-6 by AIgG in vitro. The increase in mRNA expression was directly related to the quantity of IL-6 released in the supernatant by MC. These results suggest that binding of AIgG or immune complexes to MC in vivo may induce IL-6 production by MC, thus leading to proliferation of MC.

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To analyze DR2 haplotypes as recognized by alloreactive T cells, lymphocytes from a DR7; DQw2 homozygous donor were cocultured with irradiated lymphocytes that were DRw15, DR7; DQw6, DQw2 heterozygous. In this report, we focus on two HLA-DQ-specific T-cell clones obtained from this priming. These two clones (c3518 and c3523) responded to the positive control (original stimulator) and five of 66 panel donors. Three of these donors typed DRw15, DR7; DQw6, DQw2, as did the positive control. One stimulatory donor typed DRw15, DR7; DQw6, DQw9 and one stimulatory donor typed DRw14, DR7; DQw5, DQw2. Oligonucleotide typing revealed that recognition by the clones depended on the simultaneous presence of the DQB1*0602 gene on one haplotype and DRB1*0701 or DQA*0201 on the other. The hypothesis that c3518 and c3523 recognize an HLA class II product that results from the combination of two different HLA haplotypes was further confirmed in family studies. In three families, it was shown that the DRw15, DR7; DQw6, (DQw2 or DQw9)-positive individuals were recognized, whereas the cells carrying either DRw15; DQw6, DR7; DQw2, or DR7; DQw9 were nonstimulatory. Our results can be explained in two ways: (a) the T cells recognize a class II dimer that results from trans-complementation of DQA1*0101 and DQB1*0602, and (2) the T cells recognize a DR7-derived peptide that is presented by DQw6.

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An important criterion for the selection of donors for bone marrow transplantation is the grade of matching for HLA between donor and recipient. For patients that lack an HLA-identical sibling, an extending pool of unrelated volunteers for bone marrow donation is available. From these donors the best matched candidate can be selected by serological typing, followed by a mixed lymphocyte culture (MLC). Oligonucleotide genotyping for HLA class II antigens is considered to be valuable for the prediction of MLC reactivity. We investigated whether this typing method, in combination with serological typing, would cover the recognition of all MLC stimulatory determinants. One hundred thirty-six combinations of HLA-A, -B, and -DR serologically identical individuals were tested in the MLC. Additional typing for HLA-DRB and HLA-DPB by oligonucleotide genotyping made it possible to evaluate the influence of these genes on MLC reactivity. Combinations that were matched for HLA-DRB gave significantly lower responses than those that were mismatched. Nevertheless, in the matched combinations responses were observed to 94% relative response index. These responses could all be attributed to HLA-DP, since all combinations that were identical by HLA-DPB genotyping were negative in the MLC. In conclusion, with the combined use of serology and oligonucleotide genotyping, responder-stimulator combinations can be selected that are identical for all MLC stimulatory determinants.

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The identification of 19 different HLA-DPB1 sequences implicates the existence of more DP specificities than can be typed for with cellular methods. How many of the DP beta sequences can be specifically recognized by T cells, and which of the polymorphic regions can contribute to the specificity of allorecognition, is not known. In order to investigate the distribution and the immunological relevance of recently described DPB1 alleles, we have typed a panel of 98 randomly selected Dutch Caucasoid donors for the HLA-DPB1 locus by oligonucleotide typing. Comparison of the typing results with primed lymphocyte typing (PLT) defined DP specificities shows an extremely good correlation. Moreover, additional alleles could be defined by oligonucleotide typing reducing the number of DP blanks in the panel. By selecting the appropriate responder stimulator combinations we were able to show that distinctive PLT reagents against oligonucleotide defined specificities DPB1*0401, DPB1*0402, DPB1*0901, and DPB1*1301 can be generated. To investigate in more detail which part of the DP molecule is responsible for the specificity of T-cell recognition, T-cell clones were generated against HLA-DPw3. The clones were tested for the recognition of stimulators carrying DPB1 alleles which had been defined by oligonucleotide typing and sequence analyses and which differed in a variable degree from DPB1*0301. The recognition patterns demonstrated that differences of one amino acid in polymorphic regions situated either in the beta sheets or alpha helix of the hypothetical model of the HLA class II molecule can eliminate T-cell recognition. Furthermore, sequence analyses revealed a new DPB1 allele designated DPB1*Oos.

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To study the stimulatory capacity of HLA-DR beta III in the mixed lymphocyte culture (MLC) assay, an MLC matrix between 13 Dw18 homozygous typing cells (HTCs) was analysed. Six of these HTCs were positive for the HLA-DR beta III allele LB-Q1 as defined by T cell clones. Seven HTCs were positive for LB-Q4. The MLC responses between Dw18 HTCs, matched for LB-Q, were significantly lower than the responses between the mismatched combinations. Considering the fact that HLA-DP can induce proliferation in MLC, we then analysed the DP matched and mismatched combinations separately. The influence of HLA-DR beta III mismatches was in our experiments comparable to the influence of mismatches for HLA-DP. Surprisingly, 15 out of 22 DR beta III and DP matched combinations still showed positive MLC reactions.

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LB-Q1 and LB-Q4 are two subtypes of DRw52, defined by proliferative T-cell clones. These subtypes represent a polymorphism of the DR beta III gene. Similar subtypes of DRw52 can be defined by oligonucleotide typing, serology and RFLP analysis. In the present study we compared these typing techniques on a panel of 22 HLA-D homozygous, DRw52-positive typing cells. All typing techniques correlated very well. Three subtypes of DRw52 could be identified. Our results show that typing for cellularly defined structures can be done with a variety of noncellular techniques. This observation has important implications for matching in unrelated bone marrow transplantation and for disease association studies.

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