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The mechanism of deposition of IgA in the renal mesangium in primary IgA-nephropathy is poorly understood. It has been suggested that membrane receptors for IgA on mesangial cells (MC) of the kidney may be involved. To obtain more insight in the occurrence of the myeloid receptor for IgA (CD89) on MC, both in situ and in culture, rabbit and goat polyclonal antibodies and mouse monoclonal antibody against recombinant CD89 were raised. Kidney sections from five control subjects and five patients with primary IgA-nephropathy failed to be positive for CD89 in the mesangium, using our polyclonal and monoclonal antibodies. Also, five primary human MC cultures assessed for CD89 expression showed no protein expression of CD89. Furthermore, reverse transcription-PCR failed to detect mRNA expression of CD89 in the cultured MC. It was demonstrated that all five human primary MC bound human IgA in a dose-dependent manner, which was not inhibitable by blocking monoclonal anti-CD89 antibody (My43). In contrast, binding of IgA to U937 cells was blocked efficiently by My43. Finally, incubation of human MC with either human or rat IgA led to increased interleukin-6 production, whereas only human IgA, but not rat IgA, was able to bind to human CD89. Therefore, it is concluded that human MC do not express CD89 (to a significant extent). These results strongly suggest that binding of IgA to human MC occurs via an IgA receptor distinct from CD89.

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The IgA Fc receptor (FcR; CD89) is expressed on several types of cells of the myeloid cell lineage. We investigated whether different sizes of heat-aggregated IgA (aIgA) bind to CD89 and subsequently induce cellular activation. As a model we used the murine B cell line IIA1.6 transfected with CD89 or IIA1.6 cells transfected with CD89 as well as with the FcR gamma chain to study the binding of IgA to CD89. When these cells expressing CD89 were incubated with monomeric IgA, no significant binding of IgA to the cells was detectable by fluorescence-activated cell sorter analysis; however, incubation of the cells with aggregated IgA resulted in 93 +/- 2% positive cells. Incubation of the cells with different sizes of IgA-containing aggregates revealed optimal binding with aggregates containing five to six molecules of IgA per aggregate. No difference was observed between the binding to CD89 of both IgA1- or IgA2-containing aggregates. Furthermore, the binding of aIgA was found to be CD89-specific, since the binding of IgA was completely inhibited by the CD89-specific monoclonal antibody My43 and no detectable binding occurred to the IIA1.6 parent cell line. Activation studies using interleukin-2 (IL-2) production as a marker, showed that the FcR gamma chain is necessary to induce cellular activation. Only cells transfected with both CD89 and the FcR gamma chain (CD89+/gamma +) enhance the IL-2 production 10-12-fold upon stimulation with aggregates of IgA. Furthermore, triggering of CD89 only results in increase of intracellular calcium concentration ([Ca2+]i) in cells co-expressing FcR gamma chain. Mutation of the tyrosine residues in the FcR gamma chain immunoreceptor tyrosine-based activation motif of the FcR gamma chain abolishes this increase in [Ca2+]i, indicating association and involvement of the FcR gamma chain in CD89-mediated signaling.

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CD32 (Fc gamma RII) is the most abundantly distributed class of IgG Fc receptors in the human body. In this study, we analyzed the effect of transforming growth factor (TGF)-beta 1, a cytokine with strong immunosuppressive function, on the expression and function of CD32 on freshly isolated peripheral blood monocytes and three human monocytic cell lines, U937, THP-1 and Mono mac-6. We found that TGF-beta 1 down-regulates CD32 expression on monocytes and all monocytic cell lines in a dose- and time-dependent fashion. A mean down-regulation of CD32 expression on THP-1 cells of 54 +/- 3.2% after 24 h was found at a concentration of 1 ng/ml TGF-beta 1. At the mRNA level, TGF-beta 1 induced a twofold down-regulation of CD32. Cross-linking of CD32 induced an increase in the concentration of intracellular Ca2+, which was reduced by 50% by TGF-beta 1, suggesting a decreased downstream signaling mediated by the receptor.

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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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IgA is the predominant immunoglobulin in human secretions and the second most important immunoglobulin in the circulation on a quantitative basis. The clearance of IgA is dependent on the function of at least three types of receptors. One of these receptors recognizes the Fc portion of the IgA molecule, Fc alpha R, which has been cloned recently. Fc alpha R, also designated CD89, is found on a number of cells, including human glomerular mesangial cells, and monocytes. In this study we analysed the effect of TGF-beta 1, a cytokine with strong immunosuppressive function, on the expression of CD89 on freshly isolated monocytes. We found that TGF-beta 1 down-regulates CD89 expression on human peripheral blood monocytes in a dose-dependent fashion. Optimal down-regulation occurred at a concentration of 5 ng/ml. The down-regulation of CD89 by TGF-beta 1 is linear in time, with a mean down-regulation of 34 +/- 13% after 24 h. Also at the mRNA level, CD89 expression was down-regulated by TGF-beta 1, suggesting regulation of CD89 at the transcriptional level. Monocytes pre-treated with TGF-beta 1 displayed a reduced response to IgA, as measured by IL-6 production by monocytes, in contrast to monocytes pre-treated with medium alone. These results suggest an important role for TGF-beta 1 in the regulation of CD89. This down-regulation may have direct consequences for the handling of IgA by human monocytes.

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Impairment of renal function in various types of glomerular disease is associated with tubulointerstitial changes. The mechanism of mononuclear cell infiltration in the interstitium is not fully understood. Recently, monocyte chemoattractant protein-1 (MCP-1) has been identified as a monocyte-specific chemotactic factor. We analyzed the presence of MCP-1 in renal biopsies from patients with various forms of glomerular disease and demonstrated that MCP-1 expression is increased in renal tubular epithelial cells during disease. Further analysis showed that various cell lines of human proximal tubular epithelial cells (PTEC) produce MCP-1 in culture under serum-free conditions and that the production is inhibited by cycloheximide. IL-1 alpha and TNF-alpha enhanced the production by each cell line in a dose- and time-dependent manner as measured by radioimmunoassay. Northern blot analysis demonstrated that IL-1 alpha and TNF-alpha markedly enhanced the expression of MCP-1 mRNA. Taken together these observations support the notion that MCP-1 is synthesized de novo by PTEC. MCP-1 produced by PTEC is found to be 13 kD by gel filtration chromatography. It is chemotactically active for monocytes. We conclude that in various types of glomerular disease, MCP-1 expression in tubular epithelial cells is associated with up-regulation of MCP-1 production by PTEC. These findings raise the possibility that macrophages may accumulate in renal interstitium as a consequence of MCP-1 production by PTEC.

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Activation and degranulation of polymorphonuclear leukocytes (PMN) with release of proteolytic enzymes, such as proteinase 3 (PR3) and elastase, in the vessels of patients with Wegener's granulomatosis (WG) is thought to play an important role in the vascular endothelial cell damage. We have investigated the detachment and cytolysis of 51Cr-labeled umbilical vein endothelial cells (HUVEC) induced by highly purified, enzymatically active, PR3 and elastase. Incubation of confluent monolayers of HUVEC with 100 mU/ml of PR3 for 3 h at 37 degrees C generally resulted in 20% detachment and 30% cytolysis. Elastase (350 mU/ml) induced approximately 40% detachment and 15% cytolysis. Both PR3-mediated and elastase-mediated detachment and cytolysis were fully inhibited by alpha-1-proteinase inhibitor (alpha 1 PI), while anti-leukoprotease (ALP) only inhibited elastase-mediated endothelial damage. By selective inhibition of an azurophilic granule extract with either alpha 1PI or ALP we calculated that PR3 is responsible for 23% of the total detachment and cytolysis induced by the extract. Elastase was responsible for 60% of the detachment and 19% of the cytolysis. Detachment induced by PR3 was inhibited by three out of five IgG preparations purified from c-ANCA-positive sera of WG patients. PR3-mediated cytolysis was inhibited by each of the c-ANCA+IgG preparations and also to a limited extent by control IgG, suggesting a partial nonspecific stabilization of the endothelial cells. These studies provide evidence that besides elastase, PR3 also plays an important role in the PMN-mediated endothelial cell damage.

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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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In the present study the in vitro binding, internalization and degradation of IgA immune complexes (IC) by phagocytes was studied. As a model for IgA IC, heat-aggregated human secretory IgA (AsIgA) was prepared and resident rat peritoneal macrophages (PM phi) were used as a source of phagocytes. First, binding of 125I-AsIgA to rat PM phi was investigated. Binding of 125I-AsIgA to PM phi at 4 degrees was saturable and reached plateau values after 2 hr. At 37 degrees, degradation of membrane-bound 125I-AsIgA into trichloroacetic acid (TCA)-soluble fragments occurred. Parallel experiments with unlabelled AsIgA and 125I-labelled anti-human IgA revealed that degradation of AsIgA was preceded by internalization of AsIgA. The specificity of binding of 125I-AsIgA to PM phi was investigated using human IgG, human serum IgA, human myeloma IgA1, human sIgA and the glycoproteins asialofetuin and ovalbumin. The binding of 125I-AsIgA to rat PM phi was inhibited in the presence of sIgA and asialofetuin. In contrast IgG and ovalbumin had no effect. These results suggest that receptors with a specificity for galactose on the rat PM phi are involved in the binding of AsIgA.

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The present studies were initiated to investigate whether soluble immune complexes, upon interaction with complement, can bind to endothelial cells. Human umbilical vein endothelial cells (HUVE) were incubated with purified human 125I-labeled C1q at 4 degrees C in RPMI-0.5% bovine serum albumin and assayed for binding. Optimal binding of 125I-labeled C1q to HUVE was reached within 2 h, and saturation of binding was found at concentrations of 5 micrograms/well input. The binding of 125I-labeled C1q was inhibitable with unlabeled C1q and by the collagenous region of pepsin-cleaved C1q. No inhibition was observed with the globular heads of C1q, suggesting that C1q binds to HUVE via the collagenous region of C1q. When HUVE were first reacted with various concentrations of C1q, washed and subsequently incubated with 125I-labeled aggregated human IgM (AIgM), binding of 125I-labeled AIgM to HUVE occurred depending on the dose of C1q. Only those aggregates of IgM which react with C1q in a solid-phase C1q binding assay were able to bind to HUVE presensitized with C1q. In addition it was shown that C1q mediated binding of aggregated IgG to HUVE. Furthermore, immune complexes (IC), that were prepared with bovine thyroglobulin (BTg) and rabbit anti-BTg, bound to C1q-preincubated HUVE. These studies suggest that localization of IC on endothelium can be enhanced following interaction of the IC with complement.

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Expression of C1q receptors on the plasma membrane of thioglycollate-stimulated guinea pig peritoneal exudate macrophages increased 1.54 times as compared to unstimulated controls. A Scatchard plot of the binding of 125I-C1q to the cells revealed that the binding is a result of an increase in the number of receptors and not to an increased affinity of the receptors. Thioglycollate-activated macrophages were found to be 1.6 times more active than nonactivated macrophages in the binding of 125I-C1q at 4 degrees C. The enhanced binding of 125I-C1q by activated peritoneal macrophages was reflected in an increase in the amount of 125I-C1q degraded by these cells as compared to resident peritoneal macrophages. This suggests that stimulation of phagocytic cells leads to an increase in the expression of C1q receptors and to a concomitant increase in the uptake and degradation of C1q.

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