Production of inflammatory mediators and cytokine responsiveness of an SV40-transformed human proximal tubular epithelial cell line.

Proximal tubular epithelial cells (PTEC) play a central role in the physiology of the renal tubulointerstitium. To be able to study the relationship between tubular cells and inflammatory renal diseases the availability of cultured cells is of importance. This study describes an immortalized proximal tubular epithelial cell line which was generated using SV40 DNA. To determine whether the transformation altered the cell line, the transformed cell line was characterized phenotypically using different monoclonal antibodies directed against peptidases, which are characteristic of PTEC, such as adenosine deaminase binding protein (CD26), leucine amino peptidase and carboxy peptidase M by immunofluorescent staining and FACS analysis. All peptidases were clearly present on the parental cell line and the transformed cell line. However, the level of expression of the peptidases was lower on the transformed cell line as compared to the parental nontransfected cells. The morphology of the transformed cell line, determined using a transwell culture system and electron microscopy, showed a polarized morphology of the tubular cells, tight junctions and microvilli. The transformed cell line was compared with the parental proximal tubular epithelial cells in its ability to respond to inflammatory cytokines such as IL- 1alpha TNF-alpha, IFN-gamma. Stimulation with these cytokines resulted in enhanced production of complement components C2, C3, C4 and factor H, IL-6 and the chemokines IL-8 and MCP-1. The transformed cell line responded in a similar fashion as the parental cell line, although the amount of the different proteins produced was significantly higher in the transformed cell line. Overall, the transformed tubular cell line seems to be a suitable model to study different effects on tubular cells in relation to inflammatory kidney diseases.

Transforming growth factor-beta 1 regulates chemokine and complement production by human proximal tubular epithelial cells.

Previously it has been demonstrated that human proximal tubular epithelial cells (PTEC) are able to produce chemokines (such as IL-8 and MCP-1) and complement components (such as C2, C3, C4 and factor H), and that production of these proteins is regulated by pro-inflammatory cytokines such as interleukin-1 alpha (IL-1alpha), tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). Since TGF-beta is also expressed in the renal interstitium during inflammation, we investigated the effect of TGF-beta on the production of chemokines and complement components by PTEC in culture. Transforming growth factor-beta 1 up-regulated IL-8 production by an average of 4.17 +/- 1.0 fold. macrophage chemoattractant phagocyte (MCP-1) production, on the other hand, was down-regulated by TGF-beta 1 by an average of 2.2 +/- 0.7 fold. The production of C3 and C4 was also down-regulated after incubation with TGF-beta 1 (1.9 +/- 0.3- and 3.0 +/- 1.2-fold, respectively). All effects were dose- and time-dependent and were found to be specific for TGF-beta 1, as assessed by inhibition of the effect with a neutralizing antibody against TGF-beta 1. These data, together with the knowledge that TGF-beta, chemokines and complement components play a role in several types of renal disease, suggest that TGF-beta is involved in the regulation of local expression of chemokines and complement components by tubular cells.

Expression of the IL-2 receptor on human renal proximal tubular epithelial cells.

The high-affinity receptor for interleukin (IL)-2, IL-2R, is composed of three chains, i.e., the alpha, beta, and gamma chains. Previous studies have shown that human proximal tubular epithelial cells (PTEC) express IL-2R alpha during inflammation, such as in the kidneys after renal transplantation or in crescentic glomerulonephritis. Furthermore, in this study it is shown that PTEC in culture produce more complement C3 after stimulation with IL-2, suggesting the presence of a functional IL-2R on PTEC. In these experiments, the binding of IL-2 to PTEC was analyzed. PTEC stimulated with IL-2 exhibited very low binding of digoxigenin-labeled IL-2; however, stimulation of PTEC with IL-2, in combination with interferon (IFN)-gamma, resulted in increased binding of the digoxigenin-labeled IL-2. In addition, it was found that IL-2, together with IFN-gamma, enhanced the production of C3 by PTEC from baseline levels of 69.6 +/- 3.4 to 198.6 +/- 3.3 ng of C3 per 10(6) cells. The surface expression of IL-2R alpha and IL-2Rbeta was analyzed using monoclonal antibodies. Although unstimulated PTEC hardly showed detectable expression of these chains, stimulation of PTEC with the combination of IL-2 and IFN-gamma resulted in increases of the index of mean fluorescence for the alpha- and beta-chains of 5.5 +/- 0.5 and 9.7 +/- 0.7, respectively. Reverse transcription-PCR analysis revealed mRNA expression not only for the alpha- and beta-chains, but also for the gamma-chain, after stimulation with the combination of IL-2 and IFN-gamma. This study demonstrates that PTEC express the high-affinity IL-2R and that the IL-2R might be involved in the regulation of production of complement component C3 found during renal inflammation.

Interferon-gamma induces biosynthesis of complement components C2, C4 and factor H by human proximal tubular epithelial cells.

In a previous study the authors demonstrated that the production of complement component C4 by human kidney proximal tubular epithelial cells (PTEC) is upregulated by interferon gamma (IFN-gamma). In the present study the authors describe that PTEC in culture express both mRNA and protein of the IFN-gamma receptor complex, and that culture of PTEC with 1000 U/ml IFN-gamma for 72 h results in enhanced production not only of C4 (36.1 ng/10(6) cells), but also of C2 (10.8 ng/10(6) cells) and Factor H (17.5 ng/10(6) cells). Unstimulated PTEC produced 0.5 ng/10(6) cells, 0.5 ng/10(6) cells and 0.4 ng/10(6) cells of C2, C4 and Factor H, respectively. The upregulation of the three complement components was dose- and time-dependent and specific for IFN-gamma because the effect of IFN-gamma was abolished by a monoclonal antibody directed against IFN-gamma. Furthermore no effect of other cytokines was observed. The regulation of synthesis of C2, C4 and Factor H occurred at the transcriptional level as shown by semi-quantitative RT-PCR and dot-blot analysis. Taken together with the observation that in normal kidney tissue the tubuli express IFN-gamma receptor alpha-chain and a signal transducing protein, the present study implies that enhanced production of complement by PTEC may occur during a local immune response by in situ generation of IFN-gamma by infiltrating T-cells in the interstitium of the kidney.

Possible role for CD40-CD40L in the regulation of interstitial infiltration in the kidney.

Interstitial infiltration by mononuclear cells is a hallmark of most inflammatory kidney diseases, and the degree of infiltration is associated with disease progression. It has been demonstrated that proximal tubular epithelial cells (PTEC) are an important source of different cytokines/chemokines and thereby play a central role in the regulation of the local inflammatory response. CD40 is a cell surface receptor involved in immune regulation for which the ligand is expressed on activated T cells. By different staining methods, CD40 was found expressed in cryosections on the basolateral side of tubuli, as well as on the surface of an SV40-transformed PTEC line (PTEC-TRL) and on primary PTEC cultures. Cross linking CD40 receptor on these cultured cells, using a CD40L-transfected mouse fibroblast, resulted in strong up-regulation of the production of the chemokines IL-8, MCP-1 and RANTES. For IL-8 and MCP-1 production, the stimulation index after CD40 activation ranged from two- to sevenfold. Much stronger effects were observed for RANTES production, where levels remained undetectable (< 0.1 ng/ml) in non-stimulated cultures, whereas CD40 activation resulted in a strong production reaching 5 ng/ml in a 72-hour culture period. These data suggest that CD40L-CD40 interactions between infiltrating activated T cells and PTEC might be an important factor in the regulation of interstitial infiltration within the kidney.

Interleukin-1 alpha enhances the biosynthesis of complement C3 and factor B by human kidney proximal tubular epithelial cells in vitro.

Local production in tubular cells of complement has been shown to occur in several kidney diseases by in situ hybridization, but the regulation at the local site during an inflammation is still unknown. In the present study, we demonstrate that human proximal tubular epithelial cells (PTEC) are able to produce complement components C3 and Factor B under non-stimulated conditions in vitro. The basal production of both was increased by 0.5 ng/ml interleukin-1 alpha (IL-1 alpha) for C3: from 95.5 +/- 4.0 ng/10(6) cells to 416.5 +/- 4.9 ng/10(6), and for Factor B: from 271 +/- 7.0 ng/10(6) cells to 457.5 +/- 7.0 ng/10(6) cells. In contrast cytokines such as TNF-alpha, IFN-gamma, IL-10 and IL-15 had no detectable effect. The upregulation by IL-1 alpha was dose- and time-dependent. The response to IL-1 alpha was shown to be mediated via the IL-1 receptor, as the addition of recombinant interleukin-1 receptor antagonist inhibited the IL-1 alpha induced complement production by more than 80%. IL-1 alpha enhanced mRNA expression of both C3 and Factor B as demonstrated by RT-PCR and dot-blot analysis. This indicated that IL-1 alpha upregulated the expression of the C3 and Factor B at the transcriptional level. We hypothesize that in vivo the production of C3 and Factor B at the local site during an inflammatory response in the kidney may be regulated by IL-1 alpha produced by inflammatory cells.

Increased urinary excretion of monocyte chemoattractant protein-1 during acute renal allograft rejection.

BACKGROUND: Acute rejection is characterized histologically by infiltration of the interstitium by mononuclear cells. Monocyte chemoattractant protein 1 (MCP-1) has recently been identified as a monocyte chemotactic factor. This study examined the possible role of MCP-1 in renal transplantation. METHODS: The concentration of MCP-1 in urine and serum of 19 renal transplant patients was investigated using an inhibition radioimmunoassay. The patients were divided into a non-rejection (NRj) and a rejection (Rj) group. Normal healthy volunteers were included as controls. Immunoperoxidase staining for MCP-1 and CD14, as a marker for macrophages, was performed in renal biopsies of transplant patients with rejection and six biopsies from histologically normal kidneys, as controls. The size of urinary MCP-1 was determined by gel filtration chromatography and in a number of fractions assessed for monocyte chemotactic activity using a modified Boyden chamber assay. RESULTS: Urinary excretion of MCP-1 in the Rj group ranged between 250 ng/mmol Cr and 3148 ng/mmol Cr with a median of 612 ng/mmol Cr. This is significantly higher than the results in the NRj group, ranging between 47 ng/mmol Cr and 288 ng/mmol Cr with a median of 229 ng/mmol Cr. In the normal control group, urinary MCP-1 levels ranged between 38 ng/mmol Cr and 74 ng/mmol Cr with a median of 50 ng/mmol Cr. The fractional excretion of MCP-1, calculated on the basis of MCP-1 and creatinine clearances, was found also to be significantly higher in the Rj group as compared to the NRj group. However, there was no significant difference in the serum levels of MCP-1 between the Rj, NRj, and normal control group. The intensity of MCP-1 staining in tubular epithelial cells and the degree of CD14+ cells in the interstitium was significantly higher in renal allograft biopsies than in the normal kidneys. In addition, MCP-1 isolated from urine of renal transplant patients with rejection was filtered with apparent molecular weight of 13 kDa and 11 kDa. Both sizes are chemotactically active for monocytes. CONCLUSIONS: These data suggest that urinary excretion of MCP-1 can be used as a marker for the episodes of acute rejection. The increase of urinary excretion of MCP-1 most likely is the result of local production by tubular epithelia cells. MCP-1 produced locally may, at least in part, be responsible for the influx of macrophages into the interstitium during rejection.

Proteinase 3, the major autoantigen of Wegener's granulomatosis, enhances IL-8 production by endothelial cells in vitro.

Proteinase 3 is the major target antigen of antineutrophil cytoplasmic autoantibodies (ANCA) in Wegener's granulomatosis and is contained in the azurophilic granules of polymorphonuclear neutrophils, the dominant cell type in vascular lesions during the early stages of systemic vasculitis. This study questioned whether neutrophil lysosomal enzymes, once released at the site of inflammation, are able to potentiate the influx of additional neutrophils by enhancing the production of the chemotactic cytokine interleukin-8 (IL-8) by endothelial cells. Therefore, human umbilical vein endothelial cells in culture were incubated with varying concentrations of highly purified proteinase 3, human neutrophil elastase, and cathepsin G for different time periods. The supernatants were subsequently assessed for IL-8 antigen by using a sandwich ELISA. The presence of both proteinase 3 and elastase resulted in an increased production of IL-8, up to 15.6- and 4.2-fold, respectively, in a dose- and time-dependent fashion. Cathepsin G did not influence IL-8 production. Although the addition of an alpha 1-proteinase inhibitor completely abrogated elastase-mediated IL-8 production, it did not significantly influence the effect of proteinase 3. Both proteinase 3-and elastase-mediated production of IL-8 was inhibited by cycloheximide, indicating de novo synthesis. This was supported by the finding of increased IL-8 mRNA levels in proteinase 3-treated human umbilical vein endothelial cells by using Northern blot analysis. Taken together, the neutrophil lysosomal enzymes proteinase 3 and human neutrophil elastase may contribute to a self-perpetuating process of neutrophil recruitment in acute inflammation by increasing de novo synthesis of IL-8 by endothelial cells. The studies presented here also show that proteinase 3 mediates its effect independently of its enzymatic activity, indicating a hitherto unknown mode of action on endothelial cells.

Regulation and production of IL-8 by human proximal tubular epithelial cells in vitro.

A number of inflammatory kidney diseases are associated with interstitial nephritis and influx of leucocytes in the renal interstitium. Potentially the influx of neutrophils in the interstitium may be induced by the chemotactic cytokine IL-8. In the present study we have analysed the production of IL-8 by cultured human proximal tubular epithelial cells (PTEC) in response to a number of proinflammatory cytokines. Primary cell lines of proximal tubular epithelium obtained from ten different kidneys, and cultured under serum-free conditions, were found to produce IL-8 to different degrees from not detectable levels up to 10.8 +/- 1.5 ng IL-8 per 1 x 10(5) cells in 72 h. Gel filtration chromatography of PTEC supernatant indicated that the size of IL-8 of PTEC is 15.1 and 8.1 kD, and is chemotactically active for polymorphonuclear neutrophils (PMN). Addition of 0.5 ng/ml rIL-1 alpha or 1000 U/ml recombinant tumour necrosis factor-alpha (rTNF-alpha) to the culture media of PTEC induced an up-regulation of IL-8 production up to 6.3-fold and 3.0-fold, respectively. The up-regulation by IL-1 alpha and TNF-alpha was dose- and time-dependent. In contrast, 500 U/ml recombinant interferon-gamma (rIFN-gamma) down-regulated the production of IL-8 3.4-fold. Northern blot analysis showed that IL-1 alpha and TNF-alpha increased the expression of IL-8 mRNA, whereas IFN-gamma reduced IL-8 mRNA expression. Taken together, these experiments indicate that human PTEC are a potential source of IL-8 in the kidney, and that IL-8 produced in the proximal tubule can be induced by various proinflammatory cytokines.

Production and cytokine-mediated regulation of monocyte chemoattractant protein-1 by human proximal tubular epithelial cells.

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.

Monocyte chemoattractant protein-1 in normal and diseased human kidneys: an immunohistochemical analysis.

We conducted an immunohistochemical analysis to investigate the presence of monocyte chemoattractant protein-1 (MCP-1) in normal and diseased human kidneys and the correlation with infiltration of macrophages. A total of 50 renal biopsies were classified according to pathologic diagnosis. The distribution as well as the intensity of MCP-1 staining, and infiltration by CD68+ macrophages were evaluated in diseased versus normal kidneys. Weak staining of MCP-1 was detected in normal renal tissue, especially in tubular epithelial cells. Significant alterations in MCP-1 staining were observed in a number of diseases, in which the intensity of MCP-1 staining rather than the distribution of MCP-1-positive cells was higher. In membranous nephropathy, IgA nephropathy, and glomerulosclerosis, an association was found between the intensity of MCP-1 staining in tubular epithelial cells and interstitial infiltration of macrophages. The glomeruli in membranous nephropathy showed a stronger intensity of MCP-1 staining particularly in the glomerular visceral epithelial cells. The glomerular MCP-1 staining did not correlate significantly with the number of macrophages in the glomeruli. In conclusion, we describe increased cellular staining for MCP-1 in diseased human renal tissue, especially by tubular epithelial cells. Our observations suggest, complementary to in vitro and in vivo observations made by us and others, that specific roles may be played by MCP-1 in physiological and inflammatory processes in the kidney.