Quantification of dendritic cell subsets in human renal tissue under normal and pathological conditions.

Dendritic cells (DCs) play critical roles in immune responses and can be distinguished in two major subsets, myeloid and plasmacytoid DCs. Although the presence of DC in all peripheral organs, including the kidney, has been well documented, no accurate estimates of DC subsets in human kidneys have been reported. This study shows a detailed analysis of DC subsets in cryosections of human renal tissue. The cortex of normal kidneys contains at least two different HLA-DR(+) myeloid DC subtypes characterized by BDCA-1(+)DC-SIGN(+) and BDCA-1(+)DC-SIGN(-). The staining for DC-SIGN completely overlapped with CD68 in the renal interstitium. Unexpectedly, BDCA-2(+)DC-SIGN(-) plasmacytoid DCs are also abundantly present. Both subsets are located in the tubulo-interstitium often with a high frequency around, but rarely observed within glomeruli. Quantification of BDCA-1(+), DC-SIGN(+), and BDCA-2(+) cells in normal human renal tissue (pretransplant biopsy living donors; n=21) revealed that BDCA-1 is about four times as frequently present as BDCA-2. A preliminary cross-sectional analysis of DC in diseased kidneys, including rejection and immunoglobulin A nephropathy, revealed that the number of DC as well as their anatomical distribution might change under pathophysiological conditions. In conclusion, we show that human kidneys contain a dense network of myeloid and plasmacytoid DCs and provide the tools for phenotyping and enumeration of these cells to better understand interindividual differences in immune responses.

Maturation-resistant dendritic cells induce hyporesponsiveness in alloreactive CD45RA+ and CD45RO+ T-cell populations.

Dendritic cells (DCs) play a crucial role in the induction of antigen-specific immunity and tolerance. Considering in vivo application of DCs prior to human organ transplantation, a protocol to develop tolerogenic DCs that not only induce unresponsiveness in naive (CD45RA+) T cells, but also in alloreactive memory (CD45RO+) T cells is required. The present study shows that dexamethasone (Dex) alters the differentiation of human monocyte-derived DCs. DexDCs cocultured with allogeneic CD4+ T cells induced low proliferating and low IFNgamma producing T cells. This is caused by lack of both costimulation via CD28 and hampered production of a soluble factor, as well as additional active suppression via B7-H1 and IL-10. T cells primed by DexDCs demonstrated hyporesponsiveness upon restimulation with mature DCs seemingly via the induction of anergy, since these cells showed no enhanced apoptosis and only a limited suppressive capacity. Interestingly, not only cocultures of allogeneic CD45RA+, but also of CD45RO+ T cells with DexDCs rendered T-cell populations hyporesponsive to restimulation with mature DCs. The finding that also alloreactive memory T cells can be regulated supports the rationale of cell-based therapies to obtain allograft-specific tolerance in transplant recipients.

Rapamycin induces apoptosis in monocyte- and CD34-derived dendritic cells but not in monocytes and macrophages.

Rapamycin (Rapa), a recently introduced immunosuppressive drug, seems to be effective in preventing acute allograft rejection. Although its antiproliferative effect on T lymphocytes has been investigated extensively, its effect on the initiators of the immune response, the dendritic cells (DCs), is not known. Therefore, the effect of Rapa on monocyte- (mo-DCs) and CD34(+)-derived DCs in vitro but also on other myeloid cell types, including monocytes and macrophages, was examined. The present study shows that Rapa does not affect phenotypic differentiation and CD40L-induced maturation of mo-DCs. However, Rapa dramatically reduced cell recovery (40%-50%). Relatively low concentrations of Rapa (10(-9) M) induced apoptosis in both mo-DCs and CD34(+)-derived DCs, as visualized by phosphatidylserine exposure, nuclear condensation and fragmentation, and DNA degradation. In contrast, Rapa did not affect freshly isolated monocytes, macrophages, or myeloid cell lines. The sensitivity to Rapa-induced apoptosis was acquired from day 2 onward of mo-DC differentiation. Rapa exerts its apoptotic effect via a reversible binding to the cytosolic receptor protein FKBP-12, as demonstrated in competition experiments with FK506, which is structurally related to Rapa. Partial inhibition of Rapa-induced apoptosis was obtained by addition of ZVAD-fmk, which implies caspase-dependent and caspase-independent processes. The fact that Rapa exerts a specific effect on DCs but not on monocytes and macrophages might contribute to the unique actions of Rapa in the prevention of allograft rejection and other immune responses.

Epithelial- and endothelial-cell specificity of renal graft infiltrating T cells.

Previously, we have shown that rejecting renal allografts are infiltrated by tissue-specific T cells that in vitro kill proximal tubular epithelial cells (PTEC), while donor-derived splenocytes are not affected. In the current unique study we assessed the reactivity of graft-infiltrating T cells (GIC) for three different donor-derived cell types. Cytotoxicity of GIC was tested against PTEC, as well as donor-derived gonadal vein endothelial cells (GOVEC) and splenocytes. T cells lysed PTEC, GOVEC and splenocytes expressing a mismatched donor HLA antigen, HLA-A29(19). Interestingly, PTEC and GOVEC, not splenocytes, expressing none of the donor HLA antigens were also killed. T cell clones, obtained by limiting dilution cloning of the GIC line, could be divided into different categories: clones recognizing both PTEC and GOVEC expressing the mismatched HLA-A29(19), clones recognizing either PTEC or GOVEC expressing the mismatched HLA-A29(19) and also clones specifically recognizing PTEC or GOVEC independent of donor HLA antigen expression. In conclusion, T cell clones with specificities for either epithelial or endothelial cells exist, leaving a role for tissue-specific antigens in allograft rejection.

IL-4 and IL-13 augment cytokine- and CD40-induced RANTES production by human renal tubular epithelial cells in vitro.

Local production of cytokines by infiltrating monocytes/macrophages and Th1 and Th2 cells is of importance in renal allograft rejection. Activated Th1 cells can produce interleukin-2 (IL-2), interferon-gamma (IFN-gamma), and tumor necrosis factor (TNF), whereas Th2 cells produce IL-4 and IL-13, which inhibit Th1 cells. Furthermore, activated T cells express the costimulatory molecule CD40-ligand. During renal allograft rejection, the chemokine RANTES is detected in both infiltrating mononuclear cells and tubular epithelium. It has been shown previously that stimulation of proximal tubular epithelial cells (PTEC) with cytokines or CD40-ligand results in production of RANTES. The present study investigates the influence of Th1 and Th2 cytokines on RANTES production by activated PTEC. RANTES was not detectable in supernatants of human PTEC stimulated with IL-2, IL-4, IL-10, or IL-13 alone. Likewise, combination of these cytokines with IL-1 alpha, IFN-gamma, or TNF-alpha, respectively, did not result in detectable RANTES production. IL-2 and IL-10 had no significant effect on RANTES production by activated PTEC. IL-4 or IL-13 in combination with IL-1 alpha + IFN-gamma or IFN-gamma + TNF-alpha resulted in a two- to fourfold augmentation of RANTES production, ranging from 2.2 +/- 0.2 to 35 +/- 2 ng/ml in different cell lines. CD40-activated PTEC stimulated with IL-4 or IL-13 produced six to ten times more RANTES (ranging from 7.9 +/- 1.9 to 62 +/- 3.5 ng/ml in different cell lines) compared with CD40-activated cells alone. Because RANTES production is augmented by IL-4 and IL-13, this study suggests that during rejection, direct cellular contact between activated Th2 cells and tubular epithelial cells amplifies the local inflammatory reaction in the kidney.

Synergistic effect of IL-1alpha, IFN-gamma, and TNF-alpha on RANTES production by human renal tubular epithelial cells in vitro.

Interstitial rejection of renal allografts is associated with infiltrating mononuclear cells. Mechanisms leading to this mononuclear cell influx are still not fully resolved. The chemokine RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted) is chemotactic for monocytes and T cells. In renal allograft biopsies of patients undergoing rejection, RANTES is found in infiltrating monocytes and T cells, as well as in the tubular epithelium. This study analyzes the production of RANTES in vitro by proximal tubular epithelial cells (PTEC) after stimulation with the inflammatory cytokines interleukin-1alpha, (IL-1alpha), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha). Unstimulated PTEC or PTEC stimulated with the cytokines IL-1alpha, IFN-gamma, and TNF-alpha alone did not produce detectable amounts of RANTES. However, a combination of IFN-gamma and either IL-1alpha or TNF-alpha resulted in strong induction of RANTES production up to 2046 +/- 817 pg/ml or 2595 +/- 525 pg/ml per 1 x 10(5) PTEC, respectively. After stimulation with IL-1alpha and TNF-alpha, RANTES production was less prominent than the combination of IFN-gamma with either IL-1alpha or TNF-alpha, and only detectable in 5 of 7 PTEC lines tested. The production of RANTES was both dose- and time-dependent and was inhibited by cycloheximide, indicating that de novo protein synthesis is required. Because the production of RANTES by PTEC is more pronounced in the presence of T cell-derived IFN-gamma (in combination with either IL-1alpha or TNF-alpha), it was hypothesized that RANTES produced by PTEC presumably plays a prominent role in the amplification phase of the immune response rather than in the initiation phase.

Antigen-antibody complexes enhance the production of complement component C3 by human mesangial cells.

Deposition of immune complexes (ICX), with or without complement, occurs in various forms of glomerulonephritis. It has been reported that upregulation of complement C3 mRNA expression is found in kidneys of patients with ICX glomerulonephritis. In vitro studies have indicated that mesangial cells (MC) synthesize C3. Furthermore, MC express Fc gammaRIII receptors. This study investigates whether ICX alter C3 and factor H production by MC. MC were cultured in medium alone or in medium with insoluble heat-aggregated rat IgG (AIgG) or with insoluble ICX. Basal production of C3 and factor H was 10 +/- 1 ng/10(6) and 605 +/- 15 ng/10(6) cells, respectively. The presence of 400 microg/ml AIgG or ICX resulted in upregulation of C3 production to 999 +/- 15 ng/10(6) and 510 +/- 1 ng/10(6) cells, respectively, whereas no significant change in factor H production was observed. The upregulation of C3 was inhibitable by cycloheximide, suggesting that de novo protein synthesis was required. By reverse transcription PCR and Northern blot analysis, it was demonstrated that C3 and interleukin-6 mRNA expression was upregulated in MC after incubation with AIgG. No detectable change in factor H mRNA expression was seen. In conclusion, it is shown that incubation of MC with AIgG or ICX not only results in upregulated production of inflammatory mediators such as cytokines, but also leads to an upregulation of C3 synthesis. Therefore, it is hypothesized that ICX deposited within the mesangium may enhance the local production of C3 via interaction with Fc receptors on MC.

Tissue-specific characteristics of cytotoxic graft-infiltrating T cells during renal allograft rejection.

BACKGROUND: The alloresponse after renal transplantation was studied using alloreactive T cells generated in vivo (from renal biopsies) and in vitro (from mixed kidney lymphocyte cultures). METHODS: Tissue specificity of graft-infiltrating T cells (GIC) was investigated using donor-derived proximal tubular epithelial cells (PTEC) and splenocytes as targets in cytotoxicity assays. RESULTS: The outgrowth of cytotoxic T cells was associated with histologically proven interstitial rejection. GIC were categorized into four groups: (1) GIC cytotoxic for both PTEC and splenocytes (n=30), (2) noncytotoxic GIC (n=8), (3) GIC recognizing only splenocytes (n=1), and (4) GIC specifically recognizing PTEC (n=7). Similar tissue-specific T cells could be generated in vitro using mixed kidney lymphocyte cultures. Cytotoxicity of GIC from biopsies with moderate to severe rejection was CD8 independent, whereas cytotoxicity toward splenocytes was CD8 dependent. CONCLUSIONS: Our results show that polyclonal cytotoxic T-cell responses with tissue-specific characteristics are elicited during rejection episodes after renal transplantation.