The effect of graft-versus-host disease on skin endothelial and epithelial cell chimerism in stem-cell transplant recipients.

BACKGROUND: Recent evidence indicates that bone marrow-derived cells contribute to endothelial and epithelial cell renewal in recipients of an allogeneic stem-cell transplantation (SCT). Controversy remains on the biological significance of these donor-derived cells. METHODS: This study investigated the occurrence of endothelial and epithelial cell chimerism in relation to the conditioning regimen, time interval after SCT, and development of acute and chronic graft-versus-host disease (GVHD). Fifty-five skin biopsy samples from 35 female patients transplanted with a male donor were screened for donor-derived endothelial and epithelial cells using in situ hybridization for Y chromosomes in combination with immunohistochemical cell-marking techniques. RESULTS: Endothelial cell chimerism was found in 25% of the biopsies and increased in time after SCT. Its appearance was increased in patients with acute GVHD more than 2 weeks before biopsy. Epithelial cell chimerism was found in 85% of the biopsies. Appearance of epithelial cell chimerism was not correlated with the time interval after SCT or with tissue damage caused by GVHD. CONCLUSION: From these results, we conclude that donor-derived endothelial cell chimerism results from repair of damaged endothelium and maintenance of vascular homeostasis. In contrast, epithelial cell chimerism follows a more uniform pattern of engraftment, not influenced by tissue damage.

Renal tubular epithelial cells modulate T-cell responses via ICOS-L and B7-H1.

BACKGROUND: Renal tubular epithelial cells (TECs) play an active role in renal inflammation. Previous studies have demonstrated the capacity of TECs to modulate T-cell responses both positively and negatively. Recently, new costimulatory molecules [inducible T cell costimulator-L (ICOS-L) and B7-H1] have been described, which appear to be involved in peripheral T-cell activation. METHODS: We characterized expression and regulation of costimulatory molecules on primary human TECs and the TEC line human kidney-2 (HK-2) with reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. Immunohistochemistry was performed on human kidney biopsies. The capacity of TECs to modulate T-cell activation was studied in TEC/T-cell cultures. RESULTS: We demonstrate that TECs express ICOS-L and B7-H1 in vitro and in vivo. Stimulation with interferon-gamma (IFN-gamma) resulted in increased expression of B7-H1, whereas ICOS-L expression was marginally increased upon stimulation with CD40L, with no effect of interleukin (IL-1), IL-17, or tumor necrosis factor-alpha (TNF-alpha). Furthermore, we show that TECs are able to costimulate T cells that have received signal-1 using alphaCD3 antibodies, inducing strong IL-10 production, which was partially mediated by ICOS-L. In contrast, B7-H1 appeared to be involved in inhibition of proliferation and cytokine synthesis. In addition, TECs were able to alter the cytokine profile of fully activated T cells, which were incubated with alphaCD3 and alphaCD28 antibodies, resulting in low IFN-gamma and high IL-10 production. This activity appeared to be independent of ICOS-L and B7-H1. CONCLUSION: Interaction of tubular epithelial cells and kidney infiltrating T cells via ICOS-L and B7-H1 may change the balance of positive and negative signals to the T cells, leading to IL-10 production and limitation of local immune responses.

Interactions of the extracellular matrix proteoglycans decorin and biglycan with C1q and collectins.

Decorin and biglycan are closely related abundant extracellular matrix proteoglycans that have been shown to bind to C1q. Given the overall structural similarities between C1q and mannose-binding lectin (MBL), the two key recognition molecules of the classical and the lectin complement pathways, respectively, we have examined functional consequences of the interaction of C1q and MBL with decorin and biglycan. Recombinant forms of human decorin and biglycan bound C1q via both collagen and globular domains and inhibited the classical pathway. Decorin also bound C1 without activating complement. Furthermore, decorin and biglycan bound efficiently to MBL, but only biglycan could inhibit activation of the lectin pathway. Other members of the collectin family, including human surfactant protein D, bovine collectin-43, and conglutinin also showed binding to decorin and biglycan. Decorin and biglycan strongly inhibited C1q binding to human endothelial cells and U937 cells, and biglycan suppressed C1q-induced MCP-1 and IL-8 production by human endothelial cells. In conclusion, decorin and biglycan act as inhibitors of activation of the complement cascade, cellular interactions, and proinflammatory cytokine production mediated by C1q. These two proteoglycans are likely to down-regulate proinflammatory effects mediated by C1q, and possibly also the collectins, at the tissue level.

NF-kappaB mediated IL-6 production by renal epithelial cells is regulated by c-jun NH2-terminal kinase.

Tubular epithelial cells (TEC) play an important role in tubulointerstitial inflammation, a hallmark of most renal diseases, via production of cytokines and chemokines. In this study, the role of mitogen-activated protein kinases (MAPK) in regulation of the proinflammatory cytokine IL-6 in cultured human TEC in response to the leukocyte-derived factors IL-1, TNF-alpha, IL-17, and CD40L was investigated. IL-6 production induced by IL-1, TNF-alpha, and IL-17 was specifically inhibited by the c-jun NH(2)-terminal kinase (JNK) inhibitor SP600125, but not by a selective inhibitor of p38 MAPK, and was moderately increased when the ERK1/2 pathway was inhibited. Also for CD40L stimulation, inhibition of JNK resulted in a pronounced inhibition of IL-6 production. Although stimulation of TEC induced activation of activator protein-1 (AP-1), the down-stream target of JNK, reporter assays demonstrated that mutation of the AP-1 binding site in the IL-6 promoter did not affect gene transcription. Furthermore, IL-1-induced transcriptional activation of the IL-6 promotor was repressed by SP600125 or by co-transfection of a dominant-negative expression plasmid of c-jun even in the absence of a functional AP-1 binding site. This suggests that IL-6 production by renal epithelial cells is regulated by JNK, via a mechanism, however, independent of the AP-1 binding site. The data rather suggest that the JNK pathway may interfere with other signaling pathways, involving NF-kappaB and possibly ERK.

Meconium is a source of pro-inflammatory substances and can induce cytokine production in cultured A549 epithelial cells.

Inflammation plays an important role in the pathogenesis of meconium aspiration syndrome, and pneumonitis is one of the major characteristics. We have previously shown that meconium has chemotactic properties because of the presence of IL-8. We hypothesize that IL-8 and other proinflammatory substances in meconium may amplify inflammation in meconium aspiration syndrome, inducing endogenous cytokine production by lung epithelial cells. We measured proinflammatory substances in first-pass meconium from healthy newborns and evaluated the effect of sterile meconium on cytokine production in cultured A549 alveolar epithelial cells in vitro. IL-1beta, IL-6, IL-8, and tumor necrosis factor-alpha were measured by ELISA, and heme was measured spectrophotometrically. After incubation of meconium samples with A549 cells, cytokine concentrations in the supernatant were measured. Meconium samples contained variable amounts of IL-1beta, IL-6, IL-8, tumor necrosis factor-alpha, and heme. On stimulation of A549 cells with meconium, the IL-8 concentration in the culture supernatant significantly increased above baseline measurements, whereas tumor necrosis factor-alpha showed a variable pattern and IL-1beta or IL-6 remained unchanged. There was no quantitative relationship between the concentration of the measured cytokines and heme in meconium and cytokine release by the A549 cells after meconium exposure. Meconium contains proinflammatory substances. All samples induced IL-8 release and some induced tumor necrosis factor-alpha release in cultured A549 epithelial cells. We speculate that proinflammatory substances in meconium can induce lung inflammation in meconium aspiration syndrome in two ways: directly via cytokines and heme present in meconium and indirectly by inducing cytokine release by the epithelial lung cells.

Steroid responsiveness of renal epithelial cells. Dissociation of transrepression and transactivation.

Glucocorticoids modulate cellular and inflammatory responses via stimulation or inhibition of gene transcription. Inhibition of cytokine gene expression is mediated via repression of transcription factors, including NF-kappaB. Previously we have shown that cytokine production by renal epithelial cells is insensitive to the inhibitory action of dexamethasone. In this study we demonstrate that dexamethasone is unable to inhibit NF-kappaB activation in the renal epithelial cell line HK-2, as measured by IkappaB-alpha degradation and DNA binding activity. Transfection of an NF-kappaB-inducible reporter gene demonstrated that non-stimulated HK-2 cells contain a high level of constitutively active NF-kappaB compared with the steroid-sensitive airway epithelial cell line A549, which was not blocked by dexamethasone. Expression and nuclear translocation of the glucocorticoid receptor (GR) was comparable in both cell types. In HK-2 cells, dexamethasone stimulated expression of two glucocorticoid-responsive genes, beta(2)-adrenoreceptors and angiotensinogen. The capacity of GR to transactivate the native angiotensinogen glucocorticoid-responsive element (GRE) using chromatin-IP was not impaired. Moreover, dexamethasone activation of a GRE-driven reporter construct appeared to be equally effective, although less sensitive compared with A549 cells. In conclusion, we provide evidence that glucocorticoids are unable to repress the activity of NF-kappaB in renal epithelial cells in the presence of an intact stimulatory pathway.

Production of inflammatory mediators by renal epithelial cells is insensitive to glucocorticoids.

1. In the present study we investigated the effect of glucocorticoids on the activation of renal tubular epithelial cells, which are thought to play an important role in inflammatory processes in the kidney. 2. Activation of renal epithelial cells by IL-1, TNF-alpha or CD40L resulted in increased production of cytokines and chemokines. Both in the renal epithelial cell line HK-2 and in primary cultures of human proximal tubular epithelial cells (PTEC) production of IL-6, IL-8 and monocyte chemotactic protein 1 (MCP-1) was not inhibited by glucocorticoids, independent of the stimulus. 3. In contrast, dexamethasone strongly inhibited cytokine production by immortalized renal fibroblasts and an airway epithelial cell line (A549). 4. Stimulation of renal epithelial cells resulted in activation of NF-kappaB, a pivotal transcription factor in the regulation of cytokine genes, as was shown by IkappaB-alpha degradation and increased DNA-binding activity. In contrast to dexamethasone, addition of the NF-kappaB inhibitors pyrrolidine dithiocarbamate (PDTC) and n-tosyl-l-phenylalanine chloromethyl ketone (TPCK) completely abolished cytokine and chemokine production. 5. Renal epithelial cells express abundant levels of the functional glucocorticoid receptor alpha (GRalpha) isoform and low levels of the inhibitory beta isoform (GRbeta). 6. In conclusion, cytokine production by renal epithelial cells is insensitive to the inhibitory effects of glucocorticoids. The lack of dexamethasone-mediated inhibition was specific for renal epithelial cells and could not be explained by an increased expression of the glucocorticoid receptor beta isoform.