Morphological Expression of KIT Positive Interstitial Cells of Cajal in Human Bladder

PURPOSE: We investigated the 3-dimensional morphological arrangement of KIT positive interstitial cells of Cajal in the human bladder and explored their structural interactions with neighboring cells.MATERIALS AND METHODS: Human bladder biopsy samples were prepared for immunohistochemistry/confocal or transmission electron microscopy.RESULTS: Whole mount, flat sheet preparations labeled with anti-KIT (Merck, Darmstadt, Germany) contained several immunopositive interstitial cell of Cajal populations. A network of stellate interstitial cells of Cajal in the lamina propria made structural connections with a cholinergic nerve plexus. Vimentin positive cells of several morphologies were present in the lamina propria, presumably including fibroblasts, interstitial cells of Cajal and other cells of mesenchymal origin. Microvessels were abundant in this region and branched, elongated KIT positive interstitial cells of Cajal were found discretely along the vessel axis with each perivascular interstitial cell of Cajal associated with at least 6 vascular smooth muscle cells. Detrusor interstitial cells of Cajal were spindle-shaped, branched cells tracking the smooth muscle bundles, closely associated with smooth muscle cells and vesicular acetylcholine transferase nerves. Rounded, nonbranched KIT positive cells were more numerous in the lamina propria than in the detrusor and were immunopositive for anti-mast cell tryptase. Transmission electron microscopy revealed cells with the ultrastructural characteristics of interstitial cells of Cajal throughout the human bladder wall.CONCLUSIONS: The human bladder contains a network of KIT positive interstitial cells of Cajal in the lamina propria, which make frequent connections with a cholinergic nerve plexus. Novel perivascular interstitial cells of Cajal were discovered close to vascular smooth muscle cells, suggesting interstitial cells of Cajal-vascular coupling in the bladder. KIT positive detrusor interstitial cells of Cajal tracked smooth muscle bundles and were associated with nerves, perhaps showing a functional tri-unit controlling bladder contractility.

Localization of erythropoietin gene expression in proximal renal tubular cells detected by digoxigenin-labelled oligonucleotide probes

Erythropoietin (EPO) is the main humoral stimulus of erythropoiesis. In adult mammals, the kidney releases EPO in response to hypoxic stress. Conflicting data have suggested either renal tubular or peritubular cell origins of EPO synthesis in vivo. In situ hybridization studies were performed to define further the kidney cell type(s) capable of increasing EPO gene expression during hypoxic stimulation. EPO gene expression was stimulated in mice exposed to acute hypobaric hypoxia. Kidneys from hypoxic and control normoxic mice were obtained. Six digoxigenin-labelled oligonucleotide probes complementary to EPO exon sequences were utilized for in situ hybridization for EPO messenger RNA. Positive hybridization signals were identified in some proximal tubular cells, confined to the inner third of the renal cortex of hypoxic mouse kidney.

Complement expression in retinal pigment epithelial cells is modulated by activated macrophages

Complement activation is involved in a variety of retinal diseases. We have shown previously that a number of complement components and regulators can be produced locally in the eye, and that retinal pigment epithelial (RPE) cells are the major source of complement expression at the retina-choroidal interface. The expression of complement components by RPE cells is regulated by inflammatory cytokines. Under aging or inflammatory conditions, microglia and macrophages accumulate in the subretinal space, where they are in close contact with RPE cells. In this study, we investigated the effect of activated macrophages on complement expression by RPE cells. Mouse RPE cells were treated with the supernatants from un-activated bone marrow-derived macrophages (BM-DMs), the classically activated BM-DMs (M1) and different types of the alternatively activated BM-DMs (M2a by IL-4, M2b by immune complex and lipopolysaccharide (LPS), M2c by IL-10). The expression of inflammatory cytokines and complement genes by RPE cells were determined by real-time RT-PCR. The protein expression of CFB, C3, C1INH, and C1r was examined by Western blot. Our results show that un-stimulated RPE cells express a variety of complement-related genes, and that the expression levels of complement regulators, including C1r, factor H (CFH), DAF1, CD59, C1INH, Crry, and C4BP genes are significantly higher than those of complement component genes (C2, C4, CFB, C3, and C5). Macrophage supernatants increased inflammatory cytokine (IL-1ß, IL-6, iNOS), chemokine (CCL2) and complement expression in RPE cells. The supernatants from M0, M2a and M2c macrophages mildly up-regulated (2~3.5-fold) CFB, CFH and C3 gene expression in RPE cells, whereas the supernatants from M1 and M2b macrophages massively increased (10~30-fold) CFB and C3 gene expression in RPE cells. The expression of other genes, including C1r, C2, C4, CFH, Masp1, C1INH, and C4BP in RPE cells was also increased by the supernatants of M1 and M2b macrophages; however, the increment levels were significantly lower than CFB and C3 genes. M1 and M2b macrophage supernatants enhanced CFB (Bb fragment) protein expression and C3 secretion by RPE cells. M1 macrophages may affect complement expression in RPE cells through the STAT1 pathway. Our results suggest that under inflammatory conditions, activated macrophages could promote the alternative pathway of complement activation in the retina via induction of RPE cell CFB and C3 expression.