UROTHELIAL CELL ACTIVATION IN INTERSTITIAL CYSTITIS

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Interstitial cystitis is a poorly understood inflammatory condition occurring in the urinary bladder. We hypothesize that this condition is due to or is exacerbated by the production of cytokines by the urothelial cells of the bladder, and that the local action of immunosuppressive drugs, such as Cyclosporin A, FK506, or uromodulin, will reduce or halt this cellular activation and cytokine production and play a role in controlling the symptoms of IC. To evaluate these hypotheses, we propose these studies: Specific Aim 1: To characterize the cytokine production and expression of an activated phenotype (induced expression of cell surface antigens such as HLA-DR or intercellular adhesion molecule-1 [ICAM-1]) by urothelial cells in vitro. These studies will be performed using human cultured normal urothelial cells. These urothelial cells will be cultured in vitro with stimulants such as gamma interferon, tumor necrosis factor alpha, and interleukin 1. When activated, these cells will respond by producing cytokines and/or expressing cell surface antigens such as HLA-DR or ICAM-1. The evaluation of cell surface antigen expression will be performed by enzyme-linked immunosorbent assay (ELISA) and/or flow cytometry. The production of cytokines will be evaluated by the messenger RNA (mRNA) polymerase chain reaction (PCR) and by Northern blotting. PCR, will be used because it will allow the evaluation of multiple cytokines from very small samples. Bioactivity of the cytokines produced will be evaluated by bioassay. Interactions between urothelial cells and fibroblasts will be performed in a two-phase culture model. Either or both cell types will be stimulated and both cell types will be evaluated for cell surface antigen phenotype and cytokine production and for proliferation or growth inhibition using a dye-binding technique. Specific Aim 2: To characterize the cytokine production and expression of an activated phenotype in vivo. Tissue specimens taken from patients with interstitial cystitis and normal donors will be evaluated for cytokine production and cell surface antigen expression. These specimens will be analyzed for antigen expression by immunofluorescence staining and for cytokine production by RNA PCR analysis. Specific Aim 3: To determine if immunosuppressant drugs can affect cytokine generation and other markers of cellular activation in vitro. These studies will be performed using in vitro stimulated cultured normal urothelial cells treated with drug. The cytokine response in the presence or absence of drug (such as Cyclosporin A, FK506, or uromodulin) will be evaluated by RNA PCR or Northern blotting. Co-cultures of fibroblasts and normal urothelial cells will also be evaluated in this model with or without treatment with drug. These studies will not only further our understanding of the biology of urothelial cells and their participation in inflammatory processes, but also may identify new diagnostic criteria for a subset of interstitial cystitis patients and may yield a new method of treatment for this disease.