Therefore, we examined location of cytokine receptor CXCR4 in the rat bladder; 2) baseline bladder levels of SDF-1 and changes in response to a chemicallyinduced model of bladder inflammation; 3) CXCR4 expression changes after CYP-induced cystitis and 4) association Ibrutinib between CXCR4 and MIF in the bladder before and after CYP-induced cystitis. Our results show that both CXCR4 and SDF-1 are constitutively expressed in normal rat bladder and upregulated during CYP-induced cystitis. Using dual immunohistochemistry we show that MIF and CXCR4 are colocalized within the same cells in the urothelium and co-immunoprecipitation studies demonstrate MIF-CXCR4 associations in the bladder. These MIF-CXCR4 associations are increased during CYP-induced cystitis. The results from the present study demonstrate that CXCR4, a chemokine cell-surface receptor, is constitutively expressed in normal rat urothelium localized to basal and intermediate cells. CYP treatment also resulted in up-regulation of bladder CXCR4 mRNA and redistribution of CXCR4 to the entire urothelial area. Our findings of apical CXCR4 staining in superficial urothelial cells are in agreement with observations in colonic epithelial cells. CYP treatment although producing CXCR4 mRNA upregulation did not result in increased CXCR4 protein levels, and scoring of CXCR4 immunostaining actually showed a decrease in staining intensity following CYP treatment. A similar discrepancy between CXCR4 mRNA expression and protein levels has been reported in the rat XAV939 neurons and shown to reflect activation, increased internalization and degradation of CXCR4 receptors. Such activation, internalization and degradation of CXCR4 receptors may also account for the patchy CXCR4 immunostaining in the urothelium and may represent focal areas of CYP-induced CXCR4 response. CXCR4 mRNA expression in normal human urothelium, bladder cancer and also bladder cancer cell lines was previously reported. Addition of SDF-1 increased Matrigel invasion and cell growth but was not effective in increasing intracellular calcium in these particular urothelial cancer cells. However, other investigators using a different bladder cell line did report an increase in intracellular calcium upon stimulation with SDF-1. Taken together these results suggest that CXCR4 receptors are functional in the urothelium. There is also evidence of CXCR4 mRNA expression in other areas of the human urogenital system. We examined protein levels of SDF-1 in the bladders of both saline-treated and CYP-treated rats using ELISA. We report constitutive levels of SDF-1 in saline-treated bladders which increase after CYP treatment. Although SDF-1 immunofluorescence was readily detectable in skin keratinocytes, we were unsuccessful in detecting SDF-1 by immunohistochemistry in the bladder.