Since the levels of SDF-1 in the skin are approximately twice the levels found in bladder or spleen, we consider it likely that the levels in these organs were below the detection level for immunohistochemistry. Recently, down-regulation of SDF-1 mRNA expression in the bladder was reported afer vaginal distension. To our knowledge, our findings represent the first demonstration of SDF-1 protein levels in the bladder. Thus our findings indicate that bladder injury produced by CYP-treatment results in mRNA upregulation of the chemokine receptor CXCR4 and increased protein levels of its cognate ligand, SDF-1. In addition to several cytokines reported upregulated in the bladder after CYP treatment, changes in another chemokine and its receptor were described as a result of cyclophosphamide treatment. Therefore, chemokines likely represent important mediators of bladder injury and possible targets for ameliorating bladder inflammation. Until recently, CXCR4 was considered to bind exclusively to SDF-1. However, recent in vitro evidence showed that CXCR4 is also capable of binding MIF. In this study we Lapatinib confirm these in vitro finding since we demonstrate 1) colocalization of CXCR4 and MIF in the urothelium, both in saline Y-27632 dihydrochloride treated rats and after CYP treatment; 2) CXCR4-MIF associations are present in saline-treated bladder and increase after CYP treatment. Therefore, although, not directly tested in this study, our results suggest that MIF in the bladder may participate in bladder inflammation either through binding to CXCR4 in the urothelium or to CD74 to activate signal transduction pathways that result in the production of other inflammatory cytokines. Given that bladder MIF concentrations are approximately 30-fold greater than bladder SDF-1 concentrations, it is possible that MIF may be the primary ligand at the CXCR4 receptor in the urothelium. Moreover, CYP-treatment induced immunostaining of CXCR4 in superficial urothelial cells previously devoid of CXCR4 immunostaining. This raises the possibility that these cells will be activated by MIF present in the urine and in fact, CYP and other inflammatory stimuli increase luminal MIF release. We cannot rule out a contribution of renal or ureteral MIF release to increased urine MIF levels observed in this study after CYP-treatment, Yet our current findings of increased urine MIF with concomitant decrease in bladder MIF protein levels are consistent with earlier findings where, in animals with bladders isolated from the kidneys, we observed similar results ). Thus, based on our experimental evidence we consider likely that MIF is released into the lumen from pre-formed stores in the bladder during inflammation.