In saline-treated rats, both MIF and CXCR4 could be localized in the basal and intermediate layers of the urothelium (but not in superficial cells) (Fig 4ACC)

In saline-treated rats, both MIF and CXCR4 could be localized in the basal and intermediate layers of the urothelium (but not in superficial cells) (Fig 4ACC). SDF-1; 4) increased CXCR4-MIF associations. Conclusions These data demonstrate CXCR4-MIF associations occur in vivo in rat bladder and increase in experimental cystitis. Thus, CXCR4 represents an alternative pathway for MIF-mediated transmission transduction during bladder inflammation. In the bladder, MIF may compete with SDF-1 (cognate ligand) to activate transmission transduction mediated by CXCR4. Introduction Macrophage migration inhibitory factor (MIF) is an ubiquitous pleiotropic cytokine involved in cell proliferation and inflammation [1], [2]. MIF plays an important and unique role in inflammation since MIF stands upstream of other pro-inflammatory mediators and it can counter-regulate the anti-inflammatory effects of glucocorticoids [2]. MIF is usually implicated in animal models of inflammatory diseases, including arthritis, glomerulonephritis, acute lung injury and sepsis (for recent review [3]). Our recent experimental evidence indicates that MIF participates in bladder inflammation since: (1) MIF is usually constitutively expressed in the urothelium [4], [5]; (2) bladder MIF expression is usually upregulated in different models of experimental cystitis in animals [6], [7]; (3) MIF is usually released from your bladder during experimental cystitis [6], [8], [9] and urinary tract infections in humans [10] and finally, (4) neutralizing MIF with intravesical antibodies decreased experimental bladder inflammation [7]. Thus, based on our experimental observations, our hypothesis of a pro-inflammatory role for MIF during bladder inflammation agrees well with MIF’s pro-inflammatory role in several disease models (e.g. arthritis, Crohn’s disease) where treatment with neutralizing MIF antibodies results in decreased inflammation [11], [12]. The mechanism for MIF’s action is not completely defined and remains an active area of investigation. MIF may exert autocrine effects through binding to intracellular JAB1 [13] and also paracrine effects by binding to cell-surface receptors [14]. Until recently, complex formation between MIF and cell-surface CD74 was the only described mechanism Sesamolin for MIF-receptor conversation [15] . CD74 is usually part of the major histocompatibility class-II (MHC-II) complex; however, a small amount of CD74 can be found around the cell-surface not associated with MHC-II [16]. MIF binds to cell-surface CD74 [15] and the MIF-CD74 complex then activates transmission transduction by binding to another cell-surface receptor, CD44 [14]. We showed that MIF, CD44 and CD74 are all upregulated in the urothelium after experimental inflammation in rats [6], [17]. Therefore, all of the components are in place during bladder inflammation for MIF-activated transmission transduction to occur. Recently, however, a novel functional association between MIF and chemokine receptors CXCR2 and CXCR4 was explained in T cells in vitro [18]. Chemokines are small proteins that direct leukocyte traffic to sites of inflammation or injury [19]. CXCR4 is usually a G-protein coupled receptor for stromal cell-derived factor-1 (SDF-1/CXCL12). Although chemokines typically display a high degree of receptor promiscuity, CXCR4 was (until recently) thought to bind only to SDF-1 [19]. MIF, however, competed with the acknowledged ligand for CXCR4 (SDF-1/CXCL12) for binding to CXCR4 [18]. CXCR4 is usually expressed by normal urothelium and may be associated with bladder malignancy [20], [21]. Therefore, we hypothesized that CXCR4-MIF complex formation may also occur in the bladder (as explained occurring in JTK4 vitro [18]). Such associations, if present, would indicate another possible receptor target for MIF during cystitis, aside from the already explained MIF-CD74 association [15]. The object of the present study was to determine if there was an association between MIF and CXCR4 receptors in the bladder. Therefore, we examined: 1) location of cytokine receptor CXCR4 in the rat bladder; 2) baseline bladder levels of SDF-1 (cognate ligand for CXCR4) and changes in response to a chemically-induced (cyclophosphamide; CYP) model of bladder inflammation; 3) CXCR4 expression changes after CYP-induced cystitis and 4) association between CXCR4 and MIF in the bladder Sesamolin before and after CYP-induced cystitis. Our results show that both CXCR4 and SDF-1 are constitutively expressed in normal Sesamolin 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. Results Cyclophosphamide-induced bladder inflammation Repeated steps ANOVA showed differences between saline- and CYP-treated rats in body weight, with significant decreases observed in CYP-treated rats as early as day 3 and continuing throughout the experiment (day 8; Table 1) but remaining below a 10% weight-loss threshold established as a protocol endpoint. Table 1 Effect of cyclophosphamide on body weight (g) thead TreatmentDay 0Day 3Day 6Day 8 Sesamolin /thead Saline (N?=?10)3212.6 g3222.83263.53263.6CYP.