Abstract
PurposeMacrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine expressed by urothelial cells that mediates bladder inflammation. We investigated the effect of stimulation with thrombin, a Protease Activated Receptor-1 (PAR1) agonist, on MIF release and MIF mRNA upregulation in urothelial cells.Materials and MethodsMIF and PAR1 expression was examined in normal human immortalized urothelial cells (UROtsa) using real-time RT-PCR, Western blotting and dual immunostaining. MIF and PAR1 immunostaining was also examined in rat urothelium. The effect of thrombin stimulation (100 nM) on urothelial MIF release was examined in UROtsa cells (in vitro) and in rats (in vivo). UROtsa cells were stimulated with thrombin, culture media were collected at different time points and MIF amounts were determined by ELISA. Pentobarbital anesthetized rats received intravesical saline (control), thrombin, or thrombin +2% lidocaine (to block nerve activity) for 1 hr, intraluminal fluid was collected and MIF amounts determined by ELISA. Bladder or UROtsa MIF mRNA was measured using real time RT-PCR.ResultsUROtsa cells constitutively express MIF and PAR1 and immunostaining for both was observed in these cells and in the basal and intermediate layers of rat urothelium. Thrombin stimulation of urothelial cells resulted in a concentration- and time-dependent increase in MIF release both in vitro (UROtsa; 2.8-fold increase at 1 hr) and in vivo (rat; 4.5-fold) while heat-inactivated thrombin had no effect. In rats, thrombin-induced MIF release was reduced but not abolished by intravesical lidocaine treatment. Thrombin also upregulated MIF mRNA in UROtsa cells (3.3-fold increase) and in the rat bladder (2-fold increase) where the effect was reduced (1.4-fold) by lidocaine treatment.ConclusionsUrothelial cells express both MIF and PAR1. Activation of urothelial PAR1 receptors, either by locally generated thrombin or proteases present in the urine, may mediate bladder inflammation by inducing urothelial MIF release and upregulating urothelial MIF expression.
Highlights
Macrophage migration inhibitory (MIF), the earliest identified cyokine, was originally described as produced by activated T cells and capable of stopping the random migration of macrophages in vitro [1,2]
UROtsa cells constitutively express migration inhibitory factor (MIF) and Protease Activated Receptor-1 (PAR1) and immunostaining for both was observed in these cells and in the basal and intermediate layers of rat urothelium
UROtsa cells express MIF and Protease activated receptors (PAR) receptors Since MIF and/or PAR receptors had not been studied in UROtsa cells, we examined expression of these two proteins in UROtsa cells
Summary
Macrophage migration inhibitory (MIF), the earliest identified cyokine, was originally described as produced by activated T cells and capable of stopping the random migration of macrophages in vitro [1,2]. MIF is constitutively expressed by urothelial cells and mediates inflammation in the bladder [6,7]. Inflammatory stimuli elicit MIF release from the urothelium into the bladder lumen and upregulation of MIF expression by the bladder in general and urothelium in particular [8,9]. Released luminal MIF binds and activates receptors for MIF expressed by urothelial cells [10,11] to induce a cascade of other inflammatory cytokines to be produced by the bladder and urothelium [8,9]. Release of urothelial preformed MIF and activation of MIF production in the bladder (and urothelium in particular) by inflammatory stimuli are key elements in MIF-mediated bladder inflammation. Understanding the triggers evoking urothelial MIF release is an important component of understanding how cystitis is developed or maintained
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