Thrombin Induces Macrophage Migration Inhibitory Factor Release and Upregulation in Urothelium: A Possible Contribution to Bladder Inflammation

Macrophage 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.MIF 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.UROtsa 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.Urothelial 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

Thrombin stimulation upregulates MIF mRNA.

a<p>MeanS.E.M.,</p>b<p>Determined using CT method.</p><p>*p0.05,</p><p>**p0.01.</p

UROtsa cell constitutively express MIF and PAR receptors.

<p>Results from RT-PCR experiments showed that UROtsa cells express MIF mRNA (A) and all 4 PAR receptors (B; lanes 1–4 represent PAR1-4 respectively). MIF Western-blotting of UROtsa homogenates (3 representative samples included; C) showed a strong band at approximately 12 kDa corresponding to monomeric MIF (arrow). In addition, 2 distinct MIF bands were observed at higher molecular weight (approximately 80 and 120 kDa) corresponding to MIF binding to protein complexes as described in other systems <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015904#pone.0015904-Vera5" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015904#pone.0015904-MeyerSiegler3" target="_blank">[25]</a>.</p

MIF and PAR1 immunostaining in urothelial cells.

<p>Representative samples from MIF, PAR1 immunostaining and an overlay showing both and nuclear staining (DAPI; blue). UROtsa cells displayed MIF (A) and PAR1 (B) immunostaining simultaneously in the same cell (C; overlay). However, a number of cells were observed that displayed neither immunostaining (C; arrows) and only showed nuclear staining. Control slides where primary antisera had been omitted (D;E) showed only nuclear staining (F). In rat urothelium, MIF immunostaining was detected in basal and intermediate cells with surface cells displaying weak or no MIF immunofluorescence. PAR1 immunostaining was also observed in rat urothelium, mainly on basal cells but also in some intermediate cells (H) while surface cells showed no PAR1 staining. Overlay of the single staining panels showed that basal cells and some intermediate cells were positive for both MIF and PAR1. Calibration bar = 20 m.</p