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Thrombin induces macrophage migration inhibitory factor release and upregulation in urothelium: a possible
Pedro L Vera1, Terra E Wolfe, Alexander E Braley
1Research and Development, The Bay Pines VA Healthcare System, Bay Pines, Florida, United States of America. pvera@health.usf.edu
Purpose:
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.
Materials And Methods:
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.
Results:
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.
Conclusions:
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.
Insights
Thrombin activates urothelial Protease Activated Receptor-1 (PAR1), increasing macrophage migration inhibitory factor (MIF) release and mRNA. This suggests PAR1 activation may drive bladder inflammation by upregulating urothelial MIF.
Area of Science:
- Urology
- Immunology
- Cell Biology
Background:
- Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine.
- Urothelial cells express MIF and contribute to bladder inflammation.
Purpose of the Study:
- To investigate the effect of thrombin, a Protease Activated Receptor-1 (PAR1) agonist, on MIF release and mRNA in urothelial cells.
Main Methods:
- Real-time RT-PCR, Western blotting, and immunostaining were used to examine MIF and PAR1 expression in urothelial cells (UROtsa) and rat urothelium.
- Thrombin stimulation was applied in vitro (UROtsa cells) and in vivo (rats) to measure MIF release and mRNA levels.
- ELISA and real-time RT-PCR quantified MIF levels and mRNA upregulation, respectively.
Main Results:
- UROtsa cells and rat urothelium express both MIF and PAR1.
- Thrombin stimulation significantly increased urothelial MIF release (2.8-fold in vitro, 4.5-fold in vivo) and MIF mRNA upregulation (3.3-fold in vitro, 2-fold in vivo).
- Lidocaine treatment partially reduced thrombin-induced MIF release and mRNA upregulation in rats, indicating a role for nerve activity.
Conclusions:
- Urothelial cells express both MIF and PAR1.
- Activation of urothelial PAR1 receptors by thrombin or other proteases can induce urothelial MIF release and expression.
- This mechanism may contribute to bladder inflammation.
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