title: Thrombin induces macrophage migration inhibitory factor release and upregulation in urothelium: a possible contribution to bladder inflammation.
abstract: OBJECTIVE
  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.
  
  
  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.
authors.affiliation: Research and Development, The Bay Pines VA Healthcare System, Bay Pines, Florida, United States of America. pvera@health.usf.edu
authors.forename: Pedro L
authors.initials: PL
authors.lastname: Vera
authors.forename: Terra E
authors.initials: TE
authors.lastname: Wolfe
authors.forename: Alexander E
authors.initials: AE
authors.lastname: Braley
authors.forename: Katherine L
authors.initials: KL
authors.lastname: Meyer-Siegler
mesh_terms: D000818:Animals; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007249:Inflammation; D008263:Macrophage Migration-Inhibitory Factors / Q000378:metabolism*; D008297:Male; D051381:Rats; D044463:Receptor, PAR-1 / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013917:Thrombin / Q000378:metabolism*; D015854:Up-Regulation; D001743:Urinary Bladder / Q000378:metabolism* / Q000473:pathology; D019459:Urothelium / Q000378:metabolism*
chemical_list: D016207:Cytokines; D008263:Macrophage Migration-Inhibitory Factors; D044463:Receptor, PAR-1; D013917:Thrombin