title: Macrophage Migration Inhibitory Factor Mediates PAR-Induced Bladder Pain.
abstract: BACKGROUND
  Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is constitutively expressed in urothelial cells that also express protease-activated receptors (PAR). Urothelial PAR1 receptors were shown to mediate bladder inflammation. We showed that PAR1 and PAR4 activator, thrombin, also mediates urothelial MIF release. We hypothesized that stimulation of urothelial PAR1 or PAR4 receptors elicits release of urothelial MIF that acts on MIF receptors in the urothelium to mediate bladder inflammation and pain. Thus, we examined the effect of activation of specific bladder PAR receptors on MIF release, bladder pain, micturition and histological changes.
  
  
  METHODS
  MIF release was measured in vitro after exposing immortalized human urothelial cells (UROtsa) to PAR1 or PAR4 activating peptides (AP). Female C57BL/6 mice received intravesical PAR1- or PAR4-AP for one hour to determine: 1) bladder MIF release in vivo within one hour; 2) abdominal hypersensitivity (allodynia) to von Frey filament stimulation 24 hours after treatment; 3) micturition parameters 24 hours after treatment; 4) histological changes in the bladder as a result of treatment; 5) changes in expression of bladder MIF and MIF receptors using real-time RT-PCR; 6) changes in urothelial MIF and MIF receptor, CXCR4, protein levels using quantitative immunofluorescence; 7) effect of MIF or CXCR4 antagonism.
  
  
  RESULTS
  PAR1- or PAR4-AP triggered MIF release from both human urothelial cells in vitro and mouse urothelium in vivo. Twenty-four hours after intravesical PAR1- or PAR4-AP, we observed abdominal hypersensitivity in mice without changes in micturition or bladder histology. PAR4-AP was more effective and also increased expression of bladder MIF and urothelium MIF receptor, CXCR4. Bladder CXCR4 localized to the urothelium. Antagonizing MIF with ISO-1 eliminated PAR4- and reduced PAR1-induced hypersensitivity, while antagonizing CXCR4 with AMD3100 only partially prevented PAR4-induced hypersensitivity.
  
  
  CONCLUSIONS
  Bladder PAR activation elicits urothelial MIF release and urothelial MIF receptor signaling at least partly through CXCR4 to result in abdominal hypersensitivity without overt bladder inflammation. PAR-induced bladder pain may represent an interesting pre-clinical model of Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS) where pain occurs without apparent bladder injury or pathology. MIF is potentially a novel therapeutic target for bladder pain in IC/PBS patients.
authors.affiliation: Research and Development, Lexington Veterans Affairs Medical Center, Lexington, Kentucky, United States of America; Saha Cardiovascular Research Center, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Dimitrios E
authors.initials: DE
authors.lastname: Kouzoukas
authors.affiliation: Department of Natural Sciences, St. Petersburg College, St. Petersburg, Florida, United States of America.
authors.forename: Katherine L
authors.initials: KL
authors.lastname: Meyer-Siegler
authors.affiliation: Research and Development, Lexington Veterans Affairs Medical Center, Lexington, Kentucky, United States of America; Department of Physiology, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Fei
authors.initials: F
authors.lastname: Ma
authors.affiliation: Research and Development, Lexington Veterans Affairs Medical Center, Lexington, Kentucky, United States of America; Department of Physiology, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Karin N
authors.initials: KN
authors.lastname: Westlund
authors.affiliation: Research and Development, Lexington Veterans Affairs Medical Center, Lexington, Kentucky, United States of America.
authors.forename: David E
authors.initials: DE
authors.lastname: Hunt
authors.affiliation: Research and Development, Lexington Veterans Affairs Medical Center, Lexington, Kentucky, United States of America; Department of Physiology, University of Kentucky, Lexington, Kentucky, United States of America; Department of Surgery, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Pedro L
authors.initials: PL
authors.lastname: Vera
mesh_terms: D000818:Animals; D002461:Cell Line, Transformed; D005260:Female; D006801:Humans; D007249:Inflammation / Q000378:metabolism / Q000473:pathology; D019746:Intramolecular Oxidoreductases / Q000378:metabolism*; D008263:Macrophage Migration-Inhibitory Factors / Q000378:metabolism*; D051379:Mice; D010146:Pain / Q000378:metabolism* / Q000473:pathology; D044463:Receptor, PAR-1 / Q000378:metabolism*; D019718:Receptors, CXCR4 / Q000378:metabolism; D011971:Receptors, Immunologic / Q000378:metabolism; D018179:Receptors, Thrombin / Q000378:metabolism*; D001743:Urinary Bladder / Q000378:metabolism* / Q000473:pathology; D019459:Urothelium / Q000378:metabolism / Q000473:pathology
chemical_list: C527276:CXCR4 protein, human; C514936:CXCR4 protein, mouse; D008263:Macrophage Migration-Inhibitory Factors; D044463:Receptor, PAR-1; D019718:Receptors, CXCR4; D011971:Receptors, Immunologic; D018179:Receptors, Thrombin; C033184:macrophage migration inhibitory factor receptor; D019746:Intramolecular Oxidoreductases; C515598:MIF protein, human; C076231:Mif protein, mouse; C112908:protease-activated receptor 4