title: Substance P induces localization of MIF/alpha1-inhibitor-3 complexes to umbrella cells via paracellular transit through the urothelium in the rat bladder.
abstract: BACKGROUND
  Macrophage migration inhibitory factor (MIF) is released into the intraluminal fluid during bladder inflammation in the rat complexed to alpha1-inhibitor-3 (A1-I3; a rodent proteinase inhibitor in the alpha-macroglobulin family). The location of A1-I3 in the bladder had not been investigated. Therefore, we examined the location of A1-I3 and MIF/A1-I3 complexes in the bladder and changes due to experimental inflammation.
  
  
  METHODS
  Anesthetized male rats had bladders removed with no treatment (intact) or were injected with Substance P (SP; s.c.; saline vehicle). After one hour intraluminal fluid was removed, bladder was excised and MIF and A1-I3 levels were determined using ELISA and/or western-blotting. MIF co-immunoprecipitation determined MIF/A1-I3 complexes in the bladder. Bladder sections were immunostained for A1-I3 and MIF/A1-I3.
  
  
  RESULTS
  A1-I3 immunostaining was observed in interstitial spaces throughout the bladder (including submucosa) but not urothelium in intact and saline-treated rats. RT-PCR showed that the bladder does not synthesize A1-I3, therefore, A1-I3 in the interstitial space of the bladder must be plasma derived. In SP-treated rats, A1-I3 in the bladder increased and A1-I3 was observed traversing through the urothelium. Umbrella cells that do not show MIF and/or A1-I3 immunostaining in intact or saline-treated rats, showed co-localization of MIF and A1-I3 after SP-treatment. Western blotting demonstrated that in the bladder MIF formed non-covalent interactions and also binds covalently to A1-I3 to form high molecular weight MIF/A1-I3 complexes (170, 130 and 75-kDa, respectively, verified by co-immunoprecipitation). SP-induced inflammation selectively reduced 170-kDa MIF/A1-I3 in the bladder while increasing 170 and 130-kDa MIF/A1-I3 in the intraluminal fluid.
  
  
  CONCLUSIONS
  A1-I3 and MIF/A1-I3 complexes are resident in bladder interstitium. During SP-induced inflammation, MIF/A1-I3 complexes are released from the bladder into the lumen. Binding of MIF/A1-I3 complexes to urothelial cells during inflammation suggests these complexes participate in the inflammatory reaction through activation of receptors for MIF and/or for A1-I3.
authors.affiliation: The Bay Pines VA Healthcare System, Research & Development, Bay Pines, FL, USA. pvera@hsc.usf.edu
authors.forename: Pedro L
authors.initials: PL
authors.lastname: Vera
authors.forename: Katherine L
authors.initials: KL
authors.lastname: Meyer-Siegler
mesh_terms: D000209:Acute-Phase Proteins / Q000378:metabolism*; D000818:Animals; D003556:Cystitis / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D008263:Macrophage Migration-Inhibitory Factors / Q000378:metabolism*; D008297:Male; D021381:Protein Transport; D051381:Rats; D013373:Substance P; D014018:Tissue Distribution / Q000187:drug effects; D001743:Urinary Bladder / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D019459:Urothelium / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology
chemical_list: D000209:Acute-Phase Proteins; D008263:Macrophage Migration-Inhibitory Factors; C017894:alpha 1-inhibitor 3; D013373:Substance P