title: Intraluminal blockade of cell-surface CD74 and glucose regulated protein 78 prevents substance P-induced bladder inflammatory changes in the rat.
abstract: BACKGROUND
  Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine constitutively expressed by urothelial cells. During inflammatory stimuli, MIF is released into the lumen complexed to other proteins and these complexes can bind to urothelial cell-surface receptors to activate signaling pathways. Since MIF is complexed to alpha1-inhibitor III (A1-I3; a member of the alpha2-macroglubulin family) and glucose regulated protein 78 (GRP78) is a receptor for A1-I3 the goals of this study were to determine if substance P elicits urothelial cell-surface expression of GRP78 and to assess the functional role of CD74 (receptor for MIF) or GRP78 in substance P-induced bladder inflammatory changes.
  
  
  RESULTS
  Anesthetized male Sprague-Dawley rats received either saline or substance P (s.c.), bladders were collected 1 hour after treatment and processed for histology or protein/mRNA. The expression of GRP78 at urothelial cell-surface was determined by performing in vivo biotinylation of urothelial cell-surface proteins. Finally, in order to determine the effects of receptor blockade on substance P-induced MIF release and inflammatory changes, rats received either intraluminal antibodies to CD74, GRP78, both, or non-specific IgG (as a control). GRP78 and MIF immunostaining was simultaneously visualized in umbrella cells only after substance P treatment. Immunoprecipitation studies showed GRP78-MIF complexes increased after substance P while in vivo biotinylation confirmed substance P-induced GRP78 cell-surface expression in urothelial cells. Intraluminal blockade of CD74 and/or GRP78 prevented substance P-induced changes, including bladder edema, intraluminal MIF release by urothelial cells and production of inflammatory cytokines by urothelial cells.
  
  
  CONCLUSIONS
  GRP78 is expressed on the surface of urothelial cells after substance P treatment where it can bind MIF complexes. Blocking CD74 (receptor for MIF) and/or GRP78 prevented substance P-induced inflammatory changes in bladder and urothelium, indicating that these urothelial receptors are effective targets for disrupting MIF-mediated bladder inflammation.
authors.affiliation: The Bay Pines VA Healthcare System, Research & Development, Bay Pines, Florida, United States of America. pvera@health.usf.edu
authors.forename: Pedro L
authors.initials: PL
authors.lastname: Vera
authors.forename: Xihai
authors.initials: X
authors.lastname: Wang
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Bucala
authors.forename: Katherine L
authors.initials: KL
authors.lastname: Meyer-Siegler
mesh_terms: D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte / Q000096:biosynthesis*; D002462:Cell Membrane / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000502:physiology*; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis*; D007249:Inflammation; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D020411:Oligonucleotide Array Sequence Analysis; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013373:Substance P / Q000378:metabolism; D001743:Urinary Bladder / Q000378:metabolism / Q000473:pathology*; D000091342:Endoplasmic Reticulum Chaperone BiP
chemical_list: D000944:Antigens, Differentiation, B-Lymphocyte; D006360:Heat-Shock Proteins; D000949:Histocompatibility Antigens Class II; D013373:Substance P; C051684:GRP78 protein, rat; D000091342:Endoplasmic Reticulum Chaperone BiP; C045084:invariant chain