title: Toll-like receptor responses of normal human urothelial cells to bacterial flagellin and lipopolysaccharide.
abstract: OBJECTIVE
  We determined toll-like receptor expression in normal human urothelium and functional responses in normal human urothelial cell cultures to bacterial lipopolysaccharide via toll-like receptor-4 and to flagellin via toll-like receptor-5.
  
  
  METHODS
  Toll-like receptor protein expression was examined immunohistochemically. Toll-like receptor transcript expression was determined in freshly isolated urothelium, and in proliferating and differentiated normal human urothelial cultured cells. Lipopolysaccharide binding was assessed by flow cytometry. Functional responses of proliferating and differentiated normal human urothelial cells to lipopolysaccharide and flagellin were determined by interleukin-6 and 8 secretion, and transcription factor activation. Polymyxin B and siRNA were used to confirm the specificity of toll-like receptor-4 and 5 responses, respectively. Western blot detection of phosphorylated IκB was used to confirm toll-like receptor-4 results.
  
  
  RESULTS
  Human urothelium expressed transcripts for toll-like receptor-4 and 5. Although bladder cancer derived T24 cells responded to lipopolysaccharide, there was no lipopolysaccharide binding to normal human urothelial cells and no functional response of proliferative or differentiated normal human urothelial cells even in the presence of exogenous CD14 and MD-2 accessory proteins. In contrast, flagellin evoked a toll-like receptor-5 mediated response in proliferating but not in differentiated normal human urothelial cells, which was abrogated by toll-like receptor-5 specific siRNA.
  
  
  CONCLUSIONS
  Results suggest that human urothelium may mediate a host response to uropathogenic Escherichia coli through the detection of flagellin. The absent constitutive toll-like receptor-4 response may reflect an adaptation of urothelium toward sustaining barrier function and limiting inflammation to soluble bacterial products.
authors.affiliation: Jack Birch Unit for Molecular Carcinogenesis, Department of Biology, University of York, York, United Kingdom.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Smith
authors.forename: Claire L
authors.initials: CL
authors.lastname: Varley
authors.forename: Ian
authors.initials: I
authors.lastname: Eardley
authors.forename: Sally
authors.initials: S
authors.lastname: Feather
authors.forename: Ludwik K
authors.initials: LK
authors.lastname: Trejdosiewicz
authors.forename: Jennifer
authors.initials: J
authors.lastname: Southgate
mesh_terms: D002478:Cells, Cultured; D004847:Epithelial Cells / Q000276:immunology*; D004926:Escherichia coli / Q000276:immunology*; D005408:Flagellin / Q000276:immunology*; D006801:Humans; D008070:Lipopolysaccharides / Q000276:immunology*; D051197:Toll-Like Receptor 4 / Q000276:immunology*; D051198:Toll-Like Receptor 5 / Q000276:immunology*; D019459:Urothelium / Q000276:immunology*
chemical_list: D008070:Lipopolysaccharides; D051197:Toll-Like Receptor 4; D051198:Toll-Like Receptor 5; D005408:Flagellin