title: Tumor necrosis factor-alpha and interferon-gamma directly impair epithelial barrier function in cultured mouse cholangiocytes.
abstract: OBJECTIVE
  In primary biliary cirrhosis (PBC), cytokines from CD4+ T lymphocytes were suggested to contribute to the intralobular bile duct damage together with cellular immunity by CD8+ T lymphocytes. Recently, we reported that immunolocalization of 7H6--a tight junction (TJ)-associated protein--was significantly diminished in cholangiocytes in the PBC liver. In this study, we examined the direct effects of several cytokines--tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-2 and 4 (IL-2 and 4)--on TJ in immortalized mouse cholangiocytes. Moreover, we examined the inhibitory effect of ursodeoxycholic acid (UDCA) on cytokine-induced changes in paracellular permeability.
  
  
  METHODS
  Barrier function of TJ was evaluated by measuring transepithelial electrical resistance (TER) and 3H-inulin flux. We also performed immunostaining and immunoblotting for TJ-associated proteins--claudin-1 and -3, occludin, zonula occluden-1 (ZO-1) and 7H6.
  
  
  RESULTS
  TNF-alpha and IFN-gamma, but neither IL-2 nor IL-4, significantly decreased TER (P < 0.005). 3H-inulin flux studies confirmed IFN-alpha-induced increases in paracellular permeability of cholangiocytes (P < 0.001). In immunostaining and immunoblotting studies, TJ-associated proteins were well preserved in TNF-alpha- or IFN-gamma-treated cells. Ursodeoxycholic acid has been found to have no inhibitory effect on increased paracellular permeability induced by TNF-alpha or IFN-gamma.
  
  
  CONCLUSIONS
  These findings show that TNF-alpha and IFN-gamma disrupt barrier function of TJ in cholangiocytes without major structural changes to TJ and suggest that disruption of TJ function and subsequent leakage of the bile constituents may influence the aggravation of cholestasis in PBC.
authors.affiliation: Second Department of Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan. hanadas@med.kurume-u.acjp
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Hanada
authors.forename: Masaru
authors.initials: M
authors.lastname: Harada
authors.forename: Hironori
authors.initials: H
authors.lastname: Koga
authors.forename: Takumi
authors.initials: T
authors.lastname: Kawaguchi
authors.forename: Eitaro
authors.initials: E
authors.lastname: Taniguchi
authors.forename: Ryukichi
authors.initials: R
authors.lastname: Kumashiro
authors.forename: Takato
authors.initials: T
authors.lastname: Ueno
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Ueno
authors.forename: Motoyasu
authors.initials: M
authors.lastname: Ishii
authors.forename: Shotaro
authors.initials: S
authors.lastname: Sakisaka
authors.forename: Michio
authors.initials: M
authors.lastname: Sata
mesh_terms: D000818:Animals; D001652:Bile Ducts / Q000166:cytology*; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D004306:Dose-Response Relationship, Immunologic; D017097:Electric Impedance; D004848:Epithelium / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D015151:Immunoblotting; D007371:Interferon-gamma / Q000494:pharmacology*; D007376:Interleukin-2 / Q000494:pharmacology; D015847:Interleukin-4 / Q000494:pharmacology; D008105:Liver Cirrhosis, Biliary / Q000276:immunology* / Q000473:pathology; D051379:Mice; D019108:Tight Junctions / Q000187:drug effects / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014580:Ursodeoxycholic Acid / Q000494:pharmacology*
chemical_list: D007376:Interleukin-2; D014409:Tumor Necrosis Factor-alpha; D015847:Interleukin-4; D014580:Ursodeoxycholic Acid; D007371:Interferon-gamma