Tumor necrosis factor-alpha and interferon-gamma directly impair epithelial barrier function in cultured mouse cholangiocytes.
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.
Hanada
Shinichiro
S
Second Department of Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan. hanadas@med.kurume-u.acjp
Harada
Masaru
M
Koga
Hironori
H
Kawaguchi
Takumi
T
Taniguchi
Eitaro
E
Kumashiro
Ryukichi
R
Ueno
Takato
T
Ueno
Yoshiyuki
Y
Ishii
Motoyasu
M
Sakisaka
Shotaro
S
Sata
Michio
M
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*
D007376:Interleukin-2; D014409:Tumor Necrosis Factor-alpha; D015847:Interleukin-4; D014580:Ursodeoxycholic Acid; D007371:Interferon-gamma