Tumor necrosis factor-α and interferon-γ directly impair epithelial barrier function in cultured mouse cholangiocytes


Shinichiro Hanada, MD, Second Department of Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan.
Tel: 81 942 31 7561. Fax: 81 942 34 2623.
e-mail: hanadas@med.kurume-u.acjp


Abstract:Background/Aims: 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-α (TNF-α), interferon-γ (IFN-γ), 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-α and IFN-γ, but neither IL-2 nor IL-4, significantly decreased TER (P < 0.005). 3H-inulin flux studies confirmed IFN-α-induced increases in paracellular permeability of cholangiocytes (P < 0.001). In immunostaining and immunoblotting studies, TJ-associated proteins were well preserved in TNF-α- or IFN-γ-treated cells. Ursodeoxycholic acidhas been found to have no inhibitory effect on increased paracellular permeability induced by TNF-α or IFN-γ. Conclusion: These findings show that TNF-α and IFN-γ 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.