Tumor necrosis factor a (TNF-a), which is primarily primary biliary cirrhosis. The administration of TNFproduced by macrophages, is a cytokine with various a to patients as an anticancer agent caused cholestatic biological activities. Macrophage infiltration often acjaundice. It is known that the cholangitis induced by companies experimental cholangitis in rats, and chronic formyl peptide, 8 primary biliary cirrhosis, and primary cholangitis in humans. The pathophysiologic signifisclerosing cholangitis 9 are each accompanied by the incance of TNF-a in cholangitis is not known. We used filtration of macrophages. Such findings suggest that cultured, polarized intrahepatic bile duct epithelial cells TNF-a per se may contribute to cholangitis or cholesta-(IBDECs) from rat liver to determine whether TNF-a sis through an unfavorable effect on the intrahepatic directly affects the organization of IBDEC monolayers.
bile duct epithelial cells (IBDECs). However, the effects
The addition of recombinant TNF-a (rTNF-a) to culture of TNF-a on IBDECs have not been described. media at concentrations from 10 to 200 U/mL lacked cytotoxicity to the IBDECs as judged by trypan blue exclu-Because the immune response is regulated by a comsion and lactate dehydrogenase (LDH) release. rTNF-a plicated network of cytokines, it is difficult to ascertain transiently reduced transepithelial electrical resistance the direct effect of TNF-a in vivo. We recently have in a dose-dependent manner. During this decrease in established a full-sheet monolayer culture of IBDEC resistance, the cellular tight junctions became leaky, from normal rat liver. 10 These cells are grown on an allowing horseradish peroxidase (HRP) penetration. artificial basement membrane and are joined to neigh-rTNF-a, at concentrations up to 200 U/mL, did not detach boring cells with tight junctions similar to IBDECs in IBDECs from Matrigel, an artificial basement memvivo. Such polarized IBDECs provide an opportunity to brane. Electron microscopy and immunohistochemisexamine the direct effect of TNF-a on IBDEC. try for F-actin showed a well-preserved cell structure To investigate the changes that take place during and organization of IBDECs. Results suggest that TNFa is nontoxic to IBDECs, and that it increases the per-inflammation in such cells, we examined the effects of meability of tight junctions. TNF-a may thus disturb TNF-a on cultured IBDECs by studying their cytotoxicthe barrier function of the bile duct. (HEPATOLOGY ity, integrity of the tight junctions, and the interactions 1996;23:1602-1607.)
between the basement membrane and IBDEC.
Methods
Tumor necrosis factor a (TNF-a), a potent cytokine that is produced primarily by the macrophages, exerts This experimental protocol was approved by the Commitseveral proinflammatory activities. These include the tee on Care of Laboratory Animals of the Laboratory Re- induction of adhesion molecules, the activation of polysearch Council, Tohoku University School of Medicine.
Reagents and Monoclonal Antibodies. Dispase, epidermal morphonuclear cells and macrophages, immunologic growth factor, and ITS/ were obtained from Becton Dickinson regulation, and cytotoxic activities. 1,2 Therefore, TNF-Labware (Bedford, MA). Ham/F12 medium, collagenase, soya has been implicated in the pathogenesis of various bean trypsin inhibitor, forskolin, and bovine serum albumin disease with immunologic disorders. An enhanced were obtained from Wako Chemical Co (Osaka, Japan).
production of TNF-a has been observed in patients with Horseradish peroxidase type VI (HRP) and dexamethasone were obtained from Sigma Chemical Co. (St. Louis, MO). The monoclonal antibody raised against normal rat IBDECs was the gift of Dr. Nicholas F. LaRusso (Mayo Clinic, Rochester, Abbreviations: TNF-a, tumor necrosis factor a; IBDEC, intrahepatic bile MN). 11 Recombinant TNF-a (rTNF-a) was purchased from duct epithelial cell; HRP, horseradish peroxidase; rTNF-a, recombinant tumor necrosis factor a; TER, transepithelial electrical resistance; NBD, 7-nitrobenz-Becton Dickinson Labware. This substance has a specific ac-2-oxa-1,3-diazole; LDH, lactate dehydrogenase. tivity of 3.7 1 10 6 U/mg protein (determined by cytolysis of