CD40 activation-induced, Fas-dependent apoptosis and NF-kappaB/AP-1 signaling in human intrahepatic biliary epithelial cells.

Fas-mediated mechanisms of apoptosis are thought to be involved in the bile duct loss that characterizes diseases such as primary biliary cirrhosis (PBC). We have previously shown that activation of CD40 on hepatocytes can amplify Fas-mediated apoptosis; in the present study, we investigated interactions between CD40 and Fas in biliary epithelial cells (BEC). We report that the bile ducts in PBC liver tissue frequently express increased levels of Fas, Fas ligand (FasL), and CD40 associated with apoptotic BEC. The portal mononuclear infiltrate contains CD40L+ve T cells and macrophages, thereby demonstrating a potential mechanism for CD40 engagement in vivo. Primary cultures of human BEC also expressed Fas, FasL, and CD40 but not CD40L protein or mRNA. Activation of CD40 on BEC using recombinant CD40L increased transcriptional expression of FasL and induced apoptosis, which was inhibited by neutralizing antibodies to either Fas or FasL. Thus, CD40-induced apoptosis of BEC is mediated through Fas/FasL. We then investigated the intracellular signals and transcription factors activated in BEC and found that NF-kappaB and AP-1 were both activated after CD40 ligation. Increased functional NF-kappaB was seen early after CD40 ligation, but returned to baseline levels after 4 h. In contrast, the rapid up-regulation of AP-1 was sustained over 24 h. This study provides further functional evidence of the ability of CD40 to induce Fas/FasL-dependent apoptosis of liver epithelial cells supporting the importance of cross-talk between tumor necrosis factor (TNF) receptor family members as an amplification step in apoptosis induction. Sustained activation of AP-1 in the absence of NF-kappaB signaling may be a critical factor in determining the outcome of CD40 engagement.

The protein kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methyl-piperzine (H-7) prevents and reverses Ca(2+)-mediated injury in isolated rat hepatocyte couplets.

We have recently shown that protein kinase C (PKC) activation induces similar morphological and functional alterations in couplets to that caused by increments of intracellular Ca(2+). Since certain PKC isoforms are activated by Ca(2+), we tested whether the PKC inhibitor H-7 can counteract the alterations induced by this ion in couplets. The Ca(2+) ionophore A23187, which can mobilize Ca(2+) from extracellular and intracellular sources, decreased, in a dose-dependent manner, the percentage of couplets accumulating the fluorescent bile acid analogue cholyl-lysyl-fluorescein (CLF) in their canalicular vacuoles, i.e., in the canalicular vacuolar accumulation test (cVA of CLF), a measure of the overall capability of the couplets to secrete and retain CLF. To a similar extent, A23187 also decreased the percentage of couplets retaining CLF once secreted, i.e., in the canalicular vacuole retention test (cVR of CLF), a measure of tight junctional integrity. ATP (50 microM), another Ca(2+)-elevating compound, altered canalicular function in a similar extent to A23187. All these functional changes were prevented by H-7 in a dose-dependent manner. Canalicular dysfunction was accompanied by bleb formation and extensive redistribution of F-actin from the pericanalicular area to the cell body, which was also fully prevented by H-7; the intracellular Ca(2+) chelator, 1, 2-bis(o-aminophenoxy)-ethene-N,N,N',N'-tetraacetate tetrakis-(acetomethylester), (BAPTA/AM) (20 microM) had virtually the same preventive effects as H-7. Both H-7 and BAPTA/AM not only prevented but also reversed the decrease in cVA of CLF and blebbing induced by A23187. Thus, H-7 can both prevent and reverse Ca(2+)-mediated hepatocellular injury.

ANIT-induced disruption of biliary function in rat hepatocyte couplets.

alpha-Naphthylisothiocyanate (ANIT) induces intrahepatic cholestasis in rats, involving damage to biliary epithelial cells; our study aims to investigate whether disruption of biliary function in hepatocytes can contribute to early stages of ANIT-induced intrahepatic cholestasis. Isolated rat hepatocyte couplets were used to investigate biliary function in vitro by canalicular vacuolar accumulation (cVA) of a fluorescent bile acid analogue, cholyl-lysyl-fluorescein (CLF), within the canalicular vacuole between the two cells. After a 2-h exposure to ANIT, there was a concentration-dependent inhibition of cVA (cVA-IC50; 25 microM), but no cytotoxicity (LDH leakage or [ATP] decline) within this ANIT concentration range. There was no loss of cellular [GSH] at low ANIT concentrations, but, at 50 microM ANIT, a small but significant loss of [GSH] had occurred. Diethylmaleate (DEM) partially depleted cellular [GSH], but addition of 10 microM ANIT had no further effect on GSH depletion. Reduction in cVA was seen in DEM-treated cells; addition of ANIT to these cells reduced cVA further, but the magnitude of this further reduction was no greater than that caused by ANIT alone, indicating that glutathione depletion does not enhance the effect of ANIT. F-actin distribution (by phalloidin-FITC staining) showed an increased frequency of morphological change in the canalicular vacuoles but only a small, non-significant (0.05 < p < 0.1) increase in proportion of the F-actin in the region of the pericanalicular web. The results are in accord with a disruption of hepatocyte canalicular secretion within two h in vitro, at low, non-cytotoxic concentrations of ANIT, and the possible involvement of a thiocabamoyl-GSH conjugate of ANIT (GS-ANIT) in this effect.

Tauroursodeoxycholate and S-adenosyl-L-methionine exert an additive ameliorating effect on taurolithocholate-induced cholestasis: a study in isolated rat hepatocyte couplets.

The monohydroxy bile acid, taurolithocholate (TLC), causes cholestasis in vivo and in isolated perfused livers. It is also cholestatic in vitro and, in this study using isolated rat hepatocyte couplets, causes a reduction of the accumulation of (fluorescent) bile acid in the canalicular vacuoles (cVA) of this polarized cell preparation. The hepatoprotective bile acid, tauroursodeoxycholate (TUDCA), partially protects against the action of TLC when added at the same time. It also partially reverses the cholestatic effect if added after the cells have been exposed to TLC. A second hepatoprotective compound, S-adenosyl-L-methionine (SAMe) also not only partially protects against the action of TLC when added at the same time, but it too is able to partially reverse the cholestatic effect. Neither hepatoprotective agent is fully effective alone, but their effects are additive. In combination, a full restoration of cVA is observed in moderate cholestasis, but not in severe cholestasis. We discuss briefly some possible mechanisms involved in the additive mode of action of both hepatoprotective compounds. In summary, we show for the first time that SAMe and TUDCA can exert an additive effect in the amelioration of TLC-induced cholestasis in isolated rat hepatocyte couplets. This finding may be of possible clinical relevance.

Hepatobiliary effects of tertiary-butylhydroperoxide (tBOOH) in isolated rat hepatocyte couplets.

The organic hydroperoxide, tertiary-butylhydroperoxide (tBOOH), causes oxidative damage in a number of cell types. It is used here in an isolated rat hepatocyte couplet preparation to study adverse hepatobiliary effects of peroxidative damage in vitro. At subcytotoxic concentrations (as determined by lactate dehydrogenase release and maintenance of cytoplasmic ATP concentrations) tBOOH caused decreased accumulation of a fluorescent bile acid analogue, cholyl-lysyl-fluorescein (CLF), in the canalicular vacuole of couplets (a hepatobiliary effect; cholestasis). This was dose dependent in the range 100-200 microM. At the same concentrations it brought about release of preaccumulated CLF, suggesting that its effect was more likely to be on sealing properties of the vacuole than processes of uptake, transcytosis, and secretion. Pretreatment of tBOOH-treated couplets with the antioxidants deferoxamine mesylate (iron chelator) and dimethyl sulfoxide (free radical scavenger) resulted in the prevention of both canalicular vacuolar accumulation (cVA, which assesses canalicular function) and canalicular vacuolar retention (cVR, which assesses the retaining ability of couplets) depression at 100 microM tBOOH but not at higher concentrations. This indicates that the cholestatic effect of tBOOH has a preventable and nonpreventable phase and that free radicals are involved in these processes. By selectively generating the two types of tBOOH radical, peroxyl (tBOO.) and alkoxyl (tBO.), using suitable catalysts, we were able to determine that the peroxyl radical was most probably involved in tBOOH-induced cholestasis. This was further supported by experiments employing specific peroxyl and alkoxyl radical scavengers; only the peroxyl scavenger reduced the effect of tBOOH upon canalicular function under the conditions studied.

Urinary levels of neopterin and biopterin in autism.

The pterins, neopterin and biopterin, occur naturally in body fluids including urine. It is well established that increased neopterin levels are associated with activation of the cellular immune system and that reduced biopterins are essential for neurotransmitter synthesis. It has been suggested that some autistic children may be suffering from an autoimmune disorder. To investigate this further we performed high performance liquid chromatography analyses of urinary pterins in a group of pre-school autistic children, their siblings and age-matched control children. Both urinary neopterin and biopterin were raised in the autistic children compared to controls and the siblings showed intermediate values. This supports the possible involvement of cell-mediated immunity in the aetiology of autism.