François Ballet

The Interleukin-2 Receptor Down-regulates the Expression of Cytochrome P450 in Cultured Rat Hepatocytes

BACKGROUND & AIMS Interleukin (IL) 2 is used in advanced cancers, but its effects on cytochrome P450 remain unknown. Other cytokines down-regulate hepatic cytochrome P450, but it is not known whether this involves cytokine receptors. The aim of this study was to determine whether the IL-2 receptor is expressed on hepatocytes and whether its activation by IL-2 depresses cytochrome P450 in cultured rat hepatocytes. METHODS A monoclonal antibody specific for the rat IL-2 receptor alpha chain was used to label the receptor, whereas effects on cytochrome P450 were determined after 24 hours of culture with human recombinant IL-2 (5000 U/mL). RESULTS The presence of the IL-2 receptor in hepatocytes was shown by immunoblots, flow cytometry, and scanning confocal microscopy. IL-2 caused a 46% decrease in total cytochrome P450; a 35%, 35%, 36%, 26%, and 56% decrease in immunoreactive cytochrome P4501A1, 2B, 2C11, 2D1, and 3A, respectively; and a marked decrease in cytochrome P4503A2 and 2C11 messenger RNAs. Addition to the culture medium of the anti-receptor antibody or the tyrosine kinase inhibitor genistein prevented the IL-2-mediated decrease in cytochrome P450. CONCLUSIONS IL-2 down-regulates the expression of cytochrome P450 genes in cultured rat hepatocytes by interacting with its receptor expressed on hepatocytes.

Effects of bile acids and cholestasis on major histocompatibility complex class I in human and rat hepatocytes

BACKGROUND/AIMS Major histocompatibility complex (MHC) class I molecules, which are normally poorly expressed on the surface of hepatocytes, are overexpressed during cholestasis. The mechanisms responsible for this overexpression were examined. METHODS The expression of class I molecules, assessed by flow cytofluorimetry, and the class I messenger RNA (mRNA) transcripts, assessed by Northern blot analysis, were measured on normal human hepatocytes in primary culture. RESULTS Chenodeoxycholic acid induced an overexpression of MHC class I molecules, whereas ursodeoxycholic acid did not. The level of class I mRNA closely reflected that of the membrane protein. Moreover, cholestasis, induced in the rat by ligation-section of the common bile duct, increased the MHC class I mRNA level. Actinomycin D inhibited bile acid-induced class I transcription of rat hepatocytes in primary culture, whereas cycloheximide did not. Finally, class I mRNA expression was induced in hepatocytes by phorbol myristate acetate and by forskolin. This hyperexpression, as well as that observed with chenodeoxycholic acid, was suppressed by an inhibitor of protein kinase C and protein kinase A. CONCLUSIONS Taken together, these results suggest that chenodeoxycholic acid, as interferon, activates protein kinase C and protein kinase A, resulting in the induction of MHC class I expression.

Actin filament alteration as a potential marker for cholestasis: a study in isolated rat hepatocyte couplets

It has been suggested that modification of the pericanalicular microfilament network (F-actin) plays a role in cholestasis. The purposes of this study were to assess (i) the process of F-actin network reorganization in isolated rat hepatocyte couplets (IRHC) in order to define the optimal study conditions in this model, (ii) the effect of cholestatic and hepatotoxic but non-cholestatic compounds on F-actin distribution in IRHC. F-actin was stained with fluorescein isothiocyanate phalloidin and fluorimetric measurements were performed in single couplets using a scanning laser cytometer, ACAS 570. F-actin distribution was assessed by the ratio of canalicular area fluorescence/total couplet fluorescence (CF/TF). The organization of the F-actin filaments was restored in IRHC 3-6 h after plating. At non-cytotoxic concentrations, most cholestatic compounds induced a significant accumulation of F-actin around the bile canaliculus as indicated by increased fluorescence in the pericanalicular area and by the increased CF/TF ratio as compared to the controls. This accumulation could be a consequence of an inhibition of F-actin depolymerization or a higher organization of actin (redistribution, bundling or reorientation). Hepatotoxic but non-cholestatic compounds did not induce any change in pericanalicular F-actin. Abnormalities of pericanalicular F-actin therefore appear to be a specific marker of hepatocellular cholestasis.

Effect of bile acids on intracellular calcium in isolated rat hepatocyte couplets.

The effects of bile acids on cytosolic free calcium ([Ca2+]i) were studied in single isolated rat hepatocyte couplets using a scanning laser cytometer and the fluorescent dye, indo-1. Cholestatic bile acids, chenodeoxycholate (CDC) and taurolithocholate (TLC) increased [Ca2+]i in a dose-dependent manner. Choleretic bile acids, tauroursodeoxycholate (TUDC) and taurocholate (TC), did not induce any change in [Ca2+]i except TC at very high doses. Treatment with TUDC added concomitantly with CDC or TLC significantly decreased the rise in [Ca2+]i induced by bile acids. These results, obtained with a polarized hepatocyte model that secretes bile, confirmed that cholestatic bile acids increase [Ca2+]i and showed that TUDC inhibits this phenomenon. These data are in agreement with a key role of intracellular calcium in cholestasis.

Effect of tauroursodeoxycholate on actin filament alteration induced by cholestatic agents. A study in isolated rat hepatocyte couplets

The mechanism of the protective effect of ursodeoxycholic acid in cholestatic liver diseases remains unclear. Since there is evidence that alterations in the pericanalicular actin microfilament network play a major role in cholestasis, the aims of this study were (a) to determine the effect of the cholestatic agents, taurolithocholate (TLC) and erythromycin estolate (ERY), on F-actin distribution in isolated rat hepatocyte couplets and (b) to assess the effect of tauroursodeoxycholate (TUDC) and taurocholate on the modifications induced by these two compounds. F-actin was stained with fluorescein-isothiocyanate phalloidin and fluorimetric measurements were performed using a scanning laser cytometer ACAS 570. F-actin distribution was assessed in the couplets by the ratio of the pericanalicular area fluorescence/total couplet fluorescence (CF/TF). At non-cytotoxic concentrations, TLC (50, 100 microM) and ERY (10, 50, 100 microM) induced a significant accumulation of F-actin around the bile canaliculus as indicated by increased fluorescence in the pericanalicular area and by the increased CF/TF ratio compared with the controls. Electron microscopy studies showed significant alterations in bile canaliculi microvilli in couplets treated with 100 microM TLC. Only a few canaliculi showed an increase in pericanalicular microfilaments after treatment with 100 microM ERY. As assessed by scanning laser cytometry, TUDC prevented changes in F-actin distribution when it was added to the medium with taurolithocholate or erythromycin estolate at equimolar concentrations. However, the morphological changes observed by electron microscopy after treatment with TLC were not modified by co-treatment with TUDC. Taurocholate was ineffective. We conclude that (a) abnormalities of pericanalicular F-actin microfilaments occur in two different models of cholestasis, (b) tauroursodeoxycholate prevents the accumulation of pericanalicular F-actin detected by scanning laser cytometry but not the morphological changes of the canaliculus observed by electronic microscopy. Therefore, in these experimental conditions, the protective effect of TUDC appears to be partial.