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Effects of bile acids on biliary epithelial cells: Proliferation, cytotoxicity, and cytokine secretion
Life Sciences 72 (2003) 1401 – 1411 www.elsevier.com/locate/lifescie
Effects of bile acids on biliary epithelial cells: Proliferation, cytotoxicity, and cytokine secretion Thierry Lamireau a,b, Monica Zoltowska a, Emile Levy a,c, Ibrahim Yousef a,d, Jean Rosenbaum b, Beatriz Tuchweber a,c, Alexis Desmoulie`re b,* a
Unite´ de Recherche en Gastroente´rologie-Nutrition, Centre de Recherche, Hoˆpital Sainte-Justine, Coˆte Sainte Catherine, Montre´al, Que´bec, Canada b Groupe de Recherches pour l’Etude du Foie, INSERM E9917, Universite´ Victor Segalen Bordeaux 2, 146 rue Le´o Saignat, 33076 Bordeaux, France c De´partement de Nutrition, Universite´ de Montre´al, Montre´al, Que´bec, Canada d De´partement de Pharmacologie, Universite´ de Montre´al, Montre´al, Que´bec, Canada Received 30 July 2002; accepted 22 October 2002
Abstract Hydrophobic bile acids, which are known to be cytotoxic for hepatocytes, are retained in high amount in the liver during cholestasis. Thus, we have investigated the effects of bile acids with various hydrophobicities on biliary epithelial cells. Biliary epithelial cells were cultured in the presence of tauroursodeoxycholate (TUDC), taurocholate (TC), taurodeoxycholate (TDC), taurochenodeoxycholate (TCDC), or taurolithocholate (TLC). Cell proliferation, viability, apoptosis and secretion of monocyte chemotactic protein-1 (MCP-1) and of interleukin-6 (IL-6) were studied. Cell proliferation was increased by TDC, and markedly decreased by TLC in a dose dependant manner (50 – 500 AM). Cell viability was significantly decreased by TLC and TCDC at 500 AM. TLC, TDC and TCDC induced apoptosis at high concentrations. The secretion of MCP-1 and IL-6 was markedly stimulated by TC. TUDC had no significant effect on any parameter. These findings demonstrate that hydrophobic bile acids were cytotoxic and induced apoptosis of biliary epithelial cells. Furthermore, TC, a major biliary acid in human bile, stimulated secretion of cytokines involved in the inflammatory and fibrotic processes occurring during cholestatic liver diseases. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Cholestasis; Apoptosis; Monocyte chemotactic protein-1; Interleukin-6
* Corresponding author. Tel.: +33-557-571-771; fax: +33-556-514-077. E-mail address: [email protected] (A. Desmoulie`re). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 2 4 0 8 - 6
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Introduction Bile duct proliferation is a hallmark of human cholestatic disorders [34]. It is also observed in experimental models of cholestasis such as bile duct ligation in the rat [4,41] or multidrug resistance-2 (Mdr2) knock-out mice [27]. The increase in hepatic bile ductular structures is generally accompanied by the development of periductular fibrosis. Biliary epithelial cells (BEC) may participate in the complex cell interactions leading to these changes, as they secrete cytokines such as monocyte chemotactic protein-1 (MCP-1) which allows recruitment of inflammatory cells [25] and promotes fibrogenesis through attraction of hepatic stellate cells (HSC) [26]. BEC also secrete interleukin-6 (IL-6) which promotes HSC proliferation and collagen synthesis [8]. Bile acids (BA), the most abundant biliary components, play a major role in the maintenance of bile flow and are natural detergents that allow lipids to be digested in the intestinal lumen. BA may accumulate in the liver in patients suffering from chronic liver diseases [15], worsening histologic abnormalities [37]. Their cytotoxicity has been demonstrated in vivo and in vitro on hepatocytes [10], and they are also known to induce hepatocyte apoptosis [13]. In general, there is an association between the bile acid hyrophobicity and cellular toxicity with deoxycholic and lithocholic exerting the most toxic effects. Although BEC are continuously exposed at their apical site to millimolar concentrations of BA, the present knowledge about effects of BA on proliferation, cellular injury (including apoptosis) and cytokine secretion results mainly from studies performed on hepatocytes and colonic epithelial cells with relatively little evidence available on BEC [1,2,6]. The aim of this study was to investigate the effects of BA with various hydrophobicities on BEC proliferation, viability, apoptosis, and cytokine secretion.
Methods BEC culture and treatment We used an immortalized murine BEC line, established from H-2Kb-tsA58 transgenic mice [30]. These transgenic mice harbour the temperature-sensitive mutation of SV40 large T antigen (tsA58 mutant) under the control of an interferon-inducible mouse histocompatibility complex H-2Kb class I promoter element. The functional expression of the SV40 large T antigen can be turned on by culturing the cells in medium containing interferon-g at a temperature of 33 jC permissive for function of the tsA58 mutation. This model provides unlimited numbers of pure BEC that behave similarly to BEC from ‘‘normal’’ mice, (notably organizing themselves into duct-like structures [30]. Cells were grown in 25 cm2 flasks coated with Matrigel (Becton Dickinson, Palo Alto, CA). The culture medium consisted of a 50/50 mixture of Dulbecco’s modified Eagle’s medium with L-glutamine / Ham’s nutrient mixture F-12 with L-glutamine, non-essential amino acid solution, D-glucose (5.4 g/l), and HEPES buffer (10 mmol/l) adjusted at pH 7.40. The medium was supplemented with 100 IU/ml penicillin G and 100 Ag/ml streptomycin sulfate (both from Gibco, Grand Island, NY); 10 ng/ml epidermal growth factor, 5 Ag/ml each of insulin and transferrin, and 5 ng/ml of selenium (all from Collaborative Biomedical Products, Bedford, MA); and 32 ng/ml thyroxin, 10 ng/ml prostaglandin E1,
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40 ng/ml hydrocortisone, and 10 U/ml mouse recombinant interferon-g (all from Roche Diagnostics, Meylan, France). Fresh medium was added every 3–4 days, and cells were passaged every 14 days by digestion using dispase (Collaborative Biomedical Products) and 0.01% trypsin (Gibco). Studies were performed in 24-well plates at permissive conditions (33 jC, interferon-g), except for proliferation studies. The following sodium salts of tauro-conjugated forms of BA of increasing hydrophobicity were tested: tauroursodeoxycholate (TUDC), taurocholate (TC), taurodeoxycholate (TDC), taurochenodeoxycholate (TCDC), and taurolithocholate (TLC). All BA were obtained from Calbiochem Corp, San Diego, CA, USA. Purity was assesed by thin layer chromatograpy, liquid chromatography-electrospray tandem mass spectrometry and gas-chromatography-mass spectrometry and no impurities were detected. Proliferation studies Cells were grown for 3 days at permissive conditions, leading to approximately 60% confluence. Then BA (0 to 500 AM) were added for 72 hours to the culture medium devoid of interferon-g and epidermal growth factor, and maintained at 33 jC. During the last 4 hours, 5 ACi of [3H]Thymidine (Amersham, Buckinghamshire, England) was added to each well. Then, each well was washed, Matrigel was digested with dispase (1h30 at 37 jC), and cells were harvested. The radioactivity was measured in a liquid scintillation counter (Beckman Instruments, Mississauga, ON, Canada), and proteins were measured according to Lowry et al. [24] using bovine serum albumin as a standard. A radioactivity index (disintegrations per minute per milligram of protein) was calculated and results were expressed as a mean percentage of the BA-free control values. BEC cultured with interferon-g (10 U/ml) and epidermal growth factor (10 ng/ml) served as positive control. Viability assay Cells were grown until 100% confluence (6–7 days), then BA (0 to 500 AM) were added to the medium for 72 hours. Cells were harvested as above, washed, trypsinized to allow flow cytometry analysis, and resuspended in phosphate buffer solution with 4 AM calcein acetoxymethyl (AM) (Molecular Probes, Eugene, OR) for 45 minutes. Non fluorescent cell-permeant calcein AM is converted in live cells by an esterase into the intensely green fluorescence calcein. The percentage of fluorescent cells was assayed by flow cytometry using a FACS IV apparatus (Becton Dickinson, Bedford, MA). Apoptosis studies Cells were grown until confluence (6–7 days), then BA (0 to 500 AM) were added to the medium for 20 hours. Cells were harvested and apoptosis was assessed by measuring the cytoplasmic enrichment in histone-associated DNA fragments (mono- and oligonucleosomes) with an Enzyme-Linked Immunosorbent Assay kit in accordance with the manufacturer’s recommendations (Roche Diagnostics). Samples were read with a spectrophotometer at 410 nm wavelength, the specific enrichment of monoand oligonucleosomes was calculated for each sample, and results were expressed as a mean percentage of the BA-free control values. Incubation with camptothecin (Calbiochem, La Jolla, CA, USA), an apoptosis-inducing agent, was used as positive control, leading to apoptosis from 170% of the BA-free control value at 0.04 Ag/ml to 329% at 4 Ag/ml.
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Cytokine secretion Cells were grown until confluence (6–7 days), then BA (0 to 500 AM) were added to the medium for 72 hours. Supernatants were then collected, centrifuged to eliminate possibly detached cells, and tested for the presence of MCP-1, IL-6 or tumor necrosis factor-a (TNF-a), with an Enzyme-Linked Immunosorbent Assay kit (Biosource, Camarillo, CA). Samples were read with a spectrophotometer at 490 nm wavelength, and results were expressed in pg per mg of protein. The secretion of cytokines after stimulation of BEC with 10 8 AM phorbol myristate acetate (Sigma, St Louis, MO, USA) was used as positive control, leading to IL-6 and MCP-1 secretion increase of 129% (74.78 F 1.07 pg/ml) and of 143% (2.36 F 0.05 pg/ml), respectively, compared with the BA-free control values. Statistics All experiments were performed in triplicates and values presented as mean F SE. A non parametric Mann-Whitney test was used to compare each concentration of BA with control, i.e. cells cultured without BA. Differences were considered significant when p < 0.05.
Results Effect of BA on BEC proliferation BEC proliferation was significantly enhanced by TDC at concentrations ranging from 20 to 250 AM, up to 212% of BA-free control values for 50 AM, which was higher than the 124% obtained with positive control, i.e. medium containing interferon-g and epidermal growth factor. A slight non significant increase in proliferation was observed with TC from 50 to 500 AM and with TCDC at 50 AM. TUDC had no effect on cell proliferation. By contrast, BEC proliferation was significantly decreased by TLC down to 55% of BA-free control values at concentration as low as 50 AM and 2% of BA-free control values at 500 AM (Fig. 1). Effect of BA on BEC viability BEC viability, expressed as the percentage of fluorescent cells was not significantly influenced by BA except for TDC and TLC which decreased it at 500 AM concentration (71% and 59% respectively) (Fig. 2). Effect of BA on BEC apoptosis BEC apoptosis was induced by TCDC and TDC at 250 AM (254% and 255%, respectively, of the BAfree control values) and 500 AM (210% and 195%, respectively, of the BA-free control values). Incubation of BEC with TLC led to a dramatic induction of apoptosis at concentrations ranging from 50 AM (183% of the BA-free control values) to 500 AM (410% of the BA-free control values). TC had only a slight non
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Fig. 1. Effect of bile acids on biliary epithelial cell proliferation. Biliary epithelial cell proliferation was measured by the incorporation of [3H]Thymidine, and expressed as the proliferation index (count per minute per mg of protein). * p < 0.05; ** p < 0.01.
significant effect at 250 AM (159% of the BA-free control values). BEC apoptosis was not significantly induced by TUDC (Fig. 3). Effect of BA on cytokine secretion MCP-1 and IL-6 secretion were found to be stimulated only by TC which induced a 4-fold increase of MCP-1 secretion and a 3-fold increase in IL-6 secretion. TLC from 20 AM to 250 AM decreased MCP-1
Fig. 2. Effect of bile acids on biliary epithelial cell viability. Biliary epithelial cell viability was assessed after incubation with calcein-AM and expressed as the percentage of fluorescent cells. * p < 0.05.
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Fig. 3. Effect of bile acids on biliary epithelial cell apoptosis.
Fig. 4. Effect of bile acids on cytokine secretion by biliary epithelial cells. MCP-1 and IL-6 secretion was assessed by ELISA. * p < 0.05; ** p < 0.01.
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secretion, but had no effect on IL-6 secretion. TUDC, TCDC, and TDC had no significant effect on MCP1 or IL-6 secretion. TNFa was never detected in the supernatant of cultured BEC (Fig. 4).
Discussion This study demonstrates that specific bile acids exert potent effects on BEC. Cell proliferation was markedly dose-dependently induced by TDC from 20 to 250 AM. By contrast, TLC which is also a very hydrophobic BA, induced a dose-dependent decrease in proliferation from 50 AM to 500 AM. Thus there appears to be no association between BA hydrophobicity and the induction of cell proliferation in this model of BEC culture. We may propose that the opposite effects of TDC and TLC on cell proliferation could be related to BA-specific effects on membrane composition and signaling pathways [18]. Previous studies have already reported variable effects of BA on BEC growth. Chronic feeding of TC and TLC to normal rats induces an increase in the number of intrahepatic bile ducts [2], whereas depletion of endogenous BA, by continuous external bile drainage, is associated with a decrease in cholangiocyte proliferative capacity, which is preserved by continuous infusion of TC during bile depletion [3]. It has also been demonstrated that the cell proliferation induced by TC and TLC is limited to large cholangiocytes [1]. Indeed, cholangiocytes are functionally heterogeneous along the biliary tree in rats [22], and it can be assumed that similar differences also exist in mice. We did not observe any proliferative effect of TC and TLC, but the exact origin (small or large BEC) along the biliary tree of the BEC cell line used in our model is not known. Thus, it is difficult to attribute the lack of effect of TC and TLC to cholangiocyte size. We investigated the effect of different BA on BEC cytotoxicity and apoptosis. Induction of cytotoxicity by high concentrations of hydrophobic BA such as TDC and TLC was observed and is in accordance with earlier work showing a correlation between BA physicochemical properties and cytotoxicity in hepatocytes. At high concentrations, hydrophobic BA are able to solubilize polar lipids, such as phospholipids and cholesterol, leading to the disruption of plasma membranes [17]. Unconjugated hydrophobic BA were also able to induce damage of BEC intracellular organelles in a model of isolated bile duct fragments [6]. In contrast, BEC alterations were not seen in rat liver perfused through portal vein with cholate, chenodeoxycholate, or lithocholate. Changes of BEC apical membrane occurred only with lithocholate, while hepatocytes showed marked intracellular damage with cholate and chenodeoxycholate and profound alterations of the canalicular membrane with lithocholate [6]. BEC could be protected, in vivo, from the toxicity of hydrophobic BA by an efficient clearance of unconjugated BA reabsorbed against bicarbonate, through a transport process towards peribiliary plexus named cholehepatic shunting [16], and by the presence of biliary phospholipids which are known to protect membranes from the detergent action of BA through the formation of mixed micelles [33]. Support for the protective role of biliary phospholipid against hepatic damage caused by BA accumulation also comes from studies of conditions where there is lack of phospholipid secretion in bile leading to BA retention and severe hepatic disease. It is noteworthy that destructive damage of biliary epithelium but also ductular proliferation are characteristic histological features in the Mdr2 deficient mice [27] and in type 3 progressive familial intrahepatic cholestasis, the corresponding disease in humans, [9], where there is abscence of phospholipids in bile.
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Hydrophobic BA including TCDC also induced apoptosis of BEC. This may mediate BA-related death in bile duct epithelial cells observed in several cholestatic liver diseases. Several studies have described and characterized hydrophobic BA-induced apoptosis in hepatocytes and colorectal cells [20,36], and found that the same pathways do not seem to mediate apoptotic response. The present study did not clarify mechanisms of apoptosis and future work will determine if this form of cell death in BEC is induced by mechanisms similar to those described for other epithelial liver cells. One novel and major finding of this work is the capacity of BA to stimulate cytokine secretion by cultured BEC. In this respect we observed BA specifity since only TC (a major BA in human bile) had a striking effect by inducing a 4-fold increase in MCP-1 secretion and a 3-fold increase in IL-6 secretion. MCP-1 determines recruitment and activation of monocytes and T lymphocytes [7,42]. MCP-1 expression, confined to few HSC and BEC in normal liver, is markedly increased within portal tracts during active hepatic fibrogenesis, especially in BEC [25]. MCP-1 has also been shown to be chemotactic for HSC [26]. IL-6 is a pleiotropic cytokine, produced by a variety of cells, including epithelial cells, and exerts variable growth-inducing, growth-inhibitory and differentiation-inducing effects, depending on the target cells [19]. Serum IL-6 levels have been found to be increased in humans with chronic liver disease [11,21,40], and IL-6 expression was shown to be increased in the liver of patients with end-stage cirrhosis [28] and primary biliary cirrhosis [43]. Although knock-out mice for IL-6 variably respond to a fibrogenic challenge [12,23,29], treatment with exogenous IL-6 induces hepatic inflammation, stimulates HSC proliferation and collagen synthesis in rat [8]. The results of the present study showing that TC stimulates BEC cytokine secretion, suggest that this BA contributes to the inflammatory and fibrotic processes observed in cholestatic diseases. Besides MCP-1 and IL-6, proliferating BEC also express platelet-derived growth factor B chain [14], a major mitogen for HSC [31], and are a major source of connective tissue growth factor [38], that induces collagen synthesis in fibrogenic cells. TDC, by inducing BEC proliferation, could thus promote fibrogenesis during cholestatic diseases. Our studies also tested TUDC in BEC. This BA is known to exert hepatoprotective properties in various conditions. In fact it can improve liver enzymes and histology in numerous cholestatic diseases [35,39]. When administrated orally, UDCA decreases intestinal absorption of potentially toxic endogenous BA, and becomes predominant in the BA pool. In contrast with hydrophobic BA, UDCA has no toxic effects, and is hepatoprotective by numerous mechanisms [39]. Although previous studies reported that TUDC and UDCA inhibit ductal hyperplasia after bile duct ligation in rat [5,32], TUDC had no significant effect on basal proliferation in our model of cultured BEC. The lack of effect of TUDC on BEC viability, cytotoxicity and apoptosis in this study is in accordance with previous work [6] showing no cell abnormalities in isolated bile ductule fragments exposed to UDCA.
Conclusion This study shows that individual BA exert potent biological effects on cultured BEC. This adds strength to the hypothesis that BA could be directly involved in the bile duct damage and proliferation observed in cholestatic diseases. Our finding that TC (a major BA in humans) stimulates cytokine secretion suggests that some BA’s could enhance the fibrogenic potential of BEC.
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Effects of bile acids on biliary epithelial cells: Proliferation, cytotoxicity, and cytokine secretion Thierry Lamireau a,b, Monica Zoltowska a, Emile Levy a,c, Ibrahim Yousef a,d, Jean Rosenbaum b, Beatriz Tuchweber a,c, Alexis Desmoulie`re b,* a
Unite´ de Recherche en Gastroente´rologie-Nutrition, Centre de Recherche, Hoˆpital Sainte-Justine, Coˆte Sainte Catherine, Montre´al, Que´bec, Canada b Groupe de Recherches pour l’Etude du Foie, INSERM E9917, Universite´ Victor Segalen Bordeaux 2, 146 rue Le´o Saignat, 33076 Bordeaux, France c De´partement de Nutrition, Universite´ de Montre´al, Montre´al, Que´bec, Canada d De´partement de Pharmacologie, Universite´ de Montre´al, Montre´al, Que´bec, Canada Received 30 July 2002; accepted 22 October 2002
Abstract Hydrophobic bile acids, which are known to be cytotoxic for hepatocytes, are retained in high amount in the liver during cholestasis. Thus, we have investigated the effects of bile acids with various hydrophobicities on biliary epithelial cells. Biliary epithelial cells were cultured in the presence of tauroursodeoxycholate (TUDC), taurocholate (TC), taurodeoxycholate (TDC), taurochenodeoxycholate (TCDC), or taurolithocholate (TLC). Cell proliferation, viability, apoptosis and secretion of monocyte chemotactic protein-1 (MCP-1) and of interleukin-6 (IL-6) were studied. Cell proliferation was increased by TDC, and markedly decreased by TLC in a dose dependant manner (50 – 500 AM). Cell viability was significantly decreased by TLC and TCDC at 500 AM. TLC, TDC and TCDC induced apoptosis at high concentrations. The secretion of MCP-1 and IL-6 was markedly stimulated by TC. TUDC had no significant effect on any parameter. These findings demonstrate that hydrophobic bile acids were cytotoxic and induced apoptosis of biliary epithelial cells. Furthermore, TC, a major biliary acid in human bile, stimulated secretion of cytokines involved in the inflammatory and fibrotic processes occurring during cholestatic liver diseases. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Cholestasis; Apoptosis; Monocyte chemotactic protein-1; Interleukin-6
* Corresponding author. Tel.: +33-557-571-771; fax: +33-556-514-077. E-mail address: [email protected] (A. Desmoulie`re). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 2 4 0 8 - 6
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Introduction Bile duct proliferation is a hallmark of human cholestatic disorders [34]. It is also observed in experimental models of cholestasis such as bile duct ligation in the rat [4,41] or multidrug resistance-2 (Mdr2) knock-out mice [27]. The increase in hepatic bile ductular structures is generally accompanied by the development of periductular fibrosis. Biliary epithelial cells (BEC) may participate in the complex cell interactions leading to these changes, as they secrete cytokines such as monocyte chemotactic protein-1 (MCP-1) which allows recruitment of inflammatory cells [25] and promotes fibrogenesis through attraction of hepatic stellate cells (HSC) [26]. BEC also secrete interleukin-6 (IL-6) which promotes HSC proliferation and collagen synthesis [8]. Bile acids (BA), the most abundant biliary components, play a major role in the maintenance of bile flow and are natural detergents that allow lipids to be digested in the intestinal lumen. BA may accumulate in the liver in patients suffering from chronic liver diseases [15], worsening histologic abnormalities [37]. Their cytotoxicity has been demonstrated in vivo and in vitro on hepatocytes [10], and they are also known to induce hepatocyte apoptosis [13]. In general, there is an association between the bile acid hyrophobicity and cellular toxicity with deoxycholic and lithocholic exerting the most toxic effects. Although BEC are continuously exposed at their apical site to millimolar concentrations of BA, the present knowledge about effects of BA on proliferation, cellular injury (including apoptosis) and cytokine secretion results mainly from studies performed on hepatocytes and colonic epithelial cells with relatively little evidence available on BEC [1,2,6]. The aim of this study was to investigate the effects of BA with various hydrophobicities on BEC proliferation, viability, apoptosis, and cytokine secretion.
Methods BEC culture and treatment We used an immortalized murine BEC line, established from H-2Kb-tsA58 transgenic mice [30]. These transgenic mice harbour the temperature-sensitive mutation of SV40 large T antigen (tsA58 mutant) under the control of an interferon-inducible mouse histocompatibility complex H-2Kb class I promoter element. The functional expression of the SV40 large T antigen can be turned on by culturing the cells in medium containing interferon-g at a temperature of 33 jC permissive for function of the tsA58 mutation. This model provides unlimited numbers of pure BEC that behave similarly to BEC from ‘‘normal’’ mice, (notably organizing themselves into duct-like structures [30]. Cells were grown in 25 cm2 flasks coated with Matrigel (Becton Dickinson, Palo Alto, CA). The culture medium consisted of a 50/50 mixture of Dulbecco’s modified Eagle’s medium with L-glutamine / Ham’s nutrient mixture F-12 with L-glutamine, non-essential amino acid solution, D-glucose (5.4 g/l), and HEPES buffer (10 mmol/l) adjusted at pH 7.40. The medium was supplemented with 100 IU/ml penicillin G and 100 Ag/ml streptomycin sulfate (both from Gibco, Grand Island, NY); 10 ng/ml epidermal growth factor, 5 Ag/ml each of insulin and transferrin, and 5 ng/ml of selenium (all from Collaborative Biomedical Products, Bedford, MA); and 32 ng/ml thyroxin, 10 ng/ml prostaglandin E1,
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40 ng/ml hydrocortisone, and 10 U/ml mouse recombinant interferon-g (all from Roche Diagnostics, Meylan, France). Fresh medium was added every 3–4 days, and cells were passaged every 14 days by digestion using dispase (Collaborative Biomedical Products) and 0.01% trypsin (Gibco). Studies were performed in 24-well plates at permissive conditions (33 jC, interferon-g), except for proliferation studies. The following sodium salts of tauro-conjugated forms of BA of increasing hydrophobicity were tested: tauroursodeoxycholate (TUDC), taurocholate (TC), taurodeoxycholate (TDC), taurochenodeoxycholate (TCDC), and taurolithocholate (TLC). All BA were obtained from Calbiochem Corp, San Diego, CA, USA. Purity was assesed by thin layer chromatograpy, liquid chromatography-electrospray tandem mass spectrometry and gas-chromatography-mass spectrometry and no impurities were detected. Proliferation studies Cells were grown for 3 days at permissive conditions, leading to approximately 60% confluence. Then BA (0 to 500 AM) were added for 72 hours to the culture medium devoid of interferon-g and epidermal growth factor, and maintained at 33 jC. During the last 4 hours, 5 ACi of [3H]Thymidine (Amersham, Buckinghamshire, England) was added to each well. Then, each well was washed, Matrigel was digested with dispase (1h30 at 37 jC), and cells were harvested. The radioactivity was measured in a liquid scintillation counter (Beckman Instruments, Mississauga, ON, Canada), and proteins were measured according to Lowry et al. [24] using bovine serum albumin as a standard. A radioactivity index (disintegrations per minute per milligram of protein) was calculated and results were expressed as a mean percentage of the BA-free control values. BEC cultured with interferon-g (10 U/ml) and epidermal growth factor (10 ng/ml) served as positive control. Viability assay Cells were grown until 100% confluence (6–7 days), then BA (0 to 500 AM) were added to the medium for 72 hours. Cells were harvested as above, washed, trypsinized to allow flow cytometry analysis, and resuspended in phosphate buffer solution with 4 AM calcein acetoxymethyl (AM) (Molecular Probes, Eugene, OR) for 45 minutes. Non fluorescent cell-permeant calcein AM is converted in live cells by an esterase into the intensely green fluorescence calcein. The percentage of fluorescent cells was assayed by flow cytometry using a FACS IV apparatus (Becton Dickinson, Bedford, MA). Apoptosis studies Cells were grown until confluence (6–7 days), then BA (0 to 500 AM) were added to the medium for 20 hours. Cells were harvested and apoptosis was assessed by measuring the cytoplasmic enrichment in histone-associated DNA fragments (mono- and oligonucleosomes) with an Enzyme-Linked Immunosorbent Assay kit in accordance with the manufacturer’s recommendations (Roche Diagnostics). Samples were read with a spectrophotometer at 410 nm wavelength, the specific enrichment of monoand oligonucleosomes was calculated for each sample, and results were expressed as a mean percentage of the BA-free control values. Incubation with camptothecin (Calbiochem, La Jolla, CA, USA), an apoptosis-inducing agent, was used as positive control, leading to apoptosis from 170% of the BA-free control value at 0.04 Ag/ml to 329% at 4 Ag/ml.
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Cytokine secretion Cells were grown until confluence (6–7 days), then BA (0 to 500 AM) were added to the medium for 72 hours. Supernatants were then collected, centrifuged to eliminate possibly detached cells, and tested for the presence of MCP-1, IL-6 or tumor necrosis factor-a (TNF-a), with an Enzyme-Linked Immunosorbent Assay kit (Biosource, Camarillo, CA). Samples were read with a spectrophotometer at 490 nm wavelength, and results were expressed in pg per mg of protein. The secretion of cytokines after stimulation of BEC with 10 8 AM phorbol myristate acetate (Sigma, St Louis, MO, USA) was used as positive control, leading to IL-6 and MCP-1 secretion increase of 129% (74.78 F 1.07 pg/ml) and of 143% (2.36 F 0.05 pg/ml), respectively, compared with the BA-free control values. Statistics All experiments were performed in triplicates and values presented as mean F SE. A non parametric Mann-Whitney test was used to compare each concentration of BA with control, i.e. cells cultured without BA. Differences were considered significant when p < 0.05.
Results Effect of BA on BEC proliferation BEC proliferation was significantly enhanced by TDC at concentrations ranging from 20 to 250 AM, up to 212% of BA-free control values for 50 AM, which was higher than the 124% obtained with positive control, i.e. medium containing interferon-g and epidermal growth factor. A slight non significant increase in proliferation was observed with TC from 50 to 500 AM and with TCDC at 50 AM. TUDC had no effect on cell proliferation. By contrast, BEC proliferation was significantly decreased by TLC down to 55% of BA-free control values at concentration as low as 50 AM and 2% of BA-free control values at 500 AM (Fig. 1). Effect of BA on BEC viability BEC viability, expressed as the percentage of fluorescent cells was not significantly influenced by BA except for TDC and TLC which decreased it at 500 AM concentration (71% and 59% respectively) (Fig. 2). Effect of BA on BEC apoptosis BEC apoptosis was induced by TCDC and TDC at 250 AM (254% and 255%, respectively, of the BAfree control values) and 500 AM (210% and 195%, respectively, of the BA-free control values). Incubation of BEC with TLC led to a dramatic induction of apoptosis at concentrations ranging from 50 AM (183% of the BA-free control values) to 500 AM (410% of the BA-free control values). TC had only a slight non
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Fig. 1. Effect of bile acids on biliary epithelial cell proliferation. Biliary epithelial cell proliferation was measured by the incorporation of [3H]Thymidine, and expressed as the proliferation index (count per minute per mg of protein). * p < 0.05; ** p < 0.01.
significant effect at 250 AM (159% of the BA-free control values). BEC apoptosis was not significantly induced by TUDC (Fig. 3). Effect of BA on cytokine secretion MCP-1 and IL-6 secretion were found to be stimulated only by TC which induced a 4-fold increase of MCP-1 secretion and a 3-fold increase in IL-6 secretion. TLC from 20 AM to 250 AM decreased MCP-1
Fig. 2. Effect of bile acids on biliary epithelial cell viability. Biliary epithelial cell viability was assessed after incubation with calcein-AM and expressed as the percentage of fluorescent cells. * p < 0.05.
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Fig. 3. Effect of bile acids on biliary epithelial cell apoptosis.
Fig. 4. Effect of bile acids on cytokine secretion by biliary epithelial cells. MCP-1 and IL-6 secretion was assessed by ELISA. * p < 0.05; ** p < 0.01.
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secretion, but had no effect on IL-6 secretion. TUDC, TCDC, and TDC had no significant effect on MCP1 or IL-6 secretion. TNFa was never detected in the supernatant of cultured BEC (Fig. 4).
Discussion This study demonstrates that specific bile acids exert potent effects on BEC. Cell proliferation was markedly dose-dependently induced by TDC from 20 to 250 AM. By contrast, TLC which is also a very hydrophobic BA, induced a dose-dependent decrease in proliferation from 50 AM to 500 AM. Thus there appears to be no association between BA hydrophobicity and the induction of cell proliferation in this model of BEC culture. We may propose that the opposite effects of TDC and TLC on cell proliferation could be related to BA-specific effects on membrane composition and signaling pathways [18]. Previous studies have already reported variable effects of BA on BEC growth. Chronic feeding of TC and TLC to normal rats induces an increase in the number of intrahepatic bile ducts [2], whereas depletion of endogenous BA, by continuous external bile drainage, is associated with a decrease in cholangiocyte proliferative capacity, which is preserved by continuous infusion of TC during bile depletion [3]. It has also been demonstrated that the cell proliferation induced by TC and TLC is limited to large cholangiocytes [1]. Indeed, cholangiocytes are functionally heterogeneous along the biliary tree in rats [22], and it can be assumed that similar differences also exist in mice. We did not observe any proliferative effect of TC and TLC, but the exact origin (small or large BEC) along the biliary tree of the BEC cell line used in our model is not known. Thus, it is difficult to attribute the lack of effect of TC and TLC to cholangiocyte size. We investigated the effect of different BA on BEC cytotoxicity and apoptosis. Induction of cytotoxicity by high concentrations of hydrophobic BA such as TDC and TLC was observed and is in accordance with earlier work showing a correlation between BA physicochemical properties and cytotoxicity in hepatocytes. At high concentrations, hydrophobic BA are able to solubilize polar lipids, such as phospholipids and cholesterol, leading to the disruption of plasma membranes [17]. Unconjugated hydrophobic BA were also able to induce damage of BEC intracellular organelles in a model of isolated bile duct fragments [6]. In contrast, BEC alterations were not seen in rat liver perfused through portal vein with cholate, chenodeoxycholate, or lithocholate. Changes of BEC apical membrane occurred only with lithocholate, while hepatocytes showed marked intracellular damage with cholate and chenodeoxycholate and profound alterations of the canalicular membrane with lithocholate [6]. BEC could be protected, in vivo, from the toxicity of hydrophobic BA by an efficient clearance of unconjugated BA reabsorbed against bicarbonate, through a transport process towards peribiliary plexus named cholehepatic shunting [16], and by the presence of biliary phospholipids which are known to protect membranes from the detergent action of BA through the formation of mixed micelles [33]. Support for the protective role of biliary phospholipid against hepatic damage caused by BA accumulation also comes from studies of conditions where there is lack of phospholipid secretion in bile leading to BA retention and severe hepatic disease. It is noteworthy that destructive damage of biliary epithelium but also ductular proliferation are characteristic histological features in the Mdr2 deficient mice [27] and in type 3 progressive familial intrahepatic cholestasis, the corresponding disease in humans, [9], where there is abscence of phospholipids in bile.
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Hydrophobic BA including TCDC also induced apoptosis of BEC. This may mediate BA-related death in bile duct epithelial cells observed in several cholestatic liver diseases. Several studies have described and characterized hydrophobic BA-induced apoptosis in hepatocytes and colorectal cells [20,36], and found that the same pathways do not seem to mediate apoptotic response. The present study did not clarify mechanisms of apoptosis and future work will determine if this form of cell death in BEC is induced by mechanisms similar to those described for other epithelial liver cells. One novel and major finding of this work is the capacity of BA to stimulate cytokine secretion by cultured BEC. In this respect we observed BA specifity since only TC (a major BA in human bile) had a striking effect by inducing a 4-fold increase in MCP-1 secretion and a 3-fold increase in IL-6 secretion. MCP-1 determines recruitment and activation of monocytes and T lymphocytes [7,42]. MCP-1 expression, confined to few HSC and BEC in normal liver, is markedly increased within portal tracts during active hepatic fibrogenesis, especially in BEC [25]. MCP-1 has also been shown to be chemotactic for HSC [26]. IL-6 is a pleiotropic cytokine, produced by a variety of cells, including epithelial cells, and exerts variable growth-inducing, growth-inhibitory and differentiation-inducing effects, depending on the target cells [19]. Serum IL-6 levels have been found to be increased in humans with chronic liver disease [11,21,40], and IL-6 expression was shown to be increased in the liver of patients with end-stage cirrhosis [28] and primary biliary cirrhosis [43]. Although knock-out mice for IL-6 variably respond to a fibrogenic challenge [12,23,29], treatment with exogenous IL-6 induces hepatic inflammation, stimulates HSC proliferation and collagen synthesis in rat [8]. The results of the present study showing that TC stimulates BEC cytokine secretion, suggest that this BA contributes to the inflammatory and fibrotic processes observed in cholestatic diseases. Besides MCP-1 and IL-6, proliferating BEC also express platelet-derived growth factor B chain [14], a major mitogen for HSC [31], and are a major source of connective tissue growth factor [38], that induces collagen synthesis in fibrogenic cells. TDC, by inducing BEC proliferation, could thus promote fibrogenesis during cholestatic diseases. Our studies also tested TUDC in BEC. This BA is known to exert hepatoprotective properties in various conditions. In fact it can improve liver enzymes and histology in numerous cholestatic diseases [35,39]. When administrated orally, UDCA decreases intestinal absorption of potentially toxic endogenous BA, and becomes predominant in the BA pool. In contrast with hydrophobic BA, UDCA has no toxic effects, and is hepatoprotective by numerous mechanisms [39]. Although previous studies reported that TUDC and UDCA inhibit ductal hyperplasia after bile duct ligation in rat [5,32], TUDC had no significant effect on basal proliferation in our model of cultured BEC. The lack of effect of TUDC on BEC viability, cytotoxicity and apoptosis in this study is in accordance with previous work [6] showing no cell abnormalities in isolated bile ductule fragments exposed to UDCA.
Conclusion This study shows that individual BA exert potent biological effects on cultured BEC. This adds strength to the hypothesis that BA could be directly involved in the bile duct damage and proliferation observed in cholestatic diseases. Our finding that TC (a major BA in humans) stimulates cytokine secretion suggests that some BA’s could enhance the fibrogenic potential of BEC.
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