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Role of triiodotyronine in hepatocyte proliferation during liver injury
HEPATOLOGY go1. 34, No. 4, Pt. 2, 2001
AASLD ABSTRACTS
281A
435
436
GADOLINIUM CLORIDE, AN INHIBITOR OF KUPFFER CELLS, PREVENTS CIRRHOSIS I N D U C E D BY CCL 4 INTOXICATION IN THE RAT.
DYSREGULATED INTERLEUKIN 18 METABOLISM IN ALCOROLIC LIVER DISEASE A N D NON-ALCOHOLIC STEATOHEPATITIS. Craig J
Pablo Muriel M, Yesenia Escobar F, Cinvestav-fPN, Mexico City Mexico
McClain, University of Louisville, Louisville, KY; Daniel B Hill, University- of Kentucky, Lexington, KY; Luis S Marsano, University of Louisville, Louisville, ICY; Luis R Pena, University of Kentucky, Lexington, KY; Marcelo Kugelmas, University of Colorado, Denver, CO; Shirish Barve, University of Louisville, Louisville, KY
Background/Aims: Hepatic fibrosis is an important consequence of chronic liver disease. The replacement of normal hepatic parenchymal tissue compromises the functional capacity of the liver and disrupts the normal architecture relationships of the organ. An important feature of liver fibrosis is the deposition of connective tissue around the hepatic sinusoids. Tranformation of stellate ceils with deposition of collagen and other extracellular matrix proteins is considered a key component of the pathogenisis of liver fibrosis. Kupfer cells (KCs)activation with the release of fibrogenic mediators is know to precede stellate cell activation. Thus, it is reasonable to postulate that modulation of KCs could disrupt the sequence of events leading to organ injury by damping down the fibrogenic stimulus. In order to elucidate the role of KCs in fiver fibrosis and cirrhosis, rats were treated with GdC13 and cirrhosis was induced by chronic CC14 intoxication. Methods: Carbon tetrachloride was administered three times per week for 8 weeks to male Wistar rats treated simultaneouslywith GdCI3 (20 mg/kg, daily); appropriate controls were performed. Results: Chronic treatment with CCI+induced a 40% of mortality, while treatment with GdC13 (in addition to CC14)reduced the mortality to 6.6% (p< 0.05), indicating that this compound has an important protective effect. Control animals treated with mineral oil or GdC13 alone showed no mortality. Serum enzyme activities of alkaline phosphatase (ALP), 3,-glutamyl transpeptidase (3'-GTP) and alanine aminotrasferase (ALT) and bilirubitls concentration increased significantly by CCI+,while GdCI3prevented completely the increase in T-GTP, and partially ALP, ALT, and bilirubins (p<0.05). Liver glycogen was depleted by CCI4, an effect that GdCl3 was not capable of preventing. Moreover, gadolinium chloride by itselfdepleted it. The degree of lipid peroxidation increased about 2.5-fold by intoxication with CCL, GdC13 preserved lipid peroxidation within normal values. Hepatic collagen increased 3-fold after chronic intoxicationwith CCI+(P<0.05). Gadolinium chloride prevented partially (p<0.05) the increase in collagen as evidenced by the liver hydroxyproline content and by the histophatological analysis. Conclusions: In relation with glycogen depletion by gadolinium chloride, it it has been reported that this compound may affect the hepatocyte plasma membrane, and alterations on plasma membrane activate adenyl cyclase, increasing cAMP and inducing glycogen breakdown. The effect of GdCI3 on KCs are reported to be manifold in nature and more complex than merely a possible change in ceil number, including the induction of apoptosis, the reduction in nitric oxide synthase expression, the decrease of phagocytic activity, probably by interfering with calciumdependent cell surface interactions, the attenuation of endotoxin-induced TNF-a mRNA production and protein release, the impairment of their capacity to generate superoxide, the abolition of expression of certain cell-specific antigens and the switch of phenotypic shape with loss of cytokine expression. TNP-a and IL-10 have been related with inflammation, necrosis and fibrosis of the liver. Thus, inhibition of their expression in Kupffer cells by GdCi3 (reported by others) may explain the beneficial effects observed in this study, mainly in the prevention of fibrosis, and the partial prevention of necrosis, evidenced by ALT serum levels and by the histophatological analysis. Moreover, the reported ability"of GdC13 to impair KCs to generate superoxide is in concordance with the prevention of the increase in lipid peroxidation observed herein, which, in turn is associate with necrosis, cholestasis and fibrosis. The present results clearly indicate that KCs are needed for the production of CCh-cirrosis, since their inactivation with GdC13 prevents the disease.
437 THE EFFECT OF GADOLINIUM CHLORIDE ON KUPFFER CELL FUNCTION IN VITRO. Clair Lee, George Yeoh, John K Olynyk, University of
Western Australia, Fremantle, WA Australia Gadolinium chloride is commonly used to study the role of Kupffer cells in vivo. Whilst some studies suggest that gadolinium may kill Kupffer cells, other studies suggest that it may alter Kupffer cell function. The aim of this study was to determine the effect of gadolinium chloride on Kupffer cell viability, phagocytic capacity and turnout necrosis factor alpha production in vitro. Methods: Kupffer cells were isolated from Sprague-Dawley rats by sequential pronasecollagenase perfusion and centrifugal elutriation. Cells were plated in 12-well plates at a density of i x l 0 6 cells per well. After 24 hours, the culture medium was changed and replaced either with control medium or medium containing gadolinium chloride (0-270 uM). After a further 24 hours, cell numbers and viability were determined by trypan blue staining whilst phagocytic capacity was determined by phagocytosis of latex beads. Pulse dosing with gadolinium chloride (1 mM) was undertaken to determine if calcium channel blockage was likely to be responsible for changes in phagocytosis. To study cytokine production, lipopolysaccharide (10ng/ml) was added to control and gadolinium treated cells for 6 hours and tumour necrosis factor alpha production measured by ELISA in harvested supernatants. Results: Kupffer cell viability was not impaired by addition of gadolinium chloride at concentrations of 0-270 uM. There was a progressive reduction in both phagocytosis and turnout necrosis factor alpha production with increasing concentrations of gadolinium chloride in a dose-responsive manner. Pulse dosing with gadolinium chloride ( l m M ) was associated with a reduction in phagocytic ability. Conclusions: Gadolinm m chloride reduces phagocytosis and turnout necrosis factor alpha production in isolated Kupffer cells in a dose-responsive manner but does not alter viability. This effect may be mediated, at least in part, by effects of gadolinium chloride on calcium channel function.
Dysregulated cytoknie metabolism is increasingly recognized as a cause of liver injury and a cause of several metabolic complications in both alcoholic liver disease and non alcoholic steatohepatitis (NASH). Interleukin 18 is a critical immunomodulatory cytokine that has been shown to play an etiologic role in many forms of experimental liver injury (e.g. hepatotoxicity induced by C. parvum injection). The objectives of this study were to: 1) determine whether plasma IL-18 levels were elevated in patients with alcoholic liver disease and NASH, 2) to determine whether values were consistent over time in patients with stable liver disease, and 3) to determine if values decreased with clinical improvement of acute alcoholic hepatitis. Normal volunteers and three well characterized patient groups with liver disease were studied. The first group included nine patients with stable alcoholic cirrhosis (aI1 patients had stable liver enzymes and serum albumin concentrations for the preceding 3 months). The second group consisted of fourteen patients with stable documented NASH. The last group included ten patients with acute alcoholic hepatitis who were followed for 3 months at regular intervals. Normal volunteers had a mean plasma IL-18 concentration of 2 3 8 + 2 7 pg/ml. Patients with stable cirrhosis (384+49) and NASH (399+35) had significantly elevated plasma levels that were almost identical when patients were re-evaluated two to six weeks later (402+59 and 410 +40 respectively). Patients with alcoholic hepatitis had very elevated levels on admission, and values improved during the clinical course of disease (758+ 99 on admission, 4 7 9 + 3 6 three months later). We conclude that there is dysregutated IL-18 metabolism in both alcoholic liver disease and NASH, and we postulate that interleukin 18 may play a role in the development of these forms of liver injury. Novel methods for inhibiting IL-18 activity or blocking cleavage of the active form IL-18 are under development and may prove to be effective therapeutic intervention in these forms of liver disease.
438 ROLE OF TRIIODOTYRONINE IN HEPATOCYTE PROLIFERATION
DURING LIVER INJURY. Revital Kariv, Anat Enden, Isabel Zvibel, Shlomo BrilI, Zamir Halpern, Ran Oren, Tel Aviv Medical Center, Tel Aviv Israel Background: Triiodothyronine (T3) is considered a primary mitogen for hepatocytes. Priming has been shown to be induced by hepatic injury and is mediated via increased production of TNF-a. TNF-a causes release of IL-6, that primes hepatocytes to respond to hepatic mitogens. T3, by itself or in combination with other hepatic mitogens (HGF, KGF), has been shown to promote hepatocyte proliferation in vivo. However, T3 has no effect whatsoever on the proliferation of hepatocytes in vitro. Aim: To investigate the mechanism of triiodothyronine stimulation of hepatocyte proliferation both in vivo and in vitro. Methods: In vivo experiments: rats received injections of LPS, T3 and the combination of the two every 5 days for 3 weeks. Proliferation of hepatocytes was determined by mitotic index and PCNA staining in hematoxilin & eosin paraffin sections. In vitro: The hepatic stellate cell line HT5 was cultured in the presence of T3, IL-6 and T3 and IL-6. Culture supernatants were assessed for presence of IL-6 and HGF, measured by ELISA. Results: In vivo: injections of LPS and T3 resulted in a significant increase in the mitotic index and PCNA staining in the livers of the treated rats. In vitro: After 48h in culture, sups from hepatic stellate cells cultured with T3 and IL-6 showed a five-fold increase in HGF amounts compared to sups from stellate cells cultured with control medium alone. T3 alone had no effect on IL-6 secretion by stellate ceils. Conclusion: T3 may induce hepatocyte proliferation during injury by turning on the expression of HGF in stellate cells.
AASLD ABSTRACTS
281A
435
436
GADOLINIUM CLORIDE, AN INHIBITOR OF KUPFFER CELLS, PREVENTS CIRRHOSIS I N D U C E D BY CCL 4 INTOXICATION IN THE RAT.
DYSREGULATED INTERLEUKIN 18 METABOLISM IN ALCOROLIC LIVER DISEASE A N D NON-ALCOHOLIC STEATOHEPATITIS. Craig J
Pablo Muriel M, Yesenia Escobar F, Cinvestav-fPN, Mexico City Mexico
McClain, University of Louisville, Louisville, KY; Daniel B Hill, University- of Kentucky, Lexington, KY; Luis S Marsano, University of Louisville, Louisville, ICY; Luis R Pena, University of Kentucky, Lexington, KY; Marcelo Kugelmas, University of Colorado, Denver, CO; Shirish Barve, University of Louisville, Louisville, KY
Background/Aims: Hepatic fibrosis is an important consequence of chronic liver disease. The replacement of normal hepatic parenchymal tissue compromises the functional capacity of the liver and disrupts the normal architecture relationships of the organ. An important feature of liver fibrosis is the deposition of connective tissue around the hepatic sinusoids. Tranformation of stellate ceils with deposition of collagen and other extracellular matrix proteins is considered a key component of the pathogenisis of liver fibrosis. Kupfer cells (KCs)activation with the release of fibrogenic mediators is know to precede stellate cell activation. Thus, it is reasonable to postulate that modulation of KCs could disrupt the sequence of events leading to organ injury by damping down the fibrogenic stimulus. In order to elucidate the role of KCs in fiver fibrosis and cirrhosis, rats were treated with GdC13 and cirrhosis was induced by chronic CC14 intoxication. Methods: Carbon tetrachloride was administered three times per week for 8 weeks to male Wistar rats treated simultaneouslywith GdCI3 (20 mg/kg, daily); appropriate controls were performed. Results: Chronic treatment with CCI+induced a 40% of mortality, while treatment with GdC13 (in addition to CC14)reduced the mortality to 6.6% (p< 0.05), indicating that this compound has an important protective effect. Control animals treated with mineral oil or GdC13 alone showed no mortality. Serum enzyme activities of alkaline phosphatase (ALP), 3,-glutamyl transpeptidase (3'-GTP) and alanine aminotrasferase (ALT) and bilirubitls concentration increased significantly by CCI+,while GdCI3prevented completely the increase in T-GTP, and partially ALP, ALT, and bilirubins (p<0.05). Liver glycogen was depleted by CCI4, an effect that GdCl3 was not capable of preventing. Moreover, gadolinium chloride by itselfdepleted it. The degree of lipid peroxidation increased about 2.5-fold by intoxication with CCL, GdC13 preserved lipid peroxidation within normal values. Hepatic collagen increased 3-fold after chronic intoxicationwith CCI+(P<0.05). Gadolinium chloride prevented partially (p<0.05) the increase in collagen as evidenced by the liver hydroxyproline content and by the histophatological analysis. Conclusions: In relation with glycogen depletion by gadolinium chloride, it it has been reported that this compound may affect the hepatocyte plasma membrane, and alterations on plasma membrane activate adenyl cyclase, increasing cAMP and inducing glycogen breakdown. The effect of GdCI3 on KCs are reported to be manifold in nature and more complex than merely a possible change in ceil number, including the induction of apoptosis, the reduction in nitric oxide synthase expression, the decrease of phagocytic activity, probably by interfering with calciumdependent cell surface interactions, the attenuation of endotoxin-induced TNF-a mRNA production and protein release, the impairment of their capacity to generate superoxide, the abolition of expression of certain cell-specific antigens and the switch of phenotypic shape with loss of cytokine expression. TNP-a and IL-10 have been related with inflammation, necrosis and fibrosis of the liver. Thus, inhibition of their expression in Kupffer cells by GdCi3 (reported by others) may explain the beneficial effects observed in this study, mainly in the prevention of fibrosis, and the partial prevention of necrosis, evidenced by ALT serum levels and by the histophatological analysis. Moreover, the reported ability"of GdC13 to impair KCs to generate superoxide is in concordance with the prevention of the increase in lipid peroxidation observed herein, which, in turn is associate with necrosis, cholestasis and fibrosis. The present results clearly indicate that KCs are needed for the production of CCh-cirrosis, since their inactivation with GdC13 prevents the disease.
437 THE EFFECT OF GADOLINIUM CHLORIDE ON KUPFFER CELL FUNCTION IN VITRO. Clair Lee, George Yeoh, John K Olynyk, University of
Western Australia, Fremantle, WA Australia Gadolinium chloride is commonly used to study the role of Kupffer cells in vivo. Whilst some studies suggest that gadolinium may kill Kupffer cells, other studies suggest that it may alter Kupffer cell function. The aim of this study was to determine the effect of gadolinium chloride on Kupffer cell viability, phagocytic capacity and turnout necrosis factor alpha production in vitro. Methods: Kupffer cells were isolated from Sprague-Dawley rats by sequential pronasecollagenase perfusion and centrifugal elutriation. Cells were plated in 12-well plates at a density of i x l 0 6 cells per well. After 24 hours, the culture medium was changed and replaced either with control medium or medium containing gadolinium chloride (0-270 uM). After a further 24 hours, cell numbers and viability were determined by trypan blue staining whilst phagocytic capacity was determined by phagocytosis of latex beads. Pulse dosing with gadolinium chloride (1 mM) was undertaken to determine if calcium channel blockage was likely to be responsible for changes in phagocytosis. To study cytokine production, lipopolysaccharide (10ng/ml) was added to control and gadolinium treated cells for 6 hours and tumour necrosis factor alpha production measured by ELISA in harvested supernatants. Results: Kupffer cell viability was not impaired by addition of gadolinium chloride at concentrations of 0-270 uM. There was a progressive reduction in both phagocytosis and turnout necrosis factor alpha production with increasing concentrations of gadolinium chloride in a dose-responsive manner. Pulse dosing with gadolinium chloride ( l m M ) was associated with a reduction in phagocytic ability. Conclusions: Gadolinm m chloride reduces phagocytosis and turnout necrosis factor alpha production in isolated Kupffer cells in a dose-responsive manner but does not alter viability. This effect may be mediated, at least in part, by effects of gadolinium chloride on calcium channel function.
Dysregulated cytoknie metabolism is increasingly recognized as a cause of liver injury and a cause of several metabolic complications in both alcoholic liver disease and non alcoholic steatohepatitis (NASH). Interleukin 18 is a critical immunomodulatory cytokine that has been shown to play an etiologic role in many forms of experimental liver injury (e.g. hepatotoxicity induced by C. parvum injection). The objectives of this study were to: 1) determine whether plasma IL-18 levels were elevated in patients with alcoholic liver disease and NASH, 2) to determine whether values were consistent over time in patients with stable liver disease, and 3) to determine if values decreased with clinical improvement of acute alcoholic hepatitis. Normal volunteers and three well characterized patient groups with liver disease were studied. The first group included nine patients with stable alcoholic cirrhosis (aI1 patients had stable liver enzymes and serum albumin concentrations for the preceding 3 months). The second group consisted of fourteen patients with stable documented NASH. The last group included ten patients with acute alcoholic hepatitis who were followed for 3 months at regular intervals. Normal volunteers had a mean plasma IL-18 concentration of 2 3 8 + 2 7 pg/ml. Patients with stable cirrhosis (384+49) and NASH (399+35) had significantly elevated plasma levels that were almost identical when patients were re-evaluated two to six weeks later (402+59 and 410 +40 respectively). Patients with alcoholic hepatitis had very elevated levels on admission, and values improved during the clinical course of disease (758+ 99 on admission, 4 7 9 + 3 6 three months later). We conclude that there is dysregutated IL-18 metabolism in both alcoholic liver disease and NASH, and we postulate that interleukin 18 may play a role in the development of these forms of liver injury. Novel methods for inhibiting IL-18 activity or blocking cleavage of the active form IL-18 are under development and may prove to be effective therapeutic intervention in these forms of liver disease.
438 ROLE OF TRIIODOTYRONINE IN HEPATOCYTE PROLIFERATION
DURING LIVER INJURY. Revital Kariv, Anat Enden, Isabel Zvibel, Shlomo BrilI, Zamir Halpern, Ran Oren, Tel Aviv Medical Center, Tel Aviv Israel Background: Triiodothyronine (T3) is considered a primary mitogen for hepatocytes. Priming has been shown to be induced by hepatic injury and is mediated via increased production of TNF-a. TNF-a causes release of IL-6, that primes hepatocytes to respond to hepatic mitogens. T3, by itself or in combination with other hepatic mitogens (HGF, KGF), has been shown to promote hepatocyte proliferation in vivo. However, T3 has no effect whatsoever on the proliferation of hepatocytes in vitro. Aim: To investigate the mechanism of triiodothyronine stimulation of hepatocyte proliferation both in vivo and in vitro. Methods: In vivo experiments: rats received injections of LPS, T3 and the combination of the two every 5 days for 3 weeks. Proliferation of hepatocytes was determined by mitotic index and PCNA staining in hematoxilin & eosin paraffin sections. In vitro: The hepatic stellate cell line HT5 was cultured in the presence of T3, IL-6 and T3 and IL-6. Culture supernatants were assessed for presence of IL-6 and HGF, measured by ELISA. Results: In vivo: injections of LPS and T3 resulted in a significant increase in the mitotic index and PCNA staining in the livers of the treated rats. In vitro: After 48h in culture, sups from hepatic stellate cells cultured with T3 and IL-6 showed a five-fold increase in HGF amounts compared to sups from stellate cells cultured with control medium alone. T3 alone had no effect on IL-6 secretion by stellate ceils. Conclusion: T3 may induce hepatocyte proliferation during injury by turning on the expression of HGF in stellate cells.