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Stimulation and proliferation of primary hepatic stellate cells by cytochrome P450 2E1-derived reactive oxygen species
278A
AASLD ABSTRACTS
HEPATOLOGYOctober 200 ]
423
424
STIMULATION AND PROLIFERATION OF PRIMARY HEPATIC STELLATE CELLS BY CYTOCHROME P450 2El-DERIVED REACTIVE OXYGEN SPECIES. Natalia Nieto, Scott L Friedman, Arthur I Cederbaum, Mount
THE ROLE OF GP130 DURING ACUTE-PHASE-REACTION AND LIVER INJURY. Konrad L Streetz, Torsten Wuestefeld, Medical Sch of Hannover,
Sinai School of Medicine, New York, NY Initiation of hepatic stellate cell activation results from paracrine stimulation due to rapid, disruptive effects of liver injury on the homeostasis of neighboring cells, and early changes in extracellular matrix composition. The alcoholinducible cytochrome P450 2El (CYP2E1), is expressed mainly in hepatocytes, and acts as a potent source of reactive oxygen species in different models of liver injury. To better understand how hepatic stellate cells become activated in the presence of oxidative stress and evaluate the possibility that CYP2E1derived reactive oxygen species can initiate hepatic stellate cells activation, we co-incubated primary steUate cells with HepG2 cells, which do (E47 cells) or do not (C34 cells) express CYP2E1. Morphologic changes such as spreading and generation of cytoplasmic processes, stretching and elongation, redistribution of the lipid droplets, and loss of auto-fluorescence were more apparent in the hepatic stellate ceils co-cuhured with the E47 cells, as was increased expression of a-smooth muscle actin, and both intra- and extracellular H202 and lipid peroxidation products. Intracellnlar and secreted collagen type I protein was increased in the stellate cells/E47 system, an effect prevented by catatase and vitamin E. Stellate cells proliferation as assessed by methyl[3H]Thymidine incorporation into the DNA was 4-fold greater in the stellate cells/ E47 co-culture, and this was abrogated by catalase and vitamin E. These results show that hepatocytes containing CYP2E1 can release diffusable mediators including reactive oxygen, species, which interact with hepatic stellate cells to cause activation, phenotypic changes, collagen production, and proliferation, key events in liver fibrosis.
Hannover Germany; Andrea Graw, Medical Sch of Hannover, Hannover; K Kamino, Medical Sch of Hannover, Hannover Germany; Francois Tronche, DKFZ, Heidelberg Germany; U Betz, Univ Koln, K61n Germany; Werner Mueller, Univ K61n, K61n; Michael Manns, Medical Sch of Hannover, Hannover; Christian Trautwein, Medical Sch of Hannover, Hannover Germany Gp130 functions as the common signal-transducer for several members of the IL-6-famfly. As a result of IL-6 mediated signal transduction the acute-phasereaction (APR) is induced. To further evaluate the contribution of the APR and other gp130 dependent processes in liver injury we generated conditional gp130 knockout mice. Cre-recombinases are thereby controlled by hepatocyte (alb-cre) specific and interferon (mx-cre) sensible promoters. We first demonstrated deletion of gpl30 in early liver development (which appeared normal in alb-cre gp130 mice). To prove the function of our system we stimulated alb-cre gpl30 mice with IL-6 (9/~g). Thereby virtually no STAT3-answer (gelshift) and an almost completely abolished APR (northern-blots) were evident after stimulation. Bacterial infections are a potent inducer of the APR. As a substitute we administered lipopolysaccharides (Lps 100mg) intraperitoneally. Alb-cre gp130 loxP mice developed 7x higher transaminases (alb: 2000 U/1 vs wt: 350 U/l) after 24h. Gelshift-analyses displayed an almost complete absence of STAT3- and a diminished APl-activation. In contrast, no changes in NF-kB complex formation were evident. Northern-blot experiments revealed that the induction of APR-genes was nearly abolished in alb-cre gpl301oxP mice. We next investigated the relevance of gpl30 during CC14 (2ml/g) induced liver injury. Ahhought STAT3-activation and the induction of the APR were totally diminished only a slight increase in transaminase levels was found in alb-cre gpl301oxP mice. Previous observations reported a strong hypersensitivity of IL6 4- mice in various liver injuring models. To exclude that other cells then hepatocytes might be the target of IL6-dependent protecting pathways we also challenged mx-cre gpl301oxP mice. Also in mxcre/gpl301oxP mice no STAT3 answer and no induction of the APR was found. BrdU stainings were performed to investigate hepatocyte proliferation. This revealed a slight reduction in BrdU positive cells after 48h and a slight increase after 72h in alband mx-cre gp 130 loxP mice, which was not significant. Chronically CC14 exposure (2x weekly over 6 weeks) resulted in perivascular fibrotic changes and impaired tissue remodelling selectively in mx-cre gpl301oxP and not albcre/gp 1301oxP mice. This was associated with higher coflagene Ia mRNA expression in mx-cre compared to alb-cre animals. Our results demonstrate that in several models of liver injury gp130 predominantly mediates the APR. During Lps challenge a missing APR causes hypersensitivity. Whereas after acute toxic induced liver injury gpl30 not seems to be of major importance, after chronic liver injury gpl30 in non-hepatocytes prevents liver fibrosis and is necessary for tissue remodelling.
425
426
STIMULATION OF NAIVE CD4 LYMPHOCTI"ES BY M[tC II-EXPRESSING HEPATOCYTES: PRESENCE OF INTERLEUKIN-12 DETERMINES TIlE CHARACTER OF THE RESPONSE. Johannes Herkel, Bettina Jage-
HEPATOPROTECTIVE MECHANISMS OF YAN-GAN-WAN (PRO-LIVER) MEDICINAL HERB. Melissa D Yang, Keck School of Medicine of University of
mann, Stephan Lueth, Edgar Schmitt, Ansgar W Lohse, Johannes GutenbergUniversity Mainz, Mainz Germany Viral hepatitis is often associated with aberrant expression of class II antigens of the major histocompatibifity complex (MHC II) on hepatocytes. The mode of antigen presentation by hepatocytes may be an important factor that determines chronicity of viral hepatitis. To study the functional consequences of hepatocellular MHC II expression, we generated transgenic mice, which overexpress the class II transactivator molecule (CIITA) in their hepatocytes. Expression of the CIITA molecule directly correlates with MHC II expression. We have found that MHC II-expressing hepatocytes were able to process protein antigens and stimulate CD4 T cell lines. We then wished to examine whether CIITA transgenic hepatocytes can also prime naive CD4 T cells. Therefore, 2x105 CD4 T cells from mice that were transgenic for an Ovalbumin-specific T cell receptor (Ova-TCR) were incubated with i04 antigen presenting cells in the presence of the specific Ovalbumin peptide (5/zg/ml). CIITA transgenic hepatocytes, but not wild-type hepatocytes, were able to prime naive Ova-TCR CD4 T cells to secrete Interferon-T(1.4 ng/ml), which was less efficient than priming by professional APC (A20 B cell line: 7.9 ng/ml). In addition, CIITA transgenic hepatocytes induced a TH0/TH2 phenotype by stimulating Interleukin-4 secretion (882 pg/ml) in naive CD4 T ceils. However, addition of lnterleukin-12 to the culture markedly augmented the antigen-presentation capability of CIITA transgenic hepatocytes, inducing a strong TH1 response of a magnitude comparable to professional APC (9.2 ng/ml Interferon-T). These data indicate that hepatocytes, when expressing MHC II molecules, can function as fully apt antigen presenting cells. This finding is important, because it may explain the clinical observations that low Interleukin-12 serum levels seem to correlate with chronicity of viral hepatitis, and that Interleukin-12 therapy can help in the clearance of chronic hepatitis B infection. In summary, hepatocytes can act as functionally apt antigen presenting cells. Since hepatocytes lack classical co-stimulatory molecules, like CD40 and B7, the molecular details of hepatocellular antigen presentation need further clarification. Be that as it may, the immune response to antigen presentation by hepatocytes may be of TH0/TH2-type by default. However, presence of Interleukin-12 can result in a potent inflammatory response to antigen presented by hepatocytes. Thus, hepatocytes may initiate and maintain immunity to hepatic virus; however, lack of Interleukin-12 may determine chronicity of infection.
Southern California, Los Angeles, CA; Dennis Koop, Oregon Health Sciences University, Portland, OR; Qinggao Deng, Sean Wang, Alfonso Cardenas, Zesong Zhang, Shigang Xiong, Hidekazu Tsukamoto, Keck School of Medicine of University of Southern California, Los Angeles, CA Yan-gan-wan (Pro-Liver), a medicinal herbal prescription, contains phenols (fernfic acid, eoumaric acid, angelicin, paeonols), polyphenols (tannin), and flavonoidsand other active ingredients and has been shown to offer remarkable therapeutic effects on liver diseases in Asia for many years. However, the mechanisms by which this herb provides the hepatoprotective effects are largely unknown. [Aimsl The present study was aimed to understand whether and how Yan-gan-wanprevents acute hepatotoxicity and liver fibrosis in animal models and at the cellular levels. Methods: Male C57/B16 mice were orally administered Yah-gnu-wan(0.3rag/g) or placebo for 2 weeks and: 1) their liver CYP (2El, 2A, 3A, 4A) and reductase protein levels and activities were analyzed by Western blot analysis and enzymatic assays;2) their liver RNA examined by Affymetrixmicroarray analysis; 3) given an acute dose of CC14 to assess liver injury 16 hr later; 4) hepatocytes isolated and tested for CC14 hepatotoxicity in culture; and 5) treated with CC14for 8 weeks to assess the herb's effect on liver fibrosis. Male Wistar rats were also given Yan-gan-wan (0.3mg/g) and injected with porcine serum or saline twice a week for 8 weeks to determine its effects on immnne-mediated liver fibrosis with minimal necroinflammatory changes. The effects of the herb on LPS-stimulatedTNFa release was also examined both in vivoand in vitro. Results: The herb treatment reduced plasma ALT levels (1149..249 vs 2171..303 U/L, p=0.03), the area of centrilobular necrosis, and the number of immunostained neutrophiis (36.8--4.4 vs 58.4-+ 5.0 per HPF, p<0.02) at 16 hr after acute CCl4 treatment. Hepatocytesisolated from the herb-treated mice released 50% less ALT into the media at 10, 20, 30 rain after CC14 addition to the culture and underwent less necrosis as determined by Sytox Green (29.7--2.6 vs 44.9.. 2.30%, p<0.001). Protein levels and activities of all CYP isoforms analyzed, were unchanged by the herb treatment, suggesting that the effects were not at the level of toxin metabolism. However, microarray analysis demonstrated a 9.5 fold reduction in both CYP4A10 and 4A14 expression. CYP4Aimmunoblot confirmed this depletion by the herb. The herb treatment ameliorated liver fibrosis induced by chronic CC14 injection (1.43..0.17 vs 2.42..0.14, p<0.001 grading by reticulin stain) or porcine serum (0.83--0.49 vs 2.3..0.61), suggesting direct anti-fibrogenic effects. Plasma TNFo~levels at 2 hr after intravenous LPS administration (50 mg/kg) was redneed by 60% by the herb (6008..2177 vs 14050--849 U/ml, p=0.046). Direct addition of the herbal but not placebo extracts to cuhured rat Kupffer cells dosedependently suppressed LPS-stimulated TNFczrelease. Conclusions: Yan-gan-wan ameliorates acute CC14hepatotoxicity and liver fibrosis induced by hepatotoxic and immunological mechanisms. Its direct heptoprotective effects are not likely due to changes in toxin metabolism but to inhibition of Kupffer cell TNF~ expression. Additionally, suppressed CYP4A10 and 4A14 expression may offer protection against lipid peroxidation since they are now considered as alternative prooxidant cytochromes.
AASLD ABSTRACTS
HEPATOLOGYOctober 200 ]
423
424
STIMULATION AND PROLIFERATION OF PRIMARY HEPATIC STELLATE CELLS BY CYTOCHROME P450 2El-DERIVED REACTIVE OXYGEN SPECIES. Natalia Nieto, Scott L Friedman, Arthur I Cederbaum, Mount
THE ROLE OF GP130 DURING ACUTE-PHASE-REACTION AND LIVER INJURY. Konrad L Streetz, Torsten Wuestefeld, Medical Sch of Hannover,
Sinai School of Medicine, New York, NY Initiation of hepatic stellate cell activation results from paracrine stimulation due to rapid, disruptive effects of liver injury on the homeostasis of neighboring cells, and early changes in extracellular matrix composition. The alcoholinducible cytochrome P450 2El (CYP2E1), is expressed mainly in hepatocytes, and acts as a potent source of reactive oxygen species in different models of liver injury. To better understand how hepatic stellate cells become activated in the presence of oxidative stress and evaluate the possibility that CYP2E1derived reactive oxygen species can initiate hepatic stellate cells activation, we co-incubated primary steUate cells with HepG2 cells, which do (E47 cells) or do not (C34 cells) express CYP2E1. Morphologic changes such as spreading and generation of cytoplasmic processes, stretching and elongation, redistribution of the lipid droplets, and loss of auto-fluorescence were more apparent in the hepatic stellate ceils co-cuhured with the E47 cells, as was increased expression of a-smooth muscle actin, and both intra- and extracellular H202 and lipid peroxidation products. Intracellnlar and secreted collagen type I protein was increased in the stellate cells/E47 system, an effect prevented by catatase and vitamin E. Stellate cells proliferation as assessed by methyl[3H]Thymidine incorporation into the DNA was 4-fold greater in the stellate cells/ E47 co-culture, and this was abrogated by catalase and vitamin E. These results show that hepatocytes containing CYP2E1 can release diffusable mediators including reactive oxygen, species, which interact with hepatic stellate cells to cause activation, phenotypic changes, collagen production, and proliferation, key events in liver fibrosis.
Hannover Germany; Andrea Graw, Medical Sch of Hannover, Hannover; K Kamino, Medical Sch of Hannover, Hannover Germany; Francois Tronche, DKFZ, Heidelberg Germany; U Betz, Univ Koln, K61n Germany; Werner Mueller, Univ K61n, K61n; Michael Manns, Medical Sch of Hannover, Hannover; Christian Trautwein, Medical Sch of Hannover, Hannover Germany Gp130 functions as the common signal-transducer for several members of the IL-6-famfly. As a result of IL-6 mediated signal transduction the acute-phasereaction (APR) is induced. To further evaluate the contribution of the APR and other gp130 dependent processes in liver injury we generated conditional gp130 knockout mice. Cre-recombinases are thereby controlled by hepatocyte (alb-cre) specific and interferon (mx-cre) sensible promoters. We first demonstrated deletion of gpl30 in early liver development (which appeared normal in alb-cre gp130 mice). To prove the function of our system we stimulated alb-cre gpl30 mice with IL-6 (9/~g). Thereby virtually no STAT3-answer (gelshift) and an almost completely abolished APR (northern-blots) were evident after stimulation. Bacterial infections are a potent inducer of the APR. As a substitute we administered lipopolysaccharides (Lps 100mg) intraperitoneally. Alb-cre gp130 loxP mice developed 7x higher transaminases (alb: 2000 U/1 vs wt: 350 U/l) after 24h. Gelshift-analyses displayed an almost complete absence of STAT3- and a diminished APl-activation. In contrast, no changes in NF-kB complex formation were evident. Northern-blot experiments revealed that the induction of APR-genes was nearly abolished in alb-cre gpl301oxP mice. We next investigated the relevance of gpl30 during CC14 (2ml/g) induced liver injury. Ahhought STAT3-activation and the induction of the APR were totally diminished only a slight increase in transaminase levels was found in alb-cre gpl301oxP mice. Previous observations reported a strong hypersensitivity of IL6 4- mice in various liver injuring models. To exclude that other cells then hepatocytes might be the target of IL6-dependent protecting pathways we also challenged mx-cre gpl301oxP mice. Also in mxcre/gpl301oxP mice no STAT3 answer and no induction of the APR was found. BrdU stainings were performed to investigate hepatocyte proliferation. This revealed a slight reduction in BrdU positive cells after 48h and a slight increase after 72h in alband mx-cre gp 130 loxP mice, which was not significant. Chronically CC14 exposure (2x weekly over 6 weeks) resulted in perivascular fibrotic changes and impaired tissue remodelling selectively in mx-cre gpl301oxP and not albcre/gp 1301oxP mice. This was associated with higher coflagene Ia mRNA expression in mx-cre compared to alb-cre animals. Our results demonstrate that in several models of liver injury gp130 predominantly mediates the APR. During Lps challenge a missing APR causes hypersensitivity. Whereas after acute toxic induced liver injury gpl30 not seems to be of major importance, after chronic liver injury gpl30 in non-hepatocytes prevents liver fibrosis and is necessary for tissue remodelling.
425
426
STIMULATION OF NAIVE CD4 LYMPHOCTI"ES BY M[tC II-EXPRESSING HEPATOCYTES: PRESENCE OF INTERLEUKIN-12 DETERMINES TIlE CHARACTER OF THE RESPONSE. Johannes Herkel, Bettina Jage-
HEPATOPROTECTIVE MECHANISMS OF YAN-GAN-WAN (PRO-LIVER) MEDICINAL HERB. Melissa D Yang, Keck School of Medicine of University of
mann, Stephan Lueth, Edgar Schmitt, Ansgar W Lohse, Johannes GutenbergUniversity Mainz, Mainz Germany Viral hepatitis is often associated with aberrant expression of class II antigens of the major histocompatibifity complex (MHC II) on hepatocytes. The mode of antigen presentation by hepatocytes may be an important factor that determines chronicity of viral hepatitis. To study the functional consequences of hepatocellular MHC II expression, we generated transgenic mice, which overexpress the class II transactivator molecule (CIITA) in their hepatocytes. Expression of the CIITA molecule directly correlates with MHC II expression. We have found that MHC II-expressing hepatocytes were able to process protein antigens and stimulate CD4 T cell lines. We then wished to examine whether CIITA transgenic hepatocytes can also prime naive CD4 T cells. Therefore, 2x105 CD4 T cells from mice that were transgenic for an Ovalbumin-specific T cell receptor (Ova-TCR) were incubated with i04 antigen presenting cells in the presence of the specific Ovalbumin peptide (5/zg/ml). CIITA transgenic hepatocytes, but not wild-type hepatocytes, were able to prime naive Ova-TCR CD4 T cells to secrete Interferon-T(1.4 ng/ml), which was less efficient than priming by professional APC (A20 B cell line: 7.9 ng/ml). In addition, CIITA transgenic hepatocytes induced a TH0/TH2 phenotype by stimulating Interleukin-4 secretion (882 pg/ml) in naive CD4 T ceils. However, addition of lnterleukin-12 to the culture markedly augmented the antigen-presentation capability of CIITA transgenic hepatocytes, inducing a strong TH1 response of a magnitude comparable to professional APC (9.2 ng/ml Interferon-T). These data indicate that hepatocytes, when expressing MHC II molecules, can function as fully apt antigen presenting cells. This finding is important, because it may explain the clinical observations that low Interleukin-12 serum levels seem to correlate with chronicity of viral hepatitis, and that Interleukin-12 therapy can help in the clearance of chronic hepatitis B infection. In summary, hepatocytes can act as functionally apt antigen presenting cells. Since hepatocytes lack classical co-stimulatory molecules, like CD40 and B7, the molecular details of hepatocellular antigen presentation need further clarification. Be that as it may, the immune response to antigen presentation by hepatocytes may be of TH0/TH2-type by default. However, presence of Interleukin-12 can result in a potent inflammatory response to antigen presented by hepatocytes. Thus, hepatocytes may initiate and maintain immunity to hepatic virus; however, lack of Interleukin-12 may determine chronicity of infection.
Southern California, Los Angeles, CA; Dennis Koop, Oregon Health Sciences University, Portland, OR; Qinggao Deng, Sean Wang, Alfonso Cardenas, Zesong Zhang, Shigang Xiong, Hidekazu Tsukamoto, Keck School of Medicine of University of Southern California, Los Angeles, CA Yan-gan-wan (Pro-Liver), a medicinal herbal prescription, contains phenols (fernfic acid, eoumaric acid, angelicin, paeonols), polyphenols (tannin), and flavonoidsand other active ingredients and has been shown to offer remarkable therapeutic effects on liver diseases in Asia for many years. However, the mechanisms by which this herb provides the hepatoprotective effects are largely unknown. [Aimsl The present study was aimed to understand whether and how Yan-gan-wanprevents acute hepatotoxicity and liver fibrosis in animal models and at the cellular levels. Methods: Male C57/B16 mice were orally administered Yah-gnu-wan(0.3rag/g) or placebo for 2 weeks and: 1) their liver CYP (2El, 2A, 3A, 4A) and reductase protein levels and activities were analyzed by Western blot analysis and enzymatic assays;2) their liver RNA examined by Affymetrixmicroarray analysis; 3) given an acute dose of CC14 to assess liver injury 16 hr later; 4) hepatocytes isolated and tested for CC14 hepatotoxicity in culture; and 5) treated with CC14for 8 weeks to assess the herb's effect on liver fibrosis. Male Wistar rats were also given Yan-gan-wan (0.3mg/g) and injected with porcine serum or saline twice a week for 8 weeks to determine its effects on immnne-mediated liver fibrosis with minimal necroinflammatory changes. The effects of the herb on LPS-stimulatedTNFa release was also examined both in vivoand in vitro. Results: The herb treatment reduced plasma ALT levels (1149..249 vs 2171..303 U/L, p=0.03), the area of centrilobular necrosis, and the number of immunostained neutrophiis (36.8--4.4 vs 58.4-+ 5.0 per HPF, p<0.02) at 16 hr after acute CCl4 treatment. Hepatocytesisolated from the herb-treated mice released 50% less ALT into the media at 10, 20, 30 rain after CC14 addition to the culture and underwent less necrosis as determined by Sytox Green (29.7--2.6 vs 44.9.. 2.30%, p<0.001). Protein levels and activities of all CYP isoforms analyzed, were unchanged by the herb treatment, suggesting that the effects were not at the level of toxin metabolism. However, microarray analysis demonstrated a 9.5 fold reduction in both CYP4A10 and 4A14 expression. CYP4Aimmunoblot confirmed this depletion by the herb. The herb treatment ameliorated liver fibrosis induced by chronic CC14 injection (1.43..0.17 vs 2.42..0.14, p<0.001 grading by reticulin stain) or porcine serum (0.83--0.49 vs 2.3..0.61), suggesting direct anti-fibrogenic effects. Plasma TNFo~levels at 2 hr after intravenous LPS administration (50 mg/kg) was redneed by 60% by the herb (6008..2177 vs 14050--849 U/ml, p=0.046). Direct addition of the herbal but not placebo extracts to cuhured rat Kupffer cells dosedependently suppressed LPS-stimulated TNFczrelease. Conclusions: Yan-gan-wan ameliorates acute CC14hepatotoxicity and liver fibrosis induced by hepatotoxic and immunological mechanisms. Its direct heptoprotective effects are not likely due to changes in toxin metabolism but to inhibition of Kupffer cell TNF~ expression. Additionally, suppressed CYP4A10 and 4A14 expression may offer protection against lipid peroxidation since they are now considered as alternative prooxidant cytochromes.