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Reversion by interferon of liver fibrosis and damage induced by biliary obstruction in the rat
HEPATOLOGY Vol. 22, No. 4, Pt. 2, 1995
AASLD ABSTRACTS
277A
6 8 ] . INTERFERON-T-MEDIATED ACTIVATION OF STATlcL REGULATES MITOGENESIS OF HEPATIC STELLATE CELLS. Fabio Man'a, Goutam Ghosh Choudhurv, Hanna E. Abboud. Dept. of Medicine, University of Texas HSCSA, San Antonio TX. In human hepatic stellate cells (HSC), and in other human meseachymal cell lines, preincubation with intefferon-~ (1FN-3,) results in potentiation of DNA synthesis in response to subsequent exposure of cells to PDGF or EGF. This effect is independent of PDGF or EGF receptor early signaling or activation of mitogen-activated protein kinase (MAPK) (Hepatology 1994;20:289A). Pathways leading to gene transcription independent of MAPK activation have been recently reported. In these pathways, latent cytosolic transcription factors (known as STATs, for signal transducers and activators of transcription) are activated by tyrosine phosphorylation and translocated to the nucleus, where they bind to DNA regulatory dements. STATlct, a protein belonging to this group, is activated by several cytokines, including PDGF, EGF, and IFN-T. We investigated if STATla activation could mediate the mitogenic interaction between IFN-T and growth factors in HSC. STATla activation was determined by measuring its DNA binding activity using gel-mobility shift analysis. 48-hour incubation with IFN-T or short-term (15 rain) incubation with PDGF caused an increase in STATIa binding to m67, a regulatory element from the c-los promoter. When treatment with IFN-T was followed by PDGF, a synergistic effect on STATla activation was observed. Since m67 may bind different molecules ofthe STAT family, involvement of STATla was confirmed using monoclonal antibodies which caused a "supcrshiff' of the DNA-protein complex. Synergistic activation of STATla was also observed when incubation with IFN-T was followed by EGF, but not thrombin. Likewise, prolonged incubation with other cytokines such as IL-la or TNFct followed by PDGF did not cause synergystic activation of STATIa. Hence, only the combinations of cytokines which result in potentiated mitogenesis also synergize at the level of STATIa activation. Incubation of HSC with IFN-ff also increased the levels of STATla mRNA and protein in a time-dependent manner. This effect was not observed when cells were incubated with either 1L-let or TNFa. To confirm the role of STATIa in mesenchymal cell mitogenesis, HSC were transfected with phospborothioate antisense oligonueleotides targeting STATla mRNA. This procedure was associated with a significant reduction in DNA synthesis following a combination of IFN-T and EGF in comparison to cells transfected with "sense" oligonucleotides. These data indicate that interaction between IFN-y and growth factors at the level of STATler results in increased DNA synthesis, and establish a role for STATler in this important biologic phenomenon.
682 ANTIPROLIFERATIVE EFFECTS OF T-INTERFERON IN CARBON
683 UPREGULATED
684 ~ O N
EXPRESSION O F HUMAN THROMBIN RECEPTOR IN ACUTE AND CHRONIC LIVER DISEASE. F.Marra, R.DeFraneo, C.Grappone*, M.Pinzani, S.Milani*, G.PeUegrini*, L.F.Brass*, G. Laffi, P.Gentilini. Istituto di Medicina Interna and *Gastroenterology Unit, University of Florence, Italy; *Dept. of Medicine, University of Pennsylvania, Philadelphia, PA. Thrombin is a senne protease which is generated during tissue damage in several districts including the liver, and participates in theprocess of tissue repair. T~'ombin is mitogenic for maerophages a n d activates endothelial cells. In addition, incubation of hepatic stellate cells (HSC) with thrombin results in cell contraction, proliferation and increased secretion of monocyte chemotactic protein-l, a chemoattractant for monocytes and T lymphocytes. The action of thrombin is mediated by a specific receptor (TR) activated by a proteolytic mechanism. Aim of this study was to investigate TR expression during liver disease. TR expression was studied by immunohistochemastry using specific monoelonal antibodies directed against the ~racellular domain, and by in situ hybridization with antisense cRNA [ ~S]probes. In normal liver, staining for TR was present in the endothelial lining of the hepatic sinuso~ds. In cirrhosis x;cith chronic active hepatitis, expression of the TR was clearly upregulated in the sinusoidal endothelium, the inflammatory infiltrate, and mesenchymal cells present within the fibrous septa, particularly at the septum/lobule interface. TR expression was also upregulated in liver tissue obtained from patients with fulminant hepatitis, withlaighest expression in mesenchymal cells infiltrating necrotic areas. Upregu]ation o f TR expression was associated with increased levels of TR mRNA, as indicated by in situ hybridization experiments, mRNA expression was detected in the same cells that showed immunostalnin~ for the TIL especially in cells of the inflammatory infiltrate ana proliferating cells in the active cirrhotic septa, which likely represent activated I-ISC. Accordingly, TR expression was also shown in cultureactivated human HSC by lmmunocytochemistry. RNAse protection assay of HSC RNA showed that the message for the TR is upregutatea vy incubation in the presence of serum, thus showing that TR expression may be regulated by soluble mediators. We conclude that expression of TR is upregulated during acute and chronic liver injury. Since thrombin becomes immediately available after tissue injury, regulated TR expression may be involved in tissue remodeling and/or scarring following acute or chronic liver injury.
TETRACHLORIDE (CCI4)-INDUCED CIRRHOSIS. I Elias. G Alpini. S Gubba, S Glaser. W Robertson. H Francis, M Richards. J Phinizv and G LeSage. Dept of Medicine, Scott/White & Texas A&M University COM, Temple TX 76504. Cirrhosis induces proliferation of hepatocytes, cholangiocytes and collagen producing cells which is associated with increased genetic expression of c~-fetoprotein (c~-FP) from hepatocytes, increased secretininduced choleresis from cholangiocytes and increased collagen synthesis. Since T-interferon has been shown in vitro to have antiproliferative effects, we tested the HYPOTHESIS that T-interferon inhibits hepatic proliferation associated with cirrhosis. METHODS: Mice were treated with CCI40.1ml 0f25% CCI4 IP twice weekly and 5% alcohol in drinking water for 10 weeks to induce cirrhosis. Two weeks after finishing CCi4 treatment, T-interferon 10~ units daily IM was administered for 12 weeks. In total liver, we measured the effects ofT-interferon on: (0 liver collagen content (hydroxyproline), and morphometric determination of collagen deposition; (i0 genetic expression for collagen type III, H 3 histone (index of DNA synthesis), a-FP and secretin receptor (SR, an index of cholangiocyte proliferation); and (ii0 secretin-induced choleresis and biliary bicarbonate concentrations. RESULTS: Compared to controls, there was a marked increase in hydroxyproline content (~2.5-fold) in the liver of CCI4 treated mice, with histologic evidence for cirrhosis. In CCI4 treated mice, administration of T-interferon decreased liver hydroxyproline content (.~l.4-fold). Collagen type 1II, H 3 histone, c~-FP and SR mRNA levels were higher in cirrhotic mice compared to normals, but in each case this increase was partially inhibited by T- interferon. Secretin-induced choleresis and elevated biliary bicarbonate levels were observed in the cirrhotic mice (+1.1+_0.2 Id/min/g liver and +10.1+_1.9 mEq/L, respectively) but not in normal mice or cirrhotic mice who received T-interferon. SUMMARY/CONCLUSIONS: 1) We established a novel marine model for cirrhosis; 2) the increased genetic expression for collagen type 1II, H3 histone, c¢-FP and SR in cirrhotic mice is inhibited by T-interferon; 3) The secretin-induced, bicarbonate rich, choleresis in cirrhotic mice was ablated by T- interferon. Consistent with our hypothesis, T-interferon inhibits proliferation of hepatocytes and cholangiocytes as well as collagen deposition in murine cirrhosis.
BY INTMBPIRONOFLIV]BZFIBROM(SANDpaMaq EK]DUCMDBY BIT.lADyOBSTftUCTIONIN THUGRAT. P Muriel, MP Gonzile~ Departamento de Farmacologia y Toxlcologin, Centro de lnvestigaci6n y de Estudios Avanzados del I.P.N., M6xico, D.F.,M6xico. Fibrosis is a dynamic process associated with the continuous deposition and resorption of connective tissue, mainly collagen. Therapeutic strategies are emerging by which this dynamic process can be modulated. Since interferons are known to inhibit collagen production, the aim of this study was to investigate if the administration of interferon-~ (IFN-,~)can restore the normal hepatic content of collagen in rats with established fibrosis. Fibrosis was induced by prolonged bile duct ligation. IFN-a (100,000 IU/rat/day; sc) was adminintered to flhrotic rats for 15 days. Bile duct iigation increased liver collagen content 6fold. In addition, serum and liver markers of hepatic injury increased significantly; liver histology showed an increase in collagen deposition, and the normal architecture was lost whit large zones of necrosis being observed frequently. IFN-~ administration reversed to normal the values of all the biochemical markersmeasured and restored the normal architecture of the liver. ~ ~ ~ 0 u r ~ results demonstrate 6 / ~-~ that IFN-,~ is useful in ,/ ~-.. reversing fibrosis and liver i~ " r ~--. damage induced by billary ~" J ""~ohstruction in the rat. /' o~-~*~:~ ' ~ "~ H o w e v e r , further investigations are required . . . . . . . . . . . . . . . . . . . . . . .IFN-CC ....... //J to evaluate the therapeutic t~,ot~* c -~t , , relevance of tuterferons on ~Days after ~2'0 2~ ~o non-viral fibrosis and t i o r y obstructi~l cholestasls.
AASLD ABSTRACTS
277A
6 8 ] . INTERFERON-T-MEDIATED ACTIVATION OF STATlcL REGULATES MITOGENESIS OF HEPATIC STELLATE CELLS. Fabio Man'a, Goutam Ghosh Choudhurv, Hanna E. Abboud. Dept. of Medicine, University of Texas HSCSA, San Antonio TX. In human hepatic stellate cells (HSC), and in other human meseachymal cell lines, preincubation with intefferon-~ (1FN-3,) results in potentiation of DNA synthesis in response to subsequent exposure of cells to PDGF or EGF. This effect is independent of PDGF or EGF receptor early signaling or activation of mitogen-activated protein kinase (MAPK) (Hepatology 1994;20:289A). Pathways leading to gene transcription independent of MAPK activation have been recently reported. In these pathways, latent cytosolic transcription factors (known as STATs, for signal transducers and activators of transcription) are activated by tyrosine phosphorylation and translocated to the nucleus, where they bind to DNA regulatory dements. STATlct, a protein belonging to this group, is activated by several cytokines, including PDGF, EGF, and IFN-T. We investigated if STATla activation could mediate the mitogenic interaction between IFN-T and growth factors in HSC. STATla activation was determined by measuring its DNA binding activity using gel-mobility shift analysis. 48-hour incubation with IFN-T or short-term (15 rain) incubation with PDGF caused an increase in STATIa binding to m67, a regulatory element from the c-los promoter. When treatment with IFN-T was followed by PDGF, a synergistic effect on STATla activation was observed. Since m67 may bind different molecules ofthe STAT family, involvement of STATla was confirmed using monoclonal antibodies which caused a "supcrshiff' of the DNA-protein complex. Synergistic activation of STATla was also observed when incubation with IFN-T was followed by EGF, but not thrombin. Likewise, prolonged incubation with other cytokines such as IL-la or TNFct followed by PDGF did not cause synergystic activation of STATIa. Hence, only the combinations of cytokines which result in potentiated mitogenesis also synergize at the level of STATIa activation. Incubation of HSC with IFN-ff also increased the levels of STATla mRNA and protein in a time-dependent manner. This effect was not observed when cells were incubated with either 1L-let or TNFa. To confirm the role of STATIa in mesenchymal cell mitogenesis, HSC were transfected with phospborothioate antisense oligonueleotides targeting STATla mRNA. This procedure was associated with a significant reduction in DNA synthesis following a combination of IFN-T and EGF in comparison to cells transfected with "sense" oligonucleotides. These data indicate that interaction between IFN-y and growth factors at the level of STATler results in increased DNA synthesis, and establish a role for STATler in this important biologic phenomenon.
682 ANTIPROLIFERATIVE EFFECTS OF T-INTERFERON IN CARBON
683 UPREGULATED
684 ~ O N
EXPRESSION O F HUMAN THROMBIN RECEPTOR IN ACUTE AND CHRONIC LIVER DISEASE. F.Marra, R.DeFraneo, C.Grappone*, M.Pinzani, S.Milani*, G.PeUegrini*, L.F.Brass*, G. Laffi, P.Gentilini. Istituto di Medicina Interna and *Gastroenterology Unit, University of Florence, Italy; *Dept. of Medicine, University of Pennsylvania, Philadelphia, PA. Thrombin is a senne protease which is generated during tissue damage in several districts including the liver, and participates in theprocess of tissue repair. T~'ombin is mitogenic for maerophages a n d activates endothelial cells. In addition, incubation of hepatic stellate cells (HSC) with thrombin results in cell contraction, proliferation and increased secretion of monocyte chemotactic protein-l, a chemoattractant for monocytes and T lymphocytes. The action of thrombin is mediated by a specific receptor (TR) activated by a proteolytic mechanism. Aim of this study was to investigate TR expression during liver disease. TR expression was studied by immunohistochemastry using specific monoelonal antibodies directed against the ~racellular domain, and by in situ hybridization with antisense cRNA [ ~S]probes. In normal liver, staining for TR was present in the endothelial lining of the hepatic sinuso~ds. In cirrhosis x;cith chronic active hepatitis, expression of the TR was clearly upregulated in the sinusoidal endothelium, the inflammatory infiltrate, and mesenchymal cells present within the fibrous septa, particularly at the septum/lobule interface. TR expression was also upregulated in liver tissue obtained from patients with fulminant hepatitis, withlaighest expression in mesenchymal cells infiltrating necrotic areas. Upregu]ation o f TR expression was associated with increased levels of TR mRNA, as indicated by in situ hybridization experiments, mRNA expression was detected in the same cells that showed immunostalnin~ for the TIL especially in cells of the inflammatory infiltrate ana proliferating cells in the active cirrhotic septa, which likely represent activated I-ISC. Accordingly, TR expression was also shown in cultureactivated human HSC by lmmunocytochemistry. RNAse protection assay of HSC RNA showed that the message for the TR is upregutatea vy incubation in the presence of serum, thus showing that TR expression may be regulated by soluble mediators. We conclude that expression of TR is upregulated during acute and chronic liver injury. Since thrombin becomes immediately available after tissue injury, regulated TR expression may be involved in tissue remodeling and/or scarring following acute or chronic liver injury.
TETRACHLORIDE (CCI4)-INDUCED CIRRHOSIS. I Elias. G Alpini. S Gubba, S Glaser. W Robertson. H Francis, M Richards. J Phinizv and G LeSage. Dept of Medicine, Scott/White & Texas A&M University COM, Temple TX 76504. Cirrhosis induces proliferation of hepatocytes, cholangiocytes and collagen producing cells which is associated with increased genetic expression of c~-fetoprotein (c~-FP) from hepatocytes, increased secretininduced choleresis from cholangiocytes and increased collagen synthesis. Since T-interferon has been shown in vitro to have antiproliferative effects, we tested the HYPOTHESIS that T-interferon inhibits hepatic proliferation associated with cirrhosis. METHODS: Mice were treated with CCI40.1ml 0f25% CCI4 IP twice weekly and 5% alcohol in drinking water for 10 weeks to induce cirrhosis. Two weeks after finishing CCi4 treatment, T-interferon 10~ units daily IM was administered for 12 weeks. In total liver, we measured the effects ofT-interferon on: (0 liver collagen content (hydroxyproline), and morphometric determination of collagen deposition; (i0 genetic expression for collagen type III, H 3 histone (index of DNA synthesis), a-FP and secretin receptor (SR, an index of cholangiocyte proliferation); and (ii0 secretin-induced choleresis and biliary bicarbonate concentrations. RESULTS: Compared to controls, there was a marked increase in hydroxyproline content (~2.5-fold) in the liver of CCI4 treated mice, with histologic evidence for cirrhosis. In CCI4 treated mice, administration of T-interferon decreased liver hydroxyproline content (.~l.4-fold). Collagen type 1II, H 3 histone, c~-FP and SR mRNA levels were higher in cirrhotic mice compared to normals, but in each case this increase was partially inhibited by T- interferon. Secretin-induced choleresis and elevated biliary bicarbonate levels were observed in the cirrhotic mice (+1.1+_0.2 Id/min/g liver and +10.1+_1.9 mEq/L, respectively) but not in normal mice or cirrhotic mice who received T-interferon. SUMMARY/CONCLUSIONS: 1) We established a novel marine model for cirrhosis; 2) the increased genetic expression for collagen type 1II, H3 histone, c¢-FP and SR in cirrhotic mice is inhibited by T-interferon; 3) The secretin-induced, bicarbonate rich, choleresis in cirrhotic mice was ablated by T- interferon. Consistent with our hypothesis, T-interferon inhibits proliferation of hepatocytes and cholangiocytes as well as collagen deposition in murine cirrhosis.
BY INTMBPIRONOFLIV]BZFIBROM(SANDpaMaq EK]DUCMDBY BIT.lADyOBSTftUCTIONIN THUGRAT. P Muriel, MP Gonzile~ Departamento de Farmacologia y Toxlcologin, Centro de lnvestigaci6n y de Estudios Avanzados del I.P.N., M6xico, D.F.,M6xico. Fibrosis is a dynamic process associated with the continuous deposition and resorption of connective tissue, mainly collagen. Therapeutic strategies are emerging by which this dynamic process can be modulated. Since interferons are known to inhibit collagen production, the aim of this study was to investigate if the administration of interferon-~ (IFN-,~)can restore the normal hepatic content of collagen in rats with established fibrosis. Fibrosis was induced by prolonged bile duct ligation. IFN-a (100,000 IU/rat/day; sc) was adminintered to flhrotic rats for 15 days. Bile duct iigation increased liver collagen content 6fold. In addition, serum and liver markers of hepatic injury increased significantly; liver histology showed an increase in collagen deposition, and the normal architecture was lost whit large zones of necrosis being observed frequently. IFN-~ administration reversed to normal the values of all the biochemical markersmeasured and restored the normal architecture of the liver. ~ ~ ~ 0 u r ~ results demonstrate 6 / ~-~ that IFN-,~ is useful in ,/ ~-.. reversing fibrosis and liver i~ " r ~--. damage induced by billary ~" J ""~ohstruction in the rat. /' o~-~*~:~ ' ~ "~ H o w e v e r , further investigations are required . . . . . . . . . . . . . . . . . . . . . . .IFN-CC ....... //J to evaluate the therapeutic t~,ot~* c -~t , , relevance of tuterferons on ~Days after ~2'0 2~ ~o non-viral fibrosis and t i o r y obstructi~l cholestasls.