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Release of soluble intercellular adhesion molecule-1 into bile and serum in murine endotoxin shock
362A
AASLD ABSTRACTS
HEPATOLOGY O c t o b e r 1995
1021 RELEASE OF SOLUBLE INTERCELLULAR ADHESION
1022 LIVER ENDOTHELIAL CELL DYSFUNCTION AND INCREASED
MOLECULE-1 INTO BILE AND SERUM IN MURINE ENDOTOXIN SHOCK. H Jaeschke, NA Essani, MA Fisher, SL Vanderfecht, and CW Smith'. Drug Metabolism Research, and Drug Development Toxicology I, The Upjohn Company, Kalamazoo, MI; "Spores P. Martel Laboratory of Leukocyte Biology, Department of Pediatrics, Baylor College of Medicine, Houston, TX.
ADHESION MOLECULE EXPRESSION IN ACUTE ALCOHOLIC HEPATITIS. DB H,~j1,S Barry, B Hennig. and CI McClain. Departments of internal Medicine and Nutrition Sciences. University of Kentucky Medical Center and Lexington VAMC, Lexington, Kentucky.
Neutrophil-induced liver injury during endotoxemia is dependent on the adhesion molecules Mas-1 (CD11b/GD18) on neutrophils (Am J Physiol 261: G1051,1991) and its eounterreceptor on endothelial cells and hepatocytes, intercellular adhesion molecule1 (ICAM-1) (Hepatology 21: 1632, 1995). To investigate a potential release of a soluble form of ICAM-1 (sICAM-1), animals received 100 ttg/kg Salmonella abortus equi endotoxin (ET) with or without cotreatment with 700 mg/kg galaetosamine (Gal). In ET-sansitive mice (C3Heb/FeJ), ET did not cause liver injury hut induced a timedependent 300% increase of serum sICAM-1 (baseline: 8.5±1.0 Bg/ml) and a 615% increase in bile (baseline: 3.6+0.4 ng/min/g liver) without affecting bile flow. In Gal/ET-treated animals, which developed liver injury, the increase in both compartments was only 97% and 104%. In either case, the increase in sICAM-1 concentrations went parallel to the enhanced ICAM-1 protein expression in the liver. An endotoxin-resistant strain (C3H/HeJ) did not show elevated sICAM-1 levels in serum or bile; in contrast, the intravenous injection of murine tumor necrosis factor-alpha (TNF-a), interleukin-1 alpha (ILla) or IL-113(13 to 23 ~tg/kg) into the same strain induced a 225-354% increase in serum sICAM-1 and a 370% elevation of the biliary efllux of sICAM-1, again independent of changes in bile flow. These data indicate that cytokines are major inducers of sICAM-1 formation in vivo. Furthermore, our findings are consistent with the hypothesis that sICAM-1 is able to prevent or at least attenuate an inflammatory liver injury by either directly blocking integrin receptors on neutrophils and/or indirectly by reducing ICAM-1 molecules on target cells.
1023
MECHANISM OF THE ADHESION AND MIGRATION OF NEUTROPHILS ON SINUSOIDAL ENDOTHELIAL CELLS IN FLOW SYSTEM S SAKAMOTO, T OKANOUE, Y,YOSHITO, K,NISHIOJI, AND K KASHIMA. THIRD DEPT. of Int. Med., Kyoto Pref. Univ. of Med., Kyoto, Japan Neutrophils are considered to play a pivotal role in the pathogenesis of alcoholic hepatitis, lmmunohistchemical study demonstrated the increased expression of ICAM-1 on sinusoidal endothelial cells in inflamed lesions, however, the interaction between neutrophils and sinusoidal endothelial cells has been little investigated in vitro system. The aim of this study is to assess the role of ICAM-1 on the adherence and migration of neutrophils on cultured sinusoidal endothelial cells. In addilJon, we investigated the effects of dexamethasone (DEX) on the interaction between neutrophils and sinusoidal endothelial cells. METHODS ;Sinusoidal endothelial cells (SEC) obtained from male Wistar rats were cultured on coverslips and were stimulated with IOOU of TNF-a for 4hours. Rat neutrophills were passed on SEC through the in vitro flow chamber at a wall shear stress of 0.25 dynes/cm 2 and 37°C. The number of adherent and migrated neutrophils was observed with phase contrast microscopy. To investigate the role of ICAM-1 on neutrophil adhesion, we observed neurtophil adhesion on SEC preincubated with anti-ICAM-1 or DEX after TNFstimulation. In addition we measured ICAM-1 expression on SEC treated with TNF-a and DEX. RESULTS ; Treatment of SEC with TNF-a increased neutrophil adhesion on SEC and preincubation with antMCAM decreased not only adhesion but also migration of neutrophils on SEC. TNF-a induced overexpression of ICAM-1 on SEC in dose dependent manner. D E X suppressed ICAM-1 expression on SEC and inhibited neutrophil adhesion and migration. CONCLUSION ; Overexpression of ICAM-1 on SEC induced by TNF-sflmulation increased neutrophil adhesion and migration and pretreatment with anti-ICAM and DEX inhibited these reaction. ICAM-1 plays important role in the interaction between neutrophils and SEC.
Neutrophil (PMN) infiltration is a principle manifestation of alcoholic hepatitis (AH). However, its pathogenesis is not well defined. Ethanol metabolism produces reactive oxygen intermediates, and chronic alcohol abuse is associated with decreased levels of free radical scavengers (ie giutathione). NFKBis an oxidative stress sensitive transcription factor for TNF, IL-6, IL-8 and adhesion molecules. TNF activity is increased in AH. TNF alters endothelial tell function in vitro and induces expression of adhesion molecules (ICAM) for PMN's and IL-8 (PMN activafing/chemotactic factor). IL-6 reflects the degree of TNF activity and severity of inflammation in AH. Hyaluronic acid (HA) is a noninvasive marker of liver endothelial cell function (LEC). To evaluate cytokine activity, LEC function and adhesion molecule expression we measured serial plasma IL-6, HA, S-ICAM and IL-8 levels in 20 patients with moderate to severe acute AH. NFKB activity was determined in monocytes from AH patients and in Hep G2 cells cultured in ethanol. MONTH _.0._ 1 ~ 6 normal IL-6 57+11 34_+8 21+11 ]5+6 < 2 u/ml Bflirubin ]6.8+2.6 7.5+2.0 2.1+0.4 2.9+0.5 0.2-1.0mg/dl HA 581+39 610_+105 262_+64 316+67 43+4 S-ICAM 488+70 325+65 249+15 183+27 230+37 IL-8 493+149 200+61 189+86 87+81 10+5 p g / m l Plasma IL-6, bilirubin, and HA levels were initially very elevated indicating increased cytokine (TNF) activity and inflammation with severe hepatocellular and LEC dysfunction. S-ICAM and IL-8 levels were initially quite elevated but decreased as the patients improved clinically. NFKB activity in monocytes from patients with AH was increased. There was increased NFKB activity in Hep G2 cells cultured in ethanol. We speculate that the increased NF~B activity associated with AH results in increased expression of cytokines such as TNF, IL-6, IL-8 and adhesion molecules which play etiologic roles in LEC dysfunction, PMN adhesion with hepatic PMN infiltration and ultimately PMN-induced liver damage.
1024 RESISTANCE TO MURINE HEPATITIS VIRUS STRAIN 3 IS DEPENDENT ON PRODUCTION OF NITRIC OXIDE. M. Pone. P. Marsden. S. Sloan. L.S. Fun~. G.A. Lew. Multi Organ Transplant Program, The Toronto Hospital - University &Toronto, Toronto, ON. Murine hepatitis virus strain 3 is known to induce a strain-specific spectrum of liver disease in inbred strains of mice that is dependent upon inflammatory mediators released by macrophages. Production of nitric oxide (NO) by macrophages has been implicated in resistance to several viral pathogens, including ectromelia, vancinia and herpes simples Type 1. The present study was undertaken to define the role of NO which in mufine hepatitis virus strain 3 (MHV-3) infection. Interferon-y-induced production of NO inhibited the growth of MHV-3 in a murine macrophage cell line (RAW 264.7). The NO donor S-nitroso-N-anetylpenicillamine (SNAP) also inhibited MHV-3 replication in RAW 264.7 cells and peritoneal macrophages from both resistant and susceptible mice. Electron microscopic studies confirmed that SNAP is a potent inhibitor of viral particle formation and that it can reproduce the antiviral action iflFN-y. Macrophages isolated from resistant A/J mice produced higher levels of mRNA transcripts ofiNOS and a five-fold higher level of NO in response to interferon-y than macrophages from susceptible Balb/cJ mice. Furthermore, only macrophages from resistant A/J mice were able to restrict MHV-3 replication in response to IFN- V. In-vivo inhibition of NO production by N-monomethyl-L-arginine ( L - N M M ) resulted in loss of resistance to MHV-3 in A/J mice. These results collectively demonstrate both a strain dependent difference in production of NO to interferon-V and MHV-3 and the importance of endogenous NO in resistance to MHV-3 infection. Supported by a Program Project Grant from the Medical Research Council of Canada (MRC).
AASLD ABSTRACTS
HEPATOLOGY O c t o b e r 1995
1021 RELEASE OF SOLUBLE INTERCELLULAR ADHESION
1022 LIVER ENDOTHELIAL CELL DYSFUNCTION AND INCREASED
MOLECULE-1 INTO BILE AND SERUM IN MURINE ENDOTOXIN SHOCK. H Jaeschke, NA Essani, MA Fisher, SL Vanderfecht, and CW Smith'. Drug Metabolism Research, and Drug Development Toxicology I, The Upjohn Company, Kalamazoo, MI; "Spores P. Martel Laboratory of Leukocyte Biology, Department of Pediatrics, Baylor College of Medicine, Houston, TX.
ADHESION MOLECULE EXPRESSION IN ACUTE ALCOHOLIC HEPATITIS. DB H,~j1,S Barry, B Hennig. and CI McClain. Departments of internal Medicine and Nutrition Sciences. University of Kentucky Medical Center and Lexington VAMC, Lexington, Kentucky.
Neutrophil-induced liver injury during endotoxemia is dependent on the adhesion molecules Mas-1 (CD11b/GD18) on neutrophils (Am J Physiol 261: G1051,1991) and its eounterreceptor on endothelial cells and hepatocytes, intercellular adhesion molecule1 (ICAM-1) (Hepatology 21: 1632, 1995). To investigate a potential release of a soluble form of ICAM-1 (sICAM-1), animals received 100 ttg/kg Salmonella abortus equi endotoxin (ET) with or without cotreatment with 700 mg/kg galaetosamine (Gal). In ET-sansitive mice (C3Heb/FeJ), ET did not cause liver injury hut induced a timedependent 300% increase of serum sICAM-1 (baseline: 8.5±1.0 Bg/ml) and a 615% increase in bile (baseline: 3.6+0.4 ng/min/g liver) without affecting bile flow. In Gal/ET-treated animals, which developed liver injury, the increase in both compartments was only 97% and 104%. In either case, the increase in sICAM-1 concentrations went parallel to the enhanced ICAM-1 protein expression in the liver. An endotoxin-resistant strain (C3H/HeJ) did not show elevated sICAM-1 levels in serum or bile; in contrast, the intravenous injection of murine tumor necrosis factor-alpha (TNF-a), interleukin-1 alpha (ILla) or IL-113(13 to 23 ~tg/kg) into the same strain induced a 225-354% increase in serum sICAM-1 and a 370% elevation of the biliary efllux of sICAM-1, again independent of changes in bile flow. These data indicate that cytokines are major inducers of sICAM-1 formation in vivo. Furthermore, our findings are consistent with the hypothesis that sICAM-1 is able to prevent or at least attenuate an inflammatory liver injury by either directly blocking integrin receptors on neutrophils and/or indirectly by reducing ICAM-1 molecules on target cells.
1023
MECHANISM OF THE ADHESION AND MIGRATION OF NEUTROPHILS ON SINUSOIDAL ENDOTHELIAL CELLS IN FLOW SYSTEM S SAKAMOTO, T OKANOUE, Y,YOSHITO, K,NISHIOJI, AND K KASHIMA. THIRD DEPT. of Int. Med., Kyoto Pref. Univ. of Med., Kyoto, Japan Neutrophils are considered to play a pivotal role in the pathogenesis of alcoholic hepatitis, lmmunohistchemical study demonstrated the increased expression of ICAM-1 on sinusoidal endothelial cells in inflamed lesions, however, the interaction between neutrophils and sinusoidal endothelial cells has been little investigated in vitro system. The aim of this study is to assess the role of ICAM-1 on the adherence and migration of neutrophils on cultured sinusoidal endothelial cells. In addilJon, we investigated the effects of dexamethasone (DEX) on the interaction between neutrophils and sinusoidal endothelial cells. METHODS ;Sinusoidal endothelial cells (SEC) obtained from male Wistar rats were cultured on coverslips and were stimulated with IOOU of TNF-a for 4hours. Rat neutrophills were passed on SEC through the in vitro flow chamber at a wall shear stress of 0.25 dynes/cm 2 and 37°C. The number of adherent and migrated neutrophils was observed with phase contrast microscopy. To investigate the role of ICAM-1 on neutrophil adhesion, we observed neurtophil adhesion on SEC preincubated with anti-ICAM-1 or DEX after TNFstimulation. In addition we measured ICAM-1 expression on SEC treated with TNF-a and DEX. RESULTS ; Treatment of SEC with TNF-a increased neutrophil adhesion on SEC and preincubation with antMCAM decreased not only adhesion but also migration of neutrophils on SEC. TNF-a induced overexpression of ICAM-1 on SEC in dose dependent manner. D E X suppressed ICAM-1 expression on SEC and inhibited neutrophil adhesion and migration. CONCLUSION ; Overexpression of ICAM-1 on SEC induced by TNF-sflmulation increased neutrophil adhesion and migration and pretreatment with anti-ICAM and DEX inhibited these reaction. ICAM-1 plays important role in the interaction between neutrophils and SEC.
Neutrophil (PMN) infiltration is a principle manifestation of alcoholic hepatitis (AH). However, its pathogenesis is not well defined. Ethanol metabolism produces reactive oxygen intermediates, and chronic alcohol abuse is associated with decreased levels of free radical scavengers (ie giutathione). NFKBis an oxidative stress sensitive transcription factor for TNF, IL-6, IL-8 and adhesion molecules. TNF activity is increased in AH. TNF alters endothelial tell function in vitro and induces expression of adhesion molecules (ICAM) for PMN's and IL-8 (PMN activafing/chemotactic factor). IL-6 reflects the degree of TNF activity and severity of inflammation in AH. Hyaluronic acid (HA) is a noninvasive marker of liver endothelial cell function (LEC). To evaluate cytokine activity, LEC function and adhesion molecule expression we measured serial plasma IL-6, HA, S-ICAM and IL-8 levels in 20 patients with moderate to severe acute AH. NFKB activity was determined in monocytes from AH patients and in Hep G2 cells cultured in ethanol. MONTH _.0._ 1 ~ 6 normal IL-6 57+11 34_+8 21+11 ]5+6 < 2 u/ml Bflirubin ]6.8+2.6 7.5+2.0 2.1+0.4 2.9+0.5 0.2-1.0mg/dl HA 581+39 610_+105 262_+64 316+67 43+4 S-ICAM 488+70 325+65 249+15 183+27 230+37 IL-8 493+149 200+61 189+86 87+81 10+5 p g / m l Plasma IL-6, bilirubin, and HA levels were initially very elevated indicating increased cytokine (TNF) activity and inflammation with severe hepatocellular and LEC dysfunction. S-ICAM and IL-8 levels were initially quite elevated but decreased as the patients improved clinically. NFKB activity in monocytes from patients with AH was increased. There was increased NFKB activity in Hep G2 cells cultured in ethanol. We speculate that the increased NF~B activity associated with AH results in increased expression of cytokines such as TNF, IL-6, IL-8 and adhesion molecules which play etiologic roles in LEC dysfunction, PMN adhesion with hepatic PMN infiltration and ultimately PMN-induced liver damage.
1024 RESISTANCE TO MURINE HEPATITIS VIRUS STRAIN 3 IS DEPENDENT ON PRODUCTION OF NITRIC OXIDE. M. Pone. P. Marsden. S. Sloan. L.S. Fun~. G.A. Lew. Multi Organ Transplant Program, The Toronto Hospital - University &Toronto, Toronto, ON. Murine hepatitis virus strain 3 is known to induce a strain-specific spectrum of liver disease in inbred strains of mice that is dependent upon inflammatory mediators released by macrophages. Production of nitric oxide (NO) by macrophages has been implicated in resistance to several viral pathogens, including ectromelia, vancinia and herpes simples Type 1. The present study was undertaken to define the role of NO which in mufine hepatitis virus strain 3 (MHV-3) infection. Interferon-y-induced production of NO inhibited the growth of MHV-3 in a murine macrophage cell line (RAW 264.7). The NO donor S-nitroso-N-anetylpenicillamine (SNAP) also inhibited MHV-3 replication in RAW 264.7 cells and peritoneal macrophages from both resistant and susceptible mice. Electron microscopic studies confirmed that SNAP is a potent inhibitor of viral particle formation and that it can reproduce the antiviral action iflFN-y. Macrophages isolated from resistant A/J mice produced higher levels of mRNA transcripts ofiNOS and a five-fold higher level of NO in response to interferon-y than macrophages from susceptible Balb/cJ mice. Furthermore, only macrophages from resistant A/J mice were able to restrict MHV-3 replication in response to IFN- V. In-vivo inhibition of NO production by N-monomethyl-L-arginine ( L - N M M ) resulted in loss of resistance to MHV-3 in A/J mice. These results collectively demonstrate both a strain dependent difference in production of NO to interferon-V and MHV-3 and the importance of endogenous NO in resistance to MHV-3 infection. Supported by a Program Project Grant from the Medical Research Council of Canada (MRC).