G Szabo1, D Catalano, G Bellerose, P Mandrekar. 1. Liver Center, Division of Gastroenterology, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA. gyongyi.szabo@umassmed.edu
Abstract
BACKGROUND: The mechanisms for decreased therapeutic response to IFNalpha in chronic hepatitis C patients with alcohol are unknown. We investigated the hypothesis that IFNalpha and alcohol regulate cells both in the liver parenchyma and the immune system. METHODS: We used the hepatocellular carcinoma cells (HepG2) to determine if IFNalpha (500-10,000 U/ml) or ethanol (25-100 mM) modulates NF-kB activation alone or in combination with TNFalpha (0.1-20 microg/ml) as determined in electromobility gel shift assays. IkB levels were evaluated in the cytoplasmic extracts by western blot. Monocytes from normal donors were activated with LPS (1 microg/ml) in combination with IFNalpha or ethanol overnight and TNFalpha, IL-6, and IL-12 were measured in the supernatants. RESULTS: In HepG2 cells, both IFNalpha and acute alcohol treatment induced NF-kappaB activation and augmented TNFalpha-induced NF-kappaB binding. Pretreatment of HepG2 cells with IFNalpha resulted in the highest levels of NF-kappaB activation in response to TNFalpha or TNFalpha plus ethanol stimulation. Supershift experiments confirmed that the NF-kappaB dimer induced by TNFalpha and its combination with IFNalpha or ethanol contains RelA (p65) and involves rapid degradation of IkappaBalpha. Experiments using the proteasome inhibitor, MG132, revealed that augmentation of NF-kappaB by ethanol and IFNalpha is mediated via the proteasome pathway. We show that in normal monocytes, IFNalpha augments LPS-induced production of the inflammatory cytokines TNFalpha, IL-6, and IL-12 (p < 0.06) without further modulation by acute alcohol treatment. CONCLUSIONS: These results suggest that IFNalpha can increase HepG2 cell sensitivity to TNFalpha and ethanol-mediated activation. Augmentation of monocyte inflammatory cytokines, particularly of IL-12 production, by IFNalpha could be a key element of the antiviral response in chronic HCV. These results support the hypothesis that the therapeutic benefits of IFNalpha likely involve activation of both immune and parenchymal cells in the liver.
BACKGROUND: The mechanisms for decreased therapeutic response to IFNalpha in chronic hepatitis Cpatients with alcohol are unknown. We investigated the hypothesis that IFNalpha and alcohol regulate cells both in the liver parenchyma and the immune system. METHODS: We used the hepatocellular carcinoma cells (HepG2) to determine if IFNalpha (500-10,000 U/ml) or ethanol (25-100 mM) modulates NF-kB activation alone or in combination with TNFalpha (0.1-20 microg/ml) as determined in electromobility gel shift assays. IkB levels were evaluated in the cytoplasmic extracts by western blot. Monocytes from normal donors were activated with LPS (1 microg/ml) in combination with IFNalpha or ethanol overnight and TNFalpha, IL-6, and IL-12 were measured in the supernatants. RESULTS: In HepG2 cells, both IFNalpha and acute alcohol treatment induced NF-kappaB activation and augmented TNFalpha-induced NF-kappaB binding. Pretreatment of HepG2 cells with IFNalpha resulted in the highest levels of NF-kappaB activation in response to TNFalpha or TNFalpha plus ethanol stimulation. Supershift experiments confirmed that the NF-kappaB dimer induced by TNFalpha and its combination with IFNalpha or ethanol contains RelA (p65) and involves rapid degradation of IkappaBalpha. Experiments using the proteasome inhibitor, MG132, revealed that augmentation of NF-kappaB by ethanol and IFNalpha is mediated via the proteasome pathway. We show that in normal monocytes, IFNalpha augments LPS-induced production of the inflammatory cytokines TNFalpha, IL-6, and IL-12 (p < 0.06) without further modulation by acute alcohol treatment. CONCLUSIONS: These results suggest that IFNalpha can increase HepG2 cell sensitivity to TNFalpha and ethanol-mediated activation. Augmentation of monocyte inflammatory cytokines, particularly of IL-12 production, by IFNalpha could be a key element of the antiviral response in chronic HCV. These results support the hypothesis that the therapeutic benefits of IFNalpha likely involve activation of both immune and parenchymal cells in the liver.
Authors: Ting Zhang; Chang-Jiang Guo; Steven D Douglas; David S Metzger; Charles P O'Brien; Yuan Li; Yan-Jian Wang; Xu Wang; Wen-Zhe Ho Journal: Alcohol Clin Exp Res Date: 2005-09 Impact factor: 3.455