Cytokines, alcohol metabolizing enzymes and stress-inducible ER proteins in alcoholic liver disease

University dissertation from Stockholm : Karolinska Institutet, Department of Medical Biochemistry and Biophysics

Abstract: The release of inflammatory cytokines by Kupffer cells is believed to be a key step in the pathogenesis of alcoholic liver disease (ALD). Kupffer cells are activated not only by endotoxins [lipopolysaccharides (LPS)] but also by the lipid peroxidation products generated mainly via ethanol-inducible cytochrome P450 2E1 (CYP2E1) redox cycling. In addition, alcohol dehydrogenase class I (ADH1) is a major enzyme in the first step of ethanol metabolism, whereas induction of endoplasmic reticulum (ER) stress/heat shock proteins as a cellular adaptation in rat model of ALD has been related to the severity of liver injury and lipid peroxidation. The aims of the present studies were to better understand the role of alcohol, endotoxins, Kupffer cells, cytokines, CYP2E1, ADH1 and ER stress proteins in the pathogenesis of ALD. Using a modified competitive PCR and Western blot techniques, we evaluated zonated expression of several cytokines in the liver of rats infused intragastrically with chronic ethanol and the total enteral nutrition. Chronic ethanol treatment significantly increased perivenous expression of transforming growth factor-[beta]1 (TGF-[beta]1) and interleukin-1[beta] (IL-1[beta]) as well as panlobular expression of tumor necrosis factor-cc (TNF-[alpha]) mRNA. Chlormethiazole (CMZ), an inhibitor of CYP2E1 that has been shown to inhibit the development of ALD in this rat model, counteracted the effects of ethanol on the cytokine expression. Rats were treated in an intragastric dietary model with ethanol in a low-carbohydrate/high-fat liquid for 6 weeks. Some rats received LPS infused by minipumps intravenously for 4 weeks. Chronic endotoxin infusion alone resulted in a marked tolerance as judged by insignificant liver damage, increased hepatic expression of both proinflammatory cytokines (TNF-[alpha] and IL- 1 [beta]) and anti-inflammatory cytokines (TGF-[beta]1, IL- 10 and IL-4), as well as no influence on expression of LPS binding protein and CD14 endotoxin receptor. Additional chronic ethanol enhanced the expression of CYP2E1 protein as well as ADH1 mRNA. An increased pathology score was observed by ethanol, which might be related to an increased ratio of expression of proinflammatory over anti-inflammatory cytokines. Kupffer cell inactivation by Gadolinium chloride alleviated ethanol-induced steatosis as well as CYP2E1 induction, eliminated ED2-positive Kupffer cells, but did not affect inflammation and the expression of TNF-[alpha] and IL- 1[beta] as well as the CD 14 receptor. By a combination of direct DNA sequencing with degenerate primer-mediated PCR and RACE, we successfully cloned the cDNA of a novel ERp29 protein from rat liver. Characterization of ERp29 showed that it interacts with ER stress protein BiP/GRP78 and is widely expressed and induced by ER stress. This suggests that ERp29 is functionally related to the major ER stress proteins and may have an important function in protein maturation and the stress defence in ER. Increase of ERp29 protein after chronic LPS exposure further proposes that it may counteract LPS-induced cytotoxicity and contribute to the observed endotoxin tolerance. We found a decrease of ERp29 expression in the Cyp2e1 knockout mice, which suggests possible regulatory relationship between ERp29 and CYP2E1. In conclusion, the present studies suggest that it is important to consider the balance of pro- and anti-inflammatory cytokine expression and different kinds of Kupffer cells in the development of ALD. CYP2E1 is involved in alcohol-induced liver injury, whereas ERp29 may function as a cellular adaptive response to chronic LPS exposure.

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