Michael D. Wheeler

Essential role of tumor necrosis factor α in alcohol-induced liver injury in mice

BACKGROUND & AIMS Tumor necrosis factor (TNF)-alpha is associated with increased mortality in alcoholics, but its role in early alcohol-induced liver injury is not fully understood. Recently, it was shown that injury induced by the enteral alcohol delivery model of Tsukamoto and French was reduced by antibodies to TNF-alpha. To obtain clear evidence for or against the hypothesis that TNF-alpha is involved, we studied TNF receptor 1 (TNF-R1, p55) or 2 (TNF-R2, p75) knockout mice. METHODS Long-term enteral alcohol delivery was modified for male gene-targeted mice lacking TNF-R1 and TNF-R2. Animals were given a high-fat liquid diet continuously with either ethanol or isocaloric maltose-dextrin as a control for 4 weeks. RESULTS Ethanol elevated serum levels of alanine aminotransferase nearly 3-fold in wild-type and TNF-R2 knockout mice but not in TNF-R1 knockout mice. Likewise, ethanol caused severe liver injury in wild-type mice (pathology score, 5.5 +/- 0.6) and TNF-R2 knockout mice (pathology score, 5.0 +/- 0.4), but not in TNF-R1 knockout mice (pathology score, 0.8 +/- 0.4; P < 0.001). CONCLUSIONS Long-term ethanol feeding caused liver injury in wild-type and TNF-R2 knockout mice but not in TNF-R1 knockout mice, providing solid evidence in support of the hypothesis that TNF-alpha plays an important role in the development of early alcohol-induced liver injury via the TNF-R1 pathway. Moreover, the long-term enteral ethanol feeding technique we described for the first time for knockout mice provides a useful new tool for alcohol research.

Reduced Early Alcohol-Induced Liver Injury in CD14-Deficient Mice

Activation of Kupffer cells by gut-derived endotoxin is associated with alcohol-induced liver injury. Recently, it was shown that CD14-deficient mice are more resistant to endotoxin-induced shock than wild-type controls. Therefore, this study was designed to investigate the role of CD14 receptors in early alcohol-induced liver injury using CD14 knockout and wild-type BALB/c mice in a model of enteral ethanol delivery. Animals were given a high-fat liquid diet continuously with ethanol or isocaloric maltose-dextrin as control for 4 wk. The liver to body weight ratio in wild-type mice (5.8 ± 0.3%) was increased significantly by ethanol (7.3 ± 0.2%) but was not altered by ethanol in CD14-deficient mice. Ethanol elevated serum alanine aminotransferase levels nearly 3-fold in wild-type mice, but not in CD14-deficient mice. Wild-type and knockout mice given the control high-fat diet had normal liver histology, whereas ethanol caused severe liver injury (steatosis, inflammation, and necrosis; pathology score = 3.8 ± 0.4). In contrast, CD14-deficient mice given ethanol showed minimal hepatic changes (score = 1.6 ± 0.3, p < 0.05). Additionally, NF-κB, TGF-β, and TNF-α were increased significantly in wild-type mice fed ethanol but not in the CD14 knockout. Thus, chronic ethanol feeding caused more severe liver injury in wild-type than CD14 knockouts, supporting the hypothesis that endotoxin acting via CD14 plays a major role in the development of early alcohol-induced liver injury.

The role of Kupffer cell oxidant production in early ethanol-induced liver disease.

Considerable evidence for a role of Kupffer cells in alcoholic liver disease has accumulated and they have recently been shown to be a predominant source of free radicals. Several approaches including pharmacological agents, knockout mice, and viral gene transfer have been used to fill critical gaps in understanding key mechanisms by which Kupffer cell activation, oxidant formation, and cytokine production lead to liver damage and subsequent pathogenesis. This review highlights new data in support of the hypothesis that Kupffer cells play a pivotal role in hepatotoxicity due to ethanol by producing oxidants via NADPH oxidase.

Role of Lipopolysaccharide-Binding Protein in Early Alcohol-Induced Liver Injury in Mice

Cellular responses to endotoxins are enhanced markedly by LPS-binding protein (LBP). Furthermore, it has been demonstrated that endotoxins and proinflammatory cytokines such as TNF-α participate in early alcohol-induced liver injury. Therefore, in this study, a long-term intragastric ethanol feeding model was used to test the hypothesis that LBP is involved in alcoholic hepatitis by comparing LBP knockout and wild-type mice. Two-month-old female mice were fed a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin as control continuously for 4 wk. There was no difference in mean urine alcohol concentrations between the groups fed ethanol. Dietary alcohol significantly increased liver to body weight ratios and serum alanine aminotransferase levels in wild-type mice (189 ± 31 U/L) over high-fat controls (24 ± 7 U/L), effects which were blunted significantly in LBP knockout mice (60 ± 17 U/L). Although no significant pathological changes were observed in high-fat controls, 4 wk of dietary ethanol caused steatosis, mild inflammation, and focal necrosis in wild-type animals as expected (pathology score, 5.9 ± 0.5). These pathological changes were reduced significantly in LBP knockout mice fed ethanol (score, 2.6 ± 0.5). Endotoxin levels in the portal vein were increased significantly after 4 wk in both groups fed ethanol. Moreover, ethanol increased TNF-α mRNA expression in wild-type, but not in LBP knockout mice. These data are consistent with the hypothesis that LBP plays an important role in early alcohol-induced liver injury by enhancing LPS-induced signal transduction, most likely in Kupffer cells.

Glycine: a new anti-inflammatory immunonutrient

Abstract. The mechanism of the immunosuppressive effects of glycine and its pathophysiological applications are discussed in this review. Glycine has been well characterized in spinal cord as an inhibitory neurotransmitter which activates a glycine-gated chloride channel (GlyR) expressed in postsynaptic membranes. Activation of the channel allows the influx of chloride, preventing depolarization of the plasma membrane and the potentiation of excitatory signals along the axon. Glycine has recently been shown to have similar inhibitory effects on several white blood cells, including hepatic and alveolar macrophages, neutrophils, and lymphocytes. Pharmacological analysis using a GlyR antagonist strychnine, chloride-free buffer, and radiolabeled chloride has provided convincing evidence to support the hypothesis that many white blood cells contain a glycine-gated chloride channel with properties similar to the spinal cord GlyR. Molecular analysis using reverse transcription-polymerase chain reaction and Western blotting has identified the mRNA and protein for the β subunit of the GlyR in total RNA and purified membrane protein from rat Kupffer cells. Dietary glycine is protective in rat models against endotoxemia, liver ischemia-reperfusion, and liver transplantation, most likely by inactivating the Kupffer cell via this newly identified glycine-gated chloride channel. Glycine also prevents the growth of B16 melanomas cell in vivo. Moreover, dietary glycine is protective in the kidney against cyclosporin A toxicity and ischemia-reperfusion injury. Glycine may be useful clinically for the treatment of sepsis, adult respiratory distress syndrome, arthritis, and other diseases with an inflammatory component.

Cytochrome P450 CYP2E1, but not nicotinamide adenine dinucleotide phosphate oxidase, is required for ethanol-induced oxidative DNA damage in rodent liver.

The occurrence of malignant tumors of the upper gastrointestinal tract and liver is, based largely on epidemiological evidence, causally related to the consumption of ethanol. It is widely recognized that oxidants play a key role in alcohol‐induced liver injury; however, it is unclear how oxidants may be involved in DNA damage. We asked whether nicotinamide adenine dinucleotide phosphate oxidase, cytochrome P450 CYP2E1, or both are responsible for the production of DNA damage. The rodent Tsukamoto‐French model of intragastric ethanol infusion was used. Wistar rats, Cyp2e1‐, p47phox‐null, and hCyp2e1 transgenic mice were used. The abundance of oxidative DNA adducts, mutagenic apurinic/apyrimidinic sites, and expression of base excision DNA repair genes was determined. In rats and wild‐type mice, ethanol treatment for 4 weeks led to an increase in oxidative DNA damage and induction of expression of the base excision DNA repair genes that are known to remove oxidative DNA lesions. No increase in either of the endpoints was observed in ethanol‐treated Cyp2e1‐null mice, whereas the magnitude of response in p47phox‐null mice and transgenic hCyp2e1 was identical to that in wild types. The increase in expression of DNA repair genes was completely abolished by treatment with the P450 inhibitor 1‐aminobenzotriazole. In conclusion, the data support the hypothesis that oxidative stress to DNA is induced in liver by ethanol. Furthermore, although it was shown that nicotinamide adenine dinucleotide phosphate oxidase‐derived oxidants are critical for the development of ethanol‐induced liver injury, CYP2E1 is required for the induction of oxidative stress to DNA, and thus may play a key role in ethanol‐associated hepatocarcinogenesis. (HEPATOLOGY 2005;41:336–344.)

Adenoviral gene delivery can inactivate Kupffer cells: role of oxidants in NF‐κB activation and cytokine production

Kupffer cells play a significant role in the pathogenesis of several liver diseases; therefore, a potential therapeutic strategy would be to inactivate the Kupffer cell with a gene‐delivery system. Although recombinant adenovirus provides robust, transgene expression in parenchymal cells, whether adenovirus transduces Kupffer cells is unclear. Thus, the purpose of this study was to evaluate this possibility. In animals infected with adenovirus, Kupffer cells were identified positively to express adenoviral transgenes by immunohistochemical techniques and Western blot analysis, indicating that Kupffer cells are transduced in vivo. Indeed, isolated Kupffer cells were transduced in vitro with recombinant adenovirus in a dose‐dependent manner. Moreover, adenoviral transduction of Kupffer cells was blocked by inhibitors of αVβ5 integrin, the co‐receptor for adenovirus binding, supporting the hypothesis that adenovirus transduces Kupffer cells via an αVβ5 integrin‐dependent mechanism. Indeed, it is shown here that Kupffer cells express αVβ5 integrins. In a functional assay, infection of isolated Kupffer cells with adenovirus containing superoxide dismutase or IκBα super‐repressor blunted LPS‐induced nuclear transcription factor kappa B (NF‐κB) activation and tumor necrosis factor α (TNF‐α) production but not IL‐10 production. Moreover, superoxide production was blocked by expression of superoxide dismutase. These data support the hypothesis that LPS‐induced NF‐κB activation and TNF‐α production in Kupffer cells are oxidant‐dependent. These findings suggest that Kupffer cell‐targeted approaches may be a potential therapeutic strategy against many inflammatory diseases including early alcohol‐induced liver injury.

Kupffer cell and interleukin-12-dependent loss of natural killer T cells in hepatosteatosis.

Hepatosteatosis is associated with increased expression of tumor necrosis factor alpha (TNF‐α) and interleukin (IL)‐12, major T helper (Th) 1 cytokines, and reduced hepatic natural killer T (NKT) cell numbers. The relationship between lipid accumulation, cytokine expression, and hepatic NKT cells is not known. This study was conducted to assess the role of IL‐12 in the development of hepatic steatosis and its potential impact on liver NKT cells. Male C57Bl/6 wildtype (WT) and IL‐12‐deficient (IL‐12−/−) mice were fed a choline‐deficient diet (CDD) for 0, 10, or 20 weeks. CDD led to marked hepatosteatosis, reduced hepatic but not splenic NKT cell numbers and function, and increased hepatic expression of the Th1‐type cytokines IL‐12, interferon gamma (IFN‐γ), and TNF‐α in WT mice. The absence of IL‐12 resulted in similar CDD‐induced hepatosteatosis, but preserved hepatic NKT cells and significantly reduced hepatic IFN‐γ and TNF‐α expression. Treatment of CDD‐fed mice with lipopolysaccharide led to a significant increase in hepatic IL‐12 expression, and Kupffer cell (KC) depletion reduced liver IL‐12 expression and restored NKT cells in CDD‐induced fatty liver. Interestingly, KCs from CDD‐fed mice failed to produce increased quantities of IL‐12 upon activation in vitro when compared to similarly treated KCs from control fed mice, suggesting that secondary factors in vivo promote heightened IL‐12 production. Finally, human livers with severe steatosis showed a substantial decrease in NKT cells. Conclusion: Hepatosteatosis reduces the numbers of hepatic NKT cells in a KC‐and IL‐12‐dependent manner. Our results suggest a pivotal and multifunctional role of KC‐derived IL‐12 in the altered immune response in steatotic liver, a process that is likely active within human nonalcoholic fatty liver disease. (HEPATOLOGY 2010;51:130–141.)

Green tea extract protects against early alcohol-induced liver injury in rats

Abstract Oxidants have been shown to be involved in alcoholinduced liver injury. This study was designed to test the hypothesis that the antioxidant polyphenolic extract of green tea, comprised predominantly of epigallocatechin gallate, protects against early alcoholinduced liver injury in rats. Male Wistar rats were fed highfat liquid diets with or without ethanol (10 14 g kg 1 day 1) and green tea (300 mg kg 1 day 1) continuously for 4 weeks using an intragastric enteral feeding protocol. Mean body weight gains (~4 g/day) were not significantly different between treatment groups, and green tea extract did not the affect average concentration or the cycling of urine ethanol concentrations (0 550 mg dl 1 day 1). After 4 weeks, serum ALT levels were increased significantly about 4-fold over control values (35±3 IU/l) by enteral ethanol (114±18); inclusion of green tea extract in the diet significantly blunted this increase (65±10). Enteral ethanol also caused severe fatty accumulation, mild inflammation, and necrosis in the liver. While not affecting fat accumulation or inflammation, green tea extract significantly blunted increases in necrosis caused by ethanol. Furthermore, ethanol significantly increased the accumulation of protein adducts of 4-hydroxynonenal, a product of lipid peroxidation and an index of oxidative stress; green tea extract blocked this effect almost completely. TNFa protein levels were increased in liver by alcohol; this phenomenon was also blunted by green tea extract. These results indicate that simple dietary antioxidants, such as those found in green tea, prevent early alcoholinduced liver injury, most likely by preventing oxidative stress.

Comparison of the effect of adenoviral delivery of three superoxide dismutase genes against hepatic ischemia-reperfusion injury

The purpose of this study was to investigate the effectiveness of superoxide dismutase (SOD) overexpression in an acute model of hepatic oxidative stress. Oxidative stress was established using a warm ischemia-reperfusion model, where nearly 70% of the liver was made hypoxic by clamping the hepatic artery and a branch of the portal vein for 1 hr followed by restoration of blood flow. Animals were infected i.v. with 1 x 10(9) plaque-forming units (PFU) of adenovirus containing the transgene for cytosolic Cu/Zn-SOD (Ad.SOD1), mitochondrial Mn-SOD (Ad.SOD2), extracellular Cu/Zn-SOD (Ad.SOD3), or the bacterial reporter gene for beta-galactosidase (Ad.lacZ) 3 days prior to experiments. Ad.SOD1 and Ad.SOD2 caused a three-fold increase in SOD expression and activity in liver compared to Ad.lacZ-treated control animals. Intravenous administration of Ad.SOD3 increased SOD activity slightly in serum but not in liver. Increases in serum transaminases and pathology due to ischemia-reperfusion were blunted by Ad.SOD1 and Ad.SOD2; however, extracellular SOD had no significant effect. Moreover, lipid-derived free radical adducts (a(N) = 15.65 G and a(H)(beta) = 2.78 G) were increased by ischemia-reperfusion. This effect was blunted by about 60% in Ad.SOD1- and Ad.SOD2-infected animals, but was unaffected by Ad.SOD3. However, when high doses of Ad.SOD3 (3 x 10(10) PFU) were administered. serum SOD activity was elevated three-fold and was protective against hepatic ischemia-reperfusion injury under these conditions. These data demonstrate that adenoviral delivery of superoxide dismutase can effectively reduce hepatic oxidative stress.