Development of a new, simple rat model of early alcohol-induced liver injury based on sensitization of kupffer cells

Abstract

The continuous intragastric in vivo enteral feeding model in the rat developed by Tsukamoto and French has been very useful; however, it requires surgical expertise. Recently, we found that Kupffer cells isolated from rats treated only once with ethanol were sensitized to endotoxin 24 hours later. Accordingly, these experiments were designed to determine if a new, simple animal model of ethanol hepatotoxicity could be developed based on Kupffer cell sensitization. Female Wistar rats were given ethanol (5 g/kg body weight) once every 24 hours intragastrically. Livers were stained with hematoxylin-eosin to assess steatosis, inflammation, and necrosis, and tissue triglycerides, serum transaminases, and plasma endotoxin were measured. Kupffer cells were isolated 0 to 24 hours after one intragastric dose of ethanol daily, and intracellular Ca 2؉ ([Ca 2؉ ] i ) was measured using fura-2, while tumor necrosis factor ␣ (TNF-␣) was measured by enzyme-linked immunosorbent assay. CD14 was evaluated by Western and Northern analysis. Ethanol caused steatosis, necrosis, and inflammation in only a few weeks, and after 8 weeks, serum aspartate transaminase (AST) levels were doubled. Values were similar to levels achieved in the enteral feeding model. Triglycerides were also increased significantly by ethanol as expected, and endotoxin levels were increased to 70 to 80 pg/mL. This latter increase was prevented (F20 pg/mL) by antibiotics implicating endotoxin. In isolated Kupffer cells from untreated control rats, [Ca 2؉ ] i increased to 82 ؎ 7 nmol/L after addition of lipopolysaccharide (LPS) (100 ng/mL), and levels were elevated about twofold by ethanol given 24 hours earlier ( Although it is well known that alcoholic liver disease results from the dose-and time-dependent consumption of ethanol, mechanisms remain unclear. For a long time, a significant drawback for research in this area was the lack of appropriate animal models. For example, feeding ethanol chronically with the diet causes only fatty liver. 1 The establishment of a continuous intragastric in vivo enteral feeding model in the rat by Tsukamoto and French represented a major development in this area. 2,3 With this model, it has been shown that inactivation of Kupffer cells with gadolinium chloride (GdCl 3 ) prevented early alcohol-induced liver injury. 4 Moreover, intestinal sterilization with antibiotics (polymyxin B and neomycin) 5 or decreasing endotoxin with lactobacillus 6 prevented alcohol-induced liver injury. These experiments pointed to a role for activation of Kupffer cells by gut-derived endotoxin as a primary event in mechanisms of alcohol-induced liver injury. Although this chronic enteral ethanol model has been very useful, it is both timeconsuming and expensive, and requires surgical expertise.Recently, we found that Kupffer cells isolated from rats treated once with ethanol 24 hours earlier exhibited enhanced sensitivity to lipopolysaccharide (LPS) as a result of up-regulation of the endotoxin receptor, CD14. 7 Furthermore, Su et al. 8 showed that ...

Heme Oxygenase-1 Mediates the Anti-Inflammatory Effects of Acute Alcohol on IL-10 Induction Involving p38 MAPK Activation in Monocytes

“…The modulatory potential of acute alcohol on cytokine expression includes attenuation of TNF-␣ and augmentation of IL-10 production in monocytes (2,3,11). Ethanol generates oxidative stress, which plays an important role in immune cell activation as well as in alcoholic liver injury (14,35,36). HO-1 is an important antioxidant molecule and its up-regulation is a critical cytoprotective mechanism activated during cellular stress (8,(37)(38)(39).…”

“…Despite convincing data indicating the cytoprotective function of HO-1 in oxidative stress, the mechanisms by which HO-1 serves as a cytoprotectant remain poorly understood. LPS, a potent HO-1 activator, has a central role in alcohol-induced liver damage via activation of Kupffer cells, the resident liver macrophages (14). LPS binds to CD14 and TLR4 at the cell surface (15) and then activates the MAPK pathways, including p38, p42/p44 ERK, and JNK (15)(16)(17).…”

Bacterial translocation and changes in the intestinal microbiome associated with alcoholic liver disease

“…In �articular, TLR4, the cellular LPS receptor, plays an important role in the innate i��une res�onse to bacterial translocation. Selective intestinal decontamination with antibiotics have shown to reduce plasma endotoxin levels and prevents liver injury in ani�al �odels of ALD [31,32,22] . Moreover, mice deficient in CD14, the cellular co-receptor for LPS, are also resistant to alcohol�induced liver injury [17�] .…”

Human Immunodeficiency Virus-Related Microbial Translocation and Progression of Hepatitis C

“…14, 15 In animal models, both sensitization and tolerance of Kupffer cells has been described and alcohol related liver disease can be reduced by suppression of microbial burden with antibiotics or inhibition of effector cytokines such as TNFα. 15 Recently, it was shown that TLR4 activation by LPS upregulates chemokine secretion and sensitizes stellate cells to transforming growth factor β and the activating effects of Kupffer cells. 16 Microbial translocation has also been implicated in liver disease associated with other enteric processes, such as graft versus host disease and celiac sprue.…”