Chantal A. Rivera

Alcohol causes both tolerance and sensitization of rat Kupffer cells via mechanisms dependent on endotoxin.

BACKGROUND & AIMS Ethanol causes both tolerance and sensitization of Kupffer cells. This study was designed to evaluate temporal effects of ethanol in an attempt to understand this paradox. METHODS Rats were given ethanol (4 g/kg body wt) intragastrically, and Kupffer cells were isolated 0-48 hours later. After addition of lipopolysaccharide (LPS), intracellular calcium concentration ([Ca2+]i) was measured using a microspectrofluorometer with the fluorescent indicator fura-2, and tumor necrosis factor alpha (TNF-alpha) was measured by enzyme-linked immunosorbent assay. CD14 was evaluated by Western and Northern analysis. RESULTS Two hours after ethanol administration, the LPS-induced increase in [Ca2+]i and TNF-alpha release by Kupffer cells was diminished by 50%, and these parameters were reciprocally enhanced twofold at 24 hours. Sterilization of the gut with antibiotics blocked all effects of ethanol on [Ca2+]i and TNF-alpha release completely. Twenty-four hours after ethanol, CD14 in Kupffer cells was elevated about fivefold. CONCLUSIONS Kupffer cells isolated from rats early after ethanol exhibited tolerance to LPS, whereas sensitization was observed later. It is likely that both of these phenomena are caused by gut-derived endotoxin and that sensitization in Kupffer cells is caused by increases in CD14.

Role of endotoxin in the hypermetabolic state after acute ethanol exposure

This study investigated the role of endotoxin in the hypermetabolic state or swift increase in alcohol metabolism (SIAM) due to acute ethanol exposure. Female Sprague-Dawley rats (100-120 g) were given ethanol (5 g/kg) by gavage. Endotoxin measured in plasma from portal blood was not detectable in saline-treated controls; however, 90 min after ethanol, endotoxin was increased to 85 +/- 14 pg/ml, and endotoxin clearance was diminished by approximately 50%. Oxygen uptake in perfused livers was increased 48% by ethanol, and production of PGE2 by isolated Kupffer cells was increased similarly. These effects were blunted by elimination of gram-negative bacteria and endotoxin with antibiotics before ethanol administration. To reproduce ethanol-induced endotoxemia, endotoxin was infused via the mesenteric vein at a rate of 2 ng. kg-1. h-1. Endotoxin mimicked the effect of ethanol on oxygen uptake. The specific Kupffer cell toxicant GdCl3 completely prevented increases in oxygen uptake due to endotoxin. These findings demonstrate that endotoxin plays a pivotal role in SIAM, most likely by stimulating eicosanoid release from Kupffer cells.

Estriol sensitizes rat Kupffer cells via gut-derived endotoxin

The relationship between gender and alcohol-induced liver disease is complex; however, endotoxin is most likely involved. Recently, it was reported that estriol activated Kupffer cells by upregulation of the endotoxin receptor CD14. Therefore, the purpose of this work was to study how estriol sensitizes Kupffer cells. Rats were given estriol (20 mg/kg ip), and Kupffer cells were isolated 24 h later. After addition of lipopolysaccharide (LPS), intracellular Ca2+ concentration was measured using a microspectrofluorometer with the fluorescent indicator fura 2, and tumor necrosis factor-α was measured by ELISA. CD14 was evaluated by Western analysis. One-half of the rats given estriol intraperitoneally 24 h before an injection of a sublethal dose of LPS (5 mg/kg) died within 24 h, whereas none of the control rats died. Mortality was prevented totally by sterilization of the gut with antibiotics. A similar pattern was obtained with liver histology and serum transaminases. Translocation of horseradish peroxidase was increased about threefold in gut segments by treatment with estriol. This increase was not altered by treatment with nonabsorbable antibiotics. On the other hand, endotoxin levels were increased to 60-70 pg/ml in plasma of rats treated with estriol. As expected, this increase was prevented (<20 pg/ml) by antibiotics. In isolated Kupffer cells, LPS-induced increases in intracellular Ca2+ concentration, tumor necrosis factor-α production, and CD14 were increased, as previously reported. All these phenomena were blocked by antibiotics. Therefore, it is concluded that estriol treatment in vivo sensitizes Kupffer cells to LPS via mechanisms dependent on increases in CD14. This is most likely due to elevated portal blood endotoxin caused by increased gut permeability.The relationship between gender and alcohol-induced liver disease is complex; however, endotoxin is most likely involved. Recently, it was reported that estriol activated Kupffer cells by upregulation of the endotoxin receptor CD14. Therefore, the purpose of this work was to study how estriol sensitizes Kupffer cells. Rats were given estriol (20 mg/kg ip), and Kupffer cells were isolated 24 h later. After addition of lipopolysaccharide (LPS), intracellular Ca2+ concentration was measured using a microspectrofluorometer with the fluorescent indicator fura 2, and tumor necrosis factor-alpha was measured by ELISA. CD14 was evaluated by Western analysis. One-half of the rats given estriol intraperitoneally 24 h before an injection of a sublethal dose of LPS (5 mg/kg) died within 24 h, whereas none of the control rats died. Mortality was prevented totally by sterilization of the gut with antibiotics. A similar pattern was obtained with liver histology and serum transaminases. Translocation of horseradish peroxidase was increased about threefold in gut segments by treatment with estriol. This increase was not altered by treatment with nonabsorbable antibiotics. On the other hand, endotoxin levels were increased to 60-70 pg/ml in plasma of rats treated with estriol. As expected, this increase was prevented (<20 pg/ml) by antibiotics. In isolated Kupffer cells, LPS-induced increases in intracellular Ca2+ concentration, tumor necrosis factor-alpha production, and CD14 were increased, as previously reported. All these phenomena were blocked by antibiotics. Therefore, it is concluded that estriol treatment in vivo sensitizes Kupffer cells to LPS via mechanisms dependent on increases in CD14. This is most likely due to elevated portal blood endotoxin caused by increased gut permeability.

A choline-rich diet improves survival in a rat model of endotoxin shock

This study investigated whether dietary choline can prevent endotoxin shock. Female Sprague-Dawley rats fed chow or chow plus choline chloride (0.025-0.4%) for 3 days were given lipopolysaccharide (LPS) via the tail vein. Eighty-three percent and 56% of chow-fed rats survived after 2.5 or 5.0 mg/kg LPS, respectively. Choline increased survival in a dose-dependent manner, with maximal effects observed at 0.4%; this dose of choline prevented mortality completely after 2.5 or 5 mg/kg LPS. Choline also improved the microscopic appearance of the lungs and blunted increases in serum aspartate aminotransferase levels. Intracellular Ca2+ was monitored in liver and lung macrophages during LPS exposure. Ca2+ increases in macrophages from choline-fed rats were blunted by 40-60% compared with chow-fed controls. Feeding choline also blunted tumor necrosis factor-alpha production. Feeding glycine, which prevents macrophage activation via a chloride channel, in addition to choline was even more effective than feeding choline alone, suggesting that glycine and choline act via distinct sites. These data are consistent with the hypothesis that choline diminishes endotoxin shock by preventing macrophage activation.This study investigated whether dietary choline can prevent endotoxin shock. Female Sprague-Dawley rats fed chow or chow plus choline chloride (0.025-0.4%) for 3 days were given lipopolysaccharide (LPS) via the tail vein. Eighty-three percent and 56% of chow-fed rats survived after 2.5 or 5.0 mg/kg LPS, respectively. Choline increased survival in a dose-dependent manner, with maximal effects observed at 0.4%; this dose of choline prevented mortality completely after 2.5 or 5 mg/kg LPS. Choline also improved the microscopic appearance of the lungs and blunted increases in serum aspartate aminotransferase levels. Intracellular Ca2+ was monitored in liver and lung macrophages during LPS exposure. Ca2+ increases in macrophages from choline-fed rats were blunted by 40-60% compared with chow-fed controls. Feeding choline also blunted tumor necrosis factor-α production. Feeding glycine, which prevents macrophage activation via a chloride channel, in addition to choline was even more effective than feeding choline alone, suggesting that glycine and choline act via distinct sites. These data are consistent with the hypothesis that choline diminishes endotoxin shock by preventing macrophage activation.