The purpose of this study was to determine if exacerbation of apoptosis precedes liver injury during chronic exposure of rats to alcohol. After 7 weeks of feeding an alcohol- or dextrin-containing liquid diet, the animals were treated with gram-negative bacterial lipopolysaccharide (1 mg x kg(-1) body weight, intravenously) or sterile saline and sacrificed 3 hr after the treatment. Alanine:2-oxoglutarate aminotransferase (ALT) and lactate:NAD oxidoreductase [lactate dehydrogenase (LDH)] were measured in plasma. The caudate lobe of the liver was resected for histology, while the rest of the organ was perfused with collagenase to isolate hepatocytes, Kupffer cells (KCs), and sinusoidal endothelial cells (SECs) by centrifugal elutriation. Hepatocyte mitochondria were isolated by differential centrifugation of the cell homogenate. Reduced and oxidized glutathione (GSH and GSSG) in isolated hepatocytes and hepatocyte mitochondria, and malondialdehyde in hepatocytes were assayed. Caspase-3 activity and Fas ligand mRNA expression were determined in hepatocytes, KCs, and SECs. Plasma ALT and LDH activity, liver histology, GSH, GSSG and their ratio, and malondialdehyde content were not affected by alcohol treatment Caspase-3 activity was significantly increased in alcohol-treated rats in all three cell types, with the lowest response observed in hepatocytes and the highest in KCs. Fas ligand mRNA expression, which had the highest level in SECs, followed by KCs and hepatocytes, was not affected by alcohol administration. Lipopolysaccharide had the following effects: an increase in ALT in both pair- and alcohol-fed rats, and LDH only in alcohol-fed rats, a decrease in GSH + GSSG levels in both mitochondria and hepatocytes, an elevation of malondialdehyde content in hepatocytes, a raise in caspase-3 activity in all groups and cell types, and an augmentation of Fas ligand expression in hepatocytes and KCs, but not in SECs. These data suggest that, during chronic alcohol consumption, an exacerbated apoptosis precedes alcohol-induced liver injury.
Tumor necrosis factor (TNF) is suggested to play an important role in host defense as well as in eliciting some of the metabolic alterations in endotoxemia. Bacteria and their products are involved in triggering the production and release of TNF. Alcohol consumption is known to suppress the immune system and increase susceptibility to infections. The present study was undertaken to investigate the effect of acute ethanol administration on the ability of endotoxin to increase circulating TNF levels and to determine the relationship between blood ethanol levels and endotoxin-induced serum TNF. A 50% decrease in serum TNF levels was seen 1-1.5 h after endotoxin challenge in conscious rats with blood alcohol levels between 75-175 mg/dl. A dose-related depression of serum TNF was observed with increasing blood alcohol levels. This was accompanied by a markedly diminished hyperlactacidemia seen following endotoxin administration. These data suggest that impaired TNF release may have a role in the altered immune response of alcoholics to infection.
We investigated the effect of alcohol (ethanol) on the ability of the alveolar macrophage to produce tumor necrosis factor-alpha (TNF-alpha), superoxide anion (O2-), and nitric oxide (NO)--three critical components of pulmonary host defense. Male rats were treated with alcohol either acutely (priming dose 175 mg/100 g of body weight, followed by a 7-hr continuous intravenous infusion of 30 mg/100 g of body weight/hr) or chronically (12-14 weeks of feeding ethanol in a liquid diet). Three hours before sacrifice, the rats received an intravenous injection of saline or lipopolysaccharide (LPS; Escherichia coli, 026:B6, 100 micrograms/100 g of body weight). Alveolar macrophages (AMs) were then isolated by bronchoalveolar lavage and assessed for their in vitro capacity to produce TNF-alpha, O2-, and NO spontaneously and in response to different stimuli. Acute alcohol administration suppressed in vitro LPS-stimulated AM TNF-alpha secretion by 52%. AMs from both pair-and alcohol-fed rats secreted TNF-alpha spontaneously in culture. However, the AMs from chronic alcohol-fed group secreted 42-53% less TNF-alpha spontaneously and in response to LPS, interferon-gamma (IFN-gamma) or IFN-gamma + LPS compared with the AMs from pair-fed group. Systemic LPS treatment inhibited in vitro LPS-stimulated AM TNF-alpha secretion (50%) only in the control rats. Acute alcohol administration enhanced significantly in vitro phorbol 12-myristate 13-acetate (PMA)- and opsonized zymosan (OPZ)-induced AM O2- secretion (4-and 1.8-fold, respectively). Systemic LPS treatment primed the AMs from control rats to secrete 83% more O2- in response to PMA but not OPZ; however, in the acute alcohol-treated group, it suppressed both PMA (54%)- and OPZ (66%)-induced AM O2- release (loss of priming effect of LPS). Chronic alcohol feeding suppressed PMA-induced AM O2- secretion (40%) without affecting the OPZ-induced release. Although systemic LPS treatment had no significant effect on PMA or OPZ-induced AM O2- secretion in the pair-fed group, it enhanced the PMA-stimulated AM O2- release (88%) in the alcohol-fed group. AMs recovered from control or acute alcohol-treated rats did not secrete NO spontaneously in vitro. However, AMs from both pair and chronic alcohol-fed rats secreted NO spontaneously with AMs from chronic alcohol-fed group secreting 34% less. Both acute and chronic alcohol treatment inhibited AM NO secretion in response to IFN-gamma, LPS, and IFN-gamma + LPS significantly. Systemic LPS had no effect on AM NO production in response to different in vitro stimuli in any of the treatment groups. These data suggest that (1) both acute and chronic alcohol administration to rats inhibit AM TNF-alpha and NO secretion; (2) acute and chronic alcohol treatment have differential effects on AM O2- secretion; and (3) alcohol-induced alteration in AM TNF-alpha, O2-, and NO secretion may in part explain the increased susceptibility of alcohol-consuming individuals to pulmonary infections.
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