Kupffer cell sensitization by alcohol involves increased permeability to gut-derived endotoxin.
BACKGROUND
Studies with gut sterilization and Kupffer cell inactivation support the hypothesis that endotoxin and Kupffer cells are involved in mechanisms of alcohol-induced liver injury. Recently, we found that Kupffer cells isolated from rats treated only once with ethanol were sensitized to endotoxin 24 hr later. Moreover, we established a new, simple animal model of ethanol hepatotoxicity based on Kupffer cell sensitization. The purpose of this study was to determine the mechanisms by which alcohol sensitizes Kupffer cells to lipopolysaccharide (LPS).


METHODS
Female Wistar rats were given ethanol (5 g/kg body weight) once every 24 hr intragastrically, and ethanol concentration, ethanol elimination, and portal vein endotoxin were measured. Gut permeability was measured in isolated segments of ileum by translocation of horseradish peroxidase. Kupffer cells were isolated 24 hr after ethanol administration in vivo and were cultured in RPMI 1640 with 10% fetal bovine serum. After the addition of LPS, intracellular Ca2+ was measured by using a microspectrofluorometer with the fluorescent indicator fura-2, and tumor necrosis factor (TNF)-alpha was measured by enzyme-linked immunosorbent assay. CD14 was evaluated by Western analysis.


RESULTS
Ethanol levels exhibited a cyclic pattern in ethanol-treated rats. Similar results were obtained in groups given ethanol and antibiotics for 4 weeks. Rates of alcohol elimination were around 3.5 mmol/kg/hr in control rats. After 4 weeks of ethanol treatment with or without antibiotics, elimination rates were not changed. Translocation of horseradish peroxidase was increased about 3-fold in gut segments by treatment with ethanol. This increase was not altered by treatment with antibiotics. Moreover, portal vein endotoxin levels were increased from nearly undetectable levels to 80 pg/ml in plasma of rats treated with ethanol. As expected, this increase was prevented (<20 pg/ml) by antibiotics. In isolated Kupffer cells from rats treated with ethanol for 4 weeks, CD14, LPS-induced intracellular Ca2+, and TNF-alpha all were increased. These phenomena were blocked by antibiotics.


CONCLUSIONS
Kupffer cells isolated from rats treated with ethanol for 4 weeks exhibit sensitization to LPS. It is likely that increased permeability of the gut is a prominent event that leads to alcoholic liver injury.
Enomoto
N
N
Department of Gastroenterology, Juntendo University, Bunkyo-ku, Tokyo, Japan.
Ikejima
K
K
Yamashina
S
S
Hirose
M
M
Shimizu
H
H
Kitamura
T
T
Takei
Y
Y
Sato And
N
N
Thurman
R G
RG
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D004064:Digestive System / Q000378:metabolism*; D004731:Endotoxins / Q000097:blood / Q000378:metabolism* / Q000494:pharmacology; D000431:Ethanol / Q000097:blood / Q000493:pharmacokinetics / Q000494:pharmacology*; D005260:Female; D006735:Horseradish Peroxidase / Q000493:pharmacokinetics; D007728:Kupffer Cells / Q000187:drug effects*; D010539:Permeability; D011169:Portal Vein; D051381:Rats; D017208:Rats, Wistar; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000900:Anti-Bacterial Agents; D004731:Endotoxins; D014409:Tumor Necrosis Factor-alpha; D000431:Ethanol; D006735:Horseradish Peroxidase; D002118:Calcium