Ethanol and arachidonic acid synergize to activate Kupffer cells and modulate the fibrogenic response via tumor necrosis factor alpha, reduced glutathione, and transforming growth factor beta-dependent mechanisms.
Because of the contribution of ethanol and polyunsaturated fatty acids (PUFAs) to alcoholic liver disease, we investigated whether chronic ethanol administration and arachidonic acid (AA) could synergistically mediate Kupffer cell (KC) activation and modulate the stellate cell (HSC) fibrogenic response.


RESULTS
(1) the effects of ethanol and AA on KC and HSC were as follows: Cell proliferation, lipid peroxidation, H(2)O(2), O(2).(-), nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase activity, and tumor necrosis factor alpha (TNF-alpha) were higher in KC(ethanol) than in KC(control), and were enhanced by AA; HSC(ethanol) proliferated faster, increased collagen, and showed higher GSH than HSC(control), with modest effects by AA. (2) AA effects on the control co-culture: We previously reported the ability of KC to induce a pro-fibrogenic response in HSC via reactive oxygen species (ROS)-dependent mechanisms; we now show that AA further increases cell proliferation and collagen in the control co-culture. The latter was prevented by vitamin E (an antioxidant) and by diphenyleneiodonium (a NADPH oxidase inhibitor). (3) Ethanol effects on the co-cultures: Co-culture with KC(control) or KC(ethanol) induced HSC(control) and HSC(ethanol) proliferation; however, the pro-fibrogenic response in HSC(ethanol) was suppressed because of up-regulation of TNF-alpha and GSH, which was prevented by a TNF-alpha neutralizing antibody (Ab) and by L-buthionine-sulfoximine, a GSH-depleting agent. (4) Ethanol plus AA effects on the co-cultures: AA lowered TNF-alpha in the HSC(control) co-cultures, allowing for enhanced collagen deposition; furthermore, AA restored the pro-fibrogenic response in the HSC(ethanol) co-cultures by counteracting the up-regulation of TNF-alpha and GSH with a significant increase in GSSG and in pro-fibrogenic transforming growth factor beta (TGF-beta).


CONCLUSIONS
These results unveil synergism between ethanol and AA to the mechanism whereby KC mediate ECM remodeling and suggest that even if chronic ethanol consumption sensitizes HSC to up-regulate anti-fibrogenic signals, their effects are blunted by a second "hit" such as AA.
Cubero
Francisco Javier
FJ
Department of Medicine, Division of Liver Diseases, Mount Sinai School of Medicine, Box 1123, 1425 Madison Avenue, Room 11-76, New York, NY 10029, USA.
Nieto
Natalia
N
Department of Medicine, Division of Liver Diseases, Mount Sinai School of Medicine, Box 1123, 1425 Madison Avenue, Room 11-76, New York, NY 10029, USA.
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D016718:Arachidonic Acid / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D002492:Central Nervous System Depressants / Q000494:pharmacology*; D018920:Coculture Techniques; D024042:Collagen Type I / Q000378:metabolism; D004195:Disease Models, Animal; D004357:Drug Synergism; D000431:Ethanol / Q000494:pharmacology*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005260:Female; D005978:Glutathione / Q000378:metabolism; D055166:Hepatic Stellate Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D007728:Kupffer Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D008103:Liver Cirrhosis / Q000378:metabolism* / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D016212:Transforming Growth Factor beta / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D002492:Central Nervous System Depressants; D024042:Collagen Type I; D005231:Fatty Acids, Unsaturated; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha; D016718:Arachidonic Acid; D000431:Ethanol; D005978:Glutathione