Ethanol and Arachidonic Acid Synergize to Activate Kupffer Cells and Modulate the Fibrogenic Response via Tumor Necrosis Factor α, Reduced Glutathione, and Transforming Growth Factor β-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, H2O2, O-2(center dot-), nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase activity, and tumor necrosis factor alpha (TNF-alpha) were higher in KCethanol than in KCcontrol, and were enhanced by AA; HSCethanol proliferated faster, increased collagen, and showed higher GSH than HSCcontrol, 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 KCcontrol or KCethanol induced HSCcontrol and HSCethanol proliferation; however, the pro-fibrogenic response in HSC(ethanol)was suppressed because of upregulation 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 HSCcontrol co-cultures, allowing for enhanced collagen deposition; furthermore, AA restored the pro-fibrogenic response in the HSCethanol 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). Conclusion: 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. (HEPATOLOGY 2008;48:2027-2039.)