Effect of chronic ethanol administration on the in vitro production of proinflammatory cytokines by rat Kupffer cells in the presence of apoptotic cells

Background: Chronic ethanol consumption can lead to a variety of pathological consequences by as yet undefined mechanisms. Recently, it has been noted that alcohol-associated liver disease is often accompanied by morphological liver changes that include the increased production of apoptotic cells. Additionally, it has been demonstrated that hepatocellular uptake and removal of potentially damaging apoptotic cells is impaired after ethanol treatment. The aim of the present study was to determine whether the presence of apoptotic cells leads to Kupffer cell (KC) production and release of proinflammatory cytokines that have been linked to hepatocyte damage, such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha). Methods: Kupffer cells were isolated from female rats after an 8-week oral administration of a dextrose control or ethanol-containing fish-oil diet. The isolated KCs were cultured for up to 24 hours in the absence or presence of apoptotic or nonapoptotic hepatoma cells, or lipopolysaccharide. After incubation, media from the cultures were assayed for the presence of TNF-a and IL-6 by immunoassay detection. Also, the expression of these cytokines was measured in KC lysates by a quantitative real-time polymerase chain reaction. Results: Kupffer cells cultured for up to 24 hours in the presence of apoptotic cells produced significantly more TNF-alpha and IL-6 (80 and 60%, respectively, p < 0.05) when the cells were isolated from ethanol-fed animals compared with controls. Additionally, after as early as 4 hours in culture with apoptotic cells, mRNA levels of both cytokines were increased (2-5-fold) in KCs isolated from ethanol-fed animals compared with controls. Conclusions: The presence of apoptotic cells results in the in vitro activation of KCs. Additionally, chronic ethanol administration results in an enhanced responsiveness of KCs to produce proinflammatory cytokines indicated by the increased production of inflammatory mediators from KCs obtained from ethanol-fed animals.