Ethanol Feeding Potentiates the Pro-Inflammatory Response of Kupffer Cells to Cellular Fibronectin

Background: Excessive alcohol consumption leads to the increased extracellular matrix deposition of cellular fibronectin (cFn) in the liver, which is also implicated as an initiating event in the fibrogenic process. We propose that cFn directly stimulates Kupffer cells (KCs), which are involved in the early response to tissue damage, to produce factors that enhance the progression of alcohol-induced liver injury toward inflammation and fibrosis. Method: KCs were isolated from rats fed a control or ethanol liquid diet for 4 to 6 weeks. The effect of exogenous cFn on KC viability and the secretion of the cytokines, TNF-alpha and IL-6, as well as of matrix remodeling factors, MMP-2 and TIMP-2, was determined after 20 hours of cell culture. Results: For KCs from both control- and ethanol-fed rats, viability remained unaffected by treatment with cFn. TNF-alpha and IL-6 production were increased in KCs exposed to cFn, with cells treated with 1, 2.5, and 5 mu g/ml cFn secreting significantly higher levels of both cytokines compared with untreated cells (p < 0.05). Chronic ethanol administration resulted in a significantly enhanced secretion of IL-6 by KCs regardless of treatment with cFn. When MMP-2 protein and activity levels were measured by western blot analysis and gelatin zymography, respectively, we found that cFn stimulated a dramatic increase in both cells from ethanol- and control-fed rats, with the KCs from ethanol animals being more responsive to cFn at higher concentrations (p < 0.05). Significantly higher levels of TIMP-2, which inhibits both the activation and activity of MMP-2, were secreted by KCs treated with 5 mu g/ml cFn. Correspondingly, more pro-MMP-2 than active-MMP-2 was detected. Conclusion: Altogether, these results show that cFn stimulates KCs to produce factors that may enhance the promotion of tissue damage and that ethanol administration increases these responses.