Induction of cytokine tolerance requires internalization of Chylomicron-Bound LPS into hepatocytes

BACKGROUND: Chylomicron-bound LPS (CM-LPS) renders hepatocytes unresponsive to stimulation by proinflammatory cytokines, a process termed cytokine tolerance. We have shown that cytokine tolerance is a time- and dose-dependent process requiring functional low-density lipoprotein receptors (LDLR). Thus, we hypothesized that cytokine tolerance directly correlates with the internalization of CM-LPS complexes, and inhibition of lipoprotein binding and/or internalization inhibits the induction of cytokine tolerance in hepatocytes. MATERIALS AND METHODS: We correlated the rate of internalization of radioiodinated CM-LPS complexes with hepatocellular NO production as a measure of cytokine responsiveness. In additional studies, we used four different strategies to inhibit binding/internalization of CM-LPS via LDLR and then determined the effect of each strategy on the induction of cytokine tolerance. RESULTS: There was a strong inverse correlation between the internalization of CM-LPS and the responsiveness of hepatocytes to proinflammatory cytokines (r(2) = -0.997). Furthermore, the greater the degree of LDLR inhibition, the less susceptible hepatocytes were to the induction of cytokine tolerance by CM-bound LPS. Accordingly, cytokine tolerance induction was inhibited in hepatocytes with decreased membrane expression of LDLR as compared to control cells (69 versus 12% control; P = 0.005). Competitive inhibition of CM-LPS binding prevented internalization of CM-LPS and resulted in loss of the cytokine-tolerant phenotype. Whereas CM-LPS successfully induced cytokine tolerance in ldlr(-/-) hepatocytes, it only occurred after a prolonged pretreatment period of 8 h. CM-LPS complexes containing apolipoprotein (apo) E(2) also required a prolonged pretreatment period to induce a level of cytokine tolerance comparable to that induced by CM-LPS complexes containing either apo E(3) or E(4). CONCLUSION: Lipoprotein-bound LPS inhibits the responsiveness of hepatocytes to proinflammatory cytokines in a manner directly correlated with the internalization of LPS. Furthermore, inhibition of lipoprotein binding/internalization prevents this LPS-mediated induction of cytokine tolerance in rodent hepatocytes.