Endotoxin tolerance attenuates LPS-induced TLR4 mobilization to lipid rafts: a condition reversed by PKC activation

Abstract

Endotoxin tolerance is characterized by attenuated macrophage activation to subsequent LPS challenge and can be reversed through nonspecific protein kinase C (PKC) activation, and activation by LPS within naïve cells requires the activation of the cell surface receptors CD14 and TLR4 on lipid rafts. The effect of PKC activation and endotoxin tolerance on lipid raft receptor complex assembly is unknown and the focus of this study. Tolerance was induced in THP-1 cells through LPS pre-exposure. Naïve and tolerant cells were stimulated with LPS, with or without PMA pretreatment to activate PKC. TLR4 surface expression and LPS binding were determined by flow cytometry and immunohistochemistry. Cellular and lipid raft protein was analyzed for the presence and activation of the TLR4 complex components. Harvested supernatants were examined for TNF-α production. Total TLR4 surface expression and LPS binding were not affected by tolerance induction. LPS stimulation of naïve cells resulted in TLR4 and heat shock protein (HSP)70 lipid raft mobilization, MAPK activation, and TNF-α production. LPS stimulation of tolerant cells was associated with attenuation of all of these cellular events. Although PKC activation by PMA had no effect on naïve cells, it did result in reversal in tolerance-induced suppression of TLR4 and HSP70 lipid raft mobilization, MAPK activation, and TNF-α production. In addition, the effects associated with PMA were reversed with exposure to a myristoylated PKC-ζ pseudosubstrate. Thus, endotoxin tolerance appears to be induced through attenuated TLR4 formation following LPS stimulation. This complex formation appears to be PKC-dependent, and restoration of PKC activity reverses tolerance.