Synergistic Effect Of TLR4 And TLR2 Agonists On The Expression Of SR-A And Its Role In The Endotoxin Tolerance Of RAW264.7

Pre-exposure of macrophage to LPS leads to hypersensitivity to secondary LPS stimulation, which is known as endotoxin tolerance. As TLR (Toll-like receptor) 4 is the major signaling receptor for LPS, many investigations have focused on changes in the TLR4 signaling pathways as a mechanism of endotoxin tolerance. Although gene deletion studies indicate that the SR-A (macrophage scavenger receptor A) protects mice from LPS-induced endotoxemia, its role in endotoxin tolerance is still unknown. Here we reported that LPS could induce the expression of SR-A on the mouse macrophage cell line RAW264.7 in both dose- and time-dependent manners, which was correlated well with inflammatory cytokines suppression in RAW264.7 upon secondary stimulation of LPS. Over expression of SR-A in RAW264.7 suppressed both TNF-αand NF-κB activation, demonstrating the involvement of SR-A in endotoxin tolerance of RAW264.7. In addition, LPS-pretreated RAW264.7 could bind and internalize more FITC-LPS than that of the un-pretreated cells, and both SR-A ligand fucoidan and anti-SR-A 2F8 could completely suppress the LPS-induced binding and internalization of FITC-LPS by RAW264.7. The results suggested that LPS would promote RAW264.7 to bind and internalize FITC-LPS by inducing SR-A expression. Furthermore, we also found that over expression of SR-A?1-49 in RAW264.7 could also suppress both TNF-αand NF-κB activation, implying that binding of LPS to SR-A could inhibit the activation of RAW264.7 by LPS. Although TLR4 was the signaling receptor of LPS, inhibition of TLR4 signaling with MTS510 didn't suppress SR-A expression on RAW264.7 stimulated by LPS. In contrast, p38 specific inhibitor SB203580 could completely suppress the expression of SR-A induced by LPS. Our results demonstrated that p38, but not TLR4, dependent up-regulation of SR-A was also involved into endotoxin tolerance through binding and internalization of excess LPS.It was reported that sLPS (Escherichia coli O111:B4 LPS from Sigma) was able to induce the expression of SR-A on murine macrophage cell line RAW264.7. However, many...