β2-glycoprotein I, lipopolysaccharide and endothelial TLR4 : three players in the two hit theory for anti-phospholipid-mediated thrombosis

The thrombogenic effect of \u3b22-glycoprotein I (\u3b22GPI)-dependent anti-phospholipid antibodies (aPL) in animal models was found to be LPS dependent. Since \u3b22GPI behaves as LPS scavenger, LPS/\u3b22GPI complex was suggested to account for in\ua0vitro cell activation through LPS/TLR4 involvement being LPS the actual bridge ligand between \u3b22GPI and TLR4 at least in monocytes/macrophages. However, no definite information is available on the interaction among \u3b22GPI, LPS and endothelial TLR4 in spite of the main role of endothelial cells (EC) in clotting. To analyse at the endothelial level the need of LPS, we investigated the in\ua0vitro interaction of \u3b22GPI with endothelial TLR4 and we assessed the role of LPS in such an interaction. To do this, we evaluated the direct binding and internalization of \u3b22GPI by confocal microscopy in living TLR4-MD2 transfected CHO cells (CHO/TLR4-MD2) and \u3b22GPI binding to CHO/TLR4-MD2 cells and human umbilical cord vein EC (HUVEC) by flow cytometry and cell-ELISA using anti-\u3b22GPI monoclonal antibodies in the absence or presence of various concentrations of exogenous LPS. To further investigate the role of TLR4, we performed anti-\u3b22GPI antibody binding and adhesion molecule up-regulation in TLR4-silenced HUVEC. Confocal microscopy studies show that \u3b22GPI does interact with TLR4 at the cell membrane and is internalized in cytoplasmic granules in CHO/TLR4-MD2 cells. \u3b22GPI binding to CHO/TLR4-MD2 cells and HUVEC is also confirmed by flow cytometry and cell-ELISA, respectively. The interaction between \u3b22GPI and TLR4 is confirmed by the reduction of anti-\u3b22GPI antibody binding and by the up-regulation of E-selectin or ICAM-1 by TLR4 silencing in HUVEC. \u3b22GPI binding is not affected by LPS at concentrations comparable to those found in both \u3b22GPI and antibody preparations. Only higher amount of LPS that can activate EC and up-regulate TLR4 expression are found to increase the binding. Our findings demonstrate that \u3b22GPI interacts directly with TLR4 expressed on EC, and that such interaction may contribute to \u3b22GPI-dependent aPL-mediated EC activation. At variance of monocytic cells, we also showed a threshold effect for the action of LPS, that is able to enhance anti-\u3b22GPI antibody EC binding only at cell activating concentrations, shown to increase TLR4 expression. This in\ua0vitro model may explain why LPS behaves as a second hit increasing the expression of \u3b22GPI in vascular tissues and triggering aPL-mediated thrombosis in experimental animals