Background Beta2 glycoprotein I has been suggested as LPS scavenger protein and the complex b2GPI-LPS is uptaken by monocytes. In vivo/in vitro indirect data suggest the role of Toll Like Receptor (TLR) in aPL-mediated endothelial cell (EC) activation. Furthermore TLR4 has recently been suggested as a component of a multi-protein complex on the EC surface involved in beta2GPI/anti- beta2GPI antibody-induced signalling pathway. However, what is/are the molecule(s) eventually involved in this interaction is still an open question.

Objectives To investigate the role of endothelial TLR4 in beta2GPI/anti- beta2GPI antibody interaction.

Methods i) direct binding and internalization of beta2GPI was evaluated by confocal microscopy in living CHO/TLR4 cells; ii) anti- beta2GPI antibody binding and activation (as adhesion molecule up-regulation) were investigated in TLR4- and AnnexinA2-silenced HUVEC. Receptor expression was evaluated both at mRNA and protein level by real time RT-PCR and western blotting, respectively.

Results incubation of living CHO/TLR4 cells, but not TLR4-negative CHO Mock cells, with beta2GPI-FITC resulted in a strong membrane signal shortly after the addition of the protein. After about 15 min the fluorescent signal was detected within the cytoplasm as granules, progressively more abundant in the following 20-30 min of observation, when membrane signal faded. Immunofluorescence studies on fixed cells showed the interaction between beta2GPI and TLR4 both on the cell membrane and in intracytoplasmic granules. beta2GPI/LPS complex induced EC activation evaluated as ELAM upregulation, while LPS alone or in combination with BSA had no effect.

TLR4 and AnnexinA2 silencing significantly down-regulated mRNA and protein expression in HUVEC. Both tlr4 and annexinA2 siRNA transfection strongly inhibited anti-beta2GPI antibody binding to HUVEC. On the other hand aPL-induced ELAM/ICAM-1 up-regulation was significantly decreased in tlr4 but not annexinA2 silenced cells. This observation is in line with the fact that AnnexinA2 lacks an intracytoplasmic tail and needs co-receptors to induce cell signalling. No synergic effects were observed in tlr4 and annexinA2 simultaneously silenced HUVEC.

Conclusions Our findings strongly support the binding of beta2GPI to TLR4 on EC offering the rationale for the TLR4 involvement in EC signalling and activation by aPL. At the same time, the interaction of beta2GPI with LPS and in turn with TLR4 may represent the molecular basis to explain the “two hit” theory in APS.

Disclosure of Interest None Declared