The Activation Of THP-1 Cells By Anti-β2GPI/β2GPI Complex And Its Signal Transduction Pathway

Objective:Our previous study has shown that anti-β2GPI/β2GPI complex could stimulate blood monocytes and monocytic cell line THP-1 to express tissue factor (TF), which becomes one of mechanisms of thrombosis in antiphospholipid syndrome (APS). Toll-like receptor 4 (TLR4) and annexin A2 (ANX2) may be involved in these processes as receptors. In this study, we mainly investigate whether anti-β2GPI/β2GPI complex can activate THP-1 cells to express some proinflammatory molecules (IL-6, IL-8 and TNF-α), thereby contributing to the pathological process in APS.Methods:①The expression TF, IL-6, IL-8 and TNF-αmRNAs in THP-1 cells was detected by Real-time PCR after stimulating with anti-β2GPI/β2GPI complex. The protein levels of IL-6, IL-8 and TNF-αsecreted from cells were analyzed using immuno fluorescence method. The TF activity in cells was detected by the commercial Kit.②The binding of receptors (TLR4 and ANX2) on THP-1 cells to (32GPI was analyzed withβ2GPI-Affi-Gel columns.③The expression of TLR4, MyD88 (myeloid differentiation factor 88) and MD-2 (myeloid differentiation protein 2) in THP-1 cells induced by anti-β2GPI/β2GPI complex was investigated by Real-time PCR and western blotting.④The special inhibitor of TLR4, TAK242, and MD-2 inhibitor, paclitaxel, were used in assays to analyze whether the effects of anti-β2GPI/β2GPI on THP-1 cells activation, including IL-6, IL-8 and TNF-αexpression, could be inhibited.Results:①The TF expression at both mRNA and its activity in THP-1 cells was Significantly up-regulated with treatment of anti-β2GPI/β2GPI complex (100μg/mL), compared with untreated cells (p<0.05). The expression of IL-6, IL-8 and TNF-αin cell was also increased, in the manner of time-dependence, with the maximal levels at 2 h of stimulation (p<0.0\ vs control).②The TLR4 and ANX2 on THP-1 cell membrane could bind toβ2GPI-Affi-Gel columns.③After THP-1 cells were stimulated with anti-β2GPI/β2GPI (100μg/ml) complex, the TLR4, MD-2 and MyD88 expression (both mRNA and protein) in cells was significantly increased (p<0.05 vs control).④Both TAK2425 (μM/L) and paclitaxel (1μmol/L) could inhibit the stimulating effects of anti-β2GPI/β2GPI on THP-1 cells, decreasing the expression of IL-6, IL-8 and TNF-α(P<0.01 vs on inhibitors).Conclusions:1. Anti-β2GPI/β2GPI complex can increase not only TF, but also IL-6, IL-8 and TNF-αexpression in monocytic cell line THP-1 cells, suggesting that antiphospholipid antibodies may contribute to the pathological process in APS by enhancing some proinflammatory molecules expression.2. The TLR4, as the co-receptor of ANX2, can mediate anti-β2GPI/β2GPI complex activating THP-1 cells.3. The expression of TLR4 and its adaptor molecules, MyD88 and MD-2, can be up-regulated when anti-β2GPI/β2GPI complex induced THP-1 cell activation, suggesting that TLR4 and their signal transduction pathway can be the targets for the prevention and treatment of thrombosis in APS.4. The TLR4 inhibitor (TAK242) and MD-2 inhibitor (paclitaxel) can intervene with the effects of anti-β2GPI/β2GPI on THP-1 cells activation, decreasing IL-6, IL-8 and TNF-αexpression, indicating the new way for therapy of thrombosis in APS.