Equipe d'Accueil 2216, Université de Brest, France.
J Immunol. 2010 Aug 1;185(3):1550-7. doi: 10.4049/jimmunol.1000526. Epub 2010 Jul 2.
During the antiphospholipid syndrome, beta2-gpI interacts with phospholipids on endothelial cell (EC) surface to allow the binding of autoantibodies. However, induced-pathogenic intracellular signals suggest that beta2-gpI associates also with a receptor that is still not clearly identified. TLR2 and TLR4 have long been suspected, yet interactions between TLRs and beta2-gpI have never been unequivocally proven. The aim of the study was to identify the TLR directly involved in the binding of beta2-gpI on EC surface. beta2-gpI was not synthesized and secreted by ECs in vitro, but rather taken up from FCS. This uptake occurred through association with TLR2 and TLR4 which partitioned together in the lipid rafts of ECs. After coimmunoprecipitation, mass-spectrometry identification of peptides demonstrated that TLR2, but not TLR4, was implicated in the beta2-gpI retention. These results were further confirmed by plasmon resonance-based studies. Finally, siRNA were used to obtain TLR2-deficient ECs that lost their ability to bind biotinylated beta2-gpI and to trigger downstream phosphorylation of kinases and activation of NFkappaB. TLR4 may upregulate TLR2 expression, thereby contributing to beta2-gpI uptake. However, our data demonstrate that direct binding of beta2-gpI on EC surface occurs through direct interaction with TLR2. Furthermore, signaling for anti-beta2-gpI may be envisioned as a multiprotein complex concentrated in lipid rafts on the EC membrane.
在抗磷脂综合征中，β2-GPI 与内皮细胞（EC）表面的磷脂相互作用，允许自身抗体结合。然而，诱导的致病细胞内信号表明，β2-GPI 还与尚未明确鉴定的受体结合。TLR2 和 TLR4 早已被怀疑，但 TLRs 与β2-GPI 之间的相互作用从未被明确证实。本研究旨在确定直接参与β2-GPI 在 EC 表面结合的 TLR。β2-GPI 不是由体外 EC 合成和分泌的，而是从 FCS 中摄取的。这种摄取是通过与 TLR2 和 TLR4 结合来实现的，TLR2 和 TLR4 一起分布在 EC 的脂筏中。共免疫沉淀后，肽的质谱鉴定表明 TLR2 而非 TLR4 参与了β2-GPI 的保留。这些结果通过基于等离子体共振的研究进一步得到证实。最后，使用 siRNA 获得 TLR2 缺陷的 EC，这些 EC 丧失了结合生物素化的β2-GPI 和触发下游激酶磷酸化和 NFkappaB 激活的能力。TLR4 可能上调 TLR2 的表达，从而促进β2-GPI 的摄取。然而，我们的数据表明，β2-GPI 在 EC 表面的直接结合是通过与 TLR2 的直接相互作用发生的。此外，针对抗β2-GPI 的信号可以被设想为集中在 EC 膜上脂筏中的多蛋白复合物。
