Sorice Maurizio, Longo Agostina, Capozzi Antonella, Garofalo Tina, Misasi Roberta, Alessandri Cristiano, Conti Fabrizio, Buttari Brigitta, Riganò Rachele, Ortona Elena, Valesini Guido
Sapienza University of Rome, Viale del Policlinico 155, 00161 Rome, Italy.
Arthritis Rheum. 2007 Aug;56(8):2687-97. doi: 10.1002/art.22802.
To investigate the association of beta(2)-glycoprotein I (beta(2)GPI) with lipid rafts in monocytic cells and to evaluate the proinflammatory and procoagulant effects of anti-beta(2)GPI binding to its target antigen on the monocyte plasma membrane.
Human monocytes were fractionated by sucrose density-gradient centrifugation and analyzed by Western blotting. Immunoprecipitation experiments were performed to analyze the association of beta(2)GPI with lipid rafts and the possible interaction of beta(2)GPI with annexin A2 and Toll-like receptor 4 (TLR-4). Monocytes were then stimulated with affinity-purified anti-beta(2)GPI antibodies from patients with the antiphospholipid syndrome (APS). Interleukin-1 receptor-associated kinase (IRAK) phosphorylation and NF-kappaB activation were evaluated by immunoprecipitation and transcription factor assay, respectively. Supernatants from monocytes were tested for tumor necrosis factor alpha (TNFalpha) and tissue factor (TF) levels by enzyme-linked immunosorbent assay.
We found beta(2)GPI and its putative receptor annexin A2 in lipid raft fractions of human monocytes. Moreover, there was an association between beta(2)GPI and TLR-4, suggesting that it was partially dependent on raft integrity. Triggering with anti-beta(2)GPI antibodies induced IRAK phosphorylation and consequent NF-kappaB activation, which led to the release of TNFalpha and TF.
Anti-beta(2)GPI antibodies react with their target antigen, likely in association with annexin A2 and TLR-4, in lipid rafts in the monocyte plasma membrane. Anti-beta(2)GPI binding triggers IRAK phosphorylation and NF-kappaB translocation, leading to a proinflammatory and procoagulant monocyte phenotype characterized by the release of TNFalpha and TF, respectively. These findings provide new insight into the pathogenesis of APS, improving our knowledge of valuable therapeutic targets.
研究β2糖蛋白I（β2GPI）与单核细胞中脂筏的关联，并评估抗β2GPI与其靶抗原结合对单核细胞质膜的促炎和促凝作用。
通过蔗糖密度梯度离心法分离人单核细胞，并进行蛋白质免疫印迹分析。进行免疫沉淀实验以分析β2GPI与脂筏的关联以及β2GPI与膜联蛋白A2和Toll样受体4（TLR-4）的可能相互作用。然后用来自抗磷脂综合征（APS）患者的亲和纯化抗β2GPI抗体刺激单核细胞。分别通过免疫沉淀和转录因子测定评估白细胞介素-1受体相关激酶（IRAK）磷酸化和核因子κB（NF-κB）激活。通过酶联免疫吸附测定法检测单核细胞上清液中的肿瘤坏死因子α（TNFα）和组织因子（TF）水平。
我们在人单核细胞的脂筏组分中发现了β2GPI及其假定受体膜联蛋白A2。此外，β2GPI与TLR-4之间存在关联，表明其部分依赖于脂筏完整性。用抗β2GPI抗体触发可诱导IRAK磷酸化并随后激活NF-κB，从而导致TNFα和TF的释放。
抗β2GPI抗体与其靶抗原反应，可能与膜联蛋白A2和TLR-4相关，存在于单核细胞质膜的脂筏中。抗β2GPI结合触发IRAK磷酸化和NF-κB易位，导致分别以TNFα和TF释放为特征的促炎和促凝单核细胞表型。这些发现为APS的发病机制提供了新的见解，增进了我们对有价值的治疗靶点的认识。
