Anti–β₂‐glycoprotein I antibodies induce monocyte release of tumor necrosis factor α and tissue factor by signal transduction pathways involving lipid rafts

Abstract

Objective. To investigate the association of ␤ 2 -glycoprotein I (␤ 2 GPI) with lipid rafts in monocytic cells and to evaluate the proinflammatory and procoagulant effects of anti-␤ 2 GPI binding to its target antigen on the monocyte plasma membrane.Methods. Human monocytes were fractionated by sucrose density-gradient centrifugation and analyzed by Western blotting. Immunoprecipitation experiments were performed to analyze the association of ␤ 2 GPI with lipid rafts and the possible interaction of ␤ 2 GPI with annexin A2 and Toll-like receptor 4 (TLR-4). Monocytes were then stimulated with affinity-purified anti-␤ 2 GPI antibodies from patients with the antiphospholipid syndrome (APS). Interleukin-1 receptor-associated kinase (IRAK) phosphorylation and NF-B activation were evaluated by immunoprecipitation and transcription factor assay, respectively. Supernatants from monocytes were tested for tumor necrosis factor ␣ (TNF␣) and tissue factor (TF) levels by enzyme-linked immunosorbent assay.Results. We found ␤ 2 GPI and its putative receptor annexin A2 in lipid raft fractions of human monocytes. Moreover, there was an association between ␤ 2 GPI and TLR-4, suggesting that it was partially dependent on raft integrity. Triggering with anti-␤ 2 GPI antibodies induced IRAK phosphorylation and consequent NF-B activation, which led to the release of TNF␣ and TF.Conclusion. Anti-␤ 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-␤ 2 GPI binding triggers IRAK phosphorylation and NF-B translocation, leading to a proinflammatory and procoagulant monocyte phenotype characterized by the release of TNF␣ and TF, respectively. These findings provide new insight into the pathogenesis of APS, improving our knowledge of valuable therapeutic targets.

Receptors involved in cell activation by antiphospholipid antibodies

“…TLR2 and TLR4 are both expressed on platelets [53] and on monocytes [54]. Sorice et al [55] have identified that TLR4, β2GP1 and annexin A2 form an activation cluster in plasma membrane microdomains of human monocytes after LPS or aPLA ligation. This study however did not evaluate the association of β2GP1 with other TLRs in lipid rafts in monocytic cells, and in particular because TLR2 and CD14 are required for aPLA recognition by monocytes and EC [34].…”

“…Among the important signaling intermediates that play a central role in this process are proximal mediators of innate responses, such as NF-kB [80][81][82], p38-MAP kinase [83,84], ERK [85] and IRAK [55] as well as TRIF, MyD88 and TRAF6 [86]. In addition, the PI3K/Akt pathway seems to play a specific role in platelet activation by aPLA.…”

The role of TLR4 in pathophysiology of antiphospholipid syndrome‐associated thrombosis and pregnancy morbidity

“…Similar results were also found in monocytes. Sorice et al (2007) observed a preferential association of b2GPI dimers and ANXA2 within lipid rafts in the plasma membrane of monocytes. Both human anti-b2GP antibodies and LPS could recruit TLR4 to lipid rafts bound to b2GPI, triggering a TLR4-mediated signalling cascade with consequent IRAK phosphorylation and NF-jB activation, and resulting in the release of TNF-a, as well as of TF, which may contribute to the pathogenesis of thrombosis in patients with APS.…”

“…Recent studies have shown that affinitypurified human anti-b2GPI antibodies induce thrombosis in mice (Galli et al, 2003;Arad et al, 2011). Anti-b2GPI antibodies may promote thrombosis through b2GPI-dependent activation of ECs by increasing the expression of adhesion molecules (E-selectin SELE, vascular cell adhesion molecule 1 [VCAM1], intercellular adhesion molecule 1 [ICAM1]) and tissue factor (TF; coagulation factor III, FIII, F3) and enhancing the expression, synthesis, and/or secretion of proinflammatory cytokines (interleukin (IL)-1b [IL1B], I-6, IL8, monocyte chemotactic protein 1 [MCP1; chemokine (C-C motif) ligand 2, CCL2], tumour necrosis factor-a [TNF-a; TNF]) and chemokines, and eventually lead to a pro-inflammatory and procoagulant monocyte phenotype characterized by the release of TNF-a and TF, respectively (Meroni et al, 2001;Conti et al, 2003;Riboldi et al, 2003;Sorice et al, 2007). TF plays a critical role in APS pathogenic processes.…”

“…Activation of the MAPK pathway and NF-kB is an essential late step in TLR4 signal transduction pathways. Several research groups working independently have shown the integral role for p38MAPK and NF-jB in the transduction cascade of aPLinduced cell activation (Dunoyer-Geindre et al, 2002;Bohgaki et al, 2004;Simoncini et al, 2005;Sorice et al, 2007), while there is both in vitro and in vivo evidence that direct inhibitors of the two transcription factors interfere with aPL-induced pathogenesis (Montiel-Manzano et al, 2007;Vega-Ostertag et al, 2007). Therefore, inhibitors of NF-jB and p38MAPK may be a worthwhile approach in the APS setting.…”