Anti-β2GPI/β2GPI induced TF and TNF-α expression in monocytes involving both TLR4/MyD88 and TLR4/TRIF signaling pathways
Introduction
The antiphospholipid syndrome (APS) is an autoimmune disorder of acquired hypercoagulability and characterized by the presence of antiphospholipid (aPL) antibodies in the plasma of patients with vascular thrombosis, recurrent complications of pregnancy, or both (Miyakis et al., 2006). The prothrombotic status has been related to the induction of a proinflammatory and procoagulant phenotype induced by aPL that reacts with serum phospholipid binding proteins, among them, β2-glycoprotein I (β2GPI) is described as the major antigenic target (Tanne et al., 2008, Willis et al., 2012). Anti-β2GPI antibodies are found frequently in the plasma of the patients, suggesting their important roles in APS. Growing evidences suggest that anti-β2GPI antibodies could stimulate monocytes and vascular endothelium to express tissue factor (TF) activity and exert procoagulant effects (Nojima et al., 2008, Seshan et al., 2009). Moreover, anti-β2GPI antibodies could activate endothelial cells, resulting in an enhanced expression and secretion of procoagulant cytokines, such as TNF-α and IL-1β, which are beneficial to the thrombus formation of the syndrome (Mehdi et al., 2010, Mulla et al., 2009).
Stimulation of endothelial cells or monocytes by aPL has been shown to be mediated by intracellular pathways dependent on some receptors which help ligand recognition and binding. However, the cell surface receptors involving in the interaction between anti-β2GPI/β2GPI and blood monocytes, and the signal transduction pathways leading to TF and cytokines expression are not thoroughly understood. Previous studies have suggested that annexin A2 (ANX2), a member of a large family of annexin proteins, is the main specific receptor for β2GPI on certain cell surfaces. ANX2 is richly expressed on the surface membrane of many cells and mediates the pathogenic effects of aPL in vivo and in vitro (Romay-Penabad et al., 2009, Zhang and McCrae, 2005, Zhou et al., 2007). However, ANX2 does not span the cell membrane. Thus it unlikely involves in intracellular signaling transduction. On the other hand, some infectious agents could not only trigger the production of autoantibodies cross-reacting with phospholipid binding proteins, but also enhance the secretion of inflammatory molecules that potentiate the thrombogenic effects of aPL (Blank et al., 2004, Shoenfeld et al., 2006). In this regard, it has been proposed that transmembrane proteins of toll-like receptors (TLRs) may exist to associate with ANX2 on cell surfaces to lead to intracellular signal transduction (Hurst et al., 2010).
The TLRs are a family of integral membrane proteins that recognize conserved pathogen-associated molecular patterns. These receptors thereby function as the first line of defense against pathogens and are essential factors in the innate immune response (Ostuni et al., 2010). Among human TLRs, TLR4 has been reported to mediate aPL-induced endothelial cell or monocytic cell activation (Pierangeli et al., 2007, Sorice et al., 2007). Like most TLRs, TLR4 is structurally characterized by extracellular leucine-rich repeats (LPRs) and Toll/IL-1 receptor (TIR) signaling. Once binding to ligands, TLR4 can trigger both MyD88-dependent and TRIF-dependent (MyD88-independent) signal transduction pathways. Furthermore, TLR4 could induce activation of AP-1 and NF-κB, leading to alteration of cell function (McGettrick and O’Neill, 2010, Ostuni et al., 2010).
Our previous study has demonstrated that certain aPL (mainly anti-β2GPI) with its antigen could induce monocyte TF activation in APS, and ANX2 and TLR4 on cell surface could function as mediators participating in anti-β2GPI/β2GPI complex-stimulated TF expression in monocytes (Zhou et al., 2009, Zhou et al., 2004, Zhou et al., 2011). In the present study, we mainly investigated whether MyD88 and TRIF are involved in anti-β2GPI/β2GPI-induced, TLR4-mediated monocyte activation and explored the relationship among β2GPI, ANX2 and TLR4.
Section snippets
Monocytes
Peripheral blood mononuclear cells (PBMCs) were isolated from blood buffy coats of healthy volunteers as described previously (Zhou et al., 2004). After washed twice and resuspended in Macrophage SFM medium (Gibco BRL, Grand Island, NY, USA), 5 × 104 cells were inoculated into each well of 96-well tissue culture plates and incubated at 37 °C in a humidified incubator supplemented with 5% CO2 for 1 h to allow monocytes to adhere. Non-adherent cells were removed by washing 3 times with medium. The
Anti-β2GPI/β2GPI complex induces TF and TNF-α expression in monocytes
Treatment of blood monocytes or THP-1 cells with anti-β2GPI (10 μg/ml)/β2GPI (100 μg/ml) complex significantly enhanced TF and TNF-α expression at the mRNA level (Fig. 1A and B) compared to untreated cells (p < 0.05). The TF activity and TNF-α protein levels were also increased (Fig. 1C and D). The effects of anti-β2GPI/β2GPI complex on blood monocytes seemed stronger than those on THP-1 cells, almost comparable to those of LPS (500 ng/ml). However, TF and TNF-α expression did not increase in cells
Discussion
Antiphospholipid syndrome is an acquired autoimmune disease which differs from most other systemic autoimmune diseases by its propensity to develop thrombosis. aPL antibodies, which bind to a range of cellular targets such as platelets, monocytes and endothelial cells, could up-regulate TF. As it is reported, the concentration of physiologically active TF in non-cytokine-stimulated blood from healthy individuals cannot exceed and is probably lower than 20 fM (Butenas et al., 2005).
Disclosure
None.
Acknowledgments
This work was supported by National Natural Science Foundation of China (no. 30971301) to Hong Zhou, Sci-tech Innovation Team of Jiangsu Province (no. LJ201116) to Jinchuan Yan and Student's Scientific Research of Jiangsu University (no. 10A107) to Hongxiang Xie.
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