Toll-like receptor 2 mediates the activation of human monocytes and endothelial cells by antiphospholipid antibodies

Abstract

IntroductionThe antiphospholipid antibody syndrome (APS) is characterized by clinical manifestations such as arterial or venous thromboembolism and/or recurrent pregnancy complications, as well as the presence of antiphospholipid antibodies (aPLAs). 1 Endothelial cells, monocytes, and platelets are targeted by aPLAs, and inflammatory activation of these cells has been proposed as a pathogenic mechanism. aPLAs are composed of heterogenous auto-antibodies that recognize plasma proteins bound to phospholipid surfaces. One antigenic target of aPLAs is the plasma phospholipid-binding protein ␤2-glycoprotein 1 (␤2GP1), which has been detected on the surface of endothelial cells and monocytes. 2,3 Patients with persistent aPLAs only occasionally present with thrombotic episodes, and sometimes bacterial or viral infections are associated with the clinical manifestations. 4 These observations suggest that the presence of aPLAs alone is not sufficient to promote thrombosis. Most likely, a priming factor of infectious or inflammatory origin is needed. The implication of infection is particularly obvious in catastrophic APS, a rare but fatal subset of APS, which presents with characteristic features comparable with those occurring in septic shock. 5,6 Stimulation of endothelial cells by aPLAs has been shown to be mediated by intracellular pathways dependent on NF-B, 7,8 p38-MAPK, 9,10 myeloid differentiation factor 88, and TNF receptor-associated factor 6. 11 The latter mediates signaling by members of the TLR family. 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. 12 Among the 10 TLRs present in humans, TLR4/MD2 forms homodimers and recognizes lipopolysaccharide (LPS), 13 whereas TLR2 heterodimerizes with TLR1 or TLR6 and recognizes bacterial triacylated or diacylated lipopeptides. 14 TLRs work with accessory proteins, which help in ligand recognition and binding. One of these, CD14, functions as an accessory protein for both TLR4 and TLR2. 13,15,16 We demonstrated previously that TLR2 is required for the activation of mouse embryonic fibroblasts by aPLAs. 17 In contrast, in a mouse thrombosis model, the absence of functional TLR4 reduced the prothrombotic effect of aPLAs. 18 Monocytes constitutively express TLR2, TLR4, and CD14,19,20 whereas endothelial cells express TLR4 and very low levels of TLR2 and CD14. 17,21,22 Expression of TLR2 by human endothelial cells is strongly increased after activation by inflammatory stimuli such as TNF, LPS, or IL-1␤. 23,24 The principal aim of the present work was to investigate the respective role of TLR2 and TLR4 in human cell activation by aPLAs. We used 3 cell types for our studies: monocytes, endothelial cells, and human embryonic kidney 293 (HEK293) cells expressing either TLR2 or TLR4. Cell activation by aPLAs was inhibited by antibodies to TLR2 or CD14, but not by antibodies t...

Receptors involved in cell activation by antiphospholipid antibodies

“…While the nature of possible first-hit conditions is still elusive, accumulating evidence, although controversial, suggest that the responses of EC, monocytes or platelets to aPLA require interactions with innate immune receptors, co-receptors and accessory proteins. As discussed above, whereas under resting conditions EC abundantly express TLR4 molecules, but little TLR2 proteins, EC activation by TNF or TLR4 ligands increases TLR2 levels [52] and aPLA-associated response of EC [34]. While the exact role of TLR4 or TLR2 in EC activation by aPLA remains to be defined, and may likely depend on the relative expression of these TLRs by EC, we propose that a first hit, such as an infectious insult, induces TLR2 up-regulation by EC through a TLR4-dependent mechanism.…”

“…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]. By using the proximity ligation assay, a technique that generates information concerning the strength of the protein-protein interactions, aPLA were found to interact more strongly with TLR2 on monocytes and cytokine activated EC than with TLR4 [34].…”

“…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]. By using the proximity ligation assay, a technique that generates information concerning the strength of the protein-protein interactions, aPLA were found to interact more strongly with TLR2 on monocytes and cytokine activated EC than with TLR4 [34]. The pathogenesis of APS is further complicated by the more recent findings that show that TNF production induced by aPLA-activated monocytes could be dependent on activation of TLR7 and TLR8 [36,56,57].…”

“…Moreover, ex vivo studies suggest a role for TLR2 [32][33][34], CD14 [34], GPIbα [35] and TLR8 [36] in aPLA-activated monocytes and EC. Below the role of each receptor/co receptor is described in more detail.…”

Toll-like receptor 2-mediated innate immune response in human nonparenchymal liver cells toward adeno-associated viral vectors

“…For prestimulation treatment, HUVECs were incubated with 100 ng/mL of recombinant HumaXpress tumor necrosis factor a (TNFa; Humanzyme, Chicago, IL) as described. 7 Production of rAAV, Empty Capsids, and MockInoculum. rAAV, empty capsids and mock-inoculum were produced in HEK293 cells (ATCC number: CRL-1573).…”