Role of TLR‑4 in anti‑β2‑glycoprotein I‑induced activation of peritoneal macrophages and vascular endothelial cells in mice.

Abstract

Anti-phospholipid syndrome (APS) is a systematic autoimmune disease that is associated with presence of antiphospholipid antibodies (aPL), recurrent thrombosis, and fetal morbidity in pregnancy. Toll-like receptor-4 (TLR-4), a member of TLR family, is known to have a fundamental role in pathogen recognition and activation of innate immunity. The β2-glycoprotein I (β2GPI), a protein circulating in the blood at a high concentration, is able of scavenging lipopolysaccharide (LPS) and clear unwanted anionic cellular remnants, such as microparticles, from the circulation. Our previous study demonstrated that TLR-4 and its signaling pathways contribute to the upregulation of pro-coagulant factors and pro-inflammatory cytokines in monocytes induced by anti-β2GPI in vitro. The present study aimed to define the roles of TLR-4 in vivo. C3H/HeN mice (TLR-4 intact) and C3H/HeJ mice (TLR-4 defective) were stimulated with an intraperitoneal injection with anti-β2GPI-immunoglobulin G(IgG), then peritoneal macrophages and vascular endothelial cells (VECs) were extracted from treated mice, and analyses were conducted on the expression profiles of pro-inflammatory cytokines and adhesion molecules. The results demonstrated that the expression of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6, in the peritoneal macrophages, and adhesion molecules, including intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, in VECs of C3H/HeN mice (TLR-4 intact) were significantly higher than those of C3H/HeJ mice (TLR-4 defective). The phosphorylation levels of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) p65 in peritoneal macrophages and VECs from C3H/HeN mice stimulated with anti-β2GPI–IgG were significantly increased compared with those from C3H/HeJ mice (TLR-4 defective). The isotype control antibody (NR-IgG) had no such effects on peritoneal macrophages and VECs. Furthermore, the inhibitors of TLR-4, p38 MAPK and NF-κB may significantly reduce the anti-β2GPI–IgG-induced TNF-α, IL-1β and IL-6 mRNAs expression in the peritoneal macrophages from TLR-4 intact mice. The results indicated that a TLR-4 signal transduction pathway is involved in anti-β2GPI–IgG-induced activation of peritoneal macrophages and VECs. This study has provided a basis for subsequent investigations to elucidate the pathological mechanisms underlying anti-phospholipid syndrome.