The Exploration Of Roles Of Tlr2 On β₂gPI/Anti-β₂GPI Complex Stimulated Mice Peritoneal Macrophages

Objective:The antiphospholipid syndrome(APS) is characterized by clinical manifestations such as arterial or venous thrombosis, recurrent pregnancy complications, as well as the presence of antiphospholipid antibodies(APL). The main antigenic target of APL is the plasma phospholipid-binding protein beta-2 glycoprotein I(β2GPI), and the antibodies(anti-β2GPI) are significant pathogenic autoantibodies of APS.The interaction of anti-β2GPI with membrane-bound β2GPI could activate monocytes, platelets and endothelial cells. Moreover, toll-like receptors(TLRs) are significant involved in the process of cell activation.Many studies found that TLR4 plays a major role when β2GPI induce cell activated and expressed active factors, however, it has not been obviously determined that whether TLR2 can participate in cell activation and the mechanism in APS. In the present study, we focused on whether TLR2 could induce β2GPI/anti-β2GPI complex activated murine peritoneal macrophages, and the relative importance of TLR2 and TLR4 in the process.Methods:(1)Flow cytometric analysis(FCM) was used to detect F4/80 and CD11 b,which were macrophages surface markers, in order to identify the purity of peritoneal macrophages isolated from BALB/c mice.(2)After different treatments involving β2GPI/anti-β2GPI complex,Pam3CSK4(agonist of TLR2), lipopolysaccharide(LPS),β2GPI/anti-β2GPI/TAK-242(inhibitor of TLR4),β2GPI/anti-β2GPI/anti-m TLR2-Ig G(inhibitor of TLR2),β2GPI/anti-β2GPI/TAK-242/anti-m TLR2-Ig G disposed on murine peritoneal macrophages in vitro, FCM and quantitative reverse transcription PCR(q RT-PCR) was used to detect protein and m RNA expression of TLR2, respectively.(3)The mRNA and protein levels of IL-6, IL-1β and TNF-α in murine macrophages from different groups were measured by q RT-PCR,Western blot, and immunofluorescence to determine whether TLR2 could induce β2GPI/anti-β2GPI complex promote macrophages expressing inflammatory factors.(4)To view whether the effect of MCP-1 and TF expression could be intervened, their m RNA expressions induced by β2GPI/anti-β2GPI complex in murine macrophages pretreated with inhibitors of TLR2 and TLR4 were observed by q RT-PCR. Meanwhile, the secretion of MCP-1 and the activity of TF were detected by ELISA and TF activity kit, respectively.(5)Western blot was used to explore the levels of NF-κB p65 and its phosphorylation induced by β2GPI/anti-β2GPI complex in murine macrophages, and inhibitors of TLR2 and TLR4 were utilized to investigate whether TLR2 could activate murine macrophages via NF-κB signal pathway.Results:(1)The positive rate of F4/80 and CD11b(BALB/c mice peritoneal macrophages surface markers) detected by flow cytometry was about93%, therefore, the purity of isolated macrophages was high.(2)The β2GPI/anti-β2GPI complex, Pam3CSK4 and LPS could increase protein and m RNA levels of TLR2 in mice peritoneal macrophages(p<0.05 vs control). The inhibitory effect of anti-m TLR2-Ig G, or TAK-242, or combination of both on the protein expression of TLR2 was no statistical significance. However, anti-m TLR2-Ig G could block the effect of above stimuli on the expression of TLR2 m RNA(p<0.05 vs β2GPI/anti-β2GPI), which was weaker than that of TAK-242, and the combination effect did not show much stronger inhibitory effects.(3)The β2GPI/anti-β2GPI complex, Pam3CSK4 and LPS could obviously enhance the m RNA expression of inflammatory factors involving IL-6, IL-1β and TNF-α, which could be inhibited by anti-m TLR2-Ig G. Nevertheless, the inhibitory effect of anti-m TLR2-Ig G was weaker than that of TAK-242, and the combination effect was not much stronger. The trend of protein levels of IL-6, IL-1β and TNF-α was similar to their m RNA levels.(4)MCP-1 and TF m RNA level stimulated by β2GPI/anti-β2GPI complex,Pam3CSK4 and LPS in mice peritoneal macrophages in vitro were significantly increased. Anti-m TLR2-Ig G could attenuate the effects,while the depression effect of TAK-242 was not much stronger, and the combination of both showed the strongest inhibitory effect. The secretion level of MCP-1 and TF activity were in accordance with their m RNA expression.(5)Expression of p-NF-κB p65 was enhanced by β2GPI/anti-β2GPI complex, Pam3CSK4 and LPS in macrophages. The blocking effect of anti-m TLR2-Ig G on macrophages stimulated by β2GPI/anti-β2GPI complex was weaker than that of TAK-242, and the combined use of both showed the maximum blocking effect.Conclusions:(1)In the process of β2GPI/anti-β2GPI complex activated murine peritoneal macrophages, the expression of TLR2 was increased, while TLR2 m RNA level was decreased by inhibitors of TLR2 and TLR4. It indicated that TLR2 played a certain role on the pathological process of APS.(2)The β2GPI/anti-β2GPI complex obviously enhanced the expression of inflammatory factors, MCP-1 and TF in murine peritoneal macrophages, and the effect could be blocked by TLR2 inhibitor,whereas the inhibitory effect of TLR4 inhibitor was much stronger,suggesting that TLR2 was partly involved in the activation of macrophages mediated by β2GPI/anti-β2GPI complex.(3)TLR2 inhibitor could inhibit the activation effect of NF-κB signaling molecule induced by β2GPI/anti-β2GPI complex in murine peritoneal macrophages. But the inhibitory effect was weaker than that of TLR4 inhibitor, and the combination of both showed much stronger inhibitory effect. It advised that both TLR4 and TLR2 were involved in the pathological process of APS via NF-κB signal pathway, while the effect of TLR2 was weaker than that of TLR4.