OxLDL Antibody Inhibits MCP-1 Release In Monocytes/macrophages By Regulating Ca²⁺/K⁺ Channel Flow

Atherosclerosis is a chronic inflammatory disease induced by imbalance of lipid metabolism-hyperlipaemia.Oxidation of low-density lipoprotein(oxLDL)induces macrophage release of monocyte chemoattractant protein-1(MCP-1)and overload uptake of lipid which lead to the foam cell formation and deposition of fatty plaque.Recently,oxLDL peptide vaccine and its antibody adoptive transferring have shown a significantly preventive or therapeutic effect in atherosclerotic animal model.Moreover,in vitro,anti-oxLDL antibody(BI-204)exhibits an inhibitory effect on the release of MCP-1 induced by oxLDL in monocyte/macrophages.However,the molecular mechanism of the reduction of MCP-1 by anti-oxLDL antibody is still obscure.Here,we report that oxLDL induced MCP-1 release in monocytes/macrophages through their Toll-like receptor 4(TLR-4)and ERK pathway.Using blocking antibodies against CD36,TLR-4,SR-AI and LOX-1 to interact with monocytes/macrophages,only TLR-4 antibody was found to have an inhibitory effect on MCP-1 release triggered by oxLDL.In contrast,administration of the inhibitors of p38 MAPK,JNK and ERK respectively,all resulted in inhibitory effect on oxLDL-induced MCP-1 release.In addition,the release of MCP-1 triggered by oxLDL was regulated by calcium channel and potassium channel.The application of calcium channel inhibitor nifedipine,or potassium channel(potassium ion efflux channel)inhibitor glyburide,both restrained MCP-1 release induced by oxLDL in monocytes/macrophages.Furthermore,under fluorescence confocal microscope,oxLDL was found to enhance the influx of extracellular calcium and the following intracellular calcium[Ca2+]i oscillation.Using patch clamp to observe the ion current of cellular membrane,we verified that oxLDL stimulation elicited a reduction of inward rectifier channel(Kir)current.Furthermore,JNK and ERK inhibitors reduced oxLDL-induced[Ca2+]i oscillation and Kir channel depression.While the recombinant monoclonal antibody against oxLDL(BI-204)exhibited an obvious interfering with oxLDL-induced MCP-1 release and restored the activity of Kir.These anti-inflammatory effect of BI-204 was through its binding to FcyR?B on cellular membrane.Noticeably,oxLDL antibody specifically inhibited oxLDL-induced ERK activation but showed no effects on JNK and p38 MAPK activation.To summarize,oxLDL is through TLR-4 to activate cellular MAPK signaling,while the activation of JNK or ERK by this inhibits the activity of Kir and increases the influx of calcium ion,leading to the release of MCP-1 to the outside of the cells.While anti-oxLDL antibody(BI-204)reduces the activation of intracellular ERK signaling through FcyRIIB,thereafter restores the influx of potassium ion through Kir channel and reduces the influx of calcium ion,which at last inhibits the release of MCP-1 induced by oxLDL.Thus,our results reveal the molecular mechanism of oxLDL mAb inhibits inflammatory reaction in atherosclerotic animal model,which provide evidences for anti-antigen antibody treatment.