IgG在CpG DNA诱导巨噬细胞活化与JNK和p38通路中的作用

The roles of IgG in CpG DNA activated macrophages and induction of JNK and p38 pathways

目的:探究免疫球蛋白G(IgG)在CpG DNA诱导巨噬细胞活化过程中的调节作用及其对信号通路的影响。方法:体外培养小鼠骨髓源巨噬细胞,采用流式细胞仪和免疫荧光技术对巨噬细胞表面标志物和吞噬功能进行鉴定。IgG、CpG DNA以及IgG联合CpG DNA分别处理巨噬细胞后,采用流式细胞技术检测巨噬细胞表面活化分子及胞内细胞因子,免疫蛋白印迹检测核因子(nuclear factor,NF)-κB和MAPK信号通路相关蛋白磷酸化。结果:体外培养巨噬细胞表面标志物CD11b和F4/80的表达率约99.7%,巨噬细胞对FITC-IgG吞噬比例约70%;IgG上调CpG DNA活化巨噬细胞表面CD40、CD80和CD86的表达(P <0.05);IgG促进CpG DNA诱导巨噬细胞内JNK和p38磷酸化,但对ERKp44/42和NF-κBp65的磷酸化没有明显作用;IgG联合CpG DNA诱导巨噬细胞分泌大量的细胞因子肿瘤坏死因子(tumor necrosis factor,TNF)-α。结论:IgG在CpG DNA诱导巨噬细胞活化与MAPK信号通路JNK和p38磷酸化中起促进作用,通过促使表达上调共刺激分子CD40、CD80和CD86与分泌TNF-α进一步活化巨噬细胞。

Objective:This study aims to investigate the effect of IgG on activating signaling pathway of macrophages induced by CpG DNA.Methods:Bone-marrow derived macrophages were cultured in vitro and their surface markers and phagocytic functions were identified by flow cytometry and immunofluorescence technique.After the treatment of macrophages with IgG,CpG DNA and IgG plus CpG DNA,the activated molecules on the surface of macrophages were detected by flow cytometry,and the protein phosphorylation was detected by immuno-protein Western blot.Results:The expression rate of CD11 b and F4/80 on the surface of macrophage in vitro was about 99.7%,and the proportion of macrophage to FITC-IgG was about 70%;IgG up-regulated the expression of CD40,CD80 and CD86 on the surface of macrophage induced by CpG DNA(P<0.05);IgG promotes the phosphorylation of JNK and p38 in macrophages induced by CpG DNA,but it has no effect on the phosphorylation of ERK and NF-κB;CpG DNA induces macrophages to secrete TNF-α,and combination of IgG with CpG DNA enhanced this process.Conclusion:CpG DNA activates macrophages and increases the phosphorylation of JNK and p38,and the combined treatment of CpG DNA and IgG highly induced this process;CpG DNA increased the co-stimulation molecule of CD40,CD80 and CD86 on the surface of macrophages,while the co-therapy of IgG and CpG DNA highly up-regulates this expression.