MRP8/MRP14诱导腹腔巨噬细胞释放炎性细胞因子

Expressions of inflammatory cytokines in mouse peritoneal macrophages induced by MRP8/MRP14 in vitro

目的探讨MRP8/MRP14诱导小鼠腹腔巨噬细胞细胞因子表达效应及其作用机制。方法 Luminex x MAP液相芯片系统检测重组MRP8/MRP14蛋白诱导腹腔巨噬细胞6种细胞因子/趋化因子的水平变化;MRP14不同结构域融合蛋白刺激细胞,检测TNF-α、IP-10和IL-6表达;Western blot检测MRP8/MRP14刺激细胞p38 MAPK、JNK和ERK激酶磷酸化变化;细胞预先用p38 MAPKs、JNK、ERK激酶抑制剂、TLR4和RAGE受体拮抗剂预处理,之后给予MRP8/MRP14蛋白刺激,检测TNF-α、IP-10和IL-6表达。结果 MRP8/MRP14能显著诱导TNF-α、IP-10和IL-6的表达,与对照组相比,蛋白表达水平分别升高约98.2、378.6和6.3倍(P<0.01),MRP8/MRP14不能诱导IL-2、IL-5和IFN-g的表达;MRP14全长及其结构域EFhand-1、EFhand-2及EFhand-1+2融合蛋白能够诱导TNF-α、IP-10和IL-6的表达(P<0.01),CT末端结构域不具有诱导活性;MRP8/MRP14刺激细胞后1 h p38 MAPK、JNK及ERK激酶发生显著磷酸化变化,持续至2 h;与单纯MRP8/MRP14组相比,p38 MAPK抑制剂SB203580显著抑制TNF-α、IP-10和IL-6的表达(P<0.05);JNK抑制剂SP600125显著抑制TNF-α和IP-10的表达(P<0.05),对IL-6的表达无影响;ERK及其上游MEK1/2的抑制剂PD98059和U0126显著抑制IL-6的表达(P<0.05);TLR4抑制剂TAK242抑制了MRP8/MRP14诱导的TNF-α、IP-10和IL-6的表达(P<0.05),而RAGE中和性抗体仅部分抑制MRP8/MRP14诱导的IL-6的表达(P<0.05)。结论 MRP8/MRP14能够诱导小鼠腹腔巨噬细胞TNF-α、IP-10和IL-6的表达;MRP蛋白以包含有钙离子结合基序的结构域具有诱导细胞因子表达的活性;TNF-α和IP-10的表达与TLR4受体及其下游的p38 MAPKs、JNK通路有关,IL-6的表达则同时由TLR4和RAGE受体介导,继而激活下游的p38 MAPKs和ERK信号通路。

Objective To investigate MRP8/MRP14-induced expression of inflammatory cytokines in mouse peritoneal macrophages and explore the mechanism.Methods Tumor necrosis factor-α （TNF-α）,interferon-γ inducible protein 10 （IP-10）,interleukin-2 （IL-2）,IL-6,IL-5 and interferon-γ （IFN-γ） proteins in the culture supernatants of mouse peritoneal macrophages treated with recombinant MRP8/MRP14 were quantified using Luminex x MAP system.TNF-α,IP-10 and IL-6 levels were detected in the culture supernatants of the peritoneal macrophages after treatment with different domains of MRP14.Western blotting was used to detect the phosphorylation of p38 MAPK,JNK and ERK in the cells after MRP8/MRP14 treatment.The effects of p38 MAPK,JNK,ERK inhibitors,TLR4 or RAGE receptor antagonists on MRP8/MRP14-induced expressions of TNF-α,IP-10 and IL-6 were tested.Results MRP8/MRP14 significantly increased TNF-α,IP-10 and IL-6 levels in mouse peritoneal macrophages by 98.2,378.6 and 6.3 folds （P〈0.01）,respectively,but did not obviously affect IL-2,IL-5 and IFN-α levels.MRP14 protein and its calcium binding motifs such as EF hand-1,EF hand-2,EF hand-1＋2,but not CT terminal domain,all induced TNF-α,IP-10 and IL-6 expressions （P〈0.01）.Phosphorylation of p38 MAPK,JNK and ERK were detected by Western blotting in the cells at 1 h after MRP8/MRP14 stimulation and sustained to 2 h.Compared with MRP8/MRP14,SB203580 （p38 MAPK inhibitor） significantly inhibited TNF-α,IP-10 and IL-6 expression （P〈0.05）,and SP600125 （JNK inhibitor） inhibited the expression of TNF-α and IP-10 （P〈0.05） but not IL-6,PD98059 and U0126 （ERK and MEK1/2 inhibitor） reduced IL-6 expression （P〈0.05）.TNF-α,IP-10 and IL-6 levels were inhibited by TAK242 （P〈0.05）; IL-6 level in the cells was also partially inhibited by RAGE neutralizing antibody （P〈0.05）.Conclusion MRP8/MRP14 can induce the expression of TNF-α,IP-10 and IL-6 in mouse peritoneal macrophages.MRP14 protein,which contain calcium binding motifs,has the biological activity of inducing cytokine expression.TNF-α and IP-10 expressions are related with TLR4 and its downstream p38 MAPKs and JNK; IL-6 is regulated by both TLR4 and RAGE and their downstream p38 MAPKs and ERK.