CCK-8对LPS作用下巨噬细胞B7.1和B7.2表达及其协同刺激功能的影响

目的探讨八肽胆囊收缩素(CCK-8)对LPS活化的巨噬细胞B7.1和B7.2表达及其协同刺激功能的影响。方法用CCK-8(10-12~10-6)mol.L-1和(或)脂多糖(LPS)孵育小鼠腹腔巨噬细胞,采用流式细胞术分析细胞表面B7.1和B7.2含量的变化,用免疫磁珠从小鼠脾细胞分离CD4+T细胞,按4∶1数量比与腹腔巨噬细胞[预先用LPS、CCK-8和(或)抗B7.1抗体、抗B7.2抗体、CCK1R拮抗剂CR1409、CCK2R拮抗剂CR2945孵育24h]共同体外培养,同时加入ConA5mg.L-1,采用3H参入法测定CD4+T细胞增殖反映巨噬细胞的协同刺激活性。结果CCK-8可以下调LPS诱导的巨噬细胞的B7.1和B7.2表达,抑制LPS活化的巨噬细胞的协同刺激活性。CCK-8的作用呈剂量依赖性,最大效应剂量在(10-7~10-9)mol.L-1之间。CR1409及CR2945均能逆转CCK-8的上述作用,且CR1409的作用较CR2945更明显。抗B7.1抗体和抗B7.2抗体可减轻LPS活化的巨噬细胞协同刺激活性。结论CCK-8通过下调LPS诱导的巨噬细胞B7.1和B7.2表达而抑制其协同刺激活性,该作用由CCK1R及CCK2R介导,其中CCK1R起主要介导作用。

Effect of CCK-8 on B7. 1 and B7. 2 expressions and costimulatory activity of LPS-activated murine macrophages

Aim To investigate in vitro effects of CCK-8 on the expressions of B7.1 and B7.2 and the costim-ulatory activity for T lymphocytes in LPS-activated macrophages.Methods Mouse peritoneal macrophages were isolated and incubated with LPS and/or CCK-8(10-12~10-6)mol·M-1 for indicated times.The B7.1 and B7.2 expressions of murine peritoneal macrophages were analyzed by flow cytometry.CD4+ T cells were isolated from mouse spleen by using immunomagnetic beads, and cultured with 1/4 numbers of macrophages which were pretreated with LPS, CCK-8 and/or anti-B7.1 antibody, anti-B7.2 antibody, CCK1R antagonist CR1409, CCK2R antagonist CR2945 for 24 h. ConA was added into the culture medium to stimulate CD4+T cell proliferation.The proliferation was determined by measuring[3H]-TdR incorporation in a β-scintillation counter.Results LPS-induced B7.1 and B7.2 expressions and costimulatory activity of peritoneal macrophages were inhibited by CCK-8 in a dose-dependent manner, with the maximal effects occurred at the concentrations from 10-7 mol·L-1 to 10-9 mol·L-1. Both CR1409 and CR2945 reversed the effect of CCK-8 on costimulation, and the role of CR1409 was more significant. Anti-B7.1 antibody and anti-B7.2 antibody inhibited the modulatory role of LPS on costimulatory activity.Conclusion CCK-8 inhibited LPS-induced macrophage costimulatory activity by down-regulating B7.1 and B7.2 expressions, which was mediated by CCK1R and CCK2R. CCK1R might be the major receptor responsible for the modulation of CCK-8 on costimulation.