抑制中性粒细胞PD-L1表达对脓毒症T淋巴细胞功能的影响

目的: 探讨脓毒症状态下中性粒细胞对T淋巴细胞功能的影响及相关机制。方法: 采用脂多糖(lipopolysaccharide，LPS)刺激中性粒细胞，模拟脓毒症状态下的体外模型。分离人外周血中性粒细胞并分离出淋巴细胞，分为对照组、LPS组、中性粒细胞组和LPS+中性粒细胞组4个组。采用流式细胞术检测淋巴细胞凋亡、增殖、活性和炎症细胞因子浓度，以及中性粒细胞表面程序性死亡配体1（PD-L1）的表达；流式细胞术检测使用不同浓度PD-L1抗体后的淋巴细胞活性。结果： 与对照组相比，中性粒细胞组和LPS+中性粒细胞组淋巴活性明显降低，凋亡明显增加，细胞增殖明显降低，CD69和CD71表达百分比、平均荧光强度、γ干扰素和IL-2的释放显著降低(P均<0.01)；与LPS组相比，LPS+中性粒细胞组能显著抑制淋巴细胞增殖、活化和细胞因子的释放(P均<0.05)；与对照组相比，LPS组PD-L1表达明显增加(P<0.05)；与对照组相比，中、高浓度组淋巴细胞存活百分比明显增加(P均<0.05)，且呈浓度依赖性。 结论： 脓毒症状态下中性粒细胞通过PD-L1/PD-1通路途径抑制T淋巴细胞的功能。

The effect of suppressing PD-L1 expression of neutrophils on immunoactivity of T lymphocytes during sepsis

Objective: To investigate the effect of neutrophils on T lymphocytes during sepsis and its potential mechanism. Methods: Sepsis model in vitro was established by co-culturing T lymphocytes with lipopolysaccharide(LPS). Neutrophils were isolated by density gradient centrifugation and T lymphocytes were isolated by anti-CD3 magnetic beads from human peripheral blood. T lymphocytes were divided into 4 groups, including control group, LPS group, neutrophil group and LPS+ neutrophil group. The apoptosis, proliferation, activation and secretion of T lymphocytes were assessed by flow cytometry. Phenotype programmed death ligand 1(PD-L1) on neutrophil was detected in control group and LPS group. Anti-PD-L1 antibody was added into mixed culture of neutrophils with T cells and the apoptosis of T cells were assessed. Results: Compared with the control group, neutrophil group and LPS + neutrophil group showed significantly lower immunoactivity of T lymphocytes, higher proportion of apoptosis, lower expression levels of CD69 and CD71, as well as secretion of interferon-γ(IFN-γ) and IL-2(all P<0.05). Compared with LPS group, the proliferation of T cells, secretion of IFN-γ and IL-2 were decreased in LPS+ neutrophilgroup (all P<0.05). The ratio of PD-L1 in LPS group was significantly higher than that of the control group (P<0.05). The proportion of viable lymphocytes increased with higher concentrations of PD-L1 monoclonal antibody (all P<0.05). Conclusion: Neutrophils suppressed the immunoactivity of T lymphocytes through PD-L1/PD-1 pathway during sepsis.   ［Key words］sepsis; neutrophil; T lymphocyte; programmed death-ligand 1