脓毒症患者外周血自然杀伤细胞的表型和功能及其临床意义

Phenotype and functions of natural killer cells in septic patients and its clinical significance

目的：通过检测脓毒症患者外周血自然杀伤细胞（NK细胞）的表型和功能，探讨其在脓毒症免疫功能紊乱中的可能作用机制。方法采用回顾性研究方法，选择2011年8月至2013年8月安徽省立医院重症医学科收治的59例全身炎症反应综合征（SIRS）患者和65例脓毒症患者，在患者进入重症监护病房（ICU）48 h内抽取外周血，用流式细胞仪检测NK细胞的表型和功能。以同期28例健康体检者的血样本为对照。结果 SIRS和脓毒症患者外周血CD3-CD56＋NK细胞比例及计数均正常，与健康对照组均无明显差异〔细胞比例：0.102±0.019、0.102±0.108比0.106±0.018，F＝0.018，P＝0.982；细胞计数（＆#215;106/L）：182.46±65.98、172.97±63.51比179.25±60.44，F＝0.349，P＝0.706〕。NK细胞脱颗粒作用试验显示，健康对照组、SIRS组和脓毒症组CD107表达及γ-干扰素（IFN-γ）分泌无明显差异〔CD107：0.135±0.050、0.140±0.058、0.128±0.070，F＝0.583，P＝0.560；IFN-γ（kU/L）：14.36±4.74、12.49±4.21、13.45±5.04， F＝1.616，P＝0.202〕。抗体依赖细胞毒作用（ADCC）试验显示，健康对照组、SIRS组和脓毒症组CD107表达无差异（0.574±0.166、0.643±0.165、0.581±0.157，F＝0.808，P＝0.448）；但与健康对照组比较，SIRS组NK细胞分泌IFN-γ的能力显著增加（kU/L：40.5±13.2比28.4±9.6，P＝0.001），而脓毒症组NK细胞分泌IFN-γ的能力显著降低（kU/L：19.8±6.7比28.4±9.6，P＜0.01）。与SIRS组比较，脓毒症组只有NK细胞表面抑制性受体CD158e（KIR 3DL1）表达明显升高（0.203±0.057比0.079±0.021，t＝15.762，P＜0.001）；而两组间其他表型均无明显差异。与SIRS组比较，脓毒症组基础血清IFN-γ分泌水平显著降低（kU/L：0.280±0.040比0.310±0.038，t＝3.390，P＝0.009），IL-12的产生也明显减少（ng/L：0.15±0.03比0.30±0.08， t＝32.832，P＜0.001）。结论 NK细胞表型和功能检测结果表明，脓毒症患者NK细胞功能受到损害，产生IFN-γ的能力降低。IFN-γ介导的免疫功能紊乱可能为脓毒症NK细胞功能低下的主要原因，为临床改善以NK细胞为基础的免疫干预治疗奠定基础。

Objective To investigate the possible mechanism of natural killer cells（NK cells）in immune dysfunction in sepsis by monitoring the phenotype and function of periphery NK cells in patients with sepsis. Methods A retrospective study was conducted. The patients with systemic inflammatory response syndrome（SIRS,n=59）or sepsis（n=65）admitted to Department of Critical Care Medicine of Anhui Provincial Hospital from August 2011 to August 2013 were enrolled. Blood samples were collected within 48 hours after intensive care unit（ICU）admission,the phenotype and function of periphery NK cells were determined by flow cytometry. Twenty-eight healthy people served as controls. Results The proportion and number of peripheral blood CD3-CD56＋NK cells in SIRS and sepsis groups were normal,and no statistical difference was found when compared with those of the healthy control group〔cell proportion：0.102±0.019,0.102±0.108 vs. 0.106±0.018,F=0.018,P=0.982;cell number（＆#215;106/L）：182.46±65.98, 172.97±63.51 vs. 179.25±60.44,F=0.349,P=0.706〕. It was shown by NK cell degranulation detection that there was no significant difference in the expression of CD107 and interferon-γ（IFN-γ）secretion〔CD107：0.135±0.050,0.140±0.058,0.128±0.070,F=0.583,P=0.560;IFN-γ（kU/L）：14.36±4.74,12.49±4.21, 13.45±5.04,F=1.616,P=0.202〕among healthy control group,SIRS group,and sepsis group. It was shown by antibody dependent cytotoxic effect（ADCC）test that there was no difference in the expression of CD107 among healthy control group,SIRS group,and sepsis group（0.574±0.166,0.643±0.165,0.581±0.157,F=0.808,P=0.448）. When compared with healthy controls,the secretion of IFN-γwas increased in SIRS patients（kU/L：40.5±13.2 vs. 28.4±9.6,P=0.001）,while reduced in sepsis patients（kU/L：19.8±6.7 vs. 28.4±9.6,P〈0.01）. Compared with SIRS group,only NK cell surface inhibitory receptors CD158e（KIR 3DL1）expression in sepsis group was significantly increased（0.203±0.057 vs. 0.079±0.021,t=15.762,P〈0.001）,and there were no significant differences in the other phenotype between the two groups. Compared with SIRS group,the IFN-γproduction of the sepsis group was significantly lowered（kU/L：0.280±0.040 vs. 0.310±0.038,t=3.390,P=0.009）,and the level of IL-12 was also significantly decreased（ng/L：0.15±0.03 vs. 0.30±0.08,t=32.832,P〈0.001）. Conclusion It was showed by NK cell phenotype and function assay that the function of NK cells in patients with sepsis was impaired and led to a poor production of IFN-γ. The IFN-γmediated immune dysfunction may be a main reason for the disorder of NK cell function,which laid the foundation of the clinical immune intervention practice to improve to NK cell function.