正常人外周血中性粒细胞表型和功能鉴定

Phenotype and function of neutrophils in normal human peripheral blood

目的正常人外周血中性粒细胞的表型和功能鉴定。方法采用密度梯度离心法和红细胞裂解方法分离正常人外周血中性粒细胞,利用多重免疫荧光染色和流式细胞术分析中性粒细胞表面标记,及其在中性粒细胞亚群中的分布特征。通过pHrodo^(TM) Green E.coli BioParticles^(TM)吞噬实验检测中性粒细胞吞噬功能。利用Wright-Giemsa染色和免疫荧光染色,分别在光学显微镜和激光共聚焦显微镜下,观察体外刺激中性粒细胞产生中性粒细胞胞外陷阱(NET)的形态学特征。结果正常人外周血中性粒细胞高表达表面标记CD66b、CD11b和CD16,且CD16分子表达百分率高于CD11b。中性粒细胞可根据CD16分子表达密度不同分为CD16 hi、CD16 int和CD16 lo三个亚群,其中CD16 hi细胞亚群的百分率远高于CD16 int细胞亚群和CD16 lo细胞亚群。中性粒细胞亚群均表达抑制性分子程序性死亡配体1(PD-L1),但在三个亚群的表达无明显差异;抑制性分子CD300LD在CD16 hi细胞亚群的表达明显高于其在CD16 int细胞亚群和CD16 lo细胞亚群中的表达。中性粒细胞亚群均高表达黏附分子CD62L,且三个亚群无明显差异。pHrodo^(TM) Green E.coli BioParticles^(TM)在CD16 int亚群上的平均荧光强度(MFI)值高于其在CD16 hi和CD16 lo亚群上的MFI值,且其在CD16 hi亚群的MFI值高于CD16 lo亚群。Wright-Giemsa染色结果显示,正常人外周血中性粒细胞多呈现分叶核形态;经佛波酯(PMA)刺激后,细胞核不再呈现分叶状态,并产生网状结构。激光共聚焦结果显示,中性粒细胞经PMA刺激后,中性粒细胞弹性蛋白酶(NE)释放增加,并呈现网状结构。结论正常人外周血中性粒细胞存在异质性,不同中性粒细胞亚群表面标记分子的表达存在差异,可能发挥不同的免疫功能;PMA可刺激中性粒细胞产生NET,不同中性粒细胞亚群产生NET能力的差异则有待进一步研究。

Objective To investigate the phenotypic and functional characteristics of normal human peripheral blood neutrophils.Methods Normal human peripheral blood neutrophils were isolated through density gradient centrifugation followed by red blood cell lysis,and their surface markers and distribution characteristics in different neutrophil subsets were detected using multi-color immunofluorescence staining and flow cytometry.The phagocytic activity of neutrophils was evaluated with pHrodo^(TM) Green E.coli BioParticles^(TM).The morphological characteristics of neutrophils undergoing the formation of neutrophil extracellular traps(NET)in vitro were examined via Wright-Giemsa staining and immunofluorescence staining,and were observed using standard light microscopy and confocal microscopy,respectively.Results CD66b,CD11b and CD16 were all highly expressed on normal human peripheral blood neutrophils and the percentage of CD16 positive neutrophils was higher compared to CD11b positive neutrophils.Neutrophils could be classified into three subsets based on the density of CD16 expression:namely,CD16 hi neutrophil,CD16 int neutrophil and CD16 lo neutrophil.The proportion of the CD16 hi neutrophil was the highest among the three subsets.The inhibitory molecule programmed cell death ligand 1(PD-L1)was expressed on all neutrophil subsets without significant differences;however,the expression level of another inhibitory molecule,CD300LD,was markedly higher in the CD16 hi neutrophil compared to CD16 int or CD16 lo neutrophil.Each neutrophil subset demonstrated high levels of adhesion molecule CD62L without notable differences.Notably,the mean fluorescence intensity(MFI)value for pHrodo^(TM) Green E.coli BioParticles^(TM) in the CD16 int neutrophil was the highest among three subsets;and that in CD16 hi neutrophil was higher compared to CD16 lo neutrophil.Results of Wright-Giemsa staining showed that normal peripheral blood neutrophils typically possessed lobulated nuclei;upon stimulation with phorbol 12-myristate 13-acetate(PMA),the nuclei of these neutrophils formed a network structure while losing their lobulated nuclear morphology.Laser confocal microscopy results showed an increased release of neutrophil elastase(NE)with network formation in neutrophils after PMA stimulation.Conclusions Normal human peripheral blood neutrophils are heterogenous population with different expression of surface marker molecules,which might exert different immune functions.NET formation can be induced by PMA stimulation,and the differences of NET formation in different neutrophil subsets need to be further studied.