[Phenotype and function of neutrophils in normal human peripheral blood]

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.