Ultrastructure of cytokine induced human natural killer cells

Abstract

Background & Aim Natural Killer (NK) cells account for 3.66-26.74% of lymphocytes in the human peripheral blood. They secrete exosomes which express both NK-cell markers and cytokines. It has been shown that Interkin-2 (IL-2) stimulated the proliferation of natural killer cells, a sub-class of lymphocytes in human. In this study, the NK cells were isolated and cultured under different period of time, the ultrastructure of the highly purified activated NK cells were analyzed by transmission electron microscopy (TEM).The increase in the number of the NK cells were measured by flow cytometry. Methods, Results & Conclusion Methods The peripheral blood mononuclear cells (MNCs) were isolated by Ficoll centrifugation followed by phosphate buffer saline (PBS) with 10% Acid Citrate Dextrose (ACD). The MNCs were cultured according to the protocol of Cellex Natural Killer Cell Kit (Japan). The NK cells at Day 0, and cultured at Day 3, 5, 7 and 14 were sampled and fixed with 2.5% glutaraldehyde for 24 hours at 4°C, and processed for routine TEM examination. Samples at Day 0 and Day 14 were quantified by flow cytometry. Results The resting NK cell at Day 0 was about 3.5um with a round nucleus of about 1.7um in diameter. Electron-dense granules (EDGs) of varying sizes were observed (100-200nm) in figure 1. The NK cells started to proliferate after cytokine activation cultured at Day 3. The average size of the NK cells (6um) and nuclei (4um) increased in sizes when compared with Day 0. Membrane-bounded vesicles were observed at the proximity of Golgi area (Fig. 2). Electro-dense granules of similar sizes as at Day 0 were observed at Day 5. Micro-vesicles (150-200nm) and exosomes (50-100nm) were found associated with plasma membrane of NK cells at Day 7 (Fig. 3). The average size (280-330nm) and number of the EDGs increased at Day 14 when compared with Day 0 and Day 5 (Figs. 4). The average number of activated NK cells increased from 304 cells/ul at Day 0 to 1796 cells/ul at Day 14 as measured by flow cytometry. Conclusion An increase in number of NK cells after Interkin-2 (IL-2) activation was confirmed in the present study. With a better understanding of the characteristics of exosomes, micro-vesicles and electron-dense granules as observed in the activated NK cells is crucial as a new therapeutic tool in the future.