Natural killer (NK) cells are of special interest among a multitude of microvesicle (MV) source cells. NK cells are a lymphocyte subpopulation performing contact cytolysis of virus-infected cells and tumor cells. Each of the NK cell populations has a unique receptor repertoire on its surface and, thus, unique functions. During their contact with a target cell, the most common mechanism of cytolysis is an exocytosis of lytic granules. However, some indirect evidence suggests that MV with CD56 phenotype and leukocyte-derived MV with various phenotypes are present in the peripheral blood plasma.This research is aimed to study the phenotype, composition and cytotoxic activity of microvesicles produced by NK cells. The analysis of receptor expression showed that MV, as well as source cells of the NK-92 cell line, had a similar CD56 molecule expression profile. The expression profile in MV differs from the same in source cells by higher CD119 and CD11b expression and by lower CD18 expression. Culturing of NK-92 cells in the presence of PMA, IL-1β, TNFα, IFNγ resulted in alterations of cell phenotypes and MV. Immunoblots revealed a change of perforin and granzyme B (GrB) in MV. The analysis of the cytotoxic activity of NK-92 cells in a natural killer in vitro assay employing K562 target cells demonstrated that MV obtained from TNFα-activated cells of the NK-92 cell line increased the cytotoxicity of the same TNFα-activated NK-92 cells regarding cytotoxicity levels. This coincides with the previously revealed increased content of GrB in MV obtained from TNFα-activated cells of the NK-92 cell line. To sum up depending on the cytokine NK-92 cells produce MV that differ in their phenotype, composition and activity. Any changes in MV composition can result in changes in their functional activity: in particular, changes can increase the cytotoxic activity of NK cells of the NK-92 cell line. Thus, besides a well-known and proved way for GrB delivery to a target cell, we can suggest an additional way – the transportation of GrB within MV.

Microvesicles (MVs) are plasma extracellular vesicles ranging from 100 (150) to 1000 nm in diameter. These are generally produced by different cells through their vital activity and are a source of various protein and non-protein molecules. It is assumed that MVs can mediate intercellular communication and modulate cell functions. The interaction between natural killer cells (NK cells) and endothelial cells underlies multiple pathological conditions. The ability of MVs derived from NK cells to influence the functional state of endothelial cells in inflammatory conditions has yet to be studied well. In this regard, we aimed to study the effects of MVs derived from NK cells of the NK-92 cell line stimulated with IL-1β on the phenotype, caspase activity, proliferation and migration of endothelial cells of the EA.hy926 cell line. Endothelial cells were cultured with MVs derived from cells of the NK-92 cell line after their stimulation with IL-1β. Using flow cytometry, we evaluated changes in the expression of endothelial cell surface molecules and endothelial cell death. We evaluated the effect of MVs derived from stimulated NK cells on the proliferative and migratory activity of endothelial cells, as well as the activation of caspase-3 and caspase-9 therein. It was established that the incubation of endothelial cells with MVs derived from cells of the NK-92 cell line stimulated with IL-1β and with MVs derived from unstimulated NK cells, leads to the decrease in the proliferative activity of endothelial cells, appearance of the pan leukocyte marker CD45 on them, caspase-3 activation and partial endothelial cell death, and reduced CD105 expression. However, compared with MVs derived from unstimulated NK cells, a more pronounced effect of MVs derived from cells of the NK-92 cell line stimulated with IL-1β was found in relation to the decrease in the endothelial cell migratory activity and the intensity of the CD54 molecule expression on them. The functional activity of MVs is therefore mediated by the conditions they are produced under, as well as their internal contents.

Extracellular vesicles that are shed from the plasma membrane contain a wide range of molecules, among which are proteins, lipids, nucleic acids, and sugars. The cytotoxic proteins of natural killer cells play a key role in the implementation of their cytolytic functions. One of the important steps in understanding the distant communication of cells is the determination of the proteome of microvesicles. This study was aimed at the protein profiling of the microvesicles produced by the NK-92 natural killer cell line. 986 proteins with a variety of functions were identified in the lysate of microvesicles using the MALDI-TOF mass spectrometric analysis. With automated methods of functional analysis applied, it has been shown that the largest protein groups are hypothetical proteins, proteins with unknown functions, and domains. The most representative groups are also comprised by transcription regulators; intracellular signaling proteins; RNA translation, transcription, processing, and utilization regulators; receptors; protein processing and proteolysis regulators; amino acid metabolism enzymes, as well as transport proteins and transport regulators. Minor functional groups are represented by vitamins and mineral metabolism enzymes, membrane and microdomain-forming proteins, hormones, hemostatic regulators, regulators of sensory systems, specific mitochondrial and Golgi apparatus proteins, and extracellular signaling proteins. An intermediate position is occupied by various functional groups, including cytoskeleton and motor proteins; proteins of centrioles; ion channels and their regulators; proteins of the ubiquitin-proteasome pathway of protein degradation; lipid, steroid, and fatty acid metabolism enzymes; nucleic acid base and carbohydrate metabolism enzymes, as well as energy metabolism enzymes and other proteins involved in intermediate metabolism; proteins of the immune response and inflammation; antigens and histocompatibility proteins; cytokines and growth factors; regulators of apoptosis, autophagy, endocytosis, and exocytosis; regulators of the cell cycle and division; regulators of proliferation, cell differentiation, and morphogenesis; regulators of cell adhesion and matrix metabolism; nuclear transport proteins; transposition proteins; DNA replication and repair proteins, as well as inactive proteins. The data obtained expand the existing knowledge of the distant communication of cells and indicate new mechanisms of interaction between natural killer and target cells.

Микровезикулы естественных киллеров линии NK-92 влияют на фенотип и функции эндотелиальных клеток линии EA.Hy926