Резюме. Микровезикулы (МВ)-субклеточные структуры размером от 100 до 1000 нм, продуцируемые клетками в состоянии покоя и активации. МВ могут передавать молекулы клеткам-мишеням, регулировать физиологические процессы, участвовать в патологиях. Микровезикулы лейкоцитарного происхождения, в частности МВ NK-клеток, остаются наименее изученной популяцией МВ. NK-клетки способны изменять функциональную активность эндотелиальных клеток (ЭК), участвуют в регуляции ангиогенеза. Недостаточно изучена способность МВ NK-клеток влиять на функциональное состояние ЭК. Целью настоящего исследования явилось изучение влияния МВ, образуемых естественными киллерами линии NK-92, на фенотип, активность каспаз, пролиферацию и миграцию ЭК линии EA.Hy926. ЭК культивировали в присутствии МВ клеток линии NK-92. При помощи проточной цитофлуориметрии оценивали изменение фенотипа ЭК, передачу внутриклеточного белка из МВ в ЭК, относительную гибель ЭК. При помощи Western blot analysis оценивали экспрессию гранзима B в NK-клетках и их МВ, появление гранзима B в ЭК, экспрессию каспаз, Erk, AKT в ЭК. Также оценивали пролиферацию и миграцию ЭК в присутствии МВ клеток линии NK-92. Установлено значимое различие протеомных профилей клеток линии NK-92 и образуемых ими МВ. Контакт ЭК с МВ клеток линии NK-92 сопровождается развитием следующих событий: 1) экспрессией в ЭК гранзима В; 2) активацией каспазы-9, каспазы-3 и частичной гибелью ЭК; 3) появлением на ЭК панлейкоцитарного маркера CD45; 4) снижением экспрессии CD105 и повышением экспрессии CD34 и CD54; 5) ингибированием миграции ЭК. Передача эндотелиальным клеткам Erk, но не AKT, в составе МВ клеток линии NK-92 в концентрации в 10 раз ниже концентрации, вызывающей гибель ЭК, способствует повышению пролиферации ЭК.
Membrane extracellular microvesicles serve as carriers of a wide range of molecules, the most important among these are proteins, lipids, and nucleic acids. Cytotoxic proteins of natural killer cells play a key role in the realization of their cytolytic functions. An important stage in understanding of the distant communication of cells and mechanisms of its regulation is analysis of the proteome composition of microvesicles. We studied the proteomic composition of microvesicles produced by NK-92 natural killer cells. Granzyme A, a specific protein of cytotoxic cells, has been identified in the microvesicles by QTOF-mass spectrometry. It was shown that heat shock proteins, components of the ubiquitin-proteasome system, enzymes of protein biosynthesis and energy metabolism, nuclear and serum proteins, as well as cytoskeleton proteins are associated with the microvesicles.
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.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.