The Role of Cytokines in Maintaining the Dynamics of Cell-Cell Interaction between Natural Killer Cells and Trophoblast Cells.

We analyzed the effect of individual cytokines that are secretory products of placenta typical of the uteroplacental bed. The proinflammatory cytokines IL-6, IFNγ, and IL-1ß increased the expression of TGFßR2 molecule by trophoblast cells, while VEGF and PLGF increased the expression of CD45, CD29, and CD54 adhesion molecule by trophoblast cells. The antiinflammatory cytokine IL-4 increased LeptinR expression by trophoblast cells. PMA and TNFα also enhanced the adhesion of NK cells to trophoblast cells. Our findings suggest that NK cells involved CD11a, CD11b, and CD18 molecules during their transmigration through trophoblast, as well as during their transendothelial migration.

Microvesicles Produced by Natural Killer Cells Regulate the Formation of Blood Vessels.

We studied the effect of microvesicles derived from cells of the NK-92 cell line on the formation of tube-like structures by endothelial cells of the ЕА.Hy926 cell line. Microvesicles were isolated by differential centrifugation and their size was controlled by granulometric analysis using dynamic light scattering method. The effect of microvesicles produced by NK cells on angiogenesis was evaluated by cultural methods. In the course of the research, a model of co-culturing of microvesicles and endothelial cells on extracellular matrix Matrigel was developed. It was found that microvesicles derived from NK-92 cells promoted elongation of tube-like structures formed by endothelial ЕА.Hy926 cells. Microvesicles produced by NK cells can modulate functional activity of endothelial cells by affecting their ability to form blood vessels.

Methodological Approaches to Assessing the Size and Morphology of Microvesicles of Cell Lines.

Morphological properties and the size of microvesicles were assessed using atomic force microscopy, electron microscopy, and granulometric analysis. As these methods require significant numbers of microvesicles, we chose microvesicles derived from cell lines for our research.

Trophoblast cell influence on peripheral blood natural killer cell proliferation and phenotype in non-pregnant women and women in early pregnancy.

Natural killer (NK) cells are the main population of leukocytes in decidua during the first trimester of pregnancy. NK cells can have contact with trophoblast cells during pregnancy, which raises the possibility of mutual influence. This research aimed to evaluate the proliferation and phenotype of peripheral blood NK cells in the presence of trophoblast cells of the JEG-3 cell line. We showed that trophoblast cells of the JEG-3 cell line (American Type Culture Collection (ATCC), USA) produced TGFß. However, co-culturing of NK and trophoblast cells did not change the SMAD2/3 to pSMAD2/3 ratio within NK cells. These data indicate that the canonical signaling pathway from TGFß is not activated, but do not preclude activation of SMAD-independent signaling pathways through the effect of TGFß and/or other cytokines. We established that trophoblast cells inhibited both constitutive and IL-2-induced expression of Ki-67 proliferation marker by NK cells in vitro in both pregnant and non-pregnant women. Constitutive and induced Ki-67 expression by peripheral blood NK cells was increased in pregnant women compared with non-pregnant women. The influence of trophoblast cells on Ki-67 expression by NK cells was more pronounced in the presence of other mononuclear cells than in their absence. In the presence of trophoblast cells and IL-2, the number of NK cells with the CD16+CD57- phenotype in peripheral blood mononuclear cells (PBMCs) was increased in pregnant and non-pregnant women, compared with culturing with IL-2 only. This might reflect a decrease in the number of NK cells at the terminal stage of differentiation. We also revealed the increased content of NK cells with the CD16-CD56bright phenotype in PBMCs of pregnant women when incubated with trophoblast cells and IL-2, compared with culturing with trophoblast cells only. Our results suggest that NK cells need contact interactions with trophoblast cells and additional cytokine stimulation (IL-2, cytokines of other mononuclear cells) to acquire the CD56bright phenotype.

Role of Caspases in the Cytotoxicity of NK-92 Cells in Various Models of Coculturing with Trophoblasts.

Studies of interactions between natural killer (NK) cells and trophoblasts and identification of conditions for the NK cells to perform their cytotoxic function are of fundamental and practical importance for understanding their role in the development of pathological processes and complications during pregnancy. In this study, we examined changes in the content of caspases and studied activation of these enzymes in Jeg-3 trophoblasts in various models of their coculturing with NK-92 cells and demonstrated the necessity of direct contact between these cell populations for the activation of caspase-8 and caspase-3 in the trophoblasts. Contact coculturing of the two cell lines resulted in the appearance of the cytotoxic protein granzyme B in Jeg-3 cells that was accompanied by a decrease in the content of this enzyme in NK-92 cells. Distant coculturing of NK-92 and Jeg-3 cells did not trigger initiator and effector caspases characteristic for the apoptosis development in Jeg-3 cells. The observed decrease in the content of procaspases in the trophoblasts may be associated with alternative non-apoptotic functions of these enzymes.

Changes in expression of Ki-67, CD16 and CD56 by natural killer cells from peripheral blood mononuclear cells in the setting of recurrent miscarriage after in vitro culturing in the presence of trophoblast cells and IL-2.

The aim of this research was to assess the proliferative activity of Natural Killer Cells (NK cells) from Peripheral Blood Mononuclear Cells (PBMCs) in the presence of trophoblast cells in women with a history of recurrent miscarriages. We examined the peripheral blood of women with recurrent miscarriage in the proliferative (n = 12) or secretory (n = 13) phase of their menstrual cycle, and pregnant women with a history of recurrent miscarriage at 6-7 weeks of their current pregnancy (n = 14). Controls were fertile non-pregnant women in the proliferative (n = 11) or secretory (n = 13) phase of their menstrual cycle, and pregnant women at 6-7 weeks of a physiologically normal pregnancy (n = 20). We used IL-2 as a factor maintaining PBMCs viability during long-term culturing. We established that culturing in the presence of IL-2 contributed to an increase in the number of CD56+CD16- NK cells and to a decrease in the number of CD56+CD16+ NK cells from PBMCs compared with these numbers before culturing in both healthy women and in women with recurrent miscarriage. After culturing of PBMCs in the presence of trophoblast cells and IL-2 (compared with culturing without trophoblast cells), the intensity of Ki-67 expression by NK cells was reduced in the whole NK cell population (CD3-CD56+), and in the CD56+CD16- and CD56+CD16+ populations of NK cells in women with recurrent miscarriage and in healthy controls. The intensity of CD56 expression was reduced in the presence of trophoblast cells and IL-2 in non-pregnant women with recurrent miscarriage in the secretory versus the proliferative phase of the menstrual cycle.

Interaction of NK Cells, Trophoblast, and Endothelial Cells during Angiogenesis.

We studied changes in angiogenesis during contact interaction of natural killer cells and endothelial cells in the presence of secretory products of trophoblast cells activated by various cytokines. Activated trophoblast regulates angiogenesis by producing soluble factors that affect endothelial cells either directly or indirectly through activation of proangiogenic activity of natural killer cells. A stimulating effect of the trophoblast supernatants activated by IL-1ß and an inhibitory effect of trophoblast supernatants activated by IL-6 and TGFß for the formation of tube-like structures by endothelial cells were revealed. During contact culturing, natural killer cells increased the length of tube-like structures formed by endothelial cells. The trophoblast activated by IL-1ß affects angiogenesis both directly through the production of proangiogenic factors and indirectly through activation of the proangiogenic potential of natural killer cells. Trophoblast activated by IFNγ affects angiogenesis only by stimulating the proangiogenic potential of natural killer cells. Under conditions of contact interaction of natural killer cells and endothelial cells, soluble factors of trophoblast activated by IL-6 or TGFß attenuated the angiogenesis-stimulating effect of natural killer cells.

Cytotoxic Activity of Peripheral Blood NK Cells towards Trophoblast Cells during Pregnancy.

We evaluated cytotoxic activity of peripheral blood NK cells towards trophoblast cells. NK cells either isolated or in the composition of mononuclear cell fraction, caused death of trophoblast cells. In women with physiological pregnancy, the cytotoxic effect of NK cells present in mononuclear cell fraction preincubated with IL-2 was lower than in nonpregnant women, who had never been pregnant previously, and in fertile women. Cytotoxic activity of isolated NK cells preincubated with IL-2 in fertile women was lower than in nonpregnant women, who had never been pregnant previously.

Mass-Spectrometric Analysis of Proteome of Microvesicles Produced by NK-92 Natural Killer Cells.

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.

Interactions of NK Cells and Trophoblast Cells. Methodological Aspects.

NK cells present in different organs differ by their functional characteristics, in particular, proliferative activity. For studying tissue-resident NK cells, tissue-specific microenvironment should be reproduced. In case of decidual NK cells, this microenvironment is created by trophoblast cells. We developed a method for evaluation of proliferative activity of peripheral blood NK cells in the presence of trophoblast cells. Proliferative activity of peripheral blood NK cells was evaluated by the expression of protein Ki-67 after culturing with JEG-3 trophoblast cells. This method allows evaluating the functional state of NK cells in microenvironment specific for the decidua.

Effect of Cytokines on the Formation Tube-Like Structures by Endothelial Cells in the Presence of Trophoblast Cells.

Despite ample data on cytokine secretion in the uteroplacental interface, the influence of microenvironment cells, in particular, trophoblast cells on angiogenesis and the role of cytokines in this process remain poorly studied. We studied the influence of cytokines on the formation of tube-like structures by endothelial cells in the presence of trophoblast cells and showed that trophoblast cells suppressed the angiogenic potential of endothelial cells. Antiangiogenic cytokines IFN-γ, IL-10, TNF-α, and TGFß via modulation of trophoblast cells stimulated the formation of tube-like structures by endothelial cells. In the co-culture of endothelial and trophoblast cells, the effects of cytokines changed and they gained additional regulatory functions.

Effect of THP-1 Cells on the Formation of Vascular Tubes by Endothelial EA.hy926 Cells in the Presence of Placenta Secretory Products.

We studied the effect of THP-1 cells on the formation of vessel-like structures by endothelial cells in the presence of placenta-conditioned media. Addition of THP-1 cells to endothelial cells cultured in the presence of media conditioned by first-trimester placentas led to an increase in the length of cell tubes and reduced their number in comparison with endothelial cell monoculture. In the presence of media conditioned by third-trimester placentas, THP-1 cells did not affect the length and number of cell tubes formed by endothelial cells. When evaluating the formation of vessel-like structures by endothelial cells in co-culture, marked decrease in the length of cell tubes in the presence of media conditioned by first-trimester placentas vs. third-trimester placentas was noted. No differences in the length and number of cell tubes formed by endothelial cells co-cultured with THP-1 cells in the presence of placental factors from women with preeclampsia and uncomplicated pregnancy were found. These findings can reflect the peculiarities of the influence of macrophages on the formation of blood vessels by endothelial cells in the placenta.

Effect of Factors Secreted by the Placenta on Phenotype of THP-1 Cells Cultured on a 3D Scaffold.

We studied the effects of secretory products of the placenta obtained from women with normal pregnancy and preeclampsia on the expression of surface markers by THP-1 cells cultured on a 3D Matrigel scaffold. Secretory products of third trimester placentas obtained from women with normal pregnancy reduced the relative number of THP-1 cells expressing CD54 and CD14 molecules and expression of CD14 and CD95 molecules by THP-1 cells in comparison with the effect of secretory products first trimester placentas. In parallel, the intensity of CD49d expression by THP-1 cells increased in the presence of secretory products of third trimester placentas in comparison with the first trimester. No differences in the expression of the studied molecules by THP-1 cells under the effect of placentas from women with physiological pregnancy and patients with preeclampsia were found.

Detection of Antibodies In Vitro Binding to Endothelial Cells in the Sera from Women with Normal Pregnancy and Preeclampsia.

Activity of serum antibodies in vitro binding to endothelial cells in women with normal pregnancy and preeclampsia was studied. Flow cytometry detected peculiarities of antibody binding to endothelial cells in health and disease. Detection of antiendothelial antibodies in trimesters II and III can be diagnostically important in preeclampsia.

Proliferative and Migration Activity of JEG-3 Trophoblast Cell Line in the Presence of Cytokines.

TNFα inhibited proliferation and did not inhibit migration JEG-3 trophoblast cells, IL-1ß stimulated both cell proliferation and migration, IL-6 and IL-8 stimulated only cell migration, IFNγ has either stimulating or inhibitory effect on proliferation of trophoblast cells depending on its concentration. Cytokines IL-10 and IL-4 stimulated only migration of trophoblast cells. VEGF, PlGF, and TGFß stimulated both proliferation and migration, and bFGF only migration of trophoblast cells.

Effect of Monocyte-Like THP-1 Cells on the Formation of Vascular Tubes by EA.Hy926s Endothelial Cells in the Presence of Cytokines.

The interaction of endothelial cells with cells of the microenvironment, including monocytes/ macrophages, and extracellular matrix during angiogenesis is controlled by cytokines. The stimulating effect bFGF, IL-8, and VEGF on the formation of capillary-like structures by endothelial cells was demonstrated in both monoculture and in co-culture with THP-1 cells; in the latter case, the effects of bFGF and VEGF were more pronounced. IL-8 reduced branching of vascular tubes in co-culture in comparison with monoculture of endothelial cells. Placental growth factor PlGF had no effect of tube formation by endothelial cells in monoculture, but in co-culture with THP-1 cells this cytokine in high concentrations exhibited proangiogenic activity. TGFb inhibited the formation of vascular tubes by endothelial cells and its antiangiogenic potential was more pronounced in co-culture with THP-1 cells.

Detection of microparticles of leukocytic origin in the peripheral blood in normal pregnancy and preeclampsia.

Microparticles are microvesicles forming during cell activation and as a result of apoptotic cell death. Normal pregnancy is associated with apoptosis induction in active immune system cells, present in the decidual tissue. Preeclampsia is associated with activation of the peripheral blood leukocytes and more intense apoptosis of the trophoblast cells. As a result, the number of microparticles in the peripheral blood is changing in normal gestation and in preeclampsia. The content of the leukocytic microparticles in the peripheral blood is evaluated in normal pregnancy and preeclampsia. The content of neutrophilic and monocytic microparticles is higher than normally in preeclampsia, this indicating activation of these cells. The number of microparticles formed by NK cells is low in preeclampsia, which can reflect the incompetence of immunological tolerance mechanisms under these conditions.

Effect of factors produced by the placenta on cytokine secretion by THP-1 cells cultured on a 3D scaffold.

We compared the effects of soluble products from the placenta obtained from women with normal pregnancy and pregnancy complicated by preeclampsia on cytokine secretion by THP-1 cells cultured on a 3D Matrigel scaffold. In the presence of soluble products from all placentas, the cells actively secreted IL-8, MCP-1, and soluble forms of CD14, TNFRI, and TNFRII receptors. Secretion of VEGF was below the spontaneous level. Secretion of IL-6 by THP-1 cells after incubation with soluble products of the placentas obtained during weeks 9-11 of physiological pregnancy and 38-39 of pregnancy complicated by preeclampsia surpassed the spontaneous level. In the presence of soluble factors of trimester I placentas, secretion of IL-6 and soluble form of TNFRI receptor was higher than in the presence of trimester III placental factors. Secretion of IL-6 by THP-1 cells was higher, while secretion of soluble TNFRII receptor was lower in the presence of placentas from women with preeclampsia in comparison with physiological pregnancy.

[Role of microparticles in intercellular communication].

Adaptive reactions involving different body systems are essential for human living activity. These adaptive reactions are based both on contact cell interactions and distant cell-to-cell transfer of secreted molecules. Transfer of bioactive substances is realized both on system and local levels. Currently, there is lack of information about mechanisms of transport in the intercellular space. Secretion of biologically active molecules within microparticles is considered to be one of the possible modes of signal transduction. Microparticles are microvesicles generated on the cell membrane surface. They may contain molecules derived from membrane, cytoplasm and nucleus. Blood plasma microparticles are established to be involved in blood coagulation, inflammation and immune response. In this review we summarize current concepts of microparticles as signal carriers participating in intercellular communication.

Effect of placenta secretory products on migration activity of endothelial EA.Hy926 cells.

We studied the influence of factors secreted by the placenta in physiological and preeclampsia-complicated pregnancy on migration activity of endothelial EA.Hy926 cells. It was found that migration of endothelial cells was more intensive in the presence of secretory factors from trimester I placentas in comparison with trimester III placentas and was lower in the presence of placental factors in preeclampsia in comparison with physiological pregnancy.