First trimester pregnancy decidual natural killer cells contain and spontaneously release high quantities of granulysin

Citation Veljkovic Vujaklija D, Gulic T, Sucic S, Nagata K, Ogawa K, Laskarin G, Saito S, Haller H, Rukavina D. First trimester pregnancy decidual natural killer cells contain and spontaneously release high quantities of granulysin. Am J Reprod Immunol 2011; 66: 363–372 Problem Granulysin (GNLY) is a novel cytolytic protein lytic against a variety of tumor cells and microbes. The role of GNLY during pregnancy has not been extensively explored. The aim of this study is to examine GNLY expression and distribution in the first trimester pregnancy peripheral blood (PB) and decidua, the ability of decidual and PB natural killer (NK) cells to secrete GNLY spontaneously, and the role of antigen‐presenting cells (APC) in the regulation of GNLY expression in decidual NK cells. Method of study GNLY expression was analyzed using cell permeabilization method, flow cytometry, and immunohistochemistry. GNLY secretion by purified NK cells was detected by ELISA method. Results GNLY is abundantly expressed at the maternal–fetal interface in the first trimester pregnancy. Decidual T lymphocytes express significantly higher levels of GNLY (58%) then PB T lymphocytes (11%). Over 85% of decidual CD56⁺ cells express GNLY and when cultured spontaneously release high quantities of GNLY. Decidual APC participate in the control of GNLY expression in CD56⁺ cells. Conclusion Abundant expression of GNLY in the decidual immunocompetent cells and the capacity of decidual CD56⁺ cells to spontaneously secrete high quantities of GNLY point to important protective and immunomodulatory role that this molecule could play at the maternal–fetal interface.     

Altered phenotype of HLA-G expressing trophoblast and decidual natural killer cells in pathological pregnancies

BACKGROUND: The interaction between decidual natural killer (NK) cells and alloantigens expressed on fetal trophoblast cells are thought to be essential for successful implantation and placentation. Consequently, a disturbed interaction during the first trimester of pregnancy might well lead to a subsequent pregnancy failure. METHODS: We investigated the expression of HLA-G and NK cell markers in tissue sections from recurrent miscarriage (n = 9) and ectopic tubal pregnancies (n = 5), and two hysterectomy specimens of healthy pregnancy as well as decidual biopsies (n = 9) were used as controls. RESULTS: We show in normal pregnancy not only a decrease, but also a morphological change in CD56+ NK cells upon interaction with HLA-G-expressing trophoblasts. The cells appear to be transitioning from a blast-like (activation) state into a state of apoptosis. The number of CD16+ NK cells was low. In contrast, in recurrent miscarriage tissue a sustained NK cell marker expression of both CD56 and CD16 was paralleled by a decreased expression of HLA-G. No morphological changes from the blast-like stage were apparent. Finally, in ectopic pregnancies HLA-G expression in the absence of decidual NK cells was associated with a disturbed trophoblast differentiation. CONCLUSIONS: In pathological pregnancies we show an in-situ altered phenotype of trophoblast and NK cells.     

Decidual natural killer cell tuning by autologous dendritic cells

PROBLEM: Dendritic cells (DC)/natural killer (NK) cells interactions in the deciduas of early human pregnancies were analyzed in vitro. METHOD OF STUDY: Phenotype, cytokine expression and/or cytolytic mediators' expression were measured by flow cytometry in NK and DC from the freshly isolated decidual mononuclear cells or after their purification and co-culture in vitro. Proliferation of 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE)-labeled CD56(+) cells was analyzed by flow cytometry after the co-culture with CD1a(+) or CD83(+) DC. RESULTS: Decidual CD1a(+) cells show less mature phenotype with no expression of CD197, lower expression of CD80 and CD86 and higher expression of CD206 and CD195 in comparison to CD83(+) cells. Interleukin (IL)-15, interferon-gamma and tumor necrosis factor-alpha productions were higher in immature than mature DC, whereas IL-10 and IL-18 were equally produced in both subpopulations. Immature DC increase perforin, FasL and TRAIL protein expression and proliferation of NK cells, but decrease their intracellular IL-15 production. Mature DC caused less efficient proliferation of NK cells, and did not affect cytokine and cytolytic mediator expression. CONCLUSION: These results suggest that decidual CD1a(+) cells regulate and shape NK cell function more profoundly than CD83(+) cells in decidua.     

IL-22 secreted by decidual stromal cells and NK cells promotes the survival of human trophoblasts

Interleukin-22 (IL-22) has been implicated as an important immune regulator in many physiologic and pathological processes, but little is known about the IL-22 in the fetal-maternal interface. In this study, we demonstrated that co-culture of decidual stromal cells (DSCs) and decidual natural killer (dNK) cells resulted in increased secretion of IL-22, compared to culture of DSCs or dNK cells alone. The trophoblast cell line, HTR8/SVneo, expresses IL-22 receptor α1 (IL-22R1). Combinant human (rh) IL-22 significantly promoted the proliferation and viability, and inhibited the apoptosis of HTR8/SVneo cells. By Western blotting and immunohistochemistry, we confirmed that villi expressed IL-22R1, and the villi from unexplained spontaneous miscarriage patients expressed reduced levels of IL-22R1 than those from normal early pregnancy. These findings indicate that the IL-22 secreted by DSCs and dNK might promote the survival of trophoblasts and participate in the maintenance of pregnancy by binding to the IL-22R1. The reduced level of IL-22/IL-22R1 in villi might be involved in the occurrence of spontaneous miscarriage.     

Synergistic activation of human natural killer cell cytotoxicity by histamine and interleukin-2

Interleukin-2 (IL-2) is recognized as a major activating signal for human natural killer (NK) cells. The presence of monocytes alters NK cell responsiveness to IL-2. Thus, while IL-2 effectively augments NK cell cytotoxicity (NKCC) in monocyte-depleted NK effector cells, it is relatively ineffective or even suppressive for NK cells in monocyte-containing, NK-cell-enriched mononuclear cell fractions. Here we report that concomitant treatment with IL-2 and the biogenic amine histamine synergistically augmented NKCC against K562 and Daudi target cells of CD3-/CD16+ human NK cells in the presence but not in the absence of monocytes. Addition of peripheral-blood monocytes, recovered by countercurrent centrifugal elutriation, to purified NK cells abrogated IL-2 induced NK cell activation, reconstituted the synergistic, NK-activating effects of histamine and IL-2, and strongly reduced baseline NKCC. The effects of histamine on baseline NKCC and on NK cell responsiveness to IL-2 were related to counteraction of monocyte-mediated NK cell suppression. By contrast, histamine did not affect baseline or IL-2-induced NKCC in mixtures of NK cells and monocytes when the latter cells were recovered after adherence. The effect of histamine on NK cell responsiveness to IL-2 was mediated by H2-type histamine receptors, as judged by mimicry exerted by the specific H2 receptor agonist dimaprit, but not by an H2-receptor-inactive derivative of this compound, N-methyldimaprit, and blocking by the H2 receptor antagonist ranitidine. Experiments in which monocytes and nonadherent NK cells were separately pretreated with ranitidine prior to histamine treatment suggested that NK-regulatory effects of histamine were mediated by H2 receptors on monocytes. Our data suggest that histamine may provide an important signal in the regulation of NK cell responsiveness to IL-2.     

Expression of ADAMTS-5/implantin in human decidual stromal cells: regulatory effects of cytokines

BACKGROUND: The restricted expression of ADAMTS-5 (A Disintegrin And Metalloproteinase with ThromboSpondin repeats-5) to the maternal-fetal interface in mice has led to this novel metalloproteinase being assigned the trivial name 'implantin'. METHODS: As a first step in determining whether ADAMTS-5 also contributes to the implantation process in humans, we have examined the spatiotemporal expression of this ADAMTS subtype in the endometrium during the menstrual cycle and pregnancy by immunohistochemical analysis. A quantitative competitive PCR (QC-PCR) strategy and western blotting were subsequently used to determine whether interleukin (IL)-1beta and transforming growth factor (TGF)-beta1, two cytokines involved in the formation of the maternal-fetal interface, were capable of regulating ADAMTS-5 messenger RNA (mRNA) and protein levels in primary cultures of stromal cells isolated from first trimester decidual tissues. RESULTS: ADAMTS-5 expression in the stroma of the human endometrium correlates with decidualization of this cellular compartment in vivo. IL-1beta was found to increase (P < 0.05) whereas TGF-beta1 decreased (P < 0.05) ADAMTS-5 mRNA and protein levels in decidual stromal cell cultures in a concentration- and time-dependent manner. These regulatory effects were attenuated by function-perturbing antibodies directed against either cytokine. CONCLUSIONS: ADAMTS-5 expression is restricted to decidualized stromal cells of the human endometrium in vivo and is subject to regulation by cytokines in vitro.     

TGF-{beta} signaling of human T cells is modulated by the ancillary TGF-{beta} receptor endoglin

Transforming growth factor beta (TGF-beta) inhibits T cell activation and alters differentiation of naive T cells into effector cells. Although four main cell-surface proteins can interact with TGF-beta, only the signaling receptors type I (TGF-betaR type I) and type II (TGF-betaR type II) have so far been described on T cells. The aim of the present study was to investigate the expression of the ancillary receptor endoglin (CD105) by T cells and its role in TGF-beta-mediated signal transduction and function. CD105 expression was analyzed on resting and activated human CD4(+) T cells by flow cytometry, western blot, immunoprecipitation, proliferation and SMAD-responsive reporter gene assays. CD4(+) T cells constitutively expressed CD105 in memory T cells and partially also in naive T cells; however, surface expression is regulated and is increased following TCR engagement, which induced serine/threonine phosphorylation of CD105. In contrast to the suppressive signal mediated by the TGF-beta, cross-linking of CD105 substantially enhanced T cell proliferation, indicating that CD105 by itself mediates signal transduction. Furthermore, CD105 cross-linking induced SMAD-independent signaling via ERK kinase phosphorylation. The present study demonstrates that CD105 is expressed on the surface by activated CD4(+) T cells and CD3 regulated by post-translational means. Furthermore, CD105 acts as a regulatory receptor, counteracting TGF-beta-mediated suppression.     

Effect of human endometrial stromal cell-derived conditioned medium on uterine natural killer (uNK) cells' proliferation and cytotoxicity

Citation Chen Y, Zhuang Y, Chen X, Huang L. Effect of human endometrial stromal cell‐derived conditioned medium on uterine natural killer (uNK) cells’ proliferation and cytotoxicity. Am J Reprod Immunol 2011; 65: 589–596 Problem Human endometrial stromal cells are involved in the regulation of immune cell proliferation, apoptosis, differentiation, and function. In the endometrium, uNK cells are in close contact with stromal cells. The aim of the study was to investigate the effects of human endometrial stromal cells on uNK‐cell proliferation and uNK‐cell cytotoxicity. Method of study The conditioned medium was derived from the endometrial stromal cells in the proliferative phase, secretory phase, and early pregnancy. The effects of stromal cell‐derived conditioned medium on uNK‐cell proliferation and cytotoxicity were detected by mitochondrial lactate dehydrogenase‐based MTS staining and flow cytometry. Results The stromal cell‐derived conditioned medium in both secretory phase and early pregnancy significantly promoted uNK‐cell proliferation. Compared with the control group, the uNK‐cell cytotoxicity were significantly reduced by conditioned medium in the proliferative, secretory, and decidua groups, but there were no significant differences among these different physiological stages in the inhibiting ability. Conclusion Human endometrial stromal cells may be involved in the regulation of uNK‐cell functions through influencing proliferation and cytolytic activity.     

Role of interleukin-18 in human natural killer cell is associated with interleukin-2

Human natural killer (NK) cells constitute an important cellular component of innate immunity, capable of killing infected and transformed cells. The proliferation and activation of NK cells are regulated by various cytokines. Interleukin-18 (IL-18) promotes NK cell activation; however, whether the effects of IL-18 on NK cell are associated with other cytokines is still unknown. In this study, we observed that IL-18 induced NK cell apoptosis and inhibited NK cell expansion in the presence of low concentrations of interleukin-2 (IL-2), while high concentrations of IL-2 overcame these effects of IL-18, and high concentrations of IL-2 promoted the stimulatory activity of IL-18 on NK cells. At a low concentration of IL-2, IL-18 induced NK cell apoptosis in part through activation of the FasL/Fas- and TNFα/TNFR-mediated death receptor signaling by enhancing FasL expression and inhibiting c-FLIP_long expression. However, high concentrations of IL-2 strongly blocked IL-18-induced NK cell apoptosis through alleviating IL-18-induced FasL expression and activation of Fas-mediated death signaling and increasing anti-apoptosis molecule (Bcl-X_L). These results reveal that the effects of IL-18 on human NK cell are associated with IL-2 concentration and suggest the importance of IL-2 level in cytokine immunotherapy.     

Role of interleukin-18 in human natural killer cell is associated with interleukin-2

Human natural killer (NK) cells constitute an important cellular component of innate immunity, capable of killing infected and transformed cells. The proliferation and activation of NK cells are regulated by various cytokines. Interleukin-18 (IL-18) promotes NK cell activation; however, whether the effects of IL-18 on NK cell are associated with other cytokines is still unknown. In this study, we observed that IL-18 induced NK cell apoptosis and inhibited NK cell expansion in the presence of low concentrations of interleukin-2 (IL-2), while high concentrations of IL-2 overcame these effects of IL-18, and high concentrations of IL-2 promoted the stimulatory activity of IL-18 on NK cells. At a low concentration of IL-2, IL-18 induced NK cell apoptosis in part through activation of the FasL/Fas- and TNFα/TNFR-mediated death receptor signaling by enhancing FasL expression and inhibiting c-FLIP(long) expression. However, high concentrations of IL-2 strongly blocked IL-18-induced NK cell apoptosis through alleviating IL-18-induced FasL expression and activation of Fas-mediated death signaling and increasing anti-apoptosis molecule (Bcl-X(L)). These results reveal that the effects of IL-18 on human NK cell are associated with IL-2 concentration and suggest the importance of IL-2 level in cytokine immunotherapy.     

Functional subsets of mouse natural killer cells

Summary:  Human natural killer (NK) cells can be divided into two phenotypically distinct functional subsets based on their cell surface expression of CD56 (CD56^bright and CD56^dim). As mouse NK cells do not express CD56, comparable mouse NK cell subsets have proven difficult to identify. Recently, we have found that mouse NK cells can be subdivided by the expression of CD27. The CD27^hi and CD27^lo mouse NK cell subsets show some intriguing similarities to but also some distinct differences from the human CD56 NK cell subsets in terms of their function and phenotype. Extending our knowledge of mature NK cell heterogeneity between the species will be critical to further our understanding of the pathological role of NK cells in immune responses.     

Catecholamines induce alterations of distribution and activity of human natural killer (NK) cells

Catecholamines have been suggested to be responsible for altered cellular immunity after stress. This study was performed to determine the effects of adrenaline and noradrenaline on lymphocyte subpopulations and NK cell functions. Subjects were given a subcutaneous injection of either NaCl, adrenaline (5 µg/kg), or noradrenaline (10 µg/kg). Catecholamine concentrations, subsets of peripheral blood lymphocytes, NK activity, and antibody-dependent cellular cytotoxicity (ADCC) were analyzed before (baseline) and 5, 15, 30, 60, and 120 min after injection. There were no differences between groups in the distribution of CD2⁺ and CD8⁺ lymphocytes over time. However, CD3⁺ and CD4⁺ T cells decreased significantly 5 to 60 min after injection of adrenaline. In contrast, NK cell numbers (CD16⁺, CD56⁺) increased significantly 5 min after injection of adrenaline and noradrenaline, reached the highest values 15 to 30 min postinjection, and subsequently declined to baseline values 60 (noradrenaline) and 120 (adrenaline) min, respectively, after injection. Similar alterations for NK activity and ADCC were observed after administration of both catecholamines. These data suggest that both sympatheticadrenal hormones are similarly potent modulators of natural immunity and provide further evidence that catecholamines might be responsible for the observed alterations in immune functions after phases of acute stress.     

Potentiation of human natural killer cell activity by recombinant interleukin-2 towards multidrug-resistant human epidermoid carcinoma

Relatively little is understood about the antigen recognition and target structures used by natural killer (NK) cells. The purpose of this study was to analyze the relationship of a multidrug-resistant cell line expressing the P-glycoprotein (GP170 surface glycoprotein) derived from the parent non-drug-resistant malignant tumor cell line for susceptibility to lysis by either NK or lymphokine-activated killer cells. Our results demonstrate no significant difference in NK activity against either the non-drug-resistant or drug-resistant malignant cell line; Interleukin-2 induces a significant increase in cytolytic activity toward the multidrug-resistant cell line. These data suggest that malignant cells refractory to treatment or occurring after successful treatment are susceptible to immunotherapeutic intervention.     

Down-regulation of interleukin-2 receptor expression on natural killer cells/large granular lymphocytes by interleukin-4.

It has recently been demonstrated that IL-4 inhibits IL-2 receptor expression on T cells. Studies have also shown that IL-4 can inhibit IL-2-induced natural killer cell (NK) cytotoxicity, and that IL-4 in combination with IL-2 and large granular lymphocyte (NK/LGL) cells suppresses Ig synthesis. Therefore, we examine whether IL-2 receptor expression on NK/LGL cells is affected with or without IL-4, using fluorescent receptor analysis. Our results demonstrate that IL-4 inhibits/down-regulates the expression of IL-2 receptors on either phytohemagglutinin or IL-2-stimulated NK/LGL cells.     

Human decidual stromal cells express HLA-G: Effects of cytokines and decidualization

BACKGROUND: Decidual stromal cells (DSC) are the main cellular componentof the decidua, the maternal tissue in close contact with fetaltrophoblast. Although of mesenchymal origin, DSC exert numerousimmune functions that seem to be relevant for the immunologicalrelationship between the mother and fetus. HLA-G, an antigenpreferentially expressed by trophoblast, appears to participatein the immune tolerance by the mother of the semiallogeneicfetus. METHODS AND RESULTS: We show by flow cytometry, fluorescence microscopy, westernblotting and RT–PCR that DSC isolated and maintained inculture express HLA-G weakly but consistently. We also detectedthis antigen by flow cytometry in fresh DSC. Interleukin (IL)-10,a cytokine associated with normal pregnancy, increased the expressionof HLA-G by DSC (P < 0.00001), whereas IL-2, a cytokine involvedin spontaneous abortion, showed no effect. Decidualization byprogesterone and cAMP also up-regulated the expression of HLA-Gby DSC (P < 0.001). Interferon , a cytokine implicated inthe vascular remodelling of the decidua necessary for embryoimplantation, also increased the expression of HLA-G by DSC(P < 0.05). CONCLUSIONS: Our results suggest the existence of a network in which hormonestogether with cytokines regulate the expression of HLA-G byDSC, and that may be of relevance in the maintenance of maternal–fetaltolerance.     

Pentoxifylline suppresses interleukin-2-mediated activation of immature human natural killer cells by inhibiting endogenous tumor necrosis factor-alpha secretion

We recently reported that immature human peripheral blood-derived natural killer (NK) cells, the free NK subset, can be activated by interleukin-2 (IL-2) to become killer cells and to undergo proliferation. Activation by IL-2 is dependent on endogenous secretion of tumor necrosis factor-alpha (TNF-) by the free cells. Because pentoxifylline (PTX) inhibits TNF- synthesis and secretion in monocytes, we hypothesized that PTX may also inhibit TNF- secretion by NK cells and thus would inhibit IL-2-mediated activation of free cells. The free NK cells were separated from purified NK cells by flow cytometry and cell sorting of non-target binding cells. IL-2-mediated secretion of TNF- by the free cells was inhibited by PTX. In the presence of PTX, IL-2-mediated activation of free cells into cytotoxic function, proliferation, and recruitment of binder and killer cells was markedly inhibited. Also, PTX inhibited IL-2-triggered upregulation of the expression of CD69, CD25, ICAM-1, and p75TNF-R on the cell surface. These findings demonstrate that PTX has a marked suppression on IL-2-mediated activation of immature free NK cells and that the suppression is due, in large part, to PTX-mediated inhibition of endogenous TNF- secretion. The implication of these findings in the clinical use of PTX for therapy is discussed.