IL-18 is present at the maternal-fetal interface and enhances cytotoxic activity of decidual lymphocytes

PROBLEM: Perforin expressing uterine natural killer (uNK) cells are under complex cytokine influence. The aim of the study was to investigate the presence and role of interleukin (IL)-18 on NK cytolytic potential at maternal-fetal (M-F) interface. METHOD OF STUDY: Peripheral blood cells and decidual tissue were obtained from elective pregnancy termination of normal human 6-10-week-old pregnancies. Perforin expression and cytolytic activity of peripheral blood (PBL) and decidual lymphocytes (DL) were analyzed by flow cytometry. IL-18 positive decidual adherent cells (DAC) were detected by the same method. Interleukin-18 and IL-18 receptor (IL-18R) expression on the trophoblastic cells was detected by immunohistology using biotinylated anti-IL-18 and IL-18R monoclonal antibodies. RESULTS: The IL-18 added in a dose of 10 ng/mL up-regulates perforin expression and cytolytic activity of DL. Simultaneous stimulation with IL-18 and IL-12 enhanced DL cytolytic activity, while IL-18 combined with IL-10 or IL-15 did not show this effect. Cytolytic activity of PBL was up-regulated by IL-18 as well, and this effect was enhanced by the addition of IL-12 and IL-15. Interleukin-18 did not affect perforin-protein expression in cultured PBL. Approximately 20% of DAC were IL-18 positive and these cells were mostly human leukocyte antigen (HLA)-DR negative. IL-18R positive cells were found on syncytiotrophoblast cell layer, interstitial tissue cells of villi and fetal blood cells. There was no detectable IL-18 staining on trophoblast cell layer on villi, but strong staining of fetal blood cells in villous vessels. CONCLUSION: These are first results showing IL-18R expression, but not IL-18 expression on villous trophoblastic cells, as well as enhancement of perforin expression and NK cytolytic potential of DL under the influence of IL-18. IL-18 in concert with other cytokines and hormones could play an important role in the regulation of cytolytic potential of first trimester pregnancy decidual and peripheral blood NK cells.     

Progesterone directly and indirectly affects perforin expression in cytolytic cells

PROBLEM: Decidual lymphocytes (DL) expressing the cytolytic molecule perforin represent approximately 55% of DL in the first trimester of human pregnancy. Progesterone dominates this phase of pregnancy and controls the production of uterine cytokines and growth factors. The aim of this study was to investigate the role of progesterone and progesterone-induced blocking factor (PIBF) on perforin expression in DL and peripheral blood lymphocytes (PBL). METHOD OF STUDY: Perforin expression was analyzed in PBL and DL incubated either in culture medium or with decidual adherent cells (DAC) and peripheral blood adherent cells (PBAC) and their supernatants with or without progesterone or PIBF. Perforin was detected by flow cytometry in PB and in decidual first trimester pregnancy lymphocytes. RESULTS: Progesterone in high concentrations directly affects perforin expression in DL but not in PBL. Progesterone in a concentration dependent manner indirectly blocks perforin expression in DL and PBL cultured with adherent cells or their supernatants. PIBF blocked upregulation of perforin expression of DL cultured with DAC, but none of those cultured with PBAC. Similarly, PIBF was inefficient when PBL or DL were cultured with PBAC. CONCLUSION: Progesterone present in a high concentration locally at the maternal-fetal interface modulates perforin expression in the first trimester pregnancy DL.     

Progesterone induced blocking factor (PIBF) mediates progesterone induced suppression of decidual lymphocyte cytotoxicity

PROBLEM: Progesterone induced blocking factor (PIBF) is a mediator of progesterone that blocks peripheral blood lytic natural killer (NK) activity. Progesterone or PIBF stimulated decidual macrophages block up-regulation of perforin expression in decidual lymphocytes (DL). Therefore, we investigated whether progesterone regulates cytotoxicity of DL. METHOD OD STUDY: Decidual mononuclear cells were cultured with progesterone. PIBF, progesterone and anti-PIBF antibody or in the medium only. Cytolytic activity of non-adherent DL was measured by PKH-26 (red) 2 hr cytolytic assay and flow cytometry. Perforin positive DL were detected by immunofluorescency and PIBF-positive cells by immunohistology. RESULTS: Progesterone and PIBF, in a dose-dependent manner decreased cytotoxicity of DL against K-562 targets, and perforin egzocytosys was blocked. Anti-PIBF antibodies reversed the progesterone mediated reduction in cytolytic activity of DL. PIBF positive cells were found in first trimester pregnancy decidua. CONCLUSION: The results indicate possible role for PIBF, as a mediator of progesterone in regulation of DL cytolytic activity at the maternal-foetal (M-F) interface.     

Decidual macrophages are the population of decidual adherent cells which regulates perforin expression in cytolytic cells.

PROBLEM: We have shown that addition of decidual adherent cells (DAC) to the culture of decidual lymphocytes (DL) prevents the downregulation of perforin expression in these cells. Because DAC are a mixture of various cell populations, the aim is to analyze immunophenotypic characteristics of DAC and to determine which cell population is involved in the regulation of perforin expression. METHOD OF STUDY: First trimester pregnancy decidual cells were obtained by enzymatic tissue digestion. Decidual cells and peripheral blood lymphocytes (PBL) were centrifuged on Ficoll-Hypaque density gradient and cultured overnight to obtain adherent cells, which were analyzed by flow cytometry and immunocytochemically. RESULTS: Almost all peripheral blood adherent cells (PBAC) (ca 90%) expressed monocyte/macrophage markers but only 10-20% of DAC. The rest of DAC expressed markers of stromal cells. HLA-DR depleted population of DAC (stromal cells only) could not prevent downregulation of perforin expression in cultured DL and PBL. CONCLUSION: Decidual macrophages are involved in the regulation of perforin expression in DL.     

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.     

Progesterone-induced blocking factor inhibits degranulation of natural killer cells.

PROBLEM: During the first trimester of pregnancy, nonclassical (CD3-, CD56+, CD16-, perforin [P]bright+) natural killer (NK) cells comprise the major decidual lymphocyte population. These cells, in spite of their high perforin content, exert a low cytolytic activity. Peripheral blood lymphocytes of healthy pregnant women produce progesterone-induced blocking factor (PIBF), which inhibits NK activity. PIBF-producing cells are likely to be present in decidua and might contribute to low decidual NK activity. METHOD OF STUDY: Decidual cells obtained from elective pregnancy termination were double labeled for CD56 and PIBF. We tested the effect of PIBF on perforin liberation by activated peripheral blood NK cells. RESULTS: Sixty percent of decidual lymphocytes were CD56 + and expressed PIBF at the same time. PIBF-treated and untreated peripheral blood NK cells were incubated with K-562 cells, and perforin content of target conjugated NK cells was detected with immunocytochemistry. PIBF treatment of peripheral blood lymphocytes significantly reduced lysis of K-562 cells. Among target bound lymphocytes in PIBF-treated samples, we found a significantly (P < 0.01) higher rate of P+ cells than in untreated samples. CONCLUSIONS: These data suggest that PIBF inhibits cytotoxicity of NK cells via a block of degranulation, and since decidual NK cells are PIBF+, it cannot be ruled out that this effect of PIBF contributes to low decidual NK activity.     

Modulation of perforin expression in the decidual and peripheral blood cytotoxic lymphocytes in culture.

PROBLEM: Perforin (P) is a cytolytic molecule located in intracellular granules of cytotoxic lymphocytes both in the peripheral blood and decidua of pregnancy. The aim was to analyze the kinetics of P expression during in vitro culture and modulation of P expression by adherent cells, their supernatants and mitogen (PHA) stimulation. METHOD OF STUDY: P (intracellular antigen) was detected by flow cytometry in the suspension of first trimester pregnancy peripheral blood lymphocytes (PBL) and decidual lymphocytes (DL). RESULTS: A decrease of the percentage of P+ cells was obtained after 1 hr incubation and was prevented by addition of 30% of decidual adherent cells (DAC) or their supernatants. Upregulation of P expression was obtained when, in addition to adherent cells, DL and PBL were stimulated by PHA. DAC present in the culture in physiological concentrations prevent downregulation of P expression. CONCLUSION: DAC located in the vicinity of decidual cytotoxic lymphocytes, owing to their unique ability to produce a wide range of substances on demand, contribute to the high level of P expression in the decidua of pregnancy.     

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.     

Impaired susceptibility of human trophoblast to MHC nonrestricted killer cells: implication in the maternal-fetal relationship

Mammalian pregnancy has frequently been termed "Nature's allograft," and there is developing evidence that the placental trophoblast cells in their key position at the maternal fetal interface are responsible for escape mechanisms to the maternal immune system. In this paper, we show impaired susceptibility of human trophoblastic tumor cells to major histocompatibility complex (MHC) nonrestricted cytotoxicity systems such as natural killer (NK) cells and lymphokine-activated killer (LAK) cells. LAK cells were induced by the culture of peripheral blood mononuclear lymphocytes (PBL) in recombinant interleukin-2 (rIL-2). Among 21 cultured cell lines derived from various tissues and organs tested, five choriocarcinoma-derived cell lines gave decreased levels of LAK lysis. Cold-target inhibition study and trinitrophenyl (TNP) modification experiment clearly indicated that the impaired sensitivity of trophoblast cells to LAK lysis is due to the decrease of a common target molecule recognized by LAK effector cells. It is suggested that the impaired susceptibilty of trophoblast to MHC nonrestricted killer cells should be functional for the survival of the semiallogeneic fetus.     

Human recombinant IL-4 decreases the emergence of non-specific cytolytic cells and favours the appearance of memory cells (CD4+CD45RO+) in the IL-2-driven development of cytotoxic T lymphocytes against autologous ovarian tumour cells.

As IL-4 and IL-6 have also been reported to promote the development of T lymphocytes such as IL-2, we investigated their role in the development of specific cytotoxic T lymphocytes (CTL) against autologous ovarian tumours in mixed lymphocyte tumour cultures (MLTC). Peripheral blood lymphocytes (PBL) from five ovarian carcinoma (OC) patients were incubated with autologous OC cells at a PBL:OC cell ratio of 20:1 in IL-2 alone (50 U/ml for the first week and 200 U/ml thereafter) or with IL-4 (100 U/ml) and/or IL-6 (5 U/ml). Neither IL-4 nor IL-6 improved lymphocyte proliferation consistently. In contrast, IL-4 reduced significantly the development of LAK activity as assayed against Daudi cell line, and decreased modestly the emergence of natural killer (NK) activity as assayed against K562. This property was not shared by IL-6. The prevention of the development of non-specific cytolytic activity (LAK and NK activities) was much stronger when the MLTC was started with IL-4 in the absence of IL-2 during the first week in culture. A concomitant drop in NKH-1 expression (CD56) was observed. By inhibiting the emergence of non-specific cytotoxicity, IL-4 provided better evidence of the specific cytolytic activity directed at ovarian cells. In parallel, a significant increase in the generation of memory cells (CD4+CD45RO+) was observed with IL-4. In conclusion, in this model, IL-4 added before IL-2 decreases significantly the emergence of non-specific cytotoxic cells, and promotes the generation of memory cells. These properties may be of interest in the design of strategies aimed at obtaining tumour-specific cells for investigational and immunotherapeutic purposes.     

弓形虫感染对人早孕母胎界面细胞因子转录水平的影响

研究弓形虫感染时IL-4、IL-10在绒毛滋养层细胞和IFN-γ在蜕膜NK细胞中mRNA水平的变化,从而探讨弓形虫感染致不良妊娠的分子免疫机制。选取正常妊娠5~10周妇女行人工流产术后的绒毛及蜕膜组织14例,利用绒毛组织分离滋养层细胞和蜕膜组织分离蜕膜NK细胞。分别将绒毛滋养层细胞和蜕膜NK细胞平均分为实验组和对照组,加入弓形虫培养和单独培养48 h后,采用实时荧光定量聚合酶链式反应(real-time PCR)检测滋养层细胞IL-4、IL-10 mRNA表达水平和蜕膜NK细胞IFN-γmRNA表达水平。结果:实验组和对照组IL-4、IL-10、IFN-γmRNA平均表达水平分别为(0.05±0.02)、(0.06±0.03)、(0.33±0.09)和(0.31±0.13)、(0.18±0.06)、(0.08±0.03),三者之间均具有显著性差异(P<0.05、P<0.01、P<0.05);实验组IFN-γ/IL-10、IFN-γ/IL-4的比值分别为(3.96±0.84)和(3.21±0.41),对照组则分别为(0.60±0.21)、(0.78±0.25),两组间同样均具有显著性差异(P<0.01、P<0.01)。早孕期弓形虫感染可致母胎界面Th1型细胞因子表达增高,Th2型细胞因子表达降低,从而打破正常妊娠状态下二者的平衡,可能是早孕期弓形虫感染导致不良妊娠的重要分子机制。     

早孕妇女外周血和蜕膜组织中自然杀伤细胞亚群的变化

目的和方法：用流式细胞仪检测正常非孕及早孕妇女外周血和蜕膜自然杀伤细胞表面标记ＣＤ５６和ＣＤ１６分子,探讨早孕外周血与蜕之间自然杀伤细胞亚群的差异以及雌、孕激素对外周血自然杀伤细胞的影响。结果：蜕膜以ＣＤ５６细胞为主要淋巴细胞,早孕外周血则以ＣＤ５６ＣＤ１６和ＣＤ１６为主。孕酮在一定范围内增加,可使外周血自然杀伤细胞各亚群的百分率明显增加;在早孕期、雌、孕激素水平的急剧升高可能使外周血ＣＤ５６细胞     

Cytokine secretion patterns of NK cells and macrophages in early human pregnancy decidua and blood: implications for suppressor macrophages in decidua

PROBLEM: Local immune modulation has been shown to be of considerable importance for the maintenance of successful pregnancy. We have previously reported the secretion of interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and IL-10 in human decidua from early normal pregnancy. The aim of this study was to investigate the cellular source of cytokine secretion in the decidua, and compare this to secretion patterns in peripheral blood. METHOD OF STUDY: Decidual tissue and peripheral blood was collected from 20 women undergoing surgical abortion during first trimester pregnancy. Monocytes/macrophages and NK cells were enriched by immunomagnetic cell separation and cytokine secretion was detected by enzyme-linked immunosorbent spot-forming cell assay. RESULTS: Decidual and peripheral monocytes/macrophages and NK cells spontaneously secrete IFN-gamma, IL-4 and IL-10. The number of IL-10 secreting cells was significantly higher in decidual macrophages compared with decidual non-monocytic cells as well as compared with blood monocytes/macrophages. These differences were not seen for IFN-gamma or IL-4. CONCLUSIONS: Our results indicate that decidual macrophages subserve important suppressive functions in the pregnant uterus.     

人早孕期蜕膜基质细胞对蜕膜NK细胞分泌IL-22的调节作用

目的:探讨蜕膜基质细胞(Decidual stromal cells,DSCs)与蜕膜NK细胞(dNK)共培养后IL-22的分泌水平。方法:收集早孕蜕膜组织,分离蜕膜基质细胞(DSCs)及蜕膜免疫活性细胞(Decidual immunocytes,DICs),磁珠分选蜕膜CD56brightCD3-NK细胞,再与DSC按不同比例直接接触共培养(dNK∶DSC为1∶1、1∶2、1∶3)24小时,收集上清。ELISA检测上清中IL-22的表达。结果:与对照组相比,DSC能上调dNK分泌IL-22。结论:蜕膜基质细胞可以促进蜕膜NK细胞分泌IL-22。     

孕早期人母-胎免疫耐受机制的研究

目的：探讨孕早期母胎免疫耐受调节机制。方法;应用流式细胞仪分析（ＦＡＣＳ）蜕膜及孕妇外周血淋巴细胞亚群;采用改进的乳酸脱氢酶释放试验测定蜕膜淋巴细胞细胞对Ｋ５６２的自然杀伤活性;同时以滋养鲺蜕膜淋巴细胞共同培养,研究平体免疫系统对滋养细胞抗原免疫应管状态。结果：蜕膜组织中Ｄ５６＾＋ＮＫ细胞含量高于孕妇及对照组外周血ＣＤ５６＾＋细胞比例（Ｐ〈０．０５）,蜕膜组织中ＮＫ细胞杀伤活性明显低于孕妇及对照组     

妊娠子宫微环境中uNK细胞NKG2A和NKG2D及其配体表达的研究

目的：研究妊娠子宫微环境中子宫自然杀伤细胞（uNK细胞）NKG2A和NKG2D及其相应配体的表达，探讨NKG2A与NKG2D的不平衡表达在母胎免疫耐受形成中的作用。方法：选择30例孕6-9周的正常妊娠妇女,分离其新鲜蜕膜组织，除去绒毛,分离蜕膜和外周血单个核细胞，采用流式细胞仪测定NK细胞的数量及NKG2A与NKG2D的表达；采用RT-PCR技术检测滋养层组织NKG2A与NKG2D配体人类白细胞抗原-E(HLA-E)、主要组织相容性复合体-Ⅰ类分子相关蛋白A(MICA)mRNA的表达结果：妊娠子宫蜕膜淋巴细胞中NK细胞约占70%,流式细胞分析的结果显示，子宫自然杀伤细胞NKG2A的表达显著高于外周血NK细胞，分别为97.86%±1.75%与33.35%±10.92%（〖AKx-D〗±s），两者差异显著（P<0.05），在滋养层细胞中检测到其配体HLA-E的表达；而与外周血相比，uNK细胞表面NKG2D的表达与之较为相近，分别为93.21%±4.52%与97.80%±1.72%，但两者仍有显著差异（P<0.05）。在滋养层组织未检测到其相应配体MICA mRNA的表达结论：蜕膜中的淋巴细胞主要为NK细胞，其免疫学表型与外周血NK细胞有较大的区别，妊娠期子宫自然杀伤细胞表面高表达抑制性受体NKG2A，同时滋养层组织表达相应的配体人类白细胞抗原-E，这可能是维持母胎界面免疫耐受的重要因素。     

The role of gamma/delta T cells in progesterone-mediated immunomodulation during pregnancy: a review.

PROBLEM: To determine if pregnancy is recognized by the immune system and if inadequate recognition of fetal antigens might result in failed pregnancy. METHOD OF STUDY: Review of literature and current data. RESULTS: In the decidua gamma/delta TCR positive cells significantly increase in number. A subset of gamma/delta T cells reacts with nonpolymorphic Class I or Class I like molecules. Trophoblast recognition is mediated by the V gamma 1 subset which recognize a conserved mammalian sequence on the trophoblast. Almost all gamma/delta T cells in the decidua are activated and use the V delta 1 chain, whereas the majority of human peripheral gamma/delta lymphocytes expresses V gamma 9/V delta 2 TCR. Peripheral gamma/delta T cells of healthy pregnant women preferentially use V gamma V delta 1 chains, on the other hand, those of recurrent aborters use the V gamma 9V delta 2 combination. Signaling via the V gamma 1.4V delta 1 receptor induces a Th2 type response, whereas activation of the lymphocytes via the V gamma 9V delta 2 receptor results in increased IL-12 production and natural killer (NK) activity. In the presence of progesterone, activated lymphocytes synthesize the progesterone induced blocking factor (PIBF), which inhibits NK activity and exerts an anti abortive effect in vivo. Decidual CD56+ and gamma delta+ cells are to a high extent the same population. CONCLUSION: All decidual CD56+ cells express PIBF, thus it cannot be excluded that local production of this substance contributes to low decidual NK activity and thus to the success of the pregnancy.