Pathogenetic Implication of Interleukin-2 Expressed in Preeclamptic Decidual Tissues: A Possible Mechanism of Deranged Vasculature of the Placenta Associated with Preeclampsia

PROBLEM: The purpose of this study is to clarify whether IL-2 expressed in the decidua in preeclampsia affects the angiogenesis of the placenta. METHOD OF STUDY: We investigated the angiogenic substances released from human trophoblasts obtained from early pregnancy that had been pretreated with either IL-2, non-activated lymphocytes from peripheral blood mononuclear cells (PBMCs), decidual lymphocytes, or these lymphocytes activated by lymphokine (LAK cells). Angiogenic activity was determined by evaluating the ability of growth-promotion of cultured human microvascular endothelial cells (HMvECs) using MTT assay. RESULTS: Trophoblasts pretreated with IL-2 or non-activated lymphocytes, irrespective of their origin, released angiogenic factor similar to those without pretreatment. However, trophoblasts pretreated with LAK cells released less angiogenic factor compared with those without pretreatment. CONCLUSIONS: Interleukin-2 (IL-2) expressed in preeclamptic decidua might reduce the angiogenic substances arising from trophoblasts by inducing LAK cells from decidual lymphocytes, which might be relevant to deranged vasculature of the placenta, a characteristic histology in preeclampsia.     

Osteopontin facilitates invasion in human trophoblastic cells via promoting matrix metalloproteinase-9 in vitro

Successful implantation of embryo and placentation depend on proper trophoblast proliferation and differentiated into specialized invasive trophoblast. However, little is known about the regulatory factors and mechanisms in trophoblast proliferation and differentiation. Osteopontin (OPN) is a member of the small integrin-binding ligand N-linked glycoprotein family and participates in cell adhesion and invasion. It has been identified that OPN is highly expressed in invasive trophoblasts in human placenta. In this study, we demonstrated that OPN is constitutively expressed in highly invasive phenotype of human choriocarcinoma cell lines of JAR and JEG-3 cells, and OPN could promote trophoblast proliferation and invasion, partly through promoting MMP-9 secretion. Inhibition of OPN will compromise the abilities of proliferation and invasion in JAR and JEG-3 cell lines. Our data showed that the expression of OPN in trophoblast may participate in placentation, OPN expression defects may be involved in gestational trophoblastic diseases.     

Altered Expression of Human Leukocyte Antigen G (HLA-G) on Extravillous Trophoblasts in Preeclampsia: Immunohistological Demonstration With Anti-HLA-G Specific Antibody “87G” and Anti-cytokeratin Antibody “CAM5.2”

PROBLEM: Human leukocyte antigen-G (HLA-G) is suggested to be at play in the materno-fetal immune relationship during pregnancy. In the light of current concept that disruption of the materno-fetal immune relationship could account for several complications of pregnancy, including preeclampsia, we asked whether the expression of HLA-G protein on the trophoblasts is altered in preeclampsia. METHOD: The presence of HLA-G protein in the extravillous trophoblasts in placenta obtained from five preeclamptic patients and seven uncomplicated pregnant women was determined by means of an immunohistochemical technique. RESULTS: All of the extravillous trophoblasts, which were stained for cytokeratin, were stained for HLA-G protein in every woman with an uncomplicated pregnancy. In contrast, clusters of extravillous trophoblasts were insularly devoid of the staining for HLA-G in all the preeclamptic patients. CONCLUSION: The attenuated expression of HLA-G protein on the extravillous trophoblasts could be at play in the pathophysiology of preeclampsia.     

A homodimeric complex of HLA-G on normal trophoblast cells modulates antigen-presenting cells via LILRB1

In healthy individuals, the non-classical MHC molecule HLA-G is only expressed on fetal trophoblast cells that invade the decidua during placentation. We show that a significant proportion of HLA-G at the surface of normal human trophoblast cells is present as a disulphide-linked homodimer of the conventional beta(2)m-associated HLA-I complex. HLA-G is a ligand for leukocyte immunoglobulin-like receptors (LILR), which bind much more efficiently to dimeric HLA-G than to conventional HLA-I molecules. We find that a LILRB1-Fc fusion protein preferentially binds the dimeric form of HLA-G on trophoblast cells. We detect LILRB1 expression on decidual myelomonocytic cells; therefore, trophoblast HLA-G may modulate the function of these cells. Co-culture with HLA-G(+) cells does not inhibit monocyte-derived dendritic cell up-regulation of HLA-DR and costimulatory molecules on maturation, but did increase production of IL-6 and IL-10. Furthermore, proliferation of allogeneic lymphocytes was inhibited by HLA-G binding to LILRB1/2 on responding antigen-presenting cells (APC). As HLA-G is the only HLA-I molecule that forms beta(2)m-associated dimers with increased avidity for LILRB1, this interaction could represent a placental-specific signal to decidual APC. We suggest that the placenta is modulating maternal immune responses locally in the uterus through HLA-G, a trophoblast-specific, monomorphic signal present in almost every pregnancy. See accompanying commentary: (http://dx.doi.org/10.1002/eji.200737515).     

HLA-G mRNA差异表达对细胞膜表面HLA-G异构体分子表达的影响

目的 探讨HLA-G异构体(HLA-G1～6)mRNA差异表达对HLA-G分子在细胞表面表达的影响.方法 通过RT-PCR方法分析卵巢癌细胞株HO-8910、H0-8910PM、OVCAR-3,白血病细胞株Jurkat、K562、HL60、MUTZ-1,绒癌细胞株JEG-3、JAR内HLA-G异构体mRNA的表达种类,采用流式细胞术分析上述细胞株细胞表面及细胞内HLA-G分子的分布及表达水平.结果 阳性对照JEG-3细胞内表达HLA-G1～6 mRNA,阴性对照JAR不表达HLA-G1～6 mRNA.HLA-G1 mRNA在HO-8910、HO-8910PM、OVCAR-3、MUTZ-1、Jurkat细胞内表达.除阳性对照JEG-3外,其他细胞均不表达HLA-G2 mRNA;表达HLA-G3 mRNA的细胞有HO-8910、HO-8910PM、K562、HL60、MUTZ-1、OVCAR-3、Jurkat;表达HLA-G4 mRNA的细胞有HO-8910、HO-8910PM、HL60、Jurkat;表达HLA-G5mRNA的细胞为Jurkat.FACS分析显示在JEG-3、HO-8910PM、Jurkat细胞膜表面表达HLA-G分子,而除JAR细胞外,其他细胞内均表达HLA-G分子.结论 HLA-G2、-G3、-G4、-G5、-G6均不能在细胞表面表达,而HIJA-G1分子则能在特定的细胞表面表达.     

Effects of Notch2 and Notch3 on Cell Proliferation and Apoptosis of Trophoblast Cell Lines

Aims: To investigate the effect of Notch2 and Notch3 on cell proliferation and apoptosis of two trophoblast cell lines, BeWo and JAR.Methods: Notch2 and Notch3 expression in BeWo and JAR cells was upregulated or downregulated using lentivirus-mediated overexpression or RNA interference. The effect of Notch2 and Notch3 on cell proliferation was assessed by the CCK-8 assay. The effect of Notch2 and Notch3 on the apoptosis of BeWo and JAR cells was evaluated by flow cytometry using the Annexin V-PE Apoptosis kit. Lentivirus-based overexpression vectors were constructed by cloning the full-length coding sequences of human Notch2 and Notch3 C-terminally tagged with GFP or GFP alone (control) into a lentivirus-based expression vector. Lentivirus-based gene silencing vectors were prepared by cloning small interfering sequences targeting human Notch2 and Notch3 and scrambled control RNA sequence into a lentivirus-based gene knockdown vector. The effect of Notch2 and Notch3 on cell proliferation was assessed by the CCK-8 assay. And the effect of Notch2 and Notch3 on the apoptosis of BeWo and JAR cells was evaluated by flow cytometry using the Annexin V PE Apoptosis kit.Results: We found that the downregulation of Notch2 and Notch3 gene expression in BeWo and JAR cells resulted in an increase in cell proliferation, while upregulation of Notch3 and Notch2 expression led to a decrease in cell proliferation. Moreover, the overexpression of Notch3 and Notch2 in BeWo and JAR cells reduced apoptosis in these trophoblast cell lines, whereas apoptosis was increased in the cells in which the expression of Notch3 and Notch2 was downregulated.Conclusions: Notch2 and Notch3 inhibited both cell proliferation and cell apoptosis in BeWo and JAR trophoblast cell lines.     

First trimester human endovascular trophoblast cells express both HLA-C and HLA-G.

PROBLEM: In human pregnancies, trophoblasts, in contrast to placental connective tissue and the fetus itself, come into direct contact with the maternal allorecognizing system at special sites. Villous syncytiotrophoblasts washed around by maternal blood lack HLA class I proteins, whereas extravillous trophoblasts, which deeply invade maternal uterine tissues, express high amounts of HLA-G and also HLA-C, the latter to a lesser degree, however. A subpopulation of extravillous trophoblasts, the endovascular trophoblast, enters maternal spiral artery lumen and, like syncytiotrophoblast, comes into direct contact with maternal blood. Less is known about HLA class I distribution on this endovascular trophoblast subpopulation. METHOD OF STUDY: A comparative immununohistochemical analysis was done on decidual cryo-sections containing trophoblast-invaded spiral arteries using different anti-HLA class I monoclonal antibodies (mAbs) and a peroxidase-labeled streptavidinbiotin detection system. RESULTS: MAbs W6/32 (anti-HLA-A, -B, -C, -G), HCA2 (anti-HLA-A, -G) G233 and 87G (both anti-HLA-G) resulted in strong positivity on endovascular trophoblasts. L31 (anti-HLA-C) and HC10 (anti-HLA-B, -C) revealed clear positivity, whereas TU149 (anti-HLA-B, -C, some -A) produced a heterogeneous staining pattern, faintly positive on some endovascular trophoblastic cells and negative on others. MAb LA45 (anti-HLA-A, -B) did not bind to any endovascular trophoblast, neither did BFL.1 (anti-HLA-G) nor 16G1 (anti-HLA-G, soluble). CONCLUSION: This study shows that trophoblastic cells belonging to the endovascular subpopulation express considerable amounts of HLA-G and slightly less HLA-C.     

Role of the HLA-G immune checkpoint molecule in pregnancy

The non-classical HLA class I molecule HLA-G is expressed in trophoblasts where it contributes to maternal-fetal tolerance. HLA-G has been implicated in the control of trophoblast invasion, uterine vascular remodeling, and maintenance of a local immunosuppressive state. Understanding HLA-G biology at the maternal-fetal interface is therefore a critical issue in reproduction. In this regard, we review here: (i) the effects of HLA-G on decidual leucocytes and stromal cells, (ii) the contribution of trogocytosis in HLA-G expression on decidual cells, (iii) its interaction with the ILT2, ILT4 and KIR2DL4 receptors, (iv) the link between HLA-G polymorphism and pregnancy disorders, and (v) the expression of newly-described HLA-G isoforms 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.     

膜结合型HLA-G1～G4分子的克隆表达及对NK细胞杀伤功能的影响

目的 探讨膜结合型HLA-G1～G4异构体的表达及对NK细胞杀伤功能的影响.方法 通过基因克隆及转染,分别建立稳定表达HLA-G1～G4抗原的人绒癌JAR细胞株.采用RTPCR、流式细胞术、Western blot及免疫细胞化学法分析、鉴定转染细胞中HLA-G的mRNA及蛋白表达.通过加载HLA-G高亲和性KIPAQFYIL抗原肽,观察对HLA-G表达的影响.LDH释放法检测HLA-G1～G4表达对NK细胞杀伤活性的影响.结果 RT-PCR、Western blot及免疫细胞化学结果显示,HLA-G1～G4/pVITRO2-mcs重组质粒成功转染HLA-G表达阴性的人绒癌JAR细胞株.FACS分析显示HLA-G1抗原能在JAR-HLA-G1细胞株表面表达,HLA-G2～G4抗原不能有效到达细胞表面.体外杀伤试验发现表达HLA-G1～G4抗原的细胞均能抑制NK细胞的杀伤活性(P＜0.05);加载HLA-G高亲和性KIPAQFYIL抗原肽对HLA-G表达无明显影响,对NK细胞杀伤抑制程度也未见明显改变.结论 HLA-G1～G4能够明显抑制NK细胞的杀伤活性,提示不同膜结合型HLA-G异构体分子均能作为免疫耐受分子,具备免疫调节功能.     

复发性自然流产患者绒毛及蜕膜组织中白细胞介素8的表达

目的:研究复发性自然流产患者绒毛及蜕膜组织中白细胞介素8(interleukin-8,IL-8)的表达。方法:应用免疫组化法及图像分析技术,对30例复发性自然流产患者绒毛、蜕膜组织中IL-8的蛋白表达进行定位及半定量分析;H- E染色后光镜下观察绒毛及蜕膜组织的形态学变化。结果:在绒毛组织中,IL-8蛋白定位于绒毛上皮细胞的细胞质内,且病例组的表达明显高于对照组;在蜕膜组织中,病例组蜕膜细胞的胞质内可见IL-8蛋白表达,且高于对照组;H- E染色可见病例组绒毛组织的滋养层变薄,细胞变性甚至坏死、嗜酸性增强,绒毛中轴纤维化程度增强;蜕膜组织中蜕膜细胞失去细胞间连接,部分蜕膜细胞解体、核消失。结论:病例组绒毛及蜕膜组织中IL-8蛋白表达的增强与复发性自然流产有关,IL-8可能参与复发性自然流产的病理过程。     

层粘连蛋白和纤维粘连蛋白在正常绒毛膜葡萄胎、绒毛膜癌组织中的表达

目的探讨层粘连蛋白和纤维粘连蛋白在正常绒毛膜和滋养细胞肿瘤中的作用。方法用免疫组织化学方法观察层粘连蛋白和纤维粘连蛋白在正常绒毛膜、葡萄胎和绒毛膜癌组织中的表达。结果在正常绒毛膜组织中,二种蛋白有广泛表达,如绒毛上皮基膜、毛细血管内皮基膜、绒毛间质、滋养细胞柱和蜕膜。在葡萄胎,层粘连蛋白分布于绒毛上皮基膜和蜕膜组织,在基膜中表达强度有所不同;纤维粘连蛋白分布于绒毛和蜕膜内,呈弱阳性表达。在侵蚀性葡萄胎和绒毛膜癌,层粘连蛋白和纤维粘连蛋白在细胞质中均呈强阳性表达,在细胞间质内呈阳性或弱阳性表达。结论层粘连蛋白和纤维粘连蛋白与孕早期绒毛膜和蜕膜的形成及滋养细胞肿瘤的发生、侵润密切相关。     

人母-胎界面滋养细胞来源的CXCL16促进蜕膜γδT细胞产生IL-10及TGF-β

目的:探讨人早孕滋养细胞通过CXCL16/CXCR6途径对蜕膜γδT细胞分泌细胞因子功能的影响。方法:临床收集正常早孕妇女绒毛组织及蜕膜组织各10例,体外分离蜕膜γδT细胞和滋养细胞,原代培养滋养细胞12、24、48、72、96小时,ELISA检测培养上清中CXCL16的浓度;RT-PCR检测蜕膜γδT细胞中CXCR6的表达;建立人早孕蜕膜γδT细胞与滋养细胞的共培养体系,同时加或不加CXCL16的中和性抗体,或者γδT细胞与滋养细胞间接共培养,48小时后流式细胞术检测γδT细胞中IL-10和TGF-β的表达。结果:人早孕滋养细胞能够分泌CXCL16,且其浓度呈现时间累积效应;蜕膜γδT细胞则表达CXCR6。与滋养细胞直接共培养后,γδT细胞表达IL-10和TGF-β显著增高,且明显高于间接共培养组,加入CXCL16的中和性抗体后,IL-10和TGF-β的增加效应被部分抵消。结论:人早孕滋养细胞通过分泌CXCL16促进表达CXCR6的蜕膜γδT细胞产生IL-10和TGF-β,从而可能有利于妊娠期母-胎界面Th2型偏移,进而有利于正常妊娠的维持。     

Expression of functional chemokine receptors of human placental cells

PROBLEM: Chemokine receptors of placental trophoblasts possibly act as co-receptors or alternative receptors of maternal fetal infection by HIV. To clarify their possible expression and the physiological roles of chemokines on human placentae, we studied chemokine chemokine receptor expression and the effects of exogenous chemokines on choriocarcinoma cell lines. MATERIALS AND METHODS: Placental samples were obtained from 13 placentae of various gestational ages. Villous tissue was mechanically dissected from samples. Trophoblasts were enriched by anti-human chorionic gonadotropin (hCG)-coated magnetic beads. Human choriocarcinoma cell lines (JAR, BeWo, JEG-3) were maintained in RPMI 1640 media supplemented with 10% FCS. Expression of chemokine receptors was studied by RT-PCR. The effects of MIP-1alpha, RANTES, MCP-1 on hCG production were estimated by EIA. Effects of chemokines on proliferation of choriocarcinoma cell lines were examined by MTT assay. RESULTS: We observed mRNA expression of CCR-1, 2, 3, 4, 5 and CXCR-1, 2, 4 in 1st trimester placental villi, CCR-I, 2, 4 and CXCR-1, 2. 4 in 2nd trimester placental villi, CCR-1, 2, 4 and CXCR-4 in 3rd trimester placental villi. Using MACS enriched trophoblasts, we observed identical results. A choriocarcinoma cell line BeWo expressed CCR-1, 3, 4 and CXCR-1, 2, 4 while JEG-3 and JAR expressed CCR-1, 3, 4, 5 and CXCR-1, 2, 4. Expression of the CCR-5 and CXCR-4 protein in choriocarcinoma cell lines and MACS-enriched trophoblats were confirmed by flow cytometry. Chemokine MCP-3, MIP-1alpha, RANTES mRNA were expressed by the 1st, 2nd and 3rd trimester placental samples and the three choriocarcinoma cell lines examined. MCP-1 was expressed by 1st and 2nd trimester placental villi. Administration of chemokines up-regulated proliferation (10(-1) - 10 ng/mL) and hCG production (10(-1) - 10(-2)ng/ mL) of the three choriocarcinoma cell lines examined. CONCLUSIONS: Our results suggest possible roles of chemokines/chemokine receptors on placental physiology and their involvement in HIV transmission as alternative receptors.     

Sperm with damaged membranes are profoundly at risk to have caspases 8, 9 and 3 activated

PROBLEM:  It is almost dogma that IL-2 is not expressed at the M–F interface during normal pregnancy. However, recent results by ours and others clearly showed that IL-15-Th1 type cytokine which shares many similarities with IL-2 is expressed at the interface. IL-15 can affect cytolytic activity of maternal decidual lymphocytes which heavily infiltrate maternal decidua during the first trimester pregnancy. These cells are in a direct contact with trophoblastic cells. IL-18 is a recently discovered Th1 type cytokine with many interesting functions. The aim is to examine IL-18 distribution at the interface and its potential in up-regulating peripheral blood (PB) and decidual lymphocytes (DL) cytotoxicity. Th1 activated lymphocytes are LAK cells and they can kill by both Perforin and Fas pathways in non MHC restricted manner.
METHODS:  PBL and DL were obtained from elective pregnancy termination of pregnancy. IL-18 and IL-18R expression was detected by flow cytometry and immunohistology and cytolytic potential by cytotoxicity against K-562 (NK sensitive) and P815 (NK resistant) cell lines.
RESULTS:  IL-18 positive cells were found in the suspension of Decidual adherent cells and IL-18 R expressions at the trophoblastic cells of villi. Both IL-15 and IL-18 are increasing cytolytic potential of PBL and DL against NK sensitive cell line. Decidual lymphocytes are activated cells with the potential of killing NK resistant but LAK sensitive lines and this is mediated by both perforin and Fas pathways.
CONCLUSIONS:  Physiological and pathophysio- logical role(s) of cytolytic pathways and Th1 cytokines (IL-15 and IL-18) at the interface will be discussed.     

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.