The involvement of HLA-E and -F in pregnancy

The human MHC class I genes, HLA-E, -F and -G are referred to as non-classical or class Ib genes and are distinguished from their close relatives (the classical HLA class I genes) by expression patterns and low allelic polymorphism. To date, most studies that relate these molecules to the immunology of pregnancy have concerned only HLA-G. However, recent advances have suggested potential unique roles as well for HLA-E and HLA-F in pregnancy. A notable advance was the observation that all three proteins are expressed on the surface of extravillous trophoblast that has invaded the maternal decidua. Given this expression site, possibly the only cell type in human development where this occurs, it is logical to hypothesize that all three antigens, each with its own unique receptor-ligand interaction(s), contribute collectively to enable the growth of the developing child. In this review, we examine and discuss the accumulated data on expression and function of HLA-E and HLA-F and attempt to relate what is known to the involvement of HLA-E and -F in human pregnancy.     

人类白细胞抗原-E在正常妊娠、妊娠期高血压及子(疒间)前期绒毛或蜕膜组织中的表达

目的 探讨人类白细胞抗原-E(human leukocyte antigen-E,HLA-E)在正常妊娠、妊娠期高血压和子(疒间)前期绒毛或蜕膜组织中的表达与意义. 方法 选择早、晚期正常妊娠及妊娠期高血压、轻度子(疒间)前期、重度子(疒间)前期患者各6例,应用免疫组化法分别检测HLA-E在绒毛及蜕膜的表达. 结果HLA-E在早、晚期正常妊娠细胞滋养细胞和合体滋养细胞中的表达均较弱分别为0.246±0.031和0.252±0.045,t=2.85,P>0.05);而在侵入蜕膜的绒毛外滋养细胞和腺上皮细胞中强表达,且在妊娠晚期的表达较早期增强(0.308±0.057和0.270±0.035,t=3.79,P<0.05).在正常足月妊娠、妊娠期高血压、轻度子(疒间)前期及重度子痢前期绒毛的平均吸光度分别为0.252±0.045、0.227±0.032、0.210±0.019和0.214±0.023(F=13.18,P<0.05);而在上述各组蜕膜中表达的平均吸光度分别为0.308±0.057、0.211±0.017、0.196±0.013和0.187±0.014(F=33.91,P<0.05).HLA-E在妊娠期高血压、轻度子(疒间)前期和重度子瘌前期绒毛和蜕膜的表达分别与正常足月妊娠比较,均显著减少(P均<0.05). 结论 HLA-E的表达在正常妊娠早期可能与胚胎植入有关,在晚期参与妊娠的维持,而在妊娠期高血压及子(疒间)前期中的低表达可能与该疾病的发生有关.     

Expression of Human Leukocyte Antigen-G during Normal Placentation and in Preeclamptic Pregnancies

Objectives. This study was undertaken to investigate the expression pattern of human leukocyte antigen-G (HLA-G) during normal placentation and determine whether altered expression of HLA-G is associated with severe preeclampsia. Methods. We investigated HLA-G protein levels in first (n = 27), second (n = 7), and third trimester placentas (n = 10) from normal pregnancies, and determined HLA-G levels in term placentas from normal (n = 15) and severe preeclamptic pregnancies (n = 14) using real-time RT-PCR and western blot analysis. Results. In normal placentas, HLA-G protein expression reached a peak level at gestational weeks 6 and 7, then gradually decreased from week 8 to third trimester (p < 0.05). In preeclamptic placentas, both HLA-G mRNA and protein levels were decreased significantly in comparison with normal term placentas (p < 0.05). Conclusion. HLA-G may contribute to placentation during early and mid-term pregnancy, and participate in maintaining gestation during term pregnancy. The reduced level of HLA-G may be associated with pathogenesis of preeclampsia.     

人类白细胞相关抗原-G及其各亚型mRNA在正常妊娠及重度子前期患者胎盘组织中的表达

目的研究人类白细胞相关抗原-G(HLA-G)及其各个亚型mRNA在正常妊娠及重度子前期患者胎盘组织中的表达。方法选取2005-01-2005-06第四军医大学唐都医院10例重度子前期患者(研究组)及10例正常妊娠胎盘组织(对照组),应用荧光定量RT-PCR比较两组间HLA-G及其各个亚型mRNA(HLA-G1、G2、G3、G4、G5、G6)的表达。结果与对照组相比,重度子前期患者胎盘组织中总HLA-GmRNA显著降低,以HLA-G1、HLA-G3降低为主,差异有统计学意义(P<0.05)。结论HLA-G1与HLA-G3在胎盘组织中的低表达可能与妊娠期高血压疾病的发病和病理生理过程有关。     

人类白细胞抗原G、E在人胎盘组织中的表达及其意义

目的:探讨人类白细胞抗原G、E(HLA-G、E)在人胎盘组织中的表达及意义。方法:用原位杂交及免疫组化法分别检测HLA-G、E mRNA及蛋白质在人正常早孕绒毛组织、晚孕胎盘组织中的表达。结果:在人正常早孕绒毛组织中的绒毛细胞滋养细胞、合体滋养细胞及绒毛外滋养细胞(EVCT)内均有HLA-G、E mRNA及HLA-E蛋白质表达,而HLA-G蛋白质仅在EVCT内表达;晚孕胎盘组织中HLA-G、E mRNA及蛋白质表达于蜕膜板内的EVCT及羊膜上皮细胞。结论:HLA-E表达于人正常早孕绒毛组织中所有类型的滋养细胞及晚孕胎盘组织中的EVCT和羊膜上皮细胞。抗人HLA-G单克隆抗体4H84仅能检测出人胎盘组织中EVCT及羊膜上皮细胞内的HLA-G蛋白。     

HLA-G,pre-eclampsia,immunity and vascular events

Pre-eclampsia, one of the main complications in pregnancy, is characterised by shallow cytotrophoblast invasion of decidua as well as by vascular endothelial cell dysfunction, leading to a poor perfusion of placenta. A striking feature of pre-eclamptic pregnancies is that expression of HLA-G protein is reduced in term placentas compared with normal pregnancy. How such HLA-G deficient expression may be related to the pre-eclamptic pathology is unknown. Here, we review the major structural characteristics of HLA-G and some of its functions that have been recently characterised. Soluble HLA-G1 isoform down-regulates both CD8(+) and CD4(+) T cell reactivity. HLA-G also modulates innate immunity by binding to several NK and/or decidual receptors, inducing particular cytokine secretion. HLA-G was shown to be less susceptible to human cytomegalovirus-derived US protein down-modulation. Finally, soluble HLA-G1 down-regulates endothelial cell proliferation and migration. In view of these different HLA-G properties, we will briefly discuss how defective HLA-G function may contribute to the low trophoblast invasion and vascular abnormalities observed in pre-eclamptic placentas.     

表皮生长因子受体在子宫内膜、早孕蜕膜及滋养细胞中的表达

目的了解表皮生长因子受体（ＥＧＦＲ）在子宫内膜细胞分化及发育中的作用。方法采用免疫组织化学及逆转录聚合酶链反应（ＲＴＰＣＲ）技术测定５８例正常子宫内膜（３２例增生期,２６例分泌期）与２６例早孕蜕膜。结果ＥＧＦＲ存在于正常子宫内膜、早孕蜕膜腺体及间质细胞的细胞膜、核膜及胞浆内,分布均匀。ＥＧＦＲ还位于早孕蜕膜腺体及间质细胞核内。正常子宫内膜ＥＧＦＲ的表达,腺体部分高于间质部分（Ｐ＜０．０５）,ＥＧＦＲ在增生期及分泌期子宫内膜腺体的表达差异无显著性（Ｐ＞０．０５）,但ＥＧＦＲ在早孕蜕膜组织中的表达明显高于增生期及分泌期（Ｐ＜０．０５）。ＥＧＦＲ在间质细胞的表达,早孕蜕膜高于分泌期内膜,而增生期内膜则表达最低（Ｐ＜０．０５）。ＥＧＦＲｍＲＮＡ的表达从弱到强依次为增生期、分泌期、蜕膜、滋养细胞,增生期与分泌期比较,差异无显著性（Ｐ＞０．０５）,早孕蜕膜较分泌期及增生期明显增加（Ｐ＜０．０５）,滋养细胞ＥＧＦＲｍＲＮＡ的表达明显高于增生期、分泌期及早孕蜕膜（Ｐ＜０．０５）。结论ＥＧＦＲ存在于各期子宫内膜中,其表达在正常月经周期中无明显变化,但在早孕蜕膜、滋养细胞中的表达明显高于增生期及分泌期内膜     

Differential expression of human placental PAPP-A2 over gestation and in preeclampsia

IntroductionPregnancy Associated Plasma Protein A2 (PAPP-A2) is a pregnancy related insulin-like growth factor binding protein-5 (IGFBP-5) protease, known to be elevated in preeclampsia. As the insulin-like growth factors are important in human implantation and placentation, we sought to determine the expression pattern of PAPP-A2 over human gestation in normal and preeclamptic pregnancies to evaluate its role in placental development and the pathogenesis of preeclampsia.MethodsPlacental basal plate and chorionic villi samples, maternal and fetal cord blood sera were obtained from preeclamptic and control pregnancies. Formalin-fixed tissue sections from across gestation were stained for cytokeratin-7, HLA-G, and PAPP-A2. PAPP-A2 immunoblot analysis was also performed on protein lysates and sera.ResultsPAPP-A2 expression is predominately expressed by differentiated trophoblasts and fetal endothelium. Chorionic villi show strong expression in the first trimester, followed by a progressive decrease in the second trimester, which returns in the third trimester. PAPP-A2 expression is not impacted by labor. PAPP-A2 is increased in the basal plate, chorionic villi and maternal sera in preeclampsia compared to controls, but is not detectable in cord blood.DiscussionPAPP-A2 is differentially expressed in different trophoblast populations and shows strong down regulation in the mid second trimester in chorionic villous samples. Both maternal sera and placental tissue from pregnancies complicated by preeclampsia show increased levels of PAPP-A2. PAPP-A2 levels are not altered by labor. Additionally, PAPP-A2 cannot be detected in cord blood demonstrating that the alterations in maternal and placental PAPP-A2 are not recapitulated in the fetal circulation.     

Differential expression of human leukocyte antigen-G (HLA-G) messenger RNAs and proteins in normal human prostate and prostatic adenocarcinoma

Human leukocyte antigen-G (HLA-G) is a major histocompatibility complex class Ib gene expressed in normal organs and in some tumors. The glycoproteins encoded by this gene are best known for their immunosuppressive properties. Because isoform-specific expression of HLA-G in male reproductive organs has not been reported, we investigated HLA-G1, -G2, -G5, -G6 mRNAs and proteins in four-to-five samples of normal prostate glands, prostates with benign prostatic hyperplasia and prostate adenocarcinomas using RT-PCR and immunohistochemistry. All tissues contained HLA-G1, -G2, -G5 and -G6 specific mRNAs, but only HLA-G5 protein was detectable. In normal prostate glands, HLA-G5 protein was prominent in the cytoplasm of tubuloglandular epithelia and in glandular secretions. Staining was reduced in samples of benign prostatic hyperplasia but remained localized to the cytoplasm of glandular epithelia and secretions. In prostatic adenocarcinomas, HLA-G5 protein was detectable mainly in the secretions. Thus, HLA-G5 but not HLA-G1, -G2 or -G6 is produced in the normal prostate and is present in prostatic secretions. In addition, normal cellular localization is disturbed in benign and malignant prostatic adenocarcinomas. The results are consistent with this molecule may influencing female immune receptivity to sperm and suggest that such immunosuppression could be disturbed in men with prostatic adenocarcinomas.     

Review: The ADAM metalloproteinases – Novel regulators of trophoblast invasion?

During pregnancy, the extravillous trophoblast (EVT) invades the maternal decidua and remodels spiral arteries reaching as far as the inner third of the myometrium. This process is mandatory to a successful pregnancy since EVTs regulate spiral artery remodeling to achieve maximal vasodilation and thus an adequate nutrient supply to the embryo or communicate with maternal leukocyte populations to guarantee acceptance of the allogeneic conceptus. To achieve this, EVTs undergo a remarkable and unique differentiation process, which yields different phenotypes such as proliferative cell column trophoblasts or growth-arrested, invasive interstitial or endovascular cytotrophoblasts. Matrix metalloproteinases have long been seen as imperative to trophoblast invasion because of their ability to degrade extracellular matrix and therefore allow cellular movement in foreign tissues. However, global gene expression analysis reveals that EVTs also express various members of distintegrin and metalloproteinases (ADAMs). These proteases are associated with the process of proteolytic shedding and activation of surface proteins including growth factors, cytokines, receptors and their ligands rather than extracellular matrix breakdown. While ADAM12 has been associated with chromosomal abnormalities as well as preeclampsia or intrauterine fetal growth restriction, the function of ADAMs in trophoblasts remains elusive. In this article, we review the diverse invasive trophoblast phenotypes, EVT-associated protease systems and related open questions. In addition, we examine recent information about relevant ADAM members and their putative implications for EVT biology.     

DNA microarrays detect the expression of apoptosis-related genes in preeclamptic placentas

AIMS: To investigate the expression of apoptosis-related genes in preeclamptic placentas and the possible mechanism of the regulation process. METHODS: Complementary DNA microarrays were employed to compare gene expression profiles of five preeclamptic and five normal placentas. RESULTS: Among the 368 genes detected over 35% showed an over 2-fold difference of expression between preeclamptic placentas and normal placentas. Many genes involved in cell cycle or apoptosis were more highly expressed in preeclamptic placentas than in normal placentas. The expression of many immune-activation genes in preeclamptic placentas was also higher than that in normal placentas. Additionally, many cytokine receptor/kinase genes were also induced in preeclamptic placentas. CONCLUSIONS: The change in expression of cell apoptosis-related genes in placentas might be involved in the pathogenesis of preeclampsia, while the activation of the immune system might be one cause of this change.     

Correlation of Cyr61 and CTGF in placentas from the late pre-eclamptic pregnancy

It is thought that a shallow invasion of the maternal decidua by extravillous trophoblasts (EVT) is associated with the development of pre-eclampsia. Here, we focus on the expression of the proangiogenic proteins Cyr61 and CTGF in the human placenta during normal pregnancy compared with that in the late pre-eclamptic placenta. Cyr61 and CTGF are expressed in the extravillous trophoblast and in endothelial cells. We found the expression of Cyr61 was significantly decreased in pre-eclamptic placentas compared with matched controls. In contrast, the CTGF expression level was upregulated in pre-eclamptic placentas. There was a negative correlation between Cyr61 and CTGF. These results suggest that decreased Cyr61 and overexpressed CTGF may play a part in the development of pre-eclampsia.     

人白细胞抗原-G mRNA各亚型在重度子(癎)前期胎盘组织中的表达

目的　探讨人白细胞抗原-G(human leukocyte antigen-G,HLA-G)各亚型在早发型、晚发型重度子(癎)前期患者以及正常妊娠产妇胎盘组织中表达的差异. 方法　使用巢式RT-PCR方法检测11例早发型重度子(癎)前期患者、14例晚发型重度子(癎)前期患者以及8例正常分娩产妇胎盘组织中HLA-G各亚型mRNA表达的差异情况. 结果 与正常对照组相比,HLA-G1亚型在早发型和晚发型重度子(癎)前期患者的胎盘组织中表达明显减少(中位数:早发型组1.37,P<0.05;晚发型组24.90,P<0.05;正常对照组46.67,P<0.05).HLA-G2亚型在晚发型重度子(癎)前期患者的胎盘组织中表达水平较高(中位数:早发型组0;晚发型组21.59,P<0.05;正常对照组5.39).HLA-G5亚型在早发型以及晚发型重度子(癎)前期患者的胎盘组织中表达水平增高(中位数:早发型组19.23;晚发型组3.65;正常对照组1.33). 结论 HLA-G膜结合型亚型mRNA在胎盘组织中表达减少以及HLA-G可溶性亚型mRNA在胎盘组织中表达的增高,可能在子(癎)前期的发病机制中起到一定的作用.     

A study of human leukocyte antigen G expression in hydatidiform moles

OBJECTIVE: Human leukocyte antigen G (HLA-G) is a nonclassic major histocompatibility gene normally expressed only in extravillous trophoblasts throughout pregnancy. It may be responsible in part for the successful evasion of the hemiallogenic trophoblasts from maternal immune surveillance. We investigated whether HLA-G is expressed in molar pregnancies. STUDY DESIGN: We examined 5 complete hydatidiform mole specimens and 5 partial hydatidiform mole specimens to determine whether HLA-G is expressed by immunohistochemistry and by RNA in situ hybridization analysis. RESULTS: We found that both the protein and RNA of HLA-G is expressed in complete and partial hydatidiform moles. CONCLUSION: HLA-G RNA and protein are expressed in molar pregnancies. HLA-G expression is independent of embryonic development and may therefore be an integral part of placental development. Furthermore, expression of HLA-G in the complete hydatidiform mole, a naturally occurring androgenote, confirms expression of the paternal allele of HLA-G. Imprinting of HLA-G is therefore unlikely to play a role in protecting fetal trophoblasts from maternal immune rejection.     

ADAM17 regulates TNFα production by placental trophoblasts

Increased trophoblast TNFα production is an important component of placental dysfunction in preeclampsia. However, the mechanism of increased TNFα production in the preeclamptic placenta is largely unknown. ADAM17 is a metallopeptidase that functions as a TNFα converting enzyme. In this study, we examined ADAM17 expression in placentas from normal and preeclamptic pregnancies and found increased ADAM17 expression in preeclamptic placentas compared to those from normal placentas, p < 0.05. Since hypoxia/oxidative stress is an underlying pathophysiology in the preeclamptic placenta, we further determined if hypoxia/oxidative stress could modulate ADAM17 expression and subsequently induce TNFα production in placental trophoblasts. Trophoblasts were isolated from normal term placentas and treated with cobalt (II) chloride (CoCl₂), a hypoxia mimetic agent, at different concentrations. Our results showed that CoCl₂ induced a dose-dependent increase in TNFα production that is associated with enhanced ADAM17 expression. Trophoblast expressions of HO-1 (a sensor of cellular oxidative stress) and caspase-3 (an indicator of apoptosis) in response to CoCl₂ stimulation were also examined. We further found that metallopeptidase inhibitor GM6001 and ADAM17 siRNA could block CoCl₂ induced TNFα production, demonstrating the role of ADAM17 in TNFα production in placental trophoblasts. These results suggest that oxidative stress-induced increased ADAM17 expression could contribute to the increased TNFα production in preeclamptic placentas.     

Nuclear localisation of the endocannabinoid metabolizing enzyme fatty acid amide hydrolase (FAAH) in invasive trophoblasts and an association with recurrent miscarriage

Endocannabinoids are lipid signalling molecules that are related to the major psychoactive component in marijuana, delta-9-tetrahydrocannabinol and are increasingly recognized as being important in implantation and development of early embryos. The endocannabinoid anandamide, is metabolized by the enzyme fatty acid amide hydrolase (FAAH), and insufficient levels of this enzyme have been implicated in spontaneous miscarriage in women and implantation failure in mice. We screened placental bed biopsies and placental tissue from 45 women with recurrent miscarriage and 17 gestation-matched women with normal pregnancies for the expression of FAAH by immunohistochemistry. Unexpectedly, the enzyme appeared to be localised to the nucleus of trophoblasts and this was confirmed by western blotting of sub-cellular fractions and confocal microscopy. FAAH was expressed in the cytoplasm of large decidual stromal cells and significantly more women with recurrent miscarriage (73%) expressed FAAH in these cells than women with normal pregnancy (31%). FAAH was also expressed in the nucleus of extravillous trophoblasts that had invaded the decidua from 67% of women with recurrent miscarriage but was not expressed by these cells in any women with normal pregnancies. In contrast, FAAH was expressed in extravillous trophoblasts that had migrated out of the villi but that had not yet invaded the decidua in both normal pregnancies and in cases of recurrent miscarriage. FAAH was also present in the nucleus of a small number of villous trophoblasts in some specimens. FAAH appears to be over expressed in trophoblasts that have invaded the decidua, as well as in large decidual stromal cells in many cases of recurrent miscarriage. This may reflect inadequate control of the cannabinoid system in the uterus of women who experience recurrent miscarriages. The functional significance of the unexpected nuclear localisation of FAAH in trophoblasts is not yet clear.     

Lipocalin2 enhances the matrix metalloproteinase-9 activity and invasion of extravillous trophoblasts under hypoxia

Objectives

The invasion of extravillous trophoblasts (EVTs) to the decidua and spiral arteries in early pregnancy is a crucial step for a successful pregnancy; however, its mechanisms are not fully understood. Lipocalin2 (LCN2), a multifunctional secretory protein known as neutrophil gelatinase-associated lipocalin (NGAL), reportedly enhanced invasiveness via the activation of matrix metalloproteinase-9 (MMP-9) in several cancer cells. In this study, the expression and function of LCN2 in early placenta were analyzed.

Methods

Early placental tissues between 7 and 10 weeks of gestation were obtained from normal pregnant women who underwent elective termination. The expression of LCN2 was examined using immunostaining and RT-PCR. EVTs isolated from these placental tissues and a choriocarcinoma cell line (JAR) were used to investigate the effects of LCN2 on proliferation, invasion potential, and MMP-9 activity under hypoxia using a WST-1 assay, Matrigel invasion assay, and gelatin gel zymography, respectively.

Results

The immunohistochemical expression of LCN2 was observed in the cytoplasm of EVTs, cytotrophoblasts and the decidua, but not in syncytiotrophoblasts. The addition of recombinant LCN2 did not affect proliferation, but enhanced the invasiveness (500 ng/mL, p < 0.01) and MMP-9 activity of primary cultured EVTs and JAR in a dose-dependent manner. Silencing LCN2 using shRNA reduced the invasiveness (p < 0.01) and MMP-9 activity of JAR. In addition, the hypoxic condition (2% O₂) increased LCN2 expression (p < 0.01), MMP-9 activity, and invasive ability (p < 0.01).

Conclusions

LCN2 was involved in the invasiveness of EVTs, especially under hypoxia, via increased MMP-9 activity.