TNF-alpha/TNFRI in primary and immortalized first trimester cytotrophoblasts

During the first trimester of pregnancy endogenous expression of tumour necrosis factor (TNF)-alpha has been detected in villous, as well as in proliferating and invading extravillous, trophoblasts suggesting that the protein could be involved in trophoblast differentiation. To gain insights into the putative role of the TNF-alpha signalling pathway, we investigated expression of its receptors, TNFR I and II, in first trimester placentae and early trophoblasts, and studied the influence of the cytokine on cell proliferation and apoptosis. ELISA and RT-PCR revealed secretion/expression of TNFRI protein/mRNA in immortalized ED27 cells and purified first trimester cytotrophoblasts, while soluble TNFRII was undetectable in cell culture supernatants. In agreement, immunohistochemical analyses of first trimester placentae showed that TNFRI is localized to the villous cyto- and syncytiotrophoblast, to the proliferating cytotrophoblasts of the cell islands and cell columns, as well as to extravillous cells invading decidual tissue. TNFRII, however, was absent in early trophoblast populations. Interleukin (IL)-1 and phorbol 12-myristate 13-acetate (PMA) induced shedding of TNFRI from ED27 and primary cells suggesting that under inflammatory conditions the soluble receptor protein may protect from cytotoxic effects of TNF-alpha. Upon incubation with increasing amounts of TNF-alpha no significant changes in DNA-content or cell numbers were found, suggesting that the cytokine does not augment proliferation of primary cytotrophoblasts. High doses of TNF-alpha, however, provoked growth arrest in ED27 cells as evaluated by cell counting, but did not induce necrosis/apoptosis as was assessed by TUNEL assay. In first trimester cells addition of elevated amounts of TNF-alpha resulted in the appearance of TUNEL-positive cells and an increase in caspase-3 enzyme activity suggesting that the TNF-alpha-dependent apoptotic cascade is executed in a portion of the early cytotrophoblasts.     

Expression of gap junctional connexins 26, 32 and 43 in bovine placentomes during pregnancy

Gap junctional connexins (Cx) are induced in the endometrium during implantation in rodents, the human receptive window, and in the decidua Cx26 and Cx43 expression increases in response to trophoblast invasion. In contrast, this gap junctional response and decidualization is absent in non-invasive epitheliochorial placentae of pigs and horses. Bovine (syn)epitheliochorial placentation represents an intermediate type of trophoblast invasion, since it is characterized by the continuous migration and fusion of trophoblast giant cells (TGC) with uterine epithelial cells. Therefore the objective of the present study was to investigate the expression of Cx26, Cx32, and Cx43 in placental tissues during bovine pregnancy, to determine if Cx expression patterns correlate with the depth of trophoblast invasion. Cx26, Cx32, and Cx43 proteins were detected by immunohistochemistry and corresponding specific mRNAs were shown by RT-PCR and localized in tissue sections by in situ hybridization. Cx26 protein was detected at the feto-maternal contact interface and as cytoplasmic staining in TGC. Cx26 mRNA was located in maternal epithelium and in TGC. Cx32 protein expression was observed in the maternal epithelium exclusively on the tips of maternal septa, whereas Cx32 mRNA was detected in all maternal epithelial cells and single TGC. Cx43 protein and mRNA were coexpressed in TGC. Cx43 protein was present in maternal septal stroma and to a lesser extent in chorionic villous mesenchyme, while Cx43 mRNA was associated with the vasculature. In the course of gestation, expression of Cx26, Cx32, and Cx43 did not change. In conclusion, the intermediate invasive status of bovine trophoblast is supported by the fact that TGC coexpress Cx26, Cx32, and Cx43, which may be important for trophoblast migration (invasion), and fusion with maternal epithelial cells. Cx32 could be involved in the control of invasion.     

Immunohistochemical localization of HLA antigens and placental proteins (alpha hCG, beta hCG CTP, hPL and SP1 in villous and extravillous trophoblast in normal human pregnancy: a distinctive pathway of differentiation of extravillous trophoblast

Immunohistochemical localization of HLA antigens and placental proteins (alpha hCG, beta hCG CTP, hPL and SP1) in villous and extravillous trophoblast at various stages of normal human gestation were studied, using hysterectomy specimens. In the chorionic villi, the capacity for synthesizing placental proteins seemed to develop in parallel with the morphological change from mononuclear cells to multinucleated syncytiotrophoblast and no villous trophoblast expressed HLA antigens. In contrast, extravillous trophoblast, including the multinucleated trophoblastic cells at the deciduomuscular junction, expressed HLA-A, -B, and -C, and their capacity for synthesizing placental proteins did not seem to correspond with the degree of morphological change: the location of alpha hCG, beta hCG CTP and SP1 was restricted to mononuclear trophoblast in the superficial decidua, while hPL was present extensively in extravillous trophoblast. These findings strongly suggest that extravillous trophoblast possesses many distinctive biological features and differentiates in an independent manner. Mononuclear trophoblast forming the cell columns was also positive for HLA-A, -B, and -C, and no placental protein was demonstrated in these cells; this, together with previous morphological observations, may indicate the germinative nature of these cells.     

Differential expression of the receptor tyrosine kinase EphB4 and its ligand Ephrin-B2 during human placental development

Normal placentation involves the development of an utero-placental circulation following the migration of the extravillous cytotrophoblasts into the decidua and invasion of the spiral arteries, which are thereby transformed into large vessels of low resistance. Given the documented role of the receptor tyrosine kinase EphB4 and its ligand ephrin-B2 in the establishment of the embryonal vascular network, we hypothesized that these molecules are also instrumental in the development of the human placenta. Monitoring the expression during placental development revealed that in first trimester and term placentae both molecules are equally expressed at the RNA level. In contrast, the protein levels were significantly reduced during gestation. Immunohistochemistry revealed a distinct localization of the EphB4 and ephrin-B2 proteins. EphB4 was predominantly expressed in the villous syncytial trophoblast layer and in a subset of intravillous capillaries. Prominent expression was also observed in the extravillous cytotrophoblast giant cells. In contrast, ephrin-B2 expression was detected in the villous cytotrophoblast and syncytial trophoblast cell layers, as well as initially in all intravillous capillaries. Strong expression was also observed in extravillous anchoring cytotrophoblast cells. Hypoxia is a major inducer of placental development. In vitro studies employing trophoblast-derived cell lines revealed that predominantly ephrin-B2 expression is induced by hypoxia, however, in an Hif-1alpha independent manner. These experiments suggest that EphB4 and ephrin-B2 are instrumental in the establishment of a functional placental structure and of the utero-placental circulation.     

Analogous and unique functions of connexins in mouse and human placental development

Here, we review the expression, localization and the possible role of the different connexin isoforms in placental function and development in mice and men. Connexin gene deletion in mice has shown that Cx26 is responsible for transplacental uptake of glucose in the labyrinth, and Cx31 as well as Cx31.1 for trophoblast cell lineage development. In the human placenta, it appears that Cx43 is required for the fusion process of cytotrophoblastic cells leading to the formation of the syncytiotrophoblast. Thus Cx26 and Cx43 serve different species-specific functions in the functionally analogous placental compartments, mouse labyrinth and human villous trophoblast. However, like Cx31 in the mouse, Cx40 plays a critical role in the switch from a proliferative to an invasive phenotype of the trophoblast cells invading the endometrium. Both connexin channels seem to have similar functions in analogous compartments of the placentas. Taken together, connexins are important in regulating trophoblast cell differentiation in both species. In mouse, connexin channels are specifically involved in passive transport of molecules across the placental barriers.     

Expression of Fas-ligand in first trimester and term human placental villi

The expression of Fas-ligand (FasL) on trophoblast cells is thought to play a role in immune regulation during human pregnancy. However, there are some discrepancies in the published data concerning the cell types expressing FasL in the placental villi. Therefore, we examined the expression of FasL on cryosections of first trimester and term placental tissue with three different anti-sera against FasL, which are in common use. By immunohistochemistry, all three anti-sera principally gave the same staining result. In the first trimester of pregnancy, villous cytotrophoblast cells underlying the syncytium, as well as all extravillous trophoblast cells of cell columns and cell islands, gave a clear, mainly membrane-located staining, whereas the syncytiotrophoblast, which forms the borderline to the maternal blood flow, only gave a spot-like reaction in distinct areas. The same result was obtained with term placental villi; however, in this tissue, the staining of the villous cytotrophoblast cells was less pronounced. From our results, we suggest that in placental villi, an important role of FasL in immune regulation is not very conclusive because this molecule is mainly expressed on trophoblast with no access to maternal blood or tissue. This is in contrast to the uterine part of the placenta, where FasL expressing trophoblast cells are in close contact with apoptotic maternal leukocytes.     

Expression of interleukin-27 by human trophoblast cells

Cytokines produced at the fetal-maternal interface play a key role in regulating maternal tolerance to the fetus and successful pregnancy. Previously, we showed that EBV-induced gene 3 (EBI3), an interleukin (IL)-12 p40 homologue, was expressed at very high levels by syncytiotrophoblasts and extravillous trophoblasts throughout human pregnancy. EBI3 was recently shown to associate with a novel ligand, p28, to form a new heterodimeric cytokine with important immunoregulatory functions, IL-27. In this study, we investigated whether EBI3 expression by trophoblast cells is associated with that of p28 to form IL-27. We found that genes encoding IL-27 (EBI3 and p28) and its receptor (IL-27R and gp130) were expressed in the placenta at various stages of pregnancy. Co-immunoprecipitation experiments performed from placental lysates, and ELISA of culture supernatants from placental explants, showed that IL-27 heterodimer was produced and released from placental cells. In situ studies of placentae of first, second and third trimester of pregnancy, and of choriocarcinomas, demonstrated that syncytiotrophoblast cells co-expressed EBI3 and p28. Similarly, extravillous trophoblast cells invading the decidua were found to co-express both subunits of IL-27. These data suggest that IL-27 may be part of the cytokine network regulating local immune responses and angiogenesis during human pregnancy.     

Expression of endothelial nitric oxide synthase by extravillous trophoblast cells in the human placenta

Previous reports have documented the expression of endothelial nitric oxide synthase (eNOS) expression by the syncytiotrophoblast layer of the villus in the human placenta. In contrast, the underlying villous cytotrophoblast cells do not express the enzyme. Because extravillous cytotrophoblasts have not been as extensively investigated, our objective was to test whether these cells express eNOS. Using both a mouse monoclonal and a rabbit polyclonal antibody, we demonstrated immunoreactive eNOS in trophoblast cell columns emanating from anchoring villi in second trimester placentae. Cytokeratin positive trophoblast cells lying beneath remnant anchoring villi, lining decidual blood vessels and scattered throughout the basal plate of normal term and pre-eclamptic placentae also expressed immunoreactive eNOS. By Western analysis, the monoclonal and polyclonal antibodies were shown to be absolutely and relatively specific for eNOS, respectively. The finding of immunoreactive eNOS expression by extravillous trophoblast cells was substantiated by in situ hybridization. Using riboprobes generated from a bovine eNOS cDNA, we demonstrated specific hybridization in the endothelium of blood vessels in the umbilical cord, thus validating the in situ hybridization methodology, as well as specific hybridization in the extravillous trophoblast cells of the basal plate in normal term placenta. In conclusion, several different populations of extravillous trophoblast cells in the basal plate of the human placenta express eNOS.     

Spatial and temporal patterns of expression of messenger RNA for insulin-like growth factors and their binding proteins in the placenta of man and laboratory animals

To better understand the role of the insulin-like growth factors (IGF-I and -II) and their binding proteins (IGFBPs 1-6) in placental development and function, it is important to review similarities and differences between species in expression of the respective mRNAs. In human placenta, IGF-II mRNA is expressed in chorionic mesoderm and first trimester villous cytotrophoblast, but not in syncytiotrophoblast. In contrast, in rhesus monkey placenta, IGF-II mRNA is expressed in syncytiotrophoblast but not in chorionic mesoderm. IGFBP-3 mRNA is present in the chorionic mesoderm of placental villi from both these species and may modulate IGF-II action through a paracrine mechanism. In rodent placentae, IGF-II mRNA is expressed both in fetal mesoderm and in the trophoblast of the placental labyrinth. In guinea pig, where IGFBP-5 mRNA is expressed in the marginal and interlobular syncytium and IGF-II mRNA in the labyrinth, interaction between IGF-II and IGFBP-5 mRNA may be involved in vascularization of the placenta by fetal vessels. In sheep placenta, IGF-II mRNA is expressed, not in the trophoblast layer, but in the fetal mesoderm immediately adjacent to it. In the basal plate of human, rhesus monkey and baboon placentae, extravillous trophoblasts express IGF-II mRNA and uterine decidual cells IGFBP 1-6 mRNAs. The inference is that there is interaction between IGF-II and IGFBPs at the maternal-fetal interface of the primate placenta during trophoblast invasion and decidualization. IGFBP-1 expressed by the decidua may also interact with alpha(5)beta(1)integrin expressed by the extravillous trophoblast. The placentae of rodents are also of the invasive type. Glycogen cells of the mouse placenta are analogous with human extravillous trophoblast and express IGF-II mRNA. However, expression of IGFBP mRNAs in the mouse, as in the guinea pig, is confined to non-decidualized endometrium and myometrium. IGF-II mRNA is strongly expressed by trophoblasts invading uterine vessels in human and guinea pig placentae. Interactions probably occur between IGF-II expressed by these trophoblasts and IGFBPs expressed in the vessel walls. However, it is possible that IGFBPs expressed by maternal vessels are associated with processes that are independent of trophoblast invasion. Thus, IGFBP-3 mRNA is highly expressed in the maternal blood vessels of the non-deciduate sheep placenta. Findings to date highlight the diversity in the expression of the IGF system among placentae of man and different laboratory animals, and even between closely related species. Comparative studies will continue to be required to understand the functional role of IGFs and IGFBPs in each species.     

肝素结合性表皮生长因子在早孕绒毛及蜕膜组织中的表达

目的　探讨肝素结合性表皮生长因子 (HB- EGF)在早孕绒毛及蜕膜组织中的表达 ;评估 HB- EGF在人类胚泡着床和早期胎盘组织发育中的作用。　方法　采用免疫组化法检测 18例早孕绒毛及蜕膜组织中的 HB- EGF的表达及定位。　结果　全部早孕绒毛及蜕膜组织标本均有 HB-EGF表达 ,早孕绒毛滋养细胞表达高于蜕膜组织 ,HB- EGF阳性产物定位于早孕绒毛及蜕膜组织中的细胞膜上和细胞浆内 ,呈棕黄色。　结论　人类早孕绒毛及蜕膜组织表达 HB- EGF可能与胚泡着床、胎盘形成和维持胎盘功能有关。     

Messenger RNA for membrane-type 2 matrix metalloproteinase, MT2-MMP, is expressed in human placenta of first trimester

An intimately regulated cell surface activation of matrix metalloproteinases (MMPs) is believed to be of critical importance for the control of trophoblast invasion. A histological investigation of the expression and localization of three different MMPs, the membrane-type matrix metalloproteinases 1 and 2 (MT1-MMP, MT2-MMP) and matrix metalloproteinase 2 (MMP-2/gelatinase A) was performed by in situ hybridization on consecutive sections from human placentae of first trimester pregnancies. Cytokeratin immunostaining identified trophoblast cells. Both normal and tubal implantation sites were studied. We observed a high degree of coexpression of MT2-MMP, MT1-MMP and MMP-2 mRNAs in single extravillous cytotrophoblasts that had invaded the endometrium and tubal wall. Furthermore, mRNAs for all three genes were also seen in cytotrophoblasts of cell islands. In contrast to this coexpression pattern, MT2-MMP expression was absent from cell columns and decidual cells, in which signals for MT1-MMP and MMP-2 mRNAs were seen. The present data on the cellular expression of MT2-MMP mRNA in placenta extend our knowledge of the proteolytic events that take place during early pregnancy. The data suggest that MT2-MMP, capable of activating MMP-2 in vitro, is involved in the invasion of extravillous cytotrophoblast, possibly related to the physiological activation of MMP-2.     

Glycodelin A and differentiation of first trimester trophoblast cells in vitro

Aim The glycoprotein, glycodelin A (GdA) is a main product of the maternal decidua in the first trimester of pregnancy and is secreted into the amniotic fluid. The purpose of this study was to investigate the effect of GdA on secretion and surface markers of isolated first trimester trophoblasts in vitro.Methods Cytotrophoblasts were prepared from human first trimester placentae and incubated with varying concentrations of GdA or transfected separately with the expression plasmid of GdA. Supernatants were assayed for human chorionic gonadotropin (hCG) protein concentrations. Expression of human placental lactogen (hPL), mucin 1 (MUC1) and the Thomsen–Friedenreich (TF) epitope was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry.Results Glycodelin A induced a reduced expression of hPL compared with unstimulated controls. Expression of MUC1 was not affected by GdA. Freshly isolated trophoblast cells showed no TF expression but became positive for this antigen after 96 h of cultivation. GdA-stimulated trophoblast cells inhibited TF expression after 96 h of cultivation. GdA plasmids induced a significantly higher hCG production in transfected cells than in cells transfected with the empty plasmid.Conclusions The results obtained in this study suggest that GdA is involved in the differentiation of trophoblast cells. The treatment of GdA plasmid transfected trophoblast cells stimulated hCG production in isolated trophoblast cells and inhibited hPL and TF expression, suggesting a functional link between hCG and GdA.     

Interferon-gamma enhances mRNA and surface expression of class I antigen on human extravillous trophoblast

Human trophoblast cells with immunocytochemical characteristics of the extravillous population have been isolated from 1st trimester placentae. Treatment of these cells with IFN-gamma increases the expression of Class I antigens at both the cell surface and mRNA level. A similar increase in Class I antigens is also found in JEG-3 choriocarcinoma cells after treatment with IFN-gamma. The possibility that aberrant production of IFN-gamma may upset the fetal-maternal equilibrium in vivo is discussed.     

Expression pattern of the cell cycle promoter cyclin e in benign extravillous trophoblast and gestational trophoblastic lesions: correlation with expression of Ki-67.

In this study, a specific monoclonal antibody was used to immunohistochemically investigate correlated expression of the cell cycle promoter cyclin E and the proliferation marker Ki-67 in benign extravillous trophoblast and gestational trophoblastic lesions. Our data show that cyclin E is expressed in the normal extravillous trophoblast, with strongest levels of expression in the cell columns of anchoring villi. Differences could be observed in expression of Ki-67 in both normal extravillous trophoblast and gestational trophoblastic lesions. In the extravillous trophoblast of the cell columns, expression of cyclin E started more distal compared with Ki-67 and was maintained (with less intensity) into the deeper layers of interstitial trophoblast. In the benign trophoblastic lesions (exaggerated placental site [EPS] and placental site nodule [PSN]) and in the trophoblast proliferations on the surface of hydropic villi of hydatidiform moles (HM), the percentage of cells expressing cyclin E was higher than of those expressing Ki-67. The same observation could be made for a case of placental site trophoblastic tumor (PSTT). In contrast, choriocarcinomas (N=8), which are definitely malignant tumors, showed an opposite pattern, with a much higher percentage of strongly Ki-67-positive cells compared with cyclin E-positive cells. We conclude that cyclin E is expressed in benign extravillous trophoblast and gestational trophoblastic lesions, where a ratio cyclin E/Ki-67<1 characterizes choriocarcinomas, whereas PSTT and the benign lesions (HM, EPS, PSN) show expression of cyclin E in a higher percentage of cells than Ki-67 (cyclin E/Ki-67 ratio >1).     

Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening

The aim of this immunohistochemical and cytochemical study was to select specific antibodies to establish an efficient purification protocol for first trimester trophoblast and for subsequent purity screening of isolated trophoblast cells. The reactivity of antibodies to various cytokeratin filaments, glycoprotein CD9, fibroblast specific antigen (FSA), common leukocyte antigen CD45RB and macrophage antigens CD163, CD68 and CD14 were studied on cryosections of placental tissue. Among the cytokeratins tested, cytokeratin 7 was the only keratin filament type, which was not expressed in placental mesenchymal cells, but in all trophoblast subpopulations. Since anti-CD9, in addition to mesenchymal cells, also strongly labels extravillous cytotrophoblast cells, whereas the antibody to FSA only reacts with mesenchymal cells, anti-FSA is suitable as a depletion antibody for mesenchymal cells. Among the macrophage markers anti-CD163 was the most specific for Hofbauer cells. CD45RB was expressed on maternal and fetal leukocytes as well as on Hofbauer cells. Isolated first trimester placental cell preparations that have been collected from a density gradient contained up to 45 per cent non-trophoblast cells. Immunocytochemistry using antibodies to CK7, FSA, vimentin, CD45RB and CD163 demonstrated that subsequent immunodepletion with antibodies to CD45RB and FSA increased the purity of the trophoblast preparation to greater than 98 per cent.According to this study trophoblasts from first trimester placentae should be identified by cytokeratin antibodies specific for the isoform 7. Purification of isolated trophoblasts by density gradient alone does not result in a sufficient degree of purity.     

Increased expression of interleukin-1 beta is associated with persistence of the disease and invasion in complete hydatidiform moles (CHM)

Complete hydatidiform moles (CHM), a post-conceptual pathologic condition of the placenta, have a high prevalence rate (12/1,000 deliveries) in Kerala, India. This study addresses the expression of IL-1 alpha and beta by immunohistochemistry in relation to persistence and invasion of the disease. Mild to moderate expression of IL-1 alpha in the villous cytotrophoblasts, syncytiotrophoblasts and decidua of the first trimester in the normal placenta and all gestational ages in the molar placenta were observed. IL-1 beta expression was observed in the extravillous trophoblasts, syncytiotrophoblasts and decidua in both the normal and molar placentae and also in the villous cytotrophoblasts and the stromal Haufbaur cells in molar placentae. Strong expression of IL-1 beta in the placenta suggests its involvement in placental physiology supporting earlier reports. Higher expression of IL-1 beta correlated well with the invasive and persistent nature of the tumour and holds potential as a marker of persistence and invasion in CHM.