Phagocytosis of Apoptotic Trophoblast Cells by Human Endometrial Endothelial Cells Induces Proinflammatory Cytokine Production

Citation Peng B, Koga K, Cardenas I, Aldo P, Mor G. Phagocytosis of apoptotic trophoblast cells by human endometrial endothelial cells induces proinflammatory cytokine production. Am J Reprod Immunol 2010; 64: 12–19 Problem Apoptosis is a normal constituent of trophoblast turnover in the placenta; however in some cases, this process is related to pregnancy complications such as preeclampsia. Recognition and engulfment of these apoptotic trophoblast cells is important for clearance of dying cells. The aim of this study was to show the cross talk between human endometrial endothelial cells (HEECs) and apoptotic trophoblast cells in an in vitro coculture model and its effect on cytokine production by HEECs. Method of study Fluorescent‐labeled HEECs were cocultured with fluorescent‐labeled apoptotic human trophoblast cells. Confocal microscopy and flowcytometry were used to show the interaction between these two types of cells. Cytokine profiles were determined using multiplex analysis. Results HEECs are capable to phagocytose apoptotic trophoblasts. This activity is inhibited by the phagocytosis inhibitor cytochalasin B. Phagocytosis of apoptotic trophoblast cells induced the secretion of the proinflammatory cytokines interleukin‐6 and monocyte chemoattractant protein‐1 by HEECs. Conclusion This study provides the first evidence that HEECs have an ability to phagocytose apoptotic trophoblasts. Furthermore, we demonstrated an inflammatory response of HEECs after phagocytosing the apoptotic trophoblast cells. This event may contribute to the inflammatory response in both normal pregnancy and pathologic pregnancy such as preeclampsia.     

Dimethylarginine dimethylaminohydrolase (DDAH) regulates trophoblast invasion and motility through effects on nitric oxide

BACKGROUND: Invasion of trophoblast into the uterine environment is crucial for establishing a successful pregnancy. Physiological production of nitric oxide (NO) by extravillous trophoblasts results in significant pro-invasive effects. NO synthesis is competitively inhibited by methylated arginine analogues such as asymmetric dimethylarginine (ADMA) but not the enantiomer symmetric dimethylarginine (SDMA). Within cells, the concentration of ADMA is regulated by the activity of the enzyme dimethylarginine dimethylaminohydrolase (DDAH). The aim of this study was to examine DDAH expression and function in trophoblasts. METHODS AND RESULTS: DDAH-1 and DDAH-2 messenger RNA and protein were demonstrated in first trimester placental tissue, primary extravillous trophoblasts and extravillous trophoblast-derived cell lines. DDAH activity was demonstrated in both cells and tissue. Overexpression of DDAH-1 in trophoblasts led to a number of significant changes, including an 8-fold increase in enzymatic activity, a 59% decrease in production of ADMA (but not SDMA), a 1.9-fold increase in NO and a 1.6-fold increase in cyclic guanosine monophosphate (cGMP) production. Functional assays showed that increased DDAH activity led to significantly increased cell motility and invasion in response to hepatocyte growth factor (HGF). CONCLUSIONS: DDAH may play an important role in the regulation of extravillous trophoblast function via its effects on ADMA and NO production.     

Wide-ranging DNA methylation differences of primary trophoblast cell populations and derived cell lines: implications and opportunities for understanding trophoblast function

Difficulties associated with long-term culture of primary trophoblasts have proven to be a major hurdle in their functional characterization. In order to circumvent this issue, several model cell lines have been established over many years using a variety of different approaches. Due to their differing origins, gene expression profiles and behaviour in vitro, different model lines have been utilized to investigate specific aspects of trophoblast biology. However, generally speaking, the molecular mechanisms underlying functional differences remain unclear. In this study, we profiled genome-scale DNA methylation in primary first trimester trophoblast cells and seven commonly used trophoblast-derived cell lines in an attempt to identify functional pathways differentially regulated by epigenetic modification in these cells. We identified a general increase in DNA promoter methylation levels in four choriocarcinoma (CCA)-derived lines and transformed HTR-8/SVneo cells, including hypermethylation of several genes regularly seen in human cancers, while other differences in methylation were noted in genes linked to immune responsiveness, cell morphology, development and migration across the different cell populations. Interestingly, CCA-derived lines show an overall methylation profile more similar to unrelated solid cancers than to untransformed trophoblasts, highlighting the role of aberrant DNA methylation in CCA development and/or long-term culturing. Comparison of DNA methylation and gene expression in CCA lines and cytotrophoblasts revealed a significant contribution of DNA methylation to overall expression profile. These data highlight the variability in epigenetic state between primary trophoblasts and cell models in pathways underpinning a wide range of cell functions, providing valuable candidate pathways for future functional investigation in different cell populations. This study also confirms the need for caution in the interpretation of data generated from manipulation of such pathways in vitro.     

Endometrial stromal cell decidualization inhibits human chorionic gonadotrophin and human placental lactogen secretion by co-cultured trophoblasts

We have developed an in-vitro co-culture system to examine theinteraction between purified first trimester cytotrophoblastsand purified non-pregnant human endometrial stromal cells (ESC).ESC decidualization is an important step in endometrial maturationand may modulate embryo implantation. In order to investigatethe effects of ESC decidualization on trophoblast function,we examined human chorionic gonadotrophin (HCG), human placentallactogen (HPL), progesterone and oestrogen secretion by trophoblastsco-cultured in contact with ESC, either with or without decidualizationinduced by progesterone. Decidualized ESC inhibited basal HCGand HPL secretion for 3 days during the culture for HCG, andfor 5 days during the culture for HPL (P < 0.01 and P <0.03 respectively). After 5 days of co-culture, decidual transformationof ESC as indicated by prolactin production occurred in thecontrol cultures due to progesterone and oestradiol secretionby the co-cultured trophoblasts, but no significant differencesin HCG or HPL secretion were observed between the two groups.Although the type of trophoblast used in the present study isfar from implantation, our results clearly demonstrated thatHCG and HPL secretion by trophoblasts was inhibited by the presenceof co-cultured decidualized ESC, and suggested that ESC decidualizationmay regulate trophoblast function at the human fetal-maternalinterface.     

Heparin prevents programmed cell death in human trophoblast

Heparin is used clinically for the prevention of pregnancy complications associated with prothrombotic disorders, especially antiphospholipid antibody syndrome. Recent studies have suggested that heparin may exert direct effects on placental trophoblast, independently of its anticoagulant activity. We now demonstrate that heparin abrogates apoptosis of primary first trimester villous trophoblast in response to treatment with the pro-inflammatory cytokines interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha. This multifunctional glycosaminoglycan also inhibited apoptosis induced by other agents, including staurosporin, broad-spectrum kinase inhibitor and thrombin. Furthermore, heparin attenuated caspase-3 activity, a hallmark of apoptosis, in human first trimester villous and extravillous trophoblast cell lines treated with peptidoglycan, a Toll-like receptor-2 agonist isolated from Staphylococcus aureus. The ability of heparin to antagonize cell death induced by such diverse apoptotic signals suggested that it acts as a survival factor for human trophoblast. We demonstrate that heparin, like epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF), elicits phosphorylation of the EGF receptor and activation of the phosphatidyl inositol 3-kinase (PI3K)-, the extracellular signal-related kinase 1/2 (ERK1/2)- and the c-Jun NH2 terminal kinase (JNK)-signal transduction pathways in primary villous trophoblast. In summary, we have demonstrated that heparin activates multiple anti-apoptotic pathways in human trophoblast. Our results suggest that heparin may be useful in the management of at-risk patients, even in the absence of an identifiable thrombophilic disorder.     

An in vitro model of human placental trophoblast deportation/shedding

Deportation of trophoblast shed from the placenta into the maternal circulation was first described over 100 years ago. Despite this, little is known about the quantity or nature of the shed and deported trophoblasts. Neither do we have a clear understanding of the fate of deported trophoblasts nor do we have a clear understanding of their effects on the maternal physiology. This deficiency is largely due to the inaccessibility of deported trophoblasts in vivo. This study aimed to produce a model that would allow us to study deported trophoblasts. We devised a system for culturing placental explants of 12-week gestation in cell culture inserts with a stainless steel mesh bottom that allowed the ready harvesting of shed/deported trophoblasts. Immunohistochemical and morphologic investigations demonstrated that these in vitro shed/deported trophoblasts are similar to those found in vivo and that apoptotic, necrotic and viable trophoblasts were shed from the explants. Inhibiting caspases induced a change from predominantly apoptotic to predominantly necrotic trophoblast shedding. We have devised an in vitro model that allows the collection of shed/deported trophoblasts which will significantly enhance our ability to study these cells. Our preliminary investigations confirm that apoptosis plays an important role in trophoblast shedding/deportation.     

Calcitonin gene-related peptide stimulates human villous trophoblast cell differentiation in vitro

Calcitonin gene-related peptide (CGRP) is a multifunctional peptide present in both maternal and fetal circulations in pregnancy. Its receptors have been identified on human trophoblast cells, but the role of CGRP in trophoblast differentiation remains unknown. This study was designed to determine the effect of CGRP on the differentiation of villous trophoblasts isolated from normal human term placentae. The morphological and functional differentiation of the trophoblast cells were assessed by desmosomal protein immunofluorescent staining and the quantification of hCG, estrogen and progesterone secretion. Results showed that (i) exposure of villous trophoblast cells to CGRP led to a dose-dependent increase in intracellular cyclic adenosine monophosphate (cAMP) accumulation; (ii) immunofluorescent staining with antidesmosomal antibody was identified at the boundaries between aggregated cytotrophoblast cells, and these stainings disappeared when cells fused to form syncytiotrophoblast cells; (iii) the formation of multinucleated syncytiums in primary cultured cytotrophoblasts was stimulated by CGRP as evidenced by the changes in antidesmosomal staining; (iv) CGRP increases trophoblast hCG secretion in a time- and dose-dependent manner, and this secretion was blocked by CGRP antagonist, CGRP(8-37), and (v) both 17beta-estradiol (E(2)) and progesterone concentrations in the culture medium were increased by CGRP, and these increases were dose dependent. These observations suggest that CGRP may be involved in the morphological and functional differentiation of trophoblast cells, and these actions might be attributed to CGRP-induced intracellular cAMP accumulation.     

Schistosome Egg Antigens Elicit a Proinflammatory Response by Trophoblast Cells of the Human Placenta

Schistosomiasis affects nearly 40 million women of reproductive age. Many of these women are infected while pregnant and lactating. Several studies have demonstrated transplacental trafficking of schistosome antigens; however, little is known regarding how these antigens affect the developing fetus and placenta. To evaluate the impact of schistosomiasis on trophoblasts of the human placenta, we isolated primary trophoblast cells from healthy placentas delivered at term. These trophoblasts were placed in culture and treated with Schistosoma japonicum soluble egg antigens (SEA) or plasma from S. japonicum-infected pregnant women. Outcomes measured included cytokine production and activation of signal transduction pathways. Treatment of primary human trophoblast cells with SEA resulted in upregulation of the proinflammatory cytokines interleukin 6 (IL-6) and IL-8 and the chemokine macrophage inflammatory protein 1α (MIP-1α). Cytokine production in response to SEA was dose dependent and reminiscent of production in response to other proinflammatory stimuli, such as Toll-like receptor 2 (TLR2) and TLR4 agonists. In addition, the signaling pathways extracellular signal-regulated kinase 1/2 (ERK1/2), Jun N-terminal protein kinase (JNK), p38, and NF-κB were all activated by SEA in primary trophoblasts. These effects appeared to be mediated through both carbohydrate and protein epitopes of SEA. Finally, primary trophoblasts cocultured with plasma from S. japonicum-infected pregnant women produced increased levels of IL-8 compared to trophoblasts cocultured with plasma from uninfected pregnant women. We report here a direct impact of SEA on primary human trophoblast cells, which are critical for many aspects of a healthy pregnancy. Our data indicate that schistosome antigens can activate proinflammatory responses in trophoblasts, which might compromise maternal-fetal health in pregnancies complicated by schistosomiasis.     

Effect of heparin and fractionated heparin on trophoblast invasion

BACKGROUND: Heparin can significantly reduce pregnancy complications in women with certain thrombophilias, such as antiphospholipid syndrome. Recent reports suggest that heparin may act by mechanisms other than anticoagulation. However, the effect of heparin on trophoblast biology in the absence of thrombophilia has not been extensively investigated. Therefore, this study aimed to evaluate trophoblast invasion, using an established cell line and primary extravillous trophoblasts (EVTs), following exposure to heparin and fractionated heparin. METHODS: An EVT cell line (SGHPL-4) was used to study invasion in the presence of hepatocyte growth factor (HGF) and varying concentrations of fractionated and unfractionated heparin. These experiments were repeated using first trimester primary EVTs. RESULTS: Both forms of heparin significantly reduced HGF-induced invasion in the SGHPL-4 cell line. This suppression of invasion appeared to be dose-dependent for fractionated heparin. In primary EVT cells, fractionated heparin also demonstrated significant suppression of invasion. CONCLUSIONS: Heparin has the potential to reduce trophoblast invasion in cell lines and first trimester EVT cells. This article highlights the need for further evaluation of these medications in vitro and in vivo, especially when used in the absence of thrombophilic disorders.     

Cytotrophoblast stem cell lines derived from human embryonic stem cells and their capacity to mimic invasive implantation events

BACKGROUND: An effective embryonic-maternal interaction is crucial for successful human pregnancy. Failure of this process is a major cause of infertility and can lead to placental dysfunction resulting in recurrent miscarriage, fetal retardation and pre-eclampsia. Research is severely constrained by ethical and practical considerations; therefore, we aimed to generate cytotrophoblast stem (CTBS) cell lines from human embryonic stem cells (HESCs). METHOD: Beta-HCG was used as a marker of viable trophoblast cells. In defined culture, embryoid bodies were generated from HESCs and selected for trophoblast enrichment by rounds of cellular aggregation and disaggregation. Distinct CTBS cell lines were isolated and characterized. Spheroid cytotrophoblast bodies were generated and their interaction with luteal-phase endometrial stroma was analysed by real-time image analysis. RESULTS: Three CTBS cell lines were derived, which were maintained in the absence of residual HESCs, fibroblast feeder cells or extracellular matrix. CTBS cells displayed typical cytotrophoblast and syncytiotrophoblast characteristics and exhibited further differentiation to invasive endovascular cell phenotype. One cell line was generated with constitutive expression of enhanced green fluorescent protein (eGFP). Spheroid trophoblast bodies mimicked closely the early invasive stages of implantation when incubated with human endometrial stromal preparations in vitro. CONCLUSION: These human CTBS cell lines are a significant new model for investigating human placentation and may have considerable potential in cell therapy applications.     

Expression of uPAR in human trophoblast and its role in trophoblast invasion

Placental trophoblast cells differentiate into invasive trophoblasts or syncytiotrophoblasts. Abnormal trophoblast invasion results in pregnancy-associated disease and abortion. uPAR is a cell membrane-bound glycosylated protein, involved in physiological and pathological processes. However, uPAR expression in villi during threatened abortion and its role in trophoblast differentiation are unclear. We determined that, uPAR expression in the villi was reduced in threatened abortion patients than that in normal pregnancy. uPARsiRNA inhibited the potential for trophoblast migration and invasion in explants culture and HTR8/SVneo cells. It also enhanced forskolin-induced fusion of HTR8/SVneo cells. Overall, this study provides a possible reason for abortion.     

Platelet-endothelial cell adhesion molecule-1 is expressed by a subpopulation of human trophoblasts: a possible mechanism for trophoblast-endothelial interaction during haemochorial placentation

The terminal event in the establishment of the haemochorial placenta in the human is the invasion of trophoblasts into the maternal vessels, a process in which trophoblasts interact directly with the vascular endothelium and degrade the vascular basement membrane and the tunica elastica of the vessels. To further understand this heterotypic cellular interaction, we investigated the expression by human trophoblasts of the vascular cell adhesion molecule platelet- endothelial cell adhesion molecule-1 (PECAM-1) as a possible mediator of the adhesive interaction between trophoblasts and endothelium. In vitro, human trophoblasts were found to express PECAM-1 mRNA and protein. Indirect immunofluorescence indicated a diffuse staining pattern, which was most intense in a subpopulation of trophoblast cells. Co-incubation of trophoblasts with endothelial cells showed interaction between these two cell types with strong expression of PECAM-1 at points of trophoblast-endothelial cell contact, suggesting that this cell adhesion molecule participates in this heterotypic cell interaction. Immunohistochemical localization of PECAM-1 in chorionic villi and first trimester implantation sites showed that, in vivo, only extravillous interstitial and endovascular trophoblasts were positive. In first trimester placentae, villous trophoblast and extravillous trophoblast in other locations than around or within the decidual vessels did not express this molecule. In term placentae, villous trophoblast did not express these adhesion molecules except for two specimens examined. This study demonstrates that PECAM-1 is expressed by a subset of human trophoblasts in vitro and in vivo. Its tissue localization suggests that PECAM-1 is important in mediating the adhesive interaction between trophoblasts and maternal vascular endothelium during the process of haemochorial placentation. Regulation of PECAM-1 expression by human trophoblasts may play a critical role in normal and abnormal vascular invasion during implantation and placentation.      

Normal in-vivo development of marmoset monkey embryos after trophectoderm biopsy

A procedure was developed to remove trophectoderm cells fromday-8 blastocysts of the marmoset monkey. Using micro-manipulativetechniques, a tear was made in the zona pellucida opposite theinner cell mass which facilitated the controlled herniationof trophectoderm cells as the blastocysts developed in vitro.After 24 h (day-9 blastocysts) and 48 h (day-10 blastocysts)of culture {small tilde}20% and {small tilde}50% respectivelyof the blastocyst had herniated. The herniated trophectodermwas cut off by freehand and the biopsied blastocysts transferredto recipients. Normal offspring were born but pregnancies couldbe established from day-10 blastocysts only if the recipientswere treated with human chorionic gonadotrophin during earlypregnancy. One pregnancy was established after the transferof three frozen biopsied day-10 blastocysts. Biopsies of 30–50cells from day-10 blastocysts could be readily grown in vitroas trophoblast vesides to in excess of 1000 cells but biopsiesof <20 cells from day-9 blastocysts formed a monolayer ofbinucleated and multinucleated cells with limited cell replication.Assuming human trophectoderm cells have a similar capacity tothe marmoset to grow in vitro, the application of this techniqueto human blastocysts would provide sufficient cells on whichthe pre implantation diagnosis of a genetic disorder could bemade.     

Identification of committed placental stem cell lines for studies of differentiation

Trophoblasts provide a model to investigate fundamental mechanisms of stem cell differentiation, but the availability of trophoblast stem cell lines is limited. Here we report the development of an RT-PCR-based lineage-specific profile as a method to identify the lineages of placental trophoblast cells routinely and specifically. This profiling method was used to analyze the mouse SM10 and rat HRP-1 cell lines, isolated from a region of the placental labyrinth, but of previously unidentified lineage. Using this profile, the expression of trophoblast stem cell markers was detected in the SM10 and HRP-1 cells. In contrast, no expression of a marker of differentiated labyrinthine trophoblast was detected. Additionally, both cell lines expressed labyrinthine trophoblast-specific genes and did not express lineage-specific markers of spongiotrophoblasts or trophoblast giant cells. Our results suggest that SM10 and HRP-1 cell lines are trophoblast stem cell-like cell lines that can be maintained in undifferentiated but committed state in cell culture. These cell lines express labyrinthine-specific genes and are committed to differentiate solely into functional labyrinthine trophoblasts. Our profiling method provides a new technique to identify stem cells and their lineage-specific differentiation. This method additionally indicates that SM10 and HRP-1 cell lines provide new systems for future studies of stem cell differentiation, allowing investigation of basic mechanisms of differentiation, which may provide insights into the biophysics of development of a specialized system. This method should also prove to be useful for identification of other stem cell lines and examination of lineage-specific commitment.     

Enhanced monocyte binding to human cytomegalovirus-infected syncytiotrophoblast results in increased apoptosis via the release of tumour necrosis factor alpha

We have shown that monocytes bound to intercellular adhesion molecules (ICAM-1) on syncytialized placental trophoblasts (ST) induce trophoblast apoptosis, and that ST infection by human cytomegalovirus (HCMV) up-regulates ICAM-1. We hypothesize that the focal loss of trophoblast seen in HCMV-infected placenta is mediated by increased adherence of monocytes at sites of infection. We find that ST cultures (differentiated from primary cytotrophoblasts) increase monocyte binding when infected with HCMV. Monocyte adhesion was inhibited by antibodies to ICAM-1 and its ligand leukocyte function-associated molecule (LFA-1) on monocytes. When co-cultured with adhering monocytes, infected ST cultures had higher levels of apoptosis than infected cultures alone. Although trophoblast apoptosis clustered around adhering monocytes, it occurred only in non-infected cells. Blocking monocyte binding with ICAM-1 and LFA-1 antibodies reduced the rate of apoptosis to that of the infected culture. Co-cultures incubated with TNFalpha antibody and EGF inhibited both monocyte- and HCMV-induced apoptosis but did not block binding. We conclude that HCMV stimulates ST culture expression of ICAM-1, which binds to LFA-1 on monocytes that release TNFalpha, thereby inducing apoptosis of neighbouring uninfected trophoblasts. The above data indicates that trophoblast loss associated with HCMV infection can be caused by increased monocyte adhesion to ST.     

Surface antigens of human trophoblasts

Single cells and cell aggregates of human trophoblasts were studied by immunofluorescence and mixed haemadsorption for the presence of different surface antigens. Results indicate that fresh trophoblast cells do not express HL-A in detectable amounts but carry human chorionic gonadotropin (HCG) and Con-A receptors. Fresh cells also reacted with antisera to B₂-microglobulin but after outgrowth to a monolayer, this molecule was detected at a low frequency from different individuals; the reactions were weak. In contrast, the outgrowth cells seemed to carry HCG. It was not possible to demonstrate any significant adsorption of maternal immunoglobulin to fresh trophoblast cells. Surface antigens of trophoblast cells in suspension were slightly redistributed by antibodies whereas the cells in aggregate showed no signs of redistribution.