Human placental trophoblast cells express alpha-tocopherol transfer protein

Alpha-tocopherol transfer protein (alpha-TTP), a 30 kDa cytosolic protein first described to be present in the liver and important for alpha-tocopherol trafficking, plays a major role in maintaining alpha-tocopherol levels in plasma, while alpha-tocopherol is known as the major lipid-soluble antioxidant. Expression of alpha-TTP has not only been described in animal model liver, but also in diverse other tissues such as rat brain or pregnant mouse uterus, the latter finding stressing the importance of alpha-TTP for embryogenesis and foetal development. In this study, we report the identification of alpha-TTP in human liver by anti-human alpha-TTP monoclonal antibodies made in rat and the cellular localization of alpha-TTP in term human placenta. By immunohistochemistry, intense staining of alpha-TTP was seen in syncytiotrophoblast as well as in villous and invading extravillous cytotrophoblast, while basal decidual cells showed slighter, but present staining of alpha-TTP. Foetal vessel endothelium remained unstained. It is therefore suggested that alpha-TTP may play a major role in supplying alpha-tocopherol to the foetus prior to delivery and is likely involved in maintaining adequate alpha-tocopherol levels in the foetus.     

Murine ectoplacental cone-derived trophoblast cells express chemokine receptors

Chemokine receptors (CCRs) have been shown to regulate T cell migration and differentiation as well as the establishment of Th1/Th2 bias. Furthermore, T cells and T cell products are essential to trophoblast development. Thus, postulating that chemokines as well as their receptors may be expressed by trophoblast to move T cells into an interaction with the feto-placental unit, we examined whether CCRs are expressed during the early stages of ectoplacental cone (EPC) formation. For this, murine EPC-derived trophoblast were examined for their ability to express CCRs constitutively or inducible by interferon-γ (IFN-γ). Immunofluorescence experiments on EPC-derived trophoblast cells showed that CCR3, CXCR4 and CCR5 are significantly expressed. IFN-γ accelerated the mobilization of intracellular pools of CCR molecules during early cell culture periods (2–6 h) and, in most cases, increased their expression on EPC-derived trophoblast cells. CCR activity could be detected in the culture supernatants of these cells, inversely proportional to cell surface expression, suggesting the existence of rapid endocytosis and recycling mechanisms. This finding indicates that the level of intracellular CCRs may partly be determined in the extracellular matrix, an event that could play an important role towards neutralization of specific T cell/trophoblast interactions during early stages of pregnancy and protect the fetus against harmful maternal immune responses.     

Progesterone-induced blocking factor (PIBF) and trophoblast invasiveness

Controlled trophoblast invasion is a key process during human placentation and a prerequisite for successful pregnancy. Progesterone is one of the factors to regulate trophoblast invasiveness. Progesterone-induced blocking factor (PIBF) is a progesterone-induced molecule expressed by the trophoblast, and also by tumors. The distribution of PIBF within the first-trimester decidua coincides with sites of trophoblast invasion. Another molecule that has been implicated in the control of trophoblast invasiveness is placental leptin. Leptin inhibits the secretion of progesterone by cytotrophoblast. The aim of this work was to investigate the possible interaction of PIBF and leptins in regulating trophoblast invasion. Paraffin-embedded sections from normal first-trimester placentae, partial moles, complete moles, and choriocarcinomas were reacted with PIBF, leptin, and leptin receptor specific antibodies. PIBF-deficient trophoblast cells were generated using siRNA and leptin receptor was detected on Western blot analysis. The lysates of PIBF-treated cells were used for detecting leptin expression in a protein array. PIBF was expressed in both normal first-trimester villous trophoblast and in partial mole. Compared with this, PIBF expression was markedly decreased in complete mole and absent in choriocarcinoma. Neither leptinR nor leptin were detected in partial mole, whereas both of these molecules were present in complete mole and choriocarcinoma. Leptin receptor expression was upregulated in PIBF-deficient cells, while leptin expression was decreased in PIBF-treated cells. These data suggest that PIBF affects the expression of leptin and its receptor, and that PIBF expression is inversely related to trophoblast invasiveness.     

Cyclooxygenase is involved in the effects of progesterone-induced blocking factor on the production of interleukin 12

OBJECTIVE: Immunologic effects of progesterone are mediated by the progesterone-induced blocking factor. Progesterone-induced blocking factor inhibits natural killer cytotoxic activity and arachidonic acid release from mononuclear cells. The relationship between increased prostaglandin synthesis and increased cytotoxic activity of the lymphocytes is still unclear; therefore we investigated the effect of progesterone-induced blocking factor-neutralizing antibody, as well as simultaneous indomethacin treatment, on interleukin 12 production. STUDY DESIGN: Pregnancy lymphocytes were treated with anti-progesterone-induced blocking factor antibody or lipopolysaccharide as a positive control in the presence or absence of indomethacin. Interleukin 12 production by peripheral blood mononuclear cells was detected by immunocytochemical examination. The 2-tailed Student t test was used for statistical evaluation. RESULTS: Neutralization of progesterone-induced blocking factor, as well as lipopolysaccharide treatment, resulted in an increased expression of interleukin 12 that was corrected by simultaneous indomethacin treatment. CONCLUSION: Progesterone-induced blocking factor reduces the expression of interleukin 12 via the inhibition of arachidonic acid metabolism. This results in lowered cytotoxic natural killer activity, which favors a normal pregnancy outcome.     

Production and characterization of a novel monoclonal antibody against progesterone-induced blocking factor (PIBF)

Progesterone-induced blocking factor (PIBF) has been described as an active factor intimately involved in regulation of the immune response in pregnancy. It has been shown that PIBF biased the cytokine balance to Th2-type in pregnancy and inhibited the activity of NK cells. The biological roles of PIBF would be better defined if methods for its detection and measurement in biological fluids are available. However, so far, reliable antibodies have not been developed to be used as specific probes. A monoclonal antibody designated as MAB 3A6 was produced and characterized. MAB 3A6 reacts specifically with PIBF. It can detect this protein in biological fluids when tested by immunoblot and recognizes PIBF expressed on the surface of lymphocytes of pregnant women stimulated in vitro with progesterone. The characteristics of MAB 3A6 makes it the possible basis for development of a clinically applicable assay to assess the presence and concentration of PIBF in biological samples.     

Production and characterization of a novel monoclonal antibody against progesterone-induced blocking factor (PIBF)

Progesterone-induced blocking factor (PIBF) has been described as an active factor intimately involved in regulation of the immune response in pregnancy. It has been shown that PIBF biased the cytokine balance to Th2-type in pregnancy and inhibited the activity of NK cells. The biological roles of PIBF would be better defined if methods for its detection and measurement in biological fluids are available. However, so far, reliable antibodies have not been developed to be used as specific probes. A monoclonal antibody designated as MAB 3A6 was produced and characterized. MAB 3A6 reacts specifically with PIBF. It can detect this protein in biological fluids when tested by immunoblot and recognizes PIBF expressed on the surface of lymphocytes of pregnant women stimulated in vitro with progesterone. The characteristics of MAB 3A6 makes it the possible basis for development of a clinically applicable assay to assess the presence and concentration of PIBF in biological samples.     

Human sperm cells express CD44

OBJECTIVE: To determine whether CD44 is expressed by human sperm cells. DESIGN: Prospective study. SETTING: Assisted conception unit and university research laboratory. PATIENT(S): Fourteen normal fertile donors. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Enzyme-linked immunoassay was used to measure the concentration of CD44 in samples of human sperm cells. Immunocytochemistry using a fluorescent antibody system was used to confirm and localize the expression of CD44 on sperm membranes. Western blotting was used to identify CD44 protein in extracts of lysed sperm cells. RESULT(S): The presence of CD44 was identified in suspensions of pure sperm cells by ELISA. The mean sperm count was 66 million per milliliter (range, 60 million-75 million per milliliter) and the mean concentration of CD44 was 0.013 ng per 10(6) sperm (range, 0.012 ng-0.015 ng per 10(6) sperm). Use of a fluorescently labeled antibody to CD44 confirmed the presence of CD44 on sperm membranes. CD44 expression was mostly localized in the acrosome region. Western blotting using a specific monoclonal antibody to CD44 identified a variant of CD44 with an approximate molecular weight of 73 kDa. CONCLUSION(S): The expression of CD44 on the surface of human sperm membranes was confirmed by three independent methods.     

Vascular endothelial growth factor is a chemoattractant for trophoblast cells

A role for angiogenic growth factors in trophoblast invasion has been postulated. Directional motility (chemotaxis) is an important function of trophoblast cells. We have previously shown that vascular endothelial growth factor (VEGF) increases the random movement of trophoblast cells although placental growth factor (PlGF) has no effect. Heparin inhibited this effect of VEGF. Motility of trophoblast cells has been proposed to be mediated by a nitric oxide (NO) pathway. We hypothesized that VEGF but not PlGF would be chemotactic for trophoblast cells. Chemotaxis of a first trimester extravillous trophoblast cell line, SGHPL-4, and primary isolates of first trimester and term trophoblast cells was measured using a Boyden chamber. Initial experiments to optimize the time of the experiment and identify a positive control were performed. Subsequent experiments ran for 20 h, used 0.5 per cent FBS or 10 ng/ml PDGF as negative and positive controls and were performed in triplicate. VEGF (1, 10 and 100 ng/ml+/-1 microg/ml heparin or +/-100 microM L-NAME) and PlGF (1, 10, 100 ng/ml) were tested. The chamber was placed in a 5 per cent CO(2) in air, 37 degrees C incubator. The number of cells in the lower chamber were counted. There was a dose dependent increase in chemotactic motility of the SGHPL-4 cell line and term trophoblast cells in response to VEGF. PlGF had no effect on the movement of the first trimester trophoblast cell line but did increase the motility of the term trophoblast cells in a dose dependent manner. Heparin increased the cellular motility of both cell types alone. It also further enhanced the chemoactivity of VEGF on the term trophoblast cells but not the cell line. L-NAME did not affect the VEGF-stimulated motility of the first trimester cell line. However, in the term trophoblast cells L-NAME increased the directional cellular motility in the absence of, or in the presence of low concentrations of VEGF. In conclusion, the first trimester and term trophoblast cells appeared to respond differently to the various factors tested in the present study that may reflect differential cellular function as gestation progresses.     

Human trophoblast interferon: pattern of response to priming and superinduction of purified term trophoblast and choriocarcinoma cells

We recently described a beta interferon response of primary cultures of term human trophoblast exposed to poly(I:C). The response pattern has now been studied further with priming and superinduction both of normal placental cell types and JAR, JEG-3 and BeWo choriocarcinoma cells. Pre-treating placental trophoblast cells, fibroblasts and macrophages with human interferon generally led to increased yields of interferon after poly(I:C) induction, whereas choriocarcinoma cells did not respond to priming. All the cells showed the superinduction phenomenon although to varying degrees. The combination of priming and superinduction conditions led to the production of very high yields of interferon in placental fibroblast cultures. The combined procedure also produced more interferon in macrophage cultures than priming or superinduction alone. Combined superinduction and priming of normal trophoblast did not produce higher yields than those obtainable by superinduction alone. These data might help to provide additional insight into the cellular control mechanisms of sensitivity of normal and malignantly transformed cells to interferon. Furthermore, the large quantity of trophoblast interferon produced by superinduction could be used for studies of its anti-viral and immunomodulatory effects.     

Human trophoblast cells cultured in modified medium and supported by extracellular matrix

This paper describes a culture procedure which consistently yields 80 to 90 per cent trophoblast from human first-trimester placentae. The trophoblast cells are selected and maintained but there is no increase in proliferation. The cultured cells are found to resemble extravillous rather than villous trophoblast in their immunocytochemical characteristics. This technique provides a means of obtaining human trophoblast cells with a sufficient degree of homogeneity and viability to be used for in vitro experiments.     

缺氧对滋养细胞VEGF分泌与基因表达调控作用的研究

目的:研究原代培养人早孕绒毛滋养细胞VEGF分泌、基因表达的特点,以及缺氧的调控作用。方法:原代培养早孕绒毛滋养细胞,研究正常和缺氧(氯化钴化学诱导)条件下细胞培养上清液中VEGF含量(ELISA法)以及滋养细胞VEGF mRNA表达(RT-PCR方法)特点。结果:(1)免疫组化技术证实培养细胞角蛋白阳性,波形蛋白阴性,纯度达95%以上;(2)正常培养的滋养细胞在培养12h后分泌产生的VEGF明显增加,至培养结束时的72h达到最高点;缺氧诱导滋养细胞大量分泌产生VEGF的时间提前至缺氧培养后6h,持续至72h无下降,VEGF水平明显高于正常,最高达到2200ng/ml,较正常上升47%;(3)滋养细胞VEGF mRNA表达于培养12h时开始明显上升,至48h时达峰值;缺氧则使VEGF mRNA表达显著上升的时间提前至6h,约12h达峰值。结论:早孕绒毛滋养细胞中存在VEGF基因转录并分泌至细胞外;缺氧诱导VEGF转录增强、分泌增加。     

Miscarriage in the first trimester according to the presence or absence of the progesterone-induced blocking factor at three to five weeks from conception in progesterone supplemented women

PURPOSE: To determine if the failure to detect the immonomodulatory protein progesterone induced blocking factor (PIBF) at three to five weeks of seemingly normal pregnancies in women supplemented by extra progesterone is associated with a higher miscarriage rate. METHODS: Progesterone-induced blocking factor expression by lymphocytes was measured by an immunocytochemistry technique. The serum beta human chorionic gonadotropin (hCG) and/or ultrasound were also deemed appropriate so that by these criteria there was no evidence of a poor pregnancy. The minimum progesterone dosage was 200 mg twice daily vaginal suppositories. RESULTS: Progesterone-induced blocking factor was detected in 17/39 (43.5%) of pregnant patients at this early time. There were three miscarriages by 12 weeks in this group (17.6%). The miscarriage rate was 6/21 (28.5%) in those where it was not detected. CONCLUSIONS: There was insufficient power to show significance. However there seems to be a trend for higher rates of miscarriage when PIBF is absent so these preliminary data encourage continuation of the study.     

Human placental multipotent mesenchymal stromal cells modulate trophoblast migration via Rap1 activation

Introduction

Little is known about the interaction between human placental multipotent mesenchymal stromal cell (hPMSC) and trophoblast. We hypothesize that hPMSCs produce hepatocyte growth factor (HGF) which may interact with trophoblasts and regulate their migration during placentation.

Methods

hPMSCs were isolated from term placentas and conditioned medium was collected after 2 days of culture in hypoxic (<1% O₂) or control (20% O₂) conditions. Selective agonist and inhibitor or siRNA for protein kinase A (PKA) or Rap1 were combined with Rap1-GTP pull down assays, flow cytometry, integrin β1 activation assays and adhesion and migration studies to investigate HGF signaling effects in trophoblasts. The hPMSC abundance and HGF level in preeclamptic placentas were compared with gestational age-matched controls.

Results

HGF was expressed by hPMSCs and was decreased in hypoxia. HGF induced cAMP production and Rap1 activation in trophoblasts, which in turn activated integrin β1. The HGF and PKA activator 6-Bnz-cAMP induced Rap1 activation with increased trophoblast adhesion and migration. The alterations were inhibited by PKA inhibitor H89 or Rap1 siRNA. HGF and cAMP expression were reduced in preeclamptic placentas. hPMSC number was decreased in preeclamptic placenta compared to controls (0.68 ± 0.1% vs. 1.32 ± 0.5%; P = 0.026). hPMSC conditioned medium enhanced trophoblast migration which was inhibited by c-Met blocking antibody, but migration was reduced by conditioned medium from hPMSC cultured in hypoxia.

Conclusions

hPMSCs secrete HGF and increase trophoblast cAMP production. The cAMP effector PKA modulates adhesion and migration of trophoblast via signaling to Rap1 and integrin β1.     

Human amniotic fluid transferrin stimulates progesterone production by human trophoblast cells in vitro

OBJECTIVE: During pregnancy transferrin plays a key role as an iron transport protein to serve the increased fetal demands of iron. Transferrin is also present in relatively high concentrations in amniotic fluid [6], showing a different glycosylation compared with serum transferrin. The biological function of human amniotic fluid transferrin (hAFT) is still unknown. In addition trophoblast cells also synthesise transferrin. Transferrin synthesised by the trophoblast shows a special glycosylation. We found identical carbohydrate structure of hAFT and trophoblast transferrin. We investigated the influence of hAFT on the progesterone-, cortisol- and hCG-release of trophoblasts in culture compared with the influence of human holo- and apo-serum transferrin on the release of these hormones. MATERIAL AND METHODS: Cytotrophoblast cells were prepared from human term placentae by standard trypsin-DNAse dispersion of villous tissue followed by a percoll gradient centrifugation step. When placed in culture, the trophoblasts were incubated with varying concentrations (50-300 micrograms/ml) of human amniotic fluid- and serum-transferrin. Unstimulated cells of each placenta used as controls. Culture supernatants were assayed for progesterone, hCG and cortisol by enzyme-immunometric methods. RESULTS: Our results show, that the release of progesterone increased in hAFT-treated cell cultures compared to untreated cell cultures. Holo- and apo-serumtransferrin did not show any effect on the progesterone release by trophoblast cells in vitro. Neither hAFT nor holo- and apo-serum transferrin had any effect on the cortisol- and hCG-release in vitro. CONCLUSIONS: Progesterone is a marker for differentiation of trophoblasts in syncytiotrophoblasts. Only hAFT stimulates the progesterone production. We suggest, that hAFT can modulate the endocrine function of trophoblast cells in culture by regulating progesterone production.