Term Trophoblast Cells Research Articles

The inability of placental alkaline phosphatase antibodies to block immunoglobulin-G binding to cultured term trophoblast cells and to microvillous membranes

The inability of placental alkaline phosphatase antibodies to block immunoglobulin-G binding to cultured term trophoblast cells and to microvillous membranes

Human trophoblast interferons enhance major histocompatibility complex class I antigen expression on human term trophoblast cells in culture

The expression and regulation of major histocompatibility complex class I (MHC class I) antigens by virus-induced human trophoblast interferons (tro-IFNs) were examined in term trophoblast cultures. Flow cytometry studies using fluorescence monoclonal antibodies against MHC class I antigens revealed that isolated cytotrophoblasts can express MHC class I antigens. The expression of these antigens increased with stimulation of trophoblast cultures with tro-IFN-α and -β. One hundred IU tro-IFN-α and -β/ml induced no significant higher levels of MHC class I antigens as compared with the control, whereas 1000 IU tro-IFN-α and -β/ml did. The tro-IFN-enhanced expression of MHC class I antigens may be important as it increases the efficiency of local and viral antigen presentation, cytotoxicity by T cell response and local inflammatory processes, thereby preventing virus spread from mother to fetus.

Prolonged hyperglycemia in vitro affects glucose transporter protein gluti and glucose uptake in cultured term trophoblast cells

The role of clavulanic acid, an unstable antibiotic produced by Streptomyces clavuligerus, in biomass accumulation and production of clavulanic acid in batch cultures of the organism was examined. The organism was grown in a medium containing either 20 g/l lysine, 1 g/l lysine or 1 g/l lysine supplemented with degraded clavulanic acid as nitrogen sources. Biomass accumulation was highest in cultures grown with supplemented degraded clavulanic acid and reached a maximum of 2.2 g/l, compared with 1.5 g/l when lysine only was used. The yield coefficient for clavulanic acid production was again highest in cultures grown with supplemented degraded clavulanic acid, with a Yp/x value of 2 mg/g compared with Yp/x value of 1.5 mg/g in 20 g/l lysine. No clavulanic acid was produced in cultures containing non-supplemented 1 g/l lysine. Non-degraded clavulanic acid was added at 60 h to non-producing cultures of the organism containing 1 g/l lysine only. Clavulanic acid concentration immediately decreased on addition from 0.04 g/l over a period of 20 h, then remained constant at 0.02 g/l for a further 30 h until the end of the cultivation. This suggests that the rate of degradation was equivalent to the rate of production of clavulanic acid following a period of initial additive degradation. These results indicate that clavulanic acid is both produced and degraded in cultures of S. clavuligerus and that the products of degradation are used by the organism, resulting in further production of the antibiotic.

D 2-dopamine agonists inhibit adenosine 3′ :5′-cyclic monophosphate (cAMP) production in human term trophoblastic cells

We previously reported that dopamine (DA) acted via D 2-dopamine receptors on human trophoblastic cells to inhibit basal and hormone-stimulated secretion of human placental lactogen (hPL). We also described that these DA effects were coupled with inhibition of calcium influx. The present study examines the interaction of placental D 2-dopamine receptor with adenylate cyclase (AC). Incubations of isolated human term trophoblastic cells with R(-)-propylapomorphine (NPA), (±)-PPHT, and bromocriptine (3 different D 2 agonists) led to time- and dose-dependent inhibitions of cAMP production as determined by measuring the conversion of [2- 3H]-ATP into [2- 3H]-cAMP. The maximal inhibition was reached after 15 min of incubation and was 33±1 (SE)%, 29±3% and 31±1% for bromocriptine (10 −5 M), NPA (10 −7 M) and (±)-PPHT (10 −8 M) respectively. However, the time- and dose-dependent curves were biphasic with NPA and (±)-PPHT and the inhibition of cAMP production was abolished at higher agonist concentrations or after time incubations longer than 15 min. These inhibitions were receptor specific since they were reversed by spiperone and haloperidol, two specific-dopamine antagonist, and by butaclamol (mix D 2/D 1-dopamine antagonists) but not by α- and β-adrenergic, D 1- and D 4-dopaminergic, and 5-HT 2-serotonergic antagonists. The results reported here suggest that human placental D 2 receptors interact with AC to inhibit its activity. Also, bromocriptine seems a better agonist for the characterization of dopaminergic effects on human placenta.

Ca 2+ channels in human term trophoblast cells in vitro. A study using the Ca 2+-sensitive dye fura 2

The ability of elevated extracellular [Ca 2+ ] to induce a cytosolic Ca 2+ transient in trophoblast cells cultured from human near term placentae has been investigated using single cell microspectrofluorometry. Cells in early culture, similar to cytotrophoblast cells, were found to be relatively insensitive to extracellular [Ca 2+ ], but in contrast syncytiotrophoblast-like cells from older cultures demonstrated a marked increase in cytosolic [Ca 2+ ]. These responses were shown to be saturable, and could be significantly depressed by reducing the electrical component of the electrochemical gradient with the use of elevated extracellular [K + ]. There was no evidence for the existence of voltage activated Ca 2+ channels. We conclude that trophoblast in vitro can express functional Ca 2+ channels which may be related to the requirement of the fetus for calcium in vivo .

Comparison of the intracellular pathways of immunoglobulin-G and low density lipoprotein in cultured human term trophoblast cells

Trophoblast cells were cultured on microporous membrane filters. After incubation at different times with gold-conjugated ligands, the cells were processed for electron microscopy. Gold particles indicating the presence of both IgG and LDL appeared in a time-dependent manner in coated pits and coated vesicles. LDL-gold appeared primarily within lysosomes whereas approximately 50% of the internalized IgG-gold appeared within vesicles (diameters ranging from 35 to 80 nm) near the basal regions of the cell. These vesicles may be the protective mechanism which prevents IgG breakdown during transcytosis across trophoblast cells, thus allowing transport of the intact molecule to the fetus.

Detection of Antitrophoblast Antibodies in the Sera of Patients With Anticardiolipin Antibodies and Fetal Loss

AbstractWomen with anticardiolipin antibodies (ACLA) are at increased risk for fetal loss. One potential explanation for this outcome is that sera from these individuals contain antibodies reactive with trophoblast cells, which are involved in the establishment of the uteroplacental vasculature and maintenance of placental blood fluidity. To examine this hypothesis, we compared the incidence of trophoblast-reactive antibodies in 27 patients with AC LA and a history of fetal loss with that in 29 normal pregnant women. Sera from 20 patients, but only one control, contained trophoblast-reactive antibodies (P < .001). These antibodies were not directed against major histocompatibility class I antigens, and reacted with both term and first-trimester trophoblast cells. In most cases, sera from which AC LA were adsorbed by cardiolipin-containing liposomes maintained reactivity against cells. In addition, patient Ig fractions immunoprecipitated an ~62-kD protein from the trophoblast cell surface, stimulated the release of arachi-donic acid and thromboxane A2 by trophoblasts, and inhibited the binding of prourokinase to trophoblast urokinase receptors. These observations show that sera from women with AC LA and a history of fetal loss contain antitrophoblast antibodies. These antibodies may be serologically distinct from ACLA, and may contribute to the pathogenesis of fetal demise.

Detection of antitrophoblast antibodies in the sera of patients with anticardiolipin antibodies and fetal loss

Women with anticardiolipin antibodies (ACLA) are at increased risk for fetal loss. One potential explanation for this outcome is that sera from these individuals contain antibodies reactive with trophoblast cells, which are involved in the establishment of the uteroplacental vasculature and maintenance of placental blood fluidity. To examine this hypothesis, we compared the incidence of trophoblast-reactive antibodies in 27 patients with ACLA and a history of fetal loss with that in 29 normal pregnant women. Sera from 20 patients, but only one control, contained trophoblast-reactive antibodies (P &lt; .001). These antibodies were not directed against major histocompatibility class I antigens, and reacted with both term and first-trimester trophoblast cells. In most cases, sera from which ACLA were adsorbed by cardiolipin- containing liposomes maintained reactivity against cells. In addition, patient Ig fractions immunoprecipitated an approximately 62-kD protein from the trophoblast cell surface, stimulated the release of arachidonic acid and thromboxane A2 by trophoblasts, and inhibited the binding of prourokinase to trophoblast urokinase receptors. These observations show that sera from women with ACLA and a history of fetal loss contain antitrophoblast antibodies. These antibodies may be serologically distinct from ACLA, and may contribute to the pathogenesis of fetal demise.

A role for extracellular calcium in the regulation of placental lactogen release by angiotensin-II and dopamine in human term trophoblastic cells.

We previously reported that angiotensin-II (AII) stimulated and dopamine (DA) inhibited the release of human placental lactogen (hPL) from trophoblastic cells. The mechanisms of action involved in these endocrine regulations are poorly known. In this study, we investigated the role of Ca2+ as a potential cellular mediator of the effects of AII and DA. Incubation of freshly isolated human term trophoblastic cells with DA led to a dose-dependent inhibition of 45Ca2+ influx, with a maximum of 55 +/- 5% and an EC50 of 10 +/- 3 mumol/L. This DA-inhibited Ca2+ influx was reversed by spiperone, a D2-dopamine receptor antagonist. Preincubation of cells with pertussis toxin completely blocked the inhibitory effect of DA on placental 45Ca2+ influx. Nifedipine (10(-5) mol/L), like DA, inhibited 45Ca2+ influx (41 +/- 3% inhibition). Moreover, nifedipine decreased hPL release (57 +/- 10%; EC50, 0.25 +/- 0.09 mumol/L). Coincubation of DA and nifedipine did not enhance the inhibitory effects of these agents on either 45Ca2+ influx or hPL release. The incubation of trophoblastic cells with [Sar1]AII, a potent agonist of AII, led to a dose-dependent stimulation of 45Ca2+ influx. The maximal stimulation was 221 +/- 37% of the control value, with an EC50 of 50 +/- 15 nmol/L. This stimulation was inhibited by coincubation with the AII antagonist [Sar1,Ala8]AII. [Sar1]AII-stimulated Ca2+ influx was blocked by preincubation with pertussis toxin. Bay K 8644 also stimulated 45Ca2+ influx (238 +/- 41% of the control). Moreover, Bay K 8644 stimulated hPL release. The maximal stimulation was 180 +/- 22% of the control value, with an EC50 of 0.40 +/- 0.30 mumol/L. Coincubation of Bay K 8644 and AII did not led to additional stimulation of either 45Ca2+ influx or hPL release. These results suggest that Ca2+ influx is one mechanism that mediates AII and DA regulation of hPL release in human term trophoblastic cells.

A role for extracellular calcium in the regulation of placental lactogen release by angiotensin-II and dopamine in human term trophoblastic cells

A role for extracellular calcium in the regulation of placental lactogen release by angiotensin-II and dopamine in human term trophoblastic cells

Transport of human IgG1 and IgG3 monoclonal anti-D across term trophoblast cells in culture

Transport of human IgG1 and IgG3 monoclonal anti-D across term trophoblast cells in culture

Binding of GP120 to human term trophoblast cells in culture

Binding of GP120 to human term trophoblast cells in culture

Characterization of cell types within a chorionic gonadotropin-secreting, mechanically dissociated human placental cell population in perifusion.

A cell perifusion system was established to examine human placental endocrine regulation by locally synthesized peptides. First-trimester and term trophoblast cells were mechanically dissociated. Cells were plated on microcarrier beads and cultured for 7-14 days. Cells on beads were loaded in chambers, perifused with culture media and effluent was assayed for chorionic gonadotropin (hCG). Mechanical dissociation of placental tissue produced cell preparations with 85-95% viability. Staining with Masson trichrome, cytokeratin and beta-hCG antibodies suggested that greater than 50% of the cells were trophoblast. Perifused trophoblast cells secreted hCG in a continuous non-pulsatile fashion, independent of exogenous hormonal stimuli. hCG secretion from first-trimester trophoblast cells remained stable in static culture for 14 days. GnRH perifusion (10(-8) M) for 15-120 s transiently increased hCG secretion from first-trimester trophoblast cells. Longer GnRH exposure stimulated greater hCG secretion. Each of 3 consecutive pulses of 8-bromo-cyclic adenosine-3',5'-monophosphate (cAMP, 10(-9) M) administered at 2-hour intervals stimulated transient hCG secretion from first-trimester and term placental cells. cAMP stimulated hCG secretion more potently from first-trimester than from term placental cells.

The modulation of placental lactogen release by opioids: a role for extracellular calcium

We previously reported that kappa opioids stimulated the release of human placental lactogen (hPL) from trophoblastic cells and that this effect was prevented by co-incubation with naloxone. We also reported that adenylate cyclase was not directly involved in this process. In order to understand the post-receptor events mediating hPL release by opioids in the human placenta, we studied the role of extracellular calcium. Human trophoblastic cells obtained by trypsin digestion were cultured for 48 h in Ham's F-10 medium supplemented with 10% fetal bovine serum (FBS), 200 U/ml penicillin, and 200 μg/ml streptomycin. 45Ca 2+ influx was then measured by filtration on glass-fiber filters. We observed a time- and dose-dependent stimulation of 45Ca 2+ influx by ethylketocyclazocine (EKC) with an EC 50 of 0.5 nM and a maximal stimulation of 196% over control. This effect was completely blocked by naloxone, a non-specific opioid antagonist, and by nor-binaltorphimine, a specific kappa antagonist. We also demonstrated that U-50, 488 (kappa agonist) had the same stimulatory effect as EKC (221 ± 25% of control). D-Ala 2,NMe-Phe 4,Gly-ol 5)-enkephalin (DAGO) (mu agonist) slightly stimulated Ca 2+ influx (128 ± 5% of control, p > 0.05) whereas D-Ser 2,Leu, Thr 6)-enkephaIin (DSLET) (delta agonist) had no effect. Pre-incubation of trophoblastic cells with pertussis toxin (PTX) did not affect the EKC-induced 45Ca 2+ influx, suggesting that this placental opiate effect is not coupled with PTX-sensitive G proteins. The role of extracellular calcium in the regulation of hPL release by kappa opiates was confirmed by experiments in which ionomycin (ionophore) and Bay K 8644 (calcium-channel agonist) mimicked the stimulatory effect of EKC on hPL release. Also, without extracellular Ca 2+, the basal (control) and EKC-stimulated hPL release were significantly lower than with 1.8 mM Ca 2+. Moreover, nifedipine (calcium-channel antagonist) inhibited both basal and EKC-stimulated hPL release. These results suggest that calcium influx is one mechanism mediating opioid-stimulated hPL release by human term trophoblastic cells.

Luteinizing hormone-releasing hormone (LHRH)- and (hydroxyproline9)LHRH-stimulated human chorionic gonadotropin secretion from perifused first trimester placental cells.

(Hydroxyproline9)LHRH [(Hyp9)LHRH] has been isolated from human, sheep, rodent, and frog hypothalamus. (Hyp9)LHRH is the major LHRH moiety in fetal rat hypothalamus. This study compared 1) synthetic LHRH-stimulated hCG secretion from term trophoblast vs. first trimester placental cells and 2) the ability and specificity with which synthetic LHRH and (Hyp9)LHRH could stimulate hCG secretion from 8- to 12-week gestation placenta. Physically dissociated cells from multiple placentae were pooled, plated on microcarrier beads, and perifused in 1.5-ml chambers (1.5 x 10(6) cells/chamber). Effluent fractions were analyzed for hCG. Each chamber was its own control. Basal hCG secretion did not depend upon exogenous LHRH stimulation. The amplitude of LHRH-stimulated hCG pulses was greater from first trimester placental than term trophoblast cells (mean +/- SEM, 6.99 +/- 1.47 vs. 0.50 +/- 0.05 mIU/ml perifusate; peak minus basal; n = 4 chambers; P less than 0.01). LHRH and (Hyp9)LHRH (10(-9) M) increased hCG secretion from first trimester placental cells (5.02 +/- 1.29 vs. 8.64 +/- 1.61 and 4.36 +/- 0.58 vs. 7.44 +/- 1.01 mIU/ml; n = 15 and 9, respectively; P less than 0.01). At the concentrations used, LHRH and (Hyp9)LHRH seemed to stimulate hCG secretion equipotently (P greater than 0.05). Simultaneous perifusion with an LHRH antagonist, (Nal-Glu)LHRH blocked the hCG secretory response to LHRH or (Hyp9)LHRH (equimolar 10(-9) M concentrations; n = 5; P less than 0.05). (Nal-Glu)LHRH alone (10(-9) M) did not affect hCG secretion (n = 5; P greater than 0.05). The results suggested that first trimester placental cells are more responsive to LHRH than are term trophoblast cells. (Hyp9)LHRH is a potential physiological secretagogue of hCG.

Localization of transforming growth factor-beta at the human fetal-maternal interface: role in trophoblast growth and differentiation.

We examined the localization of transforming growth factor (TGF)-beta in first-trimester and term human decidua and chorionic villi and explored the role of this factor on the proliferation and differentiation of cultured trophoblast cells. Two antibodies, 1D11.16.8, a mouse monoclonal neutralizing antibody capable of recognizing both TGF-beta 1 and TGF-beta 2 and CL-B1/29, a rabbit polyclonal antibody capable of recognizing TGF-beta 2, were used to immunolocalize TGF-beta in fixed, paraffin-embedded, or fixed, frozen sections of placenta and decidua, providing similar results. Intense labeling was observed in the extracellular matrix (ECM) of the first-trimester decidua and cytoplasm of term decidual cells. Syncytiotrophoblast cell cytoplasm as well as the ECM in the core of the chorionic villi of both first-trimester and term placentas exhibited a moderate degree of labeling. Strong cytoplasmic labeling was observed in the cytotrophoblastic shell of the term placenta. To examine the role of TGF-beta on trophoblast proliferation and differentiation, early passage cultures of first-trimester and primary cultures of term trophoblast cells were established and characterized on the basis of numerous immunocytochemical and functional markers. These cells expressed cytokeratin, placental alkaline phosphatase, urokinase-type plasminogen activator, and pregnancy-specific beta glycoprotein, but not factor VIII or 63D3; they also produced hCG and collagenase type IV. Exposure of first-trimester trophoblast cultures to TGF-beta 1 significantly inhibited proliferation in a dose-dependent manner. An antiproliferative effect was also noted in the presence of TGF-beta 2. These effects were abrogated in the presence of the neutralizing anti-TGF-beta antibody (1D11.16.8) in a concentration-dependent manner. In a 3-day culture, exogenous TGF-beta 1 stimulated formation of multinucleated cells by the first trimester as well as term trophoblast cells. Addition of neutralizing anti-TGF-beta antibody to first-trimester trophoblast cells stimulated proliferation beyond control levels in a 24-h culture and reduced formation of multinucleated cells in a 3-day culture, indicating the presence of endogenous TGF-beta activity. These results indicate that TGF-beta produced at the human fetal-maternal interface plays a major regulatory role in the proliferation and differentiation of the trophoblast.

Insulin Stimulates Synthesis and Release of Human Chorionic Gonadotropin by Choriocarcinoma Cell Lines

Recent studies have shown that insulin regulates placental lactogen, progesterone, and estrogen production from human trophoblast cells. This study was performed to examine whether insulin also regulates the production of hCG by this type of cell. After 24-36 h of preincubation, JEG-3 and JAR cells (2-3 x 10(5) cells/ml.well) or human term trophoblast cells (1 x 10(6) cells/ml.well) were exposed to the test hormone in serum-free Dulbecco's Modified Eagle's Medium for 24-96 h. Secretion of hCG from JEG-3 cells was stimulated by human insulin, human proinsulin, or porcine insulin in a dose-dependent manner, with lowest effective doses of 6.7, 96, and 53 mg/L, respectively. Time-course studies showed that hCG secretion peaked at 72-96 h with insulin exposure; in contrast, no decernable peak was seen without insulin in serum-free media. Exposure of JEG-3 cells for 24 h to 209 mg/liter insulin stimulated hCG synthesis, with 40 +/- 3% more immunoreactive intracellular hCG (P less than 0.05). Cells grown in the presence of insulin and [35S]methionine had 47 +/- 21% more labeled intracellular hCG and 56 +/- 13% more immunoprecipitable [35S]methionine-hCG secreted into the medium than the control cultures (P less than 0.05). During this time period, human placental lactogen release and total trichloroacetice acid-precipitable [35S]methionine protein were not increased. The insulin-induced stimulation of hCG synthesis was inhibited by cycloheximide. Additionally, insulin did not significantly affect total intracellular protein during 24-96 h of incubation. Insulin also increased hCG release from JAR cells, but not from human term trophoblast cells. A mouse monoclonal antibody to the IGF-I receptor inhibited the stimulation of insulin in JEG-3 cells. We conclude that insulin stimulates the synthesis and secretion of hCG from JEG-3 cells and JAR cells, and that hCG regulation in choriocarcinoma cells differs from that in primary human placental trophoblast cells. The effect of insulin on JEG-3 cells may be mediated in part through the insulin-like growth factor-I receptor.

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.

Vectorial transcytosis of immunoglobulin G by human term trophoblast cells in culture

Trophoblast cells were grown on filters that allow access to apical and basal surfaces of cells. Using this experimental system, IgG transport was shown to be specific and to occur primarily in the apical to basal direction. This transport was time- and temperature-dependent, with approximately 10% of added IgG appearing on the basal side within a 60-min incubation at 37 °C. Other substances such as heparin were transported only minimally, whereas horseradish peroxidase was transported to the same degree in both directions. Greater than 90% of the transported IgG was precipitable by trichloroacetic acid and 81% was capable of binding to protein G. Such a rapid transport of large amounts of IgG in trophoblast cells is consistent with a receptor-mediated process of transcytosis.

Identification and characterization of receptors for granulocyte colony-stimulating factor on human placenta and trophoblastic cells.

Since radioiodination of human granulocyte colony-stimulating factor (G-CSF) is difficult, we synthesized a mutein of human G-CSF that retains full biological activity and receptor-binding capacity for at least 2 weeks after radioiodination. Receptors for human G-CSF were characterized in the plasma membrane fraction from the human term placenta (human placental membranes) and trophoblastic cells by using the 125I-labeled mutein of human G-CSF (KW-2228). The specific binding of 125I-labeled KW-2228 to placental membranes was pH-dependent, with maximal specific binding at pH 7.8; it increased linearly with protein to 3.7 mg of protein per ml and was both time- and temperature-dependent, with maximal binding at 4 degrees C after a 24-hr incubation. When we examined the ability of hematopoietic growth factors to inhibit 125I-labeled KW-2228 binding, we found that KW-2228 and intact human G-CSF inhibited 125I-labeled KW-2228 binding, whereas erythropoietin or granulocyte-macrophage colony-stimulating factor did not. Scatchard analysis revealed a single receptor type with a Bmax of 210 fmol/mg of protein and a Kd of 480 pM. The human G-CSF receptors on human placental membranes were shown to consist of two molecular species of 150 kDa and 120 kDa that could be specifically cross-linked to 125I-labeled KW-2228. Human trophoblastic cells, T3M-3, also possessed a single receptor for G-CSF with a Bmax of 533 receptors per cell and a Kd of 390 pM. Thus we have identified the receptor for human G-CSF on human placental membranes and trophoblastic cells, and the presence of this receptor in these membranes suggests that human G-CSF plays some role in the feto-placental unit during human development.