Human Syncytiotrophoblast Cells Research Articles

Upregulation and release of soluble fms-like tyrosine kinase receptor 1 mediated by complement activation in human syncytiotrophoblast cells.

Antiangiogenic molecule soluble fms-like tyrosine kinase receptor 1 (sFLT1) released from trophoblast cells is associated with pregnancy-specific hypertensive disorder pre-eclampsia. Cause of elevated sFLT1 in pre-eclampsia patients is not well understood. Despite evidence of excess systemic and placental complement activation in pre-eclampsia patients, its role in pathophysiology is not clear. If the complement activation plays a role in upregulation and secretion of sFLT1 is not known. Human trophoblast cells were isolated from term placentas and allowed to syncytialize. Complement was activated in vitro at sublethal levels on syncytiotrophoblast cells. Effect of complement activation on expression and release of sFLT1 was assessed by comparing its levels in these cells with and without complement activation. Sublethal level of complement activation on syncytialized human trophoblast cells induced upregulation of sFLT1 mRNA and protein. Complement also induced secretion of sFLT1 in a manner depending on degree of activation. Anaphylatoxins C3a induced upregulation but not the release of sFLT1. Release of terminal membrane attack complex (MAC) was associated with sFLT1 secretion. Complement activation plays a major role in both the expression and secretion of sFLT1 from syncytial trophoblast cells. The terminal MAC complex is involved in its secretion. Increased levels of sFLT1 in pre-eclampsia patients may be due to complement-induced upregulation and secretion.

Membrane potential regulates mitochondrial ATP-diphosphohydrolase activity but is not involved in progesterone biosynthesis in human syncytiotrophoblast cells

ATP-diphosphohydrolase is associated with human syncytiotrophoblast mitochondria. The activity of this enzyme is implicated in the stimulation of oxygen uptake and progesterone synthesis. We reported previously that: (1) the detergent-solubilized ATP-diphosphohydrolase has low substrate specificity, and (2) purine and pyrimidine nucleosides, tri- or diphosphates, are fully dephosphorylated in the presence of calcium or magnesium (Flores-Herrera 1999, 2002). In this study we show that ATP-diphosphohydrolase hydrolyzes first the nucleoside triphosphate to nucleoside diphosphate, and then to nucleotide monophosphate, in the case of all tested nucleotides. The activation energies (Ea) for ATP, GTP, UTP, and CTP were 6.06, 4.10, 6.25, and 5.26kcal/mol, respectively; for ADP, GDP, UDP, and CDP, they were 4.67, 5.42, 5.43, and 6.22kcal/mol, respectively. The corresponding Arrhenius plots indicated a single rate-limiting step for each hydrolyzed nucleoside, either tri- or diphosphate. In intact mitochondria, the ADP produced by ATP-diphosphohydrolase activity depolarized the membrane potential (ΔΨm) and stimulated oxygen uptake. Mitochondrial respiration showed the state-3/state-4 transition when ATP was added, suggesting that ATP-diphosphohydrolase and the F1F0-ATP synthase work in conjunction to avoid a futile cycle. Substrate selectivity of the ATP-diphosphohydrolase was modified by ΔΨm (i.e. ATP was preferred over GTP when the inner mitochondrial membrane was energized). In contrast, dissipation of ΔΨm by CCCP produced a loss of substrate specificity and so the ATP-diphosphohydrolase was able to hydrolyze ATP and GTP at the same rate. In intact mitochondria, ATP hydrolysis increased progesterone synthesis as compared with GTP. Although dissipation of ΔΨm by CCCP decreased progesterone synthesis, NADPH production restores steroidogenesis. Overall, our results suggest a novel physiological role for ΔΨm in steroidogenesis.

Serotonin Production by Human and Mouse Trophoblast: Involvement in Placental Development and Function

Serotonin Production by Human and Mouse Trophoblast: Involvement in Placental Development and Function

Selective regulation of peroxisome proliferator-activated receptors on fatty acid binding protein-4 in human syncytiotrophoblast cells

To observe the selective regulation of peroxisome proliferator-activated receptors (PPAR) on fatty acid binding protein-4 (FABP4) in human syncytiotrophoblasts. Cultivate normal human syncytiotrophoblast cells, and put in the specific antagonists and agonists of PPAR each subtypes receptors, then observe the different expression of FABP4 mRNA and protein. Pretreated the human syncytiotrophoblast cells with the agonists (GW7647, GW0742) and antagonists (GW6471, GSK0660) of PPARα and PPARβ receptors, the expression of the FABP4 was not significantly change (P > 0.05). However pretreated with PPAR γ agonists (rosiglitazone, 1×10(-9), 1×10(-8), 1×10(-7) and 1×10(-6) mol/L), the expression of FABP4 mRNA and protein could be dose dependent-promoted significantly (mRNA: 1.27 ± 0.12, 1.45 ± 0.14, 1.57 ± 0.14, 1.72 ± 0.12, protein:1.10 ± 0.08, 1.37 ± 0.09, 1.60 ± 0.13, 1.79 ± 0.14; P < 0.05), furthermore, the promotion can be dose dependent-reversed by specific antagonists GW9662 (mRNA:0.92 ± 0.06, 0.77 ± 0.06, 0.64 ± 0.05, 0.55 ± 0.05, protein:0.91 ± 0.03, 0.78 ± 0.06, 0.70 ± 0.07, 0.55 ± 0.06; P < 0.05). In normal human syncytiotrophoblast cells, FABP4 is a target factor of PPARγ. PPARγ regulated the expression of FABP4 mRNA and protein selectively. And the regulation will not be influenced by the other two PPAR subtypes.

Heterogeneous Pathways of Maternal-fetal Transmission of Human Viruses (Review)

Several viruses can pass the maternal-fetal barrier, and cause diseases of the fetus or the newborn. Recently, however, it became obvious, that viruses may invade fetal cells and organs through different routes without acute consequences. Spermatozoa, seminal fluid and lymphocytes in the sperm may transfer viruses into the human zygotes. Viruses were shown to be integrated into human chromosomes and transferred into fetal tissues. The regular maternal-fetal transport of maternal cells has also been discovered. This transport might implicate that lymphotropic viruses can be released into the fetal organs following cellular invasion. It has been shown that many viruses may replicate in human trophoblasts and syncytiotrophoblast cells thus passing the barrier of the maternal-fetal interface. The transport of viral immunocomplexes had also been suggested, and the possibility has been put forward that even anti-idiotypes mimicking viral epitopes might be transferred by natural mechanisms into the fetal plasma, in spite of the selective mechanisms of apical to basolateral transcytosis in syncytiotrophoblast and basolateral to apical transcytosis in fetal capillary endothelium. The mechanisms of maternal-fetal transcytosis seem to be different of those observed in differentiated cells and tissue cultures. Membrane fusion and lipid rafts of high cholesterol content are probably the main requirements of fetal transcytosis. The long term presence of viruses in fetal tissues and their interactions with the fetal immune system might result in post partum consequences as far as increased risk of the development of malignancies and chronic pathologic conditions are discussed.

Antibody-dependent enhancement of HIV-1 infection in human term syncytiotrophoblast cells cultured in vitro.

We examined if Fc receptor-mediated antibody-dependent enhancement (FcR-ADE) or complement-mediated antibody-dependent enhancement (C'-ADE) of virus infection can contribute to increasing replication of HIV-1 in human syncytiotrophoblast (ST) cells. Here we report that both FcR-ADE and C'-ADE may result in enhanced virus release from HIV-1-infected ST cells. We show that FcR-ADE of HIV-1 infection in ST cells is mediated by FcRIII and other FcR(s) belonging to undetermined Fc classes and does not require CD4 receptors, whereas C'-ADE uses both CD4 and CR2-like receptors. FcR-ADE seems to be more efficient in enhancing HIV-1 replication than C'-ADE. While FcR-ADE leads to increased internalization of HIV-1, C'-ADE does not result in enhanced endocytosis of the virus. In addition, antibodies mediating FcR-ADE are reactive with the gp120 viral envelope antigen, whereas antibodies involved in C'-ADE react with the viral transmembrane glycoprotein gp41. Data suggest that both FcR-ADE and C'-ADE may contribute to the spread of HIV-1 from mother to the fetus.

Cytomegalovirus Infection of Human Syncytiotrophoblast Cells Strongly Interferes with Expression of Genes Involved in Placental Differentiation and Tissue Integrity

The principle route of acquisition of cytomegalovirus (CMV) for the fetus is believed to be via the placenta. We subjected purified cytotrophoblast cells obtained from full-term placentas to CMV infection and examined placental gene expression using microarray analyses. Cytotrophoblast cells purified from term placentas differentiated in vitro into a multinucleated syncytium that could be productively infected with CMV, with peak virus titers of approximately 10 plaque-forming units (PFU)/mL identified in supernatants at late time points postinoculation. Infected syncytiotrophoblast cells expressed CMV-specific transcripts and proteins, as demonstrated by Northern blot and immunofluorescence assays. Microarray analyses revealed that CMV infection strongly and reproducibly altered trophoblast gene expression, elevating expression of mitotic cell cycle genes, and repressing expression of genes associated with trophoblast differentiation, particularly those associated with formation and stabilization of the extracellular matrix. We conclude that purified, differentiated syncytiotrophoblasts are permissive for CMV replication. Infection of these cells induces significant perturbations in trophoblast transcription. An improved understanding of the molecular events that occur during CMV infection of trophoblasts could provide insights into interventions that might prevent or minimize congenital transmission.

Regulation of Vitamin D hydroxylases gene expression by 1,25-dihydroxyvitamin D 3 and cyclic AMP in cultured human syncytiotrophoblasts

Human placenta synthesizes and metabolizes 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3/calcitriol] through the activity of 25-hydroxyvitamin D 3-1α-hydroxylase (CYP27B1) and 1,25(OH) 2D 3-24-hydroxylase (CYP24A1), the two key enzymes for Vitamin D metabolism. In this study, calcitriol rapidly generated intracellular cAMP accumulation in cultured human syncytiotrophoblast cells, which in turn enhanced hCG secretion, a marker of trophoblast endocrine activity. The effects of 1,25(OH) 2D 3 upon the expression of CYP27B1 and CYP24A1 were also investigated. 1,25(OH) 2D 3 and activators of the PKA signaling system decreased the expression of CYP27B1, whereas increased CYP24A1 gene transcription. The use of a selective inhibitor of PKA (H-89) prevented the effects of calcitriol on CYP27B1 gene and hCG secretion, but not on CYP24A1 transcription. Addition of ZK 159222, a Vitamin D receptor (VDR) antagonist, blocked the calcitriol-mediated upregulation of 24-hydroxylase gene expression but did not affect calcitriol-induced downregulation of CYP27B1 gene or hCG stimulation. In addition, our study also demonstrated a role of calcitonin on Vitamin D hydroxylases gene regulation in placenta. The overall data suggest that calcitriol downregulates CYP27B1 expression via a cAMP-dependent signaling pathway, whereas upregulates 24-hydroxylase gene expression through a VDR-dependent mechanism.

LPS induces secretion of chemokines by human syncytiotrophoblast cells in a MAPK-dependent manner

The maintenance of pregnancy depends on the nature and magnitude of the immune responses induced within the placenta. An elevated proinflammatory response in the intervillous space (IVS) is associated with adverse pregnancy outcomes. It is becoming more apparent that the syncytiotrophoblast (ST) cells, which are in direct contact with maternal blood, are capable of contributing to the local immune environment in response to maternal hematogenous infections or exposure to proinflammatory stimuli. In this study, we investigated mechanisms by which ST might recruit maternal immune effectors to the IVS in response to bacterial infections. To assess this, primary trophoblasts were isolated from fresh term placentas and stimulated with lipopolysaccharide (LPS). LPS induced time-dependent expression and secretion of IL-8, macrophage inflammatory protein (MIP)-1α and MIP-1β from ST cells and an upregulation of ICAM-1. The stimulation also resulted in the activation of ERK1/2 mitogen-activated protein kinase (MAPK) but not p38 or JNK1/2. Inhibition of ERK1/2 lead to a reduction in the secretion of MIP-1β and IL-8 suggesting that their production is at least partly dependent on ERK1/2 activation. Results from this study reveal a potential mechanism by which differentiated ST cells modulate the local maternal immune responses during an intrauterine bacterial infection. Such responses could contribute to the clearance of the infection but also pathological features observed in intrauterine infections of the placenta.

Proteomic Analysis of Apical Microvillous Membranes of Syncytiotrophoblast Cells Reveals A High Degree of Similarity with Lipid Rafts

Brush borders (microvilli) are cell membrane specialized structures that function mainly as high-throughput absortive/secretory areas. It has been well-established that brush borders are particularly rich in membrane lipids characteristic to lipid rafts. Here, we report 57 proteins identified from microvillous membranes (MVM) isolated from human syncytiotrophoblast cells using an experimental method that avoids the use of nonionic detergents. About 60% of the proteins reported here have been described previously as lipid-raft specific. Well-known lipid raft-markers such as Annexin A2 and alkaline phosphatase were identified. Cytoskeleton structural constituents and proteins related with the control and modulation of the cytoskeletal architecture as well as the regulation of the interaction of cytoskeletal constituents with the cell membrane and particularly with lipid raft domains were found (Ezrin, IQGAP1 and 2, EBP50). Other proteins identified include signal transduction molecules, such as Ras-related protein Rab-1B and Rab-7, and ADP-ribosylation factor 1. Several proteins harbor putative post-translational modifications that favor its localization in the lipid-raft environment, such as GPI (alkaline phosphatase and 5'-nucleotidase) and myristoylation (BASP1 and MARCKS). On the whole, this extensive description demonstrates from the protein composition point of view that brush border membranes are indeed highly enriched in lipid raft microdomains.

Intracellular pH homeostasis in cultured human placental syncytiotrophoblast cells: recovery from acidification

Resting or basal intracellular pH (pHi) measured in cultured human syncytiotrophoblast cells was 7.26+/-0.04 (without HCO3-) or 7.24+/-0.03 (with HCO3-). Ion substitution and inhibitor experiments were performed to determine whether common H+-transporting species were operating to maintain basal pHi. Removal of extracellular Na+ or Cl- or addition of amiloride or dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H2DIDS) had no effect. Acidification with the K+/H+ exchanger nigericin reduced pHi to 6.25+/-0.15 (without HCO3-) or 6.53+/-0.10 (with HCO3-). In the presence of extracellular Na+, recovery to basal pHi was prompt and occurred at similar rates in the absence and presence of HCO3-. Ion substitution and inhibition experiments were also used to identify the species mediating the return to basal pHi after acidification. Recovery was inhibited by removal of Na+ or addition of amiloride, whereas removal of Cl- and addition of H2DIDS were ineffective. Addition of the Na+/H+ exchanger monensin to cells that had returned to basal pHi elicited a further increase in pHi to 7.48+/-0.07. Analysis of recovery data showed that there was a progressive decrease in DeltapH per minute as pHi approached the basal level, despite the continued presence of a driving force for H+ extrusion. These data show that in cultured syncytial cells, in the absence of perturbation, basal pHi is preserved despite the absence of active, mediated pH maintenance. They also demonstrate that an Na+/H+ antiporter acts to defend the cells against acidification and that it is the sole transporter necessary for recovery from an intracellular acid load.

Regulation of 25-hydroxyvitamin D 3 1α-hydroxylase, 1,25-dihydroxyvitamin D 3 24-hydroxylase and vitamin D receptor gene expression by 8-bromo cyclic AMP in cultured human syncytiotrophoblast cells

In vitro differentiation of human trophoblast cells is a dynamic process accompanied by increasing intracellular levels of cyclic AMP (cAMP). Signaling through cAMP in this tissue is central to hormone expression and cytodifferentiation. In the present study, we analyzed transcriptional regulation of key enzymes involved in vitamin D endocrine system during in vitro syncytiotrophoblast formation. Total RNA was isolated from human trophoblast cells and subjected to reverse transcription, polymerase chain reaction and Southern blot analysis using specific primers and radiolabeled probes. During syncytium formation 25-hydroxyvitamin D 3 1α-hydroxylase (CYP27B1) was decreased while vitamin D receptor ( VDR) gene remained unaffected. No 1,25-dihydroxyvitamin D 3 24-hydroxylase (CYP24) transcription signal was detected. Nevertheless, incubations in the presence of 8-bromo cAMP (1.5 mM) resulted in CYP24 induction and CYP27B1 inhibition, respectively. The overall data showed that cultured human syncytiotrophoblasts express key enzymes involved in vitamin D metabolism, as well as VDR. The results support previous findings that human placenta is a source of 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2D 3), which synthesis is regulated by common growth and developmental factors. The data also suggest a tissue-dependant differential regulation of CYP27B1 gene expression by cAMP.

Placental macrophage contact induces complete replicative cycle of human immunodeficiency virus in latently infected syncytiotrophoblast cells: role of interleukine‐6 and tumor necrosis factor‐β

The phenotypic mixing between human immunodeficiency virus type 1 (HIV‐1) and vesicular stomatitis virus (VSV) has been exploited to assay the susceptibility of human term syncytiotrophoblast cells to penetration by various strains of HIV‐1. VSV (HIV‐1IIIB) and VSV (HIV‐1Ba‐L) pseudotypes were found to enter syncytiotrophoblasts. Infection of syncytiotrophoblasts was mediated by envelope glycoproteins of IIIB and Ba‐L strains of HIV‐1. Although certain strains of HIV‐1 could enter syncytiotrophoblasts, the cells did not exhibit permissiveness for HIV‐1. The next studies tested the possibility that placental macrophages might induce replication of HIV‐1 carried in syncytiotrophoblast cells and that infected syncytiotrophoblasts would be capable of transmitting virus into neighbouring macrophages. For this purpose, the macrophage‐tropic Ba‐L strain of HIV‐1 was used. Interactions between syncytiotrophoblasts and macrophages activated HIV‐1 from latency in syncytiotrophoblast cells, which delivered HIV‐1 to cocultured macrophages. The stimulatory effect of coculture on HIV‐1 gene expression was mediated by marked tumor necrosis factor‐α and interleukin‐6 release from macrophages, an effect caused by contact between the different placental cells. Results suggest an interactive role for the syncytiotrophoblast layer and placental macrophages in the dissemination of HIV‐1 among placental tissue.

Expression and activity of 25-hydroxyvitamin D-1 alpha-hydroxylase are restricted in cultures of human syncytiotrophoblast cells from preeclamptic pregnancies.

The human placenta synthesizes 1,25-dihydroxyvitamin D(3) and expresses the vitamin D receptor. Because preeclampsia (PE) is associated with low circulating levels of maternal 1,25-dihydroxyvitamin D(3) and IGF-I, it is possible that alterations in calcium metabolism seen in PE could occur at the level of the fetoplacental unit. In this study, the patterns of gene expression and enzyme activity of 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-hydroxylase) and the abundance of IGF-I mRNA in placentas from normal (NT) and PE-complicated pregnancies were investigated. Cultured syncytiotrophoblast cells from preeclamptic placentas had only one tenth the activity of 1 alpha-hydroxylase and did not respond to IGF-I, when compared with NT cultures. Similarly, the levels of 1 alpha-hydroxylase mRNA in syncytiotrophoblast cells from PE placentas under basal and IGF-I-stimulated conditions were significantly reduced. In contrast, IGF-I mRNA levels were found to increase during the differentiation process, with no differences between NT and PE cultures. These results support the role of placenta as a contributor to the abnormalities observed in calcium metabolism in PE.

Expression and Activity of 25-Hydroxyvitamin D-1 -Hydroxylase Are Restricted in Cultures of Human Syncytiotrophoblast Cells from Preeclamptic Pregnancies

Expression and Activity of 25-Hydroxyvitamin D-1 -Hydroxylase Are Restricted in Cultures of Human Syncytiotrophoblast Cells from Preeclamptic Pregnancies

Polar Expression and Phosphorylation of Human Leptin Receptor Isoforms in Paired, Syncytial, Microvillous and Basal Membranes from Human Term Placenta

The hormone leptin (OB) and its receptor (OB-R) are key homeostatic regulators of mammalian body weight. Two predominant isoforms of OB-R are expressed by alternative splicing: the long form, OB-RL, with full signalling capacity is highly expressed in the hypothalamus and the short, signalling-defective form, OB-Rs, is ubiquitously expressed.In a previous study we detected expression of OB-RL and OB-Rs in human syncytiotrophoblast cells using in situ hybridization and immunohistochemistry (Bodner et al., 1999).The aim of this study was to investigate leptin receptor isoform expression and phosphorylation in paired, syncytial, microvillous and basal membranes from human term placenta by Western blot analysis. Both the OB-RL and the OB-Rs isoforms were detected in the syncytial membrane preparations. The OB-RL isoform was observed exclusively in microvillous membranes, whereas the OB-Rs isoform was found in both microvillous and basal membrane preparations. No significant differences were observed between syncytial membranes from normal and type 1 diabetic pregnancies. To test the phosphorylation capacity of the OB-R isoforms, microvillous and basal membrane vesicles loaded with ATP were stimulated with leptin and the phosphorylation status of the OB-R at the tyrosine 985 (Y985) was determined. A single band at the molecular weight corresponding to the molecular weight of the OB-RL isoform was detected exclusively in the ATP-loaded microvillous vesicles.We conclude that the long form OB-RL is expressed exclusively in the microvillous membrane of the syncytiotrophoblast and is capable of being phosphorylated, suggesting that it has signal transduction capacity.

Human herpesvirus 6 variant a infects human term syncytiotrophoblasts in vitro and induces replication of human immunodeficiency virus type 1 in dually infected cells.

Human herpesvirus 6 (HHV-6) and human immunodeficiency virus type 1 (HIV-1) may interact during transplacental transmission of HIV-1. The placental syncytiotrophoblast layer serves as the first line of defense of the fetus against viruses. Patterns of replication of HHV-6 variant A (HHV-6A) and HIV-1 were analyzed in singly and dually infected human term syncytiotrophoblast cells cultured in vitro. For this purpose, the GS strain of HHV-6A and the Ba-L and IIIB strains of HIV-1 were used. HHV-6A replication was restricted at the level of early gene products in singly infected syncytiotrophoblasts, whereas no viral protein expression was found in cells infected with HIV-1 alone. Coinfection of syncytiotrophoblast cells with HHV-6A and HIV-1 resulted in production of infectious HIV-1. In contrast, no enhancement of HHV-6A expression was observed in cell cultures infected with both viruses. Uninfected syncytiotrophoblast cells were found to express CXCR4 and CCR3 but not CD4 or CCR5 receptors. Infection of syncytiotrophoblasts with HHV-6A did not induce CD4 expression and had no influence on chemokine receptor expression. Activation of HIV-1 from latency in coinfected cells was mediated by the immediate-early (IE)-A and IE-B gene products of HHV-6A. Open reading frames U86 and U89 of the IE-A region were able to activate HIV-1 replication in a synergistic manner. The data suggest that in vivo double infection of syncytiotrophoblast cells with HHV-6A and HIV-1 could contribute to the transplacental transmission of HIV-1 but not HHV-6A.

The expression of endothelial nitric oxide synthase (eNOS) mRNA in human trophoblast and its regulation by leukemia inhibitory factor (LIF) in vitro.

Objective: Nitric Oxide (NO) as a signaling molecule with pleiotropic biologic activities, including vasodilatation, neurotransmission, immunomodulation, antimicrobial and antitumor activities. NO is produced as a free radical during the enzymatic conversion of L-arginine to L-citruline by a family of nitric oxide synthases (NOS). The objective of this study was to examine the expression of the endothelial NOS (eNOS) gene in human cytotrophoblast and syncytiotrophoblast cells and possible regulation of these gene by cytokine LIF.

Pseudotypes of vesicular stomatitis virus‐bearing envelope antigens of certain HIV‐1 strains permissively infect human syncytiotrophoblasts cultured in vitro: Implications for in vivo infection of syncytiotrophoblasts by cell‐free HIV‐1

Intrauterine infection of the fetus is clearly an important mode of vertical transmission of human immunodeficiency virus type 1 (HIV-1). The syncytiotrophoblast layer of the human placenta must be traversed by HIV-1 in order to reach underlying cells and fetal capillaries. Although HIV-1 has been detected in the syncytiotrophoblast layer in situ, there is conflicting evidence regarding infection of syncytiotrophoblast cells with cell-free virus. The phenotypic mixing between HIV-1 and vesicular stomatitis virus (VSV) has been exploited to assay the susceptibility of human term syncytiotrophoblast cells to penetration by various strains of HIV-1. VSV(HIV-1(IIIB)) and VSV(HIV-1(Ba-L)) pseudotypes were found to enter syncytiotrophoblast cells. In contrast, VSV pseudotyped with envelope glycoproteins of RF, MN, or Ada-M strains of HIV-1 did not infect syncytiotrophoblasts. Plating efficiency of VSV(HIV-1(IIIB)) and VSV(HIV-1(Ba-L)) was 10-fold lower on syncytiotrophoblasts than on T-cells and macrophages, respectively. Incubation of VSV(HIV-1(IIIB)) and VSV(HIV-1(Ba-L)) viruses with appropriate HIV-1 neutralizing sera before infection strongly inhibited entry of pseudotyped VSV into syncytiotrophoblast cells. These findings demonstrated that infection of syncytiotrophoblasts with VSV(HIV-1) pseudotypes was mediated by Env from IIIB and Ba-L strains of HIV-1. Monoclonal antibodies (MAb) to CD4, CXCR4, CCR5, and CCR3 were tested for their ability to block VSV(HIV-1) infection of syncytiotrophoblast cells. Neither the anti-CD4 nor the anti-CXCR4, anti-CCR5, and anti-CCR3 MAb had any inhibitory effect on infection of syncytiotrophoblast cells with VSV(HIV-1) pseudotypes. Results from this study suggest that cell-free HIV-1 can enter syncytiotrophoblasts and the susceptibility of these cells to penetration by the virus is strain dependent. Pseudotype infection merely demonstrates that the first steps in HIV-1 replication are possible in syncytiotrophoblast cells.

Automated quantitation of cell-mediated HIV type 1 infection of human syncytiotrophoblast cells by fluorescence in situ hybridization and laser scanning cytometry.

Infection of human placental syncytiotrophoblast cells with HIV requires direct contact with infected leukocytes. In vitro investigations into mechanisms regulating placental HIV transmission and into the development of therapeutic interventions have been hampered by difficulties inherent in quantitating HIV levels in cocultures of infected lymphocytes and adherent multinucleated syncytiotrophoblast cells. Here, we have used fluorescence in situ hybridization (FISH) for the direct detection of HIV-1 RNA within syncytiotrophoblast cells combined with laser scanning cytometry (LSC) to quantitate HIV levels exclusively in the syncytiotrophoblast cells. HIV-1-infected lymphocytic MOLT-4 cells were cocultured with primary human syncytiotrophoblast cells. Lymphocytic cells were identified with an anti-vimentin antibody and Cy5. HIV RNA was localized by in situ hybridization, using a digoxigenin-labeled riboprobe detected by Oregon Green, and nuclei were stained with 7-aminoactinomycin D. The three-color cocultures were analyzed by LSC to remove unwanted cell populations and quantitate HIV expression levels. The total HIV RNA level (green fluorescence integral) in each colony was normalized for cell size by dividing by the total DNA content (red fluorescence integral). The nuclear-normalized fluorescence integral was 2.3 times higher in infected cocultures than in uninfected cultures. When cocultures were incubated with 10 microM AZT, the green/red fluorescence integral value was significantly lower than that of cocultures incubated in the absence of AZT, corresponding to a 78% reduction in fluorescence. Laser scanning cytometry can be used to quantitate cell-mediated HIV infection in syncytiotrophoblast cells and should allow drug assessment studies and studies aimed at understanding the mechanism of virus entry into trophoblast cells to be carried out.