The characterization of fibrocyte-like cells: a novel fibroblastic cell of the placenta.

The placenta is a highly vascularized organ thus angiogenesis is a key process in placental development. The contribution that different cells in the villous stroma play in placental angiogenesis is largely unknown. In this study we identified a novel stromal cell type in sections of term placenta which is morphologically fibroblastic and expressing the fibroblast marker TE-7 but also positive for the monocytic markers CD115 and CD14 and designated these cells as fibrocyte-like cells. Populations of fibrocyte-like cells from the placenta were isolated by two methods: culture of adherence-selected placental cells and, for higher purity, by CD45 fluorescence activated cell sorting (FACS). Fibrocyte-like cell conditioned medium increased endothelial tubule-like structure formation 2-fold versus control medium. Both pro-angiogenic growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) and the anti-angiogenic factor soluble-Flt were found in the conditioned medium. Neutralizing antibodies against VEGF and b-FGF reduced endothelial cell tubule-like structures to control levels. These data suggests that fibrocyte-like cells, a previously unidentified cell of the villous stroma, may play an important role in the regulation of placental angiogenesis.

The contribution of apoptosis-inducing factor (AIF) to villous trophoblast differentiation.

Apoptosis is postulated to be a delayed but important part of the differentiation of placental villous cytotrophoblasts (CT) into functional syncytiotrophoblast (ST). This hypothesis is based on the observation that the externalization of phosphatidylserine and the activation of caspase 8 are required for trophoblast differentiation. In contradiction to this hypothesis we have previously found that differentiation occurs in the presence of both broad spectrum and caspase 8 specific inhibitors. Apoptosis-inducing factor (AIF) is a mitochondria-associated protein which is known to translocate to the nucleus and induce caspase-independent nuclear condensation, phosphatidylserine externalization and cell death. Thus AIF nuclear translocation may result in the apoptotic-like features associated with trophoblast differentiation and may be an obligatory event for differentiation to proceed. AIF translocation was assessed in isolated primary trophoblasts by optical section microscopy of antibody stained cells. We found AIF to be strongly expressed in the villous trophoblast and that small amounts of AIF were localized to the nucleus of the cells. Significantly, inhibitors of AIF translocation (calpeptin and zFA-fmk) blocked translocation but not differentiation of the cells. We conclude that AIF translocation is not involved in CT differentiation in isolated cell culture.

Caspase activation is not required for villous cytotrophoblast fusion into syncytiotrophoblasts.

The villous trophoblast renews itself by fusion of individual stem cells (cytotrophoblasts, CT) with a functional syncytium (syncytiotrophoblast, ST). The literature indicates that fusion occurs with limited activation (proteolytic cleavage) of caspase-8 in CT and is inhibited either by blocking caspase-8 synthesis or inhibiting activation with a caspase-8-specific inhibitor, zIETD. We challenge part of this evidence: inhibition of differentiation with caspase-8 inhibitors. Br-cAMP-stimulated differentiation of isolated CT into multinucleated syncytia in culture is not blocked with three different low molecular weight inhibitors of caspase-8: broad caspase inhibitors zVAD-fmk and qVD-OPh and the caspase-8-specific inhibitor zIETD-fmk. Syncytialization was determined by desmoplakin staining of intracellular boundaries surrounding >2 nuclei and by diffusion within fused cells of long-lived cytoplasmic staining from half of original CT to the unstained half. Differentiation of isolated CT into hCGß-secreting syncytiotrophoblast was also not blocked by the inhibitors nor was upregulation of hCGß secretion blocked in ST-stripped and regenerated 5 day explant cultures. The ratio of CT to ST nuclei present was also not changed in explant cultures by caspase inhibitors. The effectiveness of caspase inhibitors was demonstrated by their ability to completely block TNFα-induced apoptosis. We conclude that activation of caspases in general, and caspase-8 in particular, is not required for villous CT differentiation into ST. However, another role of intact caspase-8 (proform) in CT differentiation remains possible.

Human cytomegalovirus interacts with toll-like receptor 2 and CD14 on syncytiotrophoblasts to stimulate expression of TNFalpha mRNA and apoptosis.

Placentae from newborns with congenital human cytomegalovirus (HCMV) infection often display shallow implantation, chronic villitis and disruptions of the syncytiotrophoblast. Little is known about how HCMV infection induces inflammation in the placenta and loss of the trophoblast. We propose that the inflammation is initiated with innate defense responses of mature syncytiotrophoblast (ST) to virus. Previously we have shown that ultraviolet irradiation-inactivated (UV-) HCMV interacts with toll-like receptor 2 (TLR2) on primary placental cytotrophoblasts (CT) differentiated into ST-like cells thereby stimulating the release of tumor necrosis factor alpha (TNFalpha) and inducing apoptosis of neighboring cells (Chan et al, J. Pathol. 210: 111, 2006). We now determine whether known co-factors of the interaction of HCMV and TLR2 (TLR1 and CD14) bind to UV-HCMV to stimulate expression of TNFalpha and apoptosis in ST-like cells but not CT. We show that CT both fail to express detectable TLR1 and express much less CD14 than ST and that ST express CD14 but not TLR1 both in vivo and in cultured cells. The interaction of UV-HCMV and HCMV with CD14 on the surface of ST-like cells increases TNFalpha expression and induces apoptosis in the population. Antibody to CD14 also inhibits infectious HCMV induction of HCMV immediate early (HCMV IE) expressing ST-like cells. We conclude that primary villous CT express low levels of CD14 and no TLR1 but that ST strongly expresses CD14 which acts upstream of TLR2 to collect even transcriptionally inactive virus particles to stimulate TNFalpha expression and villous trophoblast damage.

In vitro differentiation of villous trophoblasts from pregnancies complicated by intrauterine growth restriction with and without pre-eclampsia.

Highly purified (>99.99%) primary villous cytotrophoblasts from uncomplicated pregnancies and pregnancies complicated with intrauterine growth restriction (IUGR) alone, IUGR with pre-eclampsia (IUGR-PE) and PE alone were cultured for 5days and the extent of differentiation into syncytiotrophoblasts measured in terms of syncytialisation and secretion of chorionic gonadotropin (hCG) and placental lactogen (hPL). Three separate phenotypes were observed: (1) normal and IUGR-PE cells showed low syncytialisation and secretion of hCG and hPL, (2) IUGR cells showed the highest levels of syncytialisation and secretion and (3) PE cells showed high syncytialisation but low secretion. These results strongly suggest IUGR, IUGR-PE and PE to be distinct conditions in which villous cytotrophoblasts are either exposed to different environments or are genetically different.

Endogenous tumor necrosis factor alpha mediates enhanced apoptosis of cultured villous trophoblasts from intrauterine growth-restricted placentae.

Tumor necrosis factor alpha (TNFalpha) has been implicated in the abnormally high levels of trophoblast apoptosis seen in placentae from pregnancies complicated by small births. We examined the hypothesis that at physiological (35-50 mmHg) oxygen tensions, the production of TNFalpha stimulates the apoptosis of placental trophoblasts associated with infants that are intrauterine growth-restricted (IUGR). Highly purified cytotrophoblasts (CT) from IUGR-complicated pregnancies spontaneously underwent a higher rate of apoptosis after 24 h of culture at a normoxic (for villous CT) tension of 38 mmHg than did CT from normal placentae. Real-time PCR analysis of TNFalpha mRNA revealed approximately threefold higher levels in IUGR trophoblasts after culturing at a pO2 of 38 mmHg. A higher level of TNFalpha receptor p55 (which mediates apoptosis) was found in IUGR CT by western blot analysis at pO2 of <10, 38, and 140 mmHg. Neutralizing antibody to TNFalpha significantly inhibited the apoptosis of IUGR trophoblasts cultured at 38 mmHg and addition of TNFalpha significantly elevated apoptosis of normal and IUGR trophoblasts but less in IUGR cells cultured at <10 mmHg. We conclude that at physiological oxygen tensions (38 mmHg), villous CT from IUGR pregnancies, when compared with uncomplicated pregnancies, undergo more TNFalpha-induced apoptosis both because of elevated expression of TNFalpha and TNF receptor p55.

Placental alkaline phosphatase (PLAP) staining and human chorionic gonadotropin (hCG) production in cultures of fresh and cryopreserved cytotrophoblasts isolated by CD9/MHC class I/MHC class II immunoelimination.

We have further characterized villous trophoblasts isolated by trypsinization and purified by elimination of CD9/MHC class I/MHC class II expressing cells. The cells isolated were >99.99% cytotrophoblasts by criteria of cytokeratin (positive) and vimentin (negative) expression. Purified cells directly after isolation (fresh) were compared with cryopreserved and thawed cells (frozen) for production of human chorionic gonadotropin (hCG) and expression of placental alkaline phosphate (PLAP) after 4 h of culture. We found that fresh cells may adhere slightly more strongly than frozen cells, contained approximately 8-fold more PLAP-positive cells (indicating syncytial fragments) after adherence but neither preparation would secrete hCG until day 4 of culture. We conclude that the cells isolated by cell elimination were cytotrophoblasts with only a small number of PLAP (<0.2% of the fresh population plated) positive cells and that both populations shared the property of very low hCG production until cultured past day 4. We speculate that cells isolated by other methods (accompanying paper) may be contaminated by even more syncytial fragments, detectable by PLAP staining and by production of hCG in the first 48 h of culture.

Multiple anti-apoptotic pathways stimulated by EGF in cytotrophoblasts.

Epidermal growth factor (EGF) reduces apoptosis in primary cytotrophoblast (CT) in culture through two separate pathways: the extracellular signal related kinase (ERK) 1/2 and phosphatidyl inositol 3-kinase (PI-3 kinase) paths. Whether other pathways are involved in survival signalling is unknown. We here show that the c-Jun NH2 terminal kinase (JNK) and the mitogen activated kinase (MAPK) p38 are also activated by EGF as seen by increases in JNK and p38 phosphorylation. However, inhibition of JNK phosphorylation with the specific inhibitor SP600125 increases apoptosis in a manner refractory to the addition of EGF but inhibition of p38 phosphorylation with its specific inhibitor SB 203580 does not increase apoptosis. EGF also activates sphingosine kinase-1 (SPHK-1), which converts sphingosine to sphingosine-1-phosphate, and its inhibition with dimethyl sphingosine (DMS) increased trophoblast death. Inhibition of SPHK-1 also did not affect EGF stimulated phosphorylation of PI-3 kinase, Akt, ERK1/2 or p38 but inhibition of PI-3 kinase with a specific inhibitor LY294002 partly (40%) inhibited the EGF-stimulated increase in SPHK-1 activity. We conclude that, in addition to the PI-3 kinase and ERK1/2 pathways, EGF acts through its receptor to stimulate JNK, p38 and SPHK-1 pathways, but that the JNK and SPHK-1, and not the p38, pathways are involved in suppressing apoptosis. This information provides evidence that EGF stimulates survival along multiple pathways that differ in trophoblast and other cell types.

Preparation and functional characterization of villous cytotrophoblasts free of syncytial fragments.

Recent studies suggest that purified villous cytotrophoblasts are largely contaminated by mononucleated syncytial fragments and therefore unsuitable for studies of trophoblast differentiation. We assessed highly purified (>99.99 per cent) populations of villous trophoblasts for fragment contamination using the syncytial markers placental alkaline phosphatase (PLAP, by immunohistochemistry) and exteriorized phosphatidyl serine (ePS, by flow cytometric analysis). The preparations contained from 4-46 per cent syncytial fragments. However, we find that PLAP negative cells preferentially adhere to tissue culture surfaces and that all preparations were <2 per cent PLAP positive after routine plating and washing procedures. A second purification procedure eliminated dead (propidium iodide permeable) cells and separated viable syncytial fragments (ePS-positive) from viable cytotrophoblasts (ePS-negative) by two colour fluorescence activated cell sorting (FACS). Viable ePS-positive cells were ultrastructurally apoptotic, adhered poorly in culture and those that adhered rapidly underwent apoptosis. Viable ePS-negative cells contained large heterochromic nuclei and cytoplasmic structures, adhered strongly in culture and remained viable. The latter population (putative true villous CT) differentiated into syncytialized cells when cultured with EGF. We conclude that villous CT can be routinely purified, are viable in culture and can undergo syncytial fusion without extensive preformed syncytium.

Villous trophoblasts cultured on semi-permeable membranes form an effective barrier to the passage of high and low molecular weight particles.

An effective in vitro model of the placental villous syncytium cultured on semi-permeable substrata is essential for studies of infectious pathogen transmission from mother to fetus. Current models using amniotic membranes or thinner artificial membranes show significant leakage, suggesting disruption of tight junctions or the presence of gaps between syncytial units. Such disruption and discontinuity of trophoblast cultures are probably the result of high stromal cell contamination, poor viability and lack of proliferation in culture. We have successfully cultured confluent layers of tight-junctioned syncytium on semi-permeable insert membranes using highly viable purified cytotrophoblasts and an alternating multiple seeding and differentiation technique. Using criteria including transepithelial diffusion of high and low molecular weight substances, electrical resistance and directional secretion of the matrix metalloproteinase, MMP-9, we demonstrate that these cultures form effective and functional physical barriers that can be maintained for up to 1 month.

Monocytes adhering by LFA-1 to placental syncytiotrophoblasts induce local apoptosis via release of TNF-alpha. A model for hematogenous initiation of placental inflammations.

Placental inflammations (villitis) are accompanied by loss of the syncytiotrophoblast, which is the cellular barrier separating maternal blood from fetal tissue in the villous placenta. We propose that syncytiotrophoblast loss is mediated by adhesion of activated maternal monocytes. This hypothesis was tested with a co-culture model of peripheral blood monocytes and placental syncytiotrophoblasts. We find that LPS-activated monocytes adhere to interferon-gamma (IFN-gamma)-treated syncytiotrophoblasts via monocyte LFA-1 for >48 h, during which time the monocytes induce trophoblast apoptosis and subsequent damage of the trophoblast layer. Optimal monocyte-mediated syncytiotrophoblast death requires both lipopolysaccharide (LPS) and IFN-gamma and is inhibited by either anti-tumor necrosis factor (TNF) antibody or epidermal growth factor. Syncytiotrophoblast damage is largely limited to culture surfaces in the vicinity of bound monocytes. These results show that activated maternal monocytes bound to the placental barrier can induce focal damage mediated by the inflammatory cytokine TNF-alpha and suggest a route for maternal leukocyte infiltration into the fetal stroma.

Polarized release of matrix metalloproteinase-2 and -9 from cultured human placental syncytiotrophoblasts.

The large increase in placental surface area and fetal villous vascular development in the third trimester of pregnancy requires degradation and reformation of the placental basal lamina. Degradation is carried out by matrix metalloproteinases (MMPs) secreted by adjacent cells. Although the gelatinases, MMP-2 and MMP-9, which are released by extravillous cytotrophoblasts (CTs) are believed to play crucial roles in early placental expansion, neither has been reported in third trimester villous trophoblasts nor has appropriate (basolateral) release of any MMP by the highly polarized syncytiotrophoblast (ST) been demonstrated. We demonstrated villous trophoblast expression of both MMP-2 and MMP-9 by in situ immunohistochemistry and by Western blot analysis and zymography of lysates and culture supernatants of highly purified villous CTs. We also found that epidermal growth factor (EGF)-stimulated CT differentiation into ST and stimulation by the phorbol diester, PMA, both increase MMP-9 secretion. The direction of MMP release was determined with confluent cultures of ST on porous membranes. We found that >90% of MMP-2 and MMP-9 were released from the basolateral surface. We conclude that villous STs express and release gelatinases from their basolateral surfaces in a regulated manner and suggest that such polarized release may be important to villous tissue remodeling.

The role of Bcl-2 expression in EGF inhibition of TNF-alpha/IFN-gamma-induced villous trophoblast apoptosis.

The inflammatory cytokines tumour necrosis factor alpha (TNF-alpha) and immune interferon gamma (IFN-gamma) stimulate villous cytotrophoblast apoptosis while epidermal growth factor (EGF) protects. We hypothesize that TNF-alpha, IFN-gamma and EGF regulate apoptosis in part by modulating cellular expression levels of the anti-death gene bcl-2. While Bcl-2 is reported to be strongly expressed in villous syncytiotrophoblasts, it is not known whether the protein is expressed in cultured villous cytotrophoblasts (CT) and, if so, whether it is functional. We show by Northern blot analysis that bcl-2 mRNA is expressed in cultured CT and by immunoblot analysis that the protein is strongly expressed in highly purified first trimester and term villous cytotrophoblasts. The expression levels of Bcl-2 protein were the same in first trimester and term cytotrophoblasts. Culture with TNF-alpha/IFN-gamma and EGF did not alter expression of either Bcl-2 protein or of the pro-apoptotic Bcl-2 family member Bak. Double label flow cytometric analysis that measured apoptosis and Bcl-2 content simultaneously showed that cells expressing low levels of Bcl-2 underwent TNF-alpha/IFN-gamma-induced apoptosis at a higher frequency than cells expressing lower levels. We conclude that Bcl-2 is expressed in cytotrophoblasts, that its expression is constitutive and that modulation of its expression levels does not mediate cytokine and growth factor regulation of apoptosis in these cells.

Placental trophoblasts resist infection by multiple human immunodeficiency virus (HIV) type 1 variants even with cytomegalovirus coinfection but support HIV replication after provirus transfection.

Whether cell-free human immunodeficiency virus type 1 (HIV-1) can productively infect placental trophoblasts (which in turn could transmit the virus into the fetal circulation) is controversial but essential to know for the evaluation of alternative routes (such as cell-mediated infection or trophoblast damage). We have addressed infection factors such as cell purity, source, culture methods, and activation states as well as virus variant and detection methods to conclusively determine the outcome of trophoblast challenge by free virus. Pure (> 99.98%) populations of trophoblasts from 11 different placentas were challenged at a multiplicity of infection (MOI) as high as 6 with five different HIV-1 variants, three of which are non-syncytium-forming, macrophage-tropic isolates from infected infants, with and without coinfection with cytomegalovirus; these preparations were monitored for productive infection for up to 3 weeks after challenge by five different criteria, the most sensitive of which were cocultivation with target cells that can detect virus at an MOI of 10(-7) and HIV DNA PCR that detects 30 virus copies per 10(5) cells. Infection was never detected. However, molecularly cloned T-cell (pNL4-3)- and macrophage (pNLAD8)-tropic provirus plasmids, when transfected into primary trophoblasts, yielded productive infections, indicating that trophoblasts do not suppress late-stage virus replication and assembly. Because of the purity of the trophoblast preparations, the extended length of the infection culture period, the number of trophoblast preparations and virus types examined, the sensitivity of the bioassays and molecular detection assays, and the observations that trophoblasts can support virus replication from provirus, the results of this study strongly argue that free virus cannot infect primary villous trophoblasts.

ICAM-1-mediated adhesion of peripheral blood monocytes to the maternal surface of placental syncytiotrophoblasts: implications for placental villitis.

Accumulation of maternal monocytes in the villous/intervillous space (villitis) is associated with increased risk of perinatal morbidity and mortality and may initiate in utero transmission of cell-associated infectious agents such cytomegalovirus and HIV-1. We have developed an in vitro model of trophoblast syncytialization and have investigated the adhesive interactions between this tissue and peripheral blood monocytes. We show that monocytes strongly adhere to cultured syncytiotrophoblasts (STs) and that treatment with the inflammatory cytokines interferon-gamma, tumor necrosis factor-alpha, and interleukin-1 alpha greatly increase the number bound. Pretreatment of STs with these cytokines upregulated apical expression of intercellular cell adhesion molecule (ICAM)-1 but not E-or L-selection, ICAM-2 or -3, or various integrins. ICAM-1 expression was cytokine concentration dependent, significantly increased within 6 hours of treatment, peaked after 24 hours, and remained undiminished for 48 hours after cytokine removal from the cultures. Adhesion of monocytes to STs was inhibited > 80% by antibody to ICAM-1 or its cognate ligand LFA-1. ICAM-1 was detected immunohistochemically only in rare foci on intact term placental villi. These results suggest that villous trophoblast expression of ICAM-1 occurs only during an immune inflammatory reaction and that aberrant expression of this molecule may be an important pathological feature in those immunoinflammatory disorders of the placenta characterized by an excessive accumulation of leukocytes in the intervillous/villous space such as spontaneous abortion, perinatal hematogenous infections, and villitis of unknown etiology.

Epidermal growth factor inhibits cytokine-induced apoptosis of primary human trophoblasts.

In the placenta, as in other organs, the development and maintenance of the differentiated phenotype depend on a balance between cell proliferation, maturation, and death. We are interested in the mechanisms that regulate the survival and differentiation of placental trophoblasts and have recently demonstrated that the inflammatory cytokines tumor necrosis factor alpha (TNF alpha) and gamma interferon (IFN gamma) act in concert to induce apoptotic cell death in normal cytotrophoblasts in culture. In this report we show that exposure to epidermal growth factor (EGF), a 6,700 dalton polypeptide that is abundantly expressed in maternal and fetal tissues, blocks the in vitro TNF/IFN-induced cytotoxicity of human cytotrophoblasts and syncytiotrophoblasts from normal term placentas. This antagonistic effect is dose-related (10-10 M EGF, half-maximal) and proceeds via the interruption of an early step in the cytokine-induced apoptotic response. These observations suggest a novel role for EGF in normal placental development and indicate that the interplay between EGF, TNF alpha, and IFN gamma may determine the rate of trophoblast growth and renewal during gestation.

Analysis of the synergistic stimulation of mouse macrophage proliferation by macrophage colony-stimulating factor (CSF-1) and tumor necrosis factor alpha (TNF-alpha).

Tumor necrosis factor alpha (TNF-alpha) more than doubles tritiated thymidine ([3H]TdR) uptake in mouse macrophages stimulated by macrophage colony-stimulating factor (CSF-1). However, nothing is known of how TNF-alpha affects this increase or even whether it is manifested by increased cellular proliferation. Here we characterize the effects of TNF-alpha on CSF-1-stimulated proliferation of both primary cells (bone marrow-derived macrophages, BMMs) and a cloned growth factor-dependent macrophage cell line (S1). We show that the TNF-alpha-induced increase in [3H]TdR uptake of CSF-1-stimulated macrophages is directly proportional to an increase in the DNA content of the culture and that the effects of TNF-alpha are direct and independent of cell number. TNF-alpha decreases the population doubling time of log-phase growing macrophages having quite different growth rates to the same (approximately 30%) extent: the doubling time of BMMs decreases from 24 to 17 h and that of S1 cells from 17 to 13 h. TNF-alpha exerts its effects on log-phase growth by increasing to the same proportion CSF-1-stimulated proliferation at all concentrations of CSF-1; that is, TNF-alpha does not shift, but rather amplifies, the CSF-1 dose-response curve. Although TNF-alpha alone does not stimulate macrophage proliferation, its presence in S1 cell cultures coming to quiescence after withdrawal of CSF-1 greatly increases subsequent CSF-1-stimulated [3H]TdR uptake as the cells reenter the cycle. Finally, we show that both human and mouse TNF-alpha increase CSF-1-stimulated log-phase growth and reentry of quiescent cells into the cycle equally on a molar basis (half-maximal stimulation of approximately 0.3 nM). The latter observation argues that the growth-stimulatory effects of TNF-alpha are mediated via the 55-60-kd TNF receptor. We conclude that TNF-alpha acts directly on growth-competent macrophages to decrease significantly the population doubling time in a manner that enhances the mitogenic effects of CSF-1.