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a Reproductive Molecular Research Group, Department of Obstetrics and Gynaecology, University of Cambridge, Rosie Maternity Hospital, Cambridge CB2 2SW, United Kingdom
b Research Group in Human Reproductive Immunobiology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1, 5, or ß1 by isolated trophoblast. These results identify fetal endothelial cells and all cells of the trophoblast lineage as targets for the action of LIF in human placenta. Although its effects on trophoblast are not yet clear, LIF appears to mediate interactions between maternal decidual leukocytes and invading trophoblast. LIF may also play a critical role in controlling angiogenesis in the placental villi, since human fetal endothelial cells express LIF-R, and mice lacking a functional LIF receptor gene show altered vascular development in the placenta.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There is now clear evidence that abnormalities in the process of trophoblast invasion, which occurs primarily over the first trimester of pregnancy, may result in clinical pathology later in gestation [2]. Pre-eclampsia and intrauterine growth retardation are associated with inadequate invasion and/or premature trophoblast differentiation, whereas continued invasion through the uterine wall, as seen in placenta percreta, may be lethal [3]. Elucidation of the factors that control trophoblast migration and differentiation is therefore important in understanding these clinical problems.
Leukemia inhibitory factor (LIF) is a secreted glycoprotein, originally identified through the induction of differentiation in the myeloid leukemia cell line M1 and by its ability to prevent differentiation of embryonic stem cells [4, 5]. It exhibits a wide spectrum of biological activities and belongs to a family of ligands that includes interleukin-6 (IL-6), IL-11, oncostatin M, ciliary neurotrophic factor, and cardiotrophin [6]. Ligand binding to a low-affinity receptor (LIF-R) causes association of the ligand/receptor complex with another membrane-bound protein, gp130, resulting in signal transduction [7]; gp130 participates in the ligand/receptor complexes of all this family of ligands, accounting in part for their overlapping activities [8].
LIF plays an important role in implantation and placentation in several species [9]. LIF-R mRNA is expressed by preimplantation embryos (mouse, human, cow, and sheep), and exogenous LIF increases the number of embryos developing to the blastocyst stage in vitro [10–12]. Direct evidence for the role of LIF comes from mice lacking a functional LIF gene. Although viable, LIF-/- female mice are unable to support implantation, even though the blastocysts implant normally when transplanted to pseudopregnant recipients [13]. Infusion of LIF by osmotic pump into the uterine lumen at this time restores normal implantation, indicating that maternal LIF is critical for implantation. A further role in placental development has been revealed through the use of mice in which the LIF receptor has been deleted. Homozygous LIF-R-/- embryos implant, but die within 24 h of birth [14]. The placentas of these mice are abnormal, with alterations in the appearance of the labyrinthine layer, indicating an important role for LIF in placental growth [15].
Recently, LIF, LIF-R, and gp130 mRNA have been reported in the human placenta and decidua by Northern blotting [16]. LIF immunoreactivity has been reported in trophoblast, but the identity of the target cells that express LIF-R in the placenta is unknown. Here, we report the detailed localization of LIF and LIF-R mRNA and protein by in situ hybridization and immunocytochemistry throughout pregnancy. The effect of LIF on thymidine incorporation and integrin expression by first-trimester trophoblast, identified as a target for LIF by this analysis, was also investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study was approved by the ethical committee of Addenbrookes Hospital NHS Trust, Cambridge, UK. First-trimester trophoblast and decidua (7–12 wk gestation, n = 5) were obtained as a result of elective terminations of pregnancy. Term material (n = 5) was obtained from normal vaginal deliveries at the Rosie Maternity Hospital, Cambridge, UK. Placentas from the second trimester (18–22 wk gestation, n = 3) were obtained from fetuses with anencephaly. Tissue samples for immunohistochemistry and in situ hybridization were fixed in formalin and processed for routine histology by embedding in paraffin. Trophoblast and decidual cells were prepared from fresh samples immediately after biopsy (see below).
In Situ Hybridization
All laboratory chemical reagents were from Sigma (Poole, UK) unless otherwise indicated. In situ hybridization was carried out using sections cut from paraffin-embedded tissue cut onto slides coated with 3-aminopropyltriethoxy-silane. The protocol used in this study was based on that described previously [17]. Radiolabeled RNA probes corresponding to LIF and LIF-R were prepared from cDNA inserts cloned into Bluescript II KS (Stratagene Ltd., Cambridge, UK). The preparation of these probes has been described previously [18, 19]. The LIF cDNA probe consists of 477 base pairs (bp) corresponding to bases 207–681 [20]. The LIF-R cDNA corresponds to bases 2831–3289 from the initiation codon, giving a 458-bp probe [21]. Single-stranded sense and antisense probes were prepared by transcription with radiolabeled [33P]UTP, using a MAXIscript in vitro transcription kit (Ambion, Dallas, TX). After in situ hybridization, the sections were counterstained with hematoxylin and photographed using a Leica DMRB/E microscope (Milton Keynes, Bucks, UK.
Immunohistochemical Staining
Formalin-fixed paraffin sections were stained using a protocol based on that described previously [17]. After dewaxing, sections were heat-treated by boiling for 1 min in 0.01 M sodium citrate pH 6.0. The sections were blocked in 10% rabbit serum in PBS (Vector Laboratories, Peterborough, UK), for 30 min and then incubated for 30 min with goat polyclonal antibody to LIF-R (R&D Systems, Oxford, UK) at 5 µg/ml in PBS for 1 h. This antibody bound to a single polypeptide of 160 kDa in Western blots of placental extracts (data not shown). Binding to sections was visualized by incubation with biotinylated rabbit anti-goat IgG (1: 200; Vector Labs) for 1 h, followed by streptavidin-conjugated horseradish peroxidase for 10 min (Zymed, San Francisco, CA). For the anti-cytokeratin, anti-CD68, and anti-CD45 mouse antibodies, blocking was also carried out with 10% rabbit serum. Sections were then incubated with primary antibody, either anti-cytokeratin antibody diluted 1:200 (Dako, Copenhagen Denmark), anti-CD45 antibody diluted 1:50 (Dako) or anti-CD68 antibody diluted 1:50 (Dako). These were detected with biotinylated rabbit anti-mouse (Vector) as previously described [17]. Sections were counterstained with hemalum and mounted in Depex (BDH, Poole UK).
Isolation and Culture of Trophoblast Cells
Trophoblast cells were isolated from chorionic villi of first-trimester placentas by trypsin digestion and centrifugation through Lymphoprep (ICN Biomedicals, Thame, UK) as previously described [22]. The purity of the trophoblast preparations was greater than 90% when cytospins of the cells were stained with a panel of antibodies to identify trophoblast [23]. Purified trophoblast was plated onto 35-mm culture dishes precoated with fibronectin (Collaborative Biomedical Research Products, Bedford, MA) at 20 µg/ml for 45 min. The cells were grown overnight in Ham's F-12 (ICN Biomedicals), 20% fetal calf serum (FCS; Gibco BRL, Uxbridge, UK), glutamine, and antibiotics at 37°C in 5% CO2.
To measure [3H]thymidine uptake, trophoblast that had been cultured overnight on fibronectin was recovered by trypsinization and seeded into fibronectin-coated flat-bottomed Falcon 96-well plates (Becton Dickinson, Oxford, UK). The cells were cultured in Molecular and Cellular Development medium 131 (ICN Biomedicals) and 1% human AB serum in a volume of 200 microliters at 1 x 105 cells per well, with the appropriate dilution of recombinant human LIF added at this point (R&D Systems). Thymidine incorporation was determined after 72 h by adding [3H]thymidine (Amersham, Bucks, UK), 1 µCi/well, to the cultures 16 h before harvesting. All cultures were performed in quadruplicate and repeated with four separate samples.
Immunofluorescent Staining and Flow Cytometry
Trophoblast cultured overnight was recovered by trypsinization for 5 min, followed by washing in Ham's F-12. The cells were resuspended at 1 x 106 per tube for staining in FACScan tubes (Becton Dickinson), and washed twice in ice-cold 0.1% BSA in PBS (PBS/BSA). All antibodies used for staining were resuspended in PBS/BSA. The proportion of trophoblast in the preparations was monitored by immunofluorescence staining with monoclonal antibody BC-1, a monoclonal antibody (mAb) directed to an epitope present only on EVT [23]. After incubation on ice for 30 min, the cells were washed twice in PBS/BSA and stained for 30 min with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Becton Dickinson) diluted 1 in 15. After the indirect stain, cells were washed and incubated with 90 µl of mouse IgG at a concentration of 200 µg/ml for 15 min followed by 10 µl of phycoerythrin-conjugated Leu-M3 (CD14) mouse mAb (Becton Dickinson) to label macrophages. After 30 min, the cells were again washed twice, fixed in 2% paraformaldehyde in PBS, and stored in the dark at 4°C until analyzed. Surface expression of integrins by trophoblast was measured by staining with mouse mAbs to the integrin subunits ß1 (clone MCA667; Serotec, Oxford, UK), 5 (clone CP12L; Oncogene Sciences, Uniondale, NY), and 1 (clone CMCA1133; Serotec). Leu-19 (CD56) mouse mAb (Becton Dickinson) was used as a negative control, to set the threshold on the immunofluorescence histograms above which the cells were considered positive for antibody binding.
The cells were analyzed using a FACScan flow cytometer (Becton Dickinson).
Isolation and Culture of Decidual Natural Killer (NK) Cells
Cell suspensions containing a heterogeneous mixture of all types of decidual cells or purified decidual NK cells were isolated from finely minced decidua as previously described [22]. For whole-cell suspensions, the minced tissue was incubated for 10 min at 37°C in 0.25% trypsin, 0.02% EDTA in PBS (ICN Biomedicals). An equal volume of RPMI with 20% FCS was added, and the digest was filtered through muslin and layered onto Lymphoprep (ICN Biomedicals). After centrifugation at 600 x g for 20 min, the cells were resuspended at 1 x 106 cells/ml in RPMI/10% FCS supplemented with antibiotics, 2 mM L-glutamine and cultured in 24-well plates for 48 h with or without the addition of different cytokines. Purified decidual NK cells were isolated from these total decidual preparations. After 24 h in culture, nonadherent cells (lymphocytes) were collected, leaving behind stromal glandular and macrophage cells. T cells were then removed by resuspending the lymphocytes in 4 ml Dulbecco's Modified Eagle medium (DMEM) containing 0.5% human gamma globulins (Sigma) and 1 mM EDTA. The cells were added to a 25-ml flask coated with anti-CD3 antibody (Applied Immune Sciences, Santa Clara, CA) and incubated at room temperature for 1 h. The nonadherent CD3-NK cell population was recovered, washed in PBS, and cultured as for the whole decidual preparations. The purity of the decidual NK preparations was assessed by fluorescent-activated cell-sorting (FACS) using CD56 antibody, and more than 95% cells were determined to be positive for CD56 antigen. Interleukin-1ß (IL-1ß) was purchased from R&D Systems; interleukin 2 (IL-2) and interferon 
(IFN) were from Boehringer Mannheim (Lewes, East Sussex, UK). Supernatants from the cells were removed and centrifuged at 400 x g for 5 min to remove cells and debris. The supernatant was stored frozen at -20°C until assayed for LIF by ELISA (R&D Systems) according to the manufacturers' instructions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Localization of LIF-R mRNA expression was examined in placental tissue throughout pregnancy by in situ hybridization with an antisense RNA probe. The LIF-R probe hybridized strongly to villous cytotrophoblast and syncytiotrophoblast, although the signal was patchy (Fig. 1, A–E). Examination of villi at high power also showed hybridization to endothelial cells lining fetal vessels (labeled "n" in Fig. 2, A and B). This pattern of LIF-R mRNA expression persisted in the placental villi throughout gestation (data not shown). Hybridization with a sense control probe labeled to the same specific activity showed no specific hybridization signal (Fig. 1, D and E; Fig. 2, C and D). LIF-R mRNA was also detected in scattered cells (labeled "e") in the maternal decidua (Figs. 1C and 3, A–E). These were identified histologically as EVT (Fig. 3, A–E). This was confirmed by staining serial sections with cytokeratin to identify EVT (Fig. 3A). In addition to invading interstitial EVT, strong hybridization was also seen in endovascular trophoblast lining maternal blood vessels and in placental giant cells (data not shown). Hybridization to decidual glandular epithelium was weak or undetectable (Fig. 3, B and C).
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In situ hybridization with an antisense probe homologous to the mRNA encoding LIF failed to detect any significant hybridization to placental trophoblast at any stage of gestation (Fig. 1, G and H). However, some signal was detected over scattered cells in the villous mesenchyme (Fig. 2, E and F). On the basis of their morphology and distribution, these are likely to be Hofbauer cells, which are fetal placental macrophages, although they were not identified by double staining. In contrast, there was strong hybridization to cells within the decidua, whose distribution matched that of maternal leukocytes as revealed by staining of serial sections for CD45 (Fig. 1, F–H, and Fig. 3, F–H). Detailed examination by a placental pathologist (A. King) confirmed silver grains over a subset of leukocytes with histological characteristics of NK cells (Fig. 3, G and H). These cells have prominent cytoplasmic granules and a characteristic distribution; they comprise 70% of decidual leukocytes. Glandular epithelium in the decidua showed little or no hybridization with the LIF probe (Fig. 3, G and H), although this probe detects LIF mRNA strongly in this cell type in nonpregnant endometrium during the luteal phase (data not shown).
Immunohistochemistry
In order to confirm expression of LIF-R protein, immunohistochemistry was carried out with a goat polyclonal antibody raised against the LIF-R polypeptide. This antibody recognizes a single polypeptide of 160 kDa in placental extracts using Western blotting (data not shown). In first-trimester villi, strong immunostaining of villous cytotrophoblast and syncytiotrophoblast was observed (Fig. 4B). Endothelial cells lining fetal capillaries in the villi also showed LIF-R immunoreactivity (Fig. 4H), supporting the in situ data in Figure 2B. Control sections incubated with nonimmune goat IgG at the same concentration showed no staining (data not shown).
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In first-trimester decidua, LIF-R immunoreactivity was detected in all the EVT cell types, including endovascular trophoblast and placental giant cells (Fig. 4, E and F). Some staining was also seen in the glandular epithelium, but this was weak (Fig. 4, B and D). Interestingly, endothelial cells lining blood vessels in the myometrium stained positive for LIF-R, whereas those in the decidua did not (data not shown). A similar pattern of LIF-R immunoreactivity was seen in samples from throughout pregnancy. These results are summarized in Table 1.
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Production of LIF by Decidua
Since in situ analysis showed that LIF mRNA is expressed primarily in the decidua, the production of LIF protein by various cell types isolated from first-trimester decidua, as well as purified trophoblast, was measured by ELISA. No significant secretion of LIF was detected in culture supernatants from purified trophoblast, decidual stromal cells, or decidual NK cells cultured alone. Nor was any LIF secretion detected by NK cells treated for 24 h with either IL-2, IFN, or IL-1ß (data not shown). However, when whole decidual cell suspensions were cultured, without separation of individual cell types, appreciable quantities of LIF secretion were detected (50 ± 9.4 pg/ml).
Analysis of Effects of LIF on Trophoblast Proliferation and Integrin Expression
Because LIF-R appeared to be expressed on all EVT populations, the possible role of LIF in trophoblast migration was examined by FACS analysis of the expression of the integrin subunits 1, 5, and ß1. These integrins change in expression as trophoblast differentiates from the villous to extravillous phenotype. Freshly isolated trophoblast was plated overnight on fibronectin with or without recombinant LIF added at 20 ng/ml. The cells were then double-stained with phycoerythrin-conjugated anti-CD14 (to allow macrophages to be gated out), and each of the mAbs for the integrin subunits in order to measure integrin expression. The cell population representing trophoblast was identified by staining with BC-1, which showed that trophoblast purity was greater than 90%. There was no apparent difference in the mean fluorescent intensity of the staining for these integrins on trophoblast after treatment with or without LIF for 24 or 48 h (data not shown).
In many cell types, LIF can act to promote proliferation, as well as having effects on differentiation. We therefore sought to investigate whether LIF had any effect on DNA synthesis by trophoblast as assayed by uptake of [3H]thymidine. Freshly isolated EVT was cultured in the presence of LIF for 72 h, and the effect on [3H]thymidine incorporation was determined. No significant effect was seen over a dose range of 0.1–20 ng/ml of LIF (p = 0.7; data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Northern blotting has previously shown that LIF mRNA is present in decidua throughout gestation, however at a lower level than is seen in secretory endometrium [16, 24]. Secretion of LIF has also been demonstrated from both chorionic villous and decidual explants, with 5- to 10-fold higher levels from decidua [25]. Our analysis of the site of LIF mRNA expression in placenta would support this since only a few scattered cells in the villous mesenchyme (probably Hofbauer cells) expressed LIF mRNA. There was no detectable LIF mRNA expression in any trophoblast cells. Although there is one report of LIF immunoreactivity in trophoblast [25], other investigators did not detect LIF secretion from trophoblast cultured in vitro [26]. In the decidua, there was strong LIF mRNA expression among the CD45+ leukocytes. Seventy percent of these cells are CD56+ uterine NK cells [27], which can be identified histologically, and most of the NK cells appeared to express LIF mRNA (Fig. 3). These decidual NK cells almost certainly correspond to the strongly LIF immunopositive cells reported in the decidual stroma but not identified by Cullinan et al. [24]. Little or no expression of LIF mRNA was seen in the glandular epithelium of the decidua, even though adjacent CD45+ leukocytes were strongly positive (Fig. 1, G and H, and Fig. 3, G and H). In nonpregnant endometrium, the glandular epithelium expresses abundant LIF mRNA and immunoreactivity in the luteal phase of the cycle before implantation, with little apparent expression by the leukocytes that are also present in secretory-phase endometrium [18, 24]. Thus LIF expression by the glandular epithelium is dramatically down-regulated after implantation, whereas expression by NK cells is up-regulated in the decidua. Total cell suspensions isolated from decidua were found to secrete significant amounts of LIF, as has been reported for decidual explants [25]. However, decidual NK cells (the apparent source in vivo) purified and cultured alone did not produce LIF. This finding was unexpected in view of the in situ results, and because we have previously demonstrated LIF mRNA expression in purified NK cells sorted by flow cytometry [28]. Stimulation by exogenous IL-2, IL-1ß, or IFN failed to induce LIF production by isolated NK cells. Either the LIF mRNA is not translated, or, more likely, interaction with other cells of the decidual stroma is required for LIF production by these NK cells.
LIF-R and gp130 mRNA have previously been detected in the placenta by Northern blotting [16]. This analysis has now shown that LIF-R mRNA and immunoreactivity are localized primarily to the trophoblast lineage. Since gp130 immunoreactivity has been reported in the villous cytotrophoblast and syncytiotrophoblast [16], our results indicate that in these cells at least, all the receptor subunits are present to allow LIF signal transduction. No significant LIF-R mRNA was detected in the glandular epithelium of the decidual samples analyzed. This is similar to nonpregnant endometrium, in which LIF-R mRNA is expressed throughout the menstrual cycle only by surface epithelium lining the lumen, but not by the glandular epithelium [24].
Recently, mRNA transcripts encoding soluble forms of LIF-R (sLIF-R) have been found in the placenta, and soluble LIF-R polypeptide was detected in conditioned medium from a choriocarcinoma cell line [29]. The cDNA probe and antibody used in this study cannot distinguish between soluble and membrane-bound forms of LIF-R. It is therefore not clear how much sLIF-R is produced by the placenta in vivo.
LIF can either enhance cell differentiation or inhibit it and promote proliferation instead, depending on the cell type [4]. In the placenta, trophoblast proliferation occurs in the villous cytotrophoblast and in the EVT at the base of the columns [30]. Although all trophoblast expressed LIF-R mRNA and protein, LIF was found to have no effect on the proliferation of first-trimester trophoblast in vitro, as assayed by [3H]thymidine incorporation. The isolation procedure used produces a mixed villous and EVT population containing some proliferating (Ki67+) cells; however, after overnight culture they all acquire an extravillous phenotype (HLA class 1+ and c-erbB2+) and cease to proliferate [1]. LIF has been shown to cause cultured primordial germ cells to de-differentiate and resume proliferation [31]. However, LIF alone was not able to stimulate trophoblast in this way, even though the level of LIF used (20 ng/ml) has previously been shown to alter trophoblast function [26].
LIF has been reported to decrease metalloproteinase secretion and increase fetal fibronectin secretion by first-trimester trophoblast. This suggests that it inhibits trophoblast invasion and promotes differentiation into sessile EVT types [26, 32]. Pregnancies complicated by pre-eclampsia or intrauterine growth retardation are characterized by shallow trophoblast invasion, and may result from failure to express the correct spectrum of integrins for trophoblast invasion [33]. Individual growth factors such as epidermal growth factor have been shown to modulate the integrin profile of freshly isolated trophoblast, and this correlates with alterations in cell migration [34]. Although LIF has been reported to alter the integrin profile of some tumor cell lines [35], the results from this study indicate that LIF does not regulate trophoblast invasion by this mechanism.
In addition to trophoblast, endothelial cells in the fetal villi expressed LIF-R mRNA and protein, indicating a second site of action for LIF (Figs. 2B,C and 4H). The placenta undergoes profound angiogenesis throughout gestation. LIF has previously been shown to be a potent regulator of angiogenesis, opposing the effects of angiogenic factors such as vascular endothelial growth factor [36]. It is likely that the correct pattern of capillary growth and branching in the placenta depends on complex interactions between angiogenesis-promoting and -controlling agents. Of particular interest is the observation that the fetal vascular component is apparently disrupted in the placentas of fetuses lacking a functional LIF receptor gene [15]. Our results now identify fetal endothelial cells as a target for LIF in human placenta.
There are now data that indicate important roles for LIF at several stages of the reproductive process, including preimplantation embryo development, implantation itself, and growth and differentiation of the placenta. Human preimplantation embryos coexpress the mRNAs for LIF-R and gp130 at the blastocyst stage, and LIF has also been reported to dramatically improve embryo development in vitro [12, 18]. In human and murine endometrium, LIF expression is maximal at the time of implantation [18, 37]. In mice, this maternal expression of LIF has been shown to be essential for implantation [13]. The findings from this study identify all cells of the trophoblast lineage as targets for the action of LIF in human placenta.
As EVT expressing LIF-R invades the decidua, it encounters uterine NK cells that express LIF. Since LIF reduces metalloproteinase expression in trophoblasts, this provides a means by which decidual NK cells could regulate trophoblast invasion in vivo. Fetal endothelial cells were identified as a second site of LIF-R expression, in which it may be involved in controlling vascular development, as has been shown in the mouse placenta.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Andrew Sharkey, Department of Obstetrics and Gynaecology, University of Cambridge, Box 223, Rosie Maternity Hospital, Robinson Way, Cambridge CB2 2SW, UK. FAX: 44 1223 215327; ams{at}mole.bio.cam.ac.uk
Accepted: September 16, 1998.
Received: October 28, 1997.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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