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Genomic imprinting is the phenomenon whereby mono-allelic expression of certain genes occurs depending on their parental origin. The observation that imprinting only occurs in placental mammals has led to the suggestion that it may play a role in this form of reproduction. In the present study we have investigated the pattern of expression of the human PEG3 gene in the early to term placenta, as well as the uterus and ovary, using RT-PCR, northern blot and in situ hybridization. A comparison is made with the expression of Peg3 in the mouse by histochemical staining in betageo knock out mice. We have demonstrated high levels of PEG3 in the human placenta and have localized the signal to the layer of villous cytotrophoblast cells. In contrast, the pattern of expression of Peg3 in the mouse placenta is less restricted, the message being present in all trophoblast populations. Thus, expression of PEG3/Peg3 in the human and mouse placenta is not directly comparable. We have also detected PEG3 message in the ovarian stroma. We have sequenced the human PEG3 gene from exon 3 to exon 9. By utilizing a polymorphism detected in exon 9, we have established that only the paternal allele is expressed in human placenta. Human PEG3 is therefore maternally imprinted as in mouse.

During implantation, the uterine decidua is invaded by extravillous trophoblast (EVT) cells whose function is to destroy the walls of the uterine spiral arteries in order to provide an adequate blood flow to the fetus. These EVT cells express an unusual combination of HLA class I molecules-HLA-C, HLA-E and HLA-G. The decidua is infiltrated by a population of natural killer (NK) cells with a distinctive phenotype of CD56(bright)CD16(-)CD3(-). These cells are particularly numerous in the decidua basalis at the implantation site where they come into close contact with invading EVT cells. These NK cells express a variety of receptors (CD94/NKG2, KIR and ILT) which are known to recognize HLA class I molecules. We believe that interaction between these NK cells and EVT cells provides the controlling influence for implantation.

In this paper definitive evidence that the classical class I product, HLA-C, is expressed on the surface of normal trophoblast cells is provided. HLA-C transcripts were sequenced from cDNA isolated from first trimester trophoblast cells obtained by flow cytometric sorting. Both paternal and maternal alleles were transcribed. HLA-C proteins were demonstrated by biochemical analysis and found on the cell surface in association with beta(2)-microglobulin. Upregulation of cell surface HLA-C but not HLA-G expression after interferon (IFN)-gamma treatment was demonstrated by flow cytometric analysis. Immunohistology has confirmed HLA-C is expressed by all extravillous subpopulations in vivo. The question of whether trophoblast HLA-C molecules interact with decidual NK cells expressing killer Ig-like receptors (KIR) has also been addressed. Our results demonstrate that extravillous trophoblast expresses at least two HLA class I molecules, HLA-G and HLA-C on the cell surface.

The immunological relationship beween the mammalian fetus and its mother during pregnancy has been considered similar to that between a transplanted allograft and its recipient ever since Medawar (1953) first proposed the concept of the ‘fetus as an allograft’ in the early 1950s. Based on this analogy, it has been assumed that implantation of the fetal placenta in the uterus would be controlled similarly by a maternal immune response mediated by T-cells recognizing paternally-derived alloantigens expressed by the placenta. Surprisingly, recent evidence suggests that implantation might involve predominantly a novel allogeneic recognition system based on natural killer cells rather than T-cells (Loke & King, 1995). The cellular and molecular basis of this local immune interaction between the fetal placenta and maternal uterus is now the focus of intense research interest. Since aberrant implantation can cause a variety of clinical problems, including miscarriage, intrauterine growth retardation and pre-eclampsia, an understanding of the immunological mechanism by which this process is controlled could lead to the development of regimens to improve fetal growth and development.

Mice in which the gene that encodes the receptor (R) for leukemia inhibitory factor (LIF) has been deleted show abnormal growth and development of the placenta. This indicates that LIF plays an important role in placental development. The expression of LIF-R and LIF was examined in human trophoblast and decidua using in situ hybridization and immunocytochemistry. LIF-R mRNA and immunoreactivity was localized in villous and extravillous trophoblast throughout pregnancy, and in endothelial cells of the fetal villi. Strong expression of mRNA encoding LIF was detected in decidual leukocytes, which are abundant at the implantation site. Extravillous trophoblast, which invades the maternal decidua, therefore expresses LIF-R as it moves past decidual leukocytes, which express LIF mRNA. The effect of LIF on cultured human trophoblast was examined in vitro. Recombinant human LIF had no effect on [3H]thymidine incorporation by purified extravillous trophoblast, nor on expression of integrins alpha1, alpha5, or beta1 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.

There is considerable interest in human HLA-G arising from the observation that it is expressed selectively on the surface of extravillous trophoblast, the fetal cell population directly in contact with the mother. We investigated several aspects of the molecular biology of this unusual molecule. Limited polymorphism at the nucleotide level, and even more restricted variation at the amino acid level, was found in our Caucasian population. A further unusual aspect of HLA-G is the occurrence of alternatively spliced mRNAs. Spliced messages that could give rise to either membrane-bound or soluble proteins have been reported and six of these alternative forms were detected in all first trimester and term placentae, highly purified villous and extravillous trophoblast and the cell lines, JEG-3 and 221-G. An additional novel splice variant involving loss of part of the 3′-untranslated region was observed with two alleles. Using a sensitive RNase protection assay higher levels of the membrane-bound RNAs as compared to the soluble forms were detected in first trimester and term placentae as well as in JEG-3. Contrary to previous findings our term samples taken from the maternal aspect showed higher levels of both mRNA species when compared to first trimester placenta. The question of imprinting was addressed through the detection of heterozygotes both in placental tissue and, more tellingly, in the purified trophoblast cells. There was no evidence of imprinting. In addition we did not find mRNA for HLA-G in human two to eight-cell embryos or in blastocyst or in sperm samples1.

During the luteal phase and the early months of pregnancy, there is a dense mucosal infiltration of CD56+ natural killer (NK) cells. These uterine NK cells have a phenotype (CD56bright, CD16-, mCD3-) which distinguishes them from peripheral blood NK cells (CD56dim, CD16bright, mCD3-). The uterine NK cells are in close association with extravillous trophoblast (EVT) cells which infiltrate into the decidua and maternal spiral arteries. This subpopulation of trophoblast expresses two human leukocyte antigen (HLA) class I molecules, HLA-G and HLA-C. Circulating NK cells express receptors for HLA class I molecules. We have recently found evidence that similar receptors are present on decidual NK cells belonging to both the Killer Inhibitory Receptor (KIR) and CD94 families. The repertoire of NK receptors expressed varies between different women. The findings indicate that decidual NK cells do have receptors for trophoblast HLA class I molecules. Experiments are underway to determine the effects of this interaction on NK cell function.

During the process of implantation in humans, fetal trophoblast cells invade and migrate into the maternal decidua. During this migration, trophoblast cells destroy the wall of the maternal spiral arteries, converting them from muscular vessels into flaccid sinusoidal sacs. This vascular transformation is important to ensure an adequate blood supply to the feto-placental unit. Both cell-cell and cell-matrix interactions are important for trophoblast invasion of the decidual stroma and decidual spiral arteries. Cell-matrix adhesions are mediated by specific receptors, mostly belonging to the family of integrins. Signals transduced to the cells from the matrix via integrins could play a pivotal role in the control of cellular behaviour and gene expression, such as metalloproteinases that facilitate matrix degradation and tissue remodelling. This review focuses on the role of integrins and extracellular matrix in trophoblast cell migration, trophoblast invasion of the decidual spiral arteries and matrix degradation by trophoblast during implantation.

The maternal leukocytes present in human decidua during the early weeks of pregnancy are composed of CD56+ NK cells (approximately 80%), CD3+ T cells (approximately 10%), and CD14+ macrophages (approximately 10%). These leukocytes are present in large numbers at the implantation site where trophoblast invades. The migrating extravillous trophoblast (EVT) cells express HLA class I antigens HLA-G and HLA-C. To examine whether EVT or other allogeneic cells are capable of inducing a local proliferative response in maternal decidual leukocytes, we have assayed [3H]thymidine incorporation by decidual leukocytes co-cultured with irradiated trophoblast or peripheral blood lymphocytes (PBL). No proliferative response was seen in decidual leukocytes either to trophoblast or to PBL as stimulators. In addition trophoblast did not stimulate a proliferative response in PBL. These findings indicate that extravillous trophoblast is not capable of initiating a classical immune response at the implantation site.

At the time of implantation, the extracellular matrix proteins laminin and fibronectin are abundant in the decidua and are distributed pericellularly around each individual stromal cell. First trimester human trophoblast expresses both laminin and fibronectin receptors, specifically the alpha 1 beta 1, alpha 5 beta 1, alpha 6 beta 1 and alpha 6 beta 4 integrin heterodimers. In this study we have demonstrated that in-vitro adhesion of first trimester human trophoblast to purified extracellular matrix proteins and to purified decidual stromal cell monolayers can be inhibited by monoclonal antibodies directed against appropriate integrin subunits and by synthetic peptides containing an arginine-glycine-aspartic acid sequence. Monoclonal antibodies (mAbs) to the alpha 5 and beta 1 integrin subunits and a synthetic peptide significantly inhibited adhesion to fibronectin. Binding of trophoblast to laminin was blocked with mAbs to the alpha 6 and beta 1 but not alpha 1 and beta 4 integrin subunits. Similarly, integrin-mediated adhesion to monolayers of decidual stromal cells could be blocked with mAbs to the alpha 5, alpha 6, beta 1 and beta 4 integrin subunits. Integrin-mediated signal transduction in normal and malignant trophoblast was investigated by Western blotting. A 115 kDa protein was the major tyrosine phosphorylated protein detected in trophoblast after binding to laminin or fibronectin. The profile of tyrosine phosphorylated proteins differed for malignant trophoblast.

During the process of placental implantation, sessile villous trophoblast cells migrate from the villi into the decidua as isolated motile extravillous trophoblast cells. There is differential expression of the epidermal growth factor-receptor (EGF-R) and c-erbB2 proteins on villous and extravillous trophoblast populations. Using monoclonal antibodies to EGF-R and c-erbB2, we have obtained highly purified populations of villous and extravillous trophoblast by fluorescence activated cell sorting. These cells were examined by the reverse transcriptase-polymerase chain reaction (RT-PCR) using nested internal primer pairs for the following cytokines: CSF-1, GM-CSF, TNF-alpha, TGF-beta1, IFN-gamma, IL-2, LIF and also for LIF-receptor. TNF-alpha and TGF-beta 1 were present in all trophoblast populations. GM-CSF and CSF-1 were only found in some samples, with preferential expression of CSF-1 in villous populations. IFN-gamma, IL-2 and LIF mRNA were not found, although all samples contained LIF-receptor mRNA. These cytokines (CSF-1, TGF-beta, TNF-alpha and GM-CSF) are likely to influence trophoblast growth and differentiation in an autocrine manner, since their receptors are also present on trophoblast. These results illustrate a quick and simple method to analyse for the presence of cytokine and other transcripts in trophoblast subpopulations during early pregnancy.

At the time of implantation, the extracellular matrix proteins laminin and fibronectin are abundant in the decidua and are distributed pericellularly around each individual stromal cell. First trimester human trophoblast expresses both laminin and fibronectin receptors, specifically the alpha 1 beta 1, alpha 5 beta 1, alpha 6 beta 1 and alpha 6 beta 4 integrin heterodimers. In this study we have demonstrated that in-vitro adhesion of first trimester human trophoblast to purified extracellular matrix proteins and to purified decidual stromal cell monolayers can be inhibited by monoclonal antibodies directed against appropriate integrin subunits and by synthetic peptides containing an arginine-glycine-aspartic acid sequence. Monoclonal antibodies (mAbs) to the alpha 5 and beta 1 integrin subunits and a synthetic peptide significantly inhibited adhesion to fibronectin. Binding of trophoblast to laminin was blocked with mAbs to the alpha 6 and beta 1 but not alpha 1 and beta 4 integrin subunits. Similarly, integrin-mediated adhesion to monolayers of decidual stromal cells could be blocked with mAbs to the alpha 5, alpha 6, beta 1 and beta 4 integrin subunits. Integrin-mediated signal transduction in normal and malignant trophoblast was investigated by Western blotting. A 115 kDa protein was the major tyrosine phosphorylated protein detected in trophoblast after binding to laminin or fibronectin. The profile of tyrosine phosphorylated proteins differed for malignant trophoblast.

The present in-vitro study using an enzyme-linked immunosorbent assay has identified the cell types responsible for colony stimulating factor-1 (CSF-1) production at the first trimester human placental uterine interface. The major sources were observed to be decidual stromal cells and decidual CD56 + natural killer (NK) cells, but decidual CD3 + T cells did not produce CSF-1, reflecting functional differences between these two decidual lymphoid populations. Of a variety of cytokines tested, only interleukin-2 (IL-2) was found to augment CSF-1 secretion by decidual NK cells. Trophoblast cells also secreted CSF-1, but the amounts were small relative to decidual stromal cells and NK cells. Therefore, most of the CSF-1 present at the implantation site appears to be maternally derived. Co-culture of decidual NK cells on a monolayer of irradiated trophoblast did not augment CSF-1 secretion by decidual NK cells, indicating that the production of this cytokine is not stimulated by contact with fetal trophoblast. CSF-1 was found to increase [ 3 H]thymidine uptake by trophoblast cultured on laminin for 72 h, but no such response was seen in trophoblast cultured on fibronectin, indicating that these extracellular matrix proteins have differential effects on the response of trophoblast to this cytokine.

Trophoblast cells from normal first trimester pregnancies have been shown to express the nonclassical Class I molecule, HLA-G, which is nonpolymorphic and has a heavy chain of 40 kDa. These HLA-G-expressing trophoblast cells infiltrate into maternal decidua, which contains abundant uterine-specific CD56bright natural killer (NK) cells. We believe HLA-G may act as a protective molecule against decidual NK lysis and thus allow trophoblast survival in the maternal tissues. To test this hypothesis, we have constructed HLA-G and HLA-A2 transfectants using LCL 721.221 HLA-null cells. We observed that both of these antigens protected target cells from NK effectors isolated from decidua or peripheral blood, although the effect of HLA-G is not as marked as that of HLA-A2. Our results, therefore, show that in this experimental system expression of a nonclassical Class 1 HLA molecule is also correlated with NK resistance in the same way as a classical Class I HLA molecule.

Summary This review focuses on HLA-G as an example of a cell surface molecule expressed by human trophoblast. The background behind the elucidation and localization of this non-classical HLA Class I antigen is surveyed. The function of this antigen is not known. We have put forward the hypothesis that it may protect trophoblast from cytolysis from the unusual population of large granular lymphocytes (LGL) which populate the pregnant uterus. Alternatively, this antigen could trigger LGL to secrete cytokines which have an influence on trophoblast growth and differentiation. It would appear that reproduction involved immunological parameters which have yet to be defined.

Trophoblast cells from normal first trimester pregnancies have been shown to express the nonclassical class I molecule, HLA-G, which is nonpolymorphic and has a heavy chain of 40 kD. In this study, in situ hybridization experiments were performed using a biotinylated HLA-G specific oligonucleotide on sections of normal placenta including the implantation site. HLA-G mRNA was identified in all extravillous trophoblast populations including the cytotrophoblast cell columns, interstitial trophoblast, endovascular trophoblast, and placental bed giant cells. In addition, villous cytotrophoblast and villous mesenchymal cells also contained HLA-G transcripts, but villous syncytiotrophoblast was always negative.

The present study has demonstrated that human extravillous trophoblast cells isolated from first trimester placentae can bind granulocyte-macrophage colony-stimulating factor (GM-CSF). We have used a technique which incorporates a phycoerythrin (PE)-conjugated GM-CSF in association with a monoclonal antibody against trophoblast (BC-1) in single and double flow cytometric analysis. Trophoblast cells are also observed to show increased DNA synthesis in response to GM-CSF as assayed by [3H]thymidine incorporation and proliferative activity as demonstrated by double immunocytochemical staining of cells with a trophoblast cytokeratin marker (PKK1) and for the nuclear proliferation antigen (Ki-67). These findings provide evidence that human extravillous trophoblast expresses receptors for GM-CSF and responds to this cytokine.

Freshly isolated decidual large granular lymphocytes (LGL) show natural killer (NK) activity against K562 cells but not against normal human trophoblast. We now show that these decidual LGL proliferate in vitro in response to recombinant interleukin-2 (rIL-2) and that these rIL-2-stimulated cells acquire a broad cytolytic potential that is characteristic of lymphokine-activated killer (LAK) cells. Both fetal fibroblasts and JEG-3 choriocarcinoma cells are resistant to lysis by freshly isolated decidual effectors but are readily killed by IL-2-stimulated decidual LGL. The ability to kill these target cells is acquired after only 18 hr exposure to rIL-2. rIL-2-activated decidual LGL also kill cultured normal trophoblast cells but much lower levels of cytolysis were seen even after the effectors had been stimulated with rIL-2 for 4-6 days. The preferential killing of malignant over normal human trophoblast cells raises questions about the potential role of IL-2-activated decidual LGL in the control of unduly invasive or malignant trophoblast populations in vivo.

The main population of lymphocytes found in the human decidua during early pregnancy are NK-like cells with a distinctive phenotype, CD56bright CD16- CD3-. These cells are in close association with invading trophoblast that may be their in vivo target. We have examined three aspects of decidual NK function in vitro: cytotoxicity, proliferation, and cytokine production. The functional assays indicate uterine lymphocytes differ fundamentally from both PBL and even from classical circulating NK cells. Their role in the establishment of normal pregnancy remains unknown.

The remodelling of the maternal uterine spiral arteries during pregnancy, known as physiological change, is critical for the normal growth and development of the fetus. Controversy has surrounded the part played by fetal trophoblast in the transformation of these spiral arteries. To address this debate, a histological and immunochemical comparison of blood vessels from the implantation sites of human pregnancies of early gestation with uterine tissue where trophoblast was absent was performed. Results showed that true physiological change, with the features of medial necrosis and deposition of fibrinoid material, only occurred in the presence of trophoblast. In addition, it was found that subpopulations of trophoblast contribute differently in the process. Interstitial trophoblast-mediated destruction of the arterial media precedes replacement of the endothelial cells by endovascular trophoblast.

Although human decidual lymphocytes have been widely studied, their function and possible interaction with trophoblast are still unclear. Here we show that whereas human early (EDL) and term (TDL) decidual lymphocytes were unable to kill human trophoblast by necrosis (assessed by the 51Cr-release assay) or apoptosis (DNA fragment assay), TDL but not EDL decreased trophoblast adhesion to a plastic substrate as determined by a [3H]thymidine assay. This effect, however, was not selective for trophoblast, as TDL also decreased the adhesion to plastic of human decidual stromal cells and HeLa cells. Our results suggest that TDL may play a role in placental detachment during parturition by decreasing trophoblast or decidual stromal cell adhesion.

A monoclonal antibody to HLA-G has been generated by immunizing HLA-A2.1/human beta 2-microglobulin (beta 2 m) double transgenic mice with murine L cells transfected with both human beta 2 m and HLA-G. This monoclonal antibody, designated as G233, has been found not to cross-react with other HLA class I antigens when tested on numerous cell lines by flow cytometry. With immunohistology, all populations of extravillous trophoblast (cell columns, interstitial trophoblast, endovascular trophoblast, placental bed giant cells) were stained. An extensive range of adult and fetal tissues was also tested but none reacted with monoclonal antibody G233, including those previously reported to express HLA-G mRNA, indicating that the protein has a highly restricted distribution. Failure to detect HLA-G in the fetal thymus raises the question as to how T-cell tolerance to this antigen is induced. Immunoprecipitation of trophoblast surface proteins with monoclonal antibody G233 revealed a heavy chain of 39 kDa and a light chain of 12 kDa, indicating that HLA-G expressed on the surface of trophoblast is complexed with beta 2 m. However, sequential immunoprecipitation with monoclonal antibody W6/32 followed by monoclonal antibody G233 continued to detect a residual band of 39 kDa, suggesting that trophoblast surface HLA-G may also occur as free heavy chains not associated with beta 2 m. Immunoprecipitation followed by two dimensional gel electrophoresis showed that monoclonal antibody G233 recognizes several isoforms of HLA-G from trophoblast similar to the characteristic spot array previously described for HLA-G. This monoclonal antibody G233 will be highly useful in future experiments to elucidate the function of HLA-G.

Flow cytometric data were used to compare the phenotype of term decidual lymphocytes and peripheral blood lymphocytes. Unlike peripheral blood lymphocytes, a significant percentage of CD3+, CD4+, CD8+ and CD16+ term decidual lymphocyte populations expressed the CD69 activation marker. The relative proportions of CD38 in CD3+, CD4+ and CD8+ populations were more than twice as large in term decidual lymphocytes as in peripheral blood lymphocytes. As reported for early decidual lymphocytes, the expression of CD38 and CD69 by term decidual lymphocytes suggests that these cells are also regionally activated. However, term decidual lymphocytes showed no spontaneous cytotoxicity against normal trophoblast or its tumoral counterpart, JEG cells. After stimulation with interleukin-2, these lymphocytes became cytotoxic, as did peripheral blood lymphocytes. The relevance of this latter result to the immune control of the physiological and pathological invasion of the decidua by the trophoblast is discussed.

Human placental implantation involves invasion of the uterus by placental trophoblast cells. The extent of this invasion is tightly controlled. Insufficient invasion will lead to inadequate blood supply to the fetoplacental unit, and overextensive invasion will result in placenta percreta (1). The mechanisms that control this trophoblast invasion are not known. The decidua is likely to provide an important element of this control because implantation over areas deficient in decidua, such as over a previous cesarean section scar or in the fallopian tubes, generally shows a significantly greater degree of trophoblast invasion. It is unclear, however, which component of the decidua is important in this regard.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a classical haematopoietic cytokine which has been implicated in placental growth and development. In this study, we investigated the production of GM-CSF in human first trimester pregnancy by the predominant uterine lymphocyte population of decidual CD56+ NK cells (large granular lymphocytes) and the factors that influence this production using enzyme-linked immunosorbent assays (ELISAs) and bioassays, supplemented by immunocytochemistry. We have also investigated and compared production of GM-CSF by human first trimester trophoblast and by JEG-3 and JAR choriocarcinoma cells. Our data show that appreciable amounts of GM-CSF are produced in first trimester maternal decidua and that a significant component of this secretion was from decidual large granular lymphocytes (LGL). Production of GM-CSF by LGL was constitutive and considerably greater than that of freshly isolated peripheral blood leukocytes. GM-CSF secretion by decidual LGL could be enhanced by co-culture on a monolayer of decidual stromal cells, and could also be increased in a dose-dependent manner by stimulation with interleukin-1 (IL-1) or IL-2. IL-4, IL-6, tumour necrosis factor alpha (TNF alpha), transforming growth factor beta (TGF beta), interferon alpha (IFN alpha) and IFN gamma individually had no effect on GM-CSF secretion, although IL-4, TGF beta and IFN alpha all inhibited the action of IL-2. IFN gamma had no effect on the IL-2-induced GM-CSF secretion, but did antagonize the action of IL-1. Normal human first trimester trophoblast was also found to produce GM-CSF, although no production whatsoever was seen by JEG-3 or JAR choriocarcinoma cells. These results suggest that GM-CSF from uterine lymphocytes, and from trophoblast itself, may influence placental growth and development in both a paracrine and an autocrine manner.

Trophoblast invasion of the maternal uterus during human placentation is a highly complex process involving proliferation, migration and cell differentiation. Tyrosine kinase receptors are known to be involved in such functions. The epidermal growth factor-receptor (EGF-R) and c-erbB2 proto-oncogene products are two closely related tyrosine kinase receptors which are expressed in high amounts in human placenta. In this study, we have investigated the expression of EGF-R and c-erbB2 proteins by different trophoblast populations using immunohistochemistry. EGF-R was expressed by proliferative villous cytotrophoblasts, but not by non-proliferative, invasive, extravillous cytotrophoblasts. In contrast, c-erbB2 was expressed by invasive extravillous trophoblasts (EVT) but not by villous cytotrophoblasts. Expression of both receptors was seen only by the terminally differentiated placental bed giant cell and villous syncytiotrophoblast populations. This precise spatial expression suggests that EGF-R plays an important role in trophoblast proliferation whereas c-erbB2 may be important for trophoblast invasion and differentiation. The surface expression of these receptors on normal isolated first trimester trophoblasts and on choriocarcinoma cell lines was also investigated by flow cytometry, and compared with that of the c-erbB2-overexpressing mammary tumour cell line BT474. Isolated trophoblasts were shown not to overexpress c-erbB2, and no differences in either EGF-R or c-erbB2 expression were seen between normal and malignant trophoblasts.

Kit ligand, or stem cell factor, is a recently identified growth factor, which binds to and activates the c-kit proto-oncogene, and which has been shown to act synergistically with other haematopoietic growth factors in the bone marrow. We have previously shown that several isoforms of kit ligand, which arise due to alternative splicing, are expressed in human placenta. In order to elucidate the role of c-kit and its ligand during human placental development we have investigated the expression of c-kit and kit ligand in human first trimester and term placenta as well as in pregnant and non-pregnant endometrium, by immunocytochemistry and flow cytometric analysis. In non-pregnant endometrium no expression of kit ligand was seen. By contrast, in first trimester decidua, kit ligand was strongly expressed by the arterial media of maternal blood vessels. Kit ligand was also expressed throughout pregnancy by invasive fetal extravillous trophoblast, and by fetal fibroblasts within the placental villi. c-kit was found to be expressed on Hofbauer cells within the chorionic villi, and by decidual macrophages at all stages in pregnancy. c-kit was also detected on the small CD56dim subset of uterine large granular lymphocytes which form the major leukocyte population in human first trimester decidua. Our results suggest that kit ligand may be involved in the regulation of fetal macrophages, and in particular in signalling between invading extravillous trophoblast which expresses kit ligand, and maternal leukocytes bearing the c-kit receptor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a classical haematopoietic cytokine which has also been implicated in placental growth and development. In this study we have performed a detailed immunohistological localization of the low affinity GM-CSF receptor (GM-CSF-Rα) in human first trimester implantation site and non-pregnant endometrium. We have also investigated receptor expression and GM-CSF binding in vitro by normal first trimester trophoblast using flow cytometric analysis and compared this with JEG-3 and JAR choriocarcinoma cells. In the first trimester, the GM-CSF-R was found to be present on villous cytotrophoblast and all populations of extravillous trophoblast. Expression by villous syncytiotrophoblast was weak or absent, but this increased markedly by term. GM-CSF-R were also expressed by fetal Hofbauer cells within the mesenchyme of the chorionic villi and by uterine glandular epithelium and decidual macrophages within maternal decidua. GM-CSF-R was not expressed by glands in proliferative phase endometrium but began to appear during the secretory phase, suggesting hormonal regulation of the receptor on uterine glandular epithelium. Flow cytometric comparison of normal isolated first trimester trophoblast and JEG-3 and JAR choriocarcinoma cells revealed two- to threefold higher surface expression of GM-CSF-R by choriocarcinoma cells and higher binding capacity for rhGM-CSF than normal trophoblast. These results suggest that GM-CSF may regulate growth and development of human trophoblast. GM-CSF may also influence placental development and function by acting via decidual and fetal macrophages, and uterine glandular epithelium, which are the other cell populations to express the receptor.

The process of placental implantation involves a series of transformations of trophoblast from a single polarized epithelial layer resting on a basement membrane (villous trophoblast), to cellular aggregates (trophoblast columns) which ultimately disperse to invade uterine decidua as individual cells (interstitial trophoblast). Such tissue re-modelling is associated with changes in the constituents of the extracellular matrix and in the expression of matrix receptors by the cells, the most relevant being the family of integrins which bind to laminin and fibronectin. In this study we show, by immunohistology and flow cytometry, a gradual loss of laminin receptors with the concomitant acquisition of fibronectin receptors as trophoblast is transformed from the villous phenotype, through the cell columns, into the extravillous population. The pattern of staining for the alpha 5, alpha 6, beta 1 and beta 4 subunits indicates that the integrins expressed by trophoblast are predominantly the alpha 5 beta 1 and the alpha 6 beta 4 heterodimers. We have also shown that isolated trophoblast cells assume a flattened, sessile phenotype when cultured on laminin but exhibit a more spreading, motile morphology when plated on fibronectin. In addition, numerous multinucleated giant cells are observed on a fibronectin substrate. Our data suggest that the relative expression of laminin and fibronectin receptors may determine the morphology and behaviour of trophoblast during the process of implantation.

Human decidual large granular lymphocytes are capable of killing both normal trophoblast and choriocarcinoma cells after stimulation with recombinant interleukin-2 (rIL-2) in vitro. We now show that pre-treatment of normal trophoblast and JEG-3 choriocarcinoma cells with interferon-gamma (IFN-gamma) protects these target cells from killing by decidual effectors, although pre-treatment with interferon-alpha (IFN-alpha) had no such effect. In contrast, JAR choriocarcinoma cells were readily killed before and after exposure to both IFN-gamma and IFN-alpha. This protective effect might be due to upregulation of non-classical trophoblast HLA Class I molecules, as IFN-gamma but not IFN-alpha enhances Class I surface expression in both normal trophoblast and JEG-3 choriocarcinoma cells. JAR cells, however, are constitutively and inductively negative for Class I expression.

Human implantation is accompanied by extensive invasion of uterine tissue by trophoblast cells. Besides anchoring the placenta to the uterus, these cells also perform the important function of converting decidual spiral arteries from highly resistant vessels into flaccid sinusoidal tubes that are no longer responsive to vasoconstrictive influences, thus allowing the maintenance of an adequate blood flow to the fetoplacental unit under all physiological conditions (1). Normal pregnancy depends on this vascular adaptation by trophoblast, without which a variety of obstetrical problems, such as preeclampsia, intrauterine growth retardation, or still birth, will result (2). The factors that control this trophoblast invasion are not known, but are likely to be contributed by uterine cells or matrix proteins encountered en route by trophoblast during its migration. In this chapter we focus on the potential immunological or paraimmunological processes that could take place between trophoblast and the population oflarge granular lymphocytes (LGL) present in decidua because there are many characteristics of these two cell types that suggest they are able to interact with each other.

This paper describes a new Mab BC-1 which is directed at a cell surface antigen expressed by extravillous cytotrophoblast and not by villous cytotrophoblast, so it is useful for distinguishing between the two populations. The antigen recognised by BC-1 is trypsin-resistant, which allows it to be used for flow cytometric analysis of living trophoblast isolated by enzymic disaggregation. However, BC-1 is not trophoblast-specific but cross-reacts with some other tissues, in particular endothelial cells and peripheral blood monocytes. The nature of this antigen has not been established.

This review discusses the three important developments that are likely to have the greatest impact on our understanding of human reproductive immunology: the establishment of the trophoblast major histocompatibility complex class I antigen as HLA-G; the identification of the trophoblast-lymphocyte-crossreactive protein as a complement regulatory protein; and elucidation of the lineage and possible functions of decidual large granular lymphocytes.

In the formation of the hemochorial placenta, an essential feature is the elaboration of waves of migratory trophoblast which invade through the basal decidua to reach as far as the myometrium (Robertson, 1987). The functional significance of this migration is not known nor is it understood which factors influence this pattern of behavior. Control is likely to be multifactorial, and the different facets are addressed by other authors in this volume. The concern of this chapter is the role played by trophoblast antigens in the interaction between invasive trophoblast and uterine tissues. The hypothesis of the placenta being antigenically neutral has, in recent years, been modified by observations that the extravillous migratory population of trophoblast does in fact express antigens, although the exact nature of these molecules is unclear. The MHC-related and blood group-related antigens expressed by invasive trophoblast populations will be reviewed, and their role in trophoblast growth and survival within the uterine environment will be examined.

Leukocyte populations were studied at all stages of the menstrual cycle using a panel of monoclonal antibodies to natural killer cells, T cells, B cells, and macrophages on frozen sections of endometrium. At the time of implantation (mid-secretory phase) the majority of leukocytes appear to be Leu19+, CD16-, Leu7-, CD2+, CD3-, CD5-, CD7-, and HLA DR- with the morphology of large granular lymphocytes. The numbers of these cells showed a marked increase in the mid-secretory phase. These cells exhibited similar phenotypic characteristics to those found in decidua. These findings, therefore, suggest that the recruitment of these large granular lymphocytes to the uterus is under hormonal control and is not a local response to the presence of invading trophoblast.

A panel of monoclonal antibodies directed at fucosylated and sialylated carbohydrates on the Type 1 and Type 2 blood group precursor chains was used in an immunohistological study of trophoblast subpopulations. Formalin-fixed paraffin-embedded sections of normal trophoblast throughout pregnancy, hydatidiform moles and gestational choriocarcinoma were examined. Sialyl-Le x was localized in extravillous trophoblast populations in normal and molar pregnancies and choriocarcinoma tumour cells. After neuraminidase treatment, Le x was identified in similar cell populations. Although Le a was identified in syncytial trophoblast cells at the maternofetal interface, no staining was seen for sialyl-Le a . Villous trophoblast did not stain with any of the monoclonal antibodies used. Our results underline the divergent differentiation of trophoblast into villous and invasive extravillous subpopulations. The analogy with tumour cells is discussed.

The susceptibility of cultured first trimester human trophoblast cells to lysis by NK cells has been studied. Trophoblast cells are resistant to lysis by NK cells from both peripheral blood and decidua. However, decidual cells extracted by enzymatic disaggregation do exhibit cytotoxicity against the NK-sensitive cell line K562. The relevance of these findings to successful implantation of the blastocyst is discussed.

Laminin was localized by immunohistology to stromal cells of the human non-pregnant endometrium as well as to similar cells of the decidua. Only small amounts of laminin were detected during the proliferative phase, but significant quantities had accumulated by the mid-secretory phase which persisted in decidua if pregnancy occurred. This cyclical variation suggested hormonal control of laminin production. The presence of laminin could also be demonstrated in decidual cells in culture, most of which was intracellular or pericellular with very little liberated into the culture supernatant. Human first trimester trophoblast cells were observed to attach preferentially to laminin-coated surfaces in vitro. The role of laminin in trophoblast-decidua interaction in vivo is discussed.