Trophoblast Research 8:331-337, 1994
T R O P H O B L A S T CELL S U R F A C E M O L E C U L E S : HLA-G AND REPRODUCTION - A Review -
Y.W. Loke, Ashley King, and Gill Chumbley Research Group in Human Reproductive Immunobiology Department of Pathology University of Cambridge Tennis Court Road Cambridge CB2 1QP, United Kingdom
H u m a n trophoblast cells express a wide variety of surface molecules which could have important functions. These are summarized in Table 1. This list is by no means exhaustive but serves to illustrate the different families of molecules present. It is quite clear from an examination of the Table that may of these, such as receptors and adhesion molecules, are ubiquitous and can be found in other cell types beside trophoblast. Interest in them, therefore, centers on the question as to whether they serve a similar function in trophoblast as in other cells. There are, however, some which appear to be restricted to trophoblast. Elucidation of their functions, therefore, would contribute more significantly to our knowledge of trophoblast biology. The two which stand out in this regard are the MHC antigen, HLA-G, and the enzyme placental alkaline phosphatase (PLAP). For this review, I will focus on HLA-G because its identification has completely transformed our conceptual view of the human placentaluterine relationship. The story of HLA-G began in the early 1980's (cf., Loke, 1989; Loke and King, 1991) when it was observed that extravillus trophoblast expresses an HLA Class I antigen in contrast to villus trophoblast which does not (Redman et al., 1984). This finding puzzled reproductive immunologists because it indicates that of the two interfaces between placental and maternal tissues, the villus trophoblast population bathed in maternal blood is devoid of HLA antigen while the extravillus population in contact with maternal uterine tissues expresses HLA Class I molecules. In terms of maternal recognition of the placenta, it seems that the systemic immunity encounters no classical allogeneic signals but the local uterine immune system can potentially by stimulated by fetally-derived HLA Class I. There is, thus, intense interest in the nature of this extravillus trophoblast Class I molecule. Initial investigations of this trophoblast Class I antigen revealed it to differ from a classical HLA Class I molecule in several important respects. The trophoblast antigen is found to be a non-polymorphic glycoprotein with a heavy chain of only 39KD molecular weight associated with 132 microglobulin (Ellis et al., 1986) and the antigen appears to be located predominantly inside the cell rather than on the cell surface (Grabowska et al., 1990a). Interestingly, in situ hybridization with a full length HLA Class I cDNA probe showed that villus cytotrophoblast cells contains Class I mRNA
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although it does not express the protein (Hunt et al., 1990). Thus, while immunocytochemistry with an anti-Class I Mab distinguishes between negative and positive trophoblast populations, mRNA localization recognizes three phenotypes: 1) villus syncytiotrophoblast (mRNA-, protein-), 2) villus cytotrophoblast cells(mRNA § protein-), and 3) extraviilus trophoblast cells (mRNA § protein§ The control of trophoblast Class I expression, therefore, appears to occur at different cellular levels according to the population. Subsequent investigations eventually established this unusual extravillus trophoblast Class I antigen to be a product of the non-classical Class I locus, HLA-G. Ellis and her colleagues (1990) observed that a clone isolated from a cDNA library from the BeWo choriocarcinoma cell line had nucleotide sequences with a high degree of homology to HLA-G, and polymerase chain reaction amplification demonstrated that similar sequences could be found in normal term chorionic plate trophoblast cDNA. Evidence that HLA-G codes for an expressed protein was provided by Kovats et al. (1990) who reported that immunoprecipitation of normal first trimester extravillus trophoblast with W6/32 resulted in a similar array of molecules on two-dimensional gels as found in HLA-G transfectants. Analyses from several individuals showed that the heavy chain of HLA-G is not obviously polymorphic. Recently, we have constructed a 23-mer HLA-G specific oligonucleotide and have confirmed by in situ hybridization and by Northern blotting the first trimester extravillus trophoblast cells, including cells of the cytotrophoblast columns, interstitial trophoblast, and endovascular trophoblast cells all contain abundant HLA-G transcripts (Chumbley et al., 1993). Table I Human Trophoblast Cell Surface Molecules MHC Antigens
Blood Group Related Carbohydrates
Complement Regulatory Proteins
MCP (CD46) DAF (CD55)
Fc~R Transferrin R GM-CSFR
Cadherin Ig Superfamily Integrin
c-fms (CSF-1R) c-erbB-1 (EGFR)
Defined by mAbs
FDO 161 G Trop 14 GB 17, 25
Trophoblast HLA-G and Reproduction
Wei and Orr (1990) found that HLA-G transcripts are present only in extraembryonic tissues such as the placenta and placental membranes after analyzing a variety of human tissues, and Kovats et al. (1990) have shown that the amount of HLA-G expressed by third trimester trophoblast is greatly reduced compared to first trimester. Taken together, these observations would seem to suggest that HLA-G has a pregnancy related role to play, possibly in early placental development. However, there have since been other reports that HLA-G may also be expressed by cells of the fetal eye, fetal thymus (Shukla et al., 1990), and fetal liver (Houlihan et al., 1992) so that the tissue distribution of this antigen may not be as restricted as originally thought. However, the expression of HLA-G in fetal thymus and liver would mean that maternal T cells are tolerant to this organ specific molecule. N o w that it is generally accepted that the HLA Class I antigen expressed by extravillus trophoblast is HLA-G, current research is directed at elucidating its function. There are immunologists who are of the opinion that non-classical HLA Class I antigens, such as HLA-G, have no function and are merely vestigial structures left behind by evolution. Many of these features, like lack of polymorphism and low surface expression, are considered to be those of a gene in decline (Lawlor et al., 1990). However, it could be argued that the identical HLA-G structure in all individuals has been conserved for some important placenta related function. The nucleotide sequences of this gene have now been analyzed from four clones derived from cell lines with unrelated HLA haplotypes and a remarkably high degree of homology has been noted (Pook et al., 1991). If HLA-G were inactive, some degree of variation would be expected as a result of random mutation. Thus, there seems to have been strong evolutionary pressures for HLA-G to retain the same structure in different individuals which is in contrast to the selective forces driving the generation of polymorphism in the classical HLA Class I antigen. Interestingly, studies of the Class I genes of the New World tamarin monkey have shown that its Class I sequences are more closely related to human non-classical HLA-F and HLA-G than to classical HLA-A,B,C (Watkins et al., 1991). Furthermore, the tamarin HLA-F homologue has a higher rate of nonsynonymous compared to synonymous substitutions at the antigen-recognition site, but this is not found in the HLA-G homologue. This suggests that our ancestral HLA-F had been selected for variability and was once functional for T cell recognition, whereas our ancestral HLA-G had not been selected for this function and could be conserved for a different purpose in humans. HLA-G, therefore, could have an organ-specific and species-specific function. If it is accepted that HLA-G does have a function, the next question that arises, at least with respect to immunology, is what kind of maternal lymphocytes are likely to recognize this monomorphic molecule. Analysis of the leukocyte populations in the h u m a n uterus reveals the surprising finding that this organ has very few T lymphocytes and virtually no B lymphocytes (Loke et al., 1992). Instead, it is infiltrated by large numbers of large granular lymphocytes (LGL) with the unusual phenotype of CD56bris h~, CD16", CD3-. They comprise over 70% of the bone marrow derived cells in decidua (King et al., 1991). These LGL vary in number through the menstrual cycle, being particularly numerous at the secretory phase of the non-pregnant endometrium and, if pregnancy occurs, remain at a high level in decidua (King et al., 1989). By immunohistochemical examination of the implantation site, LGL can be seen to be in close contact with infiltrating trophoblast so that there is both a temporal as well as a spatial relationship between these two cell types. Because of this, we have suggested that decidual LGL are potential effectors against extravillus trophoblast and
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could control the extent of trophoblast invasion into the uterus (King and Loke, 1990). Furthermore, we have recently put forward the proposal that the trophoblast HLA-G antigen provides a degree of protection against decidual LGL attack (King and Loke, 1991). The rationale of this hypothesis is based on the "missing self" concept of Natural Killer (NK) cell recognition (Ljunggren and Karre, 1990) which states that, unlike T cells which kill target cells bearing "non-self" antigens, NK cells will lyse target cells which have low or absent HLA Class I. The NK recognition system, therefore, is very different from the T cell recognition system and probably represents a more primitive innate defense mechanism which preceded the evolution of the more sophisticated T cell adaptive immune response. We believe that decidual LGL belongs to this innate defense system and the expression of the monomorphic HLA-G by trophoblast is a means of evading destruction by these effectors. The above hypothesis is testable in vitro. We have observed that up regulation of trophoblast HLA Class I by IFN-y (Grabowska et ai., 1990b) does partially protect trophoblast from decidual LGL cytolysis (King and Loke, 1992) while treatment with IFN-(z which has no effect on trophoblast Class I expression (Chumbley et al., 1991) is not protective, thereby providing indirect support that the amount of trophoblast Class I expression and susceptibility to decidual lysis is inversely proportional. We have now used a more direct approach by constructing HLA-G transfectants and have observed that these cells are less susceptible to lysis by decidual LGL than their HLA-null parental cell line (Chumbley et ai., 1994). So far, we have discussed only one possible function for HLA-G, that of a protected molecule against decidual LGL cytolysis. There are other possibilities such as HLA-G triggering decidual L,GL to synthesize cytokines which could influence trophoblast growth and differen~tiation. Alternatively, HLA-G may act as a cell-cell adhesion molecule with signal transduction capabilities. All these functions are now being investigated in our laboratory. Because of the unusual characteristics of trophoblast HLA-G and decidual LGL, the overall conclusion is that the human placental uterine relationship is not the same as the graft-host relationship and, therefore, cannot be explained by the laws of transplantation immunity. Instead, we firmly believe that reproduction involves a much more primitive defense system and it is cellular interactions within this system that will influence the development of the placenta. SUMMARY This review focuses on HLA-G as an example of a cell surface molecule expressed by h u m a n 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 d i f f e r e n t i a t i o n i It would appear that reproduction involved immunological parameters which have yet to be defined.
Trophoblast HLA-G and Reproduction
ACKNOWLEDGEMENTS We gratefully acknowledge grants from East Anglian Regional Health Authority, Sir Halley Stewart Trust, Isaac Newton Trust, Medical Research Council, Wellcome Trust and Special Programme of Research Development and Research Training in Human Reproduction, World Health Organization. REFERENCES
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Trophoblast HLA-G and Reproduction
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