Mol. Hum. Reprod. Advance Access originally published online on December 5, 2005
Molecular Human Reproduction 2005 11(10):695-698; doi:10.1093/molehr/gah196
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Editorial |
Does ‘soluble’ HLA-G really exist? Another twist to the tale
Abstract
HLA-G is thought to play a key role in implantation by modulating cytokine secretion to control trophoblast invasion and to maintain a local immunosuppressive state. It differs from other class I molecules in that the gene can be alternatively spliced to produce four membrane-bound (G1, G2, G3 and G4) and three soluble isoforms (G5, G6 and G7). The soluble isoforms have recently attracted attention as their levels may be diagnostic of poor trophoblast invasion in miscarriage or pre-eclampsia and the implantation potential of IVF embryos. Although the expression and function of HLA-G2, G3, G4 and G7 has previously been a matter of debate, until now it has been generally accepted that soluble HLA-G5 and HLA-G6 are both expressed and secreted by trophoblast. However, Blaschitz et al. (2005)
have reinvestigated this question and come to the surprising conclusion that they are not. They have shown that trophoblast only expresses the membrane-bound HLA-G1 isoform and not soluble HLA-G5 and -G6. Furthermore, although soluble HLA-G could be found in trophoblast culture supernatants, it appears not to be derived by alternative splicing but by the cleavage of HLA-G1. The source of the soluble HLA-G may not matter from a diagnostic perspective, but these findings, if confirmed, have important implications for our understanding of the biology of HLA-G.
It is now more than 20 years since it was first discovered that the extravillous cytotrophoblast, which forms the interface between placental and maternal tissue, has a unique pattern of class I major histocompatibility complex (MHC) antigen expression (Redman et al., 1984). It was shown that, although these cells expressed class 1 MHC antigens, they were not classical HLA-A or HLA-B antigens which are the primary stimuli for T cell-mediated graft rejection. Subsequent protein analysis revealed an MHC molecule distinct from classical class I in that it was non-polymorphic (Ellis et al., 1986), and sequencing showed it to be the non-classical class I molecule HLA-G (Ellis et al., 1990; Kovats et al., 1990). The nature of HLA-G and its tissue distribution strongly suggested that it might play a key role in preventing the trophoblast from being recognized as foreign and rejected by the mother’s immune system.
There is now considerable evidence that this is the case. HLA-G does not stimulate classical T cell responses but instead suppresses the activation of both CD4- and CD8-positive T cells, possibly through the induction of apoptosis (reviewed in Le Bouteiller et al., 2003). However, the actions of HLA-G are not confined to T cells, as it also interacts with cells of the innate immune system by binding to receptors expressed on natural killer (NK) cells and macrophages, both of which are found in the decidua. Although HLA-G may inhibit NK cell-mediated lysis, its primary role is thought to be the modulation of cytokine secretion by these cells to control trophoblast invasion and maintain a local immunosuppressive status (Le Bouteiller et al., 2003). It is now recognized that extravillous cytotrophoblast do not express HLA-G in isolation but it is co-expressed with HLA-E and HLA-C. These molecules also interact with NK cells and are involved in prevention of NK cytotoxicity and cytokine production as well (Trundley and Moffett, 2004).
HLA-G has membrane-bound and soluble isoforms
One key difference between HLA-G and other class I molecules is that the HLA-G gene can be alternatively spliced to produce a number of different isoforms. To date, seven HLA-G mRNA transcripts have been identified, which encode four membrane-bound isoforms (G1, G2, G3 and G4) and three soluble isoforms (G5, G6 and G7) (Ishitani and Geraghty, 1992; Paul et al., 2000). HLA-G1 is the full length isoform encoding the complete molecule, i.e. the 
1, 2 and 3 domains, the transmembrane region and the intracellular region of the class I heavy chain. The other HLA-G isoforms are alternatively spliced shorter transcripts lacking regions complementary to one or more entire exons. Thus, HLA-G2 lacks exon 3, corresponding to the 2 domain; HLA-G3 lacks exon 3 and 4 and thus, only has the 1 domain; and HLA-G4 lacks exon 4 and hence, the 3 domain. HLA-G5 and-G6 are equivalent to HLA-G1 and -G2, respectively, but are highly unusual in that, due to an incomplete splicing process, they retain intron 4 which contains a stop codon. This prevents the transcription of the anchoring transmembrane region, resulting in the expression of soluble proteins. Hence, they are also known as soluble HLA-G1 and HLA-G2. A further splice variant of HLA-G (HLA-G7) has also been reported (Paul et al., 2000). This isoform contains intron 2, which also has a stop codon, so that the resulting G7 protein would be a soluble HLA-G comprised of only the 1 region.
Soluble HLA-G in normal pregnancy
The existence of soluble forms of HLA-G extends the potential for its action. Not only could it be acting locally at the interface between extravillous cytotrophoblast and the maternal immune cells in the decidua, but it may also enter the maternal blood stream and act systemically to control maternal immune responses. Soluble HLA-G has been found in the serum of women in early pregnancy (Pfeiffer et al., 2000). However, the levels did not differ from those in preovulatory women, suggesting that soluble HLA-G was predominantly of maternal origin and may be a product of activated monocytes. Furthermore, soluble HLA-G has also been reported to be present in the circulation of men (Rebmann et al., 1999).
Evidence for a placental contribution comes from the observation that soluble HLA-G levels were found to increase during the early stages of gestation in twin pregnancies (Pfeiffer et al., 2000). It is unlikely that this is derived from the extravillous cytotrophoblast as the only form of these cells which comes into contact with maternal blood is the endovascular cytotrophoblast in the spiral arteries, and it is debatable whether there are sufficient of these to contribute significantly to a circulatory pool. However, the syncytiotrophblast, which forms the placental interface with the maternal blood and was previously thought to be class I MHC negative, has now been shown to express message for soluble HLA-G5 and-G6 and secrete soluble HLA-G in culture (Solier et al., 2002). It might be expected that if the syncytiotrophoblast is secreting soluble HLA-G into the maternal circulation, levels would increase with gestation, as for other placental products. However, this does not appear to be the case as Hunt et al. found that although soluble HLA-G levels were raised in 80% of pregnant compared to nonpregnant women, these levels did not differ significantly between trimesters (Hunt et al., 2000).
Soluble HLA-G as a marker of pregnancy failure or implantation success
If soluble HLA-G has a role in pregnancy survival, then its expression may be altered in conditions with poor trophoblast invasion such as miscarriage and pre-eclampsia. It has been shown that there is reduced HLA-G expression on the invasive cytotrophoblast in the deciduas of women who miscarry (Emmer et al., 2002), although others disagree (Patel et al., 2003), and that circulating soluble HLA-G levels are lower in pregnancies that undergo abortion at 9 weeks compared to normal pregnancy (Pfeiffer et al., 2000). Similarly, expression of HLA-G by extravillous cytotrophoblast in the implantation sites of term placentas from pre-eclamptic women is reduced compared to normal pregnancy (Goldman-Wohl et al., 2000), and there is a corresponding decrease in soluble HLA-G in both the placentas and circulation of pre-eclamptic women (Yie et al., 2004). These observations point the way to the use of serum soluble HLA-G measurements as a possible diagnostic tool for these disorders. However, it must be emphasized that reduced soluble HLA-G expression may be a consequence rather than the cause of these conditions.
Recently, these investigations have been extended to the beginnings of pregnancy, with a number of studies reporting that human IVF embryos secrete soluble HLA-G, and that the levels secreted in the early cleavage stages (day 2–3 postfertilization) are indicative of the potential of the embryo to implant (Fuzzi et al., 2002; Sher et al., 2004; Noci et al., 2005; Sher et al., 2005a, 2005b; Yie et al., 2005). IVF Pregnancy rates were found to be much higher if at least one embryo known to be secreting soluble HLA-G was transferred to the mother, compared to cases where none of the embryos produced soluble HLA-G. However, two other reports do not support these findings. Van Lierop and coworkers were unable to detect soluble HLA-G in follicular fluid or embryo culture supernatants from eight-cell, blastocyst and late blastocyst stages (Van Lierop et al., 2002), and Noriko et al. found no soluble HLA-G in 106 culture supernatants from day 3 embryos and blastocysts (Noriko et al., 2004). The reason for these discrepancies is not known, but may be due to differences in the specificity of the antibodies used in the enzyme-linked immunosorbent assays (ELISAs) to measure soluble HLA-G. This is an important issue to resolve, as if the positive findings are confirmed they have enormous implications for IVF treatment. Measuring soluble HLA-G could provide a much more objective way of selecting the ‘best’ embryos for transfer than is currently possible on the basis of morphology alone. Not only should it increase IVF success rates but it should also allow the number of embryos that are transferred to be reduced, possibly to single embryos, and thereby, avoid the problems associated with multiple pregnancies.
Is soluble HLA-G5 and -6 really secreted by native trophoblast?
There has been considerable debate about the function of the truncated isoforms of HLA-G, and in particular, whether they are expressed on the cell surface. Riteau et al. have reported that HLA-G2, -G3 and -G4 can be expressed on the surface of transfected cells (Riteau et al., 2001), thereby, protecting these cells from lysis by both NK and cytotoxic T lymplocyte (CTL) effector cells and in this way may contribute to fetal survival (Menier et al., 2000). However, we (Bainbridge et al., 2000), and others (Mallet et al., 2000; Ulbrecht et al., 2004) have found that although these truncated forms may be expressed inside the cell, there is no evidence for them reaching the cell surface. Similarly, although it has been possible to demonstrate the presence of HLA-G7 in extracts of transfected cells by western blotting, it could not be detected as a secreted protein (Paul et al., 2000). As no monoclonal antibodies specific for these isoforms are currently available, it is not possible to determine whether they are expressed on the surface of trophoblast or human embryos and have a functional role.
In contrast, until now it has been generally accepted that soluble HLA-G5 and HLA-G6 isoforms are both expressed and secreted by trophoblast. However, given the conflicting evidence which is emerging, Blaschitz et al. (2005) in their article, have reinvestigated this question and come to the surprising conclusion that they are not.
In a comprehensive and well-controlled study, they have examined the distribution of soluble HLA-G5 and -G6 isoforms in human first trimester and term placentas both in situ and in vitro, using immunohistochemistry, western blotting, RT–PCR and ELISA. Their main findings were as follows:
- Using a combination of antibodies which either react with all HLA-G isoforms or only with soluble HLA-G5 and HLA-G6 (intron-4 peptide-specific antibodies), they have shown that the expression of HLA-G is predominantly restricted to extravillous cytotrophoblast, and that the only isoform expressed is HLA-G1 and not HLA-G5 or -G6.
- Using western blotting, they found that tissues containing trophoblasts only express HLA-G1 proteins and not the HLA-G5 and -G6 isoforms.
- Although HLA-G5 and -G6 transcripts could be detected in isolated first trimester trophoblast preparations and term placenta, levels were extremely low.
- Although high levels of HLA-G could be found in trophoblast culture supernatants, no soluble forms containing the intron-4 derived peptide could be found.
Western blot analysis of these trophoblast culture supernatants suggested that the HLA-G present was derived not from secretion of the soluble forms but by cleavage of the full length membrane-bound HLA-G1 from the cell surface by metalloproteinases, as has been previously reported (Park et al., 2004); a mechanism which could be responsible for the generation of soluble classical class 1 molecules which are found in the serum of most individuals (Demaria et al., 1994). They, therefore, concluded that the ‘soluble’ HLA-G derived from native trophoblast is not derived by alternative splicing but by the cleavage of HLA-G1. This would not have been recognized previously as the ELISA systems used by others would not discriminate between the different forms.
This article brings into question the specificity of some of the antibodies and methods previously used to study soluble HLA-G and challenges the whole concept of soluble HLA-G5 and -G6 secretion. Although it can be argued from the diagnostic perspective that it does not matter how the soluble HLA-G is derived as long as it distinguishes between the normal and abnormal state, these findings, if confirmed, clearly have very important implications for our understanding of the biology of HLA-G. They will hopefully promote new debate and collaborations between groups working in this field to reconcile conflicting data and develop clinical assays with a sound scientific basis.
Nuffield Department of Obstetrics and Gynaecology, Oxford University, John Radcliffe Hospital, Headington, Oxford, UK
Acknowledgements
The author is member of ‘EMBIC’, a European Network of Excellence (www.embic.org) within the 6th Framework Programme of the European Union (LSHM-CT-2004-512040).
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