1 Department of Blood Transfusion and Transplantation Immunology, 2 Department of Obstetrics and Gynaecology and 3 Department of Pathology, University Medical Centre Nijmegen, 6500 HB, Nijmegen and 4 Department of Obstetrics and Gynaecology, Academic Medical Centre Amsterdam, 1105 AZ Amsterdam, The Netherlands
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Abstract |
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Key words: ectopic pregnancy/HLA-G/human/recurrent miscarriage/trophoblast
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Introduction |
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Of particular interest is the MHC-derived (non-classical) class Ib molecule HLA-G. HLA-G is expressed at the placental interface in particular by the trophoblast cells and it is the major HLA molecule with prolonged and significant levels of protein expression. HLA-G is most likely co-dominantly expressed (Hiby et al., 1999) and differs from the classical class I molecules such that it shows low polymorphism at the protein level, a short cytoplasmatic tail, extensive post-transcriptional alternative splicing and protein expression which is highly restricted.
Early studies using HLA-G-specific mRNA probes revealed the presence of HLA-G message in both first trimester villous and extravillous cytotrophoblasts, whereas villous syncitiotrophoblasts were negative (Yelavarthi et al., 1991). Staining with antibodies specific for class I molecules confirmed the presence of class I proteins in all extravillous, and absence in villous, trophoblast populations (Shorter et al., 1993
). Antibodies were generated against various HLA-G epitopes resulting in recognition of overlapping trophoblast populations (McMaster et al., 1995
; Yang et al., 1996
; Loke et al., 1997
; McMaster et al., 1998
). Reactivity against villous core components appears to be the major difference of the putative HLA-G-specific antibodies. With regard to the restricted expression, it has recently been reported that HLA-G protein might be expressed in malignant tissue (Cabestre et al., 1999
) and thymus (Mallet et al., 1999
) as well.
In-vitro studies have shown that the cytotoxic reactivity of uterine NK and T cells against HLA-G expressing target cells of various origin was inhibited (Rouas Freiss et al., 1997; Le Gal et al., 1999
; Rajagopalan and Long, 1999
). This suggests that the interaction between HLA-G and immunocompetent cells at the placental interface could be critical in determining the outcome of pregnancy. In this regard, it may be helpful to look for deviations in these interactions by studying early pregnancy disorders of unknown aetiology.
It has been estimated that 1.0% of reproducing couples will suffer from recurrent unexplained terminations of early pregnancies (Regan, 1992). In case of ectopic pregnancy most implantation occurs in the tubal duct (Solima and Luciano, 1997
). Both disorders occur during implantation/placentation when trophoblast proliferation and differentiation is most crucial. It is therefore quite conceivable that an altered expression of HLA-G might be associated with such pregnancy failure.
To assess the validity of this hypothesis, we investigated HLA-G protein expression in combination with the distribution of maternal NK cells in tissue obtained from recurrent spontaneous miscarriage and ectopic pregnancy.
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Materials and methods |
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A group of nine women with a history of recurrent spontaneous miscarriages was studied; a population in part previously described, but without overlap in material studied (Nelen et al., 1997, 1998
; Emmer et al., 2000
). A miscarriage was defined as a spontaneous pregnancy loss before 16 weeks menstrual age. Only women with a history of at least two consecutive spontaneous idiopathic miscarriages before 16 weeks menstrual age with the same partner were included. Women were excluded in cases of chromosomal rearrangement in either partner, Lupus coagulant, anti-cardiolipin antibodies, or medication used to treat disorders other than vitamin B6 and B12 deficiencies, diabetes mellitus, polycystic ovarian syndrome or thyroid dysfunction. Women received folic acid supplementation (0.5 mg daily) over a period of at least 2 months preceding conception. In these nine women, trophoblastic tissue samples were collected after spontaneous miscarriage or curettage of non-vital pregnancies diagnosed by ultrasonography. These nine women had experienced on average five miscarriages (range three to nine) with the index pregnancy being the most recent pregnancy. The mean menstrual age of this miscarriage tissue was 8.9 weeks (range 4.314.6).
We finally obtained tissue from five ectopic tubal pregnancies by means of tubal extirpation at a mean menstrual age of 6.9 weeks (range 5.68.3).
The Institutional Review Board of the University Medical Centre Nijmegen approved the study and women gave written informed consent, before participation.
Immunohistochemisty
To reveal HLA-G expression and NK cell distribution in the tissues, we employed an immunohistochemical method using formaldehyde-fixed tissue slides and HLA-G and NK cell-specific monoclonal antibodies. Tissues were dissected and snap-frozen in liquid nitrogen or fixed for 24 h in 4% neutral buffered formaldehyde and embedded in paraffin using an automated tissue processor (Tissue-Tek® VIP150; Sakura) under standard conditions for surgical biopsies. Sections of 4 µm were cut, mounted on SuperFrost/Plus glass slides (Menzel-Gläser, Germany) and dried overnight at 37°C. HLA-G expression and NK cell antigens were demonstrated by standard immunohistochemical procedures using antibodies described in Table I.
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We set out to compare the staining pattern of the CAM5.2 antibody, a pan-trophoblast marker to the staining pattern of the HLA-G-specific monoclonal antibodies 4H84, 87G, 16G1 and BFL.1. Because of the extremely small size of the (miscarriage) tissue, we had no choice but to use formaldehyde-fixed preparations. It thus appeared that only the 4H84 antibody gave significant staining of trophoblast cells with acceptable levels of background staining. The 4H84 antibody was induced against a peptide derived from the -1 domain and should therefore recognize all HLA-G isoforms (McMaster et al., 1998
). Staining intensity of the different antibodies was scored independently by two pathologists. The following subjective index was used: , no staining; +, some cells positive some negative; ++, strongly positive; +++, very strongly positive; absence of cell populations was scored as not present. Digital images were recorded using a Leica Dialux transmission microscope (Leica, Wetzlar, Germany) equipped for digital image capture with a 3CCD colour video camera (DXC-950P; Sony, Tokyo, Japan) and a computer-interfaced frame grabber (Intellicam; Matrox Electronic Systems, Dorval, QC, Canada). To quantify differences in HLA-G expression, the luminosity of the digital images was analysed by means of Adobe® PhotoShop® 5.5 software. In images of serial sections stained with 4H84 and CAM5.2, rectangular selections were superimposed, inverted to negative and the average luminosity determined. Luminosity ratios of 4H84/CAM5.2 were calculated to exclude effects of background staining.
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Results |
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HLA-G and NK cell expression in recurrent miscarriage tissue
In all preparations where extravillous trophoblast cells were present, HLA-G was expressed in tissue obtained from recurrent miscarriage (Figure 2, Table III
). At places where the fetal villi anchored to the decidua, a cell column of proliferating cytotrophoblast can be seen in which we observed a gradient of increasing staining intensity for 4H84 towards the decidua. Upon contact of the cell column with the decidua, cells migrate into the decidua and form a population of intermediate extravillous trophoblast that differentiate into intramural and endovascular trophoblast cells, all staining for 4H84. Staining of the placental bed giant cells could not be elucidated due to their absence caused by the nature of the miscarriage material. Thus the 4H84 staining pattern of the observed extravillous trophoblast populations in miscarriage tissue resembled that of the hysterectomy preparation. However, when looking at the staining intensity we observed a marked difference. For this purpose we compared in serial sections the ratio of staining intensities of 4H84 to CAM5.2 between both miscarriage and hysterectomy tissue. While the CAM5.2 staining intensity was similar between the preparations (compare Figure 1A
frame C with Figure 2
frame C), remarkably, in material obtained after miscarriage, the ratio between 4H84 and CAM5.2 staining at the anchoring site of the villi appeared decreased as compared with the hysterectomy preparation (compare Figure 1A
staining ratio of frames B and C to Figure 2
staining ratio frames B and C). We observed this phenomenon in all preparations where anchoring villi were present. In an effort to quantify this decrease, we analysed the luminosity of the (inverted) digital images by means of Adobe PhotoShop 5.5 software. This resulted in a mean luminosity ratio of 4H84/CAM5.2 staining of 0.91 in recurrent miscarriage (n = 6) and 1.11 (n = 11) in hysterectomy preparations and decidual biopsies resulting in a significant difference in the ratio of 0.20 (T-test P < 0.018). This clearly suggests a decreased expression of HLA-G in tissue obtained after recurrent miscarriage.
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Whilst DJ130c staining of lymphocytes in hysterectomy material was restricted, in miscarriage tissue lymphocytes localized in decidua basalis were abundantly stained (Figure 2, frame E). Most surprisingly, in three out of nine tissue samples we also found CD16+ (CD56) cells that had infiltrated the chorionic villi. Whereas in the hysterectomy preparation 1B6 staining of decidual lymphocytes was low, in contrast, in miscarriage tissue both 1B6 and DJ130c strongly stained the decidual NK cells. This altered NK cell phenotype could point to a disturbed interaction between trophoblast and NK cells and might be caused by the decreased expression of HLA-G.
HLA-G and NK cell expression in ectopic pregnancies
We observed that HLA-G was expressed on all extravillous trophoblast cell populations in ectopic pregnancy (Table IV). Concerning expression of HLA-G in ectopic trophoblast, due to the absence of a decidual layer it was difficult to compare the morphology of the `tubal placental bed' with that of an uterine pregnancy (compare Figure 3
, frame A with Figure 1A
, frame A). Although we could recognize structures similar to the anchoring villi, we also observed marked differences. Most striking was the observation that pre- and end-stages of trophoblast differentiation seem to be present in close proximity to each other. Multinucleate giant cells, positive for 4H84 (Figure 3
, frames A and B see arrowheads), resembling the placental bed giant cells in the hysterectomy preparations were found intermingled with the highly proliferative cytotrophoblast of the cell column (Figure 3
, frames A and B area marked `cc'). In analogy with the placental bed giant cells, these multinucleate giants cells could represent an end stage in trophoblast differentiation. To find these pre- and end-stage trophoblast cells at the same place suggests a deranged regulation of trophoblast differentiation in ectopic pregnancy. Furthermore there were 4H84+ cells that could be identified as endovascular trophoblast cells present in lacunae that resembled blood vessels. This could indicate that although trophoblast differentiation was changed, these cells are in part still able to effect their putative function, possibly through the expression of HLA-G.
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Discussion |
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Although we observed no differences in the distribution of HLA-G over the different populations of trophoblast cells, we here present data suggesting that HLA-G expression intensity was decreased in trophoblast tissue from first trimester recurrent miscarriage. This decrease in HLA-G protein was paralleled by the unusual presence of CD16+ lymphocytes. Expression of the CD16 marker is limited in healthy late secretory endometrium and during normal early pregnancy (Geiselhart et al., 1995; Quenby et al., 1999
) and therefore the presence of CD16 in miscarriage tissue might indicate a disturbed interaction between NK cells and the semi-allogeneic trophoblast cells. The limited variation of the HLA-G antigen binding site formed by the
-1 and
-2 domains suggests that the HLA-G molecule should always be recognized by the putative receptor on maternal effector cells such as NK cells (Hiby et al., 1999
). Inadequate signalling is most likely due to the absence or expression of sub-threshold levels of HLA-G or its putative receptor. This is in part confirmed by a recent study on the in-vitro interaction between HLA molecules expressed by tumour cells and NK cells, in which tumour cells lacking appropriate HLA class I expression induce infiltration, cytotoxic activation and transcription of IFN-
in NK cells (Glas et al., 2000
). Obviously, HLA-G is not the sole ligand for uterine NK cells. HLA-E is another class I molecule expressed on placental tissues, that interacts with these cells (King et al., 2000
). We have no data on its expression level in the tissues studied but it is clear that its expression is influenced by HLA-G expression levels. The HLA-G leader peptide is considered a very strong binder to HLA-E and stabilizes the molecular complex (Lee et al., 1998
). Thus HLA-G also indirectly interacts with uterine NK cells via HLA-E. The increase in expression of the CD16 antigen in miscarriage tissue is in concert with a flow cytometric analysis of endometrial biopsies (Lachapelle et al., 1996
) which found an increase in the CD16+ uterine NK cells in late secretory endometrium of women with a history of recurrent miscarriage. Another immunohistochemical study showed that (non-pregnant) endometrial biopsies from women who subsequently miscarried had elevated numbers of CD16+ cells (Quenby et al., 1999
). Regarding the CD16 expression in some chorionic villi in miscarriage tissue, it could be argued that this reflects non-specific binding to the Fc
receptor-bearing Hofbauer cells of fetal origin. We never saw any non-specific staining of the chorionic villi with our (IgG1) control antibodies. This supports our view that these cells are of maternal origin and have infiltrated the fetal villi. An important issue to be resolved in future studies is whether changed HLA-G expression and NK cell phenotype are related to inadequate trophoblast invasion or the result of embryonic/fetal death. However, this will be difficult, as trophoblast function and embryonic/fetal viability are intimately linked (Cross, 2001
).
The expression of HLA-G in ectopic trophoblastic tissue as described in this study is in tune with previous reports (Loke et al., 1997; Goldman-Wohl et al., 2000
; Rabreau et al., 2000
) and could indicate that the expression of HLA-G is regulated autonomically. The lack of NK cells at the tubal implantation site as demonstrated by this study and by Proll et al. suggests that recruitment of NK cells is not affected by HLA-G (Proll et al., 2000
). However, the reported deranged differentiation of trophoblast in ectopic pregnancy is in concert with the unlimited spontaneous trophoblast growth, resulting in tubal rupture. The observation that blood-filled lacunae and endovascular trophoblast are present in tubal pregnancies might indicate (functional) remodelling of tubal blood vessels by trophoblast cells. In this regard it is interesting that, in a recent study, s.c. injection of HLA-G-expressing chorion carcinoma cells into nude mice led to the formation of similar blood-filled lacunae instead of neo-vascularization found after injection of non-trophoblastic tumour cells (Grummer et al., 1999
). This supports the current view that HLA-G expression on trophoblast cells might play a role in the remodelling of spiral arteries and the establishment of a haemochorial placenta (Colbern et al., 1994
; Hara et al., 1996
). Finally, it stresses the need for strict regulation of trophoblast differentiation, presumably imposed by decidual NK cells or stromal cells.
In the present study, we have shown that in normal pregnancy the expression of the common NK cell antigen CD56 was decreased upon interaction with HLA-G-expressing trophoblast cells. In contrast, we have found that a decreased HLA-G expression in trophoblastic tissue obtained from women with a history of recurrent miscarriages was paralleled by an increased expression of both peripheral and uterine NK cell antigens. Finally, we have shown in ectopic pregnancies that expression of HLA-G was switched on in the absence of a decidual layer, associated with disturbed trophoblast differentiation. These findings are in concert with the hypothesis that bi-directional regulation of infiltrating fetal cells and maternal immune response is needed for successful pregnancy. The full extent of this relationship should be further elucidated.
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Acknowledgements |
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Notes |
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References |
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Submitted on February 5, 2001; resubmitted on September 17, 2001; accepted on November 29, 2001.