Molecular Human Reproduction, Vol. 7, No. 2, 185-194,
February 2001
© 2001 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
Fgl2 prothrombinase expression in mouse trophoblast and decidua triggers abortion but may be countered by OX-2
1 Department of Medicine, Pathology & Molecular Medicine, and Department of Obstetrics & Gynecology, McMaster University, 1200 Main St West, Room 3V39, Hamilton, Ontario, Canada L8N 3Z5, and 2 CIHR Group on Mechanisms of Organ Injury, Toronto Hospital, University of Toronto, Toronto, Ontario, Canada
Abstract
Spontaneous abortion of normal karyotype embryos in mice and in humans is associated with an increase in uterine T helper (Th) 1 type proinflammatory cytokines, tumour necrosis factor (TNF)-
, interferon-
and interleukin (IL)-1, and a deficiency of Th2/3 type cytokines, IL-4, IL-10, and transforming growth factor (TGF)-ß2. In mice, Th1 cytokines up-regulate a novel prothrombinase, fgl2, which via thrombin, leads to activation of polymorphonuclear leukocytes that terminate the pregnancy. Here we show that Th1 cytokines up-regulate fgl2 mRNA in fetal trophoblast and secondary decidua of CBA/JxDBA/2 and CBA/JxBALB/c matings, and promote fibrin deposition. This pattern is accompanied by a high rate of abortion. However, the spontaneous abortion rates in abortion-prone CBAxDBA/2 matings and in low abortion rate CBAxBALB/c matings were significantly lower than that expected from the frequency of implantations with high levels of fibrin and fgl2 mRNAhi. As the glycoprotein OX-2 occurs in the pregnant rat uterus and can deviate cytokine responses to Th2/3, we investigated OX-2 in pregnant CBA/J mice. We found OX-2 mRNA was present at the same sites as fgl2 mRNA, but was reduced in response to Th1 cytokines. Furthermore, anti-OX-2 raised the abortion rate to predicted levels, while recombinant OX-2 dramatically reduced the abortion rate. Fgl2 prothrombinase may provide a mechanism explaining pregnancy loss, and conversely, successful pregnancy may be due in part to OX-2-dependent activation of maternal tolerance mechanisms at the feto–maternal interface.
abortion/Fgl2 prothrombinase/mouse embryo/OX-2
Introduction
In humans, ~50% of otherwise unexplained recurrent pregnancy loss may be attributed to chromosome abnormalities in embryonic trophoblast (Coulam et al., 1996
; Stern et al., 1996). Immunological factors have been implicated in the recurrent loss of normal embryos, in particular, a T helper (Th)-1>Th2 cytokine pattern in blood and endometrium with deficient Th2/3 cytokines in early pregnancy decidua, infiltration of endometrium by classical natural killer (NK) cells, and procoagulant states (Lea et al., 1992; Chao et al., 1995; Lachapelle et al., 1996; Coumans et al., 1999; Yamamoto et al., 1999a, b; Lim et al., 2000; Younis et al., 2000). In the CBAxDBA/2 mating model of murine recurrent pregnancy loss, the role of infiltrating NK cells and macrophages appears to be production of the proinflammatory cytokines, interleukin (IL)-1, tumour necrosis factor (TNF)-, and interferon (IFN-) (for reviews, see Clark et al., 1999a; Clark, 1999a). Cytokine production in mice appears to be under the regulation of trophoblast-recognizing Th1 and Th2/3-type 
T and NKT cells (Arck et al., 1999; Clark, 1999b). The mechanism of abortion involves direct cytokine-triggered up-regulation of a novel mouse fibrinogen-related prothrombinase, fgl2, originally described as the basis for liver necrosis in MHV-3-virus-induced hepatitis (Ding et al., 1997; Clark et al., 1998). Thrombin generated via fgl2 leads to fibrin deposition, complement activation, and activation of polymorphonuclear leukocytes (PMNL) that appear to terminate the blood flow to the developing placenta (Heinz and Loos, 1983; Clark et al., 1998, 1999ab). This is important, since trophoblast is resistant to direct killing by most effector cells of 'classical' allograft rejection, and lymphokine-activated killer cells, the only type of effector cell capable of lysing culture-conditioned trophoblast, are not found in human miscarriage decidua (Vassiliadou and Bulmer, 1998; Clark, 1999a)
The frequency of chromosomal abnormalities in rodent embryos is low, <6% (de Boer et al., 1991), and laboratory mice provide a useful model in which to search for mechanisms that might account for the 30–50% loss of normal karyotype human embryos in sporadic or recurrent miscarriage patients (Coulam et al., 1996; Stern et al., 1996). In this paper, we investigate the site of cytokine-mediated up-regulation of fgl2 at individual implantation sites in abortion-prone CBAxDBA/2 mated mice, and relate this to the observed rate of failure. A discrepancy between the predicted and observed rates of abortion led to investigation of the role of expression of OX-2, a tolerance signal that promotes generation of IL-10 and TGF-ß-producing antigen-specific T cells (Gorczynski et al., 1997, 1998). Similar CD4–8– double negative (DN) suppressor cells developing in pregnancy decidua 4–5 days after implantation have been implicated in reducing the abortion rate in the CBAxDBA/2 mating system (Arck et al., 1997a; Clark et al., 1999a). CBAxBALB/c matings have a low spontaneous abortion rate (compared with CBAxDBA/2 matings) and this is explained by activation of maternal CD8+ suppressor T (Ts) cells earlier in pregnancy, at the time of implantation or before (Clark et al., 1994a; Arck et al., 1996; Chaouat and Menu, 1997; Clark, 1999a). These CD8+ cells are V1+ Ts which produce IL-10 and an inhibitor of NK cell activation in response to progesterone, and inhibit the Th1 response at the outset, unless inactivated by 'stress' (Arck et al., 1996; Clark, 1999b). In this mating combination, the frequency of implants showing fgl2 up-regulation proved to be low. Nevertheless, a similar discrepancy between the expected and observed rate of resorptions was noted and could be partially abrogated by anti-OX-2.
Materials and methods
Mice
CBA/J female, DBA/2 male and BALB/c male mice, aged 6–8 weeks, were obtained from the Jackson Laboratory, Bar Harbor, Maine, USA, and housed under clean cage (barrier air flow) conditions at a controlled temperature (22°C) and 12 h light:12 h dark cycle with food (Lab Diet, PMI Feeds Inc, St Louis, MO, USA) and sterilized water ad libitum. After the females had reached 8–10 weeks, they were mated overnight with a male (one male to four females), and the morning of sighting a vaginal plug was designated as day 0.5 of pregnancy. Pregnant females were injected on day 7.5, where appropriate, with 0.1 ml phosphate-buffered saline (PBS) containing 2000 IU TNF- and 1000 IU IFN- (Clark et al., 1998, 1999b). In certain experiments, the mice were injected at different times during gestation with 0.2 ml containing 200 µg monoclonal rat anti-OX-2 Tg6 monoclonal antibody (Ragheb et al., 1999) (bioSPARK; CAN/LAB, Mississauga, Ontario, Canada); in some studies, hybridoma culture supernatant was generated in our own laboratory (given in two doses of 200 µl i.p., total dose 200 µg antibody) to reduce the risk of bacterial (endotoxin) contamination which could boost abortion rates. Polyclonal rabbit anti-fgl2-peptide immunoglobulin G (IgG) neutralizing antibody was also given (Clark et al., 1998) in daily i.p. injections (22 µg/day IgG containing 6 µg/day fgl2-specific IgG) beginning on day 6.5 of gestation. The recombinant OX-2 protein OX-2:Fc, was made in a Baculovirus system using Fc mutated to prevent Fc receptor binding and complement activation, as described elsewhere (Gorczynski et al., 1999a), and from a dose–response study and testing on different days of gestation, was given i.p. in a single 200 µl injection at a dose of 35 µg. Abortion (resorption) rates were evaluated on day 14.5 of gestation. All animals were handled strictly according to institutional (CCAC) guidelines.
For histological studies, the whole uterus containing embryos was isolated from euthanized mice on day 8.5 of gestation and placed in 4% buffered paraformaldehyde (freshly prepared). After 24 h, the fixed uteri were transferred to 70% ethanol until processing. The initial step in processing was to cut across the uterine horn between implantation sites, which were obvious bulbous swellings. The individual implantation sites were then oriented with their lumen perpendicular to the plane of a standard cassette and kept under 70% ethanol until processing through 100% ethanol and 100% xylene for paraffin embedding. All of the implantation sites from an individual mouse were placed in the same cassette. Serial sections were then cut and affixed to Aptex®-coated glass slides. Every 10th section was stained with haematoxylin and eosin to allow identification of which sections contained embryo tissue; not all implantation sites in a particular section would have embryo tissue. Based upon the light microscopic survey, sets of slides containing serial sections were selected for further processing.
In-situ hybridization and immunohistochemistry
Sections of mouse uterus were stained for fibrin using a purified immunoglobulin fraction of rabbit anti-human fibrinogen (Dako, Cedarlane Laboratories, Hornby, Ontario, Canada) (Ding et al., 1997). This antibody is known to react with mouse fibrinogen, and fibrin can be recognized as focal granular deposits and strands within tissues (Ding et al., 1997). After reacting with biotinylated mouse anti-rabbit IgG and streptavidin–peroxidase, 3,3'-diaminobenzidine chromogen reagent was used to label the tissue sections. Haematoxylin was used as a counterstain. Adjacent sections were processed by in-situ hybridization.
In-situ hybridization for fgl2 mRNA was carried out using digoxigenin-11-UTP-labelled cRNA probes (sense and anti-sense) synthesized with T7 and T3 RNA polymerase after subcloning of a 650 bp fragment of murine fgl2 cDNA representing nucleotides 846 (TGTGACA...) to 1495 (...ATGAATC) that belong to the second exon of fgl2 (Ding et al., 1997), into the EcoRI site of pBluescript (Strategene, La Jolla, CA, USA). Riboprobes to detect murine OX-2 mRNA were prepared by subcloning a 501 kb fragment of OX-2, into the PBK vector with T3 promoter (for sense, using primer 5'-CCGTCGACCAAGTGGAAGTG-3') and T7 promoter (for antisense, using primer 5'-ACGGATCCTTTGTCCAGACTCTGCTT-3') to generate a 586 bp strand, using T3 and T7 RNA polymerases as described for fgl2 (Chen et al., 1997). The digoxigenin-UTP-labelled probe concentration was determined by immunoenzymatic reaction with chemiluminescent detection, and the probes were stored at –80°C. Tissue sections were deparaffinized in 100% xylene, 100% alcohol, followed by prehydration in 50% formamide in 2x SCC at 22° C for 1 h. Hybridization mixture was 50% deionized formamide, 5% dextran sulphate, 250 µg/ml salmon sperm DNA, and 2 µg digoxigenin-labelled cRNA probe/ml in 2x sodium chloride/sodium citrate (SCC). This mixture was denatured by heating in an 85°C water bath for 5 min followed by chilling on ice for 1 min, and was then placed on the tissue sections and incubated at 42°C overnight. Post-hybridization washing in serial dilutions of 2x sodium chloride/sodium citrate (SSC; 17.53 g NaCl + 8.82 g sodium citrate in 1 l DH2O pH 7.0) was followed by incubation of 3% blocking reagent (normal goat IgG) followed, after a brief wash, by alkaline phosphatase-conjugated polyclonal goat anti-digoxigenin IgG Fab'2 fragments at a 1:500 dilution in Tris–HCl buffer (pH 7.5). After two washes of 5 min in fresh buffer, 5-bromo-4-chloro-3-indoxyl-phosphate (BCIP) and Nitroblue Tetrazolium (NBT) were added and incubated for 2 h. The sections were washed, counterstained 5 min with Methyl Green, dehydrated and mounted in Permount for viewing.
Photographs were taken at x25, x100, x250, and x400 magnification using Kodak 100 ASA colour print film and an automatic exposure metered system (WILD Photoautomat MPS45).
Statistical analysis
Tissue sections were examined and scored without knowledge of the status of the donor mouse. Photographs were taken to provide an objective record. The significance of differences between groups was determined by 
2 with Yates' correction and/or by Fisher's exact test (where appropriate).
Results
Fgl2 expression in cytokine-enhanced abortions
The proportion of implantations which abort spontaneously in DBA/2-mated CBA/J females ranges from 20–40%, depending on the level of 'stress' and bacterial flora in the animal colony (Hamilton and Hamilton, 1987; Muzikova and Clark, 1995; Arck et al., 1996, 1997a, b; Clark et al., 1998, 1999a, b); in our colony, the spontaneous rate of abortions was 23%. An injection of 1000 IU IFN- + 2000 IU TNF- was given i.p. on day 7.5 of gestation to augment the endogenous level of these cytokines and boost the rate of abortions in our mice (Clark et al., 1998). To test whether fgl2 expression was enhanced by cytokine treatment prior to the onset of grossly visible resorptions that first appear on day 9.5 of gestation, 18 h after i.p. injection of cytokines or PBS, on day 8.5 of gestation, the uteri were removed and processed for histology, as described above. Figure 1A and B show the most common pattern of in-situ hybridization for fgl2 mRNA in cytokine-treated mice on day 8.5 of gestation, one day before the usual time of onset of resorptions. Figure 1A shows lack of staining using the control 'sense' probe for fgl2 mRNA and Figure 1B shows intense hybridization with the 'anti-sense' probe in maternal uterine endometrium surrounding a zone of primary decidua in which the embryo is embedded; there was also intense staining in the mesometrial zone which extended up towards the embryo (secondary decidua). In addition, there was staining of fetal trophoblast.
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A different pattern of hybridization to antisense probe shown by some implants is shown in Figure 1C
. The rim of staining around the primary decidual zone seen in Figure 1B was not evident, and there was only a low level of staining in the mesometrial secondary decidua zone. Trophoblast did not show the intense labelling seen after cytokine treatment. This pattern of staining was more common in control (non-cytokine-treated) mice (see below), and was denoted fgl2lo, as distinct from the pattern shown in Figure 1B which we denoted as fgl2hi. Staining with the control 'sense' probe generated a null result similar to Figure 1A (data not shown).
A higher power view of the staining in Figure 1B is shown in Figure 2. Figure 2A1 shows intense staining of the cytoplasm of trophoblast cells by 'antisense' probe, but not of adjacent decidua; Figure 2A2 is the control result with 'sense' probe. Figure 2B shows the staining in the peripheral uterine lining adjacent to uterine muscle, and Figure 2C shows the basal zone near the site of attachment of the mesometrium. It can be seen that vascular endothelium was labelled, but it is also evident that cells other than vascular endothelium contained increased levels of fgl2 mRNA reactive with the 'anti-sense' probe.
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To test for the presence of bioactive procoagulant activity, we stained the tissues with antibody to fibrinogen/fibrin (Ding et al., 1997
). Fibrin is distinguished by the presence of focal clumps and strands. Figure 3 shows the result from the fgl2hi phenotype illustrated in Figure 1B. First it can be seen that in trophoblast (Figure 3A1 and 3A2), there was deposition of fibrin strands and granules. A similar result was seen in the basal zone and secondary decidua (Figure 3 B1 and B2). Fibrin deposition correlated with the pattern of staining for fgl2 mRNA. The beginning of a PMNL infiltration was seen in mesometrial decidua near the trophoblast at this time point (detail not shown). By contrast, in implantation sites showing the fgl2lo pattern (Figure 4A,B), there was only background staining of fibrinogen in decidua and trophoblast (Figure 4C,D); strands of fibrin and granular deposits were not prominent. Figure 4E–H shows another fgl2lo embryo with a similar pattern of anti-fibrinogen/fibrin staining. We denoted the staining patterns illustrated in Figure 4 as fibrinlo as distinct from the fibrinhi pattern illustrated in Figure 3. These data supported the idea that functional prothrombinase activity increased at sites where fgl2 mRNA was up-regulated.
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Correlation of increased fgl2 expression and fibrin deposition with subsequent resorptions
Fgl2hi was seen at 71% (12 out of 17) of individual implantation sites from cytokine-injected CBAxDBA/2 or CBAxBALB/c-mated mice (n = 3) where both trophoblast and decidua could be assessed for the two markers, and 76% (13/17) of sites showed the fibrinhi staining pattern, with no difference detected between the two mating combinations. Increased fgl2 and fibrin expression corresponded to the 70–80% rate of abortion that has been reported previously for cytokine-injected mice of the high and low abortion phenotypes (Clark et al., 1998
, 1999b).
In two non-cytokine injected CBA/JxDBA/2 mated mice, 67% (12 out of 18) of individual implantation sites showed the fgl2 mRNAhi pattern and 47% (nine out of 19) were scored as fibrinhi. In two CBAxBALB/c matings, 33% (five out of 15) of implants were fgl2hi and 27% (four out of 15) fibrinhi. Although the data were consistent with the lower spontaneous abortion rate in non-cytokine treated CBAxDBA/2 mated mice, and particularly in matings with BALB/c as explained above, the actual spontaneous abortion rates in the two kinds of matings measured on day 14.5 of gestation were only 21% (six out of 29) and 8.5% (four out of 47) respectively. The discrepancies between the observed rate of loss and that expected from the expression patterns of flg2 and fibrinin for the two mating combinations were too large to attribute to chance (P < 0.05 by 2).
Role of the OX-2 molecule in preventing fgl2-dependent abortions
Double-negative (DN) Ts cells that produce IL-10 and TGF-ßs are generated when alloantigen is injected into the portal vein, and these cells mediate antigen-specific transplantation tolerance (Gorczynski et al., 1997, 1998). The OX-2 molecule is up-regulated on hepatic antigen-presenting cells by alloantigen, and is a crucial immunomodulatory molecule required for generation of these Ts cells (Gorczynski et al., 1998). OX-2 has also been detected at the feto–maternal interface by immunohistochemistry in pregnant rats (Bukovsky et al., 1984). To determine whether OX-2 was expressed in the uterus of allopregnant CBA mice, we carried out in-situ hybridization for OX-2 mRNA using sections adjacent to those used to stain fgl2 mRNA.
Figure 5 illustrates three of the four patterns obtained: (i) fgl2hi-only; (ii) OX-2hi only; and (iii) fgl2hiOX-2hi. There were 38 evaluable, i.e. both trophoblast and decidua hybridized for both fgl2 and OX-2 mRNA: CBAxDBA/2 and CBAxBALB/c implantation sites in control and cytokine-treated mice: 24% were double positive, 26% OX-2hi-only, 37% fgl2hi-only, and 13% double negative. As shown in the left-hand 2x2 comparison in Table I, there was an increase in the percentage of fgl2hi implantation sites in cytokine-treated mice, but this was not statistically significant. However, as shown in the central 2x2 comparison, cytokine treatment significantly reduced OX-2 expression. Adjustment for a slight imbalance in strains in the cytokine-treated group, of one extra BALB/c mating combination, did not alter the significance or lack of significance of the findings from the 2x2 comparisons. There was a trend towards a negative correlation between expression of fgl2 and OX-2 which did not reach statistical significance, as shown in the right-hand 2x2 comparison. The reduced frequency of OX-2hi implantation sites in cytokine-treated mice was seen in two independent experiments. These data suggest that the level of OX-2 expression could be a major determining factor in the tolerance of the embryo in the presence of high fgl2, particularly where implantations expressed mRNA for both molecules.
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To test whether OX-2 expression might be involved in rescuing fgl2hi implantation sites from proceeding to embryo death in the situation where both OX-2 and fgl-2 molecules are expressed, we injected control CBAxDBA/2 and CBAxBALB/c mated mice with the same anti-OX-2 monoclonal antibody that blocks induction of transplantation tolerance. Figure 6
shows that injection of sterile hybridoma culture supernatant on or after day 8.5 increased the spontaneous abortion rate to that expected if all fgl2hi sites in CBAxDBA/2 had proceeded to resorption. A similar result is shown using purified monoclonal antibody. The increase in abortion rate was not due to a toxic effect of anti-OX-2 on the embryo because co-administration of anti-fgl2 to neutralize prothrombinase activity abrogated the boost in abortion rates. More importantly, a single injection of 35 µg of OX-2:Fc on day 8.5 of gestation reduced the abortion rate in CBAx DBA/2 mated mice to 2.1%. Lower doses of OX-2:Fc were less effective (24 µg gave 13% abortions, 12 µg gave 20%); injection of 35 µg 1 day earlier (day 7.5) was less effective than a day 8.5 administration. Injection of anti-OX-2 into CBAxBALB/c mated mice also increased the abortion rate to 22%, approaching the percentage expected from the frequency of fgl2hifibrinhi implantation sites, but the number of mice was too small for the difference to achieve statistical significance. More important was the fact that anti-OX-2 did not boost abortions to the 50–70% range, as would have been expected if the antibody acted via an embryotoxic effect, independent of the level of Th1 cytokines in decidua.
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Discussion
The direct prothrombinase, fgl2, has been localized in pregnant CBA/J mice by in-situ hybridization on trophoblast and in decidua at the site of formation of the placenta. There is a basal level of activity that seems important since anticoagulation with heparin or hirudin that can prevent abortions leads to retroplacental haemorrhages if given in excess (Clark et al., 1999b). Other studies (Knackstedt et al., 2000), have shown by immunohistochemistry, fgl2 protein in the same sites as those which stained with anti-sense probe for fgl2 mRNA. More importantly, the deposition of strands and granules of fibrin when fgl2 was up-regulated (as shown in this study) indicate that the fgl2 was bioactive. Absence of such deposition of fibrin in the basal state likely reflects fibrinolysis. Indeed, a delicate balance between clotting and anti-clotting mechanisms appears to be an essential part of haemochorial placentation. With excessive anticoagulation with heparin (that generates anti-thrombin III) or with hirudin (a direct anti-thrombin), retroplacental haemorrhages, fetal and maternal death can be observed (Clark et al., 1999b).
Previous studies have shown that both TNF- and IFN- must be present for spontaneous abortions to occur in mice (Clark et al., 1998). If interferon response factor (IRF) 1–/– females are mated to IRF1+/+ males, the trophoblast possesses the IRF1 gene needed to respond to IFN- but the mother does not. These pregnancies prove resistant to TNF-- and IFN--induced abortions. Therefore, the cytokines must act on maternal cells, probably vascular endothelium, rather than on trophoblast (Clark et al., 1998). The data in this paper showing up-regulation of fgl2 on trophoblast in response to cytokines was unexpected. However, this result would be compatible with the low abortion rate in pregnant IRF–/– mice if fgl2 must be up-regulated on both fetal trophoblast cells and on decidua for abortions to occur. When the two tissue meet, which is at the zone of spontaneous cleavage, there would be a summation of fgl2 activity of the two tissues, and the highest probability of clotting leading to necrosis by PMNL and complement activation (Heinz and Loos, 1983; Clark et al., 1998, 1999a; Xu et al., 2000).
Fgl2 was originally described on the endothelium/Kuppfer cells lining hepatic sinusoids in the situation of MHV-3-induced hepatitis (Ding et al., 1997), and we did see fgl2 mRNA increase in maternal uterine vascular endothelium in the present study. An unexpected finding in our study of fgl2 in pregnancy was expression of fgl2 in cells other than vascular endothelium. Expression by trophoblast was easily demonstrated, and indeed, there appeared to be mRNA for fgl2 in cells of the embryo. The meaning of this expression is currently being tested using fgl2 knock-out mice. Lack of fgl2 expression in primary decidua (at the anti-mesometrial pole of the implantation site) was also apparent. Indeed, only certain cell populations bordering the primary decidual zone and in secondary decidua expressed fgl2. Macrophages which may express fgl2 (Ding et al., 1997), are excluded from the primary decidual zone (Clark et al., 1999a), and accumulate in secondary decidua along with NK-lineage cells in the CBAxDBA/2 system (Baines et al., 1997; Clark et al., 1999a). However, there are too few macrophages to account for the intense band of staining that is shown in Figures 1B and 2. Furthermore, fgl2-dependent abortions occur at the expected rate in cytokine-treated mice in which macrophages have been depleted (Clark et al., 1998). The identity of the fgl2+ cells in decidua remains to be determined.
Both TGF-ß and IL-10 are known to inhibit PMNL-mediated vascular injury and clotting (Hayward et al., 1997; Pintavorn and Ballerman, 1997). It is thought that V1+ T cells producing these cytokines are present only in secondary decidua, so that if such cytokines account for protection of primary decidua, there must be another source (Clark et al., 1999a, Clark, 1999a, b; Chaouat et al., 1999). Indeed, trophoblast cells can produce IL-10 (Roth et al., 1996; Chaouat et al., 1999). However, the present data indicate that an OX-2 expression at the feto–maternal interface may play an important role in preventing fgl2-dependent abortions. Indeed, the most striking effect of administration of TNF- + IFN- to boost abortion rates was a reduction in OX-2 mRNA expression.
OX-2 is a glycoprotein with 77% homology to the T cell co-stimulatory protein B7 (Chen et al., 1997; Borriello et al., 1998), is found on B7+ antigen-presenting cells, and is thought to act as a tolerance rather than a co-stimulatory signal that deviates cytokine production away from Th1 (e.g. IL-2, IFN-) and towards Th2/3 (e.g. IL-4, IL-10, TGF-ß)-producing T cells that mediate tolerance via suppression (Gorczynski et al., 1997, 1998, 1999ab). A neutralizing antibody specific for murine OX-2 that blocks transplantation tolerance also boosted the abortion rate, and anti-fgl2 abrogated the increased rate of abortion produced by anti-OX-2, suggesting that OX-2 was likely acting by activating mechanisms that inhibit fgl2 expression and/or activity. The magnitude of protection afforded by anti-fgl2 alone was slightly less than that obtained previously (9.5 versus 4% resorptions) due perhaps to use of antibody from rabbit no. 4 which produced a less potent neutralizing antibody than rabbit no. 2 used previously. Anti-fgl2 is slightly less than effective against high rates of cytokine-triggered abortion (e.g. 13% with anti-fgl2 + TNF- + IFN- compared with 4.5% with anti-fgl2 in non-cytokine-treated mice) (Clark et al., 1998), and a similar relationship was evident in these experiments (i.e. 19% with anti-fgl2 + anti-OX-2, 9.5% with anti-fgl2 but no anti-OX-2). Nevertheless, the reduction in abortion rate by anti-fgl2 was sufficient to make the point. We used in our studies a 'standard' 100 µg test dose of monoclonal anti-OX-2. Although the half-life of IgG anti-OX-2 is 2–3 days in non-pregnant mice, the full 100 µg dose was required on day 8.5 or 9.5 of pregnancy, and this would suggest either a steep dose–response curve, or rapid clearance of anti-OX-2 in pregnant mice, possibly via placental release of a soluble OX-2. The kinetics of OX-2 expression during pregnancy are currently being measured to test the latter idea.
The interpretation we have given to the effect of antiOX-2 was strongly supported by the result of administration of OX-2:Fc, which almost completely abrogated abortions when given on day 8.5 of gestation. On day 8.5, DN V Ts cells that are activated to produce IL-10 and TGF-ß2 are first detected (Lea et al., 1992; Arck et al., 1997ab). It is tempting to speculate that this putatively protective Ts cell response of the mother can prevent embryo execution triggered by cytokine up-regulation of fgl2 prothrombinase. However, OX-2 can also act via macrophages and ß T cells. We have recently developed a method for detecting the ligand for OX-2 (OX-2L) using FITC-tagged OX-2:Fc. More that 80% of activated T cells are positive, but 20% of activated ß T cells and 50% of F4/80+ macrophages also stained (Gorczynski et al., 2000). OX-2Fc was able to activate the OX-2L+ subpopulation of small-sized activated F4/80+ macrophages to inhibit T cells recognizing them. Further data will therefore be required to determine exactly how OX-2 prevents loss of putatively doomed mouse embryos.
Our mouse data provide compelling evidence that maternal tolerance mechanisms can indeed prevent immunologically triggered 'rejection' of the 'fetal allograft' in mice. As OX-2 is a co-stimulatory molecule, there must be a separate antigen molecule to engage the V1.1 T cell receptor. The nature of that antigen is a matter for speculation. HSP-60 could represent the T cell receptor ligand. However, it is also important to note that in spite of blocking OX-2, the abortion rate of CBAxBALB/c mated mice was still less than that of CBAxDBA/2 mated mice. A similar abortion rate of ~50% in both mating combinations can be achieved by injecting anti-CD8 monoclonal antibody on day 6.5–7.5 of gestation, and this treatment abrogates protection against abortion achieved by priming CBA/J females with BALB/c splenocytes before mating to DBA/2 males ((Clark et al., 1994ab; Chaouat and Menu, 1997). These CD8+ T cells are now thought to represent V1+ cells, to circulate in blood rather than localize in decidua, and to produce IL-10 and a progesterone-inducible NK cell blocking factor that prevents the initial infiltration of Th1-cytokine-producing NK cells and macrophages into implantation sites (Clark, 1999ab). The immunogenetic basis for activating these cells in mice has recently been reviewed (Clark, 1999a). It is not known if OX-2 plays a role in promoting their development.
A number of papers have shown a similarity between abortions (resorptions) in the CBAxDBA/2 model and pregnancy loss in humans, especially recurrent unexplained pregnancy loss. There is also evidence for a contribution by the blood coagulation and inflammation systems. Miscarriage of chromosomally normal human embryos have been associated with a significant increase in frequency of decidual vasculitis, thrombosis, villous infarcts, and intervillositis (Salafia et al., 1993). Other authors (Labarrere and Faulk, 1991) have speculated on a role for placental tissue factor which can activate coagulation factors VII, V, and X to create a prothrombinase complex. Tissue factor may be present in mouse tissue, but no neutralizing antibody exists. The dramatic anti-abortion effect of anti-fgl2 in the mouse (Clark et al., 1998) suggested that fgl2, rather than tissue factor, might be important in humans. Preliminary results using the in-situ hybridization methods described in this paper have shown fg12 mRNA in trophoblasts and decidua of first trimester human pregnancy with up-regulation of expression in four out of five karyotypically normal recurrent miscarriage tissue biopsies (but in only one out of six) karyotypically abnormal biopsies, compared with successful pregnancy material from elective terminations (P = 0.045 Fisher's exact test) (Clark et al., 1999c). This finding has yet to be confirmed in a larger sample, and whether fgl2 is up-regulated prior to the onset of miscarriage also remains to be determined. We also have preliminary data for expression of OX-2 mRNA and protein in human placental trophoblasts. In human pregnancy decidua, the maternal lymphomyeloid composition differs significantly from the mouse, and a subset of CD56+ cells, possibly NK-T cells, seem to be a key source of TGF-ß2-related immunosuppressive activity (Lea et al., 1995; Mincherva-Nilsson et al., 1997; Clark et al., 1999a). It remains to be determined whether OX-2 alters cytokine expression by these cells in a manner that rescues implantations with putative high levels of fgl2.
Acknowledgments
We thank the Medical Research Council (MRC) of Canada (now renamed CIHR, Canadian Institutes for Health Research) for research grant support. We thank Dr J.M.Phillips, Dept. of Pathology, Hospital for Sick Children, for helpful comments during preparation of the figures. Drs Levy, Gorczynski, and Clark have licensed the fgl2 and OX-2 technology to Transplantation Technologies Inc for consideration in the event of useful future application to humans.
Notes
3 To whom correspondence should be addressed. E-mail: clarkd{at}fhs.McMaster.ca 
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Submitted on June 2, 2000; accepted on November 10, 2000.
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