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Biology of Reproduction 66, 1612-1620 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Control of Expression of Major Histocompatibility Complex Genes in Horse Trophoblast1

S.J. Bacon2,,a, S.A. Ellisb, and D.F. Antczaka

a James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 b Institute for Animal Health, Compton Laboratory, Compton, Newbury, United Kingdom RG20 7NN


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In most mammals, the fetus limits its presentation of paternal antigens to the mother by suppressing the cell-surface expression of proteins of the major histocompatibility complex (MHC) on trophoblast. In the horse, however, functional, polymorphic MHC class I antigens are expressed at high levels on the invasive trophoblast cells of the chorionic girdle between Days 32 and 36 of pregnancy, although not on the adjacent noninvasive trophoblast of the chorion and allantochorion membranes. In this study, the control of MHC class I gene expression was investigated in invasive and noninvasive horse trophoblast, and the MHC class I loci expressed by invasive trophoblast were identified. Northern blot hybridization of Day 33–34 conceptus tissue revealed both transcriptional and posttranscriptional regulation of cell-surface MHC class I expression in horse trophoblast. The invasive MHC class I-positive trophoblast showed levels of steady-state mRNA nearly as high as those in lymphoid tissues from adult horses, whereas noninvasive MHC class I-negative trophoblast also contained transcripts for MHC class I, but at lower levels similar to those present in adult horse nonlymphoid tissue. We also cloned and sequenced polymerase chain reaction products from the transmembrane and cytoplasmic regions of MHC class I transcripts in chorionic girdle and lymphocytes, and determined that horse invasive trophoblast appears to transcribe the same MHC class I loci transcribed in lymphocytes, including both polymorphic and nonpolymorphic loci. These data from the horse demonstrate that functional alloantigen presentation by trophoblast can be a normal part of early pregnancy.

conceptus, developmental biology, early development, implantation, placenta, trophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The expression of major histocompatibility complex (MHC) proteins is tightly controlled at the interface between mother and fetus in mammalian pregnancy. In human placenta, the cell-surface expression of MHC class I molecules by the fetal trophoblast is limited to loci of low polymorphism: human leukocyte antigen G (HLA-G), HLA-E, and HLA-C [1–5]. Restricting cell-surface expression to these less polymorphic loci is assumed to prevent the immune-mediated rejection of placental tissues. This restriction may be particularly important in humans since hemochorial placentation exposes trophoblast directly to the mother's bloodstream. The expression of HLA-G and HLA-E antigens by the trophoblast may also inhibit cytolysis by natural killer cells [6–8]. HLA-G expression in particular is also associated with suppressing the activity of CD4+ T lymphocytes [9], although it is possible for fetuses to survive pregnancy without functional HLA-G protein [10]. Trophoblast is one of the few normal cell types that is known to restrict the expression of MHC class I molecules, an ability it shares with some carcinomas and virally infected cells [11–13]. The MHC class II genes are unexpressed in trophoblast cells [14]. Multiple levels of control, both transcriptional and translational, appear to regulate MHC antigen expression in trophoblast tissue in humans [15–19].

In pregnancy, the intimacy of the contact between maternal uterine tissue and fetal trophoblast varies widely across mammalian taxa. Unlike human or rodent placentation, equid placentation is relatively noninvasive. In horses, the invasive trophoblast perforates the uterine epithelium but settles in the uterine stroma before ever reaching the maternal bloodstream. Beginning on or near Day 32 of pregnancy, MHC class I protein is detectable on the surface of the proliferating invasive trophoblast (the chorionic girdle, Fig. 1). These invasive mononucleated trophoblast cells penetrate the uterine stroma, become binucleated, and organize into eCG-secreting structures called endometrial cups. Binucleated endometrial cup trophoblast cells lose both cell-surface protein and message for MHC class I by Day 45 of pregnancy [20–22]. Thus, in equids, there is natural temporal variation in MHC class I expression within a single trophoblast cell lineage. There is also spatial variation: whereas invasive chorionic girdle is MHC-class I positive, the flanking noninvasive allantochorion and chorion trophoblast cells do not bear immunodetectable class I protein (Fig. 1, [20, 22]).



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  		FIG. 1. The horse conceptus on Day 33 of gestation. Photograph (A) and schematic diagram (B) of horse fetus and its extraembryonic membranes showing both invasive (chorionic girdle) and noninvasive (allantochorion and chorion) trophoblast tissues. C) Immunohistochemical labeling of a frozen section of chorionic girdle and chorion membranes using a monoclonal antibody reactive to horse MHC class I antigen [20, 22] shows that the invasive chorionic girdle expresses high levels of MHC class I protein, whereas the neighboring noninvasive chorion trophoblast has negligible cell-surface expression. Frozen section taken from area of conceptus indicated by white box in A.

In addition to being under tight spatial and temporal control, the MHC class I antigens on the surface of the chorionic girdle induce a strong antipaternal cytotoxic antibody response in the pregnant mare [23–25]. Over 90% of mares mount a response by Day 60 of pregnancy, a reaction that is more pronounced and widespread in horses than in any other species studied. In humans, maternal sensitization to paternal antigens occurs in only about 15% of primiparous women and seldom more than 60% of multiparous women [26, 27]. At least five MHC class I loci may be transcribed in the horse [28, 29]. At least two are polymorphic, and the remaining class I loci that have been identified (represented by the sequences A1, C1, and E1), appear nonpolymorphic and encode putative nonclassical MHC genes [28]. The number of genes transcribed/expressed varies with haplotype as is seen in some other species such as cattle [30]. In the horse, it is not yet known if the nonpolymorphic genes are expressed as protein, but the polymorphic equine MHC class I genes are known to be expressed on trophoblast tissue [24, 31]. Thus, the horse offers a model system in which many aspects of the immune relationship between mother and fetus can be studied: the temporal and spatial patterns of expression of MHC class I genes in the placenta, the level at which this expression is controlled in the cell, and the maternal response to paternal antigens on the conceptus.

To explore the control mechanisms underlying the pattern of MHC class I protein expression in horse trophoblast, we have characterized the levels of steady-state mRNA for the two molecules that associate into a functional MHC class I molecule on the cell surface—the MHC class I heavy chain and the light chain, ß2-microglobulin (ß2m)—in both MHC class I protein-positive and protein-negative trophoblast tissues. In vitro cultures of the noninvasive (and normally MHC class I negative) trophoblast were also established to explore the control of MHC class I expression in cells that have lost contact with underlying fetal membranes. In addition, we have identified the particular set of MHC class I genes transcribed in the invasive chorionic girdle trophoblast, based on sequencing cloned cDNAs of amplified transmembrane and cytoplasmic domains of class I transcripts. Finally, the transcription of MHC class II genes in horse trophoblast was also explored.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Collection

Tissues were obtained from horses in the Equine Genetics Center herd at Cornell University. All animal care was performed in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee at Cornell University. After artificial insemination, conceptus tissues were collected by nonsurgical uterine lavage from lightly sedated mares between Days 17 and 33 of pregnancy. Briefly, the mare's cervix was digitally dilated, and a 12-mm bore endotracheal tube with a cuff (Bivona Veterinary Products, Gary, IN) was passed through it. The cuff was inflated in the uterine lumen, and sterile PBS containing 20 mg/L kanamycin sulfate (Sigma, St. Louis, MO) was infused into the uterus by gravity flow. When the rate of flow slowed noticeably or the mare showed signs of mild discomfort, the catheter was disconnected from the saline supply and held over a sterile beaker, into which the contents of the uterus drained. Typically, one such infusion (1–1.5 L of saline) recovered the conceptus. The conceptus (usually ruptured by the procedure, but otherwise intact) was transferred to the laboratory in saline at 4°C.

Conceptus tissues from nine pregnancies (five equine leukocyte antigen-A3 (ELA-A3) homozygotes, one ELA-A2 homozygote, and three that were heterozygotes or of unknown ELA type) were dissected under a stereoscopic microscope (Nikon Instrument Group, Melville, NY) at 150x magnification. Tissue to be used for in vitro culture or RNA extraction was rinsed well in sterile saline containing 0.2 U/ml penicillin-streptomycin (Gibco BRL, Grand Island, NY) and frozen in liquid nitrogen. These rinses, and the fact that no trophoblast had yet invaded the maternal epithelium, minimized the possibility of contamination with maternal epithelial cells known to be positive for MHC class I mRNA [22]. All fresh trophoblast tissues (both chorionic girdle and allantochorion) were collected as membranes; allantochorion to be cultured was further bead purified as described subsequently. Somatic tissues from adult mares were gathered at necropsy and frozen in liquid nitrogen within 1 h after the mares were killed. Lymphocytes from adult horses were isolated from peripheral blood as previously described [32].

Bead Purification and In Vitro Culture of Allantochorion Cytotrophoblast

Allantochorion trophoblast from Day 32–34 conceptuses was chopped into small pieces, placed in PBS with 0.2 U/ml penicillin-streptomycin (Gibco BRL), and washed by centrifugation at 300 x g for 10 min at 4°C. The cell pellet was resuspended in 0.25% trypsin with 1 mM EDTA (Gibco BRL) and digested to dissociate the trophoblast cells and free them from their underlying basement membrane. After trypsinization, the cells were washed in Dulbecco modified Eagle medium (DMEM) (Gibco BRL), counted, and then incubated with first-stage antibody (trophoblast-specific antibody 102.1 [33]) at 4°C with rotation for one-half hour. Sheep {alpha} mouse immunoglobulin G-coated Dynabeads (Dynal Inc., Oslo, Norway) were then used according to manufacturer's instructions to enrich by magnetic selection for those cells labeled with the antitrophoblast antibody 102.1. Bead-purified allantochorion trophoblast cells were plated in DMEM (Gibco BRL) with 10% fetal calf serum (Hyclone, Logan, UT) and incubated at 37°C in 8% CO2. Those to be used for immunohistochemical staining (n = 5) were grown to confluence in Lab-Tek tissue culture chamber slides (Nunc, Inc., Naperville, IL) and harvested after varying numbers of days in culture. The slides were rinsed in PBS, fixed in acetone for 10 min, and frozen at -20°C until use. Those cultures to be used for RNA analysis (n = 2 ELA-A3 homozygotes) were trypsinized, washed, and snap frozen as cell pellets.

Northern Blotting

Total RNA from conceptus tissues was extracted with the Qiagen RNeasy kit (Qiagen Inc., Chatsworth, CA). RNA from adult tissues was extracted using the single-step method [34]. Five micrograms of total RNA per lane was run on a 1.4% agarose formaldehyde gel and transferred to a Magna Charge nylon membrane (Micron Separations Inc., Westborough, MA) by a standard blotting procedure. The blots were hybridized with the probes described subsequently in a solution of 5x saline-sodium phosphate-EDTA buffer, 50% formamide (Fisher, Pittsburgh, PA), 0.5% SDS (Fisher), 5x Denhardt solution (Sigma), and 100 µg/ml denatured salmon sperm DNA (Gibco BRL) at 65°C for 12–16 h. The blots were then washed at 65°C for 30 min in the following sequence of wash solutions: 2x saline-sodium citrate (SSC), 0.1% SDS; 2x SSC; 1.5x SSC; 1x SSC; 0.5x SSC. After the blots were exposed to a Fuji BAS III-S phosphorimaging plate, the hybridization signal was quantitated on a BAS1000 Bio-imaging analyzer (Fuji Medical Systems USA, Stamford, CT). Hybridization signals were standardized within blots by comparison with the fluorescence intensity of the pretransfer 18S RNA bands with analysis performed on a Macintosh computer using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the internet at http://rsb.info.nih.gov/nih-image/). The blots were then exposed to x-ray film. The intensity with which each RNA sample bound to the radiolabeled probe was expressed as a percentage of the intensity with which lymphocytes bound the same probe on the same blot.

DNA Probes

The DNA probes used in Northern blotting included cDNA clones for a polymorphic horse MHC class I gene (clone 8–9, GenBank M95409), horse ß2m (GenBank X69083), and the horse MHC class II gene DQß (GenBank L33910). A 573-base pair reverse transcription-polymerase chain reaction (RT-PCR) product was used as a probe for the eCG ß subunit gene, eCGß. (GenBank S41704; forward primer: 5'-GCA CCA GCA AAG ATC AGT AGT GTA-3'; reverse primer: 5'-GTT CAA GAA GTC TTT ATT GGG AGG-3'). Probes were labeled with [32P]dCTP by random hexamer priming (Stratagene, La Jolla, CA).

RT-PCR Amplification

Complementary DNA was synthesized from 3 µg of total RNA using oligo(dT) primers and 200 U Superscript II reverse transcriptase (Life Technologies, Rockville, MD), and was treated with RNase (Life Technologies) before PCR amplification. For cDNA amplification, 2 µl of the cDNA reaction mixture was placed in a total reaction volume of 25 µl (PCR buffer with 1.5 mM MgCl2, 0.2 mM dNTPs, 1 U Taq polymerase; Perkin Elmer Applied Biosystems, Branchburg, NJ) and 1 µM primer specific for the genes tested (Table 1). PCR products were electrophoresed on a 1.25% agarose gel, stained with ethidium bromide, and visualized under UV light with a CCD camera (UVP, Upland, CA) using Scion Image version 1.60 (NIH Image).


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  		TABLE 1. Primers used in reverse-transcriptase polymerase chain reaction amplification of equine conceptus tissue cDNA

Sequencing MHC Class I Transcripts in Chorionic Girdle and Lymphocytes

Breeding stallions and mares homozygous for two common horse MHC haplotypes were maintained in the experimental horse herd at the Baker Institute at Cornell University. Tissue typing of horses to ascertain MHC haplotypes was performed using a panel of antisera characterized in international workshops [39]. Chorionic girdle trophoblast and peripheral blood lymphocytes were isolated from horses homozygous for the equine MHC haplotypes ELA-A2 and ELA-A3. Total RNA was isolated from these tissues and reverse-transcribed into cDNA as described previously, using primers that appear to amplify all horse MHC class I genes with equal efficiency. PCR primers to the {alpha}3 domain of the horse MHC class I molecule (positions 850–867 on clone 8–9) and to the 3' untranslated region (positions 1115–1132 on clone 8–9) (Integrated DNA Technologies, Coralville, IA) amplified the transmembrane and cytoplasmic domains of MHC class I cDNA transcripts. The amplification conditions were 94°C 1 min, 50°C 1 min, 72°C 1 min for 34 cycles, and a 10-min extension at 72°C. PCR products were purified from a 2% low melting point agarose gel with warm phenol, then treated with kinase and T4 DNA polymerase (New England Biolabs, Beverly, MA) to prepare them for M13 phage ligation. M13 phage (Gibco BRL) were linearized with SmaI (Promega, Madison, WI) and treated with calf intestine alkaline phosphatase to prevent religation. The PCR product was inserted into M13 phage (New England Biolabs) using T4 DNA ligase (New England Biolabs) and was used to transform competent MV1190 bacteria grown on 2XYT medium (Sigma). After single-stranded DNA was isolated, it was sequenced using a modification of the Sanger dideoxy method (USB Sequenase Version 2.0 sequencing kit; Cleveland, OH). A total of 123 clones (59 from lymphocytes of 2 MHC homozygous adults, 64 from chorionic girdle of 2 MHC homozygous conceptuses) were sequenced to ensure detection of all transcribed genes.

Immunohistochemistry

Indirect immunohistochemical staining of cultured trophoblast tissues was performed as described previously [40].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
All Conceptus Tissues Tested Transcribed mRNA for MHC Class I and ß2m Genes

RT-PCR was used to determine if MHC class I and ß2m genes were transcribed in horse conceptus tissues. Transcripts for horse MHC class I and ß2m genes were found in all conceptus tissues tested (Fig. 2). These results were probably not due to contamination by maternal lymphocytes, since no T-cell receptor ß chain transcripts were detected in any trophoblast tissues. Because MHC class I and ß2m genes were transcriptionally active in both class I antigen-expressing (chorionic girdle) and -nonexpressing (allantochorion and chorion) tissues, we performed Northern analysis to quantitate the relative levels of steady-state mRNA for class I and ß2m in horse trophoblast, and to compare the levels of message for these genes with those in horse lymphoid and other somatic tissues.



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  		FIG. 2. MHC class I and ß2m transcripts in horse lymphocyte and conceptus tissues measured by RT-PCR. Horse conceptus cDNA amplified using primers to horse MHC class I and ß2m. Control primers: T cell receptor ß chain (TCRß) and hypoxanthine guanine phosphoribosyltransferase (HPRT) genes. Adult horse lymphocytes and cultured mouse L cells were used as positive and negative control tissues, respectively. RT-PCR products were electrophoresed through a 1.5% agarose gel and stained with ethidium bromide

Invasive Trophoblast Contains High Levels of MHC Class I and ß2m mRNA

By Northern blot analysis, MHC class I antigen-positive tissue (invasive chorionic girdle trophoblast) had steady-state levels of mRNA for MHC class I and ß2m as high as or higher than those in adult peripheral blood lymphocytes (representative blot, Fig. 3). MHC class I and ß2m transcripts were also detected in the antigen-negative allantochorion and chorion cytotrophoblast membranes, but they were present at approximately one tenth of the steady-state levels in chorionic girdle or lymphocytes. Thus, both MHC class I antigen-positive and antigen-negative conceptus tissues transcribed MHC class I heavy and light chain genes, but in antigen-negative tissues, the mRNA was present at a small fraction of the levels in antigen-positive tissues. These data are summarized in Figure 4.



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  		FIG. 3. MHC class I and ß2m transcripts in horse lymphocyte and conceptus tissues measured by Northern blotting. Representative Northern blot autoradiographs (top) and RNA gels (bottom) of lymphocyte and conceptus RNA samples probed with horse MHC class I cDNA and a ß2m clone



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  		FIG. 4. Quantitation of Northern hybridization of horse conceptus tissue. Phosphorimage signals of equine conceptus tissue RNA hybridized to radiolabeled equine MHC class I cDNA (A) and ß2m plasmid clone DNA (B). Values are expressed as a percentage of the hybridization signal intensity of control (stallion) lymphocyte RNA. Individual RNA samples from 9 pregnancies include stallion control lymphocyte (L{Phi}, n = 9), chorionic girdle (c. girdle, n = 9), allantochorion (alc, n = 11, two samples run twice), chorion (n = 8), and cultured allantochorion (alc culture, n = 3, including one conceptus sample run twice)

Although the hybridization intensity of MHC class I and ß2m probes was comparable within each of the tissues tested, different patterns were shown when these same tissues were probed for other mRNA transcripts. In particular, whereas the MHC class I and ß2m probes hybridized with almost equal intensity to chorionic girdle and lymphocyte RNA, a cDNA probe for eCG hybridized to chorionic girdle but not to lymphocyte RNA (Fig. 5). Endometrial cup, a heterogeneous mix of syncytial (binucleated) trophoblast and maternal fibroblasts, glands, and lymphocytes [41] showed the most intense signal for eCG. The signal in the lane containing chorionic girdle RNA from a Day 33 conceptus (lane 2) was 10 times less intense, and RNA from a Day 32 chorionic girdle (lane 5) bound to the eCG probe even more weakly. No hybridization to the eCG probe was detected in the noninvasive allantochorion or chorion RNA lanes. An equine MHC class II DQß gene probe applied to duplicate samples hybridized only with RNA from those tissues containing lymphocytes. No RNA samples from pure trophoblast (chorionic girdle, allantochorion, chorion) hybridized to the equine MHC class II probe.



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  		FIG. 5. MHC class II and eCG transcripts in horse lymphocyte and trophoblast tissues measured by Northern blotting. Representative Northern blot autoradiographs (top) and RNA gel (bottom) of horse conceptus and lymphocyte RNA hybridized to probes for the eCG ß subunit (eCGß) and the MHC class II gene DQß. Blots were generated from duplicate RNA gels.

Allantochorion Trophoblast Expressed MHC Class I Antigen after In Vitro Culture

Immunohistochemical studies of frozen sections of chorionic girdle and allantochorion have demonstrated a sharp demarcation between expression of MHC class I antigen on the rapidly proliferating chorionic girdle and the lack of expression on the allantochorion ([20, 22], also Fig. 1C). To explore the control of MHC class I antigen expression in the allantochorion trophoblast, in vitro cultures were established and labeled with monoclonal antibodies (mAbs) to trophoblast and MHC molecules. After 3 days of in vitro culture, the allantochorion was labeled by the antitrophoblast antibody 102.1 (Fig. 6A) but not by mAb to horse MHC class I antigen (Fig. 6C). But after 14 days of culture, the allantochorion trophoblast cells were immunopositive for MHC class I (Fig. 6D). The increase in cell-surface expression of MHC class I antigen was accompanied by an increase in the level of mRNA in this tissue. RNA from allantochorion cultured for at least 2 wk hybridized more strongly to the MHC class I and ß2m probes than did RNA from fresh allantochorion (Figs. 3 and 4).



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  		FIG. 6. Photomicrographs of in vitro cultures of trypsinized and immunopurified allantochorion labeled with mAbs to trophoblast and MHC class I antigens on Days 3 and 14 of culture (magnification x1250). Day 34 allantochorion cytotrophoblast cells were purified using mAb 102.1 and anti-mouse Ig-coated magnetic beads and then cultured for 3 days (A, C) or 14 days (B, D). A, B) Monoclonal Ab 102.1 specific for trophoblast [33]. C, D) Monoclonal Ab CZ4 specific for MHC class I [31]. Arrows point to mAb-coated magnetic beads still bound to the allantochorion; triangles in C point to the outline of a trophoblast cell cluster that was not labeled with the anti-class I MHC mAb

MHC Class I Transcription in Adult Somatic Tissues

To place these patterns of transcription in a larger context, the MHC class I and ß2m probes were hybridized to total RNA from adult horse tissues: lymph node, kidney, and brain. These additional tissues, all of which contain cells that are presumed capable of presenting antigen to cytotoxic T cells, had mRNA levels for ß2m comparable to those in the allantochorion and chorion cytotrophoblast, and levels of mRNA for MHC class I that appeared intermediate between the antigen-negative allantochorion and the antigen-positive chorionic girdle (Figs. 7 and 8).



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  		FIG. 7. MHC class I and B2m transcripts in horse conceptus and adult somatic tissues measured by Northern blotting. Representative Northern blot autoradiographs of RNAs from selected conceptus and adult somatic tissues hybridized to probes for equine MHC class I and ß2m

Chorionic Girdle Transcribes Polymorphic MHC Class I Genes

The transmembrane and {alpha}3 domains of the equine MHC class I genes contain locus-specific features that distinguish the polymorphic (classical) genes from the nonpolymorphic (nonclassical) genes [29]. The 3' end of the gene was thus amplified in preference to the 5' (peptide-binding region) end. Within the polymorphic genes, the 3' end is relatively conserved and does not generally allow assignment to a particular gene or allele. Few horse class I haplotypes have been studied in detail, but the limited data available suggest that haplotypes generally express two polymorphic genes. No features have been identified that readily distinguish these and allow assignment to a locus. The A2 haplotype expresses two polymorphic genes, 8–9 and 1–29, that have been sequenced and are known to be expressed [35]. The A3 haplotype has thus far been shown to express only one polymorphic gene, B2 [28] however these data suggest that a second polymorphic gene may also be present.

In the present study MHC class I gene transcription was compared between chorionic girdle and lymphocyte mRNA in horses expressing the well-characterized A2 or A3 haplotypes [28, 35]. Based on sequences of the transmembrane and cytoplasmic domains of the MHC class I transcripts, horse invasive trophoblast appears to express the same complement of genes as lymphocytes. All of the MHC class I loci transcribed in lymphocytes were also represented in chorionic girdle trophoblast, including transcripts from both polymorphic and nonpolymorphic loci (Table 2). ELA-A2 haplotype tissue samples contained transcripts from the polymorphic loci and A1, but no C1 transcripts were detected. Levels of A1 and C1 transcripts appeared to be much higher in lymphocytes than in chorionic girdle.


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  		TABLE 2. MHC class I sequences detected in cloned RT-PCR products of horse chorionic girdle and lymphocyte cDNAs taken from four MHC homozygous individuals


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Invasive equine trophoblast expressing a high density of MHC class I antigen on its surface contained high levels of steady-state mRNA for the MHC class I heavy and ß2m light chains. The levels of message were much higher than those in noninvasive, antigen-negative allantochorion trophoblast, and in fact rivaled levels found in lymphocytes. Although Northern hybridization measures steady-state mRNA levels and not the rate of gene transcription, the two have been found to correlate well for MHC class I and ß2m in the mouse [42], a species in which most developmental regulation of MHC class I expression has been found to occur by transcriptional activation [43]. Consistent within-tissue patterns of steady-state mRNA levels for MHC class I and ß2m suggest that expression of these molecules is coordinately regulated in horse trophoblast. In invasive trophoblast, the expression of MHC class I and ß2m genes appears to be controlled at the level of transcription.

In the antigen-negative allantochorion, both transcriptional and posttranscriptional controls appear to operate. The low levels of steady-state message for MHC class I in fresh allantochorion trophoblast are consistent with transcriptional regulation of the gene, but the lack of immunodetectable protein on the cell surface suggests that posttranscriptional controls may operate as well. Our results clarify the previous finding of Maher et al. [22] of inconsistent hybridization signals in allantochorion trophoblast by in situ hybridization. Northern blotting reveals consistent, but low, levels of mRNA for MHC class I in fresh allantochorion trophoblast. Ellis et al. [44] have found that in the bovine placentome at term, antigen-negative trophoblast shows intermediate levels of mRNA for MHC class I, suggesting both transcriptional down-regulation of the gene and a posttranscriptional block. In the horse, it is not known if the protein is translated and retained inside the cell, but by analogy with human trophoblast (in which antigen-negative villous trophoblast cells contain intracellular MHC class I mRNA [16] and heavy chain [17]), it may be. Because the assembly, peptide loading, and export of MHC class I heavy and light chains involve numerous accessory molecules, there are several possible checkpoints for a posttranslational block. MHC class I antigen could be kept from stable cell-surface expression by a lack of ß2m light chain, a lack of proteasome-cleaved peptides, or a lack of transporters associated with antigen processing (TAP) 1 or TAP2 peptide transporters. In the horse, ß2m transcripts are clearly evident in both invasive and noninvasive trophoblast tissue. These data mirror results in humans, in which ß2m transcripts are present in antigen-negative tissue [3]. Because the horse ß2m gene is being transcribed in both MHC class I protein-positive and protein-negative tissue, it is unlikely that cell-surface expression of MHC class I in protein-negative tissues is being limited by a lack of ß2m light chain.

Although the expression of MHC class I genes can be controlled transcriptionally or translationally in horse trophoblast, MHC class II genes appear to be transcriptionally silent. In Northern blots, no horse trophoblast RNA hybridized to the equine MHC DQß class II probe, although horse lymphocytes showed an intense hybridization signal. These data are consistent with what is known for mouse [45], human [14], and rat [46], species in which the trophoblast is negative for both MHC class II message and cell-surface protein. This transcriptional silence appears to be caused by the lack of expression of a transacting factor (class II transactivator, or CIITA) responsible for both constitutive and interferon gamma-induced transcription of the MHC class II genes [47, 48]. Rats, mice, and humans lack trophoblastic CIITA, and transfection of this transacting factor is sufficient to restore cell-surface MHC class II expression [49].

In invasive horse trophoblast, MHC class I transcription and cell-surface expression increase during trophoblast proliferation and invasion. These data are consistent with what is known in the human, in which HLA-G cell-surface protein is displayed by the invading mononuclear extravillous cytotrophoblasts [50]. It is tempting to relate the temporal up-regulation of MHC class I genes in vivo to cellular differentiation and to loss of cell contact with the underlying fetal membranes, as Hunt et al. [16] have suggested. In vitro results are consistent with this interpretation: in humans and mice, and now in the horse, there is a strong association between cellular differentiation and the onset of MHC class I expression. Whether spontaneous or retinoic acid-induced, cellular differentiation is correlated with an increase in MHC class I expression. In mouse cells, retinoic acid acts through a member of the nuclear hormone receptor superfamily, retinoid X receptor beta (RXR beta or H-2RIIBP), to bind upstream of the enhancer A region and activate mouse MHC class I genes [51]. Human early-gestation cytotrophoblast stem cells also up-regulate MHC class I protein in culture as they take on an invasive phenotype [50]. In the horse, the noninvasive and normally antigen-negative equine allantochorion trophoblast up-regulates MHC class I message and protein when it is displaced from its normal context, either when contact with the maternal uterine epithelium is poor in vivo [20, 52] or on culturing (Fig. 6). These results are consistent with the hypothesis that MHC class I expression is up-regulated as a cell's three-dimensional environment is modified, as when in vivo the cells proliferate and stratify, lose contact with the basement membrane, and invade.

We compared MHC class I gene transcription between chorionic girdle and lymphocytes in horses expressing ELA-A2 and ELA-A3 haplotypes. Based on sequences of the transmembrane and cytoplasmic domains of MHC class I mRNA transcripts, all of the MHC class I loci transcribed in lymphocytes were also represented in chorionic girdle trophoblast, including both polymorphic and nonpolymorphic loci. The sequence data are also consistent with the idea that there are haplotype- and tissue-specific differences in the expression of particular MHC class I loci. ELA-A2 haplotype tissue samples contained transcripts from the polymorphic loci and A1, but no C1 transcripts were found. In addition, levels of A1 and C1 transcripts appear to be much higher in lymphocytes than in chorionic girdle. The level of message for the A1 gene has always been found to be very high in lymphocytes [28], but the significance of these locus- and tissue-specific differences in the level of message will only be elucidated by future studies of cell-surface-expressed antigen. One interesting feature is that whereas the two polymorphic genes in the A2 haplotype (8–9, 1–29) are transcribed at similar levels, the B2 gene is almost exclusively transcribed in the A3 haplotype. This reflects previous data [28] suggesting that the A3 haplotype expresses only one polymorphic gene. Although it would be interesting to investigate the second gene further, its identity and expression characteristics are not pertinent to this study. We cannot exclude the possibility that the frequency with which different clones were detected could reflect a cloning artifact in which certain sequences were favored. This underlines the importance of future studies of cell-surface expression. What has been clearly shown here is that on both haplotypes, every MHC class I gene transcribed in lymphocytes was also transcribed in invasive chorionic girdle trophoblast.

The transcription of both polymorphic and nonpolymorphic MHC class I loci on invasive trophoblast and the high levels of cell-surface expression of the polymorphic antigens set the horse apart. Maternal and paternal MHC antigens are both expressed on horse trophoblast [24], and the mare regularly produces cytotoxic antibodies to the paternal alloantigen shortly after chorionic girdle trophoblast invasion [23, 24]. Yet, pregnancy in the horse is not routinely terminated by a maternal antifetal immune response, highlighting the multiple mechanisms in place to redirect maternal immunity. For example, there is evidence for systemic down-regulation of antipaternal cytotoxic T lymphocyte responses in pregnant mares [53]. Local, intrauterine factors are also very likely to play an important role in horse pregnancy. MHC class I expression by invasive trophoblast in the horse may in fact be integral to redirecting uterine immunity during pregnancy. By comparing the maternal-fetal interface across mammalian taxa [54] rather than restricting inquiry to one or two species, it may be possible to reveal the general mechanisms by which the allogeneic fetus of eutherian mammals avoids immune-mediated rejection during pregnancy. The present data suggest that diverse mechanisms protect the fetus from the mother's immune system.



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  		FIG. 8. Intensity of Northern phosphorimage signals for equine conceptus tissue RNA hybridized to radiolabeled probes for equine MHC class I (A) and ß2m (B). Values are expressed as a percentage of the phosphorimage hybridization signal intensity of control (stallion) lymphocyte RNA. RNA samples include control lymphocyte (L{Phi}, n = 9), chorionic girdle (c. girdle, n = 9), allantochorion (alc, n = 11), chorion (n = 8), brain (n = 4), kidney (n = 4), and lymph node (l. node, n = 4)


   ACKNOWLEDGMENTS
 
The authors thank Mr. Wayne Gottlieb for technical assistance and Mr. James Hardy for assistance with horse breeding. The authors gratefully acknowledge the contributions of Drs. Francesca Stewart and William Donaldson to Figure 1.


   FOOTNOTES
 
First decision: 20 August 2001.

1 Supported by National Institutes of Health grants HD-34086 and HD-15974 and by the Dorothy Russell Havemeyer Foundation, Inc. Back

2 Correspondence: S. Bacon, Department of Biological Sciences, Clapp Laboratory, Mount Holyoke College, South Hadley, MA 01075.FAX: 413 538 2548; sbacon{at}mtholyoke.edu Back

Accepted: December 19, 2001.

Received: July 16, 2001.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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