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BOR - Papers in Press, published online ahead of print November 27, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.008912
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BIOLOGY OF REPRODUCTION 68, 1413–1421 (2003)
DOI: 10.1095/biolreprod.102.008912
© 2003 by the Society for the Study of Reproduction, Inc.

Pregnancy

A Chemokine, Interferon (IFN)-{gamma}-Inducible Protein 10 kDa, Is Stimulated by IFN-{tau} and Recruits Immune Cells in the Ovine Endometrium1

Kentaro Nagaokaa, Akiharu Sakaia, Hisashi Nojimaa, Yoshihito Sudaa, Yuichi Yokomizob, Kazuhiko Imakawa2,a, Senkiti Sakaia, and Ronald K. Christensonc

a Laboratory of Animal Breeding, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan b National Institute of Animal Health, Tsukuba, Ibaraki 305-0856, Japan c Reproduction Research Unit, USDA-ARS, U.S. Meat Animal Research Center, Clay Center, Nebraska 68933-0166


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Proper distribution of immune cells in the uterus is a prerequisite for successful implantation and subsequent placentation, but biochemical signals that govern such events have not been well characterized. In the present study, the cDNA of a chemokine, interferon (IFN)-{gamma}-inducible protein 10 kDa (IP-10), was identified from a cDNA subtraction study between uterine endometrial tissues from Day 17 pregnant and Day 15 cyclic ewes. The effect of IFN-{tau} on IP-10 expression and the involvement of IP-10 in the recruitment of immune cells were then investigated. Northern blot analysis revealed that large amounts of IP-10 mRNA were present during conceptus attachment to maternal endometrium and early placentation. IP-10 mRNA was localized to monocytes distributed in the subepithelial stroma of pregnant but not cyclic uteri. This finding was supported by the discovery of IP-10 mRNA expression in monocytes but not in lymphocytes, uterine epithelial cells, or stromal cells. Moreover, the expression of IP-10 mRNA by the monocytes was stimulated by IFN-{alpha}, IFN-{gamma}, and IFN-{tau} in a dose-dependent manner, but the expression of IP-10 mRNA by the endometrial explants was most stimulated by IFN-{tau}. In a chemotaxis assay, migration of peripheral blood mononuclear cells was stimulated by the addition of IFN-{tau} stimulated-endometrial culture medium, and the effect was significantly reduced by neutralization with an anti-IP-10 antibody. These results suggest that endometrial IP-10 regulated by conceptus IFN-{tau} regulates recruitment and/or distribution of immune cells seen in the early pregnant uterus.

cytokines, immunology, implantation, trophoblast, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pregnancy establishment is a process that involves the coordinated development of the conceptus and the maternal system to prevent luteolysis and promote the receptivity of the uterus for conceptus attachment and subsequent placentation. Although such communication undoubtedly plays a role in uterine receptivity for the conceptus, the importance of the maternal immune system in ruminants often is overlooked. Immune cells such as CD4+, CD8+, and CD45R+ are present in the ovine uterus [1, 2]. The populations of these immune cells within uterine and jugular venous blood do not change during the estrous cycle; however, the percentage of endometrial CD45R+ cells in early pregnant ewes is greater than that in cyclic ewes [1, 3]. In humans and mice, the redistribution of immune cells during early pregnancy is considered important; in particular, the increase in number of uterine natural killer (NK) cells at the implantation site is necessary for successful implantation [4, 5]. The recruitment of immune cells, possibly NK cells, within the uterus may be one of the maternal responses required for pregnancy establishment. However, the mechanisms by which immune cells are recruited to an implantation site have not been well characterized.

Interferons (IFNs) or antiviral activity associated with IFNs are present in the pregnant mammalian uterus [6–9]. However, a role for IFNs during the period of early pregnancy has not been elucidated in species other than ruminants. In ruminants, IFN-{tau} produced by the developing trophectoderm between Gestational Days 9 and 23 has been implicated in the process of maternal recognition of pregnancy [6, 10–12]. IFN-{tau} prevents luteolysis at least in part by inhibiting estrogen receptor expression, thus preventing estrogen stimulation of the oxytocin receptor and impeding the pulsatile release of endometrial prostaglandin F2{alpha} [13–15]. In addition, IFN-{tau} regulates lymphocyte proliferation [16–19] and cytokine production [20, 21], which suggests that IFN-{tau} may play a role in ruminants in adjusting the uterine environment to make it suitable for conceptus growth and differentiation.

During the course of studies to determine unique molecules associated with ovine pregnancy, we identified IFN-{gamma}-inducible protein 10 kDa (IP-10) from cDNA subtraction analysis using Day 17 pregnant and Day 15 cyclic ovine endometrium (unpublished results). This factor, a member of the C-X-C chemokine family, is induced in a variety of cell types, including macrophages, fibroblasts, astrocytes, keratinocytes, epithelial cells, and endothelial cells, and regulates multiple aspects of inflammatory and immune responses primarily through chemotactic activity toward subsets of leukocytes [22–26]. IP-10 targets preferentially NK cells and activates T cells of the Th1 phenotype [27, 28] through the C-X-C chemokine receptor 3 (CXCR3). The majority of genes induced by IFN-{gamma} result from the activation of the IFN-stimulated response element (ISRE) and IFN-{gamma}-activated site (GAS), but regulation of IP-10 gene transcription stimulated by IFN-{gamma} results from ISRE [29]. IFN-{tau} also stimulates the transcription of uterine genes through the ISRE and GAS [30], suggesting the possibility that in ruminants IFN-{tau} may be able to regulate IP-10 expression during early pregnancy.

The present study was undertaken to gain better insight into the recruitment of immune cells by the endometrial chemokine IP-10, which might be regulated by a conceptus factor, IFN-{tau}. To elucidate a possible role of IP-10 and to determine the mechanism by which its expression is regulated in the uterus, we first examined spatiotemporal expression of IP-10, IFN-{gamma}, and CXCR3 mRNA during early pregnancy. We next demonstrated the ability of IFNs to regulate IP-10 expression in vitro and then established that supernatants derived from endometrium cultured with IFN-{tau} induced the migration of peripheral blood mononuclear cells (PBMCs).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Tissue Preparation

Whiteface crossbred ewes were maintained at the U.S. Meat Animal Research Center (Clay Center, NE). The protocol for sheep experimentation has been reviewed and approved by the animal care committees at the USDA. Animal care and estrous synchronization procedures were previously described [31]. Uteri from cyclic ewes on Day 15 (n = 6) and pregnant ewes on Days 14 (n = 3), 17 (n = 6), 20 (n = 3), 25 (n = 3), and 30 (n = 3) were removed immediately after slaughter. Three uteri from Day 15 cyclic ewes and three uteri from Day 17 pregnant ewes were frozen immediately for subsequent in situ hybridization studies. Endometrial and conceptus tissues collected from the remaining pregnant ewes (n = 3 for each day examined) were frozen and stored at -70°C and used for RNA extraction. At the University of Tokyo farm, whole blood, from which PBMCs were obtained, was collected from three cyclic ewes, and these samples were used for IFN dose response and chemotaxis assays. Whole uteri (n = 3) were obtained from three additional cyclic ewes, and endometrial explants were cultured to examine the stimulatory effect of IFNs on IP-10 production. The use of sheep has been approved by the animal care committee at the University of Tokyo. Animal care and estrous synchronization were performed accordingly [32].

In Vitro Culture and RNA Extraction

Primary monocytes and lymphocytes were isolated from Day 15 cyclic ewes using a slightly modified previously described method [33]. PBMCs were separated from EDTA-treated blood (80 ml) by density gradient centrifugation (800 x g at 20°C for 30 min, OptiPrep; Nycomed, Roskilde, Denmark) and were suspended in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS; Sigma, St. Louis, MO), 40 units/ml of penicillin, and 40 µg/ml of streptomycin and anti-pleuropneumonia-like organisms (Invitrogen, Carlsbad, CA). PBMCs counted and adjusted to 3 x 107 cells/ml were plated in six-well coaster plates (3 ml/well), which were incubated at 37°C in a 5% CO2 and 95% air atmosphere for 2 h. The adherent cells (monocytes) were separated from nonadherent cells (lymphocytes) and then cultured in the fresh RPMI 1640 medium with same supplements. To determine effective doses of IFNs on IP-10 expression, monocytes were treated with 102, 103, or 104 IU/ml recombinant human (rh) IFN-{alpha} (Sigma), rhIFN-{gamma} (Upstate Biotechnology, Lake Placid, NY), or recombinant bovine (rb) IFN-{tau} (Katakura Industries Co., Tokyo, Japan). Treatments at each dose point were replicated in triplicate. After 20 h at 37°C in a 5% CO2 atmosphere, culture media were harvested and stored at -70°C, and cells were immediately processed for total RNA extraction. Ovine uterine epithelial and stromal cells [34] (a gift from Dr. Bazer, Texas A&M University, College Station, TX) were cultured in Dulbecco modified Eagle medium (DMEM; Sigma) supplemented with 40 units/ml of penicillin, 40 µg/ml of streptomycin, and 10% FCS.

Endometrial tissues (approximately 600 mg wet weight/culture dish) from Day 15 cyclic ewes were cultured in 20 ml DMEM supplemented with 40 units/ml of penicillin and 40 µg/ml of streptomycin, which were treated with 102 IU/ml rhIFN-{alpha}, rhIFN-{gamma}, or rbIFN-{tau}. These concentrations of IFNs had been determined by the dose-response experiments with monocytes. After 20 h at 37°C in a 5% CO2 atmosphere, culture media and endometrial tissues were frozen separately and stored at -70°C until subsequent Western and Northern blot analyses, respectively. Using Isogen (Nippon Gene, Tokyo, Japan), total RNAs were extracted from endometrial and conceptus tissues and from epithelial and stromal cells, PBMCs, lymphocytes, and monocytes.

Molecular Cloning of Ovine IP-10

Complementary DNA library construction and subtraction experiments were completed using Day 17 pregnant and Day 15 cyclic endometrial RNAs according to the method described previously [35]. From numerous cDNA subtraction studies, a cDNA fragment encoding ovine IP-10 was identified, and the full length IP-10 cDNA was subsequently obtained using 5' rapid amplification of cDNA ends (RACE), 3'-RACE, and full-length polymerase chain reaction (PCR) methodologies [36]. The PCR products of full-length IP-10 cDNA were ligated into a pCRII vector (Invitrogen) and then subjected to an automated sequence analysis using a Perkin-Elmer sequencer (model ABI Prism 377 XL; Roche Molecular Systems, Branchburg, NJ). Nucleotide sequences of ovine IP-10 cDNA were analyzed with the Genetyx software program (Software Development Co., Tokyo, Japan).

Probe Generation and Semiquantitative Analysis by Reverse Transcription PCR

To generate cRNA probes specific for ovine IFN-{tau}, IP-10, CXCR3, and G3 phosphate dehydrogenase (G3PDH), respective cDNAs were generated from ovine endometrial or conceptus RNA using reverse transcription (RT) PCR. Total RNA samples were first reverse transcribed with SuperScriptII (Invitrogen) and oligo-dT primers (20 µl reaction volume) and subjected to PCR amplification with primers shown in Table 1. Each RT-PCR-derived fragment was subcloned into the pCRII vector and then subjected to an automated sequence analysis. Sequence comparisons performed using the BLAST network program (National Center for Biotechnology Information, Bethesda, MD) confirmed correct ovine cDNAs. For Northern blot analysis, digoxigenin (DIG)-labeled cRNA probes [36] were generated from these cDNA constructs using T7 or SP6 RNA polymerase (Promega, Madison, WI).


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  		TABLE 1. Oligonucleotide primers used for PCR

Amounts of uterine IFN-{gamma} and CXCR3 mRNA were determined from PCR amplification using oligonucleotide primers (Table 1). Each reaction consisting of primer pairs for IFN-{gamma}/G3PDH or CXCR3/G3PDH was run with RT template (1 µl) and AmpliTaq Gold (0.625 U; Roche Molecular Systems) in a final volume of 25 µl. Ratios of primer pairs that gave each PCR product within the linear range had been determined: 6:1 for IFN-{gamma}:G3PDH and 5:2 for CXCR3:G3PDH. Both IFN-{gamma} and CXCR3 PCRs consisted of 40 cycles at 95°C for 1 min, 57°C for 1 min, and 72°C for 1 min followed by a final extension at 72°C for 5 min. Following agarose gel electrophoresis and visualization with ethidium bromide, PCR products were quantified using an image analysis system (ATTO Corporation, Tokyo, Japan) equipped with the Quantity One (v3.0 software; PDI, Inc., Huntington Station, NY).

Northern Blot Analysis

Total RNA was used for the determination of mRNAs for IP-10 and IFN-{tau}, but poly(A)+ RNA was used to examine CXCR3 mRNA. Poly(A)+ RNA was obtained from total RNA that had been isolated from PBMCs according to the manufacturer's instructions (TaKaRa, Tokyo, Japan). RNAs isolated from various tissues and cell types were separated by electrophoresis on a 1.0% agarose-formaldehyde gel and transferred to a nylon membrane (Biodyne-B; Pall, East Hills, NY). The membrane was prehybridized in the hybridization buffer containing 5x saline sodium citrate (SSC), 50% formamide, 50 mM PBS, 7% SDS, 0.1% N-lauryl sarcosine, 50 µg/ml salmon sperm DNA (ssDNA), and 2% blocking reagent (Roche Diagnostics, Mannheim, Germany) at 65°C for 1.5 h and then hybridized with cRNA probe in fresh hybridization buffer at 63°C for 12 h. After hybridization, the membrane was washed once with 2x SSC and 0.1% SDS at 65°C for 30 min, washed twice with 0.1x SSC and 0.1% SDS at 65°C for 30 min, and then incubated with RNase-A (20 µg/ml) at 37°C for 1 h. The membrane was incubated in the blocking buffer (1% blocking reagent) at room temperature for 1 h followed by the addition of anti-DIG antibody (1:10 000 dilution; Roche Diagnostics). The membrane was treated three times with the washing solution containing 100 mM maleic acid (pH 7.5), 150 mM NaCl, and 0.3% Tween 20 for 10 min each and finally rinsed in 100 mM Tris-HCl (pH 9.5) and 100 mM NaCl. The chemiluminescent reaction was performed in the latter solution containing CSPD reagent (1:100 dilution; Roche Diagnostics), and the membrane was exposed to x-ray film. Northern blots were then quantified by scanning denstometry using an ES-2000 Epson-Scanner (Seiko Epson Corporation, Nagano, Japan) and Quantity One software (PDI).

In Situ Hybridization

In situ hybridization was performed according to a method previously described with slight modification [37]. Frozen tissues were sectioned (10 µm), mounted onto silan-coated slides, and fixed in 4% paraformaldehyde in PBS. Slide sections were pretreated sequentially with 0.2 N HCl and 20 µg/ml proteinase K in Tris-HCl (pH 7.6), 4% paraformaldehyde, and 0.2% glycine. Sections were then prehybridized in the solution containing 50% formamide, 5x SSC, 1x Denhardt solution, 100 µg/ml heparin, 10 mM dithiothreitol, 10% dextran sulfate, and 0.1 mg/ml denatured tRNA and ssDNA, followed by hybridization with DIG-labeled antisense or sense cRNA probes at 45°C for 16 h. After hybridization and washing once with 4x SSC at 42°C for 20 min, sections were incubated with RNase-A (10 µg/ml) at 37°C for 30 min and then washed twice with 2x SSC at 65°C for 30 min and twice with 0.1x SSC at 65°C for 30 min. The slides were blocked with a blocking reagent (Boehringer Mannheim) and incubated with an anti-DIG alkaline phosphatase-conjugated antibody (Boehringer Mannheim). The signals were detected with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (Promega).

Western Blot Analysis

After in vitro culture of endometrial tissues, the culture media were analyzed for the presence of IP-10 by using Western blot analysis. Culture medium (40 µl) was boiled for 5 min in the SDS sample buffer, electrophoresed on 15% SDS-polyacrylamide gels under reducing conditions, and transferred onto nitrocellulose membranes (Immobilon; Millipore, Bedford, MA). The membranes were blocked with Block Ace (Dainippon Pharmaceutical, Osaka, Japan) at room temperature for 1 h and then incubated with a mouse monoclonal antibody to human IP-10 (Genzyme/Techne, Minneapolis, MN) at 4°C for 12 h. After incubation, membranes were washed three times (10 min each) in Tris-buffered saline (TBS) with Tween 20, incubated with donkey anti-mouse IgG conjugated with horseradish peroxidases (Amarsham Pharmacia Biotech, Buckinghamshire, U.K.) at room temperature for 1 h, and washed three times (15 min each) in TBS-Tween 20. The bands were detected by SuperSignal West Femto Maximum Sensitivity Substrate kit (Pierce, Rockford, IL).

In Vitro Chemotaxis Assay

Migration of PBMCs was assessed in a 96-well modified Boyden chamber (NeuroProbe, Cabin John, MD) using polyvinylpyrrolidone-free polycarbonate membrane (5 µm pore size; NeuroProbe), which had been coated with 10 µg/ml bovine plasma fibronectin (Wako Junyaku, Osaka, Japan) for 2 h before use. The assay was performed as described [38] with minor modifications. DMEM without FCS and the supernatants from endometrial cultures treated or not treated with IFN-{tau} were added to the bottom wells of the chemotaxis chamber, and PBMCs (5 x 106 cells/ml) in DMEM without FCS were added to the top wells of the chamber. After the chambers were incubated at 37°C in a 5% CO2 atmosphere for 2 h, the membranes were removed, washed with PBS, fixed, and stained with Dif-Quick (Kokusai Shiyaku, Kobe, Japan). The number of cells that migrated to the lower surface was microscopically counted in six randomly chosen high-power fields. For the blocking experiments, the supernatants from endometrium stimulated with IFN-{tau} were preincubated at 37°C for 1 h with 30 µg/ml of anti-IP-10 or control mouse IgG (Sigma) before addition to the top chamber. Chemotaxis assays at each treatment were replicated in triplicate.

Statistical Analysis

Light intensity (RT-PCR) and optical density (Northern blots) measurements were subjected to least squares (LS) ANOVA using the general linear models procedures of the Statistical Analysis System (version 6.0; SAS Institute, Cary, NC). The model used in the LS-ANOVA included day and replicate as sources of variation. The light intensity and optical density from G3PDH PCR products and G3PDH mRNA, respectively, were used as covariates for RT-PCR and Northern blot analyses. The LS means (LSMs) and SEMs illustrated in the figures were derived from this analysis. When a significant effect of day of pregnancy was detected (P < 0.05), the data for amounts of IP-10, IFN-{gamma}, or CXCR3 mRNAs were analyzed by LS regression analysis. In these analyses, day was considered a continuous and independent source of variation, and replicate was a dependent source.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of IP-10 cDNA

Ovine IP-10 cDNA fragments were obtained from endometrial tissue collected on Day 17 of pregnancy, and a full-length ovine IP-10 cDNA was cloned using 5'-RACE and 3'-RACE. The resulting ovine cDNA encompasses 1172 base pairs with an open reading frame corresponding to 102 amino acids (GenBank accession AB070717). Comparative analysis of the amino acid sequences among various species is shown in Figure 1A. Four cysteine residues are conserved within this chemokine family, and the first two cysteines are separated by a single amino acid, C-X-C. This cysteine motif was found in amino acid residues deduced from ovine IP-10 cDNA. The C-X-C chemokines are subdivided into two classes depending on the presence of the glutamate-leucine-arginine (ELR) motif preceding the first two cysteines. Similar to IP-10s of other species, the ovine chemokine lacked the ELR motif and showed a high degree of similarity to human IP-10, 82.7% and 75.5% at the nucleotide and amino acid levels, respectively (Fig. 1B).



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  		FIG. 1. A) Alignment of amino acid sequences of ovine, human, and mouse IP-10. Among these species, four cysteine residues were conserved, but the ELR motif preceding the first two cysteines was absent. B) Homology comparison of IP-10

Changes of IP-10, CXCR3, IFN-{tau}, and IFN-{gamma} mRNA Expression During Early Pregnancy

Uterine IP-10 mRNA levels on Days 14, 17, 20, 25, and 30 of pregnancy were examined by Northern blot analysis (Fig. 2A). Consistent with the result from cDNA subtraction and IP-10 cDNA cloning experiments, a single transcript (approximately 1.1 kilobases in size) for IP-10 mRNA was detected in uterine endometrium of pregnant ewes. The expression of endometrial IP-10 mRNA was much higher on Days 14, 17, 20, and 25 of pregnancy than that in cyclic ewes (P < 0.05). In addition, expression of endometrial CXCR3 mRNA, a receptor for IP-10, was higher on Days 17 and 20 in pregnant ewes (P < 0.05, Fig. 2B). Expression of IFN-{tau} mRNA was detected in the conceptus, and the level of this expression was higher in the Day 14 conceptus than that on Days 17 and 20 (P < 0.05, Fig. 3A). Expression of IFN-{gamma} mRNA was detected in the uterine endometrium of both pregnant and cyclic ewes and was higher on Days 14, 17, 20, and 25 in pregnant ewes than that in cyclic ewes (P < 0.05, Fig. 3B).



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  		FIG. 2. Levels of IP-10 mRNA expression in the ovine uterus during early pregnancy. A) Left: Northern blot analysis of IP-10 mRNA in the uterus of pregnant ewes (n = 3 for each day) and cyclic ewes (Day 15, n = 3) and in the Day 17 conceptus (Con, n = 3). Right: Densitometric analysis of Northern blot analysis of IP-10 mRNA. Bars represent LSM ± SEM. An asterisk indicates a significant difference (P < 0.05) when compared with the value from Day 15 cyclic uteri. Regression analysis on IP-10 mRNA and day of pregnancy was significant, y = -3.06x2 + 129.18x - 1092.50 (R2 = 0.869, P < 0.01). B) Left: Semiquantitative PCR of CXCR3 mRNA and G3PDH mRNA in the uterus of pregnant (n = 3 for each day) and cyclic (Day 15, n = 3) ewes. Right: Densitometric analysis of semiquantitative PCR products for CXCR3 mRNA and G3PDH mRNA. Bars represent LSM ± SEM. An asterisk indicates a significant difference (P < 0.05) when compared with the value from Day 15 cyclic uteri. Regression analysis of CXCR3 mRNA and day of pregnancy was significant, y = -0.50x2 + 19.85x + 79.34 (R2 = 0.9257, P < 0.01)



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  		FIG. 3. Levels of IFN-{tau} and IFN-{gamma} mRNA in the ovine conceptus and uterus during early pregnancy. A) Northern blot analysis of IFN-{tau} mRNA in the conceptuses of pregnant ewes (n = 3 for each day). B) Top: Semiquantitative PCR of IFN-{gamma} mRNA and G3PDH mRNA in the uterus of pregnant (n = 3 for each day) and cyclic (Day 15, n = 3) ewes, and in the Day 17 conceptus (Con, n = 3). Bottom: Densitometric analysis of semiquantitative PCR products for IFN-{gamma} mRNA and G3PDH mRNA. Bars represent LSM ± SEM. An asterisk indicates a significant difference (P < 0.05) when compared with the value from Day 15 cyclic uteri. Regression analysis of IFN-{gamma} mRNA and day of pregnancy was significant, y = -0.64x2 + 26.24x + 112.31 (R2 = 0.9987, P < 0.01)

Localization of IP-10 mRNA

To determine a cellular source(s) of IP-10 in the sheep endometrium, in situ hybridization was performed on frozen sections of uterine tissues prepared from Day 15 cyclic and Day 17 pregnant ewes (Fig. 4A). IP-10 mRNA was detected in the subepithelial stroma but not in the luminal and glandular epithelium of the pregnant uterus. At higher magnification, the signal appeared to be present in immune cells. In the cyclic animals, endometrial expression of IP-10 mRNA was not detected. In addition, Northern blot analysis was performed to identify tissue and/or cell types expressing IP-10 mRNA. IP-10 mRNA was found in RNA extracted from monocytes but not in RNA from lymphocytes, epithelial cells, or stromal cells (Fig. 4B).



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  		FIG. 4. A) In situ hybridization analysis of IP-10 mRNA in the ovine uterus. DIG-labeled antisense ovine IP-10 in Day 17 pregnant ewes (a and b), sense IP-10 in Day 17 pregnant ewes (c), and antisense IP-10 in Day 15 cyclic ewes (d). le, Luminal epithelium; ge, glandular epithelium; st, subepithelial stroma; tr, trophoblast. Bars = 40 µm (a, c, and d) or 10 µm (b). B) Northern blot analysis of IP-10 mRNA (n = 3) in RNA isolated from monocytes (M), lymphocytes (L), epithelial cells (E), and stromal cells (S)

Effect of IFN-{tau} on IP-10 Expression

Because monocytes appeared to be the source of IP-10 mRNA among cells examined (Fig. 4B), we investigated various IFNs for their dose responses and their ability to stimulate IP-10 mRNA in monocytes. Monocytes isolated from ewes were cultured in vitro for 20 h in the presence or absence of 102–104 IU/ml of rhIFN-{alpha}, rhIFN-{gamma}, or rbIFN-{tau}. All IFNs stimulated the expression of IP-10 mRNA in ovine monocytes; however, the effective doses were different for each IFN (Fig. 5A); rbIFN-{tau} at a dose of 102 IU/ml stimulated the expression of IP-10 mRNA more effectively than the other IFNs examined.



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  		FIG. 5. Effect of several IFNs on IP-10 mRNA levels. A) Dose-response experiments for the expression of IP-10 mRNA in IFN-stimulated monocytes (n = 3). B) Effect of several IFNs on IP-10 mRNA levels in the endometrial explants from cyclic ewes. Left: Northern blot analysis of IP-10 mRNA in the endometrial explants stimulated by 102 IU/ml rhIFN-{alpha}, rhIFN-{gamma}, or rbIFN-{tau}. Right: Densitometric analysis of Northern blots of IP-10 mRNA and G3PDH mRNA. Bars represent LSM ± SEM. An asterisk indicates a significant difference (P < 0.05) when compared with the value from endometrial explants cultured without IFN. C) Western blot analysis of IP-10 in the culture medium from endometrial explants stimulated by IFNs

In cyclic ewes, we then investigated whether IFNs could affect endometrial IP-10 (mRNA and protein) production. Endometrial explants from Day 15 cyclic ewes were cultured in vitro for 20 h in the presence or absence of 102 IU/ml rhIFN-{alpha}, rhIFN-{gamma}, or rbIFN-{tau}. Total RNA was extracted from IFN-stimulated endometrial tissues and examined for IP-10 mRNA by Northern blot analysis. Minute levels of IP-10 mRNA were detected in controls and in rhIFN-{alpha}- and rhIFN-{gamma}-stimulated samples, but high levels of IP-10 mRNA were found only after stimulation with rbIFN-{tau} (Fig. 5B). Western blot analysis confirmed that endometrial IP-10 production was stimulated by rbIFN-{tau} (Fig. 5C).

Effect of IP-10 on PBMC Migration

To ascertain whether endometrium-derived IP-10 was biologically active, we tested the ability of IFN-{tau}-stimulated endometrial culture medium to recruit PBMCs in an in vitro chemotaxis assay. Presence of mRNA for the IP-10 receptor CXCR3 was confirmed in RNA extracted from PBMCs using Northern blot analysis (Fig. 6A). Medium from endometrial tissue culture that had been stimulated by IFN-{tau} exerted significant chemotactic effect on PBMCs, whereas the medium obtained from the endometrium cultured without IFN-{tau} was ineffective (Fig. 6B). Immunoneutralization experiments revealed that an anti-IP-10 antibody reduced the chemotactic activities of culture medium from IFN-{tau}-stimulated endometrium by 60–70%, indicating that the chemotactic activity exhibited by the culture medium from IFN-{tau}-stimulated endometrium was due mostly to IP-10 (Fig. 6B).



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  		FIG. 6. Effect of IFN-{tau}-stimulated endometrial culture medium on migratory activity of PBMCs. A) Northern blot analysis of CXCR3 mRNA in PBMCs. Lane T: total RNA, 10 µg; lane M: poly(A)+ RNA, 2 µg. B) PBMCs were challenged with medium from IFN-{tau}-treated or untreated endometrial cultures in the presence or absence of an anti-IP-10 antibody. Bars represent LSM ± SEM. An asterisk indicates a significant difference (P < 0.05) when compared with the migration of PBMCs with IFN-{tau}-untreated endometrial culture medium. Double asterisks indicate a significant difference (P < 0.05) when compared with the migration of PBMCs with IFN-{tau}-treated endometrial culture medium in the absence of an anti-IP-10 antibody


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Ovine IP-10, originally identified from cDNA subtraction analysis of pregnant and cyclic uterine endometrium, was present in the pregnant uterus and to a much lesser degree in the cyclic endometrium. IP-10 is a member of the C-X-C chemokine family, which regulates multiple aspects of inflammatory and immune responses primarily through the receptor for IP-10, CXCR3, and/or chemotactic activity of subsets of leukocytes [28]. The number of immunocompetent CD45R+ cells in the early pregnant uterus is greater than that in the cyclic uterus [1]. In this study, we demonstrated that medium from endometrium cultured in the presence of IFN-{tau} contained IP-10 and stimulated the migration of PBMCs, and up to 70% of this stimulation could be inhibited by the use of an anti-IP-10 antibody (Fig. 6). These results suggest that immune cells seen in the early pregnant uterus could be recruited by endometrial IP-10 that had been stimulated by conceptus factor IFN-{tau}. An immunological effect of IFN-{tau} has long been suspected partially because of the observation that IFN-{tau} regulates lymphocyte proliferation and cytokine production in vitro [16–21]. So far this effect has not been proven in vivo, but the findings from this study represent an initial step toward understanding the role that IFN-{tau} plays in utero other than the prevention of luteolysis.

In the dose-response experiment using monocytes, all IFNs tested (rhIFN-{alpha}, rhIFN-{gamma}, and rbIFN-{tau}) stimulated IP-10 mRNA expression, but the effective doses differed among IFNs. IFN-{tau} effectively stimulated IP-10 expression in monocytes at a dose of 102 IU/ml, whereas rhIFN-{gamma} at the same dose was less effective (Fig. 5A). It is unclear whether this low level of stimulation of IP-10 mRNA expression in sheep by rhIFN-{gamma} reflects the fact that IFN-{gamma} is highly species specific compared with other IFNs [39]. A high dose of rhIFN-{gamma} (104 IU/ml) stimulated the expression of IP-10 mRNA in monocytes. Thus, the low stimulatory effect of IFN-{gamma} on IP-10 production was not solely due to a loss of activity. One question to be addressed is whether degrees of IP-10 mRNA expression are reflections of particular recombinant IFNs; however, the possibility of variable activities exhibited by such IFNs was not specifically addressed in the present investigation.

In endometrial explant cultures, only rbIFN-{tau} increased the production of IP-10 mRNA and protein (Fig. 5, B and C). This finding raised the question of whether IFN-{tau} was the only stimulator of endometrial IP-10 expression in utero during early pregnancy in sheep. The pattern of endometrial IP-10 mRNA expression appeared to have followed that of conceptus IFN-{tau} mRNA during early pregnancy, and changes in IP-10 expression (Fig. 2A) were similar to changes in other endometrial chemokines such as monocyte chemoattractant protein (MCP)-1 and MCP-2 [40]. Although IFN-{gamma} protein was not specifically assessed in this study, the only IFN-like activity so far characterized in the ovine uterus during early pregnancy is that of IFN-{tau}. In the present study, endometrial IFN-{gamma} mRNA could only be detected after 40 cycles of RT-PCR in both pregnant and cyclic animals, with an abundance about 1.5 times higher in pregnant animals than in cyclic ones (Fig. 3B). The increase in endometrial IP-10 expression in pregnant ewes was much greater than that in cyclic ewes (Fig. 2A). Thus, rather than IFN-{gamma}, IFN-{tau} appears to be the IFN that regulates endometrial IP-10 expression during early pregnancy in sheep.

IP-10 mRNA was detected by in situ hybridization analysis in the subepithelial stroma, and the signal appeared to be localized in immune cells (Fig. 4A). In human and mouse, IP-10 is secreted by monocytes [22, 23], and in the present study IP-10 was likewise expressed in monocytes but not in lymphocytes, epithelial cells, or stromal cells (Fig. 4B). Transcripts of MCPs, another group of endometrial chemokines, are also localized in the subepithelial storma, but MCP-positive cells are eosinophils [40]. These observations suggest that IP-10 in the subepithelial stroma is produced by resident macrophages and/or macrophages that have been recruited to that region. Migrating cells move along concentration gradients established by chemokines, and chemotactic factors are often associated with cell-surface macromolecules such as extracellular matrices [41]. These results and the observations that endometrial IP-10 production in vitro was enhanced by IFN-{tau} and that IP-10 expression was highest on Gestational Day 17 suggest that IP-10 is produced by macrophages in the subepithelial region, which stimulates migration of CXCR3-expressing immune cells toward or near the site of conceptus implantation to the maternal endometrium.

In mice and humans, IP-10 recruits cytotoxic cells such as NK and Th1 cells through CXCR3 [27, 28], and the number of immune cell subsets (uterine NK cells) and/or NK lytic activity also increase dramatically during early pregnancy [5, 42]. Segerson and Beetham [43] revealed that during early pregnancy the ovine endometrium contains NK-like cells with lytic activity. Endometrial IP-10 may affect the population of NK-like cells during the peri-implantation period, although the function of these cells and/or lytic activity during early pregnancy has not been clarified in sheep. The immune system during pregnancy is thought to be biased toward the humoral immune response [44, 45]. However, the increase in NK activity during the early pregnancy and the induction of abortion by an antibody to CD8+ in a cell-depletion experiment in vivo [46] indicate that the presence of NK and CD8+ T cells (included in cytotoxic cells) may be a prerequisite for immunological tolerance of the conceptus. Endometrial IP-10, most likely regulated by conceptus IFN-{tau}, could change local immunomodulatory signals and increase the cell-mediated immune response in early pregnancy, resulting in prevention of early spontaneous abortion until the formation of a functional placenta. These observations support the hypothesis that the relative balance between these beneficial and detrimental immune responses is of paramount importance to implantation and the establishment of pregnancy.

In the present investigation, we determined the spatiotemporal expression of IP-10 and IFN-{gamma} mRNA during early pregnancy in the ovine uterus and revealed the ability of conceptus IFN-{tau} to regulate endometrial IP-10 expression in vitro. These findings suggest that endometrial IP-10 regulated by IFN-{tau} plays an important role in the recruitment and/or redistribution of immune cells during early pregnancy in ruminants. Further investigations are required to better characterize the physiological role(s) of the immune cells recruited by endometrial IP-10.


   ACKNOWLEDGMENTS
 
We thank Dr. Fuller W. Bazer (Texas A&M University) for generously providing ovine epithelial and stromal cells and Dr. L. Weber for critical reading of the manuscript. We also appreciate the technical assistance of D. Sypherd, the U.S. MARC sheep crew and the Experimental Station for Bio-Animal Science crew, and the University of Tokyo for husbandry of animals. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.


   FOOTNOTES
 
1 This work was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by the Program for Promotion of Basic Research Activities for Innovative Bioscience (BRAIN), and by a Grant-in-Aid of Recombinant Cytokines Project from the Ministry of Agriculture, Forestry and Fisheries of Japan. K.N. was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. Back

2 Correspondence: Kazuhiko Imakawa, Laboratory of Animal Breeding, Faculty of Agriculture, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. FAX: 81 3 58418180; akaz{at}mail.ecc.u-tokyo.ac.jp Back

Received: 8 July 2002.

First decision: 4 August 2002.

Accepted: 30 October 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Segerson EC, Matterson PL, Gunsett FC. Endometrial T-lymphocyte subset infiltration during the ovine estrous cycle and early pregnancy. J Reprod Immunol 1991 20:221-236[CrossRef][Medline]
  2. Lee CS, Meeusen E, Gogolin-Ewens KJ, Brandon MR. Quantitative and qualitative changes in the intraepithelial lymphocyte population in the uterus of nonpregnant and pregnant sheep. Am J Reprod Immunol 1992 28:90-96
  3. Alder RG, Shelton JN. Lymphocyte subpopulations in lymph and blood draining from the uterus and ovary in sheep. J Reprod Immunol 1990 17:27-40[CrossRef][Medline]
  4. Guimond MJ, Luross JA, Wang B, Terhorst C, Danial S, Croy BA. Absence of natural killer cells during murine pregnancy is associated with reproductive compromise in TgE26 mice. Biol Reprod 1997 56:169-179[Abstract]
  5. King A. Uterine leukocytes and decidualization. Hum Reprod Update 2000 6:28-36[Abstract/Free Full Text]
  6. Martal J, Lacroix MC, Loudes C, Saunier M, Wintenberger-Torres S. Trophoblastin, an antiluteolytic protein present in early pregnancy in sheep. J Reprod Fertil 1979 56:63-72[Abstract/Free Full Text]
  7. Aboagye-Mathiesen G, Toth FD, Juhl C, Norskov-Lauritsen N, Petersen PM, Ebbesen P. Purification and initial characterization of human placental trophoblast interferon induced by polyriboinosinic.polyribocytidylic acid. J Gene Virol 1990 71:3061-3066[Abstract/Free Full Text]
  8. Lefevre F, Martinat-Botte F, Guillomot M, Zouari K, Charley B, La Bonnardiere C. Interferon-gamma gene and protein are spontaneously expressed by the porcine trophectoderm early in gestation. Eur J Immunol 1990 20:2485-2490[Medline]
  9. Ashkar AA, Croy BA. Interferon-{gamma} contributes to the normalcy of murine pregnancy. Biol Reprod 1999 61:493-502[Abstract/Free Full Text]
  10. Imakawa K, Anthony RV, Kazemi M, Marotti KR, Polites HG, Roberts RM. Interferon-like sequence of ovine trophoblast protein secreted by embryonic trophectoderm. Nature 1987 330:377-379[CrossRef][Medline]
  11. Roberts RM, Cross JC, Leaman DW. Interferons as hormones of pregnancy. Endocr Rev 1992 13:432-452[Abstract/Free Full Text]
  12. Roberts RM, Leaman WW, Cross JC. Role of interferons in maternal recognition of pregnancy in ruminants. Proc Soc Exp Biol Med 1992 200:7-18[CrossRef][Medline]
  13. Flint APF, Parkinson TJ, Stewart HJ, Vallet JL, Lamming GE. Molecular biology of trophoblast interferons and studies of their effects in vivo. J Reprod Fertil Suppl 1991 43:13-25[Medline]
  14. Spencer TE, Becker WC, George P, Mirando MA, Ogle TF, Bazer FW. Ovine interferon-{tau} inhibits estrogen receptor up-regulation and estrogen-induced luteolysis in cyclic ewes. Endocrinology 1995 136:4932-4944[Abstract]
  15. Spencer TE, Bazer FW. Ovine interferon-{tau} suppresses transcription of the estrogen receptor and oxytocin receptor genes in the ovine endometrium. Endocrinology 1996 137:1144-1147[Abstract]
  16. Newton GR, Vallet JL, Hansen PJ, Bazer FW. Inhibition of lymphocyte proliferation by ovine trophoblast protein-1 and a high molecular weight glycoprotein produced by the peri-implantation sheep conceptus. Am J Reprod Immunol 1989 19:99-107
  17. Pontzer CH, Bazer FW, Johnson HM. Antiproliferative activity of a pregnancy recognition hormone, ovine trophoblast protein-1. Cancer Res 1991 51:5304-5307[Abstract/Free Full Text]
  18. Skopets B, Li J, Thatcher WW, Roberts RM, Hansen PJ. Inhibition of lymphocyte proliferation by bovine trophoblast protein-1 (type I trophoblast interferon) and bovine interferon-alpha I1. Vet Immunol Immunopathol 1992 34:81-96[CrossRef][Medline]
  19. Assal-Meliani A, Charpigny G, Reinaud P, Martal J, Chaouat G. Recombinant ovine trophoblastin (roTP) inhibits ovine, murine and human lymphocyte proliferation. J Reprod Immunol 1993 25:149-165[CrossRef][Medline]
  20. Tuo W, MacMillan H, Gunter N, Bazer FW, Brown WC. Upregulation of interleukin-4 and IFN-gamma expression by IFN-tau, a member of the type I IFN family. J Interferon Cytokine Res 1999 19:79-87
  21. Emond V, Asselin E, Fortier MA, Murphy BD, Lambert RD. Interferon-tau stimulates granulocyte-macrophage colony-stimulating factor gene expression in bovine lymphocytes and endometrial stromal cells. Biol Reprod 2000 62:1728-1737[Abstract/Free Full Text]
  22. Luster A, Unkeless J, Ravetch J. {gamma}-Interferon transcriptionally regulates an early-response gene containing homology to platelet proteins. Nature 1985 315:672-676[CrossRef][Medline]
  23. Ohmori Y, Hamilton TA. A macrophage LPS-inducible early gene encodes the murine homologue of IP-10. Biochem Biophys Res Commun 1990 168:1261-1267[CrossRef][Medline]
  24. Sauty A, Dziejman M, Taha RA, Iarossi AS, Neote K, Garcia-Zepeda EA, Hamid Q, Luster AD. The T cell-specific CXC chemokines IP-10, MIG, and I-TAC are expressed by activated human bronchial epithelial cells. J Immunol 1999 162:3549-3558[Abstract/Free Full Text]
  25. Albanesi C, Scarponi C, Sebastiani S, Cavani A, Federici M, De Pita O, Puddu P, Girolomoni G. IL-4 enhances keratinocytes expression of CXCR3 agonistic chemokines. J Immunol 2000 165:1395-1402[Abstract/Free Full Text]
  26. Huang D, Han Y, Rani MR, Glabinski A, Trebst C, Sorensen T, Tani M, Wang J, Chien P, O'Bryan S, Bielecki B, Zhou ZL, Majumder S, Ransohoff RM. Chemokines and chemokine receptors in inflammation of the nervous system: manifold roles and exquisite regulation. Immunol Rev 2000 177:52-67[CrossRef][Medline]
  27. Taub DD, Lloyd AR, Conlon K, Wang JM, Ortaldo JR, Harada A, Matsushima K, Kelvin DJ, Oppenheim JJ. Recombinant human interferon-inducible protein 10 is a chemoattractant for human monocytes and T lymphocytes and promotes T cell adhesion to endothelial cells. J Exp Med 1993 177:1809-1814[Abstract/Free Full Text]
  28. Farber JM. Mig and IP-10: CXC chemokines that target lymphocytes. J Leukoc Biol 1997 61:246-257[Abstract]
  29. Majumder S, Zhou LZ, Chaturvedi P, Babcock G, Aras S, Ransohoff RM. p48/STAT-1alpha-containing complexes play a predominant role in induction of IFN-gamma-inducible protein, 10kDa (IP-10) by IFN-gamma alone or in synergy with TNF-alpha. J Immunol 1998 161:4736-4744[Abstract/Free Full Text]
  30. Stewart DM, Johnson GA, Vyhlidal CA, Burghardt RC, Safe SH, Yu-Lee LY, Bazer FW, Spencer TE. Interferon-tau activates multiple signal transducer and activator of transcription proteins and has complex effects on interferon-responsive gene transcription in ovine endometrial epithelial cells. Endocrinology 2001 142:98-107[Abstract/Free Full Text]
  31. Imakawa K, Tamura K, Lee RSF, Ji Y, Kogo H, Sakai S, Christenson RK. Temporal expression of type I interferon receptor in the peri-implantation ovine extra-embryonic membranes: demonstration that human IFN{alpha} can bind to this receptor. Endocr J 2002 49:195-205[Medline]
  32. Aida H, Yamaguchi H, Katsumura M, Imakawa K, Sakai S. Identification of interferon-tau at the maternal-fetal interface in Shiba goats. J Reprod Dev 1999 45:249-257[CrossRef]
  33. Domenech A, Goyache J, Llames L, Jesus Paya M, Suarez G, Gomez-Lucia E. In vitro infection of cells of the monocyte/macrophage lineage with bovine leukaemia virus. J Gen Virol 2000 81:109-118[Abstract/Free Full Text]
  34. Johnson GA, Burghardt RC, Newton GR, Bazer FW, Spencer TE. Development and characterization of immortalized ovine endometrial cell lines. Biol Reprod 1999 61:1324-1330[Abstract/Free Full Text]
  35. Akopyants NS, Fradkov A, Diatchenko L, Hill JE, Siebert PD, Lukyanov SA, Sverdlov ED, Berg DE. PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori. Proc Natl Acad Sci U S A 1998 95:13108-13113[Abstract/Free Full Text]
  36. Kuraishi T, Sun Y, Aoki F, Imakawa K, Sakai S. The poly(A) tail length of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of milk. Biochem J 2000 347:579-583[CrossRef][Medline]
  37. Kanai Y, Kanai-Azuma M, Noce T, Saido TC, Shiroishi T, Hayashi Y, Yazaki K. Identification of two Sox17 messenger RNA isoforms, with and without the high mobility group box region, and their differential expression in mouse spermatogenesis. J Cell Biol 1996 133:667-681[Abstract/Free Full Text]
  38. Gasperini S, Marchi M, Calzetti F, Laudanna C, Vicentini L, Olsen H, Murphy M, Liao F, Farber J, Cassatella MA. Gene expression and production of the monokine induced by IFN-{gamma} (MIG), IFN-inducible T cell {alpha} chemoattractant (I-TAC), and IFN-{gamma}-inducible protein-10 (IP-10) chemokines by human neutrophils. J Immunol 1999 162:4928-4937.[Abstract/Free Full Text]
  39. Pestka S, Launger JA. Interferons and their actions. Annu Rev Biochem 1987 56:727-777[CrossRef][Medline]
  40. Asselin E, Johnson GA, Spencer TE, Bazer FW. Monocyte chemotactic protein-1 and -2 messenger ribonucleic acids in the ovine uterus: regulation by pregnancy, progesterone, and interferon-{tau}. Biol Reprod 2001 64:992-1000[Abstract/Free Full Text]
  41. Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 1994 76:301-314[CrossRef][Medline]
  42. Yu Z, Croy BA, Chapeau C, King GJ. Elevated endometrial natural killer cell activity during early porcine pregnancy is conceptus-mediated. J Reprod Immunol 1993 24:153-164[CrossRef][Medline]
  43. Segerson EC, Beetham PK. High-density ovine endometrial cells exhibit natural killer activity during early pregnancy. Theriogenology 2000 54:1207-1214[CrossRef][Medline]
  44. Lin H, Mosmann TR, Guilbert L, Tuntipopipat S, Wegmann TG. Synthesis of T helper 2-type cytokines at the maternal-fetal interface. J Immunol 1993 151:4562-4573[Abstract]
  45. Wegmann TG, Lin H, Guilbert L, Mosmann TR. Bidirectional cytokine interactions in the maternal-fetal relationship: is successful pregnancy a TH2 phenomenon?. Immunol Today 1993 14:353-356.[CrossRef][Medline]
  46. Athanassakis I, Bleackley RC, Paetkau V, Guilbert L, Barr PJ, Wegmann TG. The immunostimulatory effect of T cells and T cell lymphokines on murine fetally derived placental cells. J Immunol 1987 138:37-44[Abstract]



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