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J. Biol. Chem., Vol. 278, Issue 31, 29048-29056, August 1, 2003
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Regulation of Blastocyst Migration, Apposition, and Initial Adhesion by a Chemokine, Interferon 
-inducible Protein 10 kDa (IP-10), during Early Gestation*
From the
Laboratory of Animal Breeding, Faculty of
Agriculture, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657,
Japan, the ¶R&D Department, Higeta Shoyu Co.,
Ltd, 2-8 Chuo, Choshi, Chiba 288-8680, Japan, the
||Korea Research Institute of Bioscience &
Biotechnology, Oun-dong 52, Yusong-ku, Daejon 305-333, Korea, and the
**United States Meat Animal Research Center, USDA-ARS,
Clay Center, Nebraska 68933-0166
Received for publication, January 16, 2003 , and in revised form, April 23, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For a pregnancy to be established, initial apposition and adhesion of the blastocyst to maternal endometrium must occur in a coordinated manner; however, a key factor(s) that mediates the trophoblast cell migration and attachment to the apical surface of the endometrium has not been identified. In this study, we examined the effect of an endometrial chemokine, interferon-
-inducible protein 10 kDa (IP-10), on conceptus migration to
the endometrial epithelium. We first studied endometrial IP-10 mRNA
expression, which was localized in the subepithelial stromal region, and
detected the protein in the uterine flushing media during early pregnancy.
Expression of IP-10 mRNA by the endometrium of cyclic animals was stimulated
by the addition of a conceptus factor interferon-tau (IFN-
).
Immunofluorescent analysis revealed that IP-10 receptor, CXCR3, was localized
in the trophoblast cells, to which biotinylated-recombinant caprine IP-10
(rcIP-10) bound. Chemotaxis assay indicated that rcIP-10 stimulated the
migration of trophoblast cells, and the effects of rcIP-10 were neutralized by
the pretreatment with an anti-IP-10 antibody. Adhesive activity of trophoblast
cells to fibronectin was promoted by rcIP-10, and the effect was inhibited by
the use of anti-IP-10 antibody. Further adhesion experiments demonstrated that
binding of trophoblast cells to fibronectin was completely inhibited by a
peptide of the Arg-Gly-Asp (RGD) sequence, which binds to integrins
5
1, V1,
V3, and
V5, whereas non-binding peptide containing
Arg-Gly-Glu (RGE) had minimal effects. More importantly, rcIP-10 promoted the
adhesion of trophoblast cells to primary cells isolated from endometrial
epithelium. Furthermore, rcIP-10 stimulated the expression of integrin
5, V, and 3 subunit mRNA
in trophoblast cells. These findings suggest that endometrial IP-10 regulates
the establishment of apical interactions between trophoblast and epithelial
cells during early gestation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Implantation requires closely harmonized processes of apposition, attachment, and adhesion of the conceptus to the maternal endometrium and is completed when an initial formation of the placenta between the conceptus and endometrium is established. In the ruminant ungulates, interferon-
(IFN-),1 secreted
by the trophoblast cells, plays a role in the process of maternal recognition
of pregnancy (1,
2). When IFN- production
is highest between days 15 and 18 of gestation, the elongated conceptus
reaches a point of attachment in utero, and fuses with endometrial
epithelial cells, which undergoes the developmental changes, resulting in the
formation of a noninvasive synepitheliochorial placenta
(3). When the conceptus
attachment begins, physical interactions are established between the tips of
the endometrial microvilli and the trophoblast cells, which involve changes in
the expression of cell adhesion molecules and extracellular matrix (ECM)
proteins
(4–6).
Integrins are a large family of cell surface receptors that mediate the
attachment of cells possessing ECM. The expression of integrins on endometrial
luminal epithelium changes during the estrous cycle, and such expressions on
uterine surface and embryonic trophectoderm also change during gestation. In
addition, the increase in integrin expressions on the trophectoderm during the
early pregnancy is required for initial and/or firm adhesion of the conceptus
to endometrial cells. However, a key factor(s) that mediates the trophoblast
cell migration and attachment to the apical surface of endometrium has not
been identified.
Recently, we demonstrated that a chemokine, interferon--inducible
protein 10 kDa (IP-10), was expressed in the pregnant ovine endometrium, and
the endometrial tissue expression of IP-10 in vitro was stimulated by
the addition of conceptus factor IFN-
(7). Chemokines are a family of
structurally related low molecular mass cytokines and play a key role in the
migration and activation of different subpopulations of leukocytes
(8). Four subfamilies,
designated C, CC, CXC, and CX3C chemokines, are identified based on
the relative positions of conserved cysteine residues
(9). IP-10, belongs to the CXC
chemokine subfamily, is induced in a variety of cell types
(10), and regulates multiple
aspects of inflammatory and immune responses primarily through chemotactic
activity toward subsets of leukocytes
(11). IP-10 targets the
receptor CXCR3 and activates several subsets of leukocytes, NK cells, Th1
lymphocytes, eosinophils and dendritic cells
(12–15).
Several chemokines have been discovered in the fetal and maternal annexes
during the early pregnancy in humans
(16–18),
mice
(19–21),
and ruminants (7,
22,
23). These chemokines are
thought to play an important role in cellular movement and positioning of
leukocytes, which infiltrate into the subepithelial stromal regions of the
uterus. It has been suspected that distribution and/or redistribution of
leukocytes, regulated by chemokines expressed in utero in a
spatial-temporal manner, is necessary for the establishment of immunological
environments suitable for implantation and subsequent development.
It is known that non-hematopoietic tumor cells exhibit specific patterns of
metastasis to other organs, suggesting that tumor cells do not migrate
randomly. One explanation for this phenomenon is that the migration of tumor
cells may be determined by interactions between particular chemokines and
specific receptors at target organs
(24,
25). It has been reported that
some chemokine receptor transcripts are detected in human and mouse
non-hematopoietic trophoblast cells
(26–28).
These observations lead us to postulate that interactions between chemokines
and their receptors modulate trophoblast migration to the endometrium.
Moreover, chemokines may stimulate the integrin-dependent adhesion of
trophoblast cells to the apical surface of endometrium because chemokines are
also known as physiological activators of rapid integrin-dependent leukocytes
arrest on endothelial cells
(29). However, it is unclear
whether endometrial IP-10, stimulated by conceptus factor IFN-, modulates
the migration and adhesion of trophoblast cells to the endometrium in
ruminants.
In the present study, we assessed a putative role that IP-10/CXCR3 plays within the uterus during early pregnancy in ruminants. To study a role of IP-10/CXCR3 for engagement of the conceptus and endometrium, temporal expression of IP-10 and CXCR3 within the uterus of Shiba goats was examined. Whether the interaction between IP-10 and CXCR3 could mediate trophoblast cell migration and adhesion to the apical surface of endometrium was then determined. Examination is extended to identify which integrin subunits and ECMs were involved in the trophoblast cell adhesion to the endometrium through the IP-10/CXCR3 signaling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals and Tissue Preparation—Shiba goats were maintained at the University of Tokyo farm and the protocol for goat experimentation was reviewed and approved by the Animal Care Committee at the university. Uteri from cyclic goats on days 14 (n = 9) and 17 (n = 3) and pregnant goats on days 14 (n = 6), 17 (n = 9), and 20 (n = 3) were removed by hysterectomy under isoflurane anesthesia. Whole uteri from day 14 cyclic and days 14 and 17 pregnant animals (n = 3 each) were frozen for in situ hybridization analysis (7). Uteri from days 14 and 17 pregnant and days 14 and 17 cyclic animals were subjected to uterine flushing procedure for the isolation of conceptuses and/or collection of flushed media. PBS (15 ml) was gently flushed into the uterus, and the uterine flushing media collected were concentrated (Macrosep, Pall Corporation, East Hills, NY) and subjected to the Western blot analysis for the detection of IP-10. Conceptus tissues collected from days 14 (n = 3, whole conceptus), 17 (n = 3, approximately one half of conceptus, 200 mg each) and 20 (n = 3, trophoblast region only) of pregnant animals were subjected to in vitro culture, the remaining days 17 and 20 conceptus tissues were frozen for subsequent RNA extraction and immunostaining. The in vitro culture media were subjected to Western blot analysis for the determination of IFN-
. Uteri from day 14 cyclic animals (n = 3)
were used for epithelial cell isolation. Endometrial explants from day 14
cyclic goats (n = 3) were cultured to examine the stimulatory effect
of IFN- on IP-10 production. Endometrial tissues of goats (n = 3
for each day examined) from which conceptuses had been removed were frozen and
subjected to RNA extraction.
In Vitro Culture—Whole conceptuses (day 14, n = 3)
and minced conceptuses (days 17 and 20, 
200 mg/culture dish, n =
3 each) were cultured under conditions essentially described previously
(30).
The procedures for endometrial epithelial cell dissociation and subsequent characterization were previously described (31). Uterine horns from three day 14 cyclic goats were filled with 0.76% EDTA-PBS and preincubated at 37 °C for 1 h. The endometrial epithelium was scraped off by a surgical blade and then incubated at 37 °C for 8 min in 0.1% collagenase (Sigma). The cell suspension was passed through a nylon mesh (70 µm) and subjected to density gradient centrifugation. Collected epithelial cells were resuspended in Dulbecco's modified Eagle's medium and Ham's F-12 (DMEM/Ham's F-12, Sigma) supplemented with 40 units/ml of penicillin, 40 µg/ml, of streptomycin and 10% fetal calf serum. The cells were then plated in 24-well dishes coated with swine-skin type I collagen (Nitta gelatin, Osaka, Japan) and subjected to the adhesion assay when the cells had grown to confluent.
Dissociated cells were also prepared from day 17 goat conceptuses. These conceptuses (n = 3) were dissected into small pieces and incubated in 0.2% collagenase (Sigma) at 37 °C for 30 min. The cells were passed through a nylon mesh (70 µm) and resuspended in DMEM with antibiotics, which were immediately used for chemotaxis or adhesion assays. In addition, bovine B cells (KU-1, Ref. 32) and caprine trophoblast clone cells (HTS-1, Ref. 33) were cultured in DMEM supplemented with 10–20% fetal calf serum and antibiotics.
Endometrial tissues (600 mg wet weight/culture dish) from day 14
cyclic goats (n = 3) were cultured in 20 ml of DMEM supplemented with
antibiotics, and were treated with 5 nM recombinant bovine
IFN- (rbIFN-, Katakura Industries, Inc., Tokyo, Japan). After 24 h
at 37 °C and 5% CO2 atmosphere, endometrial tissues were frozen
separately and stored at –70 °C for subsequent RNA extraction and
Northern blot analyses.
Cloning of Caprine IP-10 and CXCR3 cDNAs—Through the use of RT-PCR, caprine IP-10 and CXCR3 cDNAs were amplified from caprine endometrial RNA and subcloned into a pSTBlue vector (TaKaRa, Tokyo, Japan), which was then subjected to an automated sequence analysis using a PerkinElmer sequencer (model ABI Prism 377 XL, Roche Applied Science). Nucleotide sequence comparisons of caprine IP-10 and CXCR3 were performed using the BLAST network program (National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD) and GenBankTM accession numbers were obtained (AB099892 [GenBank] and AB099893 [GenBank] , respectively).
Transfection of CXCR3 Expression Plasmid—The nucleotide sequence encoding the open reading frame of caprine CXCR3 was PCR-amplified and cloned into a mammalian expression vector pcDNA3.1 (Invitrogen Corp., Carlsbad, CA). This plasmid was transfected into KU-1 and HTS-1 cells by using TransfastTM (Promega, Madison, WI) according to the manufacturer's protocol. Two days after transfection, cells were used for chemotaxis or adhesion assay.
Production of Recombinant Caprine IP-10 and the Antibody—The nucleotide sequence encoding the mature region of caprine IP-10 (cIP-10) cDNA was PCR-amplified and cloned into an expression plasmid, pET-14b (Novagen, Madison, WI), which consisted of a histidine tag at the N-terminal side of cIP-10. This expression vector, pET-14b-cIP-10, was used to transform Escherichia coli BL21-SI (Invitrogen) cells, which were grown overnight. These cells were harvested, resuspended in 50 mM NaH2PO4, 500 mM NaCl, and 10 mM imidazole (pH 7.4), and disrupted on ice by sonication. After cell debris and insoluble proteins were removed by centrifugation, recombinant cIP-10 (rcIP-10) in the supernatant was examined using SDS-PAGE. Recombinant cIP-10 was purified using a nickel-chelating column (Hi-trap Chelating HP, Amersham Biosciences) on the chromatography system (AKTA, Amersham Biosciences). The protein was eluted from the column with a linear gradient of imidazole (20–500 mM), which was then dialyzed against PBS to remove imidazole.
200 µg of rcIP-10 proteins in PBS were mixed with Freund's complete adjuvant (Sigma) and injected sc into rabbits (n = 2). Recombinant proteins were injected two more times biweekly. A titer was checked 1 week after the last injection, and blood was collected on the following day. Anti-caprine IP-10 antibody was purified from antiserum using Hitrap Protein G column (Amersham Biosciences).
SDS-PAGE and Western Blot Analysis—After determining protein
concentrations using protein assay kit II (Bio-Rad, Hercules, CA), culture
media (3–8 µl, 10 µg of protein/lane, n = 3),
concentrated uterine flushing media (10 µg of protein/lane, n = 3
for each day), or recombinant IP-10 (50 or 200 ng/lane) were subjected to
Western blot analysis as described previously
(7). In brief, protein samples
were boiled for 5 min in the SDS sample buffer, electrophoresed on 10 or 15%
SDS-PAGE gels under reducing conditions, and the gel was stained with
Coomassie Blue. For Western blot analysis, proteins in the gel were
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 rabbit polyclonal
antibody to ovine IFN- (1:5000, a gift from Dr. Bazer), histidine tag
(Sigma), or caprine IP-10 (1:1000) at 4 °C for 12 h. After incubation,
membranes were washed three times in TBS-Tween 20, incubated with donkey
anti-rabbit IgG-conjugated with horseradish peroxidases (Amersham Biosciences)
at room temperature for 1 h, and washed three times in TBS-Tween 20. The bands
were detected using ECL Western blotting detection reagents (Amersham
Biosciences).
RT-PCR Analysis—Using Isogen (Nippon Gene, Tokyo, Japan), total RNAs were extracted from endometrial and conceptus tissues and cultured cells (n = 3 each). Total RNA samples were first reverse-transcribed with SuperScriptII (Invitrogen) and oligo(dT) primers (20 µl of reaction volume). Amounts of CXCR3, XCR1 and the integrin receptor mRNAs were determined from PCR amplification using oligonucleotide primers (Table I). Each reaction consisting of primer pairs was run with RT template (1 µl) and AmpliTaq Gold (0.625 units; Roche Applied Science) in a final volume of 25 µl. All PCR reactions consisted of 40 cycles at 94 °C for 1 min, 60 °C for 1 min and 72 °C for 1 min, followed by the final extension at 72 °C for 12 min. Following agarose gel electrophoresis and visualization with ethidium bromide, PCR products were quantified by using an image analysis system (ATTO Corporation, Tokyo, Japan) equipped with the Quantity One (v3.0 software; PDI, Inc., Huntington Station, NY).
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Northern Blot Analysis—Northern blot analysis was performed as described previously (7). DIG-labeled cRNA probes were generated from IP-10 and CXCR3 cDNA plasmids by using T7 or SP6 RNA polymerase (Promega). Total RNAs were used for the determination of IP-10 mRNA whereas poly(A)+ RNA was used to examine CXCR3 mRNA. Poly(A)+ RNA was obtained from total RNA that had been isolated from PBMCs using Oligotex-dT30 (TaKaRa) according to the manufacturer's instructions. Total RNAs (20 µg/lane, n = 3 each) or poly (A)+ RNAs (2 µg/lane, n = 3 each) were separated by electrophoresis and transferred to a nylon membrane (Biodyne-B; Pall Corporation). The membrane was hybridized with cRNA probe at 63 °C for 12 h. After hybridization, the signals were detected using the DIG detection systems (7).
Immunofluorescence—Frozen conceptus tissues sectioned (10 µm, n = 3 for each day examined) were mounted onto silan-coated slides and fixed in acetone. Nonspecific binding was blocked by a treatment with Block Ace at room temperature for 1 h, and slides were then incubated with a mouse monoclonal antibody to human CXCR3 (10 µg/ml, R&D Systems Inc., Minneapolis, MN) or normal mouse IgG (Sigma) at 4 °C for 12 h. After the primary antibody incubation, slides were treated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (15 µg/ml, Jackson ImmunoResearch Laboratories Inc., West Grove, PA) at room temperature for 1 h. Nuclei were stained with propidium iodide (2 µg/ml, Sigma). The slides were examined and digital images were captured on a confocal laser scanning microscope (FV300, OLYMPUS, Tokyo, Japan).
Protein Biotinylation and Detection—Recombinant cIP-10 protein was biotinylated by EZ-link biotinylation reagent (Pierce). As a control, recombinant GST and recombinant caprine lymphotactin-GST protein2 were also labeled with biotin. Proteins were incubated with 1 ml of 0.3 mg/ml EZ-link biotinylation reagent in PBS on ice for 2 h. After incubation, samples were dialyzed to remove the free biotin and measured their protein concentrations. For labeling efficiency conformation, biotinylated proteins (2 µg/lane) were resolved on 15% SDS-PAGE gels under reducing condition and visualized by streptavidin-horseradish peroxidases (Amersham Biosciences).
Frozen conceptus tissues sectioned (10 µm, n = 3 each) were mounted onto silan-coated slides and fixed in acetone. Slide sections were blocked with Block Ace at room temperature for 1 h and then incubated with biotinylated protein (100 µg/ml) at room temperature for 1 h. After incubation, slides were incubated with streptavidin-horseradish peroxidases (1:5000, Amersham Biosciences) at room temperature for 1 h. The biotinylated protein bound to the tissues was visualized with DAB solutions (Sigma). Tissue sections were then counterstained with hematoxylin.
Chemotaxis Assay—Migration of KU-1, HTS-1, or primary trophoblast cells was assessed in a 96 well-modified Boyden chamber (Neuro-Probe, 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. The assay was performed as described (7). In brief, DMEM-0.1% bovine serum albumin (without phenol red or fetal calf serum) supplemented with indicated concentrations of rcIP-10 was added to the bottom wells of the chemotaxis chamber, whereas cells (5 x 106 cells/ml) in DMEM, 0.1% bovine serum albumin (without phenol red and fetal calf serum) were added to the top wells of the chamber. After the chambers were incubated at 37 °C and 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 at six randomly chosen high power fields. For the blocking experiments, before addition to the bottom chamber, rcIP-10 was preincubated at 37 °C for 1 h with 30 µg/ml of anti-IP-10 antibody or control rabbit IgG (Sigma). Three replicates were performed for each treatment, and three independent experiments were done for each treatment.
Adhesion Assay—24-well plates were coated with type I collagen (Nitta gelatin) or fibronectin at a concentration of 10 µg/ml at room temperature for 2 h, or plated with caprine epithelial cells. Following three washes with PBS, the plates were blocked with 1% bovine serum albumin at room temperature for 30 min. HTS-1 or primary trophoblast cells were labeled with the intracellular fluorescent dye, 4 µM calsein-AM (Molecular Probes Inc., Eugene, OR) at 37 °C for 30 min. Following three washes with PBS, cells were incubated with the indicated concentrations of rcIP-10 at 37 °C for 1 h, and the cells were then added to each well, and the plates were incubated at 37 °C for 1 h. After incubation, unbound cells were removed by three washes with PBS, and the remaining cells were treated with 1% Triton X-100, 10% ethanol in PBS. Fluorescence of cells was measured using a fluorescence reader (excitation filter 485 nm and emission filter 535 nm) (ARVOTM SX 1420 Multilabel Counter, PerkinElmer Life Sciences Inc.). For the blocking experiments, rcIP-10 protein had been preincubated at 37 °C for 1 h with 30 µg/ml of anti-IP-10 antibody or control rabbit IgG (Sigma). To further investigate the involvement of IP-10 on cell adhesion to fibronectin, the Gly-Arg-Gly-Asp-Ser-Pro-Lys (GRGDSPK, Sigma) synthetic peptide at a concentration of 50 mM, its control, Arg-Gly-Glu-Ser (RGES, Sigma), or 5 mM EDTA was preincubated with cells and rcIP-10 protein. Three replicates were examined for each treatment, and three independent experiments were performed for each substrate and treatment.
Statistical Analysis—Measurements of optical density (Western blot) and light intensity (RT-PCR) were subjected to least squares (LS) ANOVA, which employed the general linear models procedures of the Statistical Analysis System (version 6.0; SAS Institute, Cary, NC). The light intensity from G3PDH PCR products was used as covariates for RT-PCR analyses. In chemotaxis assays, the number of cells migrated or attached with a treatment was calculated as the number relative to the cells without any treatment, which was then analyzed statistically as aforementioned. The model used in the LS-ANOVA included treatment and replicate as sources of variation. The least square means (LSMs) and S.E. illustrated in the figures were derived from this analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of rcIP-10 Protein and the Antibody—Recombinant cIP-10 (rcIP-10) was expressed in E. coli transformed with pET-14b-cIP-10 plasmid, purified using His tag systems, and confirmed by SDS-PAGE (Fig. 1A). Antibody to rcIP-10, purified from the antiserum using Hitrap protein G column, detected the specific band at
14 kDa on Western blot analysis
(Fig. 1B). To check
the biological activity of rcIP-10, in vitro chemotaxis assay was
also carried out: bovine B-cell line, KU-1, transiently transfected with the
caprine CXCR3 cDNA was used as target cells. CXCR3 mRNA expression on the
transfected cells was examined by Northern blot analysis
(Fig. 1C, left
panel). The chemotaxis assay demonstrated that CXCR3 transfectants
consistently responded to 1–20 ng/ml rcIP-10, but their responses
declined at higher rcIP-10 concentrations, resulting in a characteristic
bell-shaped dose response curve (Fig.
1C, right panel). Moreover, immunoneutralization
experiments revealed that an anti-rcIP-10 antibody reduced the chemotactic
activity of rcIP-10 (Fig.
1D).
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Expression of IP-10 within Goat Uterus during Early
Pregnancy—The production of IFN- protein by caprine
conceptuses was confirmed in the uterine flushing media and was the highest on
day 17 of pregnancy (Fig.
2A). Northern blot analysis revealed that IP-10 mRNA
level in the caprine endometrium began to increase from day 17 of pregnancy,
and that the conceptus factor IFN- stimulated IP-10 mRNA level by day 14
cyclic endometrium in vitro (Fig.
2B). IP-10 protein was detected in the uterine flushing
media, adjusted to 10 µg proteins/lane, from days 14 and 17 cyclic and
pregnant animals (n = 3 for each day examined)
(Fig. 2C). It appeared
that the uterine flushing media from day 17 pregnant animals possessed more
IP-10 than those of other days examined. In situ hybridization
revealed that IP-10 mRNA was observed in the subepithelial stroma regions of
pregnant endometrium, and the intensity was greater in day 17 pregnant
endometrium than that in day 14 cyclic or pregnant endometrium
(Fig. 2D).
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Functional Expression of CXCR3 Receptor in Trophoblast Cells—The expression of CXCR3 mRNA was observed in days 14, 17, and 20 caprine endometria (n = 3) and day 17 conceptus tissues (n = 3) during early pregnancy (Fig. 3A). Further confirmation of its expression in days 17 and 20 conceptuses (2 µg poly(A)+ RNA/lane, n = 3 for each day) was obtained by Northern blot analysis (Fig. 3B). To define the cellular origin of CXCR3 receptor in caprine conceptus tissues, immunofluorescence analysis was performed using frozen conceptuses and anti-CXCR3 antibody. A patchy fluorescence associated with the trophoblast layer was found and the localization was clearly different from nuclei (Fig. 3C). A dull autofluorescence was found on the trophoblast when the control mouse immunoglobulin was used.
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To obtain more evidence on the significance of CXCR3 expression in the trophoblast cells, binding experiment of rcIP-10 protein to caprine trophoblast cells was performed. Recombinant cIP-10 protein along with recombinant GST and lymphotactin-GST were biotinylated, and the efficiency of biotinylation was confirmed (Fig. 4A). Lymphotactin belongs to the C chemokine family, and its expression, similar to IP-10, was observed in the goat endometrium during early pregnancy.2 However, unlike CXCR3, the expression of lymphotactin receptor, XCR1, mRNA was not detected in the conceptus (Fig. 4B). For these reasons, lymphotactin/XCR was used as a negative control. The presence of CXCR3 was observed in the trophoblast cells incubated with only biotinylated rcIP-10 but not with biotinylated GST or lymphotactin-GST (Fig. 4C).
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Recombinant cIP-10 Stimulation on the Migration of CXCR3-expressing Trophoblast Cells—It was suspected that IP-10 from the endometrium might interact with trophoblast cells expressing CXCR3 during early pregnancy. To study whether trophoblast cell migration was affected by endometrial IP-10 expression, in vitro chemotaxis assay was performed. Initially, trophoblast clone HTS-1 cells, which do not exhibit CXCR3 expression, were subjected to the chemotaxis study. HTS-1 transfected with the empty plasmid did not respond to rcIP-10 protein (Fig. 5A, left panel), however, HTS-1 transfected with CXCR3 consistently responded to 1–20 ng/ml rcIP-10, but their responses declined as rcIP-10 concentrations increased, resulting in a characteristic bell-shaped dose response curve (Fig. 5A, right panel). Moreover, immunoneutralization experiments revealed that an anti-rcIP-10 antibody reduced the chemotactic activity of rcIP-10 (Fig. 5B). In the next step, primary trophoblast cells expressing CXCR3 on day 17 of pregnancy were subjected to the chemotaxis assay. The migration of trophoblast cells was stimulated by rcIP-10 protein, and the chemotaxis activity was neutralized with the use of an anti-IP-10 antibody (Fig. 5C).
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Effect of rcIP-10 on the Adhesion of Trophoblast Cells to Fibronectin and Endometrial Epithelial Cells—In addition to the migration activity of trophoblast cells to IP-10, adhesion assay was performed to determine whether IP-10 could increase the adhesion activity of trophoblast cells. Primary trophoblast cells bound to the plates coated with or without collagen I or fibronectin, but the binding rate of rcIP-10-stimulated cells to the fibronectin-coated plates was considerably higher than that of others (Fig. 6A). In the blocking experiment, rcIP-10 enhanced the adhesion of the trophoblast cells to fibronectin, which was inhibited by the anti-IP-10 antibody (Fig. 6B). The RGD peptide is known to inhibit cell adhesion occurring through RGD sites of fibronectin whereas the RGE peptide serves as an inactive control. RGD-treated cells, not RGE, decreased the adhesion of rcIP-10-stimulated cells to fibronectin (Fig. 6B). EDTA, known as an inhibitor of integrin signaling, also decreased the adhesion of trophoblast cells to fibronectin (Fig. 6B).
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To obtain further evidence on IP-10-CXCR3 interactions, HTS-1 cells transfected with or without CXCR3 cDNA were examined for adhesive activity. In HTS-1 cells transfected with CXCR3, but not without CXCR3, the adhesion of cells to fibronectin was stimulated by rcIP-10 protein and inhibited by the use of anti-IP-10 antibody in the blocking experiment (Fig. 6C). The addition of RGD peptides and EDTA abolished the adhesion of CXCR3-transfected cells to fibronectin (Fig. 6C). Further decline of adhesion activity exhibited with RGD and EDTA treatments resulted from the observation in which cell adhesion occurred even in mock-transfected cells, and from the definition in which the degree of nonspecific adhesion was considered as 100% of attached cells. In this study, nonspecific and CXCR3 transfected cells to fibronectin were further inhibited by RGD or EDTA treatment. These results indicated that HTS-1 transfected with CXCR3 cDNA could reflect primary trophoblast cells in both cell adhesion and migration models.
Furthermore, whether trophoblast cells activated by IP-10 could bind to the endometrial epithelial cells was investigated. Fig. 6D shows that the adhesion of CXCR3 expressing HTS-1 to primary epithelial cells from the caprine endometrium was increased by rcIP-10 protein, and the adhesive activity was inhibited by the use of the IP-10 antibody in the neutralizing experiment. EDTA-treated trophoblast cells lost the adhesion activity to epithelial cells. Interestingly, the adhesion activity of RGD peptide-treated cells was reduced but the activity was higher than the EDTA-treated ones (Fig. 6D).
Expression of Integrin Subunits in Trophoblast Cells Stimulated by
IP-10 —To define which integrin subunit was involved in the
IP-10-induced trophoblast cell adhesion to fibronectin, the expression of
integrin subunits, major candidates to bind fibronectin, was examined in HTS-1
cells stimulated by rcIP-10. Transcripts for integrin subunits,
5, V, 1,
3, and 5, were detected by using RT-PCR. The
expression of integrin 5, V, and
3 subunits mRNA in trophoblast cells were stimulated by
rcIP-10, and the stimulation was diminished by the neutralization with the
anti-IP-10 antibody (Fig. 7).
The expression of integrin 1 and 5 subunits
mRNA was not influenced by rcIP-10.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, the presence of several chemokines and/or their receptors in the conceptus and maternal endometrium has been recognized during early pregnancy. These chemokines are suspected to play a role in the processes of implantation, but a definitive role that chemokines play during early pregnancy has not been identified. In the present study, we detected IP-10, mRNA and protein, in the subepithelial stroma and uterine lumen, respectively, and its receptor CXCR3 localized in the conceptus is functioning during early gestation. These results indicate that other than the regulation of immune cell distribution, a chemokine present in the pregnant uterus plays an important role in the regulation of trophoblast cells for their migration and adhesion to the endometrial epithelial cells. This work therefore represents the demonstration of a trophoblast cell mobilization in the early stage of conceptus implantation to the maternal endometrium.
The expression of CXCR3 mRNA in the conceptus tissues was detected using
RT-PCR and Northern blot analysis. It is known that conceptus tissues during
peri-implantation period consist of several different cell types, to which
maternal leukocytes bind (27),
therefore, the determination of the cellular origin of CXCR3 mRNA was
required. The expression of a leukocyte marker CD45 mRNA was detected in RNA
samples extracted from conceptuses, but its level was lower than that in blood
leukocyte samples (data not shown). However, immunofluorescent and ligand
binding analyses revealed that CXCR3 receptors were localized in the
trophoblast cells but not in leukocytes, suggesting that the caprine
trophoblast had the ability to respond to IP-10 produced from the endometrium.
Northern blot analysis revealed that the expression of CXCR3 mRNA in the
conceptus tissues declined from day 17 to day 20 of pregnancy. These results
suggest the possibility that trophoblastic CXCR3 was regulated by a factor(s)
other than endometrial IP-10 during early pregnancy. It was reported that the
removal of IL-2 from CXCR3+ T cell cultures results in rapid loss of cell
surface CXCR3 (34), and
recombinant IFN- could stimulate the expression of chemokine receptor,
CCR3, in the human trophoblast cells
(26). Other than these
observations, however, the regulation of chemokine receptor expression has not
been definitively elucidated. Therefore, further investigation is required to
determine the regulation of CXCR3 expression in the caprine trophoblast
cells.
Implantation is completed through a number of steps in which the trophoblast cells undergo a series of distinct interactions with maternal endometrium. It is initiated by close proximity between trophoblast and endometrial epithelial cells, promoted by not only closure of the uterine lumen but also activation of blastocyst migration. In chemokines and their receptor interactions, cells possessing a chemokine receptor move into the direction of increasing concentrations of a chemokine, which typically is a soluble molecule that could diffuse away from the site of production where its concentration is highest (35, 36). In this study, IP-10 mRNA was not found in the conceptus, but was detected in the subepithelial stroma, and the protein was present in the uterine flushing media, providing evidence that IP-10 could have diffused into the pregnant uterine lumen. These results and the observation in which migration of isolated primary trophoblast cells was regulated by IP-10 suggest that endometrial IP-10 establishes its concentration gradient in the pregnant uterus, and trophoblast cells could sense concentration differences and migrate toward the endometrium.
In ruminant ungulates, elongated conceptus occupies almost entire uterine horn during the initial attachment stage; days 17–18 and days 18–19 pregnancy in sheep and goats, respectively. This association or close proximity between two organisms, which could be regulated through the IP-10/CXCR3 system, may increase chances of conceptus attachment to the caruncular regions of the uterus. It has been noted that apical epithelial surfaces are normally non-adhesive, yet during implantation, the transition to pre-receptive state such as a decline in mucin Muc-1 is required in these cells (5, 37, 38). Initial adhesion of the trophoblast cells involves physical adhesion and may interact with the appropriate ECM on the endometrium. In the present investigation, trophoblast cell adhesion to non-, collagen I- or fibronectin-coated plates was observed, and the adhesion to the fibronectin-coated plates was most stimulated by the addition of IP-10. Fibronectin belongs to a member of ECM molecules identified at the conceptus-endometrium interface, which is capable of serving as a ligand for the receptor, integrin family. Fibronectin is also implicated in murine trophoblast outgrowth and adhesion processes, both of which could be blocked with RGD-containing peptides in vivo (39) and in vitro (40). The blocking with RGD peptides abolished the IP-10 stimulated adhesion activity of trophoblast cells to fibronectin, and EDTA, an inhibitor of integrin signaling, also abolished the adhesion activity. These results were further confirmed by the observations that trophoblast cells stimulated by IP-10 increased the adhesion to endometrial epithelial cells, which was reduced by the treatment with the RGD peptide. These results indicated that IP-10 could stimulate the expression or activation of integrins on the trophoblast cell surface and promote the trophoblast cell adhesion to one of ECM, fibronectin.
In this study, IP-10 induced the expression of integrin
5, V, and 3 subunits in
caprine trophoblast cells. Our results agree with previous reports that the
presence of 51,
V1, V3
and V5 integrins on the trophoblast cell
surface is required for successful interaction between the trophoblast and
endometrium (4,
41–43).
In addition, 51,
V1, V3,
and V5 integrin isoforms recognize ECM
ligands that contain the RGD sequence. Although the in vivo data are
not available, these results from the in vitro study suggest that
trophoblast cell migration is regulated by endometrial IP-10, which could
control the expression of some integrin isoforms,
51, V1,
V3, and
V5, on the apical surface, facilitating the
adhesion of trophoblast cells to endometrial ECM ligands. Interestingly, the
effect in RGD blocking of the trophoblast cell adhesion to the epithelial
cells was lower than that to the fibronectin. These results suggest that
although IP-10 could stimulate the adhesion of trophoblast cell to particular
RGD-containing ECM, other adhesion molecule(s) was also present at the sites
of interactions between trophoblast and epithelial cells in utero. A
molecule, L-selectin, expressed on the human trophoblast cells recently
described by Genbacev et al.
(44), could also be playing a
role in the establishment of initial attachment to the endometrium.
Involvement of other adhesion molecule(s) predicted in this study might be
L-selectin. Based on these observations, we propose that the production of
endometrial chemokines, stimulated by a conceptus factor(s), is required for
the expression of suitable adhesion molecules on trophoblast cells, resulting
in the progression to successful implantation.
In conclusion, we identified a cell mobilization pathway potentially functioning in the early stage of embryonic implantation to the endometrium. For successful implantation in goats and other ruminant ungulates, the endometrial IP-10 and trophoblastic CXCR3 might regulate the trophoblast apposition and adhesion to the endometrium through the stimulation of integrin expression, recognizing the RGD-containing ECM, on the trophoblast cell surface. Since the initial apposition and adhesion of the conceptus to the endometrium occurs universally among mammals, the presence of IP-10 or other chemokines with similar functions and their receptors is prerequisite for engagement of the conceptus to the endometrium.
|
FOOTNOTES |
|---|
* This work was supported by a Grants-in-aid for Scientific Research (13556050 and 14206032) (to K. I.) from the Japan Society for the Promotion of Science and by the Program for Promotion of Basic Research Activities for Innovative Bioscience (BRAIN). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by Research Fellowships of the Japan Society for the Promotion of
Science for Young Scientists.
To whom correspondence should be addressed: Laboratory of Animal Breeding,
Faculty of Agriculture, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo
113-8657, Japan. Tel.: 81-3-5841-5382; Fax: 81-3-5841-8180; E-mail:
akaz{at}mail.ecc.u-tokyo.ac.jp.
1 The abbreviations used are: IFN, interferon; DMEM, Dulbecco's modified
Eagle's medium; PBS, phosphate-buffered saline; ECM, extracellular matrix;
LSM, least square means; IP-10, interferon -inducible protein 10
kDa.
2 K. Imakawa, unpublished data.
|
ACKNOWLEDGMENTS |
|---|
We thank Drs. Fuller W. Bazer (Texas A&M University) and Chikashi Tachi (Mitsubishi-Kagaku Co. Ltd., Japan) for generously providing an anti-ovine IFN-
antibody and caprine trophoblast HTS-1 cells, respectively. We also
thank Dr. Hitomi Takahashi (National Institute of Livestock and Grassland
Science, Japan) and Tamako Matsuhashi for the isolation and characterization
of uterine epithelial cells. We thank the technical assistance of the
Experimental Station for Bio-Animal Science crew, 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. |
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