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Spatial and Temporal Expression of Ligands for CXCR3 and CXCR4 in Human Endometrium

Departments of Obstetrics and Gynecology (K.K., T.N., N.D., H.H.) and Pathology and Applied Neurobiology Research Institute (S.F.), Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan

Address all correspondence and requests for reprints to: Kotaro Kitaya, M.D., Ph.D., Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-hirokoji-agaru, Kamigyo-Ku, Kyoto 602-8566, Japan. E-mail:kitaya{at}koto.kpu-m.ac.jp.

	Abstract

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In this study, we investigated the expression of ligands for CXCR3 (Mig, IP-10, and I-TAC) and CXCR4 (SDF-1) in the human endometrium throughout the menstrual cycle. By immunohistochemistry, immunostaining for Mig and IP-10 was found in the surface epithelia, glandular epithelia, and stroma with some menstrual cycle-dependent fluctuation. By contrast, immunostaining for I-TAC or SDF-1 was not detected. ELISA demonstrated that the concentrations of Mig and IP-10 were higher in the secretory phase than in the proliferative phase, but I-TAC and SDF-1 was detected in only a few samples. Endometrial Mig and IP-10 concentrations strongly correlated with the number of endometrial natural killer cells. Progesterone significantly induced Mig secretion and tended to induce IP-10 secretion from the cultured endometrial stromal cells, whereas 17ß-estradiol had no significant effect. Neither I-TAC nor SDF-1 was detected in the supernatant of cultured endometrial stromal cells in the presence or absence of 17ß-estradiol or progesterone. The results suggest that Mig and IP-10 may be involved in the recruitment of natural killer cells or other phenomena in the human endometrium.

	Introduction

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DURING THE MID- to late secretory phase, human endometrium is densely infiltrated by diverse leukocyte subsets (1). Unlike other mucosal tissues, the distinct leukocyte subset in the endometrium at this phase is natural killer (NK) cells. Endometrial NK cells accumulate in the stroma, preferably around glands and vessels as single cells or aggregates (1). These NK cells are capable of producing a wide range of molecules such as interferon (IFN)-, macrophage colony-stimulating factor, and vascular endothelium growth factor that are important for the embryo implantation and early placentation (2, 3, 4). Moreover, they are postulated to regulate trophoblast invasion (5) through the interaction between their surface NK receptors (6) and major histocompatibility class I molecules (human leukocyte antigen-C, -E, and -G) expressed on the surface of the invading trophoblasts (7, 8, 9).

The number of endometrial NK cells fluctuates during the menstrual cycle; they are sparse in the proliferative phase but acutely increase after ovulation and account for 70–80% of all the endometrial leukocytes in the mid- to late secretory phase (1). Interestingly, more than 95% of endometrial NK cells have the phenotypes of CD3^neg, CD16^neg, and CD56^bright, which comprise less than 0.5% of the peripheral blood leukocytes (1). The number of endometrial CD16^neg CD56^bright NK cells in the secretory phase is decreased in the patients with unexplained recurrent miscarriages (10) and in vitro fertilization-embryo transfer failure (11), indicating that their postovulatory increase is essential for successful pregnancy.

Although the mechanism underlying the increase of endometrial NK cells at the implantation period remains unknown, selective recruitment from circulating peripheral blood, seen in other mucosal systems, is a possible theory. Chemokines are a group of small peptides that induce the migration of leukocytes into normal, inflammatory, and tumor tissues by binding to a family of seven-transmembrane G protein-coupled receptors (12). Chemokines are classified into four subgroups (C, CC, CXC, and CX₃C). CXC chemokines are characterized by the first two cysteines being separated by a single amino acid. Recent studies have shown that three CXC chemokine receptor (CXCR) 3 ligands, monokine induced by IFN (Mig), IFN-inducible protein-10 (IP-10), and IFN-inducible T-cell -chemoattractant (I-TAC), and one CXCR4 ligand, stromal cell-derived factor (SDF)-1, have a selective chemoattractive activity especially for peripheral blood CD16^neg CD56^bright NK cells (13, 14, 15, 16, 17, 18, 19, 20).

These chemokines therefore may be involved in the selective recruitment of NK cells into the endometrium. However, their expression in the nonpregnant endometrium has not been fully elucidated. Previously one study demonstrated the presence of IP-10 in the human endometrium (21), but its spatial and temporal expression pattern remains unknown. In the present study, we examined the expression of these chemokines in the human endometrium. We propose a hypothesis on their possible biological functions in the human reproduction.

	Materials and Methods

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Samples

This study was approved by the Kyoto Prefectural University of Medicine Institutional Review Board. Informed consent was obtained from each patient before the operation.

Endometrial samples were obtained from 45 fertile women aged 29–43 yr who had undergone hysterectomy for leiomyoma or carcinoma in situ of the uterine cervix. The patients had regular menstrual cycles ranging from 28 to 35 d. None of them had received any hormonal treatment at least 3 months before the operation or showed any pathological findings in the uterus such as polyps, tumors, or endometritis. Full-thickness endometrial pieces were dissected within 15 min after hysterectomy. According to the standard criteria (22), they were classified as 14 of the proliferative phase (P), 11 of the early secretory phase (ES), nine of the midsecretory phase (MS), and 11 of the late secretory phase (LS). Decidual samples were obtained from two women who had undergone selective termination during the first-trimester pregnancy (7 wk and 8 wk).

Tissue preparation

We used 39 endometrial samples (eight P, 11 ES, nine MS, and 11 LS) to evaluate in vivo expression of chemokines. After being washed in PBS, a portion of each sample was fixed overnight in a 4% paraformaldehyde [in phosphate buffer (pH 7.3)] and embedded in paraffin. The remainder was homogenized in TRIzol Reagent (Life Technologies Inc., Gaithersburg, MD). Total protein was extracted according to the manufacturer’s protocol and preserved in lysis buffer (2 µM aprotinin, 50 µM leupeptin, 125 µM bestatin, and 25 µM pepstatin A, Nakarai, Kyoto, Japan) at –80C until measurement.

Culture of endometrial stromal cells

Six endometrial samples of the LS phase were used for the isolation of the endometrial stromal cells. Endometrium was minced into approximately 1-mm³ pieces and incubated in phenol-red free DMEM (Life Technologies Inc.) containing 0.5% collagenase (Sigma, St. Louis, MO) for 1 h at 37 C. The cell supernatant was then collected and passed through a 40-µm nylon mesh (Becton Dickinson, San Jose, CA). The cells were incubated in culture medium (phenol-red free DMEM containing 10% charcoal-stripped fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml amphotericin B) at 37 C in 5% CO₂. As previously described (23), the cells contained more than 95% stromal cells (vimentin+) and approximately 2% macrophages (CD14+) after one passage. After 48 h culture, more than 99% were stromal cells.

Isolated endometrial stromal cells were cultured in 6-well plates (Becton Dickinson) with 4 ml fresh culture medium supplemented with 10^–6 M of 17ß-estradiol or progesterone (Sigma) dissolved in dimethylsulfoxide for 48, 72, and 96 h at 37 C in 5% CO₂. Dimethylsulfoxide was also used as a vehicle control. The culture supernatant was collected by centrifugation and frozen at –80 C until measurement.

Immunohistochemistry

Paraffin-embedded samples were cut into 4-µm sections, dewaxed in xylene, and rehydrated in a graded series of ethanol. Sections were immersed in 3% hydrogen peroxide for 5 min at room temperature to remove endogenous peroxidase activity and then incubated with PBS containing 10% fetal calf serum (JRH Biosciences, Lenexa, KS) for 10 min at room temperature to suppress nonspecific antibody binding.

In a moist chamber, sections were incubated with antihuman Mig monoclonal antibody (clone 49106, mouse IgG₁, 1:25 dilution, R&D Systems, Minneapolis, MN), antihuman IP-10 monoclonal antibody (clone 33036, mouse IgG₁, 1:100 dilution, R&D Systems), antihuman I-TAC monoclonal antibody (clone 87328, mouse IgG_2A, 1:5 dilution, R&D Systems), antihuman SDF-1 monoclonal antibody (recognizing both SDF-1 and SDF-1ß, clone 79014, mouse IgG₁, 1:2 dilution, R&D Systems), or antihuman CD45 (leukocyte common antigen) monoclonal antibody (1:50 dilution, Nichirei, Tokyo, Japan) overnight at 4 C. Sections were washed in PBS three times and incubated with a universal LSAB kit (Dako, Kyoto, Japan) according to the manufacturer’s instruction. Sections were washed three times, developed with diaminobenzidine (Dako), and counterstained with Meyer’s hematoxylin solution. The specificity of immunostaining was confirmed by the immunoabsorption test using recombinant proteins (R&D Systems).

Immunostaining using anti-CD3 (T-cell marker, Nichirei), anti-CD56 (NK cell marker, Nichirei), anti-CD68 (macrophage marker, Immunotech, Marseille, France), and anticytokeratin 7 (trophoblast marker, Dako) antibodies was performed by a similar protocol. The sections were subjected to microwave pretreatment for antigen retrieval before immersion in 3% hydrogen peroxide.

Under a light microscope, the number of CD56+ round cells (NK cells) was counted in 10 nonoverlapping high-power fields that were selected randomly. The counting was done in triplicate by two independent observers who were blinded to the study. The mean number was calculated for statistical analysis.

ELISA

The amounts of the chemokines in the homogenized endometrium and supernatant of cultured endometrial stromal cells were measured with Quantikine ELISA kits (R&D Systems). The minimal detectable concentration for Mig, IP-10, I-TAC, and SDF-1 was 5, 5, 15, and 5 pg/ml, respectively. Assay measuring SDF-1ß is not currently available. Total protein amount in the homogenized endometrium was measured with a DC protein assay kit (Bio-Rad Laboratories, Hercules, CA). The assay was done in triplicate, and the mean concentration (chemokine amount/total protein amount) was calculated.

Coefficients of variation were less than 8% (intraassay) and 10% (interassay) for Mig and less than 7% (intraassay) and 11% (interassay) for IP-10. Coefficients of variation could not be determined for I-TAC or SDF-1 because it was detected in only a few samples (see Results).

Statistical analysis

The chemokine concentration of each group was compared by one-way ANOVA with post hoc test (Sheffe’s F test) using Statcel software (OMS, Tokorozawa, Japan). The number of CD56+ round cells in each group was compared by Kruskal-Wallis test. The correlation between the chemokine concentration and the number of CD56+ cells was analyzed by Spearman’s correlation coefficient by rank test. P < 0.05 was considered significantly different.

	Results

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Mig

Immunostaining for Mig was found in the surface epithelia, glandular epithelia, and stroma (Fig. 1, A–D and G). The surface epithelia were constantly stained throughout the menstrual cycle, whereas the immunoreactivity in the glandular epithelia was weak in the P phase, faint in the ES phase, and intense in the MS to LS phase (Fig. 1, A–D). The immunoreactivity in the stroma was distinct in the vascular and perivascular areas (Fig. 1, B and D). The overall immunoreactivity increased from the P phase toward the LS phase. In addition, punctate immunostaining was observed in the stroma in occasional samples (one in the MS phase and four in the LS phase) of 39 samples examined (Fig. 1E). By serial section staining, most of the stromal punctate immunostaining coincided with CD56+ cells (Fig. 1F). Immunoreactivity for Mig in CD3+ cells and CD68+ cells was rarely seen.

View larger version (130K): [in this window] [in a new window]   FIG. 1. Localization of Mig, IP-10, I-TAC, and SDF-1 in human endometrium. Arrows indicate the immunostaining. Scale bars, 100 µm. Immunostaining for Mig (A–E, G), IP-10 (I–L), I-TAC (M), and SDF-1 (N, O) CD56 (F) and cytokeratin-7 (P). Immunoabsorption test (H) confirmed the specificity of the immunostaining. P phase (A, I). ES phase (B, J). MS phase (C, K, M, N). LS phase (D–F, L). First-trimester pregnancy decidua (O, P). Photos E and F were overexposed to identify punctuate stromal staining.

View this table: [in this window] [in a new window]   TABLE 1. Concentration of Mig, IP-10, I-TAC, and SDF-1 (picograms/milligram protein) in the human endometrium, and the number of NK (CD56+) cells in 10 nonoverlapping endometrial stromal areas

View larger version (16K): [in this window] [in a new window]   FIG. 2. Correlation between the number of endometrial NK cells and the concentrations of endometrial Mig (A, rs = 0.80) and IP-10 (B, rs = 0.76).

View larger version (18K): [in this window] [in a new window]   FIG. 3. Concentrations of Mig (A–C) and IP-10 (D–F) in the supernatant of the cultured endometrial stromal cells.

Immunostaining for IP-10 was found in the surface epithelia, glandular epithelia, and stroma (Fig. 1, I–L). The surface and glandular epithelia were constantly stained throughout the menstrual cycle. Similar to Mig, the immunoreactivity in the stroma mainly showed a vascular and perivascular pattern (Fig. 1, I and L). Nonperivascular cells were stained focally and diffusely especially in the MS to LS phase (Fig. 1, K and L).

The IP-10 concentration in the lysis buffer was below the minimal detectable concentration. The IP-10 concentration (mean ± SD) in the homogenized endometrium (Table 1) was 63.2 ± 25.6 pg/mg protein in P (n = 7), 95.8 ± 55.1 pg/mg protein in ES (n = 9), 370.2 ± 218.3 pg/mg protein in MS (n = 8), and 470.9 ± 298.1 pg/mg protein in LS (n = 10). It was significantly higher in MS than P (P < 0.05) and significantly higher in LS than P or ES (P < 0.005). There was a strong positive correlation (rs = 0.76) between the endometrial IP-10 concentration and the number of endometrial CD56+ cells (Fig. 2B).

IP-10 was not detected in the culture medium without cells but was detected in the control culture medium in three of six samples after 48 h and in all samples after 72 h (Fig. 3D). After a 96-h culture, progesterone significantly induced IP-10 secretion (Fig. 3F), compared with 17ß-estradiol (P = 0.046, 1- to 5.9-fold) but not compared with the control (P = 0.055, 1.2- to 4-fold). 17ß-Estradiol had no significant effect, compared with the control (Fig. 3E).

I-TAC

We could not detect immunostaining for I-TAC with a specific antibody at a 1:10 dilution in the endometrium (Fig. 1M) and first-trimester pregnancy decidua. Even at a 1:5 dilution, only the background signal was seen. I-TAC in the homogenized endometrium was below the minimal detectable concentration in 27 (five P, eight ES, seven MS, and seven LS) of 30 samples examined. Immunoreactivity for I-TAC was detected in only three samples: one in MS and two in LS (Table 1). We could not calculate the correlation between the endometrial I-TAC concentration and the number of endometrial NK cells. I-TAC in the supernatant of cultured endometrial stromal cells was below the minimal detectable concentration after 96 h in all samples examined, in the presence or absence of 17ß-estradiol or progesterone.

SDF-1

We could not detect immunostaining for SDF-1 with a specific antibody at a 1:5 dilution in the endometrium from each menstrual phase (Fig. 1N). Even at a 1:2 dilution, only the background signal was seen. By contrast, the immunoreactivity for SDF-1 was observed in the first-trimester pregnancy decidua, By serial section staining, the staining for SDF-1 was detected in the cytokeratin-7-positive cells in the decidual vessels (intravascular trophoblasts) but not in the decidual stroma (Fig. 1, O and P). We measured only SDF-1 concentration in the homogenized endometrium because an assay measuring SDF-1ß is not currently available. SDF-1 in the homogenized endometrium was below the minimal detectable concentration in 25 (four P, seven ES, six MS, and eight LS) of 26 samples examined. Immunoreactivity for SDF-1 was detected in only one sample in MS (Table 1). We could not calculate the correlation between the endometrial SDF-1 concentration and the number of endometrial NK cells. SDF-1 in the supernatant of cultured endometrial stromal cells was below the minimal detectable concentration after 96 h in all samples examined, in the presence or absence of 17ß-estradiol or progesterone.

	Discussion

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There are two possible explanations for the increase in endometrial NK cells during the secretory phase: one is in situ proliferation, and the other is selective recruitment from circulating peripheral blood.

Endometrial NK cells are, at least in part, likely to be proliferating in the endometrium because some of the population expresses proliferation-associated nuclear marker Ki-67. The fact that the expression rate of Ki-67 in endometrial NK cells is higher in the secretory phase than in the proliferative phase (1) supports this idea. Female sex steroids, especially progesterone, are likely to be involved in this phenomenon. We recently found that female sex steroids do not have a direct effect on the proliferation of endometrial CD16^neg CD56^bright NK cells in vitro (24). However, progesterone stimulated the endometrial stromal cells to produce IL-15, a cytokine that has a strong proliferating effect on peripheral blood and endometrial CD16^neg CD56^bright NK cells (25, 26, 27, 28). In vivo expression of IL-15 in the human endometrium also corresponds temporally and spatially to the numerical fluctuation of endometrial NK cells (26). These findings suggest that IL-15 is involved in the in situ proliferation of endometrial NK cells.

However, it may be difficult to explain their acute increase during the secretory phase (5- to 10-fold, compared with the proliferative phase) only by in situ proliferation. Several studies demonstrated that peripheral blood CD16^neg CD56^bright NK cells are specifically attracted by ligands for CXCR3 and CXCR4 (19, 20). Of these chemokines, we found that Mig and IP-10 were constitutively expressed in the endometrium. The protein levels of Mig and IP-10 in the endometrium were higher in the secretory phase than in the proliferative phase and had a strong positive correlation with the numerical fluctuation of endometrial NK cells, suggesting that they may be potential chemoattractants for these NK cells. We recently demonstrated that potential chemoattractants for endometrial NK cells, macrophage inflammatory protein-1ß (CCR5 ligand), 6Ckine, and macrophage inflammatory protein-3ß (CCR7 ligands) are expressed in the human endometrium (23, 29, 30). Although there are some differences between their expression patterns, Mig and IP-10 may cooperate with these chemokines in the selective recruitment of endometrial NK cells.

By contrast, I-TAC and SDF-1 were not detected or were detected at a very low level. In the pregnant endometrium (decidua), an in situ hybridization study demonstrated that the transcripts for Mig, IP-10, and I-TAC were mainly localized in the stroma with individual variability, whereas the transcript for SDF-1 were occasionally expressed in the blood vessels or leukocytes (31). On the other hand, another study using RT-PCR and immunohistochemistry showed the presence of Mig and IP-10 and the absence of I-TAC and SDF-1 in the maternal component (20). Our findings in the nonpregnant endometrium supported the latter (studies on the pregnant endometrium).

The presence of Mig and IP-10 and the absence of I-TAC in the endometrium are intriguing because the genes encoding three CXCR3 ligands are all located in the same locus on chromosome 4 in humans and are commonly activated by IFN in a wide range of cells (15, 32, 33). However, the expression of CXCR3 ligands in the human endometrium is likely to be more tightly regulated. We found that progesterone is a positive stimulator for the expression of Mig and IP-10 (but not of I-TAC or SDF-1) in the endometrial stromal cells, whereas 17ß-estradiol has no significant effect. Progesterone, rather than IFN, may be a key regulator in the endometrial expression of Mig and IP-10.

The immunoreactivity for Mig was also detected in endometrial NK cells, although we could not determine whether Mig was produced or incorporated by these leukocytes. Given that the transcript for Mig is not expressed in the endometrial leukocytes (31), Mig protein may be selectively incorporated by these NK cells.

Recent studies have demonstrated that chemokines have diverse biological functions other than leukocyte migration. CXC chemokines are subdivided by the presence or absence of glutamate-leucine-arginine motif in the N terminus, which precedes the first two cysteines. CXCR2 ligands (including epithelial neutrophil-activating protein-78, growth-regulated oncogene-, and IL-8) contain an glutamate-leucine-arginine motif and have angiogenic activity, whereas CXCR3 ligands lack this motif and display an angiostatic activity (34). The expression of both CXCR2 and CXCR3 ligands in the endometrium (35, 36, 37, 38) suggest that these chemokines may play a role not only in leukocyte recruitment but also in the endometrial vasculature.

In the ovine endometrium, IP-10 was shown to be expressed in subepithelial stromal cells and involved not only in the leukocyte recruitment but also in the migration and attachment of the embryo (39, 40). We demonstrated that IP-10 is constantly expressed in the surface epithelial cells in the human endometrium. IP-10 may play an important role in embryo implantation.

Human endometrial perivascular cells are distinct from nonperivascular stromal cells. Endometrial perivascular cells express -smooth muscle actin and have the contractile activity, indicating that these cells are characteristic of myofibroblasts (41, 42). They show marked proliferation in response to progesterone and spread in the decidual stroma during the first-trimester pregnancy (43). CXCR3 ligands can stimulate the proliferation of myofibroblast-like renal perivascular cells (44). The localization of Mig and IP-10 around the endometrial vessels is suggestive of their involvement in the process of decidualization.

In this study, we demonstrated that Mig and IP-10 were constitutively expressed, but I-TAC or SDF-1 was not detected or was detected at a very low level. The fluctuation of temporal and spatial expression of Mig and IP-10 suggests the involvement of these CXCR3 ligands, not only in the recruitment of NK cells but also in the other phenomena, such as decidualization, in the human endometrium.

	Footnotes

 
Abbreviations: CXCR, CXC chemokine receptor; ES, early secretory phase; IFN, interferon; IP-10, IFN-inducible protein-10; I-TAC, IFN-inducible T-cell -chemoattractant; LS, late secretory phase; MS, midsecretory phase; NK, natural killer; P, proliferative phase; SDF, stromal cell-derived factor.

Received July 24, 2003.

Accepted February 16, 2004.

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