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Expression of Macrophage Inflammatory Protein-1ß in Human Endometrium: Its Role in Endometrial Recruitment of Natural Killer Cells

Department of Obstetrics and Gynecology (K.K., T.N., T.O., H.K., H.H.) and Department of 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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Human endometrium is infiltrated by natural killer (NK) cells throughout the menstrual cycle. The number of endometrial NK cells is low in the proliferative phase, but acutely increases after ovulation, and reaches a peak in the late secretory phase, suggesting that endometrium recruits these leukocytes selectively from circulating peripheral blood. We investigated the expression of macrophage inflammatory protein (MIP)-1ß, a potential chemoattractant for NK cells, in the endometrium. RT-PCR and ELISA revealed that MIP-1ß is expressed in the endometrium throughout the menstrual cycle at both the message and protein levels. MIP-1ß expression is stronger in the secretory phase endometrium than in the proliferative phase endometrium. Immunohistochemistry revealed that MIP-1ß is localized in the surface epithelial cells, glandular epithelial cells, and perivascular stromal cells throughout the menstrual cycle. Stromal cells in a wider perivascular area became immunoreactive in the secretory phase. There was a strong correlation between the endometrial MIP-1ß concentration and the number of endometrial NK cells. Progesterone significantly induced MIP-1ß secretion from cultured endometrial stromal cells, whereas 17ß-estradiol had a weak effect. These results suggest that endometrial MIP-1ß may be involved in the recruitment of NK cells from circulating peripheral blood.

	Introduction

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HUMAN ENDOMETRIUM CONTAINS a wide variety of leukocytes, including T cells, B cells, and macrophages. Natural killer (NK) cells, however, occupy more than 70% of the endometrial leukocytes (1). Peripheral blood NK cells are generally classified as the main population (90%) of CD16^bright CD56^dim NK cells that display strong cytolytic activity against their target cells and a minority (10%) of CD16^neg CD56^bright NK cells that show weak cytolytic activity (2). By contrast, more than 95% of endometrial NK cells are CD16^neg CD56^bright NK cells, there being few endometrial CD16^bright CD56^dim NK cells. These NK cells accumulate as single cells or aggregates around endometrial glands and vessels, although their function at the implantation site remains unknown.

Although the other leukocyte populations are almost constant, the number of endometrial NK cells fluctuates with the menstrual cycle; their number is low in the proliferative phase, but acutely increases after ovulation, and reaches a peak in the late secretory phase. In the menstrual period, they are shed with other endometrial components (1). Such numerical fluctuation suggests that these leukocytes may be recruited selectively from circulating peripheral blood into the human endometrium.

Chemokines are a group of small peptides that play important roles in the migration of inflammatory cells into normal, inflammation, and tumor tissue (3). According to amino acid sequences, chemokines are classified into four groups (C, CC, CXC, and CX₃C). Recent studies have demonstrated that macrophage inflammatory protein (MIP)-1ß (4), a CC chemokine, is a strong chemoattractant for peripheral blood NK cells, especially for CD16^neg CD56^bright NK cells (5). In this study, we investigated the expression and localization of MIP-1ß in the human endometrium and its involvement in endometrial recruitment of NK cells.

	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 all of the patients before sample collection.

Endometrial samples were obtained from 58 fertile women aged 31–46 yr who had undergone hysterectomy for leiomyoma or carcinoma in situ of uterine cervix. Full thickness endometrial pieces were dissected from each uterus. Endometrial samples were collected within 15 min after hysterectomy. Patients had regular menstrual cycles ranging from 28–35 d. None of them had received any hormonal treatment or showed any pathological findings such as polyps, tumors, or endometritis. Endometrial samples were dated following the patients’ menstrual history and standard histological criteria (6). They were classified as 7 of the early proliferative phase (EP; d 4–7), 9 of the mid-proliferative phase (MP; d 8–11), 12 of the late proliferative phase (LP; d 12–14), 9 of the early secretory phase (ES; d 15–18), 10 of the mid-secretory phase (MS; d 19–23), and 11 of the late secretory phase (LS; d 24 onward).

Tissue preparation

Fifty-two endometrial samples (7 EP, 9 MP, 6 LP, 9 ES, 10 MS, and 11 LS) were used to evaluate in vivo expression of MIP-1ß. After being washed in PBS, a part of the sample was fixed overnight in a 4% paraformaldehyde [in phosphate buffer (pH 7.3)]. The remainder was homogenized in a TRIZol reagent (Life Technologies, Inc., Gaithersburg, MD). Tissue protein and RNA were then isolated according to the manufacturer’s instructions. The protein was preserved in lysis buffer containing 2 µM aprotinin, 50 µM leupeptin, 125 µM bestatin, and 25 µM pepstatin A (Nakarai, Kyoto, Japan). The RNA precipitate was preserved in diethylpyrocarbonate-treated water. These were frozen at -80 C until measurement.

Culture of endometrial stromal cells

Six endometrial samples of LP were used for the isolation and culture 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 and Co., San Jose, CA). The cells were incubated in culture medium (phenol-red free DMEM containing 10% charcoal-stripped fetal bovine serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml amphotericin B) at 37 C in 5% CO₂. After one passage, immunocytochemistry confirmed that more than 95% of cells were positive for vimentin (stromal cell marker). The cells were cultured in six-well plates (Becton, Dickinson and Co.) with 4 ml fresh culture medium supplemented with 10^-6 M 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 cell supernatant was collected by centrifugation and frozen at -80 C until measurement.

RT-PCR

Two micrograms of total RNA were converted to cDNA with 1 µg of oligo deoxythymidine primers, by a RT kit (Life Technologies, Inc.) in a final volume of 20 µl. One microliter of cDNA solution was amplified with 0.5 µM human MIP-1ß specific primers: upper, 5'-AGC CTC ACC TCT GAG AAA ACC-3'; and lower, 5'-GCA ACA GCA GAG AAA CAG TGA C-3' (7) in a final volume 50-µl solution containing Taq DNA polymerase (Life Technologies, Inc.). Each cycle consisted of 60 sec at 94 C, 45 sec at 61 C, and 90 sec at 72 C. As an internal control, human glyceraldehyde 3-phosphate dehydrogenase (G3PDH) mRNA, which is constantly expressed in the human endometrium throughout the menstrual cycle (8), was simultaneously amplified with an RT-PCR G3PDH amplimer set (CLONTECH Laboratories, Inc., Palo Alto, CA) under the same PCR condition. We confirmed that the amplified products were gene transcripts for MIP-1ß and G3PDH by sequence analysis.

The optimal condition for semiquantitation was determined as previously described (9). In brief, 6 samples (3 of the proliferative phase and 3 of the secretory phase) were subjected to PCR for 22–36 cycles. Ten microliters of PCR products were subsequently electrophoresed on a 1.5% agarose gel, stained with ethidium bromide, and then verified as a band on a UV transilluminator (Funakoshi, Tokyo, Japan). The images on the UV transilluminator were input into a computer, and the band intensity was measured using densitometrical software, NIH Image 1.61 (National Institutes of Health, Bethesda, MD). The band intensity increased exponentially up to 28 cycles and almost reached a plateau at more than 29 cycles. We set the optimal condition for semiquantitative RT-PCR at 26 cycles. Amplification was triplicated for each sample, and the mean MIP-1ß/G3PDH transcript ratio was calculated. Intraassay and interassay coefficients of variation were less than 6% and less than 8%, respectively.

ELISA

MIP-1ß protein concentration in homogenized samples and cell supernatant was measured with a Quantikine colorimetric sandwich ELISA kit (R&D Systems, Minneapolis, MN) and a Plate Reader 2550 (Bio-Rad Laboratories, Inc., Hercules, CA) according to the manufacturer’s protocol. The assay used coated murine antihuman MIP-1ß monoclonal antibody and soluble antihuman MIP-1ß polyclonal antibody. The assay was confirmed by the manufacturer to not cross-react with several chemokines related to MIP-1ß, including MIP-1, growth-regulated oncogene (GRO)-, GRO-ß, GRO-, macrophage chemoattractant protein-1, and regulated upon activation, normal T cell expressed and secreted. Total protein concentration in homogenized samples was measured with a DC protein assay kit (Bio-Rad Laboratories, Inc.) and the mean MIP-1ß protein/total protein concentration was calculated. The assay was done in triplicate. Intraassay and interassay coefficients of variation were less than 5% and less than 9%, respectively.

Immunohistochemistry

Fixed samples were embedded in paraffin and cut into 4-µm sections. After being deparaffinized in xylene and rehydrated in a graded series of ethanol, sections were immersed in 3% hydrogen peroxide for 5 min to block endogenous peroxidase 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 goat antihuman MIP-1ß polyclonal antibody (1:50 dilution; R&D Systems) overnight at 4 C. Sections washed in PBS three times were incubated with a LASB kit (DAKO Corp., Kyoto, Japan). Sections were washed and developed with diaminobenzidine (DAKO Corp.) and observed under a light microscope. The specificity of immunostaining was confirmed by the immunoabsorption test using recombinant human MIP-1ß (R&D Systems).

Immunohistochemistry for CD56 (NK cell marker) was performed by a similar protocol with specific antihuman CD56 polyclonal antibody (1:50 dilution; Zymed Laboratories, Inc. Corp., San Francisco, CA). The only exception was that the sections were subjected to microwave pretreatment in distilled water for 5 min before immersion in 3% hydrogen peroxide.

Under a light microscope, the number of round cells stained was counted in 10 nonoverlapping stromal areas (x400 magnification). The counting was done in triplicate by two independent observers who are indifferent to the study, and the mean number was calculated.

Statistics

The amounts of MIP-1ß transcript and protein of each group were compared by one-way ANOVA using Statcel software (OMS, Tokorozawa, Japan). The number of CD56-positive cells in each menstrual cycle phase was compared by Kruskal-Wallis test. Multiple comparisons were done with post hoc test (Sheffe’s F test). The correlation between endometrial MIP-1ß protein concentration and the number of the endometrial NK cells was analyzed by Spearman’s correlation coefficient by rank test. A P value less than 0.05 was considered significantly different.

	Results

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Expression of MIP-1ß gene transcripts in human endometrium

The bands consistent with transcripts for MIP-1ß (555 base pairs) and G3PDH (983 base pairs) were observed in all samples examined (Fig. 1A). The amplified products were confirmed to be transcripts for MIP-1ß and G3PDH by sequence analysis. As the quantity of the products at 26 cycle amplification increased in an MIP-1ß transcript and G3PDH transcript concentration-dependent manner (Fig. 1B), we measured the ratio of MIP-1ß to G3PDH transcript under this condition. The mean value ± SD at each phase was 0.59 ± 0.09 in EP, 0.71 ± 0.13 in MP, 0.68 ± 0.27 in LP, 1.07 ± 0.15 in ES, 1.23 ± 0.16 in MS, and 1.31 ± 0.21 in LS (Fig. 1C). The ratio of MIP-1ß to G3PDH transcript was significantly higher in ES than in EP, MP and LP, respectively (P < 0.01); was significantly higher in MS than in EP, MP, and LP, respectively (P < 0.0001); and was significantly higher in LS than in EP, MP, and LP, respectively (P < 0.0001).

View larger version (20K): [in this window] [in a new window]   Figure 1. Expression of MIP-1ß gene transcript in human endometrium. A, Representative electrophoresis of PCR products. Lane 1, Molecular size marker; lane 2, mid-proliferative phase endometrium; lane 3, late secretory phase endometrium. B, A standardization set for amplification efficiency using mid-secretory phase endometrium. The quantity of the products at 26 cycle amplification increased in an MIP-1ß transcript (open squares) and G3PDH transcript (closed squares) concentration-dependent manner. C, Densitometrical MIP-1ß/G3PDH transcript ratio in human endometrium at each menstrual phase. *, P < 0.01 vs. EP, MP, and LP; **, P < 0.0001 vs. EP, MP, and LP; ***, P < 0.0001 vs. EP, MP, and LP.

MIP-1ß protein in lysis buffer was below the detectable level. The MIP-1ß concentration (the mean ± SD) was 8.8 ± 12.5 pg/mg protein in EP, 31.1 ± 16.3 pg/mg protein in MP, 55.6 ± 38.9 pg/mg protein in LP, 140.9 ± 51.6 pg/mg protein in ES, 162.1 ± 61.3 pg/mg protein in MS, and 171.0 ± 61.5 pg/mg protein in LS (Fig. 2). Although there was considerable variation in MIP-1ß concentration between samples in the secretory phase, it was significantly higher in MS than in EP and MP (P < 0.005), and significantly higher in LS than in EP (P < 0.001), MP (P < 0.001), and LP (P < 0.01).

View larger version (16K): [in this window] [in a new window]   Figure 2. MIP-1ß protein concentration in human endometrium at each menstrual phase. *, P < 0.005 vs. EP and MP; **, P < 0.001 vs. EP and MP, and P < 0.01 vs. LP.

Immunostaining for MIP-1ß was found in the surface and glandular epithelial cells throughout the menstrual cycle (Fig. 3). In addition, perivascular stromal cells were also stained throughout the menstrual cycle. Stromal cells in a wider perivascular area became immunoreactive in ES. The number of immunoreactive stromal cells increased toward MS and LS. There was no marked difference in immunostaining pattern between functionalis zone and basalis zone.

View larger version (173K): [in this window] [in a new window]   Figure 3. Localization of MIP-1ß protein in human endometrium. Arrows indicate the immunostaining. Scale bars, 100 µm. A, Proliferative phase. B, Early secretory phase. C, Mid-secretory phase. D, Late secretory phase. E, Immunoabsorption test confirmed the specificity of the immunostaining. F, Stroma shows positive vimentin staining.

CD56+ round cells were scattered in the endometrial stroma. The mean number of NK cells in 10 stromal areas was 42.7 in EP, 32.7 in MP, 52.8 in LP, 115.8 in ES, 152.7 in MS, and 172.1 in LS (Fig. 4C). The number of endometrial NK cells significantly varied with the menstrual cycle (P < 0.001) and was significantly higher in the secretory phase than in the proliferative phase (P < 0.001). It was also significantly higher in MS than in ES (P = 0.03), and higher in LS than in ES (P = 0.01). There was a strong positive correlation (rs = 0.80) between the endometrial MIP-1ß protein concentration and the number of endometrial NK cells (Fig. 5).

View larger version (62K): [in this window] [in a new window]   Figure 4. Immunohistochemistry for endometrial NK cells. A, Late proliferative phase. B, Mid-secretory phase. C, Number of endometrial NK cells at each menstrual phase. *, P < 0.001 vs. EP, MP, and LP; **, P < 0.001 vs. EP, MP, and LP, and P < 0.05 vs. ES.

View larger version (15K): [in this window] [in a new window]   Figure 5. Strong positive correlation between the endometrial MIP-1ß protein concentration and the number of endometrial NK cells (rs = 0.80).

MIP-1ß protein was not detected in the culture medium without cells, but was detected in the control culture medium (Fig. 6). Progesterone significantly (8- to 10-fold) induced MIP-1ß secretion from endometrial stromal cells at each culture time, whereas 17ß-estradiol had a weak effect (1.2- 1.5-fold). Progesterone induced MIP-1ß secretion in a timedependent manner.

View larger version (16K): [in this window] [in a new window]   Figure 6. Secretion of MIP-1ß protein by cultured endometrial stromal cells. MIP-1ß concentration in the culture medium of the control (filled bars), 17ß-estradiol-added (open bars), and progesterone-added endometrial stromal cells (shaded bars). *, P < 0.01 vs. control group; **, P < 0.05 vs. control group.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

There may be two possible mechanisms explaining the increase of endometrial NK cells; one is proliferation in the endometrium, and the other is selective recruitment from peripheral blood NK cells. Endometrial NK cells are, at least in part, likely to be proliferating in situ, because 20–70% of them express proliferation-associated nuclear marker Ki-67 (12, 13). Recently, we and other groups reported that the expression of IL-15, a cytokine that has a strong proliferative effect on CD56^bright NK cells (14), in the human endometrial stroma corresponds to the number of endometrial NK cells throughout the menstrual cycle and first trimester pregnancy (15, 16, 17). IL-15 therefore may be a candidate for the proliferation of these NK cells. However, it is difficult to explain their drastic increase only by in situ proliferation mechanism.

MIP-1ß is one of the CC chemokines that was initially identified in an activated mouse macrophage cell line (4). To date, at least seven MIP-1ß cDNAs are cloned in humans (18). In protein sequences, MIP-1ß is similar to MIP-1, which was known to show a strong chemoattractive activity for CD56^bright NK cells (19). MIP-1ß also displays a strong chemoattractive activity for CD16^neg CD56^bright NK cells because its specific receptor CCR5 is brightly expressed on this NK cell population (20). We proved that MIP-1ß is expressed in the human endometrium throughout the menstrual cycle at both the transcript and protein level.

MIP-1ß was expressed in all samples examined. The high level of variability in MIP-1ß protein expression was seen between secretory phase endometrial samples, despite our using the full thickness endometrial pieces obtained immediately after hysterectomy. It is likely that this is not due to sampling and methodological problems, but is due to individual variability. The expression level of the MIP-1ß was low in the proliferative phase but significantly higher in the secretory phase, which corresponded to the numerical fluctuation of the endometrial NK cells. The finding indicates that endometrial expression of MIP-1ß is regulated by female sex steroids, especially by progesterone, similar to other chemokines such as epithelial neutrophil-activating peptide 78 and GRO- (21, 22). Using an in vitro culture system, we found that progesterone was a positive regulator of MIP-1ß production of endometrial stromal cells.

The endometrial MIP-1ß protein concentration strongly correlates with the number of endometrial NK cells. MIP-1ß may therefore be a potential chemoattractant for the recruitment of peripheral blood NK cells (especially CD16^neg CD56^bright NK cells) into the human endometrium. Endometrial NK cells are known to be distributed in the stroma, preferably around the endometrial vessels and glands (1). The localization of MIP-1ß in the endometrium corresponds to the spatial distribution of endometrial NK cells. MIP-1ß may be involved not only in extravasation of NK cells but also in their migration in the endometrial stroma.

In this study, we demonstrate that MIP-1ß is expressed in the human endometrium and its fluctuation with the menstrual cycle corresponds to the number of endometrial NK cells. These results suggest that MIP-1ß may be involved in the recruitment of NK cells into the endometrium from circulating peripheral blood.

	Footnotes

 
Abbreviations: EP, Early proliferative phase; ES, early secretory phase; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; GRO, growth-regulated oncogene; LP, late proliferative phase; LS, late secretory phase; MIP, macrophage inflammatory protein; MP, mid-proliferative phase; MS, mid-secretory phase; NK, natural killer.

Received June 25, 2002.

Accepted December 18, 2002.

	References

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