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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-2447
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 5 2932-2940
Copyright © 2005 by The Endocrine Society

Central Role of Interleukin-15 in Postovulatory Recruitment of Peripheral Blood CD16(–) Natural Killer Cells into Human Endometrium

Kotaro Kitaya, Takeshi Yamaguchi and Hideo Honjo

Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan

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


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
A large number of CD16(–) natural killer (NK) cells appear in the human endometrium after ovulation. One possible explanation for this phenomenon is recruitment from peripheral blood (PB) CD16(–) NK cells. In this study, we examined whether IL-15 is involved in postovulatory recruitment from PB CD16(–) NK cells. The IL-15 receptor {alpha} chain was expressed on PB CD16(–) NK cells but not on CD16(+) NK cells. In an in vitro migration assay, recombinant human IL-15 enriched PB CD16(–) NK cells. Endometrial soluble protein fraction in the secretory phase, but not in the proliferative phase, also enriched these NK cells. Neutralization of IL-15 in the secretory phase endometrium with anti-IL-15 monoclonal antibody significantly lowered the enrichment of PB CD16(–) NK cells. In contrast, neutralization of other potential chemokines, including stromal cell-derived factor-1 or macrophage inflammatory protein-1{alpha}, had no significant effect. The IL-15 concentration in the endometrial soluble protein fraction was higher in the secretory phase than in the proliferative phase, with a peak in the midsecretory phase. These results support the idea that endogenous IL-15 in secretory phase endometrium plays a central role in postovulatory recruitment of PB CD16(–) NK cells into the human endometrium.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
NATURAL KILLER (NK) CELLS, which comprise 10–20% of peripheral blood (PB) lymphocytes, are characterized as CD3(–) CD56(+) granular lymphocytes. Human NK cells are further subdivided into two distinct subsets; one is CD16(+) NK cells, which show high cytolytic activity against target cells, and the other is CD16(–) NK cells, which show high cytokine production. In humans, CD16(+) NK cells comprise approximately 85–90% of PB NK cells, whereas CD16(–) NK cells comprise the remaining 10–15% (1).

After ovulation (in the secretory phase), many NK cells appear in the human uterus, mainly in the functional layer of the endometrium. Endometrial NK cells accumulate in the stroma, preferably around glands and vessels as single cells or aggregates (2). In contrast to PB NK cells, the main subset of endometrial NK cells is CD16(–) NK cells, which account for 70–80% of the endometrial lymphocytes in the secretory phase. When implantation occurs, the number of endometrial CD16(–) NK cells further rises in the decidualized endometrium during the first trimester of pregnancy. In contrast, these NK cells are shed with other endometrial cells during menstruation.

Endometrial CD16(–) NK cells are likely to play important roles in embryo implantation and early placentation. They produce a wide range of molecules, such as interferon-{gamma}, macrophage colony-stimulating factor, and vascular endothelium growth factor, which are important for endometrial vasculature and trophoblast growth (3, 4, 5). Moreover, they are likely to regulate trophoblast invasion into first trimester pregnancy decidua (6), through the interaction between their surface NK receptors (7) and major histocompatibility class I molecules, including human leukocyte antigen-C, -E, and -G, expressed on the surface of the invading trophoblasts (8, 9, 10).

The mechanism underlying the postovulatory rise of endometrial CD16(–) NK cells remains unexplained. One possible explanation for the analogy of other mucosal tissues is selective recruitment from PB CD16(–) NK cells across the endometrial vessels. Chemokines are a group of peptides that induce leukocyte migration via activation of adhesion molecules expressed on the surface of the leukocytes. Recent studies demonstrated that two chemokines, stromal cell-derived factor (SDF)-1 and macrophage inflammatory protein (MIP)-1{alpha}, are strong potential chemoattractants for selective recruitment of PB CD16(–) NK cells into first trimester pregnancy decidua (11, 12). SDF-1 and MIP-1{alpha} are expressed in invading trophoblasts and cytotrophoblasts (fetal components), but they are not expressed or are expressed at a very low level in the decidual/endometrial cells (maternal components) (12, 13, 14). The essential molecule(s) involved in the rise of CD16(–) NK cells in the nonpregnant endometrium during the secretory phase, therefore, remains undetermined.

IL-15 is a cytokine that can induce an intense proliferation of PB and endometrial CD16(–) NK cells in vitro (15, 16, 17). IL-15 has been shown to be a chemoattractant for PB NK cells as well as for T cells (18, 19), although it remains unclear whether IL-15 preferably attracts CD16(+) NK cells or CD16(–) NK cells. IL-15 is expressed in the human endometrium, and its expression in the perivascular stromal cells is higher in the secretory phase than in the proliferative phase (20, 21, 22, 23). Moreover, progesterone enhances the expression of IL-15 in the cultured endometrial stromal cells (21, 22, 23). In mice, NK cells are also present in the placenta, preferably in the decidua basalis, mesometrial triangle, and transient pregnancy-associated mural structures at each implantation site called mesometrial lymphoid aggregates of pregnancy (24). The mice lacking the IL-15 gene are fertile, but they are defective of these placental NK cells, decidual cellularity, and modification of decidual spiral arteries (25), suggesting that IL-15 and placental NK cells may play a role in placental development in mice.

These findings suggest that IL-15 may be a chemokine involved in the selective recruitment of PB CD16(–) NK cells into the endometrium. Several studies demonstrated that CD16(–) NK cells are decreased in secretory phase endometrium in the patients with unexplained recurrent miscarriages (26) and in vitro fertilization embryo transfer failure (27). The optimal rise of endometrial CD16(–) NK cells in the secretory phase is thus essential for successful human reproduction. In this study, we examined the effect of recombinant IL-15 and endogenous IL-15 in the secretory phase endometrium on enrichment of PB CD16(–) NK cells in vitro.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Reagent and antibodies

Recombinant human (rh)-IL-15 was purchased from R&D Systems (Minneapolis, MN). The following mouse antihuman monoclonal antibodies for chemokines were also purchased from R&D Systems: anti-IL-15 (MAB647, IgG1), anti-MIP-1{alpha} (MAB270, IgG1), anti-MIP-1ß (MAB271, IgG2B), antiregulated upon activation, normal T cell expressed and secreted (MAB278, IgG1), antimonokine induced by {gamma}-interferon (MAB392, IgG1), antiinterferon-inducible protein-10 (MAB266, IgG1), anti-SDF-1 (MAB310, IgG1), and anti-IL-15R{alpha} (MAB1471, IgG2B). The following fluorescent mouse antihuman monoclonal antibodies were purchased from BD PharMingen (San Jose, CA): fluorescein isothiocyanate-conjugated anti-CD16 (3G8, IgG1), phycoerythrin-conjugated anti-CD56 (MY31, IgG1), Simultest Leucogate (anti-CD45-fluorescein isothiocyanate/anti-CD14-phycoerythrin), PerCP-conjugated anti-CD3 (SK7, IgG1), allophycocyanin-conjugated goat antimouse Ig, and isotype-matched mouse IgG.

Samples

Endometrial samples were obtained from 44 fertile women aged 32–43 yr who had undergone hysterectomy for leiomyoma and carcinoma in situ of uterine cervix. Endometrial samples obtained from the patients with submucosal leiomyoma and endometriosis were excluded in this study. None of these women had received any hormonal treatment or showed any pathological findings such as endometrial polyps, tumors, or endometritis. There were no noteworthy differences in clinical background, such as age, parity, or body mass index between leiomyoma groups and carcinoma in situ of uterine cervix group. Following the standard histological dating criteria (28), endometrial samples were confirmed to be in the proliferative phase (n = 13), early secretory phase (n = 11), midsecretory phase (n = 10), and late secretory phase (n = 10). PB samples were drawn from the patients at the operation day or from healthy volunteers. This study was approved by the Kyoto Prefectural University of Medicine, Institutional Review Board (Kyoto, Japan). Informed consent was obtained from every patient.

Endometrial samples were immediately washed in PBS. After being weighed on a scale (range, 0.7–1.8 g wet weight), approximately 0.3 g sample was homogenized in 1 ml lysis buffer containing 2 µM aprotinin, 50 µM leupeptin, 125 µM bestatin, and 25 µM pepstatin A (Nakarai, Kyoto, Japan), and the soluble protein fraction was retrieved for the subsequent experiments. A part of them was preserved at –80 C until measurement.

Isolation of PB lymphocytes

Heparinized PB was overlaid onto Ficoll-Paque (Amersham Pharmacia, Uppsala, Sweden) and centrifuged at 400 x g for 30 min. The cells at the interface were removed, washed twice in PBS, and resuspended in the assay medium [RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD) containing 1% fetal calf serum (JRH Biosciences, Lenexa, KS)]. The cells were then incubated in a 25-ml culture flask (BD Biosciences, San Jose, CA) at 37 C in humidified 5% CO2 for 1 h to adhere monocytes to plastic walls. The nonadherent cells were removed, washed twice in PBS, and resuspended in the assay medium. By flow cytometric analysis, the cells were confirmed to contain more than 95% of CD45(+) CD14(–) lymphocytes.

In vitro migration assay

The cell suspension was adjusted at a concentration of 1–2 x 106 cells/ml, and 100-µl cell suspension was loaded into Transwell membrane (polycarbonate, 6.5-mm diameter, 5-µm pore size, Corning-Costar, Cambridge, MA). The membrane was then put on the bottom well filled with 600 µl rhIL-15 or endometrial soluble protein fraction (ESPF). For neutralization, rhIL-15 or ESPF was preincubated with each specific antichemokine antibody or control mouse IgG. The bottom wells filled with assay medium alone (control) were used to detect spontaneous migration. The wells at the same condition were prepared as triplicates. After incubation for 3 h at 37 C in 5% CO2, the membrane was removed, and the cells that migrated to the bottom wells were collected. In preliminary tests, intraassay variability for the number of the lymphocytes migrating was less than 12%.

Flow cytometry

The initial or migrated PB lymphocytes were incubated with 20% fetal calf serum and then incubated with fluorescent anti-CD3, anti-CD16, and anti-CD56 antibodies. After being washed twice in PBS, the cells were analyzed by a FACS Caliber and Cell Quest software (BD Biosciences). To determine the surface expression of IL-15R{alpha}, the cells were further incubated with anti-IL-15R{alpha} monoclonal antibody and washed in PBS, followed by incubation in allophycocyanin-conjugated goat antimouse Ig and washing twice. The quadrant marker was determined on a dot gram, which showed that as much as 98–99.99% of corresponding fluorescent mouse IgG-stained cells were contained within the lower left quadrant.

ELISA

The IL-15 amounts in the ESPF were measured with a Quantikine ELISA kit (R&D Systems) according to the manufacturer’s protocol. Total protein amount in the ESPF was measured with a DC protein assay kit (Bio-Rad Laboratories, Hercules, CA). The assay was done in triplicate, and the mean IL-15 amounts/ESPF amounts were calculated. Intraassay and interassay variability were less than 7 and 10%, respectively.

Statistics

The data were analyzed using Statcel software (OMS, Tokorozawa, Japan). The comparison of two groups was done by Student’s t test. The comparison of more than three groups was done by multiple comparisons (one-way ANOVA with post hoc Sheffe’s F test). P < 0.05 was regarded as significantly different.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Surface expression of IL-15R{alpha} on PB and endometrial CD16(–) NK cells but not on PB and endometrial CD16(+) NK cells

Using flow cytometry, we examined the surface expression of IL-15R{alpha} on freshly isolated PB and endometrial NK cells, which has not yet been studied. Representative data are shown in Fig. 1Go.



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  		FIG. 1. Surface expression of IL-15 receptor {alpha} (IL-15R{alpha}) on freshly isolated PB and endometrial NK cells. PB and endometrial NK cells were analyzed by flow cytometry. NK cells were determined as CD3(–) CD56(+) lymphocytes and further subdivided by CD16 expression. Solid lines indicate the cells labeled with anti-IL-15{alpha}. Broken lines indicate the cells labeled with negative control IgG2B. A, Representative data of surface expression of IL-15R{alpha} on PB NK cells. IL-15R{alpha} was partly expressed on PB CD16(–) NK cells but was not expressed on PB CD16(+) NK cells. B, Representative data of surface expression of IL-15R{alpha} on endometrial NK cells. IL-15R{alpha} was partly expressed on endometrial CD16(–) NK cells.

 
Of the PB NK cells, 80–90% were CD16(+) NK cells with some CD16(–) NK cells remaining (Fig. 1AGo). IL-15R{alpha} was partly expressed on PB CD16(–) NK cells (18.1% ± 5.3%, mean ± SD, n = 22) but was not expressed on PB CD16(+) NK cells. There was no significant difference (P > 0.69) in the expression level of IL-15R{alpha} on PB CD16(–) NK cells among the patients who underwent hysterectomy (18.3 ± 5.0%, n = 8), the healthy volunteer women (19.2 ± 6.6%, n = 9), and the healthy volunteer men (16.6 ± 2.8%, n = 5). We examined whether there was a menstrual cycle-dependent fluctuation in the expression level of IL-15R{alpha} on PB CD16(–) NK cells in the same women (n = 5), but there was no significant difference between the proliferative (16.1% ± 6.4%) and secretory (17.1% ± 6.0%) phases.

Few of the endometrial NK cells were CD16(+) NK cells, more than 95% being CD16(–) NK cells (Fig. 1BGo). IL-15R{alpha} was partly expressed on endometrial CD16(–) NK cells (8.4 ± 2.0%, n = 8). We could not evaluate its detailed expression level on endometrial CD16(+) NK cells because of the limited number of these NK cells. There was no significant difference (P = 0.15) in the expression level of IL-15R{alpha} on endometrial CD16(–) NK cells between the proliferative phase (7.4 ± 1.9%, n = 4) and the secretory phase (9.4 ± 1.6%, n = 4). On the other hand, in the same women, the expression level of IL-15R{alpha} on the PB CD16(–) NK cells is significantly higher (P = 0.00013) than that on endometrial CD16(–) NK cells.

Enrichment of PB CD16(–) NK cells by rhIL-15

To examine which subset of PB NK cells migrated preferably in response to IL-15, we performed an in vitro migration assay using recombinant protein and neutralizing antibody. The percentage of lymphocytes that migrated spontaneously into the control medium was 2.0 ± 0.7% (mean ± SD, n = 6) of the initial lymphocytes (loaded onto Transwell membrane). The percentage of lymphocytes that migrated in the presence of rhIL-15 was 6.0 ± 2.6% of the initial lymphocytes and was significantly (P < 0.0001) higher than the percentage of lymphocytes migrating spontaneously. The percentage of lymphocytes that migrated in the presence of both rhIL-15 and anti-IL-15 monoclonal antibodies was 2.1 ± 0.8% of the initial lymphocytes and was comparable with the percentage of lymphocytes migrating spontaneously. These results showed that rhIL-15 can induce the migration of PB lymphocytes.

The NK cell subsets were classified by flow cytometry. Figure 2AGo shows representative data of five independent experiments. NK cells accounted for 12.9 ± 2.4% (mean ± SD, n = 5) of freshly isolated initial PB lymphocytes. The percentage of CD16(+) NK cells migrating spontaneously was 10.7 ± 2.2% of the lymphocytes that migrated. The percentage of CD16(+) NK cells that migrated in the presence of rhIL-15 was 6.0 ± 2.5% of the lymphocytes that migrated, which were not significantly different (P = 0.070) from the percentage of CD16(+) NK cells migrating spontaneously. The percentage of CD16(+) NK cells that migrated in the presence of both rhIL-15 and anti-IL-15 antibodies was 10.9 ± 3.1% of lymphocytes that migrated, which was comparable with the percentage of CD16(+) NK cells migrating spontaneously. In contrast, the percentage of CD16(–) NK cells migrating spontaneously was 1.8 ± 0.5% of lymphocytes that migrated. The percentage of CD16(–) NK cells that migrated in the presence of rhIL-15 was 6.0 ± 1.2% of lymphocytes that migrated, which was significantly (P < 0.0001) higher than the percentage of CD16(–) NK cells migrating spontaneously. The percentage of CD16(–) NK cells that migrated in the presence of both rhIL-15 and anti-IL-15 antibody was 1.7 ± 0.3% of lymphocytes that migrated, which was comparable with the percentage of CD16(–) NK cells migrating spontaneously.



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  		FIG. 2. Enrichment of human PB CD16(–) NK cells by rhIL-15 in an in vitro migration assay. A, Representative data on expression of CD16 and CD56 on initial PB lymphocytes, and PB lymphocytes migrating in control medium alone, in the presence of rhIL-15, and in the presence of rhIL-15 + anti-IL-15 monoclonal antibody. PB CD16(–) NK cells were enriched by rhIL-15, and the effect was inhibited by anti-IL-15 antibody. B, Mean and SD of five independent experiments. C, Dose-dependent effect of rhIL-15 on enrichment of human PB CD16(–) NK cells.

 
The results showed that rhIL-15 enriched PB CD16(–) NK cells preferably but not PB CD16(+) NK cells (Fig. 2BGo). Enrichment of PB CD16(–) NK cells was seen at the concentration of more than 5 ng/ml and reached the maximal level at 10 ng/ml (Fig. 2CGo).

Enrichment of PB CD16(–) NK cells by endogenous IL-15 in secretory phase endometrium

Next, we examined whether human endometrium has a potential to induce the migration of PB lymphocytes using ESPF. The percentage of lymphocytes that migrated into the ESPF in the proliferative phase was 2.3 ± 0.4% (mean ± SD, n = 5) of the initial lymphocytes, which was comparable (P = 0.76) with that of lymphocytes migrating spontaneously (Fig. 3AGo). The percentage of lymphocytes that migrated into the ESPF in the secretory phase was 7.3 ± 2.5% of the initial lymphocytes, which was significantly (P = 0.0028) higher than the percentage of lymphocytes migrating spontaneously (Fig. 3AGo). The results showed that ESPF in the secretory phase, but not in the proliferative phase, can induce the migration of PB lymphocytes.



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  		FIG. 3. Enrichment of PB CD16(–) NK cells by endogenous IL-15 in the secretory phase, but not by that in the proliferative phase. A, Enrichment of PB lymphocytes by ESPF in the secretory phase, but not by that in the proliferative phase. B, Representative data on expression of CD16 and CD56 on initial PB lymphocytes, and migrated PB lymphocytes in control medium alone, in the presence of ESPF, in the presence of ESPF with control mouse IgG, anti-IL-15 monoclonal antibody, anti-SDF-1 monoclonal antibody, or anti-MIP-1{alpha} monoclonal antibody.

 
Of the lymphocytes that migrated into the ESPF in the secretory phase, 16.6 ± 2.5% were NK cells. We examined which subset of NK cells migrated preferably into secretory phase endometrium. Figure 3BGo shows representative data of five representative experiments. The percentage of CD16(+) NK cells that migrated into the ESPF of the secretory phase was 8.9 ± 2.6%, which was comparable with the percentage of CD16(+) NK cells migrating spontaneously. In contrast, the percentage of CD16(–) NK cells that migrated into the ESPF in the secretory phase was 7.9 ± 2.2%, which was significantly (P < 0.0001) higher than the percentage of CD16(–) NK cells migrating spontaneously. These results showed that endogenous protein in secretory phase endometrium has a potential to enrich PB CD16(–) NK cells and reduce PB CD16(+) NK cells.

We simultaneously examined whether endogenous IL-15 in secretory phase endometrium plays a role in the migration of PB CD16(–) NK cells using a neutralizing antibody. We compared the effect of IL-15 with the effect of several chemokines known to be selective chemoattractants for PB CD16(–) NK cells and expressed in the human endometrium (14, 15, 16, 29, 30, 31, 32), although it was difficult to examine all at the same time because of the limited sample size. Figure 3BGo shows the results of representative experiments that are summarized in Table 1Go. The percentages of CD16(+) NK and CD16(–) NK cells that migrated into the ESPF in the secretory phase was 9.2 ± 2.3% and 4.0 ± 1.1%, respectively, when neutralized with anti-IL-15 monoclonal antibody. Neutralization with anti-IL-15 antibody significantly (P = 0.0063) lowered the percentage of CD16(–) NK cells compared with control mouse IgG, but not (P = 0.81) the percentage of CD16(+) NK cells. On the other hand, neutralization with anti-MIP-1ß monoclonal antibody, antimonokine induced by {gamma}-interferon monoclonal antibody, and antiinterferon-inducible protein-10 monoclonal antibody lowered the percentage of PB CD16(–) NK cells that migrated, but no significant differences (P > 0.38) were found in this small sample size. In contrast, anti-SDF-1, anti-MIP-1{alpha}, and antiregulated upon activation, normal T cell-expressed, and secreted monoclonal antibodies had no effect.


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  		TABLE 1. Percentage of CD16(–) NK cells in the migrated PB lymphocytes into the ESPF in the secretory phase preincubated with or without antichemokine monoclonal antibodies

 
Menstrual cycle-dependent fluctuation of IL-15 concentration in human endometrium

To examine whether human endometrium contains sufficient amounts of endogenous IL-15 for the induction of the selective migration of PB CD16(–) NK cells, we measured the IL-15 concentration in the ESPF (Fig. 4Go). IL-15 concentration (mean ± SD) was 73.3 ± 21.6 pg/mg protein in the proliferative phase (n = 8), 200.7 ± 36.4 pg/mg protein in the early secretory phase (n = 8), 385.6 ± 110.6 pg/mg protein in the midsecretory phase (n = 7), and 269.7 ± 68.1 pg/mg protein in the late secretory phase (n = 7). There was no significant difference in IL-15 concentration between the leiomyoma group and carcinoma in situ group at each phase. It was significantly higher in the early secretory phase than in the proliferative phase (P = 0.0075), significantly higher in the midsecretory phase than in the proliferative phase (P < 0.0001), early secretory phase (P = 0.0002), and late secretory phase (P = 0.027), and significantly higher in the late secretory phase than in the proliferative phase (P < 0.0001).



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  		FIG. 4. Menstrual cycle-dependent fluctuation of IL-15 concentration in ESPF. IL-15 and protein amounts in the ESPF were measured with a commercial ELISA kit, and IL-15 concentration was expressed as IL-15 amounts in soluble protein amounts (mean and SD, picograms per milligram of protein).

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
The postovulatory rise of endometrial CD16(–) NK cells involves female sex steroids, especially progesterone. Endometrial CD16(–) NK cells, however, lack the expression of the receptors and the proliferative responses to estrogen and progesterone (17, 33, 34). Therefore, it is unlikely that progesterone directly stimulates the in situ proliferation of endometrial CD16(–) NK cells. IL-2 and IL-15 are the cytokines that can stimulate the proliferation of endometrial CD16(–) NK cells in vitro (15, 16, 17, 35). Although these two cytokines share the same molecular structure and signaling receptors (common ß- and {gamma}-chains), their genetic mapping, sequence, and tissue localization are quite different (36, 37). Although IL-2 is not expressed in the human endometrium (38), IL-15 is expressed in the endometrium at a higher level in the secretory phase than in the proliferative phase (20, 21). In the human endometrial stroma, the expression of IL-15 is faint in the proliferative phase but strong in the secretory phase and first trimester pregnancy (20). These findings suggest that IL-15 is a strong candidate for in vivo proliferation of endometrial CD16(–) NK cells.

Although it is known that IL-2 receptor {alpha}-chain is not expressed on the surface of PB CD16(–) or PB CD16(+) NK cells (39), it remains unclear whether IL-15R{alpha}, the IL-15-specific binding site, is expressed on PB NK cells. IL-15R{alpha} was recently reported to be partly expressed on CD8(+) T cells but not on CD4(+) T cells (40). We demonstrated that IL-15R{alpha} is partly expressed on freshly isolated PB CD16(–) NK cells but not on PB CD16(+) NK cells, suggesting that IL-15 selectively affects PB CD16(–) NK cells. The expression level of IL-15R{alpha} on PB CD16(–) NK cells is higher than that on endometrial CD16(–) NK cells. If endometrial CD16(–) NK cells are recruited from PB CD16(–) NK cells, IL-15R{alpha} may be down-regulated in the process.

We found that rhIL-15 can enrich PB CD16(–) NK cells but not PB CD16(+) NK cells. Its maximal effect was observed at the concentration of 10 ng/ml, which resembles the finding that the maximal effect of IL-15 on in vitro proliferation of PB and endometrial CD16(–) NK cells was observed at a similar concentration (15, 16). Furthermore, ESPF in the secretory phase attracted more PB CD16(–) NK cells than those in the proliferative phase. The effect was significantly reduced by neutralization with anti-IL-15 monoclonal antibody. These findings ensure that secretory phase endometrium is capable of attracting PB CD16(–) NK cells via action of endogenous IL-15.

Human PB CD16(–) NK cells express a combination of chemokine receptors (CCR5, CCR7, CXCR3, and CXCR4) on their surface, which are distinct from other leukocyte populations (29). If endometrial CD16(–) NK cells originate from PB CD16(–) NK cells, the expression of the chemokines binding to these receptors in the endometrial vascular areas are required. Of these potential chemokines, we previously demonstrated that MIP-1ß, MIP-3ß, 6Ckine, monokine induced by {gamma}-interferon, and interferon-inducible protein-10 are expressed in the vascular area of the secretory phase endometrium (14, 30, 31, 32). By neutralization study, multiple chemokines including MIP-1ß, monokine induced by {gamma}-interferon, and interferon-inducible protein-10 induced selective enrichment of PB CD16(–) NK cells, but their effects were less than IL-15. Although these chemokines may be involved in the selective recruitment of PB CD16(–) NK cells into the endometrium, our findings support the idea that IL-15 plays a central role in this phenomenon.

Despite the wide distribution of IL-15 transcripts in various organs, many studies have pointed to the difficulty of detecting IL-15 at the protein level under a physiological condition. One possible explanation for this discrepancy is that IL-15 synthesis is regulated at various points of translation (41, 42, 43). We and others (20, 21) previously reported that endometrial stromal cells secrete soluble form IL-15 in vitro. However, it remains unclear whether human endometrium contains IL-15 enough to exert its role. In this study, IL-15 was detected at the level of more than 1 pg/mg (ng/g) in the soluble endometrial protein fraction. As anticipated by previous studies (20, 21), IL-15 concentration was higher in the secretory phase than in the proliferative phase, with a peak in the midsecretory phase. The expression of IL-15 is distinct in the vascular and perivascular areas in the secretory phase endometrium (20), indicating that human endometrium contains sufficient amounts of endogenous IL-15 for selective recruitment of PB CD16(–) NK cells.

In conclusion, we demonstrate that endogenous IL-15 in secretory phase endometrium is a strong candidate for postovulatory recruitment of PB CD16(–) NK cells into the human endometrium. Recent studies found that a substantial number of CD16(–) NK cells infiltrated not only the human endometrium but also the synovial fluid in patients with inflamed joints (44, 45) or the liver in patients with chronic hepatitis (46), where high concentration of IL-15 is detected (47, 48). CD16(–) NK cells seen in the endometrium by their dense surface expression of CD56, another marker of NK cells, compared with PB CD16(–) NK cells (1). Recently, IL-15 was shown to enhance surface expression density of CD56 on PB CD16(–) NK cells in vitro without affecting their CD16 expression (49). These reports and our data support the idea that IL-15 expressed in the endometrial vascular areas in the secretory phase may function as a chemoattractant for PB CD16(–) NK cells in the initial step. Once PB CD16(–) NK cells extravasated into the stroma, IL-15 in the endometrial stroma may be involved in their proliferation and bright surface expression of CD56. Some of these NK cells may be further attracted to several chemokines expressed in the endometrial epithelia and localize in the subepithelial stroma.


   Footnotes
 
This work was supported in part by Grant-in-Aid for Scientific Research 16790964 from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

First Published Online February 15, 2005

Abbreviations: ESPF, Endometrial soluble protein fraction; MIP, macrophage inflammatory protein; NK, natural killer; PB, peripheral blood; rh, recombinant human; SDF, stromal cell-derived factor.

Received December 13, 2004.

Accepted February 3, 2005.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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