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BOR - Papers in Press, published online ahead of print September 7, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.045971
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BIOLOGY OF REPRODUCTION 74, 35–40 (2006)
DOI: 10.1095/biolreprod.105.045971
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Potential Selectin L Ligands Involved in Selective Recruitment of Peripheral Blood CD16(–) Natural Killer Cells into Human Endometrium1

Takeshi Yamaguchi 3, Kotaro Kitaya 2 3, Nobue Daikoku 3, Tadahiro Yasuo 3, Shinji Fushiki 4, and Hideo Honjo 3

Department of Obstetrics and Gynecology,3 Department of Pathology and Applied Neurobiology Research Institute,4 Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan

ABSTRACT

Unique CD16(–) natural killer (NK) cells appear in the human cycling endometrium and acutely increase in number after ovulation. Selective recruitment from peripheral blood (PB) CD16(–) NK cells is a potential mechanism for the postovulatory increase of these NK cells. The interaction between selectin L, an adhesion molecule playing a critical role in leukocyte extravasation, and its ligands may be involved in this phenomenon. We investigated the menstrual cycle-dependent fluctuation of selectin L expression on PB CD16(–) NK cells and selectin L ligand expression in the human endometrial endothelium. The expression of selectin L on PB CD16(–) NK cells was constantly high throughout the menstrual cycle compared with other PB CD16(+) NK cells and non-NK lymphocytes. Among eight selectin L ligands examined, podocalyxin-like, mucosal addressin cell adhesion molecule-1 (MADCAM1) and chondroitin sulfate proteoglycan 2 (CSPG2) were localized in the endometrial endothelium. Semiquantitative score of immunostaining intensity in the endometrial endothelium for MADCAM1 was highest in the late secretory phase, whereas that for CSPG2 peaked throughout the secretory phase. There was a strong positive correlation between the number of endometrial NK cells and the semiquantitative score for CSPG2. Three active isoforms of CSPG2 mRNA were detected in the human endometrium. These findings support the idea that the interaction between selectin L and selectin L ligands functions in the postovulatory selective recruitment of PB CD16(–) NK cells into the human endometrium.

endometrium, immunology, implantation, natural killer cells, selectin L, selectin L ligand, uterus

INTRODUCTION

The cycling endometrium of women of reproductive ages is infiltrated by a unique leukocyte subset, CD16(–) natural killer (NK) cells, which comprise less than 0.5% of leukocytes in peripheral blood and other mucosal tissues [1]. The number of other endometrial leukocyte subsets, including T cells, macrophages, NKT cells, and CD16(+) NK cells, is almost constantly low throughout the menstrual cycle, but the number of endometrial CD16(–) NK cells fluctuates; it is low in the proliferative phase but acutely increases after ovulation and peaks in the mid- to late secretory phase. When the embryo is successfully implanted into the endometrium, the number of endometrial CD16(–) NK cells increases further in the decidualized endometrium during the first trimester of pregnancy, whereas they are shed during menstruation [1].

The proportion of endometrial CD16(–) NK cells in the secretory phase is lower in the patients with unexplained recurrent miscarriages [2] and in vitro fertilization-embryo transfer failure [3] than in fertile women, indicating that an optimal postovulatory increase of these NK cells is essential for a successful pregnancy. Although the mechanism underlying the postovulatory rise of endometrial CD16(–) NK cells remains undetermined, recent accumulating studies found that both human endometrium and peripheral blood (PB) CD16(–) NK cells express molecules that are required for extravasation of these NK cells, supporting the potential of selective recruitment from PB CD16(–) NK cells across the endometrial vessels [4–14]. We recently demonstrated a central role of interleukin-15 in the postovulatory selective recruitment of PB CD16(–) NK cells into the human endometrium, along with its proliferative effect on endometrial CD16(–) NK cells [12, 15].

For leukocyte extravasation, tethering/rolling on the endothelial cells is an essential initial step [16]. One distinct characteristic of PB CD16(–) NK cells from other leukocyte subsets is the high surface expression of selectin L [17], which is a key adhesion molecule involved in tethering/rolling of leukocytes on endothelial cells [18]. If the selective recruitment of PB CD16(–) NK cells into secretory phase endometrium occurs, the interaction between selectin L on PB CD16(–) NK cells and selectin L ligands on endometrial endothelial cells is likely to be important for the tethering/rolling of these NK cells.

Two selectin L ligands, sialyl Lewis a and sialyl Lewis x, have been reported in the endometrial surface epithelial cells, and their expression level is stronger in the secretory phase than in the proliferative phase, but few investigators have referred to the expression of selectin L ligands in the endometrial vessels [19–21]. The aim of this study is to characterize the expression of selectin L ligands in the human endometrium, especially in the endometrial endothelium. We investigated the localization of eight selectin L ligands in the endometrial endothelium, as well as the menstrual cycle-dependent surface expression of selectin L on PB CD16(–) NK cells.

MATERIALS AND METHODS

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 30 fertile women aged 36 to 45 yr who had undergone hysterectomy for subserous leiomyoma (n = 19) or cervical carcinoma in situ (n = 11). They had regular menstrual cycles ranging from 28 to 35 days and were not receiving any hormonal treatment. None of these samples showed any pathological findings such as polyps, tumors, or endometritis. Full-thickness endometrium was collected within 15 min after hysterectomy and washed immediately in phosphate-buffered saline (PBS; Takara Biomedical, Otsu, Japan). Following the standard criteria [22], nine, seven, eight, and six endometrial samples were classified into the proliferative phase, early secretory phase, midsecretory phase, and late secretory phase, respectively. PB samples were drawn from seven patients on the operation day or from 17 healthy volunteers and immediately heparinized.

Flow Cytometry

PB samples were 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 incubated with 20% fetal calf serum (FCS; JRH Biosciences, Lenexa, KS). The cell suspension (107 cells/ml) was then incubated with 20-µl PerCP-conjugated anti-CD3 monoclonal antibody (SK7; BD Pharmingen, San Diego, CA), 20-µl fluorescein isothiocyanate-conjugated anti-CD16 monoclonal antibody (3G8; BD Pharmingen), 20-µl phycoerythrin-conjugated anti-CD56 monoclonal antibody (MY31; BD Pharmingen), and 20-µl allophycocyanin-conjugated anti-selectin L monoclonal antibody (Dreg56; BD Pharmingen) for 15 min at room temperature. After being washed twice in PBS, the cells were analyzed by a FACS Caliber and CellQuest software (BD Pharmingen). The lymphocyte gate was determined with Simultest Leucogate (BD Pharmingen). The quadrant marker was determined on a dotgram that showed that 98%–99.99% of corresponding fluorescent mouse control IgG (BD Pharmingen)-stained cells were located in the lower left quadrant. NK cells—CD3(–) CD56(+) lymphocytes—were subdivided into CD16(–) subset and CD16(+) subset and analyzed [12].

Immunohistochemistry

Endometrial samples were fixed overnight in a 4% paraformaldehyde (in phosphate buffer, pH 7.3) (Nakarai Tesque, Kyoto, Japan), embedded in paraffin (Nakarai Tesque), and cut into 4-µm sections. After being deparaffinized in xylene (Nakarai Tesque) and rehydrated in a graded series of ethanol (Nakarai Tesque), sections were immersed in 3% hydrogen peroxide (Nakarai Tesque) for 5 min to eliminate endogenous peroxidase activity and then incubated with PBS containing 10% FCS for 10 min to block nonspecific antibody binding. In a moist chamber for 30 min at room temperature, sections were incubated with one of the following primary antibodies: anti-sialyl Lewis a monoclonal antibody (KM231, 1:500 dilution; Calbiochem, San Diego, CA), anti-sialyl Lewis x monoclonal antibody (KM93, 1:500 dilution; Chemicon, Temecula, CA), anti-6-sulfo sialyl Lewis x monoclonal antibody (G72, 1:10 dilution; kindly presented by Dr. Reiji Kannnagi, Aichi Cancer Center, Department of Molecular Pathology, Nagoya, Japan), anti-sulfo Lewis a monoclonal antibody (F2, 1:5 dilution; Monosan, Uden, The Netherlands), anti-selectin P ligand (SELPLG) monoclonal antibody (PL2, 1:5 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), anti-podocalyxin-like (PODXL) polyclonal antibody (K-19, 1:5 dilution; Santa Cruz Biotechnology), anti-mucosal addressin cell adhesion molecule-1 (MADCAM1) polyclonal antibody (K-19, 1:5 dilution; Santa Cruz Biotechnology), and anti-chondroitin sulfate proteoglycan 2 (CSPG2) monoclonal antibody (2B1, 1:20 dilution; Seikagaku Corporation, Tokyo, Japan). The specificity of the antibodies was confirmed by immunostaining on positive control slides (uterine cervix and placenta). The irrelevant control isotype-matched mouse or goat immunoglobulin (Santa Cruz Biotechnology) was used to exclude the background staining. The section for immunostaining with anti-6-sialyl sulfo Lewis x antibody was subjected to microwave pretreatment for antigen retrieval in citrate buffer solution (Dako ChemMate; Dako, Kyoto, Japan) for 5 min twice before immersion in 3% hydrogen peroxide. For the detection of the endometrial endothelium and endometrial NK cells, serial sections were stained with anti-human CD34 monoclonal antibody (Nu-4A1; Nichirei Corporation, Tokyo, Japan) and anti-human CD56 (1B6; Nichirei Corporation) monoclonal antibody, respectively.

After being washed in PBS three times, sections were incubated with a streptavidin-biotin complex detection kit (LSAB+ kit; Dako). Sections were washed and developed with diaminobenzidine (Dako), rinsed in distilled water, and counterstained with hematoxylin (Nakarai Tesque).

Under a light microscope (Olympus, Tokyo, Japan), the sections were observed by two persons blinded to the study design. The immunostaining intensity for selectin L ligands in the endometrial endothelium was evaluated and semiquantitatively scored as three levels: 0 = no staining, 1 = faint staining, 2 = distinct staining. Evaluation was done with respect to the vessels identified by CD34 staining (range 7-s2236). The semiquantitative score (= the sum of the immunostaining intensity levels/the number of the identified vessels) was calculated. The number of endometrial NK cells was counted in 10 nonoverlapping stromal areas (x400 magnifications) selected randomly, as previously described [6].

Reverse Transcription-Polymerase Chain Reaction

A portion of some endometrial samples (n = 9; 3 in the proliferative phase, two each in the early secretory phase, midsecretory phase, and late secretory phase) were homogenized in a TRIzol reagent (Invitrogen, Carlsbad, CA). The RNA fraction was isolated according to the manufacturer's instructions. Two micrograms of total RNA were converted to cDNA with 1 µg of oligo deoxythymidine primers (Invitrogen) by a reverse transcription kit (Invitrogen) in a final volume of 20 µl. The primers of CSPG2 mRNA isoforms were adopted from a previous study [23]: Primer-A, 5\'-GGCTTTGACCAGTGCGATTAC-3' Primer-B, 5\'-TCAACATCTCATGTTCCTCCC-3' Primer-C, 5\'-TTCTTCACTGTGGGTATAGGTCTA-3'; Primer-D, 5\'-CCAGCCATAGTCACATGTCTC-3'.

Primer-B and -C were paired for detection of V0 splice form (405-bp size amplicon). Primer-A and -C were paired for detection of V1 splice form (336-bp size amplicon). Primer-B and -D were paired for detection of V2 splice form (498-bp size amplicon). Primer-A and -D were paired for detection of V3 splice form (429-bp size amplicon).

One microliter of cDNA solution was amplified with 0.5-µM primer pairs in a final volume 50-µl solution containing Taq DNA polymerase (Invitrogen). Each cycle consisted of 60 sec at 94°C, 45 sec at 60°C, and 90 sec at 72°C. As an internal control, human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, which is known to be constantly expressed in the human endometrium throughout the menstrual cycle [24], was simultaneously amplified with a human GAPDH control amplimer set (BD Biosciences, Franklin Lakes, NJ) under the same PCR condition. PCR products were confirmed by a sequence analysis system AB310 (Applied Biosystems, Tokyo, Japan). Ten microliters of PCR products were subsequently electrophoresed on an agarose gel (Amersham-Pharmacia), stained with ethidium bromide (Nakarai Tesque), and verified as a band on the UV transilluminator (Funakoshi, Tokyo, Japan). Amplicon of total RNA without reverse transcription was used for a negative control. Human placental tissue was used for a positive control [23].

Statistics

The comparison among the groups was done with one-way ANOVA with the post hoc Sheffe F-test. The correlation between number of CD56+ cells and the semiquantitative score of the immunostaining intensity in endometrial endothelium for selectin L ligands was analyzed by the Spearman correlation coefficient by a rank test. A P value less than 0.05 was regarded as significantly different.

RESULTS

Surface Expression of Selectin L on Freshly Isolated PB and Endometrial NK Cells

PB NK cells—CD3(–) CD56(+) lymphocytes—made up 8.5%–17.4% of PB lymphocytes (n = 24), which was similar to the result of our previous study [12]; 80%–90% of PB NK cells were CD16(+) NK cells with some CD16(–) NK cells remaining. Selectin L was expressed brightly on most PB CD16(–) NK cells, whereas it was expressed dimly on only a part of PB CD16(+) NK cells and non-NK lymphocytes (Fig. 1A). The expression rate of selectin L was significantly higher (P < 0.0001) on PB CD16(–) NK cells (91.0% ± 3.5%, mean ±SD, n = 24) than on either PB CD16(+) NK cells (23.5% ± 11.2%) or PB non-NK lymphocytes (35.0% ± 6.9%) (Fig. 1B). There was no significant difference (P = 0.57) in the expression rate of selectin L on PB CD16(–) NK cells among the patients who underwent hysterectomy (89.8% ± 3.3%, n = 7), the volunteer women (91.4% ± 3.7%, n = 11), or the volunteer men (91.8% ± 3.5%, n = 6). There was no significant menstrual cycle-dependent fluctuation (P = 0.86) in the expression rate of selectin L on PB CD16(–) NK cells in the same individual in the group of volunteer women (91.0% ± 4.7% in the proliferative phase and 90.7% ± 4.4% in the secretory phase, n = 7). Similarly, there was no significant difference (P = 0.78) in the expression rate of selectin L on PB CD16(+) NK cells among the patients who underwent hysterectomy (24.4% ± 11.1%, n = 7), the volunteer women (21.7% ± 10.1%, n = 11), and the volunteer men (25.7% ± 14.7%, n = 6). There was no significant menstrual cycle-dependent fluctuation (P = 0.81) in the expression rate of selectin L on PB CD16(+) NK cells in the same individual in the group of volunteer women (29.0% ± 22.2% in the proliferative phase and 30.9% ± 20.2% in the secretory phase, n = 7).


Figure 1
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  		FIG. 1. Surface expression of selectin L on PB lymphocytes. PB lymphocytes were divided into NK cells—CD3(–) CD56(+) lymphocytes—and non-NK cells. NK cells were further subdivided into CD16(–) and CD16(+) subsets. A) Representative histogram of selectin L expression on PB CD16(–) NK cells, CD16(+) NK cells, and non-NK lymphocytes. Solid lines indicate the lymphocytes stained with anti-selectin L antibody. Broken lines indicate the lymphocytes stained with control mouse IgG. B) The mean ± SD of expression rate of selectin L on PB CD16(–) NK cells, CD16(+) NK cells, and non-NK lymphocytes

Localization of Selectin L Ligands in Human Endometrium

Using immunohistochemistry, we examined the localization of eight selectin L ligands in the human endometrium. The immunostaining for sulfo Lewis a (Fig. 2A) was not detected in any endometrial samples examined but was detected in the uterine cervical epithelium (Fig. 2A, inset), as previously described [25]. Similarly, SELPLG was not detected in the endometrium (data not shown). The immunostaining for sialyl Lewis x (Fig. 2B), sialyl Lewis a (Fig. 2C), and 6-sulfo sialyl Lewis x (Fig. 2D) was detected in the surface epithelium and glandular epithelium but not in the stroma (including endothelial cells). In the epithelium, the immunoreactivity showed a membranous and heterogeneous pattern. There was no marked menstrual cycle-dependent fluctuation in the immunostaining intensity in these selectin L ligands. In contrast, the immunostaining for PODXL (Fig. 2E–H), MADCAM-1 (Fig. 2I–L), and CSPG2 (Fig. 2M–P) was detected in the surface epithelium, glandular epithelium, and stroma.


Figure 2
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  		FIG. 2. Localization of selectin L ligands in the human endometrium. The photos indicate the immunohistochemistry for sulfo Lewis a (A), sialyl Lewis x (B), sialyl Lewis a (C), 6-sulfo sialyl Lewis x (D), PODXL (E–H), MADCAM1 (I–L), and CSPG2 (M–P). The photos indicate the endometrium in the proliferative phase (E, I, and M), in the early secretory phase (F, J, and N), in the mid-secretory phase (A–D, G, K, and O), and in the late secretory phase (H, L, and P), respectively. The inset in A indicates the immunostaining in the human uterine cervical epithelium (a positive control). Bold arrows indicate the distinct staining in the endometrial endothelium. Thin arrows indicate the faint staining in the endometrial endothelium. Broken arrows indicate absence of staining in the endometrial endothelium. Bars = 100 µm

The immunoreactivity for PODXL was membranous, cytoplasmic, and heterogeneous in the epithelium. The immunostaining intensity was generally weaker in the stroma than in the epithelium. Few endothelial cells were stained in the proliferative phase and the early secretory phase (Fig. 2, E and F), whereas most of the endothelial cells identified by CD34 staining in serial sections were stained faintly in the mid- to late secretory phase (Fig. 2, G and H).

The immunoreactivity for MADCAM1 was membranous, cytoplasmic, and homogeneous in the epithelium (Fig. 2I–L). The immunostaining intensity was generally weaker in the stroma than in the epithelium but exceptionally distinct in the endothelial cells in the late secretory phase (Fig. 2L). The endothelial cells were stained faintly in the proliferative phase and the early to mid-secretory phase (Fig. 2I–K). The immunostaining in the epithelial cells was evident in both the apical and the basal site.

The immunoreactivity for CSPG2 was membranous, cytoplasmic, and homogeneous in the epithelium. The immunostaining intensity in the stroma was equivalent to that in the epithelium, which elevated from the proliferative phase to the secretory phase (Fig. 2M–P). Consistent with this, the immunoreactivity in the endothelial cells was faint in the proliferative phase (Fig. 2M) but distinct in the secretory phase (Fig. 2N–P), which peaked in the mid- to late secretory phase (Fig. 2, O and P). The immunostaining in the epithelial cells was evident in both the apical and the basal site.

Semiquantitative Scoring of Immunostaining Intensity in the Endometrial Endothelium for Selectin L Ligands and Its Correlation with the Number of Endometrial NK Cells

Figure 3 shows the results of the semiquantitative scoring of the immunostaining intensity in the endometrial endothelium for PODXL, MADCAM1, and CSPG2. Throughout the menstrual cycle, the score for PODXL was relatively low compared with the scores for MADCAM1 and CSPG2. The score for MADCAM1 was significantly higher in the late secretory phase than in the other phases (P < 0.0001). The scores for CSPG2 were significantly higher in the early, mid-, and late secretory phase than in the proliferative phase (P < 0.0001).


Figure 3
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  		FIG. 3. Semiquantitative scoring of immunostaining intensity in the endometrial endothelium for selectin L ligands. A) Menstrual cycle-dependent fluctuation of semiquantitative scores (mean ± SD) of immunostaining intensity in the endometrial endothelium for PODXL, MADCAM1, and CSPG2. B) Correlation of semiquantitative scores of immunostaining intensity in the endometrial endothelium for PODXL, MADCAM1, and CSPG2 with the number of endometrial NK cells in 10 stromal high-power fields

The mean number of endometrial NK cells in 10 nonoverlapping stromal areas (x400 magnifications) was 29.5 in the proliferative phase (n = 9), 66.5 in the early secretory phase (n = 7), 117.25 in the midsecretory phase (n = 8), and 139.5 in the late secretory phase (n = 6). Figure 3B shows the relationship between the number of endometrial NK cells and the semiquantitative scores in the endometrial endothelium for PODXL, MADCAM1, and CSPG2 in each endometrial sample. The number of endometrial NK cells showed a moderate positive correlation with the scores for PODXL (P < 0.001, rs = 0.792) and MADCAM1 (P < 0.001, rs = 0.842) and a strong positive correlation with the score for CSPG2 (P < 0.001, rs = 0.902).

Expression of CSPG2 mRNA Isoforms in Human Endometrium

Three isoforms of CSPG2 mRNA (V1, V2, and V3) can be generated by alternative splicing of V0 isoform [23]. Of these four, V0, V1, and V3 isoforms were expressed in the human endometrium in all samples examined, whereas V2 isoform was not detected (Fig. 4).


Figure 4
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  		FIG. 4. Expression of CSPG2 mRNA isoforms (V0, V1, V2, and V3) in the human endometrium in the proliferative (P) and secretory (S) phases. GAPDH mRNA was used an internal control in the PCR analysis. Human placental tissue was used as a positive control

DISCUSSION

We confirmed that surface expression of selectin L was constantly higher on human PB CD16(–) NK cells than that on either PB CD16(+) NK cells or non-NK lymphocytes, which was consistent with previous findings [4]. In the same individual, there was no significant menstrual cycle-dependent fluctuation in the expression rate of selectin L on PB CD16(–) NK cells, suggesting that selectin L expression on these NK cells is independent of the effect of the ovarian steroids.

Of eight selectin L ligands examined, we detected six selectin L ligands in the human endometrium, whereas the immunostaining for sulfo Lewis a, which is expressed in several human mucosal tissues including uterine cervical epithelial cells [25], and that for SELPEG, which is expressed on the surface of the leukocytes or bone marrow-derived cells [26], was not detected in the human endometrium at any stage of the menstrual cycle.

Immunostaining was detected for sialyl Lewis a, sialyl Lewis x, and 6-sulfo sialyl Lewis x in the human endometrial epithelium but not in the endometrial stroma including endothelium [19, 20]. The 6-sulfo sialyl Lewis x is a selectin L ligand expressed on the high endothelial venules in the human peripheral lymph nodes [27], where CD16(–) NK cells are enriched [28, 29]. The presence of 6-sulfo sialyl Lewis x in the peripheral lymph node vessels and its absence in the endometrial vessels indicates a difference in the mechanism underlying the recruitment of PB CD16(–) NK cells between these two types of vessels. Recent studies showed that the interaction between selectin L on trophoblasts and selectin L ligands, especially 6-sulfo sialyl Lewis x, on endometrial epithelium plays a critical role in embryo implantation in humans [21]. We confirmed that sialyl Lewis a, sialyl Lewis x, and 6-sulfo sialyl Lewis x are expressed in the human endometrial epithelium but not in the endometrial endothelium. Judging by their localization, these sialyl Lewis antigens may be involved in the embryo attachment but not in the recruitment of PB CD16(–) NK cells into the human endometrium. It is likely that embryo and lymphocytes do not recognize shared L-selectin ligands in the process of adhesion in their own elements.

On the other hand, immunostaining for PODXL [30], MADCAM1 [31], and CSPG2 [32, 33] was detected in the endometrial endothelium. PODXL is a sialomucin expressed on high endothelial venules in peripheral lymph nodes. MADCAM1 is an immunoglobulin superfamily adhesion molecule whose main ligand is integrin {alpha}47ß [34], but MADCAM1 also binds to selectin L via a mucin-like domain [31]. CSPG2 is a proteoglycan that binds to selectin L using its specific glycosaminoglycan chains [33].

By semiquantitative scoring, the immunostaining intensity for PODXL in the endometrial endothelium was relatively low compared with that for MADCAM1 and CSPG2. The immunostaining intensity in the endometrial endothelium for MADCAM1 was higher in the late secretory phase than in the other phases, whereas that for CSPG2 was high throughout the secretory phase. Judging by the menstrual cycle-dependent fluctuation pattern of these three selectin L ligands, CSPG2 is likely to be more compatible with the menstrual cycle-dependent fluctuation pattern of endometrial CD16(–) NK cells. This assumption is supported by the correlation between the number of endometrial NK cells and the semiquantitative score. Three active isoforms of gene transcripts for CSPG2 were found in the endometrium. Interestingly, CSPG2 was shown to bind to and inactivate several chemokines, such as CXCL10, CXCL12, and CCL5 [35], which are expressed in the human endometrium or decidua [7, 11, 36]. Although the recombinant proteins of these chemokines show chemotactic activity for PB CD16(–) NK cells in an in vitro migration assay [4], the endogenous protein in the human endometrium of these chemokines had low chemotactic activity if any for PB CD16(–) NK cells [11]. This may result from CSPG2 binding and inactivation of these chemokines. Recent work in T cells suggests that chemokine receptor trapping at the immunological synapse impedes the distractive signaling by other chemokines [37]. PB CD16(–) NK cells may also select effective chemoattractive signaling in the course of the interaction with endothelial cells.

In the secretory phase, circulating blood passes through the helical flow in the spiral arteries in the functional layer of the endometrium. The vascular wall shear stress in the curved blood flow is over twice as large as that in the straight blood flow [38]. Another study suggested that the adhesive property of selectin L increases with the increase in shear stress [39]. Under the shear stress in the sequential curved flow in endometrial spiral arteries, PB CD16(–) NK cells, which highly express selectin L on their surface, may become more adhesive to the endometrial endothelium. Such an unusual hemodynamic microenvironment may also contribute to selectin L-dependent selective recruitment of PB CD16(–) NK cells into secretory phase endometrium. Since human endometrium contains a small population of lymphocytes other than CD16(–) NK cells, these lymphocytes may also migrate using selectin-L-dependent system.

In this study, we characterized the localization of selectin L ligands in the human cycling endometrium. Three selectin L ligands (PODXL, MADCAM1, and CSPG2) were detected in the endometrial endothelium. Surface expression of selectin L on PB CD16(–) NK cells was constantly high throughout the menstrual cycle. The interaction between selectin L ligands on the endometrial endothelium and selectin L on PB CD16(–) NK cells may function in the selective recruitment of these NK cells into the endometrium.

FOOTNOTES

1 Supported by the Grand-in-Aid for Scientific Research (No. 16790964) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Back

2 Correspondence: Kotaro Kitaya, Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan. FAX: 81 75 212 1265; kitaya{at}koto.kpu-m.ac.jp Back

Received: 25 July 2005.

First decision: 22 August 2005.

Accepted: 7 September 2005.

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