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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-1742
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 5 2835-2842
Copyright © 2005 by The Endocrine Society

Menstrual Cycle Hormones Induce Changes in Functional Interactions between Lymphocytes and Decidual Vascular Endothelial Cells

Marianne J. van den Heuvel, Julie Horrocks, Siamak Bashar, Suzanne Taylor, Suzanne Burke, Kota Hatta, Jennifer E. Lewis and B. Anne Croy

Department of Pediatrics (M.J.v.d.H.), University of Western Ontario, London, Ontario, Canada, N6C 2V5; Departments of Mathematics and Statistics (J.H.) and Biomedical Sciences (S.Ba., S.T., S.Bu., K.H., J.E.L.), University of Guelph, Guelph, Ontario, Canada N1G 2W1; and Department of Anatomy and Cell Biology (B.A.C.), Queen’s University, Kingston, Ontario, Canada K7L 3N6

Address all correspondence and requests for reprints to: Dr. M. J. van den Heuvel, Department of Pediatrics, Child Health Research Institute, 800 Commissioner’s Road East, University of Western Ontario, London, Ontario, Canada, N6C 2V5. E-mail: mvandenh{at}uwo.ca.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
During the secretory phase of the menstrual cycle, a natural killer (NK) cell subset expressing cluster of differentiation (CD)56bright appears in the decidualizing uterus and remains until onset of menses. If pregnancy occurs, decidual NK cells increase to become the predominant uterine lymphocytes of early pregnancy. To elucidate mechanisms of CD56bright cell recruitment to the uterus, an in vitro adhesion assay was used to assess the effect of the menstrual cycle, as well as cycle-associated hormones on adhesive properties of human lymphocytes. Adhesion of human peripheral blood lymphocytes to pregnant mouse lymph nodes and Peyer’s Patches tissue sections was constant throughout the cycle. When uterine tissue was used as the substrate, adhesive CD56+ cells were found only in decidua basalis. Adhesion increased at the LH surge. Adhesion was mediated through both L-selectin and {alpha}4-integrin-dependent mechanisms. Furthermore, we observed increased adhesive function in CD56+ cells from male donors which had been cultured with estradiol or LH compared with cell aliquots cultured without additives. Lymphocytes adherent to mouse uterine tissue were predominantly CD56bright, suggesting that peripheral NK cells may be actively recruited to the uterus in an important, brief endocrine-regulated fashion at the time of ovulation to establish the decidual NK population of early pregnancy.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
UTERINE IMMUNE CELL populations undergo remarkable changes during the course of each ovulatory cycle. Leukocytes constitute 5% of uterine stromal cells in the proliferative phase of the cycle but increase to 25% of total cells in the late secretory phase, primarily as a result of a large influx of natural killer (NK) cells (1, 2). This migration is concurrent with decidualization, a process whereby endometrial fibroblasts transform, under the influences of estrogen and progesterone (P4), into decidual cells (3, 4). In decidualized tissue, 70% of lymphocytes are CD56bright, CD16null, a phenotype which constitutes less than 1% of circulating blood lymphocytes and about 10% of total peripheral NK cells, which are predominantly CD56dim, CD16+ (5). CD56bright cells are detectable in the uterus 2–3 d after the LH surge, until LH+12 d, then undergo apoptosis before the onset of menses. Loss of decidual (d)NK cells has been attributed to declining P4 levels (3). In the event of pregnancy, dNK cells increase rapidly and distribute broadly throughout decidua. As the fetus develops, dNK cells are found in close proximity to fetally derived extravillous trophoblast invading the decidua (3). This suggests dNK cells have roles in implantation and establishment of the placenta.

Murine uterine (u)NK cells are derived from circulating precursors. The iv infusion of cell suspensions derived from any lymphoid organ of wild-type mice reconstitutes uNK cells in impregnated, alymphoid mice, with maximal reconstitution from splenocytes of pregnant donors (6). Lymphocyte homing depends on a multi-step cascade of interactions with vascular endothelial cells and occurs in response to conditions such as fever, inflammation, wounding, or infection. This process is well described in secondary lymphoid tissues (7, 8). Initial rolling and tethering of circulating lymphocytes is mediated by transient binding of L-selectin and {alpha}4ß7-integrin homing receptors expressed on lymphocytes to endothelial ligands [peripheral node addressin (PNAd) and mucosal addressin cell adhesion molecule-1 (MadCAM-1), respectively] on specialized high endothelial venules (HEV). Extravasation is mediated by chemokine signals from the site of injury or inflammation. The binding of chemokines to ligands expressed on lymphocyte surfaces stimulates G protein-dependent activation of leukocyte function assisted molecule-1 (LFA-1), which results in firm adhesion to its endothelial ligand intercellular adhesion molecule-1 (ICAM-1). The final step is transendothelial migration (9).

Evidence that L-selectin and {alpha}4-integrin may orchestrate lymphocyte trafficking to decidualized mouse uterus is accumulating. In previous studies, we demonstrated dynamic, pregnancy-associated endothelial surface changes in murine lymphoid and decidualized uterine tissues but not in other tissues. These changes were recognized by L-selectin and/or {alpha}4-integrin receptors on human lymphocytes (10). When the mouse tissue donor was ovariectomized and treated with either 17ß-estradiol (E2) or P4, gains in adhesion were particularly pronounced on decidual substrate. Complementary gains in adhesive capacity were found in the lymphocytes from these pregnant or hormone-treated mice compared with virgin or sham-treated ovariectomized controls. This suggests independent, but coordinated, hormonal regulation of adhesion pathways in decidual endothelium and lymphocytes (6, 10). At gestation day (gd) 8 of murine pregnancy, Kruse et al. (11, 12) identified {alpha}4-integrin+, LFA-1+ NK cells in decidua basalis (DB), close to vascular cell adhesion molecule-1 (VCAM-1) expressing endothelium. VCAM-1, an alternate ligand for {alpha}4-integrin, is up-regulated by proinflammatory cytokines such as TNF{alpha} and IL-1, in concert with transcription factors such as the interferon (IFN)-related factor 1 in vascular endothelium (13, 14) and IFN-related factor 2 in muscle (15). Human CD56+ dNK cells express {alpha}4-integrin and LFA-1, whereas the {alpha}4-integrin ligand, VCAM-1, is expressed on decidual blood vessels in the human uterus (16, 17, 18). Both E2 and P4 are reported to stabilize expression of VCAM-1, ICAM-1, and E-selectin in primary vascular endothelial cell cultures (19, 20). E2 is also reported to directly stimulate VCAM-1 expression by endothelial cells in culture (21). Although these effects would enhance extravasation of lymphocytes, they are insufficient to induce specific homing of pre-dNK cells to the uterus because lymphocytes require specific chemotactic signals to activate the homing receptors expressed constitutively on their surfaces.

The present study was undertaken to define the effects of the normal hormonal fluctuations during the menstrual cycle on functional interactions between human lymphocytes and endothelium. Because our previous experiments used human lymphocytes purchased in quantity and the current study required serial donations from specific donors, we optimized the adhesion assay for use with smaller lymphocyte samples. Using this optimized protocol, we found that the functional ability of human blood lymphocytes, and the CD56bright subset in particular, to interact with uterine endothelium was dynamically altered at the LH surge or in association with high physiological levels of E2 and LH.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Human subjects and blood sampling

Healthy male volunteers of legal age donated either 15 ml, thrice, over several weeks [cohort A (n = 4)] or up to 50 ml blood once [cohort D (n = 4)]. Nonpregnant female subjects of legal and reproductive age (23–45 yr), having regular menstrual cycles between 26 and 33 d in length, using no form of hormonal birth control for at least 1 yr and in good health were recruited to donate serial blood samples. Based on the experiment for which their blood was used, they were divided into two cohorts, B (n = 7) and C (n = 12). All subjects were informed about the risks of participation in this study and provided an informed written consent (Ethics Committee, Office of Research, University of Guelph). About half of the female donors were multiparous (n = 3, cohort B; and n = 6, cohort C). The others had never attempted to become pregnant. Female donors provided either 25 ml (cohort B) or 20 ml (cohort C) venous blood in sterile blood collection tubes containing the anticoagulant acid citrate dextrose (ACD), which was immediately layered onto an equal volume of warmed [room temperature (RT)] Histopaque 1.077 (Sigma, Mississauga, Ontario, Canada) and centrifuged (400 x g; 30 min, at RT). The lymphocytes at the Histopaque/plasma interface were collected, washed thrice, counted, and adjusted to 2.5 x 107 cells/ml in 37 C RPMI 1640 (Sigma) with no additives.

Mice and tissue dissections

C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME), 7–8 wk old, were used for timed matings, with the morning of the copulation plug designated gd 0. Nonpregnant controls were virgin females who had never been paired with males. All procedures were performed under approved animal use protocols (Animal Care Committee, University of Guelph). Virgin mice and mice at gd 6–8 were euthanized, and the following tissues were collected: a pool of 10–12 lymph nodes (LN) from sc and intermuscular sites, a pool of 10–12 Peyer’s Patches (PP), and uteri. Implantation sites from pregnant uteri were trimmed and cut in the median sagittal plane and placed cut-face-down into biopsy cryomolds (Fisher Scientific, Mississauga, Ontario, Canada). All tissues were embedded in OCT (Miles Laboratories, Fisher Scientific), flash frozen in liquid nitrogen-cooled isopentane, and stored at –20 C. Tissues were used within 2 wk of collection.

Antibodies

For assays involving LN or uterine tissue substrates, cell aliquots were incubated (30 min, at RT) with function-blocking monoclonal antibody specific for human L-selectin (CD62L; BD PharMingen, Mississauga, Ontario, Canada). When PP was used as the tissue substrate, lymphocyte aliquots were pretreated (30 min, at RT) with a function-blocking antibody to {alpha}4-integrin (HP2/1; Coulter Immunology, Hialeah, FL; 10 µg/ml) (see Ref.23). After treatment, cells were washed in RPMI 1640 and readjusted to 5 x 107 cells/ml. In some experiments, aliquots of 5 x 106 lymphocytes were prelabeled with CD56-phycoerythrin (PE) (Immunotech, Beckman Coulter, Mississauga, Ontario, Canada) to detect NK cells at 1:100 for 20 min at RT, then washed with RPMI 1640 and resuspended in 100 µl. Preliminary assessment of labeled and unlabeled cells in bright-field microscopy indicated that no change in adhesion occurred due to labeling (data not shown). In some experiments, endothelial cells in frozen mouse tissues were labeled with 100 µl of 1:100 dilution Alexa488-conjugated isolectin from griffonia simplicifolia (Molecular Probes, Burlington, Ontario, Canada) (22).

To characterize populations in the starting cell suspension before application onto frozen sections, flow cytometric analysis was conducted using the following panel of mouse antihuman antibodies (Caltag Laboratories, Burlingame, CA): CD4 R-PE-8 fluorescein isothiocyanate (FITC) (1:100), CD19 FITC (1:10), CD33-4D3 R-PE (1:10), CD34 FITC (1:10) and isotype controls; mouse IgG2a FITC/R-PE (1:100), mouse IgG1 FITC (1:10), mouse IgG1 R-PE (1:10). To detect NK cells, CD56-PE (Immunotech, Beckman-Coulter) (1:100) was used. All antibody dilutions were optimized in preliminary studies (data not shown).

Flow cytometry

Cells suspended in PBS were labeled for 30 min at RT, then washed twice with PBS containing 2% BSA and 0.01% Na azide. Analyses of cell data were performed on a FACScan using CellQuest software (Becton Dickinson, San Jose, CA) immediately after the last wash, without the use of fixative. The lymphocyte gate was defined by forward and side scatter properties (data not shown).

Assay of cell adhesion under shear to frozen tissue sections

Twelve-micron sections of frozen mouse tissue on cover slips were placed on rubber stoppers on a table, rotating at 80 rpm in a cold chamber, and immediately overlain with 100 µl of either untreated human lymphocytes or lymphocytes treated with antibody to L-selectin, {alpha}4-integrin and/or prelabeled with CD56-PE. Rotation was increased to 112 rpm and continued for 30 min at 4 C. After incubation, slides were gently rinsed in PBS, then fixed 30 min in 3% glutaraldehyde at 4 C or, if fluorescent labeling was done, in 2% paraformaldehyde for 20 min at 4 C, then rinsed in PBS for 10 min, then in ddH2O and mounted with AquaPolymount (Polysciences, Inc., Washington, PA). The sections were assessed for lymphocyte adhesion by microscopy. Two hundred HEV were counted per blind encoded slide of LN or PP or 50 high-power fields (HPF, x400) in uterine tissues by two independent researchers.

Optimization of experimental procedures

Preliminary experiments were performed to determine the lowest number of cells necessary to detect significant differences in lymphocyte adhesion between untreated and prelabeled cells. Replicate experiments used 107, 5 x 106, 2.5 x 106, and 106 cells per 100 µl of test suspension from four different male donors (cohort A). These cells were applied to sections of LN from both virgin and pregnant animals in a standard adhesion assay. One aliquot of cells was pretreated with anti-CD62L to block L-selectin-mediated adhesion.

To assess the effects of anticoagulants on adhesive properties of isolated peripheral blood lymphocytes (PBL), 10 ml blood/subject was collected from cohort A into each of four different anticoagulants. These were ACD, EDTA, sodium citrate, and heparin. Lymphocytes were isolated and used simultaneously as described above.

Because fever range temperature promotes adhesion of PBL (23), the effect of blood storage temperature on the adhesive properties of PBL was assessed. Whole-blood samples (collected in ACD) from each member of cohort A were exposed to the following conditions: 1) 4 h at 20 C, 2) 4 h at 37 C, 3) 4 h at 4 C, or 4) 3.5 h at 4 C, then warmed back to 37 C for 30 min. Lymphocytes were isolated as described above. Control lymphocytes came from freshly drawn blood taken from the same donor 4 h after the initial 40-ml donation.

Effect of the menstrual cycle on adhesion of human PBL

Seven female volunteers (cohort B) were recruited to donate 25 ml blood thrice weekly through an entire menstrual cycle (12 samples collected per subject). Subjects maintained basal body temperature charts to monitor their cycles. White blood cells were isolated and 2.5 x 106 cells, either untreated or pretreated with anti-CD62L or anti-HP2/1, were applied to frozen tissue sections of LN and PP from virgin and pregnant (d 8) mice. The women started their donations together, on various cycle days. At completion of the experiment, they submitted their temperatures charts. All cycles were normalized by arbitrarily setting the day of temperature rise as cycle d 14. Results were correlated with the phase of the cycle as indicated by temperature shift (indicating that ovulation had occurred) and confirmed by analysis of individual serum hormone levels (moderate estrogen and low P4 levels indicate proliferative phase, and high levels of P4 and estrogen indicate the secretory phase).

Role of LH surge in determining level of adhesion

Data from the menstrual cycle study indicated that the periovulatory period was a time of change in lymphocyte-endothelial interaction. Therefore, cohort C women were recruited to donate blood on d 8, day of LH surge, and d 20 of their cycles. In addition to basal body temperature charts, cohort C used a commercial LH detection kit (Clearplan; Novartis, Mississauga, Ontario, Canada) from d 11 of their cycle until the LH surge. Two cell aliquots were prepared from each sample for use in adhesion assays, one not labeled and one pretreated with anti-CD62L. Similarly, two aliquots of cells were prepared from a second group of six subjects; both aliquots were labeled with anti-CD56-PE, and one was also pretreated with anti-CD62L. Serum from each of these samples was assayed by ELISA for E2 (DRG Instruments, Biophase Diagnostic Laboratories, Mississauga, Ontario, Canada), LH, and P4 concentrations (Biocheck, Inc., Burlingame, CA).

Role of ovarian steroids in stimulating adhesive ability in CD56bright cells

To determine the effect of ovarian steroids on adhesive capability of CD56bright cells and to avoid endogenous female hormone effects on NK cell adhesiveness, male donors from cohort D were recruited to donate up to 50 ml venous blood. Freshly isolated lymphocytes were adjusted to 7 x 106 cells/ml serum-free RPMI 1640. One milliliter of cell suspension was placed in individual wells of a 12-well plate containing 1 ml serum-free RPMI 1640 containing either no additives, E2 (Sigma), P4 (Sigma), or LH (Dr. A. F. Parlow, National Hormone and Peptide Program, Torrance, CA), such that the final concentrations were E2: 100, 200, 400, 800 pg/ml; P4: 1, 4, 8, 16 ng/ml; and LH: 10, 50, 100, 200 ng/ml. After 4 h of culture at 37 C, 5% CO2, cells were aspirated, washed with serum-free 37 C RPMI 1640, and labeled with anti-CD56-PE as above. Cell numbers were adjusted to 5 x 107 cells/ml, and 100 µl of labeled cells was used immediately in an adhesion assay.

Statistical analyses

Results depict the mean ± SEM. To analyze experimental effects of cell number, blocking, temperature, or anticoagulant on lymphocyte adhesion, either a one-way or two-way ANOVA was used, as indicated in figure legends. Further comparison of means was conducted using Bonferroni’s or Tukey’s procedure to adjust for multiple comparisons. We used Bonferroni when a fixed number of comparisons were made, and Tukey’s procedure to adjust for all pair-wise comparisons. Because some data points were missing in the time course study, due to loss of tissue substrate during processing, the mixed linear model (a generalization of repeated-measures ANOVA) was used to analyze the time course studies to test for differences over time and between conditions (untreated cells vs. function-blocking antibody) as well as for associations between adhesion and hormone concentrations. A post hoc paired t test was used to determine whether mean adhesion in the periovulatory period was different from the rest of the cycle. Analyses were performed using SigmaStat and SAS. All analyses passed the Kolmogorov-Smirnoff test for normality (with Lillefor’s correction) as performed by SigmaStat (Point Richmond, CA).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Adhesion substrate

Because frozen murine tissues were used to assess adhesion of human lymphocytes as a model of trafficking, it was important to establish the adhesive partner cell in the tissue. Figure 1Go illustrates adhesion of fluorescently labeled CD56+ cells and endothelial cells. It is apparent that the NK cells selectively adhere to vascular endothelium specific to the DB, because adhesion was nonexistent in nondecidualized areas of the same tissue.



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  		FIG. 1. Photomicrograph of results of Stamper-Woodruff adhesion assay. Human lymphocytes (5 x 106 in 100 µl) were prelabeled with anti-CD56-PE, then applied to decidua from a C57BL/6J mouse at d 7 of gestation under rotation, to mimic shear forces in blood vessels. Murine endothelium was labeled with Alexa 488 conjugated isolectin (bright green). Nonadherent cells were rinsed off; the tissues were fixed and mounted. The number of CD56+ cells (bright red cells) per HPF were counted (x400). Size bar, 50 µm.

 
Reliability of assay

A limited number of lymphocytes can be purified from 25 ml blood (~1–10 x 107). Therefore, a dose response study was conducted to determine the lowest number of cells that could be used per tissue section while still retaining statistical differences between untreated cells and cells treated with function-blocking antibody to L-selectin. Results are shown in Fig. 2Go. A two-way ANOVA showed a significant interaction between the presence of blocking antibody and number of lymphocytes applied. We subsequently tested for significant differences between the blocked and unblocked conditions at each of the six lymphocyte concentrations. Using Bonferroni’s procedure to adjust for multiple comparisons, a P value < 0.00833 on an individual test is required to show statistical significance, to maintain an overall type-I error rate of 0.05. The minimum number of applied lymphocytes with which a significant increase in adhesion between untreated cells and cells pretreated with antibody to L-selectin could be detected was 2.5 x 106 cells per tissue section (P = 0.0033). Differences between normal and blocked cells at concentrations of 5 x 106 and 107 cells were likewise significant (P < 0.0001). Thus 2.5 x 106 cells were used in each adhesion assay throughout the study, permitting more experimental conditions using a single blood sample.



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  		FIG. 2. Adhesion outcomes (number of adherent lymphocytes) after application of varying numbers of cells from the same donors to constant tissue (LN from a pregnant mouse) within single experiments. Solid bars depict the mean number of adherent cells ± SE when 105–107 lymphocytes were applied to frozen sections of LN. Each solid bar is paired with an open bar, which represents the mean number of adherent lymphocytes pretreated with a function-blocking antibody to L-selectin. The minimum number of applied lymphocytes required to detect a significant difference from control cells was determined to be 2.5 x 106 (dark bar). *, Significant difference in adhesion between untreated and treated cells applied in equal number.

 
The effect of the anticoagulant on the adhesive properties of the isolated lymphocytes was investigated with four anticoagulants for each of three subjects (data not shown). Although lymphocytes isolated from blood containing ACD, the anticoagulant used in leukopacks employed in our previous studies, appeared superior in adhesion to lymphocytes isolated from sodium citrate, EDTA, or heparin, the difference was not significant (one-way ANOVA). ACD was selected as the anticoagulant for these experiments.

The results of the temperature assay were analyzed by one-way ANOVA, which found significant differences between the various treatments (P = 0.01). Bonferroni’s procedure, which requires P < 0.0125 to maintain an overall significance level of 0.05, was used to determine which treatments differed from fresh lymphocytes (data not shown). Holding whole blood at 37 C results in adhesive ability significantly greater than the freshly isolated lymphocytes (P = 0.0048). Storage at RT has no effect on adhesion (P = 0.8106), whereas chilling blood reduces the effectiveness of the lymphocytes isolated from it to recognize and bind to ligands expressed on mouse endothelium, although the difference is not significant (P = 0.6720). Chilled blood that is then rewarmed to 37 C demonstrates a rebound in adhesion; but with a Bonferroni adjustment, this is not significantly different from fresh lymphocytes (P = 0.0311). Thus cells were kept warm after their collection.

Effect of the menstrual cycle on functional adhesive properties of lymphocytes

Lymphocytes from cohort B women appeared to increase in adhesiveness on LN or PP from virgin mice or pregnant mice at d 10–12 of the cycle (data not shown). However, when data from this periovulatory period (d 10–12 of the cycle, day of temperature shift, which occurs post ovulation, was set as d 14) were pooled and compared with the rest of the cycle, human lymphocytes collected at the periovulatory phase were not found to be more adherent to LN (P = 0.0529, t test) than those from the proliferative and luteal phases of the menstrual cycle. Treatment of the lymphocytes with function-blocking antibodies to L-selectin or {alpha}4-integrin resulted in a significant reduction in adhesion (P < 0.0005, mixed linear model F test, data not shown), which was constant over the menstrual cycle, regardless of the tissue used.

This experiment indicated that the periovulatory period was of potential interest, so LH detection was incorporated into our protocol to focus on the effect of the LH surge on the modification of lymphocyte-endothelial interactions. Lymphocytes from women in cohort C, collected at cycle d 8, day of LH surge, and cycle d 20 were applied either untreated, blocked, labeled with anti-CD56-PE, or labeled and blocked, to LN and uteri from pregnant mice only. Mean adhesion of blood lymphocytes and CD56bright cells to LN peaked at periovulation, but there was no change in mean adhesion (data not shown). When uterine tissue was used as the substrate, a significant increase in both lymphocyte (Fig. 3AGo) and CD56bright cell (Fig. 3BGo) adhesion was observed on the day of LH surge compared with proliferative (d 8) and midsecretory (d 20) lymphocytes (P = 0.0004 and P = 0.0092, mixed linear model, F test). Blocking with anti-L-selectin resulted in a significant decline in adhesion at all time points (P = 0.0004, mixed linear model, F test), but did not completely eliminate adhesion of CD56+ cells, indicating that other adhesion molecules are involved in mediating lymphocyte-endothelial adherence. The LH-associated gain in adhesion to uterus was L-selectin-dependent because there was no change in adhesion in the blocked samples at the LH surge compared with other time points.



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  		FIG. 3. Adhesion of lymphocytes from three time points in the menstrual cycle to LN and uterus from pregnant B6 mice. Solid bars represent the mean number of adherent lymphocytes ± SE (A) or CD56+ cells (B) per HPF in decidua from gd 6–8 B6 mice. Open bars show adhesion of lymphocytes pretreated with a function-blocking antibody to L-selectin (n = 6), which was significantly less than the untreated cells (P = 0.0004, mixed linear model, F test). *, Significant increase in adhesion of untreated cells at LH compared with untreated cells at cycle d 8 and 20 as determined by a mixed linear model, P = 0.0004 (A) and P = 0.0092 (B).

 
Cytometric analysis of PBL

The histogram of Fig. 4Go summarizes mean percentages (±SE) of CD4, CD8, CD19, CD56dim, and CD56bright cells within the lymphocyte gate during the monitored cycles of cohort C donors. There was considerable variation in lymphocyte subset percentages between subjects, and inconsistent variations from subject to subject over the course of the cycle, with some subjects showing an increase in the luteal phase, some a decrease, and others no change. Thus we found no significant changes in lymphocyte populations over the course of a menstrual cycle.



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  		FIG. 4. The relative percentages of lymphocyte subsets over the course of a menstrual cycle. As analyzed by flow cytometry, the numbers of CD4, CD8, CD19, CD33, and CD56dim cells remained stable. Each bar represents the mean of three to six samples.

 
Hormonal profile of subjects

Serum concentrations of E2, LH, and P4 for cohort C donors are summarized as mean ± SE in Table 1Go. The ELISA kits provided expected reference values for phases of the menstrual cycle as shown next to each of the means of the measured values. All subjects were within the normal range of hormone values for each phase of the cycle, with the exception of two subjects who had high preovulatory levels of P4 and one subject who had consistently low levels of P4.


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  		TABLE 1. Serum hormone concentrations in cycling blood donors (n = 6)

 
To assess whether the hormone concentrations were correlated with adhesion to LN and uterus, a statistical mixed linear model was applied. Estrogen was significantly associated with adhesion to uterus (P = 0.0149) but not LN. No association between P4 and adhesion in either uterus or LN was found. Finally, LH was significantly associated with adhesion in uterus (P = 0.0002) and in LN (P = 0.0186).

Effect of reproductive hormones on adhesion of CD56+ cells

To address directly the effects of cycle hormones on lymphocyte adhesive function, NK cells from four male donors of cohort D were cultured in RPMI 1640 media with no additives, RPMI 1640 with E2, RPMI 1640 with P4 or RPMI 1640 with LH. Figure 5Go summarizes these experiments. Adhesion of CD56-expressing cells increased after culture, compared with adhesion of fresh cells. Cells cultured with hormones, especially E2 and LH but also low levels of P4, demonstrated greater adherence to vascular endothelium than did cells cultured without hormone additives (Fig. 5Go). Furthermore, flow cytometric analysis of cells, before and after culture, showed a consistent and dramatic increase in CD56 expression in all samples, regardless of additives to culture media (not shown).



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  		FIG. 5. Adhesion of cultured lymphocytes of male donors (n = 4) to uterine endothelium. Peripheral blood lymphocytes were cultured with concentrations of E2 varying from 0–800 pg/ml (A), P4 varying from 0–8 ng/ml (B), LH varying from 0–200 ng/ml (C), or LH varying from 0–200 ng/ml with 800 pg/ml E2 (D) for 4 h before assay for adhesion to frozen sections of mouse uterus. The gray bar in D shows the effect of 800 pg/ml alone on adhesion (mean ± SEM), without added LH. Data points represent mean values ± SE. Open circles denote the mean number of adherent CD56+ cells pretreated with anti-L-selectin; closed circles show mean adhesion of untreated NK cells. *, Increased adhesion relative to control sample with no added hormones, P < 0.005 (one-way ANOVA); #, increased adhesion relative to control sample with no added hormones, P < 0.05 (one way ANOVA); {diamondsuit}, differs from samples pretreated with antibody to L-selectin P < 0.008 (one-way ANOVA).

 
There was an approximately linear relationship between adhesion and E2 concentration (P = 0.0071, mixed linear model) (Fig. 5AGo). For every 1-pg/ml increase in E2, there is an accompanying increase of 0.038 adherent cells per HPF. There was also an increase in adhesion between P4 levels of 0 and 2 ng/ml (P = 0.0022, mixed linear model) (Fig. 5BGo). Specifically, adhesion is estimated to increase by 15.8 cells per HPF for every nanogram increase in P4. At P4 levels greater than 2 ng/ml, there is a significant decreasing relationship between adhesion and P4 (P = 0.0403, mixed linear model). In particular, adhesion is estimated to decrease by 3.6 cells per HPF for every 1 nanogram increase in P4. There was an increasing relationship of LH with adhesion (P = 0.0055) from 0–100 ng/ml (Fig. 5CGo). LH concentrations higher than 100 ng/ml did not induce further increases in adhesion; rather, adhesion plateaued. Finally, there was no additive effect of combining various dosages of LH with 800 pg/ml E2 (Fig. 5DGo).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The highly proliferative and abundant population of CD56bright, CD16dim NK cells that inhabits the human uterus within days after the LH surge is thought to arise from uterine recruitment of circulating blood and in situ proliferation (2, 24). Here, we describe significant increases in adhesion of CD56bright blood lymphocytes taken at the LH surge of fertile females, to L-selectin ligands in decidualized mouse uterus but not to ligands in either LN or PP, indicating a tissue-specific effect. Furthermore, lymphocytes from male donors exhibited enhanced adhesive capacity after incubation with E2, low preovulatory levels of P4, or LH compared with cell aliquots cultured without additives.

Blood CD56bright cells constitute less than 1% of peripheral blood lymphocytes and differ in function from the CD56dim subset by producing greater amounts of IFN-{gamma}, TNF-ß, granulocyte macrophage-colony stimulating factor (GM-CSF), IL-10, and IL-13 when stimulated by IL-12, 15, 18, or phorbol 12-myristate 13-acetate (25) and by exhibiting reduced natural cytotoxicity in the unstimulated state (26, 27). The uNK cell populations differ from peripheral blood subsets in several species (2, 4, 28), particularly in terms of gene transcription (29). In primates, dNK cells express the phenotype and share the cytokine and comparatively nonlytic characteristics of peripheral CD56bright, CD16null cells (28, 30, 31). It is attractive to postulate that a subset of blood CD56bright cells homes to the uterus and undergoes terminal differentiation in response to uterine stromal factors such as IL-15 (5, 32, 33). We speculate that major differences between blood and dNK cells can be attributed to these uterine events, because circulating NK cells are immature.

Migration of leukocytes to inflammatory sites is induced by chemokines, secreted by cells at the site, which bind to seven-transmembrane G protein couples receptors (34). Hormonal effects on expression of chemokines and their receptors have been described; E2 induces increased production of inflammatory chemokines in dendritic cells and monocytes (35). Administration of exogenous estrogens during fertility treatment results in increased numbers of dNK cells and in glandular IL-8/CXCL8 production (36). P4 induces monokine induced by interferon {gamma}/CXCL9, whereas E2 inhibits its production (37). Investigations of menstrual cycle effects on production of chemokines or their ligands reveal up-regulation of endometrial transcripts for CXCR1, CCR5, CCR2B and, to a lesser extent, CXCR4 in the luteal phase (38) and increased expression of CXCL10 (39) and CXCL9 but not CXCL11 or CXCL12 (40) in decidua. Circulating CD56bright cells express high levels of CXCR3, the ligand for CXCL9, 10, and 11 and moderate levels of CXCR4, the ligand for CXCL12, and demonstrate migration in response to these signals (41). Thus this subset is well-equipped to respond to cyclic signals from dendritic and stromal cells in the uterus.

Here, we examined the effect of the menstrual cycle on the functional adhesiveness of peripheral blood lymphocytes, the source of dNK cell progenitors suggested from mouse models. Mouse, rather than human, uterine tissue was used as the substrate to eliminate differences in stage of cycle in the uterus and because the assay requires use of relatively fresh substrate tissue (less than 2 wk of storage at –20 C). We found significant enhancement of adhesive capacity of lymphocytes only at LH surge and only when decidua was used as substrate. When only CD56bright cells were enumerated, it was apparent that they constituted the majority of adherent cells and thus could be preferentially recruited. Because antibody-blocked lymphocytes showed no alteration in adhesion, any changes in adhesive capacity were mediated by activation of L-selectin, presumably by circulating factors.

Direct response of CD56bright cells to menstrual cycle hormones is debatable; neither rat nor human lymphocytes express {alpha}-forms of the receptor for estrogen (ER) or P4 (42, 43, 44, 45), but mouse NK cells do (46, 47, 48). It is reported that dNK cells express ERß1, but these are noncirculating, terminally differentiated cells (49). Our findings of a positive relationship between estrogen and adhesion to decidua, a stronger association between LH levels and adhesion, and a biphasic effect of P4 are suggestive of complex hormonal influences in trafficking potential. It appears that rising E2 before the LH surge creates a trafficking window enabling CD56bright cell egress from circulation. After ovulation has occurred, predominantly high P4 levels close the window, and CD56bright cells cease trafficking. A critical role for E2 on NK cell trafficking to the uterus is unlikely because a study of mice genetically devoid of ER{alpha} or ERß displays no alteration in recruitment of uNK cells to the pregnant uterus (50). It has been reported that lymphocytes of pregnant women, as well as of women in the luteal phase of the menstrual cycle, express the LH receptor but at lower levels than found in ovarian tissue (51). Therefore, it is quite plausible that peripheral blood lymphocytes are directly activated during the LH surge.

The enrichment of CD56bright NK cells in the uterine environment is well documented (1, 2, 52). However, the means by which this enrichment occurs is still not understood. Decidua contains no HEV, the usual site of lymphocyte trafficking to lymphoid tissues. Due to frequent histological observations of mouse NK cells transiting decidual arteries, we speculate that these arteries, which thin and elongate as decidualization progresses, are the site of NK cell invasion.

Menstrual cycle variation has also been described in human blood NK cell lytic function (53, 54, 55) and in number of NK cells per milliliter of blood (55). A significant increase in the number of CD56+ cells at the periovulatory period reported for 39 women between the ages of 20 and 29 was postulated to be due to LH and not to E2, P4, or testosterone (55). We observed larger differences in NK cell populations between individuals in our study group than within individuals. Although there appeared to be an increase in the percentage of CD56bright subset (relative to the total lymphocyte population) at LH surge in our cohort B of seven patients between the ages of 25 and 38, the difference was not significant. We did not enumerate the total NK cells per milliliter blood.

Although it is attractive to postulate that adhesion is representative of NK cell recruitment to the decidualizing uterus, this is difficult to prove in women. Loss of this NK cell population with each menstrual cycle indicates that renewal occurs. It is generally accepted, and these data support the hypothesis, that lymphocyte recruitment is from the peripheral blood. It would appear from our work that either a threshold level of E2 or a surge of LH activates adhesion molecules on the dNK precursor cell surface, enabling a single wave of NK cells or precursor cells to leave the circulation and enter the uterus. This is consistent with a model in which NK cells localize to the DB at LH+3, proliferate until approximately LH+12, then start to disappear, as previously described (1, 2). Furthermore, modulation of NK cell lytic activity by LH and ß human chorionic gonadotropin at physiological levels has been reported, suggesting a direct interaction between the NK cell and the polypeptide hormones (54). Complete understanding of the migration and role of dNK cells is expected to have broad clinical impact in women’s reproductive health areas such as infertility, gestational complications (i.e. recurrent spontaneous abortion, preeclampsia), and pathologies such as endometriosis.


   Acknowledgments
 
We thank our subjects for their cooperation throughout this study and for their generosity of time and spirit. Dr. A. F. Parlow of the National Hormone and Peptide Program (Torrance, CA) kindly provided LH for culture experiments. We are grateful to Dr. Sharon Evans (Buffalo, NY) for helpful discussions.


   Footnotes
 
This work was supported by awards from Natural Sciences and Engineering Council, Canada, Ontario Ministry of Agriculture, Food and Rural Affairs. M.J.v.d.H. is supported by the Ontario Women’s Health Postdoctoral Scholar Award.

First Published Online February 1, 2005

Abbreviations: ACD, Acid citrate dextrose; d, decidual; CD, cluster of differentiation; DB, decidua basalis; E2, 17ß-estradiol; ER, estrogen receptor; FITC, fluorescein isothiocyanate; gd, gestation day; HEV, high endothelial venules; HPF, high-power field(s); ICAM-1, intercellular adhesion molecule-1; IFN, interferon; LFA-1, leukocyte function associated molecule-1; LN, lymph nodes; MadCAM-1, mucosal addressin cell adhesion molecule-1; NK, natural killer; P4, progesterone; PBL, peripheral blood lymphocytes; PE, phycoerythrin; PNAd, peripheral node addressin; PP, Peyer’s Patches; RT, room temperature; u, uterine; VCAM-1, vascular cell adhesion molecule-1.

Received August 31, 2004.

Accepted January 21, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. King A, Burrows T, Verma S, Hiby S, Loke YW 1998 Human uterine lymphocytes. Hum Reprod Update 4:480–485[Abstract/Free Full Text]
  2. Bulmer JN, Morrison L, Longfellow M, Ritson A, Pace D 1991 Granulated lymphocytes in human endometrium: histochemical and immunohistochemical studies. Hum Reprod 6:791–798[Abstract/Free Full Text]
  3. King A 2000 Uterine leukocytes and decidualization. Hum Reprod Update 6:28–36[Abstract/Free Full Text]
  4. Hunt JS, Petroff MG, Burnett TG 2000 Uterine leukocytes: key players in pregnancy. Semin Cell Dev Biol 11:127–137[CrossRef][Medline]
  5. Loke YW, King A 2000 Immunological aspects of human implantation. J Reprod Fertil Suppl 55:83–90[Medline]
  6. Chantakru S, Miller C, Roach LE, Kuziel WA, Maeda N, Wang WC, Evans SS, Croy BA 2002 Contributions from self-renewal and trafficking to the uterine NK cell population of early pregnancy. J Immunol 168:22–28[Abstract/Free Full Text]
  7. Springer TA 1995 Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu Rev Physiol 57:827–872[CrossRef][Medline]
  8. Butcher EC, Williams M, Youngman K, Rott L, Briskin M 1999 Lymphocyte trafficking and regional immunity. Adv Immunol 72:209–253[Medline]
  9. Butcher EC, Picker LJ 1996 Lymphocyte homing and homeostasis. Science 272:60–66[Abstract]
  10. Chantakru S, Wang WC, van den Heuvel M, Bashar S, Simpson A, Chen Q, Croy BA, Evans SS 2003 Coordinate regulation of lymphocyte-endothelial interactions by pregnancy-associated hormones. J Immunol 171:1132–1145
  11. Kruse A, Merchant MJ, Hallmann R, Butcher EC 1999 Evidence of specialized leukocyte-vascular homing interactions at the maternal/fetal interface. Eur J Immunol 29:1116–1126[CrossRef][Medline]
  12. Kruse A, Martens N, Fernekorn U, Hallmann R, Butcher EC 2002 Alterations in the expression of homing-associated molecules at the maternal/fetal interface during the course of pregnancy. Biol Reprod 66:333–345[Abstract/Free Full Text]
  13. Lechleitner S, Gille J, Johnson DR, Petzelbauer P 1998 Interferon enhances tumor necrosis factor-induced vascular cell adhesion molecule 1 (CD106) expression in human endothelial cells by an interferon-related factor 1-dependent pathway. J Exp Med 187:2023–2030[Abstract/Free Full Text]
  14. Ochi H, Masuda J, Gimbrone MA 2002 Hyperosmotic stimuli inhibit VCAM-1 expression in cultured endothelial cells via effects on interferon regulatory factor-1 expression and activity. Eur J Immunol 32:1821–1831[CrossRef][Medline]
  15. Jesse TL, LaChance R, Iademarco MF, Dean DC 1998 Interferon regulatory factor-2 is a transcriptional activator in muscle where It regulates expression of vascular cell adhesion molecule-1. J Cell Biol 140:1265–1276[Abstract/Free Full Text]
  16. Haynes MK, Wapner RL, Jackson LG, Smith JB 1997 Phenotypic analysis of adhesion molecules in first-trimester decidual tissue from chorion villus samples. Am J Reprod Immunol 38:423–430
  17. Ruck P, Marzusch K, Kaiserling E, Horny HP, Dietl J, Geiselhart A, Handgretinger R, Redman CW 1994 Distribution of cell adhesion molecules in decidua of early human pregnancy. An immunohistochemical study. Lab Invest 71:94–101[Medline]
  18. Burrows TD, King A, Loke YW 1995 The role of integrins in adhesion of decidual NK cells to extracellular matrix and decidual stromal cells. Cell Immunol 166:53–61[CrossRef][Medline]
  19. Cid MC, Kleinman HK, Grant DS, Schnaper HW, Fauci AS, Hoffman GS 1994 Estradiol enhances leukocyte binding to tumor necrosis factor (TNF)-stimulated endothelial cells via an increase in TNF-induced adhesion molecules E-selectin, intercellular adhesion molecule type 1, and vascular cell adhesion molecule type 1. J Clin Invest 93:17–25
  20. Aziz KE, Wakefield D 1996 Modulation of endothelial cell expression of ICAM-1, E-selectin, and VCAM-1 by ß-estradiol, progesterone, and dexamethasone. Cell Immunol 167:79–85[CrossRef][Medline]
  21. Winkler M, Kemp B, Hauptmann S, Rath W 1997 Parturition: steroids, prostaglandin E2, and expression of adhesion molecules by endothelial cells. Obstet Gynecol 89:398–402[CrossRef][Medline]
  22. Sahagun G, Moore SA, Fabry Z, Schelper RL, Hart MN 1989 Purification of murine endothelial cell cultures by flow cytometry using fluorescein-labeled griffonia simplicifolia agglutinin. Am J Pathol 134:1227–1232[Abstract]
  23. Frey M, Packianathan NB, Fehniger TA, Ross ME, Wang WC, Stewart CC, Caligiuri MA, Evans SS 1998 Differential expression and function of L-selectin on CD56bright and CD56dim natural killer cell subsets. J Immunol 161:400–408[Abstract/Free Full Text]
  24. King A, Balendran N, Wooding P, Carter NP, Loke YW 1991 CD3– leukocytes present in the human uterus during early placentation: phenotypic and morphologic characterization of the CD56++ population. Dev Immunol 1:169–190[Medline]
  25. Cooper MA, Fehniger TA, Turner SC, et al 2001 Human natural killer cells: a unique innate immunoregulatory role for the CD56(bright) subset. Blood 97:3146–3151[Abstract/Free Full Text]
  26. Nagler A, Lanier LL, Cwirla S, Phillips JH 1989 Comparative studies of human FcRIII-positive and negative natural killer cells. J Immunol 143:3183–3191[Abstract]
  27. Ellis TM, Fisher RI 1989 Functional heterogeneity of Leu 19"bright"+ and Leu 19"dim"+ lymphokine-activated killer cells. J Immunol 142:2949–2954[Abstract]
  28. Slukvin II, Watkins DI, Golos TG 2001 Phenotypic and functional characterization of rhesus monkey decidual lymphocytes: rhesus decidual large granular lymphocytes express CD56 and have cytolytic activity. J Reprod Immunol 50:57–79[CrossRef][Medline]
  29. Koopman LA, Kopcow HD, Rybalov B, Boyson JE, Orange JS, Schatz F, Masch R, Lockwood CJ, Schachter AD, Park PJ, Strominger JL 2003 Human decidual natural killer cells are a unique NK cell subset with immunomodulatory potential. J Exp Med 198:1201–1212[Abstract/Free Full Text]
  30. Jokhi PP, King A, Loke YW 1997 Cytokine production and cytokine receptor expression by cells of the human first trimester placental-uterine interface. Cytokine 9:126–137[CrossRef][Medline]
  31. Saito S, Nishikawa K, Morii T, Enomoto M, Narita N, Motoyoshi K, Ichijo M 1993 Cytokine production by CD16-CD56bright natural killer cells in the human early pregnancy decidua. Int Immunol 5:559–563[Abstract/Free Full Text]
  32. Kitaya K, Yasuda J, Yagi I, Tada Y, Fushiki S, Honjo H 2003 IL-15 expression at human endometrium and decidua. Biol Reprod 63:683–687
  33. Verma S, Hiby SE, Loke YW, King A 2000 Human decidual natural killer cells express the receptor for and respond to the cytokine interleukin 15. Biol Reprod 62:959–968[Abstract/Free Full Text]
  34. Kim CH, Broxmeyer HE 1999 Chemokines: signal lamps for trafficking of T and B cells for development and effector function. J Leukoc Biol 65:6–15[Abstract]
  35. Bengtsson AK, Ryan EJ, Giordano D, Magaletti DM, Clark EA 2004 17ß-Estradiol (E2) modulates cytokine and chemokine expression in human monocyte-derived dendritic cells. Blood 104:1404–1410[Abstract/Free Full Text]
  36. DeLoia JA, Stewart-Akers AM, Brekosky J, Kubik CJ 2002 Effects of exogenous estrogen on uterine leukocyte recruitment. Fertil Steril 77:548–554[CrossRef][Medline]
  37. Daikoku N, Kitaya K, Nakayama T, Fushiki S, Honjo H 2004 Expression of macrophage inflammatory protein-3ß in human endometrium throughout the menstrual cycle. Fertil Steril 81(Suppl 1):876–881
  38. Dominguez F, Galan A, Martin JJ, Remohi J, Pellicer A, Simon C 2003 Hormonal and embryonic regulation of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in the human endometrium and the human blastocyst. Mol Hum Reprod 9:189–198[Abstract/Free Full Text]
  39. Kai K, Nasu K, Nakamura S, Fukuda J, Nishida M, Miyakawa I 2002 Expression of interferon-{gamma}-inducible protein-10 in human endometrial stromal cells. Mol Hum Reprod 8:176–180[Abstract/Free Full Text]
  40. Kitaya K, Nakayama T, Daikoku N, Fushiki S, Honjo H 2004 Spatial and temporal expression of ligands for CXCR3 and CXCR4 in human endometrium. J Clin Endocrinol Metab 89:2470–2476[Abstract/Free Full Text]
  41. Campbell JJ, Qin S, Unutmaz D, Soler D, Murphy KE, Hodge MR, Wu L, Butcher EC 2001 Unique subpopulations of CD56+ NK and NK-T peripheral blood lymphocytes identified by chemokine receptor expression repertoire. J Immunol 166:6477–6482[Abstract/Free Full Text]
  42. Stewart JA, Bulmer JN, Murdoch AP 1998 Endometrial leucocytes: expression of steroid hormone receptors. J Clin Pathol 51:121–126[Abstract]
  43. Mansour I, Reznikoff-Etievant MF, Netter A 1994 No evidence for the expression of the progesterone receptor on peripheral blood lymphocytes during pregnancy. Hum Reprod 9:1546–1549[Abstract/Free Full Text]
  44. Ogle TF, Dai D, George P, Mahesh VB 1997 Stromal cell progesterone and estrogen receptors during proliferation and regression of the decidua basalis in the pregnant rat. Biol Reprod 57:495–506[Abstract]
  45. King A, Gardner L, Loke YW 1996 Evaluation of oestrogen and progesterone receptor expression in uterine mucosal lymphocytes. Hum Reprod 11:1079–1082[Abstract/Free Full Text]
  46. Curran EM, Berghaus LJ, Vernetti NJ, Saporita AJ, Lubahn DB, Estes DM 2001 Natural killer cells express estrogen receptor-{alpha} and estrogen receptor-ß and can respond to estrogen via a non-estrogen receptor-{alpha}-mediated pathway. Cell Immunol 214:12–20[CrossRef][Medline]
  47. Sakazaki H, Ueno H, Nakamuro K 2002 Estrogen receptor {alpha} in mouse splenic lymphocytes: possible involvement in immunity. Toxicol Lett 133:221–229[CrossRef][Medline]
  48. van den Heuvel M, McBey BA, Hahnel AC, Croy BA 1996 An analysis of the uterine lymphocyte-derived hybridoma cell line GWM 1–2 for expression of receptors for estrogen, progesterone and interleukin 2. J Reprod Immunol 31:37–50[CrossRef][Medline]
  49. Henderson TA, Saunders PT, Moffett-King A, Groome NP, Critchley HO 2003 Steroid receptor expression in uterine natural killer cells. J Clin Endocrinol Metab 88:440–449[Abstract/Free Full Text]
  50. Borzychowski AM, Chantakru S, Minhas K, Paffaro VA, Yamada AT, He H, Korach KS, Croy BA 2003 Functional analysis of murine uterine natural killer cells genetically devoid of oestrogen receptors. Placenta 24:403–411[CrossRef][Medline]
  51. Lin J, Lojun S, Lei ZM, Wu WX, Peiner SC, Rao CV 1995 Lymphocytes from pregnant women express human chorionic gonadotropin/luteinizing hormone receptor gene. Mol Cell Endocrinol 111:R13–R17
  52. Flynn L, Byrne B, Carton J, Kelehan P, O’Herlihy C, O’Farrelly C 2000 Menstrual cycle dependent fluctuations in NK and T-lymphocyte subsets from non-pregnant human endometrium. Am J Reprod Immunol 43:209–217
  53. Sulke AN, Jones DB, Wood PJ 1985 Variation in natural killer activity in peripheral blood during the menstrual cycle. Br Med J (Clin Res Ed) 290:884–886
  54. Sulke AN, Jones DB, Wood PJ 1985 Hormonal modulation of human natural killer cell activity in vitro. J Reprod Immunol 7:105–110[CrossRef][Medline]
  55. Yovel G, Shakhar K, Ben-Eliyahu S 2001 The effects of sex, menstrual cycle, and oral contraceptives on the number and activity of natural killer cells. Gynecol Oncol 81:254–262[CrossRef][Medline]



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