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Originally published online as doi:10.1189/jlb.0308164 on July 15, 2008

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(Journal of Leukocyte Biology. 2008;84:1065-1074.)
© 2008 by Society for Leukocyte Biology

CD56bright cells increase expression of {alpha}4 integrin at ovulation in fertile cycles

Crystal G. Peralta*, Victor K. Han*,{dagger}, Julie Horrocks{ddagger}, B. Anne Croy§ and Marianne J. van den Heuvel{dagger},1

Departments of
* Anatomy and Cell Biology and
{dagger} Pediatrics, University of Western Ontario, London, Ontario, Canada;
{ddagger} Mathematics and Statistics, University of Guelph, Guelph, Ontario, Canada; and
§ Anatomy and Cell Biology, Queens University, Kingston, Ontario, Canada

1 Corresopndence: Department of Pediatrics, University of Western Ontario, 4910 Line 36, R R 5, Stratford, ON, Canada N5A 6S6. E-mail: mheuvel{at}quadro.net


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ABSTRACT
 
Leukocyte content of human endometrium changes rapidly after ovulation, particularly as a result of gains in CD56bright uterine NK (uNK) cells. We have proposed that uNK precursor cells are found within the blood CD56bright pool and are recruited to decidualizing endometrium through functional changes in their adhesion molecules and chemokine receptors. This study sought to quantify alterations in adhesion molecules, cytokines, chemokines, and receptors induced in circulating CD56+ cells of fertile and infertile women by ovulation. Blood was drawn from 12 fertile volunteers and six female-infertility patients at Menstrual Cycle Day (d) 5 and on the day following the preovulatory surge of luteinizing hormone (LH). CD56bright, CD56dim, and CD56+CD3+ cell subsets were isolated and evaluated by flow cytometry, quantitative PCR, or Western blotting. In CD56bright cells from fertile but not infertile women, {alpha}4 integrin increased between d5 and the preovulatory LH surge. CD56dim and NKT cells did not show a change in {alpha}4 integrin but differed significantly between fertile and infertile donors, and infertile donors had reduced homing molecule expression in CD56dim and NKT cells, and at ovulation, their NKT cells showed elevated cytokine production. None of the circulating CD56+ cell subsets had transcripts for receptors for estrogen, progesterone, LH, or prolactin. Thus, immunological events associated with the LH surge induce alterations in all subsets of CD56+ cells, and the unique induction of {alpha}4 integrin in CD56bright cells of fertile women constitutes a potential method to promote uterine homing.

Key Words: human • NK cells • adhesion molecules • chemokine receptors • reproductive immunology


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INTRODUCTION
 
A dramatic shift in human uterine lymphocyte subsets occurs immediately after ovulation. Over the 12 days that follow the ovulation-inducing surge in luteinizing hormone (LH) and under the influence of estrogen and progesterone, the stromal cells of the endometrium develop into plump, secretory cells known as decidua. This tissue becomes increasingly enriched in CD56+CD16–CD3– uterine NK (uNK) cells and then depleted prior to the onset of menses [1 ]. In the event of conception and implantation at 8–10 days postovulation [2 ], uNK cell numbers increase further and peak toward the end of the pregnancy’s first trimester [3 ]. The functions of uNK cells in human decidua are not well defined, but reduced numbers are associated with implantation failure [4 ], early spontaneous abortion [5 ], and intrauterine growth restriction [6 ]. In mouse models, uNK cells secrete angiogenic factors and contribute to maintenance of endometrial decidua but do not appear to influence implantation [7 8 9 ].

Although uNK cells are highly proliferative in early decidua, the origin of their progenitors remains unclear. Two nonmutually exclusive pathways could contribute to the cyclic, hormone-driven appearances of uNK cells. These are endometrial in situ differentiation [10 ] or homing of progenitors or precursors from blood [11 ]. Our investigations focus on the latter possibility. In humans, most blood NK cells express CD56 weakly and CD16 at moderate levels (CD56dimCD16+) [12 , 13 ]. Human uNK cells are distinguished from them by tenfold higher expression of CD56, lack of CD16, limited lytic ability, high angiogenic potential, and expression of killer Ig-like receptors (KIR) [9 , 14 , 15 ]. A minor blood NK cell subset comprising less than 10% of all NK cells expresses tenfold higher CD56 (CD56bright) but lacks expression of CD16, CD69, and KIR. Although circulating NK cells display minor differences in gene transcription, uNK cells differ significantly from the blood subsets in global gene transcription [16 ], perhaps reflecting terminal differentiation of homed NK cells, which differentiate from CD34dim CD45RA {alpha}4β7hi bone marrow-derived hematopoietic stem cells (HSC). CD34dim CD45RA {alpha}4β7hi cells are highly enriched in follicular areas of lymph node relative to blood and under IL-15 stimulation, become CD56bright CD16neg [17 , 18 ]. This subset is also highly enriched in inflamed tissues such as pleural infections, autoimmune arthritis, and pancreas [19 , 20 ]. However, as cultured blood CD56bright cells become CD56dim in the presence of IL-2 [21 ], and CD56dim cells gain CD56 in the presence of IL-12 [22 ] or TGF-β [23 ], some plasticity is present in the developmental potential of both CD56+ cell subsets.

HSC expressing CD56 and CD122 have been identified in suspensions of human endometrium [24 ] but were not localized to basal tissue that would be retained at menses. In mouse models, transplantable progenitors of uNK cells occur in all lymphoid organs but not in uterine segments [11 ]. In adhesion assays using frozen sections of decidua from mice from Gestation Days 5–15 as substrate and human NK cells or an indicator cell line as adherent cells, we found that the decidual substrate became progressively more adherent up to Gestation Day 11, the time at which the NK cell population in the mouse uterus peaks and starts to decline [25 ]. In humans, a recruitment mechanism to populate the decidua with NK cells is supported by changes, which are attractive to lymphocytes, to endometrial stroma during the progesterone-dominant, secretory phase of the menstrual cycle [26 27 28 29 ]. Adhesive function of the CD56bright cell subset but not other CD56+ cells increases in an L-selectin and {alpha}4 integrin-dependent manner within a 3-day period following the preovulatory LH surge [30 ]. This functional alteration was also found in CD56bright cells of infertile women who had successful embryo transfers during fertility treatment but was not found in women who did not achieve pregnancy, demonstrating that hormonal treatment did not alter adhesive function of this subset [31 ]. This suggests that synchronous changes having the potential for uterine recruitment align between some circulating lymphocytes and decidualizing endometrium [31 ].

Three blood CD56+ cell subsets are known (CD56bright cells, CD56dim NK cells, and CD56+CD3+ NKT cells), and each uniquely displays an array of chemokine receptors, which directs their ability to respond to trafficking signals. CD56bright cells preferentially express CCR5, CCR7, CX3CR1, CXCR3, and CXCR4 and migrate in response to CCR7 ligands (MIP-3β, secondary lymphoid tissue chemokine), CXCR3 ligands [IFN-inducible protein 10 (IP-10), IFN-inducible T cell {alpha} chemoattractant (I-TAC)], and to a lesser extent, CCR5 ligand (RANTES) [13 ]. Among CD56+ cell subsets, CD56bright cells alone express the chemokine receptors necessary for migration into secondary lymphoid tissues [32 ] and decidua. CXCR3’s ligand CXCL11 (I-TAC) is not expressed in cycling human endometrium, and CXCL9 (monokine induced by IFN-{gamma}) and CXCL10 (IP-10) are highly expressed. The latter is induced further in culture, suggesting it may play a major role in CD56bright cell recruitment during the menstrual cycle secretory phase [28 ]. In contrast, CXCL12 (stromal cell-derived factor 1), the ligand for CXCR4, is not detected in cycling endometrium [28 ]. It is strongly expressed by trophoblast cells that invade into decidua in early pregnancy and may direct uNK cell chemotaxis [9 , 33 34 35 36 ]. Here, we ask if normal menstrual cycles alter the expression of trafficking-associated molecules on circulating CD56+ cell subsets from fertile and infertile women.


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MATERIALS AND METHODS
 
Blood donors
With approval from the University of Western Ontario Research Ethics Board for Health Sciences Research Involving Human Subjects, 12 fertile, female volunteers (identified as having had at least one successful pregnancy without medical intervention) between 20 and 40 years of age (mean 32.7 years) with regular menstrual cycles, not using hormonal birth control, and sero-negative for HIV and Hepatitis B and C were recruited for blood donations. Each participant was instructed in the use of ovulation indicators (Ovulation Indicator Sticks, Life Brand Shopper’s Drug Mart, Toronto, Ontario, Canada). Blood was drawn on Cycle Day 5 (d5) and on the day of ovulation (LH), determined as day following LH surge. For flow cytometric analysis, 3 mL blood was collected using EDTA-containing Vacutainer blood collection tubes. For RNA experiments, 20 mL blood was collected using acid citrate dextrose (ACD)-containing Vacutainer tubes. For protein isolation experiments, 250 mL blood was collected using citrate phosphate dextrose adenine-containing blood collection bags. Six additional women (mean age 35.2 years) undergoing ovulation induction for unexplained female infertility (including two patients with repeated implantation failure using donor sperm) were recruited to donate two blood samples of 100 mL (in ACD) at d5 and on the day following, a natural LH surge detected by ELISA (n=3) of sera or therapeutic injection of β human chorionic gonadotrophin (βhCG; n=3). These women, whose pituitary hormones were not suppressed during treatment, attended the clinic daily for monitoring of follicle-stimulating hormone, LH, and estradiol levels and for ultrasonic verification of oocyte development. Hormone therapy was administered on an individualized basis.

NK cell subset isolation
Mononuclear cells were separated from plasma by a 30-min density gradient centrifugation after layering 1:1 onto Histopaque® 1077 (Sigma-Aldrich, Oakville, Ontario, Canada) at 400 g, 4°C. After aspiration and washing (3x10 min, 200 g) in PBS (pH 7.2), the cells were enumerated and resuspended in cold autoMACS running buffer (Miltenyi Biotec, Auburn, CA, USA) at 107 cells/80 µL for separation by an autoMACS cell sorter (Miltenyi Biotec). In a three-step procedure, mononuclear cells were labeled (15 min, 4°C, 20 µL CD56 Multisort MicroBeads per 107 cells), washed, and separated using the positive-selection, double-sensitive (posselds) separation program, and then positive cells were stripped of beads and relabeled with 50 µL CD3 MicroBeads per 107 cells for 15 min at 4°C and then sorted using the positive selection (possel) program. The positive fraction, NKT cells, was held on ice. The negative fraction was washed, counted, resuspended in 50 µL running buffer, and labeled with 50 µL CD16 MicroBeads for 30 min at 4°C. Labeled cells were separated using the possel program, and positive (CD56dimCD16+) and negative (CD56brightCD16–) fractions were pelleted for further use.

Flow cytometry
All antibodies and their isotype controls were purchased from Beckman Coulter (IOTest, Fullerton, CA, USA) and titered to optimize conditions prior to initiation of the study. Isotype control antibodies with the same conjugation were used to set negative gates.

To assess the efficiency of microbead separation of subsets, cell pellets were resuspended in 100 µL PBS and incubated for 15 min at room temperature with 10 µL of a combination of CD16-FITC, CD56-PE, CD3-phycoerythrin (PE)-Texas Reel (ECD), and 5 µL CD45-PC7. After washing, cells were reconstituted in PBS and assessed by cytometry with a Coulter XL-MCL cytometer and analyzed with CXP software (Beckman Coulter).

To assess homing receptor expression, 100 µL EDTA-anticoagulated blood was incubated with 5 µL each anti-CD3-ECD, anti-CD16-PC5, and anti-CD56 PC7 and 5 µL anti-{alpha}4 integrin-FITC and anti-lymphocyte function-associated antigen 1 (LFA-1)-PE or anti-L-selectin-FITC and anti-CXCR4-PE or anti-CXCR3-FITC and anti-CXCR4-PE and incubated 20 min. RBC lysis was performed with ImmunoPrep solution (Beckman Coulter), and cells were washed and resuspended in PBS containing 2% BSA and analyzed as above.

Protein detergent fractionation
Cytosolic and membrane proteins from the collected subsets of NK cells were obtained by detergent fractionation [37 ]. Pellets of CD56bright (2x106 cells/pellet), CD56dim (107 cells/pellet), and NKT (5x106 cells/pellet) cells were suspended in 100 µL ice-cold Digitonin buffer and permeabilized by gentle agitation on ice for 10 min and then centrifuged (10 min, 500 g). Supernatants (cytosolic proteins) were stored at –80°C. The remaining pellet was resuspended in 100 µL ice-cold Triton X-100 extraction buffer and agitated further on ice for 30 min and then centrifuged (10 min, 500 g). The supernatants (membrane-bound proteins) were removed and stored at –80°C. Additional detail is provided in Supplement 1.

Western blotting
A Protein Clean-Up Kit (Amersham Biosciences, Pittsburgh, PA, USA) was used to precipitate membrane and cytosolic proteins and remove detergents, salts, lipids, and nucleic acids according to the manufacturer’s recommendations. Cytosolic and membrane protein samples were quantified in triplicate using the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA), in which samples diluted in 200 µL BCA assay working reagent were incubated for 30 min at 37°C and then assessed for absorbance at 560 and 620 nm (Thermo LabSystems Multiskan Ascent Plate Reader, Thermo LabSystems, Franklin, MA, USA).

Depending on availability, a minimum of 5 µg to a maximum of 10 µg each protein sample was diluted in 1x SDS gel-loading buffer. Heated samples of membrane and cytosolic proteins were loaded into alternate lanes of 12% polyacrylamide gels. Prestained SDS-PAGE standard broad-range ladder (Bio-Rad, Hercules, CA, USA) was loaded in the first and last lanes to confirm transfer, and Magic Mark Western blotting protein standard (Invitrogen, Carlsbad, CA, USA) was used for determination of molecular weight after chemiluminescence. After electrophoreses at 100 V and transfer to polyvinylidene fluoride membranes (Bio-Rad), membranes were equilibrated in 1x TBS and blocked for 2 h in 3% carnation milk in TBS-Tween 20 (0.1%). The blots were then cut in horizontal strips for large, medium, and small protein detection and labeled overnight at 4°C with primary antibodies at optimized dilutions (Table 1 ). After incubation with HRP-conjugated secondary antibodies, proteins were detected by 5 min incubation in SuperSignal® West Femto maximum sensitivity substrate (SuperSignal West Femto, Pierce) and exposure to Kodak scientific imaging film (Eastman Kodak Co., Rochester, NY, USA). Images were taken using Alpha Innotech FluorChemTM 8000 imaging system (Alpha Innotech, San Leandro, CA, USA) for densitometry analysis with FluorChemTM imaging software (Alpha Innotech). Spot volume and density within each band were measured, and then, each result was normalized to its cytosolic or membrane control GAPDH or CD45 by dividing the integrated density volume (IDV) obtained by the Alpha Innotech FluorChem program by the IDV of CD45 or GAPDH to account for differences in loaded amounts.


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  		Table 1. Conditions for Primary and Secondary Antibodies Used for Western Blotting

After chemiluminescent detection, membrane strips were rinsed thrice in 1x TBS-Tween 20 (0.1%) for 10 min each. Blots were stripped with Restore Western Blot stripping buffer (Pierce) for 20 min at room temperature, washed in TBS (3x10 min), assessed for chemiluminescent signal by exposure to a fresh piece of film, and then reprobed with another size-appropriate antibody. Blots were stripped no more than three times.

Extraction of mRNA and RT-PCR
The µMACS mRNA isolation kit was used to extract mRNA from fractionated subsets of human CD56bright and CD56dim subsets according to the manufacturer’s instructions. Briefly, pellets not exceeding 107 cells were lysed with 1 mL lysis-binding buffer, vortexed 3 min, and then centrifuged (3 min, 4000 g). Lysates were applied to lysate clear columns and centrifuged (3 min, 13,000 g). Then, 50 µL Oligo-µdT beads were added to the cleared lysate and applied to prepared MACS Type µ columns in a µMACS separator magnet. Magnetically labeled mRNA was eluted with 120 µL elution buffer warmed to 68°C. RNA was quantified by spectrophotometry at 260 nm (BioPhotometer, Eppendorf, Westbury, NY, USA), and its integrity was assessed by electrophoreses on a 1% agarose gel.

After RT of the mRNA, conventional PCR was used to amplify transcripts of interest as summarized in Table 2 . Primers were designed using Primer 3 software (Whitehead Institute for Biomedical Research, Cambridge, MA, USA). Amplifications started with a 3-min template denaturation step at 94°C, followed by 35 cycles of denaturation for 20 s at 94°C and a combined primer annealing/extension at gene-specific primer temperatures for 30 s. All samples were amplified in triplicate and compared with the housekeeping gene GAPDH. Distilled water, in place of template cDNA, was used as a negative control in all reactions. For analysis of reproductive hormone receptors on NK cell subsets, human ovary and placenta cDNA were used as controls (U.S. Biologicals, Swampscott, MA, USA). PCR products were electrophoresed on a 1% agarose gel, bands excised, and cDNA was purified using QIAquick gel extraction kit (Qiagen, Mississauga, Ontario, Canada), and product identities were confirmed by sequencing (Sequencing Facility, Robarts Research Institute, London, Canada).


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  		Table 2. Primers and Product Features of Genes Studied

Statistical analysis
All data were assessed for normality and constant variance. Outliers (>2 SD) were removed prior to analysis. Western blot densitometry was analyzed by two-way repeated measures ANOVA on ranks using SigmaStat Analysis software (Systat Software Inc., Point Richmond, CA, USA). A P value of <0.05 was considered significant. Subsequent multiple pair-wise comparisons were performed using the Holm-Sidak method.


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RESULTS
 
Blood donors
The fertile group varied in age from 22 to 40, and the infertile group ranged in age from 26 to 40. There was no significant difference between the ages of the two groups of women (P=0.18; Student’s t-test).

Flow cytometric analyses of subsets
An analysis of homing-related proteins by flow cytometry on whole blood samples is summarized in Table 3 as mean percentage (±SEM) of CD56+ cells, which express each marker. Fertile and infertile groups had similar numbers of cells in each lymphocyte subset measured (not shown). The lymphocyte gate was determined by forward- and side-scatter characteristics and by CD45+ expression, and then, CD56-expressing subsets were gated on expression of CD56 and CD16. Next, expression of {alpha}4 integrin and LFA-1, L-selectin and CXCR4, or CXCR3 and CXCR4 was assessed within NK cell subsets. More than 97% of all CD56bright and CD56dim cells expressed LFA-1 and {alpha}4 integrin. In contrast, although >92% of CD56bright cells expressed L-selectin and CXCR3, fewer than 50% of CD56dim cells expressed these molecules. Finally, an average of 13% of CD56bright and <3% of CD56dim cells expressed CXCR4.


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  		Table 3. Summary of Flow Cytometric Assessment of Homing Molecule Expression

Assessment of MACS separation of CD56+ subsets
The degree of subset purity was established in five flow cytometry experiments (Fig. 1 ). Although numbers and proportions of CD56+ cells varied greatly between individual donors, an average recovery of 92% CD56+ cells was achieved in the first separation. Subsequent selection with CD3 microbeads enriched the NKT population to 75–80% purity, with contaminants evenly represented by T cells and NK cells. The remaining NK cells were fractionated by expression of CD16, resulting in populations enriched to 85% CD16+ NK cells. The remaining CD56bright cells, resulting from successive depletions of the starting population rather than a positive selection for a specific marker, were enriched 60-fold, with CD16+ cells constituting the major contaminating cells. We isolated on average 1.78 ± 0.32 x 106 mononuclear cells per mL blood. From this, we isolated an average of 0.83 ± 0.15 x 106 CD56+ cells per mL (<5% of collected lymphocytes) comprised of 0.05 ± 0.01 x 106 CD56bright cells/mL (0.3% of collected lymphocytes), 0.35 ± 0.1 x 106 CD56dim cells/mL (2% of collected lymphocytes), and 0.13 ± 0.04 x 106 NKT cells/mL (0.8% of collected lymphocytes). Thus, the isolation procedure resulted in the loss of ~35% of CD56+ cells.


Figure 1
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  		Figure 1. Quantitative evaluation of separation by flow cytometric analyses. The flow chart depicts sequential separation of three CD56+ subsets by magnetic beads. Subsequent subset purity was assessed by flow cytometry. The images are from one of five experiments done, each with similar results.

Western blotting and densitometry
All CD56bright, CD56dim, and NKT membrane protein samples were assessed for CD45, a stably expressed pan-leukocyte, membrane-bound protein (Fig. 2 ). Cytosolic samples were assessed for GAPDH (Fig. 3 ), a housekeeping protein confined to cytosol. These markers were used as loading controls for each sample as a means of compensating for variation in the total protein used in loading the gel, as 10 µg protein was not available from each sample. Our blots showed that CD45 was found exclusively in the membrane compartment, and GAPDH was found only in the cytosolic compartment, confirming effective separations (top row, right column, in Figs. 2 and 3 , respectively).


Figure 2
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  		Figure 2. Densitometric analyses of Western blots of membrane proteins. Images of Western blot membranes were assessed by Alpha Innotech fluorochrom for IDV of chemiluminescence-tagged antibodies. Within each histogram, the mean IDV from membrane proteins of each of these cell types at d5 (open bars) or the day following LH surge (LH, hatched bars) for fertile (white bars) and infertile (gray bars) is shown. IDV for each protein, LFA-1, {alpha}4 integrin, L-selectin, CXCR3, and CXCR4, was normalized to the amount of CD45 found within that sample. Two-way repeated measures ANOVA with Holm-Sidak post-test for multiple comparisons was used to analyze data (n=6 for each datapoint). A P value of 0.05 was considered significant.


Figure 3
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  		Figure 3. Densitometric analyses of Western blots of cytosolic proteins. Images of Western blot membranes were assessed by Alpha Innotech fluorochrom for IDV of chemiluminescence-tagged antibodies. Within each histogram, the mean IDV from membrane proteins of each of these cell types at d5 (open bars) or the day following LH surge (LH, hatched bars) for fertile (white bars) and infertile (gray bars) is shown. IDV for each protein, TGF-β, TNF-{alpha}, and IFN-{gamma}, was normalized against IDV of GAPDH for each sample. Two-way repeated measures ANOVA with Holm-Sidak post-test for multiple comparisons was used to analyze data (n=6 for each datapoint). A P value of 0.05 was considered significant.

Figure 2 presents plots of the mean ± SEM IDV relative to that of CD45 (shown in top row) from Western blotting. Expression of CX3CR1, CCR5, and CCR7 did not differ significantly between the two time-points or between fertile and infertile women in any of the CD56+ cell subsets, although samples from infertile women had consistently higher values (not shown).

For fertile women, amounts of LFA-1 (Fig. 2 , row 2) and CXCR3 (Fig. 2 , row 5) were not significantly different between d5 and LH surge in CD56bright cells, CD56dim cells, or NKT cells. An increase in expression of the {alpha}4 integrin (P=0.031) was found in CD56bright cells from fertile women at LH (row 3) but did not change at LH in the other two cell subsets. Expression of L-selectin (row 4) did not change between time-points in CD56bright cells or CD56dim cells but was reduced significantly at LH in NKT cells (P=0.007). Expression of CXCR4 (row 6) was unchanged in CD56bright cells and CD56dim cells from fertile women but increased at LH in NKT cells (P=0.018).

In infertile women, expression of LFA-1, {alpha}4-integrin, L-selectin, CXCR3, and CXCR4 was constant between d5 and LH in CD56bright, CD56dim, and NKT cells (Fig. 2 , gray bars). CD56dim and NKT cells differed between fertile and infertile women with more differences at d5 than at ovulation. At d5, less LFA-1 was expressed by infertile than fertile woman (P<0.001) in CD56dim cells, and in NKT cells, LFA-1 expression was elevated in infertile relative to the fertile women (P=0.016). Also at d5, there was significantly less L-selectin in CD56dim cells (P=0.019) and NKT cells (P=0.001) of infertile compared with fertile women. Expression of CXCR3 in CD56dim cells and in NKT cells was significantly lower in infertile women at d5 (P=0.007; P=0.011, respectively) and ovulation (P=0.001; P=0.004, respectively) relative to the corresponding cells of fertile women. Expression of CXCR4 was lower at d5 in CD56dim cells from infertile compared with fertile women (P=0.039) but did not differ in CD56bright or NKT cells.

NKT cells of infertile but not fertile donors appear to be activated
The cytosolic compartment of each cell sample was assessed for GAPDH expression as a loading control (Fig. 3 , top row). Three cytokines, representative of cytokines produced by NK and NKT cells, were measured as cytosolic proteins—TGF-β, TNF-{alpha}, and IFN-{gamma}. Levels of TGF-β did not change between d5 and LH for either group of women in any of the subsets studied. However, NKT cells from infertile patients expressed significantly more TGF-β at d5 (P=0.002) and at LH (P=0.007) than NKT cells of fertile women (Fig. 3 , row 2). TNF-{alpha} was expressed most strongly in the CD56bright cells of fertile and infertile donors but did not differ significantly between any of the sample groups. Quantification of IFN-{gamma} was limited to CD56bright cells, as it could not be detected in most CD56dim or NKT cell samples. Higher levels were measured in infertile relative to fertile women, particularly at d5, but the differences were not statistically significant.

Changes in protein expression are not mediated through changes in transcription or through hormone receptors
Next, we asked if the observed differences in protein expression arose from differences in gene transcription. Quantitative PCR was conducted on cDNA, generated from mRNA extracted from purified CD56bright and CD56dim subsets, and collected at d5 and day of ovulation (n=7 fertile women) for LFA-1, {alpha}4 integrin, L-selectin, CXCR3, and CXCR4. Figure 4A presents the mean number (±SEM) of copies of GAPDH per µL cDNA in each sample. There was no significant change in GAPDH between d5 and LH surge in CD56bright or CD56dim cells (P=0.109; P=0.456, respectively; repeated measures one-way ANOVA). Further ANOVA analyses revealed no significant change in CD56bright or CD56dim fractions in expression of LFA-1 (Fig. 4B , CD56bright: P=0.306; CD56dim: P=1.00), {alpha}4 integrin (Fig. 4C , CD56bright: P=0.618; CD56dim: P=0.586), L-selectin (Fig. 4D , CD56bright: P=0.224; CD56dim: P=0.609), and chemokine receptor CXCR3 (Fig. 4E , CD56bright: P=0.977; CD56dim: P=0.977) or CXCR4 (Fig. 4F , CD56brightP=0.300; CD56dim: P=0.236).


Figure 4
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  		Figure 4. Quantitative evaluation of mRNA transcripts in human CD56Bright and CD56Dim NK cells isolated at d5 and LH by real-time RT-PCR. Histograms showing the mean expression (relative to GAPDH) of each gene in human CD56Bright and CD56Dim cells isolated at cycle d5 and LH by quantitative RT-PCR. Genes include GAPDH (A), LFA-1 (B), {alpha}4 integrin (C), L-selectin (D), CXCR3 (E), and CXCR4 (F). Bars depict the mean and SE for each data point. Two or more independent experiments were performed for each gene. Data were analyzed by one-way repeated measures ANOVA. A P value of 0.05 was considered significant.

To determine whether binding of ovulatory hormones could be responsible for the shifts in expression of trafficking proteins by blood CD56+ cells, transcripts of specific hormone receptors were sought. For this experiment, CD56bright and CD56dim cells were collected at d5 and LH surge from seven subjects, pooled. cDNA was prepared, and standard RT-PCR was conducted using human ovarian cDNA as the positive control. Each sample generated a similar abundance of GAPDH transcripts (not shown). As shown in Figure 5 , expression of ER{alpha}, ERβ, PR (A and B form), LHR, and PRLR was found in ovary but not in CD56bright or CD56dim cells.


Figure 5
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  		Figure 5. Investigation of hormone receptor expression in NK subsets. Each panel shows a representative image of electrophoresed PCR product using primers against ER{alpha} (expected product 243 bp), ERβ (230 bp), PR (519 bp for A form; 213 bp for B form), LHR (342 bp), and PRLR (180 bp). Each gel was loaded identically with 100 kb molecular weight ladder, ovary-positive control, CD56dim, CD56bright, and water in place of cDNA template-negative control. One representative example from five replicate experiments is shown.


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DISCUSSION
 
This study developed from the hypothesis that an ovulation-associated change occurs in circulating progenitors of human uNK cells and that this enables recruitment of these cells to decidualize endometrium for terminal differentiation. Mature uNK cells are associated with the promotion of angiogenesis and trophoblast invasion in implantation sites [9 , 38 39 40 ] and thus, potentially contribute to pregnancy success. Adult neoangiogenesis associated with wounds and tumors involves a trap formed by CXCL12 that correctly positions a population of circulating CXCR4+, vascular endothelial growth factor (VEGF), and placental growth factor (PlGF)-producing "recruited bone marrow-derived circulating cells" around growing vessels to promote angiogenesis [41 , 42 ]. The bone marrow-derived cells, which have been identified as cell mixtures containing myeloid cells, also appear to aid in recruitment of circulating endothelial progenitor cells to the newly active angiogenic site. As uNK cells are CXCR4+ cells that produce VEGF and PlGF, it is likely that they act similarly. As unusually high levels of uNK cells and angiogenesis are found in the endometria of some patients with recurrent miscarriage [43 ] and patients with breakthrough bleeding when receiving progestin-based hormone replacement therapy [44 ], understanding fully how the uNK cell population is established in the uterus has major implications beyond pregnancy.

As the significance value used in our analyses was set at {varsigma} = 0.05, the probability of a Type I error in our analyses is minimal. However, as a result of the sample size used in our studies, the power of our analyses was usually too small to preclude Type II errors. Thus, we are confident of our positive results but concede that additional differences in molecule expression may exist, which could be detected in a larger population. We found that the subset of CD56bright cells from fertile women had increased expression of key adhesion molecules (LFA-1, {alpha}4 integrin, and L-selectin) and chemokine receptors at LH, with {alpha}4 integrin reaching statistical significance. This relative gain in {alpha}4 integrin is consistent with our previous studies using assays of functional adhesion to decidual tissue sections in vitro. An opposite trend was induced at LH in CD56dim cells from fertile women except for CXCR3, which was a highly expressed membrane protein at d5 and LH. Our flow cytometry indicated ~40% of CD56dim cells express CXCR3 compared with 100% of CD56bright cells. Thus, the Western blot results suggest that CXCR3+ CD56dim cells express CXCR3 at higher abundance relative to CD45 than do CD56bright cells. NKT cells from fertile women had remarkable LH-induced changes—a significant reduction in L-selectin and a concurrent increase in CXCR4. Loss of L-selectin+ NKT cells could be a result of loss of this cell subset from circulation or to L-selectin shedding in response to inflammatory ovulatory signals. The latter would inhibit a cell’s ability to tether to endothelium and enter tissue. This seems unlikely, as L-selectin sheds selectively from activated but not unactivated lymphocytes [45 ], and the cytokine profiles of CD56+ cells from fertile women showed stable production of TNF-{alpha}, TGF-β, and IFN-{gamma}. As transcription of adhesion molecules in CD56bright and CD56dim subsets was constant at both menstrual cycle times studied, changes in adhesive function of CD56bright cells cannot be attributed to increased gene transcription or to significantly up-regulated protein, leaving structural modifications of these molecules in response to activation as the most likely explanation [46 , 47 ]. Other physical changes, such as induction of podosomes or cytoskeletal rearrangements for palpation of endothelial cells that affect transendothelial cell diapedesis, may be induced in CD56+ cells by LH [47 ].

In infertile women, no ovulation-associated changes occurred in expression of trafficking-related proteins or cytokines in any NK cell subset, but expression of these molecules relative to CD45 was similar to that of fertile women at d5. Of particular note is the lack of change in {alpha}4 integrin on CD56bright cells at ovulation. This supports our previous observation that CD56bright cells collected at LH from infertile women function differently in adhesion assays than those from fertile women, despite universal detection of {alpha}4 integrin on CD56+ cells by flow cytometry. CD56dim and NKT subsets also differed between the infertile and fertile populations, particularly at d5. For CD56dim cells, infertile women had statistically less LFA-1, L-selectin, CXCR3, and CXCR4 at d5 and less CXCR3 at LH (Fig. 2) . NKT cells from infertile women also expressed less L-selectin at d5 and less CXCR3 at d5 and LH but expressed more LFA-1 than NKT cells from fertile women. NKT cells from infertile donors also produced more TGF-β than those of fertile women, suggestive of a dampening of cell responsiveness [23 ]. Collectively, these data suggest that all CD56+ cell subsets from infertile donors have a lower propensity for extravasation at either menstrual cycle time-point than CD56+ cells from fertile women.

There is mounting evidence that some immune cells express hormone receptors. Using a variety of techniques such as binding assays [48 ], nuclear assays [49 ], immunostaining [50 ], RT-PCR [51 ], and flow cytometry [52 ], CD8+ T cells have been shown to express ER, and ER protein and transcripts have also been detected in the monocyte/macrophage lineage [50 , 52 53 54 ] and in follicular dendritic cells [55 ]. PR has been demonstrated in stromal cells of the murine thymus, which involutes during pregnancy [56 ], and in mast cells of human airways [57 ] but not in blood or uterine lymphocytes [58 59 60 61 ]. LHR has been detected in polymorphonuclear leukocytes by ligand-binding assays and by RT-PCR [62 ] in mononuclear lymphocytes, including T cells using RT-PCR and Western blotting [63 ] and in decidual macrophages [64 ]. We used RT-PCR of RNA extracted from purified populations of CD56dim, CD56bright, and NKT cells to determine whether the changes observed at LH were mediated through hormone receptors. We were unable to amplify transcripts for ER{alpha}, ERβ, PR, PRIR, and LHR by conventional RT-PCR from CD56bright or CD56dim subsets using pooled cDNAs successful for amplification of housekeeping and adhesion molecule genes and primers successful for receptor amplifications from control human ovarian tissue-derived cDNA. Thus, we conclude that there is little likelihood for direct hormonal action on circulating CD56+ cells.

There is growing consensus that successful implantation requires the presence of inflammatory as well as anti-inflammatory cytokines [65 , 66 ] and that the LH surge induces an inflammatory state within the ovary [67 ], up-regulating cAMP and PGs to induce follicle rupture. This localized inflammation is accompanied by increased follicular IL-6 and IL-8 [68 ]. The question of whether the ovulatory uterus behaves as a secondary lymphoid tissue or as inflamed tissue remains unanswered. If LH induces cAMP and downstream inflammatory events in follicular cells, might it not also induce a mild, systemic inflammation? This notion is supported by a report of transiently exacerbated gingival inflammation, which is worsened during the ovulatory period, as well as during pregnancy [69 ]. CD56bright cells preferentially home to inflamed synovial fluid and tissue as well as in pleural fluid of patients with pleural infections [19 ]. Further, these extracirculatory cells are more responsive to cytokines in culture, producing significantly more IFN-{gamma}.

In our study, the infertile donors were undergoing controlled ovarian hyperstimulation protocols for fertility treatment. This is known to induce an inflammatory state [70 ], which is intensified after injection of βhCG to induce ovulation. We found higher IFN-{gamma} in the CD56bright subset of infertile women at d5 and significantly elevated TGF-β in their NKT cells. Strong expression of CXCR4 on CD56bright cells was also found at d5, suggestive of activation by TGF-β, and was reduced at LH when expression of this cytokine by NKT cells also declined. As IFN-{gamma} produced by uNK cells [71 ] and cultured blood NK cells [72 ] is down-regulated by TGF-β in the presence or absence of stimulatory cytokines, NKT cells do not appear to be able to regulate circulating CD56bright cells during ovarian hyperstimulation protocols. Induction of TGF-β in NKT cells of infertile women may contribute to dysregulation in cytokine production that limits the ability of innate lymphocytes to modify homing molecules and chemokine receptors in response to chemokine stimuli from the periovulatory uterus. Thus, it appears that increased, inflammatory cytokine production early in the cycle may render the uNK cell precursors unable to respond to extravasation signals at ovulation

This study has shown that the menstrual cycle induces multiple changes on circulating CD56+ cells of healthy women. Changes in surface molecules of CD56bright cells are consistent with menstrual cycle changes in adhesive function of these cells previously reported but suggest a greater importance. Differential effects of the menstrual cycle on blood CD56+ cell subsets are reported and quantified here for the first time. These endocrinologically regulated effects are indirectly mediated and not accomplished via classical hormone receptor binding. A key finding was that infertile women after ovarian stimulation do not experience the same changes in blood CD56+ cells as do fertile women during normal cycles. These data predict reduced diapedesis of CD56+ cells into decidualizing endometrium and thus, may compromise positioning of CD56+ cells within the decidua as reported in pregnancy complications [6 , 73 ].


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ACKNOWLEDGEMENTS
 
This study was supported by Natural Sciences and Engineering Research Council of Canada/Canadian Institutes of Health Research Collaborative Health Research Project Grants Program and Canada Chairs Research Chairs Program Awards to V. K. H. and B. A. C.

Received March 6, 2008; revised May 19, 2008; accepted June 5, 2008.


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