NKG2A and NKG2D during the implantation phase

The effect of mifepristone on the peripheral blood natural killer cell’s cytotoxicity and expression of CD94/NKG2A and NKG2D during the implantation phase Xiu-Ying Chen, M.M., Ya-Ling Zhuang, M.M., Li Li, M.M., Wu-Wen Zhang, M.M., and Li-Li Huang, M.D. Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China

Objective: To investigate the effect of mifepristone on peripheral blood natural killer cell’s (pbNK) cytotoxicity and the expression of the inhibitory receptor CD94/NKG2A and the activated receptor NKG2D on pbNK cells. Design: In vitro study. Setting: University hospital and research laboratory. Patient(s): Twenty healthy nonpregnant women. Intervention(s): Detected the cytolytic activity of pbNK to K562 target cells; measured the expression of CD94/ NKG2A and NKG2D on pbNK. Main Outcome Measure(s): Cytotoxicity of pbNK was detected by Methyl thiazolyl tetrazolium. The expression of CD94/NKG2A and NKG2D receptor on pbNK cells were detected by flow cytometry. Result(s): The NK cell cytotoxicity and the expression of inhibitory receptor CD94/NKG2A during the proliferative phase (81.71  11.5, 86.6  9.0) was significantly higher than the secretory phase (60.16  19.2, 60.15  31.0). The NK cells cytotoxicity, after being treated with mifepristone and the expression of inhibitory receptor CD94/NKG2A on pbNK cells treated with 200 nmol/L mifepristone, were significantly increased. Mifepristone had no effect on the expression of activating receptor NKG2D. Conclusion(s): These data suggest that Mifepristone maybe exert its anti-implantation function by increasing NK cytotoxicity. The increasing NK cytotoxicity of mifepristone is not related to CD94/NKG2A and NKG2D. In the secretory phase down-regulated CD94/NKG2A, NKG2D, and NK cytotoxicity may benefit with embryo implantation. (Fertil Steril 2010;93:2615–20. 2010 by American Society for Reproductive Medicine.) Key Words: Mifepristone, peripheral blood, natural killer cells, secretory phase, cytotoxicity

A fetus is considered to be a natural semiallograft, because half of the fetus histocompatibility antigens come from the father, and trophoblasts share common mechanisms and antigens with tumor cells (1, 2) and viruses (3). Throughout the implantation process, the embryo is not rejected by the maternal immune system for the existence of complicated immunologic regulation. There are two interfaces between maternal immune cells and the fetus. The first interface is between maternal immune cells and the fetal trophoblast in the deciduas. The second immune interface of pregnancy involves interactions between circulating maternal immune cells and the syncytiotrophoblast. The immune interactions at the maternal–fetal interface play an important role in implantation biology. Sergeant et al., argue that maternal–fetal systemic immune interactions in humans might be predominantly natural killer (NK) cell instead of T-cell mediated (4). Natural killer cells are CD3()CD56(þ) and/or CD16(þ) cytotoxic lymphocytes. Natural killer cells comprise 5% to 15% of lymphocytes in peripheral blood (5–7). They are a key component of innate immunity, particularly crucial during the early phase of immune responses against certain viruses, parasites, and microbial pathogens, and participate partly in

Received May 8, 2009; revised September 5, 2009; accepted September 8, 2009; published online November 2, 2010. X.-Y.C. has nothing to disclose. Y.-L.Z. has nothing to disclose. L.L. has nothing to disclose. W.-W.Z. has nothing to disclose. L.-L.H. has nothing to disclose. Reprint requests: Li-Li Huang, M.D., Women’s Hospital, School of Medicine, Zhejiang University, 1 Xueshi Road, Hangzhou, Zhejiang 310006, PR China (FAX: 0086-571-87061878; E-mail: [email protected]).

0015-0282/$36.00 doi:10.1016/j.fertnstert.2009.09.015

the allograft rejection. Natural killer cells can discriminate between normal cells and abnormal cells (such as virus-infected or tumor cells) by using a repertoire of cell surface activating and inhibitory receptors. The receptors on NK cells control their activation, proliferation, and effector functions, and in most instances the inhibitory signals override the triggering ones (8). Human CD94/NKG2A is an ITIM-containing inhibitory receptor expressed by NK cells and recognizes HLA-E. CD94/NKG2A appears to function as the failsafe inhibitory receptor because it is expressed on the vast majority of human NK cells (9). Studies found CD94/NKG2A can inhibit NKG2D-mediated Vav1 phosphorylation (10). The NKG2D receptor is one of the best-characterized activating receptors expressed on NK cells (11). CD94/NKG2A andNKG2D can play a crucial role in cytotoxicity, cytokine synthesis, and innate and adaptive immune responses (12). In the first weeks of pregnancy, there is increased expression of inhibitory receptors (various killer cell immunoglobulin-like receptors including CD94/NKG2A) among peripheral NK cells, reaching a maximum within the third month of gestation, with a subsequent decline to basal levels by the end of pregnancy (13). An imbalance between inhibitory and activating receptor expression was also found in women with implantation failures (14). Mifepristone (RU486), an 11b-dimethyl-amino-phenyl derivative of norethindrone, is a potent progesterone and glucocorticoid receptor antagonist. When it was first found, mifepristone is used as an abortion drug. Recently, mifepristone has been used as an emergency contraceptive, at gradually reduced dosages. For the past few years, investigations showed that daily low-dose mifepristone could inhibit the development of the endometrium to

Fertility and Sterility Vol. 93, No. 8, May 15, 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.

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achieve the purpose of contraception, so-called ‘‘endometrium contraception’’ (15–17). Some studies found that mifepristone has multiple effects on the immune system. It will suppress normal lymphocyte proliferation and down-regulation interleukin-2 receptors (IL-2R). Mifepristone exhibits both agonist and antagonist effects on the release of T-lymphocyte-derived lymphokines (18). Researchers also found that mifepristone may affect some of the markers of endometrial receptivity, disrupt the attachment of the human blastocyst to the in vitro endometrial construct, and reduces pregnancy rates (19, 20). In our previous study (21), we found that low-dose mifepristone increases the number of CD56(þ)NK cells and the percentage of CD3()CD56(þ)CD16()NK subset in receptive endometrium. This suggested that mifepristone have an effect on the uNK cells. In the previous study, some researchers found that peripheral blood NK activity had a negativite influence on the success of pregnancy. Mifepristone is an oral contraceptive drug. It may have some effect on the peripheral blood cells. So we wanted to know if mifepristone influences the fetal implantation by effect on the pbNK. The present study aimed to investigate the effect of mifepristone on pbNK cytotoxicity, as well as the expression of activating receptor NKG2D and inhibitory receptor CD94/NKG2A on NK cells.

MATERIALS AND METHODS Subjects Twenty peripheral blood samples were obtained from healthy women donors: 10 at the proliferative phase (7th–10th day of the menstrual cycle) and 10 at the midsecretory phase (19th–24th day of the menstrual cycle). The menstrual cycle phases were defined by self-reporting by subjects. The women (25–35 years, mean age 27.8  3.764 and body mass index between 19 and 25 kg/m2) had regular menstrual cycles (25–30 days), normal genital system anatomy, and endocrine indexes, and had not received hormone or immunosuppressant therapy for the last 3 months. All women gave written and informed consent. This study took place with the approval of the Women’s Hospital, School of Medicine Zhejiang University Ethics Committee.

Cell Isolation and Culture Human peripheral blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation using Ficoll-Paque plus (Shanghai Chemical Reagent Factory, Shanghai, China) according to the manufacturer’s instructions. The isolated PBMCs were directly collected for MTT and FCM assay. Part of the PBMCs from the implantation phase were resuspened into a six-well culture plate at a total of 106 cells/well with PRMI 1640 (Sigma, St. Louis, MO, USA) medium containing 10% fetal bovine serum (Gibco/BRL, Grand Island, NY, USA) and treated with various concentrations of mifepristone (0, 200, and 1,800 nmol/L). Cells were incubated at 37 C in a humidified 5% CO2 incubator for 24 hours before phenotype analysis by flow cytometry and MTT. A total of 200 nmol/L mifepristone is lower than the inhibition ovulation serum mifepristone concentration 232.7 nmol/L (22); 1,800 nmol/L mifepristone is approximately blood peak when using 10 mg mifepristone as an emergency contraceptive.

for 4 hours. The supernatant was removed and 150 mL dimethyl sulfoxide was added to each well and agitated for 10 minutes to fully liquefy crystals. Absorbance was detected at 490 nm by an automatic ELISA reader (630 nm as a reference wavelength) (24, 25). The data of MTT assay was shown as an average optical density (OD) value. Mitochondrial dehydrogenase enzyme from viable cells to cleave the tetrazolium rings of MTT and form a dark blue formazan crystal, which is largely impermeable to cell membranes, thus resulting in its accumulation within healthy cells. The number of surviving cells is directly proportional to the level of the formazan product created. Dimethyl sulfoxide is added to dissolve the purple formazan crystals to a colored solution. The color was read by an automatic ELISA reader. The number of living cells can be conjectured through the OD value. Through the formula we know the cytotoxicity of the NK cells. The percentage of cytolysis was calculated as follows: [1-(experimental group OD – effector cell OD) / target cell OD]  100% (26).

Flow Cytometric Assay The following monoclonal antibodies were used for triple immunofluorescent staining: fluorescein isothiocyanate (FITC)-conjugated antihuman CD16, phycoerythrin (PE)-conjugated antihuman CD56, PE-Cy5-conjugated antihuman CD3, FITC-conjugated antihuman NKG2D, the relevant isotopic controls (Ebioscience, San Diego, CA, USA), and PE-conjugated mouse monoclonal antihuman CD94/NKG2A (R&D system, Minneapolis, MN, USA). The PBMC were washed with phosphate-buffered saline and incubated with FITC, PE, or PE-Cy5-conjugated monoclonal antibodies against CD3, CD56, CD16, NKG2D, and CD94/NKG2A, or the relevant isotype control in the dark at 4 C for 30 minutes for direct immunofluorescence. All analysis was conducted on a Beckman-Coulter Epics Altra Flow cytometer (Beckman-Coulter, Fullerton, CA, USA). Gating on the CD3CD16þ population within the lymphocyte gate assessed the proportion of CD94/NKG2A-positive subsets. Gating on the CD3CD56þ population within the lymphocyte gate assessed the NKG2D-positive subsets.

Statistical Analysis Date were expressed as mean  standard deviation. Statistical comparisons among different phases of the menstrual cycle were performed using independent T test. The comparison among different concentrations of mifepristone was determined by two-way analysis of variance followed by a Student–Newman–Keuls test. The dates of expression of NKG2D on pbNK were skewed distribution; statistical comparisons were determined by several related sample NPar tests. The level of significance was set at P<.05. Statistical analyses were performed by using SPSS software, version 16.0 (SPSS, Chicago, IL, USA).

RESULTS pbNK Cytotoxicity Assay During the Menstrual Cycle To examine the variety of NK cell cytotoxicity from the peripheral blood during the menstrual cycle, we measured the NK cell cytotoxicity in PBMCs from 20 healthy donors by MTT. We found that PBMCs from the proliferative phase of the menstrual cycle had significantly higher cytotoxicity percentage than from the secretory phase (81.71  11.5 vs. 60.16  19.2; P¼.007) (Table 1).

Methyl thiazolyl tetrazolium Assay

Proportions of CD94/NKG2A and NKG2D Receptors on pbNK

Natural killer cell-mediated cytotoxicity was evaluated by MTT assay using human erythroleukemic K562 cells as targets. In brief, 100 mL K562 were mixed with 100 mL PBMC at effector:target ratios 10:1 in flat-bottom 96-well plates. Some wells were left without cells, to serve as a control for minimum absorbance. A total of 100 mL target and effector cells were cultured in 100 mL medium, respectively, for control; the final volume will be 200 mL per well. After a 24-hour culture (23) at 37 C in a humidified incubator with 5% CO2, freshly prepared and filtered 10 mL metrizamide (5 mg/ mL; Sigma) was added to each well, and the cells were continuously cultured

The activation of an NK cell is regulated via the integration of negative and positive signals mediated by a variety of inhibitory and activating receptor expressed at the NK cell surface. We detected the expression proportions of CD94/NKG2A and NKG2D receptors on pbNK from the proliferative phase and secretory phase of the menstrual cycle. Results showed that the expression proportion of inhibitory receptor CD94/NKG2A on the pbNK from the secretory phase is lower than that from the proliferative phase (60.15  31.0

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TABLE 1 The pbNK cells cytotoxicity and the receptor CD94/NKG2A and NKG2D expressed on the pbNK cells from the healthy women during the proliferative phase and the secretory phase of the menstrual cycle. Proliferative phase (N [ 10)

Secretory phase (N [ 10)

P

81.7  11.5 86.62  9.04 8.73  8.27

60.1  19.2 60.15  30.92 3.61  3.62

.007 .018 .114

MTT(%) CD94/NKG2A NKG2D Note: Results are expressed as mean  SD.

Chen. Mifepristone affects the peripheral blood natural killer cells cytotoxicity. Fertil Steril 2010.

vs. 86.6  9.0; P¼.018). The expression proportion of NKG2D from the secretory phase and proliferative phase had no significant differences (P¼.114) (Fig. 1 and Table 1).

pbNK Cytotoxicity Assay after Treatment with Mifepristone After treating the pbNK cells with mifepristone for 24 hours, the mifepristone group had significantly increasing cytotoxicity compared with the control group (P¼.003), but there was no significant difference between the different concentrations of mifepristone groups (P¼.602) (Table 2).

Proportions of CD94/NKG2A and NKG2D Receptors on pbNK after Mifepristone Treatment The receptor CD94/NKG2A and NKG2D expression on the pbNK from the secretory phase (days 19–24 of the menstrual cycle) was analyzed by flow cytometric assay. The expression proportion of inhibitory receptor CD94/NKG2A on peripheral NK cells in the 200 nmol/L mifepristone group was higher than the control group (55.2  16.0 vs. 50.7  16.4; P¼.013), and also higher than the 1,800 nmol/L mifepristone group (55.2  16.0 vs. 49.6  17.7; P¼.003). There was no significant difference between the control group and the 1,800 nmol/L mifepristone group (P¼.436). No significance difference was found of the expression proportion of activating receptor NKG2D on pbNK among the three groups (Fig. 2 and Table 2).

DISCUSSION Mifepristone is a potent progesterone and glucocorticoid receptor antagonist. The resulting metabolites of mifepristone, like progesterone, are able to bind to the progesterone receptor, effectively limiting or preventing progesterone effects in the target cells. In recent years several clinical trials have shown that a single dose of mifepristone(10 mg) is effective for emergency contraception when given within 120 hours from unprotected coitus, causing mild or no side effects (27). However, the information on immunomodulatory effects of mifepristone as a contraceptive is still limited. Some researchers demonstrated (28) that progesterone induces caspase-dependent pbNK cell death, which is reversed by antiprogestin mifepristone. In our previous study (21), we found that low-dose mifepristone increases the number of CD56(þ)NK cells and the percentage of CD3()CD56(þ)CD16()NK subset in receptive endometrium. A majority of circulating maternal NK cells in blood are CD56dim cells, and they have direct contact with chorionic villi at the intervillous space. Therefore, how the developing fetus evades NK cytotoxicity from these cells may determine the reproductive outcome. In the present study, we found Fertility and Sterility

that in healthy women from the proliferative phase to the secretory phase of the menstrual cycle, the cytotoxicity of NK cells from peripheral blood was significantly reduced. However, when cultured with different concentration of mifepristone in vitro, the NK cell cytotoxicity was significantly elevated, and there was no significant difference of NK cell cytotoxicity between the 200 nmol/L mifepristone group and the 1,800 nmol/L mifepristone group. This suggested that a decreased NK cytotoxicity in the secretory phase of the menstrual cycle might be important to protect early pregnancy against immune aggression. Further, mifepristone may have a role of anti-implantation by increasing NK activity. A total of 200 nmol/L mifepristone has the same effect on NK cell cytotoxicity as 1,800 nmol/L mifeprisotne. Mifepristone is a potent progesterone and glucocorticoid receptor antagonist, which binds with high affinity to the receptors, thus exerting its effect. Because the cell contains a limited number of receptors, with the increasing of ligand concentration, the receptor will saturate. At this time even if the concentration of ligand increased, the combination of ligand and receptor will not increase. So the cytotoxicity of pbNK cells does not always increase with the increase of mifepristone dosage. The minimum effective dosage for contraception needed to be established. Souza (29) also had the similar result that NK cytotoxicity was higher in the follicular (5th–8th day of the menstrual cycle) than in the luteal phase (22nd–26th day of the menstrual cycle). It was reported that NK cell cytotoxicity in nonpregnant women who later experienced subsequent abortion with normal chromosomes was significant higher than that in women with subsequent live births (30). High preconceptional peripheral NK cytotoxicity has been found to be negatively associated with subsequent pregnancy success in women with recurrent spontaneous abortion (RSA) (31) and in patients with unexplained infertility (9). Most importantly, down-regulation of NK cells in women with RSA is associated with a favorable pregnancy outcome (32). Numerous practitioners are offering immunomodulation therapy based on the evidence provided by Coulam et al. (32) and Beer et al. (33) that the peripheral blood NK cell absolute count or percentage can affect IVF or pregnancy outcome. In addition, researchers have found that low levels of NK cell activity in the peripheral blood were reported to be present in most successful cases in women with immunotherapy for RSA (32) and infertility (33). The activation of an NK cell to kill a target cell is regulated via the integration of negative and positive signals mediated by a variety of inhibitory and activating receptors expressed at the NK cell surface (34). In the present study, we detected the effect of mifepristone on the expression of receptors CD94/NKG2A and NKG2D on the NK cells in the vitro model. We found

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FIGURE 1 Comparative analysis of CD94/NKG2A and NKG2D expression on human pNK cells. The expression of CD94/NKG2A (A) and NKG2D (C) on the pNK cells from the proliferative phase of the menstrual cycle. The expression of CD94/NKG2A (B) and NKG2D (D) on the pNK cells from the secretory phase of the menstrual cycle. Sample were analyzed by flow cytometry , and the proportion of NKG2A was calculated in gated CD3()CD16(þ) population, the proportion of NKG2D was calculated in the gated CD3()CD56(þ) population.

Chen. Mifepristone affects the peripheral blood natural killer cells cytotoxicity. Fertil Steril 2010.

that the expression of inhibitory receptor CD94/NKG2A on the peripheral blood NK cells from the proliferative phase and the secretory phase of the menstrual cycle were significantly reduced. After culture with 200 nmol/L mifeptistone, the expression of inhibitory receptor CD94/NKG2A on pbNK cells is higher than that in the control group. But there is no significant

difference in the expression of activating receptor NKG2D. This suggested that the evaluated expression of inhibitory receptor CD94/NKG2A on pbNK cells on the secretory phase of menstrual cycle may be a benefit for the embryo implantation. The increased NK cytotoxicity of mifepristone is not related to CD94/NKG2A and NKG2D. There is a variety of activating

TABLE 2 The cytotoxicity of pbNK cells and the expression of CD94/NKG2A and NKG2D on pbNK cells from the secretory phase after culture with mifepristone in vitro.

MTT(%) CD94/NKG2A NKG2D

Control group (N [ 10)

200 nmol/L mifepristone (N [ 10)

1,800 nmol/L mifepristone (N [ 10)

P

37.9  33.0 50.7  16.4b 1.3  2.2

51.7  26.0a 55.2  16.0 1.4  1.4

55.1  37.5a 49.6  17.7b 3.0  6.0

.003 .006 .436

Note: Values given as mean  SD. a P<.05 mifepristone treated groups compared with the control group. b P<.05 control group and 1,800 nmol/L mifepristone group compared with 200 nmol/L mifepristone group. Chen. Mifepristone affects the peripheral blood natural killer cells cytotoxicity. Fertil Steril 2010.

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FIGURE 2 Comparative analysis of CD94/NKG2A and NKG2D expression on human secretory phase pNK cells after culture with difference concentration Mifepristone. The expression of CD94/NKG2A (A) and NKG2D (D) on the pNK cells after culture with 0 nmol/L mifepristone. The expression of CD94/NKG2A (B) and NKG2D (E) on the pNK cells after culture with 200 nmol/L mifepristone. The expression of CD94/NKG2A (C) and NKG2D (F) on the pNK cells after culture with 1,800 nmol/L mifepristone. Sample were analyzed by flow cytometry, and the proportion of NKG2A was calculated in gated CD3()CD16(þ) population, the proportion of NKG2D was calculated in the gated CD3()CD56(þ) population.

Chen. Mifepristone affects the peripheral blood natural killer cells cytotoxicity. Fertil Steril 2010.

and inhibitory receptors that govern NK cell activity, such as natural cytotoxicity receptors (e.g., NKp30, NKp44, and NKp46), NKG2D, NKRP1, CD16, activating killer cell Ig-like receptors in humans (activating KIRs), LFA-1, DNAM, and members of the CD2 family. Activation of NK cells requires the action of pro-inflammatory cytokines (such as IL-12, IL-15, IL-18, IL-21, and interferon [IFN]-ab) in combination with differential engagement of cell surface receptors. In addition, some researchers found that the initiation of NK cell immune responses might result from signals originating from one or more adhesion or costimulatory molecules, rather than from a unique NK receptor (35). So, we presume that the effect of mifepristone on the cytotoxicity of pbNK cells may be through some factors other than CD94/NKG2A. Mcmahon et al. (36) found that most of pathogen-specific murine CD8(þ)T cells initiated expression of the inhibitory CD94/NKG2A heterodimer when they responded to intracellular pathogens, and binding of the CD94/NKG2A receptor by its ligand did not significantly inhibit CD8(þ)T-cell effector functions. They suggest that CD94/NKG2A-mediated inhibition of T cells may be limited to particular circumstances. Also, some researchers considered that CD94/NKG2A is a potential activating receptor(s) and could induce responses to normal

Fertility and Sterility

bystander cells (37). We presume that CD94/NKG2A may exert an action of activating receptors induced by mifepristone. McGrath et al. (38) found that the expression of CD94 and NKG2A by peripheral blood NK cells did not differ throughout the menstrual cycle either in infertile or fertile women. Other studies reported that there is no difference in the expression of NKG2D receptors between spontaneous abortion and normal control (39). In addition, some researcher found that the triggering of NK cells in the process of tumor cell lysis maybe depend on the concerted action of natural cytotoxicity receptors and NKG2D. In some instances, it may uniquely depend upon the activity of natural cytotoxicity receptors or NKG2D only (40). In conclusion, in this study we found that in the secretory phase mifepristone increased the cytotoxicity of pbNK cells. These results suggest that mifepristone may be an antinidation drug. The activation of an NK cell is regulated via the intergration of negative and positive signals mediated by a variety of inhibitory and activating receptors expressed at the NK cell surface. In the present study, we concluded that down-regulated CD94/NKG2A and NK cytotoxicity may benefit embryo implantation in the secretory phase. Mifepristone may exert its anti-implantation function by increasing NK cytotoxicity. The increased NK cytotoxicity of mifepristone is not

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related to CD94/NKG2A and NKG2D. As this is an in vitro model, which is far less complicated than in vivo, further research is needed

concerning the mechanism by which the cytotoxicity of NK cells is changed by mifepristone.

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Mifepristone affects the peripheral blood natural killer cells cytotoxicity

Vol. 93, No. 8, May 15, 2010