mifepristone [14]. The exact mechanisms responsible for immune modulation by mifepristone in uNK cells, however, are not fully understood. Progesterone modulates local immune responses at the maternal-fetal interface [15]. Recently, classical progesterone receptor (PR) expression was demonstrated in peripheral blood NK cells. These cells mediate the reduction of interferon (IFN)-c secretion and the proapoptotic effects of progesterone [16]. However, human uNK cells have no PR expression [17] and progesterone cannot directly affect the biological activity of human uNK cells [18]. In contrast, the glucocorticoid receptor (GR) was identified in both uterine and peripheral blood NK cells [17,19]. Quenby’s study [20] showed that 29 women with a history of recurrent miscarriage had elevated levels of uNK cells. Oral prednisolone suppressed uNK cells from 14 to 9% between the first experimental biopsy before which the drug had not been administered and the second in which it had. These results indicated that glucocorticoid therapy has potential applications in treatment of female reproductive disorders. In light of their immunosuppressant activity, it has been suggested that glucocorticoids might assist in preventing immune rejection of the implanting embryo [21]. These results raise the possibility that the GR is involved in the regulation of uNK cell function. Therefore, the aim of the present study was to investigate whether mifepristone acts as a glucocorticoid or progesterone antagonist to modulate the cytotoxicity of uNK cells. We also studied the role of the mitogen-activated protein kinase (MAPK) pathway as a potential mechanism underlying the effects of mifepristone on human uNK cells. Materials and Methods Tissue collection The study was approved by the Ethics Committee of Women’s Hospital, School of Medicine, Zhejiang University, China. Informed consent was obtained from all patients before tissue collection. Decidual tissue was obtained from 25 women aged 21– 36 years (gestational age 5–7 weeks) who had undergone first- trimester surgical termination of pregnancy. All subjects had an ultrasound scan to confirm a viable intrauterine pregnancy and gestational age. None of them had received any hormone or immunosuppressant treatment for the previous three months. Preparation of uNK cells Decidual cells were isolated from tissue as previously described [22]. Briefly, decidual samples were finely minced into approxi- mately 1–3 mm3-size pieces and digested with collagenase type I (0.1%, Gibco BRL, Gaithersburg, MD, USA) and DNase I (0.01%, Applichem, Darmstadt, Germany). The cellular superna- tant was consecutively filtered through 100- and 40-mm mesh screens. The cells that passed through the mesh screens were collected, resuspended and plated in phenol red-free RPMI-1640 medium supplemented with 10% charcoal-stripped fetal bovine serum (FBS; Gibco BRL), 100 IU/mL penicillin (Sigma, St. Louis, MO, USA), 100 mg/mL streptomycin (Sigma) and 1% L- glutamine (Gibco BRL). The supernatant containing decidual lymphocytes was collected. Decidual mononuclear cells were isolated by density-gradient centrifugation using Ficoll-Paque PLUS (Shanghai Chemical Reagent Factory, Shanghai, China) according to the manufacturer’s instructions. Mononuclear cells were recovered from the interface and washed twice in phosphate- buffered saline (PBS). Uterine NK cells were purified with a human NK cell isolation kit (Miltenyi Biotec, Camberley, UK) and an AutoMACS instrument (Miltenyi Biotec). Cell viability was Mifepristone Augments uNK Cell-Cytotoxicity consistently $95% as tested by trypan blue exclusion. Isolated cells underwent suspension culture in phenol red-free RPMI-1640 medium (Gibco BRL) containing 10% charcoal-stripped FBS (Gibco BRL), 100 IU/mL penicillin (Sigma), 100 mg/mL strepto- mycin (Sigma), 1% L-glutamine (Gibco BRL), and 200 IU/mL recombinant human interleukin (IL)-2 (Primegen Bio-Tech, Shanghai, China). Flow cytometry To confirm the purity of isolated uNK cells, the cells were washed with PBS and incubated with phycoerythrin (PE)-cyanine 5 (Cy5), PE, and fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies against CD3, CD56, CD16 or the relevant isotype control (eBioscience, San Diego, CA, USA) in the dark at 4uC for 30 min. For intracellular perforin analysis, the cells were collected, fixed with 2% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then stained intracellularly with FITC- conjugated antibodies for perforin or the isotype control (eBioscience). Analyses were then conducted on a Beckman- Coulter Epics Altra Flow cytometer (Beckman-Coulter, Fullerton, CA, USA). Purity of isolated cells was confirmed to be .95% in each experiment, as shown previously [22]. Cytotoxicity assay Uterine NK cell-mediated cytotoxicity was evaluated with the MTS assay using the NK cell-sensitive cell line K562 (American Type Culture Collection, Manassas, VA, USA) as the target [23,24]. The purified uNK cells were cultured in culture medium for 24 h in a 96-well U-bottom plate (BD Biosciences, Franklin Lakes, NJ, USA) in the absence or presence of different concentrations of progesterone (0.01, 0.10, 1, and 10 mM; Sigma-Aldrich), cortisol (0.01, 0.10, 1, and 10 mM; Sigma- Aldrich), or mifepristone (0.065, 0.2, and 1 mM; Sigma-Aldrich). To investigate whether mifepristone affects uNK cell-mediated cytotoxicity by acting as a glucocorticoid antagonist, we incubated purified uNK cells with or without 1.0 mM cortisol in the presence or absence of 1.0 mM mifepristone for 24 h. Cells were then incubated with K562 cells for 4 h at an effector:target cell ratio of 10:1. Following co-culture, the viability of the target K562 cells was determined by the MTS assay according to the manufactur- Figure 1. Analysis of uNK cell-mediated cytotoxicity to target cells (K562) using the MTS assay. Uterine NK cells were treated with different concentrations of progesterone or cortisol for 24 h, then uNK cell-mediated cytotoxicity was evaluated by the MTS assay using the NK cell-sensitive cell line K562. doi:10.1371/journal.pone.0036413.g001 PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e36413