Mifepristone Augments uNK Cell-Cytotoxicity Figure 3. Effects of cortisol on mifepristone-induced uNK-cell cytotoxicity and perforin expression. Isolated uNK cells were treated with cortisol (1.0 mM) 6 and mifepristone (1.0 mM) for 24 h. A, a representative flow cytometry result for perforin expression in different groups. B, results of uNK-cell cytotoxicity in different groups. C, data summary of flow cytometry results for perforin expression. The value is the percent of perforin- positive cells in the total number of uNK cells. Experiments were independently repeated 5 independent experiments. Data were analyzed using ANOVA and expressed as means 6 SEM. *, P,0.05. doi:10.1371/journal.pone.0036413.g003 Results Cortisol increases uNK cell-mediated cytotoxicity but not progesterone Human uNK cells were isolated by immunomagnetic separa- tion and treated with different concentrations of progesterone or cortisol. As shown in Figure 1, treatment with progesterone from 0 (control) to 10.0 mM did not change uNK cell-mediated cytotoxicity towards K562 cells. In contrast, concentrations of cortisol $1.0 mM caused a significant decrease in the effective cytotoxicity of uNK cells. While the uNK cell-mediated cytotox- icity without cortisol was 62.362.7%, extents of cytotoxicity after cortisol treatments were 56.263.1% and 55.164.0% at doses of 1 mM and 10 mM, respectively (P,0.05 for both). Mifepristone increases uNK cell-mediated cytotoxicity, an effect reversed by cortisol It has been reported that when mifepristone was administered at a dose of 1 mg/day, endometrial development appeared to be slightly retarded and the steady plasma level of mifepristone was found to be 65 nmol/L. When the serum mifepristone concen- tration reached 232.7 nmol/L, ovulation was inhibited. 1000 nmol/L is the steady plasma level of mifepristone with 10 mg/day that achieved the purpose of emergency contraception [30]. We found that, 65 and 200 nmol/L mifepristone had no significant influence on human uNK-cell cytotoxicity in vitro. Compared with control group, human uNK-cell cytotoxicity (73.1664.27% vs. 62.2464.39%, P,0.05) (Fig. 2B) significantly increased in 1000 nmol/L (1.0 mM) mifepristone group. Uterine NK cells were then treated without or with mifepristone (1.0 mM) in the presence or absence of 1.0 mM cortisol. Mifepristone without cortisol increased uNK cell-mediated cytotoxicity (62.362.7% vs. 73.264.3%, P,0.05) and this effect was reversed by cortisol (73.264.3% vs. 66.962.9%, P,0.05; Fig. 3B). PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e36413