Mifepristone increases the cytotoxicity of uterine natural killer cells by acting as a glucocorticoid antagonist via ERK activation

Abstract

Background: Mifepristone (RU486), a potent antagonist of progesterone and glucocorticoids, is involved in immune regulation. Our previous studies demonstrated that mifepristone directly augments the cytotoxicity of human uterine natural killer (uNK) cells. However, the mechanism responsible for this increase in cytotoxicity is not known. Here, we explored whether the increased cytotoxicity in uNK cells produced by mifepristone is due to either anti-progesterone or anti-glucocorticoid activity, and also investigated relevant changes in the mitogen-activated protein kinase (MAPK) pathway.

Methodology/principal findings: Uterine NK cells were isolated from decidual samples and incubated with different concentrations of progesterone, cortisol, or mifepristone. The cytotoxicity and perforin expression of uNK cells were detected by mitochondrial lactate dehydrogenase-based MTS staining and flow cytometry assays, respectively. Phosphorylation of components of the MAPK signaling pathway was detected by Western blot. Cortisol attenuated uNK cell-mediated cytotoxicity in a concentration-dependent manner whereas progesterone had no effect. Mifepristone alone increased the cytotoxicity and perforin expression of uNK cells; these effects were blocked by cortisol. Furthermore, mifepristone increased the phosphorylation of ERK1/2 in a cortisol-reversible manner. Specific ERK1/2 inhibitor PD98059 or U0126 blocked cortisol- and mifepristone-induced responses in uNK cells.

Conclusions/significance: These results suggest that mifepristone acts as a glucocorticoid antagonist to augment uNK cell-mediated cytotoxicity via ERK activation, which may be caused by increased perforin expression. These observations may reveal an important mechanism by which mifepristone upregulates the cytotoxicity of uNK cells.

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.

Figure 2. Analysis of uNK-cell cytotoxicity and perforin expression after treatment with different concentrations of mifepristone.

The purified uNK cells were incubated with 0, 65, 200, and 1000 nmol/L mifepristone for 24 h. Then, they were subjected to analysis of uNK-cell cytotoxicity and perforin expression. A, a representative flow cytometry profiles of perforin expression was shown. Uterine NK-cell cytotoxicity (B) and perforin (C) expression after treatment with different concentrations of mifepristone were evaluated. Values are expressed as means ± SEM. The influence of mifepristone was evaluated by 4 independent experiments. P-values referred to One-way analysis of variance, n = 6, * P<0.05 vs. control group.

Figure 3. Effects of cortisol on mifepristone-induced uNK-cell cytotoxicity and perforin expression.

Isolated uNK cells were treated with cortisol (1.0 µM) ± and mifepristone (1.0 µM) 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 ± SEM. *, P<0.05.

Figure 4. Effects of mifepristone on the phosphorylation level of MAPK in uNK cells.

A, a representative immunoblot is shown after the treatment of uNK cells with mifepristone (1.0 µM) for 0, 15, 30, 60, and 120 min. Uterine NK cells were isolated and cultured in the medium without IL-2 for 12 h as a negative control of MAPK/ERK activation. In the drug treatment group, uNK cells were cultured in the medium supplemented with IL-2. Uterine NK cells were cultured in the medium with IL-2 and IL-15 for 15 min as a positive control. Immunodetection of MAPK members used specific antibodies for phosphorylated and total proteins of ERK1/2, P38 and JNK. B, densitometric scans of triplicate blots are shown. Experiments were independently repeated 3 times in each group. Data were analyzed using ANOVA and expressed as means ± SEM. *, P<0.05.

Figure 5. Effects of mifepristone on the ERK signaling pathway in uNK cells.

Western blot of p-ERK and total ERK in uNK cells treated with mifepristone ± cortisol. To detect p-ERK and total ERK, total cell lysates were harvested after a 30 min treatment with mifepristone (1.0 µM) ± cortisol (1.0 µM). Uterine NK cells were isolated and cultured in the medium without IL-2 for 12 h as a negative control of MAPK/ERK activation. In the drug treatment groups, uNK cells were cultured in the medium supplemented with IL-2. Uterine NK cells were cultured in the medium with IL-2 and IL-15 for 15 min as a positive control. A typical blot (A) and densitometric scans of triplicate blots (B) are shown. Data were analyzed using ANOVA and expressed as means ± SEM. *, P<0.05.

Figure 6. The increased cytotoxicity of uNK cells by mifepristone was inhibited by PD98059 or U0126.

Before the treatment with cortisol ± mifepristone, uNK cells were pretreated with PD98059 (A) or U0126 (B) for 30 min. They were then treated with mifepristone ± cortisol for 24 h. Cells stimulated with IL-15 were used as positive control. The cytotoxicity of uNK cells was detected by the MTS assay. Data were analyzed using ANOVA and expressed as means ± SEM. *, P<0.05.

Figure 7. Upregulation of perforin expression by mifepristone was inhibited by PD98059 or U0126.

Before the treatment with cortisol ± mifepristone, uNK cells were pretreated with PD98059 (A) or U0126 (B) for 30 min. They were then treated with mifepristone ± cortisol for 24 h. Cells stimulated with IL-15 were used as positive control. Perforin expression in uNK cells was examined by flow cytometry. The value is the percent of perforin-positive cells in the total number of uNK cells. Data were analyzed using ANOVA and expressed as means ± SEM. *, P<0.05.