ERα signaling antagonizes GC-induced apoptosis
Corticosteroids used to treat asthma mediate their effects by binding the glucocorticoid (GC) receptor (GR) [36]. A major anti-inflammatory effect of GC is the ability to induce apoptosis, clearing inflammatory cells from sites of exposure and the circulation [37, 38]. Our finding that the level of circulating Th2 cells correlated with total daily dose of ICS in women, but not men, suggested that aspects of female biology may influence GC responses. Since estrogen can inhibit apoptosis of Th2 cells [39-41], we first hypothesized that estrogen receptor (ER) activation would decrease Th2 cell sensitivity to GC-induced apoptosis. Using primary in vitro differentiated Th2 cells, we examined the effect of various concentrations of the GC dexamethasone (DEX) on apoptosis. We observed that DEX treatment (0.1-1µM, 48 hours) induced Th2 cell apoptosis determined by Annexin V positivity and 7AAD negativity (Annexin V+/7AAD-, Fig. 2A). Concomitant exposure to DEX and the ERα selective agonist propyl pyrazole triol (PPT) resulted in significantly less apoptosis at all concentrations, with the greatest reduction at 0.1 µM DEX (Fig. 2A). Similar results were observed when β-estradiol (E2) was used, particularly at 0.1µM DEX (Fig. 2B). The effect of ERα on DEX-induced apoptosis was also confirmed by staining with fluorochrome-labeled inhibitor of caspase (FLICA, Fig. S3). Apoptosis occurs when pores are formed in the mitochondrial membrane, allowing release of cytochrome c [42]. B cell lymphoma-2 (BCL-2) inhibits pore formation [43] and has been shown to be induced by ER signaling [41]. We found that Th2 cells treated with DEX alone exhibited a small but significant increase in BCL-2 mRNA, likely an attempt by the Th2 cells to counteract apoptosis [44]. Addition of both DEX and PPT, however, resulted in significantly higher levels of BCL-2 mRNA than DEX alone (~1.8-fold at 0.1µM DEX; Fig. 2C). These results suggest that combined signaling of GC and ERα increases the level of the anti-apoptotic factor BCL-2, leading to enhanced Th2 cell survival. The other major effect of GC action is their ability to suppress type 2 cytokine production [45, 46]. We found that Th2 cells treated with DEX exhibited suppression of constitutive IL-5 and IL-13 mRNA levels. Unlike apoptosis, suppression of constitutive cytokine levels was not antagonized by PPT (Fig. 2D) suggesting multiple mechanisms by which ERα signaling influences GC action.