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.