The Inhibitory Effect of Corticosteroids on GATA-3 Nuclear Localization in Primary T Lymphocytes Ex Vivo and In Vivo
Treatment with FP ex vivo demonstrated a concentration-dependent decrease in the direct interaction between phospho-GATA-3 and importin-α in PBMCs from patients with asthma (Figure 6A and 6B), which was significantly inhibited at 10−12 M FP (p<0.001, ANOVA and Newman-Keuls test) and completely attenuated by 10−8 M FP (p<0.001, ANOVA and Newman-Keuls test).
10.1371/journal.pmed.1000076.g006 Figure 6  Fluticasone propionate impairs GATA-3 interaction with importin-α and GATA-3 nuclear localization in vivo and ex vivo.
(A and B) Co-immunoprecipitation analysis of PBMCs from steroid-naïve asthma patients treated with FP in vitro demonstrated impaired interaction between GATA-3 and importin-α measured at 60 min. Each bar represents the mean±SEM of at least three independent experiments; *** p<0.001 compared with control as determined by ANOVA/Newman-Keuls analysis. (C and D) Co-immunoprecipitation analyses of PBMCs from steroid-naïve asthma patients treated with inhaled FP (500 µg via a spacer) in vivo demonstrated decreased association between GATA-3 and importin-α. The individual values for each treatment are presented graphically. (E) Representative Western blot showing that importin-α expression was unaffected by inhalation of FP. Blot is representative of gels from three participants.   Our previous T cell line studies indicated that 10−12 M FP suppresses IL-4 and -5 gene expression and attenuated the interaction of GATA-3 with importin-α (see Figures 1D and 2). This concentration is close to peak plasma levels obtained from asthmatic patients treated with inhaled FP (500 µg) [27]. Inhaled FP (500 µg) treatment of seven steroid-naive asthma patients significantly reduced GATA-3–importin-α interaction in vivo in a time-dependent manner. This produced a >90% decrease in GATA-3–importin-α association at 2 h (median [95% CI], 13,494 [6,828–17,829] versus 879 [597–1,165]; p<0.05 Friedman's analysis). However, this did not reach significance using Wilcoxon's post-test analysis (W = 6.00) probably due to low numbers of participants. Similar results were observed when GATA-3–importin-α association was measured (Figure 6C and 6D). The lower dose of FP (100 µg) was not effective. The attenuated interaction of GATA-3 did not result from the defective recycling of importin-α, as a significant decrease in the abundance of importin-α in the cytoplasmic pool was not detected (Figure 6E).
We further examined whether inhaled FP could affect cellular localization of GATA-3 in peripheral blood T cells. Treatment with inhaled FP (500 µg) for 2 h significantly increased GR nuclear translocation (Figure 7A) and concomitantly decreased the number of nuclear GATA-3 immunoreactive peripheral blood T cells (37%±4.2% versus 58.2%±4.95%, p = 0.016, W = 28.0, Wilcoxon's rank test) compared with placebo as measured by immunocytochemistry (Figure 7A and 7B). This was confirmed by Western blotting, which also indicated that this effect was both time- and dose-dependent (Figure 7C and 7D). Thus, inhaled FP (500 µg) induced significant loss in nuclear GATA-3 at 2 h (median [95% CI], 0.40 [0.27–0.53] versus 0.14 [0.11–0.19], p<0.05, W = 21.00, Wilcoxon's rank test) (Figure 7C) and cytoplasmic GATA-3 levels were enhanced by inhaled FP in a dose-dependent manner (median [95% CI], 0.0032 [0.0026–0.0039] versus 0.658 [0.592–0.720], p<0.05, W = −21.00, Wilcoxon's rank test) (Figure 7D). In addition, FP (500 µg) inhibited p38 MAPK phosphorylation in primary T cells in vivo at 2 h in samples from two patients (Figure 7E).
10.1371/journal.pmed.1000076.g007 Figure 7  Inhaled fluticasone propionate impairs GATA-3 nuclear localization in PBMCs.
(A) Representative immunocytochemistry of showing the effect of inhaled FP (500 µg) on GR and GATA-3 nuclear localisation. (B) Nuclear GATA-3 immunoreactivity in PBMCs from seven steroid-naïve asthma patients 2 h following inhaled FP treatment (100 or 500 µg via spacer). The median and interquartile ranges for each treatment are presented as a box-and-whiskers plot (n = 7); * p<0.05 Wilcoxon's rank test compared with placebo. (C) Immunoblotting analyses of PBMCs demonstrated a time-dependent decrease in nuclear expression of GATA-3, and increased cytoplasmic GATA-3 expression after inhalation of FP. (D) Immunoblotting analyses of PBMCs demonstrated a dose-dependent decrease in nuclear expression of GATA-3, and increased cytoplasmic GATA-3 expression 2 h after inhalation of FP. Histone H1 and MEK-1 immunoblotting confirmed equivalent total protein loading for the nuclear and cytoplasmic fractions respectively. Quantification of the densitometry data in (C) and (D) is shown as a box-and-whiskers plot of results from n = 6 participants for which data were available. *p<0.05 compared to control. (E) Western blot analyses of PBMCs demonstrated a time-dependent decrease in dual phosphorylation (threonine-180 and tyrosine-182) of p38 MAPK after inhalation of FP (500 µg). The results shown in (E) are representative of samples from two participants.   Taken together, our data suggest that inhaled FP reduces nuclear localization of GATA-3 in vivo by acutely inhibiting phospho-GATA-3–importin association. This effect may be direct, through competition for importin-α or associated molecules, or secondary to an effect on p38 MAPK-mediated GATA-3 phosphorylation via rapid induction of MKP-1. The combination of these two interacting effects can result in complete suppression of GATA-3 nuclear import and thus Th2 cytokine gene expression.