Glucocorticoid Receptor Translational Isoforms Contribute to Distinct Glucocorticoid Responses of Neutrophils and Eosinophils

Abstract

Neutrophils, but not eosinophils, have been suggested to mediate steroid resistance in asthma and COPD, the mechanisms of which are unknown. We determined whether the recently described glucocorticoid receptor (GR) translational isoforms underlie the distinct steroid sensitivity of neutrophils and eosinophils. Neutrophils and eosinophils from deidentified human blood samples were isolated to 97% purity by gradient centrifugation, immunomagnetic column separation, and fluorescence-activated cell sorting. Sensitivity of isolated cells to glucocorticoids was examined by measuring apoptotic markers such as annexin-V and caspase 3 activation. GR translational isoforms were determined using Western blot analyses. The pro-apoptotic GR-A isoform was five-fold higher in eosinophils than in neutrophils, whereas the non-apoptotic GR-D isoform was five-fold higher in neutrophils compared to eosinophils (n=9, Student's t-test, p<0.05). The ratio of GR-A/D in eosinophils was more than ten-fold higher than that in neutrophils (p<0.05). Dexamethasone (1 uM, 16 hr) increased eosinophil apoptosis by almost 50%; in contrast, neutrophil apoptosis decreased by approximately 40% in the presence of dexamethasone (p<0.05 for both). These distinct steroid responses in eosinophils and neutrophils were blocked by RU486 (1 uM, 16 hr), a selective GR antagonist, indicating that selective GR isoforms play a role in the different steroid sensitivities of eosinophils and neutrophils. GRβ, a splice variant of GR previously implicated in steroid resistance, was undetectable in either cell type. Neutrophils and eosinophils have distinct GR translational isoforms. The GR-D isoform in neutrophils likely mediates their resistance to steroids and may underlie steroid-resistant diseases characterized by neutrophil-predominant inflammation.