Methods: Primary human bronchial smooth muscle cells were transfected with pEGFP-hGRa plasmid which expresses green fluorescent protein (GFP) fused with human GRa. The transfected cells were treated with EBC from patients with mild persistent asthma (n=11), nonasthmatic controls (n=6) and buffer for 60 min and GR function was assessed by stimulating the cells with Dex for 30 minutes. Cells were fixed and GR-GFP translocation from cytoplasm to nucleus (nuclear/cytoplasmic ratio, N/C) was analyzed as a measure of GR function.
Results: EBC from asthmatic subjects abolished dex induced GR translocation, but EBC from nonasthmatics and control (saline) had no effect. The N/C ratio measured as percent of control (N/C) was 92.2 ± 14.4 in asthmatics prior to stimulation with dex and 97.5 ± 11.2 after stimulation (p=0.908). In contrast, dex induced significant increase in N/C ratio in cells treated with nonasthmatic EBC (100.7 ± 16.9 before, and 141.1 ± 9.6 after, p<0.001).
Conclusions: EBC from mild persistent asthmatics impairs GR translocation to the nucleus suggesting that those with mild asthma have a component of steroid resistance. There are multiple inflammatory mediators in EBC that are likely responsible for this effect. This novel observation has important implications in the understanding of the pathophysiology of asthma.