2 Inhaled long - acting β 2 - agonists enhance glucocorticoid receptor nuclear translocation and 3 efficacy in sputum macrophages in COPD 4

24 Background: Combination inhaled therapy with long-acting β2-agonist (LABA) and corticosteroid 25 is beneficial in treating asthma and chronic obstructive pulmonary disease (COPD). 26 Objective: In asthma, LABAs enhance glucocorticoid receptor (GR) nuclear translocation in the 27 presence of corticosteroids. Whether this biological mechanism occurs in COPD, a relatively 28 corticosteroid-resistant disease, is uncertain. 29 Methods: Eight patients with mild/moderate COPD participated in a double-blind, placebo30 controlled, crossover study and inhaled single-doses of; fluticasone propionate (FP)-100μg, FP31 500μg, salmeterol(SLM)-50μg and combination FP-100μg+SLM-50μg. One hour post-inhalation 32 sputum was induced, nuclear proteins isolated from purified macrophages and levels of 33 activated nuclear GR quantified using a GR-GRE ELISA-based assay. 34 Results: Nuclear GR significantly increased after FP-500μg (p<0.01), but not after FP-100μg or 35 SLM-50μg, compared to placebo. Interestingly, SLM in combination with FP-100μg increased 36 nuclear GR levels equivalent to those of FP-500μg alone. This was significantly greater than 37 either FP-100μg (p<0.05) or SLM-50μg (p<0.01) alone. In vitro in a human macrophage cell line, 38 SLM (10M) enhanced FP (10M)-induced mitogen-activated protein kinase phosphatase-1 39 (MKP-1) mRNA (5.8±0.6 vs. 8.4±1.1 x 10 copies, p<0.05) and 2xGRE-luciferase reporter gene 40 activity (250.1±15.6 vs. 103.1±23.6 fold induction, p<0.001). Addition of SLM (10M) to FP (10 41 M) significantly enhanced FP-mediated suppression of IL-1β-induced CXCL8 (p<0.05). 42 Conclusion: Addition of SLM-50μg to FP-100μg, enhanced GR nuclear translocation equivalent 43 to that seen with a five-fold higher dose of FP in sputum macrophages from COPD patients. This 44 may account for the superior clinical effects observed in COPD of combination 45 LABA/corticosteroid treatment compared to either as monotherapy. 46 47