Roflumilast n-oxide associated with PGE2 prevents the neutrophil elastase-induced production of chemokines by epithelial cells

Neutrophil chemotaxis is involved in the lung inflammatory process in conditions such as chronic obstructive pulmonary disease (COPD). Neutrophil elastase (NE), one of the main proteases produced by neutrophils, has an important role in the inflammatory process via the release of chemokines from airway epithelial cells. It was recently shown that roflumilast N-oxide has therapeutic potential in COPD. The aim of the present study was to investigate roflumilast N-oxide's effect on NE-induced chemokine production and signaling pathways in A549 epithelial cells. A549 cells were incubated with NE for 30 min, washed with PBS and then cultured for 2 h (for measurement of mRNA expression) and 24 h (for chemokine release) or for 5 to 30 min (for protein phosphorylation assays). Prior to the addition of NE, cells were also pre-incubated with prostaglandin E-2 (PGE(2)), alone and in combination with roflumilast N-oxide. Addition of NE was associated with elevated chemokine production by A549 cells and induction of the p38 alpha pathway. In contrast when combined with PGE(2), the roflumilast N-oxide had an additive effect on the inhibition of NE-induced chemokine release and p38 alpha and other kinases activation. In conclusion, we demonstrated that NE is able to increase the release of chemokines from epithelial cells via the activation of p38 alpha MAP-kinase and that roflumilast N-oxide when combined with PGE(2) lowers NE-induced kinase activation and chemokine production. (C) 2015 Elsevier B.V. All rights reserved.