AIMS: Beclomethasone/formoterol (BDP/FOR) has been reported to be more effective than its separate components in airway disease control and in airway inflammation improvement. However, BDP/FOR effects on cytokine-induced inflammation in structural cells have not been described and whether these effects occur in a cell- and mediator-dependent manner has not been fully elucidated. We sought to evaluate BDP and/or FOR effects on endothelial ICAM-1, E-selectin, IL-8 and on bronchial epithelial ICAM-1 and IL-8. Specific intracellular signaling pathways were also investigated. MATERIALS AND METHODS: Surface adhesion molecule expression and IL-8 release induced by TNF-alpha were measured by ELISA. Intracellular signaling pathways were investigated by a) EMSA and Western blot analysis to evaluate NF-κB DNA-binding and MAPK-p38 phosphorylation; b) PDTC/SB203580 as NF-κB/p38 inhibitors; c) forskolin/H-89 as PKA activator/inhibitor. KEY FINDINGS: BDP/FOR additively reduced endothelial E-selectin and IL-8 as well as bronchial epithelial ICAM-1 and IL-8. BDP/FOR and SB203580 showed the highest inhibitory effect on epithelial IL-8, whereas endothelial ICAM-1 was never affected by BDP/FOR and PDTC. TNF-alpha-induced NF-κB DNA-binding and MAPK-p38 phosphorylation were not influenced by BDP/FOR. Forskolin mimicked FOR effects; H-89 partially reversed the BDP/FOR inhibition in a mediator-dependent manner. SIGNIFICANCE: The BDP/FOR inhibition degree was related to the inflammatory mediator- and cell-type considered. FOR additively enhanced BDP effects by partially involving both dependent- and independent-PKA mechanisms. Our results might contribute to highlight the strong relationship between specific molecular pathways and different sensitivity to the corticosteroid/β2-agonist effects and to clarify the molecular mechanisms underlying the BDP/FOR anti-inflammatory activity in vivo.
The beclomethasone anti-inflammatory effect occurs in cell/mediator-dependent manner and is additively enhanced by formoterol: NFkB, p38, PKA analysis
Menegazzi, Marta;Darra, Elena;
2018-01-01
Abstract
AIMS: Beclomethasone/formoterol (BDP/FOR) has been reported to be more effective than its separate components in airway disease control and in airway inflammation improvement. However, BDP/FOR effects on cytokine-induced inflammation in structural cells have not been described and whether these effects occur in a cell- and mediator-dependent manner has not been fully elucidated. We sought to evaluate BDP and/or FOR effects on endothelial ICAM-1, E-selectin, IL-8 and on bronchial epithelial ICAM-1 and IL-8. Specific intracellular signaling pathways were also investigated. MATERIALS AND METHODS: Surface adhesion molecule expression and IL-8 release induced by TNF-alpha were measured by ELISA. Intracellular signaling pathways were investigated by a) EMSA and Western blot analysis to evaluate NF-κB DNA-binding and MAPK-p38 phosphorylation; b) PDTC/SB203580 as NF-κB/p38 inhibitors; c) forskolin/H-89 as PKA activator/inhibitor. KEY FINDINGS: BDP/FOR additively reduced endothelial E-selectin and IL-8 as well as bronchial epithelial ICAM-1 and IL-8. BDP/FOR and SB203580 showed the highest inhibitory effect on epithelial IL-8, whereas endothelial ICAM-1 was never affected by BDP/FOR and PDTC. TNF-alpha-induced NF-κB DNA-binding and MAPK-p38 phosphorylation were not influenced by BDP/FOR. Forskolin mimicked FOR effects; H-89 partially reversed the BDP/FOR inhibition in a mediator-dependent manner. SIGNIFICANCE: The BDP/FOR inhibition degree was related to the inflammatory mediator- and cell-type considered. FOR additively enhanced BDP effects by partially involving both dependent- and independent-PKA mechanisms. Our results might contribute to highlight the strong relationship between specific molecular pathways and different sensitivity to the corticosteroid/β2-agonist effects and to clarify the molecular mechanisms underlying the BDP/FOR anti-inflammatory activity in vivo.
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