Acetylcholine mediates the release of IL-8 in human bronchial epithelial cells by a NFkB/ERK-dependent mechanism

Abstract

Acetylcholine may play a role in cell activation and airway inflammation. We evaluated the levels of both mRNA and protein of muscarinic M 1, M 2, M 3 receptors in human bronchial epithelial cell line (16HBE). 16HBE cells were also stimulated with acetylcholine and extracellular signal-regulated kinase1/2 (ERK1/2) and NFkB pathway activation as well as the IL-8 release was assessed in the presence or absence of the inhibitor of Protein-kinase (PKC) (GF109203X), of the inhibitor of mitogenic activated protein-kinase kinase (MAPKK) (PDO9805), of the inhibitor of kinaseB-α phosphorilation (pIkBα) (BAY11-7082), and of muscarinic receptor antagonists tiotropium bromide, 4-Diphenylacetoxy- N-methylpiperidine methiodide (4-DAMP), telenzepine, gallamine. Additionally, we tested the IL-8-mediated neutrophil chemotactic activity of 16HBE supernatants stimulated with acetylcholine in the presence or absence of tiotropium. 16HBE cells expressed both protein and mRNA for muscarinic M 3, M 2 and M 1 receptors with levels of muscarinic M 3 receptor > muscarinic M 1 receptor > muscarinic M 2 receptor. Acetylcholine (10 μM) significantly stimulated ERK1/2 and NFkB activation as well as IL-8 release in 16HBE cells when compared to basal values. Furthermore, while the use of tiotropium, 4-DAMP, GF109203X, PDO98059, BAY11-7082 completely abolished these events, the use of telenzepine and gallamine were only partially able to downregulate these effects. Additionally, acetylcholine-mediated IL-8 release from 16HBE cells significantly increased chemotaxis toward neutrophils and this effect was blocked by tiotropium. In conclusion, acetylcholine activates the release of IL-8 from 16HBE involving PKC, ERK1/2 and NFkB pathways via muscarinic receptors, suggesting that it is likely to contribute to IL-8 related neutrophilic inflammatory disorders in the airway. Thus, muscarinic antagonists may contribute to control inflammatory processes in airway diseases.