The presence of serotonin in cigarette smoke - a possible mechanism in cigarette smoke-induced airway inflammation

Abstract

INTRODUCTION: Serotonin (5-hydrotrytamine [5-HT]) is a well-known neurotransmitter but recent evidences have suggested that it may also regulate pulmonary functions. Clinical study indicated increased 5-HT levels in the platelet of smokers, implicating a role of 5-HT in smoking-related diseases. Cigarette smoke has reported to inhibit monoamine oxidase (MAO)–A, which metabolises 5-HT, in line with the reduction of MAO-A levels in lung of smokers. Using human bronchial epithelial cells, we aimed to compare the effects of cigarette smoke and 5-HT on oxidative stress and inflammation and the signalling pathways. METHODS: We first prepared cigarette smoke medium (CSM) by bubbling smoke from two cigarettes through 20-mL phosphate-buffered saline, and measured levels of 5-HT in CSM using commercially available enzymelinked immunoassay (EIA) kits. We then cultured BEAS-2B cells and incubated cells with serotonin or CSM. MAO activity was measured by the MAO fluorometric assay kit. Intracellular reactive oxygen species (ROS) was detected by the dichlorofluorescein diacetate assay. Protein expressions of p38, ERK1/2 MAPK, NADPH oxidase 2 (NOX2) were detected by Western blot. Release of pro-inflammatory mediators interleukin 8 (IL-8) was measured by ELISA. RESULTS: We detected the presence of 5-HT in CSM. In BEAS-2B cells, CSM significantly inhibited the activity of MAO-A and MAO-B. Exogenous application of 5-HT or CSM rapidly increased ROS generation, which was accompanied by increased protein expression of NOX2. 5-HT or CSM induced phosphorylation of ERK1/2 at 5 minutes and sustained up to 30 minutes, while activation of p38 was started at 2 minutes and sustained up to 60 minutes. Exposure to 5-HT or CSM caused elevation of IL-8 release, which was attenuated by pre-treatment of MAPK inhibitors U0126 (a selective ERK1/2 inhibitor) or SB203580 (a selective p38 MAPK inhibitor) as well as ROS scavenger TEMPOL. CONCLUSION: Our data suggest that the presence of 5-HT in CSM and the CSM-induced inhibition of 5-HT metabolism may cause oxidative stress and inflammation via NOX2 and activation of ERK and p38 MAPK dependent pathways in bronchial epithelial cells. CSM and 5-HT responses share common characteristics, which suggest the importance of 5-HT–related pathways in CSM-induced epithelial cell injury.