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Acetylcholine (ACh), synthesized by choline acetyltransferase (ChAT), and muscarinic M 1 , M 2 , and M 3 receptors (MRs) are involved in fibroblast proliferation. We evaluated ChAT, MRs, and extracellular signal-regulated kinase (ERK) 1/2 and nuclear factor (NF) B activation in lung fibroblasts from patients with chronic obstructive pulmonary disease (COPD), control smokers, and controls. Human fetal lung fibroblasts (HFL-1) stimulated with interleukin (IL)-1␤, tumor necrosis factor (TNF)-␣, and cigarette smoke extracts (CSEs) were evaluated for ChAT and MR expression. We tested the effects of ACh on fibroblast proliferation and its ability to bind fibroblasts from patients with COPD, control smokers, controls, and HFL-1 stimulated with IL-1␤, TNF-␣, and CSE. ChAT, M 1 , and M 3 expression and ERK1/2 and NFB activation were increased, whereas M 2 was reduced, in COPD and smoker subjects compared with controls. IL-1␤ increased the ChAT and M 3 , TNF-␣ down-regulated M 2 , and CSE increased ChAT and M 3 expression while downregulating the expression of M 2 in HFL-1 cells. ACh stimulation increased fibroblast proliferation in patients with COPD, control smokers, and controls, with higher effect in control smokers and patients with COPD and increased HFL-1 proliferation only in CSE-treated cells. The binding of ACh was higher in patients with COPD and in control smokers than in controls and in CSE-treated than in IL-1␤-and TNF-␣-stimulated HFL-1 cells. Tiotropium (Spiriva; [1␣,2␤,4␤,5␣,7␤-7-hydroxydi-2-thienylacetyl)oxy]-9,9-dimethyl-3-oxa-9-azoniatrcyclo[3.3. [(E)-3-(4-methylphenylsulfonyl)-2-propenetrile, C 10 H 9 NO 2 C], down-regulated the ACh-induced fibroblast proliferation, promoting the MRs and ERK1/2 and NFB pathways involvement in this phenomenon. These results suggest that cigarette smoke might alter the expression of ChAT and MRs, promoting airway remodeling in COPD and that anticholinergic drugs, including tiotropium, might prevent these events.An overpresence of fibroblasts has been observed in chronic airway diseases and several factors, released by both parenchymal and inflammatory cells, can contribute to this event promoting fibroblast proliferation (Jeffery, 1998 ABBREVIATIONS: M 1 , muscarinic M 1 receptor; M 2 , muscarinic M 2 receptor; M 3 , muscarinic M 3 receptor; MR, muscarinic M 1 , M 2 , and M 3 receptor; MAPK, mitogen-activated protein kinase; ACh, acetylcholine; ChAT, choline acetyltransferase; COPD, chronic obstructive pulmonary disease; IL, interleukin; TNF, tumor necrosis factor; CS, cigarette smoke; ERK, extracellular signal-regulated kinase; NF, nuclear factor; CSE, cigarette smoke extract; C, asymptomatic nonsmoking subject(s) with normal lung function; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; ELISA, enzyme-linked immunosorbent assay; pNFB, phosphorylated NFB; tNFB, total NFB; tiotropium, Spiriva, [1␣,2␤,4␤,5␣,7␤-7-hydroxydi-2-thienylacetyl)oxy]-9,9-dimethyl-3-oxa-9-azoniatrcyclo[3.3. O; 4-DAMP, 4-diphenylacetoxy-N...

Cigarette smoke is the main cause of chronic obstructive pulmonary disease (COPD), where it can contribute to the observed airway inflammation. PGE(2) is produced within human airways, and both pro- and anti-inflammatory activities have been reported. We quantitated PGE(2) concentrations in induced sputum supernatants from different groups of subjects and correlated the obtained values to neutrophil infiltration as well as to the expression of cyclooxygenase-2 (COX-2). Cigarette smoke extract (CSE) was used to evaluate the effect of smoking on COX-2 and PGE(2) receptor expression as well as on PGE(2) release in neutrophils and alveolar macrophages (AM) obtained from normal donors. The effects of PGE(2) and of PGE receptor agonists and antagonists were evaluated on the adhesion of neutrophil to a human bronchial epithelial cell line (16HBE). PGE(2) levels, COX-2 expression, and neutrophil infiltration were significantly higher in normal smokers and COPD smokers (P < 0.0001) compared with controls and COPD former smokers. Induced sputum supernatant caused neutrophil adhesion to 16HBE that was significantly reduced, in COPD smokers only, by PGE(2) immunoprecipitation. In vitro experiments confirmed that CSE increased PGE(2) release and COX-2 and PGE(2) receptor expression in neutrophils and AM; PGE(2) enhanced the adhesion of neutrophils to 16HBE, and a specific E-prostanoid 4 (EP(4)) receptor antagonist blunted its effect. These results suggest that CSE promote the induction of COX-2 and contributes to the proinflammatory effects of PGE(2) in the airways of COPD subjects.

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