Smoke, Choline Acetyltransferase, Muscarinic Receptors, and Fibroblast Proliferation in Chronic Obstructive Pulmonary Disease

Abstract

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...

Tiotropium inhibits pulmonary inflammation and remodelling in a guinea pig model of COPD

“…However, in vitro studies have indicated that ACh can induce proliferation of lung fibroblasts [20], and collagen synthesis by these cells [22]. In addition, increased ChAT expression has recently been found in lung fibroblasts from healthy smokers and from COPD patients [15], suggesting that non-neuronal ACh may modulate fibroblast function in an autocrine fashion under these conditions. Our results demonstrated that tiotropium inhibits LPS-induced collagen accumulation in the lung and airway wall, indicating that ACh may be a key regulator of airway fibrosis.…”

“…Thus, the ACh-synthesising enzyme choline acetyltransferase (ChAT), as well as muscarinic receptors, are expressed in both structural and inflammatory cells in the lung [12]. Remarkably, increased ChAT expression was found in lung fibroblasts from smokers and COPD patients [15]. Muscarinic M 3 receptor stimulation has been shown to increase the release of neutrophil chemotactic activity by alveolar macrophages [16], induce interleukin (IL)-8 release by bronchial epithelial cells [17] and monocytes [18], and augment cigarette smoke-induced IL-8 release by ASM cells [19].…”

Prostaglandin E2 possesses different potencies in inducing Vascular Endothelial Growth Factor and Interleukin-8 production in COPD human lung fibroblasts

“…Human lung fibroblasts were isolated from surgical specimens of human bronchi as previously described [20]. To confirm the purity of the cultured fibroblasts, the recovered cells were identified by their morphology, adherent nature, expression of vimentin and types I and III collagen, and lack of expression of cytokeratin, α-smooth muscle actin, factor VIII and CD45.…”

“…Purified lung fibroblasts from COPD, HS, and C subjects were grown in a humidified atmosphere containing 5% CO 2, and passaged by trypsinization at nearly confluence onto 100 mm culture plates as previously described [20].…”

Neuro-immune interactions during development of pulmonary fibrosis

“…Stimulation of the vagus nerve in patients with depression significantly increased levels of transforming growth factor beta (TGF-β) , a substance which promotes fibrosis [13], while vagotomy eliminated the drug-induced activation of TGF-β in cerebrospinal fluid [14]. ACh can also activate M receptors to promote fibroblast proliferation in the lung [15]. Additionally, maternal nicotine exposure increased levels of collagen mRNA in pulmonary fibroblasts via α-7nAChR [16].…”