Summary Airway epithelium is emerging as a regulator of innate immune responses to a variety of insults including cigarette smoke. The main goal of this study was to explore the effects of cigarette smoke extracts (CSE) on Toll‐like receptor (TLR) expression and activation in a human bronchial epithelial cell line (16‐HBE). The CSE increased the expression of TLR4 and the lipopolysaccharide (LPS) binding, the nuclear factor‐κB (NF‐κB) activation, the release of interleukin‐8 (IL‐8) and the chemotactic activity toward neutrophils. It did not induce TLR2 expression or extracellular signal‐regulated signal kinase 1/2 (ERK1/2) activation. The LPS increased the expression of TLR4 and induced both NF‐κB and ERK1/2 activation. The combined exposure of 16‐HBE to CSE and LPS was associated with ERK activation rather than NF‐κB activation and with a further increase of IL‐8 release and of chemotactic activity toward neutrophils. Furthermore, CSE decreased the constitutive interferon‐inducible protein‐10 (IP‐10) release and counteracted the effect of LPS in inducing both the IP‐10 release and the chemotactic activity toward lymphocytes. In conclusion, cigarette smoke, by altering the expression and the activation of TLR4 via the preferential release of IL‐8, may contribute to the accumulation of neutrophils within the airways of smokers.

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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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BackgroundAltered pulmonary defenses in chronic obstructive pulmonary disease (COPD) may promote distal airways bacterial colonization. The expression/activation of Toll Like receptors (TLR) and beta 2 defensin (HBD2) release by epithelial cells crucially affect pulmonary defence mechanisms.MethodsThe epithelial expression of TLR4 and of HBD2 was assessed in surgical specimens from current smokers COPD (s-COPD; n = 17), ex-smokers COPD (ex-s-COPD; n = 8), smokers without COPD (S; n = 12), and from non-smoker non-COPD subjects (C; n = 13).ResultsIn distal airways, s-COPD highly expressed TLR4 and HBD2. In central airways, S and s-COPD showed increased TLR4 expression. Lower HBD2 expression was observed in central airways of s-COPD when compared to S and to ex-s-COPD. s-COPD had a reduced HBD2 gene expression as demonstrated by real-time PCR on micro-dissected bronchial epithelial cells. Furthermore, HBD2 expression positively correlated with FEV1/FVC ratio and inversely correlated with the cigarette smoke exposure. In a bronchial epithelial cell line (16 HBE) IL-1β significantly induced the HBD2 mRNA expression and cigarette smoke extracts significantly counteracted this IL-1 mediated effect reducing both the activation of NFkB pathway and the interaction between NFkB and HBD2 promoter.ConclusionsThis study provides new insights on the possible mechanisms involved in the alteration of innate immunity mechanisms in COPD.

Th17 cells and IL-17A play a role in the development and progression of allergic diseases. We analyzed the IL-17A levels in sputum supernatants (Ss), nasal wash (NW) and plasma (P) from Healthy Controls (HC) and children with Asthma/Rhinitis. We tested the expression of IL-17A, RORγ(t) and FOXP3 in peripheral blood T-lymphocytes from intermittent and mild-moderate asthma. The effect of Budesonide and Formoterol was tested “in vitro” on IL-17A, RORγ(t) and FOXP3 expression in cultured T-lymphocytes from mild-moderate asthma/persistent rhinitis patients, and on nasal and bronchial epithelial cells stimulated with NW and Ss from mild-moderate asthma/persistent rhinitis. Further, the effect of 12 weeks of treatment with Budesonide and Formoterol was tested “in vivo” in T-lymphocytes from mild-moderate asthma/persistent rhinitis patients. IL-17A was increased in Ss, NW and P from children with mild-moderate asthma compared with intermittent and HC. In cultured T-lymphocytes IL-17A and RORγ(t) expression were higher in mild-moderate asthma/persistent rhinitis than in mild-moderate asthma/intermittent rhinitis, while FOXP3 was reduced. Budesonide with Formoterol reduced IL-17A and RORγ(t), while increased FOXP3 in cultured T-lymphocytes from mild-moderate asthma/persistent rhinitis, and reduced the IL-8 release mediated by IL-17A present in NW and Ss from mild-moderate asthma/persistent rhinitis in nasal and bronchial epithelial cells. Finally, Budesonide with Formoterol reduced IL-17A levels in P and Ss, CD4+IL-17A+T-cells, in naïve children with mild-moderate asthma/persistent rhinitis after 12 weeks of treatment. Th17 mediated immunity may be involved in the airway disease of children with allergic asthma and allergic rhinitis. Budesonide with Formoterol might be a useful tool for its therapeutic control.

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