Giusy Daniela Albano

Chronic obstructive pulmonary disease and neutrophil infiltration: role of cigarette smoke and cyclooxygenase products

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.

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

Acetylcholine (ACh), synthesized by choline acetyltransferase (ChAT), and muscarinic M1, M2, and M3 receptors (MRs) are involved in fibroblast proliferation. We evaluated ChAT, MRs, and extracellular signal-regulated kinase (ERK) 1/2 and nuclear factor (NF) κB 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)-1β, tumor necrosis factor (TNF)-α, 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-1β, TNF-α, and CSE. ChAT, M1, and M3 expression and ERK1/2 and NFκB activation were increased, whereas M2 was reduced, in COPD and smoker subjects compared with controls. IL-1β increased the ChAT and M3, TNF-α down-regulated M2, and CSE increased ChAT and M3 expression while down-regulating the expression of M2 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-1β- and TNF-α-stimulated HFL-1 cells. Tiotropium (Spiriva; [1α,2β,4β,5α,7β-7-hydroxydi-2-thienylacetyl)oxy]-9,9-dimethyl-3-oxa-9-azoniatrcyclo[3.3.1.024], C19H22 NO4S2Br·H2O), gallamine triethiodide (C19H22N4O2S·2HCl·H2O), telenzepine [4,9-d-dihydro-3-methyl-4-[(4-methyl-1piperazinyl) acetyl]-10H-thieno [3,4-b][1,5]benzodiazepine-10-one dihydrobromide, C30H60I3N3O3], 4-diphenylacetoxy-N-methylpiperidine, PD098059 [2-(2-amino-3methoxyphenyl)-4H-1benzopyran-4-one, C16H13NO3], and BAY 11-7082 [(E)-3-(4-methylphenylsulfonyl)-2-propenetrile, C10H9NO2C], down-regulated the ACh-induced fibroblast proliferation, promoting the MRs and ERK1/2 and NFκB 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.

Cigarette smoke extract activates human bronchial epithelial cells affecting non-neuronal cholinergic system signalling in vitro.

AIMS Acetylcholine (ACh) is synthesized by Choline Acetyl-Transferase (ChAT) that exerts its physiological effects in airway epithelial cells via muscarinic receptor (MR) activation. We evaluate the effect of ACh stimulation on human bronchial epithelial cells (16-HBE) and test whether cigarette smoke extract (CSE) can modify the basal cellular response to ACh affecting the non-neuronal cholinergic system signalling. MAIN METHODS ACh stimulated 16-HBE were tested for ACh-binding, Leukotriene B(4) (LTB(4)) release and ERK1/2 and NFkB pathway activation. Additionally, we investigated all the aforementioned parameters as well as ChAT and MR proteins and mRNA expression and endogenous ACh production in CSE-treated 16-HBE. KEY FINDINGS We showed that ACh induced in 16-HBE, in a concentration-dependent manner, LTB(4) release via the activation of ERK1/2 and NFkB pathways. The addition of Tiotropium (Spiriva®), Gallamine, Telenzepine and 4-DAMP (muscarinic receptor antagonists), as well as of PD 098059 (MAPKK inhibitor) and BAY117082 (inhibitor of IkBα phosphorilation), down-regulated the ACh-induced effects. Additionally, CSE treatment of 16-HBE increased the binding of ACh, and shifted the LTB4 release from the concentration ACh 1μM to 10nM. Finally, we observed that the treatment of 16-HBE with CSE increased the expression of ChAT, M(2) and M(3) and of endogenous ACh production in 16-HBE. Tiotropium regulated the LTB4 release and ACh production in CSE treated 16-HBE. SIGNIFICANCE CSE increases the pro-inflammatory activity of human bronchial epithelial cells, and promotes the cellular response to lower concentrations of ACh, by affecting the expression of ChAT and MRs. Tiotropium might prevent pro-inflammatory events generated by ACh together with CSE.

β₂ long-acting and anticholinergic drugs control TGF-β1-mediated neutrophilic inflammation in COPD.

We quantified TGF-β1 and acetylcholine (ACh) concentrations in induced sputum supernatants (ISSs) from 18 healthy controls (HC), 22 healthy smokers (HS) and 21 COPDs. ISSs from HC, HS and COPD as well as rhTGF-β1 were also tested in neutrophil adhesion and in mAChR2, mAChR3 and ChAT expression experiments in human bronchial epithelial cells (16-HBE). Finally, we evaluated the effects of Olodaterol (a novel inhaled β(2)-adrenoceptor agonist) and Tiotropium Spiriva®, alone or in combination, on neutrophil adhesion and mAChRs and ChAT expression in stimulated 16-HBE. The results showed that 1) TGF-β1 and ACh concentrations are increased in ISSs from COPD in comparison to HC and HS, and TGF-β1 in HS is higher than in HC; 2) ISSs from COPD and HS caused increased neutrophil adhesion to 16-HBE when compared to ISSs from HC. The effect of ISSs from COPD was significantly reduced by TGF-β1 depletion or by the pretreatment with Olodaterol or Tiotropium alone or in combination, while the effect of ISSs from HS was significantly reduced by the pretreatment with Olodaterol alone; 3) mAChR2, mAChR3 and ChAT expression was increased in 16-HBE stimulated with ISSs from COPD and TGF-β1 depletion significantly reduced this effect on mAChR3 and ChAT expression; 4) rhTGF-β1 increased mAChR2, mAChR3 and ChAT expression in 16-HBE; 5) Olodaterol did not affect the expression of mAChRs and ChAT in 16-HBE. Our findings support the use of β₂ long-acting and anticholinergic drugs to control the bronchoconstriction and TGF-β1-mediated neutrophilic inflammation in COPD.

Cysteinyl Leukotriene-1 Receptor Activation in a Human Bronchial Epithelial Cell Line Leads to Signal Transducer and Activator of Transcription 1-Mediated Eosinophil Adhesion

We studied the effect of leukotriene D4 (LTD4) on a human bronchial epithelial cell line (16HBE) overexpressing the cysteinyl leukotriene (CysLT) 1 receptor (HBECysLT1R), looking at the associated signal transduction mechanisms as well as at effects on inflammatory cell adhesion. The results obtained showed that LTD4 increases the phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2 and of the signal transducer and activator of transcription 1 (STAT-1) in serine 727 (STAT-1Ser727), resulting in increased eosinophil adhesion to HBECysLT1R, associated with enhanced surface expression of intercellular adhesion molecule (ICAM) 1. Pretreatment with a CysLT1R-selective antagonist or with a selective inhibitor of protein kinase C (PKC) or with a selective inhibitor of the mitogen-activated protein kinase kinase (MEK) successfully suppressed both LTD4-induced STAT-1Ser727 phosphorylation and the associated increase in eosinophil adhesion. The use of the MEK inhibitor and of the selective CysLT1R antagonist in electrophoretic mobility shift assay experiments showed that LTD4 promotes the nuclear translocation of STAT-1 through the activation of ERK1/2 pathway. The key role of STAT-1 in leukotriene D4 transduction signaling was confirmed by RNA interference experiments, where silencing of STAT-1 expression abolished the effect of leukotriene D4 on eosinophil adhesion. In conclusion, for the first time, we provide evidence of the involvement of STAT-1 in the signal transduction mechanism of the CysLT1 receptor; phosphorylation of STAT-1, through PKC and ERK1/2 activation, causes enhanced ICAM-1 surface expression and eosinophil adhesion. Effective CysLT1R antagonism may therefore contribute to the control of the chronic inflammatory condition that characterizes human airways in asthma.

Th17 Immunity in Children with Allergic Asthma and Rhinitis: A Pharmacological Approach

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.

IL-33/ST2 axis controls Th2/IL-31 and Th17 immune response in allergic airway diseases.

IL-33 targeting ST2 receptor (T1/ST2), expressed on Th2 cell surface, regulates the production of cytokines like IL-17A and IL-31. We studied the role of IL-33/ST2 axis in IL-31 and IL-17A production in patients with allergic rhinitis (AR) and with concomitant allergic asthma and rhinitis (AAR). 20 healthy control subjects (HC), 14 AR and 17 AAR subjects were recruited and blood samples collected. IL-33, soluble ST2 (sST2), IL-17A and IL-31 plasma concentrations were measured by ELISA method. T1/ST2, IL-31 and IL-17A cellular expression were studied in peripheral blood mononuclear cells (PBMC) from HC, AR and AAR (n=6 for each group) by flow-cytometry. In vitro, we also evaluated the effect of beclomethasone dipropionate (BDP) on T1/ST2, IL-31 and IL-17A expression in CD3(+)T-cells from PBMC of AAR (n=6). Plasma levels of IL-33, IL-31 and IL-17A were significantly higher and sST2 was lower in patients with AR and AAR than in HC. IL-31 and IL-17A intracellular levels significantly increased, whereas T1/ST2 expression was significantly lower, in CD3(+)T-cells from AR and AAR compared to HC. Positive correlations were observed between plasmatic components of IL-33/ST2 axis and IL-31 in both AR and AAR and IL-17A in AAR. In vitro IL-31 and IL-17A intracellular levels decreased after BDP treatment, whereas T1/ST2 expression increased in cultured CD3(+)T-cells obtained from AAR. IL-33/ST2 axis is involved in Th2/IL-31 and Th17 immune response during the progression of allergic airway disease. In vitro BDP is able to control Th2/IL-31 and Th17 immune response in PBMC from allergic patients.

IκB kinase–driven nuclear factor-κB activation in patients with asthma and chronic obstructive pulmonary disease

BACKGROUND Nuclear factor-κB (NF-κB) is a transcriptional factor of different inflammatory patterns involved in asthma and chronic obstructive pulmonary disease (COPD) that is tightly controlled by IκB kinase (IKK) complex. OBJECTIVE We investigated the dysregulation of IKK-driven NF-κB activation in patients with asthma and COPD. METHODS We assessed IKKα and IKKβ expression and activation, their regulation by glucocorticosteroids, and their involvement in IL-8 synthesis in PBMCs isolated from asthmatic patients, healthy smokers (HSs), patients with COPD, and control subjects. PBMCs from control subjects were stimulated with TNF-α and cigarette smoke extract in the presence or absence of fluticasone propionate (FP), L-glutathione reduced, or both, and IKK activation and IL-8 release were evaluated. RESULTS IKKα activity was higher in patients with COPD and HSs than in asthmatic patients and control subjects. IKKβ activity was higher in asthmatic patients, HSs, and patients with COPD than in control subjects. In vitro FP treatment induced inhibition of both IKKα and IKKβ activity in PBMCs from asthmatic patients, patients with COPD, and HSs, although IKKβ activity was more sensitive to FP than that of IKKα. FP reduced the IL-8 released from PBMCs of asthmatic patients, patients with COPD, and HSs, although IL-8 inhibition was higher in asthmatic patients than in patients with COPD and HSs. FP reduced IKKα and IKKβ activities in TNF-α and cigarette smoke extract-treated PBMCs, with higher levels of inhibition for IKKβ than IKKα activity. L-glutathione reduced improved the downregulatory effects of FP on IKKα and IL-8 levels. CONCLUSION Based on differential activation of IKKα and IKKβ, our findings suggest a different profile in the upstream regulation of the IKK-driven NF-κB system in asthmatic patients and patients with COPD. These differences in the regulation of the inflammatory process may explain, at least in part, the different pharmacologic responses in these patients.

Increased levels of Th17 cells are associated with non-neuronal acetylcholine in COPD patients

T-lymphocytes, including Th17-cells and T-cells expressing acetylcholine (ACh), are key components of systemic inflammation in chronic obstructive pulmonary disease (COPD). We investigated whether ACh promotes Th17 cells in COPD. ACh, IL-17A, IL-22, RORγt, FOXP3 expression and AChIL-17A, AChIL-22, AChRORγt coexpression was evaluated in peripheral blood mononuclear cells (PBMC) from COPD patients (n=16), healthy smokers (HS) (n=12) and healthy control subjects (HC) (n=13) (cultured for 48 h with PMA) by flow cytometry. Furthermore, we studied the effect of Tiotropium (Spiriva®) (100 nM) and Olodaterol (1nM) alone or in combination, and of hemicholinium-3 (50 μM) on AChIL-17A, AChIL-22, AChRORγt, and FOXP3 expression in CD3+PBT-cells of PBMC from COPD patients (n=6) cultured for 48 h with PMA. CD3+PBT-cells expressing ACh, IL-17A, IL-22 and RORγt together with CD3+PBT-cells co-expressing AChIL-17A, AChIL-22 and AChRORγt were significantly increased in COPD patients compared to HC and HS subjects with higher levels in HS than in HC without a significant difference. CD3+FOXP3+PBT-cells were increased in HS than in HC and COPD. Tiotropium and Olodaterol reduced the percentage of CD3+PBT-cells co-expressing AChIL-17A, AChIL-22, and AChRORγt while increased the CD3+FOXP3+PBT-cells in PBMC from COPD patients, cultured in vitro for 48 h, with an additive effect when used in combination. Hemicholnium-3 reduced the percentage of ACh+IL-17A+, ACh+IL-22+, and ACh+RORγt+ while it did not affect FOXP3+ expression in CD3+PBT-cells from cultured PBMC from COPD patients. We concluded that ACh might promote the increased levels of Th17-cells in systemic inflammation of COPD. Long-acting β2-agonists and anticholinergic drugs might contribute to control this event.

Acetylcholine leads to signal transducer and activator of transcription 1 (STAT-1) mediated oxidative/nitrosative stress in human bronchial epithelial cell line

The induction of nitric oxide synthase (iNOS) expression via the signal transducer and activator of transcription 1 (STAT-1) is involved in the mechanism of oxidative/nitrosative stress. We investigated whether acetylcholine (ACh) generates oxidative/nitrosative stress in bronchial epithelial cells during airway inflammation of COPD and evaluated the effects of Tiotropium, a once-daily antimuscarinic drug, and Olodaterol, a long-acting β2-agonist on these mechanisms. Human bronchial epithelial cells (16-HBE) were stimulated (4h, 37°C) with induced sputum supernatants (ISSs) from healthy controls (HC) (n=10), healthy smokers (HS) (n=10) or COPD patients (n=10), as well as with ACh (from 1μM to 100μM). The activation of STAT-1 pathway (STAT-1Ser727 and STAT-1Tyr701) and iNOS was evaluated in the cell lysates by Western blot analysis as well as nitrotyrosine levels by ELISA, while reactive oxygen species (ROS) were evaluated by flow cytometry. Finally, the effect of Tiotropium (Spiriva®) (100nM), alone or in combination with Olodaterol (1nM), was tested in this model. ISSs from COPD patients significantly increased the phosphorylation of STAT-1Ser727 and STAT-1Tyr701, iNOS and ROS/Nitrotyrosine when compared with ISSs from HC or HS subjects in 16-HBE cells. Furthermore, synthetic ACh increased all these parameters in stimulated 16HBE when compared with untreated cells. Tiotropium and Olodaterol reduced the oxidative/nitrosative stress generated by ACh and ISSs. We concluded that ACh mediated the oxidative/nitrosative stress involving the STAT-1 pathway activation in human bronchial epithelial cells during COPD. β2-Long acting and antimuscarinic drugs, normally used in the treatment of COPD as bronchodilator, might be able to control these cellular events.