Maria Ferraro

Acetylcholine mediates the release of IL-8 in human bronchial epithelial cells by a NFkB/ERK-dependent mechanism.

Acetylcholine may play a role in cell activation and airway inflammation. We evaluated the levels of both mRNA and protein of muscarinic M(1), M(2), M(3) receptors in human bronchial epithelial cell line (16HBE). 16HBE cells were also stimulated with acetylcholine and extracellular signal-regulated kinase1/2 (ERK1/2) and NFkB pathway activation as well as the IL-8 release was assessed in the presence or absence of the inhibitor of Protein-kinase (PKC) (GF109203X), of the inhibitor of mitogenic activated protein-kinase kinase (MAPKK) (PDO9805), of the inhibitor of kinaseB-alpha phosphorilation (pIkBalpha) (BAY11-7082), and of muscarinic receptor antagonists tiotropium bromide, 4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), telenzepine, gallamine. Additionally, we tested the IL-8-mediated neutrophil chemotactic activity of 16HBE supernatants stimulated with acetylcholine in the presence or absence of tiotropium. 16HBE cells expressed both protein and mRNA for muscarinic M(3), M(2) and M(1) receptors with levels of muscarinic M(3) receptor>muscarinic M(1) receptor>muscarinic M(2) receptor. Acetylcholine (10 microM) significantly stimulated ERK1/2 and NFkB activation as well as IL-8 release in 16HBE cells when compared to basal values. Furthermore, while the use of tiotropium, 4-DAMP, GF109203X, PDO98059, BAY11-7082 completely abolished these events, the use of telenzepine and gallamine were only partially able to downregulate these effects. Additionally, acetylcholine-mediated IL-8 release from 16HBE cells significantly increased chemotaxis toward neutrophils and this effect was blocked by tiotropium. In conclusion, acetylcholine activates the release of IL-8 from 16HBE involving PKC, ERK1/2 and NFkB pathways via muscarinic receptors, suggesting that it is likely to contribute to IL-8 related neutrophilic inflammatory disorders in the airway. Thus, muscarinic antagonists may contribute to control inflammatory processes in airway diseases.

Beta defensin-2 is reduced in central but not in distal airways of smoker COPD patients.

Background Altered 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. Methods The epithelial expression of TLR4 and of HBD2 was assessed in surgical specimens from current smokers COPD (s-COPD; nu200a=u200a17), ex-smokers COPD (ex-s-COPD; nu200a=u200a8), smokers without COPD (S; nu200a=u200a12), and from non-smoker non-COPD subjects (C; nu200a=u200a13). Results In 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. Conclusions This study provides new insights on the possible mechanisms involved in the alteration of innate immunity mechanisms in COPD.

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

AIMSnAcetylcholine (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.nnnMAIN METHODSnACh 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.nnnKEY FINDINGSnWe 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.nnnSIGNIFICANCEnCSE 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.

Clinical Benefits of 7 Years of Treatment with Omalizumab in Severe Uncontrolled Asthmatics

Rationale. Severe asthma is characterized by inadequate symptom control and by high rate of inflammation despite high doses of steroids. Omalizumab, a recombinant humanized monoclonal anti-IgE, provides a new therapeutic strategy in severe allergic asthma. Aims. This study was aimed to assess whether long-term treatment with omalizumab improved clinical control in severe asthmatics. Methods. We investigated omalizumab effects on asthma outcomes evaluating seven severe allergic asthmatic patients who were treated for 7 years with add-on omalizumab. Number of exacerbations, use of antibiotics, additional asthma medications (systemic steroids, nebulized steroids and bronchodilators), and spirometry were analyzed before and after omalizumab treatment. Results. Omalizumab was well tolerated by all the studied patients. It improved FEV1 and FEV1/FVC ratio and reduced symptom score, asthma exacerbations, use of antibiotics, and use of nebulized steroids, bronchodilators, and oral corticosteroids. These effects were evident after 4 years of treatment and more pronounced after 7 years of treatment. Conclusions. This study underlines the utility of a long-term treatment with omalizumab to improve asthma clinical outcomes in severe asthmatics.

Comparative cytoprotective effects of carbocysteine and fluticasone propionate in cigarette smoke extract-stimulated bronchial epithelial cells

Cigarette smoke extracts (CSE) induce oxidative stress, an important feature in chronic obstructive pulmonary disease (COPD), and oxidative stress contributes to the poor clinical efficacy of corticosteroids in COPD patients. Carbocysteine, an antioxidant and mucolytic agent, is effective in reducing the severity and the rate of exacerbations in COPD patients. The effects of carbocysteine on CSE-induced oxidative stress in bronchial epithelial cells as well as the comparison of these antioxidant effects of carbocysteine with those of fluticasone propionate are unknown. The present study was aimed to assess the effects of carbocysteine (10−4xa0M) in cell survival and intracellular reactive oxygen species (ROS) production (by flow cytometry) as well as total glutathione (GSH), heme oxygenase-1 (HO-1), nuclear-related factor 2 (Nrf2) expression and histone deacetylase 2 (HDAC-2) expression/activation in CSE-stimulated bronchial epithelial cells (16-HBE) and to compare these effects with those of fluticasone propionate (10−8xa0M). CSE, carbocysteine or fluticasone propionate did not induce cell necrosis (propidium positive cells) or cell apoptosis (annexin V-positive/propidium-negative cells) in 16-HBE. CSE increased ROS production, nuclear Nrf2 and HO-1 in 16-HBE. Fluticasone propionate did not modify intracellular ROS production, GSH and HDCA-2 but reduced Nrf2 and HO-1 in CSE-stimulated 16-HBE. Carbocysteine reduced ROS production and increased GSH, HO-1, Nrf2 and HDAC-2 nuclear expression/activity in CSE-stimulated cells and was more effective than fluticasone propionate in modulating the CSE-mediated effects. In conclusion, the present study provides compelling evidences that the use of carbocysteine may be considered a promising strategy in diseases associated with corticosteroid resistance.

Oxidative stress and innate immunity responses in cigarette smoke stimulated nasal epithelial cells.

Cigarette smoke extracts (CSE) may play a significant role in diseases of the upper airway including chronic rhinosinusitis. Even short term exposure of cigarette smoke has adverse effects on mitochondrial functions and redox homeostasis in tissues which may progress to further complications associated with chronic smoking. Cigarette smoke alters toll-like receptor 4 (TLR4) expression and activation in bronchial epithelial cells. Carbocysteine is an anti-oxidant and mucolytic agent. The effects of carbocysteine on CSE induced oxidative stress and on associated innate immune and inflammatory responses in nasal epithelial cells are largely unknown. The present study was aimed to assess in CSE stimulated nasal epithelial cells (RPMI 2650) the effects of carbocysteine (10(-4)M) on: cell survival, intracellular reactive oxygen species (ROS) production, TLR4 expression, LPS binding and neutrophil chemotaxis (actin reorganization). We found that CSE increased ROS production, TLR4 expression, LPS binding and neutrophil chemotaxis and all these events were counteracted by pre-incubating CSE stimulated RPMI 2650 cells with carbocysteine. In conclusion, the present study provides compelling evidence that carbocysteine may be considered a promising therapeutic strategy in chronic inflammatory nasal diseases.

Apigenin affects leptin/leptin receptor pathway and induces cell apoptosis in lung adenocarcinoma cell line

BACKGROUNDnApigenin, a common edible plant flavonoid, is a well characterised antioxidant. The adipokine leptin exerts proliferative and anti-apoptotic activities in a variety of cell types. In cancer cells, apigenin may induce a pro-apoptotic pathway whereas leptin has an anti-apoptotic role. The purpose of the study is to investigate the role of apigenin and of leptin/leptin receptor pathway on proliferation and on apoptosis in lung adenocarcinoma.nnnMETHODSnImmunocytochemistry, flow cytometry and RT-q-RT PCR, were used to investigate the expression and modulation of leptin receptors on the lung adenocarcinoma cell line A549 in presence or absence of apigenin and of leptin, alone or combined. Clonogenic test to evaluate cell proliferation was assessed. Exogenous leptin binding to its receptors by flow cytometry, reactive oxygen species (ROS) by dichlorofluorescein diacetate analysis, cell death by ethidium bromide and apoptosis by annexin V analysis were assessed. Apoptosis was assessed also in presence of lung adenocarcinoma pleural fluids (PF) (n=6).nnnRESULTSnA549 express leptin/leptin receptor pathway and its expression is upregulated by apigenin. Apigenin alone or combined with leptin significantly decreases cell proliferation and significantly increases the spontaneous release of ROS, with augmented cell death and apoptosis, this latter also in the presence of lung adenocarcinoma PF. Leptin alone significantly increases cell proliferation and significantly decreases cell death.nnnCONCLUSIONSnThese results strongly suggest the potential utility of the flavonoid apigenin in the complementary therapeutic approach of patients with lung adenocarcinoma.

Carbocysteine regulates innate immune responses and senescence processes in cigarette smoke stimulated bronchial epithelial cells.

Cigarette smoke represents the major risk factor for chronic obstructive pulmonary disease (COPD). Cigarette smoke extracts (CSE) alter TLR4 expression and activation in bronchial epithelial cells. Carbocysteine, an anti-oxidant and mucolytic agent, is effective in reducing the severity and the rate of exacerbations in COPD patients. The effects of carbocysteine on TLR4 expression and on the TLR4 activation downstream events are largely unknown. This study was aimed to explore whether carbocysteine, in a human bronchial epithelial cell line (16-HBE), counteracted some pro-inflammatory CSE-mediated effects. In particular, TLR4 expression, LPS binding, p21 (a senescence marker), IL-8 mRNA and release in CSE-stimulated 16-HBE as well as actin reorganization in neutrophils cultured with supernatants from bronchial epithelial cells which were stimulated with CSE and/or carbocysteine were assessed. TLR4 expression, LPS binding, and p21 expression were assessed by flow cytometry, IL-8 mRNA by Real Time PCR and IL-8 release by ELISA. Actin reorganization, a prerequisite for cell migration, was determined using Atto 488 phalloidin in neutrophils by flow cytometry and fluorescence microscopy. CSE increased: (1) TLR4, LPS binding and p21 expression; (2) IL-8 mRNA and IL-8 release due to IL-1 stimulation; (3) neutrophil migration. Carbocysteine in CSE stimulated bronchial epithelial cells, reduced: (1) TLR4, LPS binding and p21; (2) IL-8 mRNA and IL-8 release due to IL-1 stimulation; (3) neutrophil chemotactic migration. In conclusion, the present study provides compelling evidences that carbocysteine may contribute to control the inflammatory and senescence processes present in smokers.

In vitro anticholinergic drugs affect CD8+ peripheral blood T-cells apoptosis in COPD

Novel pharmacological strategies are aimed at the resolution of systemic inflammation in COPD potentiating peripheral blood T-cell (PBT-cell) apoptosis. Although muscarinic acetylcholine receptors (mAChRs) M(3) and choline-acetyltransferase (ChAT) participate in the airway inflammation of COPD, their role in PBT-cell apoptosis remains unexplained. We evaluated in PBT-cells from COPD patients, smoker (S) and control (C) subjects: (1) apoptosis (by annexin V binding), (2) mAChR M(3) and ChAT expression, acetylcholine (ACh)-binding; (3) choline levels in serum and PBT-cells extracts. We tested the effects of Tiotropium (Spiriva(®)) and hemicholinium-3 (HCh-3) on apoptosis, NFκB pathway, caspases 3 and 8 activity and choline levels, in PBT-cells from COPD patients. We showed that: (1) apoptosis, mAChR M(3) and ChAT expression and the CD3+ and CD8+ ACh-binding are increased in PBT-cells from COPD patients when compared to C subjects, while CD4+/CD8+ ratio of ACh-binding to PBT cells was reduced in COPD; (2) choline levels are higher in serum and PBT-cells extracts from COPD patients than in S and C; (3) Tiotropium and HCh-3 reduced CD4+ and increased CD8+ apoptosis via caspases 3 and 8 activities and via IκB mediated mechanisms in COPD patients. This study suggests the involvement of non-neuronal components of cholinergic system in the regulation of PBT-cell apoptosis in COPD and demonstrates that Tiotropium regulates CD4+ and CD8+ PBT-cell apoptosis. It provides novel putative pharmacological targets for the resolution of systemic inflammation in COPD.