Anna Bonanno

Muscarinic receptors, leukotriene B4 production and neutrophilic inflammation in COPD patients.

Background:  Acetylcholine (ACh) plays an important role in smooth muscle contraction and in the development of airway narrowing; preliminary evidences led us to hypothesize that ACh might also play a role in the development of airways inflammation in chronic obstructive pulmonary disease (COPD).

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.

Increased prostaglandin E2 concentrations and cyclooxygenase-2 expression in asthmatic subjects with sputum eosinophilia

BACKGROUND Prostaglandin E2 (PGE2) is known to be produced within human airways, but it is not clear whether in airway diseases it can play a deleterious or a beneficial role. Recently it has been reported that PGE2 can enhance eosinophil survival in vitro. OBJECTIVE To evaluate whether the concentrations of PGE2 in asthmatic airways correlate with the number of eosinophils and can be responsible for eosinophil-enhanced survival and to identify the cyclooxygenase isoform contributing to the synthesis of PGE2 by cells present in asthmatic airways. METHODS Reversed-phase high-performance liquid chromatography and/or specific radioimmunoassay was used to measure PGE2 concentrations in induced sputum supernatants from 14 control and 30 asthmatic subjects. Correlations between concentrations of PGE2 and the number of eosinophils in induced sputum were evaluated. Expression of cyclooxygenase-2 (COX-2) in induced sputum cells was determined by immunocytochemistry, and the effect of COX-2 inhibition on PGE2 production was evaluated with the use of radiolabeled arachidonic acid. The effects on eosinophil apoptosis by PGE2 or induced sputum supernatants were studied by using peripheral blood eosinophils obtained by negative immunomagnetic selection. RESULTS PGE2 concentrations resulted in elevated samples from asthmatic subjects and directly correlated with the percentage of eosinophils and the concentrations of eosinophilic cationic protein. Immunostaining for COX-2 showed enhanced expression in macrophages of asthmatic subjects when compared with control subjects, and the use of a specific COX-2 inhibitor provided evidence that PGE2 synthesis was the result of COX-2 enzymatic activity in asthma-induced sputum cells. Supernatant from induced sputum of asthmatic subjects with high eosinophil counts caused a decreased apoptosis of peripheral blood eosinophils when compared with control subjects, and immunoprecipitation of PGE2 significantly reverted this phenomenon, suggesting that PGE2 was present in biologically relevant concentrations in induced sputum. CONCLUSIONS The results obtained suggest that COX-2 expression in alveolar macrophages from asthmatic subjects may contribute to enhanced eosinophil survival through an increased PGE2 production.

Increased airway inflammatory cells in endurance athletes: what do they mean?

Background Inflammatory cells are increased in the airways of endurance athletes, but their role in causing exercise‐induced respiratory symptoms and bronchoconstriction, or their possible long‐term consequences, are uncertain.

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.

Airway cells after swimming outdoors or in the sea in nonasthmatic athletes.

BACKGROUND Marathon runners and elite swimmers showed increased inflammatory cells in the airways at baseline. Although airway neutrophils increase further after a marathon race, the airway response to swimming is unknown. The aim of this study was to assess the effects of swimming on airway cells. To avoid the concomitant effects of chronic exposure to chlorine, the study was conducted in seven nonasthmatic swimmers [mean age (SD): 23.3 +/- 7.7 yr, training: 32 +/- 15 km.wk-1] habitually training in an outdoor pool (OP), i.e., a low-chlorine environment. METHODS Spirometry, exhaled nitric oxide (NO), induced sputum, and peripheral blood samples were obtained at baseline, after a 5-km trial in OP, and after a 5-km race in the sea (S), i.e., hypertonic airway exposure. RESULTS Airway neutrophil differential counts at baseline were higher in swimmers than in sedentary controls (N = 10), but cell counts, neutrophil elastase, and eosinophil cationic protein were unaffected by 5-km swimming. After swimming, L-selectin expression on airway cells decreased, suggesting exercise-induced cell mobilization into the airways and/or direct effects of hyperventilation on airway cells. After S, airway eosinophil differential counts increased slightly. Exhaled NO concentration was 19 +/- 6 ppb at baseline, 8 +/- 4 ppb after OP, and 21 +/- 7 ppb after S (P < 0.005 for OP vs baseline and S). CONCLUSIONS In swimmers not chronically exposed to high chlorine concentrations, data obtained at baseline suggest a direct relationship between airway neutrophilia and endurance training. The low L-selectin expression by airway cells postexercise suggests hyperventilation-induced cell recruitment or modulation of cell function. Hypertonic exposure of airways during exercise may slightly increase airway eosinophils and exhaled NO. Overall, 5-km swimming exerted smaller effects on airway cells than running a marathon.

Effects of Exercise Training and Montelukast in Children with Mild Asthma

PURPOSE Data from the general population suggest that habitual exercise decreases bronchial responsiveness, but the possible role of exercise in asthmatics is undefined. The leukotriene receptor antagonist montelukast decreases bronchial responsiveness and exercise-induced symptoms in asthmatic children. This randomized study in children with mild asthma evaluated the combined effects of aerobic training for 12 wk and montelukast or placebo on bronchial responsiveness (BHR) to methacholine, exercise-induced bronchoconstriction (EIB), inflammatory markers in exhaled breath condensate (EBC), and asthma exacerbations. METHODS Fifty children (mean age +/- SD: 10.2 +/- 2.4 yr) with mild stable asthma were randomly assigned to placebo (N = 25) or montelukast (N = 25). Before and after training, we assessed BHR and EIB and markers of airway inflammation-that is, exhaled nitric oxide (eNO), pH, and cysteinyl-leukotriene concentration-in EBC. RESULTS Training increased maximal workload and peak minute ventilation. After training, the methacholine dose causing a 20% fall in FEV1 (PD20) increased in both groups. A decreased slope of FEV1 decline at increasing methacholine dose was found only in montelukast-treated children. EIB prevalence halved after training in both groups (EIB + children, placebo group: 10 pretraining, 4 posttraining; EIB + children, montelukast group: 8 pretraining, 5 posttraining; P < 0.05 by chi on all children). Resting eNO was unaffected, whereas the pH of EBC decreased after training in both groups. Cysteinyl-leukotriene concentrations were low in most children at both times. During training, montelukast-treated children showed fewer asthma exacerbations compared with the same period of the previous year. CONCLUSIONS In children with mild stable asthma, exercise training decreased bronchial responsiveness to methacholine. Montelukast also decreased bronchial reactivity (FEV1 slope) and protected against exacerbations, suggesting a beneficial synergistic action of these two interventions in mild asthma.

Urinary leukotriene E4 in the assessment of nocturnal asthma

BACKGROUND Urinary leukotriene E4 (LTE4) is a marker of the bodys production of cysteinyl LTs, important mediators of airway inflammation. The role of the latter in nocturnal asthma is a topic of increasing interest. OBJECTIVE This investigation was aimed at determining whether nighttime attacks are associated with increased release of LTs, expressed by urinary LTE4, and the relationship between the two phenomena. METHODS Three groups were studied: group A, seven control subjects; group B, nine asthmatic patients without nocturnal attacks; and group C, nine asthmatic patients with a comparable daytime FEV1 but who were experiencing nocturnal exacerbations (morning dips in peak expiratory flow greater than 20%). Urine was collected over 24 hours in three samples (9:00 AM to 3:00 PM; 3:00 PM to 9:00 PM; and 9:00 PM to 9:00 AM). LTE4 was measured by high-performance liquid chromatography and radioimmunoassay and expressed as nanograms per millimole of creatinine. RESULTS No significant differences between urinary LTE4 were noticed within groups A and B. Conversely, in group C urinary LTE4 at night (geometric mean with 95% confidence interval; 35.16 with 28.77-42.85) was significantly higher than that of the other samples (respectively 23.12 with 17.78-30.06, p less than 0.05; and 25.18 with 21.03-30.13, p less than 0.02); it was also significantly higher than in all the samples of other groups. A significant (p less than 0.02) linear correlation was observed between morning dip in peak expiratory flow and the log urinary LTE4 in the nocturnal sample. CONCLUSION These results indicate the role of LTs in nocturnal asthma and suggest that urinary LTE4 may be a useful marker of this condition.

Hemopoietic and angiogenetic progenitors in healthy athletes: different responses to endurance and maximal exercise

The effects of endurance or maximal exercise on mobilization of bone marrow-derived hemopoietic and angiogenetic progenitors in healthy subjects are poorly defined. In 10 healthy amateur runners, we collected venous blood before, at the end of, and the day after a marathon race (n = 9), and before and at the end of a 1.5-km field test (n = 8), and measured hemopoietic and angiogenetic progenitors by flow cytometry and culture assays, as well as plasma or serum concentrations of several cytokines/growth factors. After the marathon, CD34(+) cells were unchanged, whereas clonogenetic assays showed decreased number of colonies for both erythropoietic (BFU-E) and granulocyte-monocyte (CFU-GM) series, returning to baseline the morning post-race. Conversely, CD34(+) cells, BFU-E, and CFU-GM increased after the field test. Angiogenetic progenitors, assessed as CD34(+)KDR(+) and CD133(+)VE-cadherin(+) cells or as adherent cells in culture expressing endothelial markers, increased after both endurance and maximal exercise but showed a different pattern between protocols. Interleukin-6 increased more after the marathon than after the field test, whereas hepatocyte growth factor and stem cell factor increased similarly in both protocols. Plasma levels of angiopoietin (Ang) 1 and 2 increased after both types of exercise, whereas the Ang-1-to-Ang-2 ratio or vascular endothelial growth factor-A were little affected. These data suggest that circulating hemopoietic progenitors may be utilized in peripheral tissues during prolonged endurance exercise. Endothelial progenitor mobilization after exercise in healthy trained subjects appears modulated by the type of exercise. Exercise-induced increase in growth factors suggests a physiological trophic effect of exercise on the bone marrow.