Fabio Luigi Massimo Ricciardolo

Multiple roles of nitric oxide in the airways

Nitric oxide is endogenously released in the airways by nitric oxide synthase. Functionally, two isoforms of this enzyme exist: constitutive and inducible. The former seems to protect airways from excessive bronchoconstriction while the latter has a modulatory role in inflammatory disorders of the airways such as asthma. This review explores the physiological and pathophysiological role of endogenous nitric oxide in the airways, and the clinical aspects of monitoring nitric oxide in exhaled air of patients with respiratory disease.

Cellular and molecular mechanisms in chronic obstructive pulmonary disease: an overview

In the last decade, the analysis of bronchial biopsies and lung parenchyma obtained from chronic obstructive pulmonary disease (COPD) patients compared with those from smokers with normal lung function and non‐smokers has provided new insights on the role of the different inflammatory and structural cells, their signalling pathways and mediators, contributing to a better knowledge of the pathogenesis of COPD. This review summarizes and discusses the lung pathology of COPD patients with emphasis on inflammatory cell phenotypes that predominate in different clinical conditions. In bronchial biopsies, a cascade of events takes place during progression from mild‐to‐severe disease. T lymphocytes, particularly CD8+ cells and macrophages are the prevalent inflammatory cells in the lung of healthy smokers and patients with mild COPD, while total and activated neutrophils predominate in severe COPD. The number of CD4+, CD8+ cells and macrophages expressing nuclear factor‐kappa B (NF‐κB), STAT‐4 and IFN‐γ proteins as well as endothelial adhesion molecule‐1 in endothelium is increased in mild/moderate disease. In contrast, activated neutrophils (MPO+ cells) and increased nitrotyrosine immunoreactivity develops in severe COPD. In bronchial biopsies obtained during COPD exacerbations, some studies have shown an increased T cell and granulocyte infiltration. Regular treatment with high doses of inhaled glucocorticoids does not significantly change the number of inflammatory cells in bronchial biopsies from patients with moderate COPD. The profile in lung parenchyma is similar to bronchial biopsies. ‘Healthy’ smokers and mild/moderate diseased patients show increased T lymphocyte infiltration in the peripheral airways. Pulmonary emphysema is associated with a general increase of inflammatory cells in the alveolar septa. The molecular mechanisms driving the lymphocyte and neutrophilic prevalence in mild and severe disease, respectively, needs to be extensively studied. Up‐regulation of pro‐inflammatory transcription factors NF‐κB and STAT‐4 in mild, activated epithelial and endothelial cells in the more severe disease may contribute to this differential prevalence of infiltrating cells.

Randomised double-blind placebo-controlled study of the effect of inhibition of nitric oxide synthesis in bradykinin-induced asthma

BACKGROUND Bronchoconstriction induced by bradykinin is reduced by the release of nitric oxide (NO) in the airways of guinea pigs. Inhaled NO is known to cause bronchodilatation in asthmatic patients. To find out the role of endogenous NO in airway response to bradykinin in asthma, we examined the effect of the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) on broncho-constriction after bradykinin challenge in ten patients with mild asthma. METHODS The study had a randomised, double-blind, placebo-controlled, cross-over design. Participants were studied during two phases, each consisting of 2 study days. After baseline measurements of forced expiratory volume in 1 s (FEV1) participants inhaled an aerosol of L-NMMA or saline (placebo). After 5 min, saline and doubling doses of bradykinin (from 0.25 nmol) were inhaled until FEV1 fell by at least 20% of the post-saline value. The effect of L-NMMA and placebo on airway response to doubling concentrations of methacholine (from 0.03 mg/mL) was then examined. We also assessed the effect of the inactive enantiomer of L-NMMA, D-NMMA, and placebo on bronchoconstriction after bradykinin or methacholine challenge in six of the participants. FINDINGS The geometric mean of the provocative dose producing a 20% fall in FEV1 to bradykinin was 138.0 nmol (range 48.2-475.2 nmol) after placebo and 11.2 nmol (range 0.9-51.3 nmol) after L-NMMA (p < 0.01). L-NMMA also caused a decrease in the provocative concentration of methacholine producing a 20% fall in FEV1 from 0.93 mg/mL (range 0.12-2.55 mg/mL) to 0.38 mg/mL (range 0.06-0.92 mg/mL; p < 0.01). In contrast, D-NMMA did not affect airway response to bradykinin or methacholine. INTERPRETATION The results suggest that bronchoconstriction after bradykinin inhalation is greatly inhibited by the formation of NO in airways of asthmatic patients and that NO could have a bronchoprotective role in asthma.

STAT4 activation in smokers and patients with chronic obstructive pulmonary disease

Activation of the transcription factor signal transducer and activator of transcription (STAT)‐4 is critical for the differentiation of T‐helper 1 cells/type‐1 cytotoxic T‐cells and the production of interferon (IFN)‐γ. Expression of STAT4, phospho‐STAT4, IFN‐γ and T‐box expressed in T‐cells (T‐bet) proteins in bronchial biopsies and bronchoalveolar lavage (BAL)‐derived lymphocytes, obtained from 12 smokers with mild/moderate chronic obstructive pulmonary disease (COPD) (forced expiratory volume in one second (FEV1) 59±16% predicted), 14 smokers with normal lung function (FEV1 106±12% pred) and 12 nonsmoking subjects (FEV1 111±14% pred), was examined by immunohistochemistry and immunocytochemistry. In bronchial biopsies of COPD patients, the number of submucosal phospho‐STAT4+ cells was increased (240 (22–406) versus 125 (0–492) versus 29 (0–511) cells·mm−2) when compared with both healthy smokers and control nonsmokers, respectively. In smokers, phospho‐STAT4+ cells correlated with the degree of airflow obstruction and the number of IFN‐γ+ cells. Similar results were seen in BAL (2.8 (0.2–5.9) versus 1.03 (0.09–1.6) versus 0.69 (0–2.3) lymphocytes·mL−1×103). In all smokers who underwent lavage, phospho‐STAT4+ lymphocytes correlated with airflow obstruction and the number of IFNγ+ lymphocytes. T‐bet expression was not altered in bronchial biopsies and BAL‐derived lymphocytes between the three groups. In conclusion, this study suggests that stable mild/moderate chronic obstructive pulmonary disease is associated with an active T‐helper 1 cell/type‐1 cytotoxic T‐cell inflammatory process involving activation of signal transducer and activator of transcription 4 and interferon-gamma production.

High serum levels of tumour necrosis factor‐α and interleukin‐8 in severe asthma: markers of systemic inflammation?

Background Severe asthma is characterized by elevated levels of pro‐inflammatory cytokines and neutrophilic inflammation in the airways. Blood cytokines, markers of ‘systemic’ inflammation, may be a feature of amplified inflammation in severe asthma.

Serum interleukin-17 levels are related to clinical severity in allergic rhinitis.

Background:  T helper (Th)‐17 cells are a subset of T helper lymphocytes that exert regulatory activities. Recently, it has been reported that serum interleukin (IL)‐17 levels are high in the most severe cases of birch allergy studied outside the pollen season.

Mechanisms of citric acid-induced bronchoconstriction

In asthma patients, microaspiration of acid into the lower airways (ie, airway acidification) causes such respiratory responses as cough and bronchoconstriction. The mechanism of bronchoconstriction induced by airway acidification is unknown, although evidence is emerging that increasing proton concentrations in airway tissues can activate a subpopulation of primary sensory neurons, so-called capsaicin-sensitive primary sensory neurons, that contain such neuropeptides as the tachykinins substance P (SP) and neurokinin A (NKA). Protons activate a capsaicin-operated channel/receptor, located in the afferents of capsaicin-sensitive neurons, with the subsequent opening of ion channels that are permeable to sodium, potassium, and calcium ions. This event initiates a propagated action potential that antidromically depolarizes collateral fibers and triggers neuropeptide release from nerve fiber varicosities. The tachykinins SP and NKA, released from terminals of primary sensory neurons in peripheral tissues, cause all the major signs of inflammation (neurogenic inflammation) by means of activation of NK(1) and NK(2) receptors. Exposure of the airways to acidic solutions stimulates sensory nerve endings of capsaicin-sensitive sensory neurons and causes different airway responses, including bronchoconstriction. Recently, the NK(2), and to a lesser extent the NK(1), receptors have been shown to be involved with citric acid-induced bronchoconstriction in the guinea pig, which is in part mediated by endogenously released bradykinin. Tachykinins and bradykinin, released by airway acidification, could also modulate citric acid-induced bronchoconstriction by their ability to subsequently release the epithelially derived bronchoprotective nitric oxide (NO). Further study with selective tachykinin NK(1) and NK(2) agonists demonstrated that only the septide-insensitive tachykinin NK(1) receptor releases NO. Thus, bronchoconstriction induced by citric acid inhalation in the guinea pig, mainly caused by the tachykinin NK(2) receptor, is counteracted by bronchoprotective NO after activation of bradykinin B(2) and tachykinin NK(1) receptors in airway epithelium. If a similar mechanism is involved in the pathogenesis of bronchial asthma associated with gastroesophageal reflux in the respiratory tract, new therapeutic strategies should be investigated.

Association of increased CCL5 and CXCL7 chemokine expression with neutrophil activation in severe stable COPD

Background: Increased numbers of activated neutrophils have been reported in the bronchial mucosa of patients with stable chronic obstructive pulmonary disease (COPD), particularly in severe disease. Objectives: To investigate the expression of neutrophilic chemokines and adhesion molecules in bronchial biopsies from patients with stable COPD of different severity (GOLD stages I–IV) compared with age-matched control subjects, smokers with normal lung function and never smokers. Methods: The expression of CCL5, CXCL1, 5, 6, 7 and 8, CXCR1, CXCR2, CD11b and CD44 was measured in the bronchial mucosa using immunohistochemistry, confocal immunofluorescence, real-time quantitative polymerase chain reaction (RT-QPCR) and Western blotting (WB). Results: The numbers of CCL5+ epithelial cells and CCL5+ and CXCL7+ immunostained cells were increased in the bronchial submucosa of patients with stable severe COPD compared with control never smokers and smokers with normal lung function. This was also confirmed at the level of mRNA expression. The numbers of CCL5+ cells in the submucosa of patients with COPD were 2–15 times higher than any other chemokines. There was no correlation between the number of these cells and the number of neutrophils in the bronchial submucosa. Compared with control smokers, the percentage of neutrophils co-expressing CD11b and CD44 receptors was significantly increased in the submucosa of patients with COPD. Conclusion: The increased expression of CCL5 and CXCL7 in the bronchial mucosa of patients with stable COPD, together with an increased expression of extracellular matrix-binding receptors on neutrophils, may be involved in the pathogenesis of COPD.

Nitric oxide synthase (NOS) as therapeutic target for asthma and chronic obstructive pulmonary disease.

In the respiratory tract, NO is produced by residential and inflammatory cells. NO is generated via oxidation of L-arginine that is catalysed by the enzyme NO synthase (NOS). NOS exists in three distinct isoforms: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). NO derived from the constitutive isoforms of NOS (nNOS and eNOS) and other NO-adduct molecules (nitrosothiols) are able to modulate bronchomotor tone. NO derived from the inducible isoform of NO synthase, up-regulated by different cytokines via NF-kappaB-dependent pathway, seems to be a pro-inflammatory mediator with immunomodulatory effects. The production of NO under oxidative stress conditions secondarily generates strong oxidising agents (reactive nitrogen species) that may amplify the inflammatory response in asthma and COPD. Moreover, NO can be exhaled and levels are abnormal in stable atopic asthma and during exacerbations in both asthma and COPD. Exhaled NO might therefore be a non-invasive tool to monitor the underlying inflammatory process. It is suggested that NOS regulation provides a novel target in the prevention and treatment of chronic inflammatory diseases of the airways such as asthma and COPD.

Evidence for reduction of bradykinin-induced bronchoconstriction in guinea-pigs by release of nitric oxide

1 In this study the influence of nitric oxide (NO) on the bronchoconstriction induced by bradykinin in anaesthetized and artifically ventilated guinea‐pigs pretreated with atropine was investigated. 2 Aerosol administration of bradykinin (0.1 −1 mm, 40 breaths) caused a dose‐dependent increase in lung resistance (R1): maximum increase in R1 was 2.5 fold the baseline value. Pretreatment with aerosolized NG‐nitro‐1‐arginine methyl ester (1‐NAME) or NG‐monomethyl‐1‐arginine (1‐NMMA) (1 mm, 10 breaths every 5min for 30min), NO synthase inhibitors, markedly increased the bron‐choconstrictor response to bradykinin. 1‐Arginine, but not D‐arginine, (3 mm, 10 breaths every 5 min for 30 min) reversed the hyperresponsiveness to aerosolized bradykinin caused by 1‐NAME and 1‐NMMA. 3 1‐NAME (1 mm, 10 breaths every 5 min for 30 min) increased the bronchoconstriction induced by intravenous bradykinin (1‐10nmol kg1). 1‐Arginine, but not D‐arginine, (10 breaths every 5 min for 30 min) reversed the hyperresponsiveness to intravenous bradykinin caused by 1‐NAME. 4 The increase in RL induced by capsaicin, either aerosol (10 μm, 10 breaths) or i.v. (20 nmol kg−1) was not affected by 1‐NAME (1 mm, 10 breaths every 5 min for 30 min). Acute resection of the vagi did not affect the bronchoconstriction evoked by bradykinin in guinea‐pigs, either in the absence or presence of 1‐NAME (1 mm, 10 breaths every 5 min for 30 min). 4 These results suggest that, irrespective of the route of administration, bradykinin releases NO or a related molecule which exerts a bronchodilator action that opposes the bronchoconstrictor mechanisms activated by bradykinin itself.