Role of NADPH oxidase/ROS in pro-inflammatory mediators-induced airway and pulmonary diseases

Reactive oxygen species (ROS) are products of normal cellular metabolism and are known to act as second messengers. Under physiological conditions, ROS participate in maintenance of cellular 'redox homeostasis' in order to protect cells against oxidative stress. In addition, regulation of redox state is important for cell activation, viability, proliferation, and organ function. However, overproduction of ROS, most frequently due to excessive stimulation of either reduced nicotinamide adenine dinucleotide phosphate (NADPH) by pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) or the mitochondrial electron transport chain and xanthine oxidase, results in oxidative stress. Oxidative stress is a deleterious process that leads to airway and lung damage and consequently to several respiratory inflammatory diseases/injuries, including acute respiratory distress syndrome (ARDS), asthma, cystic fibrosis (CF), and chronic obstructive pulmonary disease (COPD). Many of the known inflammatory target proteins, such as matrix metalloproteinase-9 (MMP-9), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), cyclooxygenase-2 (COX-2), and cytosolic phospholipase A(2) (cPLA(2)), are associated with NADPH oxidase activation and ROS overproduction in response to pro-inflammatory mediators. Thus, oxidative stress regulates both key inflammatory signal transduction pathways and target proteins involved in airway and lung inflammation. In this review, we discuss mechanisms of NADPH oxidase/ROS in the expression of inflammatory target proteins involved in airway and lung diseases. Knowledge of the mechanisms of ROS regulation could lead to the pharmacological manipulation of antioxidants in airway and lung inflammation and injury.     

Oxidative stress and lung inflammation in airways disease.

Oxidative stress results from an oxidant/antioxidant imbalance in favour of oxidants. A large number of studies have demonstrated that increased oxidative burden occurs in airways diseases, shown by increased marks of oxidative stress in the airspaces and systemically in these patients. There is now substantial evidence that oxidative stress plays an important role in the injurious and inflammatory responses in airways diseases such as asthma and chronic obstructive pulmonary disease (COPD). In addition to these proinflammatory mechanisms resulting from oxidative stress, protective mechanisms such as the upregulation of protective antioxidant genes also occur. At present, effective antioxidant therapy that has good bioavailability and potency is not available. Such drugs are being developed and should in the future allow the hypothesis that oxidative stress is a fundamental factor in the inflammation, which occurs in these airways diseases to be tested.     

Airway oxidative stress causes vascular and hepatic inflammation via upregulation of IL-17A in a murine model of allergic asthma

Oxidants are generated in asthmatic airways due to infiltration of inflammatory leukocytes and resident cells in the lung. Reactive oxygen species (ROS) such as hydrogen peroxide and superoxide radical may leak into systemic circulation when generated in uncontrolled manner and may impact vasculature. Our previous studies have shown an association between airway inflammation and systemic inflammation; however so far none has investigated the impact of airway oxidative inflammation on hepatic oxidative stress and Th1/Th2/Th17 cytokine markers in liver/vasculature in a murine model of asthma. Therefore, this study investigated the contribution of oxidative stress encountered in asthmatic airways in modulation of systemic/hepatic Th1/Th2/Th17 cytokines balance and hepatic oxidative stress. Mice were sensitized intraperitoneally with cockroach extract (CE) in the presence of aluminum hydroxide followed by several intranasal (i.n.) challenges with CE. Mice were then assessed for systemic/hepatic inflammation through assessment of Th1/Th2/Th17 cytokines and oxidative stress (iNOS, protein nitrotyrosine, lipid peroxides and myeloperoxidase activity). Challenge with CE led to increased Th2/Th17 cytokines in blood/liver and hepatic oxidative stress. However, only Th17 related pro-inflammatory markers were upregulated by hydrogen peroxide (H₂O₂) inhalation in vasculature and liver, whereas antioxidant treatment, N-acetyl cysteine (NAC) downregulated them. Hepatic oxidative stress was also upregulated by H₂O₂ inhalation, whereas NAC attenuated it. Therefore, our study shows that airway oxidative inflammation may contribute to systemic inflammation through upregulation of Th17 immune responses in blood/liver and hepatic oxidative stress. This might predispose these patients to increased risk for the development of cardiovascular disorders.     

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.     

Oxidants in asthma and in chronic obstructive pulmonary disease (COPD)

Experimental and clinical evidences suggest that oxidants play a role in the pathogenesis of respiratory disorders characterised by chronic airway inflammation such as asthma and chronic obstructive pulmonary disease (COPD). The respiratory system is chronically exposed to environmental pollutants, including oxidants. Exogenous sources of oxidants are particularly relevant to the pathogenesis of COPD, being cigarette smoke an extremely rich source of oxidants. In addition, the inflammatory cells recruited to the airways of patients with asthma and COPD, have an exceptional capacity to produce oxidants. Many decades of research have produced a significant amount of data indicating pro-oxidative molecular mechanisms putatively relevant in the pathogenesis of the oxidative stress which characterises these diseases, both locally and systemically. As a consequence, a drug therapy able to restore the redox imbalance in asthma and COPD would probably exert clinical and functional benefits. Indeed, currently available therapies for asthma and COPD can exert an inhibitory effect on oxidant production in the airways. However, it is unknown whether the efficacy of the treatment is somehow linked to the pharmacological modulation of the oxidant/antioxidant balance. So far, it appears that the potential role of antioxidant compounds in the treatment of asthma and COPD has not been fully explored.     

Adenosine receptors as promising therapeutic targets for drug development in chronic airway inflammation

A growing body of evidence has emerged in support of a pro-inflammatory role for adenosine in the pathogenic mechanisms of chronic inflammatory disorders of the airways such as asthma and COPD. The demonstration that adenosine enhances mast cell allergen-dependent activation, the notion that elevated levels of adenosine are present in chronically inflamed airways, and the results from exposure studies of nebulised adenosine showing dose-dependent bronchoconstriction in subjects with asthma and COPD, emphasise the importance of adenosine in the initiation, persistence and progression in these common inflammatory disorders of the airways. Adenosine exerts its manifold biological activities by interacting with at least four adenosine receptor subtypes. Selective activation or blockade of these sites is being exploited by the pharmaceutical industry in an attempt to generate novel therapies for asthma and COPD. This review article intends to address the potential role of adenosine in asthma and to put forward the idea that drugs that have been developed to selectively activate or downregulate adenosine receptor subtypes may constitute a considerable advance in the management of chronic airway inflammation.     

Anti-asthmatic effect of schizandrin on OVA-induced airway inflammation in a murine asthma model

Asthma comprises a triad of reversible airway obstruction, bronchial smooth muscle cell hyperreactivity to bronchoconstrictors, and chronic bronchial inflammation. Clinical and experimental findings have established eosinophilia as a sign of allergic disorders. In the present investigation, we evaluated the anti-asthmatic effects of schizandrin and its underlying mechanisms in an in vivo murine asthmatic model. To accomplish this, female BALB/c mice were sensitized and challenged with ovalbumin (OVA), and examined for the following typical asthmatic reactions: increased numbers of eosinophils and other inflammatory cells in bronchoalveolar lavage fluid (BALF); production of Th1 cytokines (such as tumor necrosis factor (TNF)-α in BALF); production of Th2 cytokines (such as interleukin IL-4 and IL-5) in BALF; presence of total and OVA-specific immunoglobulins (Ig)E in serum; presence of oxidative stress; hyperplasia of goblet cells in the lung; and marked influx of inflammatory cells into the lung. Our results collectively show that schizandrin exerts profound inhibitory effects on accumulation of eosinophils into the airways and reduces the levels of IL-4, IL-5, IFN-γ, and TNF-α in BALF. Additionally, schizandrin suppresses the production of reactive oxygen species (ROS) in a dose-dependent manner, and inhibits goblet cell hyperplasia and inflammatory cell infiltration in lung tissue. Thus, schizandrin has anti-asthmatic effects, which seem to be partially mediated by reduction of oxidative stress and airway inflammation, in a murine allergic asthma model. These results indicate that schizandrin may be an effective novel therapeutic agent for the treatment of allergic asthma.     

Inhibitors of ceramide de novo biosynthesis rescue damages induced by cigarette smoke in airways epithelia

Exposure to cigarette smoke represents the most important risk factor for the development of chronic obstructive pulmonary disease (COPD). COPD is characterized by chronic inflammation of the airways, imbalance of proteolytic activity resulting in the destruction of lung parenchyma, alveolar hypoxia, oxidative stress, and apoptosis. Sphingolipids are structural membrane components whose metabolism is altered during stress. Known as apoptosis and inflammation inducer, the sphingolipid ceramide was found to accumulate in COPD airways and its plasma concentration increased as well. The present study investigates the role of sphingolipids in the cigarette smoke-induced damage of human airway epithelial cells. Lung epithelial cells were pre-treated with sphingolipid synthesis inhibitors (myriocin or XM462) and then exposed to a mixture of nicotine, acrolein, formaldehyde, and acetaldehyde, the major toxic cigarette smoke components. The inflammatory and proteolytic responses were investigated by analysis of the mRNA expression (RT-PCR) of cytokines IL-1β and IL-8, and matrix metalloproteinase-9 and of the protein expression (ELISA) of IL-8. Ceramide intracellular amounts were measured by LC-MS technique. Ferric-reducing antioxidant power test and superoxide anion radical scavenging activity assay were used to assess the antioxidant power of the inhibitors of ceramide synthesis. We here show that ceramide synthesis is enhanced under treatment with a cigarette smoke mixture correlating with increased expression of inflammatory cytokines and matrix metalloproteinase 9. The use of inhibitors of ceramide synthesis protected from smoke induced damages such as inflammation, oxidative stress, and proteolytic imbalance in airways epithelia.

     

Production and degradation of oxygen metabolites during inflammatory states in the human lung

Lung represents a tissue that encounters a high oxidant burden but is also endowed with efficient protection against oxygen and reactive oxygen species (ROS). The oxidant stress experienced by the lung is enhanced by exogenous oxidant producing toxins most importantly pollutants and cigarette smoke, as well as by increased oxidant production during lung inflammation. The major oxidant generating enzymes present in human lung include NADPH oxidase, myeloperoxidase, eosinophil peroxidase and nitric oxide synthases, all of which are induced during inflammatory states. The antioxidant machinery of human lung against ROS is more versatile than often assumed. In addition to metal binding proteins, mucus components and small molecular weight antioxidants and vitamins, lung tissue possesses a highly cell specific and compartmentalized defense system containing several antioxidant enzymes with variable locations, inducibilities and kinetics. Inflammatory states like asthma, chronic obstructive lung disease (COPD) and parenchymal lung disorders have been shown to lead to serious disturbances in the oxidant/antioxidant balance of the lung with consequent oxidant mediated cell injury. Novel synthetic antioxidant mimetics may have the potential to slow or terminate the progression of lung diseases associated with free radicals.     

Gluthathione: in defence of the lung.

Oxidative stress is implicated in the pathology of numerous diseases of the lung. These include cystic fibrosis, chronic obstructive airway disease and asthma. All these conditions are characterised by an imbalance between the amounts of reactive oxygen species (ROS) and available antioxidant defences. In the lung, ROS arise from endogenous sources, such as the influx of inflammatory cells or exogenous sources, such as from air pollution and cigarette smoke. When ROS production increases the redox balance of the airways alters, and this can lead to bronchial hyperactivity and further inflammation. The lung, like many other tissues, has a range of antioxidant defences which help to maintain a balanced redox status. These antioxidants are present in the intracellular, the vascular and extracellular respiratory tract lining fluid (RTLF) compartments. The reduced glutathione (GSH) content of RTLF is particularly high and new findings are beginning to reveal the role that the RTLF GSH pool plays in defending the lung.     

Oxidative stress in patients with COPD

The lung is the organ with the highest exposure to ambient air in the entire human architecture. Due to its large surface area and blood supply, the lung is susceptible to oxidative injury in the form of myriads of reactive oxygen species (ROS) and free radicals. In order to provide defense against the oxidative burden, the lungs produce various endogenous agents called antioxidants. The antioxidant species help the lungs ward off the deleterious consequences of a wide variety of oxidants/ROS, either of endogenous or environmental origin. Several mechanisms are related to the potential connection between COPD and oxidative stress. One of the most important actions of the oxidative stress is the influence of the molecular mechanisms involved in the expression proinflammatory genes. There is plenty of evidence supporting an imbalance between oxidants and antioxidants in the lung and systemic circulation of smokers and COPD patients. Detection of the oxidative burden and evaluation of their progression and phenotypes by oxidative stress biomarkers have proven challenging and difficult. Both invasive and non-invasive techniques have provided different biomarkers which contribute to the oxidative burden of the airways. An effective wide-spectrum antioxidant therapy with bioavailability is urgently needed to control the local and systemic oxidative burst in COPD. In that direction, several antioxidant agents have been evaluated as potential candidates for the management of COPD. However, despite some encouraging results, clinical trials so far have failed to elaborately define the type of antioxidant, the regimen and the time period of treatment that may improve clinically meaningful outcomes in patients with COPD.     

Dual effects of antioxidants in neurodegeneration: direct neuroprotection against oxidative stress and indirect protection via suppression of glia-mediated inflammation

Oxidative stress, in which production of highly reactive oxygen species (ROS) and reactive nitrogen species (RNS) overwhelms antioxidant defenses, is a feature of many neurological diseases and neurodegeneration. ROS and RNS generated extracellularly and intracellularly by various processes initiate and promote neurodegeneration in CNS. ROS and RNS can directly oxidize and damage macromolecules such as DNA, proteins, and lipids, culminating in neurodegeneration in the CNS. Neurons are most susceptible to direct oxidative injury by ROS and RNS. ROS and RNS can also indirectly contribute to tissue damage by activating a number of cellular pathways resulting in the expression of stress-sensitive genes and proteins to cause oxidative injury. Moreover, oxidative stress also activates mechanisms that result in a glia-mediated inflammation that also causes secondary neuronal damage. Associated with neuronal injuries caused by many CNS insults is an activation of glial cells (particularly astrocytes and microglia) at the sites of injury. Activated glial cells are thus histopathological hallmarks of neurodegenerative diseases. Even though direct contact of activated glia with neurons per se may not necessarily be toxic, the immune mediators (e.g. nitric oxide and reactive oxygen species, pro-inflammatory cytokines and chemokines) released by activated glial cells are currently considered to be candidate neurotoxins. Therefore, study of the protective role of antioxidant compounds on inhibition of the inflammatory response and correcting the fundamental oxidant/antioxidant imbalance in patients suffering from neurodegenerative diseases are important vistas for further research. The purpose of this review is to summarize the current evidence in support of this critical role played by oxidative stress of neuronal and glial origin in neurodegenerative diseases. The mechanistic basis of the neuroprotective activity of antioxidants does not only rely on the general free radical trapping or antioxidant activity per se in neurons, but also the suppression of genes induced by pro-inflammatory cytokines and other mediators released by glial cells. We propose that combinations of agents which act at sequential steps in the neurodegenerative process can produce additive neuroprotective effects. A cocktail of multiple antioxidants with anti-inflammatory agents may be more beneficial in the prevention of neurodegenerative disease. A clearer appreciation of the potential therapeutic utility of antioxidants would emerge only when the complexity of their effects on mechanisms that interact to determine the extent of oxidative damage in vivo are more fully defined and understood.     

Adenosine in the airways: implications and applications

Adenosine in a signaling nucleoside eliciting many physiological responses. Elevated levels of adenosine have been found in bronchoalveolar lavage, blood and exhaled breath condensate of patients with asthma a condition characterized by chronic airway inflammation. In addition, inhaled adenosine-5'-monophosphate induces bronchoconstriction in asthmatics but not in normal subjects. Studies on animals and humans have shown that bronchoconstriction is most likely due to the release of inflammatory mediators from mast cells. However a number of evidences suggest that adenosine modulates the function of many other cells involved in airway inflammation such as neutrophils, eosinophils, lymphocytes and macrophages. Although this clear pro-inflammatory role in the airways, adenosine may activate also protective mechanisms particularly against lung injury. For many years this dual role of adenosine in the respiratory system has represented an enigma, and only recently it has become clear that biological functions of adenosine are mediated by four distinct subtypes of receptors (A1, A2A, A2B, and A3) and that biological responses are determined by the different pattern of receptors distribution in specific cells. Therefore, pharmacological modulation of adenosine receptors, particularly A2B, may represent a novel therapeutic approach for inflammatory diseases. Moreover, as bronchial response to adenosine strictly reflects airway inflammation in asthma, bronchial challenge with adenosine is considered a valuable clinical tool to monitor airway inflammation, to follow the response to anti-inflammatory treatments and to help in the diagnostic discrimination between asthma and chronic obstructive lung disease.     

Neurogenic inflammation and asthma

Over the past number of decades there has been considerable interest in the role of neurogenic inflammation in asthma with the identification of many biologically active neuropeptides in the lung. Whilst there is convincing evidence of neurogenic inflammation in various animal models of asthma, the evidence in humans is less clear and replicating the experimental approaches in humans has proven difficult with different studies producing conflicting results. In terms of human studies, research has focused on whether pro-inflammatory neuropeptides are elevated in the asthmatic airway, and if so, what their functional effects are. There have also been studies to assess the efficacy of tachykinin receptor antagonists in improving indices of asthma control. Information to date would suggest that neuropeptides are present in human airways and are possibly upregulated in asthma, but this effect does not appear to be specific and may occur in other inflammatory airways conditions (chronic obstructive pulmonary disease (COPD) and smoking). At present there is insufficient evidence to suggest that tachykinin receptor antagonists confer any additional benefit over inhaled corticosteroid regimes for asthmatic patients.     

Reactive nitrogen species in the respiratory tract

Endogenous Nitric Oxide (NO) plays a key role in the physiological regulation of airway functions. In response to various stimuli activated inflammatory cells (e.g., eosinophils and neutrophils) generate oxidants ("oxidative stress") which in conjunction with exaggerated enzymatic release of NO and augmented NO metabolites produce the formation of strong oxidizing reactive nitrogen species, such as peroxynitrite, in various airway diseases including asthma, chronic obstructive pulmonary diseases (COPD), cystic fibrosis and acute respiratory distress syndrome (ARDS). Reactive nitrogen species provoke amplification of inflammatory processes in the airways and lung parenchyma causing DNA damage, inhibition of mitochondrial respiration, protein dysfunction and cell damage ("nitrosative stress"). These effects alter respiratory homeostasis (such as bronchomotor tone and pulmonary surfactant activity) and the long-term persistence of "nitrosative stress" may contribute to the progressive deterioration of pulmonary functions leading to respiratory failure. Recent studies showing that protein nitration can be dynamic and reversible ("denitration mechanisms") open new horizons in the treatment of chronic respiratory diseases affected by the deleterious actions of "nitrosative stress".