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Nitric Oxide Synthase Inhibitors Attenuate Ozone-induced Airway Inflammation in Guinea Pigs
Abstract
Nitric oxide (NO) is increased in exhaled air of asthmatics. We hypothesized that endogenous NO contributes to airway inflammation and hyperresponsiveness, and that interleukin-8 (IL-8) might be involved in this mechanism. In human transformed bronchial epithelial cells in vitro, NO donors increased IL-8 production dose-dependently. In addition, tumor necrosis factor-alpha (TNF-alpha) plus IL-1beta plus interferon-gamma (IFN-gamma) increased IL-8 in culture supernatant of epithelial cells; the combination of NO synthase (NOS) inhibitors, aminoguanidine (AG) plus N(G)-nitro-L-arginine methyl ester (L-NAME) attenuated the cytokine-induced IL-8 production in epithelial cells. In guinea pigs in vivo, ozone exposure induced airway hyperresponsiveness to acetylcholine and increased neutrophils in bronchoalveolar lavage fluid (BALF), and these changes persisted for at least 5 h. Pretreatment with NOS inhibitors had no effect on airway hyperresponsiveness or neutrophil accumulation immediately after ozone, but significantly inhibited the changes 5 h after ozone. NOS inhibitors also attenuated the increases of nitrite/nitrate levels in BALF and the IL-8 mRNA expression in epithelial cells and in neutrophils in guinea pig airways 5 h after ozone. These results suggest that endogenous NO may play an important role in the persistent airway inflammation and hyperresponsiveness after ozone exposure, presumably partly through the upregulation of IL-8.
... One potential biological target for ozone or its secondary oxidation products is nitric oxide (NO), which can be converted to various reactive nitrogen oxides that might contribute to development of lung damage or inflammation (22). There is an obvious linkage to the concept that ozone-induced lung damage is related to endogenous or induced NO levels in the lung, and this relationship has been suggested by several recent studies (8,10). Exposure to ozone is associated with increased levels of mRNA for the inducible form of nitric oxide synthase (iNOS) in alveolar macrophages and alveolar type II cells (16). ...
... The ubiquitous NO may also play a role in this process. In a recent paper, Inoue et al. (8) reported that treatment of guinea pigs with inhibitors of iNOS attenuated expression of IL-8 and decreased the accumulation of neutrophils in lung lavage fluid from animals exposed to 3 parts/million (ppm) of ozone for 2 h. Exposure of rats and mice to ozone is also known to cause lung fibrosis (14). ...
... Our results would appear to be inconsistent with those of Inoue et al. (8), who found decreased ozoneinduced airway inflammation in guinea pigs treated with inhibitors of NOS activity, including less neutrophil accumulation and less IL-8 production by epithelial cells. However, it should be noted that different exposure protocols (concentration and duration of exposure) and different species were used in their study and ours, and that a combination of pharmacological NOS inhibitors was used in their study, which would have also affected other NOS isozymes. ...
Mice deficient in inducible nitric oxide synthase (iNOS; C57Bl/6Ai-[KO]NOS2 N5) or wild-type C57Bl/6 mice were exposed to 1 part/million of ozone 8 h/night or to filtered air for three consecutive nights. Endpoints measured included lavagable total protein, macrophage inflammatory protein (MIP)-2, matrix metalloproteinase (MMP)-9, cell content, and tyrosine nitration of whole lung proteins. Ozone exposure caused acute edema and an inflammatory response in the lungs of wild-type mice, as indicated by significant increases in lavage protein content, MIP-2 and MMP-9 content, and polymorphonuclear leukocytes. The iNOS knockout mice showed significantly greater levels of lung injury by all of these criteria than did the wild-type mice. We conclude that iNOS knockout mice are more susceptible to acute lung damage induced by exposure to ozone than are wild-type C57Bl/6 mice and that protein nitration is associated with the degree of inflammation and not dependent on iNOS-derived nitric oxide.
... Group B Streptococcus is a leading cause of neonatal morbidity and mortality with long term sequelae despite antibiotic treatment [1,2]. Systemic inflammation is associated with increased synthesis and combined action of pro-inflammatory mediators such as interleukin-6 (IL-6), macrophage inflammatory protein-2 (MIP-2), and inducible nitric oxide synthase (iNOS) [3][4][5][6][7][8][9][10]. ...
... Nitric oxide (NO) is a biological signaling and effector molecule that regulates the expression of other pro-inflammatory mediators involved in the early response to GBS [3,4,[7][8][9][10]. NO is derived from the conversion of L-arginine to L-citrulline by three isoforms of NOS. ...
... Another cytokine that plays a significant role in rat lung inflammatory response is macrophage inflammatory protein-2 (MIP-2). MIP-2, expressed in rat lung tissue (analogous to IL-8 in humans), belongs to the C-X-C chemokine family and binds to IL-8 receptor [4,15,16]. Recently, IL-8 upregulation during systemic inflammation has been suggested to be NO-dependent [4]. The possible involvement of the nitric oxide pathway in the regulation of cytokines such as IL-6 and MIP-2 Science, vol. ...
Group B Streptococcus (GBS) infection leading to sepsis and lung injury is a major cause of neonatal morbidity and mortality. Lung injury may result from overproduction of pro-inflammatory mediators (cytokines), caused by nitric oxide (NO). Our objective was to characterize the molecular signaling events involving the pro-inflammatory mediators interleukin-6 (IL-6) and macrophage inflammatory protein (MIP-2) in the presence of aminoguanidine (AG), an inducible nitric oxide synthase (iNOS) specific inhibitor, in lung tissue from GBS-treated young rats. Changes in iNOS mRNA, lactic acid, and rectal temperature were determined as markers of the inflammatory response. Expression and regulation of IL-6 and MIP-2 mRNA in lung tissue were studied by RT-PCR with densitometry analysis. GBS treatment of young rats induced the expression of pro-inflammatory mediators IL-6 (6-fold) and MIP-2 (3-fold) in lung tissue compared to controls. AG decreased IL-6 and MIP-2 expression. Addition of L-arginine (L-arg) reversed the AG effect on IL-6 and MIP-2 expression. These data suggest a role for the nitric oxide pathway in the overproduction of pro-inflammatory mediators IL-6 and MIP-2 during GBS-induced lung inflammation. This pathway may be responsible for the initiation of lung injury.
... [36][37][38] There is also evidence that oxygen free radicals can induce the transcription of mRNA for neutrophil-recruiting cytokines, such as the neutrophil chemoattractant IL-8, leading to subsequent IL-8 protein release and perpetuation of neutrophil recruitment. 39,40 Hypotheticallly, these events may contribute to airway remodeling and altered lung function. In line with this, there is evidence that oxygen free radicals contribute to bronchial hyperresponsiveness and that this phenomenon involves neutrophils under certain conditions in mammals in vivo. ...
... [46][47][48][49][50][51][52][53][54] Interestingly, there is evidence to support that both TNFα and IL-8 cause bronchial hyperresponsiveness in rodent airways in vivo . 40,55,56 It may be that this TNFα perpetuates additional neutrophil recruitment via the stimulation of IL-8 in bronchial epithelial cells. 39,57,58 The fact that the luminal concentration of IL-8 correlates negatively with bronchial reactivity in patients with mild asthma 19 is, indeed, compatible with neutrophils exerting a functional impact in obstructive airway disease. ...
There is increasing evidence that an exaggerated accumulation of activated neutrophils is linked to the clinical course of obstructive airway diseases, such as asthma and chronic obstructive pulmonary disease. The present review article focuses on the evidence that the T cell cytokine interleukin (IL)-17 plays a role in orchestrating the accumulation and subsequent activation of neutrophils in the airways and lungs. The mechanistic roles of neutrophils and IL-17 in obstructive airway disease are discussed. It is concluded that targeting IL-17 may consitute a potential strategy for developing novel pharmacotherapeutic interventions against obstructive airway disease.
... The present study was designed to elucidate the involvement of inducible nitric oxide synthase (iNOS) and other mediators in the acid aspiration ALI. Nitric oxide (NO) production through the iNOS system has been shown to be detrimental to the lung tissue in ALI due to various causes (2, 8, 9, 14, 19, 42, 46). Jian and coworkers have found that ALI induced by acid aspiration was attenuated by nonspecific and specific iNOS inhibitors (20). ...
... PLA 2 and other cytolytic enzymes in inflammatory lung injury account for the surfactant alteration following gastric particle instillation (15). Generation of NO through upregulation of iNOS isoform has been shown to be detrimental to the alveolar endothelial-epithelial barrier in various models of lung injury (2, 14, 19, 20, 42). Jian and coworkers (20) found that acid instilled into the rat's lung caused lung injury accompanied by increases in nuclear cell counts, neutrophil counts, albumin concentration, TNF α , interleukin-6 and nitrate/nitrite in bronchoalveolar lavage fluid. ...
Acid aspiration or intrapulmonary instillation of gastric particles causes lung inflammation leading to acute lung injury (ALI). Hypercapnia exerts different effects on ALI caused by various insults. The effects of hypercapnia on lung inflammation and injury due to acid aspiration are yet to be determined. The present study was designed to investigate the involvement of inducible nitric oxide synthase (iNOS) and other mediators in acid-aspiration-induced ALI. We also sought to evaluate the effects of hypercapnia on the lung and associated changes induced by acid aspiration. We used Spague-Dawley rats anesthetized with intraperitioneal pentobarbital (40 mg/kg). Gastric acid particles were prepared from the stomach contents of rats at necropsy. The rats were randomly assigned to receive intratracheal instillation of physiological saline solution (PSS) at pH 7.24 (Control group), PSS at pH 1.25 (Low pH, LPH group), gastric particles (GP group), and GP with low pH PSS (GPLPH group). There were 10 rats in each group. The animals were observed for 6 hrs. To evaluate the effects of hypercapnia, we carried out two series of experiments: one under normocapnia and the other under hypercapnia with alteration of CO2 fraction in inspired air. Arterial pressure (AP) was monitored from the femoral arterial catheter. Heart rate was obtained from AP traicing. We determined the blood gases and acid-base status. Lung weight to body weight (LW/BW) ratio, LW gain (LWG), protein concentration in bronchoalveolar lavage (PCBAL) and leakage of Evans blue dye tracer were measured. Plasma nitrate/nitrite, methyl guanidine (MG), myeloperoxidase (MPO), phospholipase A2 (PLA2), proinflammatory cytokines were assessed. Histopathological examination of the lung tissue was performed. We employed reverse-transcriptase polymerase chain reaction to detect the expression of iNOS mRNA. GP and GPLPH caused hypotension, decreases in PaO2, pH and SaO2, and an increase in PaCO2. The insults also elevated LW/BW, LWG, PCBAL and dye leakage, plasma nitrate/nitrite, MG, MPO, PLA2, tumor necrosis factor(alpha), interleukin-beta and interleukin-6. The lung pathology was characterized by alveolar edema and hemorrhage with inflammatory cells infiltration. Assessment of lung injury score revealed that GP and GPLPH caused ALI. Furthermore, hypercapnia significantly enhanced ALI and associated changes following LPH, GP and GPLPH. Intratracheal instillation of GP in normal or low pH PSS causes ALI accompanied with biochemical changes. The release of nitric oxide via iNOS isoform is detrimental to the lung. Hypercapnia tended to enhance ALI and associated changes induced by gastric acid instillation.
... Nitric oxide has been implicated in other forms of oxidant lung injury but it may be difficult to predict whether it has net pro-or antiinflammatory effects. For example, injury from ozone inhalational exposure, another form of oxidant insult that may share mechanisms of airway damage with chlorine, has been shown to be adversely affected by the absence of inducible nitric oxide synthase (iNOS) in mice (12) but ameliorated by administration of inhibitors of NOS activity in guinea pigs (13). ...
... Lung damage was ameliorated by inhibition of iNOS with aminoguanidine (35). Similarly, the inhibition of NOS activity has been reported to be accompanied by an attenuation of airway inflammation caused by ozone exposure of guinea pigs (13). However, ozone-induced injury is more pronounced in iNOS knockout mice, indicating the complexity related to the balance of potential pro-and antiinflammatory effects of NO in the lung (12) and, perhaps, the dependence of such effects on the species studied. ...
Exposure to chlorine gas (Cl2) causes occupational asthma that we hypothesized occurs through the induction of airway inflammation and airway hyperresponsiveness by oxidative damage. Respiratory mechanics and airway responsiveness to methacholine were assessed in A/J mice 24 hours after a 5-minute exposure to 100, 200, 400, or 800 ppm Cl2 and 2 and 7 days after inhalation of 400 ppm Cl2. Airway responsiveness was higher 24 hours after 400 and 800 ppm Cl2. Responsiveness after inhalation of 400 ppm Cl2 returned to normal by 2 days but was again elevated at 7 days. Airway epithelial loss, patchy alveolar damage, proteinaceous exudates, and inflammatory cells within alveolar walls were observed in animals exposed to 800 ppm Cl2. Macrophages, granulocytes, epithelial cells, and nitrate/nitrite levels increased in lung lavage fluid. Increased inducible nitric oxide synthase expression and oxidation of lung proteins were observed. Epithelial cells and alveolar macrophages from mice exposed to 800 ppm Cl2 stained for 3-nitrotyrosine residues. Inhibition of inducible nitric oxide synthase with 1400W (1 mg/kg) abrogated the Cl2-induced changes in responsiveness. We conclude that chlorine exposure causes functional and pathological changes in the airways associated with oxidative stress. Inducible nitric oxide synthase is involved in the induction of changes in responsiveness to methacholine.
... The increases we observed in arginine and ornithine at both 1 and 24 h suggest increased urea cycle activity (Mori et al., 1998). Nitric oxide is a potent proinflammatory mediator produced by alveolar macrophages and has been shown to be reliant on arginine (Inoue et al., 2000;Wolff and Lubeskie, 1995). ...
Controlled human exposure to the oxidant air pollutant ozone causes decrements in lung function and increased inflammation as evidenced by neutrophil influx into the lung and increased levels of proinflammatory cytokines in the airways. Here we describe a targeted metabolomics evaluation of human bronchioalveolar lavage fluid (BALF) following controlled in vivo exposure to ozone to gain greater insight into its pulmonary effects. In a two-arm cross-over study, each healthy adult human volunteer was randomly exposed to filtered air (FA) and to 0.3 ppm ozone for 2 hr while undergoing intermittent exercise with a minimum of 4 weeks between exposures. Bronchoscopy was performed and BALF obtained at 1 (n = 9) or 24 (n = 23) h post-exposure. Metabolites were detected using ultrahigh performance liquid chromatography-tandem mass spectroscopy. At 1-hour post-exposure, a total of 28 metabolites were differentially expressed (DE) (p < 0.05) following ozone exposure compared to FA-exposure. These changes were associated with increased glycolysis and antioxidant responses, suggesting a rapid increased energy utilization as part of the cellular response to oxidative stress. At 24-hour post-exposure, 41 metabolites were DE. Many of the changes were in amino acids and linked with enhanced proteolysis. Changes associated with increased lipid membrane turnover were also observed. These later-stage changes were consistent with ongoing repair of airway tissues. There were 1.37 times as many metabolites were differentially expressed at 24 hour compared to 1-hour post-exposure. The changes at 1 hour reflect responses to oxidative stress while the changes at 24 hour indicate a broader set of responses consistent with tissue repair. These results illustrate the ability of metabolomic analysis to identify mechanistic features of ozone toxicity and aspects of the subsequent tissue response.
... Thus guinea pigs were exposed to total of 4.00 ppm ozone. This concentration is higher than environmental levels, which can reach 1.04 ppm over an 8-h day (13,33), but it is known to cause acute, reproducible airway inflammation and hyperreactivity in animals (35,44,75,80,81). Similarly, in humans, ozone causes hyperreactivity (6,28,36,39,72) and inflammation in bronchoalveolar lavage (1,3,31,72). ...
Ozone causes vagally-mediated airway hyperreactivity and recruits inflammatory cells, including eosinophils, to lungs where they mediate ozone-induced hyperreactivity one day after exposure, but are paradoxically protective three days later. To test the role of newly divided eosinophils in ozone-induced airway hyperreactivity in sensitized and non-sensitized guinea pigs. Non-sensitized and sensitized guinea pigs were treated with 5-bromo-2-deoxyuridine (BrdU) to label newly divided cells and were exposed to air or ozone for 4 hours. One or three days later, vagally-induced bronchoconstriction was measured and inflammatory cells harvested from bone marrow, blood, and bronchoalveolar lavage. Ozone induced eosinophil hematopoiesis. One day post ozone, mature eosinophils dominate the inflammatory response and potentiate vagally-induced bronchoconstriction. However, by three days, newly divided eosinophils have reached the lungs where they inhibit ozone-induced airway hyperreactivity, since depleting them with AbIL-5 or a TNFα antagonist, worsened vagally-induced bronchoconstriction. In sensitized guinea pigs, both ozone-induced eosinophil hematopoiesis and subsequent recruitment of newly divided eosinophils to lungs three days later failed to occur. Thus, mature eosinophils dominated the ozone-induced inflammatory response in sensitized guinea pigs. Depleting these mature eosinophils prevented ozone-induced airway hyperreactivity in sensitized animals. Ozone induces eosinophil hematopoiesis and recruitment to lungs where three days later, newly divided eosinophils attenuate vagally-mediated hyperreactivity. Ozone-induced hematopoiesis of beneficial eosinophils is blocked by a TNFα antagonist, or by prior sensitization. In these animals, mature eosinophils are associated with hyperreactivity. Thus, interventions targeting eosinophils, while beneficial in atopic individuals, may delay resolution of airway hyperreactivity in non-atopic individuals.
... Animal models . Inoue and coworkers (25) investigated the contribution of endogenous nitric oxide to airway inflammation and hyperresponsiveness, and the potential involvement of interleukin-8. In transformed cells of the bronchial epithelium of humans, nitric oxide donors increased production of interleukin-8; production was also increased by tumor necrosis factor-␣ plus interleukin-1  plus interferon-␥ , and production was attenuated by a combination of nitric oxide synthase inhibitors (aminoguanidine and N 4 -nitro-L -arginine methyl ester [ L -NAME]). ...
Leukotriene B 4 (LTB 4 ) is a potent chemotactic compound that is synthesized from arachidonic acid, by the action of 5-lipoxygenase and leukotriene A 4 hydrolase (1-3). While 5-lipoxygenase is present to a limited extent in leukocytes (4), LTA 4 hydrolase is present ubiquitously in many tissues including the lung, kidney, spleen, and gastrointestinal tract (5). Cytosolic phospholipase A 2 (cPLA 2 ) plays a major role in releasing arachidonic acid from membrane phospholipids (6). We have cloned a human LTB 4 receptor (BLT), a G protein-coupled receptor (GPCR), by subtraction technology using retinoic acid-differentiated HL-60 cell cDNA libraries (7). In this article, we present data on the structure, signal transduction, and chemotactic activity of BLT.
... Ozone-associated lung inflammation can be characterized by polymorphonuclear leukocyte infiltration (Mudway and Kelly 2000). Ozone exposure also contributes to lung injury that may directly or indirectly affect adaptive immune responses such as T cell proliferation and response to allergen (Jakab et al. 1995;Inoue et al. 2000;Kleeberger et al. 2001;Depuydt et al. 2002;Fakhrzadeh et al. 2002;Tank et al. 2011). Lung diseases, such as asthma and COPD, have a tremendous impact on health and quality of life worldwide. ...
The objective of this study was to develop a novel risk analysis approach to assess ozone exposure as a risk factor for respiratory health. Based on the human exposure experiment, the study first constructed the relationship between lung function decrement and respiratory symptoms scores (ranged 0–1 corresponding to absent to severe symptoms). This study used a toxicodynamic model to estimate different levels of ozone exposure concentration-associated lung function decrement measured as percent forced expiratory volume in 1 s (%FEV1). The relationships between 8-h ozone exposure and %FEV1 decrement were also constructed with a concentration–response model. The recorded time series of environmental monitoring of ozone concentrations in Taiwan were used to analyze the statistical indicators which may have predictability in ozone-induced airway function disorders. A statistical indicator-based probabilistic risk assessment framework was used to predict and assess the ozone-associated respiratory symptoms scores. The results showed that ozone-associated lung function decrement can be detected by using information from statistical indicators. The coefficient of variation and skewness were the common indicators which were highly correlated with %FEV1 decrement in the next 7 days. The model predictability can be further improved by a composite statistical indicator. There was a 50 % risk probability that mean and maximum respiratory symptoms scores would fall within the moderate region, 0.33–0.67, with estimates of 0.36 (95 % confidence interval 0.27–0.45) and 0.50 (0.41–0.59), respectively. We conclude that statistical indicators related to variability and skewness can provide a powerful tool for detecting ozone-induced health effects from empirical data in specific populations.
... Generation of NO by the activated neutrophil caused alveolar injury from smoke inhalation [86] . Many laboratories using specific iNOS inhibitors and/or iNOS-knockout animals have supported the contention that NO/iNOS is responsible for the oxidative stress and endothelial damage in the ARDS/ ALI caused by endotoxin, ozone exposure, carrageenan treatment, acute hypoxia, bleomycin administration, acid aspiration and other challenges [11,[87][88][89][90][91][92][93][94][95][96] . Our laboratory further provided evidence to suggest that the NO/iNOS system is involved in the pathogenesis of ALI caused by air embolism [71] , fat embolism [10,97,98] , ischemia/reperfusion [67] , oleic acid [99] , and PMA [100] . ...
To review possible mechanisms and therapeutics for acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). ALI/ARDS causes high mortality. The risk factors include head injury, intracranial disorders, sepsis, infections and others. Investigations have indicated the detrimental role of nitric oxide (NO) through the inducible NO synthase (iNOS). The possible therapeutic regimen includes extracorporeal membrane oxygenation, prone position, fluid and hemodynamic management and permissive hypercapnic acidosis etc. Other pharmacological treatments are anti-inflammatory and/or antimicrobial agents, inhalation of NO, glucocorticoids, surfactant therapy and agents facilitating lung water resolution and ion transports. β-adrenergic agonists are able to accelerate lung fluid and ion removal and to stimulate surfactant secretion. In conscious rats, regular exercise training alleviates the endotoxin-induced ALI. Propofol and N-acetylcysteine exert protective effect on the ALI induced by endotoxin. Insulin possesses anti-inflammatory effect. Pentobarbital is capable of reducing the endotoxin-induced ALI. In addition, nicotinamide or niacinamide abrogates the ALI caused by ischemia/reperfusion or endotoxemia. This review includes historical retrospective of ALI/ARDS, the neurogenic pulmonary edema due to head injury, the detrimental role of NO, the risk factors, and the possible pathogenetic mechanisms as well as therapeutic regimen for ALI/ARDS.
... In separate experiments that exploit the effect of the NOS inhibitor nitro-L-arginine methyl ester (L-NAME) on IL-8 release by AEC II, we showed that 10 Ϫ4 M L-NAME significantly decreased the spontaneous and cytokine-induced IL-8 mRNA expression and protein production in primary cultured AEC II (25, and unpublished data). These observations are supported by the studies of Villarete and Remick (32) and Inoue et al. (13), in which blockade of endogenously generated NO significantly downregulated IL-8 release in human endothelial and transformed bronchial epithelial cells in vitro. In line with the constitutive expression of NOS3 mRNA and protein in cultured AEC II, no differences in NO x production could be detected. ...
... Consistent with our findings, a decrease of FeNO levels in exhaled air, a related marker of the same NO pathway, was observed by Liu et al. after acute exposure to O3 in 182 asthmatic children [48]. The mechanism(s) by which O3 may influence biological markers in the NO pathway might be an inhibition of NOS activity [49] or through oxidative stress. Further research is needed on the chronic effects of ambient air pollution on body fluids, particularly EBC, to better understand the associations with respiratory and cardiovascular effects. ...
This study evaluated the associations between biological markers in the nitrate-nitrite-NO pathway and four environmental exposures among subjects examined in the second survey (2003-2007) of the French Epidemiological study on Genetics and Environment of Asthma (EGEA). Total nitrite and nitrate (NO(2)(-) /NO(3)(-)) levels were measured both in plasma and in exhaled breath condensate (EBC) in 949 adults. Smoking, diet and exposure to chlorine products were assessed using standardized questionnaires. Exposure to air pollutants was estimated by using geostatistical models. All estimates were obtained with generalized estimating equations for linear regression models. Median levels of NO(2)(-)/NO(3)(-) were 36.3 μM (1st-3rd quartile: 25.7, 51.1) in plasma and 2.0 μmol/mg proteins (1st-3rd quartile 0.9, 3.9) in EBC. After adjustment for asthma, age, sex and menopausal status, plasma NO(2)(-)/NO(3)(-) level increased with leafy vegetable consumption (above versus below median=0.04 (95%CI: 0.001, 0.07)) and decreased in smokers (versus non/ex-smokers=-0.08 (95%CI: -0.11, -0.04). EBC NO(2)(-)/NO(3)(-) level decreased in smokers (-0.08 (95%CI: -0.16, -0.001)) and with exposure to ambient O(3) concentration (above versus below median=-0.10 (95%CI: -0.17, -0.03)). Cured meat, chlorine products, PM(10) and NO(2) concentrations were not associated with NO(2)(-)/NO(3)(-) levels. Results suggest that potential modifiable environmental and behavioral risk factors may modify NO(2)(-)/NO(3)(-) levels in plasma and EBC according to the route of exposure.
... We have also identified the lung as the major site that produces NO, which is toxic to the lung [28]. Several experimental studies have indicated that the release of NO via iNOS is responsible for oxidative stress and lung injury following smoke inhalation [29], exposure to ozone [30], carrageen treatment [31], acute hypoxia [32], acid aspiration [33] and endotoxaemia [34][35][36][37]. In a murine model of sepsis, Razavi et al. [38] proposed that up-regulation of iNOS in the lung caused neutrophil infiltration. ...
FES (fat embolism syndrome) is a clinical problem, and, although ARDS (acute respiratory distress syndrome) has been considered as a serious complication of FES, the pathogenesis of ARDS associated with FES remains unclear. In the present study, we investigated the clinical manifestations, and biochemical and pathophysiological changes, in subjects associated with FES and ARDS, to elucidate the possible mechanisms involved in this disorder. A total of eight patients with FES were studied, and arterial blood pH, PaO(2) (arterial partial pressure of O(2)), PaCO(2) (arterial partial pressure of CO(2)), biochemical and pathophysiological data were obtained. These subjects suffered from crash injuries and developed FES associated with ARDS, and each died within 2 h after admission. In the subjects, chest radiography revealed that the lungs were clear on admission, and pulmonary infiltration was observed within 2 h of admission. Arterial blood pH and PaO(2) declined, whereas PaCO(2) increased. Plasma PLA(2) (phospholipase A(2)), nitrate/nitrite, methylguanidine, TNF-alpha (tumour necrosis factor-alpha), IL-1beta (interleukin-1beta) and IL-10 (interleukin-10) were significantly elevated. Pathological examinations revealed alveolar oedema and haemorrhage with multiple fat droplet depositions and fibrin thrombi. Fat droplets were also found in the arterioles and/or capillaries in the lung, kidney and brain. Immunohistochemical staining identified iNOS (inducible nitric oxide synthase) in alveolar macrophages. In conclusion, our clinical analysis suggests that PLA(2), NO, free radicals and pro-inflammatory cytokines are involved in the pathogenesis of ARDS associated with FES. The major source of NO is the alveolar macrophages.
... O 3 -induced lung inflammation is characterized by polymorphonuclear leukocyte (PMN) infiltration (Mudway and Kelly 2000) and release of a number of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) (Bhalla et al. 2002;Cho et al. 2001), interleukin-6 (IL-6) (Johnston et al. 2005), and the PMN chemoattractant macrophage inflammatory protein 2 (MIP-2) (Driscoll et al. 1993). O 3 causes upregulation of inducible nitric oxide synthase (iNOS), which further contributes to lung injury Inoue et al. 2000;Kleeberger et al. 2001). Lung injury after O 3 exposure may directly or indirectly affect adaptive immune responses such as T-cell proliferation (Jakab et al. 1995), response to allergen (Depuydt et al. 2002), and upregulation of costimulatory molecules such as cellular differentiation factor 86 (CD86, B7.2) that contribute to T-cell activation (Koike et al. 2001). ...
The mechanisms underlying ozone (O₃)-induced pulmonary inflammation remain unclear. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that is known to inhibit inflammatory mediators.
We investigated the molecular mechanisms underlying interleuken-10 (IL-10)-mediated attenuation of O₃-induced pulmonary inflammation in mice.
Il10-deficient (Il10(-/-)) and wild-type (Il10(+/+)) mice were exposed to 0.3 ppm O₃ or filtered air for 24, 48, or 72 hr. Immediately after exposure, differential cell counts and total protein (a marker of lung permeability) were assessed from bronchoalveolar lavage fluid (BALF). mRNA and protein levels of cellular mediators were determined from lung homogenates. We also used global mRNA expression analyses of lung tissue with Ingenuity Pathway Analysis to identify patterns of gene expression through which IL-10 modifies O₃-induced inflammation.
Mean numbers of BALF polymorphonuclear leukocytes (PMNs) were significantly greater in Il10(-/-) mice than in Il10(+/+) mice after exposure to O₃ at all time points tested. O₃-enhanced nuclear NF-κB translocation was elevated in the lungs of Il10(-/-) compared with Il10(+/+) mice. Gene expression analyses revealed several IL-10-dependent and O₃-dependent mediators, including macrophage inflammatory protein 2, cathepsin E, and serum amyloid A3.
Results indicate that IL-10 protects against O₃-induced pulmonary neutrophilic inflammation and cell proliferation. Moreover, gene expression analyses identified three response pathways and several genetic targets through which IL-10 may modulate the innate and adaptive immune response. These novel mechanisms of protection against the pathogenesis of O₃-induced pulmonary inflammation may also provide potential therapeutic targets to protect susceptible individuals.
... Animal models . Inoue and coworkers (25) investigated the contribution of endogenous nitric oxide to airway inflammation and hyperresponsiveness, and the potential involvement of interleukin-8. In transformed cells of the bronchial epithelium of humans, nitric oxide donors increased production of interleukin-8; production was also increased by tumor necrosis factor-␣ plus interleukin-1  plus interferon-␥ , and production was attenuated by a combination of nitric oxide synthase inhibitors (aminoguanidine and N 4 -nitro-L -arginine methyl ester [ L -NAME]). ...
In the early 1990s, numerous clinical trials with antileukotriene drugs confirmed Brocklehurst's hypothesis that slow-reacting substance of anaphylaxis (SRS-A) is an important bronchoconstrictor agent in asthma (1). Bronchoprovocation studies showed that inhibition of the synthesis of leukotrienes (LTs) or antagonism of cysteinyl-LT receptors partly or wholly blocks constrictor responses to a wide range of asthma triggers, including allergen, platelet-activating factor (PAF), exercise, cold air, sulfur dioxide, adenosine 5'-monophosphate, and nonsteroidal antiinflammatory drugs (NSAIDs) (2). Multiple-dose studies of patients with allergic and nonallergic asthma commonly report 10-15% improvements in baseline lung function, with 25-60% improvements in secondary outcome measures, including symptom scores, night-time awakenings, use of rescue medication (β 2 -agonists and glucocorticoids), and days lost from work and school (3). To many researchers, the clinical improvements observed after blockade of this single family of inflammatory mediators were surprising, in view of the widely accepted model that airway narrowing is caused by numerous agents acting as components of a mediator soup. Indeed, the efficacy of antileukotriene drugs is highlighted by the relative failure in clinical trials of other mediator antagonists such as histamine H 1 antagonists, thromboxane antagonists, and PAF antagonists (4), The antibronchoconstrictor efficacy of antileukotriene drugs provided the main impetus behind their introduction as the first novel class of asthma therapy in more than 20 yr. However, clinical trials also provided surprising evidence for a hitherto unsuspected role of cysteinyl-leukotrienes in promoting persistent eosinophilia in the airway and blood of patients with asthma, and possibly influencing pathways involved in airway wall remodeling (5). A better understanding of these actions of antileukotriene drugs will influence their place in asthma management.
... Animal models . Inoue and coworkers (25) investigated the contribution of endogenous nitric oxide to airway inflammation and hyperresponsiveness, and the potential involvement of interleukin-8. In transformed cells of the bronchial epithelium of humans, nitric oxide donors increased production of interleukin-8; production was also increased by tumor necrosis factor-␣ plus interleukin-1  plus interferon-␥ , and production was attenuated by a combination of nitric oxide synthase inhibitors (aminoguanidine and N 4 -nitro-L -arginine methyl ester [ L -NAME]). ...
The members of the MAPEG superfamily have been aligned and found to be distantly related, with a common pattern of hydropathy. Figure 2A shows the multiple sequence alignments of the human members and Figure 2B the corresponding superimposed hydropathy profiles. The alignment in Figure 2A demonstrates a total of six strictly conserved residues. The Arg-51 in LTC4 synthase has been suggested to function as proton donor for the opening of the LTA4 epoxide. This arginine is found in all but the FLAP sequences in accordance with the observation that FLAP has no known enzyme activity. Also the Tyr-93 in LTC4 synthase has been suggested to function as a base for the formation of the thiolate anion of glutathione. This tyrosine is not conserved in MGST1 or MGST1-L1. Table 1 summarizes some other properties of the individual human proteins. They are all of the same size, ranging from 147 to 161 amino acids. Only FLAP differs in that its isoelectric point is more neutral than that of the other, more basic proteins. The genes encoding these proteins all reside on different chromosomes (when known) (Table 1). In addition to the human proteins, MAPEG members have been identified in plants, fungi, and bacteria. It is clearly a challenge to elucidate their role in these different phyla in relation to their defined physiological functions in humans.
... Animal models . Inoue and coworkers (25) investigated the contribution of endogenous nitric oxide to airway inflammation and hyperresponsiveness, and the potential involvement of interleukin-8. In transformed cells of the bronchial epithelium of humans, nitric oxide donors increased production of interleukin-8; production was also increased by tumor necrosis factor-␣ plus interleukin-1  plus interferon-␥ , and production was attenuated by a combination of nitric oxide synthase inhibitors (aminoguanidine and N 4 -nitro-L -arginine methyl ester [ L -NAME]). ...
... Animal models . Inoue and coworkers (25) investigated the contribution of endogenous nitric oxide to airway inflammation and hyperresponsiveness, and the potential involvement of interleukin-8. In transformed cells of the bronchial epithelium of humans, nitric oxide donors increased production of interleukin-8; production was also increased by tumor necrosis factor-␣ plus interleukin-1  plus interferon-␥ , and production was attenuated by a combination of nitric oxide synthase inhibitors (aminoguanidine and N 4 -nitro-L -arginine methyl ester [ L -NAME]). ...
... Animal models . Inoue and coworkers (25) investigated the contribution of endogenous nitric oxide to airway inflammation and hyperresponsiveness, and the potential involvement of interleukin-8. In transformed cells of the bronchial epithelium of humans, nitric oxide donors increased production of interleukin-8; production was also increased by tumor necrosis factor-␣ plus interleukin-1  plus interferon-␥ , and production was attenuated by a combination of nitric oxide synthase inhibitors (aminoguanidine and N 4 -nitro-L -arginine methyl ester [ L -NAME]). ...
... Animal models . Inoue and coworkers (25) investigated the contribution of endogenous nitric oxide to airway inflammation and hyperresponsiveness, and the potential involvement of interleukin-8. In transformed cells of the bronchial epithelium of humans, nitric oxide donors increased production of interleukin-8; production was also increased by tumor necrosis factor-␣ plus interleukin-1  plus interferon-␥ , and production was attenuated by a combination of nitric oxide synthase inhibitors (aminoguanidine and N 4 -nitro-L -arginine methyl ester [ L -NAME]). ...
... Animal models . Inoue and coworkers (25) investigated the contribution of endogenous nitric oxide to airway inflammation and hyperresponsiveness, and the potential involvement of interleukin-8. In transformed cells of the bronchial epithelium of humans, nitric oxide donors increased production of interleukin-8; production was also increased by tumor necrosis factor-␣ plus interleukin-1  plus interferon-␥ , and production was attenuated by a combination of nitric oxide synthase inhibitors (aminoguanidine and N 4 -nitro-L -arginine methyl ester [ L -NAME]). ...
... Animal models . Inoue and coworkers (25) investigated the contribution of endogenous nitric oxide to airway inflammation and hyperresponsiveness, and the potential involvement of interleukin-8. In transformed cells of the bronchial epithelium of humans, nitric oxide donors increased production of interleukin-8; production was also increased by tumor necrosis factor-␣ plus interleukin-1  plus interferon-␥ , and production was attenuated by a combination of nitric oxide synthase inhibitors (aminoguanidine and N 4 -nitro-L -arginine methyl ester [ L -NAME]). ...
There is reasonable evidence that both cross-priming and direct transfection of antigen-presenting cells (APCs) play a role in induction of immune responses by DNA vaccines. It is not known which mode is more important for priming cytotoxic T cell responses, but both are sufficient and neither alone is necessary. Hence, a rational strategy for increasing DNA vaccine potency would be to facilitate both pathways. With regard to cross-priming, a better understanding of the nature of the antigen transferred and the molecules/cells involved may suggest ways to design DNA vaccines to enhance this pathway. With respect to transfection of APCs, certain DNA formulations or delivery systems may be able to target APCs for increased DNA uptake. Other considerations include recruitment of APCs to the site of DNA injection and manipulation of these cells to ensure the proper activation state for priming immune responses. The burgeoning scientific literature in these areas indicates that much effort is currently being directed toward these goals.
... BAL. Guinea pigs were killed by exsanguination under anesthesia with an overdose of pentobarbital sodium, and BAL was performed as previously described (15). Both lungs were lavaged gently three times with saline via the tracheal cannula at a pressure of 25 cmH 2 O. ...
Goblet cell metaplasia is an important morphological feature in the airways of patients with chronic airway diseases; however, the precise mechanisms that cause this feature are unknown. We investigated the role of endogenous platelet-activating factor (PAF) in airway goblet cell metaplasia induced by cigarette smoke in vivo. Guinea pigs were exposed repeatedly to cigarette smoke for 14 consecutive days. The number of goblet cells in each trachea was determined with Alcian blue-periodic acid-Schiff staining. Differential cell counts and PAF levels in bronchoalveolar lavage fluid were also evaluated. Cigarette smoke exposure significantly increased the number of goblet cells. Eosinophils, neutrophils, and PAF levels in bronchoalveolar lavage fluid were also significantly increased after cigarette smoke. Treatment with a specific PAF receptor antagonist, E-6123, significantly attenuated the increases in the number of airway goblet cells, eosinophils, and neutrophils observed after cigarette smoke exposure. These results suggest that endogenous PAF may play a key role in goblet cell metaplasia induced by cigarette smoke and that potential roles exist for inhibitors of PAF receptor in the treatment of hypersecretory airway diseases.
... These observations are consistent with those of Li et al. (32), who found that L-NMMA similarly inhibited lung hyperpermeability induced by instilled endotoxin in rats and that endotoxin-induced infiltration of macrophages and PMNs was not affected by the NOS inhibitor. However, another study (18) found that the NOS inhibitors N G -nitro-L-arginine methyl ester and aminoguanidine reduced airway inflammation in guinea pigs exposed to 3 ppm O 3 for 2 h. The treatment effect was not observed until 5 h after exposure, and later postexposure time points were not reported. ...
We tested the hypotheses that 1) inducible nitric oxide synthase (iNOS) mediates ozone (O3)-induced lung hyperpermeability and 2) mRNA levels of the gene for iNOS (Nos2) are modulated by Toll-like receptor 4 (Tlr4) during O3 exposure. Pretreatment of O3-susceptible C57BL/6J mice with a specific inhibitor of total NOS (N(G)-monomethyl-L-arginine) significantly decreased the mean lavageable protein concentration (a marker of lung permeability) induced by O3 (0.3 parts/million for 72 h) compared with vehicle control mice. Furthermore, lavageable protein in C57BL/B6 mice with targeted disruption of Nos2 [Nos2(-/-)] was 50% less than the protein in wild-type [Nos2(+/+)] mice after O3. To determine whether Tlr4 modulates Nos2 mRNA levels, we studied C3H/HeJ (HeJ) and C3H/HeOuJ mice that differ only at a missense mutation in Tlr4 that confers resistance to O3-induced lung hyperpermeability in the HeJ strain. Nos2 and Tlr4 mRNA levels were significantly reduced and correlated in resistant HeJ mice after O3 relative to those in susceptible C3H/HeOuJ mice. Together, the results are consistent with an important role for iNOS in O3-induced lung hyperpermeability and suggest that Nos2 mRNA levels are mediated through Tlr4.
... The reason for lack of effect of L-NMMA on Rrs includes the possibility that the available NO inhibitors might be poorly selective and lacking in potency. Inoue and associates (20) reported no significant inhibitory effect of L-NMMA alone on interleukin-8 (IL-8) production in the guinea pig after ozone-induced airway inflammation. Only when given in combination with L-NAME or with aminoguanadine (NO inhibitors) was IL-8 production significantly inhibited. ...
Airway function is largely preserved during exercise or isocapnic hyperventilation in humans and guinea pigs despite likely changes in airway milieu during hyperpnea. It is only on cessation of a hyperpneic challenge that airway function deteriorates significantly. We tested the hypothesis that nitric oxide, a known bronchodilator that is produced in the lungs and bronchi, might be responsible for the relative bronchodilation observed during hyperventilation (HV) in guinea pigs. Three groups of anesthetized guinea pigs were given saline and three groups given 50 mg/kg N(G)-monomethyl-L-arginine (L-NMMA), a potent nitric oxide synthase inhibitor. Three isocapnic ventilation groups included normal ventilation [40 breaths/min, 6 ml/kg tidal volume (VT)], increased respiratory rate only (150 breaths/min, 6 ml/kg VT), and increased respiratory rate and increased volume (100 breaths/min, 8 ml/kg VT). L-NMMA reduced expired nitric oxide in all groups. Expired nitric oxide was slightly but significantly increased by HV in the saline groups. However, inhibition of nitric oxide production had no significant effect on rate of rise of respiratory system resistance (Rrs) during HV or on the larger rise in Rrs seen 6 min after HV. We conclude that nitric oxide synthase inhibition has no effect on changes in Rrs, either during or after HV in guinea pigs.
... A journal acts as a unifying force for members of a discipline, because it gives them interests in common (17). The summaries in Year in Review are presented in words that make it possible for sleep researchers to get ideas from studies of airway inflammation in asthma (18)(19)(20)(21)(22)(23)(24), for asthma investigators to get stimulated by recent advances in the molecular biology of sepsis (25)(26)(27)(28)(29)(30)(31)(32), and for critical care researchers to catch hints in reports on the regulation of the vasculature in sleep apnea (33)(34)(35)(36). And so on. ...
... In in vitro experiments, NO donors reduced the expression of IL-8 production by activated endothelial cells (53), but increased the LPSinduced expression of IL-8 in neutrophils (54). While pharmacologic NOS inhibition increased IL-8 accumulation in the cell culture supernatant of a human endothelial cell line (55), the cytokine-induced IL-8 production by epithelial cells was attenuated by a NOS inhibitor (56). In a mouse model of zymosan-induced inflammation, the early enhancement of neutrophil infiltration in iNOS-deficient mice was paralleled by increased levels of MIP-2 and KC protein, while the delayed suppression of leukocyte infiltration was paralleled by reduced MIP-2 levels in the peritoneal cavity (57). ...
Nitric oxide (NO) plays a central role in the pathogenesis of bacterial meningitis. However, the role of NO produced by endothelial NO synthase (eNOS) in meningitis is still unclear. We investigated the influence of eNOS depletion on the inflammatory host response, intracranial complications, and outcome in experimental pneumococcal meningitis. Leukocyte accumulation in the cerebrospinal fluid was more pronounced in infected eNOS-deficient mice than in infected wild type mice. This effect could be attributed to an increased expression of P-selectin, macrophage inflammatory protein-2, keratinocyte-derived cytokine, and interleukin (IL)-1beta in the brain of infected eNOS-deficient mice. However, no differences in the cerebral expression of intercellular adhesion molecule-1, tumor necrosis factor-alpha, and IL-6 as well as of neuronal NOS and inducible NOS could be detected between infected wild type and mutant mice. In addition to enhanced leukocyte infiltration into the CSF, meningitis-associated intracranial complications including blood-brain barrier disruption and the rise in intracranial pressure were significantly augmented in infected eNOS-deficient mice. The aggravation of intracranial complications was paralleled by a worsening of the disease, as evidenced by a more pronounced hypothermia, an enhanced weight reduction, and an increased death rate. The current data indicate that eNOS deficiency is detrimental in bacterial meningitis. This effect seems to be related to an increased expression of (certain) cytokines/chemokines and adhesion molecules; thus leading to increased meningeal inflammation and, subsequently, to aggravated intracranial complications.
... In separate experiments that exploit the effect of the NOS inhibitor nitro-L-arginine methyl ester (L-NAME) on IL-8 release by AEC II, we showed that 10 Ϫ4 M L-NAME significantly decreased the spontaneous and cytokine-induced IL-8 mRNA expression and protein production in primary cultured AEC II (25, and unpublished data). These observations are supported by the studies of Villarete and Remick (32) and Inoue et al. (13), in which blockade of endogenously generated NO significantly downregulated IL-8 release in human endothelial and transformed bronchial epithelial cells in vitro. In line with the constitutive expression of NOS3 mRNA and protein in cultured AEC II, no differences in NO x production could be detected. ...
The human alveolar type II epithelium-like cell line A549 expresses nitric oxide synthase type 2 (NOS2), but not NOS3, and produces nitric oxide (NO) upon appropriate stimulation. However, relatively little is known regarding the NOS2 and NOS3 expression of type II human alveolar epithelial cells (AEC II) in primary culture. We detected NOS3 mRNA in freshly isolated AEC II and after 24 h of culture. NOS3 mRNA levels were much higher in AEC II cultured for 24 h with or without interferon-gamma, interleukin-1beta, and tumor necrosis factor-alpha, compared with freshly isolated cells. Cytokine stimulation did not change the NOS3 mRNA expression level in AEC II compared with unstimulated cells. NOS3 protein expression was verified by Western blot, and measuring nitrate/nitrite revealed that the protein is active. In contrast, neither NOS2 mRNA nor protein could be detected in freshly isolated, unstimulated or cytokine-stimulated human AEC II in 24- or 72-h primary cultures, whereas A549 cells expressed NOS2 message and protein upon stimulation with proinflammatory cytokines. In situ hybridization confirmed that AEC II express NOS3, but not NOS2 mRNA in vivo. These data demonstrate that there are significant differences between primary AEC II and A549 cells in NOS mRNA expression pattern.
... However, as with many previous studies investigating the airways response to ozone (Murlas et al., 1993;Schultheis et al., 1994;Holbrook et al., 1996;Inoue et al., 2000;, anaesthesia was utilized to determine AHR, which may in¯uence vagal tone or sensory re¯exes. In the current study, we assessed both AHR and airway function changes in conscious guinea-pigs following ozone exposure. ...
The effects of ozone inhalation (90 min, 2.15±0.05 p.p.m.) and their modification by dexamethasone (20 mg kg−1) or the phosphodiesterase-4 inhibitor, rolipram (1 mg kg−1), administered (i.p.) 24 and 0.5 h before and 24 h after ozone exposure were examined in conscious guinea-pigs.
Ozone caused an early-phase bronchoconstriction (EPB) as a fall in specific airways conductance (sGaw) measured by whole body plethysmography, followed at 5 h by a late-phase bronchoconstriction (LPB) and increased respiratory rate. Rolipram did not alter this profile but dexamethasone inhibited the EPB.
Airway hyperreactivity to inhaled histamine (1 mM, 20 s) occurred at 0.5, 2, 12, 24 and 48 h after ozone inhalation, the 2 h change being abolished by rolipram and dexamethasone.
Bronchoalveolar lavage fluid (BALF) macrophages, eosinophils and neutrophils were significantly (P<0.05) elevated at 12, 24 and 48 h after ozone exposure, the 48 h influx being significantly attenuated (P<0.05) by rolipram and dexamethasone.
BALF nitric oxide (NO) metabolites decreased 0.5 h after ozone exposure by 52%, recovered at 2 h and significantly increased at 12 (101%) and 24 h (127%). The elevated NO was unaffected by rolipram or dexamethasone.
Lung oedema, measured from wet/dry weight differences, was significant 12, 24 and 48 h after ozone exposure, the latter being significantly attenuated (P<0.05) by rolipram and dexamethasone.
Ozone exposure of guinea-pigs produced features common to COPD. Although rolipram and dexamethasone did not affect the airway function changes, they inhibited the inflammation, airway hyperreactivity and oedema.
British Journal of Pharmacology (2002) 136, 735–745; doi:10.1038/sj.bjp.0704764
Importance
Exposure to ozone has been associated with cardiovascular mortality, but the underlying biological mechanisms are not yet understood.
Objective
To examine the association between ozone exposure and cardiopulmonary pathophysiologic mechanisms.
Design, Setting, and Participants
A longitudinal study involving 89 healthy adult participants living on a work campus in Changsha City, China, was conducted from December 1, 2014, to January 31, 2015. This unique quasiexperimental setting allowed for better characterization of air pollutant exposure effects because the participants spent most of their time in controlled indoor environments. Concentrations of indoor and outdoor ozone, along with the copollutants particulate matter, nitrogen dioxide, and sulfur dioxide, were monitored throughout the study period and then combined with time-activity information and filtration conditions of each residence and office to estimate 24-hour and 2-week combined indoor and outdoor mean exposure concentrations. Associations between each exposure measure and outcome measure were analyzed using single-pollutant and 2-pollutant linear mixed models controlling for ambient temperature, secondhand smoke exposure, and personal-level time-varying covariates.
Main Outcomes and Measures
Biomarkers indicative of inflammation and oxidative stress, arterial stiffness, blood pressure, thrombotic factors, and spirometry were measured at 4 sessions.
Results
Of the 89 participants, 25 (28%) were women and the mean (SD) age was 31.5 (7.6) years. The 24-hour ozone exposure concentrations ranged from 1.4 to 19.4 parts per billion (ppb), corresponding to outdoor concentrations ranging from 4.3 to 47.9 ppb. Within this range, in models controlling for a second copollutant and other potential confounders, a 10-ppb increase in 24-hour ozone was associated with mean increases of 36.3% (95% CI, 29.9%-43.0%) in the level of platelet activation marker soluble P-selectin, 2.8% (95% CI, 0.6%-5.1%) in diastolic blood pressure, 18.1% (95% CI, 4.5%-33.5%) in pulmonary inflammation markers fractional exhaled nitric oxide, and 31.0% (95% CI, 0.2%-71.1%) in exhaled breath condensate nitrite and nitrate as well as a −9.5% (95% CI, −17.7% to −1.4%) decrease in arterial stiffness marker augmentation index. A 10-ppb increase in 2-week ozone was associated with increases of 61.1% (95% CI, 37.8%-88.2%) in soluble P-selectin level and 126.2% (95% CI, 12.1%-356.2%) in exhaled breath condensate nitrite and nitrate level. Other measured biomarkers, including spirometry, showed no significant associations with either 24-hour ozone or 2-week ozone exposures.
Conclusions and Relevance
Short-term ozone exposure at levels not associated with lung function changes was associated with platelet activation and blood pressure increases, suggesting a possible mechanism by which ozone may affect cardiovascular health.
Accumulating evidence suggests that phagocytic cells of the innate immune system, including neutrophils and macrophages, play an important role in the pathogenesis of tissue injury in response to diverse toxicants. Through the release of proinflammatory and cytotoxic oxidants, cytokines, proteases, and bioactive lipids, these cells can contribute to tissue damage. More recent studies suggest that, at later times following injury, macrophages and mediators they generate also play a key role in downregulating the inflammatory response and initiating tissue repair. These divergent activities appear to be mediated by a distinct subpopulation of macrophages. It is apparent that the outcome of inflammatory responses to tissue injury depends on the balance between the pro- and anti-inflammatory actions of these cells. Elucidating the contribution of phagocytes and inflammatory mediators to tissue injury and repair may help in the design of new and effective approaches to mitigating xenobiotic-induced pathologies.
Acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) can be associated with various disorders. Recent investigation has involved clinical studies in collaboration with clinical investigators and pathologists on the pathogenetic mechanisms of ALI or ARDS caused by various disorders. This literature review includes a brief historical retrospective of ALI/ARDS, the neurogenic pulmonary edema due to head injury, the long-term experimental studies and clinical investigations from our laboratory, the detrimental role of NO, the risk factors, and the possible pathogenetic mechanisms as well as therapeutic regimen for ALI/ARDS.
Ozone is a potent oxidant and causes airway hyperresponsiveness and neutrophilia. To determine the role of p38 mitogen-activated protein kinase (MAPK) activation, we studied the effect of a p38alpha inhibitor SD-282 (Scios Inc, Fremont, CA USA) on ozone-induced airway hyperresponsiveness and neutrophilia. Balb/c mice received SD-282 (30 or 90 mg/kg i.p) or vehicle 1 h before exposure to either ozone (3 ppm, 3 h) or air. Three hours after exposure, lungs were analysed for cytokine levels and bronchoalveolar lavage was performed. Another set of mice were dosed 6 h after exposure and 1 h before assessing airway hyperresponsiveness. SD-282 (90 mg/kg) significantly inhibited ozone-induced airway hyperresponsiveness (-LogPC(150): SD-282: -1.73+/-0.14 vs. vehicle: -0.99+/-0.15, P<0.05). Bronchoalveolar lavage neutrophil numbers were time-dependently increased in vehicle-dosed, ozone-exposed mice, greatest at 20-24 h after exposure. SD-282 (30 and 90 mg/kg) significantly inhibited ozone induced neutrophil numbers at 3 h and 20-24 h after ozone SD-282 significantly inhibited ozone-induced increases in phosphorylated p38 MAPK expression, and in cyclooxygenase-2 (COX-2), interleukin-6 (IL-6) and IL-1beta but not MIP-1alpha gene expression. We conclude that p38 MAPK is involved in ozone-induced airway hyperresponsiveness and lung neutrophilia. Inhibition of p38 MAPK with small molecule kinase inhibitors may be a means of reducing ozone-induced inflammation and airway hyperresponsiveness.
The highest incidences of cancer are found in the skin, but endogenous pigmentation is associated with markedly reduced risk. Agents that enhance skin pigmentation have the potential to reduce both photodamage and skin cancer incidence. The purpose of this review is to evaluate agents that have the potential to increase skin pigmentation. These include topically applied substances that simulate natural pigmentation: dihydroxyacetone and melanins; and, substances that stimulate the natural pigmentation process: psoralens with UVA (PUVA), dimethylsulfoxide (DMSO), l-tyrosine, l-Dopa, lysosomotropic agents, diacylglycerols, thymidine dinucleotides, DNA fragments, melanocyte stimulating hormone (MSH) analogs, 3-isobutyl-1-methylxanthine (IBMX), nitric oxide donors, and bicyclic monoterpene (BMT) diols. These agents are compared with regard to efficacy when administered to melanoma cells, normal human epidermal melanocytes, animal skin, and human skin. In addition, mechanisms of action are reviewed since these often reveal issues releated to both efficacy and safety. Both dihydroxyacetone and topically applied melanins are presently available to the consumer, and both of these have been shown to provide some photoprotection. Of the pigmentation stimulators, only PUVA and MSH analogs have been tested extensively on humans, but there are concerns about the safety and side effects of both. At least some of the remaining pigmentation stimulators under development have the potential to safely induce a photoprotective tan.
This unit describes methods for establishing guinea pig models of asthma. Detailed descriptions are provided so that investigators can establish preparations in which bronchoconstriction, airways hyperresponsiveness, cough and airways inflammation can be studied.
Repr.(with corrections) Bibliogr. na konci kapitol
Acute respiratory distress syndrome (ARDS) is the most devastating form of acute lung injury (ALI) or pulmonary edema (PE). We presented the experimental studies and clinical investigations of two serious forms of ALI. Drastic and severe PE could be induced by intracranial hypertension or cerebral compression (CC). The CC-induced PE was attributed to overactivation of the medullary sympathetic mechanism. Sympathetic vasoconstriction of the systemic and pulmonary resistance and capacitance vessels caused shift of blood volume from the splanchnic vascular beds to the lung. The hemodynamic changes led to systemic and pulmonary hypertension. Consequently, left ventricular failure as evidenced by dramatic decline in aortic flow with a slow decrease in pulmonary flow resulted in pressure and volume loading in the pulmonary circulation. These changes finally produced severe alveolar flooding and sudden death. Vasodilators such as sodium nitroprusside or nitroglycerin were capable of reducing the CC-induced pulmonary pathology and hemodynamic alterations. Fat embolism syndrome (FES) is a serious clinical problem in patients suffering from long bone fractures. ARDS may develop and cause mortality. Our laboratory reported a total of 14 subjects associated with FES and died of ARDS. We also developed a simple technique to produce FES. Corn oil was mixed with distilled water to form fatty micelles. Intravenous administration of or introduction of fatty micelles in anesthetized rats or isolated perfused lungs caused severe alveolar damage. Our clinical observation and animal experimentation revealed that infusion of fatty acids caused physical phase, resulting in microvascular obstruction accompanied by pulmonary hypertension and increased capillary permeability. Thereafter, the lipases in the lung hydrolyzed the neutral fat and released free fatty acids and biochemical mediators which were toxic to the lung. Our data have suggested that nitric oxide (NO), inducible NO synthase (iNOS), phospholipase A2, free radical and inflammatory cytokines (tumor necrosis factor alpha, interleukin-1beta and interleukin-6) are involved in the biochemical phase of FES with ARDS. The alveolar macrophages are the major source of iNOS. Later study also found that neutrophil elastase and myeloperoxidase were elevated following fat embolism. N-acetylcysteine (an antioxidant), and NOS inhibitors such as Nomega nitro-L-arginine methyl ester (L-NAME), S-methylisothiourea (SMT) or L-N6 (1-iminoethyl)-lysine (L-Nil) were able to abrogate the FES or the fat embolism-induced changes.
Ozon verursacht als wichtiger Luftschadstoff pathologische Veränderungen des Respirationssystems sowie Einschränkungen der Lungenfunktion. Seine Toxizität beruht dabei auf seiner hohen Oxidationskraft mit Auslösung eines „oxidativen Stresses“. Dieser besteht auch unabhängig von Ozon bei Entzündungsreaktionen und wird als wichtige pathogene Komponente bei der Entstehung verschiedener Lungenerkrankungen wie beispielsweise dem Asthma bronchiale und der COPD postuliert. Heat Schock Proteine (HSPs) werden nach Exposition mit Stress-Faktoren wie beispielsweise Hyperthermie, Inflammation und oxidativem Stress gebildet und können daher als Parameter für zellulären Stress angesehen werden. Ambroxol wird als mukolytisches Medikament bei Atemwegserkrankungen eingesetzt. Diesem Wirkstoff werden u.a. auch antioxidative Eigenschaften zugeschrieben. Ziel dieser Arbeit ist es, die ozonvermittelte Stressantwort (HSP 32, -60, -70 und HSC 70) in der Lunge sowie eine eventuelle Protektion durch zusätzliche Ambroxolgabe zu untersuchen. Dazu wurden Sprague Dawley Ratten Ozon in unterschiedlicher Konzentration und Dauer (8h mit 1,5 bzw. 3ppm oder 12h bzw. 24h mit 0,6ppm) mit und ohne Ambroxolgabe ausgesetzt und anschließend die HSP Protein- und m-RNA-Expression im Gesamtlungengewebe mittels Western Blot und PCR bestimmt. Die zeit- und konzentrationsabhängige Zunahme der HSP 32-Expression nach Ozonexposition (360%-989,3%) wurde durch eine zusätzliche Ambroxolgabe gehemmt (119,8%). Unter Ozonexposition war vermehrt HSP 70 m-RNA (163,4%-249,8%) bei allerdings stark verminderter HSP 70 Konzentration (10,7%-53,7%) nachweisbar. Bei zusätzlich verabreichtem Ambroxol war der Proteinabfall weniger stark ausgeprägt (46,5%-69%). Die Proteinkonzentrationen von HSC 70 und HSP 60 stellen sich insbesondere bei den kürzeren Expositionszeiten (8 und 12h) vermindert dar (9,3%-55,3% bzw. 40,2%-76,3%). Unter zusätzlicher Ambroxolgabe war die Ozon-bedingte Verminderung des HSC 70 geringer ausgeprägt (61,3%), bei HSP 60 war kein eindeutiger Effekt auf die Proteinexpression nachweisbar. Eine alleinige Ambroxolgabe bewirkte eine Hemmung von HSP 70 und HSP 60, HSP 32 war nach Ambroxolapplikation vermehrt nachweisbar, während HSC 70 kaum durch Ambroxol beeinflusst wurde. Die Ergebnisse zeigen unterschiedliche Auswirkungen der Ozonexposition auf die verschiedenen HSPs. Ozon bewirkte eine erhöhte HSP 32 Expression, zudem war eine verstärkte Induktion der HSP 70 mRNA durch Ozon zu beobachten. HSC 70 und HSP 60 hingegen waren nach Ozon vermindert nachweisbar. Möglicherweise zeigt diese Hemmung eine Prioritätensetzung zugunsten anderer HSPs auf. Ambroxol verminderte die Ozonbedingte HSP 32 Stressantwort. Diese Beobachtung unterstreicht das Postulat von Ambroxol als eine potente antioxidative Substanz mit möglicher protektiver Wirkung auf das Lungengewebe. Ozone is an important air pollutant that compromises lung function and causes acute lung injury. Cellular injury induces stress related expression of several high conserved proteins. Of those proteins one important group consisits of the heat shock proteins (HSP). Ambroxol, often used in respiratory diseases, is postulated to provide antioxidant properties. To investigate the stress response in ozone-induced lung injury and the potential protection by ambroxol, we analyzed HSP´s protein and mRNA expression in rat whole lung tissue after ozone exposure, with and without ambroxol treatment. Two groups of Sprague Dawley rats (5 each) were exposed to 3ppm ozone for 8 hours. One group was treated with ambroxol, while the other was not. Two groups (6 each) of control animals were not exposed to ozone, either treated with ambroxol or not. Heat shock protein (HSP 32, -60, -70, -73kD) and mRNA expression were analyzed using specific immunoblot and competive PCR. GAPDH were used to normalize mRNA. HSP 32 protein (436%) and mRNA (536%) were significantly increased in ozone exposed animals (% of controls), while exposed animals treated with ambroxol showed dignificant reduction in protein amount (119%). HSP 60 (40,2%), -70 (10,6%) and -73 (9,3%) protein were significant decreased after ozone exposure. This decrease was inhibited by ambroxol in part (HSP 60; 75,8%, HSP 73: 61,3%, HSP 72: 46,5%). HSP 72 mRNA was elevated in ozone exposed animals (248%) with further increase in those teated with ambroxol, while HSP 60 and 73 mRNA were not significantly changed. Ozone induced lung injury results in HSP 32 expression and decreases HSP 60/72/73, while both is inhibited by ambroxol, suggesting that ambroxol may work as an potent antioxidant.
Ambient ozone has been linked to the worsening of symptoms of patients with obstructive diseases such as chronic obstructive pulmonary disease (COPD) and asthma. We investigated the role of cathepsin S on ozone-induced airway hyperresponsiveness (AHR) and inflammation, using the selective cathepsin S inhibitor, Compound A. Balb/c mice were exposed to ozone at a concentration of 3 ppm or air for 3 h, following administration by gavage of Compound A or vehicle. Bronchoalveolar lavage (BAL) was performed 3 h and 20-24 h following exposure, AHR was measured at 20-24 h only. Ozone exposure, compared to air exposure increased BAL cathepsin S levels, AHR and BAL inflammatory cells. Compound A (30 mg kg(-1) p.o.) dosing compared to vehicle dosing inhibited ozone-induced AHR (-logPC100 vehicle: -0.70+/-0.12, n=8 vs. cathepsin S inhibitor: -1.30+/-0.06, P<0.001, n=8) at 20-24 h and BAL neutrophilia at 3 h and 20-24 h (P<0.05, n=6). Ozone exposure increased levels of BAL cytokines IL-6, TNF-alpha and IFN-gamma. Compound A reduced IL-6 at 3 h and 20-24 h (P<0.05, n=5) and TNF-alpha, at 20-24 h (P<0.05, n=6). These data indicate an important role for cathepsin S in the regulation of ozone-induced AHR and neutrophil cell recruitment and suggest that cathepsin S may be a target in the treatment of oxidative stress-induced AHR and inflammation.
This is a clinicopathologic study of three subjects with irritant-induced asthma. They were pulpmill workers who had a history of multiple "gassing" episodes that occurred over a period of years. Persistent symptoms of asthma and nonspecific bronchial hyperresponsiveness and/or variable airflow obstruction occurred after at least one episode of "gassing," resulting in symptoms severe enough to require emergency room treatment. One of the three subjects had normal spirometry values before he entered the pulpmill. Bronchial biopsy done on these subjects showed changes compatible with asthma, including thickened basement membrane in two and cellular infiltration with activated eosinophils and mononuclear cells in all three. The results of immunohistology of bronchial mucosal biopsy of these subjects were compared with those of patients with allergic asthma and patients with Western red cedar-induced asthma. Subjects with irritant-induced asthma had a greater density of activated eosinophils and fewer T-lymphocytes, suggesting that cell-mediated immune mechanisms are not involved in the pathogenesis of this condition.
Raising intracellular cAMP or cGMP concentrations protects lungs from ischemia-reperfusion injury. These nucleotides are catabolized by a number of distinct phosphodiesterase (PDE) isoenzyme subfamilies. We examined the ability of PDE inhibitors of differing selectivities to protect lungs from the effects of prolonged hypothermic storage. Rat lungs were perfused with bicarbonate buffer mixed with rat blood (4:1 vol/vol, 37 degrees C), ventilated, and vascular resistance, airway compliance, and resistance, and gas exchange measured. Lungs were then flushed with, and immersed in, St. Thomas' Hospital Solution (STH) (4 degrees C) or STH containing rolipram, milrinone, zaprinast, or theophylline. After 8 h storage, function was reassessed during 40 min reperfusion. Lungs stored in STH containing rolipram or theophylline had improved function on reperfusion. After 40 min reperfusion, pulmonary compliance (Cstat) was 0.07 +/- 0.01 ml/cm H(2)O in lungs stored in STH alone. Adding rolipram (100 microM) or theophylline (3,000 microM) to the STH used for flushing and storage improved Cstat after reperfusion to 0.17 +/- 0.02 ml/cm H(2)O (p < 0.05) and 0.17 +/- 0.02 ml/cm H(2)O (p < 0. 05), respectively. Theophylline also improved the increase in perfusate PO(2) on transit through the lung after storage to 25.16 +/- 2.33 compared with 4.72 +/- 2.18 mm Hg in lungs stored in STH alone (p < 0.05). Of the selective PDE inhibitors tested, rolipram (type IV inhibitor) was most effective. However, the nonselective agent, theophylline, provided the best protection of function after storage and reperfusion of rat lungs.
Induction of pleurodesis offers benefit for patients with metastatic tumors and symptomatic malignant pleural effusions, but the best method for achieving this is still unknown. In this prospective, randomized comparison of two well-established pleurodesis procedures, 36 patients with malignant pleural effusions, expanded lungs after drainage, and expected survival of > 1 mo received either bleomycin instillation (60E) via a small-bore thoracostomy tube or thoracoscopic talc poudrage (5 g) under local anesthesia. Efficacy, safety, and cost could be evaluated for 32 treatments (17 bleomycin, 15 talc) in 31 patients. Recurrence rates of effusion with bleomycin and talc poudrage after 30 d were 41% and 13% (p = 0.12), respectively, those after 90 d were 59% and 13%, respectively (p = 0.01), and those after 180 d were 65% and 13% (p = 0.005), respectively. Neither procedure showed any major adverse effect, and both were equally well tolerated. Cost estimation favored thoracoscopic talc poudrage, both for the initial hospitalization and with regard to recurrences. In conclusion, thoracoscopic talc pleurodesis under local anesthesia is superior to bleomycin instillation for pleurodesis in cases of malignant pleural effusion.
This study was designed to investigate the mechanisms through which tumor necrosis factor (Tnf) modulates ozone (O(3))-induced pulmonary injury in susceptible C57BL/6J (B6) mice. B6 [wild-type (wt)] mice and B6 mice with targeted disruption (knockout) of the genes for the p55 TNF receptor [TNFR1(-/-)], the p75 TNF receptor [TNFR2(-/-)], or both receptors [TNFR1/TNFR2(-/-)] were exposed to 0.3 parts/million O(3) for 48 h (subacute), and lung responses were determined by bronchoalveolar lavage. All TNFR(-/-) mice had significantly less O(3)-induced inflammation and epithelial damage but not lung hyperpermeability than wt mice. Compared with air-exposed control mice, O(3) elicited upregulation of lung TNFR1 and TNFR2 mRNAs in wt mice and downregulated TNFR1 and TNFR2 mRNAs in TNFR2(-/-) and TNFR1(-/-) mice, respectively. Airway hyperreactivity induced by acute O(3) exposure (2 parts/million for 3 h) was diminished in knockout mice compared with that in wt mice, although lung inflammation and permeability remained elevated. Results suggested a critical role for TNFR signaling in subacute O(3)-induced pulmonary epithelial injury and inflammation and in acute O(3)-induced airway hyperreactivity.
Ozone is one of the most powerful oxidants available, with many applications in industry and medicine. Medically relevant features of ozone include bacterial and virucidal properties, disinfection, sterilization, circulatory stimulation, and disruption of malignant cells. Ozone therapy is administered in various ways, including intravenously, intramuscularly, and intrarectally. The latter modality is used for the treatment of colitis and hepatitis. Our aim was to examine the effect of ozone water enema on normal and inflamed rat colonic mucosa. Ozone water (20 microg/ml) was prepared via ozone generator and administered intrarectally (0.5 ml) daily. Rats were killed one, three, and seven days after rectal ozone water administration, and their colons resected, rinsed, and weighed (grams per 10 cm). Damage was assessed macro- and microscopically and tissue processed for myeloperoxidase and nitric oxide synthase activity. Rats receiving saline served as controls. In an additional experiment colitis was induced by intrarectal iodoacetamide. Ozone therapy caused no macroscopic damage. Ozone therapy induced microscopic colitis, which lasted for at least a week and was accompanied by increase in segmental weight, myeloperoxidase and nitric oxide activity, and prostaglandin E2 generation. Ozone therapy had no protective effect on inflamed mucosa. In conclusion, ozone water therapy had a deleterious effect on normal colonic mucosa, suggesting intrarectal administration be reevaluated. Ozone water enema may serve as a model of microscopic colitis.
There is evidence that T-lymphocytes may orchestrate the recruitment and activation of granulocytes in the airways and lungs; in particular there is such evidence for CD4+ lymphocytes and eosinophilic granulocytes. It is well known that the total number of lymphocytes as well as eosinophils and neutrophils is increased in the airways of patients with newly diagnosed asthma (52). Furthermore, specific blockade of CD4+ lymphocytes, either with an anti-CD4+ antibody or an anti-IL-2 receptor antibody, prevents allergen-induced recruitment of both eosinophils and neutrophils in sensitized murine airways in vivo (53,54). The number of CD4+ lymphocytes is also increased in parallel with the number of neutrophils in the airways of patients with COPD (55,56) and exposure to cigarette smoke causes recruitment of neutrophils and CD4+ lymphocytes in rodent airways (57). A recent study shows an increased number of CD4+ lymphocytes and neutrophils in bronchial tissue from patients with bronchiectasis as well (58). There is also evidence that CD4+ lymphocytes have a functional impact; the presence of CD4+ lymphocytes is related to bronchial reactivity in murine and in human airways in vivo, but it is not known whether this phenomenon depends upon orchestration of eosinophils or neutrophils or both (59–61). Furthermore, the mechanisms linking CD4+ lymphocytes to neutrophil recruitment and activation as well as to functional alterations, have remained unknown. This lack of knowledge has been in sharp contrast to the link between CD4+ lymphocytes and eosinophil recruitment; where for example the Th2 cytokine IL-5 probably plays an important role (62–65).
Ozone (O3) is an air pollutant produced by sunlight-driven reactions involving the oxides of nitrogen and volatile organic compounds. The population of many large metropolitan areas in the US is exposed to high levels of O3, particularly in the summer months. Individuals exposed to O3 levels in human experiments at higher than common ambient levels develop reversible reductions in lung function often associated with symptoms, such as airway hyperreactivity and lung inflammation. Animal models have helped characterize potential mechanisms of lung injury from O3 exposure. Defining the adverse effects of chronic exposure to ambient levels of O3 on lung function and disease have been challenging, in part due to the presence of co-pollutants, such as particulate matter. The US Environmental Protection Agency's 1997 revised standard for O3 (0.08 ppm averaged over 8 hours) is designed to provide better protection to susceptible individuals. The revised standard is being implemented following the failure of court challenges.
Nitric oxide (NO) synthase in rat brain was found to be constitutive and Ca2(+)-dependent. The enzyme in rat lung or liver (predominantly in parenchymal cells) was not constitutive, but was induced by endotoxin treatment and was Ca2(+)-independent. The NO synthases in rat brain and liver or lung are therefore distinct both in their properties and in their regulation.
The presence of nitric oxide (NO) in the exhaled air of humans has recently been described. We wanted to assess at what level exhaled NO originates in normal airways, and to determine whether airway inflammation induces changes in the levels of exhaled NO. Exhaled NO was continuously measured by chemiluminescence technique during normal tidal breathing through the nose or mouth, with a detection limit of 1 part per billion (ppb). Twelve control subjects were compared to eight patients with mild atopic asthma and rhinitis caused by occupational allergen. In control subjects, the major part of NO in exhaled air (up to 30 ppb) seemed to originate in the nasal airways, with only minor contribution from the lower airways and the oral cavity. However, in mild asthmatics, the level of exhaled NO during oral breathing, indicating the involvement of the lower airways, was increased 2-3 fold. Since increased production of NO in the lower airways may involve activated macrophages or neutrophils, we suggest that exhaled NO may be used to instantly monitor ongoing bronchial inflammation, at least when involving inducible NO synthase.
Previous work from our laboratory localized nitric oxide to the affected spinal cords of mice with experimental autoimmune encephalomyelitis, a prime model for the human disease multiple sclerosis. The present study shows that activated lymphocytes sensitized to the central nervous system encephalitogen, myelin basic protein, can induce nitric oxide production by a murine macrophage cell line. Induction was inhibited by amino-guanidine, a preferential inhibitor of the inducible nitric oxide synthase isoform, and by NG-monomethyl-L-arginine. Aminoguanidine, when administered to mice sensitized to develop experimental autoimmune encephalomyelitis, inhibited disease expression in a dose-related manner. At 400 mg aminoguanidine/kg per day, disease onset was delayed and the mean maximum clinical score was 0.9 +/- 1.2 in aminoguanidine versus 3.9 +/- 0.9 in placebo-treated mice. Histologic scoring of the spinal cords for inflammation, demyelination, and axonal necrosis revealed significantly less pathology in the aminoguanidine-treated group. The present study implicates excessive nitric oxide production in the pathogenesis of murine inflammatory central nervous system demyelination, and perhaps in the human disease multiple sclerosis.
Histochemical activity and immunoreactivity of nitric oxide synthase (NOS, EC 1.14.13.39) have been recently demonstrated in human lung epithelium. However, the molecular nature of NOS and the regulation and function of the enzyme(s) in the airway is not known. A549 cells (human alveolar type II epithelium-like), BEAS 2B cells (transformed human bronchial epithelial cells), and primary cultures of human bronchial epithelial cells all exhibited constitutive NOS activity that was calcium dependent and inhibitable by the NOS inhibitor NG-monomethyl-L-arginine. Nitric oxide production by epithelial cells was enhanced by culture in the presence of interferon gamma, interleukin 1 beta, tumor necrosis factor alpha, and lipopolysaccharide; the NOS activity expressed under these conditions showed less dependence on calcium, reminiscent of other inducible forms of NOS. Two distinct NOS mRNA species, homologous to previously identified constitutive brain (type I) and inducible hepatic (type II) NOS, were demonstrated by reverse transcription-polymerase chain reaction in all cell lines. Northern analysis confirmed the expression of inducible NOS mRNA. Cell culture with epidermal growth factor, a principal regulator of epithelial cell function, decreased inducible NOS activity by posttranscriptional action but did not affect constitutive NOS activity. The coexistence of constitutive and inducible NOS in human alveolar and bronchial epithelial cells is consistent with a complex mechanism evolved by epithelial cells to protect the host from microbial assault at the air/surface interface while shielding the host from the induction of airway hyperreactivity.
A potent leukocyte chemotactic and activating cytokine, interleukin-8 (IL-8), is produced by numerous types of cells in response to inflammatory stimuli. Accumulating evidence indicate that the transcription of IL-8 gene requires the activation of either the combination of NF-kappa B and AP-1 or that of NF-kappa B and NF-IL6, depending on the type of cells. Alternatively, the activation of NF-kappa B is indispensable for IL-8 gene activation in any types of cells examined. On the other hand, an immunosuppressant, FK506, and a glucocorticoid inhibit the gene transcription as well as the production of IL-8. Molecular analyses of IL-8 gene repression by these agents revealed that both affected the activity of the transcription factor(s) bound to the NF-kappa B site, albeit in different ways, thereby suppressing IL-8 gene transcription. Collectively, IL-8 production seems to be controlled mainly at the activation step of the transcription factor(s) bound to the NF-kappa B site.
Nitric oxide (NO) has been shown to be both an intercellular and intracellular messenger. We propose here that exogenous NO induces chemotactic locomotion of human neutrophils. Indeed, when human neutrophils were placed in a gradient of a nitric oxide donor (S-nitroso-N-acetylpenicillamine; SNAP), a directed locomotion was induced, as evidenced by experiments of chemotaxis under agarose. Degraded SNAP (i.e., SNAP solution which had previously released NO) did not induce directed locomotion. Moreover, oxyhemoglobin, a scavenger of free NO, suppressed the chemotactic effect of SNAP, whereas LY-83583, a soluble guanylate cyclase inhibitor, inhibited the SNAP-mediated chemotaxis in a dose-response manner. Other unrelated NO donors, SIN-1 and S-nitroso-cysteine--a natural S-nitroso-compound, also induced a directed locomotion of neutrophils. Taken together, these in vitro experiments indicate that exogenous NO could mediate the chemotaxis of neutrophils and thus suggest that NO could contribute to neutrophil recruitment in vivo.
Interleukin 8 (IL-8) is a recently described cytokine that functions as a potent neutrophil chemoattractant and activator. We sought to examine the link between the generation of reactive oxygen intermediates (ROI) and the regulation of IL-8 gene expression to specifically test the hypothesis that ROI would induce production of IL-8 mRNA and protein. In lipopolysaccharide-stimulated human whole blood, the OH radical scavenger dimethyl sulfoxide (Me2SO) dramatically inhibited (approximately 90%) IL-8 production, but had minimal effects on the production of tumor necrosis factor, interleukin 1 beta (IL-1), and IL-6. To determine whether NADPH-oxidase-generated free radicals were critical in the regulation of IL-8, studies were performed using blood from patients with chronic granulomatous disease. In both normal individuals and patients with chronic granulomatous disease, production of IL-8 could be initiated with lipopolysaccharide, phytohemagglutinin, or aggregated immune complexes, and this production could be inhibited by Me2SO (1% v/v). To examine if oxidant stress represents a ubiquitous mechanism for the induction of IL-8, experiments were performed in cultured cell lines. In the human hepatoma cell line Hep-G2, Me2SO dose-dependently inhibited tumor necrosis factor-stimulated IL-8 production, with a 74 +/- 1% reduction observed at a Me2SO concentration of 1%. Direct exposure to ROI demonstrated that H2O2 stimulated IL-8 production in a dose-dependent manner in Hep-G2 cells, A549 pulmonary type II epithelial cells, and human skin fibroblasts; this induction could be prevented by addition of catalase. The production of IL-8 appeared to be specific to an oxidant stress since exposure of the cells to heat shock or chemical stress did not induce expression of IL-8. These studies demonstrate that oxidant stress is an important regulator of IL-8 gene expression and support the hypothesis that low levels of ROI may serve to initiate IL-8 production which then serves to recruit neutrophils to sites of inflammation.
Gene products of all three distinct nitric oxide synthases are present in the mammalian kidney. This mosaic topography of nitric oxide synthase (NOS) isoforms probably reflects distinct functional role played by each enzyme. While nitric oxide (NO) is cytotoxic to isolated renal tubules, inhibition of NO production in vivo invariably results in the aggravation of renal dysfunction in various models of acute renal failure. We reasoned that the existing ambiguity on the role of nitric oxide in acute renal failure is in part due to the lack of selective NOS inhibitors. Phosphorothioated derivatives of antisense oligodeoxynucleotides targeting a conserved sequence within the open reading frame of the cDNA encoding the inducible NOS (iNOS) were designed to produce a selective knock-down of this enzyme. In vivo use of these antisense constructs attenuated acute renal failure in rats subjected to renal ischemia. This effect was due, at least in part, to the rescue of tubular epithelium from lethal injury. Application of antisense constructs did not affect endothelial NOS, as evidenced by a spared NO release after the infusion of bradykinin during in vivo monitoring with an NO-selective microelectrode. In conclusion, the data provide direct evidence for the cytotoxic effects of NO produced via iNOS in the course of ischemic acute renal failure, and offer a novel method to selectively prevent the induction of this enzyme.
We determined in rat lung whether ozone exposure was associated with the expression of the chemokine, cytokine-induced neutrophil chemoattractant (CINC), and of the transcription factor, NF-κB. CINC mRNA expression peaked at 2 h after cessation of ozone exposure, and returned to basal levels by 24 h. DNA-binding activity of NF-κB showed a marked increase after ozone, maximal at 2 h. Dexamethasone inhibited CINC mRNA and NF-κB expression, together with neutrophilic inflammation. Our data supports the concept that ozone leads to NF-κB activation which increases CINC mRNA expression. These series of events could lead to neutrophilic inflammation.
Nitric oxide is a messenger molecule, mediating the effect of endothelium-derived relaxing factor in blood vessels and the cytotoxic actions of macrophages, and playing a part in neuronal communication in the brain. Cloning of a complementary DNA for brain nitric oxide synthase reveals recognition sites for NADPH, FAD, flavin mononucleotide and calmodulin as well as phosphorylation sites, indicating that the synthase is regulated by many different factors. The only known mammalian enzyme with close homology is cytochrome P-450 reductase.
Overproduction of the free radical nitric oxide (NO) has been implicated in the pathogenesis of a variety of inflammatory and immunologically mediated diseases as well as complications of diabetes. In the present study we have demonstrated that aminoguanidine selectively inhibits the cytokine-inducible isoform of NO synthase which appears to be responsible for the excess production of NO linked to these disease states. By using organ, cell and enzyme-based measurements we have shown that aminoguanidine is equipotent to NG-monomethyl-L-arginine (L-NMA) as an inhibitor of the cytokine-induced isoform of NO synthase but is 10 to 100-fold less potent as an inhibitor of the constitutive isoform. Thus, aminoguanidine may be useful as a selective inhibitor of the inducible NO synthase in the treatment of disease states characterized by the pathological overproduction of NO.
In nitrinergic signal transduction, nitrogen oxide (NO) synthases (NOS) (EC 1.14.23) catalyze the conversion of L-arginine to L-citrulline and NO, which in turn activates soluble guanylyl cyclase. Macrophages were reported to contain a single isoform of NOS (type II, soluble, Ca(2+)-independent, 130-kDa) and only upon activation of the cells by interferon-gamma (INF) and lipopolysaccharides (LPS). By a mechanism involving L-type Ca2+ channels, calmodulin, and serine proteases, INF/LPS also induce a cytotoxic activation of macrophages. In RAW264.7 macrophages, NO release was detected upon activation of the cells by INF/LPS but also, although at a 20-fold lower level, in control cells. The latter constitutive NOS activity and NO release were Ca2+ dependent and were decreased in INF/LPS-activated RAW264.7 cells or with increasing passage number. RAW264.7 cells did not express soluble guanylyl cyclase, suggesting other target molecules for NO. In INF/LPS-activated cells, NOS activities and NO release were Ca2+ independent (type II) and coinduced with NADPH-diaphorase activities both in the soluble and in the particulate fractions. The NOS-II activities corresponded to a 130-kDa protein, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was not recognized in a protein immunoblot with anti-NOS-I antibody. The serine protease inhibitor tosyl-lysyl chloromethyl ketone abolished the induction of NOS-II by INF/LPS, by depleting intracellular thiol pools and interfering with protein synthesis. Induction of NOS-II by INF/LPS was transcriptionally based and, for maximal enzyme activity, required increased intracellular tetrahydrobiopterin levels, intracellular Ca2+ mobilization, and activation of non-L-type Ca2+ channels but, unlike the induction of macrophage-mediated cytotoxicity, neither L-type-Ca2+ channels nor calmodulin.
Nitric oxide (NO) conveys a variety of messages between cells, including signals for vasorelaxation, neurotransmission, and
cytotoxicity. In some endothelial cells and neurons, a constitutive NO synthase is activated transiently by agonists that
elevate intracellular calcium concentrations and promote the binding of calmodulin. In contrast, in macrophages, NO synthase
activity appears slowly after exposure of the cells to cytokines and bacterial products, is sustained, and functions independently
of calcium and calmodulin. A monospecific antibody was used to clone complementary DNA that encoded two isoforms of NO synthase
from immunologically activated mouse macrophages. Liquid chromatography-mass spectrometry was used to confirm most of the
amino acid sequence. Macrophage NO synthase differs extensively from cerebellar NO synthase. The macrophage enzyme is immunologically
induced at the transcriptional level and closely resembles the enzyme in cytokine-treated tumor cells and inflammatory neutrophils.
Inhibitory non-adrenergic non-cholinergic (NANC) nerves are thought to be important in the autonomic innervation of the gastrointestinal tract and other organ systems. The nature of their neurotransmitter is still debated. Speculation that nitric oxide (NO), formed from L-arginine in neuronal structures and other cells, could act as a neurotransmitter, is not yet supported by demonstration of its release upon nerve stimulation. Using a superfusion bioassay, we report the release of a vasorelaxant factor upon stimulation of the NANC nerves in the canine ileocolonic junction. Several pieces of evidence, including the selectivity of the bioassay tissues, chemical instability, inactivation by superoxide anion and haemoglobin, inhibition by NG-nitro-L-arginine (L-NNA) and potentiation by L-arginine all indicated that NO accounted for the biological activity of this transferable NANC factor.
Many cell types are known to synthesize nitric oxide (NO.) from L-arginine. There appear to be at least two forms of NO. synthase: an inducible, tetrahydrobiopterin- and flavin-dependent activity exemplified by the macrophage enzyme and a constitutive, Ca+(+)-dependent activity exemplified by the endothelial cell enzyme. L-NG-methylarginine inhibits NO. synthesis by both cell types. We now report that L-NG-aminoarginine and L-NG-nitroarginine are about 100-fold more potent than NG-methylarginine in blocking endothelial cell NO. synthesis. In contrast, NG-aminoarginine and NG-methylarginine are about equipotent with macrophages whereas NG-nitroarginine is much less potent. Since macrophage and endothelial cell NO. synthesis are differentially sensitive to the inhibitors, the panel of inhibitors can be used in complex biological systems to determine if macrophage-like or endothelial-like cells are the predominant source of NO.. Indeed, all three inhibitors elicit a strong pressor response in the anesthetized guinea pig, a result consistent with the view that endothelial cells continually produce vasodilatory NO(.).
Consideration of the origin of airway hyperresponsiveness appears central to understanding the origin of asthma. Subjects with and without asthma differ both in the ease with which airway narrowing is produced by inhalation of histamine or methacholine and in the ability to demonstrate a maximal response to these agents. The latter appears, on present evidence, to be due to an added mechanism in asthma rather than the absence of a potent inhibitory process. Airway hyperresponsiveness is probably acquired during life as a result of airway reactions to various stimuli, although genetic factors such as atopy are likely to predispose the person to develop hyperresponsiveness. Environmental stimuli include inhaled allergens, chemical sensitizers, airway infections, immunization, and ozone. Allergen-induced airway hyperresponsiveness occurs in association with late-phase asthmatic responses. This and ozone-induced hyperresponsiveness have been demonstrated to be associated with release of chemical mediators and the cellular phase of inflammation. Their effect does not appear to be accounted for by increase in airway epithelial permeability, decrease in airway caliber, reflex bronchoconstriction, or beta-adrenoceptor blockade. The mechanism(s) responsible for the induced hyperresponsiveness are unknown but may involve airway epithelial damage, edema in and around the airway walls, stimulation of the noncholinergic excitatory or inhibition of the nonadrenergic inhibitory systems, or a change in function of airway smooth muscle. Airway hyperresponsiveness can be transient or persistent. Transient increases in responsiveness are almost certainly associated with mediator release and inflammation. It is not known whether persistent hyperresponsiveness is due to the same process, fired, for example, by leaky mediator-releasing cells and/or to some persisting change in neurogenic or smooth muscle function.
We examined the effect of ozone (O3) on muscarinic bronchial reactivity in the guinea pig and compared reactivity determined by two different routes of agonist delivery. Reactivity before and from 4 h to 2 days after O3 exposure (3.0 ppm, 2 h) was determined by measuring specific airway resistance upon administration of intravenous acetylcholine and/or aerosolized methacholine challenge in 34 unanesthetized, spontaneously breathing animals. Before exposure, we observed more gradual and reproducible results to intravenous agonist. After exposure, hyperreactivity to parenteral agonist occurred consistently, but not to inhaled agonist. Hyperreactivity demonstrable by either route was similar in magnitude and time course within 14 h of exposure. Two days later, hyperreactivity to inhaled agonist had remitted; that to intravenous drug persisted. Our results indicate that variability in the occurrence and time course of O3-induced hyperreactivity to inhaled agonist may be a consequence of the technique employed. The consistent occurrence of hyperreactivity after O3 to parenteral agonist suggests mechanisms other than airway mucosal hyperpermeability are responsible for this hyperreactivity.
cDNA for neutrophil attractant protein-1 (NAP-1, also known as IL-8) was cloned from Con A-stimulated guinea pig spleen cells with human NAP-1 cDNA as a probe. Guinea pig NAP-1 cDNA is composed of 1433 bp with an open reading frame which encodes for a 101-amino-acid protein. Guinea pig NAP-1 had 70% amino acid sequence similarity to human NAP-1, which was much higher than a similarity between human and guinea pig monocyte chemoattractant protein-1 (MCP-1) (56%). Nucleotide sequence similarity within the coding region was 75%. To confirm its biological activity in guinea pig, recombinant guinea pig NAP-1 was expressed in COS-7 cells then purified. N-terminal sequence analysis gave two different N-termini at position 23 (Met) or 24 (Val). The two proteins showed their peak activity for guinea pig neutrophils at the concentration of 1 microgram/ml (10-7 M). Despite its high similarity to human NAP-1, the responsiveness of human neutrophils to guinea pig NAP-1 was minimum. Recombinant guinea pig NAP-1 caused strong neutrophil infiltration after intradermal injection into guinea pig skin. Since guinea pig is classified as a rodent, it was of interest to know whether human NAP-1 cDNA hybridizes to genomic DNA of other rodents such as mouse or rat, in which a NAP-1 homologue has not been found. Under low stringency conditions, human NAP-1 cDNA hybridized to human, rabbit, and guinea pig DNA, but not to mouse or rat DNA. Unlike NAP-1, human MCP-1 cDNA hybridized to genomic DNA of rabbit, guinea pig, mouse, and rat; MCP-1 cDNA have been cloned from these species. The apparent absence of a NAP-1 gene in mouse or rat makes this chemoattractant unique among the members of the protein family to which NAP-1 and MCP-1 belong.
Nitrogen oxides (NOx), regarded in the past primarily as toxic air pollutants, have recently been shown to be bioactive species formed endogenously in the human lung. The relationship between the toxicities and the bioactivities of NOx must be understood in the context of their chemical interactions in the pulmonary microenvironment. Nitric oxide synthase (NOS) is a newly identified enzyme system active in airway epithelial cells, macrophages, neutrophils, mast cells, autonomic neurons, smooth muscle cells, fibroblasts, and endothelial cells. The chemical products of NOS in the lung vary with disease states, and are involved in pulmonary neurotransmission, host defense, and airway and vascular smooth muscle relaxation. Further, certain patients with pulmonary hypertension, adult respiratory distress syndrome and asthma may experience physiologic improvement with NOx therapy, including inhalation of nitric oxide (NO.) gas. Both endogenous and exogenous NOx react readily with oxygen, superoxide, water, nucleotides, metalloproteins, thiols, amines, and lipids to form products with biochemical actions ranging from bronchodilation and bacteriostasis (S-nitrosothiols) to cytotoxicity and pulmonary capillary leak (peroxynitrite), as well as those with frank mutagenic potential (nitrosamines). Recent discoveries demonstrating the relevance of these species to the lung have provided new insights into the pathophysiology of pulmonary disease, and they have opened a new horizon of therapeutic possibilities for pulmonary medicine.
Nitric oxide (NO) is detectable in exhaled air. To elucidate whether airway epithelial cells could be a source of NO, we investigated the expression of inducible nitric oxide synthase (iNOS) by the murine lung epithelial cell line, LA-4, in response to cytokine stimulation and the ability of corticosteroids to modulate this effect. Stimulation with cytomix, a combination of interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma, elevated nitrite levels by 873% in the culture supernatants and enhanced the conversion of arginine to citrulline by 273% at 24 h. An increased number of cells stained for iNOS and an increase in iNOS mRNA was also observed. Dexamethasone decreased the cytokine-induced increase in nitrite levels, NOS activity, iNOS immunoreactivity, and mRNA but did not change the half life of iNOS mRNA. These results show that lung epithelial cells can release NO, a process which can be inhibited by dexamethasone.
1. Ozone inhalation causes airway hyper-responsiveness and airway inflammation in dogs. The purpose of this study was to determine whether these effects are associated with increases in oxygen radical production from bronchoalveolar lavage (BAL) cells. 2. Twelve randomly selected dogs were studied twice, 4 weeks apart. On each study day, acetylcholine (ACh) airway responsiveness was measured before and 1 h after ozone (3 p.p.m., 30 min) or dry air inhalation, followed by BAL. The response to ACh was expressed as the concentration causing an increase in lung resistance of 5 cmH2O l-1 s-1 above baseline. Spontaneous and phorbol myristate acetate (PMA) (2.4 mumol l-1)-stimulated oxygen radical release from washed BAL cells (4 x 10(6) cells ml-1) was measured by luminol-enhanced chemiluminescence in a luminometer at 37 degrees C. 3. Ozone inhalation caused airway hyper-responsiveness. The concentration of ACh causing an increase in lung resistance of 5 cmH2O l-1 s-1 (the 'provocative' concentration) fell from 4.68 mg ml-1 (% S.E.M., 1.43) before, to 0.48 mg ml-1 (% S.E.M., 1.60) after ozone (P < 0.0001). Spontaneous chemiluminescence area under the curve (AUC) significantly increased after ozone from 4.08 mV (10 min) (% S.E.M., 1.28) after dry air to 8.25 mV (10 min; % S.E.M., 1.29) after ozone (P = 0.007). Ozone inhalation also increased PMA-stimulated chemiluminescence AUC from 18.97 mV (10 min; % S.E.M., 1.18) after dry air to 144.03 mV (10 min; % S.E.M., 1.45) after ozone (P = 0.0001). The increase in PMA-stimulated chemiluminescence was significantly correlated with ozone-induced ACh airway hyper-responsiveness (r = 0.83, P < 0.001). 4. These results indicate that inhaled ozone increases oxygen radical release from BAL cells and suggest that oxygen radicals are important in causing ozone-induced airway hyper-responsiveness.
The importance of the potent neutrophil chemoattractant leukotriene (LT)B4 in causing ozone-induced bronchoconstriction, airway inflammation, and airway hyperresponsiveness in dogs was studied using the LTB4-receptor antagonist SC-53228. Seven dogs from random sources were studied three times, at least 2 wk apart. On each occasion, acetylcholine (Ach) airway responsiveness was measured before and 1 h after ozone (3 ppm, 30 min) or dry air inhalation, followed by a bronchoalveolar lavage (BAL). On the first day, dogs were treated with SC-53228 (0.4 mg/kg intravenously) followed by a continuous intravenous infusion of 1.2 mg/kg/h before ozone inhalation. On the other two days, diluent was infused followed by ozone or dry air inhalation. Cell counts were measured in BAL and cell activation was measured by spontaneous and by phorbol myristate acetate-stimulated (PMA) (2.4 mumol/L) oxygen radical release, measured from washed BAL cells (4 x 10(6) cells) by lucigenin-enhanced chemiluminescence. Ozone inhalation caused bronchoconstriction and airway hyperresponsiveness. SC-53228 inhibited the ozone-induced airway hyperresponsiveness (p = 0.006), but not the bronchoconstriction. Spontaneous (p = 0.004) and PMA-stimulated (p = 0.04) lucigenin-enhanced chemiluminescence were increased after ozone inhalation. The ozone-induced increases in PMA-stimulated chemiluminescence were significantly attenuated by treatment with SC-53228 (p = 0.04). These results suggest that LTB4 is involved in the pathogenesis of ozone-induced airway hyperresponsiveness, possibly through activation of airway inflammatory cell.
Nitric oxide (NO) is known to be present in measurable quantities in the exhaled air of normal subjects and at higher concentrations in asthmatic subjects not treated with glucocorticoids. We confirmed these findings by analyzing the mean mixed expired NO concentrations of 43 stable asthmatics and 90 normal subjects; NO levels were higher in the asthmatic population (13.9 parts per billion [ppb] versus 6.2 ppb, p < 0.001). Although the effects of glucocorticoids on the NO content of mixed expired air are known, it is not known if beginning systemic glucocorticoid therapy reduces exhaled NO levels in a given individual. To examine this question, seven patients needing emergency therapy for asthma underwent repeated measurements of mixed expired NO levels during their course of treatment with glucocorticoids. All patients had a reduction in mixed expired NO concentration (p = 0.002) and an accompanying improvement in airway obstruction. The decrease in exhaled NO was evident as early as 48 h after the initiation of therapy (p = 0.05). These data suggest mixed expired NO concentrations may prove useful as an index of asthma severity and treatment efficacy for an individual patient.
Products released through the L-arginine/nitric oxide biosynthetic pathway regulate soluble guanyl cyclase activity, which in turn modulates polymorphonuclear leukocyte chemotaxis. We hypothesized that inhibitors of nitric oxide synthase attenuate polymorphonuclear leukocyte chemotaxis in vitro. To test this hypothesis, unstimulated polymorphonuclear leukocytes were pretreated with buffer or the nitric oxide synthase inhibitors NG-monomethyl-L-arginine (L-NMMA), NG-nitro-L-arginine methyl ester, and L-canavanine before being exposed to three structurally unrelated chemoattractants, N-formyl-methionyl-leucyl-phenylalanine, C5a des arginine, and leukotriene B4. Polymorphonuclear leukocyte chemotaxis was quantified with a modified blind-well chamber technique. We found that L-NMMA and L-canavanine but not NG-nitro-L-arginine significantly attenuated polymorphonuclear leukocyte chemotaxis (p < 0.05). L-Arginine but not D-arginine, the nitric oxide donor sodium nitroprusside, and 8-bromo-cyclic guanosine monophosphate restored polymorphonuclear leukocyte chemotaxis attenuated by L-NMMA. Chemotaxis of polymorphonuclear leukocytes primed with lipopolysaccharide (Escherichia coli 0127:B8) or phorbol-13-butyrate was also significantly attenuated by pretreatment with L-NMMA and L-canavanine. Consistent with these observations, intracellular concentrations of cyclic guanosine monophosphate in polymorphonuclear leukocytes was decreased by L-NMMA during exposure to N-formyl-methionyl-leucyl-phenylalanine. These data indicate that nitric oxide synthase inhibitors attenuate chemotaxis of unstimulated and primed polymorphonuclear leukocytes in vitro. We suggest that the L-arginine/nitric oxide biosynthetic pathway plays an important role in regulating polymorphonuclear leukocyte emigration in vivo.
To infer possible mechanisms of acute airway inflammation and mucus hypersecretion in acute severe asthma, we performed cellular and biochemical analysis on sputum from 18 adults with acute severe asthma and compared the results with results of analysis of sputum from 12 adults with cystic fibrosis (CF). We found that in subjects with asthma neutrophils made up more than 75% of sputum cells in 10 samples whereas eosinophils made up more than 75% of cells in only three samples. Fifty percent of the subjects with asthma reported that their asthma exacerbation was precipitated by a respiratory tract infection, and these subjects had a significantly higher percentage of neutrophils in their sputum (85% +/- 6% vs 57% +/- 12%, p = 0.05). In the CF samples neutrophils made up more than 95% and eosinophils less than 1% of cells in all samples analyzed. Analysis of fluid phase chemicals in asthmatic and CF sputum samples showed that despite overall lower mean values of neutrophil elastase (27 +/- 11 micrograms/ml vs 466 +/- 121 micrograms/ml, p = 0.0001) and interleukin-8 (IL-8) (55 +/- 15 ng/ml vs 186 +/- 24 ng/ml, p = 0.0001), some of the asthmatic samples had values for these variables that overlapped those in the CF samples. In addition, the asthmatic samples were distinguished by the presence of higher tryptase (10 +/- 7 U/L vs 0.9 +/- 0.9 U/L, p = 0.0001) and interleukin-6 (1166 +/- 447 ng/ml vs 186 +/- 24 ng/ml; p = 0.0001) levels and by a higher ratio of albumin to mucin-like glycoprotein (0.8 +/- 0.5 vs 0.1 +/- 0.002, p = 0.02). DNA levels were lower in the asthmatic samples (0.5 +/- 0.3 mg/ml vs 3.5 +/- 1.2 mg/ml, p = 0.05). We conclude that neutrophils predominate more frequently than eosinophils as the major inflammatory cell in sputum from patients with asthma in acute exacerbation. We speculate that this may be because respiratory tract infections are a frequent precipitant of acute asthma. In addition, the high IL-8 levels and free neutrophil elastase activity observed in asthmatic sputum suggests that IL-8 may mediate airway neutrophilia in acute asthma and that neutrophil elastase may mediate mucin glycoprotein hypersecretion in acute asthma, as has been proposed for the mucin hypersecretion in CF.
Interleukin-8 (IL-8) has been shown to be a chemotactic factor for neutrophils, T-lymphocytes and eosinophils, but it is unknown whether the IL-8-induced inflammatory cell accumulation into the airways can cause the bronchial hyperresponsiveness (BHR) characteristic of asthma. IL-8 at a dose of 0.5 or 5 micrograms/kg was administered intranasally to guinea-pigs twice a week for 3 weeks. One day after the last administration, animals were anesthetized and artificially ventilated through tracheal cannula and lateral pressure at the cannula (Pao) was measured as an overall index of airway responses to increasing concentrations of inhaled histamine (25, 50, 100, and 200 micrograms/ml). The IL-8 treatment significantly enhanced bronchial responsiveness to histamine in a dose-dependent manner (ANOVA P < 0.01). The provocative concentration of histamine causing a 100% increase in Pao (PC100) at a dose of 0.5 and 5 micrograms/kg of IL-8 was 68.1 (GSEM 1.12) and 35.6 (GSEM 1.25) micrograms/ml, respectively. The latter was significantly (P < 0.01) lower than that in control animals treated with PBS (93.3 [GSEM, 1.14] micrograms/ml). The IL-8 treatment also induced a significant influx of neutrophils, but not eosinophils, in bronchoalveolar lavage (BAL) fluid (18.3 +/- 8.8 and 30.6 +/- 8.3% in animals treated with 0.5 and 5 micrograms/kg, respectively, of IL-8 vs 3.6 +/- 0.7% in phosphate buffered saline-(PBS)-treated animals). Furthermore, we examined the effect of the thromboxane receptor antagonist S-1452 (0.01 or 0.1 mg/kg, i.p. 24 and 1 h before anesthesia) on this IL-8 induced BHR. S-1452 significantly inhibited the BHR dose-dependently (ANOVA P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)
The concentration of nitric oxide (NO) is increased in the exhaled air of asthmatic patients and may reflect cytokine-mediated inflammation in the airways. We investigated whether allergen-induced inflammation causes an elevation in the level of exhaled NO. Of 25 patients who underwent allergen challenge, 16 developed dual early and late responses, whereas eight had a single early response. In the patients with a dual response, the maximal fall in FEV1 during the late response was 26.8 +/- 4.2% at 9 h and there was a significant increase in the level of exhaled NO (maximal increase of 59.4 +/- 9.8%) 10 h after challenge. There was a significant relationship between the size of the late response and the increase in exhaled NO (r = 0.75, p < 0.01). In patients who have a single early response, there was no significant increase in exhaled NO, with the exception of a single time point at 21 h. In five patients given a control challenge with methacholine there was no change in exhaled NO. There was no increase in exhaled NO after inhaled histamine in any of the patient groups. We conclude that the late asthmatic response to allergen is associated with elevated exhaled NO concentrations and that this provides further evidence that exhaled NO may reflect allergic inflammation in asthmatic airways, and may be a useful marker in monitoring asthma and its response to anti-inflammatory treatments. Whether endogenously produced NO plays a pathophysiologic role in the late response remains to be determined.
We investigated the role of tachykinins in ozone-induced airway hyperresponsiveness (AHR) in guinea pigs. Airway responsiveness was assessed by determining the provocative concentration (PC200) of a histamine aerosol. Ozone exposure (3.0 ppm for 2 h) caused significant AHR. For vehicle-pretreated animals, the geometric mean pre- and post-ozone PC200 values were 0.87 mg/ml (GSEM 1.33) and 0.11 mg/ml (GSEM 1.17), respectively. Tachykinin depletion by capsaicin (50 mg/kg) prevented this AHR, whereas it did not alter pre-ozone airway responsiveness. The PC200 was 0.36 mg/kg (GSEM 1.64) before ozone and 0.24 mg/kg (GSEM 1.72) after ozone for this group. Ozone also caused a significant increase in neutrophils in bronchoalveolar lavage fluid (BALF) compared with BALF from a normal control group (1.71 +/- 0.69 versus 0.07 +/- 0.02 x 10(5)/ml, respectively). Capsaicin pretreatment attenuated this neutrophil influx (0.23 +/- 0.16 x 10(5)/ml). Morphometric assessment revealed edema of the bronchiolar wall after ozone exposure, which was not observed in the capsaicin group. BAL and morphometry revealed that the degree of ozone-induced epithelial desquamation was similar in both groups. These results suggest that tachykinins may be responsible for ozone-induced AHR, possibly via neurogenic inflammation.
The regulatory signals required to induce the production of IL-8, an important neutrophil chemoattractant and activator, have yet to be clearly defined. We examined the role of nitric oxide (NO) in IL-8 regulation. The NO synthase inhibitor, (L)-NG-nitroarginine methyl ester (L-NAME), inhibited the TNF-stimulated IL-8 production in the human endothelial cell line, ECV304, in a dose-dependent manner without affecting cellular viability (TNF alone, 5.5 +/- 0.9 ng/ml; TNF + 5 mM L-NAME, 2.4 +/- 0.5 ng/ml). Moreover, exogenously added NO produced by the spontaneous NO generating compounds, S-Nitroso-N-acetyl-D,L-pennicillamine (SNAP) and Ethanamine, 2,2'-(hydroxynitrosohydrazono)bis- (DETA NONOate), induced a dose-dependent release of IL-8 from these cells. Maximal stimulation of IL-8 was found to be 1.2 +/- 0.1 ng/ml with the 1 mM concentration of SNAP and 1.6 +/- 0.1 ng/ml with the 2 mM concentration of DETA NONOate. These results provide key evidence substantiating a regulatory role of NO in IL-8 expression.
Neutrophils may play important roles in chronic airway diseases. Pseudomonas is a common pathogen in some chronic airway diseases, and expression of the neutrophil chemoattractant interleukin-8 (IL-8) is induced by Pseudomonas in various cells in vitro. Here we examine the localization of IL-8 mRNA expression after incubating human and dog bronchi with Pseudomonas supernatant in vitro. To examine IL-8 expression in recruited neutrophils, we also superfused the dog bypassed tracheal segment with Pseudomonas supernatant in vivo and measured neutrophil number and IL-8 concentration in luminal fluid; simultaneously, we introduced Pseudomonas supernatant by catheter in a peripheral airway. After 6 h, we analyzed IL-8 mRNA expression and localization in removed tissue. Unincubated bronchi showed no IL-8 mRNA expression, but incubation with Pseudomonas supernatant in vitro resulted in IL-8 mRNA expression in surface epithelial, gland duct, and a subpopulation of serous gland cells. In vivo, introduction of Pseudomonas supernatant into dog trachea and peripheral airways caused IL-8 mRNA expression in epithelial and gland duct cells but also in the recruited neutrophils. Pseudomonas lipopolysaccharide alone was without effect in vitro and in vivo. We conclude that Pseudomonas products, but not lipopolysaccharide, stimulate IL-8 expression in airways and that this expression occurs primarily in surface epithelial and gland duct cells, thus bringing the chemoattractant to the bacterial site. Furthermore, IL-8 expression in recruited neutrophils provides a potential mechanism for positive feedback of this protective antibacterial response.
Acute exposure of animals and humans to ozone results in decrements in lung function, development of airway hyperreactivity, inflammation, edema, damage to pulmonary cells, and production of several compounds with tissue damaging, fibrinogenic or fibrotic potential. The contribution of airway epithelial cells and alveolar macrophages to these processes is unclear. In this study we have directly exposed human alveolar macrophages and human airway epithelial cells to ozone in vitro and measured the cytotoxic effects of ozone, as well as the production of the inflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), and fibronectin, all of which are substantially elevated in the bronchoalveolar lavage fluid of humans exposed to ozone. Cells were grown on rigid, collagen-impregnated filter supports, and the interaction of cells with ozone facilitated by exposing them to the gas with medium below the support but no medium on top of the cells. The results show that, although macrophages are much more sensitive to ozone than epithelial cells, they do not produce increased amounts of IL-6, IL-8, or fibronectin following ozone exposure. In contrast, epithelial cells produce substantially more of all three proteins following ozone exposure, and both IL-6 and fibronectin are secreted vectorially. An immortalized human airway epithelial cell line (BEAS 2B) was used in these experiments since human airway epithelial cells are infrequently available for in vitro studies. Data from this study extend previous findings which suggest that the BEAS cell line is a useful model to study the interaction between airway epithelial cells and environmental toxicants.
Ozone (O3) is a major constituent of urban air pollution. The acute effects of the inhalation of O3 at ambient or near-ambient concentrations on bronchoalveolar lavage (BAL) end points consistent with a distal lung inflammatory response have been well documented in human subjects. Animal toxicologic studies have shown that the airway is also a major site of O3-induced injury and inflammation. To date, no studies have confirmed this finding in human subjects. Effects of O3 on the proximal airways are not adequately studied by BAL, which is primarily influenced by events occurring in the terminal bronchioles and alveoli. We hypothesized that O3 causes injury and inflammation in the airways in addition to that previously documented to occur in the distal lung. We performed isolated lavage of the left mainstem bronchus and forceps biopsy of the bronchial mucosa in a group of 14 healthy, athletic subjects 18 h after exposure to 0.20 ppm O3 for 4 h during moderate exercise in order to assess this possibility. We followed an identical protocol in a similar group of 12 subjects exposed to filtered air. The mean (SD) total cell count and the lactate dehydrogenase (LDH) concentration in the isolated airway lavage were significantly greater after O3 than after air, 13.9 (20.5) versus 4.9 (5.4) cells/ml x 10(4) and 18.9 (11.2) versus 9.6 (9.0) U/L, respectively. Morphometry (2,070 neutrophils/cm2 of tissue for O3 and 330 neutrophils/cm2 of tissue for air) demonstrated that O3 exposure induced an acute inflammatory cell influx into the airway.(ABSTRACT TRUNCATED AT 250 WORDS)
We have previously reported various inductive effects of nitric oxide on human PBMC. We describe a novel and potentially important mechanism of nitric oxide signaling-through direct activation of guanine nucleotide-binding proteins (G proteins). We have found that nitric oxide treatment of membranes isolated from fresh human PBMC enhances the ability of these membranes to hydrolyze [gamma-32P]GTP and bind [gamma-35S]GTP. In addition, treatment of whole cells with nitric oxide yielded membranes with enhanced GTPase activity. Furthermore, the GTPase activity of pure, recombinant Gs alpha, Gi alpha 1, and p21ras was greatly enhanced by nitric oxide. In support of the existence of this pathway in whole cells, we found that the G protein inhibitor, GDP-beta-S, blocked NF-kappa B translocation induced by nitric oxide or LPS in permeabilized cells. In addition, nitric oxide greatly reduced the pertussis toxin-mediated ADP-ribosylation of 45- and 41-kDa proteins in membranes of these cells. Because G proteins play a central role in many diverse signaling systems, activation by an endogenous and inducible oxidant may represent a novel signaling pathway.
To determine the histologic differences in the airways of patients who died from sudden-onset asthma and the more common slow-onset asthma, we studied seven cases of fatal asthma. The numbers of eosinophils and neutrophils, as well as extracellular deposition of their respective granule contents in the airway mucosa and submucosa, were determined and statistically analyzed. Four of the seven patients had slow-onset asthma attacks in which the time interval between onset of asthma and death was more than 2.5 h. In contrast, three patients had sudden-onset asthma in which the time interval between onset of asthma attack and death was less than 1 h. The four patients with slow-onset fatal asthma had more eosinophils (34.1 +/- 6.3 in slow-onset; 9.7 +/- 3.5 in sudden-onset; p = 0.002) and fewer neutrophils (4.8 +/- 2.0 in slow-onset; 16.8 +/- 5.4 in sudden-onset; p = 0.008) in the airway submucosa than did patients with sudden-onset fatal asthma. In addition, within the slow-onset fatal asthma group, eosinophils exceeded neutrophils in the airway submucosa (eosinophils > neutrophils, p = 0.002). By contrast, within the sudden-onset fatal asthma group, neutrophils exceeded eosinophils (neutrophils > eosinophils, p = 0.04). We suggest that sudden-onset fatal asthma is immunohistologically distinct from slow-onset fatal asthma and that it is characterized by a relative paucity of eosinophils in the face of an excess of neutrophils in the airway submucosa.(ABSTRACT TRUNCATED AT 250 WORDS)
Peroxynitrite (ONOO-) is a cytotoxic product of the rapid reaction between nitric oxide and superoxide that may initiate inflammation. Isolated perfused tracheas from guinea pigs were incubated from the mucosal side for 15 min with peroxynitrite (1 to 100 muM). Thereafter, concentration-response curves to histamine and methacholine were constructed on the preparations. Peroxynitrite (10 muM) caused a significant hyperresponsiveness; the maximal contractions in response to histamine and methacholine were enhanced by 30% and 40%, respectively. In the peroxynitrite-treated group, clear epithelial damage as well as eosinophil destruction were detected. Moreover, 3, 5, and 10 days after intratracheal instillation of peroxynitrite (100 nmol), a significant rise in pulmonary resistance to histamine of anesthetized animals was observed. It is suggested that the generation of peroxynitrite from nitric oxide superoxide radicals during inflammatory processes induces epithelial damage, mediator release, and hence airway hyperresponsiveness. These findings may have clinical implications, because airway inflammation, epithelial damage, and hyperresponsiveness are characteristic features in patients suffering from asthma.
Inhalation of the pulmonary irritant ozone is associated with an accumulation of macrophages in the lung. These cells, along with type II epithelial cells, are activated to release increased quantities of hydrogen peroxide and nitric oxide, two reactive mediators that have been implicated in tissue injury. In the present studies we determined whether pretreatment of rats with bacterially derived endotoxin, which modulates oxidant levels in tissues, could abrogate the effects of ozone on lung injury and nitric oxide production. Acute exposure of rats to ozone (2 parts per million, 3 h) resulted in nitric oxide production in the lung as measured by electron paramagnetic resonance spin trapping. This was correlated with expression of inducible nitric oxide synthase (iNOS) mRNA in the lung as determined by in situ hybridization. Particularly high levels of iNOS were evident in alveolar macrophages and type II cells. Alveolar macrophages isolated from ozone-treated rats also expressed increased iNOS mRNA and protein as measured by Northern and Western blotting, respectively, and produced more nitric oxide compared with cells from air-exposed animals. Treatment of rats with endotoxin (5 mg/kg, intravenously), 30 min prior to ozone, was found to abrogate ozone-induced increases in iNOS mRNA and protein expression, as well as nitric oxide production by alveolar macrophages. This was associated with a reduction in ozone-induced tissue injury as determined by levels of lung lavage fluid protein. Ozone inhalation also resulted in a reduction in intracellular glutathione in alveolar macrophages, an effect that was blocked by endotoxin administration. Taken together, these data provide evidence that the protective effects of endotoxin against ozone-induced injury are mediated, at least in part, by alterations in levels of lung oxidants and antioxidants.
We investigated the role of neurogenic inflammation and the subsequent mechanisms in cigarette smoke-induced airway hyperresponsiveness in guinea pigs. Exposure to cigarette smoke was carried out at tidal volume for 3 min. Airway responsiveness to histamine was determined before and after smoke exposure followed by bronchoalveolar lavage (BAL). Plasma extravasation was evaluated by measuring the extravasation of Evans blue dye in the airway. Cigarette smoke produced significant airway hyperresponsiveness and plasma extravasation, with an influx of neutrophils in BAL fluid. FK-224 (10 mg/kg i.v.), a tachykinin antagonist at NK1 and NK2 receptors, significantly inhibited these changes. The thromboxane (Tx) B2 concentration was increased in BAL fluid after smoke exposure and was significantly inhibited by FK-224. OKY-046 (10 mg/kg i.v.), a Tx synthase inhibitor, significantly inhibited airway hyperresponsiveness but had no effect on neutrophil influx or plasma extravasation. The results suggest that neurogenic inflammation and the subsequent generation of Tx in the airway are important in the development of the airway hyperresponsiveness induced by cigarette smoke.
The induction of nitric oxide (NO) production and the expression of cytokine-induced neutrophil chemoattractant (CINC-1) were studied in rat peritoneal adherent cells stimulated with insoluble immune complexes containing rabbit IgG Ab and OVA as the cognate Ag (IC). Incubation with IC at concentrations as low as 10 microg/ml induced NO production and the expression of inducible NO synthase (iNOS) protein. This was accompanied by the expression of CINC-1 mRNA and the activation of nuclear factor-kappaB (NF-kappaB). However, the expression of iNOS and CINC-1 mRNA induced by IC showed a different temporal pattern and a different sensitivity to both the antioxidant agent pyrrolidine dithiocarbamate (PDTC) and modulation by NO itself. Whereas iNOS mRNA and protein expression were blunted by PDTC and NO-generating compounds, CINC-1 mRNA expression was either enhanced or not affected by PDTC and NO donors. The time course of NF-kappaB activation was parallel to that of iNOS induction and was influenced in the same sense as iNOS induction by antioxidants, NO donors, the protease inhibitor N-tosyl phenylalanine chloromethyl ketone, and inhibitors of protein tyrosine phosphorylation reactions. These data indicate the existence in rat macrophages of a signaling mechanism triggered by Fc gammaR occupancy that leads to nuclear signaling, is initiated by protein tyrosine phosphorylation reactions, and shows specific sensitivities to antioxidants and NO. Whereas trans-activation of the iNOS gene can be fully explained by the stimulation of NF-kappaB, induction of CINC-1 mRNA expression seems influenced by additional regulatory elements.
We have isolated a full-length cDNA for an inducible nitric oxide synthase (iNOS) from guinea-pig lung. The cDNA has a 3447 bp open reading frame encoding 1149 amino acid residues. The deduced amino acid sequence is approx. 80% identical with iNOS of human epithelial cells and murine macrophages. Consensus recognition sites for cofactors are highly conserved. COS cell lysate transfected with the guinea-pig iNOS shows significant levels of nitric oxide synthase (NOS) activity, and this is inhibited by 79% by chelation of Ca2+ ions. The NOS activity is restored in a concentration-dependent manner by increasing the free Ca2+ level. The NOS activity is also inhibited by trifluoperazine, a calmodulin antagonist, which suggests that the Ca2+ dependence is due to Ca2+-dependent calmodulin binding to the enzyme. Northern blot analysis reveals that the cloned iNOS mRNA is expressed in the lung and the colon in normal guinea pigs. Stimulation in vivo by lipopolysaccharide induces the expression of iNOS in the kidney, the spleen and the colon, but in the lung the same stimulation decreases its expression. These results suggest that the cloned guinea-pig iNOS is distinct in characteristics and expression from previously described iNOS forms.