Ozone exposure enhances mast-cell inflammation in asthmatic airways despite inhaled corticosteroid therapy.

Asthmatics are recognised to be more susceptible than healthy individuals to adverse health effects caused by exposure to the common air pollutant ozone. Ozone has been reported to induce airway neutrophilia in mild asthmatics, but little is known about how it affects the airways of asthmatic subjects on inhaled corticosteroids. We hypothesised that ozone exposure would exacerbate the pre-existent asthmatic airway inflammation despite regular inhaled corticosteroid treatment. Therefore, we exposed subjects with persistent asthma on inhaled corticosteroid therapy to 0.2 ppm ozone or filtered air for 2 h, on 2 separate occasions. Lung function was evaluated before and immediately after exposure, while bronchoscopy was performed 18 h post exposure. Compared to filtered air, ozone exposure increased airway resistance. Ozone significantly enhanced neutrophil numbers and myeloperoxidase levels in airway lavages, and induced a fourfold increase in bronchial mucosal mast cell numbers. The present findings indicate that ozone worsened asthmatic airway inflammation and offer a possible biological explanation for the epidemiological findings of increased need for rescue medication and hospitalisation in asthmatic people following exposure to ambient ozone.

Airway antioxidant and inflammatory responses to diesel exhaust exposure in healthy humans.

Pulmonary cells exposed to diesel exhaust (DE) particles in vitro respond in a hierarchical fashion with protective antioxidant responses predominating at low doses and inflammation and injury only occurring at higher concentrations. In the present study, the authors examined whether similar responses occurred in vivo, specifically whether antioxidants were upregulated following a low-dose DE challenge and investigated how these responses related to the development of airway inflammation at different levels of the respiratory tract where particle dose varies markedly. A total of 15 volunteers were exposed to DE (100 microg x m(-3) airborne particulate matter with a diameter of <10 microm for 2 h) and air in a double-blinded, randomised fashion. At 18 h post-exposure, bronchoscopy was performed with lavage and mucosal biopsies taken to assess airway redox and inflammatory status. Following DE exposure, the current authors observed an increase in bronchial mucosa neutrophil and mast cell numbers, as well as increased neutrophil numbers, interleukin-8 and myeloperoxidase concentrations in bronchial lavage. No inflammatory responses were seen in the alveolar compartment, but both reduced glutathione and urate concentrations were increased following diesel exposure. In conclusion, the lung inflammatory response to diesel exhaust is compartmentalised, related to differing antioxidant responses in the conducting airway and alveolar regions.

Airway inflammation in iron ore miners exposed to dust and diesel exhaust.

The aim of the present study was to investigate if underground miners exposed to dust and diesel exhaust in an iron ore mine would show signs of airway inflammation as reflected in induced sputum. In total, 22 miners were studied, once after a holiday of at least 2 weeks and the second time after 3 months of regular work. Control subjects were 21 "white-collar" workers. All subjects completed a questionnaire regarding medical and occupational history, and underwent lung function testing and induced sputum collection. Total and differential cell counts and analyses of the fluid phase of the induced sputum were performed. Sampling of personal exposure to elemental carbon, nitrogen dioxide and inhalable dust was recorded. The average concentrations of inhalable dust, nitrogen dioxide and elemental carbon were 3.2 mg.m-3, 0.28 mg.m-3 and 27 microg.m-3, respectively. Miners had increased numbers of inflammatory cells, mainly alveolar macrophages and neutrophils, and increased concentrations of fibronectin, metalloproteinase-9 and interleukin-10 in induced sputum compared with controls. In conclusion, miners in an underground iron ore mine demonstrated persistent airway inflammation that was as pronounced after a 4-week holiday as after a 3-month period of work underground in the mine.

Different airway inflammatory responses in asthmatic and healthy humans exposed to diesel.

Particulate matter (PM) pollution adversely affects the airways, with asthmatic subjects thought to be especially sensitive. The authors hypothesised that exposure to diesel exhaust (DE), a major source of PM, would induce airway neutrophilia in healthy subjects, and that either these responses would be exaggerated in subjects with mild allergic asthma, or DE would exacerbate pre-existent allergic airways. Healthy and mild asthmatic subjects were exposed for 2 h to ambient levels of DE (particles with a 50% cut-off aerodynamic diameter of 10 microm (PM10) 108 microg x m(-3)) and lung function and airway inflammation were assessed. Both groups showed an increase in airway resistance of similar magnitude after DE exposure. Healthy subjects developed airway inflammation 6 h after DE exposure, with airways neutrophilia and lymphocytosis together with an increase in interleukin-8 (IL-8) protein in lavage fluid, increased IL-8 messenger ribonucleic acid expression in the bronchial mucosa and upregulation of the endothelial adhesion molecules. In asthmatic subjects, DE exposure did not induce a neutrophilic response or exacerbate their pre-existing eosinophilic airway inflammation. Epithelial staining for the cytokine IL-10 was increased after DE in the asthmatic group. Differential effects on the airways of healthy subjects and asthmatics of particles with a 50% cut-off aerodynamic diameter of 10 microm at concentrations below current World Health Organisation air quality standards have been observed in this study. Further work is required to elucidate the significance of these differential responses.

Clara cell protein as a biomarker for ozone-induced lung injury in humans.

Exposure to ozone (O3) impairs lung function, induces airway inflammation and alters epithelial permeability. Whilst impaired lung function and neutrophilia have been observed at relatively low concentrations, altered lung epithelial permeability is only seen after high-dose challenges. The appearance of Clara cell protein (CC16) in serum has been proposed as a sensitive marker of lung epithelial injury. Here, the use of CC16 as an injury biomarker was evaluated under a controlled exposure to O3 and the relationship between this marker of lung injury and early lung function decrements was investigated. Subjects (n=22) were exposed on two separate occasions to 0.2 parts per million O3 and filtered air for 2 h. Blood samples were drawn and lung function assessed at 2 h pre-exposure, immediately before and immediately after exposure as well as 2 and 4 h postexposure. O3 increased CC16 serum concentrations at 2 h (12.0+/-4.5 versus 8.4+/-3.1 microg x L(-1)) and 4 h postexposure (11.7+/-5.0 versus 7.9+/-2.6 microg x L(-1)) compared with air concentrations. Archived samples from O3 studies utilising the same design indicated that this increase was sustained for up to 6 h postexposure (9.1+/-2.6 versus 7.1+/-1.7 microg x L(-1)) with concentrations returning to baseline by 18 h (7.7+/-2.9 versus 6.6+/-1.7 microg x L(-1)). In these studies, the increased plasma CC16 concentration was noted in the absence of increases in traditional markers of epithelial permeability. No association was observed between increased CC16 concentrations and lung function changes. To conclude, Clara cell protein represents a sensitive and noninvasive biomarker for ozone-induced lung epithelial damage that may have important uses in assessing the health effects of air pollutants in future epidemiological and field studies.

Repeated daily exposure to 2 ppm nitrogen dioxide upregulates the expression of IL-5, IL-10, IL-13, and ICAM-1 in the bronchial epithelium of healthy human airways.

BACKGROUND: Repeated daily exposure of healthy human subjects to NO2 induces an acute airway inflammatory response characterised by neutrophil influx in the bronchial mucosa AIMS: To assess the expression of NF-kappaB, cytokines, and ICAM-1 in the bronchial epithelium. METHODS: Twelve healthy, young non-smoking volunteers were exposed to 2 ppm of NO2/filtered air (four hours/day) for four successive days on separate occasions. Fibreoptic bronchoscopy was performed one hour after air and final NO2 exposures. Bronchial biopsy specimens were immunostained for NF-kappaB, TNF-alpha, eotaxin, Gro-alpha, GM-CSF, IL-5, -6, -8, -10, -13, and ICAM-1 and their expression was quantified using computerised image analysis. RESULTS: Expression of IL-5, IL-10, IL-13, and ICAM-1 increased following NO2 exposure. CONCLUSION: Upregulation of the Th2 cytokines suggests that repeated exposure to NO2 has the potential to exert a "pro-allergic" effect on the bronchial epithelium. Upregulation of ICAM-1 highlights an underlying mechanism for leucocyte influx, and could also explain the predisposition to respiratory tract viral infections following NO2 exposure since ICAM-1 is a major receptor for rhino and respiratory syncytial viruses.

Ozone-induced bronchial epithelial cytokine expression differs between healthy and asthmatic subjects.

BACKGROUND: Ozone (O3) is a common air pollutant associated with adverse health effects. Asthmatics have been suggested to be a particularly sensitive group. OBJECTIVE: This study evaluated whether bronchial epithelial cytokine expression would differ between healthy and allergic asthmatics after ozone exposure, representing an explanatory model for differences in susceptibility. METHODS: Healthy and mild allergic asthmatic subjects (using only inhaled beta2-agonists prn) were exposed for 2 h in blinded and randomized sequence to 0.2 ppm of O3 and filtered air. Bronchoscopy with bronchial mucosal biopsies was performed 6 h after exposure. Biopsies were embedded in GMA and stained with mAbs for epithelial expression of IL-4, IL-5, IL-6, IL-8, IL-10, TNF-alpha, GRO-alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), fractalkine and ENA-78. RESULTS: When comparing the two groups at baseline, the asthmatic subjects showed a significantly higher expression of IL-4 and IL-5. After O3 exposure the epithelial expression of IL-5, GM-CSF, ENA-78 and IL-8 increased significantly in asthmatics, as compared to healthy subjects. CONCLUSION: The present study confirms a difference in epithelial cytokine expression between mild atopic asthmatics and healthy controls, as well as a differential epithelial cytokine response to O3. This O3-induced upregulation of T helper type 2 (Th2)-related cytokines and neutrophil chemoattractants shown in the asthmatic group may contribute to a subsequent worsening of the airway inflammation, and help to explain their differential sensitivity to O3 pollution episodes.

Effect of ozone on bronchial mucosal inflammation in asthmatic and healthy subjects.

Epidemiological studies suggestthat asthmatics are more affected by ozone than healthy people. This study tested three hypotheses (1) that short-term exposure to ozone induces inflammatory cell increases and up-regulation of vascular adhesion molecules in airway lavages and bronchial tissue 6 h after ozone exposure in healthy subjects; (2) these responses are exaggerated in subjects with mild allergic asthma; (3) ozone exacerbates pre-existent allergic airways inflammation. We exposed 15 mild asthmatic and 15 healthy subjects to 0.2 ppm of ozone or filtered air for 2 h on two separate occasions. Airway lavages and bronchial biopsies were obtained 6 h post-challenge. We found that ozone induced similar increases in bronchial wash neutrophils in both groups, although the neutrophil increase in the asthmatic group was on top of an elevated baseline. In healthy subjects, ozone exposure increased the expression of the vascular endothelial adhesion molecules P-selectin and ICAM- 1, as well as increasing tissue neutrophil and mast cell numbers. The asthmatics showed allergic airways inflammation at baseline but ozone did not aggravate this at the investigated time point. At 6 h post-ozone-exposure, we found no evidence that mild asthmatics were more responsive than healthy to ozone in terms of exaggerated neutrophil recruitment or exacerbation of pre-existing allergic inflammation. Further work is needed to assess the possibility of a difference in time kinetics between healthy and asthmatic subjects in their response to ozone.

Differences in basal airway antioxidant concentrations are not predictive of individual responsiveness to ozone: a comparison of healthy and mild asthmatic subjects.

The air pollutant ozone induces both airway inflammation and restrictions in lung function. These responses have been proposed to arise as a consequence of the oxidizing nature of ozone, depleting endogenous antioxidant defenses with ensuing tissue injury. In this study we examined the impact of an environmentally relevant ozone challenge on the antioxidant defenses present at the surface of the lung in two groups known to have profound differences in their antioxidant defense network: healthy control (HC) and mild asthmatic (MA) subjects. We hypothesized that baseline differences in antioxidant concentrations within the respiratory tract lining fluid (RTLF), as well as induced responses, would predict the magnitude of individual responsiveness. We observed a significant loss of ascorbate (ASC) from proximal (-45.1%, p <.01) and distal RTLFs (-11.7%, p <.05) in healthy subjects 6 h after the end of the ozone challenge. This was associated (Rs, -0.71, p <.01) with increased glutathione disulphide (GSSG) in these compartments (p =.01 and p <.05). Corresponding responses were not seen in asthmatics, where basal ASC concentrations were significantly lower (p <.01) and associated with elevated concentrations of GSSG (p <.05). In neither group was any evidence of lipid oxidation seen following ozone. Despite differences in antioxidant levels and response, the magnitude of ozone-induced neutrophilia (+20.6%, p <.01 [HC] vs. +15.2%, p =.01 [MA]) and decrements in FEV(1) (-8.0%, p <.01 [HC] vs. -3.2%, p <.05 [MA]) did not differ between the two groups. These data demonstrate significant differences between the interaction of ozone with RTLF antioxidants in MA and HC subjects. These responses and variations in basal antioxidant defense were not, however, useful predictive markers of group or individual responsiveness to ozone.

Nitric oxide (NO) in exhaled air after experimental ozone exposure in humans.

We hypothesized that ozone, a common air pollutant, potent in producing airway inflammation, would increase the production of exhaled nitric oxide (NO). If so, measurement of exhaled NO could potentially be a valuable tool in population studies of air pollution effects. Eleven healthy non-smoking volunteers were exposed to 0.2 ppm ozone (O3) and filtered air for 2h on two separate occasions. Exhaled NO and nasal NO were measured before and on five occasions following the exposures. Changes in exhaled and nasal NO after ozone exposure were adjusted for changes after air exposure. There was a slight decrease in exhaled NO (-0.6; -3.1-1.2 ppb) (median and 95% confidence interval) and of nasal NO (-57; -173-75 ppb) directly after the ozone exposure. No significant changes in exhaled or nasal NO were however found 6 or 24 h after the exposure. Within the examined group, an O3 exposure level proven to induce an airway inflammation caused no significant changes in exhaled or nasal NO levels. Hence, the current study did not yield support for exhaled NO as a useful marker of ozone-induced oxidative stress and airway inflammation after a single exposure. This contrasts with data for workers exposed to repeated high peaks of ozone. The potential for exhaled NO as a marker of oxidative stress therefore deserves to be further elucidated.

Bronchoalveolar inflammation after exposure to diesel exhaust: comparison between unfiltered and particle trap filtered exhaust.

OBJECTIVES: Air pollution particulates have been identified as having adverse effects on respiratory health. The present study was undertaken to further clarify the effects of diesel exhaust on bronchoalveolar cells and soluble components in normal healthy subjects. The study was also designed to evaluate whether a ceramic particle trap at the end of the tail pipe, from an idling engine, would reduce indices of airway inflammation. METHODS: The study comprised three exposures in all 10 healthy never smoking subjects; air, diluted diesel exhaust, and diluted diesel exhaust filtered with a ceramic particle trap. The exposures were given for 1 hour in randomised order about 3 weeks apart. The diesel exhaust exposure apperatus has previously been carefully developed and evaluated. Bronchoalveolar lavage was performed 24 hours after exposures and the lavage fluids from the bronchial and bronchoalveolar region were analysed for cells and soluble components. RESULTS: The particle trap reduced the mean steady state number of particles by 50%, but the concentrations of the other measured compounds were almost unchanged. It was found that diesel exhaust caused an increase in neutrophils in airway lavage, together with an adverse influence on the phagocytosis by alveolar macrophages in vitro. Furthermore, the diesel exhaust was found to be able to induce a migration of alveolar macrophages into the airspaces, together with reduction in CD3+CD25+ cells. (CD = cluster of differentiation) The use of the specific ceramic particle trap at the end of the tail pipe was not sufficient to completely abolish these effects when interacting with the exhaust from an idling vehicle. CONCLUSIONS: The current study showed that exposure to diesel exhaust may induce neutrophil and alveolar macrophage recruitment into the airways and suppress alveolar macrophage function. The particle trap did not cause significant reduction of effects induced by diesel exhaust compared with unfiltered diesel exhaust. Further studies are warranted to evaluate more efficient treatment devices to reduce adverse reactions to diesel exhaust in the airways.

Persistent airway inflammation but accommodated antioxidant and lung function responses after repeated daily exposure to nitrogen dioxide.

Nitrogen dioxide (NO2) is a common indoor and outdoor air pollutant that may induce deterioration of respiratory health. In this study the effects of repeated daily exposure to NO2 on airway antioxidant status, inflammatory cell and mediator responses, and lung function were examined. Healthy nonsmoking subjects were exposed under controlled conditions to air (once) and to 2 ppm of NO2 for 4 h on four consecutive days. Lung function measurements were made before and immediately after the end of each exposure. Bronchoscopy with endobronchial biopsies, bronchial wash (BW), and bronchoalveolar lavage (BAL) was carried out 1.5 h after the air exposure and after the last exposure to NO2. Repeated NO2 exposure resulted in a decrease in neutrophil numbers in the bronchial epithelium. The BW revealed a twofold increase in content of neutrophils (p < 0.05) and a 1.5-fold increase in myeloperoxidase (MPO) (p < 0.01) indicative of both migration and activation of neutrophils in the airways. After the fourth NO2 exposure, antioxidant status of the airway fluid was unchanged. Significant decrements in FEV1 and FVC were found after the first exposure to NO2, but these attenuated with repeated exposures. Together, these data indicate that four sequential exposures to NO2 result in a persistent neutrophilic inflammation in the airways, whereas changes in pulmonary function and airway antioxidants are resolved. We conclude that NO2 is a proinflammatory air pollutant under conditions of repeated exposure.

Nitrogen dioxide exposure impairs the frequency of the mucociliary activity in healthy subjects.

In previous studies, we have reported bronchoalveolar lavage (BAL) findings following single and repeated exposures to NO2. Here, we present the first data on measurements of the frequency of the mucociliary activity in healthy subjects following exposure to NO2. Twenty four healthy nonsmoking volunteers underwent fibreoptic bronchoscopy, during which mucociliary activity was measured using an in vivo method based on a two-way fibreoptic system, to obtain baseline data. Two weeks later, the subjects were reinvestigated following NO2 exposure. Eight volunteers were investigated 45 min after a 20 min exposure to 1.5 ppm NO2 (Group A); eight subjects 45 min after a 20 min exposure to 3.5 ppm NO2 (Group B); and eight subjects 24 h after a 4 h exposure to 3.5 ppm NO2 (Group C). No activity was detected in Group A or B 45 min after exposure, in contrast to the easily identifiable mucociliary activity waves at the reference bronchoscopy. In group C, a significant elevation of the frequency of the mucociliary activity waves was seen at 24 h. It is concluded that short-term exposure to NO2 in man produces a significant reduction in the mucociliary activity 45 min after exposure. The effect ceases within 24 h. Further studies are needed to evaluate the effects of repeated exposures with NO2 on mucociliary activity in man.

Differences in bronchoalveolar cell response to nitrogen dioxide exposure between smokers and nonsmokers.

We have previously reported on the bronchoalveolar inflammatory effects of the common air pollutant NO2 in nonsmokers. In this study, we have investigated these effects in tobacco smokers. Eight young nonbronchitic smokers and, as a reference group, eight healthy life-time nonsmokers were exposed to 3.5 ppm NO2 for 20 min. Bronchoalveolar lavage (BAL) was performed 3 weeks before and 24 h after exposure. The first recovered 20 ml was analysed separately and defined as the bronchial portion (BP), and the following fluid recovered as the bronchoalveolar portion (BAP). Before exposure, the smokers had significantly less CD3+ cells and more alveolar macrophages (AMs) in the BP and the BAP, as well as reduced AM phagocytosis in vitro compared to nonsmokers. After NO2 exposure, the smokers reacted with an increase of AMs and neutrophils in BAP. Nonsmokers reacted with an increase of neutrophils in BP, an increase of lymphocytes in BAP, and a tendency to reduced AM phagocytosis. In summary, young smokers and nonsmokers differed to some extent in their reactions to NO2 exposure. This is probably due to the pre-existing airway inflammation and compensatory mechanisms to oxidant stress in the smokers.

Reductions in lymphocyte subpopulations after repeated exposure to 1.5 ppm nitrogen dioxide.

In this investigation the effects of repeated exposure to 1.5 ppm NO2 on immune competent cells in bronchoalveolar lavage (BAL) fluid was studied. Special attention was focused on effects on lymphocyte subpopulations. Eight healthy subjects were exposed to 1.5 ppm NO2 every second day on six occasions. Bronchoalveolar lavage fluid was collected at least three weeks before the exposure series as reference and 24 hours after the last exposure. The results obtained were analysed using a non-parametric test for paired observations, with each subject as his own control. Significant reductions were found in the total number and percentage of T cytotoxic-suppressor cells in BAL fluid; this caused an increase in the ratio of T helper-inducer: cytotoxic-suppressor cells. The total number of natural killer cells in the BAL fluid was also reduced. The numbers of all other cell types were unchanged after exposure. No reduction of phagocytosis of opsonised yeast particles by alveolar macrophages in vitro was detected. It is concluded that repeated short term exposures to 1.5 ppm NO2, a moderate occupational concentration, induces significant effects on immune competent bronchoalveolar lymphocytes. This indicates that previous findings of changes in the lymphoid immune system induced by NO2 in animals may well be applicable to humans.

Effects of repeated exposure to 4 ppm nitrogen dioxide on bronchoalveolar lymphocyte subsets and macrophages in healthy men.

Following the basal descriptive studies of the bronchoalveolar inflammatory cell response induced by single exposure with nitrogen dioxide (NO2) in man, it was considered important to clarify the cell response to repeated exposure with NO2. This investigation was, therefore, undertaken with bronchoalveolar lavage (BAL) 3 weeks before and 24 h after six repeated exposures with 4 ppm NO2 (7 mg.m-3) in ten healthy volunteers. The exposures were performed during 20 min and every second day. Analysis of the recovered BAL fluid demonstrated that repeated exposures to NO2 caused a lung cell response different from that reported after a single exposure. Amounts of lavaged alveolar macrophages, B-cells, and natural killer (NK)-cells were decreased and the T-helper-inducer/cytotoxic-suppressor cell ratio was altered, but there was no lymphocytosis or mastocytosis as after single exposure. Lymphocyte numbers in peripheral blood were reduced after exposure. These results suggest that repeated exposure with NO2 adversely affects the immune defence. This could contribute to the increased susceptibility to airway infections reported to be associated with NO2 exposure.