INTRODUCTION

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Inhalation of ozone in normal subjects causes an inflammatory response in the airways. This is manifest as significant decreases in lung volume (1), increased bronchial reactivity to direct-acting stimuli such as methacholine (2), and increases in inflammatory cells and markers measured in both bronchoalveolar lavage fluid (BALF) (5, 6) and induced sputum (4, 7). The inflammatory response involves an increase in neutrophil numbers and various proinflammatory cytokines, for example, interleukin-8 (IL-8), and myeloperoxidase (MPO) within the airway (4).

Studies in dogs have shown that pretreatment with 1 wk of inhaled budesonide before exposure to ozone causes a significant attenuation of the neutrophil influx into BALF (8), and decreases in oxygen radical production by BALF cells from ozone-exposed dogs (9). Studies in rats have shown that dexamethasone treatment significantly reduces inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) expression in the lungs of ozone-exposed rats (10). To our knowledge, the effects of steroid treatment on the response to ozone exposure in human subjects have not previously been studied. Therefore, we studied the effects of inhaled corticosteroid treatment on ozone-induced inflammation in normal human volunteers.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects

Fifteen healthy subjects were studied (six male; mean age, 31.1 ± 2.1 yr). All had normal lung function (FEV₁, 94.7 ± 2.5% predicted value), normal bronchial reactivity (screening provocative concentration of methacholine causing a 20% reduction in FEV₁ [PC₂₀] > 64 mg/ml) and were nonatopic on skin prick testing to common aeroallergens (cat, grass pollen, Dermatophagoides pteronyssinus, and Aspergillus fumigatus). They reported no history of respiratory or allergic disease and were receiving no medications. All were nonsmokers or ex-smokers of at least 5 yr and none had suffered from a respiratory tract infection in the 8 wk before the trial. Written informed consent was obtained from all volunteers prior to entry to the study.

Study Design

Volunteers attended an initial screening visit and underwent a number of investigations in the following order: exhaled nitric oxide (NO) and carbon monoxide (CO) measurement, spirometry, skin prick testing, collection of exhaled air condensate, methacholine challenge, and sputum induction. They were randomized to receive either inhaled budesonide 800 µg or matched placebo twice daily for 2 wk. After 12 d of treatment, subjects returned to the laboratory for a further methacholine challenge, followed by sputum induction. Subjects then continued the first treatment for a further 2 d. At 14 d treatment subjects attended at 8:00 A.M. for ozone exposure. They underwent a number of baseline investigations prior to exposure (spirometry, exhaled NO, CO, and exhaled air condensate). They were then exposed in a challenge chamber to an atmosphere of 400 parts per billion (ppb) ozone for 2 h with intermittent exercise. Spirometry and exhaled NO and CO were measured immediately after the exposure and then hourly for 4 h. Exhaled air condensate, methacholine challenge, followed by sputum induction were repeated at 4 h postexposure. Subjects returned the following day at 24 h postexposure for measurement of exhaled NO and CO, bronchial reactivity, and for collection of exhaled air condensate. Subjects underwent each of the treatment arms with a 4-wk washout between treatments.

Ozone Exposure

Subjects were exposed for 2 h in a chamber (1.4 × 1.7 × 2.3 m) exercising on a cycle ergometer (Tunturi Ergometer W1; Tunturi, Piispanristi, Finland) for 20 min of each 30-min period. Work loads were 50 watts to simulate mild to moderate work. Ozone was generated by passing compressed air through an ozonator (Wallace & Tiernan Laboratory Ozonator type BA.023; Wallace & Tiernan Ltd., Tonbridge, UK). Concentrations were maintained at 0.4 ± 0.03 ppm (mean ± SD), measured using a Dasibi 1008-AH ozone monitor (Quantitech Ltd., Milton Keynes, UK).

Lung Function Measurements

FEV₁ and FVC were measured using a dry wedge spirometer (Vitalograph, Buckingham, UK). Values were expressed as percent predicted normal. Baseline values were measured after 15 min rest and taken as the highest of three readings. Single readings only were taken at other times. The level of bronchial reactivity was assessed by methacholine challenge performed according to a standardized technique (11). The PC₂₀ was determined by linear interpolation of the concentration-FEV₁ response curve.

Exhaled Breath NO and CO Levels

Exhaled breath NO and CO were measured simultaneously by a chemiluminescence analyzer (Model LR2000; Logan Research, Rochester, UK). The analyzer is sensitive to NO from 1 to 500 ppb by volume, with a resolution of 0.3 ppb. The analyzer was calibrated using certified NO mixtures (90 ppb and 436 ppb) in nitrogen (BOC Special Gases, Guildford, UK). Simultaneous CO measurements were made using a modified analyzer (EC50-MICRO Smokerlyzer CO monitor; Bedfont Scientific Ltd., Upchurch, UK) sensitive to CO from 0 to 500 ppm by volume, adapted for online recording of CO concentration and integrated with the Logan chemiluminescence analyzer. Ambient CO concentrations were made before each recording and subtracted from the highest of three readings. In addition to NO and CO, the analyzer measures CO₂ (resolution, 0.1% CO₂; response time, 200 ms) and sample pressure and volume in real time. Measurements were made by slow exhalation (5 to 6 L/min) from total lung capacity for 15 to 20 s against a mild resistance to exclude nasal contamination. The value corresponding to the plateau of the end-exhaled CO₂ reading was taken as representative of an alveolar sample. Pressure during expiration is kept constant (3 ± 0.4 mm Hg) by using a visual display of expiratory flow measured by pressure and volume sensors within the analyzer.

Exhaled Breath Condensate and Nitrite Assay

Breath condensate was collected using a glass condensing device that contained ice and water and was suspended in a second glass chamber, as previously described (12). Condensate was formed on the outer surface of the inner glass chamber that was separated from ambient air. After rinsing the mouth, subjects wearing a nose-clip breathed tidally through a mouthpiece and a system of one-way valves, which allowed inhalation of room air and exhalation into the condensing apparatus through a nonreturnable valve. During the first 3 min, no ice or water was present, to allow room air within the apparatus to be expelled. Ice and water were then added and the subject continued to breathe into the apparatus for a further 12 min. At any time the subjects felt saliva in their mouths, they were instructed to rinse their mouths again. The mouthpiece was used as a saliva trap. Approximately 1 ml of condensate was collected and stored at 70° C until further analysis. Nitrite was measured using a modification of the method of Misko and coworkers (13). Aliquots of 100 µl of samples and standards were added in duplicate to a clear-bottomed, 96-well plate (Costar UK Ltd., High Wycombe, UK). These were mixed with 10 µl of 2,3-diaminonaphthalene (DAN), 0.5 mg/ml in 0.62 M hydrochloric acid (Alexis Corp., Nottingham, UK). The plate was incubated in the dark at room temperature for 10 min. The reaction was then stopped by the addition of 10 µl of 1.4 M NaOH. The reaction product was measured immediately in a fluorescent plate reader (Biolite F1; Labtech, Uckfield, UK) with excitation at 360 nm and emission read at 460 nm. Standard curves for nitrite were made in distilled water. The limit of sensitivity of the assay is 0.1 µM. The reproducibility of exhaled breath nitrite concentrations performed on paired samples collected on two separate days from 34 normal subjects showed an intraclass correlation coefficient of 0.71.

Sputum Induction and Processing

Subjects inhaled 3.5% saline for 15 min in total, via an ultrasonic nebulizer (DeVilbiss 2000; DeVilbiss Co., Heston, UK) with a calibrated mass median aerodynamic diameter of 4.5 µm, and output of 4.5 ml/ min. The aerosol was inhaled through a tube 110 cm long with an internal diameter of 22 mm equipped with a mouthpiece, with the subject's nose clipped. Subjects discarded saliva into a bowl and mouth-washed before each expectoration. Secretions collected during the first 5 min were discarded to minimize squamous epithelial cell contamination. Subjects were encouraged to cough deeply at 5-min intervals and any other time they felt the need. Secretions expectorated over the final 10 min were kept at 4° C for not more than 2 h before processing. The whole sputum sample was processed as described previously (14). Sputum was diluted with Hanks' balanced salt solution (HBSS) containing dithiothreitol (DTT) (Sigma Chemicals, Poole, UK) and vortexed at room temperature. When homogeneous, the volume was recorded and the sample was diluted further with HBSS to a final concentration of 0.05% DTT and centrifuged at 300 × g for 10 min. The supernatant was separated and frozen at 70° C until analysis. The cell pellet was resuspended in HBSS. Total cell counts were determined on a hemocytometer slide using Kimura stain and slides were prepared using a cytospin (Shandon, Runcorn, UK) and stained with May-Grunwald-Giemsa stain. Differential cell counts were performed by a "blinded" observer. Three hundred nonsquamous cells were counted on two slides for each sample. Samples with > 50% squamous cells were considered unsatisfactory and were discarded. Differential cell counts were expressed as a percentage of lower airway cells, that is, excluding squamous epithelial cells.

MPO Assay

Sputum supernatant concentrations of MPO were measured using a commercially available kit (R&D Systems, Abingdon, UK), according to manufacturer's instructions. Standards were prepared in 0.05% DTT and all samples and standards were assayed in duplicate.

Tumor Necrosis Factor-Alpha (TNF-) Assay

TNF- concentrations were measured as previously described using an amplified sandwich enzyme-linked immunosorbent assay (ELISA) (15). Ninety-six well microtiter plates (Greiner Labortecnik Ltd., Dursley, UK) were coated with 100 µl mouse monoclonal anti-TNF- antibody (NBS Biologicals, Huntingdon, UK) at a 1:400 dilution and incubated for 2 h at 37° C. Plates were then washed with phosphate-buffered saline (PBS) containing 0.05% vol/vol Tween 20 and blocked with bovine serum albumin (BSA) 5% wt/vol for 25 min at 37° C. The plates were washed again and TNF- standards (containing 0.05% DTT) and sputum supernatant samples were added in duplicate and incubated at 4° C for 18 h. Plates were washed and incubated at room temperature for 2 h with 100 µl rabbit anti-human TNF- polyclonal antibody (Genzyme Diagnostics, West Malling, UK). After a further wash, an alkaline phosphatase-conjugated donkey anti-rabbit polyclonal IgG antibody (Jaxon/Stratech Scientific Ltd., Luton, UK) diluted 1:2,000 was added and incubated for 2 h at room temperature. Excess antibody was washed off and plates were developed with a p-nitro-phenyl phosphate assay kit (No. 50-80-00; KPL/Dynatech Laboratories Ltd., Billinghurst, UK). The optical density of the wells was read at 405 nm using a plate photometer and quantified by interpolation on a standard curve of known concentrations of human recombinant TNF- in the range 8 to 800 pg/ml (R&D Systems). The lower detection limit of the assay is 8 pg/ml.

IL-8 Assay

IL-8 concentrations were measured using an amplified sandwich ELISA as previously described (15). Ninety-six well microtiter plates were coated with 100 µl mouse monoclonal anti-human IL-8 antibody (Genzyme Diagnostics) at a concentration of 2.5 µg/ml and incubated overnight at 4° C. Plates were washed with PBS containing 0.05% vol/ vol Tween 20 and immediately blocked with BSA 1% wt/vol for 2 h at 37° C. Plates were decanted and IL-8 standards (containing 0.05% DTT) and sputum supernatant samples were added in duplicate and incubated at 37° C for 1 h. Plates were washed and incubated at 37° C for 1 h with 100 µl rabbit anti-human IL-8 polyclonal antibody (Genzyme Diagnostics). After a further wash, streptavidin-horseradish peroxidase (Genzyme Diagnostics) diluted in 0.05% PBS Tween 20 with 1% BSA was added and incubated for 15 min at 37° C. After a further wash, 100 µl tetramethylbenzidine (TMB) (Sigma Chemicals) was added and plates left for 20 min at room temperature. The reaction was stopped with 2 N sulfuric acid (Sigma Chemicals). Optical density of the wells was read at 450 nm using a plate photometer and quantified by interpolation from a standard curve constructed to known concentrations (16 to 2,000 pg/ml) of human recombinant IL-8 (Genzyme Diagnostics).

Statistical Analysis

Spirometry, exhaled NO and CO, and methacholine reactivity were analyzed using repeated measures analysis of variance (ANOVA). Where differences were found, the peak value after exposure was taken as a summary measurement and comparisons between treatments were made using a paired t test. Results of cell counts and supernatant assays were compared using a Wilcoxon signed rank test to examine differences between treatments at 4 h. The effect of ozone exposure on bronchial reactivity was calculated by comparing the difference in PC₂₀ before and after exposure in each subject. The effect is expressed as doubling doses using the formula: (log₁₀PC₂₀ preexposure  log₁₀PC₂₀ postexposure)  log₁₀ 2. Baseline measurements between groups and effects of treatment (pretreatment versus preexposure values) were compared using the appropriate paired test. Results of parametric data are expressed as mean ± SEM unless stated otherwise. Cell counts and supernatant concentrations are expressed as medians throughout. A value of p < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

All subjects completed the trial. Four subjects suffered adverse effects from budesonide treatment (one had a sore throat, two had a dry cough, and one complained of a strange taste of the inhaler). One subject complained of a sore throat with the placebo inhaler. One subject had a tight chest and sore throat after the ozone exposure that had been preceded by placebo treatment; all symptoms had resolved by the following day.

Spirometry

There was no difference in the pretreatment values for FEV₁ and FVC between treatment groups and no change in either parameter after treatment with placebo or budesonide. After ozone exposure there was a significant decrease in FEV₁ maximal immediately postexposure, in both placebo and budesonide treatment groups (mean fall placebo, 0.44 ± 0.1 L; mean fall budesonide, 0.60 ± 0.13 L; Figure 1A) but no difference between the two groups. There was also a significant decline in FVC immediately postexposure in both placebo (mean fall, 0.39 ± 0.08 L; Figure 1B) and budesonide (mean fall, 0.58 ± 0.13 L; Figure 1B) treatment groups. There was no significant difference in mean decrease in FVC between the two groups, although there was a tendency for a greater fall after budesonide treatment (p = 0.09).

View larger version (16K): [in this window] [in a new window]   Figure 1.   Changes in spirometry for placebo (open circles) and budesonide treatment (closed circles) for 2 wk, followed by ozone exposure (400 ppb for 2 h) in 15 normal subjects. Values at baseline, prior to exposure (pre-), immediately postexposure (0), and up to 24 h postexposure are given as liters for FEV1 (A) and FVC (B) and are mean ± 1 SEM. **p < 0.001, ***p < 0.0005 compared with preexposure values.

Bronchial Reactivity

There was no difference in methacholine reactivity at baseline prior to treatment (PC₂₀ placebo: 61 ± 1 mg/ml; PC₂₀ budesonide: 62 ± 1 mg/ml) and no change with either treatment (PC₂₀ placebo: 62 ± 1 mg/ml; PC₂₀ budesonide: 52 ± 1 mg/ml). There was a small but significant increase in mean reactivity to methacholine for the 15 subjects at 4 h after ozone exposure compared with preexposure values (PC₂₀ placebo: 40 ± 1 mg/ ml; PC₂₀ budesonide: 31 ± 1 mg/ml), but no difference in size of response between the two treatment groups (placebo: 0.6 ± 0.3 doubling doses; budesonide: 0.7 ± 0.3 doubling doses). Bronchial reactivity had returned to baseline values at 24 h postexposure.

Exhaled CO and NO Levels

There was no difference in the baseline values for CO and NO before either treatment, and no change in either parameter after placebo or budesonide treatment. Ozone exposure did not cause changes in either CO or NO and there were no differences between the two treatment groups at any time point (Figure 2).

View larger version (18K): [in this window] [in a new window]   Figure 2.   Changes in exhaled carbon monoxide (A) and exhaled nitric oxide (B) for placebo (open circles) and budesonide treatment (closed circles) for 2 wk, followed by ozone exposure (400 ppb for 2 h) in 15 normal subjects. Values at baseline, prior to exposure (pre-), immediately postexposure (0), and up to 24 h postexposure are given. Values for CO are parts per million and for NO, parts per billion and are mean ± 1 SEM.

Sputum Cell Counts

Two sputum samples had greater than 50% squamous cell contamination and were therefore excluded from analysis. There were no differences in the differential or absolute counts of any cell type before either treatment period and no change in cell counts after placebo or budesonide treatment (Table 1 and Figures 3, 4, and 5). There was a significant increase in differential and absolute neutrophil counts after ozone exposure in both the placebo- and budesonide-treated groups (Figures 3A and 3B), but no difference in the size of the response between the two groups. There was also a significant decrease in percentage macrophage counts after ozone exposure (Figure 4A), but no change in the absolute macrophage counts (Figure 4B). There was no significant difference between the two treatment groups at any time. There was a small but significant increase in absolute lymphocyte counts after both exposures to ozone (Figure 5B) but no difference between the two groups. There was no change in the differential lymphocyte counts (Figure 5A). There were no changes in either absolute or differential cell counts of epithelial cells or eosinophils after ozone exposure.

View larger version (16K): [in this window] [in a new window]   Figure 3.   Changes in sputum neutrophil counts after ozone exposure (400 ppb for 2 h) in 15 normal subjects. Values are shown for start of treatment period (Baseline), prior to exposure (Pre-), and 4 h after exposure (Post-) for placebo and budesonide treatment. Differential counts (A) are percentage of lower airway cells and absolute counts (B) are millions of cells per ml sputum. ***p < 0.0001. NS = not significantly different.

View larger version (17K): [in this window] [in a new window]   Figure 4.   Changes in sputum macrophage counts after ozone exposure (400 ppb for 2 h) in 15 normal subjects. Values are shown for start of treatment period (Baseline), prior to exposure (Pre-), and 4 h after exposure (Post-) for placebo and budesonide treatment. Differential counts (A) are percentage of lower airway cells and absolute counts (B) are millions of cells per ml sputum. ***p < 0.0001. NS = not significantly different.

View larger version (17K): [in this window] [in a new window]   Figure 5.   Changes in sputum lymphocyte counts after ozone exposure (400 ppb for 2 h) in 15 normal subjects. Values are shown for start of treatment period (Baseline), prior to exposure (Pre-), and 4 h after exposure (Post-) for placebo and budesonide treatment. Differential counts ( A) are percentage of lower airway cells and absolute counts (B) are millions of cells per ml sputum. *p < 0.05, **p < 0.01. NS = not significantly different.

Sputum Supernatants

There was no difference in sputum supernatant MPO concentrations between baseline values pretreatment, and no effect of budesonide treatment was seen. After ozone exposure there was a significant increase in MPO in the placebo-treated group (p = 0.01, Table 2), and a similar increase in the budesonide-treated group, although this failed to reach statistical significance (p = 0.06, Table 2). There was no difference in the size of the response between the two groups. Sputum supernatant TNF- and IL-8 were comparable at baseline pretreatment, and no treatment effects or effects of ozone exposure were seen (Table 2).

Exhaled Breath Nitrite

There was no difference between baseline concentrations of nitrite in exhaled breath condensate (placebo: 1.1 ± 0.2; budesonide: 0.8 ± 0.2 picomolar) and levels were not affected by budesonide treatment (post-placebo treatment, 0.8 ± 0.1; postbudesonide, 1.3 ± 0.2 picomolar). There were no changes in nitrite concentrations at 4 h (placebo: 1.0 ± 0.1; budesonide: 1.5 ± 0.3 picomolar) or 24 h (placebo: 1.0 ± 0.1; budesonide: 1.7 ± 0.4 picomolar) after ozone exposure in either drug treatment group.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The present study has demonstrated that exposure to 400 ppb ozone for 2 h caused a decrease in FEV₁ and FVC and an increase in methacholine reactivity in normal subjects. We have also shown a significant increase in absolute and differential neutrophil counts, and an increase in absolute lymphocyte counts in induced sputum at 4 h postexposure. None of these parameters was affected by pretreatment with inhaled budesonide 800 µg twice daily for 2 wk prior to exposure. Budesonide treatment alone did not affect any of the measured indices.

Our results are in agreement with published studies on the clinical effects of ozone exposure in normal subjects. Exposure to the same dose of ozone has been shown to produce decreases in lung function (1) and increases in reactivity to methacholine (3, 4). The former study (3) showed a similar increase in bronchial reactivity to the present study. However, in the latter study (4), the increase in methacholine reactivity was much greater than seen herein (3.0 doubling doses versus 0.6, respectively). This may be due to the time of methacholine challenge; in our study methacholine reactivity was increased at 4 h postexposure and had returned to normal at 24 h postexposure, whereas Hiltermann and coworkers (4) found increased reactivity at 12 h postexposure. It is possible that methacholine reactivity in our subjects worsened after 4 h before returning to baseline. Because the FEV₁ of all subjects had not returned to baseline before the methacholine challenge performed at 4 h post-ozone exposure, it is possible that the increase in methacholine reactivity after ozone exposure was caused by a lower starting FEV₁ rather than a direct effect of ozone.

Our data also confirm neutrophilic inflammation after ozone exposure in normal subjects, previously reported in both bronchoalveolar lavage fluid (BALF) (3, 5) and induced sputum (4, 7). It has previously been reported that methacholine challenge leads to an increase in differential neutrophil counts in asthmatic subjects (16). This is unlikely to have significantly biased the results reported herein, because all sputum inductions were preceded by methacholine challenge, hence controlling for this effect of methacholine challenge on neutrophil counts. We also found a decrease in differential macrophage counts, which is likely to represent a change in the population of cells within the airway (owing to the influx of neutrophils), rather than a real fall in macrophage numbers within the lung, since there was no change in the absolute numbers of macrophages seen.

We also demonstrated an increase in sputum supernatant MPO concentrations but no change in TNF- or IL-8. Previous studies have shown increases in granulocyte macrophage colony-stimulating factor (GM-CSF) in BALF (17) and IL-8 in bronchial fractions (18) in normal subjects after ozone exposure, but no change in TNF- concentrations (6). A previous study using induced sputum to study responses to 400 ppb ozone showed significant increases in MPO and nonsignificant increases in IL-8 (7), as seen herein.

In the present study, all parameters were measured after drug treatment prior to ozone exposure and were compared with pretreatment values. This is less rigorous than comparison with values after a control air exposure. However, the present protocol was designed to maximize patient recruitment and compliance. A 4-wk washout for steroid was needed. To put volunteers through four treatments and exposures (placebo-air, placebo-ozone, budesonide-air, budesonide- ozone) would have required a 20-wk protocol, which was considered impractical. The effects of exposure to 400 ppb ozone for 2 h in normal individuals have been well documented previously. Because the present results are in agreement with both clinical and inflammatory responses found in previous studies, the lack of air exposures as a negative control is unlikely to have significantly affected the interpretation of the study.

Treatment effects on methacholine reactivity and induced sputum were assessed at 48 h preexposure. Sputum induction alone is known to cause an increase in differential sputum counts (14, 19), apparent 24 h later. However, repeated sputum induction in normal volunteers at 48 h does not affect differential or absolute counts of any cell type (unpublished observation). In view of this it was decided to collect the preexposure sputum sample 48 h before exposure. Preexposure methacholine reactivity was not measured immediately prior to ozone exposure to prevent any residual effects of the methacholine affecting the response to inhaled ozone. For convenience this was also measured 48 h before exposure.

We also measured exhaled CO and NO after ozone exposure. NO can be detected in the exhaled air of animals and humans (20), and increased concentrations are thought to signify lower airway inflammation. Exhaled NO is increased in several inflammatory conditions including asthma (21, 22), viral respiratory tract infection (23), and bronchiectasis (24). In vivo exposure of rats to high doses of ozone causes increased production of NO from type II epithelial cells (25) and BAL macrophages (26). Exhaled CO is also a marker of airway inflammation, and particularly of oxidative stress (27). To our knowledge, there are no published data on the effect of ozone exposure on exhaled breath NO and CO concentrations in human subjects. The present study found no effect of ozone exposure on either of these measurements. There was, however, a trend for lower NO concentrations after ozone exposure, which was inhibited by budesonide treatment. It is possible that a significant effect of ozone on NO concentrations may have been found had greater numbers of subjects been studied, or that such a change may occur in a different patient group such as asthmatics who have higher baseline concentrations of exhaled NO. Although our experiment had no air exposure as a control, our lung function and cell count results are in agreement with previously published controlled exposure studies. Therefore, had there been a significant effect of this dose of ozone on exhaled NO and CO levels, it is likely to have been apparent in the present study.

From the present data, pretreatment of normal subjects with inhaled budesonide has no effect on the clinical or airways inflammatory response to ozone. Previous data from animal experiments have shown that the response to inhaled ozone can be modified by steroids. Dogs treated with inhaled budesonide for 1 wk have an attenuated response to inhaled ozone, with smaller increases in pulmonary resistance (8), reduced neutrophils and eosinophils in BALF (8), and reduced oxygen radical production from BALF cells (9), but with no significant effects on airway resistance to methacholine (8). Pretreatment with methylprednisolone abolishes the increase in bronchial blood flow in response to ozone inhalation in sheep (28). The reason for the discrepancy between the present data and those of the latter studies is not clear, but could be either species differences or the fact that larger doses of steroids were used in the animal studies.

Corticosteroids may be expected to have no effect on neutrophilic inflammation, because they are known to prolong neutrophil survival by inhibiting neutrophil apoptosis (29). Chronic obstructive pulmonary disease (COPD) is also associated with neutrophilic inflammation within the lung, with increased neutrophils in induced sputum (30). Studies investigating the effect of inhaled corticosteroids on neutrophilic inflammation in COPD have reported conflicting results. One study (31) found no effect on induced sputum neutrophils after a 2-wk treatment with either inhaled or oral corticosteroids. However, another study (32) reported significant decreases in induced sputum neutrophil counts after a longer (2-mo) course of inhaled corticosteroids. The reason for this discrepancy is unclear but may be due to differences in the length of treatment. The present data confirm that 2 wk of treatment with inhaled corticosteroids had no effect on neutrophilic inflammation in response to ozone. It is possible that a longer treatment period may have resulted in an attenuated neutrophilic response to ozone exposure. The ozone model in normal subjects may make it possible to further explore the question of steroid resistance in neutrophilic inflammation, because it may be possible to measure neutrophil survival and chemotactic factors.

In conclusion, pretreatment of normal subjects with inhaled budesonide does not prevent the clinical and inflammatory changes seen in response to inhaled ozone.