Ozone Exposure Increases Eosinophilic Airway

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Ozone Exposure Increases Eosinophilic Airway Response Induced by Previous Allergen Challenge Barbara Vagaggini, Mauro Taccola, Silvana Cianchetti, Stefano Carnevali, Maria Laura Bartoli, Elena Bacci, Federico L. Dente, Antonella Di Franco, Daniele Giannini, and Pier Luigi Paggiaro Cardiothoracic Department, Respiratory Pathophysiology Unit, University of Pisa, Pisa, Italy

We investigated whether exposure to ozone (O3) 24 hours after an allergen challenge test would increase airway eosinophilia induced by allergen in subjects with mild asthma with late airway response. Twelve subjects with mild atopic asthma participated in a randomized, single-blind study. Subjects underwent allergen challenge 24 hours before a 2 hour exposure to O3 (0.27 ppm) or filtered air. Pulmonary function was monitored during the allergen challenge and after the exposure to O3 or air. Six hours later, induced sputum was collected. After 4 weeks, the experiment was repeated with the same subjects. Allergen induced a comparable late airway response in both challenges. O3 exposure induced a significant decrease in FVC, FEV1, and vital capacity, and was associated with a significant increase in total symptom score compared with air exposure. The percentage of eosinophils, but not the percentage of neutrophils, in induced sputum was significantly higher after exposure to O3 than after exposure to air (p ⫽ 0.04). These results indicate that O3 exposure after a late airway response elicited by allergen challenge can potentiate the eosinophilic inflammatory response induced by the allergen challenge itself in subjects with mild atopic asthma. This observation may help explain the synergistic effect of air pollution and allergen exposure in the exacerbation of asthma. Keywords: ozone; allergen; asthma; eosinophils; induced sputum

The strict link between increased air pollution and hospital admissions is well known (1). Epidemiologic studies have shown that patients with asthma are at increased risk for adverse effects due to exposure to ambient levels of pollutants (2, 3). Laboratory studies have demonstrated that short-term ozone (O3) exposure, at a concentration similar to that encountered in the ambient air, causes a decrease in pulmonary function both in normal subjects and in subjects with asthma, the decrease being more pronounced in the latter group (4, 5). A neutrophilic inflammatory response in the airways of both normal subjects and subjects with asthma is evident 6 to 18 hours after short-term exposure to O3, both in bronchoalveolar lavage fluid and in induced sputum (6–9). Bascom and coworkers have reported that subjects with allergic rhinitis exposed to 0.5 ppm O3 for 4 hours had an increased influx of eosinophils in the nasal lavage fluid (10). Peden and coworkers demonstrated that subjects with asthma exposed to 0.16 ppm O3 for 7.6 hours had a significant increase of eosinophils in the bronchoalveolar lavage fluid (11). Some studies have reported an increase in eosinophilic cationic protein level both in bronchoalveolar lavage fluid and in

(Received in original form January 10, 2002; accepted in final form July 8, 2002) Correspondence and requests for reprints should be addressed to Dr. Barbara Vagaggini, Dipartimento Cardiotoracico, Ospedale Cisanello, via Paradisa 2, 56100 Pisa, Italy. E-mail: [email protected] Am J Respir Crit Care Med Vol 166. pp 1073–1077, 2002 DOI: 10.1164/rccm.2201013 Internet address: www.atsjournals.org

sputum supernatant obtained from subjects with asthma after exposure to O3 (12, 13). In the last decade, several studies have investigated the relationship between air pollutants and allergens to better understand the mechanism by which exposure to pollutants potentiates the effect of allergens in subjects with asthma. Most of the findings show that pre-exposure to O3 increases allergen-induced bronchoconstriction in subjects with asthma (14–16). There are studies showing that acute exposure to O3 or nitrogen dioxide primes the eosinophils for subsequent activation by allergens in the nose (17, 18). In contrast, there are no studies examining the inflammatory response to O3 occurring after a previous allergen challenge. Our principal objective was to investigate the change in cellular profile of the induced sputum obtained after O3 exposure in subjects with mild atopic asthma who had developed a late airway response to allergen challenge the day before. Our hypothesis was that acute airway inflammation due to allergen challenge could be increased by a subsequent exposure to O3. METHODS Subjects We studied 12 subjects with mild persistent asthma who had not received regular treatment in the last 3 months but had used short-acting, inhaled ␤2-agonists as needed. All subjects were free from upper respiratory infections for at least 6 weeks before the study. Diagnosis of asthma and assessment of asthma severity were made according to international guidelines (19). All subjects underwent a nonspecific challenge test with methacholine by means of the previously described method for evaluating bronchial hyper-reactivity (20). All had shown a dual (early and late) response to a previous allergen challenge performed 3 to 6 months before the study. The functional characteristics of the 12 subjects examined are reported in Table 1. The study was approved by the Hospital Medical Ethics Committee, and informed consent was obtained from all participants.

Study Protocol Exposures were done in a single-blind manner: subjects were not aware of the type of exposure. All subjects came to the laboratory at 8:00 a.m. on four different days (Figure 1). On Day 1, all subjects underwent the allergen challenge test. On Day 2 (24 hours after the allergen test), they were randomly exposed to air or O3 (0.27 ⫾ 0.04 ppm) for 2 hours. Four weeks later (Day 3), all subjects repeated the allergen challenge test, and on Day 4 (24 hours after the allergen challenge), they were again randomly exposed to O3 or air for 2 hours. In both allergen challenges, the same dose of inhaled allergen was used. During the exposure, subjects exercised for 20 minutes every hour on a cycloergometer at a workload predetermined to produce a ventilation rate of 25 L/minute per square meter of body surface area. Before and after each 2-hour exposure to air or O3, pulmonary function tests were performed and a questionnaire was completed. Each subject was asked to grade the severity of each symptom on a scale from 0 (no symptom) to 4 (worst symptom): cough, shortness of breath, tearing, burning eyes, throat and nose irritation, chest pain on deep inspiration, headache, dizziness, nausea, confusion, and sweating. A total symptom score was computed for each subject as the sum of all

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AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 166 2002 TABLE 1. FUNCTIONAL CHARACTERISTICS OF THE 12 SUBJECTS EXAMINED

No.

Age (yr)

Allergen Used in sBPT

Smoker (y/n)

PD20FEV1 Methacholine (lg)

Baseline FEV1 (L)

Baseline FEV1 (% predicted value)

1 2 3 4 5 6 7 8 9 10 11 12

19 18 24 24 20 23 31 26 18 37 25 21

Phleum DP DP DP DP DP Parietaria DP Phleum Phleum DP DP

N N N N N N N N N N N N

704 72 251 586 891 258 937 356 41 77 116 215

2.84 4.50 4.20 3.90 3.70 4.75 2.80 4.00 3.60 3.10 3.20 2.90

92 111 102 84 107 111 110 114 89 83 107 104

Definition of abbreviations: DP ⫽ Dermatophagoides pteronyssinus; PD20FEV1 ⫽ provocative dose of 20% fall in FEV1; sBPT ⫽ specific bronchial provocative test.

single symptom scores. Six hours after the end of the chamber exposures, pulmonary function tests were performed again, and sputum was induced by means of the hypertonic saline inhalation test.

Techniques Specific bronchial provocation test. Allergen challenge was performed with allergens (Table 1) standardized in biologic units according to a method described previously (20). Early and late airway responses were reported as the maximum percentage change in FEV1 during the first hour or between 2 and 8 hours after allergen inhalation, respectively. In each test, the same total dose of allergen was delivered. The reproducibility of the test was good (21). Challenge chamber. Subjects were exposed for 2 hours in a 9-m3 static challenge chamber made of glass and aluminum, while they exercised on a stationary cycloergometer for 20 minutes every hour. The mean (⫾ SD) air temperature was 21 ⫾ 1⬚C, and the relative humidity was 45 ⫾ 5%. Methods of O3 generation and conditions of exposure have been reported previously (9). The mean O3 concentration was 0.27 ⫾ 0.04 ppm. Hypertonic saline inhalation test. The hypertonic saline inhalation test was performed according to the method of Pin and coworkers (22), with partial modification (23). Hypertonic saline solution was inhaled

for 5-minute periods for up to 30 minutes. At 10-minute intervals, NaCl concentration was increased from 3 to 4 to 5%. Every 5 minutes after the start of nebulization, patients were asked to rinse their mouths and throats carefully to discard saliva and to try to cough sputum into a clean container; FEV1 was then measured. Nebulization was stopped after 30 minutes or when FEV1 decreased by 20% or more from baseline values. Sputum processing. Sputum was processed as soon as possible (within 2 hours) after collection, according to a method derived from Pin and coworkers (22). Two investigators, blinded to the subjects’ history and exposure, each counted at least 500 cells on each sputum slide. Macrophage, lymphocyte, neutrophil, and eosinophil counts were expressed as a percentage of the total number of inflammatory cells, excluding squamous cells. The quality of the cytospin was defined by an association of various characteristics, such as absence of debris, well-preserved cell outline, low number of damaged cells, low number of squamous epithelial cells (⬍ 20%), and cell viability greater than 60%. Interleukin-8 level in sputum supernatant. Antigenic interleukin-8 levels in sputum supernatant were quantified using a sandwich enzymelinked immunosorbent assay, as described previously (9). The lower limit of interleukin-8 detectable by this assay is 10:00 p.m. All samples were assayed in duplicate, and mean values were computed.

Statistical Analysis Maximum FEV1 decrease during early and late airway responses, FVC, vital capacity, FEV1 (as percentage of the predicted value), and total symptom score are expressed as mean ⫾ SD. Differential cell percentages in induced sputum and interleukin-8 levels in the supernatant are expressed as median and range. A paired t test was used to compare late airway response to allergen challenge before O3 exposure and before air exposure. A concordance index between the maximum percentage change in FEV1 during late airway response obtained in the two tests was computed. Paired t tests were also used to compare the FVC, FEV1, and vital capacity values obtained after O3 exposure with those obtained after air exposure. Nonparametric Wilcoxon test was used to compare cell percentages in the induced sputum and interleukin-8 concentrations in the supernatant after O3 exposure with the corresponding values obtained after air exposure.

RESULTS Figure 1. Schematic protocol schedule. On Day 1 and Day 3 (4 weeks after Day 1), allergen challenge tests were performed to obtain early and late airway responses. Pulmonary function was tested every hour between Hours 2 and 8. On Day 2 and Day 4 (4 weeks after Day 2), subjects were randomly exposed to O3 or air. Six hours after the end of the exposure, sputum was induced by means of a hypertonic saline inhalation test. HS ⫽ hypertonic saline; PFT ⫽ pulmonary function test.

The mean values of early and late airway responses to the allergen challenge were similar the day before O3 exposure and the day before air exposure (early response, maximum percentage change in FEV1: 34.2 ⫾ 7.7% versus 35.7 ⫾ 7.3%, p ⫽ 0.6; late response, maximum percentage change in FEV1: 26.9 ⫾ 10% versus 23.3 ⫾ 9.3%, p ⫽ 0.3). The concordance index between the late airway responses was good (0.72).

Vagaggini, Taccola, Cianchetti, et al.: Ozone and Allergen-induced Eosinophilia TABLE 2. MEAN VALUES (⫾ SD) OF FVC, FEV1, VITAL CAPACITY (ABSOLUTE VALUE AND PERCENTAGE OF THE PREDICTIVE VALUE), AND TOTAL SYMPTOM SCORE BEFORE AND AFTER EXPOSURE TO AIR OR OZONE

FVC before Liter Percent FVC after Liter Percent FEV1 before Liter Percent FEV1 after Liter Percent VC before Liter Percent VC after Liter Percent TSS before TSS after

Air

Ozone

4.89 ⫾ 0.8 91.3 ⫾ 8.4

4.93 ⫾ 0.87 92.6 ⫾ 0.9

5.07 ⫾ 0.9 94.5 ⫾ 11.0

4.57 ⫾ 0.92* 86 ⫾ 9.6*

3.58 ⫾ 0.67 83.3 ⫾ 10.4

3.53 ⫾ 0.62 82.4 ⫾ 10.4

3.92 ⫾ 0.74 90.7 ⫾ 9.50

3.36 ⫾ 0.84* 78.2 ⫾ 13.3*

4.95 ⫾ 0.92 90.8 ⫾ 8.4

5.01 ⫾ 0.88 92.9 ⫾ 8.9

5.14 94.2 14.3 15.4

⫾ ⫾ ⫾ ⫾

0.92 11 1.7 1.6

4.70 87.0 15.2 18.1

⫾ ⫾ ⫾ ⫾

0.93 9.1* 1.4 2.7*

Definition of abbreviations: TSS ⫽ total symptom score; VC ⫽ vital capacity. *p ⬍ 0.05 in comparison with air.

The FEV1 values measured immediately before exposure to O3 or air were not significantly different (82.4 ⫾ 10.4% and 83.0 ⫾ 10.4%, respectively), but they were significantly lower than those measured before allergen challenge 24 hours previously (97.1 ⫾ 9.5% and 95.1 ⫾ 9.5%, respectively). Exposure to O3 induced a functional response. The FVC, FEV1, and vital capacity values, measured immediately after O3 exposure, were significantly lower than those obtained after air exposure. The total symptom score obtained at the end of O3 exposure was significantly higher than that obtained at the end of air exposure (Table 2). All subjects were able to produce sputum samples of adequate quality after both O3 and air exposures. The percentage of eosinophils in the induced sputum obtained after O3 exposure was significantly greater than that in the induced sputum obtained after air exposure (27.5% [2.3–72.8%] after O3 versus 9.9% [3.5– 71.5%] after air; p ⫽ 0.04) (Figure 2). The percentage of neutrophils in induced sputum obtained after O3 exposure was not statistically different from that in induced sputum obtained after

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air exposure (35.3% [14–78%] after O3 versus 38.7% [9–87.6%] after air; p ⫽ 0.75) (Figure 2). The interleukin-8 level in sputum supernatant obtained after O3 exposure was not significantly different from that in sputum supernatant obtained after air exposure (1,629 pM [229–2,056 pM] after O3 versus 1,162 pM [301–5,505 pM] after air; p ⫽ 0.62).

DISCUSSION The results of this study show that in subjects with mild allergic asthma, exposure to O3 potentiated the eosinophilic inflammatory response obtained with a previous allergen challenge. A functional response to O3 was still present, even after allergen challenge, but the typical neutrophilic airway inflammation was not observed after O3 exposure. These data suggest that the profile of the airway inflammatory response induced by O3 exposure can change when an acute preexisting airway inflammation is present. One of the most important differences between our study and those performed in recent years is the use of a different protocol schedule. In previous studies, exposure to pollutants took place before the allergen challenge (14–17), whereas in our study, O3 or air exposure took place the day after the allergen challenge. We chose this protocol schedule to examine the effect of O3 exposure on the airways of subjects experiencing a laboratory-induced exacerbation of asthma that is similar to what individuals with allergic asthma can have during natural allergen exposure. Furthermore, whereas the aim of previous investigators was to study the relationship between pollutants and allergens in terms of functional airway response in subjects with allergic asthma, our principal purpose was to examine the profile of cellular inflammatory response in induced sputum when the pollutant exposure followed the allergen challenge. The 1-day interval between allergen challenge and O3 or air exposure was chosen to allow at least a partial recovery of the functional response before the exposure to O3 or to air. Peden and coworkers (17) showed that previous exposure of subjects with perennial allergic asthma to 400 ppb O3 for 7.6 hours significantly increased the allergen-induced release of eosinophilic cationic protein in nasal lavage fluid. Wang and coworkers (18) demonstrated that 400 ppb nitrogen dioxide alone did not alter eosinophilic cationic protein concentration in the nasal lavage fluid of subjects with seasonal allergic rhinitis, whereas the same concentration of nitrogen dioxide followed by allergen challenge increased the concentration of eosinophilic cationic protein in the nasal lavage fluid. These results suggest

Figure 2. Median values, interquartiles, 90th percentiles, and range of cell percentages in the induced sputum collected after air (hatched box plot) or O3 (open box plot) exposure.

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that pollutants may “prime” the eosinophils for subsequent activation by allergen. It is well known that inhalation of allergen results in a marked increase in eosinophils in bronchoalveolar lavage fluid, in bronchial biopsy specimens, and in induced sputum obtained at the time of or immediately after the late airway response (24, 25). It is also known that allergen inhalation produces an increase in airway permeability (26). Studies investigating the pathophysiologic effects resulting from inhalation of O3 have demonstrated that exposure to this pollutant leads to epithelial damage and increases epithelial permeability, as indicated by leakage of lactate dehydrogenase, albumin, and total protein and by the release of inflammatory cytokines such as granulocyte–macrophage colony-stimulating factor, interleukin-6, and interleukin-8 (27, 28). Other studies show that O3 elicits an expression of adhesion molecules in humans (29). Thus, O3 and allergen can have a synergistic effect in inducing airway inflammation. Eosinophils have a negative effect on the airway wall because of the release of cationic proteins, which can increase the epithelial damage, and other oxidant mediators (30). Therefore, increased sputum eosinophilia induced by O3 exposure after allergen challenge can contribute to greater damage of the airways (in comparison with allergen challenge alone) and may possibly contribute to greater clinical and functional consequences. To simplify the protocol, we did not measure bronchial hyperresponsiveness in the days after O3 or air exposure. This measurement could demonstrate the longer duration of bronchial hyperresponsiveness after allergen and O3 in comparison with allergen alone. It has been shown that the severity of eosinophilic airway inflammation induced by allergen challenge is related to the severity of the increase in bronchial hyperresponsiveness (31). It is noteworthy that the degree of late airway response we obtained after allergen challenge performed the day before O3 exposure was similar to the degree of late response we obtained after allergen challenge the day before air exposure. The late airway response to allergen and the increase in sputum eosinophils after late airway response are reproducible (21, 32). Therefore, our results cannot be due to a different degree of inflammatory response during late airway response. We did not find a significant increase in the percentage of neutrophils in the induced sputum collected after O3 exposure compared with that collected after air exposure. There was also no significant difference in interleukin-8 levels in the supernatant obtained after exposure to O3 or air. These data are not in agreement with those reported in previous studies. Experimental studies using bronchoalveolar lavage fluid and sputum induction showed that exposure to O3 in normal subjects and in subjects with asthma produces a neutrophilic inflammatory response that appears to peak between 6 and 18 hours after exposure and persists beyond 24 hours (8, 9, 33). A possible explanation for the discordance between our results using allergen and O3 exposure and those using only O3 exposure could be that shortterm exposure to O3 alone is not powerful enough to recruit eosinophils in the airways, whereas it can potentiate the allergen challenge in producing airway eosinophilic inflammation. Longer exposure to O3 can increase the percentage of eosinophils in bronchoalveolar lavage fluid (11), and this effect could be potentiated by the pre-existing airway eosinophilic inflammation induced by allergen challenge. Interleukin-8 is a fairly nonspecific marker of epithelial damage, and it is particularly involved in the recruitment of neutrophils (34). Some investigators have suggested that interleukin-8 can also be active on “primed” eosinophils, depending on the specific environment (35). Unfortunately, we were not able to show an increased level of interleukin-8 in sputum after O3

exposure. Other chemotactic mediators could be involved, as suggested by recent animal and in vitro studies (36, 37). The levels of exposure to O3 that we used in the challenge chamber were slightly higher than those measured in the ambient air during rush-hour traffic (38), but there was some concern that a low concentration of O3 would not be able to induce a measurable response for a short-term exposure (39). Other experimental studies have used similar O3 concentrations (10, 16). In conclusion, our data show that O3 exposure can potentiate the eosinophilic airway inflammatory response due to allergen inhalation in subjects with asthma. This finding may help explain the interaction between allergen and pollutant exposure, and in particular, the increased severity of asthma exacerbation due to air pollution. References 1. Schwartz J, Slater D, Larson TV, Pierson WE, Koenig JQ. Particulate air pollution and hospital emergency room visits for asthma in Seattle. Am Rev Respir Dis 1993;147:826–831. 2. Norris G, YoungPong SN, Koenig JQ, Larson TV, Sheppard L, Stout JW. An association between fine particles and asthma emergency department visits for children in Seattle. Environ Health Perspect 1999;107: 489–493. 3. Romieu I, Meneses F, Sienra-Monge JJ, Huerta J, Ruiz Velasco S, White MC, Etzel RA, Hernandez-Avila M. Effects of urban air pollutants on emergency visits for childhood asthma in Mexico City. Am J Epidemiol 1995;141:546–553. 4. McDonnell WF, Horstman DH, Hazucha MJ, Seal E, Haak ED, Salaam SA, House DE. Pulmonary effects of ozone exposure during exercise: dose–response characteristics. J Appl Physiol 1983;54:1345–1352. 5. Kreit JW, Gross KB, Moore TB, Lorenzen TJ, D’Arcy J, Eschenbacher WL. Ozone-induced changes in pulmonary function and bronchial responsiveness in asthmatics. J Appl Physiol 1989;66:217–222. 6. Scannell C, Chen L, Aris RM, Tager I, Christian D, Ferrando R, Welch B, Kelly T, Balmes JR. Greater ozone-induced inflammatory responses in subjects with asthma. Am J Respir Crit Care Med 1996;154:24–29. 7. Aris MR, Christian D, Hearne PQ, Kerr K, Finkbeiner WE, Balmes JR. Ozone-induced airway inflammation in human subjects as determined by airway lavage and biopsy. Am Rev Respir Dis 1993;148:1363–1372. 8. Fahy JV, Wong HH, Liu JT, Boushey H. Analysis of induced sputum after air and ozone exposures in healthy subjects. Environ Res 1995;70: 77–83. 9. Vagaggini B, Carnevali S, Macchioni P, Taccola M, Fornay E, Bacci E, Bartoli ML, Cianchetti S, Dente FL, Di Franco A. Airway inflammatory response to ozone in subjects with different asthma severity. Eur Respir J 1999;13:274–280. 10. Bascom R, Naclerio RM, Fitzgerald TK, Kagey-Sobotka A, Proud D. Effect of ozone inhalation on the response to nasal challenge with antigen of allergic subjects. Am Rev Respir Dis 1990;142:594–601. 11. Peden DB, Boehlecke B, Horstman D, Devlin R. Prolonged acute exposure to 0.16 ppm ozone induces eosinophilic airway inflammation in asthmatic subjects with allergies. J Allergy Clin Immunol 1997;100:802– 808. 12. Hiltermann JT, Lapperre TS, Van Bree L, Steerenberg PA, Brahim JJ, Sont JK, Sterk PJ, Hiemstra PS, Stolk J. Ozone-induced inflammation in sputum and bronchial lavage fluid from asthmatics: a new noninvasive tool in epidemiologic studies on air pollution and asthma. Free Radic Biol Med 1999;27:1448–1454. 13. Newson EJ, Krishna MT, Lau LCK, Howarth PH, Holgate ST, Frew AJ. Effects of short-term exposure to 0.2 ppm ozone on biomarkers of inflammation in sputum, exhaled nitric oxide, and lung function in subjects with mild atopic asthma. J Occup Environ Med 2000;42:270– 277. 14. Molfino NA, Wright SC, Katz I, Tarlo S, Silverman F, McClean PA, Szalai JP, Raizenne M, Slutsky AS, Zamel N. Effect of low concentrations of ozone on inhaled allergen responses in asthmatic subjects. Lancet 1991;338:199–203. 15. Jenkins HS, Devalia JL, Mister RL, Bevan AM, Rusznak C, Davies RJ. The effect of exposure to ozone and nitrogen dioxide on the airway response of atopic asthmatics to inhaled allergen. Am J Respir Crit Care Med 1999;160:33–39. 16. Jorres R, Nowak D, Magnussen H. The effect of ozone exposure on allergen responsiveness in subjects with asthma or rhinitis. Am J Respir Crit Care Med 1996;153:56–64.

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