Vol.96 (2002) 352^358

E¡ectof ozone onbronchialmucosal in£ammation inasthmatic andhealthy subjectsN. STENFORS*, J. POURAZAR*, A.BLOMBERG*,M.T.KRISHNA

w, I.MUDWAYz, R.HELLEDAY*,

F. J.KELLYz, A.J.FREWw

ANDT. SANDSTROº M*

*Department of Respiratory Medicine and Allergy,University Hospital,Ume2, Sweden; wAir Pollution ResearchGroup, School of Medicine,University of Southampton, Southampton,UK; zLung Biology,King’s College London,London,UK

Abstract Epidemiological studies suggestthat asthmatics are more affected byozone than healthy people.This studytested three hypotheses (1) that short-term exposure to ozone induces inflammatorycell increases and up-regulation ofvascular adhesionmoleculesin airwaylavages and bronchial tissue 6 h afterozone exposure inhealthy subjects; (2) theseresponses are exaggerated in subjects with mild allergic asthma; (3) ozone exacerbates pre-existent allergic airways in-flammation.

We exposed 15 mild asthmatic and 15 healthy subjects to 0?2 ppm of ozone or filtered air for 2 h on two separateoccasions. Airwaylavages 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 neutrophilincrease in the asthmatic group was on top of an elevated baseline. In healthy subjects, ozone exposure increased theexpression of the vascular endothelial adhesion molecules P-selectin and ICAM-1, as well as increasing tissue neutrophiland mastcellnumbers.The asthmatics showed allergic airways inflammation at baseline butozone did not aggravate thisatthe investigated time point.

At 6 hpost-ozone-exposure, we foundno evidencethatmild asthmaticsweremoreresponsivethanhealthy to ozonein terms of exaggerated neutrophil recruitment or exacerbation of pre-existing allergic inflammation. Further work isneeded to assess the possibilityof a difference in time kinetics between healthy and asthmatic subjects in their responseto ozone.r2002 Elsevier Science Ltd

doi:10.1053/rmed.2001.1265, available online at http://www.idealibrary.com on

Keywords asthma, pollution, ozone, airways in£ammation, neutrophil.

INTRODUCTIONOzone (O3) is an important component of modern dayair pollution and is linked to increased respiratory mor-bidity in healthy and asthmatic subjects. Exposure toozone impairs lung function, increases bronchial reactiv-ity and is associated with increased hospital admissions(1).In healthy subjects, ozone induced in£ammatory re-

sponses in bronchoalveolar lavage (BAL) have been de-tected across the ¢rst 24h post-challenge (2).We havein earlier studies shown exposure to ozone to increase

Received 7 November 2001and accepted in revised form14 November2001.Supportedby: Swedish Asthma and Allergy Association,The SwedishHeart and Lung Foundation,TheWellcome Trust and the NationalAsthma Campaign.Correspondence should be addressed to: Prof Thomas Sandstr˛m,Dept of Respiratory Medicine & Allergy,University Hospital, SE-90185Ume2, Sweden.Faxþ46-90-141369;E-mail: thomas.sandstrom@lung.umu.se

the expression of the vascular endothelial adhesion mo-lecules P-selectin and ICAM-1 in bronchial mucosa asearly as 1?5h post-exposure. At this very early timepoint, no neutrophilic in¢ltration occurred in the airwaytissue, despite evidence of oxidative stress demonstratedin BAL £uid (3,4). At a later time point,18 h post-ozone-exposure a neutrophilic in¢ltration in bronchial mucosawas observed (5).Asthma is considered an airway mucosal in£amma-

tory disease, but to the best of our knowledge, no pre-vious data are available on the early bronchial mucosaltissueresponses of asthmatic subjects exposed to ozone.A few experimental exposure studies using BAL have re-ported greater increases in neutrophils, IL-6, IL-8 andprotein18h after ozone challenge in asthmatics as com-pared to healthy volunteers (6^8). Importantly however,these previous experimental studies comparing airwayin£ammatory responses between healthy and asthmaticsubjects have used quite high ozone burden during expo-sures. Such exposure conditions areveryrare even in the

OZONE EXPOSURE OFASTHMATIC AND HEALTHYSUBJECTS 353

US. Additionally, the scienti¢c literature has been lackinginformationregarding the earlyozone inducedresponsesin bronchialmucosa and BAL in asthmatics.This study was therefore designed to evaluate the

early airway responses of healthy and mild asthmaticsubjects to a brief exposure to 0?2 ppm ozone, giving anozone burdenmore environmentally relevant than thoseused in previous studies of ozone e¡ects in asthmatics.Bronchoscopy was performed at 6h after the end ofthe exposure period to obtain bronchial mucosal biop-sies and airway lavage samples. We hypothesised thatshort-term exposure to ozonewould induce neutrophilicairways in£ammation inhealthy subjects, withup-regula-tion of relevant vascular adhesion molecules, and thateither these responseswouldbe exaggerated in subjectswith mild allergic asthma, or ozone would exacerbatepre-existent allergic airways in£ammation.

METHODS

Subjects

Fifteen healthy, non-atopic, subjects (six males, nine fe-males; mean age 24, range 19^31 yrs) and 15 asthmaticsubjects (ninemales, six females; mean age 29, range 21^48 yrs) with intermittent tomildpersistentdiseasewereincluded (9). The asthmatics were hyper-responsive tomethacholine (geometric mean PC20 2?3 mg ml

�1) withmean FEV190% (range 75^114) of predicted and at leastone positive skin prick test to common allergens. Apartfrom inhaled b2-agonists on demand, they needed noanti-asthma therapy. All subjects were never-smokersand free of airway infection for at least 6 weeks beforeand throughout the study. No anti-in£ammatory drugsor antioxidant supplements were allowed during thestudy.

Study design

Subjects were exposed to ¢ltered air or 0?2 ppm of O3

for 2h in random order in an exposure chamber (4). Ex-posures were at least 3 weeks apart. Bronchoscopy was

TABLE 1. Monoclonal antibodies used for immunohistochemical

Antibody Epitope stained

NE Neutrophil elastaseAA1 Mastcell tryptaseEG2 ECPCD3 CD3þ T-cellsEN4 EndotheliumCD62P P-selectinCD54 ICAM-1

performed 6 h after each exposure as previously de-scribed (10). Lung function tests (FEV1 and FVC) wereperformedbefore and immediately after each exposure.

Methods

During exposures, subjects exercised on a bicycle erg-ometer (VE¼20 lminm�2) alternating with rest for 15-min periods. The O3 concentration remained stable at0?2070?01 ppm (mean7SEM). The study was approvedby Ume2 University Ethics Committee; all subjects gaveinformed consent. Lung function was measured using aconventional spirometer (Vitalograph-COMPACT; Vita-lograph Ltd., Buckingham, UK). The asthmatic subjectsinhaled salbutamol, 0?2mg dry powder, prior tobronchoscopy.Mucosal biopsies were processed into gly-colmethacrylate resin, stained using monoclonal antibo-dies (Table 1) and assessed as previously described(3,4,11,12). Bronchial wash (BW) was performed by instil-ling 2� 20ml sterile saline (pH 7?4, 371C).The ¢rst BWsamplewas used for total and di¡erential cell counts andanalysis for soluble mediators. The second BW fractionwas used for antioxidant determinations (13).Bronchoalveolar lavage (BAL, 3� 60ml saline) was

then performed. BW and BAL samples were collectedseparately and immediately placed on ice. Lavage £uidwas ¢ltered to remove mucus (pore diameter 100mm,Syntab, Malm˛, Sweden) and centrifuged at 400g for15min to remove cellular components.Cell pellets werere-suspended in PBS at106 cells ml�1 for total and di¡er-ential cell counts. Cytocentrifuged specimens were pre-paredwith 5�104 non-epithelial cells per slide (Cytospin3s, Shandon Southern Instruments Inc., Sewikly, PA,USA). Di¡erential cell counts were performed afterstaining with May-Grˇnwald Giemsa (400 cells slide�1).Mastcellswere countedin�10 visual ¢elds at �160mag-ni¢cation on slides stained with acid toluidine blue, andcounterstained with Mayer’s acid haematoxylin. BW andBAL myeloperoxidase, methyl-histamine and eosinophilcationic protein concentrations were analysed by RIA(Kabi Pharmacia Diagnostics,Uppsala, Sweden).

staining of bronchial biopsies

Source

Dako,Glostrup,DenmarkDako,Glostrup,DenmarkPharmacia-Upjohn,Uppsala,SwedenDako,Glostrup,DenmarkMonosan,Uden, the NetherlandsSerotec,Kidlington,Oxford,UKDako,Glostrup,Denmark

FIG. 1. Bronchial airway neutrophil responses to ozone andcontrol air challenges in mild asthmatic and healthy controlsubjects. Individual responses and group median values areillustrated. *Po 0?05, **Po 0?01,.** *Po 0?001.

354 RESPIRATORYMEDICINE

Analyses

Statistics were performed using the Unistat (UnistatLtd. London,UK) and SPSS version10?05 (SPSS inc., Chi-cago, USA) software.Wilcoxon’s nonparametric signed-rank test was used to assess paired observations. Be-tween-group comparisons were assessed by Mann-Whitney U-test, except for lung function data that wereanalysed using a repeated measures two-way ANOVA.The degree of associationbetween neutrophil responsesto ozone in di¡erent compartments were assessed bySpearman’s rank correlation test. P-values o0?05 wereconsidered signi¢cant.

RESULTS

Lung function responses

Baseline FEV1 and FVC values did not di¡er signi¢cantlybetween the healthy and asthmatic subjects. Exposureof healthy subjects to ozone resulted in signi¢cant de-creases in FEV1 and FVC (P o 0?05). Signi¢cant decre-ments in FVC were observed in the asthmatics (P o0?05).There was no di¡erence between the two groupsin the magnitude of the FEV1 and FVC decrements(Table 2).

Cellular data

Post-air total leukocyte numbers were lower in the BWof asthmatics relative to healthy subjects (median [inter-quartile range]: 6?7 [4?0^10?7]� 104 vs. 11?1 [7?30^20?0]� 104 cellsml�1, P o 0?05). At baseline, the asth-matic group had higher neutrophil counts in BW, BAL,bronchial epithelium andbronchial submucosa comparedto the healthy group (Fig.1) but BW^MPOwas lower inthe asthmatic group (1?7 vs. 8?3 mg l�1).Ozone exposuredid not alter total leukocyte counts in BW or BAL ineither group, but in both groups there was a signi¢cantand similar increase in neutrophils in BW, but not in BAL(Fig. 1). In the asthmatic group, there was also evidence

TABLE 2. Lung function responses in healthy and mild asthmatic

Healthy Controls

Pre-Air Post-A

FVC (l) 5?1271?16 5?107FEV1 (l sec�1) 4?0570?90 4?107

Mild Asthmatics

Pre-Air Post-A

FVC (l) 5?0371?01 5?047FEV1 (l sec�1) 3?7170?75 3?747

Analysed using a repeated measures two-way ANOVA, with e

of neutrophil activation, as shown by an increased BW^MPOconcentration after ozone, increasingby1?8-fold (Po 0?01) (Fig. 2).In the healthy controls, ozone exposure increased

neutrophil numbers in bronchial epithelium and submu-cosa (respectively 0?0 [0?0^1?4] cells mm�1 post air vs.2?0 [0?0^8?0] cells mm�1 post ozone, P o 0?05;and 21?0 [13?6^54?2] cells mm�2 post air vs. 97?3

subjects exposed to air and 0?2 ppm ozone

ir Pre-Ozone Post-Ozone

1?19 5?0271?22 4?7670?97*0?92 3?9970?91 3?6970?85*

ir Pre-Ozone Post-Ozone

0?99 5?0771?00 4?9671?05*0?72 3?7670?75 3?6970?75

xpressions for time and treatment. *Po0?05.

FIG. 2. Soluble mediator responses to ozone in the bronchialairways of asthmatic and healthy subjects.Data are illustrated asthe fold-increase in concentration afterozonerelative to post airvalues. The dotted line through 1 on the y-axis illustrates nochange. **P o 0?01, when comparing non-transformed MPOconcentrations after air and ozone in the mild asthmatic group.The comparison between the fold increase in MPO seen be-tween the asthmatic and control subjects was performed usingthe Mann-Whitney U-test.

OZONE EXPOSURE OFASTHMATIC AND HEALTHYSUBJECTS 355

[52?1^122?2] cellsmm�2 postozone,Po 0?01).Therewasno change in tissue neutrophil numbers after ozone inthe asthmatic group. (Figs1and 3). Expression of P-selec-tin and ICAM-1onvascular endotheliumwasupregulatedafter ozone in healthy controls (P o 0?01; P o 0?05 re-spectively) but no signi¢cant changes in adhesion mole-cule expression were seen in the asthmatic group,although they did start from a higher baseline level ofICAM-1expression (Po 0?01) (Table 3 and Fig. 3).Mast cell numbers were elevated in the BW and BAL

of asthmatics compared with healthy subjects, (respec-tively 5?0 [4?0^10?0] % vs. 0?4 [0?3^1?1] %, P o 0?0001;and 6?0 [4?0^9?0] % vs. 1?0 [0?3^1?7] %, P ¼ 0?0001).The asthmatics also showedhighermast cell counts thanhealthy subjects in epithelium and submucosa (respec-tively 2?1 [0?0^4?0] cells mm�1 vs. 0?0 [0?0^4?0] cellsmm�1, Po 0?05; and 36?2 [13?7^58?6] cells mm�2 vs. 8?0[3?5^15?4] cells mm�2, P¼ 0?001).Mast cell numbers didnot change in BW or BAL in either group after ozone,but submucosal mast cell numbers increased in healthysubjects (8?0 [3?5^15?4] cells mm�2 post air vs. 18?3[11?7^25?2] cells mm�2 post ozone, P o 0?05) whileephithelial mast cell numbers decreased signi¢cantly inthe asthmatics (0?4 [0?0^2?1] cells mm�1 post ozone, Po 0?05).Lymphocyte numbers in BAL £uid were increased at

baseline in the mild asthmatics compared with healthycontrols (13?9 [8?8^15?6] % vs. 3?5 [3?0^8?0] % respec-tively, P o 0?05). Asthmatics also had higher baselinelymphocyte cell numbers in the epithelium (10?56 [5?2^

24?3] cells mm�1vs. 0?0 [0?0^1?2] cells mm�1; Po0?001)and submucosa (81?6 [39?7^180?6] cells mm�2 vs. 19?8[5?3^41?0]; P o 0?001). Ozone exposure did not a¡ectlymphocyte numbers in either group.Eosinophils were present in BW, BAL and submucosa

of asthmatic subjects but did not change after ozone.Noeosinophils were detected in the healthy controls. BW/BAL methyl^histamine and ECP concentrations werenot signi¢cantly a¡ected by ozone.In healthy subjects, where increases in submucosal,

epithelial and BW neutrophils were observed, only aweak association was apparent between submucosalandBW increases (Rs, 0?46,P¼ 0?05),with no signi¢cantassociation noted between the epithelial and BW re-sponse (Rs, 0?30, P¼ 0?19) but the increases in epithelialand submucosal neutrophils were strongly correlated(Rs, 0?84, P o 0?001). In asthmatic subjects, there wasno correlation between changes in neutrophil numbersin any pair of compartments.

DISCUSSIONWhen exposed to an environmentally relevant concen-tration of ozone for 2 hours, both healthy and asthmaticsubjects showed a neutrophilic airway response, buttherewas no discernible change in eosinophil or lympho-cyte numbers. In the healthy group, ozone also inducedup-regulation of the vascular endothelial adhesionmole-cules P-selectin and ICAM-1 together with increases intissue neutrophil and mast cell numbers, responseswhichwere not seen in the asthmatic group.The e¡ectsof ozone on lung function were small and similar in bothgroups, in linewith earlier work (1,8,14^16).Previous bronchoscopy studies of the e¡ect of ozone

on asthmatics airways have used higher ozone burden,i.e. higher concentration of ozone, longer exposure timeand higher total ventilation, than were employed in thepresent study (6^8). The present study con¢rms thatthe neutrophilic responses seen after higher ozone bur-den are also present at environmentally relevant ozoneconcentrations. However, the eosinophilic response re-ported in one high dose study was not apparent here(17), neither was there any evidence of the exaggeratedneutrophil response in asthmatics seen after high ozoneexposure (6^8).We have recently reported increased expression of

the vascular endothelial adhesion molecules P-selectinand ICAM-1 in the bronchial mucosa of healthy subjects1?5 h after ozone exposure, at which time there was noneutrophilic in£ammation in the bronchial mucosa (3,4).The up-regulation of P-selectin and ICAM-1 in the pre-sent studycon¢rms the importance of thesevascular en-dothelial adhesion molecules in the recruitment ofin£ammatory cells into the airways of healthy subjectsfollowing exposure to ozone.

FIG. 3. Immunohistochemical staining for neutrophils post air (A) vs. post ozone (B), P-selectin post air (C) vs. post ozone (D) andICAM-1post air (E) vs. postozone (F) in bronchial biopsies.

356 RESPIRATORYMEDICINE

Six hours after ozone exposure, BW MPO was ele-vatedin the asthmatic group. Similar responseshavepre-viously been seen at 18h post-exposure (8) and insputum 24h post-ozone-exposure (18).The fact that in-creased neutrophil numbers and MPO levels were found

in BW of the asthmatic subjects, without any overtchange in the biopsies, indicates that their acute re-sponse to ozone may be occurring more distal than theproximal biopsies examined here. The neutrophil in£uxwas also more pronounced in the proximal BW sample.

TABLE 3 Vascular endothelial adhesion molecule expression in the bronchial mucosa of healthy and mild asthmatic subjects ex-posed to air and 0?2 ppm ozone

Adhesion molecule Exposure Healthy Controls Mild Asthmatics

P-selectin (%) Post Air 35?8 (23?8^51?1) 29?4 (22?2^42?8)Post Ozone 60?0 (42?1^78?7)** 34?9 (12?5^52?8)

ICAM (%) Post Air 56?2 (40?0^63?5) 68?0 (59?1^82?8)a

Post Ozone 60?0 (46?5^75?0)* 56?2 (40?0^63?5)

Allvaluesexpressedas thepercentage of ENþstainingvesselsexpressing thenamedadhesionmolecule.Comparisonsofpost airandpostozonevaluesperformedusing theWilcoxon ^Signed-RankTest, *Po 0?05, **Po 0?01.Comparisonsofpost air (baseline)values were performed using the Mann-Whitney U-test, aPo 0?01.

OZONE EXPOSURE OFASTHMATIC AND HEALTHYSUBJECTS 357

This is consistent with the reported deposition charac-teristics and dosimetry of ozone (19), which suggest thatozone mainly deposits in the terminal conducting air-ways.To our knowledge, this is the ¢rst study evaluating the

early bronchial tissue responses in asthmatic subjectsafter exposure to ozone The asthmatic subjects re-cruited for this study all had a pre-existent allergic air-ways in£ammation with elevated numbers ofeosinophils, mast cells, T-lymphocytes and neutrophils,cells that are considered to play important roles in thepathophysiology of asthma (20). No signs of aggravationof the mucosal allergic status were detected 6 h post-ozone-exposure other than an increase in BW neutro-phils. In contrast, Peden et al. reported increases in BALeosinophils18h after exposure to 0?16 ppmozone for 7?6h.These subjectswere said to bemild allergic asthmaticswithout inhaled steroid therapy but their baseline BALeosinophil numbers were quite high, indicating that theyhad more severe asthma than the group studied here(17).The absence of any ozone-induced in£ammatory re-

sponses in bronchial tissue of the asthmatic subjectscould be due to di¡erences in basal in£ammatory airwaystatus or in the time-kinetics of in£ammatory responsesto ozone. The asthmatic group showed a more markedtissue and lavage neutrophilia at baseline compared tothe healthy subjects.This baselinemucosal in£ammationcould have activated counter-in£ammatory mechanisms(21), which might damp the in£ammatory response toozone. This damping might a¡ect both the magnitudeandkinetics of theresponse andhence delayor attenuatethe mucosal response to ozone in asthmatics. Six hourspost-exposure has previously been reported as the peakperiod of ozone-induced neutrophil in£ux into the air-ways of healthy subjects (2) with the response persistingup to 24hpost-challenge (5, 22, 23).Given thatmany epi-demiological a studies show a time lag of days betweenexposure and negative airway e¡ects (24^26), it remainspossible that in£ammatory responsesmaybe seen in thebronchial tissue of asthmatics at a later time point postexposure.

In the present study, the only signi¢cant tissue re-sponse after ozone in the asthmatic group was a de-crease in epithelial mast cell numbers post ozoneexposure. This could possibly represent cell migrationinto the airways, although no corresponding increase inmast cell numbers was observed in the BAL or BW. Al-ternatively this change could have been due to apoptosisor degranulation, although there was no increase inmethyl-histamine in BAL or BW to support this latterexplanation.In conclusion, at 6 hpost-ozone-exposurewe foundno

evidence that mild asthmatics were more responsive toozone thanhealthycontrols in terms of exaggeratedneu-trophil recruitmentor exacerbation of pre-existing aller-gic in£ammation. Further work is needed to assess thepossibility of a di¡erence in time kinetics betweenhealthy and asthmatic subjects in their response toozone.

Acknowledgements

Lena Skedebrant, Helen Bertilsson, Maj-Cari Ledin,Ann-Britt Lundstr˛m, Annika Hagenbj˛rk-Gusta¡son,Ulf Hammarstr˛m andGerd Linden for excellent techni-cal assistance.

REFERENCES1. Health e¡ects of outdoor air pollution.Committee of the Environ-

mental and Occupational Health Assembly of the AmericanThor-acic Society. Am J Respir Crit Care Med1996; 153: 3^50.

2. Schelegle ES, Siefkin AD, McDonald RJ. Time course of ozone-in-duced neutrophilia in normal humans. Am Rev Respir Dis 1991; 143:1353^1358.

3. Krishna MT, Blomberg A, Biscione GL, Kelly F, SandstromT, FrewA,Holgate S. Short-term ozone exposure upregulates P-selectin innormal human airways. Am J Respir Crit Care Med 1997; 155: 1798^1803.

4. Blomberg A, Mudway IS, Nordenhall C, Hedenstrom H, Kelly FJ,Frew AJ, Holgate S, SandstromT.Ozone-induced lung function de-crements do not correlatewith early airway in£ammatory or anti-oxidant responses.Eur Respir J1999; 13:1418^1428.

5. Aris RM,Christian D, Hearne PQ, Kerr K, Finkbeiner WE, BalmesJR. Ozone-induced airway in£ammation in human subjects as de-

358 RESPIRATORYMEDICINE

termined by airway lavage and biopsy. Am Rev Respir Dis 1993; 148:1363^1372.

6. Balmes JR, Aris RM, Chen LL, Scannell C, Tager IB, FinkbeinerW et al. E¡ects of ozone on normal and potentially sensitivehuman subjects. Part I: Airway in£ammation and responsivenessto ozone in normal and asthmatic subjects. Res Rep Health E¡ Inst1997; 78:1^37.

7. Basha MA, Gross KB, Gwizdala CJ, Haidar AH, Popovich J, Jr.Bronchoalveolar lavage neutrophilia in asthmatic and healthy vo-lunteers after controlled exposure to ozone and ¢ltered puri¢edair.Chest1994; 106:1757^1765.

8. Scannell C,Chen L, Aris RM,Tager I,Christian D, Ferrando R et al.Greater ozone-induced in£ammatory responses in subjects withasthma. Am J Respir Crit Care Med1996; 154: 24^29.

9. National Institute of Health, National Heart LaBI.Global Strategyfor AsthmaManagement and PreventionNHLBI/WHOWorkshop.95-3659.1-1-1995.

10. Salvi S, Blomberg A, Rudell B, Kelly F, Sandstrom T, Holgate ST,Frew A. Acute in£ammatory responses in the airways and periph-eral blood after short-term exposure to diesel exhaust in healthyhumanvolunteers. Am J Respir Crit Care Med1999; 159: 702^709.

11. Britten KM, Howarth PH, Roche WR. Immunohistochemistry onresin sections: a comparison of resin embedding techniques forsmallmucosal biopsies.Biotech Histochem1993; 68: 271^280.

12. Krishna MT,Madden J,Teran LM, Biscione GL, Lau LC,Withers NJ,SandstromT, Mudway I, Kelly FJ,Wally A, Frew AJ, Holgate ST. Ef-fects of 0?2 ppm ozone on biomarkers of in£ammation in bronch-oalveolar lavage £uid and bronchialmucosa of healthy subjects.EurRespir J1998; 11:1294^1300.

13. Mudway IS, Stenfors N, Blomberg A,Helleday R,Dunster C, Mark-lund SL, Frew AJ, SandstromT,Kelly FJ.Di¡erences in basal airwayantioxidant concentrations are not predictive of individual respon-siveness to ozone: a comparison of healthy andmild asthmatic sub-jects. Free Radic Biol Med 2001; 31: 962^974.

14. Holz O, Jorres RA, Timm P, Mucke M, Richter K, Koschyk S et al.Ozone-induced airway in£ammatory changes di¡er between indi-viduals and are reproducible. Am J Respir Crit Care Med 1999; 159:776^784.

15. Jorres R,Nowak D,Magnussen H.The e¡ect of ozone exposure onallergenresponsiveness in subjectswith asthma or rhinitis.Am J Re-spir Crit Care Med1996; 153: 56^64.

16. Nightingale JA, Rogers DF, Barnes PJ. E¡ect of inhaled ozone on ex-haled nitric oxide, pulmonary function, and induced sputum in nor-mal and asthmatic subjects.Thorax1999; 54:1061^1069.

17. PedenDB, Boehlecke B,HorstmanD,Devlin R.Prolonged acute ex-posure to 0?16 ppm ozone induces eosinophilic airway in£amma-tion in asthmatic subjects with allergies. J Allergy Clin Immunol1997;100: 802^808.

18. Newson EJ,KrishnaMT,Lau LC,Howarth PH,Holgate ST, FrewAJ.E¡ects of short-term exposure to 0?2 ppm ozone onbiomarkers ofin£ammation in sputum, exhaled nitric oxide, and lung function insubjects with mild atopic asthma. J Occup Environ Med 2000; 42:270^277.

19. Miller FJ,Overton JH, Jr., Jaskot RH,Menzel DB. Amodel of the re-gional uptake of gaseous pollutants in the lung. I.The sensitivity ofthe uptake of ozone in the human lung to lower respiratory tractsecretions and exercise.Toxicol Appl Pharmacol1985; 79:11^27.

20. Bousquet J, Je¡ery PK, Busse WW, Johnson M,Vignola AM. Asth-ma.Frombronchoconstriction to airways in£ammation and remo-deling. Am J Respir Crit Care Med 2000; 161:1720^1745.

21. Barnes PJ, Lim S. Inhibitory cytokines in asthma. Mol Med Today1998; 4: 452^458.

22. Devlin RB,McDonnell WF, Becker S,MaddenMC,McGeeMP, Per-ez R et al. Time-dependent changes of in£ammatory mediators inthe lungs of humans exposed to 0?4 ppm ozone for 2 hr: a compar-ison of mediators found in bronchoalveolar lavage £uid1 and18 hrafter exposure.Toxicol Appl Pharmacol1996; 138:176^185.

23. Koren HS, Devlin RB, Becker S, Perez R, McDonnell WF.Time-de-pendent changes of markers associated with in£ammation in thelungs of humans exposed to ambient levels of ozone.Toxicol Pathol1991; 19: 406^411.

24. Gielen MH, van der Zee SC, vanWijnen JH, van Steen CJ, Brunek-reef B. Acute e¡ects of summer air pollution on respiratory healthof asthmatic children. Am J Respir Crit Care Med 1997; 155: 2105^2108.

25. HiltermannTJ, Stolk J, van der Zee SC, Brunekreef B, de BruijneCR,Fischer PH etal. Asthma severity and susceptibility to airpollu-tion.Eur Respir J1998; 11: 686^693.

26. Schwartz J. Short term£uctuations in air pollution andhospital ad-missions of the elderly for respiratorydisease.Thorax1995; 50: 531^538.