850 volume 122 | number 8 | August 2014 • Environmental Health Perspectives

Research All EHP content is accessible to individuals with disabilities. A fully accessible (Section 508–compliant) 

HTML version of this article is available at http://dx.doi.org/10.1289/ehp.1307240. 

Introduction

Nitrogen dioxide (NO2), a ubiquitous 

atmospheric pollutant, is a respiratory irritant that remains a matter of concern [World 

Health Organization (WHO) European 

Centre for Environment and Health and 

WHO Regional Office for Europe 2013]. 

Indoor concentrations of NO2 often exceed 

those found outdoors, especially when 

unvented combustion appliances are used. 

Inside homes, peak levels of NO2, associated with the use of gas and solid-fuel appliances for cooking and heating, have been 

measured in the range of 80–1,100 ppb 

(150–2,090 μg/m3) (Basu and Samet 1999; 

Dennekamp et al. 2001; Kotzias et al. 2005; 

Pilotto et al. 1997). Outdoors, hourly NO2

concentrations in cities rarely exceed 200 ppb 

(380 μg/m3) (U.S. Environmental Protection 

Agency 2008), although urban levels can 

reach levels as high as 500 ppb (950 μg/m3) 

(WHO 2006), especially for short periods in 

streets with heavy traffic and in road tunnels 

(Larsson et al. 2010).

Epidemiological and controlled human 

exposure studies suggest that people with 

asthma are more susceptible to the effects of 

NO2 when compared with healthy individuals 

(Bauer et al. 1986; Belanger et al. 2006; Bylin 

et al. 1988; Hasselblad et al. 1992; Jorres and 

Magnussen 1990; Strand et al. 1996).

However, despite the extensive literature 

on NO2-induced health effects, some inconsistencies in the results of studies have been 

noted (Jarvis et al. 2010). In asthmatics, NO2

exposure without allergen challenge did not 

result in lung functional changes in most studies (Avol et al. 1988; Kleinman et al. 1983; 

Linn et al. 1986; Mohsenin 1987), and inconsistent results were found in airway responsiveness after nonspecific bronchoconstrictor 

challenges (Bylin et al. 1988; Hazucha et al. 

1983; Jorres and Magnussen 1991; Kleinman 

et al. 1983; Roger et al. 1990; Strand et al. 

1996). After allergen challenge, exposure to 

NO2 in asthmatics increased airway hyperresponsiveness (Jenkins et al. 1999; Strand 

et al. 1997, 1998; Tunnicliffe et al. 1994) 

and eosinophilic inflammation (Barck et al. 

2002, 2005).

A few studies have investigated the 

inflammatory response to a single exposure of NO2 without allergen challenge in 

asthmatics, but the findings have been 

inconsistent (Jorres et al. 1995; Solomon 

et al. 2004; Vagaggini et al. 1996).

We investigated whether repeated brief 

exposures to 200 ppb (380 μg/m3) and 

600 ppb (1,130 μg/m3) NO2, which mimic 

indoor NO2 peaks, enhance airway inflammation in asthmatics. This clinical study 

involved 19 adults with intermittent asthma 

and used a randomized double-blind protocol 

with assessment of inflammatory response in 

induced sputum.

Materials and Methods

Participants. Nineteen patients [14 men and 

5 women; median age, 29 years; age range, 

20–69 years; median body mass index (BMI), 

26 kg/m2; BMI range, 20–39 kg/m2] were 

included in the study (Table 1). All had 

intermittent asthma as defined by the Global 

Initiative for Asthma (2010) guidelines and 

were nonsmokers (18 had never smoked and 

1 had stopped smoking some 10 years earlier). 

Address correspondence to V. Ezratty, Service des 

Etudes Médicales (SEM), EDF–DRH GROUPE, 

45 rue Kleber, Site Carré Vert, 92300 Levallois–

Perret, France. Telephone: 00 33 (1) 82 24 84 43. 

E-mail: veronique.ezratty@edf.fr

Supplemental Material is available online (http://

dx.doi.org/10.1289/ehp.1307240).

We thank the following for their contributions: 

I. Poirier, H. Casterman, S. Billot-Bonef, M. BonnetBelfais, E. Pigeon, S. Maestri, B. Vandercasteele, 

L. Alavoine, L. Nardelli, A. Boulmeau, L. Dejoie, 

Y. Retory, S. de Ferrières, and D. Ormandy. All statistical analyses were performed by S. Koscielny, Service 

de biostatistiques et épidémiologie, Gustave Roussy, 

Villejuif, France.

This study was supported by the Center for 

Research & Innovation in Gas and New Energy 

Sources, CRIGEN, GDF SUEZ; the Medical Studies 

Department of EDF; and the Clinical Center of 

Investigation at Bichat Hospital, Paris, France. 

The authors’ freedom to design, conduct, interpret 

and submit for publication the research described in 

this work was not compromised by any of the funding 

bodies.

V.E., G.G., P.-A.C., and J.L. are employed by 

EDF, Service des Etudes Médicales (SEM), LevalloisPerret, France. P.M., L.R., and S.T. are employed by 

GDF-SUEZ, Center for Research & Innovation in 

Gas and New Energy Sources CRIGEN, St-Denis, 

France. The other authors declare they have no actual 

or potential competing financial interests.

Received: 18 June 2013; Accepted: 15 April 

2014; Advance Publication: 18 April 2014; Final 

Publication: 1 August 2014.

Repeated Nitrogen Dioxide Exposures and Eosinophilic Airway Inflammation 

in Asthmatics: A Randomized Crossover Study

Véronique Ezratty,1 Gaëlle Guillossou,1 Catherine Neukirch,2 Monique Dehoux,3 Serge Koscielny,4 Marcel Bonay,5

Pierre-André Cabanes,1 Jonathan M. Samet,6 Patrick Mure,7 Luc Ropert,7 Sandra Tokarek,7 Jacques Lambrozo,1

and Michel Aubier 2

1Service des Etudes Médicales (SEM), EDF, Levallois-Perret, France; 2Service de Pneumologie A, Clinical Center of Investigation, 

Inserm U700, Bichat Hospital, Université Paris Diderot, DHU FIRE, Paris, France; 3Laboratoire de Biochimie, Bichat Hospital, Paris, 

France; 4Service de biostatistiques et épidémiologie, Gustave Roussy, Villejuif, France; 5Department of Physiology, Bichat Hospital, 

Paris, France; 6Department of Preventive Medicine, Keck School of Medicine of the University of Southern California, Los Angeles, Los 

Angeles, California, USA; 7Center for Research & Innovation in Gas and New Energy Sources, CRIGEN, GDF SUEZ, St-Denis, France

Background: Nitrogen dioxide (NO2), a ubiquitous atmospheric pollutant, may enhance the 

asthmatic response to allergens through eosinophilic activation in the airways. However, the effect of 

NO2 on inflammation without allergen exposure is poorly studied.

Objectives: We investigated whether repeated peaks of NO2, at various realistic concentrations, 

induce changes in airway inflammation in asthmatics.

Methods: Nineteen nonsmokers with asthma were exposed at rest in a double-blind, crossover 

study, in randomized order, to 200 ppb NO2, 600 ppb NO2, or clean air once for 30 min on 

day 1 and twice for 30 min on day 2. The three series of exposures were separated by 2 weeks. 

The inflammatory response in sputum was measured 6 hr (day 1), 32 hr (day 2), and 48 hr 

(day 3) after the first exposure, and compared with baseline values measured twice 10–30 days 

before the first exposure.

Results: Compared with baseline measurements, the percentage of eosinophils in sputum 

increased by 57% after exposure to 600 ppb NO2 (p = 0.003) but did not change significantly after 

exposure to 200 ppb. The slope of the association between the percentage of eosinophils and NO2

exposure level was significant (p = 0.04). Eosinophil cationic protein in sputum was highly correlated with eosinophil count and increased significantly after exposure to 600 ppb NO2 (p = 0.001). 

Lung function, which was assessed daily, was not affected by NO2 exposure.

Conclusions: We observed that repeated peak exposures of NO2 performed without allergen exposure were associated with airway eosinophilic inflammation in asthmatics in a dose-related manner.

Citation: Ezratty V, Guillossou G, Neukirch C, Dehoux M, Koscielny S, Bonay M, Cabanes PA, 

Samet JM, Mure P, Ropert L, Tokarek S, Lambrozo J, Aubier M. 2014. Repeated nitrogen dioxide 

exposures and eosinophilic airway inflammation in asthmatics: a randomized crossover study. 

Environ Health Perspect 122:850–855; http://dx.doi.org/10.1289/ehp.1307240
NO2 and airway inflammation

Environmental Health Perspectives • volume 122 | number 8 | August 2014 851

Only patients who had a diagnosis of asthma 

confirmed by a positive methacholine challenge performed twice at baseline 10–30 days 

before the first exposure were included in 

the study. A positive methacholine test was 

defined as a methacholine provocative dose 

causing a 20% decrease in forced expiratory 

volume in 1 sec (FEV1) from control FEV1

(PD20 methacholine) < 4 mg. All participants had allergy to house dust mites (HDM) 

and/or pollen confirmed by a positive skinprick test done ≥ 4 weeks before their inclusion in the study. The study was performed 

outside the pollen season for those who had 

been diagnosed with pollen allergies. Of the 

19 participants, 6 had a personal history of 

atopic dermatitis and/or an atopic familial 

history. None of those six used inhaled or 

oral corticosteroids or other antiinflammatory 

therapy, and the only permitted medication 

was an inhaled β2-agonist, used as needed 

during the study period (from baseline, i.e., 

30 days before the first NO2 exposure until 

the end of the study 2 weeks after the last 

exposure). Participants who had gas stoves 

and/or unvented combustion appliances at 

home were told not to use them at baseline 

and on the days of exposures and during the 

2 days before and after NO2 exposure. All participants had to be free of airway infection for 

at least 6 weeks before baseline measurements.

The study was registered by the French 

Ministry of Health (DGS 2006/0016) and 

approved by the Ethics Committee of HotelDieu Hospital, Paris, France (project 0611398, 

registered on 28 February 2007). All participants signed informed consent forms before 

enrollment in the study.

Study design. The study had a doubleblind, crossover design, with each participant 

acting as his or her own control. For each participant, the study involved three series each of 

three exposures at rest: one series to clean air, 

one series to 200 ppb (380 μg/m3) NO2, and 

one series to 600 ppb (1,130 μg/m3) NO2. 

The order of the three series of exposures was 

randomized (Figure 1). The design for each 

series of exposures with the timing of pulmonary function testing, and sputum inductions 

from day 1 to day 3 is described in Figure 2. 

For each series, participants were exposed to 

the same level of NO2 or to clean air once for 

30 min on day 1, and twice for 30 min on 

day 2, at the same time and on the same days 

of the week. The two exposures performed 

on day 2 were separated by 1 hr. There was 

no exposure on day 3. There was an interval 

of 2 weeks between each series of exposures. 

Only the engineer in charge of injection into 

the chamber knew whether the participant 

was being exposed to NO2 or clean air.

Sputum was induced twice at baseline (10–30 days before first exposure, with 

1–3 weeks between the sputum inductions) and 

for each series of exposures, 6 hr (on day 1), 

32 hr (on day 2), and 48 hr (on day 3) after 

the end of the first exposure. Spirometry with 

flow–volume curves was carried out at baseline 

(immediately before first exposure) and daily 

from day 1 to day 3, before and immediately 

after exposures, and immediately before sputum inductions. Allergen challenge was not 

performed, either before or after the exposures.

Nitrogen dioxide/clean air exposure. The 

exposures were performed in an 8.8-m3 exposure chamber installed in the investigation 

clinical center at the Hospital Bichat in Paris 

as previously described (Ezratty et al. 2007). 

The chamber was supplied with fresh, particlefree air at a mean temperature of 25°C and 

a mean relative humidity of 32%. The air 

supply passed through HEPA and activated 

carbon filters. We used NO2 concentrated 

at 950 ppm compressed in a 20-L gas bottle 

under 150 bar pressure for the 600-ppb exposures, and a gas bottle of NO2 concentrated 

at 520 ppm under 150 bar pressure for the 

200-ppb exposures (Air Liquide SA, Paris, 

France). A mass flow meter secured the stability of the injected flow at the expected concentration (200 ppb or 600 ppb).

During exposures, NO2 concentration 

inside the exposure chamber was continuously monitored (chemiluminescence oxides 

of nitrogen analyzer, Model AC 32 M; 

Environnement SA, 78300 Poissy, France). 

The mean concentration was 581 ppb ± 3.2% 

for 600-ppb NO2 exposures and 203 ppb 

± 1.5% for 200-ppb NO2 exposures. During 

exposures to clean air, the NO2 concentration 

was < 10 ppb.

Pulmonary function and methacholine 

challenge testing. Flow-volume curves were 

obtained using a Biomedin spirometer 

(Biomedin srl, Padova, Italy) according to the 

European Community Respiratory Health 

Survey specifications to determine FEV1 and 

peak expiratory flow (PEF) (Quanjer 1993).

Methacholine challenge tests were done 

twice at baseline with an automatic inhalationsynchronized Mefar MB3 dosimeter jet nebulizer (Mefar spa, Bovezzo, Italy), as described 

elsewhere (Aubier et al. 1992; Ezratty et al. 

2007). Methacholine challenge tests at baseline were conducted 10–30 days before the 

start of the exposures to avoid any putative 

interference of the methacholine challenge 

with the effect of NO2 (Jorres et al. 1995).

Sputum induction and inflammatory 

marker measurements. We performed sputum 

Table 1. Characteristics of participants.

Participant Age (years)

History of 

atopya

Smoking 

status Sex Height (cm) Weight (kg)

BMI

(kg/m2)

Asthma 

duration (years)

FEV1 at inclusion 

(FEV1 % predicted)

PD20 

methacholine

at baseline (μg)

Percent eosinophils 

in sputum

at baselineb

1c 26 Yes N F 164 57 21 8 3.23 (110) 1,600 NA

2 29 No E M 185 90 27 11 4.13 (89) 1,490 14.73

3 29 No N M 182 87 27 10 4.43 (99) 690 11.88

4 31 No N M 161 84 33 8 2.87 (81) 3,200 6.79

5 28 No N M 174 74 25 4 3.69 (88) 1,550 1.68

6 27 No N M 180 88 28 21 3.57 (80) 1,220 0.93

7 24 No N M 178 70 22 8 3.88 (87) 1,070 4.25

8 30 No N F 159 56 22 20 3.03 (103) 500 2.68

9 29 Yes N M 168 72 26 22 3.89 (100) 3,200 1.88

10c 28 Yes N M 186 92 27 2 4.49 (96) 800 ANR

11 20 Yes N F 158 49 20 9 2.29 (76) 930 0.26

12 69 No N M 178 90 29 5 2.49 (78) 3,200 17.72

13 30 No N F 163 55 21 20 2.68 (87) 1,950 32.08

14 32 Yes N M 179 82 26 24 4.12 (92) 340 2.51

15 24 No N F 171 60 21 14 3.41 (97) 310 5.12

16 28 No N M 174 62 21 16 3.82 (91) 2,100 20.58

17 32 No N M 176 115 38 21 4.16 (100) 2,170 2.99

18c 30 Yes N M 169 89 32 21 3.75 (95) 190 NA

19 30 No N M 174 115 39 23 3.16 (76) 210 1.55

Abbreviations: ANR: available but not relevant because participant 10 did not complete the three series of exposure; E, ex-smoker; N, never-smoker; NA: not available.

aPersonal history of atopic dermatitis and/or atopic familial history. bPercent eosinophils at baseline (10–30 days before first exposure). cParticipants excluded from the analysis: participants 1 and 18 did not produce adequate sputum specimens for cell analysis at baseline (squamous cells > 20%); participant 10 did not complete the three series of exposure.
Ezratty et al.

852 volume 122 | number 8 | August 2014 • Environmental Health Perspectives

induction with an aerosol of hypertonic saline 

using the method of Pin et al. (1992). The 

sputum was analyzed within 1 hr according to 

Pizzichini et al. (1996), as described elsewhere 

(Ezratty et al. 2007). Total nonsquamous 

inflammatory cell counts were expressed as 

103 cells/mg of induced sputum. Differential 

cell counts were performed by counting 

400 cells on May Grünwald Giemsa stained 

slides by two expert observers blinded to the 

participant’s exposure. Results were expressed 

both as percentage and as number of inflammatory cells per milligram of induced sputum. 

Only samples with cell viability > 70% and 

squamous cell contamination < 20% were 

considered adequate.

Sputum supernatant concentrations of 

eosinophil cationic protein (ECP) levels were 

measured by a commercially available enzyme 

assay (CAP-FEIA; Pharmacia, St Quentin-enYvelines, France), with a lower detection limit 

of 2 ng/mL.

Follow-up during the study period. After 0, 

15, and 30 min of exposure to NO2 or clean 

air in the chamber, participants were asked 

questions relating to respiratory symptoms 

and perception of discomfort.

FEV1 and PEF were monitored twice during exposure at 15-min intervals and hourly 

for 6 hr after leaving the chamber, with a portable combined spirometer (One Flow Tester; 

Mediflux, Croissy Beaubourg, France).

During the 10–30 days between baseline 

measurements and first exposure and during 

the 2-week interval following each series of 

exposures, subjective symptoms and medications were recorded every day. Each participant measured FEV1 and PEF twice daily 

with a portable combined spirometer.

Sample size. The primary end point was 

the change in the percentage of eosinophils 

in sputum, expressed as the ratio between the 

percentage after exposure and the baseline 

percentage. When the study was designed, 

literature reports were insufficient to determine the variance of the ratio, which was 

mandatory to estimate the sample size. 

Variance was estimated after inclusion of 

the first eight participants without unblinding. Based on the variance found of 0.10, a 

sample size of 18 participants was considered 

sufficient to demonstrate a doubling of the 

percentage of eosinophils in sputum with a 

power of 80% and a significance level of 0.05 

(see Supplemental Material, Table S1). In 

addition, a doubling has been reported to be 

consistent with clinically relevant changes in 

clinical status of asthmatics (Green et al. 2002).

Statistical analysis. The parameters studied 

in sputum were the percentage of eosinophils, 

the number of eosinophils per milligram, ECP, 

the number of neutrophils per milligram, and 

the number of macrophages per milligram. All 

parameters were log-transformed to normalize 

the distributions. We used a generalized linear 

model (GLM procedure, SAS v9.4; SAS 

Institute Inc., Cary, NC, USA) for the analyses. We included in the statistical model the 

effect of the participant, the time, the dose, 

and the interaction between the time and the 

dose. The interpretation of the results was 

based on the type III sum of squares. In case 

of a significant interaction between day and 

dose, a global analysis was performed, followed 

by per-day analyses. In the global analysis, all 

the data concerning the parameter were summarized by their geometric means (GMs) 

(in case of sputum inductions, this relates to 

three sputum inductions over 3 days, one per 

day), and we tested the relation between NO2

concentration—used as a quantitative value 

(0, 200, and 600 ppb)—and the parameter 

studied. The global analysis provided the ptrend. 

To display the results in a meaningful way, we 

also analyzed the data using classes of exposure (without any preconceived idea on the 

form of the relationship): For each class of 

exposure, we estimated the least-square mean 

(LSMEAN) and its 95% confidence limits. 

The LSMEANs and the confidence limits were 

exponentiated to obtain the changes relative 

to the baseline measurements and their confidence intervals (CIs). The per-day analyses 

were reported only when the trend in the 

global analysis was significant. p-Values < 0.05 

were considered significant.

Results

Among the 19 participants, 18 completed the 

three series of exposures and were included 

in the analysis. Among those, two did not 

produce an adequate sample of sputum at all 

Figure 1. Flow diagram of the study period for each participant. Abbreviations: H0, immediately before 

1st exposure (day 1); H0’, immediately after 1st exposure (day 1); H6, 6 hr after H0’ (day 1); H32, 32 hr after 

H0’ (day 2); H48, 48 hr after H0’ (day 3). The dose order was attributed randomly; so, series A, B, and C 

corresponds to clean air, 200-ppb NO2, or 600-ppb NO2 exposure.

Sputum induction

Sputum induction

Inclusion

Visit 1

Day 1

Day 2

1 exposure to dose A

(30 min) from H0 to H0’

1 exposure to dose B

(30 min) from H0 to H0’

2 exposures to dose A

(2 × 30 min)

+ H6 sputum induction

+ H6 sputum induction

+ H32 sputum induction

+ H32 sputum induction

Series A

dose A tested

Series B

dose B tested

Series C

dose C tested

Visit 2

Day 3

Day 1

Day 2

Day 3

2 weeks

wash out

No exposure

2 exposures to dose B

(2 × 30 min)

No exposure

1 exposure to dose C

(30 min) from H0 to H0’

2 exposures to dose C

(2 × 30 min)

No exposure

+ H48 sputum induction

+ H48 sputum induction

+ H6 sputum induction

+ H32 sputum induction

+ H48 sputum induction

Day 1

Day 2

Day 3

2 weeks

follow-up

2 weeks

wash out

1–3 weeks

BASELINE
NO2 and airway inflammation

Environmental Health Perspectives • volume 122 | number 8 | August 2014 853

time points and were not included in analyses 

of sputum (Table 1).

Respiratory function—FEV1 and PEF—

measured by spirometry, did not significantly change after NO2 exposure compared 

with clean air exposure (Table 2). No major 

clinical adverse reactions, such as coughing, wheezing, or chest tightness suggesting 

asthma attacks, were observed during exposures or follow-up. During the 2 weeks after 

each exposure, subjective symptoms and peak 

flow measurements were not significantly 

different regardless of exposure.

The primary analysis tested the exposure level (dose), the time (because the study 

design included assessment at three time 

points after exposure, one each day) and 

the interaction between time and exposure 

level. The dose was significantly related to 

the change in the percentage of eosinophils, the time was not. There was a significant interaction between time and exposure 

level, meaning that the association between 

dose and effect on eosinophils was different 

according to the day. As planned in the case 

of a significant interaction, we performed 

both a global analysis, averaging the measurements over the 3 days, and per-day analyses.

In the global analysis, the slope of the 

association between the percentage of eosinophils and NO2 exposure level was significant 

(p = 0.04) (Table 3).

Compared with baseline measurements, 

the percentage of eosinophils in sputum 

increased by 57% (95% CI: 18, 109%) 

after 600 ppb NO2 (p = 0.003) but did not 

change significantly after clean air or after 

200 ppb NO2.

Similar results were found for the association between the number of eosinophils per 

milligram of sputum and NO2 exposure level. 

In the global analysis, the slope of the association was significant (p = 0.02) (Table 3). 

The number of eosinophils per milligram 

of sputum increased by 120% (95% CI: 

60, 202%) after 600 ppb NO2 (p < 0.001) 

but not after clean air or after 200 ppb 

NO2 (Table 3).

Per-day analysis showed that the slope 

of the association between the percentage of 

eosinophils and NO2 exposure level was not 

significant at day 1 (p = 0.81), but was significant at day 2 (p = 0.01) and day 3 (p = 0.03). 

A similar pattern was found for the slope of 

the association between the number of eosinophils per milligram of sputum and the level of 

exposure to NO2, which was not significant at 

day 1 (p = 0.10), close to significance at day 2 

(p = 0.06), and significant at day 3 (p = 0.03) 

(Table 3; see also Supplemental Material, 

Figure S1).

Compared with baseline measurements, 

there was no significant change of the percentage of eosinophils in sputum regardless 

of the level of exposure to NO2 at day 1. At 

day 2, the percentage and the number of 

Figure 2. Study design for each of the three series of exposures separated by 2 weeks. Abbreviations: H0, immediately before 1st exposure (day 1); H0’, immediately after 1st exposure (day 1); H6, 6 hr after H0’ (day 1); H32, 32 hr after H0’ (day 2); H48, 48 hr after H0’ (day 3). Participants were exposed at rest in a doubleblinded, randomized, crossover design to clean air, 200-ppb NO2, or 600 ppb NO2 once for 30 min on day 1, and twice for 30 min on day 2. There was no exposure 

on day 3. Time of day is expressed in military form.

0915

hours

30 min

H48

H0 H0’ H6 × 2

× 2

H26 H32

30 min 30 min

H24

< 1hr >

Day 2

Day 1

Day 3

Arrive at

hospital

0800 hours

Arrive at

hospital

0800 hours

Methacholine

Bronchial challenge

• PD20methacholine

Arrive at

hospital

0800 hours

Leave hospital

around 1730 hours

Baseline: 10–30 days 

before first exposure

Leave hospital

around 1730 hours

Leave hospital

around 1100 hours

0845

hours

0845

hours

1015

hours

1045

hours

0845

hours

0915

hours

NO2/air

NO2/air NO2/air

Induced sputum

Exposure to

• NO2

 200 ppb, or

• NO2

 600 ppb, or

• Clean air

Spirometry

• Flow volume curve

Portable spirometry

• FEV1

 + PEF
Ezratty et al.

854 volume 122 | number 8 | August 2014 • Environmental Health Perspectives

eosinophils increased significantly at 600 ppb, 

but not at 200 ppb NO2. At day 3, the number of eosinophils increased significantly 

at 200 ppb and at 600 ppb NO2, and the 

increase of the percentage of eosinophils was 

close to significance at 600 ppb NO2, but 

not significant at 200 ppb (Table 3; see also 

Supplemental Material, Figure S1).

Absolute values not baseline adjusted 

[GMs (95% CIs)] of the percentages of 

eosinophils are reported in Supplemental 

Material, Table S2. Individual plots of the 

percentage of eosinophils are displayed in 

Supplemental Material, Figure S2. In order 

to see if results were driven by a participant 

in particular, we did a sensitivity analysis: a 

series of analyses with one different participant removed for each analysis. The trend test 

that measures the relationship between the 

dose of NO2 and the increase of the percentage of eosinophils in sputum from baseline 

was significant (p < 0.05) in 10 of the tests, 

and was close to significance (p < 0.10) for the 

other participants. Moreover, the increase of 

the percentage of eosinophils in sputum compared with baseline measurements was always 

significant at 600 ppb, regardless of which 

participant was removed from the analysis (see 

Supplemental Material, Table S3).

There was a significant correlation between 

the number of eosinophils per milligram of 

sputum and ECP (in nanograms per milliliter) 

(p < 0.001) (see Supplemental Material, 

Figure S3).

Compared with baseline measurements, 

ECP in sputum increased significantly after 

exposure to 600 ppb NO2 (43%; 95% CI: 

17, 75%; p = 0.001) but not after exposure 

to clean air or 200 ppb NO2. However, the 

slope of the association between ECP and the 

level of exposure was not significant (Table 3).

Exposure to NO2 did not affect the other 

cell types (macrophages, neutrophils) measured 

in sputum (Table 3).

There was no correlation between 

methacholine challenge tests and eosinophil 

responses (data not shown).

Discussion

Our findings indicate that repeated brief 

exposures to NO2 without allergen exposure 

increase eosinophilic airway inflammation in 

participants with intermittent asthma without inducing any changes in lung function. 

Because only mild asthmatics were tested, 

the results cannot be extrapolated to healthy 

individuals.

Although we cannot exclude that repeated 

challenges with hypertonic saline could have 

potentialized the effect of NO2, the repetition of sputum inductions is not the cause of 

the effect because the effect is expected to be 

the same for each of the three series of exposures (Pavord 1998). Eosinophils in sputum 

increased according to NO2 exposure level, 

and this significant trend supports a doserelated relationship. The effect on eosinophils 

and on ECP in sputum was significant at 

600 ppb NO2. We found a strong correlation 

between ECP and eosinophils, suggesting that 

eosinophils were activated.

In participants with asthma, several studies 

have found that NO2 exposure increased 

eosinophilic inflammation in response to 

inhaled allergen, in the distal lower airways 

assessed by bronchial wash and bronchoalveolar lavage (BAL) (Barck et al. 2002), 

and in sputum (Barck et al. 2005). The three 

previous studies that have investigated effects 

of NO2 without allergen challenge in participants with asthma did not find changes in 

inflammatory cell distributions in BAL (Jorres 

et al. 1995) and in sputum (Solomon et al. 

2004; Vagaggini et al. 1996). However, these 

studies involved small numbers of participants, 

and no repetition of exposure, and, in the 

study by Jorres et al. (1995), the evaluation of 

inflammation could have been performed too 

soon after exposure.

In the present study, participants with 

asthma were exposed to two realistic NO2

concentrations: 200 ppb and 600 ppb of 

NO2, which are close to NO2 peaks likely 

to be found indoors during the use of combustion appliances for cooking and heating 

(Basu and Samet 1999; Dennekamp et al. 

2001; Kotzias et al. 2005; Pilotto et al. 1997) 

and outdoors for short periods in streets with 

heavy traffic and in road tunnels (Larsson 

et al. 2010). Exposures lasted 30 min, close 

to the average time spent cooking dinner in 

France (38 min during the week and 46 min 

the weekend in a 2003 survey) (Hébel 

2012). Furthermore, exposure to intermittent peaks of NO2 may have greater effects 

than long-term, low-level exposure (Gardner 

et al. 1979).

Exposures in the present study were 

repeated over 2 days to mimic how exposures 

take place in real life and in order to assess a 

possible cumulative effect. At day 1, after one 

exposure, there was no significant change in 

eosinophilic airway inflammation—contrary 

to day 2 and day 3, after three exposures. 

These findings suggest that inflammatory 

response to NO2 exposure may be delayed or 

that a single exposure may be insufficient to 

enhance eosinophilic airway inflammation, 

suggesting a cumulative effect of NO2. These 

results are consistent with those of Barck et al. 

(2005) who found that two to three brief 

exposures at 260 ppb NO2 were needed to 

promote an increase in the airway inflammatory response to inhaled allergens. In addition 

to the study by Barck et al. (2005), our study 

shows that NO2, without an exposure to a 

high concentration of allergens, as with an 

allergen challenge, is sufficient to enhance 

inflammation in the airways. This finding 

could be of significance because exposure 

to peaks of NO2 is common, particularly 

indoors, whereas exposure to both high concentrations of NO2 and the specific stimulus for a susceptible individual is less likely 

(Hesterberg et al. 2009).

Many cities in Europe show an increase 

in concentrations of NO2 measured close 

to traffic due to the increasing number of 

vehicles, in particular diesel vehicles. Exhaust 

emissions from diesel vehicles are lower for 

carbon monoxide, non-methanic volatile 

organic compounds, and particulate matter 

Table 2. GMs (95% CIs) of values relative to baseline of FEV1 and PEF evaluated in the 18 participants 

who completed the study.

Variable 0 ppb NO2 (clean air) 200 ppb NO2 600 ppb NO2 p-Value

FEV1/baseline 1.02 (1.00, 1.05) 1.00 (0.97, 1.03) 1.00 (0.98, 1.03) 0.41

PEF/baseline 1.05 (0.99, 1.11) 1.04 (0.98, 1.10) 1.01 (0.96, 1.07) 0.36

Baseline values were measured immediately before first exposure (H0 on day 1) for each series of exposure. Each value 

corresponds to the GM change from baseline of the six values [H0’, immediately after first exposure (day 1); H6, 6 hr 

after H0’ (day 1); H32, 32 hr after H0’ (day 2); H48, 48 hr after H0’ (day 3)] obtained by spirometry with flow-volume curves. 

The effects of exposure, regardless of the dose, on FEV1 and PEF were not significant.

Table 3. Changes relative to baseline measurements (performed 10–30 days before the first exposure) for 

parameters measured in sputum [GM % (95% CI), n = 16].

Variable 0 ppb NO2 (clean air) 200 ppb NO2 600 ppb NO2 pTrend

Percentage of eosinophils –12 (–34, 16) –5 (–28, 26) 57 (18, 109)** 0.04

Day 1 16 (–28, 86) –34 (–59, 6) 12 (–31, 79) 0.81

Day 2 –5 (–37, 44) –3 (–36, 46) 102 (32, 211)** 0.01

Day 3 –39 (–67, 13) 36 (–26, 149) 79 (–3, 230) 0.03

No. of eosinophils/mg –5 (–31, 29) 23 (–10, 68) 120 (60, 202)# 0.02

Day 1 –5 (–44, 60) –24 (–55, 29) 64 (–3, 177) 0.10

Day 2 9 (–39, 95) 23 (–31, 119) 142 (32, 344)** 0.06

Day 3 –18 (–57, 56) 99 (5, 278)* 163 (38, 398)** 0.03

ECP 21 (–1, 47) 0 (–18, 22) 43 (17, 75)** 0.23

No. of neutrophils/mg 12 (–17, 51) –9 (–32, 22) 10 (–19, 49) 0.97

No. of macrophages/mg –18 (–33, 0) –2 (–20, 21) –6 (–24, 15) 0.46

GM percentages represent all changes of the parameter for the 3 days (one sputum induction per day). 

*p < 0.05; **p < 0.01; and #

p < 0.001, compared with baseline.
NO2 and airway inflammation

Environmental Health Perspectives • volume 122 | number 8 | August 2014 855

but may be higher for NO2 (Guerreiro et al. 

2012). Although epidemiological studies on 

NO2 have several limitations in particular 

because of the potential for exposure misclassification and co-pollutant effects, our 

results provide important evidence suggesting 

that NO2 alone has a direct effect on airway 

inflammation in asthmatics.

Conclusions

To our knowledge, this is the first study to 

demonstrate that repeated peaks of NO2 at 

realistic concentrations without allergen exposure increase eosinophilic inflammation in 

the airways of asthmatics, supporting a dose–

response relationship. Although it is difficult 

to evaluate the clinical implications of these 

findings in the present study, an increased 

eosinophilic inflammation may lead to exacerbation or loss of control of asthma (Green 

et al. 2002; Jacobsen et al. 2007). Therefore, 

we cannot rule out the effects of repeated 

exposures to NO2 over a longer period of 

time or effects in subpopulations.

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