Fluticasone Propionate Protects against Ozone-Induced Airway Inflammation and Modified Immune Cell Activation Markers in Healthy Volunteers

Abstract

Ozone exposure induces airway neutrophilia and modifies innate immune monocytic cell-surface phenotypes in healthy individuals. High-dose inhaled corticosteroids can reduce O(3)-induced airway inflammation, but their effect on innate immune activation is unknown.
We used a human O(3) inhalation challenge model to examine the effectiveness of clinically relevant doses of inhaled corticosteroids on airway inflammation and markers of innate immune activation in healthy volunteers.
Seventeen O(3)-responsive subjects [>10% increase in the percentage of polymorphonuclear leukocytes (PMNs) in sputum, PMNs per milligram vs. baseline sputum] received placebo, or either a single therapeutic dose (0.5 mg) or a high dose (2 mg) of inhaled fluticasone proprionate (FP) 1 hr before a 3-hr O(3) challenge (0.25 ppm) on three separate occasions at least 2 weeks apart. Lung function, exhaled nitric oxide, sputum, and systemic biomarkers were assessed 1-5 hr after the O(3) challenge. To determine the effect of FP on cellular function, we assessed sputum cells from seven subjects by flow cytometry for cell-surface marker activation.
FP had no effect on O(3)-induced lung function decline. Compared with placebo, 0.5 mg and 2 mg FP reduced O(3)-induced sputum neutrophilia by 18% and 35%, respectively. A similar effect was observed on the airway-specific serum biomarker Clara cell protein 16 (CCP16). Furthermore, FP pretreatment significantly reduced O(3)-induced modification of CD11b, mCD14, CD64, CD16, HLA-DR, and CD86 on sputum monocytes in a dose-dependent manner.
This study confirmed and extended data demonstrating the protective effect of FP against O(3)-induced airway inflammation and immune cell activation.

Full text

Environmental Health Perspectives
•
VOLUME 116 | NUMBER 6 | June 2008
799
Research
|
Environmental Medicine
Ozone is a commonly encountered environ-
mental air pollutant. In epidemiologic investi-
gations, exposure to increased levels of
ambient air O
3
has been associated with exac-
erbations of asthma, chronic obstructive pul-
monary disease (COPD), and pneumonia,
generally 24–48 hr after exposure occurs
(Bernstein et al. 2004; Peden 2001).
Controlled chamber exposures to O
3
cause an
influx of neutrophils to the airway and a
decrease in lung function, although these two
effects do not correlate with each other, indi-
cating that separate mechanisms account for
these effects (Bernstein et al. 2004). O
3
expo-
sure also causes increased responsiveness to
allergen in allergic asthmatics (Peden 2001).
We have recently observed that O
3
exposure
can also result in increased expression of
CD11b, CD14, CD16, CD80, CD86, and
HLA-DR on airway dendritic cells (DCs),
monocytes, and macrophages (Alexis et al.
2004b). It has been suggested that the action
of O
3
on airway neutrophils, monocytes, and
macrophages accounts for much of the disease
outcomes associated with O
3
exposure. These
inflammatory events also mimic the type of
inflammation that occurs with acute viral and
bacterial infection and exacerbations of
asthma and COPD (Maneechotesuwan et al.
2007; Pauwels 2004).
Together, these observations suggest that
O
3
challenge may be a useful controlled
human disease model for screening novel anti-
inflammatory pharmaceutical agents in phase I
proof-of-concept trials. Holz et al. (2005)
tested the utility of a 0.25-ppm O
3
challenge
as a drug efficacy screen, using a single pre-
treatment dose of the established anti-
inflammatory agents fluticasone propionate
(FP) and oral prednisolone as test agents in a
randomized three-arm crossover study in
18 healthy subjects comparing the effect of
these two treatments with that of placebo on
O
3
-induced airway inflammation. Holz et al.
(2005) reported that, compared with placebo,
pretreatment with 2 mg inhaled FP and 50 mg
oral prednisolone resulted in a significant
reduction in post-O
3
sputum neutrophils per
milliliter (by 62% and 64%, respectively) and
myeloperoxidase (MPO; by 55% and 42%,
respectively). These results demonstrated that
corticosteroids do inhibit the proinflammatory
actions of O
3
.
In the present study, we sought to extend
these observations by comparing the effect of
a single administration of a high dose of
inhaled FP (2 mg) with a dose that is
employed in clinical practice for asthma and
COPD (0.5 mg) and placebo. Given the
importance that monocytes, macrophages,
and DCs likely have in the pathophysiology of
O
3
-induced exacerbations of disease, we also
examined the effect of these treatments on
expression of CD11b/CR3, mCD14,
CD16/FcγRIII, CD64/FcγRI, CD86/B7, and
HLA-DR on monocytes, macrophages, and
DCs recovered from airway sputum. Clara cell
protein 16 (CCP16) and surfactant protein D
(SP-D) are innate immune molecules and
products of airway epithelial cells (Haczku
2006) that can be released to the circulation
during lung injury (Holz et al. 2005). CCP16
is induced in the serum of subjects exposed to
O
3
challenge (Blomberg et al. 2003). We have
previously shown that SP-D levels in the lung
are significantly altered after O
3
inhalation in
mice (Kierstein et al. 2006), but whether simi-
lar changes can be detected in the human
serum is not known. Thus, we evaluated
CCP16 and SP-D for their potential utility as
serum biomarkers for assessing the effects of
inhaled corticosteroids on O
3
injury in the
respiratory tract.
Materials and Methods
Subjects. Seventeen (nine male and eight
female) nonsmoking healthy volunteers
(10 from the Center for Environmental
Medicine, Asthma and Lung Biology; 7 from
Rancho Los Amigos National Rehabilitation
Center) between 18 and 50 years of age (age,
26.4 ± 7.4 years, mean ± SD; body mass
index, 20–30 kg/m
2
) were recruited for this
Address correspondence to N.E. Alexis, Center for
Environmental Medicine, Asthma and Lung
Biology, University of North Carolina, 104 Mason
Farm Rd., Chapel Hill, NC 27599-7310 USA.
Telephone: (919) 966-9915. Fax: (919) 966-9863.
E-mail: Neil_Alexis@med.unc.edu.
We thank M. Almond, M. Herbst, H. Wells,
F. Dimeo, and L. Newlin-Clapp for their technical
assistance with this study.
This work was supported by GlaxoSmithKline and
by the National Institutes of Health (grant ES012706).
R.T.-S. is employed by GlaxoSmithKline, the
manufacturer of fluticasone proprionate. The
remaining authors declare they have no competing
financial interests.
Received 15 October 2007; accepted 27 February
2008.
Fluticasone Propionate Protects against Ozone-Induced Airway Inflammation
and Modified Immune Cell Activation Markers in Healthy Volunteers
Neil E. Alexis,
1,2
John C. Lay,
1
Angela Haczku,
3
Henry Gong,
4,5
William Linn,
4,5
Milan J. Hazucha,
1
Brad Harris,
1
Ruth Tal-Singer,
6
and David B. Peden
1,2
1
Center for Environmental Medicine, Asthma and Lung Biology, and
2
Department of Medicine, University of North Carolina, Chapel Hill,
North Carolina, USA;
3
Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA;
4
Department of Medicine,
University of California, Los Angeles, California, USA;
5
Environmental Health Service, Rancho Los Amigos National Rehabilitation
Center, Downey, California, USA;
6
GlaxoSmithKline, King of Prussia, Pennsylvania, USA
BACKGROUND: Ozone exposure induces airway neutrophilia and modifies innate immune monocytic
cell-surface phenotypes in healthy individuals. High-dose inhaled corticosteroids can reduce
O
3
-induced airway inflammation, but their effect on innate immune activation is unknown.
O
BJECTIVES: We used a human O
3
inhalation challenge model to examine the effectiveness of clini-
cally relevant doses of inhaled corticosteroids on airway inflammation and markers of innate
immune activation in healthy volunteers.
M
ETHODS: Seventeen O
3
-responsive subjects [> 10% increase in the percentage of polymorpho-
nuclear leukocytes (PMNs) in sputum, PMNs per milligram vs. baseline sputum] received placebo,
or either a single therapeutic dose (0.5 mg) or a high dose (2 mg) of inhaled fluticasone proprionate
(FP) 1 hr before a 3-hr O
3
challenge (0.25 ppm) on three separate occasions at least 2 weeks apart.
Lung function, exhaled nitric oxide, sputum, and systemic biomarkers were assessed 1–5 hr after
the O
3
challenge. To determine the effect of FP on cellular function, we assessed sputum cells from
seven subjects by flow cytometry for cell-surface marker activation.
R
ESULTS: FP had no effect on O
3
-induced lung function decline. Compared with placebo, 0.5 mg
and 2 mg FP reduced O
3
-induced sputum neutrophilia by 18% and 35%, respectively. A similar
effect was observed on the airway-specific serum biomarker Clara cell protein 16 (CCP16).
Furthermore, FP pretreatment significantly reduced O
3
-induced modification of CD11b, mCD14,
CD64, CD16, HLA-DR, and CD86 on sputum monocytes in a dose-dependent manner.
C
ONCLUSIONS: This study confirmed and extended data demonstrating the protective effect of FP
against O
3
-induced airway inflammation and immune cell activation.
K
EY WORDS: inhaled corticosteroids, innate immune markers, ozone, sputum neutrophils Environ
Health Perspect 116:799–805 (2008). doi:10.1289/ehp.10981 available via http://dx.doi.org/ [Online
28 February 2008]

study. All subjects underwent a thorough
physical examination and had no history of
cardiovascular or chronic respiratory disease
and were free of upper or lower respiratory
tract infection at least 4 weeks before study
participation. All subjects had a forced expira-
tory volume in 1 sec (FEV
1
) of at least 80%
predicted for a normal population of similar
weight and height. A positive urine pregnancy
test resulted in exclusion of female subjects
from the study. The use of prescription drugs,
over-the-counter medication (e.g., aspirin and
nonsteroidal anti-inflammatory drugs), vita-
mins, antioxidants, and dietary supplements
was not permitted for the duration of the
study. All study participants were able to pro-
duce an adequate sputum sample (≥ 1 × 10
6
total cells, ≥ 50% cell viability, ≤ 20% squa-
mous epithelial cells) as measured on their first
baseline visit (sputum with no O
3
exposure),
and all were responsive to O
3
(defined as
≥ 10% increase in total and percent sputum
neutrophils) (Holz et al. 2005) after exposure
to 0.25 ppm O
3
for 3 hr with intermittent
moderate exercise (ventilation
expiratory
=
12.5 L/min/m
2
body surface area) as measured
on the second study visit. The study was
approved by the Committee on the Protection
of the Rights of Human Subjects, School of
Medicine, University of North Carolina at
Chapel Hill, and by the Institutional Review
Board at the Rancho Los Amigos National
Rehabilitation Center. Informed written con-
sent was obtained from all subjects before their
participation in the study.
Study design. This was a double-blind,
placebo-controlled, single-dose, randomized,
three-period crossover study conducted at two
sites. Controlled O
3
exposures were performed
in comparable chamber setups at both the
University of North Carolina, Chapel Hill and
the Rancho Los Amigos facility (Alexis et al.
2004a; Gong et al. 1998). All subjects under-
went 3-hr exposures to 0.25 ppm O
3
with
intermittent moderate exercise (15 min rest,
15 min exercise at 12.5 L/min/m
2
body surface
area) at screening visit 2 and each study session
thereafter (visits 3–5). Based on FP half-life and
washout of sputum neutrophils after O
3
expo-
sure (Holz et al. 2005), O
3
exposures were sep-
arated by a minimum of 2 weeks to avoid
carryover effects. FEV
1
and forced vital capacity
(FVC) were also measured for the purpose of
assessing subject safety. Sputum induction was
performed at screening visits and at 3 hr after
the conclusion of each O
3
exposure (i.e., post-
exposure). Sputum was analyzed for total and
differential leukocyte count and fluid-phase
components and in a subset of subjects (n =7)
for cell-surface phenotypes and cell function by
flow cytometry. The study design, including
measurement time points, is depicted in
Figure 1. FP was provided as a metered dry
powder inhaler (Diskus; GlaxoSmithKline,
Research Triangle Park, NC). Each Diskus
device contained 60 × 0.5 mg doses of FP. A
matching placebo Diskus was also provided.
Subjects were randomized to receive one of
the following treatment regimens: a) 0.5 mg
FP (one inhalation of 0.5 mg FP plus three
inhalations of placebo); b) 2 mg FP (one
inhalation of 0.5 mg FP plus three inhalations
of 0.5 mg FP); and c) placebo (one inhalation
of placebo plus three inhalations of placebo).
The study staff observed each subject using
the Diskus during clinic visits to ensure that
the device was used correctly.
Pulmonary function. We used both
spirometry and impulse oscillometry (IOS) to
assess lung function status in subjects.
Spirometry was assessed at preexposure,
immediately postexposure, and then at 1-hr
intervals for 3 hr. IOS was assessed at pre-
exposure, and then hourly for 3 hr beginning
1 hr postexposure. Airway resistance and air-
way reactance were determined by IOS (Jaeger
MS-IOS and LAB Manager Software, version
4.53.2; Jaeger, Hoechberg, Germany) using
the recommended techniques of the manufac-
turer and as previously described (Singh et al.
2006). Real-time recordings of mouth pres-
sure and flow signals pulsed through 5- to
35-Hz spectrum were superimposed on trac-
ings of tidal breathing and displayed on a
computer screen. Measurements of total respi-
ratory resistance, resonant frequency (F
res
),
reactance at 5 Hz, and low-frequency reac-
tance area (area of reactance integrated from
5 Hz up to F
res
) were recorded at the 5-, 10-,
15-, and 20-min time points after the IOS test
challenge. Spirometry was performed accord-
ing to current American Thoracic Society
spirometry standards (Enright 2003).
Sputum induction and processing and
fluid-phase analyses. Subjects provided an
induced sputum sample during the screening
visit and at 3 hr post-O
3
exposure. The sputum
induction and processing methods have been
previously described in detail (Alexis et al.
2003, 2006). In brief, three 7-min inhalation
periods of nebulized hypertonic saline (3%,
4%, 5%; Devilbiss UltraNeb 99 ultrasonic neb-
ulizer; Sunrise Medical, Somerset, Somerset,
PA) were followed by expectoration of sputum
into a sterile specimen cup. Sputum cell aggre-
gates (cellular mucus plugs) were macroscopi-
cally identified and manually selected from
their surrounding fluid and treated with 0.1%
dithiothreitol (DTT; Sputolysin, Calbiochem,
San Diego, CA). Total cell counts and cell via-
bility were determined using a Neubauer
hemacytometer and trypan blue (Sigma
Chemical Co., St. Louis, MO) exclusion
staining. Differential cell counts were ana-
lyzed using the Hema-Stain-3 kit (Fisher
Diagnostics, Middletown, VA). Aliquots of
DTT-treated sputum supernatant were imme-
diately frozen and stored at –80°C for later
analysis of MPO and total protein by multi-
plex assay (Pierce Biotechnology, Rockford,
IL). All soluble factors (cytokines and
chemokines) in sputum (MPO, total protein)
were analyzed by a contract laboratory (HFL,
Fordham, UK) using validated commercial
Alexis et al.
800
VOLUME 116 | NUMBER 6 | June 2008
•
Environmental Health Perspectives
Figure 1. Schematic of the study design. Abbreviations: PLA, placebo; Spiro, spirometry; V, visit. The study
was a double-blinded, randomized, cross-over design with a 2-week washout period between visits. Except
for the first visit (screen) and last visit (follow-up), all visits included an O
3
exposure (0.25 ppm, 3 hr).
Baseline screen (V1)
O
3
challenge (V2)
Sputum, spiro, eNO, IOS
Spiro, eNO, IOS
3 randomized visits (V3, V4, V5)
Preexposure
PLA, 0.5 mg FP, or 2 mg FP
O
3
exposure (0.25 ppm)
Postexposure
Spiro, eNO, IOS
Spiro, eNO, IOS
Intermittent exercise
Follow-up (V6)
–3 weeks
–2 weeks
Spiro, eNO
Spiro, eNO, IOS
Spiro, eNO, IOS
Sputum, spiro, IOS, eNO
Systemic markers, CCP16, SP-D (
n
= 10), eNO
Systemic markers, CCP16, SP-D (
n
= 7)
7–10 days
–2 hr
–1 hr
0 hr
1 hr
2 hr
3 hr
+ 1 hr
+ 2 hr
+ 3 hr
+ 4 hr
+ 5 hr
eNO

enzyme-linked immunosorbent assay (ELISA)
kits. All compounds were validated in the pres-
ence of DTT. The limits of detection after
dilution (to minimize potential effects of DTT
and to achieve sufficient volume for measure-
ments) were 40 µg/mL for total protein
(Dojindo Molecular Technologies, Inc.,
Gaithersburg, MD) and 36 ng/mL for MPO
(Immundiagnostik, Bensheim, Germany).
For a subset of samples, remaining cells
were resuspended in Hank’s balanced salt
solution and kept on ice for immediate use in
flow cytometric assays for selected cell-surface
molecules and phagocytosis.
Systemic biomarkers. Venipuncture was
performed at 4 or 5 hr after O
3
exposures to
obtain serum for Multiplex systemic biomarker
analysis of tumor necrosis factor-α (TNF-α),
interferon-γ (INF-γ), interleukin-6 (IL-6),
IL-1β, IL-1Ra, IL-17, eotaxin, and IL-12P40
using fluorometric custom-designed validated
Multiplex kits (Pathway Diagnostics, Malibu,
CA). CCP16 and SP-D were assayed using
commercially available ELISA kits (Biovendor,
Candler, NC) according to the manufacturer’s
instructions.
Flow cytometry and immunofluorescent
staining. All flow cytometry acquisitions and
analyses (surface markers, phagocytosis) were
performed as previously described (Alexis
et al. 2000a) using a FACSort flow cyto-
meter (Becton Dickinson, Franklin Lakes,
NJ) and CellQuest Pro v5.3 software (Becton
Dickinson).
Cell-surface phenotypes. Immuno-
fluorescent staining and flow-cytometry
methodology have been described in detail in
previous publications (Alexis et al. 2003,
2006). In brief, cells (100 µL, 1 × 10
6
/mL)
were incubated with 10 µL fluorochrome-
labeled monoclonal antibodies, washed in
Dulbecco’s phosphate-buffered saline (DPBS),
fixed with 0.5% paraformaldehyde in DPBS,
and analyzed by flow cytometry within 48 hr
of fixation. Viable macrophages, monocytes,
neutrophils, lymphocytes, and DCs in spu-
tum were initially identified and gated on the
basis of light-scatter properties and positive
expression for CD45 (pan-leukocyte marker).
Cell populations were then confirmed by
positive staining with CD16 (neutrophils),
mCD14 (monocytes), HLA-DR (macro-
phages), HLA-DR/CD86 (DCs), and CD3
(lymphocytes). The acquired data were ana-
lyzed using CellQuest Pro v5.3 software, and
results were expressed as a rightward shift
from control in mean fluorescence intensity
(MFI) on histogram analysis. Control cells
were incubated with appropriately labeled iso-
typic control antibodies. Surface markers ana-
lyzed included markers of innate (CD11b/
CR3, mCD14/LPS receptor, CD16/FcγRIII,
CD64/FcγRI) and adaptive (HLA-DR/MHC
class II, and CD86/B7.2 co-receptor)
immune function. All monoclonal antibodies
were purchased from Beckman Coulter
Corporation (Miami, FL).
Phagocytosis. We analyzed phagocytosis
using fluorescein isothiocyanate–labeled IgG-
opsonized Saccharomyces cerevisiae zymosan-A
BioParticles (Molecular Probes, Eugene, OR)
as previously described (Alexis et al. 2003,
2006). All samples were analyzed by flow
cytometry within 24–48 hr of fixation in 1%
paraformaldehyde. Particle uptake was dis-
played on histograms and identified as a
rightward shift in MFI of the phagocytic
population versus autofluorescence of the
unlabeled control cells.
Exhaled nitric oxide. We measured
exhaled NO (eNO) levels preexposure, imme-
diately after exposure, and then at 1-hr inter-
vals for 4 hr according to standardized
procedures jointly recommended by the
American Thoracic Society and the European
Respiratory Society (2005) using a NIOX
NO analyzer (Aerocrine AB, Solna, Sweden).
Statistical analysis. To determine the total
number of neutrophils and fluid-phase mark-
ers (MPO, protein) in induced sputum 6 hr
postchallenge, we analyzed data following a
natural logarithmic transformation using a
mixed effects model, with period and treat-
ment fitted as fixed effects and subject as a
random effect. The suitability of the transfor-
mation was assessed by examining the model
residuals. Treatment effects were evaluated in
terms of treatment ratios and were calculated
as the antilog for the differences between the
least squares means; 95% confidence intervals
(CIs) were determined using pooled estimates
of variance for the least squares means differ-
ence and then antilogged.
For assessment of differences between spe-
cific treatment conditions (postscreen O
3
chal-
lenge vs. placebo vs. both doses of FP) for
CCP16, SP-D, systemic cytokines, and cell-
surface marker expression in the subset (n =7)
of volunteers studied at University of North
Carolina, Chapel Hill, we used nonparametric
one-way analysis of variance for repeated meas-
ures (Friedman test) and Dunn’s post hoc
analysis of specific pairs of variables. An overall
significance level of p < 0.05 was considered to
be significant. All values are expressed as mean
± SE. We used GraphPad Prism 3.1 software
(GraphPad Software, Inc., San Diego, CA) for
statistical analysis.
Results
Patient demographics and overall safety.
Seventeen volunteers participated in the study;
patient demographics are outlined in Table 1.
No serious adverse events were reported dur-
ing this study.
Effects of FP on 0.25 ppm O
3
-induced
changes in pulmonary function. O
3
exposure
caused decreases in FVC and FEV
1
during all
exposures. Decrements in FVC and FEV
1
were evident immediately after O
3
exposure
during placebo, 0.5 mg, and 2 mg FP treat-
ments but were subsiding by 1 hr post-
exposure for each treatment condition
(Table 2). Decrements in FVC and FEV
1
were minimal by 3 hr postexposure (Table 2).
Neither dose of FP had a statistically signifi-
cant effect on O
3
-induced lung function
changes compared with placebo. No consis-
tent O
3
-induced changes were observed in
IOS end points at any postexposure time
point (Table 2).
Effects of FP on 0.25 ppm O
3
-induced
changes in sputum neutrophils and fluid-
phase markers of neutrophil activation
(MPO, total protein). Analysis of percent
neutrophil levels post-O
3
challenge yielded
evidence of a statistically significant difference
for both active treatments (0.5 mg and 2 mg
FP) relative to placebo. Mean ± SE levels of
percent polymorphonuclear leukocytes
(PMNs) for placebo and 0.5 mg and 2 mg FP
were 54 ± 5.4%, 44 ± 4.5%, and 35 ± 3.6%,
respectively (Figure 2), which reflect an 18%
and 35% reduction in sputum percent PMNs
for 0.5 mg and 2 mg FP, respectively. The
data indicate a dose–response pattern.
FP also affected the relatively more variable
total number of neutrophils/mL. The mean
(95% CI) numbers of PMNs/mL were 66.05 ×
10
4
cells/mL (34.78–125.41 cells/mL),
56.87 × 10
4
cells/mL (30.15–107.27 cells/mL),
and 37.49 × 10
4
cells/mL (19.89–70.68
cells/mL) for placebo, 0.5 mg FP, and 2 mg
FP, respectively, 3 hr post-O
3
exposure.
These values reflected 14% fewer neutrophils
in sputum when subjects were pretreated
with 0.5 mg FP and statistically significantly
(p < 0.05) fewer neutrophils (43%) when
pretreated with 2 mg FP, indicating a dose–
response effect on the total number of
neutrophils per milliliter. In terms of vari-
ability, the neutrophil responses on the
O
3
/placebo visit versus the O
3
-only visit were
very similar for both percent neutrophils
(mean ± SE, 54 ± 5% vs. 55 ± 5%) and the
absolute number of neutrophils per mil-
ligram sputum [mean (95% CI), 66.05 × 10
4
cells/mL (34.78 to 125.41 cells/mL) vs.
Fluticasone propionate protects against airway inflammation
Environmental Health Perspectives
•
VOLUME 116 | NUMBER 6 | June 2008
801
Table 1. Subject demographics (
n
= 17).
Characteristic Mean ± SE
Age (years) 26.4 ± 1.8
Sex
Female 9
Male 8
Race
Caucasian 10
African American 3
American Hispanic 2
Asian 1
Other 1
Height (cm) 170 ± 2.6
Weight (kg) 78 ± 3.9

62.20 × 10
4
cells/mL (–10.17 to 312.97
cells/mL), respectively]. Other than percent
macrophages, FP exerted no statistically
significant effect on total leukocytes per milli-
liter or total and percent eosinophils, lympho-
cytes, and bronchial epithelial cells. Relative to
placebo, we observed a 24% and 48% increase
in percent macrophages with 0.5 mg and 2 mg
FP, respectively.
We observed no statistically significant
treatment effect of 0.5 mg or 2 mg FP on
MPO or total protein levels in sputum. There
was, however, borderline evidence of a differ-
ence in levels of the MPO/total protein ratio
relative to placebo for 2 mg FP. We observed,
on average, reductions of 18% and 43% in
the MPO/total protein ratio for 0.5 mg and
2 mg FP, respectively, suggesting a dose–
response relationship.
Effects of FP on 0.25 ppm O
3
-induced
changes in surface marker expression and
phagocytosis on sputum monocytes, macro-
phages, DCs, and neutrophils. Figure 3 shows
the effect of 0.5 and 2 mg pretreatments with
FP on O
3
-induced changes in the cell-surface
markers CD11b, mCD14, CD64, CD16,
HLA-DR, and CD86 on monocytes,
macrophages, and DCs. Baseline (i.e., no O
3
exposure) sputum cell-surface marker values
(MFI; mean ± SE) from a different cohort of
healthy volunteers (n = 15) were as follows: for
CD11b, 21 ± 8 macrophages, 16 ± 3 DCs; for
mCD14, 65 ± 16 macrophages, 59 ± 14
monocytes, 61 ± 11 DCs; for CD64, 5 ± 1
monocytes; for CD16, 238 ± 44, macrophages,
195 ± 28 DCs; for HLA-DR: 31 ± 5 mono-
cytes; and for CD86, 22 ± 4 monocytes (Lay
et al. 2007). Compared with the O
3
-only con-
dition in this study (data not shown), baseline
expression of these surface markers was signifi-
cantly (p < 0.05) lower, indicating that O
3
causes an up-regulation of these cell-surface
phenotypes.
In general, 2 mg FP exerted a statistically
significant effect on post-O
3
surface marker
expression relative to placebo treatment. There
was also a similar trend after the 0.5 mg dose,
which suggests a dose–response effect of FP on
O
3
-induced changes in monocytic cell-surface
markers. We also observed a significant decrease
in CD16/FcγRIII expression on neutrophils
after 2.0 mg FP compared with placebo (MFI,
406 ± 64 vs. 515 ± 72; p < 0.05). We observed
no significant drug effect of 0.5 mg or 2 mg FP
versus placebo on sputum cells as measured by
MFI (mean ± SE): for phagocytosis for
macrophages, 478 ± 76 and 606 ± 102 versus
400 ± 50; for monocytes, 348 ± 46 and
365 ± 55 versus 292 ± 39; and for neutrophils,
296 ± 48 and 418 ± 87 versus 270 ± 29.
Effects of FP on 0.25 ppm O
3
-induced
changes in serum CCP16, SP-D, eNO, and
other systemic biomarkers. To determine
whether serum levels of the airway epithelial
cell products SP-D and CCP16 would reflect
inflammatory airway changes after O
3
expo-
sure, we measured the concentration of these
molecules at baseline and after each O
3
inhalation session in a subset of seven subjects
5 hr after O
3
exposure. Our results showed
that serum CCP16 levels were statistically sig-
nificantly increased after O
3
inhalation and
that pretreatment with 2 mg FP statistically
significantly inhibited this effect compared
with placebo (Figure 4). The effects of FP on
CCP16 were dose dependent. SP-D levels were
not statistically significantly altered pre- versus
post-O
3
exposure (mean ± SE, 61 ± 6 ng/mL
vs. 55 ± 5.4 ng/mL) and were not significantly
affected by 0.5 mg FP (53 ± 5 ng/mL) or 2 mg
FP (64 ± 5 ng/mL) compared with placebo
(55 ± 5 ng/mL).
Statistical analysis of other systemic bio-
markers or eNO yielded no clear changes
induced by O
3
exposure. No significant effects
on systemic cytokines (IL-6, IL-12P40, IL-15,
IL-17, IL-1β, IL-1Ra, INF-γ, TNF-α), medi-
ators (MPO, eotaxin), or eNO (Table 2) were
observed after 0.5 mg or 2 mg FP versus
placebo. For eNO, levels at 1, 2, and 3 hr
(Table 2) postexposure were not statistically
significantly different from one another.
Discussion
Numerous laboratory studies of healthy young
individuals exposed to O
3
at a dose compara-
ble to that used in the present study have
demonstrated decrements in spirometric lung
function (Holz et al. 1999, 2005; McDonnell
et al. 1997; Nightingale et al. 2000). A study
similar to this one in terms of the cohort char-
acteristics, O
3
concentration, and ventilation
rate also reported similar postexposure decre-
ments in FVC and FEV
1
(Holz et al. 1999).
In the present study we found that pre-
treatment with therapeutic doses of FP had no
significant protective effect on spirometric
response, which is in agreement with the
finding of Nightingale et al. (2000). FP did,
Alexis et al.
802
VOLUME 116 | NUMBER 6 | June 2008
•
Environmental Health Perspectives
Figure 2. The percent sputum neutrophils after O
3
exposure for each pretreatment dose of FP (0.5 or
2 mg) or placebo.
*
p
< 0.05 compared with placebo.
90
80
70
60
50
40
30
20
10
0
Placebo 0.5 mg 2 mg
*
*
Percent neutrophils
FP
Table 2. Mean (± SE) pulmonary function, eNO, and IOS.
IOS
FEV
1
(L) FVC (L) eNO (ppb) R5 X5
F
res
(Hz)
Baseline
Pretreatment 3.76 ± 0.18 4.69 ± 0.21 12.54 ± 1.55 0.376 ± 0.008 –0.112 ± 0.003 12.27 ± 0.30
Placebo
Preexposure (0 hr) 3.84 ± 0.01 4.70 ± 0.02 11.49 ± 1.26 0.409 ± 0.007 –0.161 ± 0.002 12.03 ± 0.28
Immediately after exposure 3.52 ± 0.07 4.39 ± 0.08 13.51 ± 1.39
1 hr postexposure 3.71 ± 0.05 4.60 ± 0.06 14.24 ± 1.49 0.379 ± 0.008 –0.099 ± 0.002 11.85 ± 0.27
2 hr postexposure 13.76 ± 1.43 0.404 ± 0.04 –0.102 ± 0.01 11.87 ± 1.14
3 hr postexposure 8.94 ± 1.15 0.414 ± 0.04 –0.221 ± 0.11 12.60 ± 1.38
0.5 mg FP
Preexposure (0 hr) 3.84 ± 0.02 4.75 ± 0.03 11.65 ± 1.91 0.383 ± 0.007 –0.112 ± 0.002 12.10 ± 0.24
Immediately after exposure 3.51 ± 0.05 4.43 ± 0.05 14.80 ± 1.68
1 hr postexposure 3.69 ± 0.04 4.60 ± 0.05 15.53 ± 1.70 0.356 ± 0.007 –0.106 ± 0.002 12.01 ± 0.25
2 hr postexposure 15.85 ± 1.60 0.382 ± 0.05 –0.101 ± 0.01 12.26 ± 1.43
3 hr postexposure 12.16 ± 1.2 0.393 ± 0.05 –0.196 ± 0.08 12.99 ± 1.76
2.0 mg FP
Preexposure (0 hr) 3.73 ± 0.02 4.61 ± 0.02 10.81 ± 1.75 0.382 ± 0.008 –0.113 ± 0.003 12.23 ± 0.25
Immediately after exposure 3.51 ± 0.07 4.35 ± 0.08 13.84 ± 1.32
1 hr postexposure 3.60 ± 0.04 4.41 ± 0.05 14.87 ± 1.57 0.367 ± 0.007 –0.109 ± 0.003 11.70 ± 0.24
2 hr postexposure 13.65 ± 1.18 0.365 ± 0.04 –0.099 ± 0.01 11.47 ± 1.07
3 hr postexposure 11.88 ± 1.45 0.373 ± 0.04 –0.188 ± 0.08 11.45 ± 1.07
Abbreviations: R5, total respiratory resistance (cm H
2
O/L/sec); X5, reactance (cm H
2
O/L/sec).

however, inhibit inflammatory cell (neutro-
phils, PMNs) influx to the airways induced by
a 3-hr exposure to 0.25 ppm O
3
in a dose-
dependent manner. The lack of correlation
between spirometry and airway inflammation
after O
3
has been well documented (Blomberg
et al. 1999; Hazucha et al. 1996), so our find-
ing with FP in this regard was not surprising.
We observed a significant inhibition of
the percent PMNs present in airway sputum
after O
3
challenge with either 0.5 mg or 2 mg
FP pretreatment, and a significant reduction
and a trend for reduction in the number of
sputum neutrophils per milliliter post-O
3
with 2 mg and 0.5 mg FP, respectively.
Furthermore, we showed that serum CCP16
is a valuable systemic marker of the inflam-
matory state of the lung and is responsive to
the effects of inhaled FP. We also observed
evidence of diminished neutrophil activation
with 2 mg FP, as it decreased the expression
of CD16, a marker of neutrophil activation,
compared with placebo. This observation
coincided with a reduced MPO/total protein
ratio with 2 mg FP, supporting the notion of
reduced neutrophil activation. Taken together
with previously published results (Holz et al.
2005), our results indicate that O
3
challenge
in healthy individuals is a useful model for
screening novel anti-inflammatory agents
designed for treatment of airway diseases that
have elevated neutrophils as a principal com-
ponent of their airway inflammation. These
include a subtype of severe asthma with mini-
mal airway eosinophils (Louis et al. 2000;
Wenzel 2003; Wenzel et al. 1999), as well as
COPD during an exacerbation (Hill et al.
1999; Stockley 1998).
An important feature of our study design
was that we limited volunteer recruitment to
persons with documented responsiveness to O
3
,
defined as a minimum of a 10% increase in air-
way PMNs after a screening O
3
challenge, to
enable the assessment of FP. Nightingale et al.
(2000) failed to observe an effect when they
examined the effect of 2 weeks of treatment
with 800 µg inhaled budesonide twice daily
on O
3
-induced neutrophilia in normal volun-
teers. In our study, although there was a sig-
nificant effect of O
3
alone on percent PMNs,
a substantial number of persons examined
failed to have an absolute neutrophil response
(using neutrophils per milligram sputum as a
measure) after placebo treatment. Thus, it is
possible that Nightingale et al.’s (2000) results
were influenced by a study population that
included a high proportion of O
3
“non-
responders.” In contrast, Vagaggini et al.
(2001) examined the effect of 4 weeks of pre-
treatment with 400 µg inhaled budesonide
twice daily on O
3
-induced neutrophilia in
asthmatics, and reported a significant decrease
in airway neutrophils present 6 hr after
0.27 ppm O
3
challenge compared with
placebo pretreatment; most volunteers in the
Vagaggini et al. (2001) study appeared to be
O
3
responsive. Furthermore, the objective of
the present study was not to test the efficacy of
the O
3
model, but rather to determine
whether clinically relevant doses of FP could
be assessed to support subsequent larger stud-
ies in subjects with preexisting airway disease.
In addition to its effects on airway neutro-
philia, we have recently reported that O
3
chal-
lenge (0.4 ppm, 2 hr) causes an increase in
expression of cell-surface phenotypes CD11b,
mCD14, CD16, CD86, and HLA-DR on
sputum monocytes recovered from normal
volunteers (Alexis et al. 2004a). We also
reported an increase in the numbers of spu-
tum monocytes (in addition to neutrophils),
suggesting that O
3
exposure resulted in an
influx of activated monocytes. These data are
supported by a recent animal study showing
that O
3
enhanced the expression of interstitial
lung cell-surface molecules associated with
antigen presentation and increased the number
of antigen-presenting cells in the lung (Koike
and Kobayashi 2004). In the present study,
we found that 2 mg inhaled FP decreased the
expression of CD11b, mCD14, CD16,
CD64, CD86, and HLA-DR on sputum
monocytic cells after O
3
challenge compared
with placebo treatment. The 0.5 mg dose of
FP decreased the expression of CD86 and
HLA-DR on sputum monocytes after O
3
challenge compared with placebo. Given that
these surface molecules are involved with
mediating innate immune responses (CD11b
and mCD14), acquired immune responses
(CD16, CD64), and antigen presentation
(CD86, HLA-DR), we speculate that O
3
exposure may play a role in modifying how
airway cells respond to a number of patho-
logic agents in the airborne environment. It is
unclear whether the effect of FP on O
3
-
induced changes in monocytic cell popula-
tions is due to effects on monocytes present in
the airway when exposure began, or on the
subsequent influx of monocytes that are acti-
vated from the circulation. Decreased mono-
cytic cell influx could be mediated by an effect
of FP on production of monocyte-associated
chemotactic factors or decreased adhesion
molecule expression on endothelial cells lining
the postcapillary venules.
The use of CCP16 as a systemic marker
for injury of the epithelium has been exam-
ined by several investigators (Blomberg et al.
2003; Helleday et al. 2006). O
3
exposure is
associated with increased serum levels of
CCP16 (Blomberg et al. 2003). We likewise
observed that O
3
exposure caused an increase
in serum CCP16 and further showed that
pretreatment with either 0.5 mg or 2 mg
inhaled FP prevented the O
3
-induced increase
in CCP16. We compared CCP16 with SP-D,
an innate immune molecule produced by
type II alveolar epithelial cells and Clara cells.
We previously showed that SP-D plays a
protective role in O
3
-induced injury of the
Fluticasone propionate protects against airway inflammation
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VOLUME 116 | NUMBER 6 | June 2008
803
Figure 4. CCP16 levels (mean ± SE) in serum pre-O
3
and 8 hr post-O
3
for placebo and 0.5 and 2 mg FP.
*
p
< 0.05 compared with placebo.
#
p
< 0.05 for post-O
3
compared with pre-O
3
.
25
20
15
10
5
0
CCP16 (ng/mL)
Placebo
*
0.5 mg 2 mg
FP
#
Pre-O
3
Post-O
3
Figure 3. Expression (MFI; mean ± SE) of cell-surface phenotypes on sputum monocytic cells and DCs
after O
3
exposure with 0.5 mg FP, 2 mg FP, or placebo pretreatment. (
A
) CD11b/CR3. (
B
) mCD14.
(
C
) CD64/FcγRI. (
D
) CD16/FcγRIII. (
E
) HLA-DR. (
F
) CD86. Only results in which at least one dose of FP
resulted in a change in surface marker expression compared with placebo are shown.
*
p
< 0.05 for CD11b, mCD14, CD64, CD16, and CD86 compared with 2 mg FP and for HLA-DR and CD86 with compared with
0.5 mg FP.
40
30
20
10
0
MFI
Macrophages DCs
*
*
Macrophages DCsMonocytes
300
200
100
0
*
*
*
MFI
MFI
15
10
5
0
Monocytes
*
*
750
500
250
0
MFI
Macrophages
*
*
Monocytes
150
100
50
0
MFI
*
60
50
40
30
20
10
0
MFI
Monocytes
*
*
O
3
+ placebo
O
3
+ 0.5 mg FP
O
3
+ 2 mg FP
A B C
D E F
DCs
CD11b/CR3 mCD14 CD64/FcγRI
CD16/FcγRIII HLA-DR CD86

lung (Kierstein et al. 2006), but whether
release of this protein into the circulation
could parallel the inflammatory airway
changes was unclear. Our study showed that
CCP16 is a superior serum marker for injury
of the airway epithelium and is a more sensi-
tive biomarker for the effect of inhaled FP on
airway inflammation compared with SP-D or,
indeed, compared with the wide range of
cytokines, chemokines, and inflammatory
mediators we investigated.
The apparent discrepancy we observed
between serum SP-D and CCP16 was likely
influenced by many factors, including
changes in lung concentrations. We previ-
ously showed that intracellular SP-D mRNA
and protein expression are very sensitive to
corticosteroids, cAMP, and cytokine levels
(Cao et al. 2004) and that SP-D in broncho-
alveolar lavage fluid is subject to rapid break-
down after O
3
exposure of mice (Kierstein
et al. 2006). Although no formal comparisons
have been made between local (pulmonary)
expression of SP-D and CCP16, we speculate
that the CCP16 molecule is more resistant to
O
3
-induced breakdown than is SP-D. This is
supported by the fact that CCP16 serum
levels in a number of animal and human
studies accurately reflected the extent of
increases in capillary permeability after acute
exposure to lipopolysaccharide, chlorine, or
O
3
(Lakind et al. 2007; Michel et al. 2005).
Thus, the discrepancy we observed between
serum levels of SP-D and CCP16 after O
3
exposure may be due to different structure,
regulation of expression, and sensitivity to
O
3
-induced molecular changes. This discrep-
ancy highlights the specific importance of
CCP16 as a biomarker for lung injury and
treatment effectiveness.
Overall, our observations are consistent
with the hypothesis that FP will inhibit acute
airway inflammation due to O
3
exposure in a
dose-dependent fashion that includes the
therapeutic dose of 0.5 mg FP. Apart from
percent PMNs, however, several of our find-
ings with 0.5 mg FP did not reach statistical
significance. This was likely due to an insuffi-
cient number of subjects examined at this
dose. Subsequent studies using the 0.5 mg
dose of FP will require a larger sample size. It
is also possible that a more prolonged pre-
treatment with FP before O
3
challenge would
have resulted in a more pronounced effect of
0.5 mg FP on airway inflammation, but our
single-use administration of FP provided ade-
quate drug exposure over the challenge time.
We also note that this study was conducted in
normal healthy volunteers. We chose healthy
volunteers to avoid the potentially high varia-
tion in baseline inflammation associated with
subjects with preexisting airway disease.
Lower variability in healthy subjects would
reduce the need to examine a large cohort of
subjects in this study and allow us to attain an
initial proof of pharmacology for new anti-
inflammatory chemical entities. One cannot
rule out, however, that because asthmatics
have been reported to have an increased pul-
monary sensitivity to O
3
exposure (Alexis
et al. 2000b; Scannell et al. 1996), although
we did not observe a statistically significant
effect using a single administration of 0.5 mg
FP on airway inflammation, this might have
been observed in a cohort of asthmatics. As
noted above, Vagaggini et al. (2001) reported
a significant inhibition of O
3
-induced inflam-
mation in asthmatics with treatment of
400 µg budesonide administered twice daily.
Using endotoxin as an inflammatory stimu-
lus, we previously observed that 440 µg FP
for 2 weeks delivered via a metered dose
inhaler twice daily inhibited the effect of
endotoxin on neutrophilic airway inflamma-
tion in allergic asthmatics (Alexis and Peden
2001). Thus, implementation of a longer
treatment period in healthy individuals may
have resulted in demonstration of anti-
inflammatory efficacy of a single 500 µg dose
of inhaled FP.
Conclusion
We confirmed original observations that O
3
-
induced airway neutrophilic inflammation was
inhibited by a single administration of 2 mg
FP and extended the findings by demonstrat-
ing decreased neutrophilic inflammation with
the 0.5 mg FP treatment, as well. We also
observed that both doses of FP inhibited the
up-regulatory effect of O
3
on airway mono-
cytic cell-surface phenotypes and that 2 mg FP
inhibited serum levels of CCP16. Taken
together, these observations suggest that brief
treatments with inhaled corticosteroids by per-
sons in anticipation of exposure to air pol-
lution may offer protection against the
inflammatory effects of ambient air O
3
, partic-
ularly for those individuals with preexisting
airway disease. However, it is important to
note that inhaled corticosteroids had no pro-
tective effect on the spirometric decrements
induced by O
3
, suggesting this component of
airway function, particularly in individuals
with preexisting airway disease, remains sus-
ceptible to the modifying effects of O
3
expo-
sure. A second conclusion is that O
3
challenge
with subsequent analysis of airway sputum
and serum CCP16 is a good acute disease
model for phase I screening of novel anti-
inflammatory agents intended for use in
asthma and COPD.
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In the original manuscript published
online, Brad Harris was not included as an
author. His name has been added here.
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