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ORIGINAL ARTICLE
Endotoxin in concentrated coarse and fine ambient
particles induces acute systemic inflammation
in controlled human exposures
Behrooz Behbod,
1
Bruce Urch,
2,5
Mary Speck,
2
James A Scott,
2,3
Ling Liu,
4
Raymond Poon,
4
Brent Coull,
1
Joel Schwartz,
1
Petros Koutrakis,
1
Frances Silverman,
2,3,5,6,8
Diane R Gold
1,7
▸Additional material is
published online only. To view
please visit the journal online
(http://dx.doi.org/10.1136/
oemed-2013-101498).
For numbered affiliations see
end of article.
Correspondence to
Dr Behrooz Behbod,
Environmental Health, Harvard
School of Public Health,
401 Park Drive West, Boston,
MA 02215, USA;
bbehbod@post.harvard.edu
Received 10 March 2013
Revised 8 July 2013
Accepted 26 July 2013
To cite: Behbod B, Urch B,
Speck M, et al.Occup
Environ Med Published
Online First: [please include
Day Month Year]
doi:10.1136/oemed-2013-
101498
ABSTRACT
Background Knowledge of the inhalable particulate
matter components responsible for health effects
is important for developing targeted regulation.
Objectives In a double-blind randomised cross-over
trial of controlled human exposures to concentrated
ambient particles (CAPs) and their endotoxin and
(1→3)-β-D-glucan components, we evaluated acute
inflammatory responses.
Methods 35 healthy adults were exposed to five
130-min exposures at rest: (1) fine CAPs (∼250 mg/m
3
);
(2) coarse CAPs (∼200 mg/m
3
); (3) second coarse CAPs
(∼200 mg/m
3
); (4) filtered air; and (5) medical air.
Induced sputum cell counts were measured at screening
and 24 h postexposure. Venous blood total leucocytes,
neutrophils, interleukin-6 and high-sensitivity C reactive
protein (CRP) were measured pre-exposure, 3 and 24 h
postexposure.
Results Relative to filtered air, an increase in blood
leucocytes 24 h (but not 3 h) postexposure was
significantly associated with coarse
(estimate=0.44×10
9
cells/L (95% CI 0.01 to 0.88);
n=132) and fine CAPs (0.68×10
9
cells /L (95% CI 0.19
to 1.17); n=132), but not medical air. Similar
associations were found with neutrophil responses.
An interquartile increase in endotoxin (5.4 ng/m
3
)
was significantly associated with increased blood
leucocytes 3 h postexposure (0.27×10
9
cells/L (95% CI
0.03 to 0.51); n=98) and 24 h postexposure
(0.37×10
9
cells/L (95% CI 0.12 to 0.63); n=98). This
endotoxin effect did not differ by particle size. There
were no associations with glucan concentrations or
interleukin-6, CRP or sputum responses.
Conclusions In healthy adults, controlled coarse and
fine ambient particle exposures independently induced
acute systemic inflammatory responses. Endotoxin
contributes to the inflammatory role of particle air
pollution.
INTRODUCTION
It is well recognised that exposures to inhalable
ambient particles are associated with significant
morbidity and mortality.
1–5
Knowledge of the par-
ticulate matter (PM) components responsible for
the health effects observed in epidemiological
studies is of importance for the development of tar-
geted air pollution regulations.
While fine (PM
2.5
) particles are being widely
regulated, there is more uncertainty over the tox-
icity of coarse (PM
2.5–10
) particles.
6
Fine particles,
derived mainly from mobile and industrial emission
sources, are associated with cardiovascular out-
comes and are able to reach the alveolar region and
deposit mainly by interception.
7
Coarse particles
have been shown to be associated with respiratory
inflammation and disease as well as innate immune
responses of airway macrophages,
8
and tend to
deposit by inertial impaction in the extrathoracic
airways (nose and pharynx; above the vocal
cords).
7
Smaller coarse particles may also deposit
onto the lower ciliated thoracic airways by gravita-
tional sedimentation.
9
Gram-negative bacterial cell walls contain endo-
toxin (lipopolysaccharide), which is composed of
polysaccharide chains, a connecting core and a lipid
A unit that is responsible for its toxic effects.
10
Another ambient biological exposure that is highly
correlated with endotoxin levels is (1→3)-β-D-glucan
(hereafter glucan). These compounds are glucose
polymers that are non-allergenic water-insoluble
structural cell wall components of most fungi, as well
as some bacteria and plants. Their biological activity
is independent of cell viability and may be poten-
tiated by the degree of chemical branching and inter-
molecular association (ie, single/triple helix or
randomly coil structures).
11
Human controlled exposure studies
12–15
provide
a unique opportunity to simulate air pollution
levels like those seen regularly in cities like Beijing,
China,
16
while allowing for experimental control
of the level of exposure. We used a double-blind
randomised cross-over trial of controlled human
exposures to coarse and fine concentrated ambient
particles (CAPs) to evaluate their effects on acute
pulmonary and secondary systemic inflammatory
responses. In addition, we assessed whether expos-
ure to increased concentrations of CAPs-associated
endotoxin and glucan explained the inflammatory
response to CAPs exposure.
METHODS
Study participants
We included 35 healthy non-smokers, aged
18–60 years, with no history of cardiovascular
disease, hypertension (blood pressure >140/
90 mm Hg) or diabetes. Subjects were not receiving
Behbod B, et al.Occup Environ Med 2013;0:1–7. doi:10.1136/oemed-2013-101498 1
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treatment with cholesterol lowering medication or corticoster-
oids, and were free of respiratory tract infections for at least
3 weeks prior to exposure testing. Subjects were recruited from
the University of Toronto Campus and surrounding area. The
study was approved by the human research ethics committees of
St. Michael’s Hospital, the University of Toronto and Health
Canada. All participants provided written informed consent
before enrolling.
Study design and exposure assessment
In a double-blind randomised cross-over block design, partici-
pants were exposed to five exposures: (1) fine CAPs between
0.1 and 2.5 microns aerodynamic diameter (∼250 mg/m
3
);
(2) coarse CAPs between 2.5 and 10 microns aerodynamic
diameter (∼200 mg/m
3
); (3) second coarse CAPs (∼200 mg/m
3
);
(4) filtered air; and (5) medical air. Previous studies by our
group had assessed the acute effects of fine CAPs.
17 18
When we
began this study, the effects of coarse CAPs were not well
assessed. We added the second coarse exposure per subject to
increase the power to assess effects on outcomes. The two
coarse exposures were handled as separate treatments in the stat-
istical analyses to increase variability and power. Each exposure
lasted 130 min (120-min exposure plus an additional 10 min to
complete all test measures), followed by a minimum 2-week
washout period before the next exposure. Controlled exposures
were generated using high-flow (5000 L/min) Harvard ambient
particle concentrators.
19–21
These particle concentration systems
were used to draw ambient particles from a 1.8 m high PM
10
inlet located, 10 m from a busy 4-lane downtown Toronto street
with ∼2500 vehicles passing during the 130-min exposure.
Thus, traffic emissions were a major contributor to the ambient
PM levels at this site. Ambient particle exposures were concen-
trated and adjusted through a dilution control system to deliver
target concentrations of ∼200 mg/m
3
coarse CAPs and ∼250 mg/
m
3
fine CAPs. The CAPs air stream was delivered directly to the
subject seated inside a 4.9 m
3
Lexan and steel tube frame enclos-
ure, at rest and breathing freely (no mouthpiece) via an ‘oxygen
type’face-mask covering his/her nose and mouth. The study
design called for rest so there would be no interference with the
cardiovascular measure of flow-mediated dilation (not included
in this manuscript). The delivery system was designed so that
there were no visual cues as to the exposure type while partici-
pants were seated in the chamber.
For filtered air exposures, the coarse concentrator was run with
ahigh-efficiency particulate air (HEPA) filter placed inline to
remove the particles. For medical air exposures, compressed
breathing-grade medical air was humidified to 30% relative
humidity, passed through an inline HEPA filter and delivered to
the subject at a flow rate of 30 L/min. Medical air was selected
because it is free of gaseous and particulate pollutants and odours.
The original study design (randomised block) only included
the first four exposures (no medical air). However, interim ana-
lysis showed greater than expected physiological responses with
filtered air, which was designed to serve as the control exposure
and thus was expected to induce a negligible effect. We
hypothesised that these responses may have been due to
ambient gases such as volatile organic compounds that can pass
through the HEPA filter. Thus, a fifth exposure using medical
air was added as a second additional control, although in the
first 11 subjects it was always delivered to the subject as the last
exposure in the series (ie, not randomised), but then randomised
in later subjects. This deviation from the randomised block
design was considered in statistical analyses.
Endotoxin and glucan were both collected on polycarbonate
membrane filters during coarse and fine CAPs and filtered air
(but not medical air) exposures. Filters were placed in pyrogen-
free 15 mL French square bottles containing 5 mL of Limulus
Amebocyte Lysate reagent water. After shaking for 20 min and
mixing for 1 min with a vortex, samples were divided into two
aliquots of 2.5 mL. To measure endotoxin, the sample was soni-
cated for 30 min at 26°C and then vortexed for another 1 min.
The extract was analysed for endotoxin using Pyrochrome and
Glucashield reagents following manufacturer’s instructions
(Associates of Cape Cod, Inc. (ACC), East Falmouth,
Massachusetts, USA). To measure glucan, 0.3 N NaOH was
added to the sample, shaken on ice for 25 min and diluted with
10 mM NaOH. The extract was analysed for the amount of
glucan using Glucatell reagent (ACC).
Outcome measures
We collected induced sputum at the screening visit (median 21 days
prior to the first exposure treatment) and at 24 h postexposure. In
this study, we used sputum total cell and neutrophil counts as out-
comes. Venous blood was also collected from all study participants
∼45 min prior to, and 3 and 24 h after the start of each exposure.
We evaluated total blood leucocyte and neutrophil counts as well
as blood interleukin-6 (IL-6) and high-sensitivity C reactive protein
(hs-CRP) as markers of inflammation.
Statistical methods
We first described the baseline characteristics of the study parti-
cipants and the exposure concentrations (mass, endotoxin and
glucan) across the different treatments (coarse and fine CAPs
and filtered/medical air). To account for the within-subject cor-
relation in the outcome measures, while adjusting for daily
physiological variability within subjects, we created new vari-
ables representing change. Therefore, blood outcomes were con-
verted to: (1) 3 h post—pre change and (2) 24 h post—pre
change. Sputum measures were converted to the 24 h postscre-
ening visit change. However, since the screening visit was a
median 21 days prior to the first exposure treatment, we also
performed sensitivity analyses using 24 h postsputum measures
as a single measure. We assessed the outcome measure distribu-
tions for normality, and if skewed, we transformed the data, as
appropriate, prior to further analyses.
We used linear mixed effects models to account for the
within-subject correlation in responses between the exposure
treatments. We first examined whether, relative to filtered air,
coarse and fine CAPs and medical air exposures were independ-
ently associated with each outcome. We included the four
exposure types (treatments) as a categorical variable, and used
filtered air as the control, which was randomised for all subjects
by design. We then examined whether, accounting for the
exposure type, variations in bioaerosol (endotoxin, and glucan)
concentrations were associated with the inflammatory outcomes.
Due to the collinearity in bioaerosol concentrations, we assessed
the effect of endotoxin and glucan in separate models. Coarse
and fine CAPs-associated bioaerosols were initially grouped
together. We subsequently used interaction terms between
bioaerosol concentrations and CAPs size fraction to examine
whether associations between bioaerosol exposures and out-
comes were modified by CAPs size fraction.
All models were tested as follows: (1) unadjusted, assuming
the randomised design efficiently accounted for measured and
unmeasured confounders; (2) adjusting for exposure order
(1st–5
th
, as in table 3), to account for any potential stress or
cumulative responses; and (3) adjusting for subjects’age,
2 Behbod B, et al.Occup Environ Med 2013;0:1–7. doi:10.1136/oemed-2013-101498
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gender, ethnicity, body mass index (BMI (kg/m
2
)) and season
(categorical; four levels) of exposure treatment. Continuous cov-
ariates (age and BMI) were centred at their respective means.
Last, sensitivity analyses were performed to ensure results were
not due to any outliers, identified as the highest two/three
exposure concentrations or outcomes (sputum/blood white cell
and neutrophil counts).
RESULTS
Overall, 19 (54%) men and 16 (46%) women completed a total
of 132 controlled exposure treatments. In all, 17 (48%) were
Asian, 16 (46%) were white and 2 (6%) were black. Their mean
(IQR) age and BMI were 27 (11) years and 23 (3) kg/m
2
,
respectively.
Table 1 shows the distribution of the 132 controlled exposure
treatments. While the coarse and fine CAPs concentrations were
tightly controlled by design, there was residual variability in both
exposures ((IQR)
coarse
=35.8 mg/m
3
;IQR
fine
=52.4 mg/m
3
).
Glucan levels obtained were on average (median (IQR)) 4.6 (5.2)
times higher than ambient for coarse CAPs exposures, and
4.4 (2.2) for fine CAPs exposures. Endotoxin levels were on
average (median (IQR)) 5.8 (4.2) times higher than ambient for
coarse CAPs exposures, and 7.7 (3.9) for fine CAPs exposures. No
particulate mass, glucan or endotoxin was found in the filtered air.
We did not measure bioaerosol concentrations in medical air.
At baseline, all subjects were apyrexial and showed no signs
of infection (maximum total leucocyte counts=8.9×10
9
cells/L;
maximum blood neutrophil counts=5.9×10
9
cells/L) or inflam-
mation (maximum hs-CRP=6.9 μg/mL). With the exception of
up to a few marked responses in each outcome, blood and
sputum outcome distributions (table 2) were normally distribu-
ted. While the mean changes in blood and sputum outcomes
appeared minimal, there was variability in the responses.
However, we did not observe significant differences in subject
characteristics (age, gender, ethnicity or BMI) between those
with increased or decreased responses (results not shown).
Table 3 presents the associations between the controlled
exposure treatments and the change in total blood leucocyte
responses. While there appeared to be a response to filtered air
(the reference group) 3 h postexposure in both the unadjusted
model (intercept estimate=0.50×10
9
cells/L (95% CI 0.12 to
0.87)) and in model 1 adjusting for exposure order
(estimate=0.62×10
9
cells/L (95% CI 0.20 to 1.05)), this was no
longer statistically significant when we adjusted for potential
confounders in model 2 (estimate=0.60×10
9
cells/L (95% CI
−0.06 to 1.26)). Relative to filtered air, coarse and fine CAPs
and medical air were not significantly associated with 3 h post—
pre change blood leucocyte responses (model 2). However,
an increase in total blood leucocytes 24 h postexposure
(table 4, model 2) was significantly associated with coarse
(estimate=0.44×10
9
cells/L (95% CI 0.01 to 0.88)) and fine
CAPs (estimate=0.68×10
9
cells/L (95% CI 0.19 to 1.17)), but
not medical air (estimate=0.36×10
9
cells/L (95% CI
−0.20 to 0.93)). We performed sensitivity analyses by removing
marked responses, and found consistent results in models with
blood neutrophil responses (see online supplementary material
table 1).
Adjusting for treatment type (table 3, model 4), an interquar-
tile increase in endotoxin (5.4 ng/m
3
) was significantly asso-
ciated (estimate=0.38×10
9
cells/L (95% CI 0.09 to 0.68)) with
an increase in blood leucocytes 3 h postexposure. While an
interquartile increase in endotoxin concentration was associated
(estimate=0.37×10
9
cells/L (95% CI 0.12 to 0.63)) with higher
leucocytes 24 h postexposure (table 4, model 3), this association
was no longer significant when we adjusted for treatment type
(table 4, model 4). In a model excluding medical air exposures
to obtain the same number of observations as in model 4
(results not shown in tables), coarse (estimate=0.52×10
9
cells/L
(95% CI 0.04 to 1.00)) and fine CAPs (estimate=0.74×10
9
cells/L (95% CI 0.21 to 1.27)) remained significantly associated
with higher leucocyte levels 24 h postexposure. However, when
including endotoxin in model 4, the associations of coarse and
fine CAPs became non-significant and the respective effect esti-
mates were reduced by 40% (0.52 to 0.31×10
9
cells/L) and
35% (0.74 to 0.48×10
9
cells/L). The association between
increases in endotoxin concentration and leucocyte responses
3 h ( p for interaction=0.67) or 24 h (p for interaction=0.42)
postexposure did not vary significantly by CAPs size fraction.
Variations in glucan concentrations were not associated with
3 or 24 h postleucocyte responses. Relative to filtered air, fine
CAPs exposures were associated (estimate=−0.62 mg/mL (95%
CI −1.04 to −0.20), n=132) with lower hs-CRP responses 24 h
post-treatment (see online supplementary material table 2). This
negative association did not remain significant (estimate=
−0.23 mg/mL (95% CI −0.62 to 0.15), n=128) after we
removed four marked responses presented (−2.6, 1.4, 2.2 and
3.7 mg/mL). We did not find any associations with blood IL-6
(see online supplementary material table 3) or sputum responses
(see online supplementary material table 4).
DISCUSSION
In a double-blind randomised cross-over trial in 35 healthy
adult subjects, coarse and fine ambient particle exposures were
independently associated with an acute inflammatory response.
The endotoxin content partially explained the inflammatory
role of ambient particle exposures, and the effect did not differ
between coarse and fine particles.
While we observed significant associations with systemic
inflammatory responses (blood neutrophils), the lack of signifi-
cant corollary findings in sputum may be due to insufficient
Table 1 Exposure characteristics
Exposure*
Total (all exposures)
Treatment type
Coarse CAPs
(PM
10–2.5
) Fine CAPs (PM
2.5–0.1
) Filtered air Medical air
N Median IQR n Median IQR n Median IQR n Median IQR n Median IQR
Particulate mass concentration (μg/m
3
) 132 189.6 220.3 55 202.3 35.8 29 234.7 52.4 25 −2.5 7.2 23 0.7 7.7
β-Glucan (ng/m
3
) 80 9.0 17.7 40 13.0 28.5 25 10.5 16.3 15 0.7 0.7 0
Endotoxin (ng/m
3
) 98 4.8 5.4 51 5.4 2.9 28 7.1 7.1 19 0.4 1.0 0
*Integrated gravimetric (filter sample) 130-min exposure concentrations sampled from CAPs/filtered air airstream inlet to human chamber.
CAPs, concentrated ambient particles; n, number of observations; PM, particulate matter.
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power because of the small sample size. Nevertheless, human
exposure studies of CAPs have generally not shown consistent
results with induced sputum, with suggestions that systemic
inflammation may be more pronounced than pulmonary
responses.
22–25
CAPs represent multiple sources of ambient pol-
lutants rather than just diesel exhaust emissions. While we con-
trolled total mass levels in our study, we were limited by not
accounting for particle composition which varies with time due
to changes in source emissions and prevailing environmental
conditions. CAP composition also varies by study location, and
therefore our results from Toronto may not represent what
might be found elsewhere.
25
Ambient PM constituents may include chemicals such as
metals, organics and biological materials from bacteria, viruses
and fungi. An in vitro study of rat alveolar macrophage (AM)
cells found that endotoxin in urban air particles, but not in
diesel particles, was responsible for inducing inflammatory cyto-
kine expression.
26
Humans may be more sensitive than animals
to the effects of CAPs.
22
Becker et al (2005)
27
exposed AM cells
from healthy adult subjects aged 20–35 years to fine and coarse
particles in vitro and found that the main pro-inflammatory
response was driven by the coarse size fraction, where the
majority (∼90%) of the stimulatory material in inhalable PM is
known to be found.
28
This stimulatory material is mainly
derived from biological sources, and includes microbes and
allergens.
Alexis et al (2006)
8
exposed nine healthy subjects, on three
separate occasions, to inhale nebulised saline (0.9%, control),
coarse PM collected from local ambient air in Chapel Hill,
North Carolina, USA that was heated (20 h at 120°C) to inacti-
vate biological material, or non-heated PM. Relative to saline,
coarse PM exposure was associated with an increase in inflam-
matory polymorphonuclear leucocytes and macrophage mRNA
tumour necrosis factor (TNF)-α, an upregulation of immune
surface phenotypes on macrophages (mCD14, CD11b,
HLA-DR), and increased phagocytosis, 2–3 h postinhalation.
Biological inactivation was associated with lower mRNA TNF-α,
phagocytosis and cell surface marker responses. Analysis of
ambient coarse PM from Chapel Hill showed that it contained
30% gram-negative bacteria, with the remainder mostly com-
posed of gram-positive cocci and fungal spores (Penicillium,
Cladosporium).
We measured slightly higher levels of endotoxin in fine than
in coarse CAPs (table 1). This may be due to the fact that
Table 2 Outcome characteristics
Outcome N Mean Min
25th
Percentile 50th 75th Max IQR
Blood
Total leucocytes (# cells×10
9
/L)
Pre-treatment 132 5.5 3.5 4.7 5.3 6.2 8.9 1.5
3 h post-treatment 132 5.8 3.5 5.0 5.6 6.5 10.0 1.5
24 h post-treatment 131 5.5 3.4 4.7 5.4 6.1 9.8 1.4
3 h post-pre change 132 0.4 −1.4 −0.3 0.4 0.9 5.0 1.2
24 h post-pre change 131 0.1 −2.7 −0.6 0.1 0.5 4.6 1.1
Neutrophils (# cells×10
9
/L)
Pre-treatment 132 3.1 1.6 2.4 2.9 3.6 5.9 1.2
3 h post-treatment 132 3.5 1.6 2.6 3.2 4.0 8.4 1.4
24 h post-treatment 131 3.1 1.2 2.3 3.0 3.5 6.8 1.2
3 h post-pre change 132 0.4 −1.8 −0.1 0.2 0.7 5.7 0.8
24 h post-pre change 131 0.0 −2.7 −0.5 0.0 0.4 4.0 0.9
Interleukin-6 (pg/mL)
Pre-treatment 121 1.0 0.0 0.5 0.7 1.1 5.2 0.6
3 h post-treatment 120 0.8 0.0 0.4 0.6 0.9 5.3 0.5
24 h post-treatment 121 1.1 0.0 0.5 0.8 1.1 6.0 0.6
3 h post-pre change 120 −0.1 −2.8 −0.3 −0.1 0.0 5.2 0.3
24 h post-pre change 121 0.1 −2.6 −0.2 0.0 0.2 4.9 0.4
hs-CRP (mg/mL)
Pre-treatment 121 1.3 0.0 0.1 0.5 2.0 6.9 1.9
3 h post-treatment 120 1.2 0.0 0.1 0.4 1.9 7.1 1.8
24 h post-treatment 121 1.3 0.0 0.1 0.4 1.9 10.0 1.8
3 h post-pre change 120 0.0 −1.9 0.0 0.0 0.0 2.1 0.1
24 h post-pre change 121 0.1 −2.6 −0.1 0.0 0.1 4.2 0.2
Induced sputum
Total cells (# cells×10
5
/mL)
Screening visit 10 9.7 2.6 7.1 9.2 13.2 16.6 6.1
24 h post-treatment 38 10.8 3.2 7.4 10.0 12.7 40.6 5.3
24 h post-screening change 34 0.1 −10.2 −4.4 −0.7 5.0 19.1 9.4
Neutrophils (# cells×10
5
/mL)
Screening visit 10 3.1 1.4 1.4 2.8 4.5 6.9 3.1
24 h post-treatment 38 4.4 0.1 1.0 2.7 6.6 17.9 5.6
24 h post-screening change 34 1.2 −4.1 −1.0 −0.1 2.5 13.6 3.5
hs-CRP, high sensitivity C reactive protein.
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subjects were, by design, exposed to higher concentrations
of fine (∼250 μg/m
3
) than coarse (∼200 μg/m
3
) CAPs.
Nevertheless, a separate epigenetic analysis of our study found
coarse CAPs exposure to be associated with lowered toll-like
receptor (TLR)-4 methylation, which can recognise the
endotoxin component of coarse CAPs and trigger macrophages
to release various inflammatory cytokines.
29
Concentrated ambient endotoxin concentrations, irrespective
of CAPs size fraction, were over 17-fold greater (geometric
mean ∼34 EU/m
3
) than levels normally found in outdoor and
Table 4 Associations among 130-min controlled human exposure treatments, bioaerosols and 24-h changes in blood total leucocytes
Treatment (categorical)
24 h post—pre change in leucocytes (# cells×10
9
/L)
Unadjusted (n=132) Model 1 (n=132) Model 2 (n=132)
Estimate 95% CI Estimate 95% CI Estimate 95% CI
Coarse CAP 0.35 (−0.10 to 0.80) 0.41 (−0.04 to 0.87) 0.44* (0.01 to 0.88)
Fine CAP 0.67** (0.16 to 1.17) 0.71** (0.19 to 1.22) 0.68** (0.19 to 1.17)
Medical air 0.21 (−0.33 to 0.74) 0.35 (−0.24 to 0.93) 0.36 (−0.20 to 0.93)
Filtered air Ref Ref Ref Ref Ref Ref
Treatment (categorical)
Model 3 (n=98) Model 4 (n=98) Model 5 (n=80) Model 6 (n=80)
Estimate 95% CI Estimate 95% CI Estimate 95% CI Estimate 95% CI
Coarse CAP –– 0.31 (−0.23, 0.85) –– 0.39 (−0.20, 0.98)
Fine CAP –– 0.48 (−0.14, 1.10) –– 0.75* (0.15, 1.36)
Medical air –– –– –– ––
Filtered air –– Ref Ref –– Ref Ref
Endotoxin†(ng/m
3
) 0.37** (0.12, 0.63) 0.25 (−0.05, 0.56) –– ––
Glucan†(ng/m
3
)–– –– 0.09 (−0.06, 0.25) 0.07 (−0.08, 0.22)
Treatment group estimates are for the difference between filtered air and other treatment groups (coarse CAPs, fine CAPs or medical air) in leucocyte response (24 h postexposure
minus pre-exposure) to the treatment.
Model 1 adjusts for exposure order (1st—5th).
Models 2–6 adjust for exposure order and subject characteristics: age (continuous), male gender, white ethnicity, BMI (continuous) and season (categorical; four levels).
Note: age and BMI are centred at the mean.
*p≤0.05, **p≤0.01.
†Bioaerosol concentration (endotoxin or glucan) from coarse and fine CAPs. Estimates represent an interquartile increase in the exposure concentration (endotoxin=5.4 ng/m
3
;
glucan=17.7 ng/m
3
).
BMI, body mass index; CAPs, concentrated ambient particles.
Table 3 Associations among 130-min controlled human exposure treatments, bioaerosols and 3-h changes in blood total leucocytes
Treatment (categorical)
3 h post—pre change in leucocytes (# cells×10
9
/L)
Unadjusted (n=132) Model 1 (n=132) Model 2 (n=132)
Estimate 95% CI Estimate 95% CI Estimate 95% CI
Coarse CAPs −0.10 (−0.46 to 0.26) −0.12 (−0.49 to 0.25) −0.12 (−0.50 to 0.26)
Fine CAPs −0.04 (−0.44 to 0.37) −0.07 (−0.49 to 0.35) −0.06 (−0.49 to 0.36)
Medical air −0.39 (−0.83 to 0.05) −0.40 (−0.89 to 0.09) −0.39 (−0.89 to 0.11)
Filtered air Ref Ref Ref Ref Ref Ref
Treatment (categorical)
Model 3 (n=98) Model 4 (n=98) Model 5 (n=80) Model 6 (n=80)
Estimate 95% CI Estimate 95% CI Estimate 95% CI Estimate 95% CI
Coarse CAPs –– −0.25 (−0.72 to 0.23) –– 0.25 (−0.26 to 0.75)
Fine CAPs –– −0.37 (−0.91 to 0.17) –– 0.27 (−0.24 to 0.78)
Medical air –– –– –– ––
Filtered air –– Ref Ref –– Ref Ref
Endotoxin†(ng/m
3
) 0.27* (0.03 to 0.51) 0.38* (0.09 to 0.68) –– ––
Glucan†(ng/m
3
)–– –– 0.10 (−0.06 to 0.25) 0.07 (−0.10 to 0.24)
Treatment group estimates are for the difference between filtered air and other treatment groups (coarse CAPs, fine CAPs or medical air) in leucocyte response (3 h postexposure minus
pre-exposure) to the treatment.
Model 1 adjusts for exposure order (1st—5th).
Models 2–6 adjust for exposure order and subject characteristics: age (continuous), male gender, white ethnicity, BMI (continuous) and season (categorical; four levels).
Note: age and BMI are centred at the mean.
*p≤0.05.
†Bioaerosol concentration (endotoxin or glucan) from coarse and fine CAPs. Estimates represent an interquartile increase in the exposure concentration (endotoxin=5.4 ng/m
3
;
glucan=17.7 ng/m
3
).
BMI, body mass index; CAPs, concentrated ambient particles.
Behbod B, et al.Occup Environ Med 2013;0:1–7. doi:10.1136/oemed-2013-101498 5
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group.bmj.com on September 10, 2013 - Published by oem.bmj.comDownloaded from
indoor air (<2 EU/m
3
).
30 31
Conversely, concentrated ambient
glucan concentrations ( geometric mean=8 ng/m
3
) were just
above levels found in total unconcentrated PM in normal
indoor and outdoor air,
24
perhaps explaining why we did not
find associations with glucan exposures in our study. Levels
greater than 5 ng/m
3
of glucan are generally associated with pre-
vious mould growth or water damage;
32
a villa with excessive
mould growth had levels of up to 100 ng/m
3
.
33
On the other
hand, there may indeed be no health effects with glucan
exposures.
A review of studies on the potential effects of glucan on
airway inflammation showed mixed results.
34
This lack of con-
sistency may be due to a number of reasons, such as small
sample sizes, different exposure assessment methods, or lack of
control for potential confounders or coexposures such as endo-
toxin.
35–39
Furthermore, health effects vary by route of expos-
ure (eg, inhalation/oral) and type of glucan. The Glucatell
reagent used to measure glucan concentrations are specificto
(1→3)-β-D-glucan and, therefore, cross-reactivity with plant
glucan that have 1→4 linkages (as found in barley) does not
occur. However, factor G activation has a bias for higher
molecular weight glucan and single-helix and randomly coiled
conformers over triple-helix structures. The ELISA method has
been shown to be specific for fungi, (1→6) side-branched and
(1→3)-β-D-glucan, as well as high molecular weight glucan, and
may therefore be a better method for determining exposure to
glucan likely to have important health effects.
40
IL-6 is a cytokine that stimulates neutrophil production, the
proliferation of B-lymphocytes and the production of acute
phase proteins (APP) by the liver. CRP is an important APP that
functions as a soluble pattern recognition receptor (PRR). PRRs,
such as the family of TLRs, are found on antigen presenting
cells and identify microbial conserved structures of pathogen-
associated molecular patterns. The exposure duration (130 min)
and length of follow-up (24 h) was sufficient to elicit changes in
IL-6.
20
The lack of significant associations with IL-6 or CRP in
this study may therefore be due to a number of other reasons,
including: (1) healthy subjects may not be representative of the
population susceptive to the inflammatory effects of CAPs
exposure; (2) other cytokines may have been released by macro-
phages, such as IL-1, IL-8, TNF-αand platelet-activating factor;
and (3) there may indeed have been no effects to detect.
Our study was limited by the short exposure durations and
follow-up periods, which may not be completely representative
of the spatiotemporal variability in real-life exposures. Due to
collinearity of endotoxin and glucan exposures, we were unable
to assess any effect measure modification of the association
between endotoxin and inflammation by glucan. Furthermore,
the selection of healthy adults may limit generalisability of study
findings to susceptible subpopulation.
6
A limitation was that we
did not evaluate differential deposition and we did not do nasal
lavage (it would have been problematic to do both nasal lavage
and sputum evaluation). It is possible that the weak associations
of coarse particles with sputum leucocytes counts relate, in part,
to deposition patterns. Our primary endpoints in the main
study from which our analyses came from were cardiovascular.
Finally, we were limited in that we did not have the exposures
prior to the chamber exposure. Due to the randomisation, the
role of daily life exposures in the few days prior to each treat-
ment were not expected to have had a differential impact on the
association between exposures of varying size and health
outcomes.
Despite these limitations, our study included a carefully stan-
dardised environment with well characterised exposures and
physical activity levels. Our randomised study design enables
subjects to serve as their own controls, thereby controlling for
measured and unmeasured confounders. Circadian rhythms,
physical activity and stress must be taken into account when
analysing cytokines in peripheral blood; circadian rhythms were
controlled for by standardising the time of day when exposure
treatments were performed; subjects were seated at rest during
treatments; and stress was accounted for by adjusting for expos-
ure order in statistical analyses. Study team members and sub-
jects were both unaware of their exposure assignment in this
double-blind study, thereby preventing the introduction of bias.
CONCLUSIONS
We have shown that short duration controlled human exposures
to coarse and fine CAPs were independently associated with
acute systemic inflammatory responses in healthy non-smoking
adults. Endotoxin contributes to the inflammatory role of both
coarse and fine particle air pollution.
What this paper adds
▸In healthy adults, controlled coarse as well as fine ambient
particle exposures independently induced acute systemic
inflammatory responses.
▸Endotoxin contributes to the inflammatory role of particle air
pollution.
▸Knowledge of the particulate matter components responsible
for the health effects observed in epidemiological studies is
of importance for the development of targeted air pollution
regulations.
Author affiliations
1
Environmental Health, Harvard School of Public Health, Boston, Massachusetts,
USA
2
Gage Occupational & Environmental Health Unit, St. Michael’s Hospital, University
of Toronto, Toronto, Ontario, Canada
3
Division of Occupational and Environmental Health, Dalla Lana School of Public
Health, University of Toronto, Toronto, Ontario, Canada
4
Health Canada, Ottawa, Ontario, Canada
5
Southern Ontario Centre for Atmospheric Aerosol Research (SOCAAR), Toronto,
Ontario, Canada
6
Divisions of Occupational and Respiratory Medicine, Department of Medicine,
University of Toronto, Toronto, Ontario, Canada
7
The Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School,
Boston, Massachusetts, USA
8
Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael’s Hospital,
Toronto, Ontario, Canada
Acknowledgements The authors thank Jeffrey R Brook for support and advice in
the setup and operation of the facility. This publication was made possible by the
following grants: USEPA RD-83241601, RD-83479801, Health Canada, Environment
Canada, AllerGen NCE, and NIH P01 ES009825. Its contents are solely the
responsibility of the grantee and do not necessarily represent the official views of
the USEPA. Further, USEPA does not endorse the purchase of any commercial
products or services that might be mentioned in the publication. Dr Behbod’s
doctorate has been supported by the Harvard–Cyprus Endowment Scholarship.
Contributors BB is the primary author and performed all statistical analyses.
BU, MS, JAS and FS were involved in the study design, data collection and analysis
as well as write-up. LL, RP, BC, JS, PK and DRG were involved in study design, data
analysis and write-up.
Funding Infrastructure for CAPs exposure facility provided by Southern Ontario
Centre for Atmospheric Aerosol Research (SOCAAR) through funding from the
Canada Foundation for Innovation (CFI).
Competing interests None.
6 Behbod B, et al.Occup Environ Med 2013;0:1–7. doi:10.1136/oemed-2013-101498
Environment
group.bmj.com on September 10, 2013 - Published by oem.bmj.comDownloaded from
Patient consent Obtained.
Ethics approval St. Michael’s Hospital, the University of Toronto and Health
Canada.
Provenance and peer review Not commissioned; externally peer reviewed.
REFERENCES
1 Dockery DW, Pope A, Xu X, et al. An association between air pollution and
mortality in six U.S. cities. N Engl J Med 1993;29:1753–9.
2 Pope CA III, Thun MJ, Namboodiri MM, et al. Particulate air pollution as a predictor
of mortality in a prospective study of U.S. adults. Am J Respir Crit Care Med
1995;151:669–74.
3 Pope CA, Dockery DW. Epidemiology of particle effects. In: Holgate ST Jr, Samet JM
Jr, Koren HS Jr, Maynard RL Jr, eds. Air Pollution and Health. London: Academic
Press, 1998;673–705.
4 Samet JM, Zeger SL, Dominici F, et al. The National Morbidity, Mortality, and Air
Pollution Study. Part II: Morbidity and mortality from air pollution in the United
States. Res Rep Health Eff Inst 2000;94:5–70; discussion 71–79.
5 Schwartz J, Dockery DW, Neas LM. Is daily mortality associated specifically with fine
particles? J Air Waste Manag Assoc 1996;46:927–39.
6 U.S. EPA. Integrated Science Assessment for Particulate Matter (Final Report). U.S.
Environmental Protection Agency, Washington, DC, EPA/600/R-08/139F, 2009.
7 Heyder J, Gebhart J, Rudolf G, et al. Deposition of particles in the human
respiratory tract in the size range 0.005–15μm. J Aerosol Sci 1986;17:811–25.
8 Alexis NE, Lay JC, Zeman K, et al. Biological material on inhaled coarse fraction
particulate matter activates airway phagocytes in vivo in healthy volunteers.
J Allergy Clin Immunol 2006;117:1396–403.
9 Hinds WC. Aerosol Technology: Properties, behavior, and measurement of airborne
particles. 2nd ed. New York: John Wiley & Sons, 1999:237.
10 Rylander R. Review: Endotoxin in the environment —exposure and effects.
J Endotoxin Research 2002;8:241.
11 Young SH, Robinson V, Barger M, et al. Exposure to particulate (1→3)-β-D-glucans
induces greater pulmonary toxicity than soluble (1→3)-β-D-glucans in rats. J Toxicol
Environ Health 2003;66:25–38.
12 Ghio AJ, Hall A, Bassett MA, et al. Exposure to concentrated ambient air particles
alters hematologic indices in humans. Inhal Toxicol 2003;15:1465–78.
13 Gong H, Linn WS, Terrell SL, et al. Exposures of elderly volunteers with and without
chronic obstructive pulmonary disease (COPD) to concentrated ambient fine
particulate pollution. Inhal Toxicol 2004;16:731–44.
14 Harder SD, Soukup JM, Ghio AJ, et al. Inhalation of PM2.5 does not modulate host
defense or immune parameters in blood or lung of normal human subjects. Environ
Health Perspect 2001;109(Suppl 4):599–604.
15 Samet JM, Graff D, Berntsen J, et al. A comparison of studies on the effects of
controlled exposure to fine, coarse and ultrafine ambient particulate matter from a
single location. Inhal Toxicol 2007;19(Suppl 1):29–32.
16 Streets DG, Fu JS, Jang CJ, et al. Air quality during the 2008 Beijing Olympic
Games. Atmospheric Environment 2007;41:480–92.
17 Urch B, Brook JR, Wasserstein D, et al. Relative contributions of PM2.5 chemical
constituents to acute arterial vasoconstriction in humans. Inhal Toxicol
2004;16:345–52.
18 Urch B, Silverman F, Corey P, et al. Acute blood pressure responses in healthy
adults during controlled air pollution exposures. Environ Health Perspect
2005;113:1052–5.
19 Demokritou P, Gupta T, Ferguson S, et al. Development and laboratory
characterization of a prototype coarse particle concentrator for inhalation
toxicological studies. Aerosol Science 2002;33:1111–23.
20 Rastogi N, McWhinney RD, Akhtar US, et al. Physical Characterization of the
University of Toronto Coarse, Fine, and Ultrafine High-Volume Particle Concentrator
Systems. Aerosol Sci Technol 2012;46:1015–24.
21 Sioutas C, Koutrakis P, Godleski JJ, et al. Fine particle concentrators for inhalation
exposures—Effect of particle size and composition. J Aerosol Sci
1997;28:1057–71.
22 Ghio AJ, Kim C, Devlin RB. Concentrated ambient air particles induce mild
pulmonary inflammation in healthy human volunteers. Am J Respir Crit Care Med
2000;162(Suppl. 3 Pt 1):981–8.
23 Gong H Jr, Sioutas C, Linn WS. Controlled exposures of healthy and asthmatic
volunteers to concentrated ambient PM10 and PM2.5 in Reno, NV. In: Mathal CV,
Stonefield DH. eds. Transactions, PM-10: Implementation of Standards. Pittsburgh,
PA: Air and Waste Management Association, 1988:437–9.
24 Urch B, Speck M, Corey P, et al. Concentrated ambient fine particles and not ozone
induce a systemic interleukin-6 response in humans. Inhalation Toxicology
2010;22:210–18.
25 Holgate ST, Devlin RB, Wilson SJ, et al. Health effects of acute exposure to air
pollution. Part II: Healthy subjects exposed to concentrated ambient particles.
Res Rep Health Eff Inst 2003;112:31–50; discussion 51–67.
26 Dong W, Lewtas J, Luster MI. Role of Endotoxin in Tumor Necrosis Factor α
Expression from Alveolar Macrophages Treated with Urban Air Particles. Exp Lung
Res 1996;22:577–92.
27 Becker S, Mundandhara S, Devlin RB, et al. Regulation of cytokine production in
human alveolar macrophages and airway epithelial cells in response to ambient air
pollution particles: Further mechanistic studies. Toxicol Appl Pharmacol 2005;207:
S269–75.
28 Soukup JM, Becker S. Human alveolar macrophage responses to air pollution
particulates are associated with insoluble components of coarse material, including
particulate endotoxin. Toxicol Appl Pharmacol 2001;171:20–6.
29 Bellavia A, Urch B, Speck M, et al. DNA hypomethylation, ambient particulate
matter, and increased blood pressure: findings from controlled human exposure
experiments. J Am Heart Assoc 2013;2:e000212.
30 Jacobs RR. Endotoxins in the environment. Int J Occup Environ Health 1997;3:
S3–5.
31 Park JH, Spiegelman DL, Gold DR, et al. Predictors of Airborne Endotoxin in the
Home. Environ Health Perspect 2001;109:859–64.
32 Thorn J, Rylander R. Airways Inflammation and Glucan in a Rowhouse Area. Am J
Respir Crit Care Med 1998;157:1798–803.
33 Rylander R. The first case of sick building syndrome in Switzerland. Indoor Environ
1994;3:159–62.
34 Douwes J. (1→3)-β-D-glucans and respiratory health: a review of the scientific
evidence. Indoor Air 2005;15:160–9.
35 Bowers GJ, Patchen ML, Macvittie TJ, et al. A comparative evaluation of particulate
and soluble glucan in an endotoxin model. Int J Immunopharmacol
1986;8:313–21.
36 Cook JA, Dougherty WJ, Holt TM. Enhanced sensitivity to endotoxin induced by the
RE stimulant, glucan. Circ Shock 1980;7:225–38.
37 Fogelmark B, Goto J, Yuasa K, et al. Acute pulmonary toxicity of inhaled
(1→3)-β-D-glucan and endotoxin. Agents Actions 1992;35:50–5.
38 Fogelmark B, Sjöstrand M, Rylander R. Pulmonary inflammation induced by
repeated inhalations of beta(1,3)-D-glucan and endotoxin. Int J Exp Pathol
1994;75:85–90.
39 Young SH, Robinson VA, Barger M, et al. Modified endotoxin responses in rats
pretreated with 1–3-beta-glucan (zymosan A). Toxicol Appl Pharmacol
2002;178:172–9.
40 Milton DK, Alwis KU, Fisette L, et al. Enzyme-Linked Immunosorbent Assay Specific
for (1→6) Branched, (1→3)-β-D-Glucan Detection in Environmental Samples. Appl
Environ Microbiol 2001;67:5420–4.
Behbod B, et al.Occup Environ Med 2013;0:1–7. doi:10.1136/oemed-2013-101498 7
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doi: 10.1136/oemed-2013-101498
published online August 16, 2013Occup Environ Med
Behrooz Behbod, Bruce Urch, Mary Speck, et al.
exposures
inflammation in controlled human
ambient particles induces acute systemic
Endotoxin in concentrated coarse and fine
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