Differential Inflammatory Response to Inhaled Lipopolysaccharide Targeted Either to the Airways or the Alveoli in Man
et al. (2012) Differential Inflammatory Response to Inhaled Lipopolysaccharide Targeted
Either to the Airways or the Alveoli in Man. PLoS ONE 7(4): e33505. doi:10.1371/journal.pone.0033505
Differential Inflammatory Response to Inhaled Lipopolysaccharide Targeted Either to the Airways or the Alveoli in Man
Winfried Mo ller 0
Irene Heimbeck 0
Thomas P. J. Hofer 0
Gu lnaz Khadem Saba 0
Margot Neiswirth 0
Marion Frankenberger 0
Lo ms Ziegler-Heitbrock 0
Dominik Hartl, University of Tu bingen, Germany
0 1 Comprehensive Pneumology Center and Institute for Lung Biology and Disease - Helmholtz Zentrum Mu nchen , Neuherberg, Gauting and M u nchen, Germany , 2 Asklepios Fachkliniken M u nchen-Gauting - Center for Pulmonary Medicine and Thoracic Surgery , Gauting, Germany , 3 EvA Study-Center - Helmholtz Zentrum Mu nchen , Gauting , Germany
Endotoxin (Lipopolysaccharide, LPS) is a potent inducer of inflammation and there is various LPS contamination in the environment, being a trigger of lung diseases and exacerbation. The objective of this study was to assess the time course of inflammation and the sensitivities of the airways and alveoli to targeted LPS inhalation in order to understand the role of LPS challenge in airway disease. In healthy volunteers without any bronchial hyperresponsiveness we targeted sequentially 1, 5 and 20 mg LPS to the airways and 5 mg LPS to the alveoli using controlled aerosol bolus inhalation. Inflammatory parameters were assessed during a 72 h time period. LPS deposited in the airways induced dose dependent systemic responses with increases of blood neutrophils (peaking at 6 h), Interleukin-6 (peaking at 6 h), body temperature (peaking at 12 h), and CRP (peaking at 24 h). 5 mg LPS targeted to the alveoli caused significantly stronger effects compared to 5 mg airway LPS deposition. Local responses were studied by measuring lung function (FEV1) and reactive oxygen production, assessed by hydrogen peroxide (H2O2) in fractionated exhaled breath condensate (EBC). FEV1 showed a dose dependent decline, with lowest values at 12 h post LPS challenge. There was a significant 2-fold H2O2 induction in airway-EBC at 2 h post LPS inhalation. Alveolar LPS targeting resulted in the induction of very low levels of EBC-H2O2. Targeting LPS to the alveoli leads to stronger systemic responses compared to airway LPS targeting. Targeted LPS inhalation may provide a novel model of airway inflammation for studying the role of LPS contamination of air pollution in lung diseases, exacerbation and anti-inflammatory drugs.
Endotoxin (Lipopolysaccharide, LPS) is a constituent of the
outer membrane of Gram-negative bacteria and an important
microbial trigger that stimulates innate immunity [1,2]. The
resultant inflammatory responses are essential in early host
defence, but may also contribute to chronic disease and organ
injury . Recent evidence suggests that LPS signal transduction
starts with CD14-mediated activation of one or more Toll-like
receptors (TLRs) . One of these receptorligand complexes is
formed between the mammalian TLR4-MD2-CD14 complex and
bacterial lipopolysaccharide (LPS) . Besides TLR4, the
LPSbinding protein (LBP) plays a major role. Both LBP and CD14
control ligand presentation to the TLR4 receptor complex and
influence the amplitude of LPS responses and LPS-induced type
The human body is confronted with LPS during infection with
gram-negative bacteria. There is however also LPS contamination
of particulate matter (PM10) in air pollution , including many
working environments, such as farms, cotton production, and
organic waste management [6,7]. These various types of air
pollution were suggested to play a significant role in health effects,
including the initiation and modulation of allergic reactions
[8,9,10]. Since LPS is a potent inducer of inflammation and since
many environmental dusts, including cigarette smoke, have high
LPS contamination, it was suggested that endotoxin may play
a significant role in progression on chronic lung diseases and
exacerbation. For example, it has been reported that
approximately 30% of stable COPD patients have bacterial colonisation
in their airways . Therefore LPS challenge may serve as
a model of lung inflammation and exacerbation in COPD .
Inhalation of LPS can be used to determine the competence of
the innate immune system regarding gram-negative bacteria .
In most studies the inhalation devices provided little control over
LPS dose and site of deposition in the lung, i.e. bronchial versus
alveolar dose .
In addition, we have to consider that the airways and the
alveolar space may have different sensitivities to endotoxin
challenge. Inhaled particles, including bacteria, viruses and
aerosolized drugs have different deposition probabilities in airways
and alveoli, depending on particle size and inhalation parameters
(flow rate, tidal volume) such that for example larger particles
(.4 mm diameter) at a flow rate of 500 mL/s primarily deposit in
the airways, while smaller particles at lower flow rates penetrate to
the alveoli. The alveolar space is covered with a surfactant
monolayer and there is only a 2 mm thick barrier between air and
blood. Aerosols reaching this area may therefore more directly
interact with pneumocytes and alveolar macrophages, or can
penetrate and reach the systemic circulation, as was shown for
inhaled nanoparticles .
In contrast, the airway is covered with epithelium including the
cilia, which form a more than 10 mm thick cellular barrier
between the airway lumen and the circulation. In addition, the
airways are covered with a mucus layer of 6 mm thickness,
although there are some areas without mucus . Substances
deposited onto the mucus layer may penetrate the mucus and
reach the epithelial surface or they may be transported out of the
lung within hours by mucociliary clearance . In addition,
differences in epithelial composition and cell types may trigger
different signalling pathways causing different inflammatory
Based on these differences we hypothesized that there will be
different responses when LPS is targeted to the alveoli compared
to the airways. We show herein that targeting LPS to the airways
does lead to lower inflammatory responses compared to LPS
deposition in the alveoli. This may have significant impact on
responses to inhaled endotoxin, disease progression and severity.
Subjects and study protocol
In order to exclude bronchial hyperresponsiveness, candidate
participants were subjected to increasing doses of inhaled
methacholine. Volunteers were recruited via newspaper adverts.
Among 15 healthy non-smoking volunteers tested seven volunteers
(5 male, 2 female, age 49+/217 years, mean +/2 standard
deviation) did not show any degree of hyperresponsiveness and
were selected for participating in the LPS study (Table 1).
Respiratory symptoms were obtained using a questionnaire 
and pulmonary function parameters were measured by spirometry
and body plethysmography (Jager Masterlab, Erich Jaeger GmbH,
Hochberg, Germany) . Bronchial hyperresponsiveness (BHR)
was assessed by a methacholine challenge test according to the
guidelines for bronchial challenges of the European Respiratory
Society . Subjects included in the study showed no or only
weak responses (less than 2-fold increase of airway resistance) at
the highest dose of inhaled methacholine. The protocol was
approved by the ethical committee of the
Ludwig-MaximiliansUniversity Munich, and informed and written consent was
obtained from each subject.
The study protocol is shown in Figure 1. Twenty-four hours
prior to LPS inhalation as well as 2, 6, 24, 48, and 72 h after LPS
inhalation body temperature (BT), lung function (FEV1 and peak
expiratory flow, PEF), blood samples and exhaled breath
condensate (EBC) were assessed. In addition BT and lung function
were measured 4 and 12 hours after LPS challenge. Body
temperature was measured in the ear (tympanic thermometer,
ThermoScan IRT 4520, Thermoscan Inc., San Diego, CA, USA).
FEV1 and PEF were measured using the hand-held electronic
peak flow/FEV1 meter PiKo-1 (Ferraris Respiratory Europe Ltd.,
Hertford, UK). Inflammation parameters, such as C-reactive
protein (CRP) and neutrophil counts were determined in blood
samples. Hydrogen peroxide concentration and acidity (pH) was
measured in EBC (see below). Prior to LPS challenge all
parameters shown above were in the normal range confirming
the healthy status of the subjects. In order to exclude adaptation to
the LPS inhalation after repeated LPS challenge (tolerance)
a period of at least four weeks was required between the different
LPS inhalations and all parameters mentioned above were in the
The volunteers sequentially inhaled 1, 5 and 20 mg LPS
deposited to the airways with at least 4 weeks between the
exposures. The analysis of body temperature, blood neutrophils,
CRP and H2O2 demonstrated that 5 mg was effective in inducing
responses in all individuals (see under Results). This dose of LPS
was then deposited to the alveoli. Targeted delivery of aerosolized
LPS to the airways or to the lung periphery (alveoli) was done
between 09:00 and 11:00 by aerosol bolus inhalation using the
AKITAH device (Activaero GmbH, Gemu nden, Germany).
Previous studies have shown that the AKITAH device shows little
inter- and intra-subject variation of aerosol deposition in the lung
. LPS from von Salmonella abortus equi, S-form
(TLRgradeTM, ALX-581-009, Alexis Biochemicals) was used in all subjects.
According to experimental regional deposition data summarized
in the ICRP-66 model the parameters of the nebulizer, the particle
size and the bolus penetration were adjusted in order to optimize
regional deposition in the airways and in the alveoli, respectively.
For aerosol delivery to the airways a 100 ml bolus was inhaled to
a volumetric bolus penetration front depth of 180 ml and an 8 s
breath holding was performed at the end of inhalation. The
pressure of the jet nebulizer was 1.0 bar producing 4.5 mm
MMAD (mass median aerodynamic diameter, measured by laser
diffraction spectrometry) droplets. The inhalation flow rate was
200 ml/s. Previous deposition measurements using radiolabeled
DTPA particles revealed 80% deposition efficiency for this
inhalation maneuver. For aerosol delivery to the alveoli a 150 ml
bolus was inhaled to 800 ml bolus penetration front depth. The
pressure of the jet nebulizer was 1.8 bar, generating 3.5 mm
MMAD (mass median aerodynamic diameter) droplets. The
inhalation flow rate was 150 ml/s and one second breath holding
was performed at the end of inhalation. Previous deposition
measurements revealed 95% deposition efficiency for this
inhalation maneuver. As further illustrated in the Text S1 the
number of breaths was calculated for deposited doses of 1, 5 and
20 mg of LPS in the airways and 5 mg LPS in the lung periphery,
respectively. The different particle sizes and inhalation profiles
were chosen for preferential airway and alveolar LPS targeting
according to experimental regional deposition studies summarized
by the ICRP Publication 66 . The shallow and deep bolus
placement is illustrated in Figure 2 together with the profile of
exhaled CO2 for assessing the dead space of the lung. The
distribution in the 23 generations of the lung for the shallow and
deep bolus is illustrated in Figure S1 according to simulations
using a stochastic lung deposition model. In addition deposition
distribution was assessed in previous studies using similar aerosol
bolus targeting protocols by the inhalation of radiolabeled aerosols
and planar gamma camera imaging .
Sampling of exhaled breath condensate and analysis of
hydrogen peroxide and pH
Exhaled breath condensate was collected using the
EcoScreenII (Filt GmbH, Berlin, Germany). The EcoScreen-II allows the
non-invasive collection of volatile and non-gaseous contents in
exhaled air in two separate condensation chambers . Based on
a built-in spirometer each exhaled breath can be split into four
fractions, i.e. two sampling and two discarding fractions. These
volumes were adapted to the individual Bohr dead spaces as
indicated in Figure 2 . The first 50 ml of the exhaled breath
representing gas from the oral cavity were discarded. The
following volume up to the Bohr dead space DSB was sampled
in the first container (airway sample), while the remaining exhaled
gas up to 1 l tidal volume was sampled into the second container
(alveolar sample). The second discarding volume was set to zero.
The inhalation air was filtered and conditioned to .95% relative
humidity at room temperature. EBC was collected under these
standardized conditions during 10 minutes oral breathing using
a nose clip.
Because H2O2 is not stable over longer periods of time,
immediate analysis of the collected condensate for hydrogen
peroxide (H2O2) and pH was performed using the EcoCheck
device (Filt GmbH, Berlin, Germany). The EcoCheck is a
biosensor device for measuring H2O2 concentrations by enzymatic
peroxidase reduction. The lower detection limit was 50 nmol/l
. The variability of H2O2 in EBC during a one week survey
shows a coefficient of variation of less than 20%. The EcoCheck is
also equipped with a pH-electrode for measurement of EBC
acidity. Within 10 min after EBC collection pH was measured
after 8 min de-aeration by Argon gas for removal of dissolved CO2
The concentration of IL-6 in the plasma samples was quantified
using a customized Milliplex MAP Human Cytokine/Chemokine
Panel (# HCYTOMAG-60K, Millipore, Schwalbach, Germany).
The assay was performed according to the manufacturers
instructions. Standards and samples were analyzed in duplicates
on a Luminex 200 device (BioRad, M unchen, Germany) using the
BioPlex Manager Software (Version 5, BioRad).
Data are expressed as mean +/2 standard deviation (SD).
Although the data sample is small (n = 7) the parameters did not
show significant difference from normal distribution (according to
the Kolmogorov-Smirnov-test). Differences among study groups
and between airway and alveolar study parameters were assessed
by the two-sided t-test (Winstat for Microsoft Excel, Version
2008.1, www.winstat.com), using a significance level of p,0.05.
Spearman rank correlation analysis was performed to analyze
correlations between parameters.
Subjects and clinical response
In order to exclude bronchial hyperresponsiveness, candidate
participants were subjected to increasing doses of inhaled
methacholine. Among the 15 healthy volunteers tested there was
none responding with a more than 20% decrease of FEV1 to the
highest dose of methacholine (0. 77 mg of methacholine). Looking
at resistance we found eight volunteers, who responded to
methacholine with an increase by more than factor 2 above
baseline (0.17+/20.04 kPa*sec/L), suggesting a low level of
hyperresponsiveness. Seven volunteers, who did not show any
Figure 3. A) Systemic response parameter body temperature during 72 h after targeting different doses of LPS either to the airways (closed
symbols) or to the alveoli (open symbols). B) Increase of 12 h - body temperature with increasing LPS dose targeted to the airways in comparison to
5 mg LPS targeted to the alveoli (open symbol). Data represent mean +/2 SD (n = 7; *: p,0.05, **: p,0.01 compared to baseline; ++: p,0.01 for 5 mg
alveolar compared to 5 mg airway LPS).
degree of hyperresponsiveness, were included in the LPS challenge
study. The anthropometric and lung function data of these seven
study subjects are listed in Table 1. All were never-smokers and
had no history of lung disease. These volunteers sequentially
inhaled 1, 5 and 20 mg LPS deposited to the airways and 5 mg LPS
deposited to the alveoli with at least 4 weeks between the
exposures. There was a uniform clinical response in that all
subjects developed mild to moderate flu-like symptoms including
headache and fatigue. These symptoms increased with increasing
doses and were most pronounced with 20 mg airway and 5 mg
alveolar deposition of LPS. At night subjects tended to go to sleep
one to two hours earlier than their usual bed time and there was
complete resolution of symptoms the next morning.
Body temperature. Body temperature after LPS inhalation
peaked between 6 and 12 h and returned to baseline in all subjects
at 24 h. For both 1 mg and 5 mg airway LPS we noted only a mild
increase to below 37uC. After 20 mg of airway LPS deposition
a strong increase to an average of 38.2+/20.9uC was seen at 12 h
(p,0.01, Figure 3). 12-h body temperature significantly increased
with the airway LPS dose (p,0.001) For alveolar LPS targeting of
5 mg LPS a strong temperature response was seen (37.9+/20.7uC,
p,0.01) at 12 h. When comparing 12 h body temperature after
5 mg airway LPS (36.8+/20.3uC) to 5 mg alveolar LPS then the
response to alveolar deposition was clearly higher (p,0.01,
Figure 3B), demonstrating a stronger systemic response when the
same dose of LPS is targeted to the alveoli as compared to the
Blood neutrophils. A similar pattern of responses was seen
for blood neutrophils. Here the peak response was at 6 h and the
values were back to baseline at 48 h. LPS at 1 mg airway
deposition showed an increase from 3400+/21300/mL to 5400+/
21300/mL at 6 h (p,0.005). The peak response at 6 h increased
with increasing LPS dose (p,0.001, Figure 4A). Again the
response to 5 mg alveolar LPS exceeded the response to 5 mg
airway LPS (9700+/21000/mL versus 8200+/21700/mL,
Blood CRP. Also there was a clear increase of CRP from
1.4+/20.9 mg/L before to 3.4+/22.3 mg/L after 1 mg of airway
LPS challenge at 24 h (p,0.05) and at 72 h after LPS challenge
CRP was still significantly above baseline. The 24 h CRP peak
value significantly increased with LPS dose (p,0.001, Figure 4B)
and was 10.5+/26.2 mg/L for 5 mg airway and 37.4+/
215.9 mg/L for 20 mg airway LPS. The response to 5 mg
alveolar LPS (28.3+/211.4 mg/L) was significantly higher than
the same dose of LPS when deposited to the bronchi (p,0.01).
Blood IL-6. Serum samples taken from experiments with
alveolar and bronchial exposure to 5 mg LPS were tested for IL-6
protein levels. As shown in Figure 4C there was a moderate 2-fold
rise in IL-6 at 6 h after bronchial LPS challenge (not significant),
while after alveolar LPS deposition the response was much
stronger with a 6-fold increase (p,0.05).
Lung function (FEV1). When LPS is applied to the airways
then a local inflammation may lead to air flow limitation. We
therefore monitored FEV1 using a hand held spirometer. For 1 mg
airway LPS there was a significant decrease of FEV1 at 12 h
(97.5+/22.5% of the baseline value (p,0.05), and there was
a further decrease of 12 h FEV1 with increasing airway LPS dose
to 93.4+/24.6% and 84.8+/28.8% of the baseline value after
5 mg and 20 mg LPS, respectively (p,0.01 for both doses,
Figure 5A). This included two individuals with a decrease of
FEV1 to below 80% of the individual baseline value. Also for the
5 mg alveolar LPS deposition there was an airway response with
a decrease of the 12 h FEV1 to 91.2+/25.6% of the baseline value
(p,0.01). There was no significant difference in 12 h FEV1
decrease after 5 mg airway and 5 mg alveolar LPS challenge.
Hydrogen peroxide in EBC. LPS can trigger reactive
oxygen production by inducing assembly of the NADPH oxidase
complex. We therefore have asked whether an increase of H2O2
can be detected in exhaled breath after LPS inhalation. For this we
collected EBC samples separated into an airway and an alveolar
fraction, where the airway fraction represents about one third and
the alveolar fraction two thirds of the collected volume (see
Figure 2). In average the condensate volumes collected from
201+/225 L of exhaled air were 0.74+/20.16 mL for the airway
fraction and 2.00+/20.27 mL for the alveolar fraction.
Constitutive H2O2 levels were 226+/281 nmol/L in the airway
fraction and 86+/217 nmol/L in the alveolar fraction.
When looking at the EBC-airway fraction after LPS was
targeted to the airways then the induced H2O2 peaked at 2 h with
values of 526+/2280 81 nmol/L, 442+/2208 nmol/L and
538+/2173 nmol/L for 1 mg, 5 mg and 20 mg, respectively (all
p,0.05 compared to baseline values, see Figure 5 B).
When analyzing induced H2O2 in the same EBC-airway
fraction after 5 mg LPS dose targeted to the alveoli then we also
Figure 4. Systemic inflammatory response parameters after targeting LPS either to the airways (1, 5 and 20 mg, closed symbols) or
to the alveoli (5 mg, open symbol). A: absolute blood neutrophils (6 h after LPS challenge, baseline value = 3.4+/21.3*103/mL), B: CRP (24 h after
LPS challenge, baseline value = 1.4+/20.9 mg/L) and C: IL-6 (baseline value 2.0+/20.4 pg/mL). Data represent mean +/2 SD (n = 7; *: p,0.05, **:
p,0.01 compared to baseline; ++: p,0.01 for 5 mg alveolar compared to 5 mg airway LPS).
saw significant induction at 328+/2135 nmol/L at 2 h (p,0.05
compared to baseline EBC-H2O2). The airway response to 5 mg
LPS targeted to the airways was higher in tendency compared to
this response when targeted to the alveoli.
In the alveolar EBC fraction (data not shown) we detected much
lower 2 h values with 216+/283, 182+/2177 and 196+/
2104 nmol/L after 1 mg, 5 mg and 20 mg bronchial LPS,
respectively (all p,0.01 compared to baseline values). After
alveolar LPS challenge the alveolar EBC did not show a significant
induction of H2O2.
Most studies published on human LPS challenge used full
breath LPS inhalation and thereby cannot account for differences
in defence and immune responses in the different regions of the
respiratory tract. A novel human airway inflammation study
limiting LPS challenge to the airways used segmental endotoxin
challenge in healthy subjects during bronchoscopy . This
model gives limited systemic but only local inflammatory responses
and is invasive to the subjects. The inflammation model proposed
in our study using controlled LPS challenge to the airways or to
the pulmonary region is non-invasive and allows the
understanding of the specific inflammation responses in the different lung
Our study used aerosol bolus inhalation in order to enable
controlled LPS targeting either to the airways or to the alveoli.
When controlling LPS inhalation by the Akita device then there is
minor variation of the delivered LPS dose to either of the target
sites [22,28]. The consistent deposition among the subjects is
documented in the similar response pattern among all study
subjects and a clear dose-response relationship for parameters like
CRP and neutrophils in our study. Studies using full breath
inhalation in an uncontrolled manner have to deal with several
uncertainties, such as the site of delivery and the dose delivery to
the two major sites: the airways and alveoli. For example when
comparing our study with the report by Michel et al  5 mg LPS
deposited to the alveoli in our study gives systemic responses with
respect to neutrophils and CRP that are similar to what is achieved
with inhalation of 50 mg of LPS by Michel et al. One possible
explanation for this difference is that in that earlier study
a substantial share of the inhaled LPS has impacted at the back
of the throat and has never reached the lung. Also, the type and
preparation of LPS may determine the degree of response.
However, targeting by the bolus technique does not exclusively
deposit aerosol in either of the anatomical sites although the
delivery protocols and particle size were optimized. Although the
major fraction of inhaled aerosol is deposited either in the central
airways (generations 110) after shallow bolus inhalation or in the
alveoli (generations 1823) after deep bolus inhalation, as
illustrated in Figure S1, there is an overlapping deposition in
small bronchiolar airways and alveolar structures. As a result of
this overlap we see a high correlation of hydrogen peroxide in
exhaled breath condensate (EBC-H2O2) between the airway and
the alveolar fraction. Targeting LPS to the airways by shallow
bolus inhalation may also trigger H2O2 production in the alveoli
and vice versa.
Nevertheless, as illustrated in Figure S2, because of the smaller
surface area of the airways, there is higher LPS dose deposited per
unit surface area in the airways compared to the alveoli, from
which one might expect higher responses. With the exception of
induced H2O2 in the airways this could not be confirmed in our
study and we show herein the opposite results. Differences in
inflammatory signalling, the several micrometre thick mucus
blanket and the mucociliary clearance may protect the airway
epithelium in part from LPS challenge.
Previous studies suggested that allergy can influence responses
to inhaled LPS, with either increased or impaired responses
[30,31,32,33]. In order to exclude any impact of allergy on the
results of our study, we only invited subjects without a history of
allergy to participate. Furthermore, all candidates (n = 15) were
tested with methacholine for hyperresponsiveness and we excluded
those with an increase of airway resistance by more than factor 2
(n = 8). These individuals would have been classified as
normoresponsive, had we used a decrease of FEV1 by more than 20% as
a criterion. The use of the more stringent resistance criterion
resulted in a study population unaffected by any degree of
hyperresponsiveness and hence with lower variability.
The participants repeatedly inhaled LPS targeted to the airways
in increasing doses and finally a medium dose to the alveoli. Since
repeated LPS exposure can induce a non-responsiveness also
termed tolerance [34,35] a rest period between exposures was set
for this study. Such tolerance can last for several days but normal
responses were shown to have returned after 3 weeks .
Therefore we choose a time period of at least 4 weeks between
exposures in the present study. Baseline values for the
inflammatory markers before the exposures were constant for every
individual. Also, we always detected a robust response for
inflammatory markers like CRP (see Figure 4).
When looking at systemic responses to LPS inhalation we see
a dose dependent rise in body temperature which peaks at 12 h
and has resolved after 24 h. This is in line with a transient
induction and release of endogenous pyrogens like IL-1 and IL-6,
which are produced locally and then act on the hypothalamus
. The same dose of 5 mg LPS applied to the alveoli has
a stronger response compared to airway deposition. One might
assume that with alveolar deposition some LPS may have access to
the systemic circulation thereby inducing this enhanced response.
This would however require active transport with the help of
binding proteins, since LPS is a high molecular weight molecule
that in addition tends to form larger aggregates . Also, it has
been shown that after instillation of LPS in the mouse,
Interleukin6 levels in the arterial blood is much higher than in venous blood,
suggesting that IL-6 is released from the lung into circulation .
We have, in fact, detected a significant increase in serum IL-6 in
our volunteers with deposition of 5 mg LPS to the alveoli (see
Fig. 4C). These findings do not exclude that a transfer of LPS can
occur and it will be of interest to test whether after LPS deposition
to the alveoli blood levels of LPS are in fact higher compared to
For neutrophils we saw a much earlier peak response at 6 h and
this again was dose dependent with a higher response to 5 mg
alveolar as compared to 5 mg airway deposition. The major factor
involved in immediate rise of neutrophils is G-CSF
(granulocytecolony-stimulating factor), which can mediate release of
neutrophils from bone marrow by interference with CXCL12-CXCR4
interactions that retain these cells in bone marrow . G-CSF
can be produced by various cell types including bronchial
epithelial cells, macrophages and fibroblasts [40,41]. Therefore,
we assume that after LPS inhalation G-CSF is produced locally in
the lung and triggers release of neutrophils in the bone marrow.
We have assayed for G-CSF in serum of the volunteers before and
at 6, 24, 48 and 72 h after LPS exposure but no significant
increase of this cytokine could be detected. It may well be that
serum samples taken at earlier time points, i.e. 1 or 2 h may show
an induction of this cytokine. It has been reported that in the pig
LPS inhalation can lead to an initial blood neutropenia at an early
point in time . In our studies no change in blood neutrophil
numbers were seen as early as 2 hours post inhalation. Higher
doses of inhaled LPS may be required for a neutropenia to
CRP peaked much later at 24 h, which is in line with the
clinical experience in infection and inflammation. CRP is
synthesized in the liver and it is under control of cytokines like
IL-6 [43,44]. Hence, LPS induces a cascade of events, with local
induction of IL-6, release of IL-6 into circulation, binding to the
IL-6 receptor on hepatocytes and synthesis and release of CRP. As
shown herein IL-6 peaks at 6 h post alveolar LPS deposition in
line with this pathophysiological cascade.
When looking at local responses we studied airway constriction
and noted a dose dependent decrease of FEV1. This is best
explained by the local induction of inflammation with subsequent
thickening of the airway wall leading to a reduced width of the
airway. There also may be a contribution by smooth muscle cells
via a cytokine mediated enhancement of acetylcholine triggered
contraction . This LPS induced reduction of FEV1 has been
noted earlier and was shown to be mediated by toll-like receptor 4
Of note, in our study there also was a reduction in FEV1 after
alveolar LPS deposition. We hypothesize that this response is due
to airway deposition of some LPS as it passes through the airways.
Since only a minor fraction of the LPS is deposited in the airways
during the alveolar targeting and since there is a clear linear dose
dependence this would predict a less pronounced effect on FEV1
for 5 mg alveolar compared to 5 mg airway LPS. The decrease of
FEV1 is, however, similar for the two deposition sites at the same
5 mg dose. Therefore, we assume that also a systemic component
of the inflammatory response contributes to the transient airway
obstruction (subjects reported chest tightness).
One major defence mechanism induced by LPS is via the
induction of reactive oxygen species. LPS can induce assembly of
the NADPH-oxidase complex leading to H2O2 production .
After LPS stimulation H2O2 is typically produced by macrophages
but also by airway epithelial cells [49,50]. In the present report, we
have studied the production of H2O2 in the lung by looking at
exhaled breath condensate. For this we have collected the exhaled
breath in two fractions, one representing the airways and the other
the alveoli. Since the first 50 mL of EBC were discarded only
minor influences from the oral cavity can be expected. The airway
EBC sample was collected until the Bohr dead space and
represents the total airway volume including the transition zone
between airway and alveolar space. The alveolar sample only
originates from the alveolar space, but, since this sample has to
pass the airways during exhalation, there may be contaminations
from the airways.
In a previous study using this fractionated EBC sampling
technique we have shown that basal levels of H2O2 in EBC are
higher in the airway compartment compared to the alveoli and
this was true for non-smokers, smokers and in COPD patients
. This higher level in the airways without overt stimulation
may be due to a higher deposition of ambient particles to this
compartment (per unit surface area). Alternatively, the epithelial
cells in the airways may be better constitutive producers of H2O2
compared to the alveolar macrophages in the lung, including
differences in glutathione detoxification .
With LPS inhalation we noted a pronounced rise in H2O2
production with a peak at 2 h both, in the airway and in the
alveolar EBC. Since in-vitro 20 minutes is sufficient to generate an
optimum oxidative burst  we have determined in preliminary
studies exhaled H2O2 at 30 min and at 1 h post LPS challenge
and we noted no rise at these time points. Hence, it appears the
2 h post inhalation is the earliest time point with a significant
H2O2 induction in exhaled breath condensate. In contrast to all
other parameters there was no dose dependence at 2 h in that 1, 5
and 20 mg LPS deposited to the airways gave a similar H2O2 in
airway EBC. One explanation for this finding may be that the
sensitivity of the NADPH-oxidase system to LPS is much higher
compared to induction of neutrophilia or fever or CRP, such that
1 mg of LPS gives already a maximum response. Studies with
much lower doses of LPS will be required in order to demonstrate
dose dependence in the range below 1 mg.
When comparing airway and alveolar deposition at 5 mg of LPS
each it is apparent that the H2O2 production in both the airway
and the alveolar EBC fraction is higher with the airway LPS
deposition. This higher signal in the airway fraction is conceivable
since there is a higher area concentration of LPS at this site (see
Figure S2). On the other hand, the lower response in the alveolar
EBC fraction after alveolar LPS deposition comes as a surprise.
This absence of a response may be explained by the large surface
area and hence the low LPS dose per unit surface area. In addition
the H2O2 that is released in this area may be efficiently neutralized
by anti-oxidative mechanisms, such as the glutathione detoxifying
system [51,53]. Also, alveolar macrophages may be silenced in
their response in order to prevent damage to the lung. These cells,
for instance, show a lower TNF production after LPS compared to
blood monocytes  which may be due to lower CD14
expression by these cells. Furthermore, in the mouse model
alveolar macrophages have been shown to be silenced via
avb6mediated induction of TGF-b [55,56].
Systemic and local inflammation parameters assessed in our
study peaked at characteristic time points after LPS challenge, as
summarized in Figure 6, and most responses were LPS dose
dependent (indicated as ,D). Rank correlation analysis showed
that those parameters correlating with the LPS dose are highly
inter-correlated, but respond at different time periods after LPS
challenge. Some of these characteristic responses have also been
observed in other LPS inhalation studies [29,32,57], but most of
these studies cover only a 24 hour observation period, and none of
these studies included the topical inflammation parameter
hydrogen peroxide in exhaled breath condensate.
Our data clearly show that targeted LPS delivery by controlled
inhalation will lead to a highly reproducible inflammatory
response, with predictable peaking times for blood neutrophils,
body temperature, FEV1 impairment, CRP and H2O2 production.
Also, clinical symptoms consistently have disappeared after 24 h.
Taken together we have described herein that LPS targeted to the
airways compared to the alveoli generates significantly lower
systemic responses, but similar local H2O2 responses. The human
inflammation model proposed in our study allows controlled LPS
challenge to the airways or to the pulmonary region. Note that our
study provides inflammatory responses with respect to deposited
LPS dose in the respective lung region while all other studies
provide nebulized LPS dose. Since there is great variability in
regional particle deposition with respect to particle size, size
distribution, inhalation pattern and disease severity, the deposited
dose in the target region, the airways, is very variable and partly
unknown. In addition, as our study showed higher systemic
responses of inhaled LPS in the pulmonary region, most of the
systemic responses reported in other studies using tidal breathing
may suffer from these not wanted side effects. In addition using
shallow bolus LPS inhalation in COPD patients for studying
exacerbation one may significantly reduce risks of severe side
effects, since the site of deposition and the deposited dose are
under control. This may open the opportunity of studying new
anti-inflammatory drugs in COPD, such as steroids, b2-agonists,
or anti-MCP-1 monoclonal antibody (controlling monocyte
Exacerbations are important events in patients with asthma and
chronic obstructive pulmonary disease (COPD) . Reducing
the number, frequency and the severity of exacerbations is
therefore an important management goal identified by treatment
guidelines for both diseases. Endotoxin plays a significant role in
COPD and asthma exacerbation since approximately 30% of
stable COPD patients have bacterial colonisation in the airways
. Bacteria are believed to cause approximately 50% of the
exacerbations, alone or following virus infection. It was speculated
that inhaled LPS challenge may mimic an acute COPD
exacerbation of bacterial origin and may induce a cascade of
events resulting in NF-kappa-B induction and activation, cytokine
and chemokine production and further inflammatory cell
Estimation of deposited dose.
Deposition distribution per unit surface area.
Conceived and designed the experiments: WM MF LZH. Performed the
experiments: WM IH TPJH GKS MN MF LZH. Analyzed the data: WM
IH TPJH LZH. Contributed reagents/materials/analysis tools: WM IH
GKS MN MF. Wrote the paper: WM MF LZH.
1. Alexander C , Rietschel ET ( 2001 ) Bacterial lipopolysaccharides and innate immunity . J Endotoxin Res 7 : 167 - 202 .
2. Zhang P , Summer WR , Bagby GJ , Nelson S ( 2000 ) Innate immunity and pulmonary host defense . Immunol Rev 173 : 39 - 51 .
3. Maris NA , Dessing MC , de Vos AF , Bresser P , van der Zee JS , et al. ( 2006 ) Tolllike receptor mRNA levels in alveolar macrophages after inhalation of endotoxin . Eur Respir J 28 : 622 - 626 .
4. Rietschel ET , Zahringer U , Seydel U , Ulmer AJ ( 1999 ) Bacterial endotoxins: Bioactive conformation and specific host-recognition . Shock 12 : 21 - 21 .
5. Mueller-Anneling L , Avol E , Peters JM , Thorne PS ( 2004 ) Ambient Endotoxin Concentrations in PM10 from Southern California . Environ Health Perspect 112 : 583 - 588 .
6. Rylander R ( 2002 ) Endotoxin in the environment-exposure and effects . J Endotoxin Res 8 : 241 - 252 .
7. Liebers V , Raulf-Heimsoth M , Bruning T ( 2008 ) Health effects due to endotoxin inhalation . Arch Toxicol 82 : 203 - 210 .
8. Bolte G , Bischof W , Borte M , Lehmann I , Wichmann HE , et al. ( 2003 ) Early endotoxin exposure and atopy development in infants: results of a birth cohort study . Clin Exp Allergy 33 : 770 - 776 .
9. Gehring U , Cyrys J , Sedlmeir G , Brunekreef B , Bellander T , et al. ( 2002 ) Traffic-related air pollution and respiratory health during the first 2 yrs of life . Eur Respir J 19 : 690 - 698 .
10. Shi J , Mehta AJ , Hang J- q, Zhang H, Dai H , et al. ( 2010 ) Chronic Lung Function Decline in Cotton Textile Workers: Roles of Historical and Recent Exposures to Endotoxin . Environ Health Perspect 118 : 1620 - 1624 .
11. Wedzicha JA , Donaldson GC ( 2003 ) Exacerbations of chronic obstructive pulmonary disease . Respir Care 48 : 1204 - 1213 ; discussion 1213.
12. Kharitonov SA , Sjobring U ( 2007 ) Lipopolysaccharide challenge of humans as a model for chronic obstructive lung disease exacerbations . Contrib Microbiol 14 : 83 - 100 .
13. Arbour NC , Lorenz E , Schutte BC , Zabner J , Kline JN , et al. ( 2000 ) TLR4 mutations are associated with endotoxin hyporesponsiveness in humans . Nat Genet 25 : 187 - 191 .
14. Thorn J ( 2001 ) The inflammatory response in humans after inhalation of bacterial endotoxin: a review . Inflamm Res 50 : 254 - 261 .
15. Geiser M , Rothen-Rutishauser B , Kapp N , Schurch S , Kreyling W , et al. ( 2005 ) Ultrafine particles cross cellular membranes by nonphagocytic mechanisms in lungs and in cultured cells . Environ Health Perspect 113 : 1555 - 1560 .
16. van As A , Webster I ( 1972 ) The organization of ciliary activity and mucus transport in pulmonary airways . S Afr Med J 46 : 347 - 350 .
17. Moller W , Felten K , Sommerer K , Scheuch G , Meyer G , et al. ( 2008 ) Deposition, retention and translocation of ultrafine particles from the central airways and lung periphery . Am J Respir Crit Care Med 177 : 426 - 432 .
18. Thorley AJ , Grandolfo D , Lim E , Goldstraw P , Young A , et al. ( 2011 ) Innate Immune Responses to Bacterial Ligands in the Peripheral Human Lung - Role of Alveolar Epithelial TLR Expression and Signalling. PLoS ONE 6: e21827 .
19. Ferris BG ( 1978 ) Epidemiology Standardization Project (American Thoracic Society) . Am Rev Respir Dis 118 : 1 - 120 .
20. Quanjer PH , Tammeling GJ , Cotes JE , Pedersen OF , Peslin R , et al. ( 1993 ) Lung volumes and forced ventilatory flows . Report Working Party Standardization of Lung Function Tests, European Community for Steel and Coal. Official Statement of the European Respiratory Society. Eur Respir J 16 : 5 - 40 .
21. Sterk PJ , Fabbri LM , Quanjer PH , Cockcroft DW , O'Byrne PM , et al. ( 1993 ) Airway responsiveness . Standardized challenge testing with pharmacological, physical and sensitizing stimuli in adults . Report Working Party Standardization of Lung Function Tests, European Community for Steel and Coal. Official Statement of the European Respiratory Society. Eur Respir J Suppl 6 : 53 - 83 .
22. Brand P , Friemel I , Meyer T , Schulz H , Heyder J , et al. ( 2000 ) Total deposition of therapeutic particles during spontaneous and controlled inhalations . J Pharm Sci 89 : 724 - 731 .
23. ICRP Publication 66 ( 1994 ) Human respiratory tract model for radiological protection . A report of a Task Group of the International Commission on Radiological Protection. Ann ICRP 24 : 1 - 482 .
24. Moller W , Heimbeck I , Weber N , Khadem Saba G , Korner B , et al. ( 2010 ) Fractionated Exhaled Breath Condensate Collection Shows High Hydrogen Peroxide Release in the Airways . J Aerosol Med Pulm Drug Deliv 23 : 129 - 135 .
25. Gerritsen WB , Zanen P , Bauwens AA , van den Bosch JM , Haas FJ ( 2005 ) Validation of a new method to measure hydrogen peroxide in exhaled breath condensate . Respir Med 99 : 1132 - 1137 .
26. Horvath I , Hunt J , Barnes PJ , ATS ERS Task Force Exhaled Breath ( 2005 ) Exhaled breath condensate: methodological recommendations and unresolved questions . Eur Respir J 26 : 523 - 548 .
27. Hohlfeld JM , Schoenfeld K , Lavae-Mokhtari M , Schaumann F , Mueller M , et al. ( 2008 ) Roflumilast attenuates pulmonary inflammation upon segmental endotoxin challenge in healthy subjects: A randomized placebo-controlled trial . Pulm Pharmacol Ther 21 : 616 - 623 .
28. Scheuch G , Brand P , Meyer T , Mullinger B , Sommerer K ( 2002 ) Regional drug targeting within the lungs by controlled inhalation with the AKITA inhalation system . In: Dalby RN, Byron PR , Peart J , Farr SJ, eds. Respiratory Drug Delivery VIII. Richmond , VA, USA: Virginia Commonwealth University. pp 165 - 168 .
29. Michel O , Nagy AM , Schroeven M , Duchateau J , Neve J , et al. ( 1997 ) Doseresponse relationship to inhaled endotoxin in normal subjects . Am J Respir Crit Care Med 156 : 1157 - 1164 .
30. Nightingale JA , Rogers DF , Hart LA , Kharitonov SA , Chung KF , et al. ( 1998 ) Effect of inhaled endotoxin on induced sputum in normal, atopic, and atopic asthmatic subjects . Thorax 53 : 563 - 571 .
31. Michel O , Dentener M , Corazza F , Buurman W , Rylander R ( 2001 ) Healthy subjects express differences in clinical responses to inhaled lipopolysaccharide that are related with inflammation and with atopy . J Allergy Clin Immunol 107 : 797 - 804 .
32. Kitz R , Rose MA , Borgmann A , Schubert R , Zielen S ( 2006 ) Systemic and bronchial inflammation following LPS inhalation in asthmatic and healthy subjects . J Endotoxin Res 12 : 367 - 374 .
33. Peters M , Kauth M , Schwarze J , Korner-Rettberg C , Riedler J , et al. ( 2006 ) Inhalation of stable dust extract prevents allergen induced airway inflammation and hyperresponsiveness . Thorax 61 : 134 - 139 .
34. Beeson PB ( 1947 ) Tolerance to Bacterial Pyrogens: I. Factors Influencing Its Development . J Exp Med 86 : 29 - 38 .
35. Ziegler-Heitbrock HW , Wedel A , Schraut W , Strbel M , Wendelgass P , et al. ( 1994 ) Tolerance to lipopolysaccharide involves mobilization of nuclear factor kappa B with predominance of p50 homodimers . J Biol Chem 269 : 17001 - 17004 .
36. Dinarello CA ( 2004 ) Review: Infection, fever, and exogenous and endogenous pyrogens: some concepts have changed . J Endotoxin Res 10 : 201 - 222 .
37. Beutler B , Rietschel ET ( 2003 ) Innate immune sensing and its roots: the story of endotoxin . Nat Rev Immunol 3 : 169 - 176 .
38. Tamagawa E , Suda K , Yuan W , Li X , Mui T , et al. ( 2009 ) Endotoxin-induced translocation of interleukin-6 from lungs to the systemic circulation . Innate Immunity 15 : 251 - 258 .
39. Christopher MJ , Link DC ( 2007 ) Regulation of neutrophil homeostasis . Curr Opin Hematol 14 : 3 - 8 .
40. Levine SJ , Larivee P , Logun C , Angus CW , Shelhamer JH ( 1993 ) Corticosteroids differentially regulate secretion of IL-6, IL-8, and G-CSF by a human bronchial epithelial cell line . Am J Physiol 265 : L360 - 368 .
41. Asano S ( 1991 ) Human granulocyte colony-stimulating factor: its basic aspects and clinical applications . Am J Pediatr Hematol Oncol 13 : 400 - 413 .
42. Holst H , Edqvist LE , Kindahl H , Rylander R ( 1994 ) Hematological, Blood Biochemical , and Cytological Bronchoalveolar Lavage Studies in Prepubertal Gilts after Endotoxin Inhalation and Ingestion . Journal of Veterinary Medicine Series A 41 : 159 - 166 .
43. Mackiewicz A , Speroff T , Ganapathi MK , Kushner I ( 1991 ) Effects of cytokine combinations on acute phase protein production in two human hepatoma cell lines . J Immunol 146 : 3032 - 3037 .
44. Mortensen RF ( 2001 ) C-reactive protein, inflammation, and innate immunity . Immunol Res 24 : 163 - 176 .
45. Amrani Y , Panettieri RA ( 2003 ) Airway smooth muscle: contraction and beyond . Int J Biochem Cell Biol 35 : 272 - 276 .
46. Jagielo PJ , Thorne PS , Watt JL , Frees KL , Quinn TJ , et al. ( 1996 ) Grain dust and endotoxin inhalation challenges produce similar inflammatory responses in normal subjects . Chest 110 : 263 - 270 .
47. Arbour NC , Lorenz E , Schutte BC , Zabner J , Kline JN , et al. ( 2000 ) TLR4 mutations are associated with endotoxin hyporesponsiveness in humans . Nat Genet 25 : 187 - 191 .
48. Nauseef WM ( 2004 ) Assembly of the phagocyte NADPH oxidase . Histochem Cell Biol 122 : 277 - 291 .
49. Rochelle LG , Fischer BM , Adler KB ( 1998 ) Concurrent production of reactive oxygen and nitrogen species by airway epithelial cells in vitro . Free Radic Biol Med 24 : 863 - 868 .
50. Lavigne MC , Eppihimer MJ ( 2005 ) Cigarette smoke condensate induces MMP12 gene expression in airway- like epithelia . Biochem Biophys Res Commun 330 : 194 - 203 .
51. Rahman Q , Abidi P , Afaq F , Schiffmann D , Mossman BT , et al. ( 1999 ) Glutathione Redox System in Oxidative Lung Injury . Crit Rev Toxicol 29 : 543 - 568 .
52. Prince HE , Lape-Nixon M ( 1995 ) Influence of specimen age and anticoagulant on flow cytometric evaluation of granulocyte oxidative burst generation . J Immunol Methods 188 : 129 - 138 .
53. Battin E , Brumaghim J ( 2009 ) Antioxidant Activity of Sulfur and Selenium: A Review of Reactive Oxygen Species Scavenging, Glutathione Peroxidase, and Metal-Binding Antioxidant Mechanisms . Cell Biochem Biophys 55 : 1 - 23 .
54. Dentener MA , Bazil V , Von Asmuth EJ , Ceska M , Buurman WA ( 1993 ) Involvement of CD14 in lipopolysaccharide-induced tumor necrosis factoralpha, IL-6 and IL-8 release by human monocytes and alveolar macrophages . J Immunol 150 : 2885 - 2891 .
55. Kaminski N , Allard JD , Pittet JF , Zuo F , Griffiths MJD , et al. ( 2000 ) Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis . Proceedings of the National Academy of Sciences 97 : 1778 - 1783 .
56. Morris DG , Huang X , Kaminski N , Wang Y , Shapiro SD , et al. ( 2003 ) Loss of integrin [alpha]v[beta]6-mediated TGF-[beta] activation causes Mmp12-dependent emphysema . Nature 422 : 169 - 173 .
57. Doyen V , Kassengera Z , Dinh D , Michel O ( 2011 ) Time Course of EndotoxinInduced Airways' Inflammation in Healthy Subjects . Inflammation in press. pp 1- 6 - 6 .
58. Maris NA , de Vos AF , Dessing MC , Spek CA , Lutter R , et al. ( 2005 ) Antiinflammatory effects of salmeterol after inhalation of lipopolysaccharide by healthy volunteers . Am J Respir Crit Care Med 172 : 878 - 884 .
59. O'Byrne PM ( 2007 ) Exacerbations of asthma and COPD: definitions, clinical manifestations and epidemiology . Contrib Microbiol 14 : 1 - 11 .