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Inhalation of ultrafine carbon particles
triggers biphasic pro-inflammatory
response in the mouse lung
E. Andre
´*
,#,+
, T. Stoeger
",+
, S. Takenaka
"
, M. Bahnweg*, B. Ritter
"
, E. Karg
"
,
B. Lentner
"
, C. Reinhard
"
, H. Schulz
"
and M. Wjst
#
ABSTRACT: High levels of particulate matter in ambient air are associated with increased
respiratory and cardiovascular health problems. It has been hypothesised that it is the ultrafine
particle fraction (diameter ,100 nm) that is largely responsible for these effects. To evaluate the
associated mechanisms on a molecular level, the current authors applied an expression profiling
approach.
Healthy mice were exposed to either ultrafine carbon particles (UFCPs; mass concentration 380
mg?m
-3
) or filtered air for 4 and 24 h. Histology of the lungs did not indicate any
pathomorphological changes after inhalation.
Examination of the bronchoalveolar lavage fluid revealed a small increase in polymorpho-
nuclear cell number (ranging 0.6–1%) after UFCP inhalation, compared with clean air controls,
suggesting a minor inflammatory response. However, DNA microarray profile analysis revealed a
clearly biphasic response to particle exposure. After 4 h of inhalation, mainly heat shock proteins
were induced, whereas after 24 h, different immunomodulatory proteins (osteopontin, galectin-3
and lipocalin-2) were upregulated in alveolar macrophages and septal cells.
In conclusion, these data indicate that inhalation of ultrafine carbon particles triggers a biphasic
pro-inflammatory process in the lung, involving the activation of macrophages and the
upregulation of immunomodulatory proteins.
KEYWORDS: Air pollution, alveolar macrophages, cytokines, expression profiling
Epidemiological studies have shown that
increased levels of particulate matter (PM)
in ambient air are associated with aggra-
vation of respiratory diseases and cardiovascular
complications. A strong association has been seen
for respiratory and cardiac deaths, particularly
among elderly people [1–3]. Oxidative stress,
induced by inhaled particles, successively lead-
ing to activation of pro-inflammatory gene
transcription, is one mechanism thought to cause
the adverse health effects of ambient PM. In
particular, alveolar macrophages may be acti-
vated by particles and release cytokines and
reactive oxygen species [4].
Different hypotheses were developed to explain
which particle properties drive the adverse
health effects. These hypotheses deal with the
particle’s charge, its content of transition metals,
and its size and specific surface area. According
to the ‘‘ultrafine hypothesis of particle toxicity’’,
ambient ultrafine particles (UFP), i.e. particles
with a diameter ,100 nm, are the proportion of
particulate air pollution that mainly causes the
adverse health effects [5]. Animal studies indicate
that at high exposure levels ultrafine carbon
particles (UFCPs) and titanium dioxide particles
have a greater toxic potential than fine particles
with a diameter ranging between 0.1–1.0 mm [6,
7]. UFP induce pulmonary inflammation at a
lower mass concentration than larger particles
[8]. Although UFP comprise only 1–8% of the
mass, they present up to 99% of the number of
ambient PM [9]. Due to their small size, UFP are
known to enter the alveolar–capillary barrier [10,
11], translocate from the lung into the blood [12],
and thus have the capability to directly interact
with extrapulmonary organs.
So far, the molecular and cellular events induced
by inhalation of UFP are poorly understood. The
objective of the current study was to identify
genes regulated by the exposure to UFP in order
to elucidate pathways that are activated by these
particles and whose activation possibly leads to
adverse health effects.
AFFILIATIONS
*Ludwig-Maximilians-University,
Institute for Epidemiology,
#
GSF-National Research Center for
Environment and Health, Institute for
Epidemiology, and
"
GSF-National Research Center for
Environment and Health, Institute for
Inhalation Biology, Neuherberg,
Germany.
+
Both authors contributed equally to
this study.
CORRESPONDENCE
T. Stoeger
GSF-National Research Center for
Environment and Health
Institute for Inhalation Biology
Ingolstaedter Landstrasse 1
D-85758 Neuherberg/Munich
Germany
Fax: 49 8931872400
E-mail: tobias.stoeger@gsf.de
Received:
June 16 2005
Accepted after revision:
April 20 2006
SUPPORT STATEMENT
The present study was funded by
grant 01GS0122 from the German
National Genome Network (NGFN),
Bonn, Germany.
European Respiratory Journal
Print ISSN 0903-1936
Online ISSN 1399-3003
EUROPEAN RESPIRATORY JOURNAL VOLUME 28 NUMBER 2 275
Eur Respir J 2006; 28: 275–285
DOI: 10.1183/09031936.06.00071205
CopyrightßERS Journals Ltd 2006
c
Young, healthy BALB/cJ mice that were exposed to UFCPs
were used as a model. The present authors performed DNA
microarray analysis of whole-lung RNA from mice that were
exposed to high levels of UFP for different periods of time.
Among the affected genes, concerted upregulation of genes
encoding for molecules involved in oxidative stress and
macrophage activation were found. The current results are
the first to demonstrate that inhalation of UFCPs leads to
macrophage activation and triggers pro-inflammatory pro-
cesses in healthy mice. Furthermore, the current authors
identified the regulation of soluble immunomodulatory pro-
teins that might serve as a useful marker for inflammatory
processes induced by particle inhalation in further studies.
MATERIALS AND METHODS
Animals
Female BALB/cJ mice, established at The Jackson Laboratory
(Bar Harbor, ME, USA), were shipped to the GSF-National
Research Center for Environment and Health (Neuherberg,
Germany) at 8 weeks of age. The animals were kept at the GSF
in ‘‘isolated ventilated cages’’ (IVC-Racks; BioZone, Ramsgate,
UK) supplied with filtered air in a 12-h light/12-h dark cycle.
Specified pathogen-free status was approved by a health
certificate according to Federation of European Laboratory
Animals Science Association guidelines. Food and water were
available ad libitum. Animals were aged 10–12 weeks when
studied. Eight animals per experimental group were analysed.
Experimental protocols were in accordance with the German
Law on Animal Protection and approved by the Bavarian
Animal Research Authority (approval no. 211-2531-108/99).
Particle generation and whole-body exposure chamber
The set-up of the whole-body exposure system for rodents has
been described previously by KARG et al. [13]. Briefly, the
exposure chamber was supplied with a constant flow of
humidified air (23uC, 46% relative humidity) and loaded with
UFCPs. UFCPs were produced by an improved electric spark
generator (Model GFG 1000; Palas, Karlsruhe, Germany)
operated with ultrapure graphite electrodes in an argon atmos-
phere (,10
-6
impurities) [14]. UFCPs produced by this method
consist of o96% elemental carbon [15]. Particle number
concentration (CPC 3022A; TSI, St. Paul, MN, USA) and size
distribution (EMS 150; Hauke, Gmunden, Austria) were
continuously monitoredat the entrance of the exposure chamber.
Particle number concentration was 8610
6
cm
-3
with a count
median diameter of 49 nm. Mass concentration was measured
gravimetrically by filter sampling. The average UFCP mass
concentration in the exposure chamber was 380 mg?m
-3
.
Experimental groups
Groups of eight mice were exposed to UFCPs or clean air for 4
or 24 h, respectively. After particle or clean air exposure,
bronchoalveolar lavage (BAL) was performed on eight mice
per condition, and blood was taken from the same animals.
Lungs were taken for RNA preparation from an additional
eight animals per experimental condition. Eight further mice
were sacrificed either after exposure to UFCPs or clean air for
24 h and different organs were taken for histological
examinations.
BAL
After exposure, mice were anaesthetised by i.p. injection of a
mixture of xylazine and ketamine, and killed by exsanguina-
tion. BAL was performed by cannulating the trachea and
infusing the lungs 10 times with 1.0 mL of PBS without Ca
2+
and Mg
2+
. The BAL fluid (BALF) from lavage one and two, as
well as those from three to 10 were pooled and centrifuged
(4256gfor 20 min at room temperature). The cell-free super-
natant from lavage one and two was used for the biochemical
measurements. For each animal, the 10 cell pellets were unified
and resuspended in 1 mL of RPMI 1640 Medium (BioChrome,
Berlin, Germany) supplemented with 10% foetal calf serum
(Seromed, Berlin, Germany), and the number of living cells
was determined by the trypan blue exclusion method. The cell
differentials were performed on cytocentrifuge preparations
(May–Gru
¨nwald–Giemsa staining, 26200 cells counted).
Polymorphonuclear leukocytes were used as inflammatory
markers.
Biochemical analyses
Lactate dehydrogenase (LDH) activity was assayed spectro-
photometrically by monitoring the reduction of oxidised
nicotinamide adenine dinucleotide at 366 nm in the presence
of lactate. Total proteins were determined spectrophotomet-
rically at 620 nm applying the BioRad Protein Assay Dye
Reagent (Nr.500-0006; BioRad, Munich, Germany).
RNA purification
Frozen lungs were thawed in lysis buffer (supplied with RNA
isolation kit) and homogenised with a FastPrep FP120 cell
disrupter (BIO101/Savant; Qbiogene, Heidelberg, Germany)
for 40 s. The RNA was isolated by using the RNeasy kit
(Qiagen, Hilden, Germany). A DNase I treatment was
routinely performed. For each experimental condition, RNA
from the lungs of eight animals was prepared.
Microarray analysis
The GeneChip hybridisations were carried out by a service
provider (RZPD; Deutsches Ressourcenzentrum fu
¨r
Genomforschung GmbH, Berlin, Germany). The RNA was
quantified and equal amounts of RNA from eight lungs per
experimental condition were pooled. For sample preparation,
15 mg of total RNA were used. First-strand synthesis was
carried out by a T7-(dT)24 primer and Superscript II reverse
transcriptase (Invitrogen Life Technologies, Karlsruce,
Germany). Second-strand synthesis was performed according
to the Superscript Choice system. Biotin-labelled cRNA was
generated by an in vitro transcription reaction (BioArray
HighYield RNA Transcript Labeling Kit; Enzo, Farmingdale,
NY, USA). The fragmented cRNA was hybridised to the
murine U74Av2 GeneChip (Affymetrix, High Wycombe, UK)
representing 12,500 sequences (functionally characterised
sequences and expressed sequence tag clusters). The washing
procedure was carried out using the GeneChip Fluidics Station
(Affymetrix) according to the manufacturer’s protocol. The
hybridised cRNA was stained with R-phycoerythrin-strepta-
vidin (Molecular Probes, Karlsruhe, Germany) followed by an
antibody amplification procedure with a biotinylated anti-
streptavidin antibody (Vector Laboratories, Burlingame, CA,
USA). The chips were scanned with a GeneArray Scanner
(Hewlett Packard, Bo
¨blingen, Germany). Data were analysed
ULTRAFINE PARTICLE INHALATION IS PRO-INFLAMMATORY E. ANDRE
´ET AL.
276 VOLUME 28 NUMBER 2 EUROPEAN RESPIRATORY JOURNAL
with the Affymetrix Microarray Suite (MAS 5.0) and Microsoft
Excel software. The expression level of a single mRNA was
determined as the average fluorescence intensity among the
intensities obtained by 16-paired (perfect-matched and single
nucleotide-mismatched) probes consisting of 25-mer oligonu-
cleotides. To identify differentially expressed genes, genes that
were scored absent in the test sample (upregulated genes) or
absent in the control sample (downregulated genes) were
excluded. Genes that were differentially expressed between the
two clean air exposure times (4 and 24 h) were only included if
the comparison of the test sample to both control samples
revealed a two-fold or more change. The given fold changes for
genes with a difference call increase or decrease were used.
RT-PCR and real-time RT-PCR
For RT-PCR and real-time RT-PCR, 1 mg of total RNA was used
for the first-strand cDNA reaction using hexamer primer
(Promega Corporation, Mannheim, Germany) and Superscript
II reverse transcriptase (Invitrogen Life Technologies) at 42uC
for 50 min. PCR was performed on aliquots of this reaction in a
total volume of 25 mL. For real-time PCR, the first-strand cDNA
template, primer mix (as for RT-PCR) and SYBR Green PCR
Master Mix (Applied Biosystems) were used in a total volume
of 20 mL. Primers for ribosomal 18S mRNA were used as a
control for each template in every experiment. The reactions
were repeated with independently isolated RNA samples from
a single animal per experimental condition. Expression of
target genes was normalised to ribosomal 18S mRNA and
displayed as fold-change relative to the sample from the
control animals. The experiments were performed with the
ABI Prism 7000 SDS (Applied Biosystems).
In situ hybridisation
In situ hybridisation was performed with a single-stranded
digoxigenin-labelled RNA probe on paraffin-embedded lung
sections as described elsewhere [16]. For each experimental
condition, two mice were analysed. Sense and antisense probes
specific for osteopontin, galectin-3 and lipocalin-2 were
generated by RT-PCR and in vitro transcription.
Histology and immunohistochemistry
Lungs from exposed and control mice were fixed in buffered
formalin at an inflation pressure of 20 cmH
2
O and embedded
in paraffin. Slides from exposed and control lung tissues were
stained with polyclonal antibodies against galactin-3
(Cedarlane Laboratories Ltd, Hornby, Ontario, Canada),
osteopontin (R&D systems, Wiesbaden, Germany) and
lipocalin-2 (R&D systems). After staining with a biotinylated
secondary antibody (Vector laboratories Inc.) and streptavidin-
Vectastain Elite ABC-peroxidase reagents (Vector Laboratories
Inc.), slides were developed with diaminobenzidine (Vector
Laboratories Inc.). Negative controls tissues were stained
without primary antibody.
Analysis of protein secretion
Per assay, 50 mL of cell-free BALF were applied. Mouse-specific
enzyme-linked immunosorbent assays for osteopontin (Assay
Designs, Ann Arbor MI, USA), keratinocyte-derived chemo-
kine (KC), tumour necrosis factor (TNF)-a, interleukin (IL)-10,
IL-12p40 and IL-1b(R&D Systems) were used according to the
manufacturer’s instructions.
Statistical analyses
Values are reported as mean¡SE. ANOVA was used to
establish the statistical significance between the different
experimental groups. Tukey’s honestly significant difference
procedure was applied to differentiate significant differences
between the groups. Differences were considered significant at
p,0.05.
RESULTS
Characteristics of particles
UFCPs showed a monomodal number distribution with a
median particle size (equivalent mobility diameter) of
48.9¡1.8 nm, mean geometric SD of 1.53, and mean number
concentration of 7.7¡0.8610
6
cm
-3
. The average UFCP mass
concentration in the exposure chamber was measured as 380
mg?m
-3
(fig. 1). According to the particle spectra, 92.3% of the
generated particles were classified as ultrafine and 7.7% as
fine. It was estimated that 85% of the deposited mass in rodent
lungs was from UFPs and 15% from fine particles. This
estimation was carried out using the multiple-path particle
deposition model [17], assuming standard breathing condi-
tions. The particle mass distribution needed for this calculation
was derived from the number distribution reported above and
a particle density varying with size, calculated according to
NAUMANN [18].
BALF cell and protein parameters after inhalation of UFCP
After UFCP or clean air inhalation for 4 and 24 h, mice were
sacrificed and BAL was performed to analyse cellular
distribution and protein levels. The total number (0.45¡
0.05610
6
) of lavaged cells was unchanged after UFCP inhala-
tion (fig. 2a). Noticeably, the fraction of polymorphonuclear
cells (PMN) appeared slightly, but not significantly increased
after particle inhalation for 24 h (fig. 2b). The number of
macrophages in BALF was not increased after UFCP inhalation
(fig. 2c). LDH, a marker for cytotoxicity, was not altered, but
total protein concentration in BALF was significantly increased
after 24 h of particle inhalation (fig. 2d). This, together with the
slightly elevated PMN number, points to a very mild
0.01 0.10
Diameter × µm
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
Differential number density ×107 cm-3
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
ll
l
lll
l
l
l
l
l
l
l
ll
FIGURE 1. Average number size distribution of 77 samples of generated
ultrafine carbon particles (UFCP) in 24 h. UFCPs show a median particle size of
48.9¡1.8 nm and mean number concentration of 7.7¡0.8610
6
cm
-3
.Ofthe
generated particles, 92.3% are classified as ultra-fine (,0.1 mm).
E. ANDRE
´ET AL. ULTRAFINE PARTICLE INHALATION IS PRO-INFLAMMATORY
c
EUROPEAN RESPIRATORY JOURNAL VOLUME 28 NUMBER 2 277
inflammatory response within the lungs after UFCP inhalation
for 24 h.
Changes in gene expression after inhalation of UFCPs for
4h
According to analysis of the hybridisation signals (MAS5.0
software; Affymetrix), between 45.6% (4-h clean air) and 47.2%
(24-h clean air, 24-h UFCP inhalation) of 12,422 transcripts
represented on the chip were detected (detection: p,0.04;
signal: .30).
Among the genes that were significantly expressed (detection
p-value ,0.04; signal .30) and showed significant changes
(p,0.003) according to the Affymetrix MAS5.0 software, the
current authors identified 157 (1.3%) genes that were upregu-
lated after 4 h of inhalation of UFP and 125 (1%) down-
regulated genes. From these genes, seven were induced two-
fold or more, whereas no gene was more than two-fold
downregulated (table 1). Two genes, granzyme A and a 59–39
exonuclease, were 1.8-fold downregulated.
From the two-fold and more upregulated genes, five were heat
shock proteins (hsp; table 1). The highest induction, four-fold,
was detected for hspa1A (the mouse homologue of hsp70), a
hsp described to interact with apoptosis-inducing factor. The
other upregulated genes with chaperone activity were hsp105
(2.6-fold induction), suppression of tumorigenicity-13 (2.2-
fold induction), stress-induced phosphoprotein-1 (two-fold
induction) and osmotic stress protein 94 (two-fold induction).
A 2.2-fold induction was detected for carcinoembryonic
antigen-related cell adhesion molecule-2.
Furthermore, the mRNA for prolyl-4-hydoxylase alpha (I)-
subunit, a key enzyme in the biosynthesis of collagens was
upregulated two-fold. Another eight genes fulfilled the criteria
for an increase of expression (between 1.5-fold and two-fold
induction). Amongst them WEre hsp47 and hsp40 and two
molecules associated with electron transport, cytochrome b-
561 and PFTAIRE protein kinase-1.
Changes in gene expression after inhalation of UFP for 24 h
After UFP inhalation for 24 h, expression of 236 (1.9%) genes
was increased and 307 (2.5%) genes were decreased compared
with the two clean air controls (detection p-value ,0.04; signal
.30; p,0.003) according to the Affymetrix MAS5.0 software.
From these affected genes, 17 were induced and six repressed
two-fold or more (table 1). Noticeably, all two-fold or more
upregulated genes were related to inflammatory processes. In
particular osteopontin, lipocalin-2 (24p3) and galectin-3 have
been implicated to be important mediators of inflammation
and to play a role as an integrated part of the body’s defence
system [19–21].
The highest increase, a 5.4-fold change, was detected for serum
amyloid A3, a major acute-phase response protein secreted by
activated macrophages. A 3.8-fold induction was detected for
0.00
0.10
0.20
0.30
0.40
0.50
0.60a)
Total cell number ×106
0.0
0.2
0.4
0.8
1.0
1.2
1.4
b)
PMN %
0.6
90
91
93
95
96
98
100c)
Macrophages %
92
94
97
99
0
50
100
150
200
250
d)
Protein µg·mL-1
24 h
4 h
24 h4 h
*
FIGURE 2. Results of ultrafine carbon particle (UFCP) inhalation for 4 and 24 h in a) total cell number, b) polymorphonuclear cells (PMN), c) macrophages, and d)
protein in bronchoalveolar lavage fluid. h: control group; &: UFCP exposed subjects. Data are presented as mean¡SE from eight animals. *: p,0.05.
ULTRAFINE PARTICLE INHALATION IS PRO-INFLAMMATORY E. ANDRE
´ET AL.
278 VOLUME 28 NUMBER 2 EUROPEAN RESPIRATORY JOURNAL
the mRNA of the tumour growth factor-bmember activin B.
Although the expression level was comparatively low, a three-
fold increase was detected for prostaglandin-endoperoxide
synthase 1 (cyclooxygenase-1). The mRNA expression of
leucine-rich a-2-glycoprotein was 2.8-fold increased. Tissue
inhibitor of metalloproteinase (TIMP)-1, another factor
involved in extracellular matrix remodelling, appeared 2.4-
fold upregulated. Expression of two protease inhibitors was
2.4-fold and 2.2-fold increased: serine protease inhibitor-2,
representing the homologue of human a-antichymotrypsin
and the protein kinase-120 precursor, a member of the inter-a-
trypsin inhibitor superfamily. Additionally, the later two genes
that are involved in acute phase response and blood coagula-
tion, the mRNA for coagulation factor III (tissue factor), was
2.2-fold upregulated. Another gene involved in coagulation,
thrombospondin-1, an extracellular matrix protein that is
secreted by different cell types, including endothelial cells,
fibroblasts, smooth muscle cells and type II pneumocytes, was
2.4-fold induced by UFCP exposure. A two-fold induction was
detected for keratin complex-1 gene 19, a specific cytoskeletal
component of simple epithelia, including bronchial epithelial
cells. Another 11 genes were less than two-fold but .1.5-fold
induced (data not shown), amongst them the extracellular
matrix proteins tenascin-C and TIMP-2 and the suppressor of
cytokine signalling-3.
Molecules repressed by UFCP inhalation were cytochrome P1-
450, receptor-type protein tyrosine phosphatase, translation
initiation factor-2C, nuclear factor I/C, ryanodine receptor
RyR1 and E-selectin ligand-1. To validate the results of the
microarray experiments, the current authors performed real-
time RT-PCR on lung RNA from single animals (fig. 3). For all
genes examined, the results obtained were confirmed by
microarray hybridisation. The changes were in the same order
of magnitude as detected by expression profiling.
Inhalation of UFP leads to macrophage activation
Several genes, e.g. cytokines and cell adhesion molecules,
which were found to be upregulated in the present expression
TABLE 1 Changes in gene expression after inhalation of ultrafine particles for 4 h and 24 h
Identifier Name/description Gene symbol 4 h 24 h
Fold induction
M12571 Heat shock protein, 1A Hsp1a 4.0
#
1.2
L40406 Heat shock protein, 105 kDa Hsp105 2.6
#
1.1
AF101164 CEA-related cell adhesion molecule-2 Ceacam2 2.2
#
1.3
AW124318 Suppression of tumorigenicity-13 St13 2.2
#
1.0
U27830 Stress-induced phosphoprotein-1 Stip1 2.0
#
1.2
U16162 Prolyl 4-hydroxylase alpha(I)-subunit P4ha1 2.0
#
1.1
U2392 Osmotic stress protein-94 Osp94 2.0
#
0.9
X03505 Serum amyloid A-3 Saa3 1.1 5.4
#
X69620 Inhibin beta-B Inhbb 2.5
"
3.8
#
X13986 Osteopontin, secreted phosphoprotein Spp1 1.1 3.4
#
M34141 Prostaglandin-endoperoxide synthase-1 Ptgs1 1.0 3.0
#
AW230891 Leucine-rich alpha-2-glycoprotein-1 Lrg1 1.4 2.8
#
V00755 Tissue inhibitor of metalloproteinases-1 Timp1 1.3 2.4
#
M64086 Spi2 proteinase inhibitor (spi2/eb4) Serpina3n 1.1 2.4
#
M62470 Thrombospondin-1 Thbs1 3.0
+
2.4
#
X81627 24p3, lipocalin2 Lcn2 1.0 2.4
#
L41352 Amphiregulin Areg 1.2 2.4
#
AV300608 SH2 domain binding protein-1 Sh2bp1 0.8 2.2
#
M26071 Coagulation factor III F3 1.1 2.2
#
AF023919 PK-120 precursor itih-4 0.9 2.2
#
M15131 Interleukin-1bIL1b0.9 2.0
#
M36120 Keratin complex-1, acidic gene-19 Krt1-19 1.0 2.0
#
M35970 Expressed in nonmetastatic cells-1 Nme1 0.8 2.0
#
X16834 Galectin-3, Mac-2 Lgals3 1.0 2.0
#
Fold repression
K02588 Cytochrome P450,1a1 Cyp1a1 1.2 2.6
#
X58289 Protein tyrosine phosphatase, receptor type B Ptprb 1.6 2.4
#
Y07693 Nuclear factor I/C Nfic 1.2 2.4
#
D38216 RyR1 skeletal muscle ryanodine receptor Ryr1 1.0 2.2
#
X84037 E-selectin ligand-1, golgi apparatus protein-1 Glg1 1.4 2.0
#
AI152867 Eukaryotic translation initiation factor-2C Eif2c2 1.6 2.0
#
#
: genes induced or repressed two-fold or more after 4-h or 24-h ultra-fine carbon particle inhalation;
"
: excluded from analysis because absent in test sample (see
Methods section);
+
: excluded from analysis because fold change between clean air controls (4 h and 24 h) two-fold or more.
E. ANDRE
´ET AL. ULTRAFINE PARTICLE INHALATION IS PRO-INFLAMMATORY
c
EUROPEAN RESPIRATORY JOURNAL VOLUME 28 NUMBER 2 279
study, are known to be involved in inflammatory cell
activation. To analyse their expression and cellular distribution
in the lung, exemplary galectin-3, osteopontin and 24p3 were
chosen for in situ hybridisation experiments. Lungs of mice
exposed for 24 h to either UFCP or clean air were compared.
In lungs from clean air control animals, galectin-3 expression
was hard to detect by in situ hybridisation. Only a few single
alveolar epithelial cells were weakly labelled (fig. 4). After
inhalation of UFCP for 24 h, a positive staining of alveolar
macrophages and alveolar epithelial cells was clearly recogni-
sable. No signal was detected after hybridisation with the
galectin-3 sense probe.
In situ hybridisation experiments with an osteopontin-specific
antisense probe on mouse lung revealed positive staining of
alveolar macrophages after 24-h inhalation of UFCP (fig. 4). In
lungs from clean air control mice, after hybridisation with the
sense probe, no staining was detected (fig. 4).
Lipocalin-2 (24p3) expression was detected in alveolar wall
epithelial cells from lungs after 24-h UFCP inhalation (fig. 4).
No staining was seen in lung sections from clean air controls
and after hybridisation with the sense probe.
These results were confirmed by immunohistochemistry. In
alveolar macrophages of control animals, a slight staining was
detected by anti-osteopontin and anti-galectin-3 antibodies.
This signal increased after UFCP particle inhalation (24 h).
Both antibodies revealed a staining in bronchiolar cells.
However, this signal did not increase after UFCP inhalation.
Lipocalin-2 protein expression was detected in lung septal cells
of control animals. This signal was increased after particle
inhalation (fig. 4).
Secretion of osteopontin into BALF after inhalation of
UFCPs
Osteopontin is a secreted protein, which is found in extra-
cellular fluid and serum. To determine whether newly
expressed osteopontin protein is enriched in BALF, an
osteopontin-specific immunoassay with BALF samples from
UFCP- and clean air-exposed mice was performed (fig. 5).
BALF samples from eight animals of each experimental group
were pooled. In clean air control, 18 ng?mL
-1
osteopontin
protein was detected. After UFCP inhalation for 24 h,
osteopontin protein concentration increased to 35 ng?mL
-1
.In
concordance with the current authors’ chip and in situ mRNA
expression data, osteopontin protein is about two-fold
enriched in BALF from animals after 24-h UFCP inhalation.
No increase could be observed after UFCP inhalation for 4 h.
For further characterisation of the inflammatory response to
UFCP exposition, the secretion of cytokines and chemokines,
known as inflammatory markers, into BALF was examined.
KC, the mouse homologue of IL-8, can be detected in BALF by
ELISA. KC protein levels in BALF from control animals and
after 4-h particle inhalation did not differ. After particle
inhalation for 24 h, KC concentration increased significantly
from 9.0¡0.5 to 14.4¡0.8 pg?mL
-1
(fig. 5). TNF-acould not be
detected in BALF. IL-1bshowed a mild but not significant
increase from 1.2¡0.2 to 1.5¡0.2 pg?mL
-1
after 24-h UFCP
inhalation. IL-10 protein levels decreased from 0.7¡0.2 to
0.4¡0.2 pg?mL
-1
, again not statistically significant, while IL-
12p40 increased from 4.1¡0.4 to 5.2¡0.3 pg?mL
-1
.
DISCUSSION
UFP have been identified in epidemiological studies as an
important factor inducing adverse health effects, such as
cardiovascular complications and aggravation of respiratory
diseases [1–3]. In animal experiments, intratracheal instillation
of UFP beyond a certain mass and surface area dose has been
shown to cause acute pulmonary inflammation [22, 23].
Exposures of healthy and asthmatic subjects to 25 mg?m
-3
UFCP did not cause any detectable changes in airway
inflammation [15]; however, they caused alterations in the
expression pattern of adhesion molecules on blood cells,
indicating increased retention of leukocytes in the pulmonary
vascular bed [24]. The present study provides evidence that
inhalation of elemental carbon particles at doses .40-fold
above environmental relevant doses induces mild pro-inflam-
matory processes within 24 h of exposure. Beyond this, a
comprehensive expression profiling approach gives an insight
into early pathways leading to these processes.
Oxidative stress, caused by ambient particles deposited in the
lung, is believed to be the main factor driving inflammatory
and noxious effects [25]. Even though the mechanism of the
generation of oxidative stress is not understood, it appears to
be related to the surface properties and the large particle
surface area of UFP. Thus, cell–particle interactions in the lung
might lead to the activation of pro-inflammatory gene
transcription via the induction of nuclear import of redox-
sensitive transcription factors, such as nuclear factor (NF)-kb
and activator protein-1. Several studies suggest that the
particle-bound nonelemental carbon impurities account for
the surface reactivity of UFP. LIet al. [26] and XIA et al. [27]
observed a direct correlation between oxidative stress and the
organic carbon content of UFP, in particular the polycyclic
aromatic hydrocarbons and quinones. DICK et al. [28] showed
that the property of UFP to cause oxidant damage is related to
their ability to generate surface free radicals. In this process,
transition metals may be involved via Fenton chemistry [29].
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Lipocalin-2 Osteopontin Galectin-3
Fold induction
FIGURE 3. Validation of differential gene expression induced by inhalation of
ultrafine carbon particles (UFCPs) by real-time RT-PCR. BALB/cJ mice were
exposed to UFCP for 4 h (h) and 24 h (&). RNA preparation and RT-PCR were
carried out as described in the Materials and Methods section. Data are presented
as the mean of eight samples per experimental group.
ULTRAFINE PARTICLE INHALATION IS PRO-INFLAMMATORY E. ANDRE
´ET AL.
280 VOLUME 28 NUMBER 2 EUROPEAN RESPIRATORY JOURNAL
To mimic ambient conditions, the use of ambient UFP should
be the most realistic exposure. However, apart from problems
of nonreproducibility due to varying compositional changes in
ambient particle samples and the bias arising from sampling
artefacts, the aerosolisation of collected particles at the
ultrafine range is, at present, very problematic. Like
a) b) c)
d) e) f)
g) h) i)
j) k) l)
AM
AS
AM
AS
AS
AS
AM
SC
SC
AM
AS
AM
AM
AS
AS
SC
SC
FIGURE 4. Osteopontin (a, d, g and j), galectin-3 (b, e, h and k) and lipocalin-2 (c, f, i and l) were upregulated after inhalation of ultrafine carbon particles (UFCPs).
Paraffin-embedded lung sections of control animals (a–c) and animals after 24-h UFCP inhalation (d–l) were hybridised to Dig-labelled antisense probes (a–i) or antibodies (j–
l). In situ hybridisation with an osteopontin antisense probe revealed almost no staining in control sections; however, after inhalation, alveolar macrophages (AM) showed a
positive signal (d and g). This result was confirmed by immunohistochemistry with a polyclonal osteopontin antibody (j). According to in situ hybridisation (e and h) and
immunohistochemistry (k), galectin-3 is upregulated after UFCP particle inhalation in AM. Lipocalin-2 expression was not detected in control animals by in situ hybridisation
(c). After particle inhalation, a signal was detected by in situ hybridisation (f and i) and immunohistochemistry (l) in lung septal cells (SC). AS: alveolar space. Scale bar550 mm
(a–f), 25 mm (g–i) and 10 mm (j–l).
E. ANDRE
´ET AL. ULTRAFINE PARTICLE INHALATION IS PRO-INFLAMMATORY
c
EUROPEAN RESPIRATORY JOURNAL VOLUME 28 NUMBER 2 281
combustion-derived UFPs, which represent the major compon-
ent of urban UFPs, the PALAS-generated soot particles (UFCP)
that were used in the present study represent carbonaceous
nanoparticles. Although urban particles contain trace amounts
of organic compounds and metals, the current study focused
on the effects of pure carbon particles.
The essential advantage of laboratory-made UFCPs is the
possibility to produce defined spectra of particle sizes with
similar surface and organic content properties. Thus, UFCPs
are produced with organic mass contributions ,5% [14, 15].
PALAS soot particles are known to have similar physical
properties like freshly generated diesel soot, e.g. an electron
spin resonance-signal characteristic for organic carbon-centred
radicals [30, 31]. This feature might be important for hydroxyl
generation and downstream effects on cellular oxidative stress
[32]. Recently, BECK-SPEIER et al. [31] demonstrated the high
oxidative potential of UFCP in a cell-free system, as well as the
production of the oxidative stress marker 8-isoprostane in
exposed alveolar macrophages.
In the present study, a UFP concentration of 380 mg?m
-3
was
used. This is 10–100-fold higher than ambient levels measured
in polluted urban areas [8]. This mass concentration is in the
range (170–1660 mg?m
-3
) applied in other toxicological studies
identifying only mild inflammatory responses to UFP exposure
[33, 34]. The pro-inflammatory effects described in the current
study could also be detected less pronounced at a lower
particle burden of 180 mg?m
-3
,e.g. the release of osteopontin in
BALF increased 1.5-fold after 24-h inhalation of 180 mg?m
-3
UFCPs. Although this mass concentration is still high in
comparison to ambient UFP measured at urban sites, e.g. in
Munich (Luise-Kiesselbach-Platz, autumn 1998, 8 mg?m
-3
;E.
Karg, GSF-National Research Center for Environment and
Health, Neuherberg, Germany, personal communication), it
should be notified that the particle effects described were
already detectable after acute exposures and have been found
in young and healthy animals. Aged and compromised
animals are expected to have a stronger response to particle
exposure. According to this, ELDER et al. [35] exposed different
groups of healthy and compromised mice to 110 mg?m
-3
UFCP
for 6 h and found significant lung inflammatory response only
in aged, emphysematous animals, but not in healthy mice.
In the present study, overall histology of the lung did not give
any indication for inflammation or pathological changes.
Examination of BALF showed a small increase in PMN cell
number after UFCP inhalation (4.0¡1.2610
3
) compared with
clean air controls (2.6¡0.5610
3
), suggesting a very mild
inflammatory cellular response (fig. 2). This was reinforced
by a moderate but significant increase in BALF protein
concentration after 24 h of UFCP inhalation. These results fit
into a recently published study by FRAMPTON et al. [15] where
healthy and mildly asthmatic volunteers exposed for 2 h to
UFCP (10 mg?m
-3
and 25 mg?m
-3
) did not reveal any significant
effects on pulmonary functions or markers for airway
inflammation.
However, DNA microarray profile analysis already revealed a
remarkable upregulation of mRNA expression after 4 h of UFP
inhalation. Five out of seven significantly induced genes
encode hsp and proteins with chaperone activity. This
induction is transient and no longer detected after UFCP
inhalation for 24 h. Based on their ability to chaperone
antigenic peptides, hsp can elicit specific cellular adaptive
immune responses [36]. These immunmodulatory properties
of hsp are likely to be responsible for the initiation of the
immune response after particle inhalation. The current results
support this hypothesis, as they revealed the subsequent
induction (after 24-h UFP inhalation) of several genes known
to be regulated via the NF-kB signalling pathway. Amongst
them are galectin-3 and lipocalin-2 (24p3), whereas osteopon-
tin has recently been shown to activate NF-kBvia induction of
induced-kB phosphorylation and degradation through inhibi-
tor of NF-kb kinase. Additionally, the current authors noted
transiently increased transcript levels of the alpha (I)-subunit
of prolyl-4-hydoxylase after 4 h of particle inhalation. Prolyl-4-
hydoxylase is a key enzyme in the biosynthesis of collagens.
Collagen synthesis is increased during connective tissue
remodelling that occurs in allergic asthma. In accordance to
the induction of hsp70 by UFCP exposure that is reported in
the present findings, it was recently shown that exposure of
human alveolar epithelial cells to ultrafine carbon black
particles induced hsp70 as a result of oxidative stress [37].
0
5000
10000
15000
20000
25000
30000
35000
40000
a)
OPN pg·mL-1
0
2
4
6
8
12
14
16
18
b)
KC pg·mL-1
10
Control 4 h 24 h
*
FIGURE 5. a) Osteopontin (OPN) and b) keratinocyte-derived chemokine (KC)
concentration in bronchoalveolar lavage fluid (BALF) after particle inhalation.
Osteopontin levels in BALF pools (eight animals per experimental group) and KC
levels in unpooled BALF samples were measured by ELISA. Inhalation of ultrafine
carbon particles (UFCPs) increases osteopontin concentration from 18 ng?mL
-1
(control) to 35 ng?mL
-1
(24-h UFCP). An increase in KC concentration from
9pg?mL
-1
to 15 pg?mL
-1
was measured after 24-h particle inhalation. Data are
presented as the mean¡SE of eight separate experiments. *: p,0.05.
ULTRAFINE PARTICLE INHALATION IS PRO-INFLAMMATORY E. ANDRE
´ET AL.
282 VOLUME 28 NUMBER 2 EUROPEAN RESPIRATORY JOURNAL
The highest induction after UFCP exposure for 24 h was
detected for serum amyloid A (SAA)-3, the predominant SAA
isoform expressed extrahepatically. SAA3 is known to be
secreted by macrophages after lipopolysaccharide (LPS) treat-
ment. The upregulation of SAA3 after particle inhalation
indicates an inflammatory response, although the increase is
not as high as after LPS induction.
According to the current results, osteopontin seems to play a
central role in the induction of pro-inflammatory processes by
UFP inhalation. Osteopontin is a secreted and glycosylated
phosphoprotein that contains the arginine-glycine-aspartic
acid (RGD) integrin-binding domain. Osteopontin protein
has chemokine/cytokine-like properties and is among the
most abundantly expressed proteins in a wide range of lung
diseases, such as fibrosis, sarcoidosis and lung carcinoma [19,
38]. In the present study, in situ hybridisation experiments
revealed upregulation of osteopontin expression in alveolar
macrophages and a two-fold increase of secreted osteopontin
protein was detected by ELISA in BALF after UFP inhalation.
In accordance with the current in situ results, immunohisto-
chemistry revealed osteopontin protein expression in alveolar
macrophages.
Recently, it was shown that osteopontin is implicated in
experimental particle-induced lung disease using a titanium
dioxide exposure model in a rat. Under exposure conditions,
which resulted in fibroproliferative lung disease (long-term
exposures at particle concentrations .10 mg?m
-3
), rats had
significant increases in total lung osteopontin mRNA expres-
sion and increased levels of osteopontin protein in BALF prior
to the development of lesions [40]. Osteopontin is secreted by
activated macrophages, leukocytes and activated T-lympho-
cytes, and is present in extracellular fluid at sites of
inflammation, and in the extracellular matrix of mineralised
tissues. In the immune system, osteopontin plays a role in
chemotaxis, leading to the migration of macrophages and
dendritic cells to sites of inflammation. Osteopontin protein
interacts with a variety of cell surface receptors, including the
avb3, avb1, a41b,a8b1 and a9b1 integrins, as well as CD44.
Binding of osteopontin protein to these cell surface receptors
stimulates cell adhesion, migration, and specific signalling
functions. The major integrin-binding site of osteopontin is the
RGD integrin-binding motif, which is required for the
adherence of many cell types. ASHKAR et al. [39] have
demonstrated a differential regulation of macrophage IL-12
and IL-10 expression by osteopontin, which affects type-1
immunity. Interaction between osteopontin protein and
macrophages is mediated through avb3 integrin and CD44.
The phosphorylation-dependent interaction between osteo-
pontin protein and its integrin receptor stimulates IL-12
expression, whereas the phosphorylation-independent inter-
action with CD44 inhibits IL-10 expression by macrophages. In
the present study, IL-10 protein concentration was slightly
diminished in BALF after UFCP inhalation, as was demon-
strated by ELISA. Moreover, IL-12 concentration was raised
slightly but not significantly after particle inhalation.
To support the current hypothesis that UFP inhalation triggers
inflammatory processes,the expression of other known markers
for inflammation in relation to particle inhalation was examined.
A significant increase of KC protein concentration in BALF
was detected, although the mRNA expression was not
upregulated. KC, a CXC chemokine, is released by activated
macrophages and injured epithelial cells and acts as a
chemoattractant for neutrophils. It has also been implicated
in the accelerated release of neutrophils in response to
inflammation. In the lung, the release of matrix-bound KC
by shedding is important for the migration of neutrophils
from the interstitium to the alveolar space [41].
Lipocalin-2 (24p3, neutrophil gelatinase-associated lipocalin
(NGAL)) is upregulated after 24-h UFP inhalation in epithelial
cells of the alveolar wall. Lipocalins are small secreted proteins
that play a role in diverse biological processes through binding
of small hydrophobic molecules, encompassing retinoids, fatty
acids, prostaglandins and odorants [20]. Lipocalin-2 expression
is induced by different stimuli in diverse tissues and cell lines.
It was identified as a LPS-induced protein secreted by mouse
macrophages [42]. Recently, it was demonstrated that
lipocalin-2 was expressed in bronchial goblet cells as well as
in alveolar type II pneumocytes, and that the expression is
increased in bronchial and alveolar cells of inflamed human
lungs [42]. The increase of lipocalin-2 mRNA expression is
induced by IL-1bvia a NF-kB-dependent pathway in the
human type II pneumocyte-derived cell line A549. No
induction of lipocalin-2 transcript was seen in A549 cells after
stimulation with LPS, TNF-aand IL-6. In the mouse lung,
liocalin-2 was detected by in situ hybridisation in septal cells
after particle inhalation (fig. 4). No hybridisation signal was
detected in bronchial cells and in control animals. It remains to
be investigated in further in vitro experiments if the lipocalin-2
expression in alveolar epithelial cells is upregulated directly by
UFPs that are supposed to enter these cells because of their
small size [11, 43], or if the increased expression and secretion
of a cytokine, such as IL-1b, by alveolar macrophages is
necessary for lipocalin-2 upregulation. Even if the function of
lipocalin-2 is not known, increased serum levels of the human
homologue NGAL have been related to the clinical manifesta-
tion of cardiovascular disease [44]. Recently, a putative
function of this lipocalin as a modulator of the inflammatory
response has been suggested. Under normal circumstances,
granulocytes have a short life span and die by apoptosis. In
many chronic inflammatory responses, such as bronchial
asthma or lung fibrosis, delayed apoptosis of granulocytes
leads to their accumulation at sites of inflammation, where
they cause tissue damage via the release of toxic mediators [45].
Interestingly, osteopontin and lipocalin-2 seem to play a role in
the pathogenesis of atherosclerosis [44, 46]. People suffering
from this cardiovascular disease are one of the groups
considered particularly susceptible to the effects of airborne
particles.
Galectin-3 is a b-galactoside-binding lectin, implicated in
inflammatory responses as well as in cell adhesion. The role
of galectin-3 as an adhesion molecule for neutrophil extra-
vasation during streptococcal pneumonia has been recently
demonstrated [47]. Recently, the contribution of galectin-3 to
phagocytosis by macrophages has been shown [48]. Galectins,
in particular galectin-1 and galectin-3, play a role as regulators
of inflammatory processes. It was demonstrated that alveolar
infection with Streptococcus pneumoniae, but not with Escherichia
coli, induces the production and secretion of galectin-3 by
E. ANDRE
´ET AL. ULTRAFINE PARTICLE INHALATION IS PRO-INFLAMMATORY
c
EUROPEAN RESPIRATORY JOURNAL VOLUME 28 NUMBER 2 283
alveolar macrophages [48]. Accumulation of galectin-3 in the
alveolar space of streptococcus-infected lungs correlates with
the onset of neutrophil extravasation. In vitro assays revealed
the ability of galectin-3 to mediate the adhesion of neutrophils
to endothelial cells. These data suggest that galectin-3 is
implicated in b
2
integrin-independent neutrophil extravasation
induced by S. pneumoniae; however, not in b
2
integrin-
dependent pulmonary infection induced by E. coli. Induction
of galectin-3 expression in alveolar macrophages by UFP
inhalation might maintain neutrophil extravasation in a similar
way.
In conclusion, the current data provide evidence that inhala-
tion of ultrafine carbon particles triggers a biphasic pro-
inflammatory process in the lungs of healthy mice. After a
short exposure to ultrafine particles, heat shock proteins are
transiently upregulated and might be responsible for the
subsequent activation of macrophages. The present study
demonstrates that inhalation of ultrafine carbon particles
induces the upregulation of osteopontin and galectin-3
expression in alveolar macrophages. Osteopontin, galectin-3
and lipocalin-2 are secreted proteins and their regulation might
serve as a useful marker for inflammatory processes induced
by particle inhalation in further studies.
ACKNOWLEDGEMENTS
The authors would like to thank G. Ferron and O. Schmid for
help with the application of the rodent deposition model and
critical reading of the manuscript.
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