1 Edinburgh Lung and the Environment Group Initiative/Colt Laboratories, Department of Medical and Radiological Sciences, University of Edinburgh, Edinburgh EH8 9AG; and 2 School of Life Sciences, Napier University, Edinburgh EH10 5DT, Scotland, United Kingdom
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ABSTRACT |
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There is now considerable
evidence for an association between the levels of particulate air
pollution [particulate matter <10 µm in aerodynamic diameter
(PM10)] and various adverse health endpoints. The
release of proinflammatory mediators from PM10-exposed macrophages may be important in stimulating cytokine release from lung
epithelial cells, thus amplifying the inflammatory response. A549 cells
were treated with conditioned media from monocyte-derived macrophages
stimulated with PM10, titanium dioxide (TiO2),
or ultrafine TiO2. We demonstrate that only conditioned
media from PM10-stimulated macrophages significantly
increased nuclear factor-B and activator protein-1 DNA binding,
enhanced interleukin-8 (IL-8) mRNA levels as assessed by RT-PCR, and
augmented IL-8 protein levels, over untreated controls. Furthermore,
PM10-conditioned media also caused transactivation of IL-8
as determined by an IL-8-chloramphenicol acetyl transferase reporter.
Analysis of these conditioned media revealed marked increases in tumor
necrosis factor-
(TNF-
) and protein levels and enhanced
chemotactic activity for neutrophils. Preincubation of conditioned
media with TNF-
-neutralizing antibodies significantly reduced IL-8
production. These data suggest that PM10-activated
macrophages may amplify the inflammatory response by enhancing IL-8
release from lung epithelial cells, in part, via elaboration of
TNF-
.
particulate matter; tumor necrosis factor-; nuclear factor-
B; cytokine networking; interleukin-8
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INTRODUCTION |
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THERE ARE NOW WELL-ESTABLISHED correlations between increased exacerbations of respiratory diseases, cardiopulmonary morbidity, and mortality and the levels of particulate air pollution [particulate matter <10 µm in aerodynamic diameter (PM10)] (36, 42). Attention has focused on transition metals (4, 15, 22), ultrafine particles (43), and endotoxin (3) as components of PM10 that may be intrinsic to its toxicity. Each of these individual components of PM10 has been shown to invoke an inflammatory response after exposure in animal models (11, 28, 37, 51). A critical component of the inflammatory response to particles in the lungs is the release of cytokines from activated macrophages and lung epithelial cells, resulting in neutrophil recruitment.
Interleukin (IL)-8 is a nonglycosylated 8-kDa protein synthesized by a
variety of cells, including pulmonary epithelial cells and alveolar
macrophages. It is a C-X-C chemokine that induces the migration of
polymorphonuclear leukocytes from the bloodstream to sites of
inflammation (24). IL-8 gene regulation is controlled at
the transcriptional level by a combination of the redox-sensitive transcription factors, nuclear factor-B (NF-
B) and activator protein-1 (AP-1), as well as CCAAT/enhancer-binding protein
(C/EBP)/NF/IL-6, depending on the cell type (26,
32). Previous studies have shown induction of IL-8 mRNA
and increased protein levels in response to mediators of oxidative
stress, including hyperoxia, hydrogen peroxide (6), tumor
necrosis factor-
(TNF-
), IL-1
(25), and asbestos
(46).
The levels of environmental ultrafine particles are associated with adverse health effects, as shown by a recent human cohort study linking increased levels of ambient ultrafine particulates (range 0.01-2.5 µm) with decreased peak expiratory flow in nonsmoking asthmatics (35). The damaging effects of ultrafine particles are attributed to such features as size, free radical generation, and surface area. Additionally, these particles are less readily cleared than fine particles of the same material (11), thereby prolonging interaction with the lung epithelium and possibly potentiating the damage. In in vitro studies and after inhalation by rodents, ultrafine particles (<100 nm in diameter) have been shown to induce oxidative stress and inflammation, resulting in neutrophilia in the bronchoalveolar lavage fluid (11, 29). Furthermore, diesel exhaust and PM10 also induce the release of the proinflammatory cytokines IL-6 and IL-8 from bronchial epithelial cells (23, 40).
Many studies have focused on the direct effects of environmental
particulates on either macrophages (3) or lung epithelial cells in vitro (4, 22, 23, 40). However, whether
PM10-activated macrophages or their products cause
increases in signaling pathways leading to proinflammatory mediator
production by alveolar epithelial cells has not been studied. We
hypothesized that macrophages exposed to PM10 in close
proximity to epithelial cells release proinflammatory mediators, which
then increase IL-8 gene expression and protein release from the
epithelial cells. In this study, A549 cells were exposed to conditioned
media from peripheral blood-derived macrophages stimulated with
PM10, TiO2, or ultrafine (UF) TiO2
to determine the effect of macrophage-derived mediators on IL-8
expression by epithelial cells. The latter two particles were used as
representatives of the fine and ultrafine components of
PM10, respectively. We found that conditioned media from
PM10-stimulated macrophages induced IL-8 gene
transactivation and protein release from A549 cells and were
chemotactic for neutrophils. No effects were observed with either
TiO2- or UFTiO2-conditioned media over control
levels. Further studies suggested that increased IL-8 production was
mediated by NF-B through a mechanism partially involving TNF-
in
the conditioned media.
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MATERIALS AND METHODS |
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Environmental particles. PM10 was obtained from collection filters from the London and Edinburgh air particulate monitoring stations, as previously described (15). Filters were suspended in Dulbecco's 1× phosphate-buffered saline (PBS) (Sigma, Dorset, UK), vortexed, and sonicated for 3 min. The concentration of PM10 was determined spectrophotometrically by comparing turbidity to a standard curve of carbon black particles at an optical density of 360 nm (22). The physiochemical properties of Edinburgh and London PM10 have been previously characterized (15, 39). The level of endotoxin in PM10 was semiquantitated using the E-toxate kit (Sigma, Poole, UK) and a lipopolysaccharide (LPS) standard curve. Both fine (200 nm) and ultrafine TiO2 (20 nm) fractions were obtained from Tioxide Europe (London, UK) and Degussa-Hüls (Cheshire, UK), respectively, and suspended in RPMI containing 2% autologous serum and sonicated for 3 min. Both TiO2 and UFTiO2 were prepared to a stock concentration of 1 mg/ml.
A549 cell culture.
The type II human alveolar-like epithelial cell line A549 was grown in
DMEM containing 10% FCS, 2 mM glutamate, and 100 IU · ml
penicillin1 · 100 µg/ml
streptomycin
1. For this study, A549 cells were seeded at
a density of 110,625 cells per well in 24-well culture plates and
treated at confluency. Unless otherwise indicated, all reagents were
obtained from Sigma.
Neutrophil and monocyte isolation and treatment.
Neutrophils were isolated from venous blood obtained from healthy
volunteers as previously reported (8). In brief, sodium citrate (Sigma)-anticoagulated blood was sedimented with 6% dextran 500, and the resulting leukocyte-rich layer was separated over an
isotonic discontinuous PBS/Percoll gradient (Amersham Pharmacia Biotech, Buckingham, UK) by centrifugation. The neutrophils obtained were >98% viable as assessed by trypan blue exclusion. The
mononuclear fraction was also collected from the Percoll gradient. Four
million cells per milliliter of RPMI media without serum were plated in 24-well plates, and nonadherent cells were washed off after 1 h
with warmed 1× PBS and replaced with RPMI media containing 10% autologous donor serum. Adherent monocytes were cultured for 5 days to
allow for differentiation into macrophages. Monocyte-derived macrophages were exposed to nontoxic concentrations of TNF- (10 ng/ml), PM10 (100 µg/ml), TiO2 (100 µg/ml),
and UFTiO2 (100 µg/ml) for 24 h at 37°C in the
presence of 2% autologous serum. Conditioned media from these
macrophages were collected and stored at
20°C until further analysis.
Stimulation of A549 cells with macrophage-conditioned media.
Culture media from nonexposed macrophages and those stimulated with
TNF- (10 ng/ml), PM10 (100 µg/ml), TiO2
(100 µg/ml), or UFTiO2 (100 µg/ml) for 24 h were
centrifuged at 13,000 rpm for 5 min, and 0.5 ml was transferred onto
A549 cells grown in 24-well culture plates for 4 h. These
conditioned media were centrifuged to eliminate as much as possible any
remaining particles in the culture media. After 4 h of exposure to
A549 cells, the macrophage-conditioned media were washed with warm 1×
PBS, and DMEM containing 10% FCS was added for a further 20 h
(Fig. 1). Culture media were collected, and the levels of IL-8 were measured.
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Enzyme-linked immunosorbent assays.
Monoclonal and biotinylated anti-human IL-8, TNF-, and IL-1
antibodies were obtained from R&D Systems (Abingdon, UK). Flat-bottomed 96-well microtiter plates (EIA/RIA Plate, Costar, Cambridge, MA) were
coated with monoclonal antibodies, and cytokine levels were assessed in
the test samples according to the manufacturer's instructions. The
values were determined from a standard curve of recombinant protein
(R&D Systems).
Preparation of nuclear extracts and electrophoretic mobility
shift assays for NF-B and AP-1 binding to DNA.
Nuclear extracts of A549 cells were prepared according to the method of
Staal et al. (47). Briefly, cells were rinsed in 1× PBS,
scraped, and centrifuged. Cells were lysed by incubation on ice in
buffer A [10 mM HEPES, 10 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 0.4 mM phenylmethylsulfonyl
fluoride (PMSF), 0.2 mM NaF, 0.2 mM NaVO3, and 1 µg/ml
leupeptin] and buffer B (10% Nonidet P-40), and the nuclei
were collected by centrifugation. Nuclei were resuspended in
buffer C (50 mM HEPES, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA,
1 mM DTT, 0.4 mM PMSF, 10% glycerol, 0.2 mM NaF, and 0.2 mM
NaVO3) and agitated for 20 min at 4°C followed by
centrifugation. Nuclear protein (10 µg) was incubated with 5×
binding buffer (Promega, Southampton, UK) and either
[
-32P]-labeled NF-
B or AP-1 consensus
oligonucleotides according to the manufacturer's protocol and
electrophoresed on a 6% nondenaturing polyacrylamide gel. The NF-
B
(5'-AGT TGA GGG GAC TTT CCC AGG C-3') and AP-1 oligonucleotides (5'-CGC
TTG ATG AGT CAG CCG GAA-3') were obtained from Promega. DNA binding was
assessed by autoradiography, and quantitative analysis was performed
with a Storm 860 PhosphorImager using ImageQuant software (Molecular
Dynamics, Buckinghamshire, UK). Mutant NF-
B oligonucleotides with a
"G"
"C" substitution in the NF-
B/Rel DNA-binding motif
(Santa Cruz) and AP-1 oligonucleotide mutants with "CA"
"TG" substitution in the AP-1-binding motif (Santa Cruz
Biotechnology) were used to establish the specificity of the sample
nuclear proteins for each of these transcription factors. In selected
experiments, gel shifts were performed on nuclear extracts incubated
with [
-32P]-labeled oligonucleotides containing the
5'-flanking region of the IL-8 gene corresponding to the putative
NF-
B site (5'-GTG GAA TTT CCT-3'). Two microliters of
nonradiolabeled oligonucleotides containing the same NF-
B
DNA-binding sequence were used as the cold competitor for these
studies. Supershifts for NF-
B and AP-1 were performed using
antibodies directed against p65 and p50 (Santa Cruz Biotechnology) for
NF-
B and c-Jun and c-Fos (Santa Cruz Biotechnology) for AP-1.
Nuclear protein (10 µg), 5× binding buffer, and 2 ul (4 µg) of
undiluted anti-p50, anti-p65, anti-c-Jun, and anti-c-Fos antibodies
were incubated overnight at 4°C. The samples were then treated with
the 32P-labeled NF-
B oligonucleotide as described above.
Nonimmune rabbit serum (4 µg; SAPU, Lanarkshire, UK) was used as a
serum control.
TNF- and IL-1
neutralizing studies.
Conditioned media from particle-exposed macrophages were treated for 45 min at 37°C with either monoclonal mouse-derived anti-human TNF-
(5 µg/ml) or IL-1
(20 µg/ml) whole antibodies (R&D Systems) before treatment of A549 cells. As a control for nonspecific binding, conditioned media were incubated with either 5 or 20 µg/ml of nonimmune mouse serum (SAPU). Cells were incubated for 4 h with the antibody-treated conditioned media and washed, and 10% FCS-DMEM was added for a further 20 h. IL-8 levels were measured in the culture media. According to R&D Systems, the neutralization doses for
both anti-TNF-
and anti-IL-
antibodies required to yield one-half
maximal inhibition of the cytokine activity were ~0.04-0.08 µg/ml in the presence of 0.25 ng/ml of recombinant human TNF-
and
0.05-0.2 µg/ml in the presence of 0.05 ng/ml of recombinant human IL-1
, respectively.
Transient transfection and chloramphenicol acetyl transferase assay. The IL-8 promoter chloramphenicol acetyl transferase (CAT) reporter construct (provided by Professor R. Strieter, University of Michigan, Ann Arbor, MI) was prepared by amplifying the wild-type IL-8 consensus sequence by PCR to generate a Pst1 restriction site at the 5' end and an Xba site at the 3' end. The Pst-Xba fragment was cloned into a Promega pCAT Basic vector (Promega, Madison, WI). The pCAT Basic vector, which lacks eukaryotic promoter and enhancer sequences, was used as a mock control.
A549 cells (0.4 × 106 cells per well) were seeded into six-well plates and cultured until 70-80% confluent. Plasmid DNA transfections were performed with LipofectAmine reagent according to the manufacturer's instructions (Life Technologies). A pSV-Isolation of RNA and assessment of IL-8 mRNA by semiquantitative RT-PCR. Total RNA was isolated from cultured A549 cells using the TRIzol reagent (Life Technologies, Paisley, UK) according to the manufacturer's instructions. Two micrograms of RNA were added to a solution containing 5× reverse transcription buffer (Promega), 100 µg/ml oligo deoxythymidine, 100 mM DTT, 10 mM deoxynucleotide triphosphates (dNTPs), and RNAse inhibitor, reverse transcribed into cDNA at 37°C for 1 h using Moloney murine leukemia virus reverse transcriptase (200 U/µl), and incubated at 94°C for 10 min (Gibco-BRL, Paisley, UK). Using a thermal cycler (Hybaid, Ashford, UK), we PCR-amplified aliquots of cDNA, two and five microliters, in 47- and 50-µl reaction volumes containing PCR mix (1× TAQ polymerase buffer, 2.5 mM MgCl2, and 0.2 mM dNTPs, Promega) and TAQ polymerase (1U/µl) for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and IL-8, respectively. Conditions for PCR were as follows: for IL-8, 35 cycles of denaturation (92°C for 1 min), annealing (60°C for 1 min), extension (72°C for 1 min), and final extension for 5 min at 72°C; for GAPDH, 35 cycles of denaturation (94°C for 45 s), annealing (60°C for 45 s), extension (72°C for 90 s), and final extension for 10 min at 72°C. Oligonucleotide primers used in the PCR reactions were chosen according to the published sequence of human IL-8 cDNA (30) and GAPDH (31). The primers for IL-8 and GAPDH were synthesized by MWG-Biotech (Milton Keynes, UK). The sequence of the primers used in the PCR were as follows: IL-8 (sense 5'-ATG ACT TCC AAG CTG GCC GTG GCT-3' and anti-sense 5'-TCT CAG CCC TCT TCA AAA ACT TCT C-3'); and GAPDH (sense 5'-CCA CCC ATG GCA AAT TCC ATG GCA-3' and anti-sense 5'-TCT AGA CGG CAG GTC AGG TCA ACC-3'). PCR products were electrophoresed in 1.5% agarose containing ethidium bromide, scanned using a white/ultraviolet transilluminator (Ultra Violet Products, Cambridge, UK), and quantified by densitometry. IL-8 values were expressed as a ratio of the band intensity to GAPDH, which was used as the housekeeping gene.
Neutrophil chemotaxis. Neutrophil chemotaxis was measured with the use of a NeuroProbe 96-well chemotaxis chamber (Porvair Filtronics) and polycarbonate filters with 3-µm-diameter pores (52). Hanks' balanced salt solution (HBSS) or conditioned media from either untreated or particle-exposed macrophages were placed in the bottom wells of the chemotaxis chamber. Neutrophils (200 µl at 10 × 106/ml in HBSS containing 0.3% BSA) were added to the top wells of the chamber. Each treatment was tested in triplicate. The chamber was incubated for 45 min at 37°C, 5% CO2. The polycarbonate filter, which separates the top and bottom wells, was removed, and adherent cells were scraped off the top surface. The filter was air dried, and neutrophils in and adhering to the bottom surface were fixed and stained with Diff-Quick stain. The filter was placed in an ELISA plate reader (Dynatech MR5000) and read at an optical density of 550 nm to assess the extent of neutrophil migration induced by the conditioned media. The data were expressed as units of absorbance. In selected experiments, a dose response was determined for neutrophil migration towards recombinant human IL-8 (R&D Systems).
Statistical analysis. Data are expressed as means ± SE and were analyzed on StatView SE + Graphics (Abacus Concepts), using ANOVA followed by a Fisher paired least significant difference test for multiple comparisons. Experiments were performed in triplicate unless otherwise indicated, and P < 0.05 was considered as significant.
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RESULTS |
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PM10-conditioned media induce NF-B and AP-1 DNA
binding in A549 cells.
Because IL-8 regulation is controlled by the transcription factors
NF-
B and AP-1, we were interested in determining whether macrophage-conditioned media could stimulate the DNA binding of these
transcription factors. Nuclear extracts were prepared from A549 cells
exposed to conditioned media from particle-treated macrophages. NF-
B
DNA binding was increased in A549 cells treated for 4 h with
conditioned media from PM10- and TNF-
-exposed
macrophages by 9.5- and 12-fold, respectively (P < 0.05), relative to control (Fig. 2).
NF-
B activity was unaltered in A549 cells treated with conditioned
media from macrophages exposed to either TiO2 or
UFTiO2 (Fig. 2). In addition, PM10- and
TNF-
-conditioned media increased AP-1 DNA binding by 2.1-fold over
the negative control (Fig. 2), whereas no differences were detected
with either TiO2- or UFTiO2-conditioned media.
Supershift experiments using nuclear extracts from A549 cells treated
with PM10-conditioned media showed that the components present for NF-
B were p65 and p50 and c-Jun for AP-1 (Fig.
2C). Incubation of nuclear extracts with mutant
oligonucleotides for NF-
B and AP-1 showed no DNA binding, thereby
demonstrating specifically the involvement of NF-
B and AP-1 (Fig.
2C).
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Role of transcription factors in PM10-conditioned media
regulation of IL-8 gene.
To demonstrate that NF-B induced by PM10-conditioned
media binds to the IL-8 gene promoter, a DNA fragment of the
5'-flanking region of IL-8 gene corresponding to the putative NF-
B
site was used in electrophoretic mobility shift assays (EMSA). Figure
2D shows that treatment of A549 cells with
PM10-conditioned media (lane 3) increased the
binding of NF-
B to the specific IL-8 promoter sites. The specificity
of DNA binding was assessed by competition using excess of
nonradioactive consensus probe (lane 4).
Increased IL-8 gene expression in response to PM10
macrophage-conditioned media.
Because PM10-conditioned media increased NF-B
DNA-binding, we used RT-PCR to assess IL-8 gene transcription.
Increases in IL-8 gene expression in A549 cells after exposure to
TNF-
- and PM10-conditioned media for 4 h were
observed (Fig. 3). No effects were
observed with either TiO2- or
UFTiO2-conditioned media (data not shown). Furthermore,
IL-8 gene induction was sustained in A549 cells after 8 h of total
exposure: 4 h with TNF- and PM10-conditioned media
followed by 4 h of incubation with 10% fetal bovine serum-DMEM (data not shown).
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Effects of PM10-conditioned media on IL-8 promoter
construct-derived CAT activity.
To further investigate whether PM10-conditioned media
exerted effects on IL-8 release from A549 cells via transcriptional activation, we used a putative IL-8 promoter construct-CAT reporter assay system. A549 cells transiently transfected with an IL-8 CAT
reporter and -galactosidase were exposed to conditioned media for
4 h, followed by 12 h with complete media (16 h total). We found that PM10-conditioned media increased CAT expression
by 65% over control (Fig. 3C). No differences were observed
with either TiO2- or UFTiO2-conditioned media.
The promoterless plasmid, pCAT, did not confer any significant CAT
activity in A549 cells under control conditions or in response to
PM10-conditioned media (Fig. 3C).
Increased IL-8 production in A549 cells exposed to conditioned
media from PM10-exposed macrophages.
To study the role of macrophages in regulating the proinflammatory
response in lung epithelial cells, we measured IL-8 production from
A549 cells exposed to conditioned media from macrophages treated with
particulates. Conditioned media from both TNF-- and
PM10-exposed macrophages enhanced IL-8 production by 5.7- and 6.2-fold, respectively, in A549 cells compared with the untreated control (P < 0.05) (Fig.
4). No differences compared with the control were observed with either TiO2- or
UFTiO2-conditioned media. In comparison, we assessed
whether direct exposure of A549 cells to particulates could induce an
IL-8 response. TNF-
induced a 2.3-fold induction, whereas neither
PM10, TiO2, nor UFTiO2 enhanced IL-8 release at 100 µg/ml (Fig. 5) We
have found, however, that concentrations >100 µg/ml do elicit an
IL-8 response from A549 cells (data not shown).
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Conditioned media from PM10-exposed macrophages induce
neutrophil chemotaxis.
We also assessed whether conditioned media from particle-exposed
macrophages could induce neutrophil chemotaxis. Conditioned media from
PM10-stimulated macrophages induced a 2.3-fold increase in
neutrophil chemotaxis compared with the negative control (Fig. 6), whereas no differences in chemotactic
potential were detected in either TiO2- or
UFTiO2-treated macrophage-conditioned media compared with
untreated macrophage supernatant. In separate in vitro experiments, we
assessed neutrophil chemotaxis toward recombinant human IL-8. IL-8
induced neutrophil chemotaxis in a dose-dependent manner with a maximal
response observed at 40 ng/ml (expressed as absorbance values: control
0.552 ± 0.04 SE; IL-8 1.12 ± 0.45 SE, n = 4).
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PM10 induces TNF- and IL-1
production in
macrophages.
To begin to identify which macrophage mediators in the PM10
conditioned media were responsible for inducing IL-8 release, we chose
to assay for TNF-
and IL-1
, because release of these cytokines is
frequently described in response to inflammogenic stimuli.
PM10-conditioned media contained increased levels of TNF-
(5.5-fold) over control, a response that was not observed with
either TiO2- or UFTiO2-conditioned media (Fig.
7). Furthermore, both PM10
and LPS augmented IL-1
release from macrophages at 24 h,
compared with the negative control (Fig.
8). No differences were observed with
either TiO2 or UFTiO2.
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Effects of TNF-- and IL-1
-neutralizing antibodies on IL-8
induction by PM10-conditioned media.
To determine whether TNF-
or IL-1
in PM10-conditioned
media was responsible for inducing IL-8, conditioned media from treated macrophages were preincubated with either TNF-
- or
IL-1
-neutralizing antibodies before addition to cells. To account
for nonspecific binding, all test groups were pretreated with mouse
preimmune serum. IL-8 levels for both TNF-
(26.3%)- and
PM10 (48.1%)-conditioned media were partially decreased
using TNF-
-neutralizing antibodies compared with the negative
control (Fig. 9). In contrast,
IL-1
-neutralizing antibodies were unable to prevent IL-8 production
from A549 cells in response to PM10-conditioned media (Fig.
10). To ensure that the IL-1
antibodies were indeed neutralizing, we treated A549 cells with culture
media containing 100 pg/ml of recombinant human IL-1
(R&D Systems)
preincubated with and without IL-1
antibodies. IL-1
was able to
induce a twofold increase in IL-8 protein levels, a response that was
completely abolished in the presence of IL-1
neutralizing antibodies
(Fig. 10).
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DISCUSSION |
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Episodes of high PM10 levels have been linked to increased morbidity and exacerbations of preexisting airways diseases in patients with chronic obstructive pulmonary disease (COPD) (36, 42). Elevated IL-8 levels, detected in the lungs of COPD patients, are thought to play a major role in airway inflammation in this disease, particularly in the recruitment of neutrophils to the lungs. Increased levels of this cytokine have also been found in the bronchoalveolar lavage (BAL) of rats intratracheally instilled with PM10 (23). Furthermore, IL-8 has been shown to be upregulated in response to components of PM10, such as diesel exhaust (18) and residual oil fly ash (ROFA) (40) through mechanisms involving transition metals.
As lung macrophages are one of the first cellular lines of defense
against inhaled pollutants and are known to release proinflammatory cytokines in response to particulates, we investigated the potential role of macrophages in regulating IL-8 release from lung epithelial cells. For this study we utilized blood monocyte-derived macrophages, differentiated in vitro, as a surrogate for alveolar macrophages. Whereas direct exposure of A549 cells to PM10 at 100 µg/ml did not augment IL-8 release, we found that conditioned media
from PM10-exposed macrophages stimulated the production of
IL-8 from A549 cells. This conditioned medium was also found to contain elevated levels of both TNF- and IL-1
. Increased TNF-
and
IL-1
production from macrophages is in agreement with the work of
Becker et al. (3), who demonstrated that these cytokines
were elevated in monocytes exposed to urban ambient particles.
Furthermore, a recent report by the same group found the induction of
IL-8 mRNA expression in monocytes after exposure to ROFA particles (33). This increase was accompanied by increased tyrosine
phosphorylation, which was inhibited by both herbimycin and genistein,
thus indicating that IL-8 induction is preceded by the activation of
tyrosine kinases. Our data suggest that the indirect effects of
PM10 via mediators released from macrophages play an
important role in eliciting an IL-8 response from lung epithelial
cells. Thus PM10 may trigger an epithelial cell
inflammatory response via macrophage mediators.
Although centrifugation of the PM10-conditioned media did produce a particle pellet, there exists the possibility that the media may be contaminated with some ultrafine particles. However, given that direct exposure of A549 cells to 100 µg/ml PM10 did not stimulate IL-8 production, any remaining particles in the conditioned media would be present at a low concentration insufficient to induce IL-8 release. The average concentration of PM10 pelleted from the macrophage-conditioned media after centrifugation was 6 µg/ml. Pellets from PM10-conditioned media samples were resuspended in PBS, and the concentrations were determined spectrophotometrically using a carbon black standard curve as previously described (22).
Proinflammatory genes, such as IL-8, are regulated by redox-sensitive
transcription factors such as NF-B, AP-1, and C/EBP, which are
activated in various cell types in response to oxidants, asbestos, and
cytokines (10, 20, 21). We and others have recently
demonstrated that NF-
B is activated in lung epithelial cells in
response to PM10 via a mechanism involving transition metals (22, 23). In addition, Shukla et al.
(45) reported increased lung steady-state mRNA levels of a
number of NF-
B-related genes, including TNF-
and -
, IL-6,
interferon-
, and transforming growth factor-
, after inhalation of
PM2.5 (PM <2.5 microns in aerodynamic diameter) in mice.
In the present study, we show that IL-8 production from A549 cells is
induced by PM10-conditioned media and occurs concomitantly
with increased NF-
B and AP-1 DNA binding. Supershift experiments on
nuclear extracts from these samples revealed the components of NF-
B
to be p65 and p50 and c-Jun for AP-1. In addition, using an
oligonucleotide containing the IL-8 promoter sequence, we show
increased NF-
B DNA binding and thus specificity of NF-
B induced
by PM10-conditioned media for the IL-8 gene. We
further demonstrate by RT-PCR and through the use of an IL-8-CAT
reporter that PM10-conditioned media caused a marked
increase in IL-8 gene activation, suggesting that macrophage mediators
regulate IL-8 at the transcriptional level through signaling pathways
that may involve NF-
B and AP-1. Our laboratory has also recently
shown that lung epithelial cells virally infected with E1A demonstrate
increased DNA binding for NF-
B and AP-1 along with augmented IL-8
production (16), thereby suggesting that these cells are
somewhat more responsive to air pollution particulates.
Cytokine networking plays an important role in the lung in response to
inflammogenic stimuli (48), whereby one cell population is
dependent upon the mediators synthesized by a neighboring cell. The
inhalation of particulate air pollution into the alveolar space in the
lungs may elicit the production of cytokines from alveolar macrophages,
which may then act in a paracrine fashion to stimulate nonimmune cells
of the alveolar-capillary wall to produce effector mediators.
Standiford et al. (49) previously illustrated the
paracrine effects of macrophages on nonimmune cells by exposing A549
cells to conditioned media from LPS-exposed alveolar macrophages and
demonstrating mRNA induction of monocyte chemoattractant protein-1, a
monokine upregulated during inflammation. More recently, Barrett
et al. (1) showed increased expression of macrophage
inflammatory protein-2 (MIP-2) in MLE-15, a murine lung epithelial cell
line, after exposure to culture media from silica-treated RAW 264.7 cells. Furthermore, in vivo depletion of alveolar macrophages via
intratracheal instillation of liposomes containing dichloromethylene
diphosphonate has been shown to suppress increased TNF- and MIP-2
levels in BAL fluid, neutrophil accumulation, and whole lung tissue
NF-
B activation in rats exposed to IgG-immune complexes
(27). We showed that, in addition to enhanced
IL-8 release after 24-h exposure to PM10,
PM10-treated macrophages also produced elevated levels of
TNF-
and IL-1
. TNF-
, a proinflammatory cytokine, is
upregulated in macrophages after exposure to such inflammogenic stimuli
as LPS, asbestos, and quartz (9, 10, 51). Moreover, both
IL-1
and TNF-
, a known activator of NF-
B and AP-1 in human
lung epithelial cells (26), stimulate the production of
IL-8 in A549 cells (5, 26).
TNF- activates the IL-8 gene through stimulation of both the NF-
B
heterodimer p65/p50 and C/EBP, which, in turn, bind to a composite
enhancer element within the proximal promoter (32). We
have demonstrated that TNF-
-neutralizing antibodies were able to
significantly diminish, but not abrogate, IL-8 release from A549 cells
in response to PM10-conditioned media. Surprisingly, IL-1
-neutralizing antibodies were not able to inhibit IL-8
induction, even though increased levels of this cytokine were detected
in PM10-conditioned media. To eliminate the possibility
that the A549 cells were producing TNF-
, which in turn upregulated
IL-8, we assayed the culture media for TNF-
after stimulation with the macrophage-conditioned media. No TNF-
was detected from the A549
cells (data not shown).
Our data suggest that TNF- is one of the proinflammatory mediators
present in the conditioned media, stimulating lung epithelial cells via
a paracrine mechanism. However, the inability to completely abolish the
IL-8 response with TNF-
-neutralizing antibodies also indicates that
other mediators are being secreted by macrophages exposed to
PM10, which enhance IL-8 production from lung epithelial cells. A potential mediator secreted from activated macrophages with
the ability to induce IL-8 is soluble CD14 (sCD14). sCD14, the soluble
form of the membrane-bound receptor for the LPS-LPS-binding protein
complex, is released from activated macrophages and has been shown to
induce IL-8 production from bronchial epithelial cells in the presence
and absence of LPS (50). Thus the augmented IL-8
production by addition of macrophage-conditioned media may be mediated
by sCD14 and merits further investigation.
Ideally, the use of alveolar macrophages compared with peripheral blood-derived macrophages would have further strengthened the relevance of this study. However, previous work on the comparison between alveolar macrophages and in vitro differentiation of monocytes into macrophages has demonstrated similar morphological and functional changes (13, 17), indicating that in vitro differentiated macrophages serve as an adequate substitute. Nii et al. (34) also showed that LPS-exposed human alveolar macrophages produced significantly more membrane form of TNF than blood monocytes but similar levels to monocyte-derived macrophages.
The recruitment of immune cells to the site of injury is essential to
the inflammatory response. It is well established that neutrophil
diapedesis from the pulmonary circulation occurs from the capillary bed
(7). Sequestration of neutrophils in the lungs is
initiated by their deformability (44), which facilitates cell adhesion to the endothelium and culminates in emigration of the
cells from the vasculature. The chemotactic gradient that directs the
migratory process is important to this sequence. We show here that
conditioned media from PM10-exposed macrophages induces
neutrophil migration, demonstrating that mediators released by
activated macrophages are capable of mediating the neutrophil influx as
well as stimulating resident lung cells. The chemotactic potential of
the conditioned media could be attributed to IL-8 and TNF-, major
lung chemokines, which we found to be elevated in conditioned media. In
comparison with a dose-response curve of IL-8, the
macrophage-conditioned media gave a chemotactic response over control
equivalent to that observed for 40 ng/ml IL-8. Increased airway
neutrophilia has been observed in animals after instillation of
ultrafine particles and PM10 (11, 29).
Furthermore, Salvi and colleagues (41) recently showed
significant increases in airway neutrophils in healthy human volunteers
exposed to diesel exhaust, a constituent of PM10.
Ultrafine particle-induced toxicity has been attributed to such
features as size, chemical composition, surface area, particle number,
and free radicals (43). We have hypothesized that the ultrafine fraction of PM10 may be, in part, responsible for
some of the observed adverse effects (43). Indeed,
UFTiO2 has been previously shown to generate free radicals
(14) and elicit an inflammatory response in rats after
intratracheal instillation (11). Our present data
demonstrate that UFTiO2-conditioned media do not increase
IL-8 expression in A549 cells. Furthermore, direct stimulation of
macrophages with UFTiO2 failed to induce the release of
TNF- and IL-1
. These data suggest that the ultrafine size of
UFTiO2 alone is not sufficient to stimulate the release of proinflammatory mediators like IL-8, TNF-
, and IL-1
in this in
vitro model. Modification of the surface of ultrafine particles in vivo
with proteins and/or endogenous metals present in the lung epithelial
lining fluid could presumably alter particle reactivity, as has been
shown for asbestos (19), and this may account for the
discrepancy observed between our in vitro data and previous in vivo
results. On a mass basis, UFTiO2 would be expected to be
introduced to the macrophages in greater numbers per unit volume than
PM10 because the composition of PM10 would
contain fewer numbers of ultrafine particles. As PM10 is
the only one of the three particles that is bioactive in this system,
we interpret these data to suggest that soluble components present on
the particle surface, such as endotoxin or aromatic hydrocarbons, may
be responsible for macrophage activation. Work by Becker et al.
(3) demonstrated a role for endotoxin, not transitional
iron, in the induction of TNF-
and IL-6 from ambient air
particle-exposed alveolar macrophages. Moreover, because LPS is present
in the environment, as detected in fog droplets and urban air samples
(2, 12), and hence a component of PM10, LPS
could potentially elicit biological responses from macrophages.
Although LPS has been documented to induce IL-8 from epithelial cells
(38, 50), A549 cells have low CD14 receptor expression and
therefore do not respond to low concentrations of endotoxin. We have
measured the level of endotoxin in our PM10 and have
determined it to be 155 pg/mg particles, a concentration below the
levels required to induce an IL-8 response in A549 cells.
In summary (Fig. 11), PM10
particles are capable of directly stimulating the production of IL-8 in
both lung epithelial cells and macrophages. Our results demonstrate
that PM10 can also indirectly enhance NF-B DNA binding
and IL-8 release, triggered in part by TNF-
secreted from
PM10-exposed macrophages. This suggests that
PM10-exposed macrophages may amplify the inflammatory
cascade by regulating IL-8 release from lung epithelial cells.
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ACKNOWLEDGEMENTS |
---|
This study was supported by the United Kingdom Medical Research Council, the British Lung Foundation, and the Colt Foundation. K. Donaldson is a recipient of the British Lung Foundation Transco Fellowship in Air Pollution and Lung Disease.
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FOOTNOTES |
---|
Address for reprint requests and other correspondence: L. A. Jiménez, ELEGI/Colt Research Laboratories, Univ. of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland, UK (E-mail: ajimenez{at}srv1.med.ed.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajplung.00024.2001
Received 27 August 2001; accepted in final form 24 September 2001.
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