PM10-exposed macrophages stimulate a proinflammatory response in lung epithelial cells via TNF-alpha

L. A. Jiménez1, E. M. Drost1, P. S. Gilmour1, I. Rahman1, F. Antonicelli1, H. Ritchie1, W. MacNee1, and K. Donaldson2

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa 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-alpha (TNF-alpha ) and protein levels and enhanced chemotactic activity for neutrophils. Preincubation of conditioned media with TNF-alpha -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-alpha .

particulate matter; tumor necrosis factor-alpha ; nuclear factor-kappa B; cytokine networking; interleukin-8


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa B (NF-kappa 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-alpha (TNF-alpha ), IL-1beta (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-kappa B through a mechanism partially involving TNF-alpha in the conditioned media.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 penicillin-1 · 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-alpha (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-alpha (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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Fig. 1.   Schematic for the production of macrophage conditioned media and exposure of A549 cells. After exposure of macrophages to particulate matter <10 µm in aerodynamic diameter (PM10), TiO2, or ultrafine (UF) TiO2, the conditioned media were removed, centrifuged, and added to A549 cells for 4 h. After incubation, these media were aspirated off and the cells washed with PBS. Fresh DMEM containing 10% FCS was added to the cells for an additional 20 h.

Enzyme-linked immunosorbent assays. Monoclonal and biotinylated anti-human IL-8, TNF-alpha , and IL-1beta 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-kappa 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 [gamma -32P]-labeled NF-kappa B or AP-1 consensus oligonucleotides according to the manufacturer's protocol and electrophoresed on a 6% nondenaturing polyacrylamide gel. The NF-kappa 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-kappa B oligonucleotides with a "G" right-arrow "C" substitution in the NF-kappa B/Rel DNA-binding motif (Santa Cruz) and AP-1 oligonucleotide mutants with "CA" right-arrow "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 [gamma -32P]-labeled oligonucleotides containing the 5'-flanking region of the IL-8 gene corresponding to the putative NF-kappa B site (5'-GTG GAA TTT CCT-3'). Two microliters of nonradiolabeled oligonucleotides containing the same NF-kappa B DNA-binding sequence were used as the cold competitor for these studies. Supershifts for NF-kappa B and AP-1 were performed using antibodies directed against p65 and p50 (Santa Cruz Biotechnology) for NF-kappa 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-kappa B oligonucleotide as described above. Nonimmune rabbit serum (4 µg; SAPU, Lanarkshire, UK) was used as a serum control.

TNF-alpha and IL-1beta neutralizing studies. Conditioned media from particle-exposed macrophages were treated for 45 min at 37°C with either monoclonal mouse-derived anti-human TNF-alpha (5 µg/ml) or IL-1beta (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-alpha and anti-IL-beta 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-alpha and 0.05-0.2 µg/ml in the presence of 0.05 ng/ml of recombinant human IL-1beta , 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-beta -galactosidase control vector (Promega) was cotransfected as an internal control to normalize for transfection efficiency. Cells were exposed to conditioned media for 4 h, washed with 1× PBS, and incubated for 12 h in the presence of 10% FCS-DMEM. After incubation, cell extracts were prepared and assayed for protein content using the Bio-Rad protein reagent (Bio-Rad). Samples were normalized for protein and CAT, and beta -galactosidase activities were quantified by a CAT and beta -gal enzyme-linked immunosorbent assay (Boehringer-Mannheim). Values were reported as the ratio of CAT per beta -galactosidase.

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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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PM10-conditioned media induce NF-kappa B and AP-1 DNA binding in A549 cells. Because IL-8 regulation is controlled by the transcription factors NF-kappa 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-kappa B DNA binding was increased in A549 cells treated for 4 h with conditioned media from PM10- and TNF-alpha -exposed macrophages by 9.5- and 12-fold, respectively (P < 0.05), relative to control (Fig. 2). NF-kappa 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-alpha -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-kappa B were p65 and p50 and c-Jun for AP-1 (Fig. 2C). Incubation of nuclear extracts with mutant oligonucleotides for NF-kappa B and AP-1 showed no DNA binding, thereby demonstrating specifically the involvement of NF-kappa B and AP-1 (Fig. 2C).


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Fig. 2.   PM10-conditioned media induce nuclear factor (NF)-kappa B and activator protein (AP)-1 DNA binding in A549 cells. A549 cells were exposed to conditioned media from tumor necrosis factor (TNF)-alpha (10 ng/ml)-, PM10 (100 µg/ml)-, TiO2 (100 µg/ml)-, and UFTiO2 (100 µg/ml)-exposed macrophages for 4 h. A: nuclear extracts were prepared, and NF-kappa B and AP-1 DNA binding was assessed by a standard electrophoretic mobility shift assay (EMSA) as outlined in MATERIALS AND METHODS. Promega Hela extract (+ve) and binding buffer alone (-ve) were used as positive and negative EMSA controls, respectively. B: bands were analyzed by phosphorimaging; n = 3 experiments performed in triplicate; *P < 0.05 compared with untreated control (Cont) for NF-kappa B; #P < 0.05 compared with untreated Cont for AP-1. C: supershifts were performed on nuclear extracts from A549 cells treated with PM10-conditioned media with the use of antibodies specific for the NF-kappa B subunits p50 and p65 and for the AP-1 subunits c-Jun and c-Fos. Mutant oligonucleotides for NF-kappa B and AP-1 were also incubated with these extracts. HeLa extract (Hela), binding buffer (buffer), and nonimmune rabbit serum (serum) were included as controls. D: to test the specificity of NF-kappa B DNA binding, nuclear extracts were incubated with an interleukin (IL)-8 promoter oligonucleotide containing an NF-kappa B consensus binding site. In lanes 1 and 2, the Promega NF-kappa B consensus sequence was added to nuclear extracts from A549 cells exposed to conditioned media from nonstimulated and PM10-exposed macrophages, respectively. Nuclear extracts from PM10-conditioned media-treated A549 cells were incubated with 32P-labeled IL-8-NF-kappa B oligonucleotide (lane 3) as well as in a cold competitor reaction (lane 4).

Role of transcription factors in PM10-conditioned media regulation of IL-8 gene. To demonstrate that NF-kappa 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-kappa 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-kappa 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-kappa 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-alpha - 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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Fig. 3.   PM10-conditioned media increase IL-8 gene expression. A: RNA isolated from A549 cells exposed to conditioned media from macrophages stimulated with TNF-alpha (10 ng/ml) and PM10 (100 µg/ml) for 4 h was transcribed into cDNA, extended by PCR using IL-8 primers, and loaded onto an agarose gel according to MATERIALS AND METHODS. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B: bands were analyzed by densitometry using the UVGrab program. C: A549 cells transiently transfected with an IL-8 chloramphenicol acetyle transferase (CAT) reporter and beta -galactosidase were exposed for 4 h to conditioned media from macrophages treated with PM10, TiO2, and UFTiO2 followed by 12 h with complete media. Values are reported as the ratio of CAT/beta -gal; n = 5 experiments, performed in duplicate; * P < 0.05, compared with Cont. pCAT, promoterless plasmid vector.

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 beta -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-alpha - 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-alpha 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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Fig. 4.   Conditioned media from PM10-exposed macrophages induce IL-8 production. Media from macrophages exposed to TNF-alpha (100 µg/ml), PM10 (100 µg/ml), TiO2 (100 µg/ml), or UFTiO2 (100 µg/ml) were added to A549 cells according to MATERIALS AND METHODS. IL-8 was analyzed by ELISA; n = 3 experiments; * P < 0.05 compared with conditioned media from nonstimulated macrophages (Cont).



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Fig. 5.   The direct effects of PM10 and TiO2 on IL-8 production in A549 cells. A549 cells were stimulated for 24 h with TNF-alpha (10 ng/ml), PM10 (100 µg/ml), TiO2 (100 µg/ml), or UFTiO2. The culture media were collected and analyzed by ELISA for IL-8 release; n = 3 experiments; *P < 0.05 compared with Cont.

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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Fig. 6.   Increased neutrophil chemotaxis in response to conditioned media from PM10-exposed macrophages. Neutrophils were exposed to conditioned media from PM10 (100 µg/ml)-, TiO2 (100 µg/ml)-, or UFTiO2-exposed (100 µg/ml) macrophages for 45 min. Chemotaxis was assessed according to MATERIALS AND METHODS; n = 3 experiments; *P < 0.05 compared with neutrophils stimulated with supernatants from nonstimulated macrophages (Cont).

PM10 induces TNF-alpha and IL-1beta 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-alpha and IL-1beta , because release of these cytokines is frequently described in response to inflammogenic stimuli. PM10-conditioned media contained increased levels of TNF-alpha (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-1beta 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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Fig. 7.   PM10 induces TNF-alpha production in macrophages. Monocyte-derived macrophages were exposed to PM10 (100 µg/ml), TiO2 (100 µg/ml), and UFTiO2 (100 µg/ml) for 24 h. Culture media were collected and analyzed for TNF-alpha using a standard ELISA; n = 3 experiments done in triplicate; *P < 0.05 compared with untreated Cont.



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Fig. 8.   PM10 induces IL-1beta production in macrophages. Human monocyte-derived macrophages were exposed to lipopolysaccharide (LPS, 10 ng/ml), PM10 (100 µg/ml), TiO2 (100 µg/ml), and UFTiO2 (100 µg/ml) for 24 h. Culture media were collected and analyzed for IL-1beta by ELISA; n = 3 experiments performed in triplicate; *P < 0.05 compared with untreated Cont.

Effects of TNF-alpha - and IL-1beta -neutralizing antibodies on IL-8 induction by PM10-conditioned media. To determine whether TNF-alpha or IL-1beta in PM10-conditioned media was responsible for inducing IL-8, conditioned media from treated macrophages were preincubated with either TNF-alpha - or IL-1beta -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-alpha (26.3%)- and PM10 (48.1%)-conditioned media were partially decreased using TNF-alpha -neutralizing antibodies compared with the negative control (Fig. 9). In contrast, IL-1beta -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-1beta antibodies were indeed neutralizing, we treated A549 cells with culture media containing 100 pg/ml of recombinant human IL-1beta (R&D Systems) preincubated with and without IL-1beta antibodies. IL-1beta was able to induce a twofold increase in IL-8 protein levels, a response that was completely abolished in the presence of IL-1beta neutralizing antibodies (Fig. 10).


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Fig. 9.   TNF-alpha -neutralizing antibodies diminish IL-8 release in response to PM10 conditioned media. Conditioned media from nonstimulated macrophages or macrophages exposed to either TNF-alpha (10 ng/ml) or PM10 (100 µg/ml) were incubated for 45 min with either nonimmune mouse serum (5 µg/ml) or TNF-alpha -neutralizing antibodies (5 µg/ml) before being added to A549 cells for a further 4 h. Cells were washed and incubated with 10% fetal bovine serum in DMEM for 20 h. Culture media from A549 cells were collected and assessed for IL-8 levels; n = 3 experiments done in triplicate; *P < 0.05 compared with Cont group; #P < 0.05 compared with serum.



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Fig. 10.   The effect of IL-1beta -neutralizing antibodies on IL-8 production in response to PM10-conditioned media. Conditioned media from nonstimulated macrophages and PM10-exposed macrophages were incubated with either nonimmune mouse serum (20 µg/ml) or IL-1beta -neutralizing antibodies (20 µg/ml) and added to A549 cells for 4 h. As a control, A549 cells were exposed to RPMI containing 100 pg/ml of IL-1beta with either mouse serum or anti-IL-1beta . Cells were washed and incubated with 10% FBS in DMEM for a further 20 h, and the media assessed for IL-8 levels; n = 3 experiments done in triplicate; *P < 0.05 compared with serum group; #P < 0.05 compared with Cont. Cont, conditioned media from unstimulated macrophages; PM, conditioned media from PM10-exposed macrophages.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha and IL-1beta . Increased TNF-alpha and IL-1beta 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-kappa 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-kappa 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-kappa B-related genes, including TNF-alpha and -beta , IL-6, interferon-gamma , and transforming growth factor-beta , 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-kappa B and AP-1 DNA binding. Supershift experiments on nuclear extracts from these samples revealed the components of NF-kappa 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-kappa B DNA binding and thus specificity of NF-kappa 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-kappa B and AP-1. Our laboratory has also recently shown that lung epithelial cells virally infected with E1A demonstrate increased DNA binding for NF-kappa 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-alpha and MIP-2 levels in BAL fluid, neutrophil accumulation, and whole lung tissue NF-kappa 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-alpha and IL-1beta . TNF-alpha , a proinflammatory cytokine, is upregulated in macrophages after exposure to such inflammogenic stimuli as LPS, asbestos, and quartz (9, 10, 51). Moreover, both IL-1beta and TNF-alpha , a known activator of NF-kappa B and AP-1 in human lung epithelial cells (26), stimulate the production of IL-8 in A549 cells (5, 26).

TNF-alpha activates the IL-8 gene through stimulation of both the NF-kappa 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-alpha -neutralizing antibodies were able to significantly diminish, but not abrogate, IL-8 release from A549 cells in response to PM10-conditioned media. Surprisingly, IL-1beta -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-alpha , which in turn upregulated IL-8, we assayed the culture media for TNF-alpha after stimulation with the macrophage-conditioned media. No TNF-alpha was detected from the A549 cells (data not shown).

Our data suggest that TNF-alpha 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-alpha -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-alpha , 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-alpha and IL-1beta . These data suggest that the ultrafine size of UFTiO2 alone is not sufficient to stimulate the release of proinflammatory mediators like IL-8, TNF-alpha , and IL-1beta 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-alpha 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-kappa B DNA binding and IL-8 release, triggered in part by TNF-alpha 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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Fig. 11.   A proposed mechanism for PM10 induction of IL-8 in the lung. PM10 interacts directly with macrophages and epithelial cells in the lung, resulting in upregulation and release of IL-8 and recruitment of neutrophils. Furthermore, PM10 may act indirectly to enhance and possibly prolong inflammation by stimulating IL-8 production from epithelial cells, via NF-kappa B and AP-1 signaling pathways, through the release of TNF-alpha and soluble mediators from activated macrophages.


    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.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Barrett, EG, Johnston C, Oberdörster G, and Finkelstein JN. Silica-induced chemokine expression in alveolar type II cells is mediated by TNF-alpha . Am J Physiol Lung Cell Mol Physiol 275: L1110-L1119, 1998[Abstract/Free Full Text].

2.   Battarbee, JL, Rose NL, and Long Z. A continuous high resolution record of urban airborne particulates suitable for retrospective microscopical analysis. Atmos Environ 31: 171-181, 1997[ISI].

3.   Becker, S, Soukup JM, Gilmour I, and Devlin RB. Stimulation of human and rat alveolar macrophages by urban air particulates: effects on oxidant radical generation and cytokine production. Toxicol Appl Pharmacol 141: 637-648, 1997[ISI].

4.   Carter, JD, Ghio AJ, Samet JM, and Devlin RB. Cytokine production by human airway epithelial cells after exposure to an air pollution particle is metal-dependent. Toxicol Appl Pharmacol 146: 180-188, 1997[ISI][Medline].

5.   Coulter, KR, Wewers MD, Lowe MP, and Knoell DL. Extracellular regulation of interleukin (IL)-1 beta through lung epithelial cells and defective IL-1 type II receptor expression. Am J Respir Cell Mol Biol 20: 964-975, 1999[Abstract/Free Full Text].

6.   Deforge, LE, Preston AM, Takeuchi E, Kenney J, Boxer LA, and Remick DG. Regulation of interleukin-8 gene expression by oxidant stress. J Biol Chem 268: 25568-25576, 1993[Abstract/Free Full Text].

7.   Downey, GP, Worthen GS, Henson PM, and Hyde DM. Neutrophil sequestration and migration in localized pulmonary inflammation. Capillary localization and migration across the interalveolar septum. Am Rev Respir Dis 147: 168-176, 1993[ISI][Medline].

8.   Dransfield, I, Buckle AM, Savill JS, McDowall A, Haslett C, and Hogg N. Neutrophil apoptosis is associated with a reduction in CD16 (Fcgamma RIII) expression. J Immunol 153: 1254-1263, 1994[Abstract/Free Full Text].

9.   Driscoll, KE, Hassenbein DG, Carter J, Poynter J, Asquith TN, Grant RA, Whitten J, Purdon MP, and Takigiku R. Macrophage inflammatory proteins 1 and 2: expression by rat alveolar macrophages, fibroblasts, and epithelial cells and in rat lung after mineral dust exposure. Am J Respir Cell Mol Biol 8: 311-318, 1993[ISI][Medline].

10.   Driscoll, KE, Maurer JK, Higgins J, and Poynter J. Alveolar macrophage cytokine and growth factor production in a rat model of crocidolite-induced pulmonary inflammation and fibrosis. J Toxicol Environ Health 46: 155-169, 1995[ISI][Medline].

11.   Ferin, J, Oberdörster G, and Penney DP. Pulmonary retention of ultrafine and fine particles in rats. Am J Respir Cell Mol Biol 6: 535-542, 1992[ISI][Medline].

12.   Fuzzi, S, Mandrioli P, and Perfetta A. Fog droplets: an atmospheric source of secondary biological aerosol particles. Atmos Environ 31: 287-290, 1997[ISI].

13.   Gantner, F, Kupferschmidt R, Schudt C, Wendel A, and Hatzelmann A. In vitro differentiation of human monocytes to macrophages: change of PDE profile and its relationship to suppression of tumour necrosis-alpha release by PDE inhibitors. Br J Pharmacol 121: 221-231, 1997[Abstract].

14.   Gilmour, P, Brown DM, Beswick PH, Benton E, MacNee W, and Donaldson K. Surface free radical activity of PM10 and ultrafine titanium dioxide: a unifying factor in their toxicity? Ann Occup Hyg 41, Suppl1: 32-38, 1997.

15.   Gilmour, PS, Brown DM, Lindsay TG, Beswick PH, MacNee W, and Donaldson K. Adverse health effects of PM10 particles: involvement of iron in generation of hydroxyl radical. Occup Environ Med 53: 817-822, 1996[Abstract].

16.   Gilmour, PS, Rahman I, Hayashi S, Hogg JC, Donaldson K, and MacNee W. Adenoviral E1A primes alveolar epithelial cells to PM10-induced transcription of interleukin-8. Am J Physiol Lung Cell Mol Physiol 281: L598-L606, 2001[Abstract/Free Full Text].

17.   Hammerstrom, J. Human macrophage differentiation in vivo and in vitro. A comparison of human peritoneal macrophages and monocytes. Acta Path Microbiol Scand Sect C 87: 113-120, 1979[ISI].

18.   Hashimoto, S, Gon Y, Takeshita I, Matsumoto K, Jibiki I, Takizawa H, Kudoh S, and Horie T. Diesel exhaust particles activate p38 MAP kinase to produce interleukin 8 and Rantes by human bronchial epithelial cells and N-acetylcysteine attenuates p38 MAP kinase activation. Am J Respir Crit Care Med 161: 280-285, 2000[Abstract/Free Full Text].

19.   Jabbour, AJ, Holian A, and Scheule RK. Lung lining fluid modification of asbestos bioactivity for the alveolar macrophage. Toxicol Appl Pharmacol 110: 283-294, 1991[ISI][Medline].

20.   Janssen, YM, Barchowsky A, Treadwell M, Driscoll KE, and Mossman BT. Asbestos induces nuclear factor kappa B (NF-kappa B) DNA-binding activity and NF-kappa B-dependent gene expression in tracheal epithelial cells. Proc Natl Acad Sci USA 92: 8458-8462, 1995[Abstract].

21.   Janssen-Heininger, YM, Macara I, and Mossman BT. Cooperativity between oxidants and tumor necrosis factor in the activation of nuclear factor (NF)-kappa B: requirement of Ras/mitogen-activated protein kinases in the activation of NF-kappa B by oxidants. Am J Respir Cell Mol Biol 20: 942-952, 1999[Abstract/Free Full Text].

22.   Jimenez, LA, Thomson J, Brown DA, Rahman I, Antonicelli F, Duffin R, Drost EM, Hay RT, Donaldson K, and MacNee W. Activation of NF-kappa B by PM10 occurs via an iron-mediated mechanism in the absence Ikappa B degradation. Toxicol Appl Pharmacol 166: 101-110, 2000[ISI][Medline].

23.   Kennedy, T, Ghio AJ, Reed W, Samet J, Zagorski J, Quay J, Carter J, Dailey L, Hoidal JR, and Devlin RB. Copper-dependent inflammation and nuclear factor-kappa B activation by particulate air pollution. Am J Respir Cell Mol Biol 19: 366-378, 1998[Abstract/Free Full Text].

24.   Kunkel, SL, Standiford T, Kasahara K, and Strieter RM. Interleukin-8: the major neutrophil chemotactic factor in the lung. Exp Lung Res 17: 17-23, 1991[ISI][Medline].

25.   Kwon, OJ, Au BT, Collins PD, Baraniuk JN, Adcock IM, Chung KF, and Barnes PJ. Inhibition of interleukin-8 expression by dexamethasone in human cultured airway epithelial cells. Immunology 3: 389-394, 1994.

26.   Lakshminarayanan, V, Beno DW, Costa RH, and Roebuck KA. Differential regulation of interleukin-8 and intercellular adhesion molecule-1 by H2O2 and tumor necrosis factor-alpha in endothelial and epithelial cells. J Biol Chem 272: 32910-32918, 1997[Abstract/Free Full Text].

27.   Lentsch, AB, Czermak BJ, Bless NM, Van Rooijen N, and Ward PA. Essential role of alveolar macrophages in intrapulmonary activation of NF-kappa B. Am J Respir Cell Mol Biol 20: 692-698, 1999[Abstract/Free Full Text].

28.   Li, XY, Brown D, Smith S, MacNee W, and Donaldson K. Short-term inflammatory responses following intratracheal instillation of fine and ultrafine carbon black in rats. Inhal Toxicol 11: 709-731, 1999[ISI][Medline].

29.   Li, XY, Gilmour PS, Donaldson K, and MacNee W. Free-radical and pro-inflammatory effects of particulate air-pollution (PM10) in-vivo and in- vitro. Thorax 51: 1216-1222, 1996[Abstract].

30.   Lindley, I, Aschauer H, Seifert JM, Lam C, Brunowsky W, Kownatzki E, Thelen M, Peveri P, Dewald B, von Tscharner V, Walz A, and Baggiolini M. Synthesis and expression in Escherichia coli of the gene encoding monocyte-derived neutrophil-activating factor: biological equivalence between natural and recombinant neutrophil-activating factor. Proc Natl Acad Sci USA 85: 9199-9203, 1988[Abstract].

31.   Maier, JAM, Voulalas P, Roeder D, and Maciag T. Extension of the life-span of human endothelial cells by an interleukin-1 alpha antisense oligomer. Science 249: 1570-1573, 1990[ISI][Medline].

32.   Matsusaka, T, Fujikawa K, Nishio Y, Mukaida N, Matsushima K, Kishimoto T, and Akira S. Transcription factor NF-IL6 and NF-kappa B synergistically activate transcription of the inflammatory cytokines, interleukin-6 and interleukin-8. Proc Natl Acad Sci USA 90: 10193-10197, 1993[Abstract].

33.   Mondal, K, Stephen Haskill J, and Becker S. Adhesion and pollution particle-induced oxidant generation is neither necessary nor sufficient for cytokine induction in human alveolar macrophages. Am J Respir Cell Mol Biol 22: 200-208, 2000[Abstract/Free Full Text].

34.   Nii, A, Sone S, Orino E, and Ogura T. Induction of a 26-kDa membrane-form tumour necrosis factor (TNF)-alpha in human alveolar macrophages. J Leukoc Biol 53: 29-36, 1993[Abstract].

35.   Peters, A, Wichmann HE, Tuch T, Heinrich J, and Heyder J. Respiratory effects are associated with the number of ultrafine particles. Am J Respir Crit Care Med 155: 1376-1383, 1997[Abstract].

36.   Pope, CA, and Kanner RE. Acute effects of PM10 pollution on pulmonary function of smokers with mild to moderate chronic obstructive pulmonary disease. Am Rev Respir Dis 147: 1336-1340, 1993[ISI][Medline].

37.   Pritchard, RJ, Ghio AJ, Lehmann JR, Winsett DW, Tepper JS, Park P, Gilmour MI, Dreher KL, and Costa DL. Oxidant generation and lung injury after particulate air pollutant exposure increase with the concentrations of associated metals. Inhal Toxicol 8: 457-477, 1996[ISI].

38.   Pugin, J, Schürer-Maly CC, Leturcq D, Moriarty A, Ulevitch RJ, and Tobias PS. Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14. Proc Natl Acad Sci USA 90: 2744-2748, 1993[Abstract].

39.  Quality of Urban Air Review Group. Airborne particulate matter in the United Kingdom: third report of the Quality of Urban Air Review Group. London, 1996, p. 176.

40.   Quay, JL, Reed W, Samet J, and Devlin RB. Air pollution particles induce IL-6 gene expression in human airway epithelial cells via NF-kappa B activation. Am J Respir Cell Mol Biol 19: 98-106, 1998[Abstract/Free Full Text].

41.   Salvi, S, Blomberg A, Rudell B, Kelly F, Sandstrom T, Holgate ST, and Frew A. Acute inflammatory responses in the airways and peripheral blood after short-term exposure to diesel exhaust in healthy human volunteers. Am J Respir Crit Care Med 159: 702-709, 1999[Abstract/Free Full Text].

42.   Schwartz, J, Slater D, Larson TV, Pierson WE, and Koenig JQ. Particulate air pollution and hospital emergency room visits for asthma in Seattle. Am Rev Respir Dis 147: 826-831, 1993[ISI][Medline].

43.   Seaton, A, MacNee W, Donaldson K, and Godden D. Particulate air pollution and acute health effects. Lancet 345: 176-178, 1995[ISI][Medline].

44.   Selby, C, Drost E, Wraith PK, and MacNee W. In vivo neutrophil sequestration within lungs of humans is determined by in vitro "filterability." J Appl Physiol 71: 1996-2003, 1991[Abstract/Free Full Text].

45.   Shukla, A, Timblin C, Berube K, Gordon T, McKinney W, Driscoll K, Vacek P, and Mossman BT. Inhaled particulate matter causes expression of nuclear factor (NF)-kappa B-related genes and oxidant-dependent NF-kappa B activation in vitro. Am J Respir Cell Mol Biol 23: 182-187, 2000[Abstract/Free Full Text].

46.   Simeonova, PP, and Luster MI. Asbestos induction of nuclear transcription factors and interleukin 8 gene regulation. Am J Respir Cell Mol Biol 15: 787-795, 1996[Abstract].

47.   Staal, FJT, Roederer M, Herzenberg LA, and Herzenberg LA. Intracellular thiols regulate activation of nuclear factor kappa B and transcription of human immunodeficiency virus. Proc Natl Acad Sci USA 87: 9943-9947, 1990[Abstract].

48.   Standiford, TJ, Kunkel SL, Basha MA, Chensue SW, Lynch JP, III, Toews GB, Westwick J, and Strieter RM. Interleukin-8 gene expression by a pulmonary epithelial cell line: model for cytokine networks in the lung. J Clin Inves 86: 1945-1953, 1990[ISI][Medline].

49.   Standiford, TJ, Kunkel SL, Phan SH, Rollins BJ, and Strieter RM. Alveolar macrophage-derived cytokines induce monocyte chemoattractant protein-1 expression from human pulmonary type II-like epithelial cells. J Biol Chem 266: 9912-9918, 1991[Abstract/Free Full Text].

50.   Striz, I, Mio T, Adachi Y, Bazil V, and Rennard S. The CD14 molecule participates in regulation of IL-8 and IL-6 release by bronchial epithelial cells. Immunol Lett 62: 177-181, 1998[ISI][Medline].

51.   Xing, Z, Jordana M, Kirpalani H, Driscoll KE, Schall TJ, and Gauldie J. Cytokine expression by neutrophils and macrophages in vivo: endotoxin induces tumor necrosis factor-alpha, macrophage inflammatory protein-2, interleukin-1 beta, and interleukin-6 but not Rantes or transforming growth factor-beta 1 mRNA expression in acute lung inflammation. Am J Respir Cell Mol Biol 10: 148-153, 1994[Abstract].

52.   Yokomizo, T, Izumi T, Chang K, Takuwa Y, and Shimizu T. A G-protein-coupled receptor for leukotriene B4 that mediates chemotaxis. Nature 387: 620-624, 1997[ISI][Medline].


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