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Air Pollution Particles Produce Airway Wall Remodeling in Rat Tracheal Explants Jin Dai, Changshi Xie, Renaud Vincent, and Andrew Churg Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada; and Health Canada, Ottawa, Ontario, Canada

There is evidence that chronic exposure to high levels of ambient particulate pollutants (PM) is associated with chronic airflow obstruction, but how this occurs is not known. We exposed rat tracheal explants to Ottawa urban air particles (ECH93) or diesel exhaust particles. After 7 d in air organ culture, both types of PM increased explant procollagen and transforming growth factor (TGF)-␤1 gene expression, and markedly increased tissue hydroxyproline. For both types of particle, nuclear factor-␬B inhibitor SN50 completely blocked increased gene expression. With EHC93, procollagen expression was inhibited by the oxidant scavenger, tetramethylthiourea, and by the iron chelator, deferoxamine, but TGF-␤1 expression was not inhibited by deferoxamine. Inhibitors of extracellular signal regulated kinase and p38 kinase did not affect EHC93-induced gene expression. With diesel exhaust particles, tetramethylthiourea and deferoxamine had no effect, but extracellular signal regulated kinase and p38 inhibitors completely blocked effects on procollagen and TGF-␤1. Fetuin, an inhibitor of TGF-␤ receptor binding, prevented increases in procollagen gene expression. We conclude that two common types of PM can directly induce expression of genes involved in fibrogenesis and actual airway wall fibrosis through nuclear factor-␬B– and TGF-␤–mediated mechanisms. PM-induced airway wall remodeling may play an important role in producing airflow obstruction in individuals living in high PM regions.

There is convincing evidence that increases in the level of ambient particulate air pollutants (PM) are associated with increases in acute cardiopulmonary mortality, hospital admissions and exacerbations of respiratory and cardiac disease (1). The effects of chronic exposure to high levels of PM have been less studied, but data are now accumulating that such exposures are associated with chronic lung disease, including increased risks of lung cancer (1, 2) and chronic airflow obstruction (3, 4). Chronic airflow obstruction has also been documented in women from developing countries who are exposed to very high levels of PM created by cooking in enclosed spaces with biomass fuels (5). The anatomic basis of airflow obstruction caused by exposure to PM is unknown. However, several studies have

examined the lungs of individuals living in high-PM regions and concluded that their small airways are abnormal. Souza and coworkers (6) compared the lungs of forensic death cases from a high- (Sao Paolo) and a low-PM region of Brazil, and showed greater levels of airway wall inflammation, airway wall thickness, and airway wall pigment in the high-PM region. Pinkerton and colleagues (7) evaluated autopsy lungs of forensic cases from Fresno, California, another high-PM area. They showed high particulate loads and increased fibrosis in the walls of the membranous and respiratory bronchioles. We evaluated a series of autopsy lungs from Mexico City, yet another high-PM locale, and compared them by formal grading analysis to lungs from Vancouver, a region of generally low air pollution. The small airways in the Mexico City lungs demonstrated markedly greater levels of fibrous tissue and muscle, and, in the respiratory bronchioles, pigment (8). Airway wall remodeling of this type has been shown to be associated with chronic airflow obstruction in both cigarette smokers and individuals with asthma (9), and presumably will have the same effect in those exposed to PM. The implication of these reports is that PM can, directly or indirectly, induce airway wall remodeling. In this article, we test this hypothesis by examining the effects of two different air pollution particles, Ottawa urban air particles (EHC93) and diesel exhaust particles (DEP), on fibrogenesis in a rat tracheal explant model of the airway wall.

Materials and Methods Air Pollution Particles

(Received in original form December 26, 2002 and in revised form February 12, 2003)

EHC93 were collected by vacuuming ambient air bag-house filters at the Environmental Health Center in Ottawa. Cleaning and composition of the material is described in Ref. 10. This material has a mass mean aerodynamic diameter of 3–4 ␮m, and a count median diameter of 0.5–0.6 ␮m (11). DEP, Standard Reference Material 1650a, was obtained from the National Institute of Standards and Technology (Gaithersburg, MD). This material has a count mean (SD) diameter of 1.55 ⫾ 0.04 ␮m. For use in the tracheal explant model, both types of particle were autoclaved overnight, dispersed in culture medium (see below), and sonicated before use.

Address correspondence to: Andrew Churg, M.D., Department of Pathology, University of British Columbia, 2211 Wesbrook Mall, Vancouver, BC, V6T 2B5 Canada. E-mail: [email protected]

Tracheal Explants

Abbreviations: diesel exhaust particles, DEP; deferoxamine, DFX; Ottawa urban air particles, EHC93; extracellular signal related kinase, ERK; nuclear factor-␬B, NF-␬B; ambient particulate air pollutants, PM; transforming growth factor-␤, TGF-␤; tetramethylthiourea, TMTU. Am. J. Respir. Cell Mol. Biol. Vol. 29, pp. 352–358, 2003 Originally Published in Press as DOI: 10.1165/rcmb.2002-0318OC on March 20, 2003 Internet address: www.atsjournals.org

Tracheal explants were prepared from 250-g Sprague-Dawley rats as previously described (12). The size of each explant was ⵑ 2 ⫻ 2 mm. Dust exposure was performed by submerging the explants, epithelial side up, in a 500 ␮g/cm2 (see Discussion) suspension of EHC93 or DEP in Dulbecco’s modified Eagle’s medium without serum for 1 h at room temperature. Control explants were exposed

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only to culture medium. Explants were then transferred to Petri dishes containing Dulbecco’s modified Eagle’s medium in agarose supplemented with 1% glutamine, 1% penicillin–streptomycin– fungizone, 1 ␮g/ml insulin, 0.1 ␮g/ml hydrocortisone, 1.5⫻ amino acids, and 10% chicken serum and maintained in air plus 5% CO2 organ culture with basal feeding in an incubator at 37⬚C for 7 d.

Measurement of Hydroxyproline Hydroxyproline was measured on individual explants by highperformance liquid chromatography using the method described in reference (13).

Gene Expression of Type I Procollagen and Transforming Growth Factor-␤1 by Reverse Transcriptase–Polymerase Chain Reaction At the end of the 7-d culture period, the explants were homogenized and RNA extracted. To obtain a reliable signal, three explants were used to prepare RNA for each data point for reverse transcriptase (RT)–polymerase chain reaction (PCR) analysis. To ensure that all explants for a given data point did not come from the same animal, explants were prepared from several different animals at the time of each experiment and mixed. Each test group for RT-PCR consisted of three data points. First-strand cDNA was synthesized using superscript RNase H RT (GIBCO-BRL, Grand Island, NY) according to the manufacturer’s instructions. Briefly, 5 ␮g RNA were added to a reaction mixture of 1⫻ first-strand buffer, 200 ng oligo(dT)12–18 primer, 0.5 mM each dATP, dTTP, dGTP and dCTP, 0.1 M DTT, plus water to 49 ␮l. Two hundred units of superscript RT was added, and the reaction incubated at 42⬚C for 1 h. PCR reactions contained 1 ␮M primers, 1.5 mM Mg⫹⫹, 200 ␮M deoxynucleotide triphosphates, reaction buffer, 2.5 U of Taq DNA polymerase (PerkinElmer Cetus Instruments, Norwalk, CT) and 1 or 5 ␮l of cDNA in a final volume of 20 ␮l. The PCR temperature profile consisted of 25 or 28 cycles of denaturation at 94⬚C for 45 s, primer annealing at 60⬚C for 45 s, and extension at 72⬚C for 1.25 min, followed by an additional 5 min final extension at 72⬚C. The PCR products were size fractionated on 1.5% agarose gel and quantified from this ethidium bromide-stained gel using a gel Documentation System (Bio-Rad Laboratories, Hercules CA). Expression of malate dehydrogenase was used as control (housekeeper) gene. The primer sequences were: procollagen type I, F: 5⬘-CCA ATC TGG TTC CCT CCC AC-3⬘ (GenBank M27208), R: 5⬘-GTA AGG TTG AAT GCA CTT-3⬘; transforming growth factor (TGF)-␤1, F: 5⬘-CGA GGT GAC CTG GGC ACC ATC CAT GAC-3⬘ (GenBank X52498), R: 5⬘-CTG CTC CAC CTT GGG CTT GCG ACC CAC-3⬘; malate dehydrogenase, F: 5⬘-CAA GAA GCA TGG CGT ATA CAA-3⬘ (GenBank AF093773), R: 5⬘-TTT CAG CTC AGG GAT GGC CTC-3⬘.

Treatment with Inhibitors and Chelators Oxidant scavenger tetramethylthiourea (TMTU). TMTU is a cell permeable scavenger of active oxygen species. Explants were exposed for 1 h to 10 mM TMTU (Sigma) and then to EHC93 or DEP with TMTU in the medium. TMTU (10 mM) was also included in the agarose culture medium for 7 d. Iron chelator deferoxamine (DFX). EHC93 or DEP was incubated overnight with 10 mM DFX (Desferal; Ciby-Geigy, Basel, Switzerland), and excess DFX was removed by washing the dust in saline before use. Dust was then resuspended in culture medium and used as above.

Figure 1. Explant hydroxyproline after EHC93 or DEP treatment. Both types of PM increase explant hydroxyproline by almost 3-fold compared with control. Values are mean ⫾ SD. *P ⬍ 0.05 or less.

Extracellular signal regulated kinase (ERK) inhibitor PD98059. Explants were exposed for 1 h to 50 ␮M PD98059 (Calbiochem, La Jolla, CA)and then to EHC93 or DEP with PD98059 in the medium. PD98059 (50 ␮M) was also included in the agarose culture medium for 7 d. p38 mitogen-activated protein (MAP) kinase inhibitor SB203580. Explants were exposed for 1 h to 25 ␮M SB203580 (Sigma) and then to EHC93 or DEP with SB203580 in the medium. SB203580 was also included in the agarose culture medium for 7 d. Nuclear factor (NF)-jB inhibitor SN50. Explants were exposed to 20 ␮M SN50 (BioMol, Plymouth Meeting, PA) for 1 h, and then to EHC93 or DEP with SN50 in the medium. SN50 (20 ␮M) was also included in the agarose culture medium Inhibition of TGFb1 activity with Fetuin. Explants were exposed for 2 h to 10 ␮M bovine fetuin (Sigma) or 10 ␮M bovine serum albumin (Sigma) as a control. They were then exposed to dust with fetuin or albumin in the medium and fetuin or albumin in the agarose culture medium for 7 d.

Statistics All experiments were repeated. Representative data are illustrated. Comparisons among groups were made by ANOVA. Values of P ⬍ .05 or less were considered significant.

Results EHC93 treatment led to an almost 3-fold increase in explant hydroxyproline after 7 d in organ culture (Figure 1). EHC93 roughly doubled gene expression of procollagen (Figure 2A), and this increase could be completely abolished by treatment of the explants with NF-␬B inhibitor SN50, oxidant scavenger TMTU, or treatment of the dust with DFX (Figure 2). However, treatment of the explants with inhibitors of p38 or ERK did not prevent increased procollgen gene expression (Figure 2). Figure 3 shows gene expression data for TGF-␤1. Treatment with EHC93 again approximately doubled gene expression, and both SN50 and TMTU completely prevented the increase (Figure 3). However, DFX had no effect on

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Figure 2. (Top panels) Effects of EHC93 on procollgen gene expression. EHC93 increases procollagen gene expression, and this effect is completely blocked by the NF-␬B inhibitor SN50, the oxidant scavenger TMTU, and the iron chelator DFX. Values are mean ⫾ SD. *P ⬍ .05 or less. (Bottom panels) Effects of MAP kinase inhibitors on EHC93 induced procollagen gene expression. Neither p38 kinase inhibitor SB203580 (SB203) or ERK inhibitor PD98059 (PD98) are protective. Procol ⫽ procollagen. Values are mean ⫾ SD. *P ⬍ 0.05 or less.

TGF-␤1 gene expression. MAP kinase inhibitors SB203580 and PD98059 similarly had no effect (Figure 3). Treatment of the explants with the TGF-␤ antagonist fetuin completely inhibited the EHC93-mediated increases in procollagen gene expression (Figure 4). Albumin, used here as a control protein, had no effect. DEP produced an increase in procollagen gene expression (Figure 5) and an increase in tissue hydroxyproline similar to that seen with EHC93 (Figure 1). The increase in gene expression was completely prevented by SN50 (Figure 5). Neither TMTU nor DFX had any effect (Figure 5), but SB203580 and PD98059 completely protected against DEP-mediated increases in procollagen gene expression (Figure 5B). Similar results were seen with DEP-driven TGF-␤1 gene

Figure 3. (Top panels) Effects of EHC93 on TGF-␤1 gene expression. EHC93 increases TGF-␤1 gene expression; SN50 and TMTU are protective, but DFX is not. Values are mean ⫾ SD. *P ⬍ 0.05 or less. (Bottom panels) Effects of MAP kinase inhibitors on EHC93 induced TGF-␤1 gene expression. Neither SB203580 (SB203) or PD98059 (PD98) are protective. Values are mean ⫾ SD. *P ⬍ 0.05 or less.

expression. DEP roughly doubled gene TGF-␤1 expression and SN50 was totally protective (Figure 6). TMTU and DFX had no effect (Figure 6). SB203580 and PD98059 again completely protected against the effects of DEP (Figure 6). Treatment of the explants with fetuin inhibited DEPdriven increases in procollagen gene expression (Figure 7).

Discussion In this article we have shown that two different types of air pollution particle cause airway wall fibrosis in a tracheal explant organ system. All inhaled particles evoke an inflammatory response, and this response always confounds the question of how particles induce pathologic reactions in vivo. Tracheal explants allow evaluation of fibrosis in a simple model that is free of extrinsic inflammatory cells; thus our findings indicate that air pollution particles are, in themselves, intrinsically fibrogenic in the airways, and that they activate gene expression of two important components of the fibro-


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Figure 4. Effects of the TGF-␤ antagonist fetuin on EHC93induced procollagen gene expression. Fetuin completely blocks the effects of EHC93; albumin, used here as a control protein, is not protective. Procol ⫽ procollagen. Values are mean ⫾ SD. *P ⬍ 0.05 or less.

genic process. TGF-␤ is a powerful stimulator of fibroblast matrix production (14), and also causes airway smooth muscle proliferation (15). Increased gene expression of procollagen reflects actual matrix generation, and increased matrix is detectable in our system as increased hydroxyproline. To our knowledge, this is the first demonstration of these effects using actual air pollution particles, and these observations thus provide an explanation for the morphologic evidence of airway wall remodeling found in the small airways in residents of high-PM regions (see Introduction). The tracheal explant system is ideal for examining fibrogenesis because it maintains epithelial and mesenchymal compartments in their normal anatomic arrangement, but the system has its peculiarities; in particular, it only works when relatively high doses of particles are used. This phenomenon probably reflects the fact that tracheal epithelial cells do not take up dust very readily or very fast; previous studies from our laboratory using this model have shown that, over the course of 7 d, dust is taken up from the surface, enters the airway epithelial cells, and is slowly transported through the cells to the underlying interstitium. Even with established fibrogenic dusts, there are no major increases in procollagen production before 7 d (16); for this reason, only 7-d cultures were examined in the present study. Despite the necessity of using a high dust dose, we have consistently found that fibrogenesis in this system corresponds quite well to in vivo fibrogenesis. At the same mass dose used in this paper, asbestos, a known fibrogenic dust in vivo, is fibrogenic in the explants (16), whereas fibrous glass and compact titanium dioxide particles, substances that are not fibrogenic in vivo, are not fibrogenic in the explants (17). In addition, despite the high dose of dust used, in all our studies the explants exclude trypan blue, and the cilia can be observed to beat, indicating that the cells are viable at 7 d. Histologically they appear normal at this time. NF-␬B exists in the cytoplasm as a protein dimer complexed to an inhibitory protein, I␬B (18). Signaling path-

Figure 5. (Top panels) Effects of DEP on procollgen gene expression. DEP increases procollagen gene expression, and this effect is completely blocked by SN50; however, TMTU, and DFX are not protective. Values are mean ⫾ SD. *P ⬍ 0.05 or less. (Bottom panels) Effects of MAP kinase inhibitors on DEP-induced procollagen gene expression. SB203580 (SB203) and PD98059 (PD98) are completely protective. Procol ⫽ procollagen. Values are mean ⫾ SD. *P ⬍ 0.05 or less.

ways that activate NF-␬B phosphorylate the I␬B molecule, leading to its dissociation from NF-␬B and translocation of NF-␬B into the nucleus, where it binds to and transactivates the promoters of a wide variety of genes, largely those mediating acute inflammatory reactions (18), but also genes that ultimately lead to fibrotic reactions (17). SN50 is a synthetic peptide that specifically blocks the translocation of NF-␬B into the nucleus (19). Our finding that SN50 consistently and completely abolishes gene expression of procollagen and TGF-␤1 indicates that both EHC93 and DEP produce fibrosis through an NF-␬B–driven pathway. However, our results also show that the pathways to fibrosis differ for EHC93 and DEP. EHC93 appears to operate through a metal/oxidant mechanism, presumably related to the generation of active oxygen species using transition metals either on the particle surface or leached from

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Figure 7. Effects of fetuin on EHC93-induced procollagen gene expression. Fetuin completely blocks the effects of EHC93; albumin is not protective. Procol ⫽ procollagen. Values are mean ⫾ SD. *P ⬍ 0.05 or less.

Figure 6. (Top panels) Effects of DEP on TGF-␤1 gene expression. DEP increases TGF-␤1 gene expression and SN50 is protective, but TMTU and DFX are not. Values are mean ⫾ SD. *P ⬍ 0.05 or less. (Bottom panels) Effects of MAP kinase inhibitors on DEP induced TGF-␤1 gene expression. Both SB203580 (SB203) and PD98059 (PD98) are protective. Values are mean ⫾ SD. *P ⬍ 0.05 or less.

the particle, because both the active oxygen species scavenger TMTU and the non–redox-active iron chelator, DFX, completely block the effects of EHC93 on procollagen gene expression. Active oxygen species, but not iron, also appear to be involved in EHC93 induction of TGF-␤1 gene expression. The results for procollagen gene expression with EHC93 are consistent with our previous observations in the tracheal explant model that addition of surface iron makes ordinarily nonfibrogenic fine (0.12 ␮m) TiO2 particles or nonfibrogenic glass fibers fibrogenic (17). In that article, we specifically found that addition of iron to TiO2 led to NF-␬B activation through both IkBa-serine 32/36 phosphorylation and -tyrosine phosphorylation pathways. Ambient air pollution particles are known to activate NF-␬B, and the importance of particle transition metals, usually iron, but also vanadium, cobalt, and nickel, in medi-

ating PM reactivity, and specifically in upregulating responses known to be controlled through NF-␬B, has been shown in a number of studies. Jimenez and coworkers (20) used Edinburgh PM10 and found that NF-␬B activation in A549 cells could be prevented by pretreatment of the particles with the iron chelators DFX and ferrozine, an observation that implicates iron as a mediator of NF-␬B activation. However, Shulka and colleagues (21) found that activation of NF-␬B by Vermont PM2.5 was not affected by DFX, although it was driven by oxidants. The inflammatory reaction to PM in rats has been demonstrated by Costa and Dreher (22) to correlate specifically with the content of transition metals rather than total particle mass. In a tissue culture system, Ghio and associates (23) found that release of interleukin (IL)-8 by respiratory epithelial cells correlated with the ionizable metal content of PM samples, and similarly lavage neutrophils and protein in rats instilled with PM samples also correlated with ionizable metal content. Frampton and coworkers (24) reported an interesting natural experiment in which closure of a steel mill reduced the iron, copper, and zinc content of locally collected PM10 compared with PM10 collected during years when the mill was open. The metal-reduced PM10 showed the least oxidant generation and cellular reactivity when applied to cultured BEAS-2B cells. Quay and colleagues (25) observed that residual oil fly ash, a type of PM particle rich in vanadium, induced IL-6 in cultured human airway epithelial cells, and this effect was inhibited by both DFX and the oxidant scavenger N-acetyl cysteine. DEP also contain transition metals (26), along with a carbon core and various polyaromatic hydrocarbons. DEP have been shown to liberate active oxygen species in cellfree aqueous solution, and this process can be prevented with DFX (26). However, transition metals and oxidants do not appear to play a role in DEP-induced fibrogenesis


in our model, because DFX and TMTU had no protective effect. Rather, DEP act here by activation of MAP kinases, because the DEP but not EHC93 effects are completely abolished by the MAP kinase inhibitors PD98059 and SB203580. MAP kinases are known to both activate and modulate the activity of NF-␬B, in part by causing I␬B degradation, and in part by regulating the transcriptional activity of NF-␬B bound to promoters (27). Our findings are consistent with a number of studies that have shown that DEP mediate acute proinflammatory reactions through MAP kinase, and specifically p38 kinasemediated pathways. However, our results differ somewhat from those in the literature, because some authors also report a role for active oxygen species. Takizawa and associates (28) found that DEP activated NF-␬B and caused increased expression of IL-8, whereas Hashimoto and coworkers (29) reported that this effect was driven through a p38-mediated pathway and could be inhibited not only by SB203580, but by the antioxidant N-acetyl cysteine. Of particular interest, Hashimoto and colleagues obtained only ⵑ 75% inhibition of cytokine production with SB203580 and suggested that other MAP kinases, specifically ERK and JNK, might be involved; however, PD98059 was not protective in their system. Abe and associates (30) found that treatment of human bronchial epithelial cells with DEP led to increased gene expression and protein production of both IL-8 and TGF-␤. The IL-8 responses could be prevented by antioxidant treatment; TGF-␤ responses after antioxidant treatment were not reported. We have previously (16) attempted to demonstrate production of TGF-␤ protein in explants exposed to asbestos using a commercial enzyme-linked immunosorbent assay kit, but were unable to detect any signal. This problem most likely reflects the extremely small size of the explants. For this reason we tried a different approach in this study. Fetuin is a specific competitive inhibitor of the TGF-␤ receptor type II (31). The finding that fetuin completely abolished the increased procollagen gene expression seen with both EHC93 and DEP suggests that increased gene expression of TGF-␤1 is translated into increased and activated TGF-␤ protein, and that TGF-␤ may be the proximate driver of procollagen expression and ultimate fibrosis in this model. We have not examined exactly how NF-␬B activation leads to TGF-␤ production in this paper, and the relationship of NF-␬B activation and TGF-␤ production is in fact poorly understood. In some cell types, endogenous production of IL-1 after NF-␬B activation leads to autocrine induction of TGF-␤ synthesis (32). However, in other systems TGF-␤ is produced after NF-␬B activation, but as a part of the late/downregulatory inflammatory response (33). As noted, using the tracheal explant model we have previously shown that amosite asbestos, applied at the same mass concentration as used here, induces procollagen gene expression and increased tissue hydroxyproline (16). Amosite is a known fibrogenic dust, and it is particularly interesting that the relative increase in hydroxyproline levels is much greater for EHC93 and DEP compared with amosite. Brody and colleagues have previously shown that asbestos also induces fibrosis through induction of TGF-␤ (34, 35),

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suggesting that, at least in the explant model, fibrogenic particles may all be working through fairly similar mechanisms. Our observations thus indicate that two actual air pollutant particles, EHC93 and DEP, are fibrogenic agents in the airways. There is very little muscle in the tracheal explants, so we do not know if these particles also cause the airway smooth muscle cell proliferation seen in the smaller airways in humans chronically exposed to high levels of air pollution (6–8); but, as noted, TGF-␤ is an inducer of smooth muscle cell proliferation, so this effect appears likely. Our findings thus provide an explanation for the observations that chronic exposure to high levels of PM produces fibrosis and increased muscle in human airways. They also emphasize the idea that PM and occupationally encountered particles in many respects behave in a very similar fashion. Acknowledgments: This study was supported by grant MOP-53157 from the Canadian Institutes of Health Research.

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