Interactions between human bronchoepithelial cells and lung fibroblasts after ozone exposure in vitro

Toxicology Letters 96,97 (1998) 13 – 24

Interactions between human bronchoepithelial cells and lung fibroblasts after ozone exposure in vitro D.S. Lang *, R.A. Jo¨rres, M. Mu¨cke, W. Siegfried, H. Magnussen Krankenhaus Grosshansdorf, Zentrum fu¨r Pneumologie und Thoraxchirurgie, D-22927 Grosshansdorf, Germany

Abstract Long-term exposure to ozone has been shown to cause lung fibrosis and increased collagen synthesis by fibroblasts in experimental animals. As the bronchial epithelium appears to play a major regulatory role in inflammatory processes, we investigated whether ozone induces bronchoepithelial cells in vitro to increase gene expression of procollagens and other fibrogenic mediators in human lung fibroblasts. Membrane cultures of human airway epithelial cells (BEAS-2B) in the presence or absence of lung fibroblast (HFL-1) cultures were exposed to air or 500 ppb ozone for 1 h, followed by (co-)incubation periods of 11 and 23 h. After ozone exposure of the co-cultures, there were substantial increases of steady-state mRNA levels of both a1 procollagens type I and III as well as TGF b1 in the fibroblasts above the corresponding air control levels. In the absence of ozone, the presence of epithelial cells always caused significant decreases in the basal steady-state mRNA levels of both procollagens as compared to their absence. There were no significant effects of ozone on the secretion or gene expression of TGF b2, PDGF or IL-8 in any cell type. In contrast, co-culture condition induced altered patterns of IL-8 gene expression or of PDGF production in fibroblasts and bronchoepithelial cells, respectively, both in the absence or presence of ozone. In summary, our data demonstrate that the effect of ozone on fibroblasts was mediated by epithelial cells and that mutual regulatory interactions between the different cell types occur. Thus, our co-cultivation system in vitro appears to be able to mimic the in vivo situation providing insight into the nature of cellular interactions and modulation by ozone, which may occur in the whole organism after long-term exposure. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Co-cultivation system; Pulmonary fibrosis; Fibrogenic cytokines; Collagen expression; mRNA analysis

Abbre6iations: transforming growth factor b1, TGF b1; transforming growth factor b2, TGF b2; platelet-derived growth factor AB, PDGF AB; interleukin 8, IL-8; interleukin 1b, IL-1b; tumor necrosis factor alpha, TNF a; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; keratinocyte growth medium, KGM; hanks’ balanced saline solution, HBSS; reverse transcription-polymerase chain reaction, RT-PCR; lactate dehydrogenase, LDH. * Corresponding author. Present address. Fraunhofer Society, Department of Toxicology and Environmental Medicine, Grindelallee 117 D-20146 Hamburg, Germany. Tel.: + 49 40 458117; fax: + 49 40 41235316. 0378-4274/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274(98)00045-9

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1. Introduction Tropospheric ozone, as one of the major components of ambient air pollution, has generated increasing concerns about its potential health effects. A series of studies have shown that shortterm exposure to ozone causes epithelial inflammation and cell injury in human subjects and experimental animals (Lippmann et al., 1989; Magnussen et al., 1996). Furthermore, animal studies demonstrated the development of chronic epithelial inflammation and interstitial lung fibrosis (Chang et al., 1991; Last et al., 1993) associated with an accumulation of extracellular matrix proteins such as collagen after long-term ozone exposure. The difficulty of assessing fibrotic changes within the lung of human subjects may be partially responsible for the lack of data on ozone-induced fibrotic lesions. In this respect, in vitro exposures of human cells appear to be a reasonable approach to examine the mechanisms of ozone-induced effects. Fibrogenic cytokines, such as interleukin 1 (IL-1), tumor necrosis factor a (TNF a) (Elias et al., 1990), platelet-derived growth factor (PDGF), and members of the transforming growth factor b (TGF b) family, have been demonstrated to be major regulators of fibroblast proliferation and collagen production in vitro (Antoniades et al., 1990; Sault et al., 1991). Due to the ability of airway epithelial cells to produce most of these cytokines (Barnes et al., 1994), we hypothesized that ozone might affect lung fibroblasts via its action on the epithelial cells, which are the primary cells exposed to inhaled air pollutants, and that these effects include increased fibroblast collagen synthesis as demonstrated by animal data. For this purpose, human bronchoepithelial cells (BEAS-2B) were co-cultivated with human foetal lung fibroblasts (HFL-1) beneath them and exposed to ozone, in order to match the in vivo situation as closely as possible. BEAS-2B cells were used for the present in vitro study because they are a stable proliferative cell line, derived from normal human bronchial epithelium, which has been characterized and shown to maintain typical epithelial cell morphology (Ke et al., 1988)

as well as similar antioxidative capacities as primary epithelial cells cultured in vitro (Kinnula et al., 1994). In addition, previous studies using this cell line have characterized the secretion of inflammatory and fibrogenic mediators such as IL-6 and IL-8 (Devlin et al., 1994) following ozone exposure in vitro. These mediators were also measured in elevated concentrations in the bronchoalveolar lavage fluid of human subjects after ozone exposure (Devlin et al., 1991), thus indicating a correspondence between in vitro and in vivo results. In the fibroblasts, gene expression of a1(I) and a1(III) procollagens, the major matrix proteins involved in the development of lung fibrosis in humans (Seppa et al., 1982) was examined. In addition, we measured the gene expression and/or secretion of a series of proinflammatory and fibrogenic cytokines, including IL-8, TGF b1 and b2 and PDGF in both epithelial cells and fibroblasts, to gain insight into the mechanisms of ozone-induced responses.

2. Materials and methods

2.1. Bronchoepithelial cells The BEAS-2B cells derived from normal human bronchial epithelium and immortalized by adenovirus 12-SV 40 hybrid virus transfection were obtained from the American type culture collection (ATCC, Rockville, MD, USA). They were maintained in serum-free KGM medium (KGM Bullet-Kit, Cell Systems, Remagen, Germany), which was replaced three times weekly. After brief trypsinization of the confluent cell cultures, 0.5× 106 cells were plated onto 25 mm diameter tissueculture-treated polyester membranes with a 0.4 mm pore size (Clear transwell membrane, Costar, Bodenheim, Germany), which were inserted into six-well culture plates (Costar). Cells were maintained in 1.5 ml KGM medium on top (apical compartment) and 2.5 ml KGM medium beneath (basolateral compartment). After 2 days, cells reached confluence and the medium was replaced with KGM containing 1.2 mM calcium for an additional 4 days (one medium change after 2

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days), in order to inhibit proliferation and promote differentiation. By this method, bronchoepithelial cells grown in several layers were obtained, which were used to attain maximum mediator production. For mRNA analyses in the epithelial cells, however, it has proved to be necessary to grow the cells in subconfluent monolayers. For this purpose, 0.25× 106 BEAS-2B cells were plated on the membrane inserts and cultured for 3 additional days. Medium change with KGM containing 1.2 mM calcium was performed once, one day before exposure to inhibit cellular growth. All experiments were performed between passages 110 and 120 of the BEAS-2B cells.

2.2. Lung fibroblasts The human foetal lung fibroblast cell line HFL-1 was obtained from ATCC (Rockville, MD, USA). Cells were cultured in DMEM/ Ham’s F12 (1:1) (Biochrom, Berlin, Germany) with 10% FCS (Hyclone, Linaris, Bettingen, Germany), which was replaced every other day. Prior to exposure, confluent fibroblast monolayers were trypsinized and 0.2× 106 cells were seeded into six-well plastic culture plates in a total volume of 2 ml per well. The medium was replaced every other day. The experiments were run after 4 days, when the cells had reached 80% confluence. The experiments were performed using passages between 7 and 11 of the HFL-1 cells.

2.3. Ozone exposure In vitro ozone exposure was performed in a 60 l tissue culture incubator (BB6060, Heraeus, Hanau, Germany) at 37°C, 5% CO2, and \98% relative humidity. Ozone was generated from filtered air by ultraviolet light. The final air flow rate into the incubator was 50 l/min. Ozone concentration was monitored by an ultraviolet ozone analyzer (OML 001.4, Sorbios, Berlin, Germany) and flow was adjusted by a controller (Sipart DR20, Siemens, Berlin, Germany). When stable conditions were reached inside the chamber, exposures were started. Control cells were exposed

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simultaneously in a second tissue culture incubator, which received filtered air instead of ozone. Immediately before exposure, the medium was completely removed from epithelial cell membrane cultures and fibroblasts and both cell cultures were washed once with HBSS. A total of 1.6 ml HBSS per well was added to the fibroblasts in the six-well culture plates and the membrane supports with the epithelial cells were immediately inserted into these six-well plates. By this approach, epithelial cells but not fibroblasts were directly exposed to gas phase ozone. At the same time, the volume of HBSS in the basolateral compartment was sufficient to keep the cells hydrated. Duplicate cell cultures were exposed to 500 ppb ozone or filtered air for 1 h. Immediately after exposures, HBSS was replaced with 1.5 ml KGM medium into the apical and 2.5 ml KGM in the basolateral compartment, respectively. Subsequently, cultures were co-incubated for an additional 11 and 23 h at 37°C, 5% CO2, and \98% relative humidity. These rather long incubation periods were chosen because previous experiments have demonstrated that control as well as ozoneinduced production of fibrogenic cytokines such as PDGF was not detected before 7 h after exposure. For direct comparisons with the co-cultures, separate membrane cultures of bronchoepithelial cells alone were simultaneously exposed to ozone or filtered air under otherwise identical conditions. Additional control cultures of fibroblasts alone were exposed to filtered air only because they remained submerged in a total volume of 2 ml HBSS. After 11 and 23 h, the supernatants were collected by pooling the media from the apical and basolateral compartment. Following centrifugation, the supernatants were stored at − 80°C until required for cytokine analysis. The cells were lysed in guanidinium thiocyanate for subsequent total RNA extraction.

2.4. Cytotoxicity As a marker of cell integrity, lactate dehydrogenase (LDH) was quantified in all supernatants by NADH oxidation (COBAS MIRA, Roche, Basel, Switzerland).

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2.5. Re6erse transcription-polymerase chain reaction (RT-PCR) Total cellular RNA was purified by the singlestep acid guanidinium thiocyanate-phenol-chloroform extraction method (Chomczynski et al., 1987) and quantified by absorbance spectrometry at 260 and 280 nm. cDNA synthesis was performed with 0.2–1 mg of total RNA in a 50 ml reaction mixture, containing PCR buffer (pH 9.3), 3 mM MgCl, 10 U m/l M-MLV reverse transcriptase (Gibco, Eggenstein, Germany), 1 mM dNTP (Pharmacia, Freiburg, Germany), 1 U/ml RNAse inhibitor (Serva, Heidelberg, Germany), and 5 pM random hexamers (Pharmacia). The RT reaction was performed for 45 min at 39°C, followed by 95°C for 5 min. For amplification of the specific cDNA sequences, 2 ml aliquots of the RT products were added to 48 ml PCR reaction mix, consisting of PCR buffer (pH 9.3) with 3 mM MgCl, 0.25 mM dNTP and 1.25 U of taq polymerase (Gibco). The PCR was performed in a 96-well thermocycler (PTC-100, MJ Research, Biozym, Hessisch Oldendorf, Germany). Sense and antisense primers for the different cytokines were used as follows: IL-8 sense: 5%TCTGCAGCTCTGTGTGAAGGTGCA-3% antisense: 5%AACCCTCTGCACCCAGTTTTCCTT-3% PDGFA sense: 5%CGCAGTCAGATCCACAGCAT-3% antisense: 5%-GATCAGGAAGTTGGCGGACG3% TGF b1 sense: 5%TCTGCTGAGGAGGCTCAAGT-3% antisense: 5%-CCGTGGAGCTGAAGCAATAG3% TGF b2 sense: 5%GGCACCTCCACATATACCAG-3% antisense: 5%-CGCAGCAAGGAGAAGCAGAT3%

GAPDH sense: 5%CCATGGAGAAGGCTGGGG-3% antisense: 5%-CTAAGCAGTTGGTGGTGC-3% The a1 (I) procollagen primer pair was made to the bp 6421–6440 (sense) and bp 7519–7538 (antisense) of the a1 procollagen type I cDNA sequence (D’Alessio et al., 1988) and the a1 (III) procollagen primer pair was made to the bp 57– 76 (sense) and bp 439–458 (antisense) of the a1 procollagen type III cDNA sequence (Cole et al., 1990). All primer pairs were used at a final concentration of 0.1 pmol/ml. One cycle of PCR comprised denaturation at 94°C for 1 min, primer annealing at 56°C for 1 min, and extension at 72°C for 2 min. This cycle was repeated between 25 and 34 times. The numbers of cycles have been previously optimized for each pair of primer. Negative controls (PCR mixture without template, plasmid preparations with irrelevant cytokine cDNA inserts) and positive controls (plasmid constructs with the cDNA inserts of the corresponding cytokine) were amplified in the same PCR assays. To control for the RT step, the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was always included in the RT-PCR and all samples were normalized to the corresponding GAPDH bands. In addition, PCR product identity for IL-8 and GAPDH was confirmed by double-stranded sequencing (DyeTerminator Sequenase Sequencing System, Applied Biosystems GmbH, Weiterstadt, Germany). Ten microlitres of each PCR product were analyzed by electrophoresis on a 2% agarose gel and stained with ethidium bromide. To ensure a cycledependent increase in the reaction product, two to three subsequent cycles were analyzed simultaneously. The DNA bands were visualized on a UV illuminator and recorded in digital form (DocuGel V, MWG Biotech, Ebersberg, Germany) to assess the optical densities (OneDScan, MWG Biotech).

2.6. Protein quantification assays Concentrations of PDGF AB and TGF b2 in the supernatants were quantified by ELISA (R&D Systems, Biermann, Germany). In order to acti-

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Fig. 1. Cytotoxicity of ozone on human bronchoepithelial cells (BEAS-2B) and on human lung fibroblasts (HFL-1) is shown in co-culture and in separate cultures of either epithelial cells (epi) or fibroblasts (fibro) alone. Data are shown as arithmetic means and standard error (SEM) of LDH release by the different cell cultures 11 (left panel) or 23 h (right panel) following exposure to filtered air (open bars) or ozone (hatched and full bars). The number of independent experiments was n = 10 – 14. Asterisks indicate significant differences either between ozone and air control values at one time point or between corresponding exposures at different time points with p B0.05. a indicates significant time-dependent differences between corresponding (air) exposures for epithelial cells alone with p B0.001.

vate latent TFG b2, supernatants were incubated at 80°C for 10 min prior to measurements.

3. Results

3.1. Cellular damage 2.7. Statistical analysis Arithmetic mean values and standard errors of mean (SEM) were computed for data, which were normally distributed. PCR data, which were expressed as relative densities, were logarithmically transformed when required for normal distribution and geometric mean values and SEM were computed. Two-way analysis of variance was used for statistical comparisons of air versus ozone-induced effects in each cell type for each time point separately and between time points. Furthermore, effects of corresponding exposure conditions in co-cultures versus control cultures of each cell type alone were also analyzed accordingly. Statistical significance was assumed at pB0.05.

LDH release from epithelial cells significantly (pB0.05 each) increased by 49 and 46% of the air control values 11 and 23 h after ozone exposure, respectively (Fig. 1). However, values below 100 unit/l indicate only negligible cellular damage by ozone. Likewise, fibroblasts released low amounts of LDH. In contrast to epithelial cells, they appeared not to be further affected by ozone in the co-cultures, which thus appear to reflect mainly epithelial-derived LDH releases.

3.2. a1 Procollagens type I and III gene expression In the presence of epithelial cells, ozone exposure enhanced the procollagen gene expression in

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Fig. 2. The effects of ozone on gene expression of a1 procollagens type I (left panel) and type III (right panel) are shown in HFL-1 cells, in co-culture with and without BEAS-2B. The steady-state mRNA levels of air-(open bars) and ozone-exposed (crosshatched bars) fibroblasts are shown after the different incubation periods as geometric mean values and SEM, which have been normalized to GAPDH. The number of independent experiments at 11 and 23 h was always n =14. Asterisks indicate significant differences according to Fig. 1 with pB 0.001 (**) and pB 0.05 (*). a indicates the significant time-dependent difference between corresponding ozone-induced effects in the co-cultured fibroblasts with pB 0.05.

fibroblasts. On average, steady-state mRNA levels of a1(I) procollagen were significantly increased by 245% 11 h (pB0.001, n=14) and by 85% 23 h (p B 0.05, n= 14) after ozone as compared to filtered air exposure (Fig. 2, left panel). These ozone-induced effects were significantly (pB 0.05) different with increasing incubation period (Fig. 2, left panel, crosshatched bars). Corresponding values for a1(III) procollagen of 88% (p B0.05, n =14) and of 42% (n.s., n= 14) above air control values, respectively, were not significantly different between these time points (Fig. 2, right panel). In the absence of ozone, steady-state mRNA levels of both procollagens were significantly and consistently suppressed in fibroblasts by co-culture with epithelial cells (Fig. 2, open bars).

3.3. TGF b1 gene expression Gene expression of TGF b1 was studied in fibroblasts only. In co-culture with epithelial cells, steady-state mRNA levels of TGF b1 were significantly enhanced by 115% above corresponding air

control values 11 h (pB 0.05, n = 7) after ozone exposure (Fig. 3). In the absence of ozone, the amounts of TGF b1 transcripts in the co-cultivated fibroblasts were reduced by 50% (n.s.) of their corresponding constitutively expressed levels after 11 h but not after 23 h of co-incubation (Fig. 3).

3.4. TGF b2 gene expression and secretion Gene expression and secretion of TGF b2 were analyzed in both fibroblasts and epithelial cells after incubation periods of 11 and 23 h in three to five independent experiments (Fig. 4A, B). In each cell type, there were no consistent changes of either TGF b2 transcript levels or cytokine production in response to ozone as compared to filtered air exposures, independent of culture condition or time. Interestingly, both fibroblast-derived steady-state mRNA levels encoding for TGF b2 (Fig. 4A, right panel) and their capacity to produce this fibrogenic mediator (Fig. 4B) were on average always twice as high as those exhibited by airway epithelial cells.

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3.5. PDGF gene expression and secretion Because there were no detectable amounts of PDGF in fibroblast-derived supernatants, both PDGF A gene expression (Fig. 5, left panel) and PDGF AB production (Fig. 5, right panel) were examined in bronchoepithelial cells only. In general, both parameters appeared not to be consistently altered by ozone exposure. However, ozone-induced increases, although not significant, both in mRNA levels and in PDGF secretion above the corrresponding air controls were evident in the co-cultured cells after 11 h. In addition, the timecourse of PDGF AB secretion was substantially altered due to the co-culture conditions (Fig. 5, right panel). In the presence of fibroblasts, cytokine production has not increased by 23 h as compared to 11 h, whereas, in the absence of fibroblasts, PDGF AB concentrations at 23 h were clearly (p B0.005 each, n= 9) higher than those measured at 11 h after exposure to both filtered air and ozone. As a

Fig. 3. The effects of ozone on the steady-state TGF b1 mRNA levels are presented in the HFL-1 cells in co-culture with and without BEAS-2B cells. Raw data were normally distributed and not logarithmically transformed. They are shown as arithmetric mean values and SEM, which have been normalized to GAPDH. The number of independent experiments were n =7 (11 h) and n=10 (23 h). Asterisk indicates significant difference between ozone and air control values with pB 0.05.

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consequence, PDGF AB levels in supernatants from co-cultivated epithelial cells were significantly (pB 0.005 each) lower than in those derived from epithelial cells alone.

3.6. IL-8 gene expression In general, there were no significant ozone-induced changes in IL-8 gene expression in both airway epithelial cells (Fig. 6, left panel) and fibroblasts (Fig. 6, right panel). As an effect of co-culture, fibroblasts always expressed clearly enhanced amounts of IL-8 mRNA compared to their basal mRNA levels (n=3–4) and also related to epithelial-derived levels as well.

4. Discussion The present study demonstrates that exposure of human bronchoepithelial cells (BEAS-2B) to ozone in vitro caused enhanced gene expression of a1(I) and a1(III) procollagen as well as of TGF b1 in human lung fibroblasts (HFL-1), indicating their regulatory potential on fibroblasts. In the absence of ozone, the presence of bronchoepithelial cells not only caused a strong reduction of both procollagen steady-state mRNA levels but also enhancement of gene expression of IL-8 in the fibroblasts. These findings further support the fact that airway epithelial cells have the capacity to directly modulate fibroblast activity in our in vitro model. We did not determine collagen synthesis directly because mRNA expression by RTPCR provides a fast and sensitive method to examine early fibroblast activation. As a consequence, there are no data about modulatory effects on (post)translational activity in fibroblasts due to co-culture with epithelial cells. Exposure studies in mice have shown that ozone exposure for at least 14 days was necessary to cause modest evidence for increases in mRNA levels and enhanced production of extracellular matrix proteins by lung fibroblasts (Sun et al., 1988). In the present study, the co-cultivation system with ozone-exposed epithelial cells on top and fibroblasts beneath them was designed to mimic the in vivo situation as far as possible

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Fig. 4. The effects of ozone on both gene expression (A) and production (B) of TGF b2 in human bronchoepithelial cells (Fig. 4 A, left panel) and lung fibroblasts (Fig. 4 A, right panel) are shown for co-cultures and the corresponding separate cultures of each cell type. Data for both (co-)incubation periods after exposures to air (open bars) and to ozone (solid and crosshatched bars, respectively) are shown as geometric mean values and SEM, which have been normalized to GAPDH. The number of independent experiments were n = 3 – 5 for both parameters.

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Fig. 5. The effects of ozone on both steady-state mRNA levels for PDGF A (left panel) and PDGF AB secretion (right panel) in the bronchoepithelial cells are shown for co-cultures with and without fibroblasts 11 and 23 h after exposures to air (open bars) or ozone (full and crosshatched bars, respectively). The results of PDGF A expression of three to four independent experiments are shown as geometric mean values and SEM, which have been normalized to GAPDH. The results of PDGF AB production are presented as arithmetic means and SEM of seven to nine independent experiments. Asterisks indicate significant differences according to Fig. 1 with p B0.005.

under in vitro conditions, although pulmonary cells could be exposed only for a very short period of time as compared to in vivo exposures. When epithelial cells were present, we found a consistent inhibition in steady-state mRNA expression of both procollagens in fibroblasts, as compared to the corresponding constitutively expressed amounts of mRNA in fibroblasts alone. In this respect, it is important to note that gene expression for procollagens appeared to be constant over time in the separate control (air-exposed) fibroblast cultures, thus indicating that they were not affected by the experimental procedure including their placement into serum-free culture medium. The observed downregulation by bronchoepithelial cells is in contrast to the reported finding that both type I procollagen steady-state mRNA levels and collagen production in HFL-1 cells were increased after 4 days of incubation in 50% conditioned medium from bovine bronchoepithelial cells (Kawamoto et al., 1995). This discrepancy may be due to the con-

siderable differences in experimental design by using conditioned medium from bovine epithelial cells and by the fact that cells were derived from two different species, which may have interfered with the normal interactions between these two cell types. Furthermore, the effects were seen after an incubation period of 4 days, which is considerably longer than that used in the present study. Our data, showing a difference in the time pattern of PDGF AB secretion in the co-cultured airway epithelial cells, provide evidence also for a regulatory action of fibroblasts on epithelial cells. This is further supported by our findings that these cells had a higher capacity or inducibility to secrete and/or express TGF b2 (Fig. 4) and IL-8 (Fig. 6) than the bronchoepithelial cells under co-culture conditions. Furthermore, regulatory potential has also been demonstrated previously, where human pulmonary fibroblasts were found to stimulate eosinophils (Weller et al., 1989) or mast cells (Levi-Schaffer et al., 1994).

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Fig. 6. The effect of ozone on gene expression of IL-8 in BEAS-2B cells (left panel) and HFL-1 cells (right panel) are shown in the presence or absence of the other cell type. In three to four independent experiments, subconfluent monolayers of the BEAS-2B cells were used for mRNA analysis. The steady-state mRNA levels of air-exposed (open bars) or ozone-exposed (full and crosshatched bars, respectively) cultures were normalized to GAPDH and presented as geometric means and SEM after incubation periods of 11 or 23 h following exposure.

Not only stimulatory activity but also inhibitory capacity on fibroblast collagen production has been reported in direct co-cultures in vitro of bovine epithelial cells and HFL-1 cells (Nakamura et al., 1995). Our results indeed suggest that direct exposure to ozone completely abrogated the suppressive action of epithelial cells on fibroblasts. It may be speculated that this effect was based on reduced production of inhibitory mediators by epithelial cells because the (concomitent) production of stimulatory factors could not be demonstrated, at least not with the examined fibrogenic growth factors. It is unlikely that the loss in the inhibitory capacity of epithelial cells might be associated with the observed cellular damage by ozone because the measured LDH concentrations were negligible related to maximal LDH release of nearly 1000 units/l. TNF a and IL-1b have been shown to cause a concentration-dependent inhibition of the production of type I procollagen mRNA by 50 and 66%, respectively, by a PGE2-dependent

mechanism (Diaz et al., 1993). In the present study, we only detected very low concentrations of TNF a and no IL-1b in the supernatants, which suggest a negligible role of these mediators in our test system (data not shown). Another explanation could be the possibility that the maximum concentrations of these mediators had been achieved at earlier time points and were already reduced to background levels, when the supernatants of our co-cultures were collected. TGF b has been shown to stimulate both fibroblast gene expression and synthesis of types I and III collagen in vitro (Ignotz et al., 1987; Fine et al., 1987). TGF b1 but not TGF b2 was elevated in human pulmonary disease (Khalil et al., 1996). Likewise, in the present study, TGF b1 appeared to be linked to the ozone-induced alterations in procollagen gene expression but not TGF b2. The regulatory role of this isoform of TGF b has been confirmed by the finding that the activity of the type I collagen promotor can be increased by TGF b1 (Rossi et al., 1988).

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In addition to TGF b, platelet-derived growth factor (PDGF) isoforms have also been suggested to be essential mediators in fibrogenesis, due to their chemotactic and stimulatory actions on fibroblasts (Seppa et al., 1982; Ross et al., 1986; Lurton et al., 1995). However, our findings were based on a low number of experiments and do not allow to prove the hypothesis that PDGF AB, which was exclusively secreted by bronchoepithelial cells, was involved in the ozone-induced increases of fibroblast procollagen gene expression in our co-cultures. Although the in vitro system, which we have developed, bears obvious limitations as compared to the in vivo situation, our results are consistent with the findings in experimental animals that ozone exposure may be associated with fibrotic processes within the airways and the lung. It may be speculated that we have mimicked an initial transient phase of fibroblast activation in human lung cells. The limitations in exposure and culture time set by the in vitro conditions with two different cell types in one assay did not allow us to study progression in procollagen gene expression after prolonged exposures or more extended cocultivation periods. Furthermore, our test system used an immortalized cell line with an altered cell phenotype compared to normal cells in vivo. However, BEAS-2B cells represent a stable source of tissue, which retained typical general characteristics of bronchial epithelium. In conclusion, the co-cultivation system chosen by us using bronchoepithelial cells and lung fibroblasts in co-culture can be used as a model in vitro, which provides valuable insight into the nature of mutual cellular interactions which eventually result in airway epithelium damage and structural changes induced by air pollutants such as ozone.

Acknowledgements Supported by grants from the Projekt Umwelt und Gesundheit, Karlsruhe (PUG L93009); from the Marohn-Stiftung, Erlangen; from the Landesversicherungsanstalt (LVA) Freie und Hansestadt Hamburg.

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References Antoniades, H.N., Bravo, M.A., Avila, R.E., Galanopoulos, T., Neville-Golden, J., Maxwell, M., Selman, M., 1990. Platelet-derived growth factor in idiopathic pulmonary fibrosis. J. Clin. Invest. 86, 1055 – 1064. Barnes, P.J., 1994. Airway epithelial receptors. Eur. Respir. Rev. 23, 371 – 379. Chang, L., Miller, F.J., Ultma, J., Huand, Y., Stockstill, B.L., Grose, E., Graham, J.A., Ospital, J.J., Crapo, J.D., 1991. Alveolar epithelial cell injuries by subchronic exposure to low concentrations of ozone correlate with cumulative exposure. Toxicol. Appl. Pharmacol. 109, 219 – 234. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal. Biochem. 162, 156 – 159. Cole, W.G., Chiodo, A.A., Janeczko, R., Ramirez, F., Dahl, H.-H.H., Chan, D., Bateman, J.F., 1990. A base substitution at a splice in the COL3A1 gene causes exon skipping and generates abnormal type III procollagen in a patient with Ehlers – Danlos syndrome type IV. J. Biol. Chem. 265, 17070 – 17077. D’Alessio, M., Bernard, M., Pretorius, P.J., de Wet, W.J., Ramirez, F., 1988. Complete nucleotide sequence of the region encompassing the first twenty-five exons of the human pro-alpha1 (I) collagen gene (COL1A1). Gene 67, 105 – 115. Devlin, R.B., McDonnell, W.F., Mann, R., Becker, S., House, D.E., Schreinemachers, D., Koren, H.S., 1991. Exposure of humans to ambient levels of ozone for 6.6 hours causes cellular and biochemical changes in the lung. Am. J. Respir. Cell Mol. Biol. 4, 72 – 81. Devlin, R.B., McKinnon, K.P., Noah, T., Becker, S., Koren, H.S., 1994. Ozone-induced release of cytokines and fibronectin by alveolar macrophages and airway epithelial cells, Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10), L612 – L619. Diaz, A., Munoz, E., Johnston, R., Korn, J.H., Jimenez, S.A., 1993. Regulation of human lung fibroblast a1(I) procollagen gene expression by tumor necrosis factor a, interleukin-1b, and prostaglandin E2. J. Biol. Chem. 268, 10364 – 10371. Elias, J.A., Freundlich, B., Adams, S.L., Rosenbloom, J., 1990. Regulation of human fibroblast collagen production by recombinant interleukin-1, tumor necrosis factor and interferon g. Ann. NY Acad. Sci. 580, 233 – 244. Fine, A., Goldstein, R.H., 1987. The effect of transforming growth factor-b on cell proliferation and collagen formation by lung fibroblasts. J. Biol. Chem. 262, 3897 – 3902. Ignotz, R.A., Endo, T., Massague, J., 1987. Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor-b. J. Biol. Chem. 262, 6443 – 6446. Kawamoto, M., Romberger, D.J., Nakamura, Y., Adachi, Y., Tate, L., Ertl, R.F., Spurzem, J.R., Rennard, S.I.,

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1995. Modulation of fibroblast I collagen and fibronectin production by bovine bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 12, 425–433. Ke, Y., Reddel, R.R., Gerwin, B.I., Miyashita, M., McMenamin, M., Lechner, J.F., 1988. Human bronchial epithelial cells with integrated SV 40 virus T antigen genes retain the ability to undergo squamous differentiation. Differentiation 38, 60 – 66. Khalil, N., O’Connor, R.N., Flanders, K.C., Unruth, H., 1996. TGF b1, but not TGF b2 or TGF b3 is differentially present in epithelial cells of advanced pulmonary fibrosis: an immunohistochemical study. Am. J. Respir. Cell Mol. Biol. 14, 131 – 138. Kinnula, V.L., Yankaskas, J.R., Chang, L., Virtanen, I., Linnala, A., Kang, B.H., Crapo, J.D., 1994. Primary and immortalized (BEAS-2B) human bronchial epithelial cells have significant antioxidative capacity in vitro. Am. J. Respir. Cell Mol. Biol. 11, 568–576. Last, H.A., Gelzleichter, T.R., Pinkerton, K.E., Walker, R.M., Witschi, H.P., 1993. A new model of progressive pulmonary fibrosis in rats. Am. Rev. Respir. Dis. 148, 487– 494. Levi-Schaffer, F., Rubinchik, E., 1994. Mastcell/fibroblast interactions. Clin. Exp. Allergy 24, 1016–1021. Lippmann, M., 1989. Health effects of ozone: A critical review. J. Air Pollut. Control. Assoc. 39, 672–695. Lurton, J.M., Narayanan, A.S., Raines, E., Raghu, G., 1995. PDGF AA differentially stimulates proliferation of isogenic human lung and skin fibroblasts, Am. J. Respir. Crit. Care Med., Abstract B72. Magnussen, H., Jo¨rres, R., 1996. Ozone, nitrogen dioxide, and

.

sulfur dioxide. In: Leff, A.R. (Ed.), Pulmonary and Critical Care Pharmacology and Therapeutics. McCerau-Hill, New York, pp. 9 – 20. Nakamura, Y., Tate, L., Ertl, R.F., Kawamoto, M., Mio, T., Adachi, Y., Romberger, D.J., Koizumi, S., Gossman, G., Robbins, R.A., Spurzem, J.R., Rennard, S.I., 1995. Bronchial epithelial cells regulate fibroblast proliferation, Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13), L377 – L387. Ross, R., Raines, E.W., Bowen-Pope, D.F., 1986. The biology of platelet-derived growth factor. Cell 46, 155 – 169. Rossi, P., Karsenty, G., Roberts, A.B., Roche, N.S., Sporn, M.B., de Crombrugghe, B., 1988. A nuclear factor 1 binding site mediated the transcriptional activation of a type I collagen promotor by transforming growth factor b. Cell 52, 405 – 414. Sault, M.C., Guerret, S., Janin, A., Gosselin, B., 1991. A technique for measurement of specific collagen types. Application in coal worker’s pneumoconiosis. In: Sebastien, P.P. (Ed.), Mechanism in occupational lung disease, 203, pp. 139 – 147. Seppa, H., Grotendorst, G., Seppa, S., Schiffman, E., Martin, G.R., 1982. Platelet-derived growth factor is chemotactic for fibroblasts. J. Cell. Biol. 92, 584 – 588. Sun, J.D., Pickrell, J.A., Harkema, J.R., Mclaughlin, S.I., Hahn, F.F., Henderson, R.F., 1988. Effects of buthione sulfoximine on the development of ozone-induced pulmonary fibrosis. Exp. Mol. Pathol. 49, 254 – 266. Weller, P.F., 1989. Eosinophils and fibroblasts: the medium in the mesenchyme. Am. J. Respir. Cell Mol. Biol. 1, 267 – 268.