Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

There is a substantial increase in the prevalence of allergic airway diseases, including bronchial asthma and allergic rhinitis, all over the world, and it is becoming a serious sociomedical problem. The airway inflammation observed in such disorders is complex and is a dynamic consequence that represents the results of the activation of immune and inflammatory cells such as T cells, mast cells, eosinophils, neutrophils, and so forth.

Airway epithelial cells (AECs) are known to play a central role in the airway defense mechanism via the mucociliary system as well as mechanical barriers. AECs can produce and release biologically active compounds including lipid mediators (1), growth factors (2), and a variety of cytokines/chemokines that are important in the pathogenesis of airway disorders (3). Recent experimental studies have shown that AECs respond to fine particles derived from diesel engines (diesel exhaust [DE] particles [DEPs]) and produce such cytokines as interleukin (IL)-8 and granulocyte macrophage colony-stimulating factor (GM-CSF) upon stimulation, which might play an important role in the induction and prolongation of airway inflammation by attracting and activating inflammatory cells in the airways (7). However, these studies were performed with particles collected from diesel engines that form an aggregated complex, being very different from those particles suspended in the atmosphere. The diesel engine-derived DEPs were suspended in medium and added to cultured cells, which is also different from the in vivo situation.

From these viewpoints, we attempted to establish a new in vitro exposure system that enables the cells to be exposed to newly generated DEs. By using a constant-flow system in an incubator, the cultured airway cells can be exposed to DE in a manner similar to that of the in vivo system. To assess the impact of short-term exposure to DEPs as a first step to examine effects of DEPs on the respiratory system, we investigated the effects of DEPs on cyto- kine production by AECs in vitro.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Culture of Bronchial Epithelial Cells

We used BET-1A, a simian virus 40-transformed human bronchial epithelial cell line (a kind gift from Dr. J. E. Lechner, National Cancer Institute, Bethesda, MD). The cells (passages 12-35) were cultured in serum-free hormonally defined Ham's F12 medium (HD-F12) as reported elsewhere (3, 4). The HD-F12 contained 1% penicillin- streptomycin, 5 µg/ml insulin (GIBCO, Grand Island, NY), 5 µg/ml transferrin (GIBCO), 25 ng/ml epidermal growth factor (Collaborative Research, Lexington, MA), endothelial cell growth supplement (5 µg/ml; Collaborative Research), 2 × 10¹⁰ M triiodothyronine (GIBCO), and 10⁷ M hydrocortisone (GIBCO). The cells were cultured on collagen type I-coated 100-mm dishes (Iwaki Co., Tokyo, Japan) with 10 ml of medium and incubated in a humidified atmosphere with 5% CO₂ at 37°C. The medium was changed every 2 d. Confluent monolayers of the epithelial cells were stained with antikeratin or antivimentin. No less than 98% of the cells were positive for keratin but negative for vimentin, indicating that the cells were of epithelial cell origin as described elsewhere (3, 4).

In some experiments, BET-1A cells were cultured on double-chamber plates, which mimics an in vivo situation. Briefly, BET-1A cells were plated at a density of 1 × 10⁵ cells/well onto the upper chamber (average pore size, 3 µm in diameter), which was precoated with type I collagen (Costar Transwell, Cambridge, MA).

In Vitro Cell Exposure System to DE

As shown in Figure 1A, a 2,300-cc diesel engine (manufactured by Isuzu Motor Co., Tokyo, Japan) was operated at a speed of 1,050 rpm and 80% load with a commercial light oil (Idemitsu Kosan Co., Tokyo, Japan). The engine exhaust was introduced into a dilution tunnel of 45 cm diameter and 625 cm length. Here the exhaust was mixed at a ratio of 1:8 with temperature and humidity being controlled in clean air that was obtained after passing through a high-efficiency particular air filter and a charcoal filter as described elsewhere (10).

View larger version (22K): [in this window] [in a new window]   View larger version (38K): [in this window] [in a new window]   Figure 1.   (A) The exposure system of DE. (B) The distribution of particles trapped in the DE. Particle distribution of the DEPs was determined by an Anderson 13-step sampler (minimal measurable size: > 0.06 µm). About 90% of the particles were less than 2.5 µm (PM 2.5). Results are expressed as means ± SD of three separate experiments.

The concentration of fine particles was measured by a mass monitoring system (TEOM; Rupprecht & Patashnick Co., Inc., Albany, NY). The densities of gaseous materials, including CO, NO₂, and SO₂, were evaluated by an infrared gas analyzer (ULTRAMAT-S; Fuji Electric Co. Ltd., Tokyo, Japan), the chemiluminescent detection method (ECL-300 Type; Yanagimoto MEG. Co. Ltd, Tokyo, Japan), and a flame photometric detector (Exhaust Gas Analyzer Bex-70 HD; Best Co., Inc., Tokyo, Japan). The distribution of particles trapped in the DE was determined by an Anderson 13-step sampler (Tokyo Dylec Co., Tokyo, Japan) (minimal measurable size: > 0.06 µm).

The DE was introduced into the cell culture system (Figure 1A). The culture plates were placed in a small polystyrene container (20 × 25 × 6 cm), and the container was placed in the incubator with 5% CO₂ at 37°C. The cells were exposed to DE at different time intervals (0, 0.5, 1, 2, 4, 8, and 14 h). The gas exhaust was evacuated with a constant flow of 5 liters/min. To evaluate the effect of gases in DE on AEC proliferation, we removed more than 99.99% DEPs by using an apparatus with a glass fiber filter paper (Toyo Roshi Co., Tokyo, Japan). The concentration of endotoxin in DE was examined with an endotoxin assay kit (Toxicolor; Seikagaku Corp., Tokyo, Japan). The change of pH in the culture media during the exposure experiments to DE was monitored with a pH meter (Horiba Co., Kyoto, Japan).

Uptake of [³H]Thymidine into the Cells

First, the effect of DE on BET-1A cell survival was investigated by the uptake of [³H]thymidine into the cells. The cells (200 µl, 1 × 10⁴ cells/ml) were placed on collagen type I-coated, 96-well, flat-bottomed culture plates (Iwaki Co.) and further cultured for 24 h (when the cells were 80% confluent). After exposure to DE at different time intervals (0, 0.5, 1, 2, 4, 8, and 14 h), 3.7 × 10³ Bq [³H]thymidine diluted in phosphate-buffered saline (PBS) was added to each well, and the culture plates were further incubated for 12 h. The cells were harvested on a glass fiber filter by a cell harvester (Skatron, Inc., Oslo, Norway) and dried. The incorporation of radioactivity was measured in triplicates with a  scintillation counter (Wallac Co., Turku, Finland).

Cytokine Assay

Immunoreactivity for IL-6, IL-8, IL-10, and transforming growth factor (TGF)-1 in the culture supernatants was measured by an enzyme-linked immunosorbent assay (ELISA) kit (Biosource International, Inc., Camarillo, CA). The ELISA was carried out according to the manufacturer's instruction sheet. Each sample was assayed in triplicate. To determine the effect of antioxidants on IL-8 production by BET-1A cells, pyrrolidine dithiocarbamate (PDTC) and N-acetyl-L-cysteine (NAC) were used (purchased from Sigma Chemical Co., St. Louis, MO). PDTC and NAC were dissolved in culture medium and the pH was adjusted to 7.4. PDTC and NAC were then added to the culture plates 30 min before DE exposure.

Immunohistochemistry

For immunohistochemistry, BET-1A cells were cultured on chamber glass slides with the cell attachment factor (Cell System Corp., Kirkland, WA). The cells were exposed to DEPs for 0 min (unexposed) and 4 h. The cells were incubated another 18 h with 5% CO₂ at 37°C. Immuno- histochemistry was performed using avidin-biotin complex-peroxidase (ABC-PO) as described previously in detail (11, 12). In brief, the cells were fixed with 4% paraformaldehyde and preincubated with 10% normal goat serum (Nichirei Corp., Tokyo, Japan) for 15 min to prevent nonspecific binding. The cells were then incubated with an anti-TGF- polyclonal antibody (1:100 diluted, 10 µg/ml; R&D Systems, Minneapolis, MN) for 30 min at room temperature. After rinsing in PBS, the slides were then incubated with biotin-conjugated goat antirabbit immunoglobulin (Ig)G antibody (Nichirei) and ABC-PO solution (Nichirei) for 30 min. After rinsing in PBS, the reaction products were visualized using diaminobenzidine (Sigma). Immunoreactivity was scored as negative when the BET-1A cells lacked immunostaining, and positive when they were significantly immunostained.

Reverse Transcription/Polymerase Chain Reaction

Reverse transcription/polymerase chain reaction (RT-PCR) was used to evaluate the expression levels of IL-6, IL-8, and TGF-1 messenger RNA (mRNA). Total RNA was isolated from cultured BET-1A cells using TRIzol Reagent (GIBCO BRL, Gaithersburg, MD) according to the manufacturer's instructions. The RNA was resuspended in 100 µl of 10 mM buffer and quantitated by absorbance at 260 nm by Gene Quant (Pharmacia Biotech, Cambridge, UK). Total RNA was denatured for 5 min at 95°C. RT of total RNA into complementary DNA (cDNA) was performed by using 2 µl M-murine leukemia virus reverse transcriptase (GIBCO BRL), 20 µg of total RNA, 10 µl of random primers (40 ng/ml; Takara, Shiga, Japan), 20 µl of 5× buffer (GIBCO BRL), 20 µl of deoxynucleotide triphosphate (dNTP) (2.5 mM each; Takara), and 38 µl of double-distilled water for 60 min at 37°C. A total of 10 µl of the cDNA mixture was subjected to PCR amplification in a 0.5-µl Taq polymerase (5 U/ml; Takara), 10 µl 10 × PCR buffer (Takara), 8 µl of dNTP (2.5 mM each; Takara), and 5 µl primer for each cytokine/chemokine.

The PCR primer sets for human IL-6, IL-8, and TGF-1 were purchased from Continental Laboratory Products, Inc. (San Diego, CA). The ₂-microglobulin gene primer sets were manufactured in our laboratory (13). The sequences of the primer sets are as follows: (1) IL-6 sense: 5'-ATGAACTCCTTCTCCACAAGCGC-3', antisense: 5'-GAAGAGCCCTCAGGCTGGACTG-3'; (2) IL-8 sense: 5'-CCAAGGAAAACTGGGTGCAGAG-3', antisense: 5'-GGCACAGTGGAACAAGGACTTG-3'; (3) TGF-1 sense: 5'-CAGAAATACAGCAACAATTCCTGG-3', antisense: 5'-TTGCAGTGTGTTATCCGTGCTGTC-3'; and (4) ₂-microglobulin sense: 5'-AAGATGAGTATGCCTGCCGT- 3' ,   antisense:   5' -TCACGA-CAGAGGTACAAACT-39.

The predicted amplification sizes of the amplified IL-6, IL-8, TGF-1, and ₂-microglobulin gene products were 628, 570, 186, and 262 base pairs (bp), respectively.

The PCR mixture was amplified at 30 cycles, with denaturation at 94°C for 1 min, primer annealing at 60°C for 1 min, and extension at 72°C for 2 min, with a Gene Amp PCR DNA thermal cycler (Perkin Elmer/Cetus, Norwalk, Conn.). The gels were run in 1× TAE (Tris-HCl, acetic acid, EDTA) buffer at 50 V for 60 min at room temperature, and stained with 2.5 µg/ml ethidium bromide in autoclaved double-distilled water. The products obtained by PCR with the molecular weight markers were visualized by using an ultraviolet illuminator after being separated by electrophoresis on a 2% agarose gel (molecular biology certified agarose; Bio-Rad, Hercules, CA). The efficiency of the amplification process can be greatly affected by a number of variables, including the yield and quantity of cDNA preparation, so the overall efficiency of individual PCR reactions was first calculated by measuring the amount of the coamplified 262-bp ₂-microglobulin-specific band. Briefly, the serially diluted ₂-micro-globulin gene product and the respective gene product were analyzed by electrophoresis on a 2.0% agarose gel, and then the amount of the ₂-microglobulin-specific DNA was estimated using ethidium bromide-mediated fluorescence. If the band was equal to that of one-fourth of the ₂-microglobulin gene product, for example, the relative efficiency was 1/4 (14).

Statistical Analysis

The results were analyzed by Student's t test for comparison between the two groups and by nonparametric equivalent of analysis of variance for multiple comparison, as reported (3, 4). All data are reported as means ± standard deviation (SD) of samples.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Influence of the In Vitro Exposure on BET-1A Cells

Major components of DE in the exposure chambers were measured. Average levels of DEPs in the dilution tunnel were 2.9 ± 0.9 mg/m³. The levels of CO, NO₂, and SO₂ were 10.6 ± 0.3, 7.3 ± 0.4, and 3.3 ± 0.1 ppm, respectively.

The distribution of particles trapped in the DE is shown in Figure 1B. About 90% of the particles were less than 2.5 µm (particulate matters [PM] 2.5). The DE used in the experiments was tested for endotoxin using an endotoxin assay kit. This test showed that endotoxin in the supernatants was below the detection level (< 1,000 ng/ml). The pH of DE supernatants remained almost unchanged throughout the entire experiment. The experiments performed without operation of the engine showed that such a procedure had no effect on the cell viability, thymidine incorporation, or cytokine production as compared with cells incubated without any exposure.

Uptake of [³H]Thymidine into the Cells

First, we investigated the effect of DE on [³H]thymidine incorporation into the BET-1A cells. The confluent BET-1A cells were cultured on collagen type I-coated, 96-well, flat-bottomed tissue culture plates and exposed to DE at different time intervals (0, 0.5, 1, 2, 4, 8, and 14 h). As shown in Figure 2, DE exposure resulted in significantly decreased [³H]thymidine incorporation into BET-1A cells compared with time zero at all time points (0 min, 459.8 ± 13.0 counts per min [cpm]; 30 min, 326.6 ± 5.3 cpm; 1 h, 290.3 ± 17.1 cpm; 2 h, 215.8 ± 6.5 cpm; 4 h, 178.5 ± 13.0 cpm; 8 h, 151.7 ± 13.6 cpm; and 14 h, 205.5 ± 14.0 cpm; *P < 0.01). DE decreased uptake of [³H]thymidine incorporation into BET-1A cells in a time-dependent manner up to 2 h and thereafter remained stable. Therefore, subsequent experiments were performed at 2 h exposure to DE.

View larger version (9K): [in this window] [in a new window]   Figure 2.   Effect of DE on the uptake of [3H]thymidine into BET-1A cells. The BET-1A cells (1.0 × 104 cells/ml) were placed on collagen type I-coated 96-well culture plates and cultured for 24 h (when 80% of the cells are confluent). After exposure to DE at different intervals (0, 0.5, 1, 2, 4, 8 and 14 h), 3.7 × 103 Bq [3H]thymidine was added to each well, and the cells were harvested on a glass fiber filter by a cell harvester and dried. The incorporation of radioactivity was measured with a scintillation counter. Values show averages of three independent experiments and are expressed as means ± SD. *P < 0.01 compared with 0 min.

Cytokine Induction in Response to DE

To determine whether BET-1A cells release inflammatory cytokines in response to DE, BET-1A cells were cultured on collagen type I-coated 100-mm dishes with 10 ml medium and exposed to DE for 0 min (unexposed) and 4 h. The cells were incubated another 18 h with 5% CO₂ at 37°C. The supernatants were then harvested and assayed for IL-6, IL-8, IL-10, and TGF-1. As shown in Figure 3, DE increased IL-8 production by BET-1A cells significantly at 4 h (152.3 ± 7.6 pg/ml at *P < 0.01 compared with the data at 0 min, 25.2 ± 1.0 pg/ml), whereas production of IL-6, IL-10, and TGF-1 were not increased significantly at 4 h.

View larger version (22K): [in this window] [in a new window]   Figure 3.   Effect of DE on cytokine production by BET-1A cells. BET-1A cells were cultured on collagen type I-coated 100-mm dishes with 10 ml medium and exposed to DE for 0 min (unexposed; open bars) and 4 h (shaded bars). The cells were cultured for another 18 h. The supernatants were then collected and assayed for IL-6, IL-8, IL-10, and TGF-1. Results are expressed as means ± SD of three separate experiments. *P < 0.01 compared with 0 min for IL-8.

Time Course of IL-8 Production Induced by DE

Next, we investigated the time course of IL-8 production by BET-1A cells in response to DE. BET-1A cells were cultured on collagen type I-coated 100-mm dishes with 10 ml medium and exposed to DE at different time intervals (0, 0.5, 1, 2, 4, 8, and 14 h). The cells were incubated for an additional 18 h with 5% CO₂ at 37°C. The supernatants were then harvested and assayed for IL-8. As shown in Figure 4, DE had a significant stimulatory effect on IL-8 release from BET-1A cells in a time-dependent fashion. IL-8 release was already elevated above control value at 30 min and continued to increase until 14 h (30 min, 70.5 ± 3.5 pg/ml at *P < 0.05; 1 h, 122.2 ± 5.3 pg/ml at *P < 0.05; 2 h, 134.7 ± 6.8 pg/ml at *P < 0.05; 4 h, 152.3 ± 7.6 pg/ml at **P < 0.01; 8 h, 236.9 ± 6.8 pg/ml at **P < 0.01; 14 h, 347.3 ± 7.8 pg/ml at **P < 0.01 compared with control at corresponding time points, respectively). The exposure to DE was not continued for more than 14 h.

View larger version (11K): [in this window] [in a new window]   Figure 4.   Time course of DE-induced IL-8 production by BET-1A cells. IL-8 was measured by ELISA in supernatants from BET-1A cells cultured with medium alone (circles), exposed to DE (triangles), and exposed to the flow through gases obtained by filtering the exhausts with a glass fiber filter paper (squares) at different time intervals (0, 0.5, 1, 2, 4, 8, and 14 h). Supernatants were collected for analysis at 18 h. Results are expressed as means ± SD of three separate experiments. *P < 0.05, **P < 0.01 compared with control at corresponding time points.

To investigate whether this activity of DE was induced by particles themselves or gases or neither, we removed more than 99.99% DEPs by filtering the DE with a glass fiber filter paper and studied the activity of the flow-through gases on IL-8 production by BET-1A cells. As depicted in Figure 4, the gases showed a minimal stimulatory effect on IL-8 release up to the 1 h-exposure point (30 min, 67.2 ± 1.4 pg/ml at *P < 0.05; 1 h, 81.6 ± 6.5 pg/ml at *P < 0.05 compared with control values), but had no effect on exposure at 2 h and thereafter. These results indicate that DEPs, but not DE gases, had a significant, sustained effect on the increased production of IL-8 proteins in vitro.

Effect of the Biphasic Culture Systems on IL-8 Production Induced by DEPs

To further mimic an in vivo situation, BET-1A cells were cultured on the upper side of the double-chamber plates. Confluent BET-1A cells were exposed to DE at different time intervals (0, 2, 4, and 8 h). The exposure to DE was not continued for more than 8 h. The cells were incubated for additional 18 h with 5% CO₂ at 37°C. The supernatants were then harvested and assayed for IL-8. As shown in Figure 5, only in the lower chamber did DE significantly stimulate BET-1A cells to release IL-8 in a time-dependent fashion (2 h, 56.9 ± 1.5 pg/ml at *P < 0.05; 4 h, 70.2 ± 2.4 pg/ml at *P < 0.05; 8 h, 119.3 ± 4.3 pg/ml at *P < 0.01 compared with control values of lower-side release). There was no significant production of IL-8 in the upper chamber.

View larger version (13K): [in this window] [in a new window]   Figure 5.   Effect of the biphasic culture systems on IL-8 production induced by DEPs. BET-1A cells were cultured on the upper side of double-chamber plates until confluence and exposed to DE at different time intervals (0, 2, 4 and 8 h). Supernatants were collected for analysis at 18 h. Results are expressed as means ± SD of three separate experiments. *P < 0.05, **P < 0.01 compared with 0 min of lower-side release.

Immunohistochemistry for TGF-

To investigate whether TGF- protein is induced by DEPs, we performed immunohistochemistry. BET-1A cells were cultured on chamber glass slides coated with the cell attachment factor and exposed to DEPs for 0 min (unexposed) and 4 h. The cells were incubated another 18 h with 5% CO₂ at 37°C, then immunostained. DEP-exposed BET-1A cells were immunostained with a TGF- antibody (Figure 6A). The control (unexposed) cells did not show immunostaining with a TGF- antibody (Figure 6B).

View larger version (110K): [in this window] [in a new window]   Figure 6.   Immunohistochemistry for TGF-. Immunostaining of BET-1A cells with anti-TGF- polyclonal antibody. (Original magnification: ×40; ABC-PO method): (A) BET-1A cells were exposed to DE for 4 h and incubated with anti-TGF- polyclonal antibody. (B) The cells were exposed to DE for 0 min (unexposed) and incubated with anti-TGF- polyclonal antibody.

Cytokine mRNA Expression Induced by DE

To study the mechanisms regulating the production of cytokines induced by DE, we used a semiquantitative RT-PCR. The BET-1A cells were cultured in collagen type I- coated 100-mm dishes with 10 ml medium. After exposure to DE the cells were collected at 0, 0.5, 1, 2, 4, 8, and 14 h, and the cellular RNA was isolated for RT-PCR. As shown in Figure 7A, DE induced expression of IL-6, IL-8, and TGF-1 mRNA in a time-dependent fashion. In contrast, no induction of IL-10 mRNA was detected (data not shown). The internal control, ₂-microglobulin mRNA, was expressed intensely and equally in all experiments. The relative amplification efficiency of IL-8 mRNA to ₂-microglobulin mRNA at 0, 0.5, 1, 2, 4, 8, and 14 h exposure was 0, 1/32, 1/8, 1/4, 1/2, 1/2 and 1/16, respectively; and that of IL-6 mRNA at 0, 0.5, 1, 2, 4, 8, and 14 h exposure was 1/64, 1/64, 1/32, 1/4, 1/2, 1/16, and 1/16, respectively. The relative amplification efficiency of TGF-1 mRNA to ₂-microglobulin mRNA for the same exposure times was 1/32, 1/32, 1/8, 1/4, 1/4, 1, and 1/16, respectively.

View larger version (35K): [in this window] [in a new window]   Figure 7.   RT-PCR analysis of IL-6, IL-8, and TGF-1 mRNA obtained from BET-1A cells after DE exposure. The bottom panels of A and B are serial dilutions of 2-microglobulin gene products (1, 1/2, 1/4, 1/18, 1/16, and 1/32). (A) BET-1A cells were cultured on collagen type I-coated 100-mm dishes and exposed to DE, then the cells were collected at 0 and 30 min, and 1, 2, 4, 8, and 14 h (measured in figure as minutes), and the cellular RNA was isolated. (B) IL-6, IL-8, and TGF-1 mRNA levels from BET-1A cells after the flow-through gas exposure. We removed more than 99.99% DEPs by filtering the exhaust with a glass fiber filter paper and studied the activity of the gases on IL-6, IL-8, and TGF-1 mRNA expression.

The flow-through gases alone did not show increased cytokine mRNA expression, as shown in Figure 7B.

Inhibition of IL-8 mRNA and Protein Levels by NAC and PDTC

To determine whether antioxidants NAC and PDTC had any effect on IL-8 mRNA and protein levels in BET-1A cells, the cells were cultured at different concentrations of PDTC or NAC 30 min before exposure and then exposed to DE for another 4 h. The cells were incubated for an additional 18 h with 5% CO₂ at 37°C. The supernatants were then collected and assayed for IL-8. RT-PCR was then performed to detect the changes in IL-8 mRNA levels. IL-8 production by BET-1A cells was significantly suppressed with the addition of 1 mM NAC (37.1 ± 1.4 pg/ml at *P < 0.05 compared with DE, 152.3 ± 7.6 pg/ml), 10 mM NAC (18.4 ± 1.7 pg/ml at **P < 0.01 compared with DE), and 10⁴ M PDTC (11.8 ± 1.0 pg/ml at **P < 0.01 compared with DE) as shown in Figure 8. NAC and PDTC showed a suppressive effect on IL-8 mRNA expression in BET-1A cells (Figure 9).

View larger version (11K): [in this window] [in a new window]   Figure 8.   Inhibition of IL-8 production by NAC and PDTC. BET-1A cells were cultured with different concentrations of PDTC or NAC for 30 min and then exposed to DE for 4 h. Supernatants were then collected 18 h later and assayed for IL-8. Results are expressed as means ± SD of three separate experiments. *P < 0.05, **P < 0.01 compared with DE.

View larger version (32K): [in this window] [in a new window]   Figure 9.   Inhibition of IL-8 mRNA expression by NAC and PDTC. BET-1A cells were cultured with different concentrations of PDTC or NAC for 30 min and then exposed to DE for 4 h. RT-PCR was performed to detect changes in IL-8 mRNA levels. Lane M, molecular size marker; lane 1, DE exposure alone; lane 2, DE exposure plus 1 mM NAC; lane 3, DE exposure plus 10 mM NAC; lane 4, DE exposure plus 105 M PDTC; lane 5, DE exposure plus 104 M PDTC.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

This study demonstrates that freshly generated DE induced the production of IL-8 as well as expression of IL-6, IL-8, and TGF-1 mRNA from BET-1A cells in a time-dependent manner. Exposure of the flow-through gases alone to the cells showed minimal increase in IL-8 production at 30-min and 1 h exposure but had no sustained effect at longer exposures (over 2 h). The gases did not induce any increase in IL-8 mRNA. These findings suggest that DEPs are responsible for the increased production of this potent inflammatory chemokine in in vitro exposure.

There is increasing evidence that shows a direct link between DEPs and allergic responses in the airways. Diaz-Sanchez and colleagues reported that transnasal challenges of DEP-derived extract in humans enhanced local IgE production (15). They further showed that DEPs induced local expression of various cytokines relevant to allergic inflammation (16). Studies using in vitro systems have also been reported.

We previously reported that DEPs stimulated IL-8 and GM-CSF production by human upper and lower airway epithelial cells (8). Bayram and associates also reported that DEPs induced attenuation of ciliary activity of human bronchial epithelial cells and released proinflammatory cytokines, such as IL-8, GM-CSF, and a soluble form of intercellular adhesion molecule-1 in vitro (9). However, these studies were performed with particles collected from diesel engines. The DEPs once collected by a sampler form an aggregated complex, and the sizes of particles are quite different from those collected while suspended in the atmosphere. For the better and more accurate evaluation of the effect of DEPs, we attempted to establish a new in vitro exposure system that enables the cells to be exposed to freshly generated DE. Further, we cultured airway epithelial cells in an air-liquid interface system as well as a submerged system. This in vitro system, mimicking in vivo status, can be a useful model for the bioactivities of DEPs.

IL-8 is known as a potent chemotactic factor for eosinophils, basophils, and T lymphocytes, as well as neutrophils. Erger and Casale reported that IL-8 plays an important role in eosinophil transmigration through the endothelium and epithelium (17). DEPs clearly induced an increased IL-8 production by human AECs in the present study, and thereby can be involved in the elicitation of inflammatory responses in the airways.

TGF- is also a potent chemoattractant for monocytes and macrophages and may induce transcription of other growth factors such as IL-1, platelet-derived growth factor, basic fibroblast growth factor, tumor necrosis factor (TNF)-, and TGF-. TGF- stimulates fibroblasts to synthesize collagen, fibronectin, proteoglycans, and other proteins of the extracellular matrix. TGF- also inhibits the production of proteases and can stabilize the newly formed matrix proteins. These findings suggest that TGF- may be important in tissue repair and remodeling (18, 19). In the present study DE significantly induced TGF-1 mRNA expression in BET-1A cells. Immunohistochemistry indicated that exposure to DE resulted in an increased number of cells positively stained for TGF-. All the cells, including AECs, have high-affinity receptors that specifically bind to TGF- (20, 21). That may explain why we could not detect significant levels of TGF-1 in culture supernatants in this study.

It remains to be elucidated which substance of DE is responsible for biologic effects on the function of AECs. Ohtoshi and colleagues (8) studied the activity of benzo- (a)pyrene, one of the prototypes of aromatic hydrocarbons contained in DE. It significantly activated AECs to release GM-CSF at nontoxic concentrations (8). Takenaka and coworkers also reported that direct exposure to the aromatic hydrocarbons derived from DEPs induced B cells to produce IgE (22). Seaton and associates suggested that acidic ultrafine particles characteristic of air pollution provoke alveolar inflammation which causes both acute changes in blood coagulability and release of mediators that are able to provoke attacks of acute respiratory illness (23). Moreover, Steerenberg and coworkers reported that DEPs were phagocytosed by BEAS-2B in vitro by a conventional system (7). We also used transmission electron microscopy to investigate whether the BET-1A cells phagocytose DEPs, but DEPs phagocytosed by BET-1A cells were not observed at ultrastructural levels (data not shown).

DEPs are known to produce superoxides (O₂^·) and hydroxyradicals ( ^·OH) (24). These reactive oxygen intermediates are reported to play an important role in the intracellular signaling system for a variety of biologic responses. In the present study we show inhibition of IL-8 production by antioxidants NAC and PDTC. NAC is a known precursor for glutathione synthesis. Recent clinical and experimental studies suggest that NAC can attenuate nuclear factor (NF)-B activation and reduce epithelial damage in the lung (25). PDTC is also a potent inhibitor of NF-B activation and reduces oxidant-induced cellular injuries (28, 29). DEP-derived oxidants may be responsible for the bioactivity of DEPs. Radical scavenging agents such as NAC and PDTC can easily react with DEPs and thereby eliminate reactive oxygen intermediates, resulting in inhibition of NF-B activation (25).

Epidemiologic studies have suggested associations between concentrations of ambient particulate matters and increased incidence of respiratory symptoms and hospitalization, decreased pulmonary function, and premature mortality among the general population (30). In the present study, the average levels of DEPs measured at the dilution tunnel were 2.9 mg/m³. Preliminary experiments using lower concentrations of DEPs (~ 1 mg/m³) have shown similar biologic activities (data not shown). The actual density of DEPs exposed to the cells, however, seems very difficult to measure. It is speculated that the connecting tube, lids of culture plates, and culture media (in the case of a submerged culture system) all function as barriers. The maximal daily level of suspended particulate matter in Tokyo metropolitan areas in 1995 was 192 µg/m³ (34). The levels of CO, NO₂, and SO₂ were 10.6 ± 0.3, 7.3 ± 0.4, and 3.3 ± 0.1 ppm, respectively, and these concentrations were 10 to 200 times higher than the actual measured levels in Tokyo (34). Rykowksi and Brochu reported that one can experience such dense exposure from a passing bus (35). Our present studies were conducted to investigate acute effects of DE in a new system, and further studies by using chronic exposure to lower levels of DE for more accurate elucidation of the effects of DE on human lung health. Another study for responsible substances of DE will be required to establish a more efficient environmental control program.