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Am. J. Respir. Crit. Care Med., Volume 161, Number 1, January 2000, 280-285

Diesel Exhaust Particles Activate p38 MAP Kinase to Produce Interleukin 8 and RANTES by Human Bronchial Epithelial Cells and N-Acetylcysteine Attenuates p38 MAP Kinase Activation

SHU HASHIMOTO, YASUHIRO GON, IKUKO TAKESHITA, KEN MATSUMOTO, ITSURO JIBIKI, HAJIME TAKIZAWA, SHOJI KUDOH, and TAKASHI HORIE

First Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan; Department of Medicine and Physical Therapy, University of Tokyo School of Medicine, Tokyo, Japan; and Fourth Department of Internal Medicine, Nippon Medical School, Tokyo, Japan

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Air pollutants including diesel exhaust particles (DEPs) have been shown to enhance allergic responses. DEPs stimulate airway epithelial cells to produce various cytokines; however, the intracellular signal transduction pathway and the involvement of reduction and oxidation (redox) control in DEP-activated signaling have not been determined. In the present study, we therefore examined the role of p38 mitogen-activated protein (MAP) kinase in DEP-induced interleukin 8 (IL-8) and RANTES production by human bronchial epithelial cells (BECs) in order to clarify the intracellular signal transduction pathway that regulates IL-8 and RANTES production. In addition, we also examined the effect of a thiol-reducing agent, N-acetylcysteine (NAC), on DEP-induced p38 MAP kinase activation and cytokine production in order to clarify the redox control mechanism in DEP-induced p38 MAP kinase activation and IL-8 and RANTES production. The results showed that DEP induced IL-8 and RANTES production and the threonine and tyrosine phosphorylation of p38 MAP kinase, reflecting the activation of p38 MAP kinase in BECs. SB 203580, as the specific inhibitor of p38 MAP kinase activity, inhibited DEP-induced IL-8 and RANTES production. NAC inhibited DEP-induced p38 MAP kinase activation and IL-8 and RANTES production. These results indicate that p38 MAP kinase plays an important role in the DEP-activated signaling pathway that regulates IL-8 and RANTES production by BECs and that the cellular redox state is critical for DEP-induced p38 MAP kinase activation leading to IL-8 and RANTES production. Hashimoto S, Gon Y, Takeshita I, Matsumoto K, Jibiki I, Takizawa H, Kudoh S, Horie T. Diesel exhaust particles activate p38 MAP kinase to produce interleukin 8 and RANTES by human bronchial epithelial cells and N-acetylcysteine attenuates p38 MAP kinase activation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The prevalence rate of allergic diseases such as bronchial asthma and rhinitis is increasing in industrial areas and countries (1, 2). Epidemiologic studies of the association between environmental factors and the prevalence of allergic diseases have reported that there may be a link between the incidence of allergic disease and air pollutants including nitrogen dioxide, ozone, and suspended particulate matter and its main component, diesel exhaust particles (DEPs) (1). There are several mechanisms by which air pollutants may contribute to enhancing allergic responses. Among air pollutants, DEPs have been shown to induce the production of proinflammatory cytokines (4) and helper T cell type 2 (Th2) lymphocyte- derived cytokines (7, 8), enhance IgE production (7, 9) and adhesion molecule expression (10), and enhance airway hyperresponsiveness (11). Airway epithelial cells are the first cells to come in contact with inhaled particles. We have previously shown that DEPs can stimulate airway epithelial cells to produce interleukin 8 (IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (6), which have been shown to be involved in the production of allergic inflammation through their chemotactic activity for neutrophils and eosinophils (12, 13). Therefore, it is an important issue to clarify the mechanism of DEP-induced cytokine production by airway epithelial cells.

Many extracellular stimuli elicit specific biological responses through the activation of mitogen-activated protein (MAP) kinase cascades. The superfamily of mammalian MAP kinases has been molecularly characterized: extracellular signal-regulated kinase (Erk), c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase (14). p38 MAP kinase is activated by a variety of extracellular stimuli, including proinflammatory cytokines, environmental stresses, DNA-damaging agents, hematopoietic growth factor, and oxidative stresses (15). Among these stimuli, oxidative stress can activate the p38 MAP kinase cascade (20, 21), and we have previously shown that the cellular reduction and oxidation (redox) state is critical for p38 MAP kinase activation and p38 MAP kinase- dependent cytokine production (22). DEPs have been shown to generate reactive oxygen species (ROSs) (23) and DEPs are well known to stimulate airway epithelial cells to produce various cytokines (4); however, the intracellular signal that regulates cytokine production has not been determined. In the present study, we examined the role of p38 MAP kinase in DEP-induced IL-8 and RANTES production by human bronchial epithelial cells in order to clarify the intracellular signal transduction pathway that regulates IL-8 and RANTES production. In addition, we also examined the effect of a thiol reducing agent, N-acetylcysteine (NAC) (24), on DEP-induced p38 MAP kinase activation and cytokine production in order to clarify the redox control mechanism in DEP-induced p38 MAP kinase activation and IL-8 and RANTES production.

    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of DEPs

DEPs used in this study were previously described in detail (6, 8). Briefly, a 4JB1-type, light-dusty (2,470 cm3), four-cylinder diesel engine (Isuzu Automobile Co., Tokyo, Japan) was connected to an EDYC dynamometer (Meiden-Sya, Tokyo, Japan) and was operated using standard diesel fuel at a speed of 2,000 rpm under the load of 6 torque (kg/m). DEPs were collected on glass-fiber filters (203 × 254 mm) in a constant-volume sampler system attached to the end of the dilution tunnel. The mean diameter of the particles was 0.4 µm. Most of the particles were globular in shape.

Reagents

NAC was obtained from Sigma (St. Louis, MO). The pyridinyl imidazole SB 203580, the specific inhibitor of p38 MAP kinase activity (25) was kindly provided by SmithKline Beecham (Philadelphia, PA) and was dissolved in dimethyl sulfoxide.

Cell Culture

Transformed human bronchial epithelial cell line BET-1A was kindly provided by H. Nakamura (Yamagata University School of Medicine, Yamagata, Japan). The cells (1 × 104 cells/ml) were placed onto a collagen-coated 24-well flat-bottom tissue culture plate (Iwaki, Tokyo, Japan) for determination of cytokine production, and onto a collagen-coated tissue culture dish (Iwaki) for analysis of p38 MAP kinase phosphorylation using Ham's F-12 medium containing 1% penicillin- streptomycin, insulin (5 µg/ml; GIBCO, Grand Isle, NY), transferrin (5 µg/ml; GIBCO), epidermal growth factor (EGF, 25 ng/ml; Collaborative Research, Lexington, MA), endothelial cell growth supplement (ECGS, 15 µg/ml; Collaborative Research), 2 × 10-10 M triiodothyronine (GIBCO), and 10-7 M hydrocortisone (GIBCO) (growth medium). The cells were grown until they were subconfluent, and the growth medium was replaced with Ham's F-12 medium without insulin, transferrin, EGF, ECGS, triiodothyronine, and hydrocortisone (growth factor-free medium) for 16 h. To examine DEP-induced IL-8 and RANTES production by bronchial epithelial cells (BECs) and the effect of SB 203580 and NAC on IL-8 and RANTES production, growth factor-starved cells were preincubated either with growth factor-free medium, SB 203580, or NAC for 60 min, and then the cells were stimulated with DEPs and cultured in growth factor-free medium for the desired times at 37° C in a humidified 5% CO2 atmosphere. At the end of the culture periods, the culture supernatants for determination of IL-8 and RANTES protein were harvested and centrifuged and the supernatants were collected, filtered with a Millipore (Bedford, MA) filter, and stored at -80° C until assay.

To examine the effect of DEP-induced p38 MAP kinase phosphorylation in BECs, growth factor-starved cells were stimulated with DEPs in growth factor-free medium and cultured for the desired times as indicated. To examine the effect of NAC on DEP-induced p38 MAP kinase phosphorylation, growth factor-starved cells were preincubated either with growth factor-fee medium or NAC for 60 min, and then the cells were stimulated with DEPs in growth factor-free medium and cultured for the desired times as indicated.

Measurement of IL-8 and RANTES

The concentrations of IL-8 and RANTES in the culture supernatants from BECs were measured by commercially available enzyme-linked immunosorbent assay (ELISA) kits (Amersham International, Aylesbury, UK). ELISA was performed according to the manufacturer instructions. All samples were assayed in duplicate.

Western Blot Analysis of p38 MAP Kinase, Erk, and JNK

The threonine and tyrosine phosphorylation of p38 MAP kinase was analyzed with commercially available kits (PhosphoPlus p38 MAPK antibody kit; New England BioLabs, Beverly, MA). Analysis of threonine and tyrosine phosphorylation of p38 MAP kinase was performed with an antibody to phosphorylated threonine and tyrosine of p38 MAP kinase antibody, which is specific for active p38 MAP kinase and does not cross-react with Erk and JNK. Analysis of threonine and tyrosine phosphorylation of Erk was performed with an antibody to phosphorylated threonine and tyrosine of p42/p44 MAP kinase antibody (anti-phospho-specific p42/p44 MAP kinase antibody; New England BioLabs), which is specific for active p42/p44 MAP kinase and does not cross-react with p38 MAP kinase and JNK. Analysis of threonine and tyrosine phosphorylation of JNK was performed with an antibody to anti-phosphorylated threonine and tyrosine of JNK antibody (anti-phospho-specific JNK antibody; New England BioLabs), which is specific for active JNK and does not cross-react with p38 MAP kinase and Erk.

Analysis of p38 MAP kinase, Erk, and JNK was performed according to manufacturer instructions. Briefly, the cells that had been washed with cold Tris-buffered saline were lysed in sodium dodecyl sulfate (SDS) buffer (62.5 mM Tris-HCl [pH 6.8], 2% [wt/vol] SDS, 10% glycerol, 50 mM dithiothreitol [DTT], 0.1% [wt/vol] bromophenol blue) for 15 min on ice and sonicated for 2 s to shear DNA. The samples were heated in a boiling water bath for 5 min to denature the proteins fully before electrophoresis and then centrifuged at 12,000 × g for 5 min to remove insoluble debris. After separating proteins from cell lysate by 15% SDS-polyacrylamide gel electrophoresis (PAGE), the cell lysate containing 10 µg of protein was electrophoretically transferred to nitrocellulose membrane and the membrane was washed with 0.1% Tween 20 supplemented with Tris-buffered saline (washing buffer). To block nonspecific protein binding, the membrane was incubated with 0.1% Tween 20 supplemented with Tris-buffered saline containing 5% (wt/vol) nonfat dried skimmed milk for 3 h at room temperature. It was then incubated with specific antibody to phosphorylated threonine and tyrosine of p38 MAP kinase (affinity-purified rabbit polyclonal IgG) for analysis of p38 MAP kinase, specific antibody to phosphorylated threonine and tyrosine of Erk (affinity-purified rabbit polyclonal IgG) for analysis of Erk, or specific antibody to phosphorylated threonine and tyrosine of JNK (affinity-purified rabbit polyclonal IgG) for analysis of JNK in 0.1% Tween 20 supplemented with Tris-buffered saline containing 5% bovine serum albumin (BSA) at 4° C overnight with gentle shaking. After washing with wash buffer three times, it was incubated for 1 h with gentle shaking at room temperature with horseradish peroxidase-conjugated anti-rabbit antibody (1:2,000) and horseradish peroxidase-conjugated anti-biotin antibody (1:1,000) to detect biotinylated protein markers and then washed three times with wash buffer.

Blots were incubated with enhanced chemiluminescence solution (LumiGLO; Amersham) for 1 min at room temperature and exposed to Kodak (Rochester, NY) XAR film. To show the amounts of p38 MAP kinase, Erk, and JNK precipitated, blots were stripped and reprobed with phosphorylation state-independent p38 MAP kinase-specific antibody (affinity-purified rabbit polyclonal IgG) to determine total p38 MAP kinase levels, phosphorylation state-independent p42/ p44 MAP kinase-specific antibody (affinity-purified rabbit polyclonal IgG) to determine total p42/p44 MAP kinase levels, or phosphorylation state-independent JNK-specific antibody (affinity-purified rabbit polyclonal IgG) to determine total JNK levels, respectively.

Statistical Analysis

Statistical significance was analyzed by analysis of variance (ANOVA). A p value less than 0.05 was considered significant.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DEPs Induce IL-8 and RANTES Production

First, we examined a dose-dependent induction of IL-8 and RANTES production by BECs. To this end, the culture supernatants from BECs stimulated with various concentrations of DEPs were harvested 24 h after cultivation. The concentrations of IL-8 and RANTES in the culture supernatants from DEP-stimulated culture increased in a dose-dependent manner (Figure 1).


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  		Figure 1.   DEPs induce IL-8 and RANTES production. BECs were cultured either with medium or various concentrations of DEPs (10, 50, and 100 µg/ml) and the concentrations of IL-8 (a) and RANTES (b) in the culture supernatants were determined 24 h after cultivation. The results are expressed as means ± SD of five different experiments. *1, p < 0.05 compared with IL-8 concentrations in BECs cultured with medium; *2, p < 0.01 compared with IL-8 or RANTES concentrations in BECs cultured with medium.

DEPs Induce Threonine and Tyrosine Phosphorylation of p38 MAP Kinase

Activation of p38 MAP kinase is mediated by dual phosphorylation of threonine and tyrosine residues of p38 MAP kinase (14, 16). Therefore, BECs were stimulated with DEPs and the cell lysates were immunoblotted with a specific antibody to phosphorylated threonine and tyrosine of p38 MAP kinase to determine whether DEPs cause p38 MAP kinase activation. Amounts of phosphorylated threonine and tyrosine of p38 MAP kinase in BECs stimulated with various concentrations of DEPs were determined 60 min after stimulation. Amounts of phosphorylated threonine and tyrosine of p38 MAP kinase in BECs increased in a dose-dependent manner (Figure 2a, top panel ). To determine the time course of p38 MAP kinase activation, BECs were stimulated with DEPs (100 µg/ml) for the desired times as indicated. Amounts of phosphorylated threonine and tyrosine of p38 MAP kinase in BECs stimulated with DEPs (100 µg/ml) were increased by 30 min, sustained between 30 to 60 min, and decreased by 120 min (Figure 2b, top panel ). Equal amounts of p38 MAP kinase protein were immunoblotted with phosphorylation-independent p38 MAP kinase-specific antibody regardless of dose of DEPs and time of culture periods, indicating that DEP-stimulated increases in the threonine and tyrosine phosphorylation of p38 MAP kinase occurred in the absence of changes in p38 MAP kinase protein levels (Figures 2a and 2b, bottom panels). We also examined whether DEPs could induce the threonine and tyrosine phosphorylation of Erk and JNK in identical cell lysates. DEPs did not induce Erk and JNK phosphorylation (data not shown).


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  		Figure 2.   DEPs induce threonine and tyrosine phosphorylation of p38 MAP kinase. BECs were stimulated with various concentrations of DEPs (1, 50, and 100 µg/ml) for 60 min (a). BECs were stimulated with DEPs (100 µg/ml) for the desired times as indicated (b). The lysates from BECs were separated by 15% SDS-PAGE, transferred to membranes, and blotted with a specific antibody to phosphorylated threonine and tyrosine of p38 MAP kinase [phospho-p38 MAP kinase; upper panel of (a) and (b)]. Blots shown in the upper panel of (a) and (b) were stripped and reprobed with a phosphorylation state-independent p38 MAP kinase-specific antibody to show the amounts of p38 MAP kinase blotted [p38 MAP kinase, lower panel of (a) and (b)]. Lane P of (a) and (b) represents positive protein prepared from C-6 glioma cells stimulated with anisomycin for phosphorylated threonine and tyrosine of p38 MAP kinase. Lane N of (a) and (b) represents the negative protein prepared from C-6 glioma cells unstimulated with anisomycin. The fold increase in amounts of phosphorylated p38 MAP kinase proteins is indicated. The amounts of phosphorylated p38 MAP kinase were quantitated by NIH Image Analyzer and are presented as the amounts of phosphorylated p38 MAP kinase proteins relative to control cells treated without agonist (1.0). Three identical experiments independently performed gave similar results.

N-Acetylcysteine Inhibits p38 MAP Kinase Activation

To determine the regulatory role of the cellular redox state in DEP-induced p38 MAP kinase activation, we examined the effect of NAC on DEP-induced threonine and tyrosine phosphorylation of p38 MAP kinase. To this end, BECs that had been preincubated with or without NAC for 60 min were stimulated with DEPs and cultured for 60 min. As shown in Figure 3, amounts of phosphorylated threonine and tyrosine of p38 MAP kinase were lower in NAC-pretreated BECs (lane 4) than in NAC-untreated BECs (lane 3), indicating that pretreatment of BECs with NAC resulted in the inhibition of DEP-induced p38 MAP kinase activation.


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  		Figure 3.   NAC inhibits DEP-induced p38 MAP kinase activation. BECs that had been preincubated either with medium or NAC (10 mM) for 60 min were stimulated with DEPs (100 µg/ml) for 60 min. The lysates from BECs were separated by a 15% SDS-PAGE, transferred to membranes, and blotted with a specific antibody to phosphorylated threonine and tyrosine of p38 MAP kinase (phospho-p38 MAP kinase; upper panel ). Blots shown in the upper panel were stripped and reprobed with a phosphorylation state-independent p38 MAP kinase specific antibody to show the amounts of p38 MAP kinase blotted (p38 MAP kinase; lower panel ). The cells were cultured with medium (lane 1), NAC (lane 2), DEPs (lane 3), and DEPs and NAC (lane 4). Lane P represents positive protein prepared from C-6 glioma cells stimulated with anisomycin for phosphorylated threonine and tyrosine of p38 MAP kinase. Lane N represents negative protein prepared from C-6 glioma cells unstimulated with anisomycin. The fold increase in amounts of phosphorylated p38 MAP kinase proteins is indicated. The amounts of phosphorylated p38 MAP kinase were quantitated by NIH Image Analyzer and are presented as the amounts of phosphorylated p38 MAP kinase proteins relative to control cells treated without agonist (1.0). Three identical experiments independently performed gave similar results.

SB 203580 Inhibits DEP-induced IL-8 and RANTES Production

DEPs induced IL-8 and RANTES production, and p38 MAP kinase activation. These results suggest that DEP-induced IL-8 and RANTES production might be mediated through p38 MAP kinase. To test this possibility, BECs that had been preincubated with or without SB 203580 were cultured with DEPs, and the concentrations of IL-8 and RANTES were determined 24 h after cultivation. The concentrations of IL-8 in the culture supernatants from the cell cultured with DEPs in the presence of SB 203580 were lower than those from the cells cultured with DEPs in the absence of SB 203580 (Figure 4a), indicating that SB 203580 inhibited DEP-induced IL-8 production. Similarly, SB 203580 inhibited DEP-induced RANTES production (Figure 4b).


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  		Figure 4.   SB 203580 inhibits DEP-induced IL-8 and RANTES production. BECs that had been preincubated with either medium or SB 203580 (10 µM) for 60 min were cultured either with medium or DEPs (100 µg/ml). The concentrations of IL-8 (a) and RANTES (b) in the culture supernatants were determined 24 h after cultivation as described in METHODS. SB 203580 (10 µM) contained 0.01% dimethyl sulfoxide, which had no effect on DEP-induced IL-8 and RANTES production. The results are expressed as means ± SD of five different experiments. *1, p < 0.01 compared with IL-8 or RANTES concentrations in the culture supernatants from DEP-stimulated BECs cultured without SB 203580.

NAC Inhibits DEP-induced IL-8 and RANTES Production

NAC inhibited DEP-induced p38 MAP kinase activation, and DEP-induced IL-8 and RANTES production was mediated through p38 MAP kinase, indicating that NAC-mediated inhibition of p38 MAP kinase activation might result in the inhibition of IL-8 and RANTES production. To test this possibility, BECs that had been preincubated with or without NAC were cultured with DEPs and the concentrations of IL-8 and RANTES were determined 24 h after cultivation. The concentrations of IL-8 in the culture supernatants from the cells cultured with DEPs in the presence of NAC were lower than those from the cells cultured with DEPs in the absence of NAC (Figure 5a), indicating that NAC inhibited DEP-induced IL-8 production. Similarly, NAC inhibited DEP-induced RANTES production (Figure 5b). The total number of cells and cell viability at the end of the culture period of each experiment, determined by trypan blue exclusion assay, did not differ with culture conditions, suggesting that DEP-induced IL-8 and RANTES production and the inhibition by SB 203580 and NAC of IL-8 and RANTES production did not result from cell cytotoxicity.


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  		Figure 5.   NAC inhibits DEP-induced IL-8 and RANTES production. BECs that had been preincubated with either medium or NAC (10 mM) for 60 min were cultured either with medium of DEPs (100 µg/ml). The concentrations of IL-8 (a) and RANTES (b) in the culture supernatants were determined 24 h after cultivation as described in METHODS. The results are expressed as means ± SD of five different experiments. *1, p < 0.01 compared with IL-8 or RANTES concentrations in the culture supernatants from DEP-stimulated BECs cultured without NAC.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we examined the role of p38 MAP kinase in DEP-induced IL-8 and RANTES production by human BECs and the effect of NAC on DEP-induced p38 MAP kinase activation and IL-8 and RANTES production by human BECs. The results showed that DEP induced IL-8 and RANTES production and the threonine and tyrosine phosphorylation of p38 MAP kinase, reflecting the activation of p38 MAP kinase in BECs. SB 203580 as the specific inhibitor of p38 MAP kinase activity inhibited DEP-induced IL-8 and RANTES production. NAC, a thiol reducing agent, inhibited DEP-induced p38 MAP kinase, activation, and IL-8 and RANTES production. These results indicate that p38 MAP kinase plays an important role in the DEP-activated signaling pathway that regulates IL-8 and RANTES production by BECs and that the cellular redox state is critical for DEP-induced p38 MAP kinase activation leading to IL-8 and RANTES production.

The p38 MAP kinase pathway has been characterized (14). Activation of p38 MAP kinase is mediated by dual phosphorylation of threonine and tyrosine by MKK3 and MKK6, which are upstream regulators of p38 MAP kinase (21). p38 MAP kinase activation culminates in the phosphorylation of downstream cytosolic and nuclear factors that regulate various cellular responses (14, 26). In the present study, we showed that DEP induced the threonine and tyrosine phosphorylation of p38 MAP kinase. We propose that ROSs might be involved in DEP-induced p38 MAP kinase activation, on the basis of the following evidence: (1) the antioxidant NAC inhibits DEP-induced p38 MAP kinase activation (this study); (2) overexpression of thioredoxin, a redox control protein, negatively regulates tumor necrosis factor alpha  (TNF-alpha )-induced p38 MAP kinase activation (22); (3) NAC antagonizes ROS-induced and TNF-alpha -induced activation of apoptosis signal-regulating kinase (ASK1), which is a p38 MAP kinase kinase kinase (27, 28); (4) ROSs activate p38 MAP kinase (20, 21); and (5) DEPs have been shown to generate ROSs (23). These results support our hypothesis that ROSs might be involved in p38 MAP kinase activation in DEP-stimulated BECs. Previous studies have shown that the activation of the p38 MAP kinase cascade by ROSs is mediated through ASK1 activation (28). Therefore, the upstream kinase(s) of p38 MAP kinase activated by DEP stimulation should be clarified.

The specific inhibitor of the p38 MAP kinase signaling pathway has been identified (25), providing an effective tool for investigating the role of p38 MAP kinase in cellular signaling. In this study, SB 203580 was used as the specific inhibitor of p38 MAP kinase activity in order to elucidate the biological function of p38 MAP kinase in IL-8 and RANTES production by DEP-stimulated BECs. The present results showed that SB 203580 inhibited DEP-induced IL-8 and RANTES production by 76 and 72%, respectively. SB 203580 (10 µM) was used in this study to examine the role of p38 MAP kinase in DEP- induced cytokine production, since the previous study performed by analysis of the role of p38 MAP kinase in eliciting various cellular responses, including cytokine expression, showed that 10 µM SB 203580 almost completely inhibited the expression of cytokines (19). The present results showed that 10 µM SB 203580 partially inhibited DEP-induced IL-8 and RANTES production by BECs. These results indicate that although p38 MAP kinase, at least in part, regulates DEP-stimulated IL-8 and RANTES production by BECs, other signal(s) might be involved in the regulation of IL-8 and RANTES production by DEP-stimulated BECs. Possible signals are Erk and JNK, since these kinase have been shown to regulate cytokine production (29, 30). In the present study, we also examined the threonine and tyrosine phosphorylation of Erk and JNK in DEP-stimulated BECs by immunoblotting; however, DEPs did not induce Erk and JNK phosphorylation. In addition, PD 98059, an inhibitor of MEK-1, which is an upstream regulator of Erk (31), did not affect IL-8 and RANTES production. These results indicated that Erk and JNK were not involved in DEP-induced IL-8 and RANTES production by BECs. Another signal that may regulate IL-8 and RANTES production by BECs is mediated by ROSs. ROSs act as signaling intermediates inducing cytokine expression via the activation of nuclear transcription factors including NF-kappa B (32). The promoter of the gene encoding IL-8 and RANTES contains sequences for binding several nuclear transcription factors including NF-kappa B (33). These transcription factors participate to various extents in the inducible expression of the gene encoding IL-8 and RANTES. It has been shown that DEP-induced activation of NF-kappa B is involved in IL-8 transcription and that NAC attenuates DEP-induced NF-kappa B activation in human BECs (34). p38 MAP kinase has been implicated in the activation of multiple transcription factors, including NF-kappa B (26). Although DEP-induced p38 MAP kinase activation shown in the present study may culminate in the activation of NF-kappa B, ROSs generated by DEP may directly activate NF-kappa B and this transcription factor in turn may induce gene transcription of IL-8 and RANTES. This hypothesis might be supported by the present study, in that NAC inhibited DEP-induced IL-8 and RANTES production by 94 and 93%, respectively, indicating almost complete inhibition, whereas SB 203580 achieved only partial inhibition. Taken together, the present results indicate that parallel pathways, the p38 MAP kinase-dependent pathway and the p38 MAP kinase-independent pathway, induce IL-8 and RANTES production by DEP stimulation.

DEPs are the main component of suspended particulate matter (PM). The annual average levels of PM in 12 of the world's 20 megacities (cities with population >=  10 million) are in the range of 200-600 µg/m3, and peak concentrations are frequently above 1,000 µg/m3 (35). In the present study, p38 MAP kinase activation, and IL-8 and RANTES production, were significantly induced by DEPs at 50 and 100 µg/ml but not at 10 µg/ml. The concentrations of DEP that induced p38 MAP kinase activation and cytokine production are higher than those in the annual average levels and even in the peak concentrations. In addition, the cells utilized in the present study were an undifferentiated transformed bronchial epithelial cell line, BET-1A. The functional and structural characteristics of BET-1A cells are different from those of human bronchial epithelial cells. Therefore, further study should be undertaken in order to clarify these issues and the role of p38 MAP kinase in DEP-induced IL-8 and RANTES expression in human bronchial epithelial cells in vivo.

Bronchial asthma is regarded as an allergic inflammation of the airways. Air pollutants including DEPs have been shown to enhance allergic responses (1). Exposure of ovalbumin-sensitized mice and healthy human volunteers to diesel exhaust has been reported to enhance the number of neutrophils and eosinophils in the airway (8, 10). Previous studies have demonstrated that DEPs stimulate airway epithelial cells to produce various cytokines including IL-8 and GM-CSF (4), which play an important role in the production of airway inflammation through their chemotactic activity for these cells (12, 13). In the present study, SB 203580, as the specific inhibitor of p38 MAP kinase, and NAC inhibit IL-8 and RANTES production by DEP-stimulated BECs. It is not known, at this time, whether SB 203580 and NAC are capable of producing a beneficial effect in terms of controlling the allergy-enhancing effects of diesel exhaust. However, our understanding of signal cascades and the redox control mechanism of DEP-induced cytokine expression in airway epithelial cells indicate a strategy for a therapy to control the allergy-enhancing effect of diesel exhaust.

From the data presented here, we conclude that DEP- induced IL-8 and RANTES production by BECs is, at least in part, regulated by p38 MAP kinase and that the cellular redox state is critical for DEP-induced p38 MAP kinase activation.

    Footnotes

Correspondence and requests for reprints should be addressed to Shu Hashimoto, First Department of Internal Medicine, Nihon University School of Medicine, 30-1 Oyaguchikamimachi, Itabashi-Ku, Tokyo, 173-8610 Japan. E-mail: shuh{at}med.nihon-u.ac.jp

(Received in original form April 27, 1999 and in revised form July 14, 1999).

Acknowledgments: Supported by a grant-in-aid from the Pollution-Related Health Compensation and Prevention Association of Japan.
    References
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1. Salvi, S. S., A. Frew, and S. Holgate. 1999. Is diesel exhaust a cause for increasing allergy? Clin. Exp. Allergy 29: 4-8 [Medline].

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