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Am. J. Respir. Crit. Care Med., Volume 162, Number 3, September 2000, 1075-1080

Inhalant Corticosteroids Inhibit Hyperosmolarity-induced, and Cooling and Rewarming-induced Interleukin-8 and RANTES Production by Human Bronchial Epithelial Cells

SHU HASHIMOTO, YASUHIRO GON, KEN MATSUMOTO, IKUKO TAKESHITA, SHUICHIRO MARUOKA, and TAKASHI HORIE

First Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Inhaled corticosteroids are widely used for the treatment of bronchial asthma, and a long-term treatment with inhaled corticosteroids is effective in preventing exercise-induced bronchoconstriction (EIB). We have previously shown that hyperosmolarity, and cooling and rewarming induced interleukin-8 (IL-8) expression in human bronchial epithelial cells (BEC). However, the effect of inhalant corticosteroids on hyperosmolarity-induced, and cooling and rewarming-induced IL-8 and RANTES production has not been determined. To clarify these issues, we examined the effect of inhalant corticosteroids, beclomethasone dipropionate (BDP), and budesonide (BUD) on hyperosmolarity-induced, and cooling and rewarming-induced IL-8 and RANTES production. The results showed that BDP and BUD inhibited hyperosmolarity-induced, and cooling and rewarming-induced IL-8 and RANTES production. Because our previous studies have shown that p38 mitogen-activated protein (MAP) kinase and c-Jun-NH2-terminal kinase (JNK) regulate hyperosmolarity-induced, and cooling and rewarming-induced IL-8 and RANTES production, we examined the effect of BDP and BUD on p38 MAP kinase and JNK activation. The results showed that BDP and BUD did not inhibit hyperosmolarity-induced and cooling-induced p38 MAP kinase and JNK activation. These results indicated that inhalant corticosteroids inhibited hyperosmolarity-, and cooling and rewarming-induced IL-8 and RANTES production; however, the mechanism of inhaled corticosteroid-mediated inhibition of hyperosmolarity-induced, and cooling and rewarming- induced cytokine production remains to be clarified.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The mechanism responsible for the production of exercise-induced bronchoconstriction (EIB) and the development of a late-phase response (LPR) induced by exercise has been investigated. The changes in airway osmolarity resulting from water loss and the alteration of airway temperature resulting from heat loss are likely to contribute to the production of EIB (1, 2). EIB typically occurs 5 to 15 min after cessation of exercise, but in some instances, LPR occurs 3 to 13 h after completing the exercise (2, 3). Studies on bronchoalveolar lavage fluid (BALF) in the LPR showed an increase in eosinophils and neutrophils in human subjects (4) and in dogs (5). A variety of mediators and cytokines including interleukin-8 (IL-8) and RANTES (regulated upon activation, normal T-cell expressed and secreted) contribute to the recruitment of eosinophils and neutrophils into the airway of asthmatics through their chemotactic activities (6). We have previously shown that hyperosmolarity, and cooling and rewarming induce the expression of IL-8 and RANTES, which exhibit chemotactic activity for neutrophils and eosinophils, in human bronchial epithelial cells (BEC) (12, 13, and Hashimoto, S., Y. Gon, K. Matsumoto, I. Takeshita, K. Kujima, and T. Horie. p38 MAP kinase and c-Jun-NH2-terminal kinase regulate RANTES and IL-8 production by hyperosmolarity-stimulated human bronchial epithelial cells, unpublished data). These results indicate that IL-8 and RANTES may be involved in the development of LPR induced by exercise.

Inhaled corticosteroids including beclomethasone dipropionate (BDP) and budesonide (BUD) reduce airway inflammation and airway hyperresponsiveness, and are widely used for the treatment of bronchial asthma (14). In EIB, inhaled corticosteroids have been shown to be ineffective in preventing the early-phase response (EPR) induced by exercise when given shortly before exercise (17, 18), whereas a long-term treatment with inhaled corticosteroids reduces the prevalence of EPR induced by exercise (19). However, the efficacy of inhaled corticosteroids on the LPR induced by exercise has not been established. In addition, BDP and BUD are reported to inhibit cytokine production by BEC stimulated with proinflammatory cytokines (22); however, the effect of BDP and BUD on hyperosmolarity-induced and cooling and rewarming-induced IL-8 and RANTES production has not been determined. In the present study we therefore examined the effect of inhalant corticosteroids, BDP and BUD, on hyperosmolarity-induced and cooling and rewarming-induced IL-8 and RANTES production to clarify these issues and to determine whether there may be a potential therapeutic effect of inhaled corticosteroids in preventing development of a LPR induced by exercise.

Anti-inflammatory action of corticosteroids is shown to be mediated with the inhibition of activation of transcription factors such as activator protein-1 (AP-1) and nuclear factor kappa B (NFkappa B) (22). In addition, inhibitory effects of corticosteroids on c-Jun-NH2-terminal kinase (JNK) activation by proinflammatory cytokine have been recently described (25, 26). We have previously demonstrated that hyperosmolarity-induced and cooling and rewarming-induced cytokine production are regulated by p38 mitogen-activated protein (MAP) kinase and JNK (12, 13, and S. Hashimoto, Y. Gon, K. Matsumoto, et al., unpublished data). Therefore, we also examined whether BDP and BUD could inhibit hyperosmolarity- and cooling-induced p38 MAP kinase and JNK activation.

    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cells and Reagents

Bronchial epithelial cell lines, NCI-H292, were obtained from American Type Culture Collection (Rockville, MD). NCI-H292 were cultured in culture medium that is RPMI 1640 (Nissui Corp. Ltd., Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum (FCS, Mitsubishikasei Corp. Ltd., Tokyo, Japan), streptomycin and penicillin (Meiji Pharmaceutical Corp. Ltd., Tokyo, Japan). NaCl was obtained from Kantokagaku Pharmaceutical Co. Ltd. (Tokyo, Japan). BDP and BUD were kindly provided by GlaxoWellcome Ltd. (Tokyo, Japan) and Astra Japan Inc. (Tokyo, Japan).

Cell Cultures

The cells were placed in a tissue culture plate (Falcon 1007, Oxnard, CA) for Western blot and 24-well flat-bottomed tissue culture plate (Corning, Corning, NY) for cytokine production using culture medium at 37° C in humidified 5% CO2 atmosphere. When the cells were grown in subconfluent conditions, the culture medium was replaced with serum-free RPMI 1640 and the cells were cultured for 16 h.

Cell Culture for Western Blot and Cytokine Production

Hyperosmolar stimulation. Hyperosmolar medium was prepared by adding NaCl to isomolar RPMI 1640 without FCS (medium) as described previously (12). Hyperosmolar medium-induced p38 MAP kinase and JNK activation, and hyperosmolar medium-induced cytokine production were examined as described previously (12). Briefly, in order to examine the effect of BDP and BUD on hyperosmolarity- induced p38 MAP kinase and JNK activation, the cells that had been preincubated with either medium, BDP or BUD for 60 min, were stimulated with hyperosmolar medium for 10 min. After exposure with hyperosmolar medium, hyperosmolar medium was replaced with fresh isomolar medium, and the cells were cultured for 10 min at 37° C in humidified 5% CO2 atmosphere. For the control, the cells that had been preincubated with either medium, BDP or BUD for 60 min, were stimulated with isomolar medium instead of hyperosmolar medium for 10 min. After exposure with isomolar medium, isomolar medium was replaced with fresh isomolar medium, and the cells were cultured for 10 min at 37° C in humidified 5% CO2 atmosphere. To examine the effect of BDP and BUD on hyperosmolarity-induced IL-8 and RANTES production, the cells that had been preincubated with either medium, BDP or BUD for 60 min, were stimulated with hyperosmolar medium for 10 min. After exposure with hyperosmolar medium, hyperosmolar medium was replaced with fresh isomolar medium, and the cells were cultured at 37° C in humidified 5% CO2 atmosphere. For the control, the cells that had been preincubated with either medium, BDP or BUD for 60 min, were stimulated with isomolar medium instead of hyperosmolar medium for 10 min. After exposure with isomolar medium, isomolar medium was replaced with fresh isomolar medium, and the cells were cultured at 37° C in humidified 5% CO2 atmosphere. At 24 h after cultivation, the culture supernatants were harvested and centrifuged, and the supernatants were collected, filtrated with a Millipore filter, and stored at -80° C until assay.

Cooling, and cooling and rewarming stimulation. Cooling-induced p38 MAP kinase and JNK activation, and cooling and rewarming- induced cytokine production were examined as described previously (13). Briefly, in order to examine the effect of BDP and BUD on cooling-induced p38 MAP kinase and JNK activation, the cells that had been preincubated with either medium, BDP or BUD for 60 min, were cultured with either warmed medium (37° C) at 37° C or cold (1° C) medium on ice for 120 min. To examine the effect of BDP and BUD on cytokine production, since cooling stimulation per se did not induce cytokine production as demonstrated previously (13), the cells that had been preincubated with either medium, BDP or BUD for 60 min, were cultured with cold (1° C) medium on ice for 120 min followed by fresh warmed medium (37° C) (cooling and rewarming) at 37° C in humidified 5% CO2 atmosphere for 24 h. For the control, the cells that had been preincubated with either medium, BDP or BUD for 60 min, were cultured with warmed medium instead of cold medium for 120 min followed by fresh warmed medium at 37° C in humidified 5% CO2 atmosphere for 24 h. At 24 h after cultivation, the culture supernatants were harvested and centrifuged, and the supernatants were collected, filtrated with a Millipore filter, and stored at -80° C until assay.

Western Blot Analysis of p38 MAP Kinase and JNK

Analysis of threonine and tyrosine phosphorylation of p38 MAP kinase was performed using an antiphosphorylated threonine and tyrosine of p38 MAP kinase antibody (ab) (anti-phospho-specific p38 MAP kinase ab; New England Biolabs, Inc., Beverly, MA) which is specific for active p38 MAP kinase and does not cross react with Erk and JNK. Analysis of threonine and tyrosine phosphorylation of JNK was performed using an antiphosphorylated threonine and tyrosine of JNK ab (anti-phospho-specific JNK ab; New England Biolabs, Inc.) which is specific for active JNK and does not cross react with p38 MAP kinase and Erk. Analysis of p38 MAP kinase and JNK was performed according to manufacturer's instructions as described previously (12). Briefly, after separating proteins from cell lysate by a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the cell lysate containing 10 µg of protein was electrophoretically transferred to membrane and the membrane was incubated with specific ab to phosphorylated threonine and tyrosine of p38 MAP kinase (affinity-purified rabbit polyclonal IgG) for analysis of p38 MAP kinase or specific ab to phosphorylated threonine and tyrosine of JNK (affinity-purified rabbit polyclonal IgG) for analysis of JNK, and then it was incubated with the horseradish peroxidase-conjugated anti-rabbit IgG ab and horseradish peroxidase-conjugated antibiotin ab to detect biotinylated protein markers. Blots were incubated with enhanced chemiluminescence (ECL) solution for 1 min and exposed on Kodak XAR film (Tokyo, Japan). In order to show the amounts of p38 MAP kinase and JNK precipitated, blots were stripped and reprobed using phosphorylation state-independent p38 MAP kinase-specific ab (affinity-purified rabbit polyclonal IgG) to determine total p38 MAP kinase levels or phosphorylation state-independent JNK-specific ab (affinity-purified rabbit polyclonal IgG) to determine total JNK levels, respectively.

Measurement of IL-8 and RANTES

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

Statistical Analysis

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

    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

BDP and BUD Inhibit Hyperosmolarity-induced IL-8 and RANTES Production

First, we examined the effect of BDP and BUD on hyperosmolarity-induced IL-8 and RANTES production. BDP inhibited hyperosmolarity-induced IL-8 production in a dose-dependent manner (Figure 1a). Similarly, BUD inhibited hyperosmolarity-induced IL-8 production in a dose-dependent manner (Figure 1b). In RANTES production, the inhibition by BDP and BUD of hyperosmolarity-induced RANTES production was dose-dependent (Figure 2).


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  		Figure 1.   BDP and BUD inhibit hyperosmolarity-induced IL-8 production. BEC that had been preincubated with either medium (open circle ), various concentrations of BDP (10-8 M, 10-7 M, and 10-6 M) (bullet ), or various concentrations of BUD (10-8 M, 10-7 M, and 10-6 M) (bullet ) for 60 min were stimulated with hyperosmolar medium at 1,280 mOsm/kg · H2O (open circle , bullet ) for 10 min. After exposure with hyperosmolar medium, hyperosmolar medium was replaced with fresh isomolar medium and the cells were cultured for 24 h. For the control, BEC that had been preincubated with either medium (square ), various concentrations of BDP (10-8 M, 10-7 M, and 10-6 M) (black-square), or various concentrations of BUD (10-8 M, 10-7 M, and 10-6 M) (black-square) for 60 min were stimulated with isomolar medium at 280 mOsm/ kg · H2O (square , black-square) instead of hyperosmolar medium for 10 min. After exposure with isomolar medium, isomolar medium was replaced with fresh isomolar medium, and the cells were cultured for 24 h. The concentrations of IL-8 in the culture supernatants were determined by ELISA. The results are expressed as means ± SD of six different experiments. *1 indicates p < 0.05 compared with the cell culture with hyperosmolar medium. *2 indicates p < 0.01 compared with the cell culture with hyperosmolar medium.


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  		Figure 2.   BDP and BUD inhibit hyperosmolarity-induced RANTES production. BEC that had been preincubated with either medium (open circle ), various concentrations of BDP (10-8 M, 10-7, and 10-6 M) (bullet ), or various concentrations of BUD (10-8 M, 10-7 M, and 10-6 M) (bullet ) for 60 min were stimulated with hyperosmolar medium at 1,280 mOsm/ kg  · H2O (open circle , bullet ) for 10 min. After exposure with hyperosmolar medium, hyperosmolar medium was replaced with fresh isomolar medium and the cells were cultured for 24 h. For the control, BEC that had been preincubated with either medium (square ), various concentrations of BDP (10-8 M, 10-7 M, and 10-6 M) (black-square), or various concentrations of BUD (10-8 M, 10-7 M, and 10-6 M) (black-square) for 60 min were stimulated with isomolar medium at 280 mOsm/kg · H2O (square , black-square) instead of hyperosmolar medium for 10 min. After exposure with isomolar medium, isomolar medium was replaced with fresh isomolar medium, and the cells were cultured for 24 h. The concentrations of RANTES in the culture supernatants were determined by ELISA. The results are expressed as means ± SD of six different experiments. *1 indicates p < 0.05 compared with the cell culture with hyperosmolar medium. *2 indicates p < 0.01 compared with the cell culture with hyperosmolar medium.

BDP and BUD Inhibit Cooling and Rewarming-induced IL-8 and RANTES Production

In our previous study, cooling per se induced p38 MAP kinase and JNK activation, but not IL-8 production, and cooling and rewarming-induced IL-8 production (13). These results indicate that a transient cooling stimulation followed by thermal energy is absolutely necessary for IL-8 production. From these evidences, we examined the effect of BDP and BUD on cooling and rewarming-induced IL-8 and RANTES production. BDP inhibited cooling and rewarming-induced IL-8 production in a dose-dependent manner (Figure 3a). Similarly, BUD inhibited cooling and rewarming-induced IL-8 production in a dose-dependent manner (Figure 3b). In RANTES production, the inhibition by BDP and BUD of cooling and rewarming- induced RANTES production was dose-dependent (Figure 4).


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  		Figure 3.   BDP and BUD inhibit cooling and rewarming-induced IL-8 production. BEC that had been preincubated with either medium (open circle ), various concentrations of BDP (10-8 M, 10-7 M, and 10-6 M) (bullet ), or various concentrations of BUD (10-8 M, 10-7 M, and 10-6 M) (bullet ) for 60 min were cultured with cold (1° C) medium on ice for 120 min followed by warmed medium at 37° C (open circle , bullet ; cooling and rewarming) in humidified 5% CO2 atmosphere for 24 h. For the control, BEC that had been preincubated with either medium (square ), various concentrations of BDP (10-8 M, 10-7 M, and 10-6 M) (black-square), or various concentrations of BUD (10-8 M, 10-7 M, and 10-6 M) (black-square) for 60 min were cultured with warmed medium instead of cold medium for 120 min followed by warmed medium at 37° C (square , black-square) in humidified 5% CO2 atmosphere for 24 h. The concentrations of IL-8 in the culture supernatants were determined by ELISA. The results are expressed as means ± SD of six different experiments. *1 indicates p < 0.05 compared with the cell culture with hyperosmolar medium. *2 indicates p < 0.01 compared with the cell culture with hyperosmolar medium.


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  		Figure 4.   BDP and BUD inhibit cooling and rewarming-induced RANTES production. BEC that had been preincubated with either medium (open circle ), various concentrations of BDP (10-8 M, 10-7 M, and 10-6 M) (bullet ), or various concentrations of BUD (10-8 M, 10-7 M, and 10-6 M) (bullet ) for 60 min were cultured with cold (1° C) medium on ice for 120 min followed by warmed medium at 37° C (open circle , bullet ; cooling and rewarming) in humidified 5% CO2 atmosphere for 24 h. For the control, BEC that had been preincubated with either medium (square ), various concentrations of BDP (10-8 M, 10-7 M, and 10-6 M) (black-square), or various concentrations of BUD (10-8 M, 10-7 M, and 10-6 M) (black-square) for 60 min were cultured with warmed medium instead of cold medium for 120 min followed by warmed medium at 37° C (square , black-square) in humidified 5% CO2 atmosphere for 24 h. The concentrations of RANTES in the culture supernatants were determined by ELISA. The results are expressed as means ± SD of six different experiments. *1 indicates p < 0.05 compared with the cell culture with hyperosmolar medium. *2 indicates p < 0.01 compared with the cell culture with hyperosmolar medium.

BDP and BUD Do Not Inhibit Hyperosmolarity-induced and Cooling-induced p38 MAP Kinase and JNK Activation

p38 MAP kinase and JNK regulate hyperosmolarity-induced, and cooling and rewarming-induced IL-8 and RANTES production (12, 13, and S. Hashimoto, Y. Gon, K. Matsumoto, et al., unpublished data). From these results, the inhibition of IL-8 and RANTES production by BDP and BUD might result from the BDP- and BUD-mediated inhibition of p38 MAP kinase and JNK activation. To test this possibility, we examined the effect of BDP and BUD on hyperosmolarity-induced and cooling-induced p38 MAP kinase and JNK activation. As shown in Figure 5, BDP and BUD did not inhibit hyperosmolarity-induced p38 MAP kinase activation. Similarly, BDP and BUD did not inhibit cooling-induced p38 MAP kinase activation (Figure 6).


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  		Figure 5.   BDP and BUD do not inhibit hyperosmolarity-induced p38 MAP and JNK activation. BEC that had been preincubated with either medium, BDP (10-6 M) or BUD (10-6 M) for 60 min were stimulated for 10 min with isomolar medium at 280 mOsm/kg · H2O or hyperosmolar medium at 1,280 mOsm /kg · H2O. After exposure with isomolar medium or hyperosmolar medium, isomolar medium and hyperosmolar medium were replaced with fresh isomolar medium, and the cells were cultured for 10 min. At the end of cultivation, the lysates from BEC were separated by a 15% SDS-PAGE, transferred to membranes, and blotted with either a specific ab to phosphorylated threonine and tyrosine of p38 MAP kinase [phospho-p38 MAPK; upper panel of (a)], or a specific ab to phosphorylated threonine and tyrosine of JNK [phospho-JNK; upper panel of (b)]. Blots shown in the upper panel of (a) were stripped and reprobed using a phosphorylation state-independent p38 MAP kinase specific ab to show the amounts of p38 MAP kinase blotted [p38 MAPK; lower panel of (a)]. Blots shown in the upper panel of (b) were stripped and reprobed using a phosphorylation state-independent JNK-specific antibody to show the amounts of JNK blotted [JNK; lower panel of (b)]. The cells were cultured with isomolar medium (lane 1), BDP (lane 2), BUD (lane 3), hyperosmolar medium (lane 4), hyperosmolar medium and BDP (lane 5), hyperosmolar medium and BUD (lane 6). Lane P of (a) and (b) represent phosphorylated p38 MAP kinase and JNK control protein for positive control, respectively (New England Biolabs, Inc.). Lane N of (a) and (b) represent nonphosphorylated p38 MAP kinase and JNK control protein for negative control, respectively (New England Biolabs, Inc.). The amounts of phosphorylated p38 MAP kinase and JNK proteins were quantified by NIH image analyzer and are presented as the amounts of phosphorylated p38 MAP kinase and JNK proteins relative to control cells treated without BDP and BUD. The fold increases in amounts of phosphorylated p38 MAP kinase, Erk, and JNK proteins are indicated below. Three identical experiments independently performed gave similar results.


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  		Figure 6.   BDP and BUD do not inhibit cooling-induced p38 MAP and JNK activation. BEC that had been preincubated with either medium, BDP (10-6 M) or BUD (10-6 M) for 60 min were cultured with either warmed medium (37° C) at 37° C or cold (1° C) medium on ice for 120 min. At the end of cultivation, the lysates from BEC were separated by a 15% SDS-PAGE, transferred to membranes, and blotted either with a specific ab to phosphorylated threonine and tyrosine of p38 MAP kinase [phospho-p38 MAPK; upper panel of (a)], or a specific ab to phosphorylated threonine and tyrosine of JNK [phospho-JNK; upper panel of (b)]. Blots shown in the upper panel of (a) were stripped and reprobed using a phosphorylation state-independent p38 MAP kinase specific ab to show the amounts of p38 MAP kinase blotted [p38 MAPK; lower panel of (a)]. Blots shown in the upper panel of (b) were stripped and reprobed using a phosphorylation state-independent JNK specific antibody to show the amounts of JNK blotted [JNK; lower panel of (b)]. The cells were cultured with warmed medium at 37° C (lane 1), BDP (lane 2), BUD (lane 3), cold medium (lane 4), cold medium and BDP (lane 5), and cold medium and BUD (lane 6). Lanes P and N of (a) and (b), and fold are described in the legend to Figure 5. Three identical experiments independently performed gave similar results.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we examined the effect of inhalant corticosteroids, BDP and BUD, on hyperosmolarity-induced and cooling and rewarming-induced IL-8 and RANTES production by human BEC. The results showed that (1) BDP and BUD inhibited hyperosmolarity-induced IL-8 and RANTES production in a dose-dependent manner, (2) BDP and BUD inhibited cooling and rewarming-induced IL-8 and RANTES production in a dose-dependent manner, and (3) BDP and BUD did not inhibit hyperosmolarity-induced and cooling- induced p38 MAP kinase and JNK activation. These results indicated that BDP and BUD inhibit hyperosmolarity-induced and cooling and rewarming-induced IL-8 and RANTES production by human BEC, whereas their inhibitory actions on hyperosmolarity-induced and cooling and rewarming-induced IL-8 and RANTES production did not result from the inhibition of p38 MAP kinase and JNK activation.

Inhaled corticosteroids are well known to inhibit the production of cytokines, including IL-8 and RANTES, by BEC in response to proinflammatory cytokines (22). Furthermore, our results indicate that BDP and BUD inhibit IL-8 and RANTES production by BEC in response to hyperosmolarity, and cooling and rewarming. Taken together, inhaled corticosteroids are capable of inhibiting cytokine production by BEC in response to various environmental stresses such as hyperosmolarity, and cooling and rewarming as well as inflammatory stimuli.

The mechanism responsible for the production of EIB and the development of LPR induced by exercise have been investigated (1). Studies on BALF in the LPR showed an increase in eosinophils and neutrophils in human subjects (4) and in dogs (5). A variety of mediators and cytokines, including IL-8 and RANTES, contribute to the recruitment of eosinophils and neutrophils into the airway of asthmatics through their chemotactic activities (6). Airway epithelial cells are well known to produce various cytokines that are possibly involved in the production of inflammation of asthmatic airway (6, 7). We have previously shown that human BEC produce IL-8 and RANTES, which exhibit chemotactic activity for neutrophils and eosinophils, in response to hyperosmolarity, and cooling and rewarming (12, 13, and S. Hashimoto, Y. Gon, K. Matsumoto, et al., unpublished data). These results indicate that IL-8 and RANTES may be involved in the development of LPR induced by exercise. Inhaled corticosteroids have been shown to be ineffective in preventing the EPR induced by exercise when given shortly before exercise (17, 18), whereas long-term treatment with inhaled corticosteroids reduces the prevalence of EPR induced by exercise (19). Some asthmatic patients may experience LPR induced by exercise. There are only a few studies on the efficacy of corticosteroids on the LPR induced by exercise. Iikura and coworkers has shown that inhaled corticosteroids inhibit the LPR in 50% of bronchial asthmatics but not the EPR when given shortly before exercise (27). However, the efficacy of inhaled corticosteroids on the LPR induced by exercise has not been established. In the present study, we showed that the pretreatment of BEC for 1 h with inhalant corticosteroids resulted in the inhibition of IL-8 and RANTES production in response to hyperosmolarity, and cooling and rewarming. Although it is not known, at this time, whether inhaled corticosteroids are capable of producing beneficial effect in preventing development of a LPR induced by exercise, our results indicate that a short-term treatment of inhaled corticosteroids may have a beneficial effect in preventing development of a LPR induced by exercise.

Activation of p38 MAP kinase and JNK is mediated by dual phosphorylation of the threonine and tyrosine residues of p38 MAP kinase (28) and JNK (29), respectively. Increases in the threonine and tyrosine phosphorylation of p38 MAP kinase and JNK reflect activation state of p38 MAP kinase and JNK. Inhibitory effects of corticosteroids on JNK activation by proinflammatory cytokine have been recently described (25, 26). In our previous studies, p38 MAP kinase and JNK pathway, at least, regulated hyperosmolarity-induced and cooling and rewarming-induced IL-8 and RANTES production by BEC. From these observations, in the present study we examined the effect of BDP and BUD on hyperosmolarity-induced and cooling-induced p38 MAP kinase and JNK activation to test the possibility that BDP- and BUD-mediated inhibition of cytokine production may result from BDP- and BUD-mediated inhibition of p38 MAP kinase and JNK activation. The results showed that BDP and BUD did not inhibit hyperosmolarity-induced and cooling-induced p38 MAP kinase and JNK activation, indicating that their inhibitory actions on hyperosmolarity-induced and cooling and rewarming-induced IL-8 and RANTES production did not result from the inhibition of p38 MAP kinase and JNK activation. The promoter of the gene encoding IL-8 and RANTES contains sequences for binding several nuclear transcription factors including NF-kappa B and AP-1 (30). These transcription factors participate to various extents in the inducible expression of the gene encoding IL-8 and RANTES. p38 MAP kinase has been implicated in the activation of multiple transcription factors, including NF-kappa B (33). JNK has been implicated in the activation of multiple transcription factors, including AP-1 (34, 35). Anti-inflammatory action of corticosteroids has been shown to be mediated with the inhibition of activation of these transcription factors (22). Our results with BDP- and BUD-mediated inhibition of IL-8 and RANTES production may result from the inhibition of activation of these transcription factors; however, further studies are needed to clarify this point.

From the data presented here, we conclude that inhalant corticosteroids, BDP and BUD, inhibit hyperosmolarity- induced and cooling and rewarming-induced IL-8 and RANTES production by human BEC. Although it is not known whether inhaled corticosteroids are capable of producing beneficial effect in preventing development of a LPR induced by exercise, our results may indicate that a strategy of inhibiting IL-8 and RANTES production may apply to preventing development of a LPR induced by exercise as well as reducing the allergic inflammation of asthmatic airway.

    Footnotes

Correspondence and requests for reprints should be addressed to Dr. 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 November 23, 1999 and in revised form March 7, 2000).

Acknowledgments: Supported by a Grant-in-Aid from General Scientific Research from the Ministry of Education of Japan (10670565) and a Grant-in-Aid to Nihon University for the High-Tech Research Center from the Japanese Ministry of Education, Science, Sports and Culture.
    References
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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