INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Exercise-induced bronchoconstriction (EIB) can often occur in bronchial asthma (1, 2). The alteration of airway temperature resulting from heat loss and the change in airway osmolarity resulting from water loss are likely to contribute to the development of EIB (1, 2). EIB typically occurs 5 to 15 min after cessation of exercise, but in some instance, a late-phase response (LPR) occurs 3 to 13 h after completing the exercise (2, 3). This delayed response induced by air flow-induced bronchoconstriction and hypertonic aerosol-induced bronchoconstriction in humans and animal models of EIB have shown that various inflammatory mediators and inflammatory cells may contribute to the development of LPR (2, 4). Although evaporated water loss in the airway is generally believed to lead to hyperosmolarity in the airway mucosa (2, 8), the responsiveness of bronchial epithelial cells (BEC) that might be affected by osmotic change in the airway to hyperosmolarity during exercise has not been determined.

The mitogen-activated protein (MAP) kinases are important mediators of signal transduction from the cell membrane to the nucleus. Several subgroups of mammalian MAP kinases have been molecularly characterized: extracellular-signal regulated kinase (ERK), p38 MAP kinase and c-Jun NH₂-terminal kinase (JNK) (11). Environmental stresses such as osmotic stress, heat shock and UV irradiation, proinflammatory cytokines, and DNA-damaging agents are known to cause the phosphorylation and activation of p38 MAP kinase and JNK in a variety of types of cells (11); however, little is known about the effect of osmotic stress on p38 MAP kinase and JNK activation in BEC. Therefore, it is important to examine the effect of hyperosmolarity on BEC functions such as cytokine expression and to analyze the signal transduction pathway regulating hyperosmolarity-induced cytokine expression in BEC.

Interleukin-8 (IL-8) has been suggested to have a role in the pathogenesis of allergic inflammation of bronchial asthma (21, 22) and is well known to be expressed in BEC (23, 24). In the present study, we examined the effect of hyperosmolarity on IL-8 expression in human BEC and the role of p38 MAP kinase and JNK in hyperosmolarity-induced IL-8 expression in order to clarify the responsiveness of BEC to hyperosmolarity and its signal transduction pathway, and the mechanism responsible for the development of LPR induced by EIB.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Cells and Reagents

Bronchial epithelial cell lines, NCl-H₂₉₂, were obtained from American Type Culture Collection (Rockville, MD). NCl-H₂₉₂ were cultured in RPMI 1640 (Nissui Co. Ltd., Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Mitsubishikasei Co. Ltd., Tokyo, Japan), streptomycin, and penicillin (Meiji Pharmaceutical Co. Ltd., Tokyo, Japan). NaCl and mannitol were obtained from Kantokagaku Pharmaceutical Co. Ltd. (Tokyo, Japan) and Sigma Co. Ltd. (St. Louis, MO). The pyridinyl imidazole SB 203580, which was the specific p38 MAP kinase inhibitor (25), was kindly provided by SmithKline Beecham and was dissolved in dimethyl sulphoxide.

Cell Cultures

The cells were placed in a 24-well flat-bottomed tissue culture plate (Corning, Corning, NY) for IL-8 production, tissue culture plate (Falcon 1007; Falcon Plastics, Oxnard, CA) for Western blot analysis of p38 MAP kinase, and tissue culture plate (Falcon 1005) for Northern blot analysis of IL-8 mRNA expression using culture medium and cultured at 37° C in humidified 5% CO₂ atmosphere. When the cells were grown in subconfluent conditions, the cells were stimulated.

Cell Culture for Cytokine Expression and the Effect of SB 203580 as the Specific p38 MAP Kinase Inhibitor on It

In order to examine the effect of hyperosmolar stimulation on IL-8 expression, the cells were stimulated for 10 min with either isomolar RPMI 1640 without FCS (medium), varying hyperosmolar medium prepared by adding NaCl to isomolar medium (hyperosmolar NaCl medium), or varying hyperosmolar medium prepared by adding mannitol to isomolar medium (hyperosmolar mannitol medium). After exposure with isomolar medium or hyperosmolar medium, medium was replaced with fresh isomolar medium, and the cells were cultured. At 24 h after cultivation, the culture supernatants for the determination of IL-8 concentrations were harvested and centrifuged, and the supernatants were collected, filtrated with a millipore filter, and stored at 80° C until assay. At 6 h after cultivation, the cells for the analysis of IL-8 mRNA expression were collected and stored at 80° C. In order to examine the effect of SB 203580 on hyperosmolarity-induced IL-8 expression, the cells that had been pretreated with or without SB 203580 for 60 min were stimulated for 10 min with either isomolar medium, hyperosmolar NaCl medium, or hyperosmolar mannitol medium. After exposure with isomolar medium or hyperosmolar medium, medium was replaced with fresh isomolar medium, and the cells were cultured.

Cell Culture for Western Blot Analysis and Kinase Assay

In order to examine the effect of hyperosmolar stimulation on tyrosine phosphorylation of p38 MAP kinase and p38 MAP kinase activation and JNK activation, the cells were stimulated either with isomolar medium, varying osmolarity of hyperosmolar NaCl medium, or varying osmolarity of hyperosmolar mannitol medium for the desired times. To determine the time course of tyrosine phosphorylation of p38 MAP kinase and JNK activation, after exposure with isomolar medium or hyperosmolar medium for 10 min, medium was replaced with fresh isomolar medium, and the cells were cultured for the desired times.

Measurement of IL-8

The concentration of IL-8 in the culture supernatants from BEC were measured by commercially available enzyme-linked immunosorbent assay (ELISA) kits (Amersham Corp., Arlington Heights, IL). ELISA was performed according to the manufacturer's instruction. All samples were assayed in duplicate.

Western Blot Analysis of p38 MAP Kinase

The tyrosine phosphorylation of p38 MAP kinase was analyzed by commercially available kits (PhosphoPlus p38 MAPK Antibody Kit; New England Biolabs, Inc., Beverly, MA). Analysis of tyrosine phosphorylation of p38 MAP kinase was performed according to the manufacturer's instruction. Briefly, BEC that had been washed with cold TRIS-buffered saline were lysed in sodium dodecyl sulfate (SDS) buffer (62.5 mM TRIS-HCl at pH 6.8, 2% wt/vol SDS, 10% glycerol, 50 mM dithiothreitol (DTT), 0.1% wt/vol bromphenol 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 fully denature the proteins prior to 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), proteins were electrophoretically transferred to the 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 dry milk for 3 h at room temperature. It was then incubated with specific antibody to phosphorylated tyrosine of p38 MAP kinase (affinity- purified rabbit polyclonal IgG) at 1:1,000 dilution 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 washing buffer three times, it was incubated for 1 h with gentle shaking at room temperature with horseradish peroxidase (HRP)- conjugated antirabbit antibody (1:2,000) and HRP-conjugated antibiotin antibody (1:1,000) to detect biotinylated protein markers, and then washed three times with washing buffer. Blots were incubated with enhanced chemiluminescence solution (LumiGLO; New England Biolabs Inc., Beverly, MA) for 1 min at room temperature and exposed on Fuji medical X-ray film (AIF new RX; Fuji Film Co. Ltd., Tokyo, Japan). In order to show the amounts of p38 MAP kinase precipitated, blots were stripped and reprobed using phosphorylation-state independent p38 MAP kinase-specific antibody to determine total p38 MAP kinase levels (affinity-purified rabbit polyclonal IgG).

p38 MAP Kinase Assay

The activity of p38 MAP kinase was analyzed by commercially available kits (p38 MAP Kinase Assay Kit; New England Biolabs Inc.). The kit employs two different antibodies, anti-p38 MAP kinase antibody, which is specific for p38 MAP kinase and does not cross-react with ERK1/2 or JNK, and anti-phospho-specific ATF-2 antibody to detect p38 MAP kinase-induced phosphorylation of ATF-2. Analysis of activity of p38 MAP kinase was performed according to the manufacturer's instruction. Briefly, BEC that had been washed with ice-cold phosphate-buffered saline (PBS) were lysed with 1.0 ml of ice-cold lysis buffer (20 mM TRIS at pH 7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethyleneglycol-bis-(-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM -glycerolphosphate, 1 mM Na₃VO₄, 1 µg/ml leupeptin) plus 1 mM phenylmethyl sulfonyl fluoride (PMSF) in the 6-well plate on ice for 5 min. After sonication, the cell lysate was microcentrifuged for 10 min at 4° C, and 200 µl of cell lysate were incubated with anti-p38 MAP kinase antibody (1:100 dilution) to selectively immunoprecipitate p38 MAP kinase from cell lysates at 4° C overnight with gentle shaking. The resulting immunoprecipitate was then mixed with protein A sepharose beads. After microcentrifugation, the pellet was washed twice with lysis buffer and then washed twice with kinase buffer (25 mM TRIS at pH 7.5, 5 mM -glycerolphosphate, 2 mM DTT, 0.1 mM Na₃VO₄, 10 mM MgCl₂). The pellet was suspended in 50 µl of kinase buffer with 100 µM ATP and 2 µg of ATF-2 fusion protein, and then incubated for 30 min at 30° C. The pellet was mixed with the sample buffer consisting of 62.5 mM TRIS base, 10% glycerol, 50 mM DTT and 2% SDS, and then heated in a boiling water bath for 5 min to fully denature the protein prior to electrophoresis. Equal amounts of protein (5 µg/lane) were separated in a 15% SDS gel, and transferred to a polyvinylidene difluoride sheet (Millipore, Bedford, MA) by electroelution with a constant current of 200 mA for 90 min at room temperature. After blocking with 0.1% Tween 20 supplemented with PBS (T-PBS) containing skim milk overnight, the sheet was incubated with anti-phospho-specific ATF-2 antibody (1:1,000 dilution) at 4° C overnight. The sheet was then washed three times with T-PBS, and incubated with HRP conjugated antirabbit antibody (1:2,000) and HRP-conjugated antibiotin antibody (1:2,000) for 1 h at room temperature. After washing with T-PBS three times, the sheet was incubated with 10 ml of the ECL (enhanced chemiluminescence) solution for 1 min according to manufacturer's instructions, and exposed on Fuji medical X-ray film (AIF new RX) for 1 min.

JNK Assay

The activity of JNK was analyzed by commercially available kits (SAPK/JNK Assay Kit; New England Biolabs). The kit employs an N-terminal c-Jun fusion protein bound to sepharose beads to selectively pull down JNK from cell lysates, after which the kinase reaction is carried out in the presence of unlabeled ATP. The c-Jun phosphorylation is selectively measured using phospho-specific c-Jun antibody, which specifically measures JNK-induced phosphorylation of c-Jun. Analysis of activity of JNK was performed according to the manufacturer's instructions. Briefly, BEC that had been washed with ice-cold PBS were lysed with 1.0 ml of ice-cold lysis buffer (20 mM TRIS at pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM -glycerolphosphate, 1 mM Na₃VO₄, 1 µg/ml leupeptin) plus 1 mM PMSF in the 6-well plate on ice for 5 min. After sonication, the cell lysate was microcentrifuged for 10 min at 4° C, and 250 µl of cell lysate were mixed with 2 µg of c-Jun fusion protein beads, and then incubated at 4° C overnight with gentle shaking. After microcentrifugation, the pellet was washed twice with lysis buffer and then washed twice with kinase buffer (25 mM TRIS at pH 7.5, 5 mM -glycerolphosphate, 2 mM DTT, 0.1 mM Na₃VO₄, 10 mM MgCl₂). The pellet was suspended in 50 µl of kinase buffer with 100 µM ATP and incubated for 30 min at 30° C. The cell lysate was mixed with the sample buffer consisting of 62.5 mM TRIS base, 10% glycerol, 50 mM DTT, and 2% SDS, and then heated in a boiling water bath for 5 min to fully denature the protein prior to electrophoresis. Equal amounts of protein (5 µg/lane) were separated in a 15% SDS gel and transferred to a polyvinylidene difluoride sheet (Millipore) by electroelution with a constant current of 200 mA for 90 min at room temperature. After blocking with T-PBS containing skim milk overnight, the sheet was incubated with anti-phospho-c-Jun antibody (1:1,000 dilution) at 4° C overnight. The sheet was then washed three times with T-PBS and incubated with HRP-conjugated antirabbit antibody (1:2,000) and HRP-conjugated antibiotin antibody (1:2,000) for 1 h at room temperature. After washing with T-PBS three times, the sheet was incubated with 10 ml of the ECL solution for 1 min according to manufacturer's instructions, and exposed on Fuji medical X-ray film (AIF new RX) for 1 min.

Northern Blot Analysis

Total RNA was prepared with an RNA extraction kit (RNA zol B; Cinna Scientific, Friedswods, TX) using the acid guanidine thiocyanate-phenol-chloroform extraction methods. Total RNA (10 µg) was denatured in a solution containing formaldehyde and folmamide, and electrophoresed in a 1% agarose gel containing formaldehyde (26). Then it was capillary-transferred onto a nylon membrane (Hybond N; Amersham, Buckinghamshire, UK). The membrane was prehybridized with rapid hybribuffer (Amersham) and then hybridized with [³²P]-labeled probes for 2 h at 65° C. The probes used in this study were the PstI-PstI fragments of cDNA for -actin and the full length of cDNA for IL-8 (27), which was kindly provided by Dr. Koji Matsushima (Department of Hygiene, Tokyo University School of Medicine). After hybridization, the membrane was washed with sodium citrate and sodium chloride containing SDS and then autoradiographed with Fuji medical film (AIF new RX) at 70° C.

Statistical Analysis

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

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Induction of IL-8 by Hyperosmolar Stimulation

BEC were cultured with varying hyperosmolar NaCl medium or hyperosmolar mannitol medium and the concentrations of IL-8 in the culture supernatants from BEC were determined by ELISA at 24 h after cultivation. As shown in Figure 1, the concentrations of IL-8 increased in hyperosmolar NaCl culture at 545 mOsm/kg H₂O and greater, and the maximal concentration of IL-8 was seen at 1,545 mOsm/kg H₂O. Similarly, the concentrations of IL-8 in hyperosmolar mannitol cultures increased significantly with increasing osmolarity, and this response was in an osmolarity-dependent manner. BEC cultured with hyperosmolar NaCl at 545 mOsm/kg H₂O resulted in a 1.5-fold increase in mean IL-8 concentrations, raising their mean response to a level compatible to that of BEC cultured with hyperosmolar mannitol at 545 mOsm/kg H₂O. Similar results were obtained when BEC were cultured with increasing osmolar medium. These results indicated that there was no significant quantitative difference between the response of BEC to hyperosmolar NaCl stimulation and hyperosmolar mannitol stimulation, and BEC responded similarly to hyperosmolar conditions regardless of solutes.

View larger version (17K): [in this window] [in a new window]   Figure 1.   IL-8 production by hyperosmolarity-stimulated bronchial epithelial cells. Bronchial epithelial cell lines, NCI-H292, were stimulated with either isomolar medium (closed circles), varying osmolarity of hyperosmolar NaCl medium (open circles), or varying osmolality of hyperosmolar mannitol medium (open squares) for 10 min. Thereafter, hyperosmolar medium was replaced with fresh isomolar medium and the cells were cultured for 24 h, and the concentrations of IL-8 in the culture supernatants were determined. The results are expressed as the mean ± SD in five different experiments. Asterisks 1 indicate p < 0.05 compared with isomolar medium-induced IL-8 production. Asterisks 2 indicate p < 0.01 compared with isomolar medium-induced IL-8 production.

Tyrosine Phosphorylation of p38 MAP Kinase by Hyperosmolar Stimulation

Immunoblot studies of lysate of BEC with a specific antibody to phosphorylated tyrosine of p38 MAP kinase antibody showed that stimulation of the cells with hyperosmolar NaCl medium (545, 695, 855, 1,055, and 1,280 mOsm/kg H₂O for 10 min) caused an increase in tyrosine phosphorylation of p38 MAP kinase in an osmolarity-dependent manner (Figure 2a). In order to determine the time-course of p38 MAP kinase phosphorylation, BEC, which had been stimulated with hyperosmolar NaCl medium at 1,280 mOsm/kg H₂O for 10 min, were cultured with isomolar medium for desired times as indicated. Amounts of phosphorylated tyrosine of p38 MAP kinase protein increased at 10 min and were sustained between 20 and 30 min; thereafter, they returned to near basal levels, indicating that tyrosine phosphorylation of p38 MAP kinase was transient (Figure 2b). Figures 2c and 2d showed the results from immunoblotting of lysate of hyperosmolar mannitol-stimulated BEC with a specific antibody to phosphorylated tyrosine of p38 MAP kinase antibody (upper panels) or a p38 MAP kinase-specific antibody (lower panels). Similarly, tyrosine phosphorylation of p38 MAP kinase in BEC stimulated with hyperosmolar mannitol medium was in an osmolarity-dependent manner and transient. Lower panels of Figures 2a through 2d showed that equal amounts of p38 MAP kinase protein were immunoblotted with p38 MAP kinase-specific antibody regardless of osmolarity and time of culture periods, indicating that hyperosmolar stimulation-induced increases in tyrosine phosphorylation of p38 MAP kinase occurred in the absence of changes in p38 MAP kinase protein levels. When BEC were cultured with isomolar medium at 280 mOsm/kg H₂O for 10, 20, 30, 60, and 120 min, tyrosine phosphorylation of p38 MAP kinase was not seen at any time of culture periods (data not shown).

View larger version (75K): [in this window] [in a new window]   View larger version (62K): [in this window] [in a new window]   Figure 2.   Tyrosine phosphorylation of p38 MAP kinase in bronchial epithelial cells stimulated with hyperosmolar medium. Bronchial epithelial cell lines NCI-H292 were stimulated with hyperosmolar NaCl medium (a) and (b) or hyperosmolar mannitol medium (c) and (d ). (a) and (c) show osmolarity-dependent effects, and (b) and (d ) show time-dependent effects. The cells were stimulated with varying hyperosmolar NaCl (a) or hyperosmolar mannitol medium (c) for 10 min. The cells were stimulated with hyperosmolar NaCl medium at 1,280 mOsm/kg H2O (b) or with hyperosmolar mannitol medium at 1,280 mOsm/kg H2O (d ) for 10 min. Thereafter, hyperosmolar medium was replaced with fresh isomolar medium, and the cells were cultured for desired times. At the end of cultivation, the lysates from the cells were separated by a 10% SDS-polyacrylamide gel, transferred to membranes, and blotted with a specific antibody to phosphorylated tyrosine of p38 MAP kinase (p38 MAPK-P; upper panels of a, b, c, and d ). The blot shown in the lower panels of a, b, c, and d (p38 MAPK) was stripped and reprobed using a p38 MAP kinase-specific antibody to show the amounts of p38 MAP kinase precipitated. When the cells were stimulated with isomolar medium instead of hyperosmolar medium, tyrosine phosphorylation of p38 MAP kinase was not observed in any conditions. Lane P, positive protein prepared from C-6 glioma cells stimulated with anisomycin for phosphorylated tyrosine of p38 MAP kinase; lane N, negative protein prepared from C-6 glioma cells unstimulated with anisomycin. Three identical experiments independently performed gave similar results.

p38 MAP Kinase Activation by Hyperosmolar Stimulation

We next examined the effect of hyperosmolar stimulation on p38 MAP kinase activity and the effect of SB 203580 on it. To this end, BEC that had been preincubated with or without SB 203580 were stimulated with hyperosmolar NaCl medium at 1,280 mOsm/kg H₂O or hyperosmolar mannitol medium at 1,280 mOsm/kg H₂O for 10 min. Thereafter, the cells were cultured with isomolar medium for 10 min. Hyperosmolar NaCl medium and hyperosmolar mannitol medium induced p38 MAP kinase activation and SB 203580 inhibited hyperosmolar medium-induced p38 MAP kinase activation (Figure 3).

View larger version (20K): [in this window] [in a new window]   Figure 3.   Activation of p38 MAP kinase in bronchial epithelial cells stimulated with hyperosmolar medium. Bronchial epithelial cell lines NCI-H292 that had been cultured either with or without p38 MAP kinase inhibitor SB 203580 (10 µM) for 60 min were stimulated with hyperosmolar NaCl medium at 1,280 mOsm/kg H2O or hyperosmolar mannitol medium at 1,280 mOsm/kg H2O for 10 min. Thereafter, hyperosmolar medium was replaced with fresh isomolar medium, and the cells were cultured for 10 min. At the end of cultivation, the lysates from the cells were incubated with anti-p38 MAP kinase antibody to selectively immunoprecipitate p38 MAP kinase from the cell lysates, and the immunoprecipitates were incubated with activating transcriptive factor-2 (ATF-2) fusion protein in the presence of ATP, which allowed immunoprecipitated active p38 MAP kinase to phosphorylate ATF-2. The samples were separated by a 15% SDS-gel, transferred to membranes, and blotted with anti-phospho-specific ATF-2 antibody. The cells were cultured with isomolar medium (lane 1), SB 203580 (lane 2), hyperosmolar NaCl medium (lane 3), hyperosmolar NaCl medium and SB 203580 (lane 4), hyperosmolar mannitol medium (lane 5), or hyperosmolar mannitol medium and SB 203580 (lane 6). The concentrations of dimethyl sulfoxide used in this study were 0.01%, which was with no effect. Three identical experiments independently performed gave similar results.

JNK Activation by Hyperosmolar Stimulation

Since hyperosmolar stimulation has been shown to cause JNK activation (19, 20), we next examined whether hyperosmolar NaCl medium and hyperosmolar mannitol medium could cause JNK activation in BEC. BEC, which had been stimulated with hyperosmolar NaCl medium or hyperosmolar mannitol medium at 1,280 mOsm/kg H₂O for 10 min, were cultured with isomolar medium for desired times as indicated in order to determine the time-course of JNK activation in BEC. When BEC were stimulated with hyperosmolar NaCl medium, JNK activity increased at 10 min. Maximal JNK activation occurred at 30 min, and thereafter returned to near basal levels at 120 min, indicating that JNK activation was transient (Figure 4a). Similar results were obtained in JNK activation in hyperosmolar mannitol medium-stimulated BEC (Figure 4b).

View larger version (43K): [in this window] [in a new window]   Figure 4.   Activation of JNK in bronchial epithelial cells stimulated with hyperosmolar medium. Bronchial epithelial cell lines NCI-H292 were stimulated with hyperosmolar NaCl medium at 1,280 mOsm/ kg H2O (a) or hyperosmolar mannitol medium at 1,280 mOsm/kg H2O (b) for 10 min. Thereafter, hyperosmolar medium was replaced with fresh isomolar medium, and the cells were cultured for desired times. At the end of cultivation, JNK activity in the cells was determined using c-Jun fusion protein as substrate as described in METHODS. When the cells were stimulated with isomolar medium instead of hyperosmolar medium, JNK activation was not observed in any conditions. Three identical experiments independently performed gave similar results.

Inhibition of Hyperosmolarity-induced IL-8 Production by p38 MAP Kinase Inhibitor SB 203580

For analysis of the role of p38 MAP kinase in hyperosmolarity-induced IL-8 production, we examined the effect of SB 203580 as the p38 MAP kinase inhibitor on hyperosmolarity-induced IL-8 production. To this end, BEC that had been preincubated with or without SB 203580 were cultured with isomolar medium at 280 mOsm/kg H₂O, hyperosmolar NaCl medium, or hyperosmolar mannitol medium at 1,280 mOsm/ kg H₂O, and IL-8 concentrations in the culture supernatants were determined. As shown in Figure 5, 10 µM of SB 203580 inhibited IL-8 production by hyperosmolar NaCl medium-stimulated and hyperosmolar mannitol medium-stimulated BEC by 52.4 and 57.1%, respectively, indicating that SB 203580 partially inhibited hyperosmolarity-induced IL-8 production. About 10% of inhibition was observed at 1 µM of SB 203580 and 25 µM of SB 203580 was cytotoxic for BEC determined by trypan blue exclusion (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 5.   IL-8 production by hyperosmolarity-stimulated bronchial epithelial cells and inhibition of its expression by p38 MAP kinase inhibitor SB 203580. Bronchial epithelial cell lines NCI-H292 that had been cultured either with or without p38 MAP kinase inhibitor SB 203580 (10 µM) for 60 min were stimulated with hyperosmolar NaCl medium or hyperosmolar mannitol medium for 10 min. Thereafter, hyperosmolar medium was replaced with fresh isomolar medium, and the cells were cultured for 24 h, and the concentrations of IL-8 in the culture supernatants were determined by ELISA. The concentrations of dimethyl sulfoxide used in this study were 0.01%, which was with no effect. Asterisks indicate p < 0.01 compared with the cell cultures with hyperosmolar NaCl medium or hyperosmolar mannitol medium.

Inhibition of Hyperosmolarity-induced IL-8 mRNA Expression by p38 MAP Kinase Inhibitor SB 203580

In order to determine the level at which SB 203580 inhibited IL-8 protein production by BEC, we performed Northern blot analysis. As shown in Figure 6, IL-8 mRNA levels were up-regulated when BEC were stimulated with hyperosmolar NaCl medium (lane 3). IL-8 mRNA levels in 10 µM of SB 203580-treated BEC stimulated with hyperosmolar NaCl medium were lower than those in SB 203580-untreated BEC, and weak but apparent IL-8 mRNA signals were seen in SB 203580-treated BEC (lane 4). These results indicated that SB 203580 inhibited hyperosmolar NaCl medium-induced IL-8 mRNA expression, but SB 203580-mediated inhibition was not complete. Similar results were observed in SB 203580-mediated inhibition of hyperosmolar mannitol-induced upregulation of IL-8 mRNA expression (lane 5, hyperosmolar mannitol and lane 6, hyperosmolar mannitol and SB 203580). The levels of -actin mRNA expression did not vary significantly with culture conditions. The total number of cells and cell viability at the end of culture period of each experiment determined by trypan blue exclusion did not differ with culture conditions: Figures 1-6 suggest that the hyperosmolarity-induced IL-8 expression and the inhibition by SB 203580 (10 µM) of IL-8 expression did not result from hyperosmolarity- and SB 203580-induced cell cytotoxicity (data not shown).

View larger version (39K): [in this window] [in a new window]   Figure 6.   IL-8 mRNA expression in hyperosmolarity-stimulated bronchial epithelial cells and inhibition of its expression by p38 MAP kinase inhibitor SB 203580. Bronchial epithelial cell lines NCI-H292 that had been cultured either with or without p38 MAP kinase inhibitor SB 203580 (10 µM) for 60 min were stimulated with hyperosmolar NaCl medium or hyperosmolar mannitol medium for 10 min. Thereafter, hyperosmolar medium was replaced with fresh isomolar medium, and the cells were cultured for 6 h, and the IL-8 mRNA expression (upper panel ) and the -actin mRNA expression (lower panel ) were analyzed by Northern blot analysis. The cells were cultured with isomolar medium (lane 1), SB 203580 (lane 2), hyperosmolar NaCl medium (lane 3), hyperosmolar NaCl medium and SB 203580 (lane 4), hyperosmolar mannitol medium (lane 5), and hyperosmolar mannitol medium and SB 203580 (lane 6). The concentrations of dimethyl sulfoxide used in this study were 0.01%, which was with no effect. 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 hyperosmolar NaCl and hyperosmolar mannitol stimulation on IL-8 expression and the signal transduction pathway regulating hyperosmolarity-induced IL-8 expression in human BEC. The results showed that hyperosmolar stimulation induced IL-8 protein production concomitant with upregulation of IL-8 mRNA expression regardless of solute. Analysis of the signal transduction pathway regulating IL-8 expression, tyrosine phosphorylation and activation of p38 MAP kinase, and JNK activation were induced by hyperosmolar medium. SB 203580 as the specific p38 MAP kinase inhibitor inhibited hyperosmolarity-induced p38 MAP kinase activation and partially inhibited hyperosmolarity-induced IL-8 protein production concomitant with partial SB 203580-mediated inhibition of IL-8 mRNA expression. These results indicate that hyperosmolarity per se is a key factor inducing IL-8 expression, and this induction, at least in part, is mediated through the p38 MAP kinase-dependent pathway.

We examined tyrosine phosphorylation and activation of p38 MAP kinase and JNK activation in order to clarify the signal transduction pathway regulating IL-8 expression in BEC. Activation of p38 MAP kinase is mediated by dual phosphorylation of the threonine residues and the tyrosine residues of p38 MAP kinase (14). In addition to analysis of tyrosine phosphorylation of p38 MAP kinase, p38 MAP kinase activation was examined using ATF-2 fusion protein as substrate. The results showed that p38 MAP kinase was activated and tyrosine phosphorylated in response to hyperosmolarity. Furthermore, SB 203580, which has been shown to inhibit p38 MAP kinase activity but not extracellular-regulated kinase and JNK activity (28) inhibited hyperosmolarity-induced p38 MAP kinase activation. These results indicate that hyperosmolarity activates p38 MAP kinase and SB 203580 inhibits hyperosmolarity-induced p38 MAP kinase activation. In addition, our results showed that hyperosmolarity induced JNK activation. Therefore, we examined the effect of SB 203580 on hyperosmolarity-induced IL-8 expression in order to analyze the role of p38 MAP kinase in hyperosmolarity-induced IL-8 expression. A total of 10 µM of SB 203580 was used, since 25 µM of SB 203580 was cytotoxic for BEC. Ten micromolars of SB 203580 partially inhibited hyperosmolarity-induced IL-8 protein production as well as IL-8 mRNA expression, indicating that SB 203580 partially inhibits at the transcriptional level. Since the previous study with analysis of the role of p38 MAP kinase showed that 10 µM of SB 203580 almost completely inhibited the expression of cytokines (15, 17, 29), 10 µM of SB 203580 used in this study was thought to be sufficient. Therefore, other signaling pathway(s) might be involved in regulating IL-8 expression in BEC induced by hyperosmolarity. One possible signal that may regulate hyperosmolarity-induced IL-8 expression in BEC is JNK. The present study showed that hyperosmolar NaCl and hyperosmolar mannitol stimulation activated JNK. The activation of JNK by various stresses, including hyperosmolar stimulation has been shown (19, 20). Taken together with our results and the previous results with hyperosmolarity-induced activation of p38 MAP kinase and JNK, p38 MAP kinase, at least in part, regulates hyperosmolarity-induced IL-8 expression in BEC. However, other signals such as JNK are possibly also involved in it.

The cells utilized in this study were an undifferentiated cell line, NCI-H₂₉₂. Because functional and structural characteristics of NCI-H₂₉₂ are different from human bronchial epithelial cells, further study should be undertaken in order to clarify the role of p38 MAP kinase in hyperosmolarity-induced IL-8 expression in human bronchial epithelial cells in vivo.

The mechanism responsible for the production of EIB and the development of LPR induced by EIB has been investigated in humans (2, 6) and animal models (2, 5). With regard to the effect of hyperosmolarity on cell function, hyperosmotic activation of mast cells and basophils has been demonstrated (30, 31). However, the responsiveness of BEC to hyperosmotic stimulation during exercise and the intracellular signals leading to cell activation have not been determined. In the present study, we showed that hyperosmolarity induced IL-8 expression in BEC. IL-8 exhibits a variety of biologic activity, including neutrophil chemotactic activity and T-lymphocyte chemotactic activity (32). In addition, IL-8 has been shown to exhibit a chemotactic activity for eosinophils and induce histamine release from basophils (33). Study on cellular analysis in the bronchoalveolar lavage fluid in the LPR showed an increase in eosinophils and neutrophils in human subjects (4) and in dogs (5). Although mediators and cytokines contributing to the recruitment of inflammatory cells into airway in the LPR induced by EIB have not been determined, IL-8 produced by BEC in response to hyperosmolar stimulation shown in this study might be involved in the recruitment of inflammatory cells.

The pyridinyl imidazole compound SB 203580 was originally discovered as an inhibitor of lipopolysaccharide-induced production of interleukin-1 and tumor necrosis factor- in human monocytic cell line, THP-1 (25). This drug has been thought to be useful in the treatment of inflammatory diseases. In the present study, we showed the inhibition of IL-8 production by the specific p38 MAP kinase inhibitor, SB 203580. Therefore, the specific p38 MAP kinase inhibitor may have a potential effect on controlling hyperosmolarity-induced airway inflammations by inhibiting IL-8 expression.

From the results presented here, we conclude that hyperosmolar stimulation induces IL-8 production by bronchial epithelial cells. p38 MAP kinase dependent pathway, at least in part, regulates hyperosmolarity-induced IL-8 expression in BEC. However, other signals such as JNK may also contribute to this response. These results provide new evidence concerning the mechanism of hyperosmolar stimulation-induced airway inflammation, and provide a potential strategy for treatment of airway inflammation with the specific p38 MAP kinase inhibitor.