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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 329-335, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4762

The Role of Mitogen-Activated Protein Kinases in Eotaxin-Induced Cytokine Production from Bronchial Epithelial Cells

Chang-Hao Cui, Tetsuya Adachi, Hajime Oyamada, Yumiko Kamada, Tomoe Kuwasaki, Yoshiyuki Yamada, Norihiro Saito, Hiroyuki Kayaba and Junichi Chihara

Department of Clinical and Laboratory Medicine, Akita University School of Medicine, Akita, Japan

Address correspondence to: Junichi Chihara, M.D., Ph.D., Akita University School of Medicine, Department of Clinical and Laboratory Medicine, 1-1-1, Hondo, Akita 010-8543, Japan. E-mail: chihara{at}hos.akita-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Eotaxin is a critical chemokine eliciting migration of eosinophils and basophils in the pathogenesis of bronchial asthma. Recent studies have shown that the specific receptor for eotaxin, CCR3, is expressed in bronchial epithelial cells. Although mitogen-activated protein (MAP) kinases are involved in diverse cell functions of bronchial epithelial cells, their role in eotaxin signaling is unknown. In this study, we studied the activation and functional relevance of MAP kinases in bronchial epithelial cells stimulated with eotaxin. Eotaxin (1–100 nM) induced tyrosine/threonine phosphorylation and activation of extracellular regulated kinase (ERK) 1/2 and p38 in NCI-H292 cells and normal human bronchial epithelial cells. The phosphorylation of these MAP kinases was detectable after 30 s, and peaked at 5 min. Eotaxin stimulated production of interleukin-8 and granulocyte macrophage colony-stimulating factor. Pretreatment of Compound X (a specific CCR3 antagonist), pertussis toxin, genistein, and wortmannin reduced the MAP kinase phosphorylation and cytokine production. The eotaxin-induced cytokine production was inhibited by specific inhibitors for MAP/ERK kinase (PD98059) and p38 MAP kinase (SB202190). These results suggest that both ERK1/2 and p38 MAP kinase activated by eotaxin have a critical role in the pathogenesis of asthma.

Abbreviations: activating transcription factor, ATF • CC chemokine receptor, CCR • enzyme-linked immunosorbent assay, ELISA • extracellular signal-regulated kinase, ERK • granulocyte macrophage colony-stimulating factor, GM-CSF • interleukin, IL • c-Jun N-terminal kinase, JNK • mitogen-activated protein, MAP • myelin basic protein, MBP • normal human bronchial epithelial cells, NHBE • small airway cell basal medium, SABM • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The pathogenesis of bronchial asthma is characterized by the tissue infiltration of leukocytes, involving T lymphocytes and eosinophils (1). During an allergic response, eosinophils migrate out of the bloodstream into tissue and degranulate readily, releasing cytotoxic products such as granule proteins and reactive oxygen species. The products cause epithelial damage, resulting in enhanced bronchial hyperresponsiveness and airway obstruction. However, epithelial cells are also able to modify allergic airway inflammation by virtue of their ability to produce a variety of inflammatory mediators (2). For example, the cells stimulated with interleukin (IL)-1 or tumor necrosis factor-{alpha} (TNF-{alpha}) produce cytokines such as IL-8, granulocyte macrophage colony-stimulating factor (GM-CSF), regulated on activation, normal T cells expressed and secreted (RANTES), and eotaxin (3–7).

Mitogen-activated protein (MAP) kinase cascades are one of the most studied and elucidated signal transduction systems, and are known to participate in multiple directions of cellular programs (8). Five distinct MAP kinase cascades have been described in mammalian cells, including the extracellular regulated kinase (ERK) 1 and 2, the c-Jun N-terminal kinase (JNK), the p38 MAP kinase, ERK3, and ERK5. These MAP kinases are dual phosphorylated on tyrosine/threonine residues by distinct MAP kinase kinases, although the upstream molecules of ERK3 are yet unidentified. ERK1/2 corresponding to classical MAP kinases are activated by a variety of growth factors and play critical roles in mitogenesis. JNK and p38 are activated by cellular stress or proinflammatory cytokines that are known to induce apoptosis. Several studies have shown the functional role of MAP kinases in bronchial epithelium using their specific inhibitors (9, 10). According to these results, JNK and p38 MAP kinase play critical roles in regulating cytokine production by the cells activated by a range of stimuli.

A CC chemokine, eotaxin, plays a crucial role in eosinophil migration in tissue (11). The chemotactic response of eosinophils is mostly mediated by CC chemokine receptor-3 (CCR3), which is also preferentially expressed on Th2 cells and basophils (12, 13). CCR3 is a heterotrimeric G protein–coupled receptor, which is known to transduce signals eliciting Ca2+ influx (14, 15). Eotaxin also activates ERK1/2 and p38 MAP kinases in eosinophils, and these kinases are indispensable for eosinophil chemotaxis and degranulation (16). Recent studies have shown that CCR3 is expressed on bronchial epithelial cells (17, 18). However, the involvement and functional relevance of MAP kinases in eotaxin signaling is unclear in the epithelium.

In the present study, we investigated the functional role of ERK1/2 and p38 in bronchial epithelial cells stimulated with eotaxin. We found that ERK1/2 and p38 MAP kinase are activated by eotaxin. In addition, these kinases have a critical role in IL-8 and GM-CSF production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
The human bronchial epithelial cell line (NCI-H292) and normal human bronchial epithelial (NHBE) cells were obtained from American Type Culture Collection (Rockville, MD) and Clonetics Normal Human Cell Systems (Walkersville, MD), respectively. K562 cells transfected with CCR3 were supplied by Dr. Osamu Yoshie (Shionogi Institute for Medical Science, Osaka, Japan). Human eotaxin, the anti-human CCR3 antibody (rat IgG2a), the ß-actin primers, and the ELISA kits for IL-8 and GM-CSF were purchased from R&D Systems (Minneapolis, MN). The source of isotype-matched control rat IgG2a was Southern Biotechnology Associates, Inc. (Birmingham, AL). The FITC-conjugated goat anti-rat IgG2a antibody was obtained from Nordic Immunology (Tilburg, Netherlands). The mouse monoclonal anti–phospho-ERK antibody, rabbit polyclonal anti-ERK2 and anti-p38 antibodies, HRP-conjugated goat anti-mouse and anti-rabbit IgG antibodies, protein A/G Plus agarose, and activating transcription factor-2 (ATF-2) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The polyclonal antibodies against phospho-p38 and PD98059 were purchased from Cell Signaling Technology (Beverly, MA). The source of SB202190 was CalBiochem (La Jolla, CA). Compound X, a CCR3 antagonist, was a gift from BANYU Pharmaceutical Co. (Tsukuba, Japan). The myelin basic protein (MBP), ATP, genistein, and cyclohexamide were purchased from Sigma (St. Louis, MO). The source of pertussis toxin and wortmannin was BioMol Research Laboratories (Plymouth Meeting, PA). Enhanced chemiluminescence detection system and Hybond ECL nitrocellulose membrane were obtained from Amersham (Arlington Heights, IL). [{gamma}-32P]-ATP was purchased from NEN Life Science Products (Boston, MA).

Cell Cultures
The NCI-H292 and NHBE cells were cultured in small airway cell basal medium (SABM) supplemented with 7.5 mg/ml bovine pituitary extract, 0.5 mg/ml hydrocortisone, 0.5 µg/ml human recombinant epidermal growth factor, 0.5 mg/ml epinephrine, 10 mg/ml transferrin, 5 mg/ml insulin, 0.1 µg/ml retinoic acid, 6.5 µg/ml triiodothyronine, and 0.5 mg/ml gentamicin sulfate with amphotericin-B (Clonetics, Walkersville, MD) at 37°C in a humidified 5% CO2 atmosphere. The cells were then transferred into a 24-well tissue culture plate (Becton Dickinson Labware, Franklin Lakes, NJ) and grown until subconfluence. The culture medium was replaced with SABM depleting all supplements 24 h before each experiment.

RT-PCR Analysis
Polyadenylated RNA was extracted using a QuickPrep mRNA Purification Kit (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and reverse-transcribed by a SuperScript II preamplification System (Life Technologies, Rockville, MD). One microliter of cDNA synthesis reaction was used as a template for PCR amplification with AmpliTaq DNA polymerase (Perkin-Elmer, Foster City, CA). The CCR3 primers were 5'-TCCTTCTCTCTTCCTATCAATC-3' and 5'-GGCAATTTTCTGCATCTG-3' (17). The PCR cycle was composed of 45 s denaturation at 94°C, 45 s annealing at 55°C, and 1 min extension at 72°C for 35 cycles. The PCR products were electrophoresed with a 12.5% PhastGel and PhastSystem, and the gel was stained using a PhastGel DNA Silver Staining Kit (Amersham Pharmacia Biotech AB).

Flow-Cytometric Analysis
The harvested epithelial cells were fixed by 0.4% parabenzoquinolone for 10 min in the dark at room temperature. The cells were further treated with 0.74% n-octyl-ß-D-glucopyranoside for 5 min to facilitate cell permeabilization. Then, the cells were incubated with the anti-CCR3 or isotype-matched control antibody followed by the additional treatment with the FITC-labeled secondary antibody. The ratio of CCR3-positive cells was analyzed using a flow cytometer (FACScan; Becton Dickinson Immunocytometry Systems, San Jose, CA).

Preparation of Cytosolic Cell Extracts and Immunoprecipitation
The NCI-H292 or NHBE cells were incubated with and without each inhibitor or the CCR3 antagonist for indicated times at 37°C, followed by stimulation with eotaxin. The reaction was terminated by the addition of 9 vols of ice-cold HBSS containing 1 mM Na3VO4. The cells were lysed in a lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM Na3VO4, 1 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1% Triton X-100, 10% glycerol, 1 µg/ml of aprotinin, leupeptin, and pepstain). After 20 min on ice, detergent-insoluble materials were removed by 12,000 x g centrifugation at 4°C. The whole-cell lysates were boiled in 2x Laemmli reducing buffer for 4 min.

For immunoprecipitation, the cell lysates were prepared using the above-described lysis buffer without glycerol. After measuring the protein concentration to standardize the samples, the lysates were subjected to immunoprecipitation. The cell lysates were incubated with the appropriate antibody (1–2 µg for each sample) for 1 h, followed by incubation with 20 µl of protein A/G Plus agarose for 2 h at 4°C. The beads were washed three times with the cold lysis buffer.

Immunocomplex Kinase Assay
The immunoprecipitates were assayed for ERK1/2 and p38 by measuring the phosphotransferase activity for MBP and ATF-2, respectively. The kinase reaction to measure ERK1/2 activity was performed by incubating the immunoprecipitates in 40 µl kinase buffer (10 mM HEPES, 50 mM NaCl, 10 mM MgCl2, 100 µM Na3VO4, 500 µM dithiothreiol, 25 mM ß-glycerophosphate) containing 2.5 µM ATP, 10 µCi [{gamma}-32P]-ATP and 50 µg/ml MBP for 30 min at 30°C. In some experiments, ATF-2 (12.5 µg/ml) was used as the substrate to measure p38 activity instead of MBP. After centrifugation, the reaction was stopped by boiling the supernatant with an equal amount of 2x Laemmli buffer. The kinase reaction products were then applied to SDS-PAGE. The detection of ERK1/2 and p38 activity was performed by autoradiography.

Gel Electrophoresis and Western Blotting
SDS-PAGE was performed using Ready Gels J (BioRad, Hercules, CA). The concentration of polyacrylamide was 10–15% depending on the molecular weight of the protein in which we were interested. Gels were blotted onto Hybond membranes for Western blotting using the enhanced chemiluminescence system. Blots were incubated in a blocking buffer containing 10% bovine serum albumin in a blotting buffer (20 mM Tris-HCl, 137 mM NaCl, pH 7.6, 0.05% Tween 20) for 1 h followed by incubation in the primary antibody (0.1 µg/ml) for 1–2 h. After washing three times in the blotting buffer, blots were incubated for 30 min with a horseradish peroxidase–conjugated secondary antibody (0.04 µg/ml) directed against the primary antibody. The blots were developed with the enhanced chemiluminescence substrate according to the manufacturer's instructions. In some experiments, blots were reprobed with another antibody after stripping in a buffer of 62.5 mM Tris-HCl (pH 6.7), 100 mM 2-mercaptoethanol, and 2% SDS at 50°C for 30 min.

Measurement of IL-8 and GM-CSF
The NCI-H292 cells were suspended in SABM. After treatment with or without the inhibitors for indicated times at 37°C, the cells were stimulated with 100 nM eotaxin. The supernatants were separated by centrifugation, and the concentration of IL-8 and GM-CSF was measured by enzyme-linked immunosorbent assay (ELISA).

Statistical Analysis
Results were expressed as mean ± SD. Data were analyzed for statistical significance using ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CCR3 Expression on Bronchial Epithelial Cells
We initially tried to investigate the expression of CCR3 at mRNA levels. K562 cells expressing CCR3 were used as a positive control. As shown in Figure 1A , CCR3 mRNA was detectable by RT-PCR in both NCI-H292 and NHBE cells. Next, the protein expression of CCR3 on epithelial cells was studied. The NCI-H292 cells readily expressed CCR3 protein detected by flow cytometry (Figure 1B). The ratio of CCR3-positive cells was more than 80%.




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  		Figure 1. (A) RT-PCR analysis of CCR3 mRNA expression. The amplified products were separated on 12.5% polyacrylamide gel, and the gel was subjected to silver staining. CCR3 mRNA is expressed in both NCI-H292 and NHBE cells. Lane 1: NCI-H292 cells; lane 2: NHBE cells; lane 3: K562 cells expressing CCR3; lane 4: negative control. (B) The protein expression of CCR3 in epithelial cells detected by flow cytometry. The NCI-H292 cells readily express CCR3 at protein levels.

 
Phosphorylation of ERK1/2 and p38 by Eotaxin
Because of CCR3 expression on bronchial epithelium, it is of paramount importance to investigate the eotaxin signaling in the cells. We first examined the phosphorylation of ERK1/2 and p38 MAP kinase by eotaxin. The NCI-H292 cells were stimulated with and without various concentrations of eotaxin for 5 min. The cytosolic extracts were subjected to electrophoresis and Western blotting with the anti–phospho-ERK or anti–phospho-p38 antibody. Eotaxin (1–10 nM) induced tyrosine/threonine phosphorylation of ERK1/2 in NCI-H292 cells in a dose-dependent manner (Figure 2A) . No further increase of ERK1/2 phosphorylation was observed in the case of 100 nM eotaxin stimulation. Similar results were obtained in the p38 blot with the same level of phosphorylation by 1–100 nM eotaxin (Figure 2B). Reprobing the membranes either with the anti-ERK2 or anti-p38 antibody showed that the same amount of protein was loaded on the gels. We also examined phosphorylation of ERK1/2 and p38 MAP kinase in the NHBE cells stimulated with eotaxin. Similar results were obtained in the ERK1/2 and p38 blots with the same level of phosphorylation by 1–100 nM eotaxin (Figures 3A and 3B) .




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  		Figure 2. Phosphorylation of (A) ERK1/2 and (B) p38 by eotaxin. NCI-H292 cells were stimulated with eotaxin (1–100 nM) for 5 min. The cell lysates were subjected to electrophoresis and Western blotting with the anti–phospho-ERK or anti–phospho-p38 antibody. Eotaxin induced tyrosine/threonine phosphorylation of (A) ERK1/2 and (B) p38. Reprobing the membrane with the anti-ERK2 or anti-p38 antibody revealed that the same amount of protein was loaded on the gel. The blots are representative of three independent experiments.

 



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  		Figure 3. Phosphorylation of (A) ERK1/2 and (B) p38 in NHBE cells. After stimulating the cells with eotaxin (1–100 nM) for 5 min, the cell lysates were subjected to electrophoresis and Western blotting with the anti–phospho-ERK or anti-phospho-p38 antibody. Eotaxin induced tyrosine/threonine phosphorylation of (A) ERK1/2 and (B) p38. Reprobing the membrane with the anti-ERK2 or anti-p38 antibody revealed that the same amount of protein was loaded on the gel. The blots are representative of three independent experiments.

 
Kinetics of ERK1/2 and p38 Phosphorylation
To investigate the kinetics of ERK1/2 and p38 phosphorylation, NCI-H292 cells were incubated with medium or stimulated with eotaxin (10 nM) for 0.5, 1, 3, 5, 15, or 30 min. Western blotting with the anti–phospho-ERK antibody revealed that the tyrosine/threonine phosphorylation of ERK1/2 reached a maximum at ~ 5 min and then declined (Figure 4A) . Similar results were obtained in the dual phosphorylation of p38 MAP kinase (Figure 4B).




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  		Figure 4. Kinetics of (A) ERK1/2 and (B) p38 phosphorylation. NCI-H292 cells were incubated with medium or stimulated with 10 nM eotaxin for the indicated times. The cell lysates were subjected to electrophoresis and Western blotting with anti–phospho-ERK1/2 or anti–phospho-p38 antibody. The tyrosine/threonine phosphorylation of (A) ERK1/2 and (B) p38 MAP kinase reached a maximum at ~ 5 min, and then declined. Reprobing the membrane with the anti-ERK2 or anti-p38 antibody revealed that the same amount of protein was loaded on the gel. The blots are representative of three independent experiments.

 
Kinase Activity of ERK2 and p38 by Eotaxin
To verify that the phosphorylation was accompanied by activation, we examined the kinase activity of ERK2 by eotaxin. The cell lysates were immunoprecipitated with the anti-ERK2 antibody, followed by the immunocomplex kinase assay and autoradiography to measure MBP phosphorylation as the ERK2 activity. As shown in Figure 5A , eotaxin stimulation enhanced ERK1/2 activity. In some experiments, NCI-H292 cells were incubated with and without SB202190 and then stimulated with eotaxin. The immunoprecipitated p38 from the cell lysates was applied to the kinase assay followed by electrophoresis and autoradiography. Eotaxin enhanced the kinase activity of p38, which was inhibited by SB202190 (Figure 5B).




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  		Figure 5. Kinase activity of (A) ERK2 and (B) p38 by eotaxin. NCI-H292 cells were stimulated with eotaxin (10 or 100 nM) for 5 min. In some experiments, the cells were incubated with SB202190 for 1 h before eotaxin stimulation. The cell lysates were immunoprecipitated with the anti-ERK2 or anti-p38 antibody. The immunocomplex was subjected to in vitro kinase assay using MBP as the substrate for ERK2 and ATF-2 for p38. (A) Eotaxin stimulated MBP phosphorylation, indicating the enhanced ERK1/2 activity. (B) The p38 activity was upregulated by eotaxin, which was inhibited by SB202190. The blots are representative of three independent experiments.

 
Effect of the CCR3 Antagonist or Inhibitors on the Eotaxin-induced Phosphorylation of ERK1/2 and p38
To establish whether the phosphorylation of ERK1/2 and p38 by eotaxin was mediated through CCR3, we studied the effect of CCR3 antagonist on the MAP kinase phosphorylation. We used Compound X, which has been shown to be a selective antagonist for CCR3 (19). After incubating with the compound for 30 min, the cells were then stimulated with eotaxin (10 nM) for 5 min. Western blotting with anti–phospho-ERK or anti–phospho-p38 antibody revealed that the CCR3 antagonist caused a inhibition of ERK1/2 and p38 phosphorylation, indicating that the activation of the MAP kinases was mediated through CCR3 (Figures 6A and 6B) . To elucidate the signaling pathway between CCR3 and MAP kinases, we analyzed the effect of pertussis toxin, genistein, and wortmannin on the phosphorylation. The NCI-H292 cells were incubated with genistein (10 µg/ml) or wortmannin (10-7 M) for 1 h, or with pertussis toxin (100 ng/ml) for 2 h followed by eotaxin stimulation. All the inhibitors abrogated the ERK1/2 and p38 phosphorylation, indicating the involvement of G{alpha}i, tyrosine kinases, and phosphatidylinositol (PI)-3 kinase upstream of those MAP kinases (Figures 7A and 7B) . As expected, the MEK inhibitor PD98059 blocked the phosphorylation of ERK1/2, but not p38.




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  		Figure 6. The effect of Compound X on eotaxin-induced tyrosine/threonine phosphorylation of (A) ERK1/2 and (B) p38. The NCI-H292 cells were incubated with the compound for 1 h at 37°C followed by stimulation with eotaxin for 5 min. The cell lysates were subjected to electrophoresis and Western blotting with the anti–phospho-ERK or anti–phopho-p38 antibody. Compound X abrogated the phosphorylation of (A) ERK1/2 and (B) p38 MAP kinase in dose-dependent manners. Reprobing the membrane with the anti-ERK2 or anti-p38 antibody revealed that the same amount of protein was loaded on the gel. The blots are representative of three independent experiments.

 



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  		Figure 7. The effect of pertussis toxin, genistein, wortmannin, and PD98059 on eotaxin-induced tyrosine/threonine phosphorylation of (A) ERK1/2 and (B) p38. The NCI-H292 cells were incubated with genistein or wortmannin for 1 h, or with pertussis toxin for 2 h at 37°C followed by stimulation with eotaxin for 5 min. The cell lysates were subjected to electrophoresis and Western blotting with the anti–phospho-ERK or anti–phospho-p38 antibody. The phosphorylation of (A) ERK1/2 and (B) p38 MAP kinase after eotaxin stimulation was inhibited by pertussis toxin, genistein, or wortmannin. PD98059 completely abrogated the phosphorylation of (A) ERK1/2, but not (B) p38. Reprobing the membrane with the anti-ERK2 or anti-p38 antibody revealed that the same amount of protein was loaded on the gel. The blots are representative of three independent experiments. w, wortmannin; g, genistein; px, pertussis toxin; PD, PD98059.

 
Cytokine Production from Eotaxin-stimulated Bronchial Epithelial Cells
We measured cytokine production from NCI-H292 cells to investigate the functional role of eotaxin. The NCI-H292 cells were incubated with and without cyclohexamide (1 µg/ml) for 6 h, and then stimulated with eotaxin. The cytokine concentration in the supernatants were measured by ELISA. As shown in Figure 8A , IL-8 was produced by eotaxin in a time-dependent manner. The pretreatment of cyclohexamide did not affect the IL-8 production up to 6 h after the stimulation, whereas it partially inhibited the IL-8 production for 24 or 48 h. To elucidate the signaling pathway that elicits IL-8 production, we examined the effect of inhibitors on the function. Consistent with the results in Figures 6 and 7, Compound X (10-6 M), pertussis toxin (100 ng/ml), genistein (10 µg/ml), and wortmannin (10-7 M) abrogated the effect of eotaxin on IL-8 production (Figure 8B). Similar results were obtained regarding the GM-CSF production in NCI-H292 cells stimulated with eotaxin (data not shown).




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  		Figure 8. (A) Kinetics of IL-8 production from NCI-H292 cells, and (B) effect of inhibitors on the function. After the pretreatment with and without indicated inhibitors, the cells were stimulated with eotaxin (A) for 1 h, 6 h, 24 h, and 48 h, or (B) for 24 h. The concentration of IL-8 in the supernatants was measured by ELISA. Data are expressed as means ± SD. (A) IL-8 was produced from the NCI-H292 cells in a time-dependent manner. The early phase of IL-8 production (1–6 h) was not affected by cyclohexamide, although the partial inhibition during the late phase (24–48 h) (n = 3). (B) Compound X, pertussis toxin, genistein, and wortmannin abrogated the effect of eotaxin on IL-8 production (n = 3). *P < 0.05 versus without inhibitors (ANOVA). chx, cyclohexamide; w, wortmannin; g, genistein; px, pertussis toxin.

 
Effect of PD98059 and SB202190 on Eotaxin-induced IL-8 and GM-CSF Production
Next, we tested the functional relevance of ERK1/2 and p38 in cytokine production induced by eotaxin. The NCI-H292 cells were preincubated with PD98059 or SB202190 for 1 h and stimulated with eotaxin for an additional 24 h. The cytokine concentration in the cultured supernatant was determined by ELISA. PD98059 significantly inhibited IL-8 and GM-CSF production from NCI-H292 cells in a concentration-dependent manner (Figures 9A and 9B) . The eotaxin-induced IL-8 and GM-CSF production was also abrogated by 10 µM SB202190 (Figures 9A and 9B). To rule out the possibility that these effects were due to cytotoxicity, we checked the cell viability at the end of culture by trypan blue exclusion assay. The cell viability in the presence of PD98059 or SB202190 did not differ from that without the inhibitors (data not shown).




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  		Figure 9. The effects of PD98059 and SB202190 on (A) IL-8 or (B) GM-CSF release. After incubating NCI-H292 cells with and without the inhibitors for 1 h, the cells were stimulated with or without 100 nM eotaxin for 24 h. The concentration of IL-8 and GM-CSF in the supernatants was measured by ELISA. Data are expressed as means ± SD. The IL-8 and GM-CSF concentrations in the sample without eotaxin stimulation were 259 ± 55 pg/ml and 13 ± 2 pg/ml, respectively. PD98059 significantly inhibited (A) IL-8 and (B) GM-CSF production from NCI-H292 cells in a dose-dependent manner (n = 3). The eotaxin-induced (A) IL-8 and (B) GM-CSF production was also abrogated by 10 µM SB202190 (n = 3). *P < 0.05 versus without inhibitors (ANOVA).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we studied the involvement and the functional relevance of MAP kinases in eotaxin signaling of bronchial epithelial cells. ERK1/2 and p38 were activated through CCR3 in NCI-H292 and NHBE cells stimulated with eotaxin. We found that these molecules are indispensable for the production of IL-8 and GM-CSF. This is the first report to show essential roles of MAP kinases activated by eotaxin in the pathogenesis of asthma.

CCR3 is a major chemokine receptor responsible for regulating eosinophil trafficking, which is also expressed on basophils and Th2 cells. A recent study published by Stellato and coworkers has shown that CCR3 mRNA was detectable in TNF-{alpha}–stimulated, but not untreated, BEAS-2B or primary bronchial epithelial cells (18). In our study, unstimulated NCI-H292 and NHBE cells readily expressed the message. One possible explanation for this discrepancy is that they applied Northern blotting to detect the message, whereas we used RT-PCR. The CCR3 protein was highly expressed on the epithelial cells, which is consistent with the results of Stellato and colleagues (18). We showed that the ERK1/2 and p38 MAP kinase are activated by eotaxin in NCI-H292 and NHBE cells. Preliminarily, we also found that JNK is activated by eotaxin (unpublished data). Especially, this is the first report to show the activation of p38 MAP kinase in bronchial epithelial cells stimulated with eotaxin. The phosphorylation of MAP kinases reached peak at ~ 5 min and then declined. This pattern is different from that in eosinophils exhibiting the second peak of activation at ~ 15–30 min (16). A possible explanation for this phenomenon is that other factors produced from eotaxin-stimulated eosinophils elict secondary MAP kinase activation, which may not be the case in bronchial epithelial cells. The specific CCR3 antagonist Compound X clearly inhibited the eotaxin-induced phosphorylation of MAP kinases. This result indicates the utilization of CCR3 as the receptor for eotaxin in bronchial epithelial cells.

Although our data suggest that both ERK1/2 and p38 activation are regulated through tyrosine kinases and PI-3 kinase, the details of the eotaxin-signaling pathway in bronchial epithelial cells are unclear. In G protein–coupled receptor signaling, the activation of MAP kinase is generally mediated by Gß{gamma} (20). These subunits transduce the signal to PI-3 kinase {gamma}, which induces tyrosine phosphorylation of Shc, indicating the involvement of tyrosine kinases in this process. Possible candidates of tyrosine kinase could be Lyn and Btk/Itk (21, 22). The activation of Shc induces formation of the Grb2/Sos complex, leading to activation of the Ras-Raf-MEK-MAP kinase pathway. Although the details remain to be clarified, it has also been shown that p38 MAP kinase is activated through Gß{gamma} in the signaling of m2 muscarinic acetylcholine receptor (23). Furthermore, monocyte chemoattractant protein (MCP)-1–induced p38 activation is sensitive to PP2, a src-family tyrosine kinase inhibitor (24). In support of our findings, eotaxin induces phosphorylation of tyrosine kinases and MAP kinases in eosinophils (16, 25).

Several cytokines and chemokines, such as IL-8, GM-CSF, eotaxin, RANTES, and MCP-3/4, are produced by epithelial cells during the airway inflammation of asthma (26–29). They play a pivotal role in the recruitment of inflammatory cells into the site of allergic inflammation, leading to bronchial hyperresponsiveness. GM-CSF has priming or direct effects on eosinophil functions, such as survival, adhesion, and degranulation (1). Expression of GM-CSF is upregulated in the asthmatic bronchial epithelium and is positively correlated with bronchial hyperresponsiveness (26, 27). IL-8 also exerts a variety of cellular functions, especially cell migration. It possesses chemotactic activity not only for neutrophils but also for eosinophils (30, 31). Eosinophils do express CXCR1/2, which are IL-8 receptors, after incubation with IL-5 for 5–7 d (32). Ulfman and coworkers have recently shown that IL-8 elicits transient arrest of eosinophils in rolling on endothelial cells accompanied by the increase of pertussis toxin–sensitive Ca2+ influx (33). Thus, it is of paramount interest to investigate the mechanism of GM-CSF and IL-8 production from epithelial cells. We found that the production of IL-8 and GM-CSF was observed in bronchial epithelial cells stimulated with eotaxin. Interestingly, cyclohexamide partially inhibited the late phase (24–48 h) of IL-8 production, but not the early phase (1–6 h). These results suggest that eotaxin stimulates both release of preformed IL-8 and the de novo synthesis.

The role of MAP kinase in cytokine production has been studied. SB 203580, a specific inhibitor for p38, inhibited the production of IL-8, GM-CSF, and RANTES by IL-1ß–, TNF-{alpha}–, or platelet-activating factor (PAF)-stimulated human bronchial epithelial cells (9, 10). In the present study, we showed that PD98059 or SB202190 inhibited eotaxin-induced IL-8 and GM-CSF production in NCI-H292 cells. The ERK1/2 played a greater role than p38 MAP kinase in the function. Taken together, the contribution of ERK1/2 and p38 to the cytokine production is likely to depend on the stimuli. It is noteworthy that RANTES could not be detected in the supernatant of the eotaxin-stimulated NCI-H292 cells (data not shown).

In conclusion, we have defined the activation of ERK1/2 and p38 MAP kinase in eotaxin signaling and its essential role in cytokine production from bronchial epithelial cells. Recruitment of eosinophils from the bloodstream into the airways is an important characteristic feature of asthma and allergic diseases. Although eotaxin is a critical chemokine in eosinophil migration into the tissue, it also plays a unique role in regulating epithelial function. Therefore, the delineation of the signaling pathway of chemokines and its role in various functions of epithelial cells will help to elucidate the mechanism of allergic diseases and may shed light on developing new strategy to treat allergic disorders.


   Acknowledgments
 
This work was supported in part by Grants-in-aid for Scientific Research from the Ministry of Education and Science of Japan. C.C. was supported by Rotary Yoneyama Memorial Foundation Scholarship.

Received in original form November 1, 2001

Received in final form April 13, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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