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Clin Exp Immunol. Jul 2006; 145(1): 162–172.
PMCID: PMC1942012

Interleukin (IL)-4 and IL-13 up-regulate monocyte chemoattractant protein-1 expression in human bronchial epithelial cells: involvement of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 and Janus kinase-2 but not c-Jun NH2-terminal kinase 1/2 signalling pathways

Abstract

The Th2 cytokines interleukin (IL)-4 and IL-13 and chemokine monocyte chemoattractant protein-1 (MCP-1) are significantly involved in bronchial hyperreactivity (BHR) and remodelling in allergic asthma. Although IL-4 and IL-13 can regulate a number of chemokines from bronchial epithelium, their regulatory effect on the expression of MCP-1 is as yet unproved. We aim to investigate the intracellular signalling mechanisms of IL-4 and IL-13 regulating the expression and secretion of MCP-1 from human bronchial epithelial cells. BEAS-2B cells, derived from a human bronchial epithelial cell line, were activated with or without IL-4 and/or IL-13 for different time intervals. MCP-1 gene expression and protein secretion were measured by reverse transcription–polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Activation of signalling molecules p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and Janus kinase-2 (JAK-2) was accessed by Western blotting. IL-4 and IL-13 were found to up-regulate gene expression and significantly increase the release of MCP-1 from BEAS-2B cells. Both cytokines could activate p38 MAPK, ERK and JAK-2, but not JNK activity. Inhibition of p38 MAPK, ERK and JAK-2 activities by pretreating the cells with their corresponding inhibitors SB203580, PD98059 and AG490, respectively, significantly suppressed IL-4- and IL-13-induced MCP-1 production in BEAS-2B cells. Together, the above results illustrate that the activation of p38 MAPK, ERK and JAK-2 but not JNK is crucial for IL-4- and IL-13-induced MCP-1 release in human bronchial epithelial cells. Our findings may provide insight into the future development of more effective therapeutic agents for treating allergic asthma.

Keywords: allergy, chemokines/monokines, cytokines/interleukins, epithelial cells, signalling/signal transduction

Introduction

Allergic asthma is a chronic and allergic inflammatory disease of the airway characterized by bronchial infiltration of inflammatory cells, elevated plasma concentration of allergen-specific IgE, reversible bronchial obstruction, mucus hypersecretion and bronchial hyperreactivity (BHR) [1]. The activation and recruitment of inflammatory cells such as eosinophils, activated T lymphocytes, mast cells and macrophages into the bronchial submucosa are mediated by chemokines produced from the bronchial epithelium. Interaction between inflammatory cells and bronchial epithelial cells contribute eventually to bronchial tissue damage and remodelling of the pulmonary structure in severe allergic asthma [13].

Monocyte chemoattractant protein-1 (MCP-1) is one of the C-C chemokines which is produced spontaneously by bronchial epithelial cells and is responsible for the recruitment of inflammatory cells and pathogenesis of allergic asthma [4,5]. Increased gene expression and protein concentration of MCP-1 have been reported in bronchial tissue and bronchoalveolar lavage fluid (BALF) of asthmatic patients, respectively [6,7]. MCP-1 mediates the activation and recruitment of monocytes, mast cells, basophils, eosinophils and T helper type 2 (Th2) lymphocytes from the vascular compartment to bronchoalveolar space through the expression of CCR2 on their cell surfaces [5]. Gonzalo et al. [8] has shown that antibodies against MCP-1 profoundly reduce the number of inflammatory cells in BALF and reduce BHR drastically in a mouse model of asthma. These findings suggested that MCP-1 may mediate directly the development of BHR in allergic asthma [9].

The Th2 cytokines interleukin (IL)-4 and IL-13 have been shown to play critical roles in the pathogenesis of allergic asthma [2,10,11]. We have already found an imbalanced and predominant expression of Th2 cytokines, including IL-13, in the plasma of allergic asthmatic patients [12]. In addition to plasma levels, another study has also demonstrated induced secretion and a significantly higher concentration of IL-4 and IL-13 in BALF of allergic asthmatic patients after allergen challenge [13]. These results suggested the important role and direct function of IL-4 and IL-13 in bronchial inflammation and asthma. IL-4 and IL-13 are produced predominantly by Th2 cells [14], but also by mast cells [15], natural killer (NK) cells [16] and, as reported recently, alveolar macrophages [17,18]. In addition, IL-4 can also be secreted from basophils and eosinophils [19]. Both cytokines share only 25% homology but have overlapping signalling pathways and many biological functions via common receptor subunits, IL-4Rα and IL-13Rα1 [2,20]. The type I IL-4R complex is composed of IL-4Rα and the common γ chain, and bind exclusively to IL-4 but not IL-13. For the type II IL-4R complex, it is composed of IL-4Rα and IL-13Rα, and binds to both IL-4 and IL-13 [2,20]. They are multi-functional cytokines that can inhibit the effector functions of Th1 cells but drive the development of Th2 cells [20]. They are also critical regulators of hallmark features of allergic asthma, which include increased IgE switching and secretion from B cells [2,20], bronchial mucus hypersecretion and BHR [20,21]. Studies using murine models of allergic asthma have demonstrated that IL-13 can mediate the development of BHR and mucus hypersecretion, independently of IgE and eosinophilia, by direct action on bronchial epithelial cells [20,21]. These results underscore the need to understand the gene and protein responses evoked in the bronchial epithelium by IL-4 and, especially, IL-13.

In recent years, many intracellular signalling molecules have become promising targets for the development of therapeutic strategies in allergic asthma [22]. IL-4 and IL-13 use Janus kinases (JAKs) to initiate signalling cascades and activate the signal transducer and activator of transcription-6 (STAT6), which is a transcription factor required for bronchial epithelial cell in the development of BHR [21,23]. In addition, activation of three major mitogen-activated protein kinase (MAPK) subfamilies, including p38 MAPK, extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), have been identified and demonstrated in bronchial smooth muscle and epithelial cells upon IL-4 and IL-13 stimulation [2427]. These observations prompted us to investigate the intracellular signal transduction mechanisms for IL-4 and IL-13 modulating the expression and secretion of MCP-1 from bronchial epithelial cells.

Materials and methods

Reagents

Human recombinant IL-4 and IL-13 were obtained from PeproTec Inc. (Rocky Hill, NJ, USA). Mouse monoclonal antibodies to phospho-p38 MAPK, phospho-ERK1/2 and phospho-JNK1/2 were purchased from BD Biosciences Pharmingen (San Diego, CA, USA), and mouse polyclonal antibody to phospho-STAT-1 and rabbit polyclonal antibody to phospho-STAT-6 were obtained from BD Transduction Laboratories (San Jose, CA, USA). Mouse polyclonal antibody to phospho-JAK-2 was purchased from Upstate Inc. (New York, NY, USA), and rabbit polyclonal antibodies to p38 MAPK, ERK1/2, JNK1/2 and JAK-2 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). For inhibition assays, a mouse monoclonal antibody to human IL-4 receptor was purchased from R&D Systems Inc. (Minneapolis, MN, USA). p38 MAPK inhibitor SB203580 (SB), ERK inhibitor PD98059 (PD), JNK inhibitor SP600125, JAK-2 inhibitor AG490 and protease inhibitor phenylmethylsulphonyl fluoride (PMSF) were purchased from Calbiochem Corp. (San Diego, CA, USA). SB203580 was dissolved in distilled water and PD98059, AG490 and SP600125 were dissolved in dimethyl sulphoxide (DMSO).

Cell culture

BEAS-2B is an adenovirus 12-SV40 virus hybrid (Ad12SV40)-transformed human epithelial cell line, which was isolated from normal human bronchial epithelium obtained from autopsy of non-cancerous individuals. It was purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). The cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (Gibco laboratories, New York, NY, USA) supplemented with 10% defined fetal bovine serum (FBS) (Gibco) in 5% CO2−95% humidified air at 37°C. Before each experiment, 6 × 105 and 1·5 × 105 of BEAS-2B cells were seeded and cultured in six- and 24-well plates, respectively, overnight to a confluence monolayer.

Endotoxin-free solutions

Cell culture medium was purchased from Gibco, free of detectable lipopolysaccharide (LPS) ( < 0·1 EU/ml). All other solutions were prepared using pyrogen-free water and sterile polypropylene plasticware. No solution contained detectable LPS, as determined by the Limulus amoebocyte lyase assay (sensitivity limit 12 pg/ml; Associates of Cape Cod, MA, USA).

Assay of MCP-1 by enzyme-linked immunosorbent assay (ELISA)

MCP-1 concentration in BEAS-2B cell culture supernatants was measured using the BD OptEIA™ ELISA kit (BD Biosciences Pharmingen).

Reverse transcription–polymerase chain reaction (RT–PCR)

Total RNA was extracted using Tri-Reagent (Molecular Research Center Inc., Cincinnati, OH, USA). Extracted RNA was reverse-transcribed into first-strand complementary DNA using the first-strand cDNA synthesis kit (Amersham Biosciences Corp. (Piscataway, NJ, USA). Polymerase chain reaction (PCR) was performed in a reaction mixture containing 3 mm MgCl2, 200 µm dNTPs, 1 unit of AmpliTaq Gold DNA polymerase (Perkin Elmer, Wellesley, MA, USA) and 50 pmol of 5′- and 3′ primers (Invitrogen, Foster City, CA, USA) in PCR reaction buffer (1 min each at 94°C, 60°C and 72°C) for 18 cycles for β-actin, after an initial 12 min of denaturation at 94°C. Thirty cycles (2 min each at 94°C, 56°C and 72°C) after an initial 12 min of denaturation at 94°C was adopted for MCP-1. All RT–PCR were performed in the linear range of the PCR reaction according to the preliminary experiments. PCR primers were as follows: MCP-1 sense, 5′-AATGCCCCAGT CACCTGCTGTTAT-3′ and anti-sense, 5′-GCAATTTC CCCAAGTCTCTGTATC-3′, yielding a 427-base pairs (bp) product; β-actin sense, 5′-AGCGGGAAATCGTGCGTG-3′ and anti-sense, 5′-CAGGGTACATGGTGGTGCC-3′, yielding a 300-bp product [4]. After the amplification reaction using PTC-200 DNA Engine™ (MJ Research Inc., Waltham, MA, USA), PCR products were electrophoresed on 2% agarose gel in tris-acetate-EDTA (TAE) buffer (pH 8·0) and stained with ethidium bromide. The electrophorectic bands were documented with Gene Genius Gel Documentation System (Syngene Inc., Cambridge, UK).

5-bromo-2-deoxyuridine (BrdU) incorporation cell proliferation ELISA

The cytotoxic effect of various inhibitors on BEAS-2B cells was quantified by a colorimetric BrdU cell proliferation ELISA kit (Roche Applied Science, Penzberg, Germany). Briefly, BEAS-2B cells (3 × 104/well) were seeded onto a 96-well plate. Various inhibitors with serial concentrations were added to the cells. After 24-h incubation, BrdU (10 µM) was added to each well and incubated for 2 h. Proliferating cells took up BrdU and incorporated into DNA during DNA synthesis. The cells were fixed and denatured, and BrdU-labelled DNA was detected by peroxidase conjugated anti-BrdU antibody. After addition of tetramethylbenzidine (TMB) substrate, the proliferating cells were quantified by measuring absorbance at 450 nm with the reference wavelength at 690 nnm. The results were expressed as percentage proliferation relative to the untreated control cells.

Western blot analysis

BEAS-2B cells (1 × 107) after the preceding treatment were washed with phosphate-buffered saline (PBS) and lysed in 0·3 ml lysis buffer (20 mm Tris-HCl (pH 7·5), 150 mM NaCl, 1 mM Na2 ethylenediamine tetraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA), 1% Triton, 2·5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate and 1 mM Na3VO4, 1 µg/ml leupeptin) (Cell Signalling Technology Inc., Danvers, MA, USA), added to 1 mM PMSF. Cell debris was removed by centrifugation at 14 000 g for 15 min, and the supernatants were boiled in Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) for 5 min. An equal amount of proteins was subjected to sodium dodecyl sulphate-10% polyacrylamide gel electrophoresis (SDS-PAGE) before blotting onto a polyvinyl difluoride (PVDF) membrane (Amersham Biosciences Ltd, Amersham, Bucks, UK). The membrane was blocked with 5% skimmed milk in Tris-buffered saline with 0·05% Tween 20, pH 7·6 for 1 h at room temperature, and probed with primary mouse anti-human antibodies at 4°C overnight. After washing, the membrane was incubated with secondary goat anti-mouse antibody coupled to horseradish peroxidase (Amersham Biosciences) for 1 h at room temperature. Antibody–antigen complexes were then detected using the ECL Plus™ chemiluminescent detection system according to the manufacturer’s instructions (Amersham Biosciences).

Statistical analysis

All data were expressed as mean ± s.e.m. Differences between groups were assessed by one-way anova analysis and Student’s t-test. A probability of P < 0·05 was considered as a significant difference. All analyses were performed using the Statistical Package for the Social Sciences (SPSS) statistical software for Windows, version 10·1.4 (SPSS Inc., Chicago, IL, USA).

Results

Up-regulation and kinetics of MCP-1 protein secretion and gene expression of BEAS-2B cells upon IL-4 and IL-13 stimulation

Figure 1a,b shows the dose and time responses of IL-4- and IL-13-induced release of MCP-1. Resting BEAS-2B cells constitutively produced and secreted certain amounts of MCP-1 from 4 to 48 h. There was no significant dose-dependent change of MCP-1 level in culture supernatants of BEAS-2B cells after 4 h of stimulation with either cytokines. However, after 24 and 48 h of stimulation, both IL-4 and IL-13 could significantly up-regulate MCP-1 release in a dose- and time-dependent manner. The stimulation was optimal at concentration of 20 ng/ml for both cytokines, therefore this dose was used in subsequent experiments. As shown in Fig. 1c, IL-4 and IL-13 (20 ng/ml) could induce independently an approximately twofold increase of MCP-1 release from BEAS-2B cells. IL-4 (20 ng/ml) could increase the concentration of MCP-1 from 621 ± 41 pg/ml to 1270 ± 23 pg/ml while IL-13 (20 ng/ml) could increase the concentration of MCP-1 from 647 ± 56 pg/ml to 1274 ± 97 pg/ml in culture supernatants of BEAS-2B cells after 24 h incubation (both P < 0·05). However, both cytokines were much weaker stimulators when compared with tumour necrosis factor (TNF)-α at 20 ng/ml (from 780 ± 48 pg/ml to 13 430 ± 1996 pg/ml) and interferon (IFN)-γ at 20 ng/ml (from 682 ± 47 pg/ml to 12 590 ± 1635 pg/ml). A combination of both IL-4 and IL-13 did not synergistically up-regulate MCP-1 release from BEAS-2B cells. In concurrence with the protein release, constitutive MCP-1 mRNA expression was also observed in untreated BEAS-2B cells (Fig. 2). Both IL-4 and IL-13 could up-regulate not only the protein release but also the gene expression of MCP-1 in BEAS-2B cells continuously from 3 h to 24 h (Fig. 2a,b). The combination of IL-4 and IL-13 stimulation could enhance further the gene expression when compared with the effect of a single cytokine (Fig. 2c). These results confirmed an increased production of MCP-1 from IL-4 and IL-13-stimulated BEAS-2B cells.

Fig. 1
Dose and time responses of interleukin (IL)-4- and IL-3-induced release of monoattractant protein (MCP)-1 from BEAS-2B cells. Confluent BEAS-2B cells (1·5× 105/well) were cultured with or without (a) IL-4 (10–80 ng/ml), (b) IL-3 ...
Fig. 2
Up-regulation of monoattractant protein (MCP-1) gene expression by interleukin (IL)-4 and IL-13 in BEAS-2B cells. BEAS-2B cells (6 × 105/well) were treated with or without (a) IL-4 (20 ng/ml), (b) IL-13 (20 ng/ml) for different time intervals ...

Inhibition of IL-4 and IL-13-mediated MCP-1 release by anti-human IL-4 receptor antibody

Figure 3 shows a dose kinetic of an anti-IL-4R antibody on the neutralization of cell surface IL-4 and IL-13 receptor-mediated MCP-1 release from BEAS-2B cells. The antibody could significantly suppress IL-4 and IL-13-mediated release of MCP-1 in a dose-dependent manner at concentrations from 50 to 10 000 ng/ml (Fig. 3a,b) (all P < 0·05). These results indicate that IL-4 and IL-13 could directly mediate MCP-1 release from BEAS-2B cells through the binding and activation of IL-4 and IL-13 receptors.

Fig. 3
Inhibition of interleukin (IL)-4- and IL-13-mediated monoattractant protein (MCP-1) release by anti-IL-4R antibody. Confluent BEAS-2B cells (1·5 × 105/well) were pretreated with various concentrations of anti-IL-4 receptor antibody for ...

Activation of MAPKs and JAK-2 upon IL-4 and IL-13 stimulation

Figure 4 shows that both IL-4 and IL-13 could activate p38 MAPK, ERK, JAK-2, STAT-6 and STAT-1 but not JNK signalling pathways in BEAS-2B cells. Western blot analysis showed that phosphorylation levels of p38 MAPK and ERK in cells stimulated with IL-4 (20 ng/ml) and IL-13 (20 ng/ml) were elevated after 5 min. Peak phosphorylation levels of p38 MAPK and ERK occurred after 10 min and decreased thereafter (Fig. 4a,b). As shown in Fig. 4d, IL-4 and IL-13 could activate JAK-2. The phosphorylation level of JAK-2 increased at 5 min and remained elevated for 30 min in IL-4-treated cells, while in IL-13-treated cells the level declined to near baseline after 30 min. Other signalling molecules including STAT-1 and STAT-6 for IL-4 and IL-13 receptor-signalling were also being activated with increased phosphorylation levels observed (Fig. 4e,f).Fig. 4c shows that neither IL-4 nor IL-13 could activate the JNK signalling pathway in BEAS-2B cells.

Fig. 4
Effects of interleukin (IL)-4 and IL-13 on the activation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), Janus kinase-2 (JAK-2) and signal transducer and activator of transcription ...

Cytotoxicity of different kinase inhibitors

Figure 5 illustrates the optimal concentrations of different kinase inhibitors with minimum cytotoxicity. Neither p38 MAPK inhibitor SB203580 (Fig. 5a) nor ERK inhibitor PD98059 (Fig. 5b) exhibited significant cytotoxicity on BEAS-2B cells ( < 20% inhibition) at concentrations up to 20 µM. As shown in Fig. 5c, JNK inhibitor SP600125 could significantly suppress BEAS-2B cell proliferation in a dose-dependent manner from 5 µM to 20 µM after 24 h. There was no significant cytotoxic effect observed on JAK-2 inhibitor AG490 at concentrations from 1 to 5 µM (Fig. 5d).

Fig. 5
Cytotoxic effect of SB203580, PD98059, AG490 and dimethylsulphoxide (DMSO) on BEAS-2B cells. Confluent BEAS-2B cells (3 × 104/well) were treated with (a) SB203580, (b) PD98059, (c) SP600125, (d) AG490 and (e) DMSO solvent vehicle at various concentrations ...

Inhibition of IL-4 and IL-13-stimulated MCP-1 release by different kinase inhibitors

Figure 6 shows a dose kinetic of different inhibitors, including SB203580, PD98059, SP600125 and AG490 on suppression of cytokine-induced MCP-1 release from BEAS-2B cells. All the inhibitors could suppress IL-4- and IL-13-induced release of MCP-1 in a dose-dependent manner (Fig. 6a–d). SB203580, PD98059 and AG490 could suppress significantly and almost completely the cytokine-induced MCP-1 release from activated BEAS-2B cells at concentrations of 10 µM, 20 µM and 5 µM, respectively (all P < 0·05). At or below these concentrations, the inhibitors inhibited specific signalling pathways without any significant cytotoxicity to the cells, as shown in Fig. 5. This inhibitory effect was not due to the presence of solvent vehicle DMSO as there was no significant inhibition of MCP-1 release by DMSO at all the corresponding concentrations of inhibitors being used (Fig. 6e). As shown in Fig. 6c, SP600125 could significantly inhibit MCP-1 release from activated BEAS-2B cells at concentrations of 5–20 µM. However, at these concentrations the inhibitor would significantly inhibit the growth and proliferation of the cells from 30% to 80% (Fig. 5c). Therefore, the suppression of MCP-1 release by SP600125 may not be due to specific inhibition of JNK signalling pathway but to the cytotoxic effect.

Fig. 6
Effect of SB203580, PD98059, AG490 and dimethylsulphoxide (DMSO) on interleukin (IL)-4- and IL-13-induced release of monoattractant protein (MCP-1) from BEAS-2B cells. Confluent BEAS-2B cells (1.5 × 105/well) were pretreated with various concentrations ...

Effect of SB203580, PD98059 and AG490 on gene expression of MCP-1

As shown from Figs 46, MCP-1 release from BEAS-2B cells upon IL-4 and IL-13 stimulation was regulated by p38 MAPK, ERK and JAK-2 signalling pathways. Figure 7 illustrates further the role of these signalling molecules on the gene expression of MCP-1. In Fig. 7a,b, SB203580 and PD98059 have no effect on the IL-4- and IL-13-induced MCP-1 mRNA expression of BEAS-2B cells, whereas AG490 could suppress protein secretion and also mRNA expression of MCP-1 (Fig. 7c).

Fig. 7
Effect of SB203580, PD98059, AG490 and dimethylsulphoxide (DMSO) on interleukin (IL)-4- and IL-13-induced gene expression of monoattractant protein (MCP-1) in BEAS-2B cells. BEAS-2B cells (6 × 105/well) were pretreated with or without (a) SB203580 ...

Discussion

In the present study we used the transformed human airway epithelial cell line instead of primary epithelial cells, because it is a homogenesis clone and acts as a well-established in vitro experimental model of human airway epithelium with reproducible results. The human bronchial epithelium is one of the major sources of chemokines in allergic asthma [3]. Our recent findings support that bronchial epithelial cells are the principle effector cells for the production of several chemokines, including IL-8 and MCP-1 in an in vitro model of the eosinophil–bronchial epithelial cell co-culture system [4]. Co-culture of bronchial epithelial cells with eosinophils could significantly enhance chemokine release through intercellular interaction, which is eosinophil-dependent [4]. Wills-Karp et al. [9] and Gonzalo et al. [8] demonstrated that IL-13-induced BHR is independent of conventional allergic mediators, such as IgE and eosinophils. IL-4 and IL-13 can induce chemokine production in bronchial epithelial cells through direct interaction with their cell surface receptors [2,23]. A number of studies have shown that IL-4 and IL-13 can activate bronchial epithelial cells by directly enhancing the production of a number of C-C and C-X-C chemokines, including eotaxin-1/2/3 [27,28], regulated-upon-activation normal T-cell expressed and secreted (RANTES), MCP-3/4, IL-8, growth-related oncogene (GRO)-α[27] and recently, MIP-3α[25]. In addition to the above chemokines, we have also assessed the effect of IL-4 and IL-13 on the expression of other chemokines such as IP-10, MCP-1 and MIG by using antibody-based human cytokine protein array and human chemokine cytometric bead array (CBA) methods, as described in our previous publications [4,29]. However, concentrations of IP-10 and MIG were undetectable in culture supernatants from both untreated and cytokine-treated BEAS-2B cells, while the concentration of MCP-1 was found to be up-regulated by IL-4 and IL-13.

MCP-1 production can be increased dramatically in response to diverse inflammatory stimuli, typically by IFN-γ, TNF-α, IL-1β and bacterial LPS [4,6,7,9]. However, no report has demonstrated the direct effect of IL-4 and IL-13 on MCP-1 expression in bronchial epithelial cells. Our stimulatory and blocking experiments demonstrated and confirmed the direct effect of these two cytokines on MCP-1 mRNA synthesis and protein secretion in human bronchial epithelial cells (Figs 13). These findings are particularly relevant for bronchial allergic asthma. Increased expression and concentration of MCP-1 have been reported in the bronchial tissue and BALF of asthmatic patients, respectively [6,7]. However, our recent clinical studies showed that plasma concentration of MCP-1 in adult asthmatic patients were not increased significantly compared to normal subjects [30]. These findings may suggest a bronchial-specific elevation of the MCP-1 level, which exerts a localized effect on bronchial inflammation. A previous study has demonstrated induced expression and secretion of IL-4 and IL-13 into BALF of allergic asthmatic patients after allergen challenge [13]. Activated alveolar macrophages have been proved to produce both IL-4 and IL-13 in two recent reports [17,18]. From our present findings, we propose that an increased concentration of MCP-1 in lung tissues of asthmatic patients is the result of induced secretion of IL-4 and IL-13 from alveolar macrophages after allergen challenge. We shall investigate further the correlation between IL-4, IL-13 and MCP-1 concentrations in BALF of asthmatic patients in our clinical studies.

IL-4 and IL-13 share common receptor signalling pathways which mediate similar immune regulatory functions, including enhanced expression of MHC class II genes [20], IgE switching and production [2,20], increased expression of adhesion molecules [20] and induction of chemokines and cytokines [2428,31]. Transcription factor STAT-6 has been well reported as the primary signalling protein for most of the activities of IL-4 and IL-13 [11,23,32]. Our present intracellular mechanistic study was aimed at demonstrating the activation of STAT-6 by IL-4 and IL-13 in bronchial epithelial cells (Fig. 4e). IL-4 has been found to be more potent than IL-13 in activating STAT-6 at the same concentration [27]. This concurs with our results, that IL-4 activated STAT-6 and reached optimal levels much faster than the effect of IL-13 (Fig. 4e). Furthermore, a recent study has demonstrated activation of STAT-1 by IL-4 and IL-13 in bronchial epithelial cells [33]. This has also been our finding (Fig. 4f), although we do not have data to suggest whether STAT-6 or STAT-1 or both are essential for MCP-1 expression in bronchial epithelial cells.

Several recent studies have reported that the involvement of other signalling molecules, such as MAP kinases, are also crucial for IL-4 and IL-13 activities regarding chemokine and cytokine production in different cell types [2426,32]. However, only a few studies examining the activation of MAP kinases by IL-4 and IL-13 in bronchial epithelial cells have been published. Only one study showed that IL-4 and IL-13 could induce chemokine MIP-3α production through the activation of p38 MAPK and ERK [25]. Using Western blotting, we also confirmed these results in human bronchial epithelial cells with MCP-1 release in vitro (Fig. 4a–c). In our inhibition experiments we used small molecule inhibitors SB203580, PD98059 and SP600125 to confirm the direct involvement of p38 MAPK, ERK and JNK signalling proteins, respectively, in cytokine-induced MCP-1 expression and secretion. We demonstrated that the secretion of MCP-1 was mediated by the intracellular p38 MAPK and ERK but not JNK activities (Fig. 6a–c). Neither p38 MAPK nor ERK, however, could regulate the gene expression of MCP-1 in BEAS-2B cells (Fig. 7a.b). It was possible that protein synthesis of MCP-1 was inhibited, although the expression of mRNA was not affected. These results suggest that p38 MAPK and ERK may regulate the protein synthesis or mediate the secretory process of preformed MCP-1.

The IL-4Rα–IL-13Rα1 complex is also expected to signal through JAK-1, JAK-2 and JAK-3, initiating the phosphorylation of a variety of signalling cascades, including STAT-6 and STAT-1 [23,32]. In the presence of inhibitor for JAK-2 (AG490), we have demonstrated for the first time that JAK-2 is activated and involved in IL-4- and IL-13-stimulated MCP-1 release in bronchial epithelial cells (Figs 4d, 6d). Phosphorylation of STAT-6 or STAT-1 may be mediated by JAK-2. Gene expression of MCP-1 in BEAS-2B cells is also regulated by JAK-2, as shown in Fig. 7c. However, whether gene transcription of MCP-1 in bronchial epithelial cells is mediated by a JAK2-STAT6- or JAK2-STAT1-dependent pathway is unknown and needs further investigation.

In conclusion, we have demonstrated for the first time that IL-4 and IL-13 could up-regulate differentially mRNA and protein expressions of MCP-1 in human bronchial epithelial cells through the activation of p38 MAPK, ERK and JAK-2. Our results may provide evidence of a fundamental mechanism for the immunopathology of IL-4, IL-13 and MCP-1 in BHR and facilitating the future development of more effective therapeutic agents targeting intracellular signalling molecules, including MAP kinases and transcription factors for allergic asthma.

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