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Published ahead of print on August 7, 2008, doi:10.1165/rcmb.2008-0162OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 40, pp. 159-167, 2009
© 2009 American Thoracic Society
DOI: 10.1165/rcmb.2008-0162OC

Interleukin-13 Augments Bronchial Smooth Muscle Contractility with an Up-Regulation of RhoA Protein

Yoshihiko Chiba1, Shuji Nakazawa1, Michiko Todoroki1, Koji Shinozaki1, Hiroyasu Sakai1 and Miwa Misawa1

1 Department of Pharmacology, School of Pharmacy, Hoshi University, Tokyo, Japan

Correspondence and requests for reprints should be addressed to Yoshihiko Chiba, Ph.D., Department of Pharmacology, School of Pharmacy, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan. E-mail: chiba{at}hoshi.ac.jp


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Interleukin-13 (IL-13) is one of the central mediators for development of airway hyperresponsiveness in asthma. However, its effect on bronchial smooth muscle (BSM) is not well known. Recent studies revealed an involvement of RhoA/Rho-kinase in BSM contraction, and this pathway has now been proposed as a new target for asthma therapy. To elucidate the role of IL-13 on the induction of BSM hyperresponsiveness, effects of IL-13 on contractility and RhoA expression in BSMs were investigated. Male BALB/c mice were sensitized and repeatedly challenged with ovalbumin antigen. In the repeatedly antigen-challenged mice, marked airway inflammation and BSM hyperresponsiveness with an up-regulation of IL-13 in bronchoalveolar lavage fluids were observed. In cultured human BSM cells, IL-13 caused an up-regulation of RhoA. The IL-13–induced up-regulation of RhoA was inhibited by leflunomide, an inhibitor of signal transducer and activator of transcription 6 (STAT6). In isolated BSM tissues of naive mice, the contractility was significantly enhanced by organ culture in the presence of IL-13. Moreover, in vivo treatment of airways with IL-13 by intranasal instillation caused a BSM hyperresponsiveness with an up-regulation of RhoA in naive mice. These findings suggest that IL-13/STAT6 signaling is critical for development of antigen-induced BSM hyperresponsiveness and that agents that specifically inhibit this pathway in BSM may provide a novel strategy for the treatment of asthma.

Key Words: asthma • airway hyperresponsiveness • bronchial smooth muscle • interleukin-13 • RhoA



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Our results suggest that IL-13/STAT6 signaling is critical in development of antigen-induced bronchial smooth muscle hyperresponsiveness, and that agents which specifically inhibit this pathway may provide a novel strategy for the treatment of allergic bronchial asthma.

 
Increased airway narrowing in response to nonspecific stimuli is a characteristic feature of human obstructive diseases, including bronchial asthma. This abnormality is an important symptom of the disease, although the pathophysiologic variations leading to the hyperresponsiveness are unclear now. Several mechanisms have been suggested to explain the airway hyperresponsiveness (AHR), such as alterations in the neural control of airway smooth muscle (1), increased mucosal secretions (2), and mechanical factors related to remodeling of the airways (3). In addition, it has also been suggested that one of the factors that contribute to the exaggerated airway narrowing in individuals with asthma is an abnormality of the properties of airway smooth muscle (4, 5). Rapid relief from airway limitation in patients with asthma by β-stimulant inhalation may also suggest an involvement of augmented airway smooth muscle contraction in the airway obstruction. Thus, it may be important for development of asthma therapy to understand changes in the contractile signaling of airway smooth muscle cells associated with the disease.

Smooth muscle contraction, including that in the airways, is mainly regulated by an increase in cytosolic Ca2+ concentration in myocytes. Recently, additional mechanisms have been suggested in agonist-induced smooth muscle contraction by studies in which the simultaneous measurements of force development and intracellular Ca2+ concentration, and chemically permeabilized preparations in various types of smooth muscles, were used. It has been demonstrated that agonist stimulation increases myofilament Ca2+ sensitivity in permeabilized smooth muscles of the rat coronary artery (6), guinea pig vas deferens (7), canine trachea (8), and rat bronchus (9). Although the detailed mechanism is not fully understood, a participation of RhoA, a monomeric GTP-binding protein, in the agonist-induced Ca2+ sensitization has been suggested by many investigators (10). Moreover, an augmented RhoA-mediated Ca2+ sensitization in smooth muscle contraction has been reported in experimental animal models of diseases such as hypertension (11–13), and coronary (6, 14, 15) and cerebral (16–18) vasospasms. It is thus possible that RhoA-mediated signaling is the key for understanding the abnormal contraction of diseased smooth muscles.

We have previously reported that the RhoA-mediated Ca2+ sensitization of bronchial smooth muscle (BSM) contraction is augmented in experimental asthma models of rats (19) and mice (20). An up-regulation of RhoA has also been demonstrated in BSMs of these animal models of allergic bronchial asthma (19–21). However, the mechanism(s) responsible for the up-regulation of RhoA has yet to be elucidated. We show here that interleukin (IL)-13, one of the central mediators of allergic asthma (22–30), can directly up-regulate RhoA in BSM cells and induce an augmented contraction.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Animals
Male BALB/c mice were purchased from the Charles River Japan, Inc. (Kanagawa, Japan) and housed in a pathogen-free facility. All animal experiments were approved by the Animal Care Committee of the Hoshi University (Tokyo, Japan).

Sensitization and Antigenic Challenge
Preparation of a murine model of allergic bronchial asthma, which has an in vivo AHR (31), was performed as described previously (20). In brief, BALBc mice (8 wk of age) were actively sensitized by intraperitoneal injections of 8 µg ovalbumin (OA; Seikagaku Co., Tokyo, Japan) with 2 mg Imject Alum (Pierce Biotechnology, Inc., Rockford, IL) on Day 0 and Day 5. The sensitized mice were challenged with aerosolized OA-saline solution (5 mg/ml) for 30 minutes on Days 12, 16, and 20. A control group of mice received the same immunization procedure, but inhaled saline aerosol instead of OA challenge. The aerosol was generated with an ultrasonic nebulizer (Nihon Kohden, Tokyo, Japan) and introduced to a Plexiglas chamber box (130 x 200 mm, 100 mm height) in which the mice were placed. Twenty-four hours after the last OA challenge, mice were killed by exsanguination from the abdominal aorta under urethane (1.6 g/kg, intraperitoneally; Sigma, St. Louis, MO) anesthesia.

Histologic Examination
Airways below the main bronchi were fixed in 10% formaldehyde and embedded in Paraplast Plus paraffin (Fisher Healthcare, Houston, TX). Four-micrometer sections were obtained from blocks and mounted on silane-coated glass slides, deparaffinized with xylene and graded ethanol, and processed for hematoxylin and eosin staining.

Analyses of Bronchoalveolar Lavage Fluids
After the exsanguinations, the chest of each animal was opened and a 20-gauge blunt needle was tied into the proximal trachea. Bronchoalveolar lavage (BAL) fluid was obtained by intratracheal instillation of 1 ml/animal of phosphate-buffered saline (PBS; pH 7.5, room temperature) into the lung while it was kept located within the thoracic cavity. The lavage was reinfused into the lung twice before final collection. BAL cells were isolated by centrifugation at 500 x g. The resultant pellet was resuspended in 500 µl of 10% formaldehyde and incubated for 10 minutes. Then the cells were washed by PBS and resuspended in 500 µl of PBS. An aliquot of BAL cell suspension was used for cell counts with a hemocytometer. Differential cell count was also performed under Diff-Quik staining as previously described (32). The resultant supernatants of the lavage fluids were subjected to cytokine analyses. Various cytokines/chemokines expression profile and the level of IL-13 were measured by RayBio Mouse Cytokine Array I (RayBiotech, Inc., Norcross, GA) and an IL-13 ELISA system (R&D Systems, Minneapolis, MN), respectively, according to the manufacturers' instructions.

Determination of BSM Responsiveness
Mice were killed by exsanguination from the abdominal aorta under urethane (1.6 g/kg, intraperitoneally) anesthesia, and the airway tissues under the larynx to lungs were immediately removed. About 3 mm length of the left main bronchus (~ 0.5 mm diameter) was isolated, and epithelium was removed by gently rubbing with sharp tweezers (19, 20). Removal of the epithelium was confirmed histologically in our preliminary study. The resultant tissue ring preparation was then suspended in a 5-ml organ bath by two stainless-steel wires (0.2 mm diameter) passed through the lumen. For all tissues, one end was fixed to the bottom of the organ bath while the other was connected to a force-displacement transducer (TB-612T; Nihon Kohden) for the measurement of isometric force. A resting tension of 0.5 g was applied. The buffer solution contained modified Krebs-Henseleit solution with the following composition (in mM); NaCl 118.0, KCl 4.7, CaCl2 2.5, MgSO4 1.2, NaHCO3 25.0, KH2PO4 1.2, and glucose 10.0. The buffer solution was maintained at 37°C and oxygenated with 95% O2–5% CO2. After the equilibration period, the concentration-response curve to ACh (10–7–10–3 M in final concentration) was constructed cumulatively. A higher concentration of ACh was successively added after attainment of a plateau response to the previous concentration. In some tissue preparations, a Rho-kinase inhibitor Y-27632 (11) was administered cumulatively when a plateau response had been reached by 10–3 M of ACh. In another series of experiment, isotonic K+ solution (10–90 mM in final concentration) was cumulatively administered in the presence of atropine and indomethacin (both 10–6 M) to determine the BSM responsiveness to high K+-depolarization.

Tissue Culture of Mouse Bronchi
The left main bronchi of naive mice were isolated, and epithelium was removed as described above. The isolated tissue preparations were immediately washed by Dulbecco's modified Eagle's medium (DMEM) (Invitrogen Corp., Grand Island, NY) and were then cultured in DMEM containing 50 U/ml penicillin and 50 µg/ml streptomycin (Invitrogen) at 37°C under a 5% CO2 atmosphere in the presence or absence of recombinant mouse IL-13 (100 ng/ml; PeproTech EC, Ltd., London, UK) for 12 hours.

Intranasal Administration of IL-13
Recombinant mouse IL-13 was intranasally administered by the method previously described (32–34) with minor modification. In brief, mice were anesthetized with Avertin (2.5% 2,2,2-tribromoethanol, 0.1–0.15 ml/animal; Sigma) and allowed to breathe spontaneously. Sterile PBS (20 µl; control) or recombinant mouse IL-13 (3, 10, and 30 ng in 20 µl PBS) was intranasally instilled into the airways of each animal. Under these conditions, the reagent was successfully distributed in wide area of airways by the intranasal administration (34). The intranasal administration was repeated three times in total every 4 days. Each mouse was killed 24 hours after the last IL-13 administration.

Protein Samples of Bronchial Tissues
Protein samples of BSM tissues were prepared as previously (20). In brief, the airway tissues below the main bronchi to lungs were removed and immediately soaked in ice-cold, oxygenated Krebs-Henseleit solution. The airways were carefully cleaned of adhering connective tissues, blood vessels, and lung parenchyma under a stereomicroscopy. The epithelium was removed as much as possible by gently rubbing with keen-edged tweezers (20). Then the bronchial tissue (containing the main and intrapulmonary bronchi) segments were quickly frozen with liquid nitrogen, and the tissue was crushed to pieces by Cryopress (CP-100W, 15 s x 3; Microtec Co., Ltd., Chiba, Japan). The tissue powder was homogenized in ice-cold tris(hydroxymethyl)aminomethane (Tris, 10 mM; pH 7.5) buffer containing 5 mM MgCl2, 2 mM EGTA, 250 mM sucrose, 1 mM dithiothreitol, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 20 µg/ml leupeptin, 20 µg/ml aprotinin, 1% Triton X-100 and 1% sodium cholate. The tissue homogenate was then centrifuged (3,000 x g, 4°C for 15 min) and the resultant supernatant was stored at –85°C until use.

RNA Extraction from Bronchial Tissues
Total RNA of the mouse BSM tissue was isolated from an aliquot of the tissue powder as described above with a one-step guanidium-phenol-chloroform extraction procedure using 1 ml of TRI Reagent (Sigma) according to the manufacturer's instructions.

Cell Culture and Sample Collection
Normal human BSM cells (hBSMCs; Cambrex Bio Science Walkersville, Inc., Walkersville, MD) were maintained in SmBM medium (Cambrex) supplemented with 5% fetal bovine serum, 0.5 ng/ml human epidermal growth factor (hEGF), 5 µg/ml insulin, 2 ng/ml human fibroblast growth factor-basic (hFGF-b), 50 µg/ml gentamicin, and 50 ng/ml amphotericin B. Cells were maintained at 37°C in a humidified atmosphere (5% CO2), fed every 48 to 72 hours, and passaged when cells reached 90 to 95% confluence. Then the hBSMCs (passages 7–9) were seeded in 6-well plates (Becton Dickinson Labware, Franklin Lakes, NJ) and 8-well chamber slides (Nalge Nunc International, Naperville, IL) at a density of 3,500 cells/cm2 and, when 80 to 85% confluence was observed, cells were cultured without serum for 24 hours before addition of recombinant human IL-13 (PeproTech EC). At the indicated time after the IL-13 treatment, cells were washed with PBS, immediately collected and disrupted with 1x SDS sample buffer (250 µl/well), and used for Western blot analyses. For RNA extraction, TRI Reagent (1 ml/well) was added directly to the washed cells.

Western Blot Analyses
Protein samples were subjected to 15% (for RhoA) or 7.5% SDS-PAGE (for the others) and the proteins were then electrophoretically transferred to a PVDF membrane. After blocking with 3% skim milk (for RhoA) or 1% BlockAce (Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan) (for the others), the PVDF membrane was incubated with the primary antibody. The primary antibodies used in the present study were polyclonal rabbit anti-RhoA (1:2,500 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-STAT6 (1:1,000 dilution; Santa Cruz Biotechnology), anti–phospho-STAT6 (1:1,000 dilution; Santa Cruz Biotechnology), anti-STAT3 (1:2,500 dilution; BD Biosciences, San Jose, CA), and anti–phospho-STAT3 (1:500 dilution; BD Biosciences) antibodies. Then the membrane was incubated with horseradish peroxidase–conjugated donkey anti-rabbit IgG or sheep anti-mouse IgG (1:2,500 dilution; Amersham Biosciences, Co., Piscataway, NJ), detected by an enhanced chemiluminescent system (Amersham Biosciences) and analyzed by a densitometry system. Detection of housekeeping gene was also performed on the same membrane by using monoclonal mouse anti-GAPDH (1:10,000 dilution; Chemicon International, Inc., Temecula, CA) to confirm the same amount of proteins loaded.

Immunocytochemistry
The hBSMCs cultured on the 8-well chamber slides were subjected to immunocytochemistry. In brief, cells were fixed with 10% formaldehyde in PBS (10 min) and permeabilized by incubation with 0.5% Triton X-100 in PBS for 10 minutes. Endogenous peroxidase activity was blocked by incubation with 0.3% H2O2 in 100% methanol for 10 minutes. After blocking with 5% skim milk in PBS for 1 hour, the cells were incubated with anti-STAT6 or anti–phospho-STAT6 antibody (1:100 dilution in 1% skim milk-PBS, respectively) for 1 hour. After washing with PBS, the cells were incubated with anti-rabbit IgG (Vector Laboratories, Inc., Burlingame, CA) for 1 hour. Detection was performed by using an ABC reagent (Vector Laboratories) with 3,3-diaminobenzidine (DAB) (Sigma FAST; Sigma) according to the manufacturers' instructions. Counterstaining was also performed by hematoxylin (Vector Laboratories) before examination by light microscopy.

RT-PCR
cDNAs were prepared from the total RNA (1.0 µg) by using RevertAid First Strand cDNA Synthesis Kit (Fermentas, Inc., Hanover, MD) in a total volume of 20 µl reaction buffer containing 50 mM Tris-HCl, pH 8.3, 50 mM KCl, 4 mM MgCl2, 1 mM dNTP mixture, 1 U/µl RNase inhibitor, 10 ng/µl random 6 mers, and 10 U/µl M-MuLV reverse transcriptase. The reaction mixture was incubated for 10 minutes at 25°C followed by 60 minutes at 42°C to initiate the synthesis of cDNAs. Reverse transcriptase was inactivated at 70°C for 5 minutes. Then the RT reaction mixture (1 µl) was subjected to PCR (0.1 µM forward and reverse primers, 0.025 U/l Taq DNA polymerase, 2 mM MgCl2, 0.2 mM dNTPs) in a final volume of 11 µl. The PCR primer sets used are shown in Tables 1 (for human genes) and 2 (for mouse genes), which were designed from published sequences. The thermal cycle profile used was (1) denaturing for 15 seconds at 95°C, (2) annealing primers for 15 seconds at 55°C, (3) extending the primers for 60 seconds at 72°C, and the reaction was run for 30 cycles. The PCR products were subjected to electrophoresis on 1.2% agarose gel and visualized by ethidium bromide staining.


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  		TABLE 1. PRIMER SEQUENCES FOR RT-PCR USED IN THE PRESENT STUDY (FOR HUMAN GENES)

 

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  		TABLE 2. PRIMER SEQUENCES FOR RT-PCR USED IN THE PRESENT STUDY (FOR MOUSE GENES)

 
Statistical Analyses
All the data are expressed as the mean ± SE. Statistical significance of difference was determined by unpaired Student's t test, Bonferroni/Dunn's test, or two-way ANOVA.


   RESULTS
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 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Characteristics of Murine Model of Allergic Bronchial Asthma
In the present study, we used our well-established murine model of allergic bronchial asthma (20, 32–34). Histochemical examination revealed a marked lung inflammation (i.e., infiltration of inflammatory cells such as eosinophils and lymphocytes) 24 hours after the last antigen challenge. The inflammation was also observed in the main bronchial area that was used for the current functional study: histochemical examination of main bronchial tissues revealed a distinct infiltration of inflammatory cells into subepithelial and adventitial layers (Figure 1A). Diff-Quik staining of the tissues revealed that most of the infiltrated cells were eosinophils (data not shown). The airway inflammation was further confirmed by an increase in inflammatory cells in BAL fluids (Figure 1B). In addition to the airway inflammation, BSM responsiveness to acetylcholine (ACh) of the repeatedly antigen-challenged mice was significantly augmented as compared with that of the sensitized control group: the maximal contraction induced by ACh (Emax; g) of the OA-challenged group (0.61 ± 0.07) was significantly greater than that of the control group (0.42 ± 0.04, P < 0.05), whereas no significant change in the 50% effective concentration (EC50; –logM) was observed (5.07 ± 0.15, and 5.14 ± 0.25, respectively). On the other hand, the responsiveness to high K+ depolarization was not changed (Figure 1C).


Figure 1
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  		Figure 1. Characteristics of murine model of allergic bronchial asthma. (A) Inflammation of main bronchial tissues determined by histologic examination with hematoxylin and eosin staining. (B) Airway inflammation determined by cell counts in bronchoalveolar lavage (BAL) fluids under Diff-Quik staining. Mac: macrophages, Eos: eosinophils, Neu: neutrophils, and Lym: lymphocytes. (C) Bronchial smooth muscle (BSM) hyperresponsiveness determined by the contractile responsiveness to acetylcholine (ACh; left) and high K+ depolarization (in the presence of atropine and indomethacin, both 10–6 M; right). (D) Time-course changes in the levels of IL-13 in BAL fluids (BALF) determined by ELISA. Results are presented as mean ± SEM (B, C, and D) or as the representative photos (A) from at least five independent experiments, respectively. *P < 0.05 and ***P < 0.001.

 
Many kinds of cytokines/chemokines, such as IL-4, IL-10, IL-12, IL-13, monocyte chemotactic protein (MCP)-1 and MCP-5, were detected in BAL fluids obtained at 24 hours after the last antigen challenge by using cytokine antibody array technique (data not shown). To investigate the mechanism of BSM hyperresponsiveness, we chose to investigate IL-13 among these mediators, because its critical role in hyperresponsiveness has been suggested (24, 28, 30). Figure 1D shows the time-course changes in the expression of IL-13 in BAL fluids measured by ELISA. In the murine allergic bronchial asthma presently used, the peak expression of IL-13 was observed at 6 hours after the last antigen challenge and then the IL-13 levels were reduced. However, a significant increase in the IL-13 expression was still observed at 24 hours after the last antigen challenge.

Effects of IL-13 on RhoA Expression in Cultured hBSMCs
To investigate the direct effects of IL-13 on BSM cells, cultured hBSMCs were treated with recombinant human IL-13 under the serum-free condition as described in MATERIALS AND METHODS. The RT-PCR analyses revealed that both IL-13 receptor {alpha}1 and {alpha}2 (IL13R{alpha}1 and IL13R{alpha}2) and IL-4 receptor {alpha} (IL4R{alpha}) were expressed in the cultured hBSMC (Figure 2A). Signal transducer and activator of transcription 6 (STAT6), a major signal transducer activated by IL-13 (35–39), and other STATs including STAT1, STAT3, and STAT5B, were also detected (Figure 2A). IL-13 is thus capable of activating signal transduction in BSM cells directly. To confirm this, tyrosine phosphorylation of STAT6 in the IL-13–stimulated BSM cells was determined by immunoblotting with specific antibody against 641-phosphotyrosine-STAT6. As shown in Figure 2B, the protein expression of STAT6 was detected in the cultured hBSMCs. Although the expression level of total STAT6 protein was not affected by the IL-13 treatment, a distinct phosphorylation of STAT6 was observed when the cells were treated with IL-13 for 1 hour. The phosphorylation of STAT6 induced by IL-13 seems to be a transient reaction because the band for phosphorylated STAT6 disappeared in the cells treated with IL-13 for 3 hours. A slight but distinct phosphorylation of STAT6 was also observed again at 6 hours when cells were treated with 100 ng/ml of IL-13 (Figure 2B). The phosphorylation of STAT6 induced by IL-13 was completely blocked by co-incubation with leflunomide (250 µM; data not shown), a STAT6 inhibitor (39). Immunocytochemical examination also revealed a distinct activation of STAT6 by the IL-13 stimulation: IL-13 induced a nuclear translocation of STAT6 proteins, which were phosphorylated STAT6 (Figure 2C). On the other hand, although STAT3 is also reportedly phosphorylated by IL-13 in human peripheral blood mononuclear cells (40), STAT3 phosphorylation was not observed in the IL-13–stimulated hBSMC (Figure 2B).


Figure 2
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  		Figure 2. Effects of IL-13 on RhoA expression in cultured human bronchial smooth muscle cells (hBSMCs). (A) Expression of the receptors for IL-13 and multiple signal transducer and activator of transcriptions (STATs) determined by RT-PCR. RT–: without RT reaction. (B) IL-13 (100 ng/ml)–induced phosphorylation of STAT6 and up-regulation of RhoA protein determined by Western blotting. Although STAT3 is also expressed in hBSMCs, phosphorylated STAT3 was not detected under the condition used. (C) IL-13 (100 ng/ml)–induced activation of STAT6 determined by immunocytochemistry using antibodies against STAT6 (upper panels) and phosphorylated STAT6 (pSTAT6; lower panels). Original magnification: x160. (D) IL-13–induced upregulation of RhoA protein in association with an activation of STAT6. (Upper panel) Time-course analysis of IL-13–induced up-regulation of RhoA protein. (Lower panel) Effect of co-incubation with a STAT6 inhibitor, leflunomide, on the IL-13–induced up-regulation of RhoA protein. The photos shown in A, B, and C are representative of three independent experiments, respectively. In D, results are expressed as % of control (cells incubated without any treatment) and are presented as mean ± SEM from five independent experiments. *P < 0.05 and **P < 0.01 versus control by Bonferroni/Dunn's test. #P < 0.05 versus respective IL-13 only group by unpaired Student's t test.

 
Next, the effect of IL-13 on the expression of RhoA protein was determined in the cultured hBSMC. IL-13 at the concentration of 10 or 30 ng/ml had no significant effect on the expression of RhoA protein at any incubation periods used (Figure 2D, upper panel). However, the RhoA expression was markedly and significantly increased in an incubation time–dependent manner when the cells were incubated with 100 ng/ml of IL-13 (Figure 2D, upper panel). The peak response was observed at the 12-hour incubation, which revealed an approximately 3-fold increase in RhoA protein. The IL-13–induced increase in RhoA protein was completely blocked when the cells were co-incubated with leflunomide (250 µM; Figure 2D, lower panel), indicating that an activation of STAT6 might be involved in the increased expression.

Effects of IL-13 on RhoA Expression and Smooth Muscle Contractility in Murine Bronchial Tissues
To determine the direct effects of IL-13 on BSM contractility, bronchial tissues isolated from naive BALB/c mice were subjected to the organ culture examinations. The smooth muscle tissues of the left main bronchi isolated from naive mice were cultured in serum-free medium in the absence or presence of recombinant mouse IL-13. The culture condition (i.e., 100 ng/ml concentration of IL-13 for 12 h) was chosen because of its significant effect on hBSMC as described above. The RT-PCR analyses revealed a distinct expression of mRNAs for IL4R{alpha}, IL13R{alpha}1, and multiple STATs in the bronchial tissues of naive mice, whereas the distinct band for IL13R{alpha}2 mRNA was not detected under the PCR condition used (Figure 3A). When the bronchial tissues were cultured in the presence of IL-13 (100 ng/ml for 12 h), an up-regulation of RhoA protein was observed (Figure 3B), as in the case of hBSMCs. In these cultured tissues, ACh-induced contraction of the IL-13–treated BSMs was significantly augmented as compared with that of controls (Figure 3C, left panel). The augmented ACh-induced contraction observed in the IL-13–treated muscle strips was inhibited by Y-27632, a Rho-kinase inhibitor (11) (Figure 3D). On the other hand, the IL-13 treatment had no effect on the contraction induced by high K+ depolarization (Figure 3C, right panel).


Figure 3
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  		Figure 3. Effects of in vitro treatment with IL-13 on RhoA expression and smooth muscle contractility in murine bronchial tissues. (A) Airway expression of the receptors for IL-13 and multiple STATs determined by RT-PCR. RT–: without RT reaction, L: tissue of lungs with bronchi, and B: whole brain as positive control. The bands shown are representative of three independent experiments, respectively. (B) Up-regulation of RhoA protein induced by in vitro incubation with IL-13 (100 ng/ml, for 12 h) in cultured BSM tissues determined by Western blotting. Due to the limitation of protein content, a protein sample was prepared by collecting left main bronchi of five different animals and loaded in a single lane (5 tissues/lane), respectively. (C) BSM hyperresponsiveness induced by in vitro incubation with IL-13 (100 ng/ml, for 12 h). The BSM responsiveness to acetylcholine (ACh; left) and high K+ depolarization (in the presence of atropine and indomethacin, both 10–6 M; right) were measured. Results are presented as mean ± SEM from eight animals. *P < 0.05 versus control by Bonferroni/Dunn's test. (D) Effects of Rho-kinase inhibition on BSM hyperresponsiveness induced by in vitro incubation with IL-13. In each muscle strip, a Rho-kinase inhibitor Y-27632 was cumulatively administered after the acetylcholine (ACh; 10–3 M)-induced contraction reached to the plateau. Results are presented as mean ± SEM from eight animals. *P < 0.05 versus control by Bonferroni/Dunn's test.

 
Effects of Intranasally Administered IL-13 on RhoA Expression and Smooth Muscle Contractility in Murine Bronchial Tissues
Finally, the effects of in vivo treatment with IL-13 were examined in naive BALB/c mice. Based on the schedule for the repeated antigen challenge (see MATERIALS AND METHODS), IL-13 (3, 10, or 30 ng in 20 µl of PBS) was intranasally administered three times at every 4 days under anesthesia with Avertin (2.5% tribromoethanol). Our previous study demonstrated that the reagent was successfully distributed in wide area of the airways by the intranasal administration under these conditions (34). Twenty-four hours after the last IL-13 administration, RhoA protein expression and smooth muscle contractility of the bronchial tissues were determined. Hematoxylin and eosin staining of lung and bronchial sections revealed no histologic change in the airways of the IL-13–treated mice (data not shown). However, as shown in Figure 4A, the RhoA expression in bronchial tissues was increased in an IL-13 dose–dependent manner. A significant increase in RhoA protein level was observed when the mice were treated three times with 30 ng of IL-13. In this group of mice, the isolated main bronchial tissues showed an augmented responsiveness to ACh, but not to high K+ depolarization, when compared with the vehicle-treated control group (Figure 4B). Thus, the in vivo treatments of airways with IL-13 can cause BSM hyperresponsiveness with an up-regulation of RhoA, both of which were also observed in the allergic bronchial asthma (20).


Figure 4
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  		Figure 4. Effects of in vivo treatment with IL-13 on RhoA expression and smooth muscle contractility in murine bronchial tissues. (A) Up-regulation of RhoA protein induced by in vivo treatment with IL-13 (0, 3, 10, and 30 ng/animal in 20 µl of PBS, intranasally) in BSM tissues of naive animals determined by Western blotting. (Upper panel) Representative Western blots. The bands analyzed by a densitometer and normalized by the intensity of corresponding GAPDH band, and the data are summarized in the lower panel. Results are presented as mean ± SEM from five animals. *P < 0.05 versus 0 (vehicle for IL-13) by Bonferroni/Dunn's test. (B) BSM hyperresponsiveness induced by in vivo treatment with IL-13 (30 ng/animal in 20 µl of PBS) in naive animals. The smooth muscle responsiveness to acetylcholine (ACh; left) and high K+ depolarization (in the presence of atropine and indomethacin, both 10–6 M; right) were measured. Results are presented as mean ± SEM from five animals. *P < 0.05 versus control by two-way ANOVA.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Airway smooth muscle is an important effector tissue regulating bronchomotor tone. It has been suggested that modulation of airway smooth muscle by inflammatory mediators such as cytokines may play an important role in the development of airway hyperresponsiveness (AHR) (41), one of the characteristic features of patients with allergic bronchial asthma. In the BALB/c strain of mice that were actively sensitized and repeatedly challenged using the same procedures as in the current study, an in vivo AHR accompanied by the increased IgE production and pulmonary eosinophilia has been demonstrated (31). In this animal model of allergic bronchial asthma, an increased contractility of isolated BSM to contractile agonists has also been found (20, 42). The augmented BSM contraction induced by antigen challenge has reportedly been associated with an up-regulation of RhoA (20), a small GTPase that is involved in the agonist-induced Ca2+ sensitization of smooth muscle contraction (10, 43). An importance of RhoA and its downstream Rho-kinases was also demonstrated in contraction of human BSM (44), and the RhoA/Rho-kinase pathway has now been proposed as a new target for the treatment of AHR in asthma (45). Here, the increased BSM contractility and the up-regulation of RhoA protein observed in the murine allergic asthma were reproduced by the intranasal administration of IL-13 to naive mice. The results also demonstrated that IL-13 directly up-regulates RhoA via an activation of STAT6 in BSM cells.

The CD4+ T helper 2 lymphocytes (Th2 cells) are closely associated with disease severity, suggesting an integral role for Th2 cells in the pathophysiology of allergic asthma (46–49). Th2 cells secrete various cytokines, termed Th2 cytokines, which cause several key features of allergic asthma such as airway eosinophilia, goblet cell hyperplasia, mucous hypersecretion, and AHR. Among these Th2 cytokines, there is increasing evidence that IL-13 is a central mediator of the induction of AHR (22–30). Human IL-13 gene is located on chromosome 5q in a region that has been linked to asthma (50, 51). An increased expression of IL-13 has been demonstrated in BAL cells obtained from patients with symptomatic asthma (52, 53). In addition, overexpression of IL-13 in the airway epithelial cells of mouse using the Clara cell 10-kD protein gene promoter causes AHR to aerosolized methacholine (54). Intratracheal instillation of recombinant IL-13 to naive mice also evokes AHR to inhaled methacholine (55) and intravenously administered ACh (22). Interestingly, intranasal administration of recombinant IL-13 to the histamine H1 receptor gene-deleted mouse, which fails to develop allergen-induced AHR, also induces AHR (56). In addition, the neutralization of IL-13 by systemic administration of a soluble IL-13R{alpha}2-IgG-Fc fusion protein (22, 23) or of an antibody against IL-13 (28–30) inhibits allergen-induced AHR in sensitized mice. Mice in which targeted deletion of IL-13 was performed fail to develop AHR, but AHR is restored by the intranasal administration of recombinant IL-13 (24).

As reported in the primary cultured human airway smooth muscle cells (57–59), the expression of IL13R{alpha}1, IL13R{alpha}2, and IL4R{alpha} were currently demonstrated in the cultured hBSMCs (Figure 2A). Phosphorylation of STAT6 protein was also observed when cells were stimulated with IL-13 (Figures 2B and 2C), indicating that IL-13 is capable of activating signal transduction in BSMCs directly. On the other hand, distinct expression of IL13R{alpha}2 was also found in hBSMC (Figure 2A), in agreement with the previous study (38). IL13R{alpha}2 chain binds IL-13 with high affinity. Although a significant role of IL13R{alpha}2 chain in the IL-13–mediated signal transduction has recently been suggested in macrophages (60), the IL13R{alpha}2 chain is mainly thought to function as a decoy receptor (27, 61), as it has a short cytoplasmic tail that is devoid of signaling motifs. The lack of the effect of low concentration of IL-13 (10 and 30 ng/ml; Figure 2D, upper panel) may be explainable by the decoy effect of IL13R{alpha}2 chain expressed on hBSMCs.

In the present cell and organ culture studies, the 100 ng/ml concentration of IL-13 was chosen on the basis of the result of its effect on RhoA expression (Figure 2D). We believe that the IL-13 concentration used is not a surprisingly high concentration as compared with the disease state (~ 1 ng/ml in BALF; Figure 1D). Since the BAL fluids were obtained by washing airway lumen with PBS, the actual concentration of IL-13 in the airway lumen should be higher. In addition, the IL-13 concentration might be extremely higher near the cells that produce IL-13. A relatively high concentration of IL-13 might also be needed for the study of airway smooth muscle cells: Tliba and colleagues (62) and Deshpande and coworkers (63) used 50 to 100 ng/ml of IL-13 to determine its effect on Ca2+ signaling and contraction in human and murine tracheal smooth muscles. Lee and colleagues (37) also used 100 ng/ml of IL-13 to determine its effect on gene expression in human BSM cells.

Currently, treatment of hBSMCs with a STAT6 inhibitor leflunomide (39) abolished both the STAT6 phosphorylation (data not shown) and the RhoA up-regulation induced by IL-13 (Figure 2D, lower panel), indicating that the IL-13–induced up-regulation of RhoA protein is mediated by an activation of STAT6 in hBSMCs. Although the exact mechanism of RhoA transcription in BSM cells is not fully understood, the upstream genomic DNA sequence of human RhoA contains several STAT-binding sites—for example, –77 (from the transcription start) to –69 (score 85.6), –270 to –261 (score 86.5), –417 to –409 (score 78.8) and –518 to –510 (score 84.6)—when analyzed using the TFSEARCH program (http://mbs.cbrc.jp/research/db/TFSEARCH.html). In addition, the in vitro IL-13 treatment of BSM tissues isolated from naive mice caused an up-regulation of RhoA protein (Figure 3B) with augmented smooth muscle contractility to ACh (Figure 3C). The in vivo IL-13 treatment of the airways of naive mice by intranasal administration also induced both an up-regulation of RhoA protein (Figure 4A) and BSM hyperresponsiveness to ACh (Figure 4B). An involvement of the increased expression of RhoA protein in the augmented BSM contractility was also demonstrated by using Y-27632 (Figure 3D), a Rho-kinase inhibitor (11). Our previous study demonstrated that Y-27632, at the concentration of 10–5 M, abolished the ACh-induced, RhoA/Rho-kinase-mediated Ca2+ sensitization of contraction without affecting the Ca2+-mediated contraction in BSMs (9). As shown in Figure 3D, no significant difference in the Y-27632-insensitive contraction (the remaining ACh-induced contraction in the presence of 10–5 M Y-27632) was observed between the groups, indicating that the Y-27632-sensitive contractile component of the ACh-induced contraction, that is the RhoA/Rho-kinase-mediated Ca2+ sensitization, was augmented in the IL-13–treated BSMs. The exact mechanism(s) of the BSM hyperresponsiveness induced by the intranasal administration of IL-13 is not fully explainable now, but the results of the in vitro studies using hBSMCs and cultured mouse BSM tissues suggest, at least in part, its direct effect on airway smooth muscle cells. Taken together, IL-13, generated by antigen exposure in the airways (Figure 1D), directly acts on BSM cells and activates their STAT6, and then up-regulates RhoA protein in the cells, resulting in an augmentation of the agonist-induced, RhoA/Rho-kinase–mediated contraction of BSM. As shown in Figure 1C, however, the efficacy, but not the potency, of ACh was significantly augmented in BSM contraction of the antigen-exposed animals. In contrast, it has been suggested that the level of muscarinic receptor density is normal even in the diseased BSMs (64). Thus, unknown mechanism(s) other than the up-regulation of RhoA may also be involved in the development of antigen-induced BSM hyperresponsiveness.

In contrast to the current results, Tliba and colleagues (62) suggested that IL-13 augments the smooth muscle contractility by enhancing agonist-mediated Ca2+ signaling in murine trachea. Their colleagues Deshpande and coworkers (63) further demonstrated an involvement of CD38/cyclic adenosine diphosphate ribose (cADPR)-dependent pathway in the IL-13–induced modulation of Ca2+ signaling in human tracheal smooth muscle cells. On the other hand, Tliba and colleagues (62) also suggested a possible involvement of Ca2+-independent pathways, such as Ca2+ sensitivity of myofilaments, in the IL-13–induced augmentation of tracheal smooth muscle contractility. Thus, the mechanism(s) of action of IL-13 on airway smooth muscle may not be explained simply. The difference in the airway region used (tracheal specimens in their studies versus BSMs in the current study) may also be considerable in the effect of IL-13. In this regard, we have previously reported a regional difference in the development of smooth muscle hyperresponsiveness in the murine model of allergic bronchial asthma (42).

In conclusion, the current study clearly showed that IL-13 up-regulates RhoA protein via an activation of STAT6 in BSM cells, resulting in an augmented BSM contraction, which is one of the causes of AHR.


   Acknowledgments
 
The authors thank Yuichi Nishida, Yuka Narushima and Tomoko Minemura for their technical assistance.


   Footnotes
 
This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Originally Published in Press as DOI: 10.1165/rcmb.2008-0162OC on August 7, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form April 28, 2008

Accepted in final form July 31, 2008


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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