Direct Effects of Interleukin-13 on Signaling Pathways ... - ATS Journals

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Direct Effects of Interleukin-13 on Signaling Pathways for Physiological Responses in Cultured Human Airway Smooth Muscle Cells JOHANNE C. LAPORTE, PAUL E. MOORE, SIMONETTA BARALDO, MARIE-HÉLÈNE JOUVIN, TRUDI L. CHURCH, IGOR N. SCHWARTZMAN, REYNOLD A. PANETTIERI, JR., JEAN-PIERRE KINET, and STEPHANIE A. SHORE Physiology Program, Harvard School of Public Health, Boston, Massachusetts; Pathology Departments, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts; and Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Numerous studies have suggested an important role for the Th2 cytokines interleukin (IL)-13 and IL-4 in the development of allergic asthma. We tested the hypothesis that IL-13 and IL-4 have direct effects on cultured airway smooth muscle cells (HASM). Using RT-PCR, we showed that HASM cells express transcripts for IL-4, IL-13RI, and IL-13RII, but not for the common IL-2R chain. We then analyzed the capacity of the two cytokines to activate signaling pathways in HASM cells. Both IL-13 and IL-4 caused STAT-6 phosphorylation, but the time course was different between the two cytokines, with peak effects occurring 15 min after addition of IL-4 and 1 h after addition of IL-13. Effects on signaling were observed at cytokine concentrations as low as 0.3 ng/ml. IL-4 and IL-13 also caused phosphorylation of ERK MAP kinase. As suggested by the signaling studies, the biological responses of the two cytokines were also different. We used magnetic twisting cytometry to measure cell stiffness of HASM cells and tested the capacity of IL-4 and IL-13 to interfere with the reductions in cell stiffness induced by the -agonist isoproterenol (ISO). IL-13 (50 ng/ml for 24 h), but not IL-4, significantly reduced -adrenergic responsiveness of HASM cells, and the MEK inhibitor U0126 significantly reduced the effects of IL-13 on ISO-induced changes in cell stiffness. We propose that these direct effect of IL-13 on HASM cells may contribute at least in part to the airway narrowing observed in patients with asthma.

There is now strong evidence that the Th2 cytokines interleukin (IL)-4 and IL-13 are important in allergic asthma. Both the IL-4 and IL-13 genes are located on chromosome 5q in a region that has been linked to asthma (1, 2). In addition, increased expression of IL-4 and IL-13 has been measured in bronchoalvealor lavage (BAL) cells isolated from patients with symptomatic asthma (3, 4). Intratracheal administration of exogenous IL-13 to nonimmunized mice induces eosinophil influx in the airways, globlet cell metaplasia with mucus hypersecretion, and increased airway responsiveness to intravenous acetylcholine (5). Allergen-sensitized and challenged mice deficient in IL-4 or IL-4R show attenuation of these asthma phenotypes (6). In vivo blockade of IL-13 by intratracheal administration of a soluble IL-132–IgGFc fusion protein also reverses allergen-induced increases in airway mucous hyperplasia and airway hyperresponsiveness in sensitized mice (5, 6). A number of receptors are involved in cellular activation by IL-4 and IL-13. All of them are members of the cytokine superfamily of receptors, contain a single transmembrane do-

(Received in original form August 10, 2000 and in revised form February 12, 2001) This study was supported by HL-56383, HL-33009, HL-55301, HL-64063, and AI 40203. Dr. Laporte is the recipient of an American Lung Association fellowship. Correspondence and requests for reprints should be addressed to Stephanie Shore, Ph.D., Physiology Program, Harvard School of Public Health, 665 Huntington Ave., Boston, MA, 02115. E-mail: [email protected] Am J Respir Crit Care Med Vol 164. pp 141–148, 2001 Internet address: www.atsjournals.org

main, and share characteristic motifs in their extracellular domains: four conserved cysteines and WSXSW box. Upon IL-4 binding to IL-4R, the receptor dimerizes with either the gamma chain of the IL-2 receptor (c) to form the type I IL-4 receptor, or with the IL-13R1 to form the type II IL-4 receptor. IL-13 binding to IL-13RI also results in dimerization of this receptor with IL-4R. However, IL-13RI does not dimerize with c. IL-13 can also bind IL-13RII, but this receptor does not dimerize with any other receptor moieties, and does not appear to be capable of signaling (7). Upon dimerization of IL-4R with either c or IL-13RI, Janus-tyrosine kinases (JAK) constitutively associated with the receptors become phosphorylated and activated, and subsequently phosphorylate tyrosine residues on the IL-4R, IL13RI, and c chains. A number of signaling pathways are then activated. For example, monomers of STAT-6 bind through their SH2 domains to phosphorylated tyrosine residues of IL-4R. Once bound, STAT-6 becomes phosphorylated by JAKs, whereupon it is released from the receptor, dimerizes with other phosphorylated STAT-6 molecules, translocates to the nucleus, and induces gene transcription. A tyrosine in the I4R motif of the IL-4R, once phosphorylated, is a site of interaction with insulin receptor substrate (IRS). IRS bound to the IL-4R also becomes phosphorylated and binds to the adaptor protein Grb2. Grb2 is constitutively complexed to Sos, which activates Ras, leading to Raf activation and subsequent activation of the ERK MAP kinase pathway (8). It is possible that some of the effects of IL-4 and IL-13 in allergic asthma may result from actions of these cytokines directly on airway smooth muscle. IL-4 inhibits smooth muscle mitogenesis induced by heparin, thrombin, serum, and platelet-derived growth factor (PDGF) (9, 10). IL-4 and IL-13 have both been shown to inhibit IL-1 induced RANTES and IL-8 production in cultured airway smooth muscle cells (HASM) (11, 12). These studies suggest that receptors for both cytokines are present on HASM cells, but the nature of these receptors and the signaling pathways activated by them have not been described. To address this issue, we examined the expression of IL-13 and IL-4 receptors on HASM cells using RTPCR. We also examined the ability of IL-13 and IL-4 to induce STAT-6 and ERK phosphorylation by Western blotting. -Adrenergic hyporesponsiveness is a characteristic feature of asthma. Decreased responses to -agonists are observed both in vivo and in vitro in the airways of patients with asthma as well as in animal models of asthma (13, 14). Although the mechanistic basis for this hyporesponsiveness has not been established, it is possible that cytokines expressed in the airways of patients with asthma may contribute. In support of this hypothesis, IL-1, tumor necrosis factor- (TNF-), and IL-5 have each been shown to reduce responses to -agonists (15–21) through direct effects on airway smooth muscle. Because our results indicated that IL-4 and IL-13 receptors

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were expressed in HASM cells and could induce signaling in these cells, we sought to determine whether IL-4 and IL-13 could also influence -adrenergic responsiveness. We assessed -adrenergic responsiveness by measuring changes in cytoskeletal stiffness induced by isoproterenol (ISO) using magnetic twisting cytometry (22, 23) and by measuring ISO-induced changes in cAMP formation.

METHODS Cell Culture Human tracheas were obtained from lung transplant donors, in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings. A segment of trachea just proximal to the carina was dissected under sterile conditions, and smooth muscle cells from the trachealis isolated and placed in culture as previously described (24). Cells were plated in plastic flasks at 104 cells/cm2 in Ham’s F12 media supplemented with 10% fetal bovine serum (FBS), penicillin (103 U/ml), streptomycin (1 mg/ml), amphotericin-B (2 mg/ml), NaOH (12 mM), CaCl2 (1.7 M), L-glutamine (2 mM), and HEPES (25 mM). Medium was replaced every 3–4 d. Cells were passaged with 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (EDTA) every 10–14 d. Confluent cells were serum deprived and supplemented with 5.7 g/ml insulin and 5 g/ml tranferrin 24 h prior to use. Cells from 15 different donors studied in passages 4 to 7 were used in the studies described below.

RT-PCR HASM cells from two different donors were serum deprived and hormone supplemented for 24 h. Total RNA was isolated using RNeasy spin columns (Qiagen Inc., Valencia, CA) according to the manufacturer’s specifications. For each sample, approximately 0.5 g of total RNA was reverse transcribed using Advantage RT-for-PCR (Clontech, Palo Alto, CA), according to the manufacturer’s specifications. PCR was then performed to assess the expression by HASM cells of IL-4R, c, IL-13RI, and IL-13RII. The primers used for each receptor are described in Table 1. We also used commercial primers (Clontech) for G3PDH. RT-PCR was also performed on RNA from Jurkat cells to confirm the ability of the primers to detect the c receptor. For PCR, each 50 l reaction mixture contained 1 l (200 M) dNTPs, 5 l each primers, 5 l 10 PCR buffer, 5 l cDNA, and 0.4 l Taq polymerase (Perkin-Elmer, Foster City, CA) and 33.6 l H2O. Taq was added to the mixture after a 3 min hot start at 94 C. PCR was performed as follows: 94 C for 30 s, 60 C for 45 s, and 72 C for 1 min, and were followed by a 7 min extension at 72 C. For the IL13RI, IL-13RII, and c chain, 35 cycles of PCR were used. For the IL-4R, 30 cycles of PCR were used. The PCR products were resolved on a 1.5% agarose gel and stained with ethidium bromide.

Western Blotting Confluent HASM cells were serum deprived and treated with IL-4 or IL-13. Medium was removed, and cells were washed with phosphatebuffered saline (PBS) and then lysed in 400 l of extraction buffer (10 mM Tris–HCl buffer with 50 mM NaCl, 50 mM NaF, 10 mM D -serine, 1 M EDTA, 1 M ethyleneglycoltetraacetic acid [EGTA], 1% sodium dodecyl sulfate [SDS], 1% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride, 5 g/ml leupeptin, 1 g/ml pepstatin, 102 U/ ml aprotinin). Cells were scraped off flasks, passed through a 25- and

TABLE 1. PRIMER SETS USED TO IDENTIFY RECEPTORS Receptor IL-4R c IL-13RI IL-13RII

Primers

Size (bp)

Forward: 5 -GAGAGCAGCAGGGATGACTT-3 Reverse: 5 -TCCACCGCATGTACAAACTC-3 Forward: 5 -GACAGGCCACACAGATGCTA-3 Reverse: 5 -GTTCACTGTAGTCTGGCTGC-3 Forward: 5 -GCACCAATGAGAGTGAGAAGC-3 Reverse: 5 -GGAACAACCAAAGTATTGGCC-3 Forward: 5 -TTTCGTTTGCTTGGCTATCG-3 Reverse: 5 -TGTAATGCATGATCCAAGCC-3

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5/8-gauge needle, and sonicated for 10 s. Supernatants of cell lysates were mixed with equal volumes of loading buffer (0.062 M Tris–HCl [pH 6.8], 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.01% [wt/vol] bromophenol blue) and then were boiled for 5 min. Solubilized proteins (10 to 30 g/lane) were separated by SDS–polyacrylamide gel electrophoresis on 12% Tris–glycine gel (Invitrogen, Carlsbad, CA) under nonreducing conditions and transferred electrophorically to a nitrocellulose membrane in transfer buffer (Pierce, Rockford, IL). The membrane was blocked with 5% nonfat dry milk in Trisbuffered saline (TBS) containing 0.1% Tween-20 for 3 h at room temperature. The blots were probed with the respective antibodies. Blots were washed and subsequently incubated (1 h) in TBS containing 0.1% Tween-20 and 5% nonfat dry milk containing HRP-conjugated goat anti-rabbit immunoglobulin G (IgG) for 1 h. The proteins were visualized by light emission on film with enhanced chemiluminescent substrate (Pierce, Rockford, IL). The band visualized at approximately 117 kD was quantified using a laser densitometer. Band density values were expressed in arbitrary OD units. The anti-JAK1, -JAK2, and -JAK3 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-STAT-6 antibodies was purchased from Upstate biotechnology (Lake Placid, NY); anti-phospho-STAT6, anti-phospho-Tyk2, anti-ERK; and anti-phospho-ERK antibody were purchased from New England Biolabs Inc. (Beverly, MA).

Magnetic Twisting Cytometry We examined the effect of IL-4 and IL-13 on changes in cell stiffness induced by ISO or dibutyryl-cAMP (db-cAMP). Cells were serum deprived for 24 h, treated with IL-4 or IL-13 for 18 h, harvested with trypsin and EDTA, and resuspended in serum-free media. Cells were then plated at 20,000 cells/well on collagen I (500 ng/cm2) coated bacteriological plastic dishes (6.4 mm, 96-well Removawells, Immunlon II), and cytokines were readded to the wells. Measurements of cell stiffness were made 2–6 h later using magnetic twisting cytometry. Cumulative concentration-response curves to ISO or dibutyryl-cAMP were performed as follow: first, three to five measurements of cell stiffness were made under baseline conditions. Following these measurements, 2 l of a solution containing the ISO or dibutyryl-cAMP was added to the cell well that contained 200 l of media. After a 1 min incubation with the agent, two to four measurements of cell stiffness were again obtained. This procedure was repeated with increasing concentration of the agent. The concentration ranges used were as follow: ISO (108–105 M); dibutyryl-cAMP (104–3  103 M). Only one agonist was studied per well. Details of the methodology for magnetic twisting cytometry have been previously described (22, 23). Briefly, the principle is as follows. Ferromagnetic beads are first coated with a prescribed ligand, (Peptide 2000: Arg-Gly-Asp [RGD], Telios Pharmaceuticals, San Diego, CA) in this case, then bound to the surface of the cells through the corresponding receptor system (integrins in this case). Individual wells containing adherent cells bound to RGD-coated ferromagnetic beads in serum-free medium are placed into the magnetic twisting chamber, and held at 37 C using a circulating water bath that is built into the system. The attached beads are magnetized with a brief 1000-G pulse so that their magnetic moments are aligned in one direction, parallel to the surface on which the cells are plated. The magnetic field vector generated by the beads in the horizontal direction is measured by an in-line magnetometer. Subsequently, a much smaller magnetic field is applied in the vertical direction generating an applied torque (or twisting stress). This twisting stress (80 dynes/cm2 in this case) causes the beads to rotate as would a compass needle, but bead rotation is opposed by reaction forces developed within the cytoskeleton to which the beads are bound through the integrin molecules. Magnetic twisting cytometry uses the applied twisting stress and the resulting measured angular rotation of the magnetic beads, and expresses the ratio as cell stiffness. Results obtained in previous studies have established that changes in stiffness can be used as a proxy for force generation in these cells (22, 23, 25, 26).

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cAMP Formation

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We examined the effect of IL-4 and IL-13 on changes in cAMP formation induced by ISO (106 M) or forskolin (104 M). Cells were serum deprived for 24 h, treated with IL-4 or IL-13 (50 ng/ml for 18 h), har-

Laporte, Moore, Baraldo, et al.: IL-13 Induces -Adrenergic Hyporesponsiveness vested with trypsin and EDTA, and resuspended in serum-free media. Cells were then plated at 100,000 cells/well in 24-well plates and cytokines were readded to the wells. Cells were allowed to readhere for 4 h at 37 C, at which time the medium was replaced with PBS containing 0.1 mM IBMX (to prevent degradation of cAMP by phosphodiesterases) and 300 M ascorbic acid (to prevent oxidation of ISO). Thirty minutes later, cells were either treated with either ISO or forskolin or left untreated to measure basal cAMP formation. Cells were incubated for an additional 10 min at 37 C and then placed on ice. Ice-cold ethanol (1 ml) was added to lyse the cells. The lysate was centrifuged at 2,000  g for 15 min at 4 C, and the resulting supernatant was removed, evaporated to dryness, and stored at 20 C until assayed using a Rainen cAMP 125I radioimmunoassay kit (NEN, Boston, MA).

Reagents Tissue culture reagents and drugs used in this study were obtained from Sigma (St. Louis, MO), with the exception of amphotericin-B and trypsin-ETDA solution, which were purchased from Gibco (Grand Island, NY), IL-4 and IL-13, which were obtained from R&D Systems (Minneapolis, MN), and U0126, which was obtained from Promega. The primers used for RT-PCR analysis of IL-4R and IL13R transcript were obtained from Ransom Hill Bioscience Inc. (Ramona, CA). Dibutyryl-cAMP was dissolved at 101 M in distilled water, frozen in aliquots, and diluted appropriately in media on the day of use. Isoproterenol (101 M in distilled water) was made fresh each day. Because ISO is rapidly oxidized, dilutions of ISO in media were made immediately prior to addition to the cells. U0126 was dissolved in dimethyl sulfoxide (DMSO) and diluted such that the final concentration of DMSO in the cell well was 0.01%.

Statistics The effect of IL-4 and IL-13-induced changes in cell stiffness and cAMP formation responses to ISO was examined by repeated mea-

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sures ANOVA using treatment and experimental day as main effects. Follow up t tests were used to determine where the treatment effect lay. Changes in basal phospho-STAT-6 and phospho-ERK induced by IL-4 and IL-13 were examined by paired t tests of optical densitometry measurements. A p value 0.05 was considered significant.

RESULTS IL-4 and IL-13 Receptor Expression

To define the nature of the IL-4 and IL-13 receptors expressed by cultured HASM cells, RT-PCR was conducted using primer sets described in Table 1. Products with appropriate sizes were detected using primers for IL-13RI, IL13RII, and IL-4R in cells from each of two different donors (Figure 1). In contrast, the c chain was not detected in these cultured HASM cells, but was detected in Jurkat cells. GAPDH (used as internal standard) was expressed in all samples. Reactions performed with no cDNA or with RNA in the absence of reverse transcriptase did not result in a product using any of the primer pairs. HASM Cells Express JAKs

The IL-4 and IL-13 receptors constitutively associate with JAKs, a family of tyrosine kinases. Four members of the JAK family have been described: JAK1, JAK2, JAK3, and Tyk2. JAK1 usually associates with the IL-4R, whereas Tyk2 usually associates with IL-13RI (27). Using Western blotting, we demonstrated that HASM cells express JAK1, JAK3, but not JAK2 (Figure 2A). Tyk2 was also expressed, and its phosphorylation was increased by IL-4 or IL-13 (Figure 2B). STAT-6 Activation by IL-4 and IL-13

To determine whether IL-13 and IL-4 caused activation of STAT-6 in cultured HASM cells, we examined the time course of STAT-6 phosphorylation induced by IL-4 or IL-13 using Western blotting. Both IL-4 and IL-13 caused transient STAT-6 phosphorylation, but the time course was different for the two cytokines. Figure 3A shows representative results for a single donor. Densitometry data from experiments from a number of donors are presented in Figure 3B. For IL-4, STAT-6 phosphorylation peaked 15 min after addition of cytokine and gradually declined thereafter. For IL-13, STAT-6 phosphorylation did not peak until 1–2 h after addition of the cytokine. Neither IL-4 nor IL-13 had any effect on STAT-6 protein expression in cultured HASM cells (Figure 3C). We also examined the dose range over which IL-4 and IL-13 induced STAT-6 phosphorylation (Figure 3D). Because of the differences in IL-4 and IL-13 time course, responses were assessed at each dose after 15 min of cytokine treatment for IL-4, and after 1 h of treatment for IL-13. Both IL-4 and IL-13 caused a dose-related increase in STAT-6 phosphorylation,

Figure 1. (Upper) PCR analysis of IL-13R mRNA expression in cultured HASM cells from two donors; lane 1: 100 bp markers; lanes 2–3: G3PDH; lanes 4–5: IL-13R1; lanes 6–7: no cDNA and no RT controls using IL-13RI primers; lane 8: 100 bp markers; lanes 9–10: IL-13R2; lanes 11–12: no cDNA and no RT controls using IL-13RII primers. (Lower) PCR analysis of IL-4R and c mRNA expression in cultured HASM cells and Jurkat cells; lane 1: 100 bp markers; lanes 2–3: IL-4R; lanes 4–5: no cDNA and no RT controls using IL-4RI primers; lane 6: 100 bp markers; lanes 7–8: c in HASM cells; lanes 9–10: c in two separate isolates of Jurkat cells; lanes 11–12: no cDNA and no RT controls using c primers.

Figure 2. (A) Western blot showing JAK expression in cells from three different HASM donors. (B) Western blot showing Tyk-2 phosphorylation in response to IL-4 (10 ng/ml for 15 min) and IL-13 (10 ng/ml for 15 min) treated HASM cells. C, control.

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Figure 4. Western blots showing the time course of p42 and p44 ERK MAP kinase phosphorylation induced by IL-4 (A) or IL-13 (B). ST, standard.

recognizes both the phosphorylated p42 and p44 ERK isoforms. Figure 4 shows the time course of ERK phosphorylation for IL-4 and IL-13 in cells from a single donor. Both IL-4 and IL-13 caused a time-dependent activation of ERK (Figure 4) that peaked at 15 min after addition of cytokine. ERK phosphorylation by IL-4 and IL-13 was also observed in cells from five other donors. IL-13 But Not IL-4 Causes -Adrenergic Hyporesponsiveness

Figure 3. (A) Western blot showing representative time course of STAT-6 phosphorylation in IL-4- and IL-13-treated cells. (B) Densitometric quantification (expressed as normalized optical density units), for time course of STAT-6 phosphorylation induced by IL-4 or IL-13. All cells were treated with 10 ng/ml cytokine. Results for phospho-STAT-6 are the mean SE of data from 7 (IL-13) or 12 (IL-4) donors. (C) Western blot showing the effect of IL-13 and IL-4 on STAT-6 expression. ST, STAT-6 standard; C, control. (D) Densitometric quantification (expressed as normalized optical density units) of the dose-related effects of IL-13 and IL-4 on STAT-6 phosphorylation. Results for phosphoSTAT-6 are mean SE of data from five donors in each case. In B and D, densitometry data were normalized to a phospho-STAT-6 standard included on each blot.

which was significant at doses as low as 0.3 ng/ml and peaked at approximately 10 ng/ml. ERK MAP Kinase Activation by IL-4 and IL-13

To determine whether IL-4 and/or IL-13 could induce ERK activation in cultured HASM cells, as occurs in other cell types, we performed Western blotting using an antibody that

To determine whether IL-13 and IL-4 might be involved in the reduced -adrenergic airway responsiveness observed in human asthma, we determined their effects on changes in HASM cell stiffness and cAMP formation induced by the -agonist, ISO. Changes in cell stiffness were measured using magnetic twisting cytometry. Neither IL-4 nor IL-13 had any effect on baseline cell stiffness: 120.7 10.2, 131.7 5.4, and 116.6 9.8 dyne/cm2 in control and in IL-13- and IL-4-treated cells, respectively. In control cells, ISO caused a dose-related decrease in cell stiffness (Figure 5). In cells treated with IL-13 (50 ng/ml), the response to ISO was significantly reduced (p

0.0001) by repeated measures ANOVA (Figure 5A). IL-13 at 10 ng/ml had a small though not significant effect (data not shown). In contrast, IL-4 (50 ng/ml) had no effect on responses to ISO to decrease cell stiffness (Figure 5B). To ensure that the effect of IL-13 was not a result of nonspecific effects of the cytokine on the ability of HASM cells to decrease stiffness, we studied its effect on responses to db-cAMP. DbcAMP caused a concentration dose-related decrease in cell stiffness consistent with previous reports (18, 20, 25), but IL13 (50 ng/ml) had no effect on db-cAMP responses (Figure 6). We also measured changes in cAMP formation induced by ISO in IL-13- and IL-4-treated cells. Neither IL-13 nor IL-4 had any effect on basal cAMP formation in HASM cells: 13.3 4.26, 15.9 4.1, and 12.0 3.1 pmol/106 cells in control and in IL-13- and IL-4-treated cells, respectively. ISO caused a marked increase in cAMP formation in control cells (Figure 7). IL-13 reduced ISO-induced cAMP formation to levels only onethird the levels obtained in control cells (p 0.005). In contrast, there was no significant effect of IL-4 on ISO-induced cAMP formation. To determine whether IL-13 and IL-4 might be altering the expression or activity of adenylyl cyclase, we measured changes in cAMP formation by forskolin, which directly activates the kinase. Forskolin (104 M) caused a marked increase in cAMP formation that was not altered by either IL-13 or IL-4 pretreatment (Figure 7). To determine whether phosphorylation of ERK by IL-13 contributes to the ability of IL-13 to reduce -adrenergic hyporesponsiveness, we examined the effect of U0126 on IL-13induced changes in cell stiffness responses to ISO. U0126 is a relatively potent and selective inhibitor of the enzyme MEK that phosphorylates ERK (28). We have previously reported

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Figure 7. Effect of IL-13 (50 ng/ml for 24 h) and IL-4 (50 ng/ml for 24 h) on cAMP formation induced by isoproterenol (ISO) or forskolin (FORSK). Results are expressed as a percentage of baseline values. For isoproterenol, results are mean SE of data from a total of six wells in each case and were obtained on three experimental days in cells from three different donors. For forskolin, results are mean SE from a total of 9–10 wells in each group and were obtained on five experimental days on cells from four different donors. *p 0.005.

Figure 5. Effect of IL-13 (A; 50 ng/ml for 24 h) and IL-4 (B; 50 ng/ml for 24 h) on changes in cells stiffness induced by increasing concentrations of isoproterenol. Results are expressed as percentage baseline stiffness measured prior to addition of isoproterenol. For IL-13, the results are mean SE of data obtained on nine experimental days from 20 control wells and 18 wells treated with IL-13 from seven donors. For IL-4, the results were obtained on eight experimental days from 15 control and IL-4-treated wells from five donors. *p 0.005.

that U0126 (10 M) has no effect on cell stiffness responses to ISO in control cells (29). In cells treated with IL-13, the response to ISO was significantly greater in cells treated with U0126 (10 M) compared with cells treated with vehicle (0.01% DMSO) (p 0.02 by repeated measures ANOVA) (Figure 8). Follow-up t tests indicated that the effect of U0126 was observed at all concentrations of ISO except 108 M.

DISCUSSION Our results demonstrate that HASM cells express IL-4R, IL13RI and IL-13RII, but not c (Figure 1). JAK1, JAK3, and Tyk2 are expressed in cultured HASM cells, whereas JAK2 protein is not (Figure 2). IL-4 and IL-13 stimulation both lead to STAT-6 and ERK MAP kinase phosphorylation, but the

Figure 6. Effect of IL-13 (50 ng/nl for 24 h) on changes in cell stiffness induced by increasing concentrations of db-cAMP. Results are mean SE of data obtained on two experimental days from four control wells and four wells treated with IL-13.

time course of activation of STAT-6 differs for the two cytokines (Figure 3). IL-13 reduces the ability of ISO to decrease HASM cell stiffness (Figure 5) and to increase cAMP formation (Figure 7), whereas IL-4 does not. HASM cells expressed IL-4R, IL-13RI, and IL-13RII consistent with results obtained in other nonhematopoietic cells (30, 31). These results are likely not an artifact of culture conditions, as IL-4R and IL-13RI have also been detected by immunohistochemical staining of sections of human airway (32). These results are also consistent with reports that both IL-4 and IL-13 can induce functional changes in HASM cells. For example, Hawker and coworkers reported that IL-4 reduced HASM cell proliferation (10) and Pype and coworkers reported that it prevented MCP expression induced by IL-1 and TNF- (33). Both IL-4 and IL-13 have been reported to decrease IL-8 production by HASM cells (12). In contrast to our results (Figure 1), Hakonarson and coworkers (34) reported, using Western blotting, that IL-4R was expressed only in HASM cells treated with asthmatic serum, and not in normal cells. It is likely that the reason for the observed differences in IL-4R expression is related to the sensitivity of PCR compared with Western blotting. c mRNA was not detected in our HASM cells, consistent with observations in other nonhematopoietic cells (27, 35). We did detect c in Jurkat cells, a

Figure 8. Effect of U0126 (10 M) on responses to isoproterenol in HASM cells treated with IL-13 (50 ng/ml for 18 h). U0126 or vehicle (0.01% DMSO) was administered 2 h prior to IL-13 treatment. Results are mean SE of four to five wells in each group and were obtained on cells from 2 HASM cell donors. *p 0.05.

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cell line known to express the receptor, indicating that the lack of expression in HASM cells was not due to a problem with the primers or the reaction conditions. These results suggest that IL-13RI is the sole dimerizing partner for the IL-4R in HASM cells. Because IL-13RII has not been demonstrated to be capable of signaling (7), these results also indicate that the type II IL-4 receptor, the dimer composed of IL-4R and IL-13RI, is the signaling receptor for both IL-4 and IL-13 in HASM cells. The transcription factor STAT-6 was expressed in these cultured HASM cells, and both IL-13 and IL-4 caused transient phosphorylation of STAT-6, although with different time courses. With IL-4, STAT-6 phosphorylation peaked 15 min after addition of cytokine in most donor cells and then declined. With IL-13, phosphorylation of STAT-6 was apparent 15 min after addition of cytokine, but peak phosphorylation did not occur until 1–2 h after addition of cytokine in most donors. This difference in time course is perhaps surprising given that, as described above, both IL-4 and IL-13 must use the dimer composed of IL-4R and IL-13RI for signaling. Palmer-Crocker and coworkers have also reported differences in the time course of IL-4 and IL-13 activation in cultured human endothelial cells, which also lack c (36). They demonstrated that IL-13induced tyrosine phosphorylation of the IL-4R is slower than that induced by IL-4. One potential explanation for the slower time course of action of IL-13 is that the kinetics of IL-13 binding to IL-13RI is different than for IL-4 binding to IL-4R or that the rate at which receptor dimerization occurs following binding of IL-13 to IL-13RI is slower than the rate at which dimerization occurs following IL-4 binding to IL4R. Another possible explanation is that the presence of IL13RII (37) modulates the time course of action of IL-13. For example, IL-13RII may initially bind IL-13 and gradually donate it back to IL-13RI. It is also possible that differences in the donors used for the IL-4 and IL-13 studies might have contributed to differences in the time course of STAT-6 phosphorylation. The IL-4R contains at least eight polymorphisms in its coding region and three of these have been demonstrated to alter IL-4 signaling in other cell types (38, 39). However, we think this an unlikely explanation, as even in cells from the very same donor, the time course of STAT-6 phosphorylation induced by IL-13 and IL-4 was different (Figure 3A). In HASM cells, IL-4 and IL-13 both caused a time-dependent activation of ERK. For both IL-4 and IL-13 peak phosphorylation occurred approximately 15 min after addition of cytokines in most donors (Figure 4). It is likely that ERK phosphorylation is the result of binding of IRS to the I4R motif of the phosphorylated IL-4R, recruitment of Grb2/Sos, and consequent activation of Ras, as has been described by others (8). However, it should be noted that although IL-4 phosphorylation of IRS-1/2 is consistently observed in all cell types reported to date, activation of ERK is observed in some cells but not others (40, 41), suggesting that IRS/Grb/Sos activation is not sufficient for activation of this pathway and that some other signaling molecule may also be required. Thus, the ability of IL-4 and IL-13 to activate the ERK pathway might depend on the array of signaling molecules that was expressed by particular cell types. The ERK MAP kinase pathway has been reported to be an important regulator of HASM cell proliferation (42). Interestingly, IL-4 induced ERK phosphorylation (Figure 4), but IL-4 has been reported to inhibit HASM cell mitogenesis (10). The transient nature of the phosphorylation of ERK by IL-4 may be important in this respect, as compounds that induce mitogenesis of HASM cells cause sustained rather than transient activation of ERK (42). In contrast to the effects of IL-4, IL-13

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has been reported to promote HASM mitogenesis (30). We did not observe any striking differences in the effects of IL-13 and IL-4 on ERK phosphorylation, although we examined times points only up to 4 h (Figure 4). The effects of IL-4 and IL-13 suggest that other signaling pathways are important in the effects of these cytokines on HASM cell proliferation (42) and may be differentially affected by IL-4 and IL-13. Because the results of this study indicated that IL-4 and IL-13 could act directly on HASM cells, we wished to determine whether IL-4 and IL-13 could influence airway smooth muscle responses to -adrenergic agonists, as has been demonstrated with other cytokines such as IL-1, TNF-, and IL-5 (15–21). To assess -adrenergic responsiveness, we measured changes in cytoskeletal stiffness induced by ISO. Cytoskeletal stiffness as measured here is an index of the ability of cells to resist distortions of shape in response to shear stress applied through magnetic beads linked to the cytoskeleton via integrins. Actin and myosin form part of the cytoskeleton, and the observations that contractile agonists increase stiffness while dilating agonists decrease stiffness (18, 25) and that transfection with a tonically active myosin light chain kinase results in increased myosin phosphorylation and increased cell stiffness (26) suggest that in these cells stiffness is a proxy for force generation. Our results indicate that pretreatment with IL-13, but not IL-4, reduced ISO induced changes in cell stiffness (Figure 5). Baseline stiffness prior to addition of ISO was not different in control and IL-13-treated cells. These results indicate that IL-13 does not influence either the adhesion of the HASM cells to their substrate or the mechanical properties of the cytoskeleton, as changes in either of these properties have been shown to alter baseline stiffness (22, 25). ISO acts on 2-adrenergic receptors that couple to the stimulatory G-protein, Gs, the -subunit of which activates adenylyl cyclase to produce cAMP. Increased cAMP activates protein kinase A (PKA) which results in relaxation of airway smooth muscle through effects on K channels, Na /K ATPases, Ca2 sequestration, Ca2 sensitivity of myosin, and IP3 formation (43). The observation that IL-13 had no effect on cell stiffness responses to db-cAMP, a cell permeant analog of cAMP that directly activates PKA (Figure 6), suggests that IL-13 does not alter either the ability of cAMP to activate PKA or the targets of PKA that ultimately mediate cell relaxation. These results also suggest that the effect of IL-13 on cell stiffness responses to ISO is unlikely to be related to nonspecific effects on the ability of the cells to relax. Instead, the results suggest that IL-13 acts “upstream” of PKA activation, at the level of cAMP formation. Indeed, pretreatment with IL-13 reduced ISO-induced cAMP formation to only about onethird of the levels obtained in control cells. As with cell stiffness responses to ISO, there was no significant effect of IL-4 on ISO-induced cAMP formation. Since the data were gathered in the presence of the phosphodiesterase inhibitor IBMX, cytokine-induced changes in cAMP degradation are unlikely to have contributed to these responses. The observation that cAMP formation stimulated by forskolin, which directly activates adenylyl cyclase, was not influenced by IL-13 suggests that the effect of IL-13 is likely to be upstream of cyclase activation, at the level of the -receptor, Gs, or their coupling. IL-1 also decreases -adrenergic responsiveness of cultured HASM. The mechanistic basis for the effect of IL-1 involves COX-2-generated prostanoids (20), which appear to act by increasing basal cAMP levels, resulting in activation of PKA, and consequent phosphorylation of the -adrenergic receptor, uncoupling it from Gs. The mechanisms of action of IL-13 and IL-1 on -adrenergic responsiveness are clearly

Laporte, Moore, Baraldo, et al.: IL-13 Induces -Adrenergic Hyporesponsiveness

different, as IL-13 did not induce COX-2 expression in these cells (data not shown). We have recently reported that IL-1-induced phosphorylation of ERK is required for IL-1-induced -adrenergic hyporesponsiveness in HASM cells (28). IL-13 also induces potent ERK activation (Figure 4), and ERK phosphorylation also appears to be important for IL-13-induced changes in -adrenergic responses, as the MEK inhibitor U0126 restored the ability of IL-13-treated cells to respond to ISO (Figure 8). Because IL-4 also caused ERK phosphorylation but did not affect responses to ISO, it is clear that although phosphorylation of ERK is necessary for the changes in -adrenergic responsiveness induced by IL-13, it is not sufficient to induce these changes and other signaling pathways are also involved. It is possible that differences between IL-4 and IL-13 in the time course of STAT-6 phosphorylation lead to the observed differences in their effects on -adrenergic responsiveness. In this respect, it is important to note that gene chip experiments indicate that the panel of genes expressed by HASM cells in response to IL-13 and IL-4, although having some overlap, is not the same (44). In any event, the observation that IL-4 and IL-13 can have different effects in HASM cells is not without precedent. IL-4 has been shown to inhibit HASM cell mitogenesis induced by growth factors (10), whereas a recent preliminary report indicates that IL-13 causes mitogenesis (30). Similarly, IL-4 inhibits IL-1-induced MCP-1 and MCP-2 expression in HASM cells, but IL-13 does not (33). In summary, our results indicate that IL-13 and IL-4 receptors are expressed on HASM cells and that ligation of these receptors leads to activation of STAT-6 and ERK signaling pathways. Our results also indicate that IL-13, but not IL-4, decreases -adrenergic responsiveness in HASM cells and the effect of IL-13 is likely to be exerted upstream of adenylyl cyclase activation, at the level of -receptor, Gs, or their coupling. It is possible that these direct effect of IL-13 on HASM cells may contribute at least in part to the airway narrowing observed in patients with asthma. Acknowledgment : The authors gratefully acknowledge the help of Drs. Geoff Maksym and Ben Fabry for their help in maintaining the magnetometer in the magnetic twisting cytometry experiments.

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