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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.
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INTRODUCTION |
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INTRODUCTION
METHODS
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DISCUSSION
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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 domain, 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, IL-13RI, 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 RT-PCR. 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) through direct effects on airway smooth muscle.
Because our results indicated that IL-4 and IL-13 receptors
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.
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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 µM), 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 µg/ml insulin and 5 µg/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 µg 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 µl reaction mixture contained 1 µl (200 µM)
dNTPs, 5 µl each primers, 5 µl 10× PCR buffer, 5 µl cDNA, and 0.4 µl Taq polymerase (Perkin-Elmer, Foster City, CA) and 33.6 µl 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 IL-13RI, 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.
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Western Blotting
Confluent HASM cells were serum deprived and treated with IL-4 or
IL-13. Medium was removed, and cells were washed with phosphate-buffered saline (PBS) and then lysed in 400 µl of extraction buffer
(10 mM Tris-HCl buffer with 50 mM NaCl, 50 mM NaF, 10 mM
D-serine, 1 µM EDTA, 1 µM ethyleneglycoltetraacetic acid [EGTA],
1% sodium dodecyl sulfate [SDS], 1% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 1 µg/ml pepstatin, 10
2 U/
ml aprotinin). Cells were scraped off flasks, passed through a 25- and
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 µg/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 Tris-buffered 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-STAT-6, 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 µl of a solution containing the ISO or dibutyryl-cAMP
was added to the cell well that contained 200 µl 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).
cAMP Formation
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), harvested 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 µM 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 IL-13R 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 measures 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.
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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, IL-13RII, 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.
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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).
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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, which was significant at doses as low as 0.3 ng/ml and peaked at approximately 10 ng/ml.
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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 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.
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IL-13 But Not IL-4 Causes -Adrenergic Hyporesponsiveness
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. Db-cAMP caused a concentration dose-related decrease in cell
stiffness consistent with previous reports (18, 20, 25), but IL-13 (50 ng/ml) had no effect on db-cAMP responses (Figure 6).
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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 one-third 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).
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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-13-induced 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
that U0126 (10 µM) 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 µM) 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.
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Our results demonstrate that HASM cells express IL-4R, IL-13RI 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
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
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-13-induced 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 IL-4R. Another possible explanation is that the presence of IL-13RII (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 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). 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 one-third 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
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.
| |
Footnotes |
|---|
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: sshore{at}hsph.harvard.edu
(Received in original form August 10, 2000 and in revised form February 12, 2001).
Acknowledgments: 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.
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.
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References |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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 Q. Peng, T. Matsuda, and S. J. Hirst Signaling Pathways Regulating Interleukin-13-stimulated Chemokine Release from Airway Smooth Muscle Am. J. Respir. Crit. Care Med., March 1, 2004; 169(5): 596 - 603. [Abstract] [Full Text] [PDF]
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D. S. Faffe, T. Whitehead, P. E. Moore, S. Baraldo, L. Flynt, K. Bourgeois, R. A. Panettieri, and S. A. Shore IL-13 and IL-4 promote TARC release in human airway smooth muscle cells: role of IL-4 receptor genotype Am J Physiol Lung Cell Mol Physiol, October 1, 2003; 285(4): L907 - L914. [Abstract] [Full Text] [PDF]
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H. C. Atherton, G. Jones, and H. Danahay IL-13-induced changes in the goblet cell density of human bronchial epithelial cell cultures: MAP kinase and phosphatidylinositol 3-kinase regulation Am J Physiol Lung Cell Mol Physiol, September 1, 2003; 285(3): L730 - L739. [Abstract] [Full Text] [PDF]
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J. Reibman, Y. Hsu, L. C. Chen, B. Bleck, and T. Gordon Airway Epithelial Cells Release MIP-3{alpha}/CCL20 in Response to Cytokines and Ambient Particulate Matter Am. J. Respir. Cell Mol. Biol., June 1, 2003; 28(6): 648 - 654. [Abstract] [Full Text] [PDF]
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S. Baraldo, D. S. Faffe, P. E. Moore, T. Whitehead, M. McKenna, E. S. Silverman, R. A. Panettieri Jr., and S. A. Shore Interleukin-9 influences chemokine release in airway smooth muscle: role of ERK Am J Physiol Lung Cell Mol Physiol, June 1, 2003; 284(6): L1093 - L1102. [Abstract] [Full Text] [PDF]
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C.-D. Huang, O. Tliba, R. A. Panettieri Jr., and Y. Amrani Bradykinin Induces Interleukin-6 Production in Human Airway Smooth Muscle Cells: Modulation by Th2 Cytokines and Dexamethasone Am. J. Respir. Cell Mol. Biol., March 1, 2003; 28(3): 330 - 338. [Abstract] [Full Text] [PDF]
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S. S. An, R. E. Laudadio, J. Lai, R. A. Rogers, and J. J. Fredberg Stiffness changes in cultured airway smooth muscle cells Am J Physiol Cell Physiol, September 1, 2002; 283(3): C792 - C801. [Abstract] [Full Text] [PDF]
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Y. Shibata, T. Kamata, M. Kimura, M. Yamashita, C.-R. Wang, K. Murata, M. Miyazaki, M. Taniguchi, N. Watanabe, and T. Nakayama Ras Activation in T Cells Determines the Development of Antigen-Induced Airway Hyperresponsiveness and Eosinophilic Inflammation J. Immunol., August 15, 2002; 169(4): 2134 - 2140. [Abstract] [Full Text] [PDF]
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S. J. Hirst, M. P. Hallsworth, Q. Peng, and T. H. Lee Selective Induction of Eotaxin Release by Interleukin-13 or Interleukin-4 in Human Airway Smooth Muscle Cells Is Synergistic with Interleukin-1beta and Is Mediated by the Interleukin-4 Receptor alpha -Chain Am. J. Respir. Crit. Care Med., April 15, 2002; 165(8): 1161 - 1171. [Abstract] [Full Text] [PDF]
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M. J. TOBIN Asthma, Airway Biology, and Nasal Disorders in AJRCCM 2001 Am. J. Respir. Crit. Care Med., March 1, 2002; 165(5): 598 - 618. [Full Text] [PDF]
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S. Shore Airway smooth muscle: new tricks for an old dog Am J Physiol Lung Cell Mol Physiol, March 1, 2002; 282(3): L518 - L519. [Full Text] [PDF]
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M. M. Grunstein, H. Hakonarson, J. Leiter, M. Chen, R. Whelan, J. S. Grunstein, and S. Chuang IL-13-dependent autocrine signaling mediates altered responsiveness of IgE-sensitized airway smooth muscle Am J Physiol Lung Cell Mol Physiol, March 1, 2002; 282(3): L520 - L528. [Abstract] [Full Text] [PDF]
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 H. Akiho, P. Blennerhassett, Y. Deng, and S. M. Collins Role of IL-4, IL-13, and STAT6 in inflammation-induced hypercontractility of murine smooth muscle cells Am J Physiol Gastrointest Liver Physiol, February 1, 2002; 282(2): G226 - G232. [Abstract] [Full Text] [PDF]
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A. R. Simon, S. Takahashi, M. Severgnini, B. L. Fanburg, and B. H. Cochran Role of the JAK-STAT pathway in PDGF-stimulated proliferation of human airway smooth muscle cells Am J Physiol Lung Cell Mol Physiol, June 1, 2002; 282(6): L1296 - L1304. [Abstract] [Full Text] [PDF]
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