Division of Pulmonary Medicine, Joseph Stokes, Jr. Research Institute, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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In testing the hypothesis that
interleukin-4 receptor -subunit (IL-4R
)-coupled signaling
mediates altered airway smooth muscle (ASM) responsiveness in the
atopic sensitized state, isolated rabbit tracheal ASM segments were
passively sensitized with immunoglobulin E (IgE) immune complexes, both
in the absence and presence of an IL-4R
blocking antibody
(anti-IL-4R
Ab). Relative to control ASM, IgE-sensitized tissues
exhibited enhanced isometric constrictor responses to administered ACh
and attenuated relaxation responses to isoproterenol. These
proasthmatic-like effects were prevented in IgE-sensitized ASM that
were pretreated with anti-IL-4R
Ab. In complementary experiments,
IgE-sensitized cultured human ASM cells exhibited upregulated
expression of IL-13 mRNA and protein, whereas IL-4 expression was
undetected. Moreover, extended studies demonstrated that 1)
exogenous IL-13 administration to naïve ASM elicited augmented
contractility to ACh and impaired relaxation to isoproterenol,
2) these effects of IL-13 were prevented by pretreating the
tissues with an IL-5 receptor blocking antibody, and 3)
IL-13 administration induced upregulated mRNA expression and release of
IL-5 protein from cultured ASM cells. Collectively, these findings
provide new evidence demonstrating that the altered responsiveness of
IgE-sensitized ASM is largely attributed to activation of an intrinsic
Th2-type autocrine mechanism involving IL-13/IL-4R
-coupled release
and action of IL-5 in the sensitized ASM itself.
Th2 cytokines; immunoglobulin E; atopy; asthma; interleukin-13
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INTRODUCTION |
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THE AIRWAYS IN ALLERGIC
ASTHMA are characterized by inflammation, mucus hypersecretion,
obstruction, and constrictor hyperresponsiveness to spasmogenic
stimuli. Although the mechanistic interplay between inflammation and
the associated altered airway responsiveness remains to be elucidated,
there exists ample evidence implicating the production of CD4+ Th2
lymphocyte-derived cytokines, including interleukin (IL)-4,
IL-5, IL-10, and IL-13, in orchestrating the allergic pulmonary
response and its associated changes in airway responsiveness. In this
connection, IL-5 has been shown to regulate the growth,
differentiation, and activation of eosinophils (2, 17,
29), whereas IL-4 and IL-13 were found to play both overlapping and independent roles in regulating IgE isotype switching in B cells
and differentiation of naïve CD4+ T cells into the Th2 phenotype (1, 13, 23, 27). The overlapping biological functions of IL-4 and IL-13 are likely attributed to their commonly shared IL-4 receptor -chain (IL-4R
), which, when activated, dimerizes with other cytokine receptor moieties, subsequently leading
to the activation of various signaling molecules, including the Th2
differentiating factor, STAT6 (1, 20, 27, 30).
When one considers the etiology of the characteristic changes in airway
responsiveness in the atopic asthmatic state, it is of interest to note
that, in light of emerging new evidence, the above paradigm involving
proinflammatory Th2-dependent mechanisms has recently been somewhat
redefined. In this regard, recent studies conducted in animal models of
allergic asthma have demonstrated that phenotypic expression of airway
constrictor hyperresponsiveness may be manifested independently of
pulmonary inflammation (12, 18, 25). Moreover, recent
reports have determined that under specific conditions, including
atopic sensitization (6-9) and rhinovirus exposure
(4, 11), the airway smooth muscle (ASM) itself has the
capacity to autologously elicit proasthmatic-like changes in its
constrictor and relaxant responsiveness secondary to the induced
release and autocrine actions of various proinflammatory cytokines,
including certain Th1- and Th2-type cytokines (4, 9, 10).
In this context, in ultimately leading to altered ASM responsiveness
under conditions of atopic sensitization, immunoglobulin E
(IgE)-dependent autologous release of cytokines by ASM was found to
display a temporal pattern of sequential autocrine action, as shown by
an initial IL-5-mediated induction of the subsequent release of IL-1
in the atopic sensitized ASM (9). Given this evidence,
together with that establishing that IL-4R
-dependent signaling is
fundamentally important in eliciting the Th2 phenotype of cytokine
expression (1, 13, 20, 23, 27, 30), the present study
tested the hypothesis that atopic-dependent (i.e., IgE-mediated)
changes in ASM responsiveness are attributed to IL-4R
-coupled
activation of an intrinsic Th2 mechanism in ASM. The results provide
new evidence demonstrating that IgE sensitization of ASM activates an
endogenously expressed Th2-type autocrine mechanism that involves
induced upregulated expression of IL-13 and that the latter results in
IL-4R
-mediated release and action of IL-5 in the sensitized ASM to
produce proasthmatic-like changes in ASM responsiveness.
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MATERIALS AND METHODS |
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Animals. Twenty-one adult New Zealand White rabbits were used in this study, which was approved by the Biosafety and Animal Research Committee of the Joseph Stokes, Jr. Research Institute at Children's Hospital of Philadelphia. The animals had no signs of respiratory disease for several weeks before the study.
Preparation and IgE sensitization of rabbit ASM tissue.
After general anesthesia with xylazine (10 mg/kg) and ketamine (50 mg/kg), rabbits were killed with an overdose of pentobarbital sodium
(130 mg/kg). As described previously (7, 10), the tracheae
were removed via open thoracotomy, the loose connective tissue and
epithelium were scraped and removed, and the tracheae were divided into
eight ring segments of 6-8 mm in length. Each alternate ring was
incubated for 24 h at room temperature in either vehicle alone
(control) or IgE immune complexes, comprising 15 µg/ml human IgE and
5 µg/ml anti-IgE (goat polyclonal IgG), as previously described by
our laboratory (5). In parallel experiments, 1 h
before incubation in control or IgE-containing medium, ASM segments
were treated with either an IgG2A-type anti-IL-4
receptor blocking antibody (anti-IL-4R
Ab) or an
IgG2A-isotype control Ab (cAb). All the tissues studied
were aerated with a continuous supplemental O2 mixture
(95% O2-5% CO2) during the incubation phase.
Pharmacodynamic studies of ASM responsiveness.
After incubation of the tissue preparations, each ASM segment was
suspended longitudinally between stainless steel triangular supports in
siliconized Harvard 20-ml organ baths. The lower support was secured to
the base of the organ bath, and the upper support was attached via a
gold chain to a Grass FT.03C force transducer from which isometric
tension was continuously displayed on a multichannel recorder. Care was
taken to place the membranous portion of each tracheal segment between
the supports to maximize the recorded tension generated by the
contracting trachealis muscle. The tissues were bathed in modified
Krebs-Ringer solution containing (in mM) 125 NaCl, 14 NaHC03, 4 KCl, 2.25 CaCl · 2 H20, 1.46 MgS04 · H20, 1.2 NaH2P04, and 11 glucose. The baths were aerated
with 5% CO2 in oxygen, a pH of 7.35-7.40 was
maintained, and the organ bath temperature was held at 37°C. Passive
resting tension of each ASM segment was set at 1.5-2.0 g after the
tissue had been passively stretched to a tension of 8 g to
optimize its resting length for contraction, as previously described
(8, 9). The tissues were allowed to equilibrate in the
organ baths for 45 min, at which time each tissue was primed with a
1-min exposure to 104 M ACh. Cholinergic contractility
was subsequently assessed in the ASM segments by cumulative
administration of ACh in final bath concentrations ranging from
10
10 to 10
3 M. Thereafter, the tissues were
repeatedly rinsed with fresh buffer, and, subsequently, relaxation
dose-response curves to isoproterenol
(10
10-10
4 M) were conducted after the
tissues were half-maximally contracted with their respective
ED50 doses of ACh. The initial constrictor dose-response
curves to ACh were analyzed in terms of the tissues' maximal isometric
contractile force (Tmax) to the agonist. The subsequent
relaxation responses to isoproterenol were analyzed in terms of
%maximal relaxation (Rmax) from the initial level of
active cholinergic contraction, and sensitivity to the relaxing agent
was determined as the corresponding pD50 value (i.e.,
geometric mean ED50 value) associated with 50% of
Rmax.
Preparation and IgE sensitization of cultured human ASM cells.
Cultured human ASM cells were obtained from Clonetics (San Diego, CA).
The ASM cells were derived from two male donors, 16 and 21 yr of age,
who had no evidence of lung disease. The cells were characterized by
the manufacturer with specific markers to confirm their selective
smooth muscle phenotype and to exclude contamination with other cell
types. The cells were grown in smooth muscle basal medium (SMBM)
supplemented with 5% fetal bovine serum, insulin (5 ng/ml), epidermal
growth factor (10 ng/ml; human recombinant), fibroblast growth factor
(2 ng/ml; human recombinant), gentamicin (50 ng/ml), and amphotericin B
(50 ng/ml). The experimental protocol involved growing the
cells to confluence in the above medium. Thereafter, in separate
experiments using the same donor cell preparations, the cells were
starved in unsupplemented SMBM for 24 h, at which time the cells
were treated for 0, 3, 6, 12, and 24 h with vehicle alone, IgE
immune complexes, or exogenously-administered IL-13, each in the
absence and presence of anti-IL-4RAb. The cells were then examined
for mRNA expression of IL-4, IL-13, and IL-5, intracellular protein
expression of IL-13 and IL-5, and elaboration of IL-5 protein into the
cell culture medium, as described below.
Determination of IL-4, IL-13, and IL-5 mRNA expression in human
ASM cells.
Total RNA was isolated from the ASM cell preparations with the
modified guanidinium thiocyanate phenol-chloroform extraction method to
include proteinase K (in 5% SDS) for digestion of protein in the
initial RNA pellet, as previously described by our laboratory (10, 11). The concentration of each RNA sample was
determined spectrophotometrically. This procedure consistently produced
yields of 15-25 µg of intact RNA from each T-75 flask of
cultured human ASM cells. To analyze for mRNA expression of the IL-4,
IL-13, and IL-5 genes, we used a RT-PCR protocol that included
human-specific primers for these genes, as well as for the
constitutively expressed -actin gene. cDNA was synthesized from
total RNA isolated from ASM cells incubated for 0, 3, 6, 12, and
24 h in control or IgE-containing medium or exposed to IL-13 in
the absence and presence of anti-IL-4R
Ab. The cDNA was primed with
oligo(dT)12-18 and extended with Superscript II RT
(Gibco BRL). The PCR was used to amplify the specific products from
each cDNA reaction, based on the published sequences of the human IL-4,
IL-13, IL-5, and
-actin genes, and including the following primer
sets: IL-4: 5'-primer: 5'-GTGCGATATCACCTTACAGG-3', 3'-primer:
5'-AACGTACTCTGGTTGGCTTA-3', product is 321 bp; IL-13: 5'-primer:
5'-TGAGGAGCTGGTCAACATCA-3', 3'-primer: 5'-TTTACAAACTGGGCCACCTC-3', product is 249 bp; IL-5: 5'-primer: 5'-GAGGATGCTTCTGCATTTGA-3', 3'-primer: 5'-GGTGTTCATTACACCAAGAA-3', product is 383 bp;
-actin: 5'-primer: 5'-GAGAAGAGCTACGAGCTGCCTGAC-3', 3'-primer:
5'-CGGAGTACTTGCGCTCAGGAGGAG-3', product is 419 bp. The cycling profile
used was as follows: denaturation: 95°C for 1 min; annealing:
52-55°C for 1 min; and extension: 72°C for 1 min, with 35, 30, 25, and 25 cycles for the IL-4, IL-13, IL-5, and
-actin genes,
respectively. The number of cycles was determined to be in the linear
range of the PCR products. The PCR reactions for the primers were
performed using equivalent amounts of cDNA prepared from 2.5 µg of
total RNA. Equal aliquots of each PCR reaction were then run on a 1.2%
agarose gel and subsequently transferred to a Zeta-probe membrane
overnight in 0.4 N NaOH. After capillary transfer, the DNA was
immobilized by ultraviolet cross-linking using a Stratalinker UV
Crosslinker 2400 at 120,000 µJ/cm2 (Stratagene).
Prehybridization in a Techne hybridization oven was conducted for
2-3 h at 42°C in 50% formaldehyde, 7% (wt/vol) SDS, 0.25 M
NaCl, 0.12 M Na2HP04 (pH 7.2), and 1 mM EDTA.
Hybridization was for 20 h at 42°C in the same solution. The
IL-4, IL-13, IL-5, and
-actin DNA levels were assayed by Southern
blot analysis using 32P-labeled probes, prepared by pooling
several RT-PCR reactions for the individual PCR fragments and purifying
them from a 1.2% agarose gel using the Qiaex II agarose gel extraction
kit. The individual PCR products were subsequently sequenced for
confirmation. Washes were as follows: 1 × 15 min in 2× SSC,
0.1% SDS; 1 × 15 min in 0.1× SSC, 0.1% SDS both at room
temperature, and 2 × 1 min at 50°C in 0.1× SSC, 0.1% SDS.
Determination of IL-13 and IL-5 intracellular proteins in ASM cells by flow cytometry. Intracellular protein expression of IL-13 and IL-5 was examined in the cultured human ASM cells with a Coulter EPICS Elite flow cytometer (Coulter EPICS Division, Hialeah, FL) equipped with a 5-W argon laser operated at 488 nM and 300-mW output. Fluorescence signals were accumulated as two-parameter fluorescence histograms, with both percent positive cells and mean channel fluorescence intensity (MFI) being recorded. Cells treated for 24 h with control or IgE-containing medium were carefully washed, scraped from the culture flasks, and then resuspended in PBS buffer. The cells were then dispersed by pipetting through a 23-gauge needle and orbital shaking, and subsequently fixed and permeabilized using reagents provided in a commercially available cell fixation/permeabilization kit (PharmMingen, San Diego, CA). The cells were then stained with mouse anti-human monoclonal antibodies to IL-13 and IL-5. To examine for nonspecific binding, the primary antibody was replaced by immunoglobulins of the same isotype following the manufacturer's protocol, with mouse IgG1 serving as a negative control. After serial washing, the cells were stained with FITC-conjugated goat anti-mouse secondary antibody. The antibody-stained cells were then evaluated by flow cytometry and analyzed with the Elite Immuno 4 statistical software (Coulter EPICS Division). Fluorescence intensities are expressed as percent positive cells as well as MFI.
ELISA measurement of IL-5 protein release. IL-5 protein levels were also assayed in the culture media of ASM cells that were exposed for varying durations up to 24 h to vehicle alone, exogenous IL-13 (20 ng/ml), or IgE immune complexes. The IL-5 protein levels were quantitatively assessed using an enzyme-specific immunoassay, as previously described (9). The latter assay was performed using a double-antibody sandwich strategy in which an ACh esterase, Fab-conjugated IL-5-specific secondary antibody, is targeted to a first IL-5-captured antibody. The enzymatic activity of the ACh was measured spectrophototometrically, and, relative to a linear standard curve, the results were used to quantify the amount of the targeted IL-5 present in the culture media.
Statistical analysis. Unless otherwise indicated, the results are expressed as mean ± SE values. Statistical analysis of the ASM constrictor and relaxation dose-response relationships was performed using ANOVA with multiple comparison of means, and analysis of the flow cytometric and ELISA data was conducted using the two-tailed Student's t-test. P values <0.05 were considered significant.
Reagents.
The human ASM cells and SMBM were obtained from Clonetics (San Diego,
CA). The IL-4, IL-13, IL-5, and -actin primers were obtained from
Integrated DNA Technologies (Coralville, IA). The IL-13 and IL-5
intracellular staining antibodies used in the flow cytometric studies
were purchased from BioSource International (Camarillo, CA). The
anti-IL-4R
antibody, the IL-5 ELISA kit, and the mouse anti-human
IL-5 primary antibody and the anti-mouse secondary antibody used in the
protein assay studies were purchased from R&D Systems (Minneapolis,
MN). ACh and isoproterenol were purchased from Sigma (St. Louis, MO).
All drug concentrations are expressed as final both concentrations.
Isoproterenol and ACh were freshly made for each experiment and were
dissolved in normal saline to prepare 10
3 M stock solutions.
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RESULTS |
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Role of IL-4R in regulating agonist responsiveness in
IgE-sensitized ASM.
To determine the role of IL-4R
activation in regulating ASM
responsiveness in the IgE-sensitized state, agonist-mediated constrictor and relaxation responses were compared in paired isolated rabbit ASM segments 24 h after exposure to IgE immune complexes (6), both in the absence and presence of an
anti-IL-4R
Ab or an isotype cAb. In agreement with our earlier
findings (6), relative to control (vehicle exposed)
tissues, the constrictor responses to exogenously administered ACh were
significantly increased in IgE-treated ASM (Fig.
1). Accordingly, the mean ± SE
Tmax values amounted to 97.8 ± 7.6 and 122.9 ± 9.0 g/g ASM wt in the control and IgE-sensitized tissues, respectively
(P < 0.01). These increased constrictor responses to
ACh were abrogated in IgE-sensitized ASM that were pretreated with a
maximally effective concentration (0.01 µg/ml) of anti-IL-4R
Ab
(Fig. 1, open squares), whereas pretreatment with an IgG2A
isotype cAb had no effect (Fig. 1, filled squares). Moreover, in
related experiments, neither anti-IL-4R
Ab nor the isotype cAb was
found to appreciably affect the ASM constrictor responsiveness to ACh
in control tissues (data not shown).
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Effects of IgE sensitization on ASM expression of IL-4 and IL-13.
We previously reported that ASM cells express IL-4R protein on their
cell surface and that IL-4R
membrane protein expression is
upregulated after exposure of the cells to high IgE-containing atopic
asthmatic serum (10). In light of this earlier evidence, together with the above results implicating a role for IL-4R
in
mediating the observed changes in agonist responsiveness in IgE-sensitized ASM, we next examined whether cultured human ASM cells
express mRNAs for IL-4 and IL-13, the endogenous ligands for
IL-4R
, and whether mRNA expression of the latter cytokines is
modulated in the IgE-sensitized state. For the mRNA analyses, Southern
blots were prepared and probed with human cDNA probes specific for the
human IL-4 and IL-13 genes, and a 419-bp
-actin probe was also
prepared as a control for gel loading (see MATERIALS AND
METHODS). There was no detectable IL-4 mRNA signal and an absent
or only faintly detected mRNA signal for IL-13 in control (untreated)
cells. Moreover, as shown by a representative experiment in Fig.
3, IL-4 mRNA expression was also
undetected in IgE-sensitized cells. In contrast, relative to the
unaltered constitutively expressed
-actin signal, IL-13 mRNA
expression was progressively enhanced at all times for up to 24 h
after exposure of the cells to IgE. Qualitatively, the temporal pattern
of upregulated IL-13 mRNA expression in the IgE-treated cells appeared
similar in three separate experiments, with distinctly increased IL-13
mRNA detected as early as 3 h after exposure of the cells to IgE
and a somewhat reduced intensity of the mRNA signal detected at 24 h (i.e., as per Fig. 3). For all three experiments, the average maximal
intensity of the IL-13 mRNA signals detected at 12 h, each
expressed as a ratio of the respective intensity of the
-actin
signal, amounted to 70.7 ± 9.1-fold above the corresponding mean
intensity ratio detected at the 0-h time point.
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Role of IL-5 in mediating IL-13-induced changes in ASM
responsiveness.
Since, under experimental conditions comparable to those described
herein, induced autocrine release and action of IL-5 were previously
implicated in mediating similar observed changes in agonist
responsiveness in atopic sensitized ASM (9), given the
above present observations, a series of studies was conducted to
further elucidate the role of IL-13 in regulating ASM responsiveness and investigate whether its action is mechanistically coupled to the
previously reported contribution of IL-5. In addressing these issues,
in our initial experiments we examined the effects of exogenous
administration of human recombinant IL-13 to naïve ASM tissues
on their agonist constrictor and relaxant responsiveness, both in the
absence and presence of pretreatment of the tissues with an
anti-IL-5RAb. As shown in Fig. 5,
exposure of tissues for 24 h to a maximally effective
concentration of IL-13 (20 ng/ml) elicited significantly increased ASM
constrictor responsiveness to ACh, wherein the Tmax values
in the IL-13-treated averaged 129.7 ± 8.6 g/g ASM, compared with
the mean Tmax value of 115.7 ± 8.7 g/g ASM obtained
in control (vehicle treated) ASM (P < 0.05). Moreover,
as demonstrated in Fig. 5, the heightened constrictor responses to ACh
were completely abrogated in IL-13-treated tissues that were pretreated
with anti-IL-5RAb (10 µg/ml), whereas an isotype cAb had no effect.
Comparably, relative to their respective controls, ASM treated with
IL-13 also exhibited significantly attenuated relaxation responses to
isoproterenol (Fig. 6), with mean ± SE Rmax values amounting to 35.69 ± 4.97 vs.
53.70 ± 6.27% in the IL-13-treated vs. control ASM, respectively
(P < 0.01). Furthermore, this impaired relaxation
responsiveness to isoproterenol was also completely inhibited in
IL-13-exposed ASM that were pretreated with anti-IL-5RAb (Fig. 6; open
squares), whereas the isotype control Ab had no effect (Fig. 6; filled
squares).
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Effects of IL-13 on IL-5 mRNA and protein expression.
Given the above observations, we next investigated whether the action
of exogenously administered IL-13 is associated with an induced altered
endogenous expression of IL-5 by the IL-13-exposed ASM. In these
studies, cultured human ASM cells were exposed to IL-13, both in the
absence and presence of IL-4RAb, for varying durations up to 24 h. Thereafter, in one series of experiments, the cells were harvested
for analysis of temporal changes in IL-5 mRNA expression. The IL-5 mRNA
signal was only faintly detected in control (vehicle-exposed) ASM
cells. In contrast, as depicted in Fig.
7, relative to the unaltered
constitutively expressed
-actin mRNA signal, IL-5 mRNA expression
was progressively enhanced in the IL-13-treated cells at all times for
up to 24 h after IL-13 exposure. Moreover, as further shown in
Fig. 7, the IL-13-induced upregulated expression of IL-5 mRNA was
largely ablated in ASM cells that were concomitantly treated with
IL-4R
Ab.
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DISCUSSION |
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It is well established that IL-4R signaling is required for
differentiation of naïve T lymphocytes into Th2
cytokine-producing cells. This phenomenon has been associated with
binding of STAT6 protein to the activated IL-4R subunit, where it
becomes tyrosyl phosphorylated, and migration of phosphorylated
dimerized STAT6 to the nucleus where, together with other transcription
factors, transcription of various IL-4R-inducible genes is activated
(14, 16, 19, 20, 22, 30). Recently, IL-4R
signaling has also been identified as a potent regulator of the characteristic airway
constrictor hyperresponsiveness seen in murine models of allergic
asthma (3, 24, 26, 28). This action of IL-4R
activation
was found to be largely mediated by IL-13 binding, as the airway
constrictor hyperresponsiveness was prevented in IL-4R
-deficient
mice and in mice receiving a soluble IL-13
2-IgG Fc fusion protein to
neutralize IL-13 (3, 28). Although these findings clearly
emphasize a crucial role for IL-4R-coupled signaling in mediating the
proasthmatic state, the mechanism of action of IL-4R activation in
regulating the induction of changes in airway responsiveness remains to
be elucidated. In this context, it is relevant to note that IL-4R
expression was recently identified in human ASM cells (10,
15) and that ASM cell expression of IL-4R
was found to be
upregulated in the atopic asthmatic sensitized state, in association
with upregulated expression of other Th2-type cytokines, including IL-5
and granulocyte-monocyte colony-stimulating factor
(10). In light of this evidence, the present study
tested the hypothesis that atopic-dependent (i.e., IgE mediated)
changes in ASM responsiveness are attributed to IL-4R
-coupled
activation of an intrinsic Th2 mechanism in ASM. The results
demonstrate that IgE sensitization of ASM activates an endogenously
expressed Th2-type autocrine mechanism that involves 1)
IgE-induced upregulated expression of IL-13 by the sensitized ASM and
2) the latter cytokine acting in an autocrine fashion to
mediate IL4R
-coupled release and action of IL-5, which evokes
proasthmatic-like changes in ASM responsiveness.
To our knowledge, the present observations are the first to demonstrate
that IgE sensitization of ASM elicits the sequential autocrine release
of IL-13 and IL-5 by the sensitized ASM itself and that this Th2-type
autocrine response contributes to the changes in ASM responsiveness
that characterize the atopic asthmatic phenotype, including heightened
agonist-mediated constrictor responsiveness and impaired
beta-adrenoceptor-mediated ASM relaxation (Figs. 1 and 2). In the
evaluation of the collection of present findings, certain noteworthy
considerations are raised. Among these, it is relevant to note that,
despite the reported presence of IL-4R in ASM cells (10,
15) and its upregulated expression in the atopic sensitized
state (10), we found no evidence for ASM cell expression
of IL-4 mRNA or protein under control or IgE-sensitized conditions. In
contrast, IL-13 mRNA expression was present and distinctly increased as
early as 3 h after incubation of the cells with IgE (Fig. 3). Of
interest, this temporal pattern of induced IL-13 mRNA expression
closely paralleled the time course of induction of IL-5 mRNA by IL-13
administration (Fig. 7). To the extent that, under experimental
conditions comparable to those described herein, the induced mRNA
expression and associated release of IL-5 protein by atopic asthmatic
serum-sensitized ASM were previously shown to elicit the same observed
changes in ASM responsiveness (9), our present results
suggested that the temporal association between the induced changes in
IL-13 and IL-5 expression may be mechanistically related. In addressing
this possibility, our extended observations demonstrated that
1) exogenous administration of IL-13 induced both an
increased expression of IL-5 mRNA (Fig. 7) and release of IL-5 protein
(Fig. 8); and 2) comparable to the effect of IgE sensitization, exogenous IL-13 administration to naïve ASM
tissues elicited proasthmatic-like changes in ASM constrictor and
relaxant responsiveness that were prevented by pretreating the tissues with an IL-5RAb (Figs. 5 and 6). These findings, together with the
observations that IgE sensitization induced the release of IL-5 protein
and that this effect was inhibited in ASM cells pretreated with
anti-IL-4R
Ab (Figs. 8 and 9), support the concept of a causal association between induced IL-13 and IL-5 expression in the
IgE-sensitized state. Accordingly, the results are consistent with the
notion that IgE-induced IL-5 release by ASM is mechanistically
dependent on the autocrine induction and action of IL-13 in the
IgE-exposed ASM.
Our collection of findings is based on studies conducted using rabbit
ASM tissues and cultured human ASM cells. Although the experiments
using these different preparations provided results that were largely
complementary in nature, the issue of potential species differences
warrants consideration. In this regard, it is relevant to note that in
earlier studies using atopic asthmatic serum sensitization of isolated
rabbit ASM (4, 6, 7, 9), we found changes in ASM
constrictor and relaxant responsiveness that, in general, were
qualitatively similar to those reported in a number of other studies
conducted on isolated human airways passively sensitized with atopic
asthmatic serum or with exogenously administered IgE (see review Ref.
21). Moreover, we found that atopic asthmatic serum
sensitization elicited qualitatively similar upregulated
expression of the low affinity receptor for IgE, FcRII (i.e.,
CD23), in both rabbit and human ASM cells (5, 6), as well
as increased release of IL-1
protein from both cell types (4). Similarly, rhinovirus inoculation of rabbit and human ASM cells was also found to provoke the release of IL-
from both cell types (4, 11). Finally, in concert with the present observations on IL-13-induced changes in agonist responsiveness in
rabbit ASM tissues, Laporte et al. (15) recently reported a similar attenuated
-adrenergic responsiveness in cultured human ASM cells treated with IL-13. Thus the findings from these earlier reports, together with those of the present study, suggest that there
exists a good concordance between rabbit and human ASM cells, at least
with respect to the effects of atopic sensitization and IL-13
administration on ASM cell function. Although it remains to be
established whether such an interspecies concordance also exists in
vivo, it is noteworthy that, in agreement with the present in vitro
observations, in vivo administration of an IL-4R antagonist in murine
models of allergic asthma was shown to prevent the induction of changes
in airway responsiveness (3, 24, 26, 28) and to inhibit
allergen-induced release of certain Th2 cytokines (notably including
IL-5) into the bronchoalveolar lavage fluid (24).
The central findings of this study lend an extended scope to the
prevailing concept of a Th2 cytokine-dependent overall mechanism underlying the pathobiology of allergic asthma. In this regard, whereas
the contemporary Th2 paradigm related to allergic asthma largely
reflects the role played by CD4+ T cells expressing the Th2 phenotype
of cytokine release, the present findings expand this model to include
an apparent Th2-type autocrine role intrinsically expressed by the ASM
itself in the IgE (atopic)-sensitized state. The ability of atopic
asthmatic serum-sensitized ASM to autologously express both Th1- and
Th2-type cytokines, as well as the pleiotropic proinflammatory cytokine
IL-1, has been previously demonstrated (4, 9, 10), and
this phenomenon was largely attributed to activation of Fc
RII
(CD23), expressed on the ASM cell surface, by the elevated IgE present
in the atopic sensitizing serum (4, 6). In light of this
previous information, together with the observations presented in the
present study, there is ample evidence to support the notion that,
notwithstanding the crucial role played by CD4+ Th2 lymphocytes, an
extended autocrine Th2-type cytokine network involving
IL-13/IL-4R
-coupled induced release and action of IL-5 also exists
in ASM that, when activated in the IgE-sensitized state, contributes to
the proasthmatic changes in ASM responsiveness.
In conclusion, the present study investigated the role and mechanism of
action of IL-4R signaling in regulating the altered agonist
responsiveness of IgE-sensitized ASM. The results demonstrate that
1) the induced proasthmatic-like changes in agonist
constrictor and relaxant responsiveness in IgE-sensitized ASM are
prevented by blocking the IL-4R in the sensitized ASM, 2)
both IL-13 and IL-5 mRNA and protein expression are upregulated in
IgE-sensitized ASM, 3) IL-13 elicits upregulated IL-5 mRNA
expression and release of IL-5 protein from ASM cells, and
4) the latter IL-13/IL-4R
-coupled induced autocrine
release of IL-5 is responsible for mediating proasthmatic changes in
ASM responsiveness. Collectively, these findings lend extended support
to the concept that, apart from the important role played by
inflammatory cells, the ASM itself constitutes a Th2-type cytokine
autocrine system that, when activated in the atopic-sensitized state,
elicits autologous proasthmatic perturbations in airway responsiveness.
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ACKNOWLEDGEMENTS |
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The authors thank M. Brown for typing the manuscript.
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FOOTNOTES |
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This work was supported in part by National Heart, Lung and Blood Institute Grants HL-31467, HL-58245, HL-61038, and HL-59906.
Address for reprint requests and other correspondence: M. M. Grunstein, Div. of Pulmonary Medicine, The Children's Hospital of Philadelphia, Univ. of Pennsylvania School of Medicine, 34th St. & Civic Center Blvd., Philadelphia, PA 19104 (E-mail: grunstein{at}emailchop.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajplung.00343.2001
Received 27 August 2001; accepted in final form 12 October 2001.
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