Division of Pulmonary Medicine, Joseph Stokes, Jr. Research Institute, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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To elucidate the role and mechanism of action of interleukin (IL)-10 in regulating airway smooth muscle (ASM) responsiveness in the atopic asthmatic state, isolated rabbit tracheal ASM segments were passively sensitized with serum from atopic asthmatic patients or nonatopic nonasthmatic (control) subjects in both the absence and presence of an anti-IL-10 receptor blocking antibody (Ab). Relative to control ASM, atopic asthmatic serum-sensitized tissues exhibited enhanced isometric constrictor responses to administered acetylcholine and attenuated the relaxation responses to isoproterenol. These proasthmatic effects were prevented in atopic asthmatic serum-sensitized ASM that was pretreated with anti-IL-10 receptor Ab. In complementary experiments, exposure of cultured human ASM cells to atopic asthmatic serum induced upregulated expression of IL-10 mRNA. Moreover, extended studies demonstrated that 1) exogenous IL-10 administration to naive ASM elicited augmented contractility to acetylcholine and impaired relaxation to isoproterenol, 2) these effects of IL-10 were prevented by pretreating the tissues with an IL-5 receptor Ab, and 3) IL-10 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 atopic asthmatic serum-sensitized ASM is largely attributed to activation of an intrinsic T helper type 2-type autocrine mechanism involving IL-10-mediated release and the action of IL-5 in the sensitized ASM itself.
interleukin; T helper type 2 cytokines; signal transduction; asthma
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INTRODUCTION |
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BRONCHIAL ASTHMA IS
CHARACTERIZED by enhanced agonist-mediated bronchoconstriction,
impaired -adrenoceptor-mediated airway relaxation, and airway
inflammation. Although the mechanism(s) underlying these
inflammation-associated changes in airway responsiveness remain to be
elucidated, substantial evidence accumulated in recent years has
implicated a crucial role for CD4+ T helper (Th) type
2-type cytokines in the pathophysiology of the airway inflammatory
response in allergic asthma and its accompanying changes in airway
responsiveness. Notably, among the Th2 cytokines, interleukin (IL)-4,
IL-13, and IL-5 are known to orchestrate various humoral and cellular
immune responses that are characteristic of allergic asthma, including
IgE synthesis and eosinophil recruitment and activation (3, 14,
16, 21). Accordingly, it is generally believed that
these cytokines modulate airway responsiveness either indirectly
through extended activation of an inflammatory cascade or directly by
acting on the atopic asthmatic serum-sensitized airway smooth muscle
(ASM) itself (4, 5, 12, 15, 26).
Of interest, the above paradigm involving Th2-dependent proinflammatory mechanisms in atopic asthma is currently being somewhat redefined in light of emerging new evidence in animal models of allergic asthma that phenotypic expression of airway constrictor hyperresponsiveness may be manifested independent of pulmonary inflammation (14a, 18, 25). In this regard, recent in vivo studies (14a, 18, 25) in allergen-sensitized mice have demonstrated that the expression and action of the Th2-type cytokine IL-10 is required for the development of airway constrictor hyperresponsiveness but not for pulmonary inflammation or eosinophilia after allergen challenge in the sensitized state. Because IL-10 is generally recognized for its immunosuppressive properties in animal models (1, 19, 28), this recent evidence suggests a potentially important role for IL-10 downstream from the pulmonary inflammatory cascade in regulating airway responsiveness in the allergic asthmatic state. In light of this consideration, the present study was designed to elucidate the role and mechanism of action of IL-10 in regulating agonist-mediated airway constrictor and relaxant responsiveness in atopic asthmatic serum-sensitized ASM. The results provide new evidence demonstrating that the altered agonist responsiveness of atopic asthmatic serum-sensitized ASM is largely attributed to activation of an endogenously expressed Th2-type autocrine mechanism involving sequentially induced IL-10-mediated release and the action of IL-5 in the atopic sensitized ASM itself.
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MATERIALS AND METHODS |
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Animals. Seventeen 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 (Philadelphia, PA). The animals had no signs of respiratory disease for several weeks before the study.
Preparation and sensitization of rabbit ASM tissue. After general anesthesia with xylazine (10 mg/kg) and ketamine (50 mg/kg), the rabbits were killed with an overdose of pentobarbital sodium (130 mg/kg). As previously described (10), the tracheae were removed via an open thoracotomy, the loose connective tissue and epithelium were scraped and removed, and the tracheae were divided into eight ring segments 6-8 mm in length. Each alternate ring was incubated for 24 h at room temperature in either 1) human serum containing >800 IU/ml of IgE obtained from allergic patients with moderate to severe asthma who demonstrated 4-5 or 6+ radioallergosorbent test (RAST)-positive specific IgE concentrations of >17.5 Phadebas RAST units/ml to Dermatophagoides pteronyssimus, D. farinae, or ragweed and who had positive skin tests to these antigens or 2) human serum from nonatopic nonasthmatic (control) individuals with normal serum IgE levels (i.e., <70 IU/ml) and negative skin test reactivity to D. pteronyssimus, D. farinae, and ragweed. In parallel experiments, 1 h before incubation in control or atopic asthmatic serum, ASM segments were treated with either an IgG1-type anti-IL-10 receptor (anti-IL-10R) blocking antibody (Ab), an IgG2-type anti-IL-10 protein neutralizing monoclonal Ab (MAb), or an IgG1-type anti-vascular cell adhesion molecule (VCAM)-1 MAb. 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 NaHCO3, 4 KCl, 2.25 CaCl2 · H2O, 1.46 MgSO4 · H2O, 1.2 NaH2PO4, and 11 glucose. The baths were aerated
with 5% CO2 in O2, 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
(10, 13). 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 acetylcholine (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 to
10
4 M) were conducted after the tissues were
half-maximally contracted with their respective doses
(ED50) of ACh. The initial constrictor dose-response curves
to ACh were analyzed in terms of the maximal isometric contractile
force (Tmax) of the tissues to the agonist. The subsequent
relaxation responses to isoproterenol were analyzed in terms of percent
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 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, 5 ng/ml of insulin, 10 ng/ml of epidermal growth factor (human recombinant), 2 ng/ml of fibroblast growth factor (human recombinant), 50 ng/ml of gentamicin, and 50 ng/ml of amphotericin B. The experimental protocol involved growing the cells to confluence in the above medium. Thereafter, in separate experiments, 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 either 10% human control serum, 10% human atopic asthmatic serum, or serum-free medium in the presence and absence of exogenously administered IL-10. The cells were then examined for the expression of IL-10 and IL-5 mRNAs as well as for the elaboration of IL-5 protein into the cell culture medium as described in Determination of IL-10 and IL-5 mRNA expression in human ASM cells.
Determination of IL-10 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 (12, 13). 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-10 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 ribosomal protein (RP) L7 gene. cDNA was synthesized from the total RNA isolated from ASM cells incubated for 0, 3, 6, 12, and 24 h with control or atopic asthmatic serum or exposed to serum-free medium in the absence and presence of IL-10. The cDNA was primed with oligo(dT)12-18 and extended with SuperScript II reverse transcriptase (GIBCO BRL). The PCR was used to amplify the specific products from each cDNA reaction based on the published sequences of the human IL-10, IL-5, and RPL7 genes and including the following primer sets: 5'-primer 5'-GTACATCCCCCACTGGAAAA-3' and 3'-primer 5'-TTTGGTACAAGGCAAGAGCC-3' (211-bp product) for IL-10, 5'-primer 5'-TATTTGTCCTGGCTGTGCCT-3' and 3'-primer 5'-CTTTCTTGGCCCTCATTCTC-3' (215-bp product) for IL-5, and 5'-primer 5'-AAGAGGCTCTCATTTTCCTGGCTG-3' and 3'-primer 5'-TCCGTTCCTCCCCATAATGTTCC-3' (157-bp product) for RPL7. The cycling profile used was as follows: denaturation at 95°C for 1 min, annealing at 52-55°C for 1.0 min, and extension at 72°C for 1.0 min, with 35, 30, and 25 cycles for the IL-10, IL-5, and RPL7 genes, respectively. The number of cycles was determined to be in the linear range of the PCR products. The PCRs for the primers were performed with equivalent amounts of cDNA prepared from 2.5 µg of total RNA. Equal aliquots of each PCR 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 with 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 Na2HPO4 (pH 7.2), and 1 mM EDTA. Hybridization was for 20 h at 42°C in the same solution. The IL-10, IL-5, and RPL7 DNA levels were assayed by Southern blot analysis with 32P-labeled probes that were prepared by pooling several RT-PCRs for the individual PCR fragments and purifying them from a 1.2% agarose gel with the QIAEX II agarose gel extraction kit. The individual PCR products were subsequently sequenced for confirmation. Washes were as follows: one time for 15 min in 2× SSC-0.1% SDS, one time for 15 min in 0.1× SSC-0.1% SDS (both at room temperature), and two times for 1 min each at 50°C in 0.1× SSC-0.1% SDS.
ELISA measurement of IL-5 protein release. IL-5 protein levels were assayed in the culture medium of ASM cells that were exposed for varying durations up to 24 h to either 10% control or atopic asthmatic serum. The IL-5 protein levels were quantitatively assessed with an enzyme-specific immunoassay as previously described (11). The latter assay was performed with a double-antibody sandwich strategy in which an acetylcholinesterase-F(ab)-conjugated IL-5-specific secondary antibody was targeted first to an IL-5-captured antibody. The enzymatic activity of the acetylcholinesterase was measured spectrophotometrically, and the results were used to quantify, relative to a linear standard curve, the amount of the targeted IL-5 present in the culture medium.
Reagents.
The human ASM cells and SMBM were obtained from Clonetics. The IL-10,
IL-5, and RPL7 primers were obtained from Integrated DNA Technologies
(Coralville, IA). The anti-IL-10R, anti-IL-10, and anti-VCAM-1
neutralizing Abs, the IL-5 ELISA kit, the mouse anti-human IL-5 primary
Ab, and the anti-mouse secondary Ab 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 bath concentrations. Isoproterenol and ACh were made fresh for each experiment and were
dissolved in normal saline to prepare 103 M stock solutions.
Statistical analysis. Unless otherwise indicated, the results are expressed as means ± SE. Statistical analysis was performed with two-tailed Student's t-test or ANOVA with multiple comparison of means where appropriate. P values < 0.05 were considered significant.
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RESULTS |
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Role of IL-10 in regulating agonist responsiveness in atopic
asthmatic serum-sensitized ASM.
To determine the role of IL-10 in regulating ASM responsiveness in the
atopic asthmatic serum-sensitized state, agonist-mediated constrictor
and relaxation responses were separately compared in paired isolated
rabbit ASM segments 24 h after exposure to serum from either
atopic asthmatic individuals or nonatopic nonasthmatic (control)
subjects in both the absence and presence of an anti-IL-10R blocking
Ab, an anti-IL-10 protein neutralizing MAb or an anti-VCAM-1 MAb. As
shown in Fig. 1, relative to
control serum-exposed tissues, the constrictor responses to exogenously
administered ACh were significantly increased in atopic asthmatic
serum-sensitized ASM. Accordingly, the Tmax values amounted
to 109.9 ± 7.9 (SE) and 131.2 ± 9.7 g/g ASM weight in the
control and atopic asthmatic serum-sensitized tissues, respectively
(P < 0.01), representing an average increase in
Tmax of ~ 20% above the control value in the atopic
asthmatic serum-sensitized ASM. These increased constrictor responses
to ACh were abrogated in atopic asthmatic-sensitized tissues that were
pretreated with a maximally effective concentration (1.0 µg/ml) of
anti-IL-10R Ab (Fig. 1), whereas pretreatment with the isotype control
anti-VCAM-1 MAb had no effect. In comparable experiments, we found that
the heightened constrictor responses to ACh were also ablated in atopic
asthmatic serum-sensitized tissues that were pretreated with 0.5 µg/ml of anti-IL-10 MAb (data not shown). Moreover, in related
experiments, neither the anti-IL-10R Ab nor the anti-IL-10 MAb was
found to appreciably affect the ASM constrictor responsiveness to ACh
in control serum-exposed tissues (data not shown).
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Induced mRNA expression of IL-10 and IL-5 in atopic
asthmatic-sensitized ASM.
Grunstein et al. (6) and Hakonarson et al.
(11) previously reported that exposure of naive
ASM to atopic asthmatic serum induces upregulated mRNA expression and
release of the Th2-type cytokine IL-5 by the atopic asthmatic
serum-sensitized tissue. In light of this earlier evidence, together
with the above results implying a role for IL-10 in mediating the
observed changes in agonist responsiveness in atopic asthmatic
serum-sensitized ASM, we next examined whether cultured human ASM cells
endogenously express mRNA for IL-10 and whether expression of the
latter is modulated in the atopic asthmatic serum-sensitized state in
temporal association with the induced changes in IL-5 mRNA expression. For the mRNA analyses, Southern blots were prepared and probed with
human cDNA probes specific for the human IL-10 and IL-5 genes, and a
157-bp RPL7 probe was also prepared as a control for gel loading (see
MATERIALS AND METHODS). As shown by a representative experiment in Fig. 3, the mRNA signals
for IL-10 and IL-5 were only faintly detected relative to the unaltered
constitutively expressed RPL7 signal, at all times after exposure of
the ASM cells to control serum. In contrast, cells exposed to atopic
asthmatic serum displayed notably increased expression of both IL-10
and IL-5 mRNAs at all times for up to 24 h after exposure to the
atopic asthmatic serum-sensitizing serum. Qualitatively, the temporal changes in IL-10 and IL-5 mRNA expression in the atopic asthmatic serum-sensitized cells appeared generally similar, with distinctly increased expression of these mRNAs detected as early as 3 h after exposure of the cells to the sensitizing serum.
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Role of IL-5 in mediating IL-10-induced changes in ASM
responsiveness.
Because, under the same experimental conditions described here, an
induced autocrine release and action of IL-5 were previously implicated
in mediating the observed changes in ASM responsiveness in the atopic
asthmatic serum-sensitized state (6, 11), given the above
present observations, a series of studies was conducted to further
elucidate the role of IL-10 in regulating ASM responsiveness and
investigate whether its action is mechanistically associated with the
previously reported contribution of IL-5. In addressing this issue,
initial experiments examined the effects of the administration of
exogenous IL-10 to naive ASM tissues on their agonist constrictor and
relaxant responsiveness in both the absence and presence of pretreatment of the tissues with an IL-5R -chain blocking Ab. As
shown in Fig. 4, exposure of tissues for
24 h to a maximally effective concentration of IL-10 (25 ng/ml)
elicited significantly increased ASM constrictor responsiveness to ACh,
where the Tmax values in the IL-10-treated tissues averaged
141.1 ± 12.6 g/g ASM compared with the mean Tmax
value of 116.9 ± 11.4 g/g ASM obtained in control
(vehicle-treated) ASM (P < 0.05). Moreover, as further
demonstrated in Fig. 4, the heightened constrictor responses to ACh
were completely abrogated in IL-10-exposed ASM that was pretreated with
anti-IL-5R Ab (10 g/ml). Comparably, relative to the respective control
tissue, ASM treated with IL-10 also exhibited significantly attenuated
relaxation responsiveness to isoproterenol (Fig.
5), with Rmax values
amounting to 54.9 ± 5.0 and 65.1 ± 4.1% in the
IL-10-treated and control ASM, respectively (P < 0.05). Furthermore, these impaired relaxation responses to isoproterenol were also completely inhibited in IL-10-exposed ASM that
was concomitantly treated with an anti-IL-5R Ab (Fig. 5).
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Effect of IL-10 on IL-5 mRNA and protein expression.
In view of the above results, we next examined whether the action of
exogenously administered IL-10 is mechanistically associated with an
induced altered endogenous expression of IL-5 by the IL-10-exposed ASM.
In these studies, cultured human ASM cells were exposed to vehicle
alone or to IL-10 for various durations up to 24 h. Thereafter, in
separate experiments, the cells were harvested for analysis of temporal
changes in IL-5 mRNA expression, and the cell culture medium was
extracted for measurement of IL-5 protein release by immunoassay (see
MATERIALS AND METHODS). IL-5 mRNA was essentially undetectable in control (vehicle-exposed) ASM cells. In contrast, as
depicted in Fig. 6, relative to the
unaltered constitutively expressed RPL7 mRNA signal, IL-5 mRNA
expression was progressively enhanced in the IL-10-treated cells at all
times for up to 24 h after IL-10 exposure. Comparably, the
IL-10-treated ASM cells also exhibited an enhanced elaboration of IL-5
protein into the cell culture medium, with increased levels of IL-5
protein detected as early as 3 h after IL-10 exposure and maximal
levels attained at 12 h (Fig. 7).
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DISCUSSION |
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Although generally recognized for its anti-inflammatory properties, the role of IL-10 in bronchial asthma is controversial. In this regard, several studies (1, 2) have reported that cellular expression of IL-10 is reduced in allergic and nonatopic asthmatic individuals, whereas other reports have demonstrated increased levels of IL-10 expression in the lungs of asthmatic subjects (17, 22, 24) as well as in the skin and peripheral blood cells of allergic individuals after allergen challenge (22). Although these apparently disparate findings regarding the expression of IL-10 in asthma remain to be explained, emerging evidence based on in vivo studies with animal models of allergic asthma suggests that in contrast to other Th2-type cytokines that are proinflammatory in nature, IL-10 may regulate the induction of airway constrictor hyperresponsiveness independent of pulmonary inflammation (18, 25). This recent evidence raises the speculation that, notwithstanding its reported anti-inflammatory properties, IL-10 may play an important role in allergic asthma by acting directly on the sensitized ASM itself. The present study addressed this possibility, and the results provide new evidence demonstrating that the altered agonist responsiveness of atopic asthmatic serum-sensitized ASM is largely attributed to activation of an endogenously expressed autocrine mechanism that involves the autologous induction of IL-10-mediated release and the action of IL-5 in the sensitized ASM itself.
To our knowledge, the present observations are the first to demonstrate
that IL-10 exerts significant effects on airway responsiveness by
acting directly on the ASM itself and, accordingly, contributes to the
changes in ASM responsiveness that characterize the atopic asthmatic
phenotype, including heightened agonist-mediated constrictor responsiveness and impaired -adrenoceptor-mediated ASM relaxation (Figs. 1 and 2). In evaluating the collection of present findings, certain issues are worthy of consideration. Among these, it is relevant
to note that IL-10 mRNA expression in ASM cells was essentially undetectable under control conditions but was distinctly increased as
early as 3 h after incubation of the cells with atopic asthmatic serum (Fig. 3). Of interest, this temporal pattern of IL-10 mRNA induction closely paralleled the time course of induction of mRNA expression of the Th2-type cytokine IL-5 by the atopic asthmatic serum-sensitized ASM cells (Fig. 3). To the extent that, under the same
experimental conditions described here, the induced mRNA expression and
associated release of IL-5 protein by atopic sensitized ASM were
previously shown to elicit the same observed changes in ASM
responsiveness as in the atopic asthmatic serum-sensitized state
(6, 13), our present results suggested that the temporal association between the induced changes in IL-10 and IL-5 expression may be mechanistically related. In addressing this possibility, our
extended observations demonstrated that 1) exogenous
administration of IL-10 to naive ASM cells induced their upregulated
expression of IL-5 mRNA (Fig. 6) and release of IL-5 protein (Fig. 7)
and 2) comparable to atopic asthmatic serum, exogenous IL-10
administration to naive ASM tissue elicited proasthmatic-like changes
in ASM constrictor and relaxant responsiveness that were prevented by pretreating the tissues with an IL-5R blocking Ab (Figs. 4 and 5).
Taken together, these results support the concept of a causal association between induced upregulated IL-10 and IL-5 expression in
the atopic sensitized state, where the release of IL-5 is
mechanistically dependent on the autocrine induction and action of
IL-10 in the sensitized ASM.
In a recent in vivo study (25), intratracheal instillation
of recombinant murine IL-10 to antigen-challenged mice was found to
produce increased bronchoalveolar lavage (BAL) fluid levels of IL-10,
whereas BAL fluid levels of IL-5 as well as of IL-4 and interferon-
were found to be reduced. In contrast, in another in vivo study with
antigen-sensitized mice, similarly elevated BAL fluid levels of IL-5
and IL-4 were detected in both wild-type and IL-10-deficient mice after
allergen challenge, and reconstitution of the IL-10 gene in the
IL-10-deficient mice failed to induce any changes in the BAL fluid
levels of these cytokines (18). Moreover, in other recent
investigations with IL-10 gene knockout in murine models of allergic
asthma, one study (14a) reported that BAL fluid levels of IL-4, IL-5,
and IL-13 were upregulated in the lungs of the IL-10 gene knockout
mice, whereas another study (27) reported that IL-5
production and lung eosinophilic infiltration were attenuated in the
IL-10-deficient mice. Although the apparent discrepancy between the
findings of some of these in vivo studies is not readily explained,
given our present in vitro observations, the general consideration is
raised that determination of BAL fluid levels of certain cytokines may
not be reflective of potential autocrine-induced changes in the
expression of such cytokines in the microenvironment of the ASM itself.
Furthermore, in this context, the possibility also exists that IL-10
exerts its cytokine-modulatory effects in a cell type-specific manner, with potential immunosuppressive actions occurring in certain cells such as peripheral blood mononuclear cells (23) and,
as demonstrated herein, cytokine-stimulatory actions manifested in other cell types (e.g., ASM).
The central findings of this study provide an extended scope to the
prevailing concept of a Th2 cytokine-dependent overall mechanism
underlying the pathobiology of allergic asthma. In this regard,
although 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 atopic asthmatic serum-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 (6, 9, 11, 12); and this phenomenon was
largely attributed to activation of the low-affinity receptor for IgE,
Fc
RII (CD23), expressed on the ASM cell, by the elevated IgE present
in the atopic sensitizing serum (7, 8). 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-10-mediated release and action of IL-5 also exists in ASM that, when
activated in the atopic asthmatic serum-sensitized state, contributes
to the proasthmatic changes in ASM responsiveness.
In conclusion, the present study investigated the role and mechanism of action of IL-10 in regulating the altered agonist responsiveness of atopic asthmatic serum-sensitized ASM. The results demonstrate that 1) the induced proasthmatic-like changes in agonist constrictor and relaxant responsiveness in asthmatic atopic serum-sensitized ASM are prevented by blocking the IL-10R in the sensitized ASM; 2) both IL-10 and IL-5 mRNA expression are upregulated in atopic asthmatic serum-sensitized ASM; 3) IL-10 elicits upregulated IL-5 mRNA expression and release of IL-5 protein from ASM cells; and 4) the latter IL-10-induced autocrine release of IL-5 is responsible for IL-10-mediated 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 asthmatic serum-sensitized state, elicits autologous proasthmatic perturbations in airway responsiveness.
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ACKNOWLEDGEMENTS |
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We 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, Division of Pulmonary Medicine, Children's Hospital of Philadelphia, Univ. of Pennsylvania School of Medicine, 34th St. and 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.
Received 20 February 2001; accepted in final form 8 June 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Borish, L.
IL-10: evolving concepts.
J Allergy Clin Immunol
101:
293-297,
1998[ISI][Medline].
2.
Borish, L,
Aarson A,
Rumbyrt J,
Cvietusa P,
Negri J,
and
Wenzel S.
Interleukin-10 regulation in normal subjects and patients with asthma.
J Allergy Clin Immunol
97:
1288-1295,
1996[ISI][Medline].
3.
Coffman, RL,
Seymour BW,
Lebman DA,
Hiraki DD,
Christiansen JA,
Shrader B,
Cherwinski HM,
Savelkoul HF,
Finkelman FD,
and
Bond MW.
The role of helper T cell products in mouse B cell differentiation and isotype regulation.
Immunol Rev
102:
5-28,
1988[ISI][Medline].
4.
Gelfand, EW,
and
Irvin CG.
T lymphocytes: setting the tone of the airways.
Nat Med
3:
382-283,
1997[ISI][Medline].
5.
Grunig, G,
Warnock M,
Wakil AE,
Venkayya R,
Brombacher F,
Rennick DM,
Sheppard D,
Mohrs M,
Donaldson DD,
Locksley RM,
and
Corry DB.
Requirement for IL-13 independently of IL-4 in experimental asthma.
Science
282:
2261-2263,
1998
6.
Grunstein, MM,
Hakonarson H,
Maskeri N,
Kim C,
and
Chuang S.
Intrinsic ICAM-1/LFA-1 activation mediates altered responsiveness of atopic asthmatic airway smooth muscle.
Am J Physiol Lung Cell Mol Physiol
278:
L1154-L1163,
2000
7.
Hakonarson, H,
Carter C,
Kim C,
and
Grunstein MM.
Altered expression and action of the low-affinity IgE receptor FcRII (CD23) in asthmatic airway smooth muscle.
J Allergy Clin Immunol
104:
575-584,
1999[ISI][Medline].
8.
Hakonarson, H,
and
Grunstein MM.
Autologously up-regulated Fc receptor expression and action in airway smooth muscle mediates its altered responsiveness in the atopic asthmatic sensitized state.
Proc Natl Acad Sci USA
95:
5257-5262,
1998
9.
Hakonarson, H,
Herrick DJ,
Gonzalez-Serrano P,
and
Grunstein MM.
Autocrine role of interleukin 1 in altered responsiveness of atopic asthmatic sensitized airway smooth muscle.
J Clin Invest
99:
117-124,
1997
10.
Hakonarson, H,
Herrick DJ,
and
Grunstein MM.
Mechanism of impaired -adrenoceptor responsiveness in atopic sensitized airway smooth muscle.
Am J Physiol Lung Cell Mol Physiol
269:
L645-L652,
1995
11.
Hakonarson, H,
Maskeri N,
Carter C,
Chuang C,
and
Grunstein MM.
Autocrine interaction between IL-5 and IL-1 mediates altered responsiveness of atopic asthmatic sensitized airway smooth muscle.
J Clin Invest
104:
657-667,
1999
12.
Hakonarson, H,
Maskeri N,
Carter C,
and
Grunstein MM.
Regulation of TH1- and TH2-type cytokines expression and action in atopic asthmatic sensitized airways smooth muscle.
J Clin Invest
103:
1077-1087,
1999
13.
Hakonarson, H,
Maskeri N,
Carter C,
Hodinka RL,
Campbell D,
and
Grunstein MM.
Mechanism of rhinovirus-induced changes in airway smooth muscle responsiveness.
J Clin Invest
102:
1732-1741,
1998
14.
Hamelmann, E,
Wahn U,
and
Gelfand GW.
Role of the Th2 cytokines in the development of allergen-induced airway inflammation and hyperresponsiveness.
Int Arch Allergy Immunol
118:
90-94,
1999[ISI][Medline].
14a.
Justice, PJ,
Shibata Y,
Sur S,
Mustafa J,
Fan M,
and
Van Scott MR.
IL-10 gene knockout attenuates allergen-induced airway hyperresponsiveness in C57BL/6 mice.
Am J Physiol Lung Cell Mol Physiol
280:
L363-L368,
2001
15.
Kelly, J.
Cytokines of the lung.
Am Rev Respir Dis
141:
765-772,
1990[ISI][Medline].
16.
Kotsimbos, AT,
and
Hamid Q.
IL-5 and IL-5 receptor in asthma.
Mem Inst Oswaldo Cruz
92:
75-91,
1997[ISI][Medline].
17.
Magnan, A,
van Pee D,
Bongrand P,
and
Vervloet D.
Alveolar macrophage interleukin (IL)-10 and IL-12 production in atopic asthma.
Allergy
53:
1092-1095,
1988.
18.
Makela, MJ,
Kanehiro A,
Borish L,
Dakhama A,
Loader J,
Joetham A,
Xing Z,
Jordana M,
Larsen GL,
and
Gelfand EW.
IL-10 is necessary for the expression of airway hyperresponsiveness but not pulmonary inflammation after allergic sensitization.
Proc Natl Acad Sci USA
97:
6007-6012,
2000
19.
Moore, KW,
O'Garra A,
de Waal Malefyt R,
Vieira P,
and
Mosmann TR.
Interleukin-10.
Annu Rev Immunol
11:
165-190,
1993[ISI][Medline].
21.
Robinson, DS,
Hamid Q,
Ying S,
Tsicopoulos A,
Barkans J,
Bentley A,
Corrigan C,
Durham SR,
and
Kay AB.
Predominant Th2-like bronchoalveolar T-lymphocyte population in atopic asthma.
N Engl J Med
326:
298-304,
1992[Abstract].
22.
Robinson, DS,
Tsicopoulos A,
Meng Q,
Durham S,
Kay AB,
and
Hamid Q.
Increased interleukin-10 messenger RNA expression in atopic allergy and asthma.
Am J Respir Cell Mol Biol
14:
113-117,
1996[Abstract].
23.
Staples, KJ,
Bergman M,
Barnes PJ,
and
Newton R.
Stimulus-specific inhibition of IL-5 by cAMP-elevating agents and IL-10 reveals differential mechanisms of action.
Biochem Biophys Res Commun
273:
811-815,
2000[ISI][Medline].
24.
Tillie-Leblond, I,
Pugin J,
Marquette CH,
Lamblin C,
Saulnier F,
Brichet A,
Wallaert B,
Tonnel AB,
and
Gosset P.
Balance between proinflammatory cytokines and their inhibitors in bronchial lavage from patients with status asthmaticus.
Am J Respir Crit Care Med
159:
487-494,
1999
25.
Van Scott, MR,
Justice JP,
Bradfield JF,
Enright E,
Sigounas A,
and
Sur S.
IL-10 reduces Th2 cytokine production and eosinophilia but augments airway reactivity in allergic mice.
Am J Physiol Lung Cell Mol Physiol
278:
L667-L674,
2000
26.
Wills-Karp, M,
Luyimbazi J,
Xu X,
Schofield B,
Neben TY,
Karp CL,
and
Donaldson DD.
Interleukin-13: central mediator in allergic asthma.
Science
282:
2258-2261,
1998
27.
Yang, X,
Wang S,
Fan Y,
and
Han Y.
IL-10 deficiency prevents IL-5 overproduction and eosinophilic inflammation in a murine model of asthma-like reaction.
Eur J Immunol
30:
382-391,
2000[ISI][Medline].
28.
Zuany-Amorim, C,
Haile S,
Leduc D,
Dumarey C,
Huerre M,
Vargaftig BB,
and
Pretolani M.
Interleukin-10 inhibits antigen-induced cellular recruitment into the airways of sensitized mice.
J Clin Invest
95:
2644-2651,
1995[ISI][Medline].