EDITORIAL FOCUS
IL-13-dependent autocrine signaling mediates altered responsiveness of IgE-sensitized airway smooth muscle

M. M. Grunstein, H. Hakonarson, J. Leiter, M. Chen, R. Whelan, J. S. Grunstein, and S. Chuang

Division of Pulmonary Medicine, Joseph Stokes, Jr. Research Institute, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In testing the hypothesis that interleukin-4 receptor alpha -subunit (IL-4Ralpha )-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-4Ralpha blocking antibody (anti-IL-4Ralpha 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-4Ralpha 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-4Ralpha -coupled release and action of IL-5 in the sensitized ASM itself.

Th2 cytokines; immunoglobulin E; atopy; asthma; interleukin-13


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -chain (IL-4Ralpha ), 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-1beta in the atopic sensitized ASM (9). Given this evidence, together with that establishing that IL-4Ralpha -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-4Ralpha -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-4Ralpha -mediated release and action of IL-5 in the sensitized ASM to produce proasthmatic-like changes in ASM responsiveness.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-4alpha receptor blocking antibody (anti-IL-4Ralpha 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 10-4 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-4Ralpha Ab. 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 beta -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-4Ralpha 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 beta -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; beta -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 beta -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 beta -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 beta -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-4Ralpha 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Role of IL-4Ralpha in regulating agonist responsiveness in IgE-sensitized ASM. To determine the role of IL-4Ralpha 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-4Ralpha 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-4Ralpha 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-4Ralpha 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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Fig. 1.   Comparison of constrictor dose-response relationships to ACh in paired control (open circle ) and immunoglobulin E (IgE)-sensitized airway smooth muscle (ASM) tissue segments in the absence () and presence of interleukin-4 receptor alpha -subunit blocking antibody (anti-IL-4Ralpha Ab, ) or an isotype control Ab (cAb, ). Note: relative to control ASM, the heightened constrictor responses to ACh were largely ablated by cotreatment of the IgE-sensitized tissues with anti-IL-4Ralpha Ab, whereas cotreatment with cAb had no effect. Data represent mean ± SE values from 6 paired experiments.

In additional studies, during comparable levels of initial sustained ACh-induced contractions in control and IgE-exposed ASM segments, averaging ~50% of Tmax, administration of the beta -adrenoceptor agonist isoproterenol produced cumulative dose-dependent relaxation of the precontracted tissues. Relative to control ASM, the Rmax responses and sensitivities (pD50 values) to isoproterenol were significantly attenuated in the corresponding IgE-sensitized tissues (Fig. 2). Accordingly, the mean ± SE Rmax values in the IgE-sensitized vs. control ASM amounted to 31.42 ± 3.74 vs. 59.01 ± 5.39%, respectively (P < 0.001), and the corresponding pD50 values for isoproterenol averaged 6.09 ± 0.03 and 6.25 ± 0.04, respectively (P < 0.05). This impaired relaxation responsiveness to isoproterenol was largely prevented in IgE-sensitized tissues that were pretreated with anti-IL-4Ralpha Ab (Fig. 2, open squares), whereas pretreatment with the isotype cAb had no effect (Fig. 2, filled squares). Moreover, in comparable experiments, neither pretreatment with anti-IL-4Ralpha Ab nor with the isotype cAb was found to appreciably affect the ASM relaxant responsiveness to isoproterenol in control tissues (data not shown).


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Fig. 2.   Comparison of relaxation dose-response relationships to isoproterenol in paired control (open circle ) and IgE-sensitized ASM tissue segments in the absence () and presence of anti-IL-4Ralpha Ab () or cAb (). Note: relative to control ASM, the attenuated relaxation responses to isoproterenol were prevented by cotreatment of the IgE-sensitized tissues with anti-IL-4Ralpha Ab, whereas cotreatment with cAb had no effect. Data represent mean ± SE values from 6 paired experiments.

Effects of IgE sensitization on ASM expression of IL-4 and IL-13. We previously reported that ASM cells express IL-4Ralpha protein on their cell surface and that IL-4Ralpha 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-4Ralpha 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-4Ralpha , 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 beta -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 beta -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 beta -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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Fig. 3.   Southern blots depicting IL-4 and IL-13 mRNA expression in cultured human ASM cells after 0-, 3-, 6-, 12-, and 24-h exposure to IgE. Constitutive expression of beta -actin mRNA was used to control for gel loading. RNA isolated from T lymphocytes was used as a positive control for detection of IL-4 cDNA. The blots were probed with human-specific IL-4, IL-13, and beta -actin 32P-labeled cDNA probes (see MATERIALS AND METHODS). Note: relative to the undetectable IL-4 signal, the mRNA signal for IL-13 was progressively enhanced at the various times after exposure of cells to IgE, whereas the intensities of the mRNA signal for the constitutively expressed beta -actin gene were essentially unaltered.

In extending the above observations, we next examined whether human ASM cells express IL-4 and IL-13 proteins and also assessed whether expression of these cytokines is modulated in cells exposed for 24 h to IgE. Using flow cytometric analysis for intracellular detection of IL-4 and IL-13, we found no evidence for expression of IL-4 protein in either control (vehicle exposed) or IgE-treated cells. In contrast, as exemplified in Fig. 4A, relatively low levels of intracellular IL-13 protein were detected under control conditions, and the expression of IL-13 was markedly upregulated in ASM cells exposed to IgE, whereas corresponding nonspecific background staining using FITC-conjugated isotype cAb remained unaltered (Fig. 4B). Based on the results from four experiments, control cells displayed mean ± SE values for percent positive staining and MFI for IL-13 of 0.41 ± 0.09 and 6.7 ± 3.1%, respectively, compared with the corresponding increased values of 58.3 ± 7.9% (P < 0.001) and 22.6 ± 6.4% (P < 0.01), respectively, obtained in IgE-treated cells.


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Fig. 4.   Comparison by flow cytometric analysis of intracellular IL-13 expression in representative control (untreated) and IgE-treated human ASM cells stained with mouse anti-human antibodies specific for IL-13 and goat anti-mouse FITC-conjugated secondary antibody used for detection of the signal. A: relative to control cells, both %positive staining and mean fluorescence intensity (MFI) for IL-13 are increased in IgE-treated cells. B: staining with FITC-conjugated isotype control IgG antibodies shows no differences in nonspecific background staining for IL-13 between control and IgE-treated conditions.

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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Fig. 5.   Comparison of constrictor dose-response relationship to ACh in paired vehicle-exposed (control, open circle ) and IL-13-treated ASM tissue segments in the absence () and presence of anti-IL-5R-Ab (). Note: relative to control ASM, the heightened constrictor responses to ACh were inhibited in IL-13-treated tissues that were cotreated with anti-IL-5R-Ab, whereas cotreatment with an isotype cAb () had no effect. Data represent mean ± SE values from 6 paired experiments.



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Fig. 6.   Comparison of relaxation dose-response relationships to isoproterenol in paired vehicle-exposed (control, open circle ) and IL-13-treated ASM tissue segments in the absence () and presence of anti-IL-5R-Ab (). Note: relative to control ASM, the attenuated relaxation responses to isoproterenol were prevented in IL-13-treated tissues that were cotreated with anti-IL-5R-Ab, whereas cotreatment with cAb () had no effect. Data represent mean ± SE values from 6 paired experiments.

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-4Ralpha Ab, 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 beta -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-4Ralpha Ab.


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Fig. 7.   Southern blots depicting temporal changes in IL-5 mRNA expression in human ASM cells after 0-, 3-, 6-, 12-, and 24-h incubation with IL-13 in the absence and presence of anti-IL-4Ralpha Ab. Constitutive expression of beta -actin mRNA was used to control for gel loading. Note: relative to the essentially unaltered beta -actin signal, mRNA expression of IL-5 was progressively enhanced at all times after exposure of cells to IL-13, and this effect of IL-13 was essentially ablated in cells concomitantly treated with anti-IL-4Ralpha Ab.

In another series of experiments, the culture medium of ASM cells exposed to IL-13 was collected for measurements of IL-5 protein release by immunoassay (see MATERIALS AND METHODS). As shown in Fig. 8, relative to the low levels of expression of IL-5 protein from control cells, IL-13-exposed cells exhibited a progressively enhanced elaboration of IL-5 protein into the cell culture medium, with markedly increased (near maximal) levels of IL-5 detected as early as 6 h after IL-13 exposure and maximal levels attained at 24 h. For comparison, also shown in Fig. 8 are results obtained in ASM cells exposed to IgE. Relative to IL-13-exposed cells, IgE-treated cells released significantly less IL-5 protein at 6 h; however, similar levels of IL-5 concentration were attained by 24 h.


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Fig. 8.   Comparison of the release of IL-5 protein into the culture media of human ASM cells after exposure to vehicle alone (control, open circle ), IgE immune complexes (), and IL-13 (20 ng/ml, ). Note: in contrast to control cells, wherein IL-5 protein levels were essentially unchanged, ASM cells exposed to either IgE or IL-13 displayed markedly induced release of IL-5. Moreover, relative to IgE-treated cells, near-maximal levels of IL-5 release were attained as early as 6 h after exposure of cells to IL-13.

Finally, in light of our observed effects of IgE and IL-13 on IL-5 release, to further substantiate the above observations implicating IL-13/IL-5-coupled autocrine signaling in mediating IgE-induced changes in ASM responsiveness, we next examined the role of IL-4Ralpha activation in regulating IgE-induced expression of IL-5. In these experiments, using flow cytometry to detect intracellular IL-5 protein, we assayed the latter in cultured human ASM cells under control (vehicle exposed) conditions and, after exposure to IgE, in the absence and presence of pretreatment of the cells with anti-IL-4Ralpha . In concert with the above results, relative to control cells, IgE-treated cells exhibited increased intracellular IL-5 protein expression, and this effect was inhibited in IgE-exposed cells that were pretreated with anti-IL-4Ralpha Ab (Fig. 9).


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Fig. 9.   Comparison by flow cytometric analysis of intracellular IL-5 protein expression in representative control (untreated) and IgE-treated human ASM cells in the absence and presence of cotreatment with anti-IL-4Ralpha Ab. Cells were stained with mouse anti-human antibodies specific for IL-5, and goat anti-mouse FITC-conjugated secondary antibody was used for detection of the signal. Note: relative to control cells, IgE-treated cells exhibited increased IL-5 protein expression, and this effect was prevented in IgE-treated cells that were concomitantly treated with anti-IL-4Ralpha Ab. In contrast, exposure of control cells to anti-IL-4Ralpha Ab had no effect on IL-5 protein expression (data not shown).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-4Ralpha 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-4Ralpha 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-4Ralpha activation was found to be largely mediated by IL-13 binding, as the airway constrictor hyperresponsiveness was prevented in IL-4Ralpha -deficient mice and in mice receiving a soluble IL-13alpha 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-4Ralpha expression was recently identified in human ASM cells (10, 15) and that ASM cell expression of IL-4Ralpha 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-4Ralpha -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 IL4Ralpha -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-4Ralpha 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-4Ralpha 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, Fcepsilon RII (i.e., CD23), in both rabbit and human ASM cells (5, 6), as well as increased release of IL-1beta protein from both cell types (4). Similarly, rhinovirus inoculation of rabbit and human ASM cells was also found to provoke the release of IL-beta 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 beta -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-1beta , has been previously demonstrated (4, 9, 10), and this phenomenon was largely attributed to activation of Fcepsilon 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-4Ralpha -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-4alpha R 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-4Ralpha -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.


    ACKNOWLEDGEMENTS

The authors thank M. Brown for typing the manuscript.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 282(3):L520-L528
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