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The Journal of Immunology, 2001, 167: 1683-1692.
Copyright © 2001 by The American Association of Immunologists

IL-13 Induces Airways Hyperreactivity Independently of the IL-4R{alpha} Chain in the Allergic Lung1

Joerg Mattes*, Ming Yang*, Ana Siqueira*, Kris Clark*, Jason MacKenzie*, Andrew N. J. McKenzie{dagger}, Dianne C. Webb*, Klaus I. Matthaei* and Paul S. Foster2,*

* Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra, Australia; and {dagger} Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The potent spasmogenic properties of IL-13 have identified this molecule as a potential regulator of airways hyperreactivity (AHR) in asthma. Although IL-13 is thought to primarily signal through the IL-13R{alpha}1-IL-4R{alpha} complex, the cellular and molecular components employed by this cytokine to induce AHR in the allergic lung have not been identified. By transferring OVA-specific CD4+ T cells that were wild type (IL-13+/+ T cells) or deficient in IL-13 (IL-13-/- T cells) to nonsensitized mice that were then challenged with OVA aerosol, we show that T cell-derived IL-13 plays a key role in regulating AHR, mucus hypersecretion, eotaxin production, and eosinophilia in the allergic lung. Moreover, IL-13+/+ T cells induce these features (except mucus production) of allergic disease independently of the IL-4R{alpha} chain. By contrast, IL-13+/+ T cells did not induce disease in STAT6-deficient mice. This shows that IL-13 employs a novel component of the IL-13 receptor signaling system that involves STAT6, independently of the IL-4R{alpha} chain, to modulate pathogenesis. We show that this novel pathway for IL-13 signaling is dependent on T cell activation in the lung and is critically linked to downstream effector pathways regulated by eotaxin and STAT6.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Asthma is clinically characterized by episodic airflow obstruction, inflammation of the airways, and enhanced bronchial reactivity (airways hyperreactivity (AHR)3) to inhaled spasmogenic stimuli (1, 2, 3, 4). The mechanisms underlying the development of AHR and diminished airflow are considered to play central roles in disease pathogenesis (1, 3, 5). Although the etiology of asthma is complex, inflammation of the airways, elicited by an inappropriate immune response to inhaled allergens, is considered a principle predisposing factor for the clinical expression and pathogenesis of this disorder (1, 2, 3, 5, 6). Indeed, disease severity directly correlates with progressive inflammation of the airways as well as the levels of airways obstruction and AHR (1, 2, 3, 5, 6).

CD4+ Th 2 lymphocytes (Th2 cells) are predominant features of inflammatory infiltrates in asthma (7, 8, 9). These cells are thought to regulate disease progression and AHR by secreting cytokines that induce the immune and pathologic responses (IgE production, mucus hypersecretion, eosinophilia, and remodeling of the airways wall) that are hallmark features of this disease (2, 3). Indeed, in murine models of experimental asthma, Th2 cells play an obligatory role in pathogenesis and the induction of AHR (10, 11, 12). In addition, the transfer of Ag-specific Th2 cell clones to naive recipients in the presence of cognate Ag is sufficient to induce fulminant disease (13, 14, 15, 16, 17, 18). Individual Th2 cytokines (e.g., IL-4, IL-5, IL-9, IL-10, and IL-13) have also been linked to the expression of defined pathophysiologic features that are characteristic of human asthma and are thought to form collaborative networks with other cells and inflammatory molecules to promote inflammation and pathogenesis (12, 15, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33).

Although clinical correlates strongly suggest that Th2 cytokines underpin the development of inflammation of the airways and AHR, the precise mechanism predisposing to alterations in smooth muscle reactivity to spasmogens has not been delineated. Evidence for the importance of IL-5 and IL-4 in the pathogenesis of asthma has been compelling. Numerous investigations show that IL-4 and IL-5 induce pathologic features of allergic disease by regulating key aspects of Th2 cell immunity and eosinophilic inflammation, respectively (10, 12, 15, 16, 19, 20, 21, 23, 24, 34, 35). However, a variety of studies have also demonstrated that pathways regulated by these cytokines, alone or collectively, are not sufficient to totally account for the development of AHR in the allergic lung (12, 21, 30). Importantly, the mechanisms regulating AHR independently of IL-4 and IL-5 are dependent on Th2 cell-operated pathways (12).

Recently, IL-13 has been identified as a potent regulator of bronchoconstriction in mouse models of allergic asthma and experimental AHR (26, 28). This cytokine is primarily secreted from Th2 cells but is also expressed by mast cells, macrophages, eosinophils, and NK cells (36, 37, 38). IL-13 shares ~30% identity with IL-4, and, in part, has similar biologic functions through utilization of the IL-4R{alpha} chain (39). IL-13 signals by binding to its primary receptor chain (IL-13R{alpha}1) (to which IL-4 does not bind) and recruiting the IL-4R{alpha} chain into the receptor complex which results in the activation of STAT6 (40, 41). Blockade of IL-13 signaling in mouse models of experimental asthma by a soluble IL-13R{alpha}2-IgGFc fusion protein, which specifically binds and neutralizes IL-13, has identified this cytokine as a key regulator of AHR and mucus production in the allergic lung (26, 28). Moreover, IL-13 alone was sufficient to induce pathognomonic features of asthma (AHR, IgE production, eosinophilia, and mucus production) when instilled into the airways of naive mice or overexpressed in the lung (18, 28). This cytokine also induces eotaxin production in the lung (18). In naive IL-4R{alpha}-deficient mice (IL-4R{alpha}-/-), IL-13 failed to induce the asthma phenotype, supporting the concept that this receptor and subsequent signaling through STAT-6 are critical for the induction of allergic disease of the lung (42, 43, 44, 45).

Although the importance of IL-13 signaling through the IL-4R{alpha} chain has been shown for the induction of AHR, eosinophilia, and mucus production in naive mice, the role of this receptor in the mechanism of IL-13-induced AHR in the allergic lung has not been delineated. Furthermore, the role of IL-13 production from disease-inducing CD4+ T cells and the interplay between other secreted Th2 cytokines for the expression of AHR and pathophysiologic responses has not been characterized. Indeed, recent investigations suggest that IL-13 can signal independently of the IL-4R{alpha} chain in murine B cells to regulate Ab production (46).

In this investigation, we use a CD4+ T cell-induced model of allergic airways inflammation to explore the role of T cell-derived IL-13 in the induction of AHR, eosinophilia, and mucus hypersecretion. Furthermore, we identify the contribution of the IL-4R{alpha} chain to these T cell driven immunopathologic processes. In contrast to studies that use transgenic T cell clones, we generated Ag-specific CD4+ Th cells in vitro after isolation from OVA-sensitized wild-type and IL-13-/- mice. IL-13-sufficient or IL-13-deficient OVA-specific T cells (IL-13+/+ and IL-13-/- T cells, respectively) were adoptively transferred to naive BALB/c mice concurrently with OVA delivery to the airways. In these investigations, we show that T cell-derived IL-13 plays a central role in the induction of AHR, eosinophilia, eotaxin production, and mucus hypersecretion in the inflamed lung. Moreover, IL-13+/+ T cells induce these features (except mucus production) of allergic disease independently of the IL-4R{alpha} chain. In addition, we show that this novel signaling pathway for IL-13 is STAT6 dependent and coupled to IL-13-induced eotaxin production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

BALB/c mice (6–12 wk) were used in all experiments and were obtained from the Special Pathogen-Free Facility or the Gene Targeting Facility (John Curtin School of Medical Research, Australian National University, Canberra, Australia). IL-13-/- (47) and STAT6-/- mice (48) were backcrossed eight and six generations with the BALB/c strain, respectively. BALB/c IL-4R{alpha}-/- mice were a gift from Dr. N. Noben-Trauth (National Institutes of Health, Bethesda, MD) (22). Mice were treated according to Australian National University Animal Welfare guidelines and were housed in an approved containment facility.

In vitro polarization of T cells

OVA-specific CD4+ T cells were derived from mice (6–8 wk of age) that were sensitized by i.p. injection with 50 µg OVA/1 mg Alhydrogel (CSL, Parkville, Australia) in 0.9% sterile saline. Seven days after sensitization, donor mice were sacrificed by cervical dislocation, and single-cell suspensions of the spleens were prepared. Erythrocytes were lysed, and the washed splenocytes were resuspended at 5 x 106 cells/ml in complete medium consisting of HL-1 (BioWhittaker, Walkersville, MD) with 10% heat-inactivated FCS, 2 mM L-glutamine, and 50 mg/L neomycin sulfate. Splenocytes were then cultured for 4 days at 37°C in the presence of 200 µg/ml OVA to generate Ag-reactive CD4+ T cells of an intermediate Th1/Th2 profile (designated Thi cells). Alternatively, T cells were polarized to the Th1 phenotype by the addition of recombinant murine IL-12 (2 ng/ml; PeproTech, Rocky Hill, NJ) and neutralizing anti-IL-4 Ab (11B11, 40 µg/ml) or the Th2 phenotype with recombinant murine IL-4 (20 ng/ml, kind gift from S. Ford and I. G. Young, John Curtin School of Medical Research) and anti-IFN-{gamma} (R46A2, 40 µg/ml) to the culture medium. CD4+ T cells were then isolated using high-gradient magnetic MiniMACS separation columns (MACS separation) (Miltenyi Biotec, Bergisch Gladbach, Germany) as described previously (49). Purified Thi, Th1, or Th2 cells were then washed and resuspended in PBS and transferred to naive recipients. Purified CD4+ T cell populations were also analyzed for Ag-specific cytokine production and intracellular cytokine profiles were determined. The purity of the enriched CD4+ cell fraction was uniformly above 96% as determined by flow cytometry (result not shown).

Ag-specific cytokine production from purified CD4+ T cell populations

CD4+ T cells (5 x 105 cells/well) were incubated with freshly isolated mitomycin C (25 µg/ml)-treated splenocytes (5 x 105 cells/well) in complete medium in the presence of 200 µg/ml OVA in 96-well plates (250 µl/well) for 96 h to determine cytokine profiles and the integrity of the polarization. Cell-free culture supernatants were then collected and stored in aliquots at -70°C until analysis.

Flow cytometric analysis of intracellular cytokines

MACS-purified CD4+ T cells were cultured at 37°C in T cell medium for 10 h in the presence of anti-CD3 (20 ng/ml; clone 2C11) and monensin (GolgiStop; BD PharMingen, San Diego, CA) according to the manufacturer’s instructions. T cells were then washed, fixed for 30 min in formaldehyde in PBS (Cytofix/Cytoperm; BD PharMingen), permeabilized with saponin (Perm/Wash; BD PharMingen), and stained with PE-conjugated anti-IFN-{gamma} and FITC-conjugated anti-IL-5 (all from BD PharMingen; kind gift from C. R. Parish, John Curtin School of Medical Research). Anti-IFN-{gamma} and anti-IL-5 were used at a 1/100 and 1/50 dilution, respectively (according to the manufacturer’s instructions). Labeled samples were analyzed on a FACScan flow cytometer (BD Immunocytometry System, Mountain View, CA). Gating of dead cells was performed using forward light scatter and side light scatter. Analysis of data was performed using CellQuest software (BD Biosciences, Mountain View, CA). Splenocytes that were obtained from nonsensitized mice served as a negative control.

Induction of allergic airways inflammation

CD4+ Th1, Th2, or Thi cells (2 x 106 cells) were injected i.v. into naive recipient wild-type, IL-4R{alpha}-/-, or STAT6-/- mice. Control mice received PBS vehicle. Twenty-four hours after the adoptive transfer, mice were exposed to an aerosol of OVA (10 mg/ml) in 0.9% saline for 30 min and then again every day for 6 days. AHR to methacholine was measured 24 h after the last aeroallergen challenge and then lung inflammation and morphology were characterized. In some experiments, T cells were labeled (as described previously (50)) with the fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, also called CFSE, provided by C. R. Parish, John Curtin School of Medical Research) before transfer to investigate the ability of these cells to traffic into the lung.

Bronchoalveolar lavage fluid and characterization of lung morphology

Bronchoalveolar lavage was performed by cannulation of the trachea and lavaging the airways with 2x 1 ml HBSS. Cytospin preparations of bronchoalveolar lavage cells were stained and the types of leukocytes were identified by morphologic criteria (20). Routinely 200 cells were counted per slide. Lung tissue representing the central (bronchi-bronchiole) and peripheral (alveoli) airways were fixed in 10% phosphate-buffered Formalin, sectioned, and stained with Alcian blue-periodic acid-Schiff for the enumeration of mucin-secreting cells or Charbol’s chromotrope-hematoxylin for the identification of eosinophils. The number of mucus-staining cells and eosinophils in the central bronchi-bronchiole area were identified by morphologic criteria (20, 51).

Preparation of lung homogenates

Lungs were excised and incubated with 1% collagenase/PBS at 37°C for 30 min, then cells were disaggregated and filtered through nylon mesh (70 µm). The filtrate was then centrifuged at 500 x g for 5 min at 4°C and the supernatant was stored at -70°C until eotaxin levels were measured. The cell pellet was resuspended in RBC lysis solution and centrifuged at 500 x g for 5 min at 4°C. The resulting pellet was cultured (1 x 106 cells/well) in complete medium in the presence of 200 µg/ml OVA in 96-well plates (250 µl/well) for 72 h. Cell-free culture supernatants were then collected and stored in aliquots at -70°C until cytokine levels were determined. In experiments where mice received fluorescently labeled T cells, lungs were perfused with PBS, then pooled (three to four lungs per group), cells were disaggregated, and the cell pellet was stained with CyChrome-conjugated anti-mouse CD4 (clone L3T4; BD PharMingen) for 30 min on ice. Flow cytometer analysis was then performed to quantify the number of transferred T cells that had homed to the lung as a percentage of the total number of pulmonary lymphocytes.

Analysis of cytokines by ELISA

IL-13 (R&D Systems, Minneapolis, MN), IL-4, IL-5, IL-10, and IFN-{gamma} (all from BD PharMingen) concentrations were determined in the supernatants from OVA-stimulated CD4+ T cells and OVA-stimulated lung homogenates by ELISA according to the manufacturer’s protocol. Eotaxin levels were measured in the supernatants from unstimulated lung homogenates by ELISA (R&D Systems).

Measurement of AHR

Responsiveness to {beta}-methacholine (methacholine) was assessed in conscious, unrestrained mice by barometric plethysmography using apparatus and software supplied by Buxco Electronics (Troy, NY). This system yields a dimensionless parameter known as enhanced pause (Penh) that reflects changes in waveform of the pressure signal from the plethysmography chamber combined with a timing comparison of early and late expiration. Pehn was used to empirically monitor airway function as described previously (51, 52). Measurement was performed essentially as previously described (51). Briefly, mice were placed in a chamber and exposed to an aerosol of water (baseline readings) and then cumulative doubling concentrations of methacholine (dissolved in water to make concentrations in solution ranging from 3.15 to 50 mg/ml). The aerosol was generated by an ultrasonic nebulizer and drawn through the chamber for 2 min at a constant flow rate. The inlet was then closed and Penh readings were taken for 3 min and averaged.

Intratracheal instillation of recombinant murine IL-13

Mice were anesthetized with 100 µl/L Saffan solution (1/4 diluted, consisting of alfaxalone and alfadolone acetate which are solubilized in saline by 20% polyoxyethylated castor oil) and intubated with an angled 22-gauge catheter needle. Afterward, 10 µg recombinant murine IL-13 dissolved in 20 µl PBS (or 20 µl PBS only for control mice) was instilled and airway reactivity to methacholine was measured 24 h later.

Inoculation of recombinant vaccinia virus (VV)

Recombinant VV were constructed as described elsewhere (53). Mice were given intranasal inocula of 107 PFU of VV-hemagglutinin (HA)-X (control virus) or VV-HA-eotaxin (encoding eotaxin) in 20 µl saline under light anesthesia 3 days after transfer of T cells. Virus growth in the lungs of mice given intranasal inocula of VV-HA-X or VV-HA-eotaxin is sustained at similar levels over 6 days (54). Eotaxin expression and viral growth is maximal after 4 days when mice were sacrificed for characterization of allergic responses. We have shown previously that the delivery of cytokines (IL-5) and chemokines (eotaxin) by this vector in the lung does not significantly effect the development of allergic inflammation or AHR (54).

Statistical analysis

The significance of differences between experimental groups was analyzed using Student’s unpaired t test. Values were reported as the mean ± SEM. Differences in mean values were considered significant if p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of recombinant IL-13 on airways reactivity in wild-type and IL-4R{alpha}-/- mice

To confirm the role of the IL-4R{alpha} chain in the mechanism of IL-13-induced AHR in naive mice, we instilled 10 µg of this cytokine into the trachea of wild-type or IL-4R{alpha}-/- mice and measured AHR 24 h later. In wild-type but not IL-4R{alpha}-/- mice, IL-13 induced a marked increase in airways reactivity to methacholine in comparison to PBS-treated controls (Fig. 1Go). These data support previous investigations that IL-13 utilizes this receptor to promote AHR (26, 28).



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  		FIGURE 1. Induction of AHR in naive mice is dependent on the IL-4R{alpha}-/- chain. Wild-type (WT) or IL-4R{alpha}-/- mice were administered recombinant IL-13 (10 µg) or control vehicle (PBS) and 24 h later airways reactivity to methacholine was determined by barometric plethysmography. IL-13 induced AHR in wild-type but not IL-4R{alpha}-/- mice. Data represent the percent increase in Penh over baseline reactivity in the absence of cholinergic stimuli (mean ± SEM, n = 4 mice per group). The maximal response to methacholine (25 mg/ml) is shown, but this result is representative of the full-dose response curve. Significant differences (p < 0.05) between IL-13 and PBS groups are shown.

 
Characterization and adoptive transfer of Th1, Th2, and Thi T cells

To explore the role of T cell-derived IL-13 in the induction of AHR, eosinophilia, and mucus hypersecretion, we used a CD4+ T cell-induced model of allergic airways inflammation. Initially, the phenotype of the CD4+ T cells that were cultured under the various conditions was characterized. Cytokine profiles were determined after Ag-specific stimulation or by intracellular cytokine staining (Fig. 2Go). CD4+ T cells cultured under conditions that favor commitment to a Th1 phenotype secreted IFN-{gamma} but no IL-4 or IL-5 when stimulated with OVA. Ag-specific stimulation of Th2 cell cultures resulted in the secretion of IL-4 and IL-5 but no IFN-{gamma}. Cells polarized to the Th1 phenotype also stained positive for intracellular IFN-{gamma}, but not for IL-5, whereas Th2 cells stained positive for IL-5 and not IFN-{gamma}. The Th population that was not artificially committed to Th1 or Th2 cells showed an intermediate phenotype (Thi), with the majority of cells secreting IFN-{gamma}, IL-4, and IL-5 when stimulated with OVA. We described the phenotype of these Thi cells as intermediate, rather than Th0, because they are Ag specific and have undergone clonal expansion. Intracellular cytokine staining of the Thi population showed that the majority of the cells expressed both IL-5 and IFN-{gamma}. Interestingly, Thi cells produced significantly more IL-13 than Th2 cells (mean ± SEM, 68.8 ± 0.8 compared with 8.2 ± 0.3 ng/ml, respectively; p < 0.005; Th1, 0.05 ± 0.002).



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  		FIGURE 2. Characterization of cytokine production by Th1 (a), Th2 (b), and Thi (c) cells. Purified CD4+ Th1, Th2, or Thi cells (5 x 105 cells/well) were recultured with freshly isolated and mitomycin C-treated splenocytes (5 x 105 cells/well) in complete medium for 96 h in the presence or absence (unstimulated) of OVA (200 µg/ml). Cell-free supernatants were assayed by ELISA for the production of IFN-{gamma}, IL-4, IL-5, and IL-13. Ag-specific cytokine assays were performed in duplicate and data (mean ± SEM) represent two independent experiments. Flow cytometric analysis of intracellular cytokines after Th populations were stimulated for 10 h with anti-CD3 in the presence of monensin. Aliquots were fixed, permeabilized, and stained for intracellular IFN-{gamma} and IL-5 as markers for Th1 and Th2 differentiation. Intracellular cytokine profiles of Th populations were obtained from two separate experiments. The number of cytokine-producing cells are indicated in each quadrant. ND, Not detected.

 
To determine the effects of these Th cell populations on the development of experimental asthma, we adoptively transferred Th1, Th2, or Thi cells to naive BALB/c mice with subsequent OVA exposure to the lung for 7 days. As Thi cells were generated in an Ag-specific manner without artificial in vitro manipulation by adding exogenous cytokines or inhibitory Abs to the culture medium, we were particularly interested in the potential of this cell to induce allergic disease of the lung. Both Thi and Th2 cells, but not Th1 cells, induced the asthma phenotype that was characterized by eosinophilic inflammation, mucus hypersecretion, and the development of AHR after OVA inhalation (data not shown). Although mucus production and eosinophilic inflammation were similar in mice that received Th2 and Thi cells, AHR was most pronounced in the presence of Thi cells (Fig. 3Go). Given that Thi cells 1) produced the greatest amounts of IL-13 when stimulated with Ag, 2) were the most potent inducers of AHR, 3) induced an equivalent inflammatory and pathophysiologic profile, and 4) were not artificially polarized, we used Thi cells in our adoptive transfer model.



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  		FIGURE 3. Thi cells are the most potent inducers of AHR when compared with Th2 and Th1 cells. Wild-type mice received 2 x 106 Th1, Th2, and Thi cells and were exposed to OVA for 7 days. Airways reactivity to methacholine was measured by barometric plethysmography. Data represent the percent increase in Penh over baseline reactivity in the absence of cholinergic stimuli (mean ± SEM, n = 6–7 per group) and are representative of two independent experiments. Significant differences (p < 0.05) between Thi and Th2 or Th2 and Th1 are shown. Th1 responses were not significantly different from those of the PBS group at any concentration of methacholine.

 
Cytokine levels in IL-13+/+ and IL-13-/- CD4+ T cells

To investigate the mechanism whereby T cells induce AHR and the role of T cell-derived IL-13 in this process, we generated IL-13+/+ and IL-13-/- T cell populations (Thi) for adoptive transfer studies. The levels of IL-4, IL-5, IL-10 and IFN-{gamma} produced after in vitro OVA restimulation were comparable between IL-13+/+ and IL-13-/- Thi cell populations (Fig. 4Go). The level of these cytokines produced from Thi cells was equivalent to that observed for Th2 cells (results not shown) except for IL-13 (see above). No cytokine production was detected in the absence of OVA (Fig. 4Go, IL-13+/+ (unstimulated)). Furthermore, stimulated T cells did not produce eotaxin (data not shown). Thus, the predominant difference in cytokine production between the two Thi cell populations in response to Ag-specific stimulation was the release of IL-13.



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  		FIGURE 4. Cytokine production by Thi cells generated from wild-type (IL-13+/+) or IL-13-/- mice. Purified CD4+ T cells (5 x 105 cells/well) were recultured with freshly isolated and mitomycin C-treated splenocytes (5 x 105 cells/well) in complete medium in 96-well plates (250 µl/well) for 96 h in the presence or absence (unstimulated) of OVA (200 µg/ml). Cell-free supernatants were assayed by ELISA for the production of IL-13 (a), IL-10 (b), IL-5 (c), IL-4 (d), and IFN-{gamma} (e). Data represent the mean ± SEM of two separate experiments conducted in duplicate. Cytokine assays were conducted on all Thi cell cultures to determine the cytokine profile of cells that were transferred. Cytokine levels were not significantly different between IL-13+/+ and IL-13-/- Thi cells.

 
Role of CD4+ T cell-derived IL-13 in the development of experimental asthma

When transferred into wild-type mice, IL-13+/+ T cells induced hallmark features of allergic asthma that included AHR, eosinophilia in the airways, blood and bone marrow compartments (data not shown), as well as mucus hypersecretion and eotaxin production (Fig. 5Go). By contrast, IL-13 -/- T cells induced an attenuated asthma phenotype in wild-type mice, with reduced (but not ablated) AHR (Fig. 5Goa), reduced accumulation of eosinophils in the airways (Fig. 5Gob), and decreased mucus production (Fig. 5Goc). Additionally, the production of eotaxin in the lung was critically dependent on IL-13 (Fig. 5God). Notably, both IL-13+/+ and IL-13-/- T cell populations homed in similar numbers to the lung compartment (11 and 8% of the total lymphocyte population, respectively; mean percentage of three to four lungs pooled per group). Further their spatial distribution within the pulmonary compartment was similar (as evidenced by fluorescence microscopy). No production of OVA-specific IgE, IgG1, IgG2b, or IgG2a was observed as a result of the transfer of IL-13+/+ or IL-13-/- T cells into wild-type mice (data not shown). These data indicate that T cell-derived IL-13 is a critical regulator of eotaxin production in the lung, pulmonary eosinophilia, mucus hypersecretion, and AHR in wild-type mice. However, alternative T cell regulated pathways also operate independently of IL-13 to induce disease.



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  		FIGURE 5. AHR and allergic disease of the lung in wild-type mice is markedly attenuated in the absence of T cell-derived IL-13. Wild-type mice received OVA-specific IL-13+/+ or IL-13-/- Thi cells (2 x 106) i.v. and were aerosolized with OVA. Pulmonary and bone marrow responses were then monitored. a, Airways reactivity to methacholine was measured by barometric plethysmography. Data represent the percent increase in Penh over baseline reactivity in the absence of cholinergic stimuli (mean ± SEM, n = 6–8 mice per group) and are representative of two independent experiments. Only the maximal increase in Penh (25 mg/ml methacholine) is shown, but these data are representative of the full-dose response curve (3–50 mg/ml methacholine). b, Number of peribronchial/perivascular eosinophils per 10 similar high-powered fields (HPF, x1000) for each group (mean ± SEM, n = 2–3 mice per group). c, Number of mucus-secreting cells per 10 similar high-powered fields (x1000) in the central bronchi-bronchiole epithelial regions (mean ± SEM, n = 3–4 mice each group). d, Eotaxin levels in lung. Lungs from respective groups (up to three per group) were taken and cells were disaggregated. Cell-free supernatants were assayed by ELISA for eotaxin. Data represent the mean ± SEM for each group. Significant differences (p < 0.05) in means are shown.

 
Role of the IL-4R{alpha} chain in the induction of T cell-induced allergic disease

The induction of AHR by the delivery of recombinant IL-13 to the lung is dependent on signaling through the IL-4R{alpha} chain in experimental models of AHR in naive (nonallergic) mice (Fig. 1Go and Ref. 28). To dissect the role of the IL-4R{alpha} chain in the mechanism of IL-13-induced AHR that is elicited by IL-13 produced from Ag-activated T cells, we transferred IL-13+/+ or IL13-/- T cells to IL-4R{alpha}-/- mice (Fig. 6Go). IL-13+/+ T cells potently induced AHR (Fig. 6Goa) that directly correlated with the accumulation of eosinophils (albeit reduced in comparison to IL-13+/+ T cell responses in wild-type mice, cf Fig. 5Gob) in the airways (Fig. 6Gob), and marked production of eotaxin (Fig. 6God). However, T cell-induced mucus production was critically dependent on the presence of the IL-4R{alpha} chain (cf Fig. 5Goc). These results suggest that experimental asthma, induced by adoptively transferred T cells, can be established in the absence of signals transduced by the IL-4R{alpha} chain. Moreover, they indicate that in the allergic lung IL-13 can signal independently of the IL-4R{alpha} chain to induce AHR, eosinophilia, and eotaxin production, but not mucus hypersecretion. Collectively, the above results raise the possibility of alternative signaling pathways for IL-13 in the mechanism of T cell-induced allergic airways inflammation and AHR.



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  		FIGURE 6. IL-13+/+ but not IL-13-/- CD4+ T cells induce AHR and allergic disease of the lung in IL-4R{alpha}-/- mice. IL-4R{alpha}-/- mice received OVA-specific IL-13+/+ or IL-13-/- Thi cells (2 x 106) i.v. and were aerosolized with OVA. Pulmonary and bone marrow responses were then monitored. a, Airways reactivity to methacholine was measured by barometric plethysmography. Data represent the percent increase in Penh over baseline reactivity in the absence of cholinergic stimuli (mean ± SEM, n = 6–8 mice per group) and are representative of two independent experiments. Only the maximal increase in Penh (25 mg/ml methacholine) is shown, but these data are representative of the full dose-response curve (3–50 mg/ml methacholine). b, Number of peribronchial/perivascular eosinophils per 10 similar high-powered fields (HPF, x1000) for each group (mean ± SEM, n = 2–3 mice per group). c, Number of mucus-secreting cells per 10 similar high-powered fields (x1000) in the central bronchi-bronchiole epithelial regions (mean ± SEM, n = 3–4 mice in each group). d, Eotaxin levels in lung. Lungs from respective groups (up to three per group) were taken and cells were disaggregated. Cell-free supernatants were assayed by ELISA for eotaxin. Data represent the mean ± SEM for each group. Significant differences (p < 0.05) between means are shown.

 
The requirement for IL-13 in the induction of allergic disease independently of the IL-4R{alpha} chain was highlighted by total abolition of AHR, eosinophilia, and eotaxin production when we transferred IL-13-/- T cells into IL-4R{alpha}-/- mice (Fig. 6Go). Although reduced in comparison to wild-type mice, both IL-13+/+ and IL-13-/- T cell populations accumulated in similar numbers (5 and 3% of the total lymphocyte population, respectively; mean percentage of three lungs pooled per group) in the lung compartment in the absence of the IL-4R{alpha} chain. Furthermore, the distribution of transferred T cells was similar in the lung tissue. The observation that IL-13+/+ T cells but not IL-13-/- T cells induced experimental asthma in IL-4R{alpha}-/- mice strongly suggests that IL-13 has a crucial role in these events and is therefore able to signal independently of the IL-4R{alpha}-/- chain in this model of T cell-induced allergic disease.

Role of eotaxin and STAT6 in the mechanism of IL-13-induced AHR independently of the IL-4R{alpha} chain

To further identify the mechanism whereby T cell-derived IL-13 may signal independently of the IL-4R{alpha} chain to induce AHR, eosinophilia, and eotaxin production, we transferred Thi cells into naive STAT6-/- mice. In contrast to experiments with IL-4R{alpha}-/- mice, the transfer of IL-13+/+ T cells did not induce AHR (Fig. 7Go), pulmonary eosinophilia, or eotaxin production (results not shown). Therefore, the alternative signaling pathway(s) for IL-13 is ultimately dependent on STAT6-regulated processes in the allergic lung.



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  		FIGURE 7. IL-13+/+ CD4+ T cells do not induce allergic disease in STAT6-/- mice. STAT6-/- mice received OVA-specific IL-13+/+ Thi cells (IL-13+/+; 2 x 106) or PBS i.v. and were aerosolized with OVA. Airways reactivity to methacholine was measured by barometric plethysmography. Data represent the percent increase in Penh over baseline reactivity in the absence of cholinergic stimuli (mean ± SEM, n = 6–7 per group) and are representative of two independent experiments. There were no significant differences (p < 0.05) in airways responsiveness between IL-13+/+ and PBS groups.

 
In our T cell-induced model of allergic airways disease, eotaxin production was critically regulated by IL-13 (Figs. 5God and 6d) and thus both factors were linked to the development of AHR and inflammation. Eotaxin production is also regulated by STAT6 (55). Therefore, to determine whether eotaxin was critical in the mechanism of T cell-induced AHR and inflammation, we transferred IL-13-/- T cells to and transiently expressed eotaxin (by the delivery of recombinant VV vectors encoding this factor) in the airways of naive IL-4R{alpha}-/- mice. At the peak of eotaxin expression and after OVA exposure to the lungs, AHR, inflammatory infiltrates, and cytokine levels were characterized (Fig. 8Go). Neither eotaxin production nor the transfer of IL-13-/- T cells to the lung, alone, induced AHR or eosinophilia (Fig. 8Go, a and b). However, transfer of IL-13-/- T cells in conjunction with the expression of eotaxin in the lung of IL-4R{alpha}-/- mice induced AHR and eosinophilic inflammation (Fig. 8Go, a and b). The recruitment of eosinophils to the airways in the presence of IL-13-/- T cells and eotaxin gene expression correlated with increased levels of IL-5 but not IL-13 in the lung compartment (Fig. 8Go, c and d).



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  		FIGURE 8. Eotaxin expression in conjunction with IL-13-/- CD4+ T cells restores allergic disease of the lung in IL-4R{alpha}-/- mice. IL-4R{alpha}-/- mice received IL-13+/+ or IL-13-/- Thi cells or PBS i.v. and were aerosolized with OVA. IL-4R{alpha}-/- mice also received VV-expressing eotaxin (VV-HA-eotaxin) or control VV (VV-HA-X), alone or in combination, with IL-13-/- Thi cells or PBS treatment. a, Airways reactivity to methacholine was measured by barometric plethysmography. Data represent the percent increase in Penh over baseline reactivity in the absence of cholinergic stimuli (mean ± SEM, n = 6–8 mice per group) and are representative of two independent experiments. Only the maximal increases in Penh (25 mg/ml methacholine) are shown, but these data are representative of the full-dose response curve (3–50 mg/ml methacholine). b, The total number of eosinophils per milliliter of bronchoalveolar lavage (mean ± SEM, n = 3–4 mice per group). IL-5 levels (c) and IL-13 levels (d) in lung. Lungs from respective groups (up to four per group) were disaggregated and cells were stimulated in the presence of OVA (200 µg/ml). Cell-free supernatants were assayed by ELISA for IL-5 and IL-13. Data represent the mean ± SEM for each group. Significant differences (p < 0.05) in means are shown.

 
Collectively, we have shown that gene transfer of eotaxin to IL-4R{alpha}-/- mice amplifies the recruitment of eosinophils to the airways and induces AHR only in the presence of OVA-specific IL-13-/- T cells. These data suggest that IL-13 may regulate eotaxin production in the lung, which subsequently promotes airways inflammation and AHR. This T cell-operated mechanism functions, in part, through the IL-4R{alpha}-/- chain but also independently of this signaling system. However, IL-13-mediated eotaxin expression, eosinophilia, and AHR are ultimately dependent on the activation of STAT6 within the lung.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IL-13 has been identified as a potential key regulator of the pathogenesis of asthma. This cytokine is present in respiratory secretions and expressed in T cells from asthmatics, and mutations in this molecule that induce enhanced signaling correlate with the presentation of the asthma phenotype (56, 57, 58). Additionally, blockade of signaling by directly neutralizing IL-13 (and not its receptor complex) results in marked attenuation of mucus production, eosinophilia, and AHR in murine models of experimental asthma (26, 28). Investigations in naive recombinase-activating gene-1-deficient mice and IL-4R{alpha}-/- mice also indicate that the mechanism by which IL-13 promotes its potent spasmogenic response in the lung was dependent on signaling through the IL-4R{alpha} chain and not resident pulmonary T or B cells (28). Although the above investigations have elegantly identified the importance of IL-13 in the mechanisms underlying the induction of AHR, they do not elucidate the pathways by which this cytokine induces its potent spasmogenic effect in the allergic lung. In this investigation, we show that 1) eotaxin production is dependent on IL-13 and STAT6 but not the IL-4R{alpha} chain; 2) mucus production is dependent on IL-13, IL-4R{alpha} chain, and STAT6; and 3) airways hyperreactivity and pulmonary eosinophilia are dependent on Thi cells/factors (but not IL-13) and eotaxin production. Collectively, our data suggest that IL-13 can signal independently of the IL-4R{alpha} chain to induce phenotypic characteristics of allergic disease.

By adoptively transferring wild-type OVA-specific CD4+ T cells (IL-13+/+ T cells) to naive mice in the presence of inhaled OVA, we were able to induce allergic airways inflammation that was characterized by lymphocytic and eosinophilic infiltrates, mucus hypersecretion, eotaxin production, and AHR (Fig. 5Go). Importantly, this model allowed for the dissection of the role of T cell-derived IL-13 in the mechanism of generation of these phenotypic characteristics of asthma that was not possible by using sensitization models. These investigations also disclosed the potential of Ag-reactive Thi cells (IFN-{gamma}, IL-4, and IL-5 producers) to induce AHR and allergic diseases of the lung. The potent induction of allergic disease by Thi cells mimics that observed when transgenic OVA-peptide-specific Th1 (IFN-{gamma}-producing) and Th2 (IL-4 and IL-5) cells were adoptively transferred together to mice in the presence of cognate Ag (17). Furthermore, these observations support the concept that the interplay between Th1 and Th2 cytokines when released from Ag-activated T cells promotes, rather than attenuates, allergic disease (17, 59). Interestingly, T cells of an intermediate phenotype (IFN-{gamma} and IL-4 producing) have been identified in respiratory secretions from asthmatics (60).

By contrasting experiments with IL-13+/+ T cells, mice that received IL-13-/- T cells developed attenuated inflammatory responses characterized by reduced eosinophilia (pulmonary and bone marrow compartments), mucus production, and AHR. Notably, however, eotaxin production in the lung was critically dependent on IL-13 liberated from T cells. These results show the central importance of T cell-derived IL-13, in association with other T cell-derived cytokines, in the fundamental mechanism regulating the induction of allergic airways disease. They also support previous investigations in sensitization models of allergic asthma that IL-13 can regulate disease pathogenesis (26, 28, 51). However, our investigations show that allergic pathways are activated by T cells independently of IL-13. It is likely that IL-4 regulates the AHR and pulmonary eosinophilia that persists in the absence of IL-13 as allergic disease does not develop in IL-4R{alpha}-/- mice in the presence of IL-13-/- T cells (Fig. 6Go). Furthermore, we have previously demonstrated that these features of allergic disease do not develop in the absence of both cytokines, while they persist independently of each other (51). It is tempting to speculate that IL-13 derived from T cells up-regulates the production of eotaxin that synergizes with other T cell-derived cytokines such as IL-5 to induce eosinophilia and AHR (13, 18, 33, 54, 61, 62, 63, 64). Indeed, IL-5 and eotaxin directly cooperate to promote eosinophil mobilization from the bone marrow and recruitment into tissues (54, 61, 65). Eotaxin also induces AHR in naive IL-5 transgenic but not normal mice (64). Furthermore, integrated signaling events among IL-13, IL-4, and IL-5 have been shown to underpin the induction of these hallmark features of allergic disease of the lung (51).

Currently, IL-13 is thought to signal through a complex consisting of the IL-13R{alpha}1/IL-4R{alpha} chains by activating STAT6 (40, 66). Furthermore, there is evidence that the IL-4R{alpha} chain is a key component in the mechanism of IL-13-induced AHR (28). To investigate the role of the IL-4R{alpha} chain in the mechanism of IL-13-induced AHR in the allergic lung, IL-13+/+ T cells or IL-13-/- T cells were transferred to mice deficient in this receptor (Fig. 6Go). In marked contrast to experiments where recombinant IL-13 was directly instilled into the airways of IL-4R{alpha}-/- mice (Fig. 1Go and Ref. 28), AHR was not attenuated when IL-13 was secreted from activated T cells. We speculate, that in contrast to naive mice, allergen-specific CD4+ T cells (Thi cells) provide a factor(s) that is critical in up-regulating alternative receptor systems in the lung, providing a mechanism for IL-13 to signal independently of the IL-4R{alpha} chain. Alternatively, the absence of IL-4R{alpha} chain in these genetically modified mice promotes the up-regulation of this receptor system in a T cell-dependent fashion. Furthermore, eosinophils expanded in the bone marrow and were recruited to the airways (albeit in reduced numbers) and eotaxin levels were not diminished in IL-4R{alpha}-/- mice. Mucus hypersecretion, however, was critically linked to IL-13 signaling through the IL-4R{alpha} chain as previously shown (26, 28, 29, 51).

The role of IL-13, rather than other T cell factors, in the induction of these features of allergic disease, independently of the IL-4R{alpha} chain, was highlighted by the transfer of IL-13-/- T cells to IL-4R{alpha}-/- mice (Fig. 6Go). All features of allergic disease were ablated in the absence of both molecules. Collectively, the above investigations demonstrate the importance of IL-13 in the generation of allergic disease and that this cytokine induces defined pathophysiologic events independently of the IL-4R{alpha} chain. It is likely that there are distinct temporal requirements for IL-4R{alpha} chain usage by IL-4 and IL-13 in the mechanisms regulating the immunopathogenesis of allergic disease. IL-4 may primarily utilize the IL-4R{alpha} chain during T cell priming and subsequently in the induction of isotype switching in B cells for the production of IgE (12, 16, 21, 28, 34). IL-13 would appear to utilize this receptor in the latter phases of the allergic response to promote AHR and inflammation (18, 26, 28, 51).

Notably, eotaxin production in the lung was critically regulated by IL-13+/+ T cells and not by the IL-4R{alpha} chain, and that increased levels of this chemokine correlated with disease severity (level of AHR, eosinophilia, and mucus production) (Figs. 5Go and 6Go). These observations suggested that IL-13-dependent production of eotaxin underpins the induction of allergic disease by T cells independently of the IL-4R{alpha} chain. There is also emerging evidence that IL-13 induces eotaxin expression in the allergic lung and that there is a distinct temporal requirement for this chemokine (by signaling through the CCR3) in the induction of AHR (18, 33, 63). To examine the interplay between T cells and eotaxin in the mechanism of induction of allergic disease, we expressed this chemokine in the airways in the absence of T cell-derived IL-13 and the IL-4R{alpha} chain. Expression of eotaxin or the transfer of IL-13-/- T cells, alone, did not induce allergic disease of the lung (Fig. 8Go). However, allergic disease was fully restored in IL-4R{alpha}-/- mice in the presence of IL-13-/- T cells and elevated levels of eotaxin (Fig. 8Go). AHR was completely re-established and this correlated with recruitment of eosinophils to the airways and increased production of IL-5 (but not IL-13) in the lung. IL-13 has been previously shown to regulate eotaxin production but here we identify this pathway, in association with factors secreted from Ag-specific T cells, as an important mechanism in the generation of allergic disease of the lung. Moreover, this pathway regulates allergic disease independently of the IL-4R{alpha} chain, highlighting the potential need to neutralize the function of IL-13, rather than solely the IL-4R{alpha} chain, to resolve asthma. This concept is further supported by the observations that Th2 responses that are not critically dependent on IL-4 (IL-5 production) can occur independently of the IL-4R{alpha} chain (67). We have also shown that aspects of allergic airways disease (AHR and eosinophilia, but not mucus hypersecretion or Ab production) persist in sensitized and aeroallergen-challenged IL-4R{alpha}-/- mice (our unpublished observations).

To further dissect the mechanism by which IL-13 induces allergic disease of the lung independently of the IL-4R{alpha} chain, we transferred IL-13+/+ T cells to STAT6-/- mice (Fig. 7Go). IL-13 is known to signal through STAT6 and eotaxin expression is also dependent on this molecule (41, 55). The induction of allergic disease of the lung in sensitization models has also been shown to be dependent on STAT6 (42, 43, 44). In STAT6-/- mice, IL-13+/+ T cells could not induce AHR (Fig. 7Go) or the hallmark features of allergic disease (result not shown). Thus, STAT6 is not only important for the priming phase of allergic disease of the lung by regulating T cell commitment and development, but also in the activation of subsequent T cell-regulated effector pathways within pulmonary tissues.

Although IL-13 is thought to signal primarily through the IL-13R{alpha}1-IL-4R{alpha} complex, we provide evidence for alternative signaling pathways for this cytokine in the allergic lung. Our data support emerging evidence in B cells where the presence of an IL-4R{alpha} chain-independent IL-13R-mediated signaling pathway for Ab (IgG2a and IgG2b) production was disclosed by using a soluble form of the IL-13R{alpha}1 chain (46). Here, we extend previous findings by showing that IL-13 signaling independent of the IL-4R{alpha} chain is critically linked to STAT6 in the allergic lung.

In conclusion, although IL-13 may utilize the IL-4R{alpha} chain to induce defined features of airways allergic disease, we have shown that this cytokine also employs alternative components of the IL-13R signaling system to modulate pathogenesis. Notably, IL-13-producing T cells operate this novel pathway and its effector mechanisms are critically regulated by STAT6 activation and linked to eotaxin production and eosinophilia. The central role of STAT6 in regulating allergic pathways identifies this molecule as a critical molecular switch for the induction of asthma. This investigation highlights the potential need to directly target IL-13, rather than the IL-4R{alpha} chain, to resolve inflammation and bronchial hyperreactivity in asthma.


   Acknowledgments
 
We thank Drs. S. P. Hogan and S. Mahalingam for helpful discussions on this manuscript. We also thank Dr. Debra Donaldson and the research support team of Genetics Institute for the gift of recombinant murine IL-13. We thank W. Damcevski and the staff from the Specific Pathogen-Free Facility (John Curtin School of Medical Research) for their invaluable assistance.


   Footnotes
 
1 This work was supported by a Human Frontiers Grant (to P.S.F.). J.M. and A.S. are supported by the German Research Association (Grant MA 2241/1) and the Coordenaçao de Aperfeiçoamento de Pessoal de Nivel Superior and Fundaçao de Amparo à Pesquisa do Estado de Sao Paulo Foundations, Brazil, respectively. Back

2 Address correspondence and reprint requests to Dr. Paul S. Foster, Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia. E-mail address: Paul.Foster{at}anu.edu.au Back

3 Abbreviations used in this paper: AHR, airways hyperreactivity; Penh, enhanced pause, VV vaccinia virus; HA, hemagglutinin. Back

Received for publication March 2, 2001. Accepted for publication June 4, 2001.


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