Introduction

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Clinical and experimental investigations have demonstrated a strong correlation between the presence of CD4⁺ T helper 2 lymphocytes (Th2 cells) and disease severity, suggesting an integral role for these cells in the pathophysiology of asthma (1). Th2 cells are thought to induce asthma through the secretion of an array of cytokines (interleukin [IL]-4, IL-5, IL-6, IL-9, IL-10, and IL-13) that activate inflammatory and resident effector pathways in the lung both directly and indirectly (5, 6). In particular, recent investigations have identified IL-13 as a potential key regulator of the pathogenic mechanism(s) underlying allergic disease. IL-13 is expressed at exaggerated levels in atopic and nonatopic asthma (7, 8), atopic dermatitis (9, 10), and allergic rhinitis (11). Furthermore, IL-13 is produced in high quantities by Th2 cells, and this cytokine regulates an array of immunopathogenic effects that are hallmark features of asthma (immunoglobulin [Ig] E production, mucus hypersecretion, eosinophil recruitment and survival, airways hyperreactivity [AHR], and the expression of CD23, adhesion systems, and chemokines [eotaxin-1]) (12). Recent investigations have also demonstrated a central role for IL-13 in the regulation of experimental asthma in mice (16). Administration of IL-13 to naive mice or chronic overexpression of this cytokine in the lung promotes mucus cell metaplasia, eosinophil accumulation, and AHR (16, 19). Furthermore, neutralization of IL-13 activity by treatment with the soluble form of the IL-13 receptor alpha 2 (IL-13R₂) chain reverses AHR and pulmonary mucus cell hyperplasia in the allergic lung of allergen-challenged mice (17).

IL-4 and IL-13 regulate similar and distinct immunopathologic functions, which can be explained by the utilization of specific subunits in their respective receptor complexes (18, 20). IL-4 binds to the IL-4R chain, which is a common component of both the IL-4R (IL-4R type 1 [IL-4R-c chains]) and the IL-13R (IL-4R type 2 [IL-4R- IL-13R1 chains]) complexes (21, 22). By contrast, IL-13 only binds to the IL-13R complex (IL-4R type 2). Signaling through either of these receptor complexes activates janus kinase 1 and signal transducer and activator of transcription (STAT)-6 (21). IL-13 has also been shown to bind to the IL-13R₂ chain, which IL-4 does not bind (28). However, this chain may primarily act as a decoy receptor or inhibitor of IL-13 function rather than participating in signaling processes (18). The differential expression of IL-4 and IL-13 receptor subchains on inflammatory cells may also explain individual effector function of these cytokines (18, 20).

Although IL-13 has an important role in the induction of the allergic phenotype in mouse models, the molecular processes and sequence of cellular events that lead to IL-13-induced manifestations of disease have not been fully elucidated. Furthermore, it is not known whether IL-13 promotes spasmogenic responses to a range of nonspecific stimuli or if this cytokine selectively upregulates cholinergic responses. Blockade of IL-13 signaling in the allergic lung inhibits AHR to acetylcholine and mucus production, and attenuates the recruitment of eosinophils. In naive IL-4R-deficient mice, IL-13 failed to induce the asthma phenotype (IgE, AHR, eosinophilia, and mucus production), whereas responses were not attenuated in recombinase-activating gene 1-deficient mice. These data in naïve mice suggest that IL-13 primarily signals through the IL-4R to induce these acute features of allergic airways disease. However, these experiments did not dissect the contribution of mucus production or tissue eosinophilia to the development of AHR. Indeed, recent investigations with IL-13-deficient mice and neutralizing monoclonal antibodies (mAbs) against IL-4 and IL-5 suggest that integrated signaling events between these molecules coordinate the development of allergic airways disease and AHR (29).

To further elucidate the mechanisms involved in IL-13- induced AHR and the requirement of mucus hypersecretion and eosinophilia for this process, we have characterized pulmonary responses in IL-4/IL-13-, IL-4R-, STAT-6-, IL-5-, eotaxin-, and IL-5/eotaxin-deficient mice after intratracheal administration of IL-13. In wild-type (WT) mice, IL-13 induced an initial AHR response (preinflammatory phase [PIP]) that occurred within 6 h (peaked at 24 h) of administration and developed independently of eosinophilic inflammation and mucus hypersecretion in the lung. The PIP response was immediately followed by an inflammatory phase (IP) (48 h) that was characterized by an amplified AHR, which correlated with the development of mucus hypersecretion and eosinophilic infiltration of the lung. IL-4R and STAT-6 were critical for the development of AHR during both responses and for mucus production and eosinophil accumulation during the IP. Notably, the IP AHR persisted (albeit diminished) in mice deficient in IL-5 and/or eotaxin where mucus secretion was not inhibited but eosinophil recruitment was attenuated. Collectively, our data imply that signals elicited through IL-4R and STAT-6 are the critical regulators of IL-13-induced AHR and that IL-5/eotaxin-regulated pathways do not have obligatory roles.

	Materials and Methods

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Mice

Seven- to nine-week-old naïve male BALB/c WT, IL-4/IL-13-, IL-4R-, STAT-6-, IL-5-, eotaxin-, and IL-5/eotaxin-deficient mice were obtained from specific pathogen-free facilities at the Australian National University. All experimental procedures complied with the requirements of the Animal Care and Ethics Committee of the Australian National University.

Intratracheal Instillation of Recombinant IL-13

Mice were anesthetized with an intravenous injection of 100 µl saffan solution (1:4 diluted in water). Mice were intubated with a 22-gauge catheter needle, through which murine recombinant IL-13 (gift from Genetics Institute, Cambridge, MA) (10 µg dissolved in 20 µl phosphate-buffered saline [PBS]) or vehicle control (PBS) was instilled. AHR, mucus production, and eosinophilia were measured at 6, 12, 24, 48, 96 h, and 8 d after instillations into the tracheas of WT mice. These parameters were also measured at 24 and 48 h (peak responses for the PIP and IP, respectively) after instillations into the tracheas of IL-4/IL-13-, IL-4R-, STAT-6-, IL-5-, eotaxin-, and IL-5/eotaxin-deficient mice.

Measurement of Airway Reactivity to Spasmogens

Airway reactivity to methacholine was assessed in conscious, unrestrained mice by barometric plethysmography, using apparatus and software supplied by Buxco (Troy, NY). This system yields a dimensionless parameter known as enhanced pause (Penh), reflecting changes in waveform of the pressure signal from the plethysmography chamber combined with a timing comparison of early and late expiration, which can be used to empirically monitor airway function. Measurement was performed as previously described (31). Briefly, mice were placed in the chamber, and baseline reading taken and averaged for 3 min. Aerosolized methacholine (concentrations in solution ranging from 3.125 to 50 mg/ml) was then delivered through an inlet into the chamber for 2 min, and readings were averaged over a period of 3 min after each dose was administered. Airways resistance to methacholine after exposure to IL-13 was also determined. Mice were anesthetized (10 mg/kg xylazine, 100 and 50 mg/kg ketamine, and every 30 min thereafter, intraperitoneally), and cannulae were placed in the trachea (to measure airflow) and jugular vein (for drug administration). Spontaneous breathing was stopped by an intravenous injection of pancuronium bromide (0.4 mg/kg initially and 0.2 mg/kg every 30 min thereafter), and the mice were ventilated (tidal volume, 8 ml/kg at 150 breaths/min) (SAR-830 ventilator; CWE Inc., Ardmore, PA). Tracheal flow was measured over the tracheal cannulae (Fleisch pneumotachograph; Fleisch, Lausanne, Switzerland), and transpulmonary pressure was measured with a differential pressure transducer. Breath-to-breath measurement of airway resistance (RL) was recorded (PMS800; Mumed, London, UK) according to the principles of Amdur and Meads (32). Methacholine was administered intravenously at increasing doses (25, 50, 100, 200, and 400 mg/kg) at 5-min intervals. Two minutes before each methacholine challenge, lungs were hyperinflated once (by delivering tidal volume twice) to prevent and reverse atelectasis.

Reactivity of Isolated Trachea to Spasmogenic Stimuli

Mice were killed by an overdose of pentobarbitone sodium administered intraperitoneally (250 mg/kg). The respiratory tract and associated alimentary tissue were rapidly excised, placed in a petri dish containing cold Krebs bicarbonate solution, and the trachea was dissected free from surrounding tissue. The trachea was cut in half to yield two 2-mm-long segments. Each segment was suspended under a resting tension of 0.5 g in organ baths containing 2 ml Krebs bicarbonate solution maintained at 37°C and sparged with 5% CO₂ in O₂. The composition of the Krebs bicarbonate solution was (in mM): NaCl 117; KCl 5.36, NaHCO₃ 25, KH₂PO₄ 1.03, MgSO₄·7H₂O 0.57, CaCl₂ 2.5, glucose 11.1, and indomethacin 0.0025. Changes in tension were recorded via an isometric force transducer (FTO3; Grass Instruments) connected to a custom-built preamplifier and data acquisition system. After a 45-min equilibration period, the viability of tissues was tested by the cumulative addition of 0.2 and 10 µM concentrations of carbachol. After the washout of carbachol and a 15-min equilibration period, preparations were exposed to a single 60-mM concentration of KCl. The peak KCl-induced contraction was used as the reference contraction for subsequent drug-induced responses. In each preparation, a cumulative concentration- effect curve was then completed to methacholine, acetylcholine, KCl, or endothelin-1. After a 30-min washout and rest period, a second curve was completed using a different spasmogen.

Characterization of Eosinophils and Mucus-Staining Cells in Lung Tissue

Lung tissues 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 enumeration of mucin-secreting cells or Charbol's chromotrope hematoxylin for identification of eosinophils. The number of mucus-staining cells and eosinophils in the central bronchi-bronchiole area was identified by morphologic criteria and quantified as previously described (31, 33).

Statistical Analysis

The significance of differences between the means of experimental groups was analyzed using Student's unpaired t test. Values were reported as the mean ± standard error of the mean (SEM). Differences in mean values were considered significant if P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Temporal Characterization of IL-13-Mediated AHR, Eosinophil Accumulation, and Mucus Secretion

Intratracheal installation of 10 µg of IL-13 into naïve WT mice induced a rapid and potent AHR response. An early-onset PIP AHR was observed between 6 and 24 h and was followed by an amplified and sustained IP AHR that peaked at 48 to 96 h (Figure 1A). The peak IP AHR response (48 h) was significantly greater than the maximal PIP (12 to 24 h) response (Figure 1A; P < 0.05), suggesting two temporally distinct responses. AHR returned to baseline levels within 8 d; however, eosinophil infiltrates and mucus production were still evident at this time. Changes in Penh in response to IL-13 directly correlated with increased RL (resistance) to methacholine. When RL was used as a measure of airway caliber, intratracheal instillation of IL-13 into naïve WT mice induced AHR. For example, at the 24-h time point, a 100 mg/kg dose of methacholine increased RL by 503 ± 79 cm H₂O/liter/s in IL-13-treated mice, but by only 252 ± 29 cm H₂O/liter/s in control mice (PBS administration).

View larger version (15K): [in this window] [in a new window]   Figure 1.   Kinetic characterization of IL-13-induced AHR, eosinophil infiltration, and mucus secretion. AHR, eosinophil, and mucus-secreting cell numbers in lung tissue of WT mice were determined at various time points after intratracheal administration of IL-13 (10 µg/20 µl PBS) or the same volume of PBS. Data are the means ± SEM from n = 4 to 10 mice per group from two individual experiments. Statistical significance of differences (P < 0.05) was determined using Student's unpaired t test. (A) AHR to methacholine (at 25 mg/ml, which gave the maximal response) challenge was measured by barometric plethysmography and the data represent the percentage increase in Penh over baseline reactivity. *P < 0.05, compared with the respective PBS control; # P < 0.05, compared with 24-h IL-13-treated mice. There was no significant difference between 12- and 24-h responses. (B) Number of peribronchial/ perivascular eosinophils per 10 similar high-powered fields (HPF) (original magnification: ×40) for each group. **P < 0.05, compared with the respective PBS control. (C) The number of mucus-secreting cells in the bronchial epithelium. Lung sections were stained with alcian blue/PAS and the numbers of mucin-secreting cells per HPF in the central bronchi-bronchiole epithelial regions were determined. Seven similar fields were counted per lung. *P < 0.05, compared with the respective PBS control.

The PIP IL-13-induced AHR was not associated with eosinophil infiltration or mucus secretion in the lung (6 to 24 h) (Figures 1A to 1C). However, 48 h after instillations, eosinophil numbers were significantly elevated in IL-13- treated mice when compared with PBS-treated animals (P < 0.05) (Figure 1B). The levels of lymphocytes, neutrophils, and macrophages did not significantly change at this time (results not shown). Similar to eosinophil infiltration, mucus levels were only significantly elevated 48 h after IL-13- administration (Figures 1B and 1C) (P < 0.005). Notably, mucus secretion and eosinophil numbers remained consistently elevated for up to 8 d after IL-13 challenge (Figures 1B and 1C). These sustained responses to a single dose of IL-13 highlight the potency of this cytokine and its potential ability to generate allergic inflammatory cascades.

Isolated Smooth Muscle Reactivity to Spasmogenic Stimuli

IL-13 may act directly on airway smooth muscle or alternatively by promoting the release of mediators from resident or infiltrating inflammatory cells that can prime for enhanced spasmogenic responses (18). To investigate the effects of IL-13 on airway smooth muscle reactivity, concentration-effect curves to a range of different spasmogens were carried out on isolated tracheal smooth muscle preparations from control and IL-13-treated mice 24 and 48 h after instillations. Methacholine induced concentration-dependent contractions in isolated tracheal preparations from both groups (Figures 2A and 2B). However, preparations from IL-13-treated mice were not more responsive to methacholine than were preparations from control mice, at either time point (Figures 2A and 2B). Similarly, IL-13 treatment had no significant influence on tissue responsiveness to acetylcholine, KCl, or endothelin-1 (Table 1).

View larger version (13K): [in this window] [in a new window]   Figure 2.   Reactivity of isolated tracheal preparations to methacholine after in vivo treatment with IL-13. Cumulative dose-response curves to methacholine were constructed in isolated tracheal preparations 24 (A) and 48 h (B) after intratracheal administration of IL-13 or PBS. Contractile responses are expressed as percent of the 60-mM KCl-induced response. No significant differences to methacholine were observed between groups. Inflammation was present in preparations at 48 h but not at 24 h.

Role of IL-4R, STAT-6, Endogenous IL-4/IL-13, and Eosinophil Regulatory Molecules in the Onset of IL-13-Induced PIP AHR

The observation that AHR was induced rapidly but was also maintained for 96 h (which correlated with the induction of pulmonary eosinophilia and mucus production) indicated that IL-13 may act via multiple pathways. The sustained effect (96 h to 8 d) of IL-13 in the lung suggested that this cytokine activated endogenous pathways that subsequently liberated mediators that maintained AHR, eosinophilia, and mucus production. Initially, we investigated the requirements for the IL-4R, STAT-6, and endogenous IL-13 and IL-4 in the induction of PIP AHR (Figure 3). Previous investigations have demonstrated that IL-13 mediated IP AHR in association with mucus secretion (> 24 h after IL-13 administration) and that this response was dependent on IL-4R (17). To determine the role of this receptor and downstream signaling processes in the IL-13- mediated PIP AHR that was independent of eosinophilia and mucus cell hyperplasia, we employed IL-4R- and STAT-6-deficient mice. IL-13-mediated PIP AHR was ablated in mice deficient in these molecules (Figure 3A). Collectively, these data suggest that IL-13 signals through the IL-4R type 2 (IL-4R and IL-13R₁ subchains) and the STAT-6 pathway to induce PIP AHR. Interestingly, AHR was not attenuated in Swiss nude mice 24 h after intratracheal administration of IL-13 (51.10 ± 1.30 [PBS] versus 115.20 ± 21.80 [IL-13 treatment] Penh [% increase of baseline] × 10; mean ± SEM at 25 mg/ml methacholine, n = 4 to 8 mice/group; P < 0.05). The level of AHR in IL-13- treated Swiss nude mice was not significantly different to that of IL-13-treated WT mice (results not shown). These findings support previous data in RAG-1-deficient mice that the mechanism for induction of AHR by the IL-4R is associated with pathways downstream of T cells (26).

View larger version (17K): [in this window] [in a new window]   Figure 3.   IL-13 induction of PIP AHR in WT mice and mice deficient in IL-4/IL-13, IL-4R, STAT-6, IL-5, eotaxin, and IL-5/ eotaxin. AHR to methacholine was determined 24 h after intratracheal administration of IL-13 (10 µg/20 µl PBS) or the same volume of PBS. Data are the means ± SEM from n = 4 to 10 mice per group from two to three experiments. Statistical significance of differences (P < 0.05) was determined using Student's unpaired t test. (A) Airways reactivity in WT and IL-4/IL-13-, IL-4R-, and STAT-6-deficient mice. *P < 0.05, compared with the respective PBS control; #P < 0.05, compared with IL-4/IL-13-, IL-4R-, and STAT-6-deficient IL-13-treated group. * P < 0.05, compared to WT IL-13-treated group. (B) Airways reactivity in WT, IL-5-, eotaxin-, and IL-5/eotaxin-deficient mice. Airways reactivity to methacholine challenge was measured by barometric plethysmography 24 h after intratracheal challenge with IL-13 and the data represent the percentage increase in Penh at 25 mg/ml methacholine over baseline reactivity in the absence of cholinergic stimuli. This concentration of methacholine induces maximal responsiveness. *P < 0.05, compared with the respective PBS control.

Next, we used IL-4/IL-13-deficient mice to determine the interplay between these cytokines for the induction of PIP AHR. The sustained effects of IL-13 on mucus hypersecretion and eosinophilia in the IP response suggested that IL-13 may generate the production of endogenous IL-13 and/or IL-4 to induce these pathophysiologic responses. Administration of IL-13 to IL-4/IL-13-deficient mice promoted PIP AHR, suggesting that endogenous IL-13 and IL-4 were not required for the induction of this AHR response. Notably, the level of AHR in these mice was significantly higher than that observed in WT mice at 24 h (Figure 3A; P < 0.05). Although eosinophils were not recruited to the airways in significant numbers during the PIP response, we observed low numbers of resident cells at baseline (Figure 1B). To determine if IL-13 may have induced PIP AHR via pathways that used eosinophils at baseline, we characterized responses to this cytokine in IL-5-, eotaxin-, and IL-5/eotaxin-deficient mice (Figure 3). No significant difference in baseline AHR levels were observed between WT, IL-5-, eotaxin-, and IL-5/eotaxin- deficient mice (Figure 3B). Furthermore, IL-13 induced PIP AHR in these factor-deficient mice to equivalent levels of that observed in WT mice (Figure 3B).

Role of IL-4R, STAT-6, Endogenous IL-4/IL-13, and Eosinophil Regulatory Molecules in the Induction of IP AHR

IL-13-induced IP AHR was associated with eosinophilic infiltration and mucus hypersecretion (Figures 1A to 1C). To identify the contribution of IL-4R and STAT-6 signaling pathways in the induction of AHR, eosinophil infiltration, and mucus secretion, IL-4R- and STAT-6-deficient mice were challenged with IL-13. As the maximal IP AHR response occurred at 48 h (Figure 1A), we measured AHR, eosinophil infiltration, and mucus secretion at this time point. Similar to the PIP AHR, IL-13-mediated IP AHR was critically dependent on IL-4R and STAT-6 (Figure 4A). Abolition of AHR in IL-4R- and STAT-6- deficient mice directly correlated with the inability of these mice to recruit eosinophils and to produce mucus in the lung in response to IL-13 administration (Figures 4B and 4C). The induction of AHR and eosinophilia during the IP was not dependent on endogenous IL-4 or IL-13 production, as these responses were not inhibited in IL-4/ IL-13-deficient mice. However, mucus production in response to IL-13 administration was, in part, dependent on the local production of IL-4 (Figure 4C; P < 0.05). AHR by 48 h was similar in both WT and IL-4/IL-13-deficient mice (Figure 4A).

View larger version (14K): [in this window] [in a new window]   Figure 4.   IL-13 induction of IP AHR, eosinophil accumulation, and mucus secretion in WT, IL-4/IL-13-, IL-4R-, STAT-6-deficient mice. AHR and eosinophil and mucus-secreting cell numbers in the lung tissue of WT, IL-4/IL-13-, IL-4R-, and STAT-6-deficient mice were measured 48 h after intratracheal administration of IL-13 (10 µg/20 µl PBS) or the same volume of PBS. Data are the means ± SEM from n = 4 to 10 mice per group from two to three individual experiments. Statistical significance of differences (P < 0.05) was determined using Student's unpaired t test. (A) Airway reactivity to methacholine challenge was measured by barometric plethysmography and the data represent the percentage increase in Penh at 25 mg methacholine over baseline reactivity in the absence of cholinergic stimuli. This concentration of methacholine induces maximal reactivity. *P < 0.05, compared with the respective PBS control. (B) Number of peribronchial/perivascular eosinophils per 10 similar high-powered fields (HPF) (original magnification: ×40) for each group. *P < 0.05, compared with the respective PBS control. (C) The number of mucus-secreting cells in the bronchial epithelium. Lung sections were stained with alcian blue/PAS, and the numbers of mucin-secreting cells per HPF in the central bronchi-bronchiole epithelial regions were determined. Seven similar fields were counted per lung. *P < 0.05, compared with the respective PBS control. # P < 0.05, compared with IL-4/IL-13 mice treated with IL-13.

Eosinophil recruitment to the lung was abolished in IL-4R- and STAT-6-deficient mice, suggesting a role for this cell in the induction of the IP AHR to IL-13. To identify the contribution of eosinophils to IL-13-mediated IP AHR and mucus secretion, IL-5-, eotaxin-, and IL-5/ eotaxin-deficient mice were administered IL-13 and responses were characterized 48 h later (Figures 5A to 5C). In IL-5-, eotaxin-, and IL-5/eotaxin-deficient mice, IL-13 induced IP AHR (Figure 5A). AHR in these mice appeared to be attenuated as compared with WT mice; however, the level of reactivity (at all concentrations of methacholine) was not significantly different between groups (Figure 5A). IL-13 administration into WT, IL-5-, eotaxin-, and IL-5/eotaxin-deficient mice promoted an eosinophil infiltration into the pulmonary compartment (Figure 5B). IL-13-mediated eosinophil accumulation in IL-5- or eotaxin-deficient mice was reduced when compared with WT mice (Figure 5B). Moreover, eosinophil numbers in IL-5/eotaxin-deficient mice were significantly reduced compared with the numbers in WT, IL-5-, and eotaxin- deficient mice (Figure 5B). In the absence of IL-5, eotaxin, and IL-5/eotaxin, IL-13-induced mucus secretion was not significantly impaired (Figure 5C).

View larger version (16K): [in this window] [in a new window]   Figure 5.   IL-13-induced IP AHR, eosinophil accumulation, and mucus secretion in WT, IL-5-, eotaxin-, and IL-5/eotaxin-deficient mice. AHR and eosinophil and mucus-secreting cell numbers in the lung tissue of WT, IL-5-, eotaxin-, and IL-5/eotaxin-deficient mice were measured 48 h after intratracheal administration of IL-13 (10 µl/20 µl PBS) or the same volume of PBS. Data are the means ± SEM from n = 4 to 10 mice per group from two to three individual experiments. Statistical significance of differences (P < 0.05) was determined using Student's unpaired t test. (A) Airway reactivity to methacholine challenge was measured by barometric plethysmography and the data represent the percentage increase in Penh at 25 mg methacholine over baseline reactivity. This concentration of methacholine induces maximal responsiveness. P < 0.05 compared with respective controls. (B) Number of eosinophils per 10 similar high-powered fields (HPF) (original magnification: ×40) for each group. *P < 0.05, compared with the respective PBS control; *P < 0.05, compared with eotaxin-deficient mice treated with IL-13; *P < 0.05, compared with IL-5-deficient mice treated with IL-13; *P < 0.05, compared with IL-5/eotaxin-deficient mice treated with IL-13. (C) The number of mucus-secreting cells in the bronchial epithelium. Lung sections were stained with alcian blue/PAS, and the numbers of mucin-secreting cells per HPF in the central bronchi-bronchiole epithelial regions were determined. Seven similar fields were counted per lung. *P < 0.05, compared with the respective PBS control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The potent spasmogenic and inflammatory actions of IL-13 have identified this molecule as a potential regulator of AHR in asthma. Although IL-13 is thought to primarily signal through the IL-13R1/IL-4R complex to initiate inflammatory responses, the subsequent cellular and molecular components employed by this cytokine to induce AHR and allergic disease in the lung has not been fully elucidated. In the present study, we have employed a mouse model system to delineate the contribution of IL-13 to the temporal induction of AHR, eosinophilic inflammation, and mucus secretion. We show that IL-13-induced AHR was characterized by a PIP and an IP. The PIP AHR response occurred independently of eosinophilic inflammation and mucus hypersecretion. By contrast, the IP AHR was associated with an amplified AHR that correlated with the recruitment of eosinophils to the airways and the production of mucus. Both the PIP and IP AHR responses were critically dependent on signaling through IL-4R and STAT-6. Notably, IP mucus secretion and eosinophil recruitment were ablated in the absence of IL-4R and STAT-6. In the absence of IL-5, eotaxin, or IL-5/eotaxin, eosinophil recruitment to the lung was attenuated in response to IL-13, whereas AHR (albeit reduced) and mucus secretion were not significantly inhibited. However, IL-13 induced eosinophil accumulation independently of both of these eosinophil regulatory molecules.

Collectively, these results suggest that IL-13 can rapidly prime for bronchoconstriction and potently activate inflammatory cascades that are sustained. Interestingly, by Day 8, IL-13-induced AHR had dissipated, whereas mucus hypersecretion and eosinophilia were still pronounced in the lung. These observations suggest that the association or dissociation between inflammation and AHR that is often observed in clinical and experimental settings will be critically dependent on the time of analyses. Our data further suggest that IL-13 may induce AHR by two pathways: one independent of inflammation and the other by promotion of inflammatory cascades. Notably, both pathways, although temporally distinct, are critically regulated by signaling through the same receptor system.

The mechanism whereby IL-13 mediates AHR has not been fully elucidated. IL-13 may contribute to AHR in a multifunctional manner by directly priming airways smooth muscle for enhanced responsiveness, by promoting the release of endogenous priming agents, or by inducing smooth muscle proliferation and/or altering matrix protein deposition (18, 34, 35). IL-13 receptor subfamilies have not been definitely shown to be present on airways smooth muscle; however, IL-13R₁ and IL-4R have been shown to be present on cardiac and vascular smooth muscle, and directly modulate noncontractile functions (36, 37). Preliminary in vitro studies using isolated airways smooth muscle preparations also indicate that IL-13 may directly modulate contractility (34). Nevertheless, in the current study the responsiveness of isolated murine airways preparations to methacholine and to a range of other spasmogens, including acetylcholine, KCl, and endothelin-1, was not augmented by exposure to IL-13. Thus, IL-13-induced AHR to methacholine cannot be readily explained by direct changes in airways smooth muscle responsiveness. These findings do not exclude, however, that IL-13 may also mediate AHR through activation and secretion of other factors derived from resident pulmonary cells (38). The abolition of IL-13-induced PIP AHR in IL-4R- and STAT-6-deficient mice suggests that IL-13-mediated signaling is through the IL-13R (IL-4R type 2 [IL-4R-IL-13R₁ chains]). This is also consistent with the fact that STAT-6 activation is downstream of IL-4R-mediated signaling and with previous investigations that have shown a critical role for IL-4R in IL-13-mediated AHR (17).

Previous investigations have demonstrated that both IL-4 and IL-13 are critically involved in airways mucus secretion (39). Moreover, chronic overexpression of IL-4 or IL-13 in the airways induces mucus hypersecretion, goblet cell hyperplasia, and airways epithelial cell hypertrophy (35, 40). The observation that mucus hypersecretion could be induced in IL-4/IL-13-deficient mice by the administration of IL-13, but not in the IL-4R- and STAT-6-deficient mice, definitively demonstrates that IL-13 (and not IL-4) is critical for the induction of this allergic response. IL-13 administration in Swiss nude mice also induced an IP mucus hypersecretion (results not shown). The observed decline in mucus secretion in IL-13-treated IL-4/ IL-13-deficient mice as compared with WT mice may be explained by the lack of baseline expression of IL-13 in the recipient animals or a reduction in residual Th2 cells (IL-13 secreting) in the pulmonary compartment because of the deficiency of IL-4.

IL-5 and eotaxin have previously been shown to be critical regulators of eosinophil trafficking and accumulation into tissue at baseline and also during inflammatory responses (41, 42). IL-13 is thought to collaborate with this pathway by regulating eotaxin expression. Furthermore, chronic overexpression of IL-13 in the lung specifically promotes eotaxin production (19). Consistent with these findings, IL-13-mediated eosinophil accumulation was significantly attenuated in IL-5-, eotaxin-, and IL-5/eotaxin- deficient mice. The level of eosinophil accumulation in eotaxin-deficient mice was significantly lower than that of IL-5-deficient mice. Interestingly, IL-13 mediated a small but significant eosinophil accumulation independently of both eotaxin and IL-5. This may be explained by IL-13 inducing the upregulation of other eosinophil-specific chemokines (43). These data confirm the importance of the interaction between IL-5 and eotaxin for localization of eosinophils in tissues and show that IL-13 is integrated into this eosinophil regulatory pathway. Interestingly, the level of IL-13-mediated eosinophil accumulation was low when compared with an allergic inflammatory response (six- to eight-fold greater) (31, 44). This may reflect the requirement for the upregulation of both eotaxin and IL-5, and the induction of synergistic interactions to promote a maximal eosinophilic response.

The contribution of eosinophils to AHR in asthma remains controversial. In some studies, eosinophils have been shown to play an obligatory role in AHR, whereas other investigations suggest that this leukocyte makes little to no contribution to the induction of AHR. These data are derived from studies that have predominantly employed neutralizing IL-5 mAbs or mice deficient in this factor (33, 44). One feature of all of these investigations is the presence of a residual eosinophil population in the pulmonary compartment of these mice after aeroallergen challenge. In this investigation, we demonstrated that IL-13 can induce a significant airways eosinophilia and AHR in the absence of IL-5. These data indicate that pathways independent of IL-5 can selectively recruit eosinophils to the lung, and support animal experimentation and recent data from clinical trials with humanized mAbs to IL-5 that AHR can occur independently of this cytokine. It is tempting to speculate that in allergen challenge models of experimental asthma (which are often employed in clinical trials), large amounts of IL-13 are released in response to airway provocation that could potentially account for the induction of the allergic phenotype (AHR and tissue eosinophilia) independently of IL-5.

Recently, we demonstrated the importance of IL-5- independent pathways for the regulation of eosinophilia and AHR in the allergic lung. We have observed that the degree of the residual pool of eosinophils after removal of IL-5 directly correlates with attenuation or persistence of AHR (unpublished observation). By employing IL-5/ eotaxin-deficient mice, we resolved tissue eosinophil accumulation after aeroallergen-challenged mice as compared with WT, IL-5-, or eotaxin-deficient mice. The eosinophil levels in the pulmonary compartment after the aeroallergen challenge of IL-5/eotaxin-deficient mice were reduced by 15- and 50-fold as compared with those of IL-5-deficient and WT mice, respectively. The level of eosinophils in the tissue of IL-5/eotaxin-deficient mice approximated nonallergic controls and responses correlated with the attenuation of AHR, suggesting a pathogenic role for low levels of tissue-dwelling eosinophils in AHR. In this context, it is interesting that we observed an attenuation in IL-13-mediated IP AHR in IL-5-, eotaxin-, and IL-5/ eotaxin-deficient mice that correlated with the reduction in eosinophil accumulation in the pulmonary compartment. These findings support the observation that eosinophils may contribute to the IP AHR response independently of IL-5. However, our data cannot exclude the possibility that IL-13-regulated eosinophilia in the IP response is only associated with and does not induce AHR. Interestingly, in allergic IL-13-deficient or IL-5-deficient BALB/c mice, AHR and eosinophilia (albeit markedly reduced in the IL-5-deficient mice) persist (40). However, blockade of both IL-5 and IL-13 conjointly results in abolition of AHR and further reduction of tissue levels of eosinophils in the allergic lung. These results suggest a close relationship between eosinophils, IL-5, and IL-13 in the mechanism regulating AHR in the allergic lung. Notably, blockade of IL-4 and IL-13 conjointly, but not alone, also ablated AHR and tissue eosinophilia, highlighting the central importance of signaling through the IL-4R chain for these responses in the allergic lung (40).

In conclusion, IL-13 can mediate both a PIP and IP asthma phenotype. The PIP response is characterized by early-onset AHR, whereas, the IP response is associated with an amplified AHR that is associated with eosinophil accumulation and mucus hypersecretion. The pathogenic effects of IL-13 are predominantly mediated through the IL-4R chain/STAT-6 signaling pathways. Furthermore, IL-13 mediates eosinophil accumulation, in part, through IL-5 and eotaxin. However, IL-13 can mediate eosinophil accumulation independently of these molecules, probably via the upregulation of other CCR3-activating chemokines. Interestingly, although IL-13-mediated pathogenic effects are restricted to IL-4R chain/STAT-6 receptor signaling pathways, these processes are temporally modulated. These findings establish the potential importance of IL-13 in the induction of key phenotypic characteristics of experimental asthma and suggest that IL-13, IL-4R chain, and STAT-6 are key targets for therapeutic intervention of this disease.