The eosinophil is a central effector cell in allergic asthma. Differentiation and function of eosinophils are regulated by the CD4 Th2 cytokines IL-3, IL-5, and GM-CSF, which all signal through a common β receptor subunit (βc). Recent therapeutic approaches targeting IL-5 alone have not ablated tissue accumulation of eosinophils and have had limited effects on disease progression, suggesting important roles for IL-3 and GM-CSF. By using a mouse model of allergic airways inflammation, we show that allergen-induced expansion and accumulation of eosinophils in the lung are abolished in βc-deficient (βc−/−) mice. Moreover, βc deficiency resulted in inhibition of hallmark features of asthma, including airways hypersensitivity, mucus hypersecretion, and production of Ag-specific IgE. Surprisingly, we also identified a critical role for this receptor in regulating type 2 immunity. Th2 cells in the lung of allergen-challenged βc−/− mice were limited in their ability to proliferate, produce cytokines, and migrate to effector sites, which was attributed to reduced numbers of myeloid dendritic cells in the lung compartment. Thus, the βc plays a critical role in allergen-induced eosinophil expansion and infiltration and is pivotal in regulating molecules that promote both early and late phases of allergic inflammation, representing a novel target for therapy.
Allergic asthma is a chronic inflammatory disease of the airways that has increased in prevalence in developed countries over recent decades (1) and has been described as an epidemic in the New England Journal of Medicine (2). Inflammatory infiltrates in the asthmatic airways are complex, and a range of cells has been associated with disease progression. These include basophils, eosinophils, effector T lymphocytes, mast cells, macrophages, neutrophils, and, more recently, T regulatory and NK-T cells (3). Of the inflammatory cells implicated in asthma, IgE-mediated degranulation of mast cells contributes to inflammatory infiltrates and acute bronchoconstriction in the early phase of allergic inflammation, whereas recruitment of CD4+ Th2 cells and eosinophils is a central feature of the late-phase response (4). Th2 cytokines (e.g., IL-3, IL-4, IL-5, IL-9, IL-13, and GM-CSF) and eosinophils are thought to play critical roles in the induction of airway hyperreactivity (AHR)3and the development of lesions that underpin chronic airway wall remodeling (5).
Indeed, the importance of eosinophils in chronic allergic disease has been elegantly highlighted both clinically and in mouse models of asthma. Reduction in tissue levels of eosinophils (∼50%) by anti-IL-5 mAb treatment in mild atopic asthmatic patients results in significantly attenuated expression of tenascin, lumican, and procollagen III in the bronchial mucosa, suggesting that eosinophils may contribute to tissue remodeling processes in chronic asthma by controlling extracellular matrix protein deposition (6, 7). These studies are supported by the temporal association between eosinophils and markers of chronic remodeling in human atopic skin, which are inhibited by i.v. infusion with anti-IL-5 mAb (8, 9). Genetic ablation of the eosinophil lineage in mice is also protective against tissue remodeling in a model of chronic asthma (10). Collectively, these studies suggest that strategies targeting eosinophils may also be effective for the resolution of at least some of the chronic manifestations of allergic asthma.
Although the above studies have identified the eosinophil as an important therapeutic target, previous attempts to inhibit accumulation of eosinophils in the airway wall of asthmatics by targeting the eosinophil-specific regulatory cytokine IL-5 have only had limited success. Notably, anti-IL-5 mAb only partially depletes tissue accumulation of eosinophils, suggesting that other molecules contribute to the regulation of this process (7, 11, 12, 13). Furthermore, we have shown that eosinophils and lesions persist in lung tissue during allergic inflammation in IL-5-deficient BALB/c mice, albeit significantly reduced (14, 15). Importantly, the elimination of eosinophils by generation of mice deficient in IL-5 and eotaxin or by genetic lineage ablation is protective against AHR or chronic tissue remodeling, respectively (10, 16). Thus, the appreciation of IL-5-independent pathways of eosinophil migration and function is of paramount importance if more complete and sustained depletion of eosinophils from the bronchial mucosa of asthmatics is to be achieved.
IL-3 and GM-CSF, which together with IL-5 are the only known mediators capable of inducing eosinophil differentiation from bone marrow progenitors, are central to the survival, migration, and activation of this granulocyte (17, 18, 19). IL-3 and GM-CSF, produced by activated T cells as well as in copious amounts by lung epithelial cells in the case of GM-CSF, share a common β receptor chain (βc) with IL-5. Indeed, signaling redundancy between these cytokines may be the critical pathway that promotes the development and recruitment of eosinophils independently of IL-5 at sites of allergic inflammation. Interestingly, limited suppression of pulmonary eosinophil numbers and AHR has been documented in a rat model of allergic asthma following partial (∼60%) attenuation of the βc using antisense oligonucleotides (20). However, the role of the βc in allergic inflammation has not been unequivocally determined by genetic deletion or direct antagonism.
Signaling through the βc by IL-3 and GM-CSF is also important in the development and function of other immune cells that play central roles in allergic inflammation. This includes dendritic cells (DC) (21), which play an integral role in the immune response to inhaled allergens (22). Importantly, IL-3 is also a critical molecular switch for the differentiation, survival, and effector function of mast cells and basophils, whereas GM-CSF is a strong activator of neutrophil and macrophage function (23, 24, 25). Thus, the central role of the βc in regulating the function of a range of immune effector cells suggests that this molecule may be a pivotal regulator of allergic inflammation.
In this investigation, we determined the role of the βc in orchestrating inflammation and its potential as a therapeutic target in a mouse model of allergic asthma. Unlike human cells, mouse cells possess an additional β receptor specific for IL-3 (βIL-3), which allows IL-3 signaling independently of the βc (26). To account for this, we used mice deficient in both βc and βIL-3 (designated as βc−/−) to mimic the signaling conditions of human cells. Mice deficient in βc or in both βc and βIL-3 possess markedly reduced basal bone marrow and peripheral blood eosinophil numbers and fail to mount an eosinophil response to parasite infestation (27, 28). In the present study, we have used βc−/− mice in an acute model of allergic airways inflammation, to show that this receptor chain is not only essential for eosinophil accumulation in tissues, but also plays a critical role in the development of hallmark features of asthma, including production of Ag-specific IgE, mucus hypersecretion, and AHR, by limiting the functional capacity of Th2 cells in the lung. Thus, βc plays a novel role in allergic disease by regulating multiple effector arms associated with the allergic inflammatory response.
Materials and Methods
Mice deficient in both βc and βIL-3 (29) on an SV129 background were supplied by L. Robb (Melbourne, Australia). These mice were backcrossed for >10 generations onto the BALB/c strain (genotyping performed by PCR). Mice deficient in both βc/βIL-3 chain are referred to as βc−/− throughout this work. Wild-type (WT) BALB/c and βc−/−-deficient mice were housed under specific pathogen-free conditions and experiments subject to approval by the Institutional Animal Care and Ethics Committee.
Induction of allergic airways disease
Mice (6–8 wk of age) were sensitized by i.p. injection of 50 μg of OVA combined with 1 mg of Alhydrogel (CSL) in 0.9% sterile saline (termed allergic mice). Control mice (nonallergic) received 1 mg of Alhydrogel in 0.9% saline. On days 12, 13, 14, and 15, all groups were challenged by intranasal instillation of 10 μg of OVA in 0.9% saline. AHR was measured 24 h after the final OVA challenge (unless otherwise stated); mice were then sacrificed by sodium pentobarbital overdose; and immunological parameters were assessed. In experiments addressing the time course of eosinophil infiltration, mice were sacrificed on days 1, 2, 3, 7, and 14 after the final OVA challenge.
Inflammatory cells in blood and bronchoalveolar lavage fluid (BALF)
Blood was collected by cardiac puncture, and smears were stained with May-Grunwald Giemsa. Differential leukocyte counts were performed based on morphological criteria (a minimum of 200 cells counted/slide), as previously described (15). The remaining blood was centrifuged (10,000 × g for 10 min), and serum was stored at −70°C until analysis. BALF was obtained by cannulation of the trachea and flushing the airways with two 1-ml vol of HBSS. Recovered cells were treated with erythrocyte lysis buffer, and differential leukocyte counts were performed on cytospins (30).
Eosinophils and mucus-secreting cells (MSC) in lung tissue
Lung tissue representing the central (bronchi-bronchiole) and peripheral (alveoli) airways was fixed in 10% phosphate-buffered formalin, sectioned, and stained with carbol chromotrope-hematoxylin for identification of eosinophils or Alcian blue/periodic acid-Schiff for enumeration of MSC (15). The mean number of eosinophils or MSC per high-powered field (×100) within 100 μm basement membrane was determined following assessment of a minimum of 10 fields.
Measurement of AHR
AHR to inhaled β-methacholine (determined by alterations in resistance, RL) was measured, as described previously (31). Responses to methacholine are expressed as the percentage change over saline control (baseline).
Determination of Ag-specific serum Igs
OVA-specific IgG1 and IgG2a were semiquantified by ELISA using reagents from BD Pharmingen. Briefly, plates were coated with either OVA (2 μg/well) for sample wells or unlabeled anti-IgG of corresponding isotype for standard wells. After blocking, wells were incubated with serum or standards (mouse IgG1 or IgG2a), followed by detection with streptavidin-HRP anti-IgG1 or anti-IgG2a. Plates were developed with tetramethyl-benzidine substrate; the reaction was stopped with 0.3 M H2SO4; and OD was determined at 450 nm using a Bio-Rad 680 Microplate reader. Relative levels of OVA-specific IgE were determined by ELISA. Plates were coated with unlabeled anti-IgE (BD Pharmingen) and blocked with 10% FCS. After incubation with serum samples, OVA-specific IgE was detected using biotinylated OVA (Pierce Biosciences; labeling performed according to manufacturer’s instructions), followed by streptavidin-HRP. Plates were developed as above, and ODs were used to calculate ELISA units relative to standardized positive and negative control serum.
T cell proliferation and cytokine production
Peribronchial lymph node (PBLN) and spleens were dispersed through 70-μm nylon mesh, treated with erythrocyte lysis buffer, and cultured at 37°C/5% CO2 in 96-well plates at 1 × 106 cells/well in animal cell culture medium (0.1 mM sodium pyruvate, 2 mM l-glutamine, 20 mM HEPES, 100 U/ml penicillin/streptomycin, 50 μM 2-ME, and 10% FBS in RPMI 1640) with 200 μg/ml OVA. Unstimulated wells contained cells and medium only. Ag-specific proliferation was measured after 72 h using the Cell-Titer 96 kit (Promega), following the manufacturer’s instructions, and calculated as percentage of proliferation in the OVA-treated compared with unstimulated cells from the same cell preparation. For cytokine analysis, cultures were incubated for 6 days and supernatants were stored at −70°C until analysis. IL-5, IL-4, IFN-γ (BD Pharmingen), and IL-13 (R&D Systems) concentrations were determined in supernatants by ELISA, according to the supplier’s recommendations.
Lymphocyte and DC profiling by flow cytometry
The phenotype of lymphocytes and DCs was determined by flow cytometry. PBLN cells were prepared, as described above. Lungs were homogenized by mechanical maceration, followed by 30-min digestion in 1 mg/ml collagenase at 37°C. Tissue was filtered through nylon mesh (70 μm) and treated with erythrocyte lysis buffer. PBLN and lung cells were plated at 1 × 106 cells/well into 96-well plates and, following 20-min incubation with Fc blocking Ab, stained with fluorochrome-conjugated Abs (BD Pharmingen) for analysis of lymphocytes (CD3, CD4, CD8, B220, and CD69) and DCs (CD11c, CD11b, GR-1, PDCA-1, MHC II, CD80, and CD86). Labeled cells were fixed in 1% paraformaldehyde and analyzed by FACS.
The significance of differences between experimental groups was analyzed using Student’s unpaired t test. Values are reported as mean ± SEM. Differences in means were considered significant if p ≤ 0.05.
Elimination of signaling through the βc induces sustained suppression of allergen-induced eosinophils
To determine whether blockade of signaling by βc could prevent eosinophilia, eosinophil recruitment into the lungs, and induction of allergic disease, we compared immune and functional responses in allergic (sensitized) and nonallergic (control) WT and βc−/− mice. Eosinophil numbers rapidly increased in the blood, pulmonary tissue, and BALF of aeroallergen (OVA)-challenged WT mice (Fig. 1). By contrast, numbers of eosinophils in the blood of βc−/− mice were reduced to levels comparable to those observed in WT nonallergic mice. Eosinophil levels in the lung tissue of allergic βc−/− mice were also profoundly reduced compared with the WT (by 10.6-fold), and this granulocyte was entirely absent from the BALF.
In a previous study, βc-deficient mice displayed an attenuated and delayed eosinophil response to parasite infestation compared with WT controls (27). These mice were deficient only in the βc, and were capable of residual IL-3 signaling using the intact βIL-3. To determine whether eosinophil expansion and mobilization were delayed in our βc and βIL-3 double-deficient mice, we characterized eosinophil expansion and migration over a time course of 2 wk. By contrast to the parasite study, eosinophil numbers in the peripheral blood, airway lumen, and lung tissue remained at baseline levels for the entire period of 2 wk after the final OVA challenge, at which time eosinophil levels in allergic WT mice had effectively returned to baseline levels (Fig. 2). These data demonstrate the pivotal role that βc plays in the regulation of eosinophil biology and indicate that signaling redundancy between the three eosinophilopoietic cytokines may account for the IL-5-independent pathways that regulate eosinophilic inflammation during allergic airways disease.
Elimination of signaling through the βc attenuates critical features of allergic airways disease
Next, we determined whether other critical pathophysiological features of allergic airways inflammation (mucus hypersecretion, AHR, and Ag-specific Ab production) were attenuated in the absence of βc and eosinophils. Although significantly reduced, mucus hypersecretion was still a feature in the lung of allergic βc−/− mice by comparison with allergic WT mice (Fig. 3,A). However, whereas the pattern of expression of MSC in WT lungs commonly presented as a high number of cells within a single highly inflamed airway, causing visible obstruction of the lumen by mucus, histological examination of allergic βc−/− mice revealed that the MSC present were disseminated throughout airway tissue, and there is no evidence of significant luminal obstruction. Aeroallergen challenge of allergic WT mice induced AHR to β-methacholine compared with the nonallergic WT (Fig. 3,B). By contrast, allergic βc−/− mice did not exhibit any significant increase in airway resistance relative to their nonallergic counterparts (Fig. 3 B).
Next, we observed the effects of βc deletion on the production of Ag-specific Igs during allergic inflammation. Elevated serum Ag-specific IgE and increased ratios of IgG1 to IgG2a are linked to Th2 immunity and allergic inflammation, whereas a dominant Ag-specific IgG2a response is associated with Th1-regulated inflammation. Aeroallergen challenge of allergic WT mice induced a significant increase in the serum concentration of OVA-specific IgE and IgG1. By contrast, serum levels of OVA-specific IgE were attenuated in βc−/− mice (Fig. 3,C). Paradoxically, despite suppression of a range of pathophysiological features of allergic disease, concentrations of OVA-specific IgG1 were significantly increased in allergic βc−/− mice (Fig. 3,D). Furthermore, very low levels of OVA-specific IgG2a (<2 ng/ml) were observed in the serum of allergic WT, but not in allergic βc−/− mice (Fig. 3 D).
Pulmonary Th2 cytokine release is impaired in the absence of βc
CD4+ Th2 cells play a central role in the induction of many features of allergic airways disease. Attenuated Ab secretion and AHR in βc−/− mice (Fig. 3) suggest defects in the T cell response to Ag, which may contribute appreciably to the attenuated allergic inflammatory response in these mice. Indeed, the ability of T cells to proliferate in response to OVA was diminished in both PBLN and spleen cultures from allergic βc−/− mice by comparison with those isolated from allergic WT mice (Fig. 4,A). Notably, βc deletion was also accompanied by a striking reduction in the ability of Ag-specific T cells to produce IL-4, IL-5, and IL-13 in cultures of PBLN cells taken from allergic βc−/− mice (Fig. 4, B–D). Only very low levels of the Th1-associated cytokine IFN-γ were detected, and there were no statistically significant differences between βc−/− and WT in Ag-challenged mice (Fig. 4 E). This effect appeared to be primarily compartmentalized to the lung, because a global reduction in Th2 cytokine responses was not observed in splenocyte cultures from βc−/− mice (data not shown). Interestingly, cells from the PBLN, but not the spleen of naive βc−/− mice also produce less IL-4, IL-5, and IL-13, but more IFN-γ in response to anti-CD3/anti-CD28 stimulation compared with WT controls (data not shown).
Collectively, these data demonstrate that signaling through the βc plays a pivotal role in the regulation of hallmark features in allergic inflammation, and suggest an important role in the manifestation of both the early (IgE-mediated) and late (AHR-, T cell-, and eosinophil-mediated) phases of the asthmatic response.
Activated lymphocytes fail to migrate to the lung in βc null mice
Activation of CD4+ Th2 cells in the PBLN and migration into the lung parenchyma play a central role in regulating the allergic inflammatory response. CD69 is an early activation marker expressed on leukocytes during inflammation (32). Thus, we characterized the profile and CD69 expression of lymphocytes in Ag-challenged βc−/− and WT mice in these compartments to determine the potential contribution of T cell activation and migration to the attenuation of allergic inflammation in βc−/− mice. Although levels of cytokine production from allergic βc−/− PBLN cells were reduced (Fig. 4, B–E), significantly more T lymphocytes (CD3+ cells) occupied this compartment by comparison with WT equivalents (Fig. 5,A). The increase in T lymphocytes could be accounted for by significant elevation of both CD4+ and CD8+ T cell numbers. Nevertheless, the level of activated T cells in this compartment is diminished in allergic βc−/− mice by contrast to WT groups (Fig. 5,B), implying that these cells were not receiving early activation signals required for migration into effector sites. Indeed, both T and B lymphocyte numbers were dramatically reduced in βc−/− lung tissue, the site where they normally orchestrate allergic inflammation (Fig. 5,C). The increase in CD4+ lymphocytes in the PBLN of βc−/− mice may thus represent retention of cells in the absence of specific migratory signals following Ag challenge. Moreover, the number of activated CD4+ T cells did not increase in response to Ag challenge in βc−/− mice (Fig. 5 D). These data suggest that defects in CD4+ T cell activation and migration occur in the absence of βc, which significantly limits the development of allergic airway disease.
βc modulates DCs in the pulmonary compartment
PBLN and airway myeloid DCs (mDCs) play pivotal roles in the activation and migration of CD4+ T cells in the lung and the onset of allergic inflammation through Ag presentation and costimulation (22). By contrast, plasmacytoid DCs (pDCs) promote tolerogenic responses and are protective against inflammatory responses to inhaled Ag (33). Because cytokines signaling through βc are important in the development and function of DCs, we used flow cytometry to profile subsets of mDCs (CD11c+CD11b+) and pDCs (CD11c+CD11b−GR-1−PDCA1+) in the PBLN and lungs of βc−/− mice. Absence of signaling through βc increased mDC numbers in the PBLN, in both naive and allergic groups, relative to the respective WT groups (Fig. 6,A). PBLN pDC numbers are also increased in naive βc−/− mice (Fig. 6,A). By contrast, significant Ag-induced expansion of mDC numbers in the lung tissue did not occur in βc−/− mice (Fig. 6,B). Moreover, a striking deficiency of pDCs in the lungs of nonallergic and allergic βc−/− mice was observed, with cell numbers significantly lower than those observed in the nonallergic WT group (Fig. 6 B).
Because mDCs are of particular importance in allergic inflammation, we compared the surface expression of MHCII and the costimulatory molecules CD80 and CD86 on this subset of DCs in WT and βc−/− mice. Lung mDCs showed significant increases in MHCII and CD86 expression in WT allergic mice compared with the nonallergic group (Fig. 6,C). However, in βc−/− mice, there was no increase in mDC expressing MHCII, CD80, or CD86 following Ag provocation (Fig. 6,C). Collectively, these data suggest defects in the expansion and maturation of mDCs in the lung microenvironment at baseline and during allergic inflammatory conditions in βc−/− mice, which may account for the defects observed in T cell activation, proliferation, pulmonary migration, and cytokine secretion (Figs. 4 and 5). Thus, βc deficiency significantly impacts on pathways that activate T cells as well as on the generation of eosinophils (Fig. 1).
Although the βc subunit of the GM-CSF/IL-3/IL-5 receptor complex has been shown to have an important role in eosinophil differentiation and function (27, 28), the present study is the first to determine the role of the βc in the regulation of eosinophils during allergic airways inflammation. We show that ablation of βc signaling abolishes Ag-induced accumulation of eosinophils in the peripheral blood, peribronchial tissue, and airway lumen of allergen-challenged mice. Importantly, suppression of eosinophilic inflammation was accompanied by attenuation of AHR, mucus hypersecretion, and notably the production of Ag-specific IgE. Reduced secretion of type 2 Abs in association with attenuation of late-phase pathological features suggested that Th2 cell as well as eosinophil function was impaired in βc−/− mice. Indeed, characterization of CD4 T cells from βc−/− mice revealed defects in activation and recruitment from lymph nodes to lung tissue. Reduced activation of Th2 cells was also reflected by their limited capacity to proliferate and to secrete effector cytokines. Interestingly, this phenomenon was primarily restricted to the pulmonary compartment and correlated with reduced numbers of mDCs expressing MHCII and costimulatory molecules in the lung tissue following Ag challenge. At baseline, βc−/− mice have been reported to have no hemopoietic abnormalities other than a reduction in eosinophil numbers (27, 28). However, our data show that DC and T cell function can be limited at the site of allergic inflammation, whereas systemic T cell responsiveness is maintained, because we did not observe a global decrease in the ability of these cells in the βc−/− spleen to secrete cytokines in response to Ag. Thus, the βc represents a single molecule capable of regulating multiple facets of the allergic response within the lung that has significant potential as a therapeutic target for allergic diseases.
Pulmonary CD4+ T cell function in asthma is critically dependent on the formation of a functional immunological synapse with mDCs (22). Within the pulmonary compartment, mDCs modulate activation and polarization of naive CD4+ T cells to a Th2 phenotype that results in the subsequent secretion of cytokines that orchestrate inflammation (34, 35). IL-3 and GM-CSF are known to play important roles in the development of DCs in vitro (21). In this study, we demonstrate the βc signaling pathway is also critical for in vivo expansion of the pulmonary DC pool during allergic inflammation. Indeed, the significant suppression of activated mDC numbers in the lung tissue of βc−/− mice is likely to be a major mechanism underlying the defects observed in Th2 cell activation, proliferation, migration and cytokine secretion, and failure to develop allergic airway disease. Interestingly, a series of recent reports have established that mediators released by mast cells provide key instructional signals for DC maturation and migration (36, 37). Because IL-3 is important for mast cell development, defects in this cell type in the absence of IL-3 signaling may represent a further indirect mechanism for the impaired development of DCs in βc−/− mice. It should be noted that alveolar macrophage (AM) can also influence T cell responses in the lung. In this regard, the increased numbers of AM at baseline in βc−/− mice may be relevant because numerous studies have emphasized the potentially important negative regulatory role of AM in pulmonary immune responses. Indeed, studies have shown that depletion of AM leads to exaggerated AHR in models of allergic inflammation (reviewed in Ref. 38). However, the role of AM might be more complicated in an inflammatory environment, because pulmonary macrophages would also be influenced by the presence of proinflammatory mediators, which are able to alter the macrophage phenotype and thus their regulation of Th cell responses (39). Thus, although no definitive conclusions can be drawn, increased numbers of AM in βc−/− mice may inhibit local T cell function and suppress development of allergic inflammation. Alternatively, impaired DC function due to βc deficiency may predominantly predispose to suppression of airway Th2 responses.
The potential compartmentalization of the suppressive effect on Th2 cell function is also intriguing. Production of Th2 cytokines by PBLN T cells from βc−/− mice was significantly suppressed after stimulation of Ag-specific cells with OVA or naive cells with anti-CD3/anti-CD28. By contrast, βc deficiency did not lead to a global suppression of Th2 cytokine production in spleen cells from allergen-challenged mice stimulated with OVA or from naive mice stimulated polyclonally. Thus, the effect of βc deficiency appears to be compartmentalized to the lung at baseline and during induction of allergic inflammation. However, more detailed studies are required to assess whether βc-deficient mice have an intrinsic defect in pulmonary T cell function, or whether the suppression of PBLN Th2 responses is a consequence of factors within the pulmonary environment in βc−/− mice, for instance DCs or macrophages.
Studies targeting individual ligands important for mature eosinophil function (e.g., IL-5) have only yielded partial ablation of tissue eosinophil numbers and shown limited improvements in disease severity, in both experimental mouse models of asthma and clinical trials (12, 13, 40, 41). Importantly, our study represents the first clinically relevant strategy, targeting a single molecule, which results in eosinophils being maintained at baseline levels following allergen provocation. The effectiveness of this approach lies in the importance of βc both for eosinophil differentiation from CD34+ progenitors, which may occur in the bone marrow or in situ in allergic tissue (42, 43), and for activation and survival of the mature eosinophil at sites of inflammation (17, 18, 19). Our study establishes not only that inactivation of both βc and βIL-3 prevents eosinophil expansion in allergic disease, but also that this suppression is maintained over time, distinct from the delayed parasite-induced eosinophil infiltration observed in mice in which only the βc chain is deficient (and the βIL-3 is intact) (27). This highlights the importance of eliminating signaling by all three eosinophilopoietic cytokines to effectively prevent eosinophil-driven inflammation.
Although the primary defect in T cell function in βc−/− mice may be associated with the role of the βc in DC development, the involvement of this receptor in eosinophil, mast cell, and basophil function may also contribute to the regulation of Th2 immunity. In response to pulmonary Ag provocation, eosinophils can internalize Ag, migrate to local lymph nodes, and secrete mediators that promote Th2 cell expansion and cytokine liberation (44, 45, 46). Indeed, eosinophil depletion has previously been associated with deficiencies in Th2 immunity (16). Furthermore, innate cells of hemopoietic origin (eosinophils, mast cells, and basophils) have recently emerged as the principal source of IL-4 in the early response to both parasite and allergen exposure, providing a critical molecular switch for type 2 inflammation (47, 48, 49). IL-3 is the dominant cytokine driving mast cell and basophil differentiation, and neutralization of this cytokine abolishes basophil IL-4 production and subsequent Th2 polarization in vitro (24, 50). Impaired mast cell and basophil function in the absence of IL-3 signaling in βc−/− mice may partially account for the defect in Th2 cell differentiation observed in the present study. However, because the experimental model used in the present study is primarily eosinophil and CD4+ Th2 cell mediated, future experiments are required using primarily mast cell-dependent models of allergic airway disease to elucidate the precise role of βc in these processes (51). Attenuation of early innate cell IL-4 production and T cell function may also explain the suppression of Ag-specific IgE production. Notably, IgE responses to parasite infestation were not suppressed in βc−/− mice, which retained IL-3 signaling via the βIL-3 (27), underscoring the importance of IL-3 in the generation of the type 2 inflammatory response.
It is important to note that alterations in lung homeostasis similar to human pulmonary alveolar proteinosis have previously been documented in GM-CSF ligand, GM-CSFα receptor subunit, and βc subunit (βc−/−) null mice, although the phenotype of the defect in βc−/− mice appears less severe with respect to surfactant accumulation in the lung (52). Although the mice used in the present study did show accumulation of macrophages in the lung and the appearance of foamy macrophages in the BALF, the presence of pulmonary alveolar proteinosis did not appear to play a role in the development of features of acute allergic airway inflammation. This is demonstrated in naive βc−/− mice, which did not significantly differ from naive WT mice in any of the parameters assessed in our characterization of allergic airway inflammation (blood, lung tissue, and BALF eosinophils (Fig. 1)) and lung MSC (Fig. 2), with the exception of airways hyperreactivity (discussed below). Notwithstanding the presence of a background inflammatory response, we still do not see the development of functional and inflammatory lesions related to allergic airways disease in these mice. Although we cannot unequivocally rule out the possibility that altered lung homeostasis in βc−/− mice contributes to the suppression of allergic inflammation, the data obtained suggest that inhibition is due to an inability of Th2 cells to secrete cytokines. The alveolar proteinosis in βc−/− mice is likely to be a dynamic effect of gene deletion throughout life. Indeed, such abnormalities have not been documented following administration of anti-GM-CSF Abs at therapeutic doses in animal models of lung inflammation (53, 54). Optimization of treatment regimes to specifically target βc in the adult mouse lung should facilitate inhibition of allergic inflammation without altering lung homeostasis.
It is interesting to note that nonallergic βc−/− mice show significantly reduced airway resistance compared with WT BALB/c. This diminution may be related to the increased amount of surfactant present in the airways of βc−/− mice (52). Previous studies have reported that the presence of surfactant in the airway lowers surface tension, a mechanism that contributes to the distal airways remaining open (55). Indeed, inhalation of synthetic surfactant diminishes AHR in clinical studies (56). The increase in surfactant in the βc−/− airway may be a factor in the reduced response to methacholine in nonallergic mice compared with the WT. Furthermore, the possibility that increased airway surfactant contributes to the lack of AHR in Ag-challenged βc−/− mice cannot be excluded. However, the concomitant reduction in the underlying inflammation governing AHR suggests that βc may have a specific role in regulating airway function. Future development of reagents to specifically target βc in the absence of effects on baseline lung homeostasis will facilitate further clarification of this point.
In conclusion, redundancy in cytokine signaling pathways has limited the effectiveness of targeting a single molecule for therapeutic intervention in asthma (e.g., IL-4, IL-5, and IgE) (57, 58). This report provides evidence that a wide spectrum of immune and pathophysiological features of asthma can be suppressed by targeting a single molecule that has a central role in allergic inflammation. Inhibition of βc function attenuated IgE production, tissue eosinophil numbers, and pulmonary Th2 cell effector function, which has important implications for suppression of both the early and late phases of allergic inflammation. Suppression of disease in the absence of βc was underpinned by the inability of mDCs to activate CD4+ T cells and their subsequent migration to effector sites in the airways. Thus, the present study enhances our understanding of the role of the βc in the regulation of molecules and cells that play pivotal roles in both early and late phases of the allergic response, and identifies this receptor chain as a clinically relevant target for suppression of disease.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by grants from the National Institutes of Health (R01-AI50744-02 to A.F.L.) and the National Health and Medical Research Council of Australia.
Abbreviations used in this paper: AHR, airway hyperreactivity; AM, alveolar macrophage; βc, common β receptor chain; βIL-3, IL-3 β receptor chain; BALF, bronchoalveolar lavage fluid; DC, dendritic cell; mDC, myeloid DC; MSC, mucus-secreting cell; PBLN, peribronchial lymph node; pDC, plasmacytoid DC; WT, wild type.