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RI, and IL-13 Are Required for Development of Airway Hyperresponsiveness after Aerosolized Allergen Exposure in the Absence of Adjuvant1
* Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206;
The Jackson Laboratory, Bar Harbor, ME 04609; and
Wyeth Institute, Cambridge, MA 02140
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Abstract |
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RI) in the development of AHR, mice with a disruption of the 
subunit of the high affinity IgE receptor (FcRI–/–) were exposed on 10 consecutive days to nebulized OVA. Forty-eight hours after the last nebulization, airway responsiveness was monitored by the contractile response of tracheal smooth muscle to electrical field stimulation (EFS). After the 10-day OVA challenge protocol, wild-type mice demonstrated increased responsiveness to EFS, whereas similarly challenged FcRI–/– mice showed a low response to EFS, similar to nonexposed animals. Further, allergen-challenged FcRI–/– mice showed less airway inflammation, goblet cell hyperplasia, and lower levels of IL-13 in lung homogenates compared with the controls. IL-13-deficient mice failed to develop an increased response to EFS or goblet cell hyperplasia after the 10-day OVA challenge. We transferred bone marrow-derived mast cells from wild-type mice to FcRI–/– mice 1 day before initiating the challenge protocol. After the 10-day OVA challenge, recipient FcRI–/– mice demonstrated EFS-induced responses similar to those of challenged wild-type mice. Transferred mast cells could be detected in tracheal preparations. These results show that FcRI is important for the development of AHR after an aerosolized allergen sensitization protocol and that this effect is mediated through FcRI on mast cells and production of IL-13 in the lung. |
Introduction |
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Mast cells may recognize allergen through the high affinity IgE receptor (FcRI), and IgE is thought to build a complex with allergen binding to this receptor. Allergen-cross-linking of receptors leads to the activation of mast cells, with the release of numerous mediators and cytokines (7, 8, 9). However, the role of mast cells in the development of AHR is somewhat controversial. When studying mechanisms of allergic airway disease in animals models, mast cell-deficient mice have variable decreases in eosinophil numbers (10, 11) after allergen challenge, but systemically sensitized and challenged mast cell-deficient mice are capable of developing a Th2 response, airway inflammation, and AHR similar to wild-type control mice (12, 13, 14, 15). In most of these studies allergen sensitization was achieved by systemically injecting the allergen in combination with an adjuvant, whereas other studies, using less potent sensitization protocols, demonstrated a more obvious role for mast cells in the development of AHR (16, 17).
To characterize the role of mast cells and FcRI in the development of AHR, we used an allergen challenge protocol in which wild-type, mast cell-deficient, and FcRI-deficient mice were challenged repeatedly with allergen via the airways without the use of an adjuvant (18). In this model allergen exposure leads to dysfunction of muscarinic receptor 2, which results in an increase in acetylcholine release after electrical field stimulation (EFS) (19). After exclusive aerosolized allergen exposure, allergen-specific IgE and IgG1 are elevated in the serum of exposed mice (20), and the presence of B cells is essential for the development of AHR in this model (21). In the present study we show that after repeated airway challenge, mast cell- and FcRI-deficient mice fail to develop increased reactivity to EFS, a response that could be reconstituted by transfer of FcRI-sufficient mast cells before the airway challenge.
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Materials and Methods |
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Female BALB/cByJ mice, C57BL/6 mice, mast cell-deficient mice ([WB/Rej-kitW/+ x C57BL6J-kitW-v/+]F1– [W/Wv] mice) and congenic WBB6F1 normal mice from 8 to 12 wk of age were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice with a disruption of the subunit of the high affinity IgE receptor (FcRI–/–; BALB/c background; provided by Drs. D. Dombrowicz and J. P. Kinet, Harvard Medical School, Boston, MA) (22, 23), IL-13-deficient mice (IL-13–/–; BALB/c background; provided by Dr. D. Umetsu, Stanford University, Stanford, CA) (24), and CD57BL/6 mice that express a transgene for green fluorescent protein (GFP) under control of a human ubiquitin C promoter (UBI-GFP) (25) (provided by Dr. P. Marrack, National Jewish Medical and Research Center, Denver, CO) were maintained and bred in the animal facility. All mice were maintained on OVA-free diets. All experimental animals used in this study were under a protocol approved by the institutional animal care and use committee of National Jewish Medical and Research Center.
Experimental protocols
Experimental groups consisted of three or four mice per group, and each experiment was performed at least twice. Mice were exposed to OVA (Sigma-Aldrich, St. Louis, MO) as described previously (18). Briefly, a solution of 1% OVA in 0.9% saline or endotoxin-depleted 1% OVA solution was delivered by ultrasonic nebulization for 20 min daily over 10 consecutive days in a closed chamber. Control mice were exposed to 0.9% saline alone following the same protocol. AHR was assessed 48 h after the last nebulization.
Administration of the soluble IL-13R2-IgGFc fusion protein (sIL-13R2-Fc)
Murine IL-13R2-hIgG fusion protein was prepared as previously described (26). In the 10-day OVA challenge protocol, IL-13R2-hIgG fusion protein was administered by i.p. injection (300 µg/mouse) 1 h before OVA challenge on days 1, 4, 7, and 10. As a control, the same amount of human IgG was injected at the identical time points.
Detection of endotoxin and removal of endotoxin from OVA solution
Endotoxin concentrations in OVA solution were assessed by Limulus amebocyte lysate (Charles Rivers Breeding Laboratories, Charleston, SC). Endotoxin depletion of OVA solutions was performed following previously described protocols (27, 28). Briefly, OVA solution (30 mg/ml in endotoxin-free saline) was mixed with 1% Triton X-114 (Sigma-Aldrich) by vigorously vortexing and was placed in an ice bath. After vortexing the chilled samples, the tubes were warmed at 37°C for 5 min to allow two phases to form. Samples were then centrifuged (10 min, 7000 rpm). After centrifugation, the detergent layer was found as an oily droplet at the bottom of the tube. The upper aqueous layer was removed carefully. Residual detergent in the aqueous phase was removed by treatment with endotoxin-free Bio-Beads SM-4 (Bio-Rad, Hercules, CA) as described previously (28). The resultant solution contained little endotoxin (1.8 ELISA unit (EU)/mg protein)
Transfer of bone marrow-derived mast cells (BMMC)
Mast cells were derived from bone marrow as previously described (29). Bone marrow was obtained from wild-type mice, FcRI–/– mice, IL-13–/– mice, and UBI-GFP mice and was cultured in IMDM (Life Technologies, Grand Island, NY) supplemented with 5% FBS (Summit Biotechnology, Fort Collins, CO), 50 µM 2-ME (Life Technologies), 2 mM glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin, 0.5 µg/ml amphotericin B, IL-3 obtained from medium conditioned by X63-AG8-653 myeloma cells transfected with a vector expressing IL-3 (30), and Kit ligand from medium conditioned by CHO cells (provided by S. Webb, National Jewish Medical and Research Center, Denver, CO). After 4 wk of culture, >98% of nonadherent cells contained granules that stained positively with toluidine blue, and >98% expressed c-Kit on their surface as determined by FACS analysis using anti-c-Kit mAb. Wild-type or FcRI–/– mice were injected with 5 x 106 BMMC i.v. either 1 day or 20 wk before staring the airway challenges and received either 10-day treatment with OVA or saline nebulization. C57BL/6 mice received 5 x 106 BMMC from UBI-GFP mice i.v. 1 day before initiating the airway challenges with either saline or OVA.
Assessment of airway smooth muscle responsiveness
Forty-eight hours after the last airway challenge, AHR was monitored as airway smooth muscle responsiveness to EFS, measured as described previously (18). Tracheas were removed, and 0.5-cm preparations were placed in Krebs-Henseleit solution suspended by triangular supports transducing the force of contractions. EFS with an increasing frequency from 0.5 to 30 Hz was applied, and the contractions were measured. The duration of the stimulation was 1 ms. Frequencies resulting in 50% of the maximal contraction (ES50) were calculated from linear plots from each individual animal and were compared between the groups. In addition, to assess postsynaptic cholinergic sensitivity of the tissues, cumulative dose-response curves to methacholine (MCh) were performed in half-log increments using concentrations ranging from 10–8 to 10–4 M. Results in the control and experimental groups at each time point were expressed in terms of the maximal contractile response as well as the EC50 for MCh.
Histology
Lungs were fixed by inflation (1 ml) and immersion in 10% formalin. Cells containing eosinophilic major basic protein (MBP) were identified by immunohistochemical staining as previously described using rabbit anti-mouse MBP (provided by Dr. J. J. Lee, Mayo Clinic, Scottsdale, AZ). The slides were examined in a blinded fashion with a Nikon microscope (Melville, NY) equipped with a fluorescein filter system. The numbers of peribronchial eosinophils in the tissues were evaluated using IPLab2 software (Signal Analytics, Vienna, VA) for the Macintosh, counting six to eight different fields per animal. For detection of mucus-containing cells in formalin-fixed airway tissue, sections were stained with periodic acid-Schiff (PAS) and H&E and were quantitated as previously described (31). Tracheal sections were stained with 1% toluidine blue (Sigma-Aldrich) to detect metachromatic mast cell cytoplasmic granules. To maximize the ability to detect mast cells stained with toluidine blue, lung tissue was fixed in formalin for <24 h. Nonetheless, this may result in some underestimation of mast cell numbers. Tracheal sections from C57BL/6 mice receiving BMMCs derived from UBI-GFP mice were deparaffinized and mounted using mounting medium with 4',6-diamido-2-phenylindole hydrochloride (Vector Laboratories, Burlingame, CA).
Assessment of IL-13 levels in lung homogenates
Lungs were weighed and completely homogenized in dilution buffer (PBS, 0.1% Triton X (Sigma-Aldrich), and proteinase inhibitor (BD PharMingen, San Diego, CA)) using a tissue homogenizer (Tissue Tearor; BioSpec, Bartlesville, OK) and then centrifuged at 14,000 rpm for 30 min. Supernatants were collected and frozen at –80°C until further analysis. Total protein content was measured by colorimetric assay (DC Protein Assay; Bio-Rad), and levels of IL-13 were assessed by ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer’s directions.
Measurement of total and OVA-specific Ab
Serum levels of total IgE and OVA-specific IgE and IgG1 were measured by ELISA as previously described (31). Briefly, 96-well plates (Immunlon 2; Dynatech, Chantilly, VA) were coated with either OVA (5 µg/ml) or purified anti-IgE (02111D; BD PharMingen). After addition of serum samples, a biotinylated anti-IgE Ab (02122D; BD PharMingen) was used as the detecting Ab, and the reaction was amplified with avidin-HRP (Sigma-Aldrich). IgG1 was detected using alkaline phosphatase-labeled anti-IgG1 (02003E; BD PharMingen). The OVA-specific Ab titers of the samples were related to pooled standards that were generated in the laboratory and expressed as ELISA units per milliliter. Total IgE levels were calculated by comparison with known mouse IgE standards (BD PharMingen). The limit of detection was 100 pg/ml for total IgE.
Statistical analysis
ANOVA was used to determine the levels of difference between all groups. Comparisons for all pairs were performed using Tukey-Kramer honestly significant difference test, and p values for significance were set at 0.05. Values for all measurements were expressed as the mean ± SEM.
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Results |
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RI–/– mice
Mice exposed to 10-day OVA challenge (in the absence of adjuvant) demonstrate increased serum levels of OVA-specific IgE and IgG1 (32). To assess the effect of 10-day OVA nebulization exposure on Ig production in FcRI–/– mice, we measured serum levels of total IgE and OVA-specific IgE and IgG1. OVA-challenged FcRI–/– mice showed increased serum levels of total IgE and OVA-specific IgE and IgG1, which were similar to those in OVA-challenged wild-type mice and significantly higher (p < 0.05) than those in saline-challenged FcRI–/– and wild-type mice (Table I).
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RI–/– mice fail to develop AHR after 10-day OVA challengeTo assess whether mast cells play a role in the development of AHR in this model, we challenged mast cell-deficient mice on 10 consecutive days with OVA. Indeed, OVA-challenged, mast cell-deficient mice demonstrated no significant difference in ES50 compared with the saline-challenged, mast cell-deficient mice (Fig. 1A), whereas the respective 10-day OVA-challenged, wild-type mice showed significantly (p < 0.05) lower ES50 values than the saline-exposed wild-type mice (Fig. 1A).
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RI–/– mice. After 10-day OVA nebulization, wild-type mice showed a significantly lower ES50 than saline-challenged animals, whereas OVA-challenged FcRI–/– mice showed a response to EFS similar to that of saline-exposed wild-type or FcRI–/– mice (Fig. 1B). Overall responses to carbachol and maximal generated force were not statistically different between any of the groups (data not shown). Lung inflammation and goblet cell hyperplasia after 10-day OVA nebulization
Following this protocol, few inflammatory cells were detectable in the bronchoalveolar lavage fluid. Lung tissue inflammation and, in particular, eosinophil accumulation were assessed using immunohistochemistry with anti-MBP. After the 10-day OVA challenge, wild-type mice demonstrated a modest increase in peribronchial eosinophil numbers compared with the saline-challenged control mice (Fig. 2). In OVA-challenged FcRI–/– mice, no such increase in tissue inflammation was found (Fig. 2). To assess goblet cell hyperplasia, slides were stained with PAS. After 10-day OVA nebulization, wild-type mice demonstrated PAS-positive cells (mean ± SEM; 15.2 ± 2.3 PAS-positive cells/mm of basement membrane; p < 0.001 compared with other groups), whereas saline-challenged wild-type and saline- and OVA-challenged FcRI–/– mice showed no PAS-positive cells (Fig. 3).
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RI-deficient mice
IL-13 has been shown to be an important mediator in the development of AHR and goblet cell hyperplasia (35, 36, 37, 38). To assess whether IL-13 is up-regulated in the 10-day challenge model, we measured levels of IL-13 in lung homogenates. Indeed, OVA-challenged wild-type mice exhibited increased levels of IL-13 in lung homogenates 48 h after the last challenge compared with levels in saline-challenged control mice (Fig. 4). However, OVA-challenged FcRI–/– mice showed no elevation in the levels of IL-13 in their lung homogenates (Fig. 5). Similarly, lung homogenates of OVA-challenged mast cell-deficient mice exhibited significantly (p < 0.05) lower IL-13 levels compared with those of OVA-challenged mast cell-sufficient mice (Fig. 5).
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To elucidate the role of IL-13 in the development of AHR after 10-day OVA challenge, we monitored tracheal smooth muscle responses to EFS in IL-13–/– and IL-13+/+ mice challenged with OVA as well as after treatment with an inhibitor of IL-13 (sIL-13R2). OVA-challenged IL-13–/– mice showed no differences in EFS compared with saline-challenged IL-13–/– and IL-13+/+ mice, and they were significantly different than the responses in OVA-challenged IL-13+/+ mice (Fig. 6). In parallel, OVA-challenged IL-13+/+ mice treated with sIL-13R2 during the challenge period normalized their EFS responses to levels seen in saline-challenged IL-13+/+ mice or IL-13–/– animals (Fig. 6). The OVA-challenged IL-13–/– and IL-13+/+ mice treated with sIL-13R2 also showed no PAS-positive cells. Importantly, in these experiments, serum Ab levels were not statistically different in OVA-challenged IL-13+/+ vs IL-13–/– mice or in sIL-13R2- vs human IgG-treated mice (Table I).
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RI-deficient mice restores their ability to develop AHR
In rodents, FcRI is expressed only on mast cells and basophils (39). We therefore investigated whether the inability of high affinity IgE receptor-deficient (FcRI–/– IL-13+/+) and IL-13-deficient mice (FcRI+/+ IL-13–/–) to develop AHR could be overcome by adoptive transfer of BMMC from wild-type (FcRI+/+ IL-13+/+) animals. Bone marrow cells from FcRI+/+ IL-13+/+, FcRI–/– IL-13+/+, and FcRI+/+ IL-13–/– mice were cultured under conditions described in Materials and Methods. After 4 wk, 98% of the cells in culture were considered to be mast cells, because they expressed c-Kit (CD117) and FcRI (in the case of FcRI+/+ donors) on their surfaces (data not shown).
Mast cells were injected i.v. into wild-type, FcRI–/–, and IL-13–/– recipients 1 day before starting the 10-day OVA challenge protocol. None of the recipients exposed to saline challenge alone showed any response to EFS (Fig. 7). The positive response to EFS was restored in FcRI–/– IL-13+/+ mice that received BMMC from FcRI+/+ IL-13+/+ or FcRI+/+ IL-13–/– mice before 10-day OVA challenge. Administration of sIL-13R2 to the FcRI–/– IL-13+/+ recipients prevented the reconstitution of EFS responsiveness after transfer of BMMC from FcRI+/+ IL-13+/+ (mean ± SEM; ES50, 4.13 ± 0.33 Hz; n = 8 from two independent experiments) and FcRI+/+ IL-13–/– donors (4.37 ± 0.27 Hz; n = 7 from two independent experiments) and OVA exposure. In contrast, FcRI–/– IL-13+/+ mice receiving autologous BMMC from FcRI–/– IL-13+/+ mice and 10-day OVA challenge showed no response to EFS. This confirms the requirement for FcRI-sufficient mast cells in the development of a positive response to EFS.
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RI–/– IL-13+/+ mice receiving BMMC from FcRI+/+ IL-13+/+ mice before 10-day OVA exposure showed a significantly (p = 0.012) lower ES50 (mean ± SEM; 3.1 ± 0.3 Hz; n = 9 from two independent experiments) compared with FcRI–/– IL-13+/+ mice receiving autologous BMMC from FcRI–/– IL-13+/+ mice before 10-day OVA exposure (4.9 ± 0.2 Hz; n = 6 from two independent experiments). Transfer of BMMC from wild-type to IL-13-deficient mice does not restore their ability to develop AHR
In addition to the need for FcRI-sufficient mast cells in the development of smooth muscle hyper-responsiveness to EFS after 10-day OVA challenge, a role for IL-13 was also identified. Mast cells are a potential source of IL-13 (40). However, in the adoptive transfer experiments described in Fig. 7, FcRI+/+ IL-13–/– recipients of FcRI+/+ IL-13+/+ BMMC failed to develop EFS responses after 10-day OVA challenge. This occurred despite the fact that these cells, in the same numbers, were capable of reconstituting the EFS response in FcRI–/– IL-13+/+ mice (Fig. 7).
Mast cells are can be detected in tracheal tissue of FcRI–/– mice
To assess whether mast cells can be detected in tracheal tissue from FcRI–/– mice, tracheal sections from 10-day OVA-challenged FcRI–/– mice and 10-day OVA-challenged FcRI–/– after transfer of BMMCs were stained with toluidine blue. In both groups mast cells were detected in the subepithelial region as well as in close proximity to smooth muscle (Fig. 8). To further assess whether transferred BMMCs migrate to tracheal tissue, green-fluorescent BMMCs from UBI-GFP mice were transferred to C57BL/6 recipients 1 day before initiating the airway challenges. After 10-day exposure to either saline or OVA, transferred green-fluorescent cells could be detected in the tracheal tissue 48 h after the last challenge (Fig. 8). In saline-exposed mice we detected 1 ± 1 GFP+ mast cells per section, whereas in OVA-exposed mice, we detected 4 ± 1.5 GFP+ mast cells (n = 4 in each group).
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Discussion |
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RI) and by implication IgE in the development of allergic airway disease, using mice deficient in FcRI due to disruption of the FcRI gene. These mice are essentially normal in all aspects of their development and maturation, have functional mast cells (22, 23), which can be found in tracheal tissue. Additionally, FcRI–/– mice are able to mount an immune response to allergen exposure, as shown by elevated levels of total IgE and allergen-specific IgE and IgG1. In these respects FcRI-deficient mice are similar to wild-type mice. In contrast, in other aspects of allergic airway disease, allergen-exposed FcRI–/– mice failed to develop increased airway reactivity, goblet cell hyperplasia, and airway inflammation. This phenotype in FcRI–/– mice is unmasked only when they are exposed to allergen, in this case via the airways, in the absence of adjuvant. This contrasts with their response to systemic sensitization (together with adjuvant), followed by airway challenge, where FcRI–/– mice develop AHR and airway inflammation similar to those exhibited by their sensitized and challenged wild-type littermates (42). Furthermore, in the 10-day OVA challenge model the heightened airway reactivity they develop is detected as a change in the response of tracheal smooth muscle to EFS, a consequence of altered muscarinic receptor 2 function and increased acetylcholine release (18) The allergen exposure protocols have been associated with differences in the approaches to detect altered airway function. After the 10-day aerosolized allergen exposure, these mice do not develop MCh-induced AHR as seen in systemically sensitized mice (42).
In rodents, FcRI has been found to be expressed only on mast cells and basophils (39). Indeed, in support of the findings in FcRI–/– mice, mast cell-deficient mice also failed to develop increased airway reactivity after 10-day OVA exposure. This finding is somewhat in contrast to previous studies using mast cell-deficient mice, which suggested that the induction of airway inflammation and AHR does not require the presence of mast cells (13, 15). Similarly, it has been shown that the presence of IgE is not mandatory for the development of allergic airway disease (43, 44). In most of these studies animals were sensitized systemically by injection of the allergen in combination with an adjuvant and then challenged via the airways, which leads to marked inflammatory changes in the lung, high serum levels of allergen-specific Abs, increased airway reactivity including sensitivity to inhaled MCh, and prominent goblet cell hyperplasia. In the present study mice were exposed to a less potent exposure protocol using aerosolized allergen alone. This resulted in little airway inflammation and less goblet cell hyperplasia than seen after systemic sensitization.
Under these conditions without adjuvant, mast cells appear to play an essential role in the development of allergic airway disease, as mast cell-deficient mice fail to develop increased airway reactivity after aerosolized allergen exposure. Similarly, mice lacking the FcRI fail to develop AHR after this exposure protocol. These findings share some features with recent reports using different systemic allergen exposure protocols (16, 45). In those reports, where mast cells and FcRI were found to be critical for the development of AHR and airway inflammation, the exposure protocol deviated from those using repeated systemic sensitization and adjuvant, conditions under which mast cells and IgE were shown not to be important (15, 43). Williams et al. (16) found that mast cell-deficient mice exhibited weaker airway responsiveness and lung tissue eosinophil inflammation after a systemic sensitization protocol, but without adjuvant. In a different study, Mayr et al. (45) described the inability of FcRI-deficient mice to develop allergic airway inflammation and AHR to MCh after systemic sensitization consisting of a single OVA injection (with adjuvant) and consecutive intranasal challenges.
IgE can bind either to FcRI or to the low affinity IgE receptor (FcRII, CD23). Whereas cross-linking of FcRI with IgE and allergen has been associated with mast cell activation, ligation of FcRII can be either activating (carrying an immunoreceptor tyrosine-based activation motif) or inhibitory (carrying an immunoreceptor tyrosine-based inhibitory motif). In allergic airway disease, FcRII has been shown to exhibit negative regulatory effects (46). In the present study cross-linking of FcRI seemed to be activating in the development of increased airway reactivity and goblet cell hyperplasia, as FcRI–/– mice failed to develop increased smooth muscle responsiveness after allergen exposure. Interestingly, transfer of FcRI+/+ BMMC to FcRI–/– animals 14 days before the assessment of airway function fully reconstituted their ability to develop increased airway reactivity, suggesting that in this model the expression of FcRI on the mast cells is important and that mast cells alone can reconstitute this response. Previously, transfer experiments with BMMCs have been performed mainly to reconstitute mast cells in mast cell-deficient mice (16, 47, 48, 49). In that approach the analysis was conducted 10 wk or more after mast cell transfer. In the present study we assessed the response to 10-day OVA challenge at 2 and 20 wk after transfer of FcRI+/+ BMMC into FcRI–/– mice. In both cases the responses were similar. After transfer of green fluorescent BMMCs derived from GFP-overexpressing mice, we were able to detect transferred cells in the tracheal tissue 14 days post-transfer, suggesting that this time period is sufficient for migration of transferred cells to the trachea. Transfer of FcRI+/+ cells reconstituted the ability of FcRI–/– mice to develop increased airway reactivity independent of the time point after the transfer was made. In other disease models, mast cell reconstitution in mast cell-deficient mice normalized the ability to mount a mast cell-dependent response 2 days (50) and 9 days (51) after the transfer. In the present study we used BMMCs cultured in a mixture of IL-3 and Kit ligand for 4 wk. Under these conditions, >98% of the cells expressed FcRI as well as c-Kit on their surface and previous studies have suggested that the overwhelming majority of cells grown under these culture conditions are mast cells (52, 53) as well as out own electron microscopy studies. A very small percentage have features of basophils (52, 53), so it cannot be completely excluded that basophils, which may also express FcRI and c-Kit on their surface, may contribute to the reconstitution of the response. The finding that FcRI+/+ mast cells alone can restore responsiveness in FcRI–/– mice does not differentiate between the activated mast cells having a direct role in the EFS response and their having an indirect effect through the release of mediators that trigger other cells necessary for the EFS response.
Activation of lung mast cells through FcRI leads to the release of a variety of mediators via degranulation. Activation also triggers cytokine transcription and protein synthesis. A significant finding was that lung homogenates from allergen-exposed FcRI–/– mice had significantly lower levels of IL-13 than those from allergen-exposed wild-type controls. Altered airway function and goblet cell hyperplasia have been closely linked to IL-13 expression in the lung (35, 36, 37, 38, 54), and IL-13 may directly act on epithelial cells to induce goblet cell hyperplasia and airway hyper-reactivity (55). As a pleiotropic cytokine, IL-13 is secreted by activated Th2 cells and exhibits immunoregulatory activities that partially overlap with those of IL-4 (56). IL-13 also facilitates the preferential recruitment of eosinophils (and T cells) to the airway tissues (57) and airway mucus secretion, which can exacerbate airway responsiveness (54, 58). Although not necessary for, or even capable of, inducing Th2 development, IL-13 plays a regulatory role in Th2 cell activation (24). Administration of IL-13 or overexpression of IL-13 in the airways induced airway eosinophilia, mucus production, and AHR to varying degrees (35, 36, 54).
In further attempts to link mast cells, FcRI, and IL-13 in the present model, we chose two approaches: using IL-13 gene-targeted mice (24, 37) and the other treatment of mice during the challenge period with the decoy receptor sIL-13R2 (35, 36, 38). Both IL-13–/– mice and sIL-13R2-treated mice had levels of serum Abs similar to those of the wild-type mice, confirming that in mice, IL-13 does not regulate serum Ab production (37, 38). Furthermore, IL-13–/– mice have fully functional mast cells that signal through FcRI, degranulate, express similar amounts of cytokines (other than IL-13) and release of lipid mediators and after activation with allergen-specific IgE and allergen (data not shown). Both approaches revealed that the presence of IL-13 was necessary for development of the heightened response to EFS and goblet cell hyperplasia after the 10-day OVA challenge.
In addition to T cells, mast cells produce and secrete IL-13 (40). In rodents it has been shown that, depending on their phenotype, mast cells express IL-13 mRNA either constitutively or only after FcRI-mediated activation (59). All of our results in the 10-day OVA challenge model are consistent with the idea that mast cells are an important source of IL-13. Mast cells may produce IL-13 after stimulation with endotoxin (33, 34). However, in the present model endotoxin contamination of OVA seemed to be irrelevant to the development of increased airway reactivity and goblet cell hyperplasia, as OVA depleted of endotoxin, induced similar degrees of airway reactivity and goblet cell hyperplasia. Interestingly, IL-13–/– mice failed to develop AHR even after adoptive transfer of IL-13+/+ mast cells, suggesting that the reconstitution solely of mast cells capable of producing IL-13 is not enough to change airway reactivity. This observation, together with the demonstration that transfer of BMMC from FcRI+/+ IL-13–/– mice to FcRI–/– IL-13+/+ recipients restores their ability to develop AHR, indicates that activation of mast cells through FcRI is directing IL-13 production in the lung, a critical element in the development of altered airway function. However, under these conditions, mast cells may not be the predominant source of the IL-13 needed for this response. This does not diminish the need for IL-13, as reconstitution of airway responsiveness after mast cell transfer (even from IL-13–/– mice) was still critically dependent on IL-13; treatment with IL-13R2 after the mast cell transfer completely inhibited the reconstitution of increased airway responsiveness. We may speculate either that the IL-13 derived from the numbers of transferred mast cells (IL-13+/+ into IL-13–/–) was insufficient to restore AHR or that other cell types are a major source of IL-13 when stimulated by factors released from activated mast cells. There are a number of other cell types that may serve as a source of IL-13 after activation by a factor(s) released from IgE-FcRI-activated mast cells, including T cells, smooth muscle cells (60), basophils, and NKT cells (61, 62). Studies assessing these potential sources are underway.
In summary, we have demonstrated essential roles for FcRI, mast cells, and IL-13 in the development of allergic airway disease after allergen exposure exclusively via the lungs in the absence of adjuvant. FcRI on mast cells mediates these effects by regulating the production of IL-13. As aeroallergen exposure via the lungs in the absence of adjuvant is a major factor in the etiology of asthma, this IgE-mast cell-IL-13 pathway may play a significant contributory role in the development, maintenance, and exacerbation of the disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Erwin W. Gelfand, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: gelfande{at}njc.org
3 Abbreviations used in this paper: AHR, airway hyperresponsiveness; BMMC, bone marrow-derived mast cell; EFS, electrical field stimulation; ES50, frequency resulting in 50% of the maximal contraction; EU, ELISA unit; GFP, green fluorescent protein; MBP, major basic protein; MCh, methacholine; PAS, periodic acid-Schiff; sIL-13, soluble IL-13; UBI, human ubiquitin C promoter.
Received for publication August 20, 2003. Accepted for publication March 15, 2004.
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References |
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