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Published ahead of print on February 25, 2003, doi:10.1164/rccm.200209-1002OC
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American Journal of Respiratory and Critical Care Medicine Vol 167. pp. 1393-1399, (2003)
© 2003 American Thoracic Society

Characterization of a Mouse Model of Allergy to a Major Occupational Latex Glove Allergen Hev b 5

Charles L. Hardy, Linda Kenins, Alexander C. Drew, Jennifer M. Rolland and Robyn E. O'Hehir

Cooperative Research Centre for Asthma; Department of Allergy, Asthma and Clinical Immunology, The Alfred Hospital;
and Department of Pathology and Immunology, Monash University, Melbourne, Australia

Correspondence and requests for reprints should be addressed to Charles Hardy, Cooperative Research Centre for Asthma, Department of Pathology and Immunology, Monash Medical School, Commercial Road, Melbourne, VIC 3004, Australia. E-mail: charles.hardy{at}med.monash.edu.au


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Allergen-specific immunotherapy is a clinically proven effective treatment for many allergic diseases, including asthma; however, it is not currently available for latex allergy because of the high risk of anaphylaxis. There is, therefore, a crucial need for an animal model of latex allergy in which to develop effective immunotherapy. Previous mouse models of latex allergy either did not characterize the allergic pulmonary immune response or used crude latex extracts, making it difficult to quantify the contribution of individual proteins and limiting their usefulness for developing specific immunotherapy. We immunized mice with recombinant Hev b 5, a defined major latex allergen, or latex glove protein extract, representing the range of occupationally encountered processed latex allergens. The immune response was compared with that seen in ovalbumin-immunized mice. Immunization with Hev b 5 or glove extract elicits hallmarks of allergic pulmonary Th2-type immune responses, comparable to those for ovalbumin, including (1) serum antigen-specific IgE, (2) an eosinophilic inflammatory infiltrate in the lung, (3) increased interleukin-5 in lung bronchoalveolar lavage fluid, and (4) mucus hypersecretion by epithelial cells in the lung airways. This mouse model will aid the development of potentially curative treatments for latex-sensitized individuals, including those with occupational asthma.

Key Words: eosinophil • inflammation • interleukins • cytokines

Latex allergy is an important medical problem, particularly among healthcare workers and patients with spina bifida. The allergic reaction to latex ranges from urticaria, conjunctivitis, and rhinitis to asthma, anaphylactic shock, and occasionally death (1, 2). Exposure occurs by cutaneous, mucosal, and parenteral routes, with aerosol inhalation and degree of exposure to latex allergens playing important roles in the development of allergy (3, 4). Allergen-specific immunotherapy is a clinically proven effective treatment for a variety of allergic diseases; however, it is not currently available for latex allergy because of the high risk of anaphylaxis (5, 6). There is, therefore, an urgent need for animal models of latex allergy in which to develop effective allergen-specific immunotherapy, as a prelude to clinical studies.

Estimates suggest that 5–15% of healthcare workers have latex allergy (3). Hev b 5 is an acidic protein of 16 kD, sharing homology with proteins in kiwifruit and potato (7, 8). Among latex-allergic healthcare workers, the frequency of reactivity to Hev b 5 ranges from approximately 60–90% depending on the assay used (8–10), thus defining Hev b 5 as a major latex allergen. In excess of 50% of latex allergic subjects expresses the asthma phenotype, with latex emerging as a major cause of occupational asthma. It has been reported that exposure to Hevea brasiliensis may induce occupational asthma in 6% of exposed individuals (11).

Previous mouse models of latex allergy that used unfractionated latex extracts (12–16), although showing allergic Th2-type immune responses, were unable to define the role of individual allergens. In another model, mice immunized with a recombinant Hev b 5 (rHev b 5)–maltose binding protein fusion protein mounted specific IgE and IgG responses and permitted identification of T- and B-cell epitopes (17, 18). However, other parameters of an allergic response, including eosinophilic inflammation of the lung and mucus hypersecretion, were not demonstrated.

We have used purified rHev b 5 lacking maltose binding protein (19) to develop a mouse model of allergy to Hev b 5 and have characterized the allergic immune response. Mice rendered allergic to ovalbumin (OVA) have been widely studied and serve as a prototypic standard against which other mouse allergy models may be evaluated. We immunized mice with rHev b 5 and compared the immune response with that seen in the well-characterized OVA asthma model. Additionally, mice were immunized with glove extract (GE) to validate the Hev b 5 model using a clinically relevant sensitizing allergen extract. Immunization with Hev b 5 or GE elicits key hall-marks of an allergic Th2-type immune response, including (1) a predominantly eosinophilic inflammatory infiltrate in the bronchoalveolar lavage (BAL) and lung tissue, (2) serum Hev b 5- and GE-specific IgE, and (3) a significant increase in mucus-producing cells in the lung airways.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Female BALB/c mice aged 7–8 weeks were obtained from Monash University Animal Services (Clayton, Melbourne, Australia) or the Walter and Eliza Hall Institute animal house (Kew, Melbourne, Australia). All experimental protocols were approved by the Monash Animal Ethics Committee.

Immunization Protocol
Mice were sensitized intraperitoneally at two sites on Days 0 and 12 with 50 µg of rHev b 5, GE, or OVA (Sigma-Aldrich, Sydney, Australia) adsorbed to 20 µl of aluminum hydroxide gel (Rehydragel HPA, Reheis, NJ) in a final volume of 200 µl; saline mixed with alum was used as a negative control. Commencing Day 24 mice were anaesthetized with 350 µl of Ketamine (5 mg/ml; Parnell Laboratories, Alexandria, NSW) and Xylazine (1 mg/ml; Troy Laboratories, Smithfield, NSW) and challenged intratracheally four times (every 2nd day) with 25 µg in 45 µl of rHev b 5, GE, OVA, or saline alone. The trachea was intubated with a 20-G catheter and guide wire, and saline or antigen was delivered via a 50 µl microsyringe (SGE International Pty. Ltd., Melbourne, Australia) attached to a blunted 20-G needle. rHev b 5 and GE immunizations were performed as independent experiments.

Expression and Purification of rHev b 5
rHev b 5 was prepared as described (19). Endotoxin was removed using a polymixin B column (Detoxi-Gel; Pierce, Rockford, IL). Endotoxin-depleted rHev b 5 failed to stimulate proliferation of naive BALB/c splenocytes in a standard tritiated thymidine incorporation assay. Hev b 5 concentration was determined using the bicinchonic acid assay (Pierce) and bovine {gamma}-globulin (Bio-Rad, Hercules, CA) as standard.

Preparation of Glove Extract
GE was prepared as described (19) and was dialyzed against 0.9% NaCl using 3,500 molecular weight cut-off dialysis tubing (Pierce). Dialyzed GE was filter sterilized, and the protein concentration was determined using the bicinchonic acid assay.

Bronchoalveolar Lavage, Differential Counts, and Tissue Sampling
Mice were killed 18 hours after the fourth intubation. After blood collection, lungs were lavaged with 0.4 ml of 1% fetal calf serum in phosphate-buffered saline (PBS) followed by three further lavages of 0.3 ml. Mediastinal lymph node and BAL cell suspensions were counted (Coulter, Luton, UK). Serum was obtained from whole blood by centrifugation for 4 minutes at 11,350 x g and stored at -20°C before analysis. BAL cell cytospots were air dried, fixed in methanol, and Giemsa stained (Merck, Kilsyth Victoria, Australia). At least 200 cells were counted per mouse, and cells were identified by morphologic criteria. The remaining BAL preparation was centrifuged at 350 x g for 4 minutes, and the BAL fluid (BALF) was collected and stored at -70°C for subsequent cytokine analysis.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Western Blot
rHev b 5 and protein markers (BenchMark; Invitrogen, Melbourne, Australia) were run on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel essentially as described (19). Gels were passively transferred to nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) for 4 hours. Membranes were probed with 6F6 monoclonal antibody and developed via enhanced chemiluminescence (SuperSignal; Pierce), as previously described (19). The purity of the rHev b 5 was confirmed by Coomassie staining and recognition by Hev b 5-specific monoclonal antibody (Figure 1) .



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  		Figure 1. Western blot of rHev b 5 with mouse monoclonal antibody 6F6. rHev b 5 was resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose and probed with 6F6. M = molecular mass markers; C = Coomassie brilliant blue-stained gel slice; membrane probed with or without 6F6 indicated by + and -, respectively.

 
Cytokine ELISA
Interleukin (IL)-4 and IL-5 cytokine ELISA was performed using the OPTEIA kit (Pharmingen, San Diego, CA). Plates were blocked with 5% skim milk powder in 0.05% Tween 20 in PBS, and all reagents diluted in 1% skim milk powder/0.05% Tween 20 in PBS. Cytokine concentrations were calculated from the standard curves using Microplate Manager (Bio-Rad Laboratories). Limits of detection were 8 pg/ml.

Detection of Latex Allergen-specific IgE
ELISA plates were coated with rHev b 5 or GE at 10 µg/ml at 4°C overnight. Serum was diluted 1:5, and IgG was depleted by incubating with Protein G Sepharose (Amersham Pharmacia, Uppsala, Sweden) for 1.5 hours at room temperature. Plates were blocked with 5% skim milk powder/0.05% Tween 20 in PBS for 1.5 hours, washed four times in 0.05% Tween 20 in PBS, and incubated with 35 µl of IgG-depleted serum for 1.5 hours. Plates were incubated in antimouse IgE–biotin (clone R35–118; Pharmingen) at 1:100 followed by streptavidin–peroxidase at 1:1,000 for 1 hour each. All antibodies and conjugates were diluted in 1% skim milk powder/0.05% Tween 20 in PBS. Plates were washed seven times between steps and additionally four times in PBS before development of reaction product. Development was for 20 minutes at 37°C using 50 µl of o-phenylenediamine (P-6912; Sigma-Aldrich) dissolved in 0.05 M phosphate citrate sodium perborate buffer, pH 5.0 (P-4922; Sigma-Aldrich). The reaction was stopped with 50 µl of 4-M HCl, and absorbance was read at 490 nm. Results are expressed as raw optical density readings minus background (no serum added).

Eosinophil and Mucus-secreting Cell Quantitation
Immediately after BAL collection, lungs were fixed in 10% neutral-buffered formalin. Paraffin-embedded sections (4 µm) were stained with hematoxylin and eosin or periodic acid-Schiff reagent. Mucus-producing cell frequency was calculated by counting periodic acid-Schiff–positive cells in eight bronchioles/mouse. Tissue-infiltrating eosinophils were quantitated in Giemsa-stained sections by counting 10 high-power fields (x1,000) from 10 similar-sized small bronchioles or 10 similar-sized blood vessels per mouse.

Statistical Analysis
Statistical analysis was performed using SAS Version 8.0 (SAS Institute, Inc., Cary, NC). Normally distributed data were analyzed using t tests and analysis of variance where appropriate. Non-normally distributed data (mucus staining data) were log transformed before calculation of geometric means and statistical analysis using analysis of variance and t-test. A p value of 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Lung Inflammation in Mice Challenged and Immunized with Hev b 5 and Glove Extract
Histologic analysis of hematoxylin and eosin–stained formalin-fixed tissue sections from Hev b 5- and OVA-immunized mice revealed a dense peribronchial and perivascular inflammatory infiltrate in the lungs (Figures 2C and 2E) . The inflammatory infiltrate consisted of mononuclear and polymorphonuclear cells, with numerous eosinophils easily identifiable by their eosinophilic cytoplasmic staining and characteristic tubular nuclei. Bronchial epithelial cells were overtly hypertrophied. Also notable were disruption of the smooth muscle layer, epithelial shedding, and particulate matter in the airway lumen. In contrast, control mice showed normal lung architecture. In particular, the respiratory epithelium was of normal size and morphology, and there was an absence of inflammatory cells in the peribronchial and perivascular regions (Figure 2A).



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  		Figure 2. Histopathology in bronchial epithelium from Hev b 5- and OVA-immunized mice. Representative hematoxylin and eosin–stained micrographs from saline control (A), Hev b 5–immunized (C), or OVA-immunized (E) mice. Periodic acid-Schiff–stained lung sections showing representative bronchioles from saline control (B), Hev b 5–immunized (D), and OVA-immunized (F) mice. Note the hypertrophied epithelium in Hev b 5 and OVA groups (arrows). Original magnification x400.

 
Compared with saline control mice there was a 28-fold increase in BAL cell numbers in Hev b 5-immunized mice, a level comparable to that seen in OVA-immunized mice (Figure 3A) . In separate experiments, a comparable 30-fold increase in BAL cell numbers was seen in GE-immunized mice (Figure 3B). Mediastinal lymph node cell numbers were also increased threefold to fivefold in Hev b 5- and GE-immunized mice compared with control subjects (data not shown). Differential counts showed that in both the Hev b 5 and OVA groups, approximately 50% of the BAL cells were eosinophils, representing a more than 3,000-fold increase in absolute eosinophil numbers in the BAL (Figure 3C). Similar results were seen in separate experiments when GE was used as immunogen (Figure 3D).



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  		Figure 3. Lung inflammation and eosinophilia in Hev b 5 and GE allergic mice. Mice were immunized and challenged intratracheally with GE or Hev b 5 and were killed the morning after the fourth intubation as described in METHODS. Total BAL counts obtained from separate experiments comparing Hev b 5 or OVA immunization (A) and GE or OVA immunization (B). Differential counts on lung inflammatory cells from experiments comparing Hev b 5 (solid bar) or OVA (striped bar) immunization (C) and GE (solid bar) or OVA (striped bar) immunization (D) with saline control mice (open bar). Representative of at least three separate Hev b 5 versus OVA or GE versus OVA experiments (n = 4–5 per group). (A and B) Horizontal bar shows mean value; (C and D) mean ± SD.

 
Hev b 5-immunized mice also had significant increases in eosinophil numbers in lung parenchyma. Eosinophil cell density was significantly greater in both peribronchial and perivascular regions of the lung of the Hev b 5 and OVA groups compared with saline control mice (p < 0.0001 in all cases; t test; Figures 4A and 4B) . Similarly, peribronchial and perivascular eosinophil density was significantly greater in GE- and OVA-immunized mice compared with saline control subjects (p < 0.0001 in all cases; t test; Figures 4C and 4D).



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  		Figure 4. Enumeration of peribronchiolar and perivascular eosinophil density in lungs of Hev b 5 and GE-immunized mice. Eosinophil density was calculated in Giemsa-stained lung sections as described in METHODS. Peribronchial (A and C) and perivascular (B and D) eosinophil density in Hev b 5–immunized (A and B) and GE-immunized mice (C and D). Saline and OVA represent negative and positive controls, respectively (n = 5 per group). The horizontal bar shows the mean value.

 
Hev b 5 and GE Immunization Induces Bronchial Mucus Hypersecretion
Mucus hypersecretion is a characteristic of allergic airways inflammation in humans (20) and is also a common feature in mouse models of asthma (21–23). Hev b 5– and OVA-immunized mice had significantly more mucus-producing goblet cells in their airways than saline control subjects (Figures 2D and 2F and Table 1) . In marked contrast, periodic acid-Schiff–stained sections from saline-treated control mice did not show evidence of mucus production (Figure 2B). The frequency of mucus-secreting cells was also determined in GE-immunized mice. Consistent with the results in the Hev b 5 experiments, there were significantly more mucus-producing cells in the airways of GE-immunized mice compared with saline control mice (Table 1).


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  		TABLE 1. Mucus-producing cell frequency in periodic acid-schiff–stained lung sections from hev b 5- or ge-immunized mice*

 
Elevated IL-5 in BALF from Hev b 5– and GE-immunized Mice
To characterize the allergic phenotype further, levels of IL-4 and IL-5 in BALF were measured. Low levels of IL-4 were detectable in all groups, although there were no differences when Hev b 5 or GE groups were compared with saline or OVA control mice (84 ± 28, 63 ± 9, and 58 ± 11 for saline, Hev b 5, and OVA, respectively, and 154 ± 53, 166 ± 28, and 137 ± 67 for saline, GE, and OVA, respectively, mean pg/ml ± SD). In contrast, levels of IL-5 were elevated approximately twofold in Hev b 5–immunized mice compared with control mice, whereas levels in OVA-immunized mice were approximately threefold greater (Figure 5A) . Similarly, mean levels of IL-5 were elevated approximately fourfold in GE-immunized mice compared with saline control mice, although the concentration varied between individual mice (Figure 5B).



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  		Figure 5. BALF IL-5 cytokine in Hev b 5 and GE allergic mice. IL-5 concentration in BALF from Hev b 5–immunized (A) and GE-immunized mice (B) (n = 4–5 per group), representative of two separate experiments. The horizontal bar shows the mean value.

 
Hev b 5 and GE Immunization Elicits Antigen-specific IgE
Serum from immunized mice was analyzed for the presence of allergen-specific IgE, a hallmark of allergic reactions. Sera from Hev b 5–immunized mice contained Hev b 5–specific IgE antibody, whereas Hev b 5–specific IgE was undetectable in saline control mice (Figure 6A) . The specificity of the reaction was demonstrated by the fact that Hev b 5–specific IgE was undetectable in sera from OVA-immunized mice. Conversely, OVA-immunized control mice produced OVA-specific IgE, whereas Hev b 5–immunized mice had undetectable OVA-specific IgE (Figure 6B).



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  		Figure 6. Detection of specific IgE in Hev b 5 and GE allergic mice. Sera were depleted of IgG and were tested at a final dilution of 1:5. Sera from saline, Hev b 5, and OVA groups were tested for the presence of Hev b 5–specific (A) and OVA-specific (B) IgE. Sera from saline, GE, and OVA groups were tested for presence of GE-specific (C) or Hev b 5-specific (D) IgE. OD 490 nm minus background (no serum applied) (n = 4–5 per group), representative of three (A and B) or two (C and D) separate experiments. The horizontal bar shows the mean value.

 
Serum from GE-immunized mice was also tested for the presence of antigen-specific IgE. As shown in Figure 6C, GE-immunized mice made GE-specific IgE, whereas OVA-immunized mice did not make GE-specific IgE, indicating the specificity of the response. Since GE is a natural source of latex to which healthcare workers and latex-allergic individuals are exposed, we determined whether GE-immunized mice made Hev b 5–specific IgE. As shown in Figure 6D, three out of five GE-immunized mice made low but detectable levels of Hev b 5–specific IgE, whereas OVA control mice did not produce such antibody.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our report is the first to describe a mouse model of pulmonary allergic inflammation to the major latex allergen Hev b 5. Mice were immunized with the latex allergen rHev b 5, a dominant latex allergen in the healthcare setting. Key traits of the latex allergic immune response were compared directly with those seen in the well-characterized OVA mouse model of experimental asthma. All sensitizations, including the control saline inoculation, were performed with alum to control for adjuvant effects. Our results show that the allergic phenotype in Hev b 5–immunized mice closely resembles that seen in OVA-immunized mice. We validated our model by immunizing mice with proteins eluted from commercial latex gloves (GE), a preparation mimicking the range of processed latex proteins occupationally encountered by humans. Immunization with GE resulted in an allergic phenotype very similar to that seen in Hev b 5– or OVA-immunized mice.

Immunization of mice with OVA is a well characterized animal model of pulmonary inflammation with eosinophilia and provides a useful benchmark by which other mouse allergy models can be assessed. Immunization of mice with rHev b 5 and GE elicited allergic symptoms superficially indistinguishable to those seen in OVA-immunized mice. In particular, there was a pronounced pulmonary eosinophilia, with approximately 50% of BAL cells being eosinophils, regardless of the immunizing antigen. This finding is consistent with those reported by others using similar immunization strategies (21, 24–26). Moreover, the distribution of eosinophils within the lungs of Hev b 5- and GE-immunized mice was very similar to that seen in the OVA groups. However, GE elicited a more pronounced peribronchiolar eosinophilic infiltration when compared with the Hev b 5 and OVA groups. Additionally, immunization with GE resulted in a greater frequency of mucus-secreting bronchial epithelial cells when compared with Hev b 5. These differences between GE and Hev b 5 may reflect the fact that GE contains a mixture of proteins, including the major latex allergens Hev b 5 and Hev b 6 (19).

Despite these superficial similarities, the immune response was specific for the immunizing antigen, as illustrated by the specificity of the IgE response. Significantly, GE-immunized mice also made IgE specific for Hev b 5, one of the most important latex allergens in healthcare workers sensitized by latex glove usage (10). This demonstrates that Hev b 5 is present in GE at sufficient levels to elicit the production of specific IgE, consistent with the finding that Hev b 5 is an abundant allergen in high-protein powdered latex gloves (19). The relative concentration of IgE specific for Hev b 5 or GE was always less than that seen when mice were immunized with OVA. In the case of Hev b 5, this may be due to the fact that Hev b 5 is less "allergenic" when compared with OVA; this idea is supported by the fact that BAL cell numbers and mucus-secreting cell frequency were lower than those seen in OVA-immunized mice. In the case of GE, this is presumably because GE immunization elicits production of IgE for a subset of proteins (allergens) present within the extract, for instance, Hev b 5 and Hev b 6. However, a plate coated with GE would presumably bind all proteins present, thus diluting the relative abundance of specific IgE-inducing target proteins such as Hev b 5 and Hev b 6. Alternatively, different molar concentrations of the immunogenic proteins may account for some of this variation.

The role of T cells secreting Th2-type cytokines in asthma and allergic diseases has been well documented (20, 27). The prototype Th2 cytokine IL-4 plays an important role in the class switching of B cells to an IgE-secreting phenotype and in the generation of Th2-type immune responses (28–30). IL-5 and the eosinophil chemokine eotaxin cooperate to promote the maturation of eosinophils and their migration into pulmonary tissue (24, 31–33), and eosinophils play a central role in asthma pathogenesis (20). Nevertheless, the finding that IL-5 blockade is ineffective at reducing clinical asthma symptoms (34, 35) and that corticosteroids can diminish eosinophilia independently of IL-5 suppression (36) highlights limitations in our understanding of factors regulating eosinophilia. IL-13 plays a central role in the pathogenesis of asthma both via its role in IgE class switching (37) and as a key regulator of mucus production and airways hyperreactivity (22, 38, 39). The cytokines IL-4, IL-5, and IL-13 act in an integrated manner to modulate allergic pulmonary inflammation (23, 33, 39, 40). The Th1 cytokine interferon-{gamma} can inhibit airways hyperreactivity (41) and pulmonary eosinophilia (42, 43) in allergen-challenged mice, although data from interferon-{gamma} knockout mice have been equivocal (44, 45). A recent study in interferon-{gamma} knockout mice suggests that interferon-{gamma} can reverse airway responsiveness even when administered after allergen challenge (46).

We demonstrated that levels of BALF IL-5 were higher in immunized mice compared with control mice. Somewhat surprisingly, no such difference could be demonstrated for IL-4 in BALF, although the level detected was comparable to that reported by others (25, 26, 47, 48). It is possible that difference in IL-4 levels would be revealed by a detailed time-course analysis of IL-4 production in BALF. Nevertheless, the findings of latex allergen-specific IgE production, eosinophilia, and mucus hypersecretion are collectively indicative of the production of the cytokines IL-4, IL-5, and IL-13, respectively.

In conclusion, we have established a mouse model for investigating traits of allergic disease using the latex allergen Hev b 5 as a model. We have already mapped T-cell epitopes for Hev b 5 (9) and Hev b 6 in humans (de Silva, personal communication), and similar dominant epitopes may be recognized in mice (49). We are currently testing this hypothesis by mapping immunodominant Hev b 5 epitopes. Knowledge of T-cell epitopes is crucial to the development of immunotherapeutic preparations, either as immunodominant peptides or mutant allergen, which downregulate the allergic response (5, 49–51). By monitoring the allergic parameters described in this report, we will be able to assess the efficacy of immunotherapy in lessening the severity of a predominantly Th2-type allergic immune response. The Hev b 5 allergic mouse model will serve as a useful tool to aid the development of allergen-specific immunotherapy for safe use in patients with latex allergic diseases, including occupational asthma.


   Acknowledgments
 
The authors acknowledge Drs. Paul Foster, Simon Hogan, Gary Anderson, Dale Godfrey, and Wayne Thomas for helpful discussions.


   FOOTNOTES
 
Funded by the Cooperative Research Centre for Asthma, Australia; the National Health and Medical Research Council, Australia; and the Alfred Research Trusts.

Received in original form September 6, 2002; accepted in final form February 19, 2003


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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