INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Airway inflammation is an important feature of asthma. Eo- sinophils are the effector cells most often associated with airway inflammation in asthmatic individuals; however, mast cells and basophils may also play an important role in the development of allergic inflammation, through IgE- and chemokine-initiated mediator release.

Mast cells are most prevalent in tissues that interface directly with the external environment, and are located in the airway epithelium throughout the respiratory tract, where they may respond rapidly to antigenic stimuli by releasing preformed mediators (1). Although the relative level of mast cells compared with other inflammatory cells in the airways is low (2), these cells have been proposed to play an important role in the pathogenesis of allergic asthma. Mast cells are present in higher numbers in the airways of symptomatic arthmatic as compared with normal subjects (2) and are increased in the airways of symptomatic as compared with asymptomatic asthmatic subjects (3), and numbers of these cells are enhanced after allergen inhalation challenge (4). Mast cells have traditionally been identified by their morphologic appearance and through metachromatic staining properties due to the binding of cationic dyes such as toluidine blue or Alcian blue to the highly acidic mast cell glycosaminoglycans. Because metachromatic staining is also observed in basophils, however, staining specific for mast cells and specific for basophils is necessary to confirm these earlier findings.

Basophils are circulating granulocytes that respond to allergic stimuli by migrating and accumulating at sites of allergic inflammation (5). It has been reported that levels of basophils are increased in the airways of asthmatic as compared with normal subjects (6), and are further increased during asthma exacerbations (7) and in response to allergen inhalation challenge (8). Identification of basophils in the studies in which these findings were made relied on morphologic and functional criteria, which cannot always discriminate mast cells from basophils. Immunohistochemical techniques have been used to identify basophils as IgE-positive, tryptase-negative cells. These studies have suggested that the number of basophils in pulmonary tissue of asthmatic individuals is increased (9), but identification of cells in asthmatic airways through detection of surface IgE may be difficult because of the expression of FcR1 on eosinophils (10), monocytes, and antigen-presenting cells (11). Specific antibodies to human basophils have only recently been characterized (12). Preliminary studies done with monoclonal antibodies indicate increased numbers of basophils in bronchial biopsy specimens from asthmatic as compared with normal subjects, which are further enhanced after allergen challenge (13, 14). The role of these cells in the development of asthmatic airway responses has not yet been elucidated.

Sputum induction is a safe, reliable, and sensitive method for measuring allergen-induced airway inflammation in atopic asthmatic subjects (15, 16), and allows serial sampling of airway secretions for examination of airway inflammatory-cell kinetics (17). We have previously demonstrated a significant increase in sputum metachromatic cells (mast cells and/or basophils) after allergen inhalation, and have shown that the numbers of these cells remain elevated for several days (17). In the present study, we used monoclonal antibodies specific for human mast cells and basophils to compare and contrast any allergen-induced changes in mast cell and basophil numbers, to compare the kinetics of these cells' respective accumulation in the airway after allergen inhalation, to compare the cellular response between isolated early responder (ER) and dual responder (DR) asthma patients, and to examine the relationship between changes in cellularity and airway physiology.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects

Nineteen subjects were selected for examination of the kinetics of allergen-induced airway basophil and mast cell accumulation (Table 1). The sample size was considered sufficient for this, since previous studies have shown that with the same methodology used in the present study, eight or more subjects can demonstrate allergen-induced airway eosinophilia as assessed with induced sputum (16). Subjects were nonsmokers with mild atopic asthma, selected because they demonstrated an allergen-induced early brochoconstrictive response of at least a 15% reduction in FEV₁ during the first 2 h after challenge. Subjects were considered isolated ERs if the late decrease in FEV₁, measured between 3 to 7 h after challenge, was less than 10% of the baseline FEV₁ (n = 5). Subjects were considered DRs if the late decrease in FEV₁ at 3 and 7 h after challenge was greater than 10% (n = 14). Signed informed consent was obtained from participants in the study, which was approved by the Ethics Committee of McMaster University Health Sciences Centre. Subjects were not exposed to sensitizing allergens and did not have asthma exacerbations or respiratory tract infections for at least 4 wk before allergen challenges. All subjects had stable asthma, with an FEV₁ above 70% predicted on all study days before challenge to the airways, and used no regular medication other than an inhaled ₂-agonist infrequently (less than twice weekly) as required to treat their symptoms. All medications were withheld for at least 8 h before each visit, and subjects were instructed to refrain from rigorous exercise and drinking tea or coffee on the mornings before visits to the laboratory.

Study Design

Each subject visited the laboratory on three consecutive days. Baseline measurements of FEV₁, the provocative concentration of methacholine causing a 20% decrease in FEV₁ (PC₂₀), and inflammatory cell counts in induced sputum were made on the day before allergen inhalation challenge. Allergen was inhaled on the following morning, and FEV₁ was measured at intervals for 7 h after challenge. Sputum samples were obtained at 7 h for evaluating the late asthmatic response. Methacholine PC₂₀ and sputum samples were obtained once again at 24 h after challenge.

Laboratory Procedures

Methacholine inhalation test. Methacholine inhalation challenge was conducted as described by Cockcroft (18). Subjects inhaled normal saline followed by doubling concentrations of methacholine phosphate from a Wright nebulizer (Roxow Medi-Tech, Montreal, PQ, Canada) for 2 min. FEV₁ was measured at 30, 90, 180, and 300 s after each inhalation, with a Collins water-sealed spirometer and kymograph (Warren E. Collins, Braintree, MA). The test was terminated when a decrease in FEV₁ of 20% of the baseline value occurred, and the methacholine PC₂₀ was then calculated.

Allergen inhalation challenge. Allergen challenge was conducted as described by O'Byrne and colleagues (19). The allergen producing the largest skin-wheal diameter was diluted in 0.9% saline for inhalation. The concentration of allergen extract for inhalation was determined from a formula described by Cockcroft and coworkers (20), using the results from the skin test and the methacholine PC₂₀. The starting concentration of allergen extract for inhalation was two doubling concentrations below that predicted to cause a 20% early decrease in FEV₁, and doubling concentrations of allergen were given until a 15% early decrease in FEV₁ was reached. The FEV₁ was measured at 10 min and then again at 20, 30, 45, 60, 90, and 120 min after allergen inhalation, and then on an hourly basis until 7 h after allergen inhalation. The early asthmatic response was taken to be the largest decrease in FEV₁ within 2 h after allergen inhalation, and the late asthmatic response was taken to be the largest decrease in FEV₁ between 3 h and 7 h after allergen inhalation.

Sputum analysis. Sputum was induced and processed according to the method described by Pizzichini and coworkers (15). Subjects inhaled 200 µg albuterol, and 10 min later inhaled 3%, 4%, and 5% saline for 7 min each. The induction was stopped when an adequate sputum sample was obtained or if the FEV₁ dropped by 20% of its baseline value. Cell plugs were selected from the sample, separated from saliva, and weighed. Samples were mixed at room temperature with four times their volume of 0.1% dithiothreitol (Sputolysin; Calbiochem Corp., San Diego, CA) on a bench rocker for 15 min, after which four times their volume of Dulbecco's phosphate-buffered saline (DPBS) (Life Technologies Inc., Grand Island, NY) was added the preparation was and mixed for an additional 5 min. The cell suspension was filtered through a 52-µm nylon gauze (BNSH Thompson, Scarborough, ON, Canada) to remove debris, and the total cell count was determined in a Neubauer hemocytometer chamber (Hausser Scientific, Blue Bell, PA) and expressed as the number of cells per milliliter of sputum. The supernatant was removed by centrifugation at 1,500 rpm for 10 min and the cells were resuspended in DPBS at 0.75 to 1.0 × 10⁶/ml. Cytospin preparations were made on glass slides, using 50 µl of cell suspension and a Shandon III cytocentrifuge (Shandon Southern Instruments, Sewickley, PA) run at 300 rpm for 5 min. Differential cell counts were obtained from the mean of two slides stained with Diff-Quik (American Scientific Products, San Diego, CA) with 400 cells counted per slide. Metachromatic cells were enumerated on slides stained with toluidine blue, from the mean of two slides with at least 2,500 cells observed on each slide.

Cytospin preparations were also made on Aptex (Sigma Chemical Co., St. Louis, MO)-coated slides, fixed for 15 min in Carnoy's solution, and stored at 70° C. Immunocytochemistry for single-labeling of mast cells and basophils was completed on separate slides within 7 d of slide preparation, since preliminary experiments showed no deterioration of staining within this period. Basophils were detected with the murine monoclonal antibody 2D7, which immunolocalizes specifically to a human basophil secretory granule (12). The 2D7 antibody was applied overnight at 4° C at a concentration of 10 µg/ml. This was followed by incubation with a biotinylated secondary antibody and by visualization through the immunoperoxidase technique (Vectastain Elite; Vector Laboratories, Burlingame, CA) with 3-amino-9-ethyl carbazole as a chromogen, resulting in a red final product. Mast cells were detected with an alkaline phosphatase-conjugated, murine monoclonal antitryptase antibody that binds specifically to mast cell tryptase (Chemicon International, Inc., Temecula, CA). The antitryptase antibody was applied overnight at 4° C at a concentration of 2 µg/ml, and was visualized with naphthol phosphate and Fast Red (DAKO Corp., Carpinteria, CA), which produced a red final product. Primary antibodies were diluted in 1.0% bovine serum albumin (BSA) (Sigma Chemical Co.) and a wash buffer made up of Tris, 0.01% thimerosal, and 0.05% Tween 20 (all from Sigma). Slides were counterstained with Mayer's hematoxylin (Sigma). The percentage of immunopositive cells was determined from a count of at least 5,000 cells made with light microscopy.

Cytospin preparations were also made on Aptex-coated slides and fixed for 10 min in periodate-lysine-paraformaldehyde for immunocytochemical staining of activated eosinophils. Slides were stained with a murine monoclonal antibody (EG2) (Kabi Pharmacia, Uppsala, Sweden) to the activated form of human eosinophil cationic protein at 1.0 µg/ml, which was applied overnight at 4° C. Protein was detected on the following day with the alkaline phosphatase-antialkaline phosphatase (APAAP) method, using rabbit antimurine secondary antibodies and mouse monoclonal APAAP tertiary antibodies (DAKO, Glostrup, Denmark). All antibodies were diluted in 1.0% BSA (Sigma) in wash buffer made up of DPBS, 0.01 M 4-(2-hydroxyethyl)-1-piperazine- N-2-ethanesulfonic acid buffer (Life Technologies), and 0.01% saponin (Sigma). Nonspecific staining with the primary and secondary antibodies were blocked by incubation with 0.75 g/ml human AB serum and 0.25 g/ ml normal rabbit serum (Sigma) diluted in wash buffer. Slides were counterstained with Mayer's hematoxylin (Sigma) The percentage of positive immunoreactive cells was determined from a count of 400 cells made with light microscopy. For each immunocytochemical stain, negative isotype controls (murine IgG₁; Sigma) were included for each slide, using the same concentration as of the primary antibody.

Statistical Analysis

Measurement of methacholine PC₂₀ were made by linear interpolation of logarithmic dose-response curves. Summary statistics are expressed as mean ± SEM, except for measurements of methacholine PC₂₀ and sputum cells, which are expressed as geometric mean and range of values. Methacholine PC₂₀ and sputum values were log-transformed to fit a normal distribution prior to analysis. The effects of allergen on methacholine PC₂₀ and sputum inflammatory cells were analyzed with repeated measures analysis of variance. Allergen-induced changes in sputum inflammatory cells in ERs and DRs were compared through two-tailed unpaired Student's t tests. Pearson's correlation coefficient was used to test for relationships of the two principal clinical markers, change in methacholine PC₂₀ and late decrease in FEV₁, to sputum basophil and mast cell counts (21).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Five subjects developed isolated early airway asthmatic responses (ER group), with a mean maximal early decrease in FEV₁ of 30.0 ± 4.2% and a mean maximal late decrease in FEV₁ of 6.0 ± 1.0% (Figure 1). Subjects with an isolated early response did not develop significant allergen-induced airway hyperresponsiveness (AHR), their methacholine PC₂₀ being 6.1 mg/ml (range: 1.0 to 21.5 mg/ml) before and 4.5 mg/ml (range: 0.26 to 16.0 mg/ml) 24 h after allergen inhalation (p = 0.13) (Table 1). Fourteen subjects developed dual airway asthmatic responses (DR group), with a mean maximal early decrease in FEV₁ of 34.1 ± 2.1% and a mean late decrease in FEV₁ of 20.4 ± 2.0 (Figure 1). These subjects developed significant allergen-induced AHR, their methacholine PC₂₀ decreasing from a mean of 2.9 mg/ml (range: 0.11 to 12.7 mg/ml) before to 0.8 mg/ml (range: 0.14 to 17.3 mg/ml) 24 h after allergen inhalation (p = 0.0001) (Table 1).

View larger version (15K): [in this window] [in a new window]   Figure 1.   Percent change in FEV1 for 7 h after allergen inhalation challenge in subjects with an isolated early asthmatic response (open circles) and in subjects with a dual asthmatic response (solid circles).

Baseline levels of sputum basophils were similar in the ER and DR groups, at a mean of 0.02 × 10⁴/ml (range: 0 to 0.30 × 10⁴/ml) and 0.02 × 10⁴/ml (range: 0 to 0.24 × 10⁴/ml), respectively. Sputum basophils increased at 7 and 24 h after allergen challenge in both the ER (p = 0.001) and DR (p = 0.000001) groups, with peak levels measured at 7 after challenge in both groups (Table 2). The allergen-induced shift in the number of basophils per milliliter of sputum was significantly greater in the DR than in the ER group at both 7 h (200-fold versus 30-fold increase, p = 0.03) and 24 h (100-fold versus seven-fold increase, p = 0.008) after allergen challenge.

Levels of sputum mast cells at baseline were the same in the ER and DR groups, at a mean of 0.01 × 10⁴/ml (range: 0 to 0.07 × 10⁴/ml) and 0.01 × 10⁴/ml (range: 0 to 0.06 × 10⁴/ml), respectively. Mast cells increased at 7 and 24 h after allergen challenge in the DR group (by fourfold and eightfold, respectively, p = 0.009), but did not change after allergen challenge in the ER group (p = 0.41) (Table 2). Peak levels of mast cells in the DR group were observed at 24 h after challenge. The allergen-induced difference in the number of mast cells per milliliter of sputum was fivefold greater in the DR than in the ER group at 7 and 24 h after allergen challenge, but these differences were not statistically significant (p = 0.14 and p = 0.08, respectively).

The allergen-induced number of basophils was approximately 70-fold and 20-fold higher than the number of mast cells measured at 7 and 24 h after challenge, respectively. Furthermore, the number of immunopositive basophils plus immunopositive mast cells measured after challenge was greater than the number of metachromatic cells (mast cells plus basophils) stained by toluidine blue (Figure 2).

View larger version (13K): [in this window] [in a new window]   Figure 2.   Comparison of allergen-induced change in numbers of immunopositive mast cells (open bars), immunopositive basophils (hatched bars), and metachromatic cells (solid bars) measured in all study subjects at baseline, 7 h, and 24 h after allergen inhalation challenge. Data are presented as geometric mean and geometric SEM. * p < 0.05, change from baseline.

There was a significant increase in the number of eosinophils per milliliter of sputum in the DR group at both 7 and 24 h after allergen challenge (p = 0.000004) (Table 2). There was also a significant increase in the number of eosinophils per milliliter of sputum in the ER group at both 7 and 24 h after allergen inhalation (p = 0.0015) (Table 2). However, the numbers of sputum eosinophils in the DR group were similar at 7 and 24 h after challenge, whereas the number of sputum eosinophils in the ER group reached a peak lower than in the DR group at 7 h, and had decreased substantially by 24 h after challenge. The allergen-induced increase in eosinophils at 24 h after challenge was not statistically different in the DR and ER groups when eosinophils were expressed either as absolute numbers per milliliter of sputum (p = 0.43) or as differential counts (p = 0.07). There was a significant increase in the number of activated eosinophils (EG2-positive cells) per milliliter of sputum in the DR group at both 7 and 24 h after allergen challenge (p = 0.00002) (Table 2). By contrast, there was no change in the number of EG2-positive cells per milliliter of sputum in the ER group at either 7 or 24 h after allergen challenge (p = 0.59) (Table 2).

There was a significant positive relationship between the allergen-induced changes in methacholine PC₂₀ and in sputum basophils at 24 h after challenge (r = 0.66, p = 0.002), and a significant negative relationship between the late asthmatic response and the change in mast cell number at 24 h after challenge (r = 0.53, p = 0.02). There was no significant relationship between sputum eosinophils and methacholine PC₂₀, or between the late asthmatic response and any cell type measured during the late response (Figure 3).

View larger version (18K): [in this window] [in a new window]   Figure 3.   Relationship between maximum percent decrease in FEV1 during the late response and the allergen-induced change in number of sputum mast cells (log-transformed) measured 24 h after allergen challenge (top panel; r = 0.53, p = 0.02), and relationship between allergen-induced change in methacholine PC20 (log-transformed) and allergen-induced change in number of sputum basophils (log-transformed) measured 24 h after allergen challenge (bottom panel; r = 0.66, p = 0.002).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study showed that the allergen-induced increase in metachromatic cells previously reported in the airways of DR atopic asthmatic subjects (17) is due to an increase in both mast cells and basophils. However, the magnitude of the increases in basophils was much greater than that of the increases in mast cells. Furthermore, the allergen-induced increases in these cells were greater in asthmatic subjects who developed a dual allergen-induced airway response than in those who developed an isolated early response.

Low numbers of mast cells and basophils were observed in the sputum before allergen challenge. Several studies have demonstrated the presence of mast cells in the unchallenged airway mucosa and sputum of asthmatic subjects (6, 9). Mast cells residing in the airway may be critical for their immediate response to antigen. Basophils, on the other hand, are circulating granulocytes that migrate to sites of inflammation in response to a stimulus. The presence of basophils in unchallenged airways of asthmatic individuals may be a response to low levels of airway inflammation. That increased numbers of sputum eosinophils are also present in subjects with mild atopic asthma as compared with normal controls (22) suggests that a mechanism may exist for recruiting these granulocytes to the airways in the absence of obvious allergic stimuli.

We found the kinetics of mast cell and basophil accumulation in sputum to be different. Peak basophil levels occurred at 7 h after allergen challenge and began to fall by 24 h after challenge, in contrast to mast cells, which were present at even higher levels at 24 h than at 7 h. The allergen-induced sputum basophilia measured in our study with a specific basophil marker was of a greater magnitude than the 20- to 100-fold increase reported with morphologic and functional criteria for basophil quantification in samples of bronchoalveolar lavage fluid (BALF) (8). Morphologic criteria may underestimate basophil numbers. We observed some sputum cells that we identified immunocytochemically as basophils through their having the size and granularity of basophils, but segmented nuclei were not apparent. Cells with this appearance have been previously observed in BALF (8).

Cells immunopositive for tryptase had the morphologic appearance of mast cells, and were considered to be mast cells on the basis of the specificity of the alkaline phosphatase-conjugated G3 antitryptase antibody for mast cell tryptase. In contrast to sputum basophils, sputum mast cells reach maximum levels at 24 h after allergen inhalation. There are conflicting reports on levels of mast cells in the airways of DR asthmatic subjects after allergen challenge, ranging from an overall increase to an overall decrease. The number of mast cells measured in BALF has been reported to decrease at 6 h (23), with no change reported in bronchial mucosal mast cells at 4 h (4). Mast cells have been reported to increase in the bronchial mucosa at 18 h (24) and 24 h (4) after allergen challenge. Similar discrepancies have been noted in studies of nasal mucosa, in which allergen-induced mast cell degranulation has been reported to cause gross underestimation of mast cells enumerated on the basis of metachromatic staining (25). This problem was overcome in the present study through the use of antibodies to mast cell tryptase, which bind to mast cells despite maximal degranulation (9). In addition, the 2D7 basophil antibody overcomes the problem of degranulation in basophils; in skin, the late phase response to allergen is associated with 2D7-positive cells that are not identified by metachromatic staining (26). Underestimation of mast cells and/or basophils by metachromatic staining was evident in the present study, since the number of mast cells plus basophils stained with specific immunologic markers was consistently greater than the number of metachromatic cells stained with toluidine blue (Figure 2).

The allergen-induced increased number of basophils in airway sputum exceeds that of mast cells. Basophils were previously suggested to represent the majority of IgE-bearing cells in BALF during the late asthmatic response (8). We found that basophils increased by approximately 200-fold from baseline values at 7 h, as compared with mast cells, which increased by approximately fourfold. The number of basophils also exceeded that of mast cells at 24 h after challenge, when basophils increased by approximately 100-fold from baseline, as compared with an eightfold increase in the number of mast cells. It may not be relevant, however, to compare the number of sputum mast cells and basophils appearing in airway secretions after allergen challenge, since basophils are granulocytes that migrate through the tissue into airway luminal secretions independently of structural changes in the airway, whereas mast cells residing in these tissues may appear in airway secretions mainly as a result of epithelial shedding.

We found that basophil numbers were increased in airway secretions after allergen inhalation, and that allergen-induced levels of basophils were significantly higher in asthmatic DRs than in asthmatic ERs. Basophils may be important for development of the allergen-induced late asthmatic response. Airway allergen challenge results in increased levels of both histamine and tryptase, probably due to mast cell activation, whereas levels only of histamine are increased 48 h later, during the late phase, as is consistent with basophil activation in the airway (27, 28). Histamine levels also remain elevated in skin chamber fluids during the late-phase response to allergen, whereas tryptase levels return to baseline values (29). The number of sputum basophils that we measured 24 h after allergen inhalation was weakly correlated with allergen-induced AHR to methacholine and with the late asthmatic response in our study, and this and the earlier finding that histamine release by peripheral blood basophils correlates with the late-phase airway response (30) suggest that basophils may in part contribute to these physiologic changes in the airways. Koshino and coworkers have reported an inverse relationship between airway responsiveness to acetylcholine and basophil numbers in bronchial biopsy specimens from asthmatic subjects (9). Numbers of allergen-induced metachromatic cells in the airway lumen, which we now know are predominantly basophils, have been reported to remain above baseline values for up to 7 d after challenge, in association with prolonged AHR to methacholine (17), supporting the idea that these cells may contribute to physiologic changes in the airway. This hypothesis is further supported by the finding that histamine-releasing factor, a cytokine present in late-phase airway fluids (31), causes basophil mediator release correlating with the severity of the late-phase nasal response (32). This is a potential mechanism by which basophils may become activated and alter airway function long after initiation of the allergic response.

Mast cells degranulate during the allergen-induced early response (4), releasing mediators such as histamine and cysteinyl leukotrienes, which are known to cause bronchoconstriction (27, 28). We found increased levels of sputum mast cells 24 h after allergen inhalation in DR but not ER asthmatic subjects, a finding previously reported in samples of bronchial mucosa (4). In addition, we and others (4) have shown that mast cell numbers measured at 24 h after allergen challenge correlate weakly but significantly with the late asthmatic response (r = 0.53, p = 0.02). Why numbers of mast cells measured in sputum at 24 h after allergen inhalation relate to a physiologic event occurring 18 h earlier is unclear, but may be partly explained by the lag time between activation of mast cells in the airway wall and appearance of the activated mast cells in airway secretions.

The late phase asthmatic response is associated with airway inflammation (17). DR asthmatic subjects have higher numbers of airway eosinophils during the allergen-induced late response than do isolated ERs (33), and these numbers can remain above baseline for 1 to 2 wk (17). We did not observe a difference between ERs and DRs in the number of allergen-induced sputum eosinophils, probably because the sample size of only five subjects in our ER group was too small to permit achieving statistical significance. The small number of subjects in the ER group was a limitation to our study. It is surprising, however, that we were able to detect differences between DRs and ERs in the allergen-induced basophilic response, suggesting that basophil number may be a more sensitive marker for distinguishing isolated ERs from asthma patients who go on to develop a late asthmatic response. Although basophils were present at much lower levels (< 2%) after allergen challenge than were eosinophils (range: 2 to 60%), the large shift from baseline suggests that basophils also play a role in the airway response to allergen.

We have previously demonstrated higher allergen-induced numbers of eosinophils in the airways of asthmatic DRs than in those of ERs (33). We now present data showing that basophil and mast cell numbers are also increased in the airways of individuals who develop a late asthmatic response after allergen inhalation, and are present at higher levels in DRs than in ERs. These findings will enhance understanding of the pathogenesis of allergic asthma and provide a rationale for choosing specific therapeutic agents for treating allergic asthma.