INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Asthma is a disease characterized by bronchoconstriction, airway hyperresponsiveness, and airway inflammation. Inhalation of allergen by sensitized subjects is an important cause of asthma, and it is characterized by biphasic changes in airway physiology known as the early- and late-phase asthmatic responses. Late asthmatic responses (LAR) are associated with transient increases in airway hyperresponsiveness (1), usually lasting several days, and increases in the numbers of activated eosinophils and metachromatic cells in the airways (2).

The predominant cell infiltrating the airways during the late response is the eosinophil, which is selectively increased in sputum (2, 3), and bronchoalveolar lavage fluid (BAL) (4), in association with the late response. Eosinophils are also in an activated state in asthma, as indicated by elevated levels of eosinophil cationic protein (ECP) in BAL fluid (5), and enhanced immunostaining with the EG2 monoclonal antibody, which recognizes the cleaved and activated form of ECP (2).

We have previously provided evidence that increases in inflammatory cell progenitors, which contribute to disease through the continued production of inflammatory effector cells, is an important aspect of allergic inflammatory responses (6, 7). Higher numbers of both circulating eosinophil/basophil colony-forming units (Eo/B-CFU) and CD34⁺ hemopoietic progenitor cells are demonstrable in the blood of atopic subjects compared with normal subjects (6, 8), and there are significantly higher numbers of progenitors in the bloodstream 24 h after allergen inhalation in atopic asthmatic subjects (9). In addition, the numbers of both Eo/B-CFU and CD34⁺ progenitors expressing the -subunit of the IL-5-receptor (IL-5R⁺) in the bone marrow of asthmatic subjects are preferentially increased 24 h after allergen inhalation (10, 11).

Inhaled glucocorticosteroids are known to improve airway hyperresponsiveness in asthmatics (12, 13), to inhibit both allergen-induced late responses and airway hyperresponsiveness (14), and to attenuate allergen-induced increases in blood eosinophils and sputum total and activated eosinophils (2). Inhaled budesonide has been shown to attenuate allergen-induced increases in bone marrow granulocyte progenitors (GM-CFU) in dogs with allergen-induced airway hyperresponsiveness and airway inflammation (17). We therefore postulated that inhaled budesonide would, as part of its activity in attenuating allergen-induced airway inflammation in allergic asthmatic subjects, also inhibit allergen-induced increases in bone marrow progenitors. The purpose of this study was to determine whether treatment with inhaled budesonide, administered for 8 d, could alter baseline or attenuate allergen-induced increases in Eo/B-CFU, total CD34⁺ hemopoietic progenitor cells, and CD34⁺IL-5R⁺ cells, as well as attenuating physiologic parameters and airway and blood eosinophilia.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects

Nine subjects with mild asthma and a previously documented early and late asthmatic response after allergen inhalation were studied (Table 1). The study was approved by the Research Advisory Group at McMaster University Medical Centre, and all subjects provided their written informed consent prior to entering the study. Subjects were atopic, as indicated by one or more positive wheal-and-flare responses to skin prick tests. All subjects were nonsmokers and none had experienced a respiratory infection during the 4 wk prior to the study. Asthmatic subjects were stable at the time of study, requiring only intermittent use of inhaled ₂-agonists with baseline FEV₁ values > 70% predicted.

Study Design

The study was carried out using a double-blind, placebo-controlled, randomized, crossover design. Subjects completed two treatment periods with either inhaled budesonide (400 µg/d) or identical placebo for 8 d. Each treatment period consisted of four visits to the laboratory. A baseline measurement of the provocative concentration of methacholine causing a 20% fall in FEV₁ (PC₂₀) was determined prior to the study. Baseline measurements of FEV₁, blood, and induced sputum differential and total cell counts were determined before treatment and on Day 6 of treatment with budesonide or placebo. Allergen challenges were performed on the morning of Day 7, and blood and sputum samples were taken at 24 h postchallenge on Day 8. An additional sputum measurement was performed at 7 h after allergen challenge. Bone marrow aspirates were performed immediately prior to allergen inhalation and at 24 h postchallenge. Each treatment period was separated by a washout period of at least 4 wk.

Methacholine Inhalation Challenge

Methacholine inhalation was performed as described in Reference 18. Subjects inhaled normal saline and then doubling concentrations of methacholine phosphate from a Wright nebulizer for 2 min. Increasing concentrations of methacholine were administered until the FEV₁ decreased by > 20% of the baseline value. Results were expressed as the provocative concentration (mg/ml) causing a 20% decrease in FEV₁ (PC₂₀).

Allergen Inhalation Challenge

Allergen inhalation challenge was performed as described by O'Byrne and colleagues (19). The concentration of allergen extract for inhalation was determined from a formula described by Cockcroft and colleagues (15), using results from the skin test and the methacholine PC₂₀. The starting concentration of allergen was chosen to be three doubling doses below that predicted to cause a 20% fall in FEV₁. Doubling concentrations of allergen were inhaled at 10-min intervals until a decrease of 15% or more occurred in the FEV₁ from baseline. Measurements of FEV₁ were performed at 10, 20, 30, 45, 60, 90, and 180 min, and then every hour for 7 h after the final inhalation. The allergen-induced early response was determined as the maximal decrease in FEV₁ between 0 and 2 h after allergen and the late response as the maximal decrease between 3 and 7 h after allergen inhalation.

Sputum Analysis

Sputum was induced and processed according to the method of Popov and colleagues (20). Subjects inhaled 3, 4, then 5% saline for 10 min each until an adequate sample was obtained or if the FEV₁ dropped 20% from baseline. Cell plugs were selected from the sample and processed using 0.1% dithiothreitol (Sputolysin; Calbiochem-Behring, San Diego, CA) and Dulbecco's phosphate-buffered saline (GIBCO, Grand Island, NY). The total number of cells obtained was recorded and expressed as absolute counts (10⁶ cells/ml). Cytospins were prepared on glass slides and differential counts were performed in a blinded fashion on slides stained with Diff-Quik (American Scientific Products, McGaw Park, IL). Mean eosinophil counts from duplicate slides were obtained (400 cells counted per slide) and expressed as absolute counts (10⁴ cells/ml).

Blood Samples

Venous blood samples were collected in ethylenediaminetetraacetic acid (EDTA)-treated tubes for total and differential WBC. Total cell counts were performed using a Neubauer hemocytometer, and differential cell counts were made from blood smears stained by Diff-Quik. Differential cell counts were performed by one investigator in a blinded fashion, and the mean of two slides was obtained (300 cells counted per slide). Eosinophils were classified using standard morphologic criteria. Results were expressed as absolute eosinophil counts (10⁴ cells/ml).

Bone Marrow Aspirate and Culture

Bone marrow aspirates were obtained from the iliac crest using a bone marrow aspiration needle (16 × 2"; Sherwood Medical, St. Louis, MO). Three milliliters of bone marrow were aspirated into a 10-ml syringe containing 1 ml sterile heparin (1,000 U/ml) (Leo Laboratories, Ajax, ON, Canada) and semisolid methylcellulose cultures of low density nonadherent mononuclear cells (NAMC) were performed. Briefly, heparinized bone marrow was diluted to 50 ml with McCoy's 5A medium (GIBCO) and separated over 65% percoll (Pharmacia, Uppsala, Sweden). The interface mononuclear-rich cell fraction was washed in McCoy's 5A medium and then incubated in McCoy's 5A medium supplemented with 15% fetal calf serum (FCS) (GIBCO), 1% penicillin/streptomycin (GIBCO) and 5 × 10⁵ M 2-mercaptoethanol (final concentration) (Sigma Chemicals, St. Louis, MO) for 2 h in plastic flasks at 37° C and 5% CO₂. Nonadherent mononuclear cell populations (NAMC; containing progenitor cells and lymphocytes) were then cultured (2.5 × 10⁵ cells per 35 × 10 mm tissue culture dish; Falcon Plastics, Oxnard, CA) in duplicate in supplemented Iscove's modified Dulbecco's medium (GIBCO) with 1% penicillin/streptomycin and 5 × 10⁵ M 2-mercaptoethanol (final concentration), 0.9% methylcellulose (Sigma), and 20% FCS, either alone or in the presence of one of the following growth factors: recombinant human IL-3 (10 or 1 ng/ml; Pharmingen, San Diego, CA), recombinant human GM-CSF (10 or 1 ng/ml; Pharmingen) recombinant human IL-5 (1 or 0.1 ng/ml; Pharmingen), or a combination of all three. Cultures were incubated for 14 d at 37° C and 5% CO₂ after which colonies were identified as Eo/B-CFU according to previously described criteria (21) and expressed as Eo/B-CFU per 2.5 × 10⁵ NAMC plated.

Immunofluorescence Staining

Bone-marrow-derived low density NAMC (1 × 10⁶/tube), isolated as described above, were resuspended in PBS plus 0.1% sodium azide (BDH Inc., Toronto, ON, Canada) and incubated for 45 min at 4° C with saturating amounts (determined in preliminary studies) of biotin-conjugated monoclonal antibodies directed against the -subunit of either IL-3R (IL-3R; 7G2), IL-5R (IL-5R; SP491), GM-CSF (GM-CSFR; 6E10), the -common subunit (c; 8E4), or IgG₁ isotype control antibody. These non-neutralizing monoclonal antibodies were all kind gifts from Dr. A. Lopez (Institute of Medical and Veterinary Science, Adelaide, South Australia) except for SP491, which was supplied by Schering Plough Research Institute (Kenilworth, NJ). The cells were then washed and stained with streptavidin-conjugated peridinin chlorophyll protein (PerCp) (Becton Dickinson Canada, Mississauga, ON, Canada), together with saturating concentrations of fluorescein isothiocyanate (FITC)-conjugated IgG₁ CD45 antibody (anti-HLE1) and phycoerythrin (PE)-conjugated IgG₁ CD34 antibody (HPCA-2), or PE-linked IgG₁ isotype control (Becton Dickinson Canada) for 30 min at 4° C. Lysis buffer (Becton Dickinson Canada) was then added to the cells and incubated for 5 min after which the cells were washed twice with PBS plus 0.1% sodium azide and finally fixed in 500 µl of PBS plus 1% paraformaldehyde (BDH Inc.). The cells were refrigerated until ready for analysis.

Flow Cytometry and Gating Strategy

Cells were analyzed using a FACScan flow cytometer equipped with an argon ion laser (Becton Dickinson Instrument Systems, San Diego, CA). Five data parameters were acquired and stored in list mode files: linear forward light scatter (FSC), linear side-angle light scatter (SSC), log FITC, log PE, and log PerCp fluorescence; each measurement contained 50,000 events. Compensation settings were established using CalBrite beads (BDIS) and confirmed using NAMC stained with anti-CD34-PE, anti-CD45-FITC or anti-IL-5R-PerCp. Off-line analysis was performed using the PC lysis software as supplied by BDIS. As previously described in detail, we used a multiparameter five sequential-gating strategy to accurately enumerate true bone-marrow-derived CD34⁺ progenitor cells that coexpressed the above-mentioned cytokine receptors (11).

Statistical Analysis

The area under the curve of both the early and late asthmatic responses was compared between treatment groups using Student's t test for paired comparisons. The numbers of CD34⁺ cells and cytokine receptor-positive progenitors were log₁₀-transformed prior to analysis and the summary statistics are expressed as the geometric mean and %SEM. A two-way, repeated-measures analysis of variance (ANOVA) was used to assess the interaction between allergen- induced increases and the effect of treatment on blood eosinophils; on sputum total cell counts and eosinophils; on bone marrow Eo/B-CFU progenitor colonies for each cytokine at each concentration; on absolute CD34⁺ cell numbers and coexpression of each cytokine receptor on CD34⁺ cells; and the effect of treatment with budesonide on baseline measurements of sputum total cell and absolute eosinophil counts, and blood eosinophils (repeated factors: preallergen versus postallergen, placebo versus budesonide) (22). Statistical significance was assumed at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Airway Responses

Inhaled budesonide attenuated both the mean maximal allergen-induced early and late asthmatic responses. The maximal fall in FEV₁ during the early response was 27.9 ± 2.8% after placebo and 14.7 ± 4.5% after budesonide treatment, and the maximal fall in FEV₁ during the late response was 23.4 ± 4.5% after placebo and 4.6 ± 1.6% after budesonide treatment (Figure 1). Budesonide treatment also significantly reduced the area under the curve of both the early (placebo, 26.9 ± 4.2 versus budesonide, 12.5 ± 3.6, p < 0.05) and late (placebo, 47.8 ± 10.6 versus budesonide, 2.8 ± 2.9, p < 0.005) responses (Figure 1).

View larger version (14K): [in this window] [in a new window]   Figure 1.   Allergen-induced bronchoconstrictor responses. Percent change in FEV1 after inhalation of allergen with placebo (open circles) or budesonide (400 µg/d) treatment (closed circles). Both the early (0 to 2 h) (p < 0.05) and the late (3 to 7 h) (p < 0.005) asthmatic responses were significantly reduced with budesonide treatment.

Sputum Eosinophils

Inhaled budesonide significantly reduced the baseline number of sputum eosinophils measured before allergen inhalation, from 11.4 ± 2.4 × 10⁴/ml before budesonide treatment to 2.9 ± 1.1 × 10⁴/ml after treatment (p < 0.005) (Figure 2). Baseline measurements of sputum eosinophils did not change significantly during placebo treatment, being 5.5 ± 1.9 × 10⁴/ml before placebo and 8.7 ± 2.5 × 10⁴/ml after placebo (Figure 2).

View larger version (23K): [in this window] [in a new window]   Figure 2.   Allergen-induced airway inflammation and blood eosinophilia. Sputum (top panel ) and blood (bottom panel ) eosinophil values before and after allergen inhalation following treatment with placebo (open bars) or budesonide (400 µg/d) (hatched bars). Baseline measurements of sputum (p < 0.005) but not blood eosinophils were affected by treatment with budesonide. Compared with the post-treatment, preallergen baseline levels, a significant increase in both sputum eosinophils at 7 h (**p < 0.01) and at 24 h post-allergen (***p < 0.005) and blood eosinophils at 24 h post-allergen (***p < 0.005) was observed. These allergen-induced increases were significantly reduced by treatment with inhaled budesonide (400 µg/d) (p < 0.05). ND = not done.

Allergen inhalation caused a significant increase in sputum eosinophils during treatment with placebo both at 7 h (p < 0.01) and at 24 h (p < 0.005) postallergen, being 64.1 ± 17.4 × 10⁴/ml at 7 h and 97.6 ± 28.7 × 10⁴/ml at 24 h (Figure 2). There was a trend for sputum eosinophils to increase 24 h after allergen inhalation during budesonide treatment, being 41.0 ± 13.0 × 10⁴/ml at 24 h, but this increase did not achieve significance (p = 0.076). Inhaled budesonide significantly attenuated the allergen-induced increases in sputum eosinophils to 10.4 ± 3.1 × 10⁴/ml at 7 h and 41.0 ± 13.0 × 10⁴/ml at 24 h (p < 0.05) (Figure 2).

Blood Eosinophils

The baseline number of blood eosinophils was unaffected by treatment with either placebo or budesonide treatment (Figure 2). However, allergen inhalation caused a significant increase in blood eosinophils during treatment with placebo, from 40.1 ± 4.5 × 10⁴/ml before allergen to 71.9 ± 9.8 × 10⁴/ml 24 h after allergen (p < 0.005). Inhaled budesonide significantly attenuated this increase (p < 0.05), from 36.2 ± 6.1 × 10⁴/ml before and 43.1 ± 6.4 × 10⁴/ml after allergen (Figure 2).

Bone Marrow-derived Eo/B Progenitors

Treatment with inhaled budesonide significantly reduced the baseline numbers of bone marrow Eo/B-CFU when grown in the presence of IL-5 at both 1 ng/ml (p < 0.05) and 0.1 ng/ml (p < 0.01) (Figure 3), but not when grown in the presence of IL-3 or GM-CSF (Table 2). Likewise, budesonide treatment suppressed the total baseline numbers of bone marrow CD34⁺ progenitor cells (p < 0.05) (Figure 4) and the numbers of CD34⁺IL-3R⁺ progenitors (p < 0.01) (Figure 5).

View larger version (25K): [in this window] [in a new window]   Figure 3.   IL-5-induced changes in bone marrow Eo/B-CFU. Bone marrow Eo/B-CFU per 2.5 × 105 NAMC were incubated with IL-5 at 1 ng/ml or 0.1 ng/ml, before (open bars) and 24 h after (hatched bars) allergen inhalation after treatment with placebo or budesonide (400 µg/d). Significant allergen-induced increases in Eo/B-CFU were seen after allergen inhalation when bone marrow cells were incubated with IL-5 at 1 ng/ml (*p < 0.05) but not at 0.1 ng/ml. These allergen-induced changes were not affected by treatment with budesonide. However, budesonide treatment caused a significant overall suppression of IL-5-responsive Eo/B-CFU numbers compared with placebo numbers at both 1 ng/ml (p < 0.05) and 0.1 ng/ml (p < 0.01).

View larger version (22K): [in this window] [in a new window]   Figure 4.   Measurement by FACS analysis of total bone marrow CD34+ progenitors. After either placebo or budesonide (400 µg/d) pretreatment bone marrow aspirates from (n = 9) dual-responder asthmatics were taken before (open bars) and 24 h after (hatched bars) allergen challenge. Because of constraints in bone marrow sample sizes, estimation of total numbers of CD34+ progenitors was performed in only n = 8 subjects. No significant increase in CD34+ cell numbers was detected after allergen challenge in both treatment groups. However, treatment with budesonide caused a significant overall attenuation in bone marrow CD34+ cell numbers compared with that in the placebo pretreatment group (p < 0.05).

View larger version (30K): [in this window] [in a new window]   Figure 5.   Total bone marrow CD34+ progenitors coexpressing cytokine receptor subunits. Bone marrow aspirates from asthmatics were taken before (open bars) and 24 h after (hatched bars) allergen challenge and IL-3R or IL-5R expression on CD34+ progenitor cell numbers was enumerated. After allergen challenge there was a significant increase in CD34+IL-5R+ (**p < 0.01) but not CD34+IL-3R+ cell numbers in both treatment groups. Budesonide treatment, however, caused a significant overall attenuation in CD34+IL-3R+ cell numbers (p < 0.01).

Inhaled allergen significantly increased the number of bone marrow Eo/B-CFU grown in the presence of IL-3 alone (10 ng/ml, p < 0.05), GM-CSF alone (10 ng/ml, p < 0.05), or IL-5 alone (1 ng/ml, p < 0.05) (Figure 3), or a combination of all three cytokines (p < 0.01) after both placebo and budesonide treatment (Table 2). In contrast to the effects on the baseline IL-5-stimulated Eo/B-CFU, treatment with inhaled budesonide did not significantly attenuate the allergen-induced increases in Eo/B-CFU in response to any cytokine (Table 2).

Inhaled allergen significantly increased the number of CD34⁺IL-5R⁺ progenitors, and again this effect was not attenuated by treatment with inhaled budesonide (Figure 5). After inhaled allergen during placebo treatment, the number of CD34⁺IL-5R⁺ progenitors increased from 45 cells/0.25 × 10⁶ WBC (%SEM, 24) to 112 cells/0.25 × 10⁶ WBC (%SEM, 33) (p < 0.01); during budesonide treatment these numbers increased from 33 cells/0.25 × 10⁶ WBC (%SEM, 11) to 71 cells/ 0.25 × 10⁶ WBC (%SEM, 18) (p < 0.01) (Figure 5). In contrast, no significant allergen-induced increase in the numbers of bone-marrow-derived CD34⁺IL-3R⁺ progenitors was detected during either placebo or budesonide treatment (Figure 5).

There was a trend for increases in the number of both CD34⁺GM-R⁺ and CD34⁺c⁺ progenitors after allergen inhalation in the placebo group only, but these changes were not significant (Figure 5 and Table 3).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study has demonstrated that treatment with a low daily dose of the inhaled corticosteroid budesonide (400 µg/d) for 1 wk, caused a selective suppression of baseline numbers of IL-5-responsive eosinophil/basophil progenitors and in the absolute numbers of CD34⁺ and CD34⁺IL-3R⁺ hemopoietic progenitors in the bone marrow of allergic asthmatic subjects. Treatment with the inhaled corticosteroid also attenuated allergen-induced early and late asthmatic responses, as well as allergen-induced increases in blood and airway eosinophils. In addition, this study has confirmed our previous findings of allergen-induced increases in bone marrow Eo/B-CFU (10) and IL-5R⁺ expression on CD34⁺ progenitor cells (11). Inhaled budesonide, however, had no effect on the allergen-induced increase in this progenitor subpopulation in the bone marrow, despite the fact that the treatment regimen did attenuate both allergen-induced blood and airway eosinophilia.

The ability of inhaled budesonide treatment prior to allergen inhalation to attenuate the magnitude of allergen-induced early and late asthmatic responses and increases in both sputum and blood eosinophils at 24 h has been previously described (2) and was an expected result. However, the lack of effect of inhaled budesonide on the allergen-induced increases in IL-5-responsive Eo/B CFU and on CD34⁺IL-5R⁺ progenitors was unexpected. This is because of our previous observations of the ability of inhaled budesonide to abolish inhaled allergen-induced increases in GM-CFU in allergic dogs (17), and to attenuate the number of circulating Eo/B-CFU in asthmatic subjects with an acute exacerbation (23). However, the dose of budesonide used in the canine study was approximately 15 times greater than that used in this study, which may explain, in part, the lack of effect of budesonide on allergen-induced increases in bone marrow eosinophil progenitors. In this study, the dose of inhaled budesonide that is effective in preventing the allergen-induced increases in both blood and airway eosinophils is not sufficient to prevent the bone marrow from increasing its production of eosinophil progenitors. However, pretreatment with inhaled budesonide did have a significant suppressive effect on the baseline number of IL-5-responsive Eo/B-CFU, the absolute number of CD34⁺ progenitors, and the absolute number of CD34⁺IL-3R⁺ progenitors demonstrating that these doses of inhaled budesonide have a systemic effect in inhibiting the turnover of specific subpopulations of bone marrow progenitor cells. It is not possible from this study to decide whether this effect on bone marrow eosinophil progenitors occurs because of inhibition of signals from the airways or a direct effect on the bone marrow, or a combination of both. It would be of interest to look at the effect of inhaled budesonide on bone marrow eosinophil progenitors in normal subjects since these subjects would likely not release a signal from the airways, and any demonstrated suppression in baseline progenitor numbers would be as a result of a direct systemic effect on the bone marrow.

It is possible that IL-3, GM-CSF, IL-5, or a combination of all three may be involved in the upregulation of the IL-5 receptor on progenitor cells, and that the level of budesonide present in the bone marrow may be sufficient to inhibit the production of these cytokines in the steady state, resulting in a lower level of IL-5-responsive cells. However, after a stimulus such as inhaled allergen, the production of these cytokines may increase to an extent that cannot be overridden by the presence of budesonide, resulting in the allergen-induced increases observed in this study. IL-3 responsiveness usually appears earlier during the differentiation process, and on more primitive hemopoietic progenitor cells than IL-5 responsiveness (24); because budesonide had a suppressive effect on the baseline levels of CD34⁺IL-3R⁺, it is possible that higher inhaled doses of budesonide or a longer treatment period may result in the inhibition of allergen-induced increases of more lineage-committed progenitors such as CD34⁺IL-5R⁺ cells. The differences in the effect of inhaled budesonide on baseline CD34⁺IL-5R⁺ cells compared with IL-5-responsive Eo/ B-CFU may potentially be explained by differences in the cell populations assayed by these two methods: the group of progenitor cells responding to IL-5 in the colony assay is very heterogenous, whereas the CD34⁺IL-5R⁺ cell is a relatively homogenous, primitive eosinophil progenitor bearing CD34, a surface receptor that is progressively lost as the progenitor matures, and not present on the colony-forming cell. It is the latter that is likely more sensitive to budesonide.

Another surprising result obtained in this study was the complete attenuation by inhaled budesonide, of the allergen-induced blood eosinophilia measured 24 h after allergen at a time when the bone marrow eosinophil progenitors were significantly increased. This suggests that inhaled budesonide may prevent maturation of the eosinophil progenitors in the bone marrow, or may prevent release of the mature cells from the bone marrow. However, there was a trend for sputum eosinophils to increase at 24 h after allergen inhalation in the budesonide treatment arm, suggesting that trafficking of some eosinophils is occurring into the airways from the bloodstream. If this is the case, it could account for the lack of increase in blood eosinophils at 24 h after challenge, and it may be that eosinophils are being released from the bone marrow into the bloodstream, even though the results suggest that they are being inhibited by budesonide.

Increases in bone marrow Eo/B-CFU were demonstrated after allergen challenge, when optimal concentrations of IL-3, IL-5, and GM-CSF either alone or in combination were used, in vitro (ex vivo). These findings suggest that there is an increase in the numbers of lineage-committed progenitor cells that are able to respond to these "eosinopoietic" cytokines after allergen challenge in asthmatic subjects. Molecular cloning of cytokine receptors has revealed that IL-3R, IL-5R, and GM-R are uniquely composed of heterodimeric structures consisting of a distinct -subunit, that binds the cognate cytokine with low affinity, and a common, shared, -subunit, which, although failing to bind the ligand itself, forms high affinity cytokine binding sites in association with the -subunit (25, 26). Therefore, the current findings showing a preferential increase in the proportion of bone marrow CD34⁺ cells expressing IL-5R-subunit in response to allergen inhalation may suggest an increase in the ability of these progenitor cells to respond more readily to IL-5, and thus differentiate terminally into mature eosinophils and basophils.

Previous studies from our laboratory in a canine model of allergen-induced airway hyperresponsiveness and airway inflammation have demonstrated that allergen inhalation increases bone marrow GM-CFU production (17) and that this is due to an as yet unidentified hemopoietic activity released into the bloodstream after allergen inhalation that stimulates the bone marrow (27). The present study raises the possibility that, after allergen inhalation by atopic asthmatics, IL-5 may represent one such hemopoietic signal, with a significant effect on IL-5-responsive progenitors induced in the bone marrow; this could then lead to increased production of eosinophils and promote eosinophilic inflammation of the airways. Alternatively, a recent study by Minshall and colleagues (28) showed that after allergen inhalation, T-lymphocytes in the bone marrow were a significant source of IL-5 in sensitized mice, suggesting that events occurring in the airways may result in local bone marrow production of IL-5, leading to increased production of eosinophils. In support of a bone marrow source of these signals, differentiating eosinophils themselves are a potent source of GM-CSF and IL-5, especially after allergen challenge (29).

The allergen-induced increase in sputum eosinophils was inhibited, but not completely attenuated, by treatment with inhaled budesonide. A considerable body of evidence now exists suggesting that IL-5 and eotaxin cooperate in mediating a rapid transfer of eosinophils from the bone marrow to the lung in response to allergen challenge (30, 31). Recent findings show that the generation of eotaxin, unlike IL-5, may not be inhibited by budesonide (31), thus preventing the complete ablation of sputum eosinophilia in response to allergen challenge. Studies in both mice and guinea pigs have shown that although eotaxin may play a predominant role in eosinophil recruitment into the lungs, IL-5 is pivotal in triggering the traffic of mature eosinophils from the bone marrow into the peripheral circulation (30). In addition, it is possible that budesonide treatment may inhibit the allergen-induced eosinophilia in the present study through attenuating allergen- induced increases in IL-5 generation.

In summary, this study has demonstrated that inhaled budesonide reduces baseline numbers of bone marrow CD34⁺ progenitors, CD34⁺IL-3R⁺ progenitors, and IL-5-responsive Eo/B-CFU in allergic asthmatic subjects, as well as allergen-induced increases in blood and airway eosinophils. However, inhaled budesonide did not significantly attenuate allergen- induced increases in bone marrow IL-5-stimulated Eo/B-CFU or CD34⁺IL-5R⁺ progenitors. This indicates a systemic anti-inflammatory property of even low doses of inhaled budesonide on bone marrow responsiveness and that, during inhaled budesonide treatment, allergen-induced increases in subpopulations of early eosinophil/basophil progenitors may be blocked from fully differentiating into mature cells, or that the mature cells are not released from the bone marrow.