This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shen, H. Articles by Inman, M. D. Search for Related Content PubMed PubMed Citation Articles by Shen, H. Articles by Inman, M. D.

The Effects of Intranasal Budesonide on Allergen-induced Production of Interleukin-5 and Eotaxin, Airways, Blood, and Bone Marrow Eosinophilia, and Eosinophil Progenitor Expansion in Sensitized Mice

Asthma Research Group, Department of Medicine, McMaster University and St. Joseph's Hospital, Hamilton, Ontario, Canada; and Department of Respiratory Medicine, Second Hospital, Zhejiang University School of Medicine, Hangzhou, China

Correspondence and requests for reprints should be addressed to Dr. Mark D. Inman, Asthma Research Group, Firestone Regional Chest and Allergy Unit, Rm. 113, St. Joseph's Hospital, 500 Charlton Ave. E., Hamilton, Ontario, Canada L8N 4A6. E-mail: inmanma{at}fhs.mcmaster.ca

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
We have previously demonstrated that allergen inhalation induces expansion of bone marrow eosinophil progenitors in sensitized mice and subjects with asthma and that the inhaled corticosteroid, budesonide, reduced baseline but not allergen-induced increase in bone marrow eosinophil/basophil progenitors (EoB-CFU) in subjects with asthma. Here, we evaluated the effects of intranasal budesonide on allergen-induced increases in interleukin (IL)-5 and eotaxin in the airway and peripheral blood, expansion of bone marrow Eo-CFU and eosinophilia in bone marrow, peripheral blood and airway, as well as airway hyperresponsiveness, in ovalbumin (OVA)-sensitized mice. Budesonide treatment attenuated allergen-induced eosinophilia in bone marrow, peripheral blood, and airways as well as allergen-induced increases in bone marrow eosinophil progenitors but not allergen-induced increases in IL-5 or eotaxin 12 h following the second of two daily exposures to allergen; at later time points treatment was associated with attenuation of IL-5, eosinophilia, Eo-CFU, and airway hyperresponsiveness. These results suggest that a component of the mechanism by which corticosteroid treatment attenuates allergen-induced airway inflammation is through suppression of bone marrow eosinophilopoiesis, and that this is likely not mediated simply through the blocking of IL-5 production at the airway.

Key Words: corticosteroids • hemopoiesis • IL-5 • eosinophils • asthma

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Asthma is a chronic inflammatory disorder of the airway in which many cells and cellular elements play a role; in particular eosinophils, and T lymphocytes and mast cells are associated with variable airflow obstruction and airway hyperresponsiveness (AHR). The degree of accumulation and activation of eosinophils in the airways correlates with clinical severity of asthma, suggesting a central role for eosinophils in asthma pathogenesis (1–3). The development of airways eosinophilia involves eosinopoiesis in the bone marrow and release into circulating blood, as well as recruitment of eosinophils from the blood into the airways (4, 5).

Bone marrow and peripheral blood eosinophilia has been documented following airway allergen challenge in mice (6) and guinea pigs (7), while increases in bone marrow and peripheral blood eosinophil progenitors have been documented following exposure of allergen to airways of subjects with atopic asthma (8–10) and sensitized mice (11, 12). These studies have demonstrated that bone marrow and peripheral blood eosinophils and their progenitors are significantly increased in association with airway eosinophilia after allergen challenge or with acute exacerbation of asthma.

Recruitment of eosinophils is regulated by cytokines that also support eosinopoiesis such as interleukin (IL)-5, IL-3, and granulocyte–macrophage colony-stimulating factor (GM-CSF) and also by chemokines such as eotaxin (13, 14). There is now evidence to suggest that IL-5 plays a prominent role in eosinophil recruitment, and a unique role in terminal differentiation and maturation of eosinophil lineage-committed progenitors (15, 16). Clinically, it has been demonstrated that there are significant correlations between T cell activation, increased concentration of IL-5 in serum and bronchoalveolar lavage fluids (BALF), and asthma severity (17–19). IL-5 has thus been considered as a potentially important eosinopoietic signal between lung and bone marrow in asthma (4, 5). Eotaxin is a CCR-3 receptor-specific, eosinophil-selective chemokine. The cells known to express CCR-3 are mainly limited to eosinophils in humans, but include neutrophils and macrophages in mice (20–22). Data from animal models suggest that cooperation occurs between eotaxin and IL-5 to allow rapid mobilization of eosinophils from a pool of bone marrow cells followed by recruitment to the airways (23, 24). It has been proposed that eotaxin provides the signal for eosinophil localization to the airways and IL-5 provides the signal for the release of a pool of eosinophils from the bone marrow (4, 14). This distinction is not clear however, as it is known that eotaxin can produce eosinophil efflux from guinea pig bone marrow (24).

Inhaled corticosteroids have been used for the management of asthma since the early 1970s and are currently considered the most effective means of reducing airway inflammation, symptoms, and morbidity in patients with asthma (25, 26). Corticosteroid treatment reduces airway and circulating eosinophilia and IL-5 levels in patients with asthma (18, 27, 28), and attenuates allergen-induced increases in blood and airway eosinophils (29). We have previously provided evidence that treatment with inhaled budesonide did not prevent allergen-induced increases in bone marrow eosinophil/basophil progenitors (EoB-CFU), but did prevent allergen-induced increases in blood and airway eosinophils in asthmatic subjects as well as reduce baseline EoB-CFU (10). Based on these studies, possible sites at which steroids may interfere with the pathogenesis of allergen-induced eosinophilic inflammation include the production of eosinopoietic mediators, expansion of the eosinophil progenitor cell population in the bone marrow, terminal differentiation of eosinophils in bone marrow, release of mature eosinophils into circulation, and recruitment of eosinophils into the airway.

We have hypothesized in the past that prevention of allergen-induced airway inflammation as a result of corticosteroid treatment is through the prevention of hematopoietic cytokine release from the airway. To test this hypothesis, we have measured the effects of intranasal treatment of sensitized mice with the corticosteroid budesonide on the time course of allergen-induced changes in eosinopoietic and chemotactic mediator levels (IL-5 and eotaxin) in bronchoalveolar lavage, serum, and bone marrow; eosinophil progenitors (Eo-CFU) in bone marrow; mature eosinophils in the bone marrow, blood, and airways; as well as the development of allergen-induced airway hyperresponsiveness.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Animals
Female BALB/c mice (10–12 wk of age) were purchased (Harlan Sprague Dawley Inc., Indianapolis, IN) and housed in specific pathogen free conditions for 1 wk prior to experimental use. All procedures were reviewed and approved by the Animal Research Ethics Board at McMaster University.

Study Design
Mice were studied in three groups: unsensitized, unchallenged, untreated (Sham/Sham); sensitized, allergen challenged, diluent treated (OVA/OVA); and sensitized, allergen challenged, budesonide treated (OVA/BUD).

Allergen Sensitization and Challenge Protocol
The ovalbumin sensitization and challenge protocol (OVA/OVA) was similar to that described previously (12). Mice were subjected to intraperitoneal injections on Days 1 and 11 and intranasal instillation on Days 11, 19, and 20. Days 1 and 11 were considered to be sensitization and Days 19 and 20 to be challenge. Intraperitoneal ovalbumin injections involved precipitating 10% aluminum potassium sulfate with 0.05% ovalbumin, adjusting to pH 6.5, centrifugation then resuspension of the pellet, followed by a 200 µl (100 µg OVA) intraperitoneal injection. Intranasal ovalbumin involved dissolving 4 mg ovalbumin in 1 ml sterile normal saline, followed by a 25 µl (100 µg OVA) intranasal instillation into anesthetized mice using a sterile pipette. Control mice were sensitized and challenged with diluent (Sham/Sham).

Budesonide Treatment
Micronized dry powder of budesonide (Astra Zeneca, Lund, Sweden) was dissolved in 70% ethanol and diluted in sterile normal saline on the experimental day. Budesonide was given by intranasal administration (25 µl each time) on Days 17, 18, 19, and 20 (Figure 1). On Days 19 and 20, budesonide was given 1 h before intranasal OVA challenge. To determine the optimal dose of budesonide, which is the lowest effective dose to inhibit airway inflammation and AHR, three groups of 10 sensitized mice were treated for 4 d with 350 µg/kg, 35 µg/kg, or 3.5 µg/kg and then challenged with OVA according to the protocol in Figure 1. The results showed that 350 µg/kg of budesonide attenuated BAL eosinophilia by 72% and completely blocked the development of AHR, but other doses had nonsignificant effects (Figure 2). Thus, the dose of budesonide used in subsequent experiments was 350 µg/kg. Mice subjected to allergen challenge without budesonide treatment (OVA/OVA) were treated with vehicle (ethanol in saline) according to the treatment schedule in Figure 1.

View larger version (8K): [in this window] [in a new window]   Figure 1. Timing of events within each treatment group. i.n., intranasal; i.p., intraperitoneal; OVA, ovalbumin; BUD, budesonide.

View larger version (20K): [in this window] [in a new window]   Figure 2. The effects of different doses of budesonide on BAL eosinophilia, airway responsiveness 24 h after airway allergen challenge (M ± SEM). Mouse numbers per group were 5–10. Eosinophil numbers in BALF and airway responsiveness were significantly increased in OVA/OVA mice (p < 0.001 and 0.01, respectively). Budesonide (350 µg/ml) attenuated BAL eosinophilia by 72% and completely blocked the development of airway hyperresponsiveness. Other doses had no effect. *p < 0.05 OVA/OVA compared with Sham/Sham mice at the same time point. p < 0.05 OVA/BUD compared with OVA/OVA mice at the same time point.

Outcome Measurements
Outcome measurements including bronchoalveolar lavage, peripheral blood (PB) and bone marrow (BM) eosinophil numbers, and BM eosinophil progenitor numbers were made 12, 24, and 48 h following the final intranasal challenge with allergen or diluent. Fluids collected from BALF, PB, and BM at these times were also assayed for IL-5 and eotaxin levels.

Bronchoalveolar Lavage (BAL)
Under anesthesia with 0.3 ml intraperitoneally of ketamine (20 mg/ml) and xylazine (2.0 mg/ml) in normal saline the chest cavity was opened and the trachea was exposed and cannulated using a blunted 18-gauge needle (n = 8–10 per group). Two injections of 250 µl phosphate-buffered saline (PBS) were injected and withdrawn through the needle. Mice were then sacrificed via cervical dislocation. The BALF was centrifuged for 10 min at 150 x g and 4° C. The supernatants were stored at -20° C for IL-5 and eotaxin assays. The cell pellet was resuspended in PBS and a total cell count performed using a hemocytometer. The cells were then diluted to an approximate concentration of 5 x 10⁵/ml with PBS and cytocentrifuge slides were prepared (Cytospin 3, Shandon Scientific, Sewickley, PA) and stained with Diff-Quik. Cell differential counts were performed based on morphological and histological criteria (400 cells counted). All cell counts were performed by one investigator, blind to the experimental condition. Cells were classified as macrophages, neutrophils, lymphocytes, or eosinophils, based on morphological criteria.

Bone Marrow Eosinophil Numbers
The right femur was isolated, the femoral head and condyles were removed, and the displaceable cells were recovered by flushing the lumen of the femur shaft with 1.5 ml HBSS (containing 30 mM HEPES, 0.25% bovine serum albumin [BSA], and 10 U/ml heparin). The BM suspension was passed through a 20-gauge needle to create a single cell suspension, and total cell counts were made using a hemocytometer. The BM fluid was centrifuged for 10 min at 150 x g and 4° C. The supernatants were stored at -20° C for IL-5 and eotaxin assay. The cell pellet was diluted to an approximate concentration of 1 x 10⁶/ml with PBS and cytocentrifuge slides were prepared and stained with Diff-Quik. Differential counts were performed based on morphological and histological criteria (400 cells counted) to determine the percentage of eosinophils. All cell counts were performed by one investigator, blind to the experimental condition.

Blood Smear and Serum
Blood samples were obtained by cardiac puncture and blood smears were prepared in duplicate. Total white blood cells were counted in heparinized blood using a hemocytometer. Serum was obtained by centrifugation for 10 min at 150 x g and 4° C and stored at -20° C for IL-5, eotaxin, and cortisol assays. Differential cell counts were performed on the blood smears using Diff-Quik stain and cells were classified as neutrophils, lymphocytes, monocytes, or eosinophils, based on morphological and histological criteria.

Bone Marrow Eosinophil Progenitor Responses
Bone marrow colony assays were performed as previously described (12). Following anesthesia and sacrifice by terminal exsanguination via cardiac puncture one femur was removed and freed of soft tissue. BM cells were flushed from the femur using 3 ml McCoys3+ buffer injected through a 25-gauge needle inserted into the lumen of the femur. To break up cell clumps, the suspension was passed through needles of 18, 20, and 22 gauge. Suspended cells were separated by density gradient centrifugation over 65% Percoll for 30 min at 150 x g, and the cells at the interface removed. The mononuclear cells were washed once with McCoys3+, then incubated overnight in plastic flasks at 37° C and 5% CO₂ to remove adherent cells. The nonadherent cells were cultured in microassay 24-well plates (Becton Dickinson, Lincoln Pk, NJ) at a concentration of 7.5 x 10⁴ cells per well. The culture medium was made up of 0.9% methylcellulose (Caledon Lab., Georgetown, Ontario), 30% fetal calf serum (GIBCO BRL, Grand Island, NY), and recombinant mouse IL-5 (R&D Systems Inc., Minneapolis, MN). An optimal concentration of IL-5, 1 ng/ml, was used for growth of eosinophil colony-forming units (Eo-CFU) (12). Following 7 d, colonies (greater than 40 cells) were counted using inverse microscopy and classified using morphological and histological criteria. To confirm the identification of colonies as Eo-CFU, selected samples were removed from the culture wells for viewing under light microscopy after staining with Diff Quik, or for identification using electron microscopy (EM). For EM, selected colonies were pooled in 2% gluteraldehyde containing 0.1 M sodium cacdylate (pH 7.4). After fixation for 2 h at 4° C, the sample was washed in 0.2 M sodium cacdylate (4° C) for 1 h, dehydrated in graded ethanol followed by propylene oxide, and embedded in Spurr's resin. The block was sectioned using a Reichert Ultracut E ultramicrotome and sections were stained for 5 min with uranyl acetate and then for 2 min with lead citrate. Stained sections were examined using a JEOL 1200EX Biosystem electron microscope.

Airway Responsiveness
Airway responsiveness was measured based on the response of total respiratory system resistance (RRS) to increasing intravenous doses of methacholine (MCh) (n = 8–10 per group). RRS was measured using the flow interrupter technique, as modified for use with mice (12, 30). Briefly, mice were anesthetized (Avertin, 240 mg/kg intraperitoneally) and the trachea was exposed and cannulated using a blunted 18-gauge needle. The needle was then attached to a ventilator (RV5, Voltek Enterprises Inc., Toronto, Canada) designed to deliver constant inspiratory flow, despite the disturbances in the respiratory impedance that occur during the methacholine (MCh) challenge. The initial pattern of ventilation was a tidal volume of 0.1 ml/kg delivered over 45 ms, with a 530-ms end-inspiratory pause and a 95-ms period of passive expiration (breathing frequency of 90 breaths/min). Heart rate and oxygen saturation were monitored via infrared pulse oxymetry (Biox 3700, Ohmeda, Boulder, CO), using a standard ear probe, placed over the proximal portion of the mouse's hind limb. After stabilizing the mouse on the ventilator, the internal jugular was cannulated using a 25-gauge needle. Paralysis was achieved using pancuronium (0.03 mg/kg intravenously) to prevent respiratory effort during measurement. The response of RRS was measured following intravenous injections of saline, then 10, 33, 100, and 330 µg/kg of methacholine (ACIC [Can], Brantford, Ontario), each delivered as a 0.2-ml bolus. To establish a constant volume history, mice were subjected to three inspirations to TLC (end-inspiratory pressure of 30 cm H₂O) followed by 60 s of 90 breaths/min ventilation prior to each dose. Upon injection, the ventilatory pattern was changed, so that the time allowed for passive expiration was extended to 1,425 ms, thus reducing the breathing frequency to 30 breaths/min. This change was to prevent dynamic hyperinflation (also termed breath stacking), which was observed during MCh challenge when mice were ventilated with shorter expiratory times. Following the peak in RRS (20–30 s) the breathing pattern was returned to 90 breaths/min. When RRS returned to baseline, the mouse was again inflated three times to TLC and ventilated for 60 s before beginning the next dose. During each MCh dosing, the airway pressure signal was converted to a digital signal (Dash 1642, Metrabyte, Staughton, MA) and recorded at 400 Hz on a PC computer. RRS was calculated as described by Ewart and coworkers (30). Evaluation of airway responsiveness was based on the peak RRS measured in the 30 s following the saline and MCh challenges. An index of airway reactivity was calculated as the slope of the straight line regression between peak RRS and the log₁₀ of the MCh dose, using the data from the 10, 33, and 100 µg/kg doses only.

IL-5 Assay
IL-5 levels in BALF, serum, and BM fluids were assayed by ELISA using a mouse IL-5 kit (PharMingen, San Diego, CA). The sensitivity of detection of the ELISA was 5 pg/ml.

Eotaxin Assay
Eotaxin levels in BALF, serum, and BM fluids were assayed by ELISA using a mouse eotaxin kit (R&D Systems, Minneapolis, MN). The sensitivity of detection of the ELISA kit was 1.9 pg/ml.

Plasma Cortisol
Serum cortisol levels were assayed by ELISA using a kit (IBL, Hamburg, Germany).

Analysis
Comparisons among Sham/Sham, OVA/OVA, and OVA/BUD mice with respect to BAL, peripheral blood, BM cell numbers, BM Eos-CFU numbers, and airway reactivity (slope of the RRS–MCh [log transformed] dose–response curve) were made using analysis of variance (ANOVA). All post hoc comparisons were carried out using Newman–Keuls test for significant effects. All comparisons were two tailed, with critical set at 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
The Effects of Allergen and Budesonide on Airway Hyperresponsiveness
Allergen challenge increased methacholine airway responsiveness measured 24 h following challenge in OVA/OVA mice when compared with Sham/Sham mice. The slope RRS–MCh was 2.92 in Sham/Sham mice and significantly increased to 4.63 in OVA/OVA mice (p < 0.05)(Figure 2). Intranasal budesonide treatment (350 µg/kg) completely prevented the development of allergen-induced airway hyperresponsiveness, the slope RRS–MCh being 2.29 in BUD/OVA mice (p < 0.05) (Figure 2). Two other doses of budesonide tested (3.5 µg/kg and 35 µg/kg) were ineffective at attenuating airway hyperresponsiveness (Figure 2). Interestingly, the lowest dose found to be effective in preventing AHR was also the lowest dose capable of attenuating allergen-induced BAL eosinophilia (Figure 2). The 350 µg/kg dose was used in all subsequent treatment groups.

The Effects of Budesonide on Baseline Levels of Outcome Measurements
Budesonide treatment (intranasally 350 µg/kg/d) for 4 d of Sham/Sham mice did not affect any outcome variables except for BM Eo-CFU numbers 48 h after the final treatment dose, which were increased from 9.0 to 18.7 colonies per 250 x 10³ nonadherent mononuclear cells (p = 0.03). Serum cortisol levels measured after 4 d of intranasal budesonide treatment (350 µg/kg/d) were 9.70 ± 1.62 ng/ml, which were significantly lower than levels in untreated mice (20.04 ± 3.28 ng/ml) (p < 0.05). Forty-eight hours after stopping this treatment, serum cortisol levels had not returned to baseline (14.3 ± 3.28 ng/ml).

The Effects of Allergen and Budesonide on IL-5 and Eotaxin Levels in BALF, Serum, and Bone Marrow
OVA challenge increased IL-5 levels in both BALF and serum, but not in bone marrow. IL-5 levels in BALF increased from 11.49 pg/ml at 12 h in Sham/Sham mice to 89.96 pg/ml in OVA/OVA mice (p < 0.05) (Figure 3) and in serum increased from 8.17 pg/ml to 67.70 pg/ml in the same mice (p < 0.005) (Figure 3). Similarly, at 24 h, IL-5 levels increased in BALF from 16.38 pg/ml to 72.19 pg/ml (p < 0.001) (Figure 3) and in serum from 4.89 pg/ml to 38.56 pg/ml (p < 0.001) (Figure 3) in Sham/Sham mice compared with OVA/OVA mice. BALF and serum IL-5 levels had returned to control levels at 48 h following OVA challenge (Figure 3). IL-5 was detectable in bone marrow, but OVA challenge did not increase IL-5 levels at any time point (Figure 3). For comparison, BALF IL-5 in sensitized but unchallenged mice was 13.59 pg/ml, which was not different than in Sham/Sham mice.

View larger version (17K): [in this window] [in a new window]   Figure 3. The effects of intranasal budesonide on IL-5 levels (pg/ml) in bone marrow, serum, and BALF after airway allergen challenge (M ± SEM). Mouse numbers per group were 5–10. *p < 0.05 OVA/OVA compared with Sham/Sham mice at the same time point. p < 0.05 OVA/BUD compared with OVA/OVA mice at the same time point.

OVA challenge increased eotaxin levels in BALF, but not in serum or bone marrow. Eotaxin in BALF at 12 h was 1.24 pg/ml in Sham/Sham mice and 4.91 pg/ml in OVA/OVA mice (p < 0.05) (Figure 4). At 24 h, the BALF eotaxin levels were also different between Sham/Sham and OVA/OVA mice, being 1.30 pg/ml and 4.13 pg/ml, respectively (p < 0.05) (Figure 4). Eotaxin was detectable in both serum and bone marrow, but did not differ significantly between Sham/Sham and OVA/OVA mice at any time point (Figure 4).

View larger version (14K): [in this window] [in a new window]   Figure 4. The effects of intranasal budesonide on eotaxin levels (pg/ml) in BALF, serum, and bone marrow fluids after airway allergen challenge (M ± SEM). Mouse numbers per group were 5–10. *p < 0.05 OVA/OVA compared with Sham/Sham mice at the same time point.

The Effects of Allergen and Budesonide on Bone Marrow Eosinophil Progenitors
Bone marrow Eo-CFU were increased at 24 h (p < 0.01) and 48 h (p < 0.01) after the second OVA challenge in OVA/OVA mice when compared with Sham/Sham mice (Figure 5), but not at 12 h following challenge.

View larger version (14K): [in this window] [in a new window]   Figure 5. The effects of intranasal budesonide on bone marrow Eo-CFU after airway allergen challenge (M ± SEM). Mouse numbers per group were 10. *p < 0.05 OVA/OVA compared with Sham/Sham mice at the same time point. p < 0.05 OVA/BUD compared with OVA/OVA mice at the same time point.

The Effects of Allergen and Budesonide on Bone Marrow, Peripheral Blood, BALF, and Lung Tissue Eosinophils
The absolute number of bone marrow eosinophils was significantly increased at both 24 h (p < 0.01) and 48 h (p < 0.001) following OVA challenge in OVA/OVA mice when compared with Sham/Sham mice (Figure 6). At both of these time points, budesonide treatment (intranasally 350 µg/kg/d) significantly attenuated the increases in bone marrow eosinophils 69% and 41%, respectively (Figure 6).

View larger version (22K): [in this window] [in a new window]   Figure 6. The effects of allergen with or without intranasal budesonide on absolute eosinophil numbers in bone marrow, peripheral blood, and BALF after airway allergen challenge (M ± SEM). Mouse numbers per group were 10. *p < 0.05 OVA/OVA compared with Sham/Sham mice at the same time point. p < 0.05 OVA/BUD compared with OVA/OVA mice at the same time point.

In BALF of OVA/OVA mice, the absolute number of eosinophils was significantly increased at 12 h after the second OVA challenge, which persisted at 24 h and 48 h following challenge when compared with Sham challenged mice (p < 0.005, 0.001, and 0.005, respectively). Budesonide treatment inhibited the BALF eosinophilia by 71.1, 70.0, and 73.7% at 12 h, 24 h, and 48 h following challenge, respectively (Figure 6).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
In this study, we have observed that treatment of mice with budesonide resulted in attenuation of allergen-induced responses, including increases in airway and circulating IL-5 levels; BM, circulating, and airway eosinophilia; increases in BM eosinophil progenitors; and airway hyperresponsiveness. Although none of these observations is by itself surprising, a comparison of the effects of budesonide on the time course of the above responses revealed an unexpected finding relating to the mechanisms by which corticosteroid treatment prevents allergen-induced eosinophilic inflammation.

It has been proposed by us and others that the allergen-induced increases in BM eosinophil production are due to distal effects of an increased production of hematopoietic cytokines by antigen-specific T cells in the airway (6, 11, 12, 31). Furthermore, based on observations that the production of numerous cytokines, including IL-5, can be suppressed following corticosteroid treatment (32–34), we have hypothesized that the prevention of allergen-induced bone marrow responses following corticosteroid treatment is likely mediated through the prevention of hematopoietic and chemotactic mediators released from these cells in the airway (35). Based on these hypotheses, we expected that attenuation of allergen-induced increases in BM progenitors as well as allergen-induced increases in mature eosinophils, both in the BM and circulation, would have been associated with an attenuation of allergen-induced IL-5 and possibly eotaxin levels in BAL and circulation. However, at the earliest time point we made measurements (12 h after the second allergen challenge) we observed that budesonide treatment had attenuated allergen-induced increases in mature eosinophil numbers in circulating blood and BALF, but that this was not associated with attenuation of either allergen-induced IL-5 or eotaxin increases observed in BALF or circulating blood. Clearly these results suggest that the antiinflammatory effects of corticosteroid treatment are not simply mediated through the prevention of IL-5 or eotaxin release from cells within the lung. It is important to note that the specific timing of events in our model is complicated by the fact that mice were exposed to two allergen challenges separated by 24 h. Thus, measurements made at the 12 h time point were actually 36 h after the first allergen exposure. Although it is likely that there are complex kinetic responses taking place, this does not affect our observation of differential effects of budesonide on cellular and mediator responses in different compartments.

Based on our observations, we propose that signaling between the lung and bone marrow in response to allergen exposure may not be simply due to distal effects of locally produced hematopoietic/chemotactic mediators. Although our current results do not specifically point to other signaling mechanisms, Minshal and coworkers have observed that following exposure of mice airways to allergen, there is an increase in CD3⁺ cells within the bone marrow and that many of these cells contain both IL-5 mRNA and protein (36). Those results suggest a mechanism by which exposure to allergen at the airway is linked to hematopoietic cytokine production at the bone marrow, possibly due to T cell trafficking. In this light, a feasible explanation for our results would be that corticosteroid treatment, although not sufficient to prevent the 12 h increase in IL-5 production at the airway, was sufficient to attenuate the production of hematopoietic mediators at the bone marrow. Unfortunately, we are not able to substantiate this explanation with our measurements of bone marrow IL-5 or eotaxin levels, which we did not observe to change at any time point following exposure to allergen in treated or untreated groups. This lack of a measurable change in BM IL-5 might be taken as further evidence that eosinophilopoiesis is not mediated through systemic changes in IL-5 levels, but rather occurs in response to local cytokine production that does not result in an overall increase in BM levels that is detectable using our gross measurement techniques. This explanation is in disagreement with an earlier claim from our institute that circulating rather than local IL-5 levels were a critical determinant of airway eosinophilia following allergen challenge (31). We would now suggest that it is not circulating IL-5 levels, but rather local bone marrow production of IL-5 or other hematopoietic cytokines that is a critical determinant of eosinophil production, release into blood, and accumulation at the airway. It has recently been reported that the recognized markers of eosinophil activation are absent in murine models of allergen challenge, suggesting that activation mechanisms may be different between mice and humans (37). Clearly, we must be aware that mechanisms involved in eosinophil production and recruitment may also differ between these species.

An alternative explanation for the suppression of BAL eosinophils, despite a lack of effect on BALF or circulating IL-5 and eotaxin levels, is that other key mechanisms were blocked. Other cytokines that are involved in eosinophilapoesis, including IL-3 and GM-CSF, may have been affected by the corticosteroid treatment. Other potential mechanisms include prevention of adhesion molecule or counter ligand upregulation.

In this study, we treated mice with budesonide delivered via intranasal instillation. This delivery route was chosen, as it has been shown to result in 48% of the instilled substance reaching the lung, and therefore potentially acting topically to prevent allergic airway responses (38). However, our observation that treatment was associated with a 50% reduction in serum cortisol levels indicates that at the doses we used, there were significant systemic effects. As discussed above, it is entirely possible that prevention of eosinophilopoiesis involves a systemic effect of corticosteroid treatment. Although lower doses of intranasal budesonide did not prevent allergic airway responses, we cannot conclude whether this was due to a decrease in topical or systemic effects. Although we did observe a smaller degree of cortisol suppression at these doses, this is not enough to conclude that this decreased systemic effect was the reason for the failure to attenuate allergic responses. Whether systemic effects are important mechanisms by which inhaled corticosteroids exert their protective effect needs to be determined, as this would have a major influence on the effectiveness of new corticosteroids with minimal systemic bioavailability.

The reduction of eosinophils in circulation and tissues, including airways and bone marrow, by systemically administered corticosteroids has been reported previously in normal subjects (39), rats (40), mice (41), and patients with asthma (32). The ability of topical corticosteroids to reduce eosinophilia in circulation and airways has also been documented in patients with asthma (10, 29, 42). Our studies, to our knowledge, for the first time demonstrate that the prevention of allergen-induced airway inflammation following corticosteroid treatment is associated with a prevention of bone marrow eosinophil progenitor expansion and terminal differentiation. The observation that the degree of attenuation of eosinophil levels in the bone marrow, peripheral blood, and BALF is of similar magnitude (50–70% in all compartments at all time points) is consistent with the hypothesis that the reduction of airway eosinophilia following corticosteroid treatment is mediated through a decreased availability of eosinophils in circulating blood. Further support for this hypothesis is that corticosteroids did not reduce the levels of eotaxin in the airway or circulation, suggesting that prevention of allergen-induced increase in eosinophil chemoattraction is not an important site of action in corticosteroid treatment of allergic airway disease. This lack of a corticosteroid effect on eotaxin production following allergen challenge is consistent with the observation that intraperitoneal dexamethasone did not suppress allergen-induced eotaxin generation in an in vivo guinea pig model (7), although an in vitro study showed that dexamethasone suppressed cytokine-induced augmentation of eotaxin mRNA and protein in human lung epithelial cells (43).

It appears based on our results that the protection against progenitor expansion was lost 48 h after exposure to allergen (see Figure 5). It is possible that the effects of budesonide had worn off by this time, allowing effector cells that had been activated in response to allergen to release hematopoietic factors. Although our cortisol measurements suggest that systemic effects of steroid treatment are apparent 48 h after stopping treatment, this does not imply that other therapeutic effects may have been lost at this time. However, it must also be considered that when we treated naive mice with budesonide, we observed an increase in bone marrow eosinophil progenitors 48 h following treatment, suggesting that the increase observed in allergen-challenged mice at this time may not have reflected a loss of protection against allergic responses, but rather a direct effect of steroid treatment. This expansion of Eo-CFU following daily budesonide treatment of nonsensitized, nonchallenged mice is similar to findings reported by other investigators. Butterfield and coworkers reported that oral prednisone (10 mg four times daily for 4 d) caused a significant drop in circulating eosinophil numbers but did not decrease eosinophil colony numbers in culture of bone marrow and peripheral blood in normal subjects (39). An in vitro study showed that addition of therapeutic concentrations of hydrocortisone to culture systems increased the eosinophil colonies in normal human bone marrow (44). We have recently confirmed this using therapeutic concentrations of budesonide added to BM mononuclear cells from subjects with asthma and normal subjects (45). Kim and coworkers confirmed that CD34⁺/CD45⁺ myeloid progenitors are present in submucosal sites of the upper airway in nasal polyposis and steroid treatment caused a rise in numbers of CD34⁺ progenitors as mature tissue eosinophils fell (46). In a previous study from our laboratory, it was observed that low doses of inhaled budesonide treatment reduced baseline bone marrow EoB-CFU, but these were subjects with asthma with an ongoing eosinophilic inflammation of the airways (10). Clearly, although a greater understanding of the effects of corticosteroids on eosinophil progenitors is required, it appears that this treatment may result in increases or decreases; these discrepancies might be explained by drug doses and also by whether or not there is an underlying eosinophilic inflammation present. Surprisingly, the Sham/Sham group displayed a decrease in Eo-CFU colonies 48 h following their second saline challenge. The reason for this is not known; the only systematic difference between this and the 12 and 24 h Sham/Sham groups is the timing since anesthesia and saline challenge.

We observed an increase in BALF IL-5 levels from approximately 12 pg/ml in unsensitized unchallenged mice to approximately 90 pg/ml following allergen challenge of sensitized mice. This 7- to 8-fold increase is comparable to increases reported by other investigators (6). In that study, the absolute maximum level of BALF IL-5 was in the order of 350 pg/ml. Differences in maximum levels between studies may reflect differences in challenge protocols or in methods for delivering and retrieving the saline used in BAL procedures.

In summary, the results from this study demonstrate that budesonide intranasal administration inhibits allergen-induced eosinophil production and mobilization from the bone marrow and subsequent trafficking through peripheral blood into the airways, as well as allergen-induced airway hyperresponsiveness. At early time points after allergen this protection occurs without any attenuation of local or systemic increases in IL-5 or eotaxin. These results suggest that a component of the mechanism by which corticosteroid treatment attenuates allergen-induced airway inflammation is through suppression of bone marrow eosinophilopoiesis, and that this is likely not mediated simply through the blocking of IL-5 production at the airway.

	Acknowledgments

 
Dr. Paul M. O'Byrne is a Medical Research Council of Canada Senior Scientist.

Supported by The Ontario Thoracic Society and MRC (Canada).

Received in original form August 30, 2000; accepted in final form August 27, 2001

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 

1. Busse WW, Calhoun WF, Sedgwick JD. Mechanism of airway inflammation in asthma. Am Rev Respir Dis 1993;147:S20–S24.[Medline]
2. Sanderson CJ. Interleukin-5, eosinophils, and disease. Blood 1992; 79:3101–3109.[Free Full Text]
3. Walker C, Kaegi MK, Braun P, Blaser K. Activated T cells and eosinophilia in bronchoalveolar lavages from subjects with asthma correlated with disease severity. J Allergy Clin Immunol 1991;88:935–942.[CrossRef][Medline]
4. Hogan SP, Foster PS, Charlton B, Slattery RM. Prevention of Th2-mediated murine allergic airways disease by soluble antigen administration in the neonate. Proc Nat Acad Sci USA 1998;95:2441–2445.[Abstract/Free Full Text]
5. Stampfli MR, Jordana M. Eosinophilia in antigen-induced airways inflammation. Can Respir J 1998;5(Suppl A):31A–35A.
6. Ohkawara Y, Lei XF, Stampfli MR, Marshall JS, Xing Z, Jordana M. Cytokine and eosinophil responses in the lung, peripheral blood, and bone marrow compartments in a murine model of allergen-induced airways inflammation. Am J Respir Cell Mol Biol 1997;16:510–520.[Abstract]
7. Humbles AA, Conroy DM, Marleau S, Rankin SM, Palframan RT, Proudfoot AE, Wells TN, Li D, Jeffery PK, Griffiths-Johnson DA, Williams TJ, Jose PJ. Kinetics of eotaxin generation and its relationship to eosinophil accumulation in allergic airways disease: analysis in a guinea pig model in vivo. J Exp Med 1997;186:601–612.[Abstract/Free Full Text]
8. Gibson PG, Manning PJ, O'Byrne PM, Girgis-Girbado A, Dolovich J, Denburg JA, Hargreave FE. Allergen-induced asthmatic responses. Relationship between increases in airway responsiveness and increases in circulating eosinophils, basophils and their progenitors. Am Rev Respir Dis 1991;143:331–335.[Medline]
9. Wood LJ, Inman MD, Watson RM, Foley R, Denburg JA, O'Byrne PM. Changes in bone marrow inflammatory cell progenitors after inhaled allergen in asthmatic subjects. Am J Respir Crit Care Med 1998; 157:99–105.[Abstract/Free Full Text]
10. Wood LJ, Sehmi R, Gauvreau GM, Watson RM, Foley R, Denburg JA, O'Byrne PM. An inhaled corticosteroid, budesonide, reduces baseline but not allergen-induced increases in bone marrow inflammatory cell progenitors in asthmatic subjects. Am J Respir Crit Care Med 1999; 159:1457–1463.[Abstract/Free Full Text]
11. Gaspar Elsas MIC, Joseph D, Elsas PX, Vargaftig BB. Rapid increases in bone marrow eosinophil production and response to eosinopoietic interleukins triggered by intranasal allergen challenge. Am J Respir Cell Mol Biol 1997;17:404–413.[Abstract/Free Full Text]
12. Inman MD, Ellis R, Wattie J, Denburg JA, O'Byrne PM. Allergen-induced increase in airway responsiveness, airway eosinophilia and bone marrow eosinophil progenitors in mice. Am J Respir Cell Mol Biol 1999;21:473–479.[Abstract/Free Full Text]
13. Lee JJ, McGarry MP, Farmer SC, Denzler KL, Larson KA, Carrigan PE, Brenneise IE, Horton MA, Haczku A, Gelfand EW, et al. Interleukin-5 expression in the lung epithelium of transgenic mice leads to pulmonary changes pathognomonic of asthma. J Exp Med 1997;185: 2143–2156.[Abstract/Free Full Text]
14. Teran LM. Chemokines and IL-5: major players of eosinophil recruitment in asthma. Clin Exp Allergy 1999;29:287–290.[CrossRef][Medline]
15. Denburg JA, Sehmi R, Upham J, Wood L, Gauvreau G, O'Byrne P. Regulation of IL-5 and IL-5 receptor expression in the bone marrow of allergic asthmatics. Int Arch Allergy Immunol 1999;118:101–103.[CrossRef][Medline]
16. Lee NA, Lee JJ. Asthma: does IL-5 have a more provocative role? Am J Respir Cell Mol Biol 1997;16:497–500.[Medline]
17. Hamid Q, Azzawi M, Ying S, Moqbel R, Wardlaw AJ, Corrigan CJ, Bradley B, Durham SR, Collins JV, Jeffery PK. Expression of mRNA for interleukin-5 in mucosal bronchial biopsies from asthma. J Clin Invest 1991;87:1541–1546.
18. Motojima S, Tateishi K, Koseki T, Makino S, Fukuda T. Serum levels of eosinophil cationic protein and IL-5 in patients with asthma without systemic corticosteroids. Int Arch Allergy Immunol 1997;114(Suppl 1): 55–59.
19. Tang C, Rolland JM, Ward C, Quan B, Walters EH. Allergen-induced airway reactions in atopic asthmatics correlate with allergen-specific IL-5 response by BAL cells. Respirology 1997;2:45–55.[Medline]
20. Gao JL, Sen AI, Kitaura M, Yoshie O, Rothenberg ME, Murphy PM, Luster AD. Identification of a mouse eosinophil receptor for the CC chemokine eotaxin. Biochem Biophys Res Commun 1996;223:679–684.[CrossRef][Medline]
21. Jose PJ, Griffiths-Johnson DA, Collins PD, Walsh DT, Moqbel R, Totty NF, Truong O, Hsuan JJ, Williams TJ. Eotaxin: a potent eosinophil chemoattractant cytokine detected in a guinea pig model of allergic airways inflammation. J Exp Med 1994;179:881–887.[Abstract/Free Full Text]
22. Ponath PD, Qin S, Post TW, Wang J, Wu L, Gerard NP, Newman W, Gerard C, Mackay CR. Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils. J Exp Med 1996;183:2437–2448.[Abstract/Free Full Text]
23. Mould AW, Matthaei KI, Young IG, Foster PS. Relationship between interleukin-5 and eotaxin in regulating blood and tissue eosinophilia in mice. J Clin Invest 1997;99:1064–1071.[Medline]
24. Palframan RT, Collins PD, Williams TJ, Rankin SM. Eotaxin induces a rapid release of eosinophils and their progenitors from the bone marrow. Blood 1998;91:2240–2248.[Abstract/Free Full Text]
25. Pauwels RA, Lofdahl CG, Postma DS, Tattersfield AE, O'Byrne P, Barnes PJ, Ullman A. Effect of inhaled formoterol and budesonide on exacerbations of asthma. Formoterol and Corticosteroids Establishing Therapy, 1998. N Engl J Med 1997;337:1405–1411.[Abstract/Free Full Text]
26. Barnes PJ, Pedersen S, Busse WW. Efficacy and safety of inhaled corticosteroids. New developments. Am J Respir Crit Care Med 1998;157:S1–S53.[Free Full Text]
27. Robinson D, Hamid Q, Ying S, Bentley A, Assoufi B, Durham S, Kay AB. Prednisolone treatment in asthma is associated with modulation of bronchoalveolar lavage cell interleukin-4, interleukin-5, and interferon-gamma cytokine gene expression. Am Rev Respir Dis 1993;148: 401–406.[Medline]
28. Jeffery PK, Godfrey RW, Adelroth E, Nelson F, Rogers A, Johansson SA. Effects of treatment on airway inflammation and thickening of basement membrane reticular collagen in asthma. A quantitative light and electron microscopic study. Am Rev Respir Dis 1992;145:890–899.[Medline]
29. Gauvreau GM, Doctor J, Watson RM, Jordana M, O'Byrne PM. Effects of inhaled budesonide on allergen-induced airway responses and airway inflammation. Am J Respir Crit Care Med 1996;154:1267–1271.[Abstract]
30. Ewart SL, Mitzner W, DiSilvestre DA, Meyers DA, Levitt RC. Airway hyperresponsiveness to acetylcholine: segregation analysis and evidence for linkage to murine chromosome 6. Am J Respir Cell Mol Biol 1996;14:487–495.[Abstract]
31. Wang J, Palmer K, Lötval J, Milan S, Lei X-F, Matthaei KI, Gauldie J, Inman MD, Jordana M, Xing Z. Circulating, but not local lung IL-5 is required for the development of antigen-induced airways eosinophilia. J Clin Invest 1998;102:1132–1141.[Medline]
32. Bentley AM, Hamid Q, Robinson DS, Schotman E, Meng Q, Assoufi B, Kay AB, Durham SR. Prednisolone treatment in asthma. Reduction in the numbers of eosinophils, T cells, tryptase-only positive mast cells, and modulation of IL-4, IL-5, and interferon-gamma cytokine gene expression within the bronchial mucosa. Am J Respir Crit Care Med 1996;153:551–556.[Abstract]
33. Schwiebert LM, Beck LA, Stellato C, Bickel CA, Bochner BS, Schleimer RP. Glucocorticosteroid inhibition of cytokine production: relevance to antiallergic actions. J Allergy Clin Immunol 1996;97:143–152.[CrossRef][Medline]
34. Mori A, Suko M, Nishizaki Y, Kaminuma O, Kobayashi S, Matsuzaki G, Yamamoto K, Ito K, Tsuruoka N, Okudaira H. IL-5 production by CD4+ T cells of asthmatic patients is suppressed by glucocorticoids and the immunosuppressants FK506 and cyclosporin A. Int Immunol 1995;7:449–457.[Abstract/Free Full Text]
35. Inman MD, Denburg JA, Ellis R, Dahlback M, O'Byrne PM. The effect of treatment with budesonide or PGE₂ in vitro on allergen-induced increased in canine bone marrow progenitors. Am J Respir Cell Mol Biol 1997;17:634–642.[Abstract/Free Full Text]
36. Minshall EM, Schleimer R, Cameron L, Minnicozzi M, Egan RW, Gutierrez-Ramos JC, Eidelman DH, Hamid Q. Interleukin-5 expression in the bone marrow of sensitized Balb/c mice after allergen challenge. Am J Respir Crit Care Med 1998;158:951–957.[Abstract/Free Full Text]
37. Malm-Erjefalt M, Persson CG, Erjefalt JS. Degranulation status of airway tissue eosinophils in mouse models of allergic airway inflammation. Am J Respir Cell Mol Biol 2001;24:352–359.[Abstract/Free Full Text]
38. Eyles JE, Williamson ED, Alpar HO. Immunological responses to nasal delivery of free and encapsulated tetanus toxoid: studies on the effect of vehicle volume. Int J Pharmaceut 2000;189:75–79.
39. Butterfield JH, Ackerman SJ, Weiler D, Eisenbrey AB, Gleich GJ. Effects of glucocorticoids on eosinophil colony growth. J Allergy Clin Immunol 1986;78:450–457.[CrossRef][Medline]
40. Blenkinsopp EC, Blenkinsopp WK. Effects of a glucocorticoid (dexamethasone) on the eosinophils of the rat. J Endocrinol 1967;37:463–469.
41. De Bie JJ, Hessel EM, Van AI, Van EB, Hofman G, Nijkamp FP, Van OA. Effect of dexamethasone and endogenous corticosterone on airway hyperresponsiveness and eosinophilia in the mouse. Br J Pharmacol 1996;119:1484–1490.[Medline]
42. Mygind N, Dahl R, Nielsen LP. Effect of nasal inflammation and of intranasal anti-inflammatory treatment on bronchial asthma. Respir Med 1998;92:547–549.[CrossRef][Medline]
43. Lilly CM, Nakamura H, Kesselman H, Nagler-Anderson C, Asano K, Garcia-Zepeda EA, Rothenberg ME, Drazen JM, Luster AD. Expression of eotaxin by human lung epithelial cells: induction by cytokines and inhibition by glucocorticoids. J Clin Invest 1997;99:1767–1773.[Medline]
44. Barr RD, Volaric Z, Koekebakker M. Stimulation of human eosinophilopoiesis by hydrocortisone in vitro. Acta Haematol 1987;77:20–24.[Medline]
45. Dorman S, O'Byrne PM, Wood LJ, Watson RM, Wasi P, Foley R, Denburg JA. Corticosteroid enhancement of IL-5 dependent eosinophil progenitor differentiation in vitro. Am J Respir Crit Care Med 2000;161:A540.
46. Kim YK, Uno M, Hamilos DL, Beck L, Bochner B, Schleimer R, Denburg JA. Immunolocalization of CD34 in nasal polyposis. Effect of topical corticosteroids. Am J Respir Cell Mol Biol 1999;20:388–397.[Abstract/Free Full Text]

This article has been cited by other articles:

				M. Lommatzsch, K. Schloetcke, J. Klotz, K. Schuhbaeck, D. Zingler, C. Zingler, O. Schulte-Herbruggen, H. Gill, P. Schuff-Werner, and J. C. Virchow Brain-derived Neurotrophic Factor in Platelets and Airflow Limitation in Asthma Am. J. Respir. Crit. Care Med., January 15, 2005; 171(2): 115 - 120. [Abstract] [Full Text] [PDF]

				R. E. Wiley, M. Cwiartka, D. Alvarez, D. C. Mackenzie, J. R. Johnson, S. Goncharova, L. Lundblad, and M. Jordana Transient Corticosteroid Treatment Permanently Amplifies the Th2 Response in a Murine Model of Asthma J. Immunol., April 15, 2004; 172(8): 4995 - 5005. [Abstract] [Full Text] [PDF]

				C. L. Hardy, L. Kenins, A. C. Drew, J. M. Rolland, and R. E. O'Hehir Characterization of a Mouse Model of Allergy to a Major Occupational Latex Glove Allergen Hev b 5 Am. J. Respir. Crit. Care Med., May 15, 2003; 167(10): 1393 - 1399. [Abstract] [Full Text] [PDF]

				M. J. Tobin Asthma, Airway Biology, and Nasal Disorders in AJRCCM 2002 Am. J. Respir. Crit. Care Med., February 1, 2003; 167(3): 319 - 332. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shen, H. Articles by Inman, M. D. Search for Related Content PubMed PubMed Citation Articles by Shen, H. Articles by Inman, M. D.