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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 883-889, (2002)
© 2002 American Thoracic Society

Original Articles

Allergen-induced Increases in Bone Marrow T Lymphocytes and Interleukin-5 Expression in Subjects with Asthma

Lorna J. Wood, Roma Sehmi, Sandra Dorman, Qutayba Hamid, Meri K. Tulic, Richard M. Watson, Ronan Foley, Parveen Wasi, Judah A. Denburg, Gail Gauvreau and Paul M. O'Byrne

Asthma Research Group, Firestone Institute for Respiratory Health, St. Joseph's Hospital and the Department of Medicine, McMaster University, Hamilton, Ontario; and Meakins-Christie Laboratories and Montreal Chest Institute Research Center, McGill University, Montreal, Quebec, Canada

Correspondence and requests for reprints should be addressed to Paul M. O'Byrne, M.D., Firestone Institute for Respiratory Disease, St. Joseph's Hospital, 50 Charlton Avenue East, Hamilton, ON, L8N 4A6, Canada. E-mail: obyrnep{at}mcmaster.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhaled allergen challenge of subjects with atopic asthmatic increases bone marrow eosinophil progenitor cells. Interleukin-5 (IL-5) specifically induces growth and maturation of eosinophils. This study examined the effect of allergen challenge on the number of bone marrow total and CD3+ cells expressing IL-5 protein and IL-5 mRNA in subjects with asthma who developed either allergen-induced isolated early responses, or early and late asthmatic responses (dual responders). At 24 hours after allergen challenge, dual responders had significantly greater blood and airway eosinophilia compared with early responders. There were significant increases in the percentage of bone marrow CD3+ cells (p < 0.005) in both groups. However, there were significant differences in the increases in bone marrow IL-5 mRNA+ (p < 0.005), CD3+ (p < 0.005), and IL-5 mRNA+ CD3+ (p < 0.005) cells between the dual and early responder groups. These results suggest that, in subjects with atopic asthma, inhaled allergen causes trafficking of T lymphocytes to the bone marrow, and that in subjects who develop late responses and greater blood and airway eosinophilia after inhalation of allergen, there is a significant increase in the ability of bone marrow cells, particularly T lymphocytes, to produce IL-5.

Key Words: asthma • bone marrow • interleukin-5 • progenitors • T lymphocytes


   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 persisting asthma, and is characterized by biphasic responses known as the early- and late-phase asthmatic responses. Late asthmatic responses are associated with transient increases in airway hyperresponsiveness (1) and increased numbers of activated eosinophils in the airways (2); however, subjects who develop isolated early asthmatic responses also develop airway eosinophilia, but to a much lesser extent than those patients developing late responses (3). The predominant cells infiltrating the airways during the late response are eosinophils. Increases in eosinophils have been demonstrated in induced sputum (2), in bronchoalveolar lavage fluid (4), and airway biopsies (5).

Another aspect of allergic inflammatory responses is the induction of increases in inflammatory cell progenitors, which contribute to disease through the continued production of inflammatory effector cells. Studies of atopic subjects have shown that there are higher numbers of both circulating eosinophil/basophil colony-forming units (Eo/B-CFU) (6) and CD34+ hematopoietic progenitor cells in the blood of atopic subjects compared with normal subjects (7). In addition, fluctuating numbers of circulating Eo/B-CFU during seasonal exposure to allergen (6) and significantly higher numbers 24 hours after allergen inhalation (8) have also been shown. Animal studies have demonstrated allergen-induced increases in bone marrow Eo/B-CFU in mice with either upper or lower airway inflammation (9, 10) and increases in granulocyte-macrophage colony-forming units in dogs (11).

We have shown that there is a significant increase in the numbers of Eo/B-CFU in the bone marrow of subjects with asthma, 24 hours after an allergen challenge (3). Furthermore, there was an increased responsiveness of these cells to interleukin-5 (IL-5), a cytokine specifically involved in eosinophil growth and maturation (12), in subjects who developed a dual asthmatic response compared with those who developed only an isolated early response after allergen inhalation. In support of these observations, Sehmi and coworkers (13) demonstrated a significant increase in the number of bone marrow–derived CD34+ cells expressing the {alpha} subunit of the IL-5 receptor (IL-5R-{alpha}) 24 hours after allergen challenge in dual responders, but not in isolated early responders. These results suggest that, in the bone marrow of those subjects who develop a greater eosinophilic airway response after allergen inhalation, a distinct phenotypic switch on progenitor cells may occur, which, in the presence of IL-5, may favor eosinophil production, thus contributing to the development of blood and tissue eosinophilia.

This hypothesis would suggest that IL-5 is present within the bone marrow and is contributing to the ongoing eosinophil production. What is not clear is whether IL-5 is produced locally within the bone marrow, or is produced elsewhere and is signaling the bone marrow via external sources. A study by Minshall and coworkers (14) has shown that there is a constitutive expression of IL-5 in the bone marrow of normal mice, which is increased after sensitization and allergen challenge. This study also demonstrated that, whereas CD34+ cells are a major source of IL-5 mRNA during unstimulated eosinophilopoiesis, it is the CD3+ T lymphocytes that are the major source of IL-5 mRNA after sensitization and allergen challenge.

The aim of this study was to determine whether the number of cells within the bone marrow of subjects with asthma that express IL-5 protein and IL-5 mRNA are increased after allergen inhalation; to determine whether differences in the number of these cells exist between subjects with asthma who develop either a dual or isolated early asthmatic response; and, finally, to colocalize IL-5 mRNA to CD3+ cells, thereby identifying T lymphocytes as a possible source of this cytokine.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Twenty-two patients with mild asthma (9 isolated early and 13 dual responders) were studied (Table 1) . Isolated early responders developed only an early fall in FEV1 of > 15% from baseline. Dual responders developed both early and late asthmatic responses (> 10% drop in FEV1 from baseline). The definitions of an early and late asthmatic response were established before the study, and the subjects were characterized as isolated early or dual responders by their airway responses to a screening allergen inhalation challenge. All subjects were nonsmokers and none had experienced a respiratory infection during the 4 weeks before the study. The subjects required only intermittent use of inhaled ß2-agonists, with baseline FEV1 values > 70% predicted. The study was approved by the Research Advisory Group at McMaster University Medical Centre (Hamilton, ON, Canada) and all subjects provided written informed consent before entering the study.


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  		TABLE 1. Subject characteristics

 
Study Design
The subjects attended the laboratory on three occasions. At the initial visit a medical history was taken and skin prick tests, spirometry, and methacholine inhalation challenge were performed, followed by sputum induction. Within 1 week, subjects returned for an allergen challenge, before which a bone marrow aspirate and blood sample were taken as baseline. A sputum sample was induced 7 hours after allergen inhalation. The third visit occurred 24 hours later, when bone marrow aspirate and blood samples were obtained and spirometry and a methacholine inhalation challenge were performed, followed by sputum induction.

Methacholine Inhalation Challenge
Methacholine inhalation was performed as described by Cockcroft and coworkers (15) and results were expressed as the provocative concentration causing a 20% decrease in FEV1 (PC20).

Skin Prick Test
The skin prick test was used, as previously described, to predict the dose of allergen required to cause a 20% fall in FEV1 (predicted allergen PC20) (16).

Allergen Inhalation Challenge
Allergen inhalation challenge was performed as previously described (17). The allergen-induced early response was determined as the maximal decrease in FEV1 between 0 and 2 hours and the late response was determined as the maximal decrease between 3 and 7 hours after allergen inhalation.

Sputum Analysis
Sputum was induced and processed according to the method of Pizzichini and coworkers (18). Mean counts from duplicate slides were obtained (500 cells counted per slide) and expressed as absolute counts (104 cells/ml).

Blood Samples
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). Results were expressed as absolute counts (104 cells/ml).

Bone Marrow Aspirate and Cytospin Preparation
Bone marrow aspirates were obtained from the iliac crest using a bone marrow aspiration needle as previously described (3). Cytospin preparations (~ 105 cells) were made on uncoated glass slides for eosinophil counts, on 3-aminopropyl-triemoxisilane (APTEX) coated slides for immunocytochemistry (IL-5 staining only), and on poly-L-lysine–coated (Sigma, St. Louis, MO) slides for in situ hybridization and double staining (IL-5 mRNA and CD3 staining). Total eosinophil counts (immature and mature forms) were performed by one investigator in a blinded fashion and the mean of two slides was obtained (1,000 cells counted per slide); results were expressed as percentages of total cells. For immunohistochemistry, the slides were fixed for 10 minutes in periodate–lysine–paraformaldehyde, followed by 10 minutes in 15% sucrose solution, and stored at -70°C until analysis. For in situ hybridization the slides were fixed in 4% paraformaldehyde for 30 minutes, washed twice in PBS, and baked overnight at 37°C. The slides were then stored at -70°C until analysis.

Immunocytochemistry
IL-5 immunoreactivity in bone marrow cell cytospins was evaluated with a monoclonal antibody specific for human IL-5 and the alkaline phosphatase–anti-alkaline phosphatase technique for visualization of positive cells. The slides were counted by one investigator in a blinded fashion (500 cells counted per slide). Results were expressed as percentages of total cells.

In Situ Hybridization
In situ hybridization was performed as previously described (19). Slides were counted by one investigator in a blinded fashion (minimum of 500 cells counted per slide), using x200 magnification, and the number of cells staining positive for IL-5 mRNA was expressed as the percentage of total cells.

Combined Immunocytochemistry and In Situ Hybridization
A combined immunohistochemistry and in situ hybridization technique was used to colocalize IL-5 mRNA–positive to CD3-positive cells as previously described (14). Cell counts were performed in a blinded fashion, with a minimum of 500 cells counted per slide. The number of CD3+ cells was expressed as a percentage of total cells. The number of cells expressing both IL-5 mRNA and CD3 was expressed as a percentage of total CD3+ cells.

Bone Marrow Eosinophil Progenitors
Increases in bone marrow eosinophil progenitor cells were assessed by flow cytometric staining for CD34+IL-5R-{alpha}+ cells as previously described (13). Results were expressed as the geometric mean and percent standard error of the mean (%SEM) of the number of CD34+IL-5R-{alpha}+ cells per 2.5 x 106 white blood cells (WBCs).

Statistical Analysis
Methacholine PC20 values were log10 transformed before analysis. Differences in logged PC20 values in blood, in bone marrow and sputum eosinophils, and in bone marrow CD34+IL-5R-{alpha}+, IL-5+, IL-5 mRNA+, CD3+, and IL-5 mRNA+CD3+ cells were analyzed by repeated measures analysis of variance. Statistical significance was accepted as p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Bronchoconstrictor Responses
The mean maximal percent fall in FEV1 during the early asthmatic response was 33.8 ± 2.8% in the dual responders and 28.3 ± 2.4% in the isolated early responders (Figure 1) . The mean maximal percent fall in FEV1 during the late asthmatic response was 19.4 ± 5.2% in the dual responders and 6.0 ± 0.8% in the isolated early responders (Figure 1).



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  		Figure 1. Allergen-induced bronchoconstriction in subjects developing isolated early responses and dual responses.

 
Airway Hyperresponsiveness
Methacholine airway hyperresponsiveness developed in the dual responders, but not in the isolated early responders, 24 hours after inhalation of allergen. The geometric mean methacholine PC20 values in the dual responders fell from 1.86 mg/ml (%SEM, 1.43) before to 0.61 mg/ml (%SEM, 1.47) after allergen (p < 0.005), whereas in the isolated early responders the values were 4.75 mg/ml (%SEM, 1.41) before and 3.83 mg/ml (%SEM, 1.56) after allergen (p = 0.35). In addition, there was a significant difference between the two groups in the allergen-induced shift in PC20 values (p < 0.05).

Sputum Eosinophils
The number of eosinophils in sputum after inhaled allergen increased significantly in the dual responders, from (18.9 ± 5.9) x 104/ml before allergen inhalation, to (140 ± 37.1) x 104/ml at 7 hours, and (77.9 ± 15.5) x 104/ml at 24 hours after allergen inhalation (p < 0.01) (Figure 2) . A smaller, but still significant, increase was also seen in the isolated early responders, from (6.1 ± 2.3) x 104/ml before allergen inhalation, to (53.0 ± 31.4) x 104/ml at 7 hours, and 28.6 ± 13.5 at 24 hours after allergen inhalation (p < 0.01) (Figure 2). There was a significant difference in the increases in sputum eosinophil numbers between the two groups (p < 0.05) (Figure 2).



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  		Figure 2. Changes in blood and bone marrow eosinophils measured before (open columns) and 24 hours after inhalation of allergen (solid columns), and in sputum eosinophils measured before and 7 and 24 hours after inhalation of allergen, in isolated early and dual asthmatic responders. Inhaled allergen significantly increased blood eosinophils (**p < 0.05) and sputum eosinophils (*p < 0.01) in dual responders, but only in sputum in isolated early responders (*p < 0.01). The increases in sputum eosinophils were significantly greater ({blacktriangleup}p < 0.05) in the dual responders compared with isolated early responders. No significant increases in bone marrow eosinophils were demonstrated in either group after inhalation of allergen.

 
Blood Eosinophils
There was a significant increase in the number of blood eosinophils in the dual responders, from (25.1 ± 3.8) x 104/ml before allergen inhalation to (40.2 ± 4.9) x 104/ml at 24 hours after allergen inhalation (p < 0.005), but not in the isolated early responders (Figure 2). There were no significant differences in the allergen-induced increases in blood eosinophils between the two groups.

Bone Marrow Eosinophils and Eosinophil Progenitors
There were no significant increases in the percentage of bone marrow eosinophils after inhalation of allergen, the values being 5.2 ± 0.6% before and 5.2 ± 0.6% 24 hours after allergen inhalation in the dual responders, and 4.1 ± 05% before and 4.7 ± 0.5% 24 hours after challenge in the isolated early responders (Figure 2).

The number of bone marrow eosinophil progenitors, as assessed by CD34+IL-5R-{alpha}+ cells, increased significantly in the dual responders from 46 (%SEM, 9) cells/2.5 x 106 WBCs before to 83 (%SEM, 18) cells/2.5 x 106 WBCs 24 hours after inhalation of allergen (p < 0.05), but not in the isolated early responders (Figure 3) . There was no significant difference in the allergen-induced increases between the two groups.



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  		Figure 3. Changes in bone marrow CD34+IL-5R{alpha}+ cells before (open columns) and after (solid columns) allergen inhalation in both isolated early and dual responders. Allergen inhalation increased only CD34+IL-5R{alpha}+ cells in dual responders (*p < 0.05).

 
Bone Marrow CD3+ and IL-5 mRNA+ Cells
The percentages of IL-5 mRNA+, CD3+, and IL-5 mRNA+ CD3+ cells significantly increased in the bone marrow of dual responders, 24 hours after inhaled allergen. The percentage of IL-5 mRNA+ cells increased from 3.9 ± 0.5% before to 10.1 ± 0.9% after allergen inhalation (p < 0.005) (Figure 4) ; the percentage of CD3+ cells increased from 5.3 ± 0.6% before to 16.6 ± 1.0% after allergen inhalation (p < 0.005) (Figure 5) ; and the percentage of IL-5 mRNA+CD3+ cells increased from 1.4 ± 0.4% before to 7.3 ± 0.7% after allergen inhalation (p < 0.005) (Figure 5). In contrast, there were no allergen-induced increases in the percentages of IL-5 mRNA+ or IL-5 mRNA+CD3+ cells in the isolated early responder group (Figures 4 and 5). There was, however, a significant increase in the percentages of CD3+ cells in this group, rising from 4.6 ± 0.4% before to 8.8 ± 1.4% after allergen inhalation (p < 0.005) (Figure 5). Finally, there were significant differences in the allergen-induced increases in IL-5 mRNA+ (p < 0.005), CD3+ cells (p < 0.005), and IL-5 mRNA+CD3+ cells (p < 0.005) (Figures 4 and 5) between the dual and isolated early responders. There were, however, no significant changes in the percentages of IL-5 protein-positive cells in either group after allergen inhalation (Figures 4 and 6) .



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  		Figure 4. Changes in bone marrow IL-5–positive (left) and IL-5 mRNA–positive (right) cells before (open columns) and after (solid columns) allergen inhalation in both isolated early and dual responder groups. Allergen inhalation increased only IL-5 mRNA-positive cells in dual responders (**p < 0.005). Pre- to postallergen changes were significantly different in the dual responder group compared with isolated early responders ({blacktriangleup}p < 0.005).

 


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  		Figure 5. Changes in bone marrow CD3+ (left) and IL-5 mRNA+CD3+ cells (right) before (open columns) and after (solid columns) allergen inhalation in both isolated early and dual responders. Pre- to postallergen differences were significant, as indicated (**p < 0.005). Pre- to postallergen changes were significantly different in the dual responder group compared with isolated early responders, as indicated ({blacktriangleup}p < 0.005).

 


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  		Figure 6. Immunocytochemical staining for IL-5 in bone marrow cells. (A) IL-5 staining before and (C) after inhaled allergen. (B) Isotype control before and (D) after inhaled allergen.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study has demonstrated an allergen-induced increase in the numbers of CD3+ cells and the expression of IL-5 mRNA in the bone marrow of subjects with asthma. Approximately 20% of the IL-5 mRNA was colocalized to CD3+ T lymphocytes within the bone marrow, suggesting that these cells are a potential source of IL-5 during allergic inflammation. In addition, the allergen-induced increase in these cells was significantly higher in the dual responders, who developed airway hyperresponsiveness, blood eosinophilia, and a greater degree of airway eosinophilia, compared with the isolated early responders.

Eosinophils develop and mature in the bone marrow from IL-5–responsive CD34+ progenitor cells (20). We have previously demonstrated allergen-induced increases in the number of CD34+IL-5R-{alpha}+ cells (13), and increased responsiveness of eosinophil/basophil colonies to IL-5 in the bone marrow of dual responders, but not isolated early responders (13), highlighting a potential role of IL-5 in bone marrow eosinophil production in allergic asthma. In this study, we again confirmed the allergen-induced increase in CD34+IL-5R-{alpha}+ in dual responders, but not isolated early responders. We have also demonstrated increases in the number of IL-5 mRNA+ cells in dual responders, but not isolated early responders, after allergen inhalation. These results suggest that, in subjects who develop a greater degree of blood and airway eosinophilia after inhalation of allergen, not only are there higher numbers of eosinophil progenitors that are able to respond to IL-5, but there are also higher numbers of cells with the ability to produce IL-5 within the bone marrow after allergen challenge.

Whereas the numbers of CD3+ cells in the bone marrow were significantly higher in both asthmatic groups after allergen inhalation, the percentage of CD3+ cells expressing IL-5 mRNA was significantly increased only in the dual responder group. This suggests that T lymphocytes are a potential source of IL-5 within the bone marrow, particularly in those subjects developing a greater degree of blood and airway eosinophilia. These data are consistent with a study by Minshall and coworkers, in which CD3+ cells in mice were shown to be a source of IL-5 in the bone marrow after sensitization and allergen challenge (14). In the same study, there was also a significant increase in bone marrow cells positive for IL-5 protein after allergen challenge, in addition to IL-5 mRNA–positive cells, suggesting that translation of the protein is occurring.

This study has used the concept that allergen-induced airway responses are dichotomized into two distinct types of responders: isolated early and dual responders. This is mainly because subjects with a greater late response have, in most studies, greater eosinophilic blood and airway responses (3, 13), as well as a greater change in airway responsiveness after inhaled allergen (1). In fact, allergen-induced late responses are a continuous variable, as demonstrated by the magnitude of the late responses in the groups in this study (Table 1). However, arbitrarily deciding on a cutoff of > 10% fall in FEV1 before beginning the study does allow a comparison of subjects, all with mild allergic asthma, with a greater or lesser allergen-induced eosinophilic response to evaluate the mechanisms of the increased production of eosinophils.

The current study demonstrated a slight, but not significant, increase in IL-5 protein-positive cells in the bone marrow after allergen inhalation. It is possible that the 24-hour time point is too early to demonstrate an increase in IL-5 protein in humans after allergen inhalation. Studies in murine models of ovalbumin-induced airway inflammation and airway hyperresponsiveness indicate that the bone marrow eosinophilia peaks 3–4 days after allergen inhalation (21, 22). These results suggest that the increases in airway eosinophils already seen in subjects with asthma 7 to 24 hours after inhalation of allergen and associated with reductions in blood eosinophils at 6–7 hours after allergen (2) are cells recruited from the blood pool or mature cells from bone marrow, whereas the persisting airway eosinophilia, lasting up to 1 week in dual responders (2), may require newly formed bone marrow cells. This would need to be addressed in studies looking at bone marrow responses in humans at later time points.

This study does not give any indication as to whether the allergen-induced bone marrow T lymphocytes are present and activated locally after allergen challenge, or are recruited to the bone marrow after challenge. Also, as IL-5 mRNA+CD3+ cells constituted approximately 20% of total IL-5 mRNA+ cells, there must be other cells present locally in the bone marrow that also express IL-5 mRNA. These may include eosinophils, which also express IL-5 mRNA (23) and IL-5 protein. Bone marrow eosinophils constitute 4–5% of total bone marrow cells, and were not increased after allergen challenge; however, it is possible that the expression of IL-5 mRNA in eosinophils may increase without an increase in the number of cells. A study by Hogan and coworkers (24) has demonstrated that bone marrow stromal cells can both transcribe and translate IL-5, and that this can be upregulated in vivo by incubation with IL-1. In addition, CD34+ cells were also shown to be a potential source of IL-5 in unsensitized mice, with a switch to T lymphocytes being the source of IL-5 after sensitization and challenge (14). One may speculate, on the basis of this and other studies, that stromal cells, CD34+ cells, and eosinophils may be the main source of IL-5 in steady state eosinophilopoiesis, but that after allergen inhalation there is an increase in allergen-specific T lymphocytes with the potential of producing IL-5, which is necessary for increased eosinophilopoiesis.

The study also has not addressed the issue of the signals that cause the increases in CD3+ cells in the bone marrow after inhaled allergen. Allergen-induced trafficking of T lymphocytes into regional lymph nodes is associated with strongly enhanced expression of the chemokines macrophage inflammatory protein (MIP)-1{alpha} and MIP-1ß mRNAs and proteins (25). Mast cells were the predominant source of MIP-1ß, whereas MIP-1{alpha} was expressed by multiple cell types. In addition, the chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 play an important role in lymphocyte trafficking through tissues, especially between peripheral blood and bone marrow (26, 27). However, the possible role of these chemokines and their receptors in lymphocyte trafficking into human bone marrow is not yet known.

Studies of mice have supported the importance of the bone marrow in allergen-induced responses. A study by Ohkawara and coworkers (22) using ovalbumin-sensitized mice demonstrated that airway eosinophilia peaked 3 days after allergen exposure and persisted for 10 to 15 days. This response was accompanied by increases in both circulating and bone marrow eosinophils that followed a similar time course. These data suggest that local inflammatory airway responses associated with allergen challenge are accompanied by an increased rate of eosinophil production, and as a consequence, bone marrow and peripheral blood are maintained at increased levels throughout the duration of the response. A second study, using a similar model of ovalbumin sensitization and airway challenge, demonstrated increases in airway responsiveness and airway eosinophilia, which resolved within 2 weeks (9). These responses were accompanied by increases in eosinophil/basophil progenitors 24 and 48 hours after challenge, but because the time course of progenitor cell expansion was shorter than that of airway inflammation it is possible that progenitor expansion may occur only briefly at the onset of the inflammatory response. In a murine model of allergic rhinitis, alterations in Eo-CFU as well as CD34+ cells in the bone marrow were seen after allergen challenge with a similar time course (28).

Taken together, the results from both animal and human models suggest that, immediately after allergen exposure, there may be a transient decrease in bone marrow eosinophils because of an efflux of mature eosinophils from the bone marrow. During the next 48 hours, concurrent with progenitor cell expansion, bone marrow eosinophil numbers are restored and ultimately increased above normal levels via an increased production rate. The increase in eosinophil production lasts for the duration of the local airway inflammatory responses, whereas progenitor cell numbers return to normal. In the current study, we did not observe any changes in the number of bone marrow eosinophils 24 hours after allergen challenge. Given the above-described model, it is possible that a steady state develops in the bone marrow eosinophils in human subjects after allergen inhalation, wherein the increased production is matched by increased egress from the bone marrow, or the increase occurs at a time point later than 24 hours after allergen inhalation.

One study has raised doubts about the importance of airway eosinophils in causing allergen-induced late responses and airway hyperresponsiveness (29). The study treated subjects with asthma with a blocking IL-5 monoclonal antibody and demonstrated a significant reduction in allergen-induced increases in blood eosinophils, but not late responses or airway hyperresponsiveness. The study used a parallel-group, double-blind, placebo-controlled design. This study was designed mainly to evaluate the tolerability of the monoclonal antibody, and may have been underpowered with the sample size entered into each treatment arm (n = 8) to reach any meaningful conclusions about the effects of the treatment on either the late response (30) or histamine airway hyperresponsiveness (31). Studies using IL-5–deficient mice have revealed that, in the absence of IL-5, eosinophils are reduced and eosinophil progenitors do not respond to IL-5; however, both IL-3 and granulocyte-macrophage colony-stimulating factor are capable of substituting for IL-5 and generating progenitor responses that can override the IL-5 deficiency and lead to a predominantly basophilic airway inflammation (28). Therefore, the importance of airway eosinophils and of IL-5 alone in the pathogenesis of allergen-induced airway responses in humans remains unresolved, given the biological redundancy of the cytokines IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor.

In conclusion, this study has demonstrated, for the first time, post-allergen inhalation increases in IL-5 mRNA in the bone marrow of subjects with asthma. In addition, the increase in IL-5 mRNA production occurred only in subjects developing a more pronounced airway eosinophilia after allergen challenge. It also demonstrates that T lymphocytes, either resident in or recruited to the bone marrow after challenge, may be an important source of IL-5 for subsequent enhanced eosinophil production. Further clarification of the contribution of these T lymphocytes, as well as the relevant eosinophilopoietic cytokines they may produce in vivo, is required.


   FOOTNOTES
 
Supported by a grant from the Canadian Institutes of Health Research.

Received in original form August 3, 2001; accepted in final form February 13, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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