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Published ahead of print on November 20, 2003, doi:10.1164/rccm.200307-1004OC
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American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 573-577, (2004)
© 2004 American Thoracic Society

Sputum CD34+IL-5R{alpha}+ Cells Increase after Allergen

Evidence for In Situ Eosinophilopoiesis

Sandra C. Dorman, Ann Efthimiadis, Irene Babirad, Rick M. Watson, Judah A. Denburg, Frederick E. Hargreave, Paul M. O'Byrne and Roma Sehmi

Firestone Institute of Respiratory Health, Asthma Research Group, St. Joseph's Healthcare, McMaster University, Hamilton, Ontario, Canada

Correspondence and requests for reprints should be addressed to Roma Sehmi, Ph.D., Firestone Institute for Respiratory Health, St. Joseph's Hospital, Luke Wing, Room L314-6, McMaster University, 50 Charlton Avenue East, Hamilton, ON, L8N 4A6 Canada. E-mail: sehmir{at}mcmaster.ca


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eosinophil lineage–committed progenitors increase in the bone marrow of subjects with asthma developing allergen-induced airway hyperresponsiveness and eosinophilia. Also, higher numbers of circulating eosinophil/basophil cfu have been demonstrated 24 hours after allergen inhalation and in bronchial and nasal biopsies of allergic individuals. These cells may undergo in situ eosinophilopoiesis, suggesting that after allergen inhalation, progenitor cells traffic from the bone marrow to the airways, providing an ongoing source of effector cells. To examine this possibility, CD34+ and CD34+IL-5R{alpha}+ cells were measured in induced sputum from allergic subjects with asthma at baseline and at 7 and 24 hours after allergen and diluent inhalation, using flow cytometry. Isolated early responders (n = 9) were contrasted to dual responders (n = 9), who develop allergen-induced sputum and blood eosinophilia and airway hyperresponsiveness, and to normal control subjects. At baseline, there were significantly fewer sputum eosinophils and CD34+ cells in normal control subjects compared with subjects with asthma. Sputum CD34+ cells increased at 7 hours after allergen inhalation in both groups of subjects with asthma, which was sustained at 24 hours in the dual responder group only, associated with sustained increases in sputum CD34+IL-5R{alpha}+ cells, eosinophils, and interleukin-5. These results indicate that eosinophil progenitors can migrate to the airways and may differentiate toward an eosinophilic phenotype.

Key Words: stem cell mobilization • asthma • airway

Activation of specific hemopoietic pathways within the bone marrow is associated with allergen-induced eosinophilic airway inflammation (1–3). In conjunction with the activation of eosinophilopoiesis, allergen inhalation challenge may also trigger the release of primitive and lineage-committed progenitors into the blood (4, 5). Evidence to support progenitor cell migration to the site of inflammation comes from recent studies on humans, where CD34 immunopositive cells have been extracted from human nasal polyp and explant tissue and shown to undergo interleukin (IL)-5–driven proliferation and differentiation into eosinophil/basophil colony-forming unit in vitro and ex vivo (6, 7), highlighting the presence of true blast cells. In addition, increased numbers of CD34+IL-5R{alpha}+ cells have been detected in asthmatic bronchial biopsy tissue (8).

It is not known whether progenitor cell numbers in the airway increase in response to allergen inhalation in subjects with asthma. Therefore, the aims of the current study were twofold. First, it was investigated whether there are time-dependent changes in the number of CD34+ and CD34+IL-5R{alpha}+ cells in the airways of subjects with asthma after allergen inhalation. Thus, sputum samples were induced before and 7 and 24 hours after allergen and diluent inhalation. Second, it was determined whether there is an association between progenitor cells and airway eosinophilia. Therefore, sputum samples were induced in dual responders, isolated early responders, and normal control subjects. Some of these results have been reported previously in an abstract (9).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study Design
Nineteen subjects with mild, atopic asthma and baseline FEV1 greater than 70% predicted participated. Postallergen, isolated early responders (n = 9) developed a fall in FEV1 greater than 20% from baseline between 0 and 2 hours, whereas dual responders developed an early and late fall (FEV1 > 15%) from baseline between 3 and 7 hours (n = 9). Subjects were nonsmokers, using inhaled ß2-agonists intermittently (not used 8 hours before each visit), and had neither respiratory infection nor altered allergen levels 2 weeks before study. Also, six subjects without atopy and asthma were enrolled. This study was approved by the Ethics Committee of McMaster University Health Sciences Centre, and subjects gave written, informed consent before study entry.

Allergen/diluent challenges were randomized with a 2-week washout period between. Subjects attended the laboratory on six occasions: at Visits 1 and 4, medical history was documented and skin-prick test sensitivity to allergen extracts (Visit 1 only), spirometry, methacholine inhalation test, and sputum induction were performed; at Visits 2 and 5, allergen/diluent challenge and sputum induction at 7 hours postchallenge were performed; at Visits 3 and 6, methacholine inhalation challenge and sputum induction were performed.

Normal control subjects visited the laboratory once, when medical history was documented and skin prick test sensitivity, spirometry, and sputum induction were performed.

Allergen–Diluent and Methacholine Inhalation Challenge
Allergen challenge was performed as described by O'Byrne and coworkers (10), and the concentration of allergen extract was determined from a formula using skin prick test and PC20 (provocative concentration of methacholine causing a 20% drop in FEV1) (11). Diluent control was physiologic saline.

Methacholine inhalation was performed as described by Cockcroft and coworkers (12).

Sputum Induction
Sputum was induced by hypertonic saline inhalation and processed according to Pizzichini and coworkers (13). Cytospins were prepared and means of duplicate slides (400 cells/slide) were expressed as absolute counts (104 cells/ml). Supernatants were separated by centrifugation and stored at -70°C. IL-5 levels were quantified using commercially available ELISAs (Pharmingen, San Diego, CA). The lower limit of detection was 4 pg/ml.

Flow Cytometry
Progenitors were stained and identified according to Sehmi and coworkers (2). True CD34+ blast cells were identified as cells with CD34high/CD45dull staining, as described previously, and expressed as absolute cell counts per milliliter of sputum sample. Cytokine receptor antibodies were biotinylated, as described previously (2). Nonneutralizing monoclonal antibodies directed against the {alpha}-subunits of IL-5R (IL-5R{alpha}; {alpha}16) was a kind gift from Dr. Jan Tavernier (Ghent University, Belgium).

Statistical Analysis
Results were expressed as mean ± SEM, except for PC20, which was expressed as geometric mean ± %SEM. PC20, IL-5, and sputum values were log10 transformed to fit a normal distribution before analysis. The allergen-induced changes from baseline in PC20, CD34+ cell numbers, CD34+IL-5R{alpha}+ cells, cytokine protein levels, and sputum eosinophils were analyzed using a repeated measures analysis of variance (between treatment: allergen vs. diluent; between group: isolated early vs. dual responders vs. normal control subjects; within group: baseline vs. 7 hours vs. 24 hours; Statistica 5.1; Statsoft, Tulsa, OK). Correlations were analyzed using Spearman's {rho}. Statistical significance was accepted as p values less than 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Airway Responses
The baseline FEV1 values were similar on all study days. Dual responders had a greater fall in FEV1 between 3 and 7 hours after inhaled allergen and a significant decrease in PC20 24 hours after inhaled allergen (p < 0.05), which was not demonstrated in isolated early responders. No changes occurred after diluent challenge in either group (Table 1) .


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  		TABLE 1. Subject allergen and diluent-induced airway responses

 
Eosinophils
Both isolated early and dual responders had significantly more eosinophils at baseline compared with normal control subjects, being 1.0 ± 0.34 x 104/ml (p < 0.01), 1.4 ± 0.53 x 104/ml (p < 0.01), and 0.02 ± 0.093 x 104/ml, respectively (Table 2) .


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  		TABLE 2. Summary of baseline data from induced sputum

 
After allergen inhalation, in dual responders, eosinophils increased from 1.6 ± 0.51 x 104/ml preallergen to 15.4 ± 5.72 x 104/ml at 7 hours (p < 0.01) and to 14.7 ± 4.03 x 104/ml at 24 hours (p < 0.001) postallergen. In addition, cell numbers increased at 7 hours (p < 0.01) and at 24 hours (p < 0.01), compared with diluent inhalation. Isolated early responder eosinophils increased from 0.78 ± 0.38 x 104/ml preallergen to 11.9 ± 8.68 x 104/ml at 7 hours (p < 0.01) and to 5.8 ± 2.12 x 104/ml at 24 hours (p < 0.005) postallergen (Figure 1) . There was a trend for a difference between the groups in the sputum eosinophil numbers at 24 hours (p = 0.07).



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  		Figure 1. Comparison of sputum eosoinophil numbers at baseline (BL) and at 7 and 24 hours after diluent (open/stippled) and allergen (hatched/solid) inhalation challenge. Sputum eosinophils significantly increased at 7 and 24 hours postallergen inhalation compared with BL and with diluent challenge in both isolated early and dual responders. *p Values less than 0.01, comparison with BL; {dagger}p values less than 0.01, comparison with diluent values.

 
CD34+ Cells
Both isolated early and dual responders had more CD34+ cells at baseline compared with normal control subjects, being 33 ± 17.3/ml, 27 ± 7.4/ml, and 0.9 ± 0.48/ml (p < 0.05), respectively (Table 2).

After allergen inhalation, dual responder CD34+ cells increased from 31 ± 11.4/ml preallergen to 79 ± 20.4/ml at 7 hours (p < 0.05) and to 114 ± 26.5/ml at 24 hours (p < 0.05) postallergen. In addition, cell numbers increased at 7 hours (p < 0.05) and at 24 hours (p < 0.05) compared with diluent inhalation (Figure 2A) . In contrast, isolated early responders showed no change in CD34+ cells postallergen compared with baseline. However, compared with diluent challenge, there was a significant increase in the numbers of CD34+ cells at 7 hours (p < 0.05) followed by a significant decrease at 24 hours (p < 0.001) (Figure 2A).



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  		Figure 2. Comparison of sputum CD34+ cells (upper panel) and CD34+IL-5R{alpha}+ cells (lower panel) at baseline (B) and at 7 and 24 hours after diluent (open/stippled) and allergen (hatched/solid) inhalation challenge. CD34+ and CD34+IL-5R{alpha}+ cells significantly increased at 7 and at 24 hours postallergen inhalation, compared with B and with diluent challenge in dual responders. In isolated early responders, CD34+ cells increased at 7 hours and decreased at 24 hours compared with diluent challenge, whereas CD34+IL-5R{alpha}+ cells were significantly increased at B and at 7 hours compared with diluent challenge. Dual responders also had significantly more CD34+IL-5R{alpha}+ cells at 24 hours compared with isolated early responders. *p Values less than 0.05, comparison with B; {dagger}p values less than 0.05, comparison with diluent values; {ddagger}p values less than 0.001, comparison between groups.

 
There was also a trend for a difference between groups at 24 hours (p = 0.086). There was a significant positive relationship between CD34+ cells and eosinophils measured by the area under the curve from baseline to 24 hours (r = 0.48; p < 0.05).

CD34+IL-5R{alpha}+ Cells
No significant differences occurred in baseline CD34+IL-5R{alpha}+ cell numbers between groups (Table 2). After allergen inhalation, dual responder CD34+IL-5R{alpha}+ cells increased from 2 ± 0.88/ml preallergen to 14 ± 4.24/ml at 7 hours (p < 0.05) and to 48 ± 14.7/ml at 24 hours (p < 0.01) postallergen. In addition, CD34+IL-5R{alpha}+ cells increased at 7 hours (p < 0.05) and at 24 hours (p < 0.05), compared with diluent inhalation (Figure 2B). In contrast, isolated early responders showed no change in CD34+IL-5R{alpha}+ cells postallergen compared with baseline. However, compared with diluent challenge, there was a significant increase in CD34+IL-5R{alpha}+ cells at baseline (p < 0.005) and at 7 hours (p < 0.005). Dual responders also had significantly more CD34+IL-5R{alpha}+ cells at 24 hours compared with isolated early responders (p < 0.001) (Figure 2B). There was a significant positive relationship between CD34+IL-5R{alpha}+ cells and eosinophils, measured by the area under the curve from baseline to 24 hours (r = 0.51; p < 0.05).

IL-5 Protein
There were no significant differences in baseline IL-5 levels between groups. Dual responders demonstrated significantly more IL-5 protein at 7 and 24 hours postallergen compared with diluent challenge. Levels increased from 6.3 ± 0.78 pg/ml preallergen to 9.8 ± 1.68 pg/ml at 7 hours (p < 0.05) and to 9.2 ± 0.94 pg/ml at 24 hours postallergen (p = 0.05) and were 6.2 ± 0.75 pg/ml prediluent to 6.7 ± 0.55 at 7 hours and 6.8 ± 0.67 pg/ml at 24 hours postdiluent. In contrast, isolated early responders showed no significant differences in IL-5 protein levels.

Dual responders also had significantly more IL-5 protein in the sputum supernatant at 24 hours compared with isolated early responders (p < 0.01) (Figure 3) . The change in IL-5 protein levels at 7 hours correlates positively with the change in IL-5R{alpha} expression at 24 hours in both groups (r = 0.49; p < 0.005).



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  		Figure 3. Comparison of sputum interleukin (IL)-5 protein levels at baseline (BL) and at 7 and 24 hours after diluent (open/stippled) and allergen (hatched/solid) inhalation challenge. IL-5 levels significantly increased at 7 and 24 hours postallergen compared with diluent challenge in dual responders only. Dual responders also had significantly more IL-5 protein at 24 hours compared with isolated early responders. {dagger}p Values less than 0.05, comparison with diluent values; {ddagger}p values less than 0.01, comparison between groups.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The novel findings of this study are: (1) CD34+ cells can be detected in human sputum; (2) the proportion of airway CD34+ cells is greater in subjects with asthma compared with normal control subjects and fluctuates after allergen inhalation challenge; (3) the proportion of CD34+ cells expressing IL-5R{alpha} increases in dual responder subjects with asthma only; and (4) this change in IL-5R{alpha} expression occurs in conjunction with increases in IL-5 protein levels. These findings indicate that in subjects with asthma, after allergen inhalation, trafficking of CD34+ progenitors to the airways occurs, with upregulation of IL-5R{alpha} expression, which, in the presence of IL-5, may mediate the in situ differentiation of progenitor cells into eosinophils.

Although changes in the levels of sputum progenitor cells would suggest the trafficking of progenitors from the peripheral circulation, one cannot disregard the possibility of in situ proliferation of progenitors. Although previous studies would suggest that a systemic source of the progenitors is likely, namely bone marrow via the blood, the current study is limited by the lack of peripheral blood data collected from the same subjects.

The development of both early- and late-asthmatic responses after allergen inhalation is known to be associated with increases in circulating eosinophils (14), greater increases of activated eosinophils in the airway (15), activation of eosinophilopoiesis in the bone marrow (6), and the development of airway hyperresponsiveness (16), when compared with isolated early responders. Contrasting these two groups allows for a useful clinical model of increased eosinophilic airway responses and associated airway functional changes. The current study suggests that the greater increases in eosinophils seen in the dual responders may in part be due to the in situ production of eosinophils by CD34+ cells. Although the number of CD34+ cells measured is small when compared with total sputum numbers, this is not surprising considering that progenitors are self-sustaining and differentiate into colonies of cells.

Robinson and coworkers (8) have examined bronchial biopsies and confirmed both the presence of true blast cells and an increase in progenitor cells in subjects with atopy and asthma and subjects with atopy but without asthma compared with normal control subjects, concluding that the increase in CD34+ cells was a feature of atopy. The data from the current study confirm these findings because both groups of subjects with asthma demonstrated significantly more CD34+ cells at baseline compared with normal control subjects and extends them, demonstrating a rapid increase in CD34+ cells, potentially reflecting an influx from the circulation as early as 7 hours and sustained for 24 hours after allergen inhalation challenge in dual responders.

In the same article, Robinson and coworkers (8) also demonstrated an increase in CD34+IL-5R{alpha} messenger RNA+ cell numbers in the airways of subjects with asthma only, compared with subjects with atopy but without asthma and normal control subjects. They concluded that the ability to respond to IL-5 was a feature of asthma. The present study was unable to show differences in the amount of IL-5R{alpha} expression at baseline between groups. However, we did not observe the intracellular messenger RNA levels.

Kim and coworkers (6) have confirmed the presence of CD34+ cells in nasal polyps and, using methylcellulose clonogenic assays, showed that nasal polyp tissue contained myeloid colony-forming cells. In addition, Cameron and coworkers (7), using an ex vivo allergen challenge model from nasal mucosa, showed an increase in eosinophils, suggesting that these cells had differentiated locally in an IL-5–dependent process. This study corroborates these findings and demonstrates an increase in CD34+IL-5R{alpha}+ cells in the airways after allergen inhalation. We have as yet not been able to show the presence of true blast cells because sputum culture is hampered by treatment with sputolysin and its lack of sterility. However, these results support the concept that local tissue differentiation and expansion of eosinophils may occur in asthma affecting humans.

IL-5 is likely to be a central cytokine in the development of tissue eosinophilia in asthma. Studies have shown that IL-5 protein levels are increased in induced sputum after allergen inhalation challenge (17, 18), and this study confirms these findings. IL-5 is also a potent eosinophilopoietin (19), and expression of membrane-bound IL-5R{alpha} is believed to be the phenotype of the earliest eosinophil lineage–committed progenitor (2). In the present study, the increase in IL-5 was positively correlated with the delayed upregulation of the IL-5R{alpha} on CD34+ cells in the dual responder group at 24 hours, supporting the hypothesis that IL-5 upregulates its own receptor and suggesting the in situ differentiation of these cells in the presence IL-5.

Recently, anti–IL-5 therapy has been developed to block eosinophil production, migration, and activation in allergic asthma. Although this therapy has been shown to be effective in reducing circulating eosinophil numbers (20, 21), it has not been shown to be equally effective in reducing bone marrow and airway tissue eosinophils. Flood-Page and coworkers (21) demonstrated a median percent reduction of only 50% for both airway tissue and bone marrow eosinophils. In a related study, Menzies-Gow and coworkers (22) extended these findings, showing that anti–IL-5 therapy had no effect on blood or bone marrow CD34+ cell numbers but decreased CD34+IL-5R{alpha} messenger RNA+ cells in the bronchial mucosa. Although eosinophilopoiesis is primarily driven by IL-5, other cytokines, including granulocyte–macrophage colony stimulating factor and IL-3, also induce eosinophil production (19). The presence of CD34+ cells in the airways capable of responding to other inflammatory cytokines may explain why anti–IL-5 could not completely abolish airways eosinophilia.

Under normal conditions of hemopoiesis, the bone marrow acts as a site for the turnover and traffic of mature leukocytes to the peripheral circulation. However, in inflammatory conditions such as atopic asthma, there is an increased release of both mature eosinophils and bone marrow eosinophil lineage–committed progenitor cells. Although the precise mechanisms that trigger progenitor cell traffic remain to be fully elucidated, this study showed for the first time that there are increased numbers of CD34+ cells in the sputum of subjects with asthma and that these cells increase after allergen inhalation. In addition, in the presence of IL-5, phenotypic changes occur in the expression of IL-5R{alpha}, together supporting a potential for local differentiation of progenitors in the airways.


   Acknowledgments
 
The authors thank George Obminski and Shauna Denis for their technical assistance and Joceline Otis for graphic production in this study.


   FOOTNOTES
 
Supported by the Canadian Institutes for Health Research and the Canadian Lung Association/Ontario Thoracic Society.

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: S.C.D. has no declared conflict of interest; A.E. has no declared conflict of interest; I.B. has no declared conflict of interest; R.M.W. has no declared conflict of interest; J.A.D. has no declared conflict of interest; F.E.H. has no declared conflict of interest; P.M.O'B. has no declared conflict of interest; R.S. has no declared conflict of interest.

Received in original form July 22, 2003; accepted in final form November 19, 2003


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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