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The Journal of Immunology, 2002, 168: 3017-3023.
Copyright © 2002 by The American Association of Immunologists

Pathogenesis of Murine Experimental Allergic Rhinitis: A Study of Local and Systemic Consequences of IL-5 Deficiency1

Hiroko Saito*, Koichiro Matsumoto*, Avram E. Denburg*, Lynn Crawford*, Russ Ellis*, Mark D. Inman*, Roma Sehmi*, Kiyoshi Takatsu{dagger}, Klaus I. Matthaei{ddagger} and Judah A. Denburg2,*

* Asthma Research Group, Division of Clinical Immunology and Allergy, Department of Medicine, McMaster University, Hamilton, Ontario, Canada; {dagger} Institute for Medical Sciences, Tokyo University School of Medicine, Tokyo, Japan; and {ddagger} John Curtin School of Medical Research, Canberra, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent studies have demonstrated an important role for IL-5-dependent bone marrow eosinophil progenitors in allergic inflammation. However, studies using anti-IL-5 mAbs in human asthmatics have failed to suppress lower airway hyperresponsiveness despite suppression of eosinophilia; therefore, it is critical to examine the role of IL-5 and bone marrow responses in the pathogenesis of allergic airway disease. To do this, we studied the effects of IL-5 deficiency (IL-5-/-) on bone marrow function as well as clinical and local events, using an established experimental murine model of allergic rhinitis. Age-matched IL-5+/+ and IL-5-/- BALB/c mice were sensitized to OVA followed by 2 wk of daily OVA intranasal challenge. IL-5-/- OVA-sensitized mice had significantly higher nasal mucosal CD4+ cells and basophilic cell counts as well as nasal symptoms and histamine hyperresponsiveness than the nonsensitized group; however, there was no eosinophilia in either nasal mucosa or bone marrow; significantly lower numbers of eosinophil/basophil CFU and maturing CFU eosinophils in the presence of recombinant mouse IL-5 in vitro; and significantly lower expression of IL-5R{alpha} on bone marrow CD34+CD45+ progenitor cells in IL-5-/- mice. These findings suggest that IL-5 is required for normal bone marrow eosinophilopoiesis, in response to specific Ag sensitization, during the development of experimental allergic rhinitis. However, the results also suggest that suppression of the IL-5-eosinophil pathway in this model of allergic rhinitis may not completely suppress clinical symptoms or nasal histamine hyperresponsiveness, because of the existence of other cytokine-progenitor pathways that may induce and maintain the presence of other inflammatory cell populations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Although the role of IL-5 in the differentiation, proliferation, and migration of eosinophils in allergic inflammation has been well documented (1, 2, 3, 4, 5, 6, 7, 8, 9), it remains unclear how critical IL-5 is to the development of clinical disease. Indeed, recent studies using anti-IL-5 mAbs in vivo in human asthmatic subjects have failed to confirm that IL-5 is both necessary and sufficient to cause lower airway hyperresponsiveness, even though it appears responsible for the development of blood and tissue eosinophilia (10, 11, 12). In animal models involving IL-5 deficiency and/or overexpression, there are only a few studies in which both pathological and clinical variables have been evaluated (13, 14, 15, 16, 17, 18, 19).

Recent reports (3, 4, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) have demonstrated an important role for the bone marrow as a source of eosinophils and other allergic inflammatory cells such as basophils, mast cells, or lymphocytes in upper or lower airway allergic inflammation. Bone marrow CD34+CD45+ progenitor cells are increased and phenotypically altered to preferentially differentiate—in response to allergen and IL-5—into eosinophils and basophils during the development of airway allergic disorders (14, 38, 39, 40, 41, 42). These progenitors migrate to airway tissues, providing a source for local expansion of eosinophils and basophils (5, 39, 40). Because it is possible that the failure of anti-IL-5 Abs to suppress asthmatic responses occurs because treatment is insufficient to control systemic progenitor responses, it becomes critical to carefully examine IL-5-dependent bone marrow responses in allergic airway disease, including whether or not IL-5 deficiency per se has any effect on this pathogenetic mechanism and the concomitant development of clinical disease. To do this, we have used a recently developed murine experimental allergic rhinitis model (14) to critically examine the pathological and clinical roles of IL-5.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and OVA sensitization

Age-matched (8- to 10-wk-old, N14.BALB/c, female and male, crossed to the BALB/c from the C57BL/6 strain for 14 generations) IL-5+/+ and IL-5-/- mice were placed into one of two groups: 1) OVA/OVA group, which was given OVA sensitization followed by 2 wk of OVA intranasal daily challenge (IL-5+/+ and IL-5-/- mice, n = 10 each), and 2) sham/sham group, which was given normal saline instead of OVA in the same schedule (IL-5+/+ and IL-5-/- mice, n = 10 each). Under pathogen-free conditions, mice in the OVA/OVA group were sensitized using OVA Ag as follows. A total of 40 µg/kg OVA (Sigma-Aldrich, St. Louis, MO) diluted by sterile normal saline with aluminum hydroxide gel (alum adjuvant, 40 mg/kg) were administered to unanesthetized animals four times by i.p. injection on days 1, 5, 14, and 21. This was followed by daily challenge with OVA diluted by sterile normal saline intranasally (20 µl of 25 mg/ml OVA per mouse) from day 22 to 35 (Fig. 1Go).



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  		FIGURE 1. Protocol for OVA sensitization and subsequent OVA intranasal challenge. Sham/sham mice were treated with diluent both during sensitization and challenge instead of OVA. In contrast, OVA/OVA mice were given daily OVA challenge intranasally from day 22 to 35 after OVA i.p. sensitization from day 1 to 21.

 
Clinical symptoms and specimens

Nasal symptoms were evaluated for each mouse in each group at the time points of days 28 and 35 by counting the number of sneezes and nasal itching motions (nasal rubbing) for 10 min after OVA intranasal provocation. Nasal histamine responsiveness (NHR)3 was also measured by determining the concentration of histamine which caused sneezing and itching and was expressed as the limiting concentration of histamine (log10 picograms per milliliter) as previously described (14). The mice in each group were euthanized by deep anesthesia using a solution that contained ketamine hydrochloride (Ketalean; Bimeda-MTC, Cambridge, Ontario, Canada) and Xylazine (Rompun; Bayer, Toronto, Ontario, Canada) diluted in normal saline, at 24-h postintranasal provocation. Nasal mucosa and bone marrow tissues were taken and immediately processed.

All procedures were performed in accordance with the ethical guidelines in the Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care and approved by the Animal Ethics Committee of McMaster University (Hamilton, Ontario, Canada).

Tissue preparation

Nasal mucosal tissues were treated by the following methods (previously described in Ref. 43). Briefly, specimens were cut into 3- to 4-mm3 pieces and fixed overnight in cold acetone containing protease inhibitors at -20°C before processing in glycolmethacrylate resin. The embedded tissues were cut into 4-µm thin sections using a microtome for ultra-thin sections (Ultra Cut; Leica Microsystems, Wetzlar, Germany) and recruited to the immunostaining procedure. Bone marrow cells were obtained from sternal or femoral bone marrow, suspended in McCoy’s 3+ culture medium, which was made from modified McCoy’s 5A medium and 15% FCS (Life Technologies, Grand Island, NY) with 1% penicillin-streptomycin and 0.35% 2-ME, as previously described (28). Total bone marrow cells, as well as mononuclear cells that were separated by density gradient centrifugation over LymphPrep (Nycomed, Oslo, Norway) for 25 min at 2000 rpm in room temperature, were diluted to a concentration of 5 x 105/ml with PBS, and cytocentrifuge slides were prepared (Cytospin 3; Thermo Shandon, Sewickly, PA) on Silane-coated glass slides. Another set of mononuclear cells, which were isolated as described from femoral bone marrow, were incubated in plastic flasks for 2 h at 37°C and 5% CO2 to remove adherent cells and then prepared for methylcellulose culture or immunofluorescence staining followed by flow cytometry analysis.

Immunostaining

For cell differential counts of bone marrow cells on cytocentrifuge slides, Diff-Quick stain (Dade Behring, Dudingen, Switzerland) was performed. To identify eosinophils and basophilic cells in the nasal mucosa, slides were fixed in acetone-methanol at room temperature for 10 min then stained in Diff-Quick; slides were then embedded in Permount (Fisher Scientific, Fair Lawn, NJ). To identify CD4+ lymphocytes in nasal mucosa and CD34+ cells in bone marrow cytocentrifuge preparations, a streptavidin-biotin complex immunostaining system method was used, using various anti-murine mAbs: anti-murine CD4 mAb (L3T4, rat IgG2a, {kappa}; BD PharMingen, Mississauga, Ontario, Canada) and anti-murine CD34 mAb (rat IgG2a, {kappa}; BD PharMingen) as previously described, with some modification (43). Briefly, glycolmethacrylate resin-embedded tissue slides and acetone-fixed cytocentrifuge slides were pretreated to inhibit endogenous peroxidase with a solution of 0.1% sodium azide and 0.3% hydrogen peroxide for 30 min, washed with TBS, then treated with TBS containing 1% Carnation skim milk (Nestle, Don Mills, Ontario, Canada) and 10% FBS (Life Technologies) for 30 min followed by incubation with saturated BSA (Sigma-Aldrich) in sterile water for 20 min and 10% normal goat serum in TBS to block nonspecific reactivity, followed by the application for 2 h of each mAb. Bound Abs were then labeled with biotinylated second-stage Abs appropriate to the isotype of each primary Ab (DAKO, Missisauga, Ontario, Canada) for 2 h, and detected using a streptavidin-biotin peroxidase detection system (StreptABComplex/HRP; DAKO). Aminoethylcarbazole (Sigma-Aldrich) was then applied as a chromogen and the sections were counterstained with Mayer’s hematoxylin (Sigma-Aldrich) to contrast with the red positive staining by aminoethylcarbazole. Negative control sections were similarly treated with the same isotype Ig Ab (DAKO) instead of the primary Ab.

Evaluation and quantitation of staining

In the lamina propria of the nasal mucosa, total numbers of cells expressing positive immunoreactivity for cellular surface markers or any of the intracellular cytokines were enumerated. The area of the nasal tissue was measured, excluding glands, using an eye piece with a grid: 10 high-power fields were randomly evaluated after the stained slides were coded by a person unconnected with the study and blinded from the investigator until all evaluations were complete. The cell count results were expressed as the number of cells per square millimeter of lamina propria. For bone marrow pathological evaluation, differential cell counts were performed of cytospin preparations of bone marrow cells after Diff-Quick stain, eosinophilic cells and basophilic cells on each slide were enumerated by light microscopy, 1000 bone marrow cells were counted, and the result was expressed as a percentage in total sternal bone marrow cells. CD34+ cells on bone marrow were also counted by light microscopy: 1000 mononuclear bone marrow cells were counted and the result was expressed as the percentage of positive cells in total mononuclear cells.

Bone marrow methylcellulose cultures

Nonadherent mononuclear cells (NAMNC) were cultured in 35 x 10-mm tissue culture dishes (Falcon Plastics; BD Biosciences Labware, Franklin Lakes, NJ) in culture medium, which was made up of 0.9% methylcellulose (Dow Chemical, Midland, Michigan), 20% FCS and Iscove’s Dulbecco’s medium (with 1% penicillin-streptomycin, 0.35% 2-ME, and 0.1% BSA) and the following recombinant mouse (rm) cytokines (R&D Systems, Minneapolis, MN): rmIL-5 (0.5, 1, 5, 10 ng/ml) with 1 x 105 NAMNC, rmIL-3 (5 ng/ml) with 5 x 104 NAMNC, or rmGM-CSF (5 ng/ml) with 2.5 x 104 NAMNC. With each batch of growth factor, dose response experiments were performed. After 6 days, colonies of >40 cells were counted using inverse microscopy and eosinophil/basophil (Eo/Baso)-CFU were classified using morphological and histological criteria (tight, compact, round refractile cell aggregates). To identify the differentiated cells from colonies as Eo/Baso-CFU, sample cells in each 10-day culture were evaluated, 3 ml of PBS was added to the sample in each culture dish, and then the sample was centrifuged at 1200 rpm for 10 min at 4°C. After the sample was resuspended in 1 ml of PBS, cytocentrifuge slides were created on Silane-coated glass slides and stained with Diff-Quick.

Immunofluorescence staining and flow cytometry analysis

Samples of 1 x 106 NAMNC derived from femoral bone marrow tissues at the time point of 24-h postintranasal provocation on day 35 were suspended in 100 µl of washing buffer, which contained PBS with 0.02% sodium azide, 0.02% EDTA, and 1% BSA, then incubated with anti-mouse CD32/CD16 Ab (Fc Block; BD PharMingen) for 15 min at 4°C to reduce Fc{gamma}II/IIIR-mediated Ab binding, which could contribute to background. This was followed by staining with saturating amounts of biotin-conjugated anti-murine IL-5R{alpha} Ab (T21, rat IgG2a, {kappa}; provided by Dr. K. Takatsu, Tokyo University, Tokyo, Japan) (44) or biotin-conjugated isotype-matched negative control for 10 min, then FITC anti-murine CD34 Ab (rat IgG2a, {kappa}; BD PharMingen) or isotype-matched negative control, and CyChrome anti-murine CD45 Ab (rat IgG2b, {kappa}; BD PharMingen) or isotype-matched negative control at each saturating concentration; samples were then incubated for 35 min at 4°C in the dark. The cells were washed with 3 ml of washing buffer, then incubated with streptavidin-conjugated PE (BD PharMingen) to label biotinized IL-5R{alpha}-positive cells for 30 min at 4°C in the dark after incubation with anti-mouse CD32/CD16 Ab for 15 min at 4°C in the dark to reduce binding by streptavidin-PE, which could contribute to background. The cells were washed with 3 ml washing buffer twice, then fixed in 500 µl of PBS plus 1% paraformaldehyde and kept at 4°C in the dark until analysis 24 h later. For analysis, FACScan (BD Biosciences, San Jose, CA) was used as a flow cytometer with flow cytometry analysis software (CellQuest from BD Biosciences and FlowJo from Tree Star, San Carlos, CA). To measure CD34+CD45+ progenitor cells a gating strategy was used as previously described (41, 42, 45), and IL-5R{alpha}-positive CD34+CD45+ progenitor cells were enumerated as a percentage of total CD34+CD45+ progenitor cells in the marrow.

Statistics

For all cell counts of stained slides, slides were read randomly and in blinded fashion. The Mann-Whitney U test and ANOVA followed by Student’s Neuman-Keuls test were used for comparison of data between groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Clinical symptoms

OVA-sensitized IL-5+/+ mice developed significant nasal symptoms of sneezing and nasal itching (rubs) during 2 wk of daily intranasal OVA challenge, as was previously reported (14). IL-5-/- mice in the OVA/OVA group had significant nasal symptoms only at day 35, the number of sneezes and rubs being significantly higher compared with the sham/sham group. IL-5+/+ mice developed sneezing significantly more quickly than IL-5-/-: means ± SE days after the first day of sensitization were 25.3 ± 0.3 for IL-5+/+ mice and 31.2 ± 0.7 for IL-5-/- mice (p < 0.0001) (Fig. 2Go). In the comparison between OVA-sensitized IL-5+/+ and IL-5-/- mice, there were significant differences in each symptom at day 28 (sneezing, p < 0.0001; itching, p < 0.0001) and in the OVA/OVA group (sneezing, p < 0.02; itching, p < 0.05).



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  		FIGURE 2. Nasal symptoms in IL-5+/+ and IL-5-/- mice. Clinical allergic nasal symptoms at days 0 (sham/sham group), 28, and 35 (OVA/OVA group) in OVA sensitization protocol. a, Sneezes. b, Nasal rubs. Each value represents mean ± SE.

 
Nasal histamine responsiveness

NHR correlated strongly with clinical symptoms (with sneezing, r = 0.691 and p < 0.0001; with itching, r = 0.811 and p < 0.0001) (Fig. 3Go). NHR in OVA/OVA IL-5+/+ or IL-5-/- mice was significantly higher than in sham/sham mice; however, in this case, too, IL-5-/- mice developed NHR, manifested by nasal symptoms, later than IL-5+/+ mice.



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  		FIGURE 3. NHR in IL-5+/+ and IL-5-/- mice. NHR is represented by the limiting concentration of histamine that caused sneezing and itching at days 0 (sham/sham group), 28, and 35 (OVA/OVA group) in OVA sensitization protocol.

 
Pathological changes in airway tissue

In IL-5+/+ mice, the numbers of allergic inflammatory cells in the nasal mucosa were increased in OVA/OVA mice (at 24-h postintranasal provocation on day 35) compared with sham/sham mice, including eosinophils (p < 0.01) and CD4+ cells (p < 0.05) but not basophilic cells (NS). In OVA/OVA IL-5-/- mice, at 24-h postintranasal provocation on day 35, significantly higher numbers of CD4+ lymphocytes (p < 0.02) and increased basophilic cell counts were observed compared with sham/sham IL-5-/- mice, without eosinophilia in the nasal mucosa, as shown in Table IGo.


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  		Table I. Pathological changes in OVA-sensitized mice1

 
Bone marrow analysis

In comparisons between the two groups, a significant increase in sternal bone marrow eosinophil counts was observed in OVA/OVA IL-5+/+ mice compared with sham/sham IL-5+/+ mice; no differences were seen between the IL-5-/- mice in the two groups (Fig. 4Go). Also, a significantly higher percentage of eosinophils in the bone marrow was detected in IL-5+/+ mice than in IL-5-/- mice in the OVA/OVA group (p < 0.01); however, there was no significant difference between IL-5+/+ mice and IL-5-/- mice in the sham/sham group. Basophilic cells and CD34+ cells also increased in the bone marrow in the OVA/OVA mice of both IL-5+/+ and IL-5-/- strains when compared with sham/sham mice. There was no significant difference in the number of basophilic cells or CD34+ cells between mice of either strain in the OVA/OVA group.



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  		FIGURE 4. Bone marrow changes in murine allergic rhinitis. The percentages of eosinophils (A), basophilic cells (B), and CD34+ cells (C) in the bone marrow. Hatched bar, result from the OVA/OVA group; open bar, result from the sham/sham group.

 
Eo/Baso-CFU analysis

In 6-day methylcellulose assays, the number of Eo/Baso-CFU that grew from NAMNC derived from murine bone marrow increased significantly in the presence of rmIL-5, rmIL-3, or rmGM-CSF in OVA/OVA, compared with sham/sham, IL-5+/+ mice (Fig. 5Go). In IL-5-/- mice, the number of Eo/Baso-CFU was higher in the OVA/OVA group than in the sham/sham group in the presence of rmIL-3 or rmGM-CSF in vitro; however, in the presence of rmIL-5 in vitro, the number of Eo/Baso-CFU did not increase significantly in the OVA/OVA group. Furthermore, in the comparison between IL-5+/+ and IL-5-/- mice, there were significantly higher numbers of Eo/Baso-CFU grown in the presence of rmIL-5 in OVA/OVA IL-5+/+ mice (p < 0.02), whereas in the presence of rmIL-3 or rmGM-CSF there were no significant differences between IL-5+/+ and IL-5-/- mice.



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  		FIGURE 5. Eo/Baso-CFU in 6-day methylcellulose cultures of murine bone marrow. Eo/Baso-CFU in the presence of 5 ng/ml each rmIL-5 (A), rmIL-3 (B), and rmGM-CSF (C). Hatched bar, result from the OVA/OVA group; open bar, result from the sham/sham group.

 
IL-5R{alpha} expression on CD34+CD45+ progenitor cells in murine bone marrow

As shown in Table IIGo, there were significant differences in IL-5R{alpha} expression on CD34+CD45+ progenitor cells between IL-5+/+ and IL-5-/- mice, in both sham/sham and OVA/OVA groups, with IL-5+/+ mice showing higher IL-5R{alpha} expression. Looking at IL-5+/+ mice across groups, the OVA/OVA group had significantly higher IL-5R{alpha} expression on CD34+CD45+ cells.


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  		Table II. IL-5R{alpha} expression on murine bone marrow CD34+CD45+ progenitor cells1

 
Eosinophil differentiation in 10-day methylcellulose bone marrow culture

As shown in Table IIIGo, sham/sham IL-5-/- mice had a significantly higher ratio of immature eosinophilic cells in 10-day cultures compared with sham/sham IL-5+/+ mice; along these lines, OVA/OVA IL-5-/- mice had significantly lower ratios of mature eosinophils compared with OVA/OVA IL-5+/+ mice.


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  		Table III. Eosinophil differentiation from murine bone marrow-derived NAMNC in 10-day methylcellulose semisolid culture assay in the presence of rmIL-5 (5 ng/ml)1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Eosinophilia has been studied as an important phenomenon in allergic disorders. There have been many studies of IL-5 as an important factor in eosinophilic inflammation, because this cytokine controls the differentiation, proliferation, and migration of eosinophils. However, other cytokines, such as GM-CSF, IL-4, and IL-13, or chemokines, such as eotaxin, also influence the inflammatory process (2, 9, 31, 46, 47, 48, 49, 50, 51, 52, 53). Whether eosinophils take part in the pathogenesis of allergic disease or behave as bystanders has been the subject of much discussion recently (10, 11, 12, 54). Views that cast doubt on the pathogenetic role of eosinophils have come from mainly clinical studies in which data have been gathered after a relatively short period of sensitization and Ag challenge, and in which protocols varied widely, making comparisons of the results difficult. In this study, for the first time, the roles of IL-5 and eosinophils in upper airway allergic inflammation were studied over a relatively protracted period.

In our model, significant nasal symptoms were surprisingly observed in IL-5-deficient animals, although these were delayed compared with wild-type mice. For a better understanding of the pathological basis for this clinical result, we measured NHR and inflammatory changes in the nasal mucosa. NHR was, like nasal symptoms, significantly higher in OVA/OVA compared with sham/sham IL-5-/- mice, but again this was significantly delayed compared with wild-type mice. The pathological changes observed in OVA/OVA IL-5+/+ mice were the same as previously reported (14): the numbers of eosinophils, basophilic cells, and CD4+ lymphocytes were increased, while IL-5-deficient mice showed no eosinophilia in either sham/sham or OVA/OVA groups and the numbers of other cellular populations, basophilic cells, and CD4+ lymphocytes were elevated and showed no significant differences when comparing OVA/OVA IL-5+/+ to IL-5-/- mice. These results demonstrate that eosinophils are not the only cells responsible for the development of NHR in allergic rhinitis; rather, nasal mucosal mast cells and basophils might be just as important in the expression of both NHR and clinical symptoms. Also, the possibility of differences between human and murine biology, including Eo/Baso function, should be considered. Although some papers have discussed comparisons of murine and human cell functions (55, 56, 57, 58), this is an area in which further work is needed to explore the contribution of these and other types of cells, such as macrophages, lymphocytes, neutrophils, and various epithelial cells, to allergic responses.

Previous reports from our group and others (3, 4, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 59) have shown that a systemic up-regulation of the bone marrow may play a pivotal role in the development and maintenance of not only lower, but also upper, airway allergic inflammation, as exemplified by asthma and allergic rhinitis, respectively. In the present study, we performed detailed examination of bone marrow cellular and molecular events, as well as function, in response to allergen sensitization and challenge in our model of experimental rhinitis. While marrow eosinophils, basophilic cells, and CD34+ cells were increased after Ag sensitization and nasal Ag challenges in wild-type mice, there was no increase of eosinophils in the marrow of IL-5-deficient mice after challenge. Nonetheless, all other inflammatory cell types were increased to the same extent as in wild-type mice, accompanied by the same nasal mucosal pathologic changes. Thus, though IL-5-/- deficiency resulted in suppression of Ag-dependent eosinophil progenitor differentiation (i.e., reduced bone marrow Eo/Baso-CFU), the actual number of progenitor cells was not lower at baseline than in wild-type mice. This defective functional response of eosinophil progenitors was attended by reduced IL-5R{alpha} expression on CD34+CD45+ bone marrow cells in IL-5 mice both before and after Ag sensitization and challenge, with defective up-regulation following Ag sensitization.

The functional consequences of reduced IL-5R{alpha} expression on CD34+CD45+ bone marrow cells in IL-5-/- mice were seen in 6-day methylcellulose assays. While the number of marrow Eo/Baso-CFU increased significantly in vitro in the presence of rmIL-5, rmIL-3, or rmGM-CSF in OVA/OVA IL-5+/+ animals, it was only in the presence of rmIL-3 or rmGM-CSF, but not IL-5 in vitro, that Eo/Baso-CFU increased in the OVA/OVA IL-5-/- mice. Related to this, in sham/sham IL-5-/- mice there was a significantly higher ratio of immature eosinophilic cells in colonies enumerated at day 10 compared with significantly higher ratios of mature to immature eosinophils in OVA/OVA IL-5+/+ mice. This poor response to IL-5 in vitro of both eosinophil colony numbers and eosinophil maturation within colonies is presumably a direct consequence of defective expression of eosinophil progenitor IL-5R{alpha} in response to Ag sensitization and nasal challenge in IL-5-deficient mice. One can speculate that IL-5 is required in vivo for the proper induction—as we have observed in human asthmatics (25)—of marrow progenitor, as well as mature eosinophil, expression of IL-5R{alpha}; this is in agreement with Tavernier’s recent demonstration that IL-5 can induce the expression of IL-5R on maturing eosinophils in vitro (60).

Because the numbers of Eo/Baso-CFU responsive to IL-3 or GM-CSF were not suppressed in IL-5-deficient mice, it appears that these latter cytokines can replace IL-5 functionally, giving rise to small numbers of eosinophils and, more importantly, to normal numbers of basophilic cells, which can probably account for the persistence of clinical symptomatology and nasal hyperresponsiveness, albeit delayed. Lantz et al. (61) have shown IL-3 dependency of basophilic responses in mice; whether upper airway inflammation and bone marrow responses are defective in IL-3-deficient mice remains to be investigated. To clarify the role of basophilic cells in the systemic and local pathogenesis of allergic rhinitis, investigations of IL-3 deficiency and/or IL-3 expression in this model would be an important future direction.

In conclusion, IL-5 is required for the development of tissue and marrow eosinophilia, the formation of Eo/Baso-CFU, and the early development of symptoms, but not for other components of the inflammatory response in murine experimental allergic rhinitis. Our findings confirm that IL-5 is required for normal bone marrow eosinophilopoiesis, in response to specific Ag sensitization during the development of experimental allergic rhinitis. However, the results also point out that the suppression of the IL-5-eosinophil pathway in the pathogenesis of allergic rhinitis (and also, by inference, asthma) may not fully suppress clinical symptoms or airway hyperresponsiveness due to the possible existence of other cytokine-progenitor pathways that may induce and maintain the presence of other inflammatory cell populations.



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  		FIGURE 6. Enumeration of bone marrow-derived CD34+CD45+ progenitor cells that express IL-5R{alpha}. Samples of bone marrow-derived NAMNC from OVA/OVA group mice were stained with CD34FITC/CD45CyChrome and either PE-linked IL-5R{alpha} or isotype control Ab. CD34+CD45+ progenitor cell populations that were analyzed by using a gating strategy were back-scattered onto a dot plot of IL-5R{alpha} vs side scatter (SSC). Quadrant statistics (i.e., data in lower right corner) are presented as the percentage of total CD34+CD45+ progenitor cells that demonstrated positive staining with anti-IL-5R{alpha} or rat IgG2a,{kappa} isotype-matched control Ab. A, Result from a mouse that is in the IL-5+/+ OVA/OVA group. B, Result from a mouse that is in the IL-5-/- OVA/OVA group.

 

   Acknowledgments
 
We thank Lynne Larocque for her help with the artwork and text.


   Footnotes
 
1 This work was supported by a grant from the Medical Research Council of Canada. H.S. is a recipient of a Medical Research Council/Canadian Thoracic Society/AstraZeneca Fellowship Award. Back

2 Address correspondence and reprint requests to Dr. Judah A. Denburg, Asthma Research Group, Division of Clinical Immunology and Allergy, Department of Medicine, McMaster University, HSC-3V46, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada. E-mail address: denburg{at}mcmaster.ca Back

3 Abbreviations used in this paper: NHR, nasal histamine responsiveness; NAMNC, nonadherent mononuclear cell; rm, recombinant mouse; Eo/Baso, eosinophil/basophil. Back

Received for publication September 14, 2001. Accepted for publication January 16, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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