Involvement of Eosinophilopoiesis in A Murine Model of

Eosinophilopoiesis in A Murine Model of Allergic Airway Eosinophilia: Involvement of Bone Marrow IL-5 and IL-5 Receptor α This information is current as of June 13, 2013.

Masafumi Tomaki, Lin-Ling Zhao, Joachim Lundahl, Margareta Sjöstrand, Manel Jordana, Anders Lindén, Paul O'Byrne and Jan Lötvall J Immunol 2000; 165:4040-4050; ; http://www.jimmunol.org/content/165/7/4040

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Eosinophilopoiesis in A Murine Model of Allergic Airway Eosinophilia: Involvement of Bone Marrow IL-5 and IL-5 Receptor ␣1 Masafumi Tomaki,* Lin-Ling Zhao,* Joachim Lundahl,† Margareta Sjo¨strand,* Manel Jordana,‡ Anders Lindeń,* Paul O’Byrne,§ and Jan Lo¨tvall2*

A

irway allergen challenge causes cellular inflammation in the airways, which is dominated by eosinophils in both humans (1–3) and mice (4). It has been suggested that eosinophils contribute to several of the clinical features of allergic asthma, including tissue damage and airway hyperresponsiveness (5– 8), which are thought to be mediated by tissue-damaging enzymes and proteins, including eosinophil cationic protein and major basic protein (9, 10). Furthermore, eosinophils also have the capacity to release substantial amounts of bronchoconstrictor mediators, such as leukotrienes (11, 12). Recently, some studies have shown that the bone marrow is stimulated in asthmatic patients (13–15). Studies in dogs (16 –18), mice (4, 19 –21), and humans (15, 22–24) suggest that increased granulocytopoiesis is induced during exacerbation of asthma-like processes, such as inflammation induced by allergen exposure. However, the degree of the contribution of the bone marrow to the

*The Lung Pharmacology Group, Department of Respiratory Medicine and Allergology, Institute of Heart and Lung Diseases, Go¨teborg University, Gothenburg, Sweden; †Department of Clinical Immunology, Karolinska Hospital, The Karolinska Institute, Stockholm, Sweden; and Departments of ‡Pathology and Molecular Medicine and §Medicine, McMaster University, Hamilton, Canada Received for publication April 1, 1999. Accepted for publication July 12, 2000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1

This work was supported in part by the Swedish Heart and Lung Foundation, the Vårdal Foundation of Sweden, GlaxoWellcome (U.K.), and the Medical Research Council (Canada). The microscopic equipment was funded by the Tornspiran Foundation. 2 Address correspondence and reprint requests to Dr. Jan Lo¨tvall, The Lung Pharmacology Group, Department of Respiratory Medicine and Allergology, Institute of Heart and Lung Diseases, Go¨teborg University, Guldhedsgatan 10A, 413 46 Gothenburg, Sweden. E-mail address: [email protected]

Copyright © 2000 by The American Association of Immunologists

airway inflammatory process, and the mechanisms by which the bone marrow is stimulated, are still poorly understood. IL-5 has been shown to be crucial for the induction of airway eosinophilia, because mice lacking IL-5 (IL-5 knockout mice) fail to develop airway eosinophilia in response to allergen exposure unless the animals are reconstituted with IL-5 (25, 26). Furthermore, treatment of wild-type mice with a neutralizing anti-IL-5 Ab strongly reduces the eosinophilic inflammatory response induced by allergen (19, 27–32). The aim of this study was to determine the kinetics and contribution of eosinophilopoiesis to the airway eosinophilia induced by airway allergen exposure and to determine the role and site of action of IL-5 in this response. To do this, we chose to use mice sensitized and airway exposed to OVA. A thymidine analog, 5-bromo-2⬘-deoxyuridine (BrdU),3 which is incorporated into DNA during the S phase of the cell cycle, was used to pulse-label newly produced inflammatory cells (33) at different time points during repeated allergen exposure. Intracellular staining of IL-5 was performed in bone marrow cells and quantified by flow cytometry. The capacity of bone marrow cells to release IL-5 protein was quantified in vitro after magnetic separation. The role and site of action of IL-5 in the allergen-induced eosinophilia were investigated by pretreatment with a neutralizing anti-IL-5 Ab, given either by airway or systemic route. To further elucidate the capacity of IL-5 to have effects in different tissues, immunocytochemical staining of the IL-5R ␣-chain in bone marrow and airways was performed.

Materials and Methods Animals This study was approved by the Animal Ethics Committee in Gothenburg, Sweden, as well as by the Animal Research Ethics Board of McMaster 3 Abbreviations used in this paper: BrdU, 5-bromo-2⬘-deoxyuridine; i.n., intranasal(ly); BAL, bronchoalveolar lavage; PLP, periodate-lysin-paraformaldehyde.

0022-1767/00/$02.00


The airway inflammation in asthma is dominated by eosinophils. The aim of this study was to elucidate the contribution of newly produced eosinophils in airway allergic inflammation and to determine mechanisms of any enhanced eosinophilopoiesis. OVAsensitized BALB/c mice were repeatedly exposed to allergen via airway route. Newly produced cells were identified using a thymidine analog, 5-bromo-2ⴕ-deoxyuridine, which is incorporated into DNA during mitosis. Identification of IL-5-producing cells in the bone marrow was performed using FACS. Bone marrow CD3ⴙ cells were enriched to evaluate IL-5-protein release in vitro. Anti-IL-5-treatment (TRFK-5) was given either systemically or directly to the airways. IL-5R-bearing cells were localized by immunocytochemistry. Repeated airway allergen exposure caused prominent airway eosinophilia after three to five exposures, and increased the number of immature eosinophils in the bone marrow. Up to 78% of bronchoalveolar lavage (BAL) granulocytes were 5-bromo-2ⴕ-deoxyuridine positive. After three allergen exposures, both CD3ⴙ and non-CD3 cells acquired from the bone marrow expressed and released IL-5-protein. Anti-IL-5 given i.p. inhibited both bone marrow and airway eosinophilia. Intranasal administration of anti-IL-5 also reduced BAL eosinophilia, partly via local effects in the airways. Bone marrow cells, but not BAL eosinophils, displayed stainable amounts of the IL-5R ␣-chain. We conclude that the bone marrow is activated by airway allergen exposure, and that newly produced eosinophils contribute to a substantial degree to the airway eosinophilia induced by allergen. Airway allergen exposure increases the number of cells expressing IL-5-protein in the bone marrow. The bone marrow, as well as the lung, are possible targets for anti-IL-5-treatment. The Journal of Immunology, 2000, 165: 4040 – 4050.

The Journal of Immunology University, Hamilton, Canada. Male BALB/c mice, 5– 6 wk old, were purchased from B&K Universal AB (Sollentuna, Sweden) and Charles River Laboratories (Ottawa, Ontario, Canada). The mice were maintained under conventional animal housing conditions and provided with food and water ad libitum.

Sensitization and allergen exposure protocol

In vivo protocol for evaluation of eosinophilopoiesis To elucidate the kinetics of cellular inflammation, we labeled newly produced inflammatory cells using BrdU. Animals were given BrdU (Boehringer Mannheim Scandinavia, Bromma, Sweden) at a dose of 1 mg in 0.25 ml of PBS i.p. twice (8 h apart, total dose 2 mg/animal) on different occasions during the repeated allergen exposure period (day 1, 3, or 5). On each day, the first injection of BrdU was performed on awake mice 1 h before the OVA exposure, and the second injection was given 7 h after the exposure. The control animals received PBS i.p. at the same time points. Three different types of treatments were given. First, to evaluate whether BrdU would influence the response to repeated i.n. OVA exposures, we exposed two groups of animals to OVA on five consecutive days (days 1 to 5), and one of these groups was injected with BrdU on two occasions, 8 h apart, on day 1. The other group was given PBS at corresponding time points. Second, to determine the time course of airway inflammation, bone marrow eosinophilia, and BrdU staining after repeated allergen exposure, these responses were evaluated in four groups of animals, exposed to either OVA or PBS on two, three, or five consecutive days. Third, to more closely determine the kinetics of the accumulation of new cells in the airways, six groups of animals were used for the examination of the effect of the timing of BrdU injection on BrdU staining in BAL and bone marrow cells. Three of these were given OVA i.n. on five consecutive days, and three were given PBS. BrdU was given i.p. to one OVA and one PBS group either on day 1, 3, or 5. Again, two injections of BrdU were given 8 h apart on each day. The groups given BrdU on day 1 are the same as in the second treatment procedure (see above). For each study group, cells were harvested 24 h after the last allergen exposure.

In vivo protocol for in vitro experiments (FACS and CD3⫹ cell enrichment) Bone marrow cells were collected a day after the third of three OVA exposures on consecutive days. These cells were washed, and RBC were eliminated by centrifugation over a 65% Percoll gradient at 1500 rpm in room temperature for 30 min. Cells were washed two to three times in PBS, and further processed for flow cytometry or magnetic separation and subsequent cytokine-release experiments (see below). Bone marrow cells from three to eight animals were pooled for further processing and analysis. We chose to collect the cells after three allergen exposures because it was clear that the bone marrow had reached an activation state at this time, evident as increased numbers of eosin staining cells.

In vivo protocol for anti-IL-5 treatment The effects of a neutralizing anti-IL-5 Ab treatment on the bone marrow and airway inflammatory responses induced by repeated allergen exposure were investigated in three series of experiments. Animals were divided into twelve groups, which were all exposed to OVA on five consecutive days. One hour before the OVA exposure, animals were given anti-mouse IL-5 mAb (clone TRFK-5; R&D Systems Europe, Abingdon, U.K.) or its iso-

type control, rat IgG1 (clone R3-34; PharMingen, San Diego, CA), by different routes (either by i.n. instillation, or by i.p. injection) and at different doses. First, six groups were pretreated i.p. with either TRFK-5 (1–100 ␮g/animal in 0.5 ml of PBS) or rat IgG1 (100 ␮g/animal in 0.5 ml of PBS). Second, four additional groups were repeatedly (total of five times) pretreated i.n. with either TRFK-5 (6 –50 ␮g/animal in 25 ␮l of PBS) or rat IgG1 (20 ␮g/animal in 25 ␮l of PBS) 1 h before each OVA exposure on five consecutive days. Third, two separate groups were pretreated i.p. with either TRFK-5 (100 ␮g/animal) or rat IgG1 (100 ␮g/ animal) and were also injected with BrdU (1 mg/animal ⫻ 2, 8 h apart, i.p.) on the same day (day 1).

Cell collection and sample processing All samples were collected 24 h after the last OVA or PBS exposure. The animals were anesthetized with a mixture of xylazine (130 mg/kg) and ketamine (670 mg/kg) i.p. When the animals were in adequately deep anesthesia, the chest was opened and the animals were bled by penetration of the heart. After tracheostomy, BAL was performed by instilling 0.25 ml of PBS through the tracheal cannula, followed by gentle aspiration. This was repeated with 0.2 ml of PBS, and fluid from both lavages was pooled and kept on ice until further processing. Approximately 0.4 ml of the instilled fluid was consistently recovered. Then, one femur was removed, the attached muscle was gently scraped off and both ends of the femur were cut open. The bone marrow cells were removed by perfusion of the femur with 1.5 ml of PBS. The bone marrow cell suspension was also kept on ice until further processing. The total cell numbers in BAL and bone marrow samples were determined using standard hematologic procedures. Cytospins of BAL and bone marrow samples were prepared and stained with MayGru¨nwald-Giemsa stain for differential cell counts. Cell differential was determined by counting 300 –500 cells using a light microscope (Zeiss Axioplan 2; Carl Zeiss, Jena, Germany). The cells were identified using standard morphological criteria, and mature vs immature eosinophils were determined by nuclear morphology, May-Gru¨nwald-Giemsa staining properties, and cytoplasmic granulation, as previously described by Lee et al. (34). The cytospin preparations for immunocytochemistry were fixed with 4% paraformaldehyde in PBS for 20 min or with periodate-lysin-paraformaldehyde (PLP) for 10 min, and then washed in 15% sucrose in PBS for 10 min. After these procedures, the preparations were air-dried overnight, and stored at ⫺80°C until further examination. BAL supernatant and serum samples were also stored at ⫺80°C.

Immunocytochemical detection of BrdU-labeled cells BrdU incorporated into cellular DNA in cytospin preparations of BAL and bone marrow was detected with immunocytochemistry, using a mouse mAb against BrdU. The paraformaldehyde-fixed cytospin preparations were washed with TBS and subjected to digestion in 0.1% trypsin and 0.1% CaCl2 in PBS at 37°C for 15 min. The slides were incubated in 4 M HCl for 15 min to denature the DNA, followed by neutralization and blocking of nonspecific binding sites with incubation buffer (0.5% BSA and 0.1% Tween 20 in PBS). The slides were then incubated with 1 U/ml of alkaline phosphatase-conjugated mouse anti-BrdU mAb (clone BMG6H8; Boehringer Mannheim) in incubation buffer at 37°C for 30 min. After washing, the alkaline phosphatase reaction was developed for 15 min, using Fast Red Substrate System (No. 1496549; Boehringer Mannheim). The slides were counterstained with Mayer’s hematoxylin for 1 min. All slides were evaluated with the light microscope (Zeiss Axioplan 2) in random fields of view by a “blinded” experimenter. Cells with any nuclear red staining were counted as BrdU-labeled cells. Three hundred cells in both BAL and bone marrow cytospin preparations were evaluated.

Immunocytochemical detection of bone marrow CD3⫹ cells CD3⫹ bone marrow cells were identified using a mAb against the specific cell surface marker CD3⑀ for mouse T lymphocytes. The PLP-fixed cytospin preparations were washed with TBS and treated with 1:20 dilution of normal rabbit serum (No. X902; Dako, Glostrup, Denmark) in incubation buffer (1% BSA and 0.1% saponin in TBS) at room temperature for 15 min. The slides were incubated with 1.7 ␮g/ml of rat anti-CD3⑀ Ab (clone CD3-12; Cedarlane Laboratories, Hornby, Ontario, Canada) or isotype control, rat IgG1 (clone R3-34; PharMingen) in incubation buffer at 4°C overnight. After washing with TBS-0.1% saponin, incubation with 1:100 dilution of rabbit anti-rat Ig (No. Z494; Dako) and 2% normal mouse serum (No. X910; Dako) was followed by a treatment with 1:50 dilution of rat monoclonal APAAP complex (No. D488; Dako) at room temperature for each 10 min, and these were repeated twice. After washing with TBS, the alkaline phosphatase reaction was developed for 15 min, using New Fuchsin Substrate System (No. K698; Dako). The slides were counterstained with Mayer’s hematoxylin for 1 min. All slides were blindly evaluated on


All mice were immunized on two different days (5 days apart) by i.p. injections of 0.5 ml alum-precipitated Ag containing 8 ␮g of OVA (SigmaAldrich Sweden AB, Tyreso¨, Sweden) bound to 4 mg of aluminum hydroxide (Sigma-Aldrich) in PBS. Eight days after the second sensitization, the animals were rapidly and briefly anesthetized using CO2 gas, and received intranasal (i.n.) administration with 100 ␮g of OVA in 25 ␮l of PBS, or PBS alone. These OVA or PBS exposures were performed on 2, 3, or 5 consecutive days (from days 1 to 2, 3, or 5). One day after the last of the OVA exposures, samples (bronchoalveolar lavage (BAL) fluid, bone marrow cells, and blood) were collected. In a preliminary experiment, we examined the efficiency of i.n. administered solutions to reach the lungs by giving Evans blue dye (n ⫽ 3). Three min after i.n. administration of 25 ␮g of Evans blue dye in 25 ␮l of PBS, we isolated trachea, bronchi, lungs, esophagus, stomach, and nose tissue. The content of dye was quantified in each organ by spectrophotometry, after extraction of dye in formamide. The relative amount of dye recovered in each organs in relation to the total amount of given dye were; 30.7 ⫾ 9.7% in trachea, bronchi, and lungs; 12.7 ⫾ 10.2% in esophagus and stomach; and 25.7 ⫾ 4.6% in nose.

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EOSINOPHILOPOIESIS IN ALLERGIC AIRWAY INFLAMMATION

the light microscope (Zeiss Axioplan 2) in random fields of view. Five hundred cells were counted.

FACS analysis of bone marrow cells

In vitro culture of CD3⫹ cells and measurements of IL-5 release Bone marrow from OVA-exposed mice (on three consecutive days) was used, and cells from three to eight animals were pooled. CD3⫹ cells were enriched from the bone marrow using a magnetic cell sorting system (MACS; Miltenyi Biotec, Germany). Bone marrow cells, depleted of RBC and granulocytes in a single cell suspension in PBS (with 2 mM EDTA and 0.5% BSA), were labeled with a biotinylated mAb directed to CD3⫹ lymphocytes (CD3⑀ 145-2C11; PharMingen) in a concentration of 0.5 ␮g/ml. After washing the cells, 10 ␮l of streptavidin magnetic microbeads (MACS)/107 cells were added, according to the manufacturer’s recommendation. The magnetic labeled CD3⫹ cells were enriched on a positive selection column over a magnetic field. The enrichment procedure resulted in ⬃90% CD3⫹ cells, according to immunocytochemical staining. The enriched CD3⫹ cell fraction and also the cell fraction depleted of CD3⫹ cells (CD3-negative) were cultured in RPMI 1640 medium (Life Technologies, Ta¨by, Sweden) complemented with 10% FCS, 1% penicillin-streptomycin, 1% sodium pyruvate, and 2 mM L-glutamine (all obtained from Sigma-Aldrich) in a concentration of 0.25 ⫻ 106 cells/200 ␮l of medium in a 96-well plate. Two different types of experiments were performed. First, the CD3-positive and -negative fractions were added directly to the wells together with calcium ionophore (end concentraction 1 ␮M) and PMA (end concentration 2 ng/ml), and incubated for 24 h in a humidified incubator at 37°C with 5% CO2. The culture media were taken off and stored at ⫺80°C until analysis of IL-5 content. Second, unseparated bone marrow cells were added to the wells in a concentration of 2 ⫻ 106 cells/ml, and adherent cells were allowed to attach to the plastic for 2 h. The wells were washed several times with medium to remove unattached cells, and the CD3-positive and -negative fractions were added and incubated as above. The culture media were then collected. IL-5 in the culture media, BAL fluid, and serum were measured using a commercial available mouse IL-5 ELISA, using the manufacturer’s instructions (Endogen, Woburn, MA). The detection limit for IL-5 was 5 pg/ml.

IL-5R ␣-chain immunocytochemistry For specific identification of the IL-5R ␣-chain, a rabbit anti-mouse polyclonal IL-5R ␣-chain Ab (No. RTIL5RACabr; Research Diagnostics, Flanders, NJ) was used. PLP-fixed BAL and bone marrow cytospin preparations were washed in TBS and treated with 1:20 dilution of normal swine serum (No. X901; Dako) in TBS supplemented with 1% BSA (TBSBSA) at room temperature for 15 min to avoid unspecific binding. The slides were incubated with 0.1 ␮g/ml of anti-IL-5R ␣-chain Ab in TBSBSA at room temperature for 1 h. After rinsing, slides were incubated with a secondary Ab, biotinylated F(ab⬘)2 of swine anti-rabbit Igs (No. E431; Dako), at a concentration of 2.1 ␮g/ml in TBS-BSA with 10% normal mouse serum (No. X910; Dako) at room temperature for 30 min. After further washing, slides were treated with 1:100 dilution of streptavidin-␤galactosidase conjugate (No. 1112481; Boehringer Mannheim) at room temperature for 30 min. The ␤-galactosidase activity was visualized by a ␤-Gal Staining set (No. 1828673; Boehringer Mannheim) at 37°C for 40 min. The slides were counterstained with Mayer’s hematoxylin for 1 min. The counterstaining was kept fairly weak to avoid interaction with the specific staining. The number of IL-5R ␣-chain-positive cells was assessed by counting of 300 cells on the light microscope (Zeiss Axioplan 2) in random fields of view. To test specificity of the immunostaining, a control peptide for competition (No. RTIL5RA-CP; Research Diagnostics) was used. To the anti-IL-5R Ab, the peptide Ag were added at a 30 ⫻ weight excess, and then preincubated at room temperature for 2 h. The Ab-peptide solution was then added at the primary step and further processed as described above. This peptide totally blocked the positive staining, arguing for the specificity of the immunostaining of IL-5R ␣-subunit.

Statistical analysis All data are expressed as mean ⫾ SEM. Statistical analysis was conducted using nonparametric ANOVA (Kruskal-Wallis test) to evaluate variance among more than two groups. If a significant variance was found, an unpaired two-group test was used to determine significant differences between individual groups. Between two groups of animals, only an unpaired two-group analysis was performed, without a preceding Kruskal-Wallis test. p ⬍ 0.05 was considered statistically significant.

Results Time course of influx of inflammatory cells into airways during repeated allergen exposure Intranasal administration of allergen (OVA 100 ␮g per day) to sensitized mice on two, three, or five consecutive days induced increases in mainly eosinophils, and to a lesser degree neutrophils in BAL, as compared with sensitized mice exposed to PBS (Table I). The number of BAL eosinophils was sequentially increased after two, three, and five consecutive days of OVA exposure. Neutrophils were significantly increased in BAL on days 3 and 4 only.

Quantification of rat IgG1 in TRFK-5-treated mice

Quantification of newly produced granulocytes in airways after repeated allergen exposure

Levels of rat IgG1 in serum and BAL fluid from TRFK-5-treated animals were measured by the ELISA method. ELISA plates (Maxisorp F96; Nunc,

Administration of BrdU i.p. to sensitized mice on day 1 of allergen exposure did not affect the numbers or the profile of inflammatory


For the double-staining procedure (CD3 surface/IL-5 intracellular), the following mAbs and corresponding isotype-matched control Abs were used: PE-conjugated anti-CD3⑀ (clone 145-2C11) and FITC-conjugated antiIL-5 (clone TRFK-5) were both purchased from PharMingen Canada (Mississauga, Ontario, Canada). Both Abs were used in saturation concentrations. Isotype-matched control Abs were run in parallel to determine unspecific binding, and this was used to define the cut-off for positively labeled cells, which was 99% of the cells labeled with respective control Ab. Surface staining was performed as follows: To block unspecific binding, cell suspensions were initially incubated at 4°C for 20 min with PBS supplemented with 5% mouse/rat sera, and thereafter incubated at 4°C for 30 min with PE-anti-CD3⑀ or isotype control Ab, and then washed twice. Subsequent intracellular staining was performed as described previously, using a cell membrane permeabilization technique, the FOG (paraformaldehyde-fixation with subsequent n-octyl-␤-D-glucopyranoside treatment)method; Ref. 35). Surface-immunostained cells were fixed in 4% paraformaldehyde at 20°C for 5 min, followed by one wash in PBS and subsequent incubation with the detergent n-octyl-␤-D-glucopyranoside (0.74%; Sigma Chemical Company, St. Louis, MO) at 20°C for another 6 min, followed by one wash. After incubation with PBS supplemented with 5% mouse/rat sera (to block intracellular unspecific staining), permeabilized cells were incubated with either FITC-anti-IL-5 or corresponding isotype-matched control Ab at 4°C for 30 min, followed by two washes. To confirm that accurate permeabilization was obtained, intracellular binding of antivimentin Abs was measured. Over 90% of cell populations subjected to permeabilization stained positive for intracellular vimentin as previously described (35). The cells were finally resuspended in PBS supplemented with 1% paraformaldehyde and analyzed using a FACScan flow cytometer equipped with an argon ion laser (Becton Dickinson Instrument Systems, Mississauga, Ontario, Canada). The instrument was calibrated with Standard Brite (Flow Cytometry Standards, San Juan, Puerto Rico) to ensure the same fluorescence level in each experiment. After compensation settings were established, a quadrant was set based on the isotype controls, and the percentage of CD3⫹/IL-5⫹ cells were calculated. Off-line analysis was performed using the PC lysis software supplied by Becton Dickinson.

Naperville, IL) were incubated with 50 ␮l of mouse anti-rat IgG1 mAb (clone B46 –2; PharMingen) at a concentration of 2 ␮g/ml in binding solution (0.1 M Na2HPO4, adjusted to pH 9.0 with 0.1 M NaH2PO4) at 4°C for 18 h. After discarding the coating solution, the remaining binding sites on the plate were blocked with 10% FBS in TBS at room temperature for 2 h. The plates were then washed with TBS containing 0.05% Tween 20, and 100 ␮l of mouse serum or BAL supernatant, undiluted or diluted between 1:2 and 1:32 with ELISA buffer (10% FBS and 0.05% Tween 20 in TBS) were added to each well. The plates were incubated at 4°C for 18 h. After washing, 100 ␮l of biotin-conjugated mouse anti-rat IgG1 mAb (clone RG11/39.4; PharMingen) at a concentration of 1 ␮g/ml in ELISA buffer was added and incubated at room temperature for 1 h. After further washing, the plates were incubated with 1:2000 dilution of alkaline phosphatase-conjugated streptavidin (No. D396; Dako). The plate-bound alkaline phosphatase activity was detected with p-nitrophenyl phosphate substrate (Sigma 104; Sigma-Aldrich) and assessed with an ELISA reader (type 349; Labsystems, Stockholm, Sweden) at 405 nm. A standard curve was obtained with serial dilutions of purified rat IgG1 (clone R3–34; PharMingen), and results were expressed as nanograms of purified rat IgG1 per milliliter of serum or BAL supernatant. The detection limit for rat IgG1 was 8 ng/ml.

The Journal of Immunology

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Table I. Numbers of inflammatory cells in BAL after repeated airway allergen exposurea

Exposure

Total No. of Cells (⫻ 104/ml BAL fluid)

OVA ⫻ 2

11.56 ⫾ 2.27

OVA ⫻ 3

22.74 ⫾ 6.82

OVA ⫻ 5

32.68 ⫾ 5.74**

PBS ⫻ 5

12.69 ⫾ 2.33

Macrophages

4.65 ⫾ 0.92* (42.86 ⫾ 5.96**) 9.27 ⫾ 1.88† (52.86 ⫾ 14.66*) 13.45 ⫾ 1.99†† (48.16 ⫾ 9.52**) 10.99 ⫾ 1.78 (88.07 ⫾ 3.67)

Neutrophils

Eosinophils

Lymphocytes

2.86 ⫾ 1.79* (21.61 ⫾ 11.06**) 1.56 ⫾ 0.57* (6.60 ⫾ 1.50†) 0.48 ⫾ 0.18† (1.58 ⫾ 0.51††) 0.48 ⫾ 0.29 (2.54 ⫾ 1.22)

2.28 ⫾ 1.17* (20.44 ⫾ 8.13) 10.55 ⫾ 4.62* (35.13 ⫾ 12.99**) 16.78 ⫾ 5.60*† (43.82 ⫾ 9.08**) 0.30 ⫾ 0.11 (2.92 ⫾ 1.20)

1.77 ⫾ 0.97 (15.09 ⫾ 7.66) 1.31 ⫾ 0.57 (5.22 ⫾ 1.22†) 1.96 ⫾ 0.42 (6.44 ⫾ 1.42) 0.92 ⫾ 0.45 (6.46 ⫾ 2.27)

a BALB/c mice sensitized to OVA were subjected to repeated allergen exposure (OVA; 100 ␮g i.n. on two to five consecutive days). Control animals were sensitized to OVA and then administered PBS i.n. on five consecutive days. BAL was performed 24 h after the last allergen exposure. Data represent mean ⫾ SEM (n ⫽ 5–9 per point). Data in parentheses represent differential counts in percentages. *, p ⬍ 0.05 and **, p ⬍ 0.01 compared with PBS ⫻ 5-exposed group; †, p ⬍ 0.05 and ††, p ⬍ 0.01 compared with OVA ⫻ 2-exposed group (Mann-Whitney U test).

Time-course of induction of bone marrow eosinophilia during repeated allergen exposure The number of bone marrow cells staining positive for eosin was markedly increased after i.n. administration of OVA (100 ␮g per day) on three (10.9 ⫾ 1.8% of total bone marrow cells, p ⬍ 0.05 compared with PBS-exposed animals) and five consecutive days (11.8 ⫾ 1.9%, p ⬍ 0.01) in the sensitized mice (Fig. 3). The number of eosin-staining cells with immature morphology was more clearly increased. Effect of timing of BrdU injection on the BrdU staining of granulocytes in BAL The number of BrdU-staining cells was significantly increased in the BAL in animals injected with BrdU on day 1 or 3, but not when BrdU was injected on day 5 (Fig. 4). In a separate group of sensitized mice, BrdU was injected on both days 1 and 3 of OVA exposure, and the relative number of granulocytes stained for BrdU in BAL was 78.0 ⫾ 4.1%.

In the bone marrow, ⬃30 – 60% of the total cells incorporated BrdU into their nuclei after BrdU injection on various timings during repeated allergen exposure (Table III). No significant difference in total bone marrow BrdU staining was induced by OVA exposure as compared with PBS exposure.

IL-5 production in bone marrow cells after repeated allergen exposure For measurements of IL-5, and for localization of IL-5-producing cells isolated from the bone marrow, we chose to examine samples 24 h after three exposures on consecutive days, because a maximal bone marrow eosinophilia was observed at this time point (Fig. 3). The concentration of IL-5 was increased in both serum and BAL fluid after repeated allergen exposure (OVA 100 ␮g on three consecutive days) in sensitized mice (233 ⫾ 87 and 152 ⫾ 26 pg/ml in serum and in BAL fluid, respectively) but undetectable in both compartments in PBS-exposed mice. CD3⫹ cells were detected in the bone marrow by immunocytochemistry, using a mAb directed against CD3⑀ (Fig. 5). The relative number of CD3⫹ cells in the bone marrow was not significantly increased in OVA- vs PBS-exposed mice (0.69 ⫾ 0.16 and 0.49 ⫾ 0.10%, respectively; p ⫽ 0.40). Flow cytometry using intracellular staining of IL-5 (Fig. 6A) revealed that OVA-exposed mice had significantly more bone marrow cells expressing both intracellular IL-5 staining and CD3 cell surface staining (Fig. 6B). However, non-CD3-cells in the same gate also showed significantly increased intracellular staining for IL-5. Furthermore, when the bone marrow cells were stimulated for 6 h with PMA and calcium ionophore, a further increase in intracellular IL-5 staining was observed, mainly in the OVA-exposed group, in both cell compartments.

Table II. The effect of BrdU treatment on cell profiles in the bone marrow and BAL after repeated airway allergen exposurea Bone Marrow

Treatment

Total no. of cells

BrdU

10.28 ⫾ 0.90

PBS

11.78 ⫾ 2.29

Immature eosinoMature eosinophilic cells phils (%) (⫻ 106/femur) (%)

BAL

Total number of cells

0.88 ⫾ 0.12 0.28 ⫾ 0.06 32.68 ⫾ 5.74 (8.93 ⫾ 1.35) (2.89 ⫾ 0.58) 0.98 ⫾ 0.28 0.40 ⫾ 0.13 24.93 ⫾ 10.32 (8.20 ⫾ 1.13) (3.27 ⫾ 0.89)

Macrophages

Neutrophils (⫻ 104/ml BAL fluid) (%)

13.45 ⫾ 1.99 0.48 ⫾ 0.18 (48.16 ⫾ 9.52) (1.58 ⫾ 0.51) 8.14 ⫾ 1.11 0.52 ⫾ 0.32 (45.24 ⫾ 9.86) (1.60 ⫾ 0.63)

Eosinophils

Lymphocytes

16.78 ⫾ 5.60 (43.82 ⫾ 9.08) 14.26 ⫾ 8.58 (45.64 ⫾ 8.84)

1.96 ⫾ 0.42 (6.44 ⫾ 1.42) 2.00 ⫾ 0.94 (7.46 ⫾ 2.31)

a BALB/c mice sensitized to OVA were subjected to repeated airway allergen exposure (OVA; 100 ␮g i.n. on five consecutive days). BrdU was given i.p. twice on the first day of OVA exposure, 1 h before and 7 h after the exposure. Vehicle control animals received PBS i.p. at corresponding time points. BAL and recovery of bone marrow cells were performed 24 h after the last of five allergen exposures. Data represent mean ⫾ SEM (n ⫽ 9 and 5 per point). Data in parentheses represent differential counts in percentages.


cells in either bone marrow or BAL after repeated OVA exposure on five consecutive days (Table II). Exposure to OVA on two, three, and five consecutive days progressively increased the number of BrdU-labeled granulocytes in a time-dependent manner (Fig. 1). The number of unlabeled granulocytes was not significantly different among these three OVAexposed groups, but increased in comparison with the PBS-exposed group. The relative number of eosinophils in relation to the total number of granulocytes was 44.4, 87.1, and 97.2% after two, three, and five consecutive days of OVA exposure, respectively, showing that a dominant portion of the granulocytes are eosinophils. Photomicrographs of BrdU-stained BAL cells from one OVA- and one PBS-exposed mouse are shown in Fig. 2.

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FIGURE 1. Time course of allergen-induced BAL granulocytes content during repeated allergen exposure (OVA; 100 ␮g i.n. on two to five consecutive days) in BALB/c mice sensitized to OVA. The relative numbers of eosinophils in relation to the total number of granulocytes were 44.4, 87.1, and 97.2% after two, three, and five consecutive days of OVA exposure, respectively. The figure shows both cells staining for BrdU (f), and unstained cells (䡺). The relative numbers of BrdU-positive cells at different time points are 27.5, 49.6, and 64.3% after two, three, and five OVA exposures, respectively. BrdU was injected twice on the first day of OVA exposure, 1 h before and 7 h after the exposure. Data are shown as mean ⫾ SEM. Kruskall-Wallis test shows significant variance among groups. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01 compared with PBS ⫻ 5-exposed group; †, p ⬍ 0.05 compared with OVA ⫻ 2-exposed group (Mann-Whitney U test).

Magnetically enriched bone marrow CD3⫹ cells (0.25 ⫻ 106 cells/well in the presence of adherent cells) from sensitized allergen-exposed mice cultured in vitro in the presence of PMA and calcium ionophore released IL-5 protein into the culture medium (185.8 ⫾ 43.6 pg/ml). Bone marrow mononuclear cells magnetically depleted of CD3⫹ cells and stimulated with PMA and calcium ionophore in the presence of adherent bone marrow cells also released IL-5 protein (106.5 ⫾ 20.5 pg/ml). Adherent bone marrow cells by themselves released much smaller quantities of IL-5 into the culture medium (24.3 ⫾ 6.0 pg/ml). Effect of anti-IL-5 Ab on bone marrow and airway eosinophilia The levels of rat IgG1 in serum and in BAL after i.p. or i.n. administration of TRFK-5 are shown in Fig. 7. Intraperitoneal administration of anti-IL-5 (TRFK-5; 1–100 ␮g/animal i.p. given on day 1) dose-dependently attenuated the increase of eosinophils in both BAL and bone marrow induced by repeated i.n. administration of allergen (OVA 100 ␮g on five consecutive days; Figs. 8A and 9A). The attenuating effect of systemically administered antiIL-5 was associated with strongly attenuated BrdU-staining of BAL granulocytes (2.8 ⫾ 1.4 vs 41.0 ⫾ 15.8 ⫻ 104 cells/ml BAL fluid in TRFK-5-treated and rat IgG1-treated mice, respectively; p ⬍ 0.05). Intranasal administration of different doses of TRFK-5 also dose-dependently attenuated the increase of eosinophils in BAL induced by repeated i.n. administration of allergen (OVA 100 ␮g on five consecutive days; Fig. 8B). This effect was associated with some degree of inhibition of the bone marrow eosinophilia induced by allergen (Fig. 9B), but only at doses causing concentration of Ab in serum exceeding ⬃50 ng/ml (Fig. 7). When comparing effects of i.p. vs i.n. administration of TRFK-5, BAL eosinophilia at doses of Ab giving similar concen-

FIGURE 2. Photomicrograph of BAL cells staining positively for BrdU in sensitized mice exposed repeatedly to PBS (on five consecutive days; A, original magnification ⫻400) or OVA (100 ␮g on five consecutive days; B, original magnification ⫻400). Positive BrdU staining is red. High magnification (⫻1000, C) of BAL cells from OVA-exposed mice shows different degrees of staining (strong staining, 1; intermediate staining, 2; weak staining, 3). In PBS-exposed mice, some BrdU staining in macrophages could be found. In OVA-exposed mice, a substantial number of BrdU-positive granulocytes could be found. These granulocytes are mainly eosinophils (Table I). The mean data for BrdU staining are shown in Fig. 1.

trations of rat IgG1 in serum and BAL can be compared. Thus 100 ␮g of i.p. TRFK-5 given once causes a similar concentration of BAL rat IgG1 compared with 6 ␮g of i.n.TRFK-5 given five times (Fig. 7B), but the effect of the i.p. treatment on BAL eosinophilia is much more prominent (Fig. 8, A and B), arguing that the systemic effect of anti-IL-5 can influence local airway eosinophilia. However, 10 ␮g of i.p. TRFK-5 given once causes similar concentration of serum rat IgG1 to 50 ␮g of i.n.TRFK-5 given five times, but the effect of the i.n. treatment on BAL eosinophilia is


slightly more prominent, arguing that there is also an effect of anti-IL-5 locally in the airways. Staining of IL-5R in bone marrow and airways Immunocytochemical staining of the murine IL-5R ␣-chain (CD␻125) was prominent in cytospins of bone marrow cells (Fig. 10A) from sensitized and OVA-exposed mice (OVA ⫻ 5), whereas no such staining could be found in BAL eosinophils from the same mice (Fig. 10B), despite prominent BAL eosinophilia. The relative number of cells in the bone marrow staining positively for the IL-5R were 37.2 ⫾ 11.4% in nonsensitized nonexposed mice, 40.6 ⫾ 3.8% in sensitized OVA-exposed mice, and 39.3 ⫾ 4.0% in sensitized PBS-exposed mice (n ⫽ 5 per group, no significant variances among groups). Furthermore, we detected no difference in the relative number of cells with weak or intense staining (intensity scale 0 –2). For example, OVA-exposed animals had 16.3 ⫾ 3.9% of bone marrow cells with high intensity staining, and the corresponding value for PBS-exposed animals was 16.5 ⫾ 3.9%.

Discussion In this study, we have demonstrated three novel findings in the mouse, showing 1) that newly produced eosinophils contribute to a substantial degree to the airway eosinophilia induced by repeated allergen exposure, 2) that IL-5 expression in both CD3⫹ and nonCD3-cells acquired from the bone marrow is increased after allergen exposure, and that these cells have the capacity to release IL-5 protein, and 3) that the expression of the IL-5R ␣-chain is more prominent in bone marrow cells than in BAL eosinophils. Furthermore, the inhibitory effect of systemically administered anti-IL-5 (TRFK-5) on eosinophilic inflammation in the airways is parallel with a strong reduction of the bone marrow eosinophil content. Also, local treatment with anti-IL-5 delivered directly to the airways revealed that anti-IL-5 has some inhibitory effects on airway eosinophilia via local mechanisms in vivo. Overall, these data sug-

FIGURE 4. Effect of timing of BrdU injection on allergen-induced BAL granulocyte BrdU staining during repeated allergen exposure (OVA; 100 ␮g i.n. on five consecutive days) in BALB/c mice sensitized to OVA. BrdU was injected either on day 1, day 3, or day 5 of OVA exposure period. The figure shows both cells staining for BrdU (f) and unstained cells (䡺). BrdU injection on day 1 resulted in the highest relative staining, whereas injection on day 5 (24 h before BAL) resulted in almost no staining of BAL cells. Data shown as mean ⫾ SEM. Kruskall-Wallis test shows significant variance among groups. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01 compared with PBSexposed group with BrdU injection on the same day as each group (MannWhitney U test).

gest that the eosinophilopoiesis is involved in the induction of airway eosinophilic inflammation induced by airway allergen exposure, and that release of IL-5 from cells within the bone marrow, acting on specific IL-5R on bone marrow cells, is involved in this process. Repeated allergen exposure results in an accumulation of eosinophils in the airways over several days, both in humans (36) and in sensitized mice (Table I). In the present experiments in a murine model of allergic airway inflammation, eosinophils are already evident in the airways 24 h after two allergen exposures on consecutive days, and after the third exposure, newly produced eosinophils significantly contribute to this inflammatory response (Table I and Fig. 1). We have proven this by detecting increased numbers of BrdU-labeled granulocytes in the airways at these time points. BrdU is incorporated into the cells by replacing thymidine, and its presence is proven by immunocytochemical staining of nuclei (33, 37, 38). Importantly, we are unable to distinguish BrdU-stained eosinophils from neutrophils, because the BrdU staining using Fast Red Substrate system is incompatible with eosin staining. However, in these experiments, the number of eosinophils in the airways is much larger than the number of neutrophils, proving that most of the BrdU-stained granulocytes are eosinophils. BrdU will stain all newly produced cells if present in the microenvironment at the time of mitosis. Furthermore, BrdU can be carried over to several new generations of cells (39). Therefore, it seems likely that the strongly BrdU-positive cells that we have detected in BAL were passing through their latest mitosis at a time of high concentrations of circulating BrdU (Fig. 2). Cells with weaker staining could be either cells produced at times of low concentrations of BrdU or cells having passed through several mitoses before having fully matured. The experiments using BrdU-injection at a single time point suggest that at least 64% of the granulocytes present in BAL after five allergen exposures on consecutive days are newly


FIGURE 3. Time course of allergen-induced relative bone marrow eosinophil content during repeated allergen exposure (OVA; 100 ␮g i.n. on two to five consecutive days) in BALB/c mice sensitized to OVA. The figure shows both eosin-staining cells with immature morphology (f) and cells with more mature morphology (䡺). A close to maximal degree of bone marrow eosinophilia was observed already after three allergen exposures. Data are shown as mean ⫾ SEM. Kruskall-Wallis test shows significant variance among groups. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01 compared with PBS ⫻ 5-exposed group; †, p ⬍ 0.05; ††, p ⬍ 0.01 compared with OVA ⫻ 2-exposed group (Mann-Whitney U test).

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Table III. BrdU staining in bone marrow cells after injection of BrdU on different time points during repeated airway allergen exposurea Exposure

OVA ⫻ 2 OVA ⫻ 3 OVA ⫻ 5 PBS ⫻ 5 OVA ⫻ 5 PBS ⫻ 5 OVA ⫻ 5 PBS ⫻ 5

BrdU Injection Day

% of BrdU-Positive Cells (%)

1 1 1 1 3 3 5 5

59.8 ⫾ 1.8 50.4 ⫾ 10.6 44.1 ⫾ 8.1 33.0 ⫾ 4.7 45.6 ⫾ 4.9 46.9 ⫾ 5.6 56.1 ⫾ 12.9 60.2 ⫾ 5.3

a BALB/c mice sensitized to OVA were subjected to repeated airway allergen exposure (OVA; 100 ␮g i.n. on two to five consecutive days). Control animals were sensitized to OVA and then administered PBS i.n. on five consecutive days. BrdU was given i.p. twice (8 h apart) either on day 1, day 3, or day 5 of the exposure period. Recovery of bone marrow cells was performed 24 h after the last allergen exposure. Data represent mean ⫾ SEM (n ⫽ 5–9 per point).

marrow that contained measurable levels of intracellular IL-5 protein (Fig. 6B). Furthermore, both bone marrow CD3⫹ cells and non-CD3 cells were able to release IL-5 protein in vitro in the presence of adherent mononuclear cells. Together these data suggest that cells expressing IL-5 protein are present in the bone marrow after repeated airway allergen exposure, confirming a previous study showing increased expression of IL-5 mRNA in the bone marrow after airway allergen exposure in the mouse (42). Our data extend this observation by showing that these cells can release IL-5 protein when stimulated in vitro. The previous study has also suggested that there is an increased total number of CD3⫹ cells within the bone marrow after airway allergen exposure, which we also have seen in C57BL/6 mice (our unpublished data) but were unable to confirm in these experiments using BALB/c mice. We observed much fewer CD3⫹ cells in the bone marrow compared with Minshall and colleagues, who used a polyclonal anti-CD3 Ab, whereas we used a monoclonal anti-CD3⑀ Ab. Despite these differences in methodology, these three studies suggest that any increase of the total number of T lymphocytes in the bone marrow after airway allergen exposure may be small. Overall, the data argue that airway allergen exposure enhances IL-5 production in both CD3⫹ cells and non-CD3 cells present in the bone marrow and, therefore, that both of these cell types may contribute to the stimulation of the eosinophilopoiesis. Also, the CD3⫹ cells are rare in the bone marrow, whereas other IL-5-producing cells are more frequent, again arguing for a role of non-CD3 cells, such as CD34⫹ cells (42) in the local IL-5 release in the bone marrow. Even though we find pronounced production of IL-5 in bone marrow cells, it is clear that IL-5 is produced in several tissue compartments, including the lungs (43– 46). Also, in these experiments, we were able to detect increased levels of IL-5 in both BAL fluid and serum after repeated allergen exposure. It is possible that increased serum level of IL-5 may originate from several tissues, including lungs, local lymph nodes, and bone marrow. However, the exact site of action of anti-IL-5 on allergen-induced airway eosinophilia has not been clearly described in several previous studies in which anti-IL-5 was administered systemically (27–32). A recent study has shown that the anti-IL-5 Ab delivered via the airway route attenuates allergen-induced eosinophilia in BAL and lung tissues (47). However, in their study it is difficult to clearly distinguish the local effect of anti-IL-5 from the systemic effect because i.n. administration of anti-IL-5 Ab resulted in a high concentration of the Ab in the circulation, which was above the reported serum concentration needed to inhibit BAL eosinophilia


produced. This may be a low estimation because when BrdU was injected both on days 1 and 3 of allergen exposure, almost 80% of the granulocytes in the airways were BrdU-positive. Importantly, the BAL eosinophilia observed during the early phase of allergen exposure is likely to involve a storage pool of eosinophils that were produced before the first allergen exposure, because a majority of BAL granulocytes after two exposures were not BrdU-positive (Fig. 1). Also, these experiments suggest that the accumulation of a majority of the newly produced eosinophils in the airways will take ⬎24 h during repeated allergen exposure because injection of BrdU 24 h before BAL resulted in few BrdUlabeled cells in BAL (Fig. 4). It is possible that some of these newly produced eosinophils may have completed their proliferation and maturation outside the bone marrow. Some studies have shown that eosinophil colony-forming activity is increased in the peripheral blood of atopic asthmatic patients during the exacerbation of asthma (22). Furthermore, the number of circulating CD34⫹ cells are increased in atopic subjects (14), and CD34⫹ cells can also be detected in the bronchial mucosa in asthmatic subjects (40). Recently, a study has suggested that a subset of eosinophils in fact may differentiate in peripheral tissue such as the nasal mucosa (41) in a process that is highly IL-5 dependent. Together, these findings suggest that some progenitors will traffic from the bone marrow, via blood, to the allergen-exposed tissue, and the eosinophil production may occur in several tissues concomitantly, especially in conditions with substantial eosinophilic inflammation. In cells taken from the bone marrow, we did not detect any increased frequency of BrdU staining in animals exposed to allergen. This argues against an overall lineage-nonspecific stimulation of the bone marrow by allergen exposure in this model (Table III), which is similar to findings in dogs (18). The stimulation of the bone marrow eosinophilopoiesis in vivo is also evident by increased numbers of eosin-staining cells with immature morphology within the bone marrow compartment (Fig. 3). This finding confirms previous experiments using C57BL/6 mice (21) as well as BALB/c mice (4, 20). The increased number of immature eosinophils is clearly evident the day after three allergen exposures, in line with the increased BrdU staining in the airways at this time point, suggesting that the eosinophilopoietic response in the bone marrow takes some days to develop. Also, these data suggest that it is possible to use this time point to evaluate mechanisms of bone marrow stimulation induced by repeated allergen exposure. After three allergen exposures, we detected increased numbers of both CD3⫹ cells and non-CD3 cells isolated from the bone

FIGURE 5. Photomicrograph (original magnification: ⫻1000) of a CD3⫹ cell (red) in the bone marrow cytospin from a sensitized mouse exposed repeatedly to allergen (OVA; 100 ␮g i.n. on three consecutive days).


(48) and also bone marrow eosinophilia in our study (Figs. 7A and 9). To elucidate more closely how an anti-IL-5 Ab (TRFK-5) would block airway eosinophilia, we treated sensitized mice with this Ab either by repeated i.n. instillation or by i.p. injection. TRFK-5 given i.p. resulted in dose-dependent concentrations of the Ab in the circulation (Fig. 7A) and dose-dependently attenuated allergen-induced BAL eosinophilia, as well as BrdU staining of BAL granulocytes. This inhibitory effect is parallel with a strong inhibition of the bone marrow eosinophilia. The importance of the circulating IL-5 for airway eosinophilia is in line with one of our previous studies, showing that airway eosinophilia can be induced in sensitized IL-5 knockout mice by systemic reconstitution with IL-5, but not by local lung reconstitution (26). Thus, systemic IL-5

FIGURE 7. The concentration of rat IgG1 in serum (A) and in BAL fluid (B) from mice treated i.p. with different doses of TRFK-5 (1–100 ␮g) on day 1, or i.n. with different doses of TRFK-5 (6 –50 ␮g/day) on five consecutive days. The concentrations of rat IgG1 in serum and BAL fluid from isotype control (rat IgG1)-treated animals are also shown (䡺). Values of rat IgG1 concentration below the detection limit (8 ng/ml) are considered to be zero. Data shown are mean ⫾ SEM.

seems to be important for the induction of airway eosinophilia, and this effect is likely to involve stimulation of eosinophilopoiesis. Intranasally administered TRFK-5 can also exert inhibitory effects on airway eosinophilia, partly via local mechanisms. For example, a high dose of locally administered TRFK-5 has a slightly more pronounced effect on BAL eosinophilia than an i.p. dose causing a similar concentration of Ab in serum (e.g., 50 ␮g ⫻ 5 i.n. vs 10 ␮g ⫻ 1 i.p.; Figs. 7 and 8). This argues that there is some effect of anti-IL-5 given locally to the airways on airway eosinophilia. In this case, the concentration of Ab was higher in BAL fluid after i.n. treatment, showing that the treatment was compartmentalized to some degree to the airways. In an attempt to understand the site of action of IL-5 at the receptor level, we stained both bone marrow and BAL cytospins for the ␣-chain of IL-5R using immunocytochemistry. Using this method, we were able to detect strong positive staining only in bone marrow cells, but not in BAL eosinophils. We could not detect IL-5Rs in lung tissue of allergen-exposed mice, using immunohistochemistry, whereas the


FIGURE 6. A, Flow cytometric scatter histogram illustrating single-cell analysis of bone marrow cell suspensions, prepared as described in Materials and Methods. The gate (R1) has the same position in both histograms to illustrate the similarities in light scatter properties of the mononuclear cell clusters. B, Relative number of intracellular staining for IL-5 (TRFK-5) in CD3-positive bone marrow cells, as well as in total cells within a gate (R1 in A), evaluated by flow cytometry. There was an increased number of both CD3-positive (f) and CD3-negative cells (䡺) staining for intracellular IL-5 within this quadrant in mice exposed to allergen repeatedly (OVA; 100 ␮g i.n. on three consecutive days). Cells exposed in vitro for 6 h to PMA (0.2 ␮M) and calcium ionophore (1 ␮M) (stim) showed enhanced intracellular staining for IL-5, mainly when harvested from OVA-exposed mice. Data are shown as mean ⫾ SEM. ⴱ, p ⬍ 0.05 compared with the same compartment of treatment (with or without stimulation) in PBS-exposed group (Mann-Whitney U test).

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staining was prominent in bone marrow tissue samples (our unpublished data). Thus, in this model, it may be that eosinophils shed most of the ␣-chain of IL-5R before reaching the airways. This confirms several previous studies showing quite low expression of IL-5R in mature eosinophils (49, 50). The high expression of IL-5R ␣-chain in the bone marrow supports the concept that IL-5 is intricately involved in eosinophilopoiesis, and also one important effect of this cytokine is localized to immature eosinophils bearing IL-5Rs. In humans, it has been shown that mRNA specific for the ␣-chain of IL-5R can be found also in airway tissue (40, 51), and it is possible that this mRNA expression leads to IL-5R expression on the cell surface. Certainly, it is likely that airway eosinophils do express small numbers of functionally active IL-5Rs, which, however, are impossible to stain by immunocytochemical techniques. This notion is supported by a previous study, showing effects of IL-5 on the survival of BAL eosino-

FIGURE 9. Effect of in vivo treatment with a neutralizing anti-IL-5 mAb (TRFK-5) vs its isotype control (rat IgG1), on bone marrow eosinophil content after repeated allergen exposure (OVA; 100 ␮g i.n. on five consecutive days) in sensitized mice when the Ab was given either i.p. (1–100 ␮g i.p.; A) or i.n. (6 –50 ␮g on five consecutive days i.n.; B). The figure shows both eosin-staining cells with immature morphology (f), and cells with more mature morphology (䡺). Data shown are mean ⫾ SEM. Kruskall-Wallis test shows significant variance among groups. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01 compared with rat IgG1-treated group (Mann-Whitney U test).

phils cultured in vitro (52). In addition, it has been reported that specific IL-5 transgenic expression in lung epithelium in mice results in pathologic changes characteristic of asthma, including eosinophil infiltration into the airways (53). However, in these animals there was a substantial leakage of IL-5 into the circulation (mean serum concentration 1696 pg/ml), and more than 20 times higher numbers of eosinophils were found in the bone marrow as compared with wildtype mice (53), supporting effects on eosinophilopoiesis by IL-5. Our finding that repeated airway administration of anti-IL-5 could attenuate allergen-induced BAL eosinophilia is in line with a role of IL-5 also at this level. Our data agree with and extend the recent study by Shardonofsky and colleagues showing that respiratory delivery of anti-


FIGURE 8. Effect of in vivo treatment with a neutralizing anti-IL-5 mAb (TRFK-5) vs its isotype control (rat IgG1) on BAL eosinophilia induced by repeated allergen exposure (OVA; 100 ␮g i.n. on five consecutive days) in sensitized mice when the Ab was given either i.p. (1–100 ␮g i.p.; A) or i.n. (6 –50 ␮g on five consecutive days i.n.; B). Data shown are mean ⫾ SEM. Kruskall-Wallis test shows significant variance among groups. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01 compared with rat IgG1-treated group (Mann-Whitney U test).


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Acknowledgments

FIGURE 10. Photomicrograph (original magnification, ⫻1000) of bone marrow (A) and BAL cytospins (B) from a mouse repeatedly exposed to allergen (OVA; 100 ␮g i.n. on five consecutive days) stained immunocytochemically for the IL-5R ␣-chain. IL-5R staining is turquoise. Substantial IL-5R staining was detected in bone marrow cytospins, but no staining could be detected in BAL cells.

IL-5 Ab is capable of inhibiting airway eosinophilia and airway hyperresponsiveness in allergic mice (47). Thus, local airway treatment with anti-IL-5 may also have beneficial effects on eosinophilic airway inflammation but larger total doses are required. In the present mouse experiments, we could detect prominent staining of IL-5R ␣-chain in ⬃16% of bone marrow cells and some staining in up to 40% of bone marrow cells, which were independent of allergen exposure. This high frequency of IL-5R staining in mice is in contrast to that in humans, in which much smaller numbers of bone marrow cells express this epitope. Furthermore, in humans this receptor also seems to be up-regulated by allergen exposure in asthmatics developing a late asthmatic reaction (15). This is in parallel with increased number of CFUs producing eosinophils in bone marrow samples taken after allergen exposure (24). Thus, allergen exposure seems to have slightly different regulatory effects in the bone marrow of allergic mice and asthmatic patients, although the models are similar in some respects because bone marrow activation can be observed in both species (24, 13–15, 20, 24). IL-5 can exert more than one important effect in the bone marrow. The first event seen when exogenous IL-5 is given by i.v. administration is that the number of eosinophils in the bone marrow is reduced, and the number of eosinophils in the circulation is increased (54). A similar effect is also seen with a single-day allergen exposure (4, 19). Recent studies using an in situ bone marrow perfusion model have shown that IL-5 stimulates mobilization of eosinophils from the bone marrow (55, 56). Thus, after allergen exposure, IL-5 seems to be involved in the release of eosinophils

We thank Carina Malmha¨ll and Susanna Goncharova for technical assistance and Drs. Mark Inman, Roma Sehmi, Malcolm Johnson, Murray McKinnon, and Bengt-Eric Skoogh for helpful discussion.

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from the bone marrow compartment into the circulation to facilitate a rapid induction of airway eosinophilia. This release of eosinophils induced by allergen can be blocked by anti-IL-5 (57). During repeated or chronic allergen exposure, the situation is more complex. IL-5 is involved in enhanced allergen-induced eosinophilopoiesis, but may also be involved in the release of more eosinophils into the circulation. However, one key effect of blocking IL-5-induced effects in these experiments seems to be the attenuation of eosinophilopoiesis because anti-IL-5 strongly blocked the increase in the numbers of bone marrow eosin-staining cells, as well as BrdU-positive BAL granulocytes, induced by repeated allergen exposure. These experiments show that a very specific chain of events occurs during repeated allergen exposure, resulting in airway eosinophilia. These events include enhanced production of IL-5 in bone marrow cells, resulting in an enhanced eosinophilopoiesis in this tissue, and a prominent contribution of newly produced eosinophils in the airway inflammatory response. To block the effects of IL-5 at the receptor level, the bone marrow can be targeted because IL-5Rs are highly expressed in this tissue. Alternative therapeutic approaches to reduce allergen-induced airway eosinophilia may be to block mechanisms that cause increased IL-5 production both in the bone marrow and peripheral tissues, or to reduce the expression of IL-5Rs.

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