help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on August 14, 2003, doi:10.1165/rcmb.2002-0311OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2002-0311OCv1
30/3/367    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sitkauskiene, B.
Right arrow Articles by Lötvall, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sitkauskiene, B.
Right arrow Articles by Lötvall, J.
American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 367-378, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2002-0311OC

Regulation of Bone Marrow and Airway CD34+ Eosinophils by Interleukin-5

Brigita Sitkauskiene, Anna-Karin Johansson, Svetlana Sergejeva, Samuel Lundin, Margareta Sjöstrand and Jan Lötvall

Lung Pharmacology Group, Department of Respiratory Medicine and Allergology, Institute of Internal Medicine, and Department of Medical Microbiology and Immunology, Göteborg University, Gothenburg, Sweden; and Department of Pulmonology and Immunology, Kaunas University of Medicine, Kaunas, Lithuania

Address correspondence to: Jan Lötvall, M.D., Ph.D., The Lung Pharmacology Group, Dept. of Respiratory Medicine and Allergology, Göteborg University, Guldhedsgatan 10A, 413 46 Gothenburg, Sweden. E-mail: jan.lotvall{at}mednet.gu.se


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The aim of this study was to evaluate the effect of a neutralizing anti–interleukin (IL)-5 monoclonal antibody (TRFK-5) on bone marrow and airway CD34+ and immature eosinophils. A focus was to determine the effect of the timing of treatment. Balb/c mice were ovalbumin-sensitized and subsequently exposed to ovalbumin for 5–10 d via airway route. Animals were treated intraperitoneally with TRFK-5 or its isotype control (50 µg) once at different time points. Newly produced eosinophils were labeled using 5-bromo-2'-deoxyuridine (BrdU). BrdU+ and CD34+ eosinophil numbers were examined by immunocytochemistry. TRFK-5 reduced bone marrow immature eosinophils within 3 d. This effect was closely related to a reduction of BrdU+ and CD34+ bone marrow eosinophils, and reduced numbers of blood eosinophils. However, bronchoalveolar lavage (BAL) eosinophilia was not attenuated to the same degree. The effect of TRFK-5 was most prominent in the extended allergen-exposure protocol, where the treatment was given in the middle of the exposure, with strongly reduced bone marrow CD34+ and immature bone marrow eosinophils, blood eosinophils as well as BAL BrdU+ eosinophils, and BAL CD34+ eosinophils. These data argue that anti–IL-5 downregulates eosinophilopoiesis within 3 d by action in the bone marrow, by inhibition of the early stages of eosinophil maturation from CD34+ cells.

Abbreviations: bronchoalveolar lavage, BAL • 5-bromo-2'-deoxyuridine, BrdU • bovine serum albumin, BSA • ethylenediaminetetraacetic acid, EDTA • interleukin, IL • ovalbumin, OVA • phosphate-buffered saline, PBS • tris-buffered saline, TBS


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Eosinophils are abundant in the airways after airway allergen exposure, which has been shown in both man and experimental animals (1–5). These cells have the capacity to release multiple bronchoconstrictor mediators such as leukotrienes and thromboxane (6), but also tissue-damaging proteins such as major basic protein and eosinophil cationic protein (7). By the release of these proteins and enzymes, the eosinophils have been suggested to be involved in several features of asthma, including acute airway narrowing, bronchial hyperresponsiveness, and airway wall remodelling (8–11).

It has previously been shown that airway allergen exposure induces enhanced eosinophil production, and increases the number of circulating CD34+ cells (12, 13). Eosinophils develop predominantly from bone marrow CD34+ progenitor cells. Late stage differentiation of eosinophils is mediated by interleukin-5 (IL-5), which acts through a membrane-bound receptor (IL-5R) and also influences certain function of mature eosinophils (13–17). Anti–IL-5 treatment primarily reduces the bone marrow eosinophil numbers, and to a lesser extent eosinophils in peripheral tissue (18–21). However, the possible regulatory effects of anti–IL-5 on CD34+ progenitor cells have not been elucidated. Also, the detailed kinetics of anti–IL-5 effects on bone marrow, blood, and airway eosinophils has not been documented. The aim of this study was to evaluate the effect of neutralizing anti–IL-5 monoclonal antibody on the bone marrow and airway eosinophils, including CD34+ and newly produced eosinophils, in relation to the timing of treatment in a mouse model of allergic inflammation.

To do this we used Balb/c mice in an ovalbumin (OVA)-induced model of allergic inflammation (20), where newly produced cells were labeled with a thymidine analog, 5-bromo-2'-deoxyuridine (BrdU), which is incorporated into DNA during the S-phase of the cell cycle (22). Animals were treated with anti–IL-5 at different time points either before or during an ongoing allergen exposure. Intranuclear BrdU and CD34 antigen on eosinophils were detected by immunocytochemistry.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
This study was approved by the Animal Ethics Committee in Gothenburg, Sweden.

Sensitization and Exposure
Male Balb/c mice, 5–6 wk old, obtained from B&K Universal (Sollentuna, Sweden), were sensitized by intraperitoneal booster injections of 0.5 ml aluminium-precipitated antigen containing 8 µg of OVA (Sigma-Aldrich Sweden AB, Tyresö, Sweden) bound to 4 mg of aluminium hydroxide (Sigma Chemical Co, St. Louis, MO) in phosphate-buffered saline (PBS) twice, 5 d apart. Eight days after the second sensitization, the different airway allergen exposure protocols were initiated. OVA solution (100 µg in 25 µl PBS) was instilled intranasally to mice briefly anaesthetized using CO2.

Treatment with Anti–IL-5
The neutralizing anti–IL-5 antibody (TRFK-5, 50 µg; R&D Systems Europe Ltd., Abingdon, UK), or its isotype control (rat IgG1, 50 µg; clone R3–34; Pharmingen, San Diego, CA) dissolved in 0.3 ml PBS was given intraperitoneally.

Treatment with BrdU
Treatment with BrdU (Boehringer Mannheim Scandinavia AB, Bromma, Sweden) was given at a dose of 1.0 mg in 0.25 ml PBS intraperitoneally twice, 7 h apart.

Protocols
Three different allergen exposure and anti–IL-5 treatment protocols were applied.

In Protocol I (Figure 1A), allergen exposure was performed on five consecutive days, and cells were collected 24 h after the last OVA exposure. Anti–IL-5 was given once on each of the allergen exposure days (1, 2, 3, 4, or 5), thus 5, 4, 3, 2, or 1 d before the harvest of sample. The isotype control was given on Day 3. BrdU was injected on Days 1 and 3 of allergen exposure.







View larger version (67K):
[in this window]
[in a new window]
  		Figure 1. The effects of timing of a neutralizing anti–IL-5 monoclonal antibody (TRFK-5; 50 µg intraperitoneally) on eosinophilia induced by allergen exposure (OVA; 100 µg intranasally on five consecutive days) in sensitized Balb/c mice. BrdU was injected on Days 1 and 3 of OVA exposure period. (A) Protocol I scheme. (B) Bone marrow and (C) BAL eosinophil staining for BrdU (BrdU+, dark part of column) and unstained eosinophils (BrdU-, open part of column). (D) Bone marrow and (E) BAL content of CD34+ eosinophil (dark part of column) and CD34- eosinophil (open part of column). Data are shown as mean ± SEM (n = 7–12 per group). Kruskall-Wallis test shows significant variance among groups. *P < 0.05, **P < 0.01 compared with rat IgG1-treated group (Mann-Whitney U test).

 
In Protocol II (Figure 5A), allergen exposure and BrdU injections were performed as in Protocol I. Anti–IL-5 or its isotype control was given on the last day of exposure (Day 5). Cells were then collected 2, 3, or 9 d after the last allergen exposure and anti–IL-5 treatment.







View larger version (59K):
[in this window]
[in a new window]
  		Figure 5. The effects of a neutralizing anti–IL-5 monoclonal antibody (TRFK-5; 50 µg intraperitoneally), given just after the end of repeated allergen exposure (OVA; 100 µg intranasally on five consecutive days) on the eosinophilia remaining up to 9 d after the end of exposure in Balb/c mice sensitized to OVA. BrdU was injected on Days 1 and 3 of OVA exposure period. (A) Protocol II scheme. (B) Bone marrow and (C) BAL eosinophil staining for BrdU (BrdU+, dark part of column) and unstained eosinophils (BrdU-, open part of column). (D) Bone marrow and (E) BAL content of CD34+ eosinophil (dark part of column) and CD34- eosinophil (open part of column). Data are shown as mean ± SEM (n = 5 per group). Kruskall-Wallis test of bone marrow samples data shows significant variance among groups. *P < 0.05, **P < 0.01 compared with rat IgG1-treated group (Mann-Whitney U test).

 
In Protocol III (Figure 6A), allergen was given in two courses, first on five consecutive days, and extended up to ten exposures, but with a 2-d rest after the first five exposures. Anti–IL-5 or its isotype control was given on the first day of the second course of allergen exposure (Day 6 of exposure). Cells were collected 1, 3, or 5 d after the anti–IL-5-treatment. BrdU was given 3 and 5 d before the harvest of sample.







View larger version (67K):
[in this window]
[in a new window]
  		Figure 6. The effects of anti–IL-5 (TRFK-5; 50 µg intraperitoneally), given in the middle of a prolonged repeated allergen exposure period (OVA; 100 µg intranasally given in total over 10 d with 2 d of rest after five exposures) on the eosinophilia in sensitized Balb/c mice. BrdU was given 3 and 5 d before collection of cells. (A) Protocol III scheme. (B) Bone marrow and (C) BAL eosinophil staining for BrdU (BrdU+, dark part of column) and unstained eosinophils (BrdU-, open part of column). (D) Bone marrow and (E) BAL content of CD34+ eosinophil (dark part of column) and CD34- eosinophil (open part of column). Data are shown as mean ± SEM (n = 5–6 per group). Kruskall-Wallis test shows significant variance among groups. *P < 0.05, **P < 0.01 compared with rat IgG1-treated group (Mann-Whitney U test).

 
Cell Collection and Processing of Samples
Animals were anaesthetized with an intraperitoneal mixture of Xylazine and Ketamine (130 and 670 mg/kg, respectively). When in adequately deep anesthesia, the chest was opened and samples of blood, bronchoalveolar lavage (BAL), and bone marrow were taken.

Blood was obtained by penetration of the right ventricle of the heart with a needle. BAL was performed through the trachea with a cannula, by instillation of 0.25 and 0.2 ml of PBS, which were then pooled 0.4 ml of BAL fluid was consistently recovered. Finally, one femur was excised and cut at the epiphyses. Bone marrow cells were removed by perfusion of the femur with 2 ml of PBS. BAL fluid and bone marrow cell suspension was kept on ice until further processing.

Cytospin of blood was obtained by taking 200 µl of blood and mixing it with 800 µl 2 mM EDTA (Sigma-Aldrich) in PBS. The red blood cells were lysed in 0.1% potassium bicarbonate and 0.83% ammonium chloride for 15 min at 4°C. The white blood cells were resuspended in PBS with 0.03% bovine serum albumin (BSA; Sigma-Aldrich).

BAL fluid with cells and bone marrow cell suspension were centrifuged at 1,000 rpm for 10 min at 4°C. Supernatants were kept, and the cell pellets were re-suspended in PBS with 0.03% BSA. Total number of cells in BAL, bone marrow, and blood was determined using standard hematologic procedures.

Cytospins of BAL, bone marrow, and blood cells were prepared and stained with May-Grünwald-Giemsa for differential cell counts. Cell differentiation was determined by counting 300–500 cells using a light microscope (Zeiss Axioplan 2; Carl Zeiss, Jena, Germany). The cells were identified by standard morphologic criteria, and bone marrow mature and immature eosinophils were determined by evaluation of nuclear morphology, staining properties, and cytoplasmic granulation, as previously described (20, 23). Cytospin preparations for immunocytochemistry were air-dried and stored at -80°C until further processing.

Immunocytochemistry of BrdU+ Eosinophils
Intranuclear incorporation of BrdU was detected by immunocytochemistry of BAL and bone marrow cytospins. Cytospins taken out of the freezer were fixed overnight in 4% paraformaldehyde, and subsequently washed with tris-buffered saline (TBS), and subjected to digestion in 0.1% trypsin and 0.1% CaCl2 in PBS at 37°C for 15 min to open the cell membrane for nuclear immunostaining. The slides were then further incubated in 4M HCl for another 15 min to denature DNA and expose BrdU. These processes were then neutralized by adding Holmes Borate-Borax buffer (1.24% H3BO3 in distilled H2O, pH 8.5) for 10 min. The samples were treated with peroxidase-blocking solution (No. S2023; Dako, Glostrup, Denmark) for 15 min. Nonspecific binding sites were blocked with 5% normal rabbit serum (No X0902; Dako) for 15 min. Subsequently, the slides were incubated with 2.5 µg/ml rat anti-BrdU antibody (clone BU1/75; No MAS250p; Harlan-Ser Lab, Loughborough, UK) or isotype control (rat IgG2a, clone R35–95; Pharmingen) in incubation buffer (0.5% BSA/TBS) for 1 h. After washing in 0.05% Tween/TBS and TBS, the slides were incubated with 1:50 dilution of rabbit F(ab')2 anti-rat Ig-HRP (No 6130–05, SBA) and 2% normal mouse serum (No X0910; Dako) for 1 h. After further washing, staining with DAB substrate-Chromogen system (No K3466; Dako) was developed for 5–10 min, by monitoring in the microscope. The slides were stained for eosinophils by using Luxol Fast Blue for 30 min, followed by counterstaining with Mayer's hematoxylin for 30 s, dehydrated, and mounted in Mountex. All slides were evaluated by light microscopy in random fields of view. Cells with nuclear brown staining together with green Luxol staining in cytoplasm were counted as BrdU+ eosinophils.

Immunocytochemistry of CD34+ Eosinophils
Cytospin preparations were fixed in 2% formaldehyde for 10 min, washed in PBS, and treated for 20 min with Glucoseoxidase solution containing 0.1% saponin in PBS, which was preheated to 37°C. The samples were incubated with Avidin-blocking solution (Dako) containing 0.1% saponin for 10 min, and after washing with TBS-saponin, the cytospins were incubated for 10 min with biotin blocking solution (Dako). After further washing, the slides were incubated with rat anti-mouse CD34–biotin-conjugated antibody (clone RAM34; Pharmingen), or isotype control (rat IgG2a biotin-conjugated; Pharmingen), diluted 1:50 in incubation buffer (0.5% BSA/TBS-0.1% saponin) for 2 h at room temperature. After washing, the samples were incubated for 45 min with peroxidase-conjugated ExtrAvidin (Sigma) diluted 1:100 in incubation buffer, and washed in TBS. Then, the staining with DAB substrate-chromogen system (No K3466; Dako) was developed for 12 min. The slides were stained with Luxol Fast Blue for 30 min as previously described. Cells with both brown and green Luxol staining were counted as CD34+ eosinophils.

Fluorescence-Activated Cell Sorter Analysis of BAL Fluid and Bone Marrow Cells
Surface staining of the CD34 antigen was performed in BAL fluid and bone marrow (positive control) samples from allergen sensitized and exposed (as in Protocol I) mice. All incubations were performed on ice and antibody dilutions were made in fluorescence-activated cell sorter buffer. Unspecific binding was blocked with 2% mouse sera (Dako) for 15 min. The cells were thereafter incubated with a monoclonal FITC-conjugated CD34 antibody (clone RAM34; BD Biosciences, Europe) or its isotype-matched control (rat IgG2a, clone R35–95; BD Bioscience) for 35 min. After washings, the cells were fixed in Cytofix/Cytoperm (BD Bioscience) for 20 min. The cells were analyzed on a FACSCalibur (Becton-Dickinson).

Statistics
Statistics were performed using standard statistical packages. Kruskal-Wallis test was used to evaluate whether any variance among more than two groups exist. If a significant variance was found, an unpaired Mann-Whitney U test was performed to determine any significant differences between individual groups. Data are shown as mean ± SEM, and a P value < 0.05 is considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experiment 1: The Effects of Timing of Anti–IL-5 on Eosinophilia in Different Tissue Compartments Induced by Allergen Exposure on Five Consecutive Days
Anti–IL-5 was given either on Day 1, 2, 3, 4, or 5 of repeated allergen exposure, in an attempt to block an ongoing inflammatory response. TRFK-5 significantly inhibited OVA-induced bone marrow eosinophilia compared with vehicle-treated mice (Table 1), but mainly the relative number of immature eosinophils when it was given on Day 1, 2, 3, or 4 of allergen exposure. Mature eosinophils in bone marrow were significantly reduced when TRFK-5 was given either on Day 1 or 2, but not at later time points (Table 1). The number of BrdU+ bone marrow eosinophils was markedly decreased in the mice treated on Day 1, 2, or 3 of allergen exposure (Figure 1B). This was significantly related to the TRFK-5 administration time point (P < 0.01, Rs = 0.8).


View this table:
[in this window]
[in a new window]
  		TABLE 1 The effect of timing of TRFK-5 treatment on eosinophil numbers in the bone marrow, blood, and BAL in Experiment 1

 
There was no significant effect on the total number of BAL eosinophils (Table 1) or BrdU+ BAL eosinophils (Figure 1C). However, in BAL of mice treated on Day 2 or 3 of exposure, the relative number of BrdU-labeled eosinophils was significantly lower than in vehicle-treated control animals (respectively 2.6 ± 0.9, 4.8 ± 1.2 versus 16.9 ± 4.2% of total cells, P < 0.05). A photomicrograph of BrdU-stained eosinophils from OVA-exposed mouse BAL and bone marrow are shown in Figure 2.



View larger version (64K):
[in this window]
[in a new window]
  		Figure 2. Photomicrograph (original magnification: x1,000) of bone marrow (A) and BAL (B) cytocentrifuge preparations for double staining for BrdU and eosinophils in Balb/c mouse repeatedly exposed to OVA (100 µg intranasally on five consecutive days). Cells with nuclear brown staining together with green Luxol staining in cytoplasma were counted as BrdU-labeled eosinophils (BrdU+ eosinophil, 1; BrdU- eosinophil, 2).

 
CD34+ eosinophils were detected by immunocytochemistry (Figure 3). The relative number of eosinophils with CD34+ staining in bone marrow were significantly reduced when TRFK-5 was given on Day 1, 2, or 3 of OVA exposure compared with vehicle-treated mice (Figure 1D). The same tendency was seen in BAL CD34+ eosinophil numbers, but without significant difference (Figure 1E).






View larger version (390K):
[in this window]
[in a new window]
  		Figure 3. Representative photomicrographs of immunocytochemistry for CD34 antigen detection in the bone marrow (original magnification: A, x400; B, x1,000) and BAL cytospin preparations (original magnification: D, x400; E, x1,000) from Balb/c mouse repeatedly exposed to OVA (100 µg intranasally on five consecutive days). The staining with rat anti-mouse CD34-biotin-conjugated antibody is brown; Luxol staining for eosinophils is green. Cells with both brown and green staining were counted as CD34+ eosinophils (CD34+ mature eosinophil, 1; CD34+ immature eosinophilic cell, 2; CD34- eosinophil, 3; other type of CD34+ cell, 4). No staining could be observed (C and F) when rat IgG2a biotin-conjugated isotype control was used.

 
Flow cytometry, using surface staining of the CD34 antigen (Figure 4), revealed that OVA-exposed mice had increased level of CD34+ cells in BAL fluid (Figure 4A), also as in the bone marrow (Figure 4C).



View larger version (52K):
[in this window]
[in a new window]
  		Figure 4. Flow cytometric scatter histogram, illustrating cell analysis of BAL (A, B) and bone marrow (C, D) cell suspensions, prepared as described in MATERIALS AND METHODS. The gate in A and C illustrates CD34-positive cells; B and D, isotype matched control.

 
Experiment 2: The Effects of Anti–IL-5 Given Just After the End of 5 d Repeated Allergen Exposure on Eosinophilia Remaining for Another 9 d
When TRFK-5 was given after the end of allergen exposure, bone marrow eosinophils were reduced on the third day after the treatment compared with vehicle-treated mice, primarily due to a decrease of immature eosinophils (2.80 ± 0.23 versus 6.25 ± 0.58% of total cells, Table 2; P < 0.01), but not of mature eosinophils. The effect of TRFK-5 on immature eosinophils was maintained 9 d after the treatment (Table 2). BrdU+ bone marrow eosinophils were reduced on the third day after the treatment (Figure 5B).


View this table:
[in this window]
[in a new window]
  		TABLE 2 The effect of TRFK-5 treatment on the eosinophilia remaining up to 9 d after the end of exposure in the bone marrow, blood, and BAL in Experiment 2

 
No significant effects were seen with TRFK-5 treatment on total BAL eosinophils (Table 2), or BrdU+ BAL eosinophils (Figure 5C).

TRFK-5 given after the end of allergen exposure reduced bone marrow CD34+ eosinophils after 3 d; an effect was maintained until nine days after the treatment (Figure 5D). No effect was observed on BAL CD34+ eosinophils (Figure 5E).

Experiment 3: Effects of TRFK-5 Given on Day 6 of an up to 10 d Extended Allergen Exposure Protocol on Eosinophilia 1, 3, and 5 d after the Treatment
After extension of the OVA exposure period, bone marrow eosinophils were significantly reduced on the third day after the TRFK-5 administration compared with vehicle-treated mice, primarily due to a decrease of immature eosinophils (3.05 ± 0.50 versus 6.08 ± 0.82% of total cells; Table 3). This effect was further pronounced 5 d after the treatment with TRFK-5 (Table 3; P < 0.05) as well as BrdU+ bone marrow eosinophils (Figure 6B).


View this table:
[in this window]
[in a new window]
  		TABLE 3 The effect of TRFK-5 treatment on an ongoing eosinophilia, with a continuous allergen exposure in the bone marrow, blood, and BAL in Experiment 3

 
Significant effect on BAL eosinophilia was found on the fifth day after TRFK-5 administration and repeated allergen exposure compared with vehicle-treated mice (Table 3) as well as BrdU+ and BrdU- eosinophils (Figure 6C).

After extension of airway allergen exposure period up to 10 d, a further increase in CD34+ eosinophil numbers were observed in both bone marrow and BAL (Figure 6D). There was no significant difference in the total CD34+ cell numbers in the bone marrow of TRFK-5 or vehicle-treated mice. A significant reduction of CD34+ eosinophils was found in the bone marrow on the fifth day after TRFK-5 administration (Figure 6D).

A significant inhibitory effect was also found on the number of CD34+ eosinophils in BAL on the fifth day after treatment and the extended exposure period (Figure 6E).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our study shows that a single dose of systemic anti–IL-5 reduces bone marrow eosinophils, especially immature eosinophils, within 2–3 d after administration. This effect is closely related to a reduction in newly produced (BrdU+) bone marrow eosinophils, and is paralleled with a reduction in blood eosinophils, but does not at the same time point affect BAL eosinophilia. Anti–IL-5 also reduces bone marrow CD34+ eosinophils within 3 d of treatment, strongly arguing for an inhibition of early stages of eosinophil maturation. The effects of anti–IL-5 were most clearly documented in the extended exposure protocol, where the treatment extensively reduced bone marrow CD34+ eosinophils, immature bone marrow eosinophils, blood eosinophils as well as BAL BrdU+ eosinophils, and BAL CD34+ eosinophils. In this protocol, it was also established that anti–IL-5 reduces the number of BrdU- eosinophils in BAL, which may reflect an inhibition of eosinophil survival. Together these data argue that anti–IL-5, in vivo, can downregulate eosinophilia by action on eosinophilopoiesis, probably by inhibition early stages of eosinophils maturation from CD34+ progenitor cells. This study extends previous studies of anti–IL-5 by documenting the effects of treatment on an established allergic eosinophilia, and especially on CD34+ eosinophils, in different compartments.

In the present experiments, we used BrdU immunocytochemistry together with Luxol Fast Blue counterstaining, which allowed us to detect newly produced eosinophils specifically. After five allergen exposures, at least 50% of eosinophils in bone marrow and almost 60% of eosinophils present in BAL are newly produced (BrdU+). Our results confirm our previous observation that repeated allergen exposure results in an accumulation of eosinophils in the airways, and that newly produced eosinophils to a substantial degree contribute to this inflammatory process (20, 24). Thus, it is clear that eosinophilopoiesis is involved and exaggerated in allergic eosinophilia in this mouse model. In addition, we have confirmed that the increased total number of eosinophils in both bone marrow and BAL is paralleled with increased number of CD34+ cells detected in the same compartments by both immunocytochemistry and flow cytometry.

In Protocol I, the anti–IL-5 was given on different days during a 5-d allergen exposure protocol. The first effect that was observed was a decrease in bone marrow immature eosinophils, which appeared when anti–IL-5 was given 2 d before cell harvest. Second, when anti–IL-5 was given 3 d before cell harvest, a decrease in bone marrow of mature eosinophils, CD34+ eosinophils, and BrdU+ eosinophils was observed. However, the most prominent effect of anti–IL-5 on all subsets of bone marrow eosinophils was detected when the treatment was given before initiation of the allergen protocol (5 d before cell harvest). However, only small and nonsignificant changes were observed on BAL eosinophils in this protocol, which most likely is due to effects of anti–IL-5 first having effect in the bone marrow, and there specifically on eosinophilopoiesis.

In Protocol II, we gave anti–IL-5 at the end of a 5-d exposure protocol, to evaluate whether, and how rapidly, anti–IL-5 would attenuate the established eosinophilia seen when allergen exposure has been terminated. In a majority of experimental studies, animals were treated just before antigen challenge was initiated. However, in a clinical setting it is likely that anti–IL-5-treatment would be initiated during an ongoing eosinophilic inflammation. Our results indicate that anti–IL-5 inhibits eosinophilopoiesis within 3 d, when a reduction in BrdU+ and CD34+ bone marrow eosinophils, and blood eosinophils, was also observed. This effect was maintained throughout the protocol, up to nine days after treatment. However, no effect was observed on BAL eosinophilia, probably because allergen exposure had been stopped, which in itself terminates the traffic of new eosinophils into airways.

In Protocol III, we extended the evaluation of effects of anti–IL-5 on allergen-induced eosinophilia by giving the antibody in the middle of a protocol extended up to 10 d. This approach gave us the possibility to determine the kinetics of the effect of the anti–IL-5 antibody treatment on an ongoing eosinophilic inflammatory process, with a continuous allergen exposure, which may better mimic a clinical situation. In this experiment, a single dose of anti–IL-5 again reduced bone marrow and blood eosinophilia 3 d after administration. In this situation also the BAL eosinophilia was significantly reduced, but this effect occurred only 5 d after anti–IL-5 treatment. The effect of anti–IL-5 on BAL eosinophils was primarily due to reduction in the number of newly produced eosinophils (BrdU+), which obviously is due to reduced production of new eosinophils, and thus due to inhibition of eosinophilopoiesis. Also, the number of BrdU- BAL eosinophils was decreased to some extent. This effect could be due to reduced survival of mature eosinophils in the absence of IL-5 activity (25–27), which is in line with our previous in vivo results (20).

The terminal differentiation of eosinophils occurs from CD34+ progenitors, normally within the bone marrow and under the influence of IL-5 (13–15, 28). In this study, we observed not only a reduction of eosinophils with anti–IL-5 treatment, but also a reduction in CD34+ eosinophils in both bone marrow and in airways. The presence of the increased number of CD34+ eosinophils in the airways suggests that airway allergen exposure induces a rapid discharge of CD34+ eosinophils from the bone marrow, allowing these cells to traffic to the airways. It is also possible that CD34+ progenitors traffic to the airways to the allergen-exposed tissue, and differentiate to some degree locally, to produce eosinophils in situ (23, 29).

Recently published clinical data argue that anti–IL-5 is ineffective in reducing clinical signs of asthma induced by allergen (30), even though sputum and blood eosoinophils were strongly reduced. It seems from our data that anti–IL-5 is able mainly to reduce the production of new eosinophils predominantly in bone marrow, and relatively smaller effects are observed on eosinophils that may be of older age (BrdU-). In fact, it has recently been shown that anti–IL-5 only partially reduce bronchial wall eosinophils (31), which may be due to relatively smaller effects on eosinophil survival (20, 26, 27). This is further supported by our finding that the effects of anti–IL-5 on airways occur much later than in bone marrow, which is paralleled with effects on CD34+ eosinophils and newly produced (BrdU+) eosinophils.

After allergen exposure, a large portion of the eosinophils in BAL have CD34 antigen on their surface. However, morphologically these cells seem to be mature (Figure 3E). It is not clear, however, whether these eosinophils also have maturated functionally.

This experiment shows that a single systemic dose of anti–IL-5 rapidly blocks eosinophilopoiesis, but needs longer time to induce prominent effects on BAL eosinophils. This effect seems to be associated with a reduction of CD34+ eosinophils, further arguing that IL-5 initially inhibits eosinophil production. Improvement of our understanding of the kinetics of allergic eosinophilia could facilitate the future development of even more powerful inhibitors of airway eosinophilia.


   Acknowledgments
 
The authors are grateful to Carina Malmhäll for technical assistance and Dr. Bengt-Eric Skoogh for helpful discussion. This work was supported in part by the Swedish Heart and Lung Foundation, and the Vårdal Foundation of Sweden. Dr. Brigita Sitkauskiene was funded by a grant of ERS for a long-term Fellowship.

Received in original form December 20, 2002

Received in final form August 6, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cui, Z. H., M. Sjöstrand, T. Pullerits, P. Andius, B. E. Skoogh, and J. Lötvall. 1997. Bronchial hyperresponsiveness, epithelial damage, and airway eosinophilia after single and repeated allergen exposure in a rat model of anhydride-induced asthma. Allergy 52:739–746.[Medline]
  2. Bousquet, J., P. Chanez, Y. J. Lacoste, G. Barneon, N. Chavanian, I. Enander, P. Venge, S. Ahlstedt, J. Simony-Lafontaine, P. Godard, and F. B. Michel. 1990. Eosinophilic inflammation in asthma. N. Engl. J. Med. 323:1033–1039.[Abstract]
  3. Kung, T. T., D. Stelts, J. A. Zurcher, A. S. Watnick, H. Jones, P. J. Mauser, X. Fernandez, S. Umland, W. Kreutner, R. W. Chapman, and R. W. Egan. 1994. Mechanism of allergic pulmonary eosinophilia in the mouse. J. Allergy Clin. Immunol. 94:1217–1224.[CrossRef][Medline]
  4. Wood, L. J., M. D. Inman, J. A. Denburg, and P. M. O'Byrne. 1998. Allergen challenge increases cell traffic between bone marrow and lung. Am. J. Respir. Cell Mol. Biol. 18:759–767.[Abstract/Free Full Text]
  5. Kroegel, C., M. C. Liu, W. C. Hubbard, L. M. Lichtenstein, and B. S. Bochner. 1994. Blood and bronchoalveolar eosinophils in allergic subjects after segmental antigen challenge: surface phenotype, density heterogeneity, and prostanoid production. J. Allergy Clin. Immunol. 93:725–734.[CrossRef][Medline]
  6. Laviolette, M., C. Ferland, J. F. Comtois, K. Champagne, M. Bosse, and L. P. Boulet. 1995. Blood eosinophil leukotriene C4 production in asthma of different severities. Eur. Respir. J. 8:1465–1472.[Abstract]
  7. Gundel, R. H., L. G. Letts, and G. J. Gleich. 1991. Human eosinophil major basic protein induces airway constriction and airway hyperresponsiveness in primates. J. Clin. Invest. 87:1470–1473.
  8. Frigas, E., S. Motojima, and G. J. Gleich. 1991. The eosinophilic injury to the mucosa of the airways in the pathogenesis of bronchial asthma. Eur. Respir. J. 4:S123–S135.
  9. Redington, A. E., and P. H. Howarth. 1997. Airway wall remodeling in asthma. Thorax 52:310–312.[Medline]
  10. Elwood, W., J. Lötvall, P. J. Barnes, and K. F. Chung. 1991. Characterization of allergen-induced bronchial hyperresponsiveness and airway inflammation in actively sensitized brown-Norway rats. J. Allergy Clin. Immunol. 88:951–960.[CrossRef][Medline]
  11. Lötvall, J., M. Inman, and P. O'Byrne. 1998. Measurement of airway hyperresponsiveness: new considerations. Thorax 53:419–424.[Free Full Text]
  12. Sehmi, R., K. Howie, D. R. Sutherland, W. Schragge, P. M. O'Byrne, and J. A. Denburg. 1996. Increased levels of CD34+ hemopoietic progenitor cells in atopic subjects. Am. J. Respir. Cell Mol. Biol. 15:645–655.[Abstract]
  13. Sehmi, R., L. J. Wood, R. Watson, R. Foley, Q. Hamid, P. M. O'Byrne, and J. A. Denburg. 1997. Allergen-induced increases in IL-5R{alpha} subunit expression on bone marrow-derived CD34+ cells from asthmatic subjects. J. Clin. Invest. 100:2466–2475.[Medline]
  14. Lanza, F., L. Healy, and D. R. Sutherland. 2001. Structural and functional features of the CD34 antigen: an update. J. Biol. Regul. Homeost. Agents 15:1–13.[Medline]
  15. Suzuki, A., D. P. Andrew, J. A. Gonzalo, M. Fukumoto, J. Spellberg, M. Hashiyama, H. Takimoto, M. Gerwin, I. Webb, G. Molineux, R. Amakawa, Y. Tada, A. Wakeham, J. Brown, I. McNiece, K. Ley, E. C. Butcher, T. Suda, J. C. Gutierrez-Ramos, and T. W. Mak. 1996. CD34-deficient mice have reduced eosinophil accumulation after allergen exposure and show a novel crossreactive 90-kD protein. Blood 87:3550–3562.[Abstract/Free Full Text]
  16. Cameron, L., P. Christodoulopoulos, F. Lavigne, Y. Nakamura, D. Eidelman, A. McEuen, A. Walls, J. Tavernier, E. Minshall, R. Moqbel, and Q. Hamid. 2000. Evidence for local eosinophil differentiation within allergic nasal mucosa: inhibition with soluble IL-5 receptor. J. Immunol. 164:1538–1545.[Abstract/Free Full Text]
  17. Shalit, M., S. Sekhsaria, and H. L. Malech. 1995. Modulation of growth and differentiation of eosinophils from human peripheral blood CD34+ cells by IL5 and other growth factors. Cell. Immunol. 160:50–57.[CrossRef][Medline]
  18. Kung, T. T., D. M. Stelts, J. A. Zurcher, G. K. Adams, III, R. W. Egan, W. Kreutner, A. S. Watnick, H. Jones, and R. W. Chapman. 1995. Involvement of IL-5 in a murine model of allergic pulmonary inflammation, prophylactic and therapeutic effect of an anti–IL-5 antibody. Am. J. Respir. Cell Mol. Biol. 13:360–365.[Abstract]
  19. Wang, J., K. Palmer, J. Lötvall, S. Milan, X. F. Lei, K. I. Matthaei, J. Gauldie, M. D. Inman, M. Jordana, and Z. Xing. 1998. Circulating, but not local lung, IL-5 is required for the development of antigen-induced airways eosinophilia. J. Clin. Invest. 102:1132–1141.[Medline]
  20. Tomaki, M., L. L. Zhao, J. Lundahl, M. Sjöstrand, M. Jordana, A. Linden, P. O'Byrne, and J. Lötvall. 2000. Eosinophilopoiesis in a murine model of allergic airway eosinophilia: involvement of bone marrow IL-5 and IL-5 receptor {alpha}1. J. Immunol. 165:4040–4050.[Abstract/Free Full Text]
  21. Minshall, E. M., R. Schleimer, L. Cameron, M. Minnicozzi, R. W. Egan, J. C. Gutierrez-Ramos, D. H. Eidelman, and Q. Hamid. 1998. Interleukin-5 expression in the bone marrow of sensitized Balb/c mice after allergen challenge. Am. J. Respir. Crit. Care Med. 158:951–957.[Abstract/Free Full Text]
  22. Reome, J. B., D. S. Johnston, B. K. Helmich, T. M. Morgan, N. Dutton-Swain, and R. Dutton. 2000. The effects of prolonged administration of 5-bromodeoxyuridine on cells of the immune system. J. Immunol. 165:4226–4230.[Abstract/Free Full Text]
  23. Lee, N. A., M. P. McGarry, K. A. Larson, M. A. Horton, A. B. Kristensen, and J. J. Lee. 1997. Expression of IL-5 in thymocytes/T cells leads to the development of a massive eosinophilia, extramedullary eosinophilopoiesis, and unique histopathologies. J. Immunol. 158:1332–1344.[Abstract]
  24. Sulakvelidze, I., M. D. Inman, T. Rerecich, and P. M. O'Byrne. 1998. Increases in airway eosinophils and interleukin-5 with minimal bronchoconstriction during repeated low-dose allergen challenge in atopic asthmatics. Eur. Respir. J. 11:821–827.[Abstract]
  25. Collins, P. D., S. Marleau, D. A. Griffiths Johnson, P. J. Jose, and T. J. Williams. 1995. Cooperation between interleukin-5 and the chemokine eotaxin to induce eosinophil accumulation in vivo. J. Exp. Med. 182:1169–1174.[Abstract/Free Full Text]
  26. Yamaguchi, Y., T. Suda, S. Ohta, K. Tominaga, Y. Miura, and T. Kasahara. 1991. Analysis of the survival of mature human eosinophils: interleukin-5 prevents apoptosis in mature human eosinophils. Blood 78:2542–2547.[Abstract/Free Full Text]
  27. Tsuyuki, S., C. Bertrand, F. Erard, A. Trifilieff, J. Tsuyuki, M. Wesp, G. P. Anderson, and A. J. Coyle. 1995. Activation of the Fas receptor on lung eosinophils leads to apoptosis and the resolution of eosinophilic inflammation of the airways. J. Clin. Invest. 96:2924–2931.
  28. Wood, L. J., M. D. Inman, R. M. Watson, R. Foley, J. A. Denburg, and P. M. O'Byrne. 1998. Changes in bone marrow inflammatory cell progenitors after inhaled allergen in asthmatic subjects. Am. J. Respir. Crit. Care Med. 157:99–105.[Abstract/Free Full Text]
  29. Robinson, D. S., R. Damia, K. Zeibecoglou, S. Molet, J. North, T. Yamada, A. B. Kay, and Q. Hamid. 1999. CD34(+)/interleukin-5Ralpha messenger RNA+ cells in the bronchial mucosa in asthma: potential airway eosinophil progenitors. Am. J. Respir. Cell Mol. Biol. 20:9–13.[Abstract/Free Full Text]
  30. Leckie, M. J., A. ten Brinke, J. Kham, Z. Diamant, B. J. O'Connor, C. M. Walls, A. K. Mathur, H. C. Cowley, K. F. Chung, R. Djukanovic, T. T. Hansel, S. T. Holgate, P. J. Sterk, and P. J. Barnes. 2000. Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyper-responsiveness, and the late asthmatic response. Lancet 356:2144–2148.[CrossRef][Medline]
  31. Flood-Page, P. T., A. N. Menzies-Gow, B. Kay, and D. S. Robinson. 2003. Eosinophil's role remains uncertain as Anti-Interleukin-5 only partially depletes numbers in asthmatic airway. Am. J. Respir. Crit. Care Med. 167:199–204.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2002-0311OCv1
30/3/367    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sitkauskiene, B.
Right arrow Articles by Lötvall, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sitkauskiene, B.
Right arrow Articles by Lötvall, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2004 American Thoracic Society.