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The CCR3 Receptor Is Involved in Eosinophil Differentiation and Is Up-Regulated by Th2 Cytokines in CD34⁺ Progenitor Cells¹

Bouchaib Lamkhioued^2,*,, Soussi Gounni Abdelilah, Qutayba Hamid, Nabil Mansour, Guy Delespesse^* and Paolo M. Renzi^*

^* Centre Hospitalier de l’Universite de Montreal, Research Center, Notre Dame Hospital, University of Montreal, Meakins-Christie Laboratories and Departments of Medicine and Pathology, McGill University, and Charles le Moyne Hospital, Montreal, Quebec, Canada

	Abstract

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The involvement of chemokines in eosinophil recruitment during inflammation and allergic reactions is well established. However, a functional role for chemokines in eosinophil differentiation has not been investigated. Using in situ RT-PCR, immunostaining, and flow cytometric analysis, we report that human CD34⁺ cord blood progenitor cells contain CCR3 mRNA and protein. Activation of CD34⁺ progenitor cells under conditions that promote Th2 type differentiation up-regulated surface expression of the CCR3. In contrast, activation with IL-12 and IFN- resulted in a significant decrease in the expression of CCR3. Eotaxin induced Ca²⁺ mobilization in CD34⁺ progenitor cells, which could explain the in vitro and in vivo chemotactic responsiveness to eotaxin. We also found that eotaxin induced the differentiation of eosinophils from cord blood CD34⁺ progenitor cells. The largest number of mature eosinophils was found in cultures containing eotaxin and IL-5. The addition of neutralizing anti-IL-3, anti-IL-5, and anti-GM-CSF Abs to culture medium demonstrated that the differentiation of eosinophils in the presence of eotaxin was IL-3-, IL-5-, and GM-CSF-independent. These results could explain how CD34⁺ progenitor cells accumulate and persist in the airways and peripheral blood of patients with asthma and highlight an alternative mechanism by which blood and tissue eosinophilia might occur in the absence of IL-5.

	Introduction

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Studies performed on respiratory secretions and biopsies of the airways in patients with asthma suggest that eosinophils play a central role in the pathogenesis and clinical expression of the disease (1, 2). It is widely believed that asthma may in part be caused by the misdirected or aberrant actions of preformed toxic proteins released by activated eosinophils that are recruited into the airways during exposure to Ag (1). The recruitment and/or activation of these cells appear to be controlled by the release of cytokines such as IL-5 and chemotactic agents such as eotaxin from Ag-stimulated T lymphocytes (3). We have previously demonstrated that IL-5 (4) and eotaxin (5) are up-regulated within the airways of patients with asthma when compared with normal controls. An association between the expression of lymphocyte Th₂ cytokines, particularly IL-5, and eotaxin has also been demonstrated (6), while eotaxin could be one of the molecular links between Ag-specific T cell activation and the recruitment of eosinophils into the airways (3). An additional link between T cell-derived IL-5, eotaxin, and eosinophil activation in asthmatic airways has been provided by evidence demonstrating that the eosinophil is the predominant site of IL-5 and eotaxin receptor expression (7, 8). It has been suggested that IL-5 and eotaxin may collaborate in the regulation of blood and tissue eosinophilia in mice (6). However, the relative contribution of eosinophil chemotactic and activating chemokines to the generation of the eosinophilia that is found in the airways of patients with asthma is poorly understood.

The most striking role of IL-5 and eotaxin has been shown in a mouse model of asthma. Of particular interest was the finding that although aeroallergen challenge of IL-5^-/- mice failed to induce eosinophilia, lung damage, and airways hyperreactivity (9), these animals still produced basal levels of eosinophils that appeared to be morphologically and functionally normal (10). In IL-5^-/- mice, IL-5-independent eosinophils that localized to helminthes-infected sites (liver, intestine, lungs, and peritoneal cavity) appeared morphologically similar to IL-5-dependent eosinophils and degranulated in the presence of worms (11). Recently, it has also been shown that eotaxin-deficient mice exhibit a marked decrease in allergen-induced eosinophil recruitment into the airways when compared with wild-type mice (12). These results and the fact that blood eosinophilia can be elicited in IL-5-deficient mice by i.v. administration of eotaxin (6) suggest that an alternative mechanism for the production of blood and tissue eosinophilia exists that is not IL-5-dependent.

Although important in cell recruitment, certain chemokines (like stromal cell-derived factor (SDF)³ 1, macrophage-inflammatory protein-1, recombinant human (rh) monokine induced by IFN-, and IFN-inducible protein-10) and chemokine receptors also appear to be involved in the proliferation and/or the differentiation of hemopoietic progenitor cells (13, 14, 15). Recently in mice, eotaxin have been associated with mast cells development from embryonic hemopoietic progenitors (16). It has been suggested that eotaxin may also be involved in the mobilization of eosinophils and their progenitors from the bone marrow (BM) and their subsequent recruitment into sites of allergic inflammation (17). This hypothesis has been strengthened by the finding of the specific receptor for eotaxin, CCR3, on eosinophils (18), basophils, (19) and Th₂-like lymphocytes (20), all of which are found in tissues undergoing allergic reactions. However, it is not known whether eotaxin affects the differentiation of hemopoietic progenitor cells.

Because eosinophils can be produced in the absence of signaling events mediated by IL-5 (10), we examined the effect of eotaxin on the differentiation of eosinophils. In this report, we provide evidence that CD34⁺ hemopoietic progenitor cells express CCR3, the receptor for eotaxin. We show that eotaxin induces the differentiation of eosinophils from CD34⁺ progenitor cells and enhances the action of known eosinophilopoietic cytokines. These results suggest another mechanism by which eotaxin may be involved in inflammatory diseases associated with tissue eosinophilia, i.e., through eosinophil differentiation from progenitor cells.

	Materials and Methods

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Mononuclear cell and CD34⁺ cell purification

Cord blood samples were obtained from Charles le Moyne Hospital (Montreal, Canada) according to the guidelines established by the Human Investigation Committee. Human umbilical cord blood mononuclear cells (CBCs) were obtained by Ficoll-Hypaque density centrifugation at 400 x g for 30 min. Monocytes were depleted by adherence to plastic flasks during at least 2 h of incubation at 37°C in RPMI 1640 supplemented with 7.5% FCS in 5% CO₂. CD34⁺ cells were purified by immunomagnetic bead selection (CD34⁺ isolation kit; Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. Cells were placed in complete medium containing RPMI 1640 supplemented with 10% FCS, 50 mM 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 µM L-glutamine, and 7 mM HEPES (Life Technologies, Gaithersburg, MD). The purity of the CD34⁺ hemopoietic progenitors ranged from 90–99% as determined by flow cytometry. Typical contaminant cells were monocytes.

Determination of receptor expression on CD34⁺ cells by immunostaining

CD34⁺ cells were positively selected from monocyte-depleted human umbilical cord blood cells by magnetic column preparation. Cells were maintained in complete medium containing either recombinant IL-3, IL-4, IL-5, GM-CSF, IFN-, or IL-12, each at a final concentration of 20 ng/ml and were incubated overnight at 37°C in a fully humidified atmosphere containing 5% CO₂ CD34⁺ cells washed and cytocentrifuged onto slides with a cytospin III (Thermo Shandon, Pittsburgh, PA). Slides were saturated with universal blocking solution for 10 min (DAKO, Carpenteria, CA), incubated with goat anti-human CCR3 Abs (Santa Cruz Biotechnology, Santa Cruz, CA) or mouse anti-CXCR4 mAb (clone 44708.111; R&D Systems, Minneapolis, MN) overnight at 4°C, and washed twice with TBS followed by an incubation for 1 h at room temperature with 5 µg/ml rabbit anti-goat IgG or horse anti-mouse IgG biotin-conjugated Abs (Vector Laboratories, Burlingame, CA). Positive cells were stained red after development with Fast Red and streptavidine-AP (Sigma-Aldrich, St. Louis, MO). Omission of the primary Ab or blocking the mAb binding with rh-eotaxin at overnight at 4°C were used as negative controls. After each incubation with Abs, slides were extensively washed with TBS. Nuclei of cells were stained for 1 min with Mayer’s hematoxylin.

Determination of receptor expression on CD34⁺ cells by in situ RT-PCR

CCR3 mRNA expression in purified CD34⁺ cells was examined by in situ RT-PCR as previously described (21). The CD34⁺ cell population was selected by sorting on a cell sorter (BD Biosciences, Mississauga, Canada). Only CD34⁺ cells were selected and the purity of CD34⁺ cells range from 95–99%. Typical contaminant cells were monocytes. CD34⁺ cells were incubated in the presence or absence of IL-4 or IL-5 (20 ng/ml) overnight at 37°C, and washed, placed in complete medium, and cytocentrifuged onto slides with a cytospin III. Cells were hybridized with 2.5 ng/µl antisense oligonucleotide to human CCR3 gene, washed, and air dried. Superscript RT (3 U/µl) in the presence of 1 mM each of dATP, dGTP, dCTP, and dTTP, and 1 U/µl RNase inhibitor was applied to cells for 2 h in a humidified chamber. Slides were washed extensively in 2 x SSC and equilibrated with PCR buffer. For the in situ amplification, the reaction mixture containing Taq polymerase, nucleotides, and 10 pmol/µl each of the 5' and 3' oligonucleotides (22) was added to the slides and the coverslips were sealed at the edge with nail polish to prevent desiccation. PCR conditions were 1 min at 94°C, 2 min at 60°C, and 3 min at 72°C for 25 cycles. After washing in TBS, the slides were incubated with alkaline phosphatase-conjugated sheep anti-digoxigenin mAb overnight at 4°C. Positive cells stained brown after development with chromogen containing 45 µl of nitroblue tetrazolium salt, 35 µl of 5-bromo-4-chlor-3-indoyl phosphate and 1 mM levamisole (Sigma-Aldrich).

Flow cytometric analysis

Cells collected from human cord blood and CD34⁺ cells were purified by immunomagnetic bead selection as described above. Human umbilical cord blood CD34⁺ (10⁶/ml) were washed once with PBS and were incubated with purified normal mouse IgG (Santa Cruz Biotechnology), 2 µg/10⁶ cells at 4°C for 20 min to block any possible nonspecific binding. FITC- or PE-conjugated mAbs or control isotype Abs were then incubated with the cells at 4°C for 30 min. After extensive washing, cells were resuspended in 1% paraformaldehyde in PBS at 4°C in the dark. Cell-associated immunofluorescence was analyzed by a FACScan flow cytometer (BD Biosciences) to determine the level of surface expression of CCR3 by CD34⁺ cells. Several mAbs were used for flow cytometry: rat anti-human CCR3 mAb FITC-conjugated (FAB155F, clone 61828.111; R&D Systems) and PE-conjugated anti-CD34 (clone BIRMA-K3; DAKO). The corresponding control isotype Abs were purchased from R&D Systems and DAKO, respectively.

Quantification of receptor expression

For immunostaining and in situ RT-PCR, positive cells were counted in a blinded fashion in a random coded order using a Zeiss Axiophot microscope (Carl Zeiss, Oberkochen, Welwyn Garden City, U.K.) at x200 magnification (5). Cells exhibiting positive mRNA signals or immunoreactivity were counted in at least three fields (counting a minimum of 400 total cells). The percentage of CCR3 and CXCR4 positive cells was calculated, and results reported as the mean percentage ± SD.

Calcium efflux assay in CD34⁺ cells

CBCs were stained with anti-CD34 mAb (DAKO). Positive cells were sorted on a cell sorter and loaded with Fluo-3 dye for 20 min at 37°C. After two washes, the cells were resuspended at a concentration of 1 x 10⁶/ml in HEPES-buffered saline containing 137 mM NaCl, 5 mM KCl, 1 mM Na₂HPO₄, 5 mM glucose, 1 mM CaCl₂, 0.5 mM MgCl₂, 1 g/L BSA, and 10 mM HEPES pH 7.4. The cells were then incubated for 10 min at 37°C, vortexed, and analyzed. Stimulation was performed by adding eotaxin (20 and 50 ng/ml), RANTES (10 and 50 ng/ml), or SDF-1 (20 and 50 ng/ml), and fluorescence changes were monitored over time for up to 200 s. In some experiments, CD34⁺ cells were preincubated at 4°C for 30 min with Abs directed against chemokine receptor (CCR3, clone 61828.111; R&D Systems) or control Abs before stimulation with eotaxin.

Chemotactic assay

CD34⁺ cell populations were selected by sorting on a cell sorter, washed, and resuspended at 10⁶/ml in RPMI medium. Migration of CD34⁺ cells in response to different concentrations of eotaxin, RANTES, and SDF was assessed in a 24-well microchemotaxis chamber (NeuroProbe, Cabin John, MD) using a polycarbonate filter (5-µm pore size) as previously described (23). CD34⁺ cells were resuspended in RPMI medium loaded into the upper chambers and tested for chemoattraction to media alone (negative control), eotaxin, RANTES, or SDF-1. The chambers were incubated at 37°C in 5% CO₂ for 120 min. In some experiments, CD34⁺ cells were preincubated at 4°C for 30 min with Abs directed against chemokine receptors (CCR3; R&D Systems) or control Abs before the chemotaxis assay.

Chemotactic effect of eotaxin on CD34⁺ cells in vivo

Male BALB/c mice were purchased from (Charles River Breeding Laboratories, St. Constant, Quebec, Canada) and used in all bioassay experiments. Donor CD34⁺ BM cells for homing experiments were obtained from femoral BM of mice. For cell sorting performed to isolate the CD34⁺ subpopulation, cells were labeled with biotinylated anti-CD34 mAb, and revealed by streptavidin-PE as recommended by the manufacturer (BD PharMingen). Sorted cells were collected in 10% FCS RPMI, whereas a fraction of the cells was restained and analyzed to verify the purity of the sorted population. Sorted CD34⁺ cells were cultured 18 h after cell sorting to release remaining bound mAbs and thereafter washed two times before adoptive transfer. After three washes, cells were resuspended in RPMI 1640 complete medium, and incubated in the presence of 10 µg/ml of the polycationic molecule, 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) overnight at 37°C (24). DAPI-stained BM cells were then harvested, washed twice in PBS, and resuspended in PBS. Trypan blue exclusion showed that >95% of the DAPI-staining cells were viable (25). Five male BALB/c mice were anesthetized by i.p. injection of somnotol (MTC Pharmaceuticals, Cambridge, Ontario, Canada). The mice were then injected i.v. with 5 x 10⁶ BM CD34⁺ cells labeled with DAPI. Increasing concentrations of eotaxin (10, 500, and 1000 pmol in 100 µl pyrogen-free isotonic saline) and saline as a control were administered into the skin 24 h after the injection, the mice were killed by an i.p. injection of a high dose of somnotol and their skin obtained for analysis. To evaluate the location of labeled BM cells within the skin and to determine whether the transferred CD34⁺ cells migrated, the skin biopsies were fixed in 4% paraformaldehyde for 2 h, and 10-µm frozen sections were examined by fluorescence microscopy.

Production of umbilical cord blood-derived eosinophils

Monocyte-depleted human cord blood cells were cultured in 6-well tissue culture plates (5 ml of cell suspension per flat-bottom well) at 37°C with 5% CO₂ for up to 4 wk. Eosinophilic differentiation was induced by the addition of rh-IL-5, rh-GM-CSF (1 ng/ml), rh-RANTES, or rh-eotaxin at different concentrations either alone or in combination. The culture medium containing these cytokines and chemokines was replaced weekly. As a control, monocyte-depleted human cord blood cells were cultured in medium alone. For the identification of eosinophils, cytospin preparations were stained as described below and cellular differential and viability counts were determined immediately. To assess the specificity of the system, neutralizing Abs to IL-3, IL-5, and GM-CSF (all from Genzyme, Cambridge, MA) or isotype control Abs were added to the cultures. The presence of eosinophils was determined by peroxidase staining as described below.

Cell counting and identification of eosinophils

Every 7 days cellular differential counts were obtained on cytocentrifuged preparations obtained from flat-bottom wells. Cells committed to eosinophil lineage were determined by cyanide-resistant eosinophil peroxidase (EPO) staining (26). Briefly, cytocentrifuged preparations were fixed with methanol-acetone (1:1) for 10 min followed by a reaction with EPO staining solution which consisted of 100 ml of PBS containing 75 mg of 3.3' diaminobenzidine tetrahydrochloride (Sigma-Aldrich), 39.2 mg of potassium cyanide, and 0.3 ml of 30% H₂O₂ for 10 min at room temperature, then counterstained with Mayer’s hematoxylin. Dark brown staining was characteristic of the eosinophil-specific cyanide-resistant peroxidase (27). After 4 wk of culture, >80% of viable cells were committed cells of eosinophilic lineage confirmed as cyanide-resistant EPO-positive cells. The remaining cells were mainly monocytes/macrophages.

Expression of EPO mRNA

The expression of EPO mRNA in CBC depleted of Monocytes was determined on days 3, 7, 14, and 21 of liquid cultures containing optimal concentrations of eotaxin (25 ng/ml) or IL-5 (1 ng/ml) using RT-PCR (28). Total RNA (2 µg) was reverse transcribed using Moloney murine leukemia virus reverse transcriptase with oligo(dT) in a 25 µl reaction and 1 µl was used for specific amplification of EPO cDNA using EPO primers (28) and Taq polymerase (Life Technologies). G3PDH was used as the standard to control for variations in RNA isolation, cDNA synthesis, and PCR performance. A sample of cDNA was subjected to sequential cycles of amplification (20, 25, 30, 35, and 40 cycles). Samples were amplified at 94°C for 1 min, 60°C for 2 min, and 72°C for 3 min. The OD obtained for each amplified fragment was plotted against the number of cycles. The amounts of PCR-generated bands increase logarithmically up to a certain number of cycles, reaching a plateau thereafter. Under these conditions, it was established when PCR were still in the exponential (quantifiable) phase. The quantification was achieved by scanning the band intensities obtained on ethidium bromide-stained agarose gels with an Instant Imager System 2000 (Amersham Pharmacia Biotech, Piscataway, NJ) (5).

Statistical analysis

Statistical comparisons were performed using a Student’s t test. A value of p < 0.05 was considered significant. Results are presented as mean ± SD.

	Results

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CCR3 mRNA and immunoreactivity on sorted CD34⁺ cells

Cord bloods obtained from six donors and enriched for CD34⁺ cells by MACS were assessed for the expression of CCR3 mRNA and protein. Photomicrographs (Fig. 1a) illustrate CCR3 protein expression among CD34⁺ cells. CCR3 mRNA and protein expression was found on 22 ± 6 and 19 ± 3, respectively, of the CD34⁺ progenitor cells derived from cord blood (Table I). Because the in vivo chemoattractant effect of eotaxin is enhanced by IL-5 (5, 6), we have evaluated the priming effect of IL-5 on the CCR3 expression. When CD34⁺ cells were pretreated with IL-5, the CCR3 expression was enhanced when compared with untreated cells (p < 0.05, Fig. 1b and Table I). No immunoreactivity was found when control Abs were used instead of the goat anti-CCR3 Abs or when the first Ab binding was blocked with rh-eotaxin (Fig. 1d).

View larger version (120K): [in this window] [in a new window]   FIGURE 1. Expression of CCR3 protein in CD34+ cells purified from human umbilical CBCs. a and b, Immunocytochemical detection of CCR3 in representative cytospin preparations of sorted CD34+ cells. a, Immunoreactivity signal (red color) for CCR3 in cytospin preparations of sorted CD34+ was observed in nontreated CD34+ cells. b, When CD34+ cells were pretreated with IL-5, the percentage of CCR3-positive cells was increased when compared with untreated cells. c, Eosinophils, which are known to express CCR3, were included as a positive control. d, No signal was obtained when the first Ab was blocked with rh-eotaxin. Nuclei of cells were stained for 1 min with hematoxylin.

CCR3 expression on CD34⁺ cells is up-regulated by Th2 cytokines

Staining of cord blood CD34⁺ cells performed using goat anti-CCR3 Abs showed that a subset of freshly isolated CD34⁺ cells expressed CCR3 and is up-regulated by IL-5 (p < 0.05, Table II). Because the expression of CCR3 could be affected by cytokines (20), we further examined by immunostaining the expression of CCR3 on CD34⁺ cells following exposure to different cytokines. As shown in Table II, exposure to Th₂ cytokines (GM-CSF, IL-3, IL-4, and IL-5) enhanced CCR3 expression on CD34⁺ cells, while activation with Th₁ cytokines (IFN- or IL-12) resulted in a significant decrease in the expression of CCR3 in cord blood-derived CD34⁺ hemopoietic progenitor cells.

The cell surface expression of CCR3 was analyzed by flow cytometry (Fig. 2). To detect CCR3 expression on CD34⁺ cells, two-color immunofluorescence staining was performed using FITC-conjugated anti-CCR3 with PE-conjugated anti-CD34 mAb as described in Materials and Methods. Flow cytometry analysis showed that CCR3 was coexpressed with CD34. The percentage of expression of CCR3 with CD34 was found to be different from donor to donor. The coexpression of CCR3 with CD34 ranged from 9–16% with an average of 13 ± 2.7. One representative experiment is shown in Fig. 2. After 24 h of incubation with cytokine-free medium, there was no significant change in CCR3⁺ cell fraction (data not shown). We observed that the number of CD34⁺ cells expressing CCR3 obtained by flow cytometry appeared lower than the number of the cells expressing CCR3 obtained by immunostaining, indicating that CCR3 my be regulated. Alternatively, the differences observed using flow cytometry vs immunostaining could be explained by the use of different anti-CCR3 Abs.

View larger version (17K): [in this window] [in a new window]   FIGURE 2. Two-color flow cytometry analysis of the cell surface expression of CCR3 on cord blood CD34+ progenitor cells. Right panel, Purified CD34+ cells were labeled simultaneously with a FITC-conjugated anti-CCR3 mAb and a PE-conjugated anti-CD34 mAb. Left panel, Double staining with PE- and FITC-labeled normal isotype rat and mouse IgG were used as negative control. The figure is consistent with the data reported in Table I, as CCR3 was commonly expressed in almost all CBC that were examined. The percentage of cells expressing CCR3 and CD34 is 16%, whereas the percentage of CD34+CCR3- is 84%.

Mobilization of intracellular calcium is an early event in the response to chemokine and cytokine signals (8). We tested the ability of eotaxin, RANTES, and SDF to induce mobilization of calcium in human CD34⁺ cells (Fig. 3). Eotaxin induced a rapid, transient flux of intracellular calcium in CD34⁺ cells that is similar to the intracellular calcium mobilization obtained with RANTES and SDF-1. Moreover, the eotaxin-induced calcium in CD34⁺ cells was completely blocked by an Ab directed against CCR3 (data not shown).

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Induction of calcium mobilization in sorted CD34+ cells by eotaxin, RANTES, and SDF. CD34+ cells sorted from cord blood were loaded with Fluo-3AM, the chemokines were added at the times indicated by the arrows and the calcium responses were analyzed as indicated in Materials and Methods. CD34+ cells showed a rapid, transient rise in intracellular calcium upon stimulation with SDF-1 (a), eotaxin (b), and RANTES (c). These results are representative of two or more independent experiments.

The expression of CCR3 on CD34⁺ cells was predictive of its functional activities that are mediated by eotaxin. In a chemotaxis assay, CD34⁺ cells migrated to eotaxin in a dose-dependent fashion (Fig. 4A). The number of migrated cells was similar to that observed with other CC and CXC chemokines such as RANTES (Fig. 4B) and SDF-1 (Fig. 4C), ligands for CCR5 and CXCR4, respectively (29, 30). Eotaxin-mediated chemotaxis was entirely due to its interaction with a protein G-coupled receptor, probably CCR3, since it was completely inhibited by pertussis toxin (Fig. 4A) and by an Ab directed against CCR3 (data not shown).

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Eotaxin is a chemoattractant for human and mouse hemopoietic progenitor cells. A, Chemotaxis assay of cord blood CD34+ cells in response to various concentration of eotaxin (10, 50, 100, 500, and 1000 ng/ml). Results are presented as the mean ± SD of three independent experiments. Results represent the average of three experiments. Data are expressed as the percent of input cells that migrated. B, Chemotactic response of cord blood CD34+ cells to different concentrations of RANTES. C, Chemotactic response of cord blood CD34+ cells to different concentrations of SDF. D, In vivo infiltration of CD34+ cells. Experimental mice received intradermal injections of eotaxin (10, 500, or 1000 pmol per site), or pyrogen-free isotonic saline. After 4 h, punch biopsies were taken and processed for histology. CD34+ cells were counted on sections (five fields per section) in areas including postcapillary venules.

Time course and kinetics of eotaxin-induced eosinophil differentiation

Cord blood cells depleted of monocytes were cultured in RPMI 1640 supplemented with 10% FCS in the presence of optimal concentrations of eotaxin (25 ng/ml), RANTES (25 ng/ml), IL-5 (1 ng/ml), or GM-CSF (1 ng/ml) for 28 days (Fig. 5A). The time course of appearance of EPO⁺ eosinophils was monitored from day 0–28. In medium, not >5% of EPO⁺ eosinophils were present. Variable numbers of eosinophils were observed in cultures performed with eotaxin, RANTES, IL-5, or GM-CSF alone. Stimulation of cord blood-derived mononuclear cells with rh-eotaxin (25 ng/ml) induced an increase in eosinophil differentiation after 1 wk of culture and reached a maximal response after 3 wk (43.2 ± 3.8%; Fig. 5). In contrast, eosinophilopoietic cytokines such as IL-5 (1 ng/ml) or GM-CSF (1 ng/ml) induced increased eosinophil differentiation that started from 1 wk of culture and peaked after 4 wk of the culture. Interestingly, RANTES, which shares the CCR3 with eotaxin, could also induce the differentiation of eosinophils and after 3 wk of culture, 34 ± 6% of total cells were EPO⁺ eosinophils. More eosinophils were found in cultures containing IL-5 or GM-CSF when compared with eotaxin or RANTES alone. Eotaxin induced the differentiation of EPO⁺ eosinophils that had nuclear features of mature (segmented) eosinophils and were morphologically similar to IL-5-differentiated eosinophils. Cyanide- resistant peroxidase staining of induced cells showed a granular pattern, and by 4 wk, cultured cells resembled mature eosinophils (Fig. 5B). Furthermore, immunostaining showed that eotaxin-differentiated eosinophils contain also the major basic protein of eosinophils that was also present in a granular pattern (data not shown). To further define the effect of eotaxin, we cultured purified cord blood CD34⁺ cells in the presence of eotaxin or IL-5. In these conditions, >50% of CD34⁺ cells were EPO⁺ eosinophils after 4 wk of culture (Fig. 5Ba) in the presence of eotaxin and were morphologically similar to IL-5-differentiated eosinophils (Fig. 5Bb). Because cytokine-responsive cells first appeared at 1 wk of culture, we evaluated whether mRNA transcripts for EPO (which is an eosinophil-specific marker) were present by using a sensitive RT-PCR assay (Fig. 5C). When CBC were stimulated with eotaxin, no mRNA transcripts for EPO could be detected on day 3, but by day 7 they were detected with increasing amounts seen by day 14, compared with the constitutively expressed transcripts for G3PDH. These data indicate that incubation with eotaxin and RANTES allow the generation of a relatively large number of eosinophils that are suitable for functional studies.

View larger version (41K): [in this window] [in a new window]   FIGURE 5. Compared effect of eotaxin, RANTES, IL-5, and GM-CSF on eosinophil differentiation from monocyte-depleted cord blood cells or sorted CD34+ cells in liquid cultures. A, Monocyte-depleted cord blood cells were isolated and incubated with IL-5 (1 ng/ml), GM-CSF (1 ng/ml), RANTES (25 ng/ml), or eotaxin (25 ng/ml) for 4 wk and cells committed to eosinophil lineage were determined by cyanide-resistant EPO staining as described in Materials and Methods. B, Morphology and phenotype of IL-5 and eotaxin-differentiated cells. Sorted CD34+ cells were incubated with eotaxin (a) or IL-5 (b) for 4 wk and eosinophils were determined by EPO staining. C, The photographic image of RT-PCR products derived from EPO and G3PDH mRNA transcripts in CD34+ cells freshly isolated at day 3 and after 7, 14, and 21 of culture in eotaxin (25 ng/ml) and IL-5 (1 ng/ml) at day 7. The isolation of total RNA, reverse transcription, PCR, and detection procedure are described under Materials and Methods. The same amount of total RNA was used for each sample loaded on the gels. The G3PDH band was used to standardize each analysis.

Because RANTES share the same receptor with eotaxin, we anticipate that that it might also exhibit differentiation effect on hemopoietic progenitor cells. The addition of increasing doses of rh-eotaxin or rh-RANTES (1, 10, 25, 50, and 100 ng/ml) to the medium was associated with increasing differentiation of eosinophils from CBC when compared with the cells incubated in medium alone (n = 10, p < 0.001 at all concentrations tested) (Fig. 6). The effect of optimal concentrations of rh-eotaxin or rh-RANTES (25 ng/ml) on the differentiation of eosinophils was less than that observed with rhIL-5 (n = 10, p < 0.05 at all concentrations of rh-eotaxin).

View larger version (16K): [in this window] [in a new window]   FIGURE 6. Dose-dependent effects of eotaxin and RANTES on eosinophil differentiation from CBCs during 3 wk of culture. Eotaxin (A) and RANTES (B) induce the differentiation of eosinophil from CBCs in a concentration-dependent manner when compared with medium alone (n = 10, p < 0.001 at all concentrations tested). The effect of eotaxin and RANTES on the differentiation of eosinophils was less than that observed with IL-5 (n = 10, p < 0.05 at all concentrations of eotaxin or RANTES). Data shown as mean ± SD.

To evaluate whether rh-eotaxin directly affects the generation of eosinophils from CBC, cells were cultured with optimal concentrations of rh-eotaxin (25 ng/ml) with or without anti-IL-3, IL-5, and GM-CSF neutralizing Abs, and the time course of appearance of EPO⁺ eosinophils was monitored during 4 wk. As shown in Fig. 7, the anti-IL-5-neutralizing Abs inhibit the differentiation of eosinophils induced by IL-5. An initial in vitro study demonstrated that eosinophil differentiation in the presence of IL-3 and GM-CSF could be also neutralized by incubation with neutralizing Abs to these cytokines (data not shown). As shown in Fig. 7, the anti-IL-3, IL-5, and GM-CSF-neutralizing Abs had no effect on the number of EPO⁺ eosinophils induced in the presence of rh-eotaxin.

View larger version (23K): [in this window] [in a new window]   FIGURE 7. Effect of anti-IL-5, anti-IL-3, and anti-GM-CSF neutralizing Abs on eotaxin-dependent generation of eosinophils from CBC. Monocyte-depleted human cord blood cells were cultured in 6-well tissue culture plates for up to 4 wk as described in Materials and Methods. Eosinophilic differentiation was induced by the addition of rh-eotaxin at different concentrations either alone or in combination with anti-IL-3, anti-IL-5, and anti-GM-CSF neutralizing Abs. Cord blood cells were also exposed to IL-5 in the presence or absence of anti-IL-5 neutralizing Abs and the number of differentiated eosinophils was determined at days 0, 7, 14, 21, and 28. Data shown as mean ± SD of five independent preparations.

The combination of rh-IL-5 (1 ng/ml) and rh-eotaxin (25 ng/ml) induced a higher percentage of eosinophils (59% of EPO⁺ cells within 2 wk) compared with rh-eotaxin or IL-5 alone (18 and 32%, respectively; Fig. 8). Culture of mononuclear cells in the presence of rhIL-5 (1 ng/ml) and RANTES (25 ng/ml) together also induced a higher percentage of eosinophils (57% within 2 wk) than RANTES or IL-5 alone (16 and 30%, respectively).

View larger version (26K): [in this window] [in a new window]   FIGURE 8. Effect of the various cytokine combinations on the differentiation of eosinophils. Monocyte-depleted human cord blood cells were cultured for 4 wk in the presence of medium, IL-5, eotaxin, and RANTES individually or in combination. A, Monocyte-depleted human cord blood cells were isolated and incubated with IL-5 (1 ng/ml), eotaxin (25 ng/ml) alone, or in combination for 4 wk and cells committed to eosinophil lineage were determined by cyanide-resistant EPO staining as described in Materials and Methods. B, Monocyte-depleted human cord blood cells were isolated and incubated in the presence of RANTES (25 ng/ml), IL-5 (1 ng/ml), or in combination, for 4 wk. Data shown as mean ± SD of five independent preparations.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

An increase in eosinophils and mast cells characterizes the inflammatory infiltrate that is present in the airways of patients with allergic asthma. These cells share a common progenitor, as demonstrated by common derivation and proliferation in clonal assays in semisolid media (31). Estimates of the number of eosinophil-basophil progenitors in the peripheral blood of patients with atopic disorders have consistently revealed levels approximately three to four times that of nonatopic individuals (32, 33). Interestingly, the level of these progenitors rises in the peripheral blood at the beginning of seasonal allergen exposure and is consistent with the concept that progenitors traffic into sites of allergic inflammation (34). Furthermore, the finding of CD34⁺CD45⁺ myeloid progenitors submucosally in nasal polyps (35) suggests that progenitors are recruited into the airways where they may differentiate within sites of allergic inflammation (in situ hemopoiesis) (36). Although these studies demonstrate an increase in the production of inflammatory progenitor cells in association with allergen inhalation, little is known of the mechanisms that lead to the release and the trafficking of cells and their progeny in response to allergen challenge. It has been shown previously that eotaxin can stimulate the mobilization of eosinophils and progenitors from the BM (17). However, there have been no reports concerning the expression of CCR3 and factors that regulate its expression on human hemopoietic progenitor cells. The detection of CCR3 in CD34⁺ progenitor cells provides a potential mechanism for progenitor cell recruitment and suggests that the increase in eotaxin release during inflammation of the airways may not only be associated with eosinophil, basophil, and Th₂ cell recruitment, but also with CD34⁺ progenitor mobilization into the tissues. In support of this hypothesis is the finding that other CD34⁺ progenitor-active chemokines, including SDF-1, have also been shown to contribute to the recruitment of CD34⁺ progenitor cells (30). Indeed, recent studies have demonstrated cell surface expression of both the SDF-1 receptor (CXCR4) and the CCR5 on CD34⁺ progenitor cells derived from peripheral blood, although different patterns of coreceptors expression could be appreciated by FACS and confocal microscopy (29). By flow cytometry, CXCR4 was found to be expressed in significant amount on circulating CD34⁺ hemopoietic progenitor cells, including more primitive subsets (CD34⁺/CD34^- and CD34⁺/Thy-1⁺ cells) (17). Furthermore, susceptibility of CD34⁺ progenitor cells to HIV infection has been characterized and the role of CCR5 and CXCR4 coreceptors in progenitor cell infection by HIV has been defined (29). Although there is no evidence suggesting the involvement of CCR3 in CD34⁺ progenitor cell susceptibility to HIV, this receptor was described as a target for HIV infection (37). The results of this study complement and extend previous investigations showing that CCR3 is present on eosinophils (18), basophils (19), and Th₂ cells (20).

A number of processes that are believed to be important in the pathogenesis of asthma have been ascribed to the activities of Th₂ cytokines. These cytokines, particularly IL-4 and IL-5, are increased in lung lavage fluid of patients with asthma and are released in increased amounts by T lymphocytes (38), eosinophils (39), and mast cells (40, 41) obtained from asthmatic patients. Th₂ cytokines seem to contribute indirectly to the inflammation of the airways. Indeed, as has been described for IL-13 (42, 43), IL-4 induces VCAM-1 expression on vascular endothelial cells (44), a key step in the recruitment of eosinophils and mononuclear cells to the airways after allergen challenge. Although IL-4 and IL-5 receptors are increased on CD34⁺ progenitor cells (36, 45, 46, 47), IL-5 seems to have little effect on the migration of eosinophils into tissues (48), but has been shown to prime human eosinophils to respond to other chemotactic stimuli such as RANTES, IL-8 (49), and eotaxin (5). Chemokine receptor expression can be modulated by cytokines known to affect T lymphocyte differentiation, such as IFN- (which promotes the Th₁ phenotype) and TGF- (which promotes the Th₂ phenotype). Indeed, TGF- has been shown to up-regulate CCR3, but decrease CCR4 expression, whereas IFN- has been shown to inhibit CCR3 but up-regulate CXCR3 expression (20). Our results show that IL-4 and IL-5 up-regulate CCR3 expression on CD34⁺ progenitor cells. It is thus possible that the amount of eotaxin released in normal airways is insufficient to lead to the recruitment of CD34⁺ cells. In contrast, priming of CD34⁺ cells with Th₂ cytokines such as IL-5 or IL-4 and the increase in CCR3 ligands production (such as eotaxin and monocyte chemoattractant protein-4) (5, 50) in the airways of asthmatics may contribute to CD34⁺ cells influx and activation. This hypothesis is consistent with results in mice where eotaxin seem to be involved in both the mobilization of eosinophils and their progenitors from the BM into the blood and their subsequent recruitment into sites of allergic inflammation (17). In addition, we find that eotaxin and IL-5 seem to act synergistically on eosinophil differentiation from progenitor cells. During allergic inflammation, in addition to its activity as a chemoattractant, eotaxin can signal to BM to increase the number of myeloid progenitor cells (17). These events may contribute to CD34⁺ progenitor cell mobilization toward sites of inflammation and further differentiation within these sites (36). Although the data presented herein do not prove a direct association between activation of CD34⁺ progenitor cells and the development of airway pathology, as a result of increased inflammatory cell differentiation from progenitor cells and recruitment into the tissues, they are consistent with the view that a feedback mechanism exists between tissues involved in allergic inflammation and distal sites such as the BM. Despite the high level expression of CCR3 mRNA and protein in CD34⁺ cells, the level of the CCR3 protein expression on the cell surface appears to be low. The reason for this discrepancy is yet to be determined, but may be explained by enhanced receptor internalization (51) in the presence of its ligands. Alternatively, posttranscriptional regulation or existence of receptor activity-modifying proteins necessary for expression of mature membrane protein (52) may exist.

It has previously been reported that the chemokines IL-8, macrophage-inflammatory protein-1, and SDF-1 enhance the recruitment of hemopoietic progenitor cells from the BM (30, 53, 54, 55). We have assessed whether eotaxin stimulates the in vitro and in vivo mobilization of CD34⁺ progenitor cells. Our experiments provide evidence that hemopoietic progenitor cells migrate in vitro and in vivo toward a gradient of eotaxin. Eotaxin, at the concentrations that have been shown to be effective in chemotaxis assays, elicited a transient elevation in the concentration of cytoplasmic calcium in CD34⁺ cells. Although the effects of eotaxin are not restricted to hemopoietic progenitor cells, as it is also a chemoattractant for human eosinophils and basophils, it elicits a rapid and direct chemotactic response in CD34⁺ progenitor cells. Accordingly, CD34⁺ cells express CCR3 constitutively and our results indicate that eotaxin elicits maximal migration of 25% of CD34⁺ cells 2 h after exposure that was completely blocked by pertussis toxin (Fig. 4). An increase in eosinophil progenitor cells has been reported in the blood of atopic asthmatics during exacerbation of asthma and in nasal polyp tissue (33, 34, 35, 36). We have shown that CD34⁺ cells express the eotaxin receptor CCR3, and can be mobilized by eotaxin from the blood into the skin (Fig. 4). Although the mediators that regulate the recruitment of CD34⁺ cells into the airways have not been identified, eotaxin, which has been detected in human asthmatic lung (5) and nasal polyps (56), is a potential candidate for this function.

Mice genetically deficient in eotaxin have been described (12), but eosinophil differentiation in these mice has not been examined. These mice have reduced numbers of circulating eosinophils and the number of eosinophils recruited into inflamed airways was also diminished. There was no reduction in the number of eosinophil progenitors in the BM. The finding that eosinophils normally account for only a small percentage of circulating or tissue-dwelling cells and that their numbers markedly and selectively increase under specific disease states indicate the existence of molecular mechanisms that regulate the selective generation and accumulation of these leukocytes. Eosinophils are generated in the BM from pluripotential stem cells. IL-3, IL-5, and GM-CSF regulate eosinophil development by binding to receptors that share a common -chain (c) and have unique -chains. In addition to c, mouse IL-3 also binds to a mouse receptor that contains a separate IL-3 chain. Of these three cytokines, IL-5 (also known as eosinophil differentiation factor) is the most specific to the eosinophil lineage (26). IL-5 also stimulates the release of eosinophils from the BM into the peripheral circulation. The critical role of IL-5 in regulating the production of eosinophils is most clearly demonstrated by genetic manipulation of mice. Overproduction of IL-5 in transgenic mice results in marked peripheral blood eosinophilia. Deletion of IL-5 gene inhibits allergen-induced peripheral blood and pulmonary eosinophilia (9). Interestingly, the baseline levels of eosinophils in the BM or blood of mice deficient in IL-5, c, GM-CSF, or both c and IL-3 are similar or only modestly reduced when compared with wild-type mice (57). This indicates the importance of other factors and/or receptors in the generation of eosinophils. Recently, it has been shown that during the development of lung allergic inflammation, the in vivo administration of eotaxin increased the number of myeloid progenitors present in the bone morrow. Furthermore, eotaxin seem to be a CSF for granulocytes and macrophages (58). We then analyzed the long-term effect of eotaxin on the differentiation of cord blood cells by growing the cells in the presence of eotaxin. In our system, we found that eotaxin alone can induce extensive differentiation of eosinophils from hemopoietic progenitor cells (Fig. 5). The differentiated cells showed the typical eosinophil morphology and expressed EPO as assessed by cyanide-resistant peroxidase staining (Fig. 5) and RT-PCR. To exclude an indirect differentiation effect by eotaxin through the activation of nonhemopoietic progenitor cells, eotaxin was also tested for its ability to stimulate the differentiation of purified CD34⁺ cells into eosinophils. We found that eotaxin induced the differentiation of CD34⁺ cells for up to 4 wk. This differentiation was also coupled with the expression of EPO and the typical morphology of eosinophils. The use of neutralizing anti-IL-5, IL-3, and GM-CSF Abs demonstrates that eosinophils can arise from a direct effect of eotaxin that does not depend on secretion of these three cytokines by differentiating progenitors. Recently, it has been shown that eotaxin may act as a GM-CSF during lung inflammation either in the presence or absence of neutralizing Abs to IL-3 or IL-5 (58). In addition, the pretreatment of CD34⁺ progenitor cells with pertussis toxin inhibited the eotaxin-induced eosinophil differentiation (data not shown). Our results suggest that eotaxin by itself could induce the differentiation of hemopoietic progenitors into eosinophils by acting directly on the progenitor cells. However, at this stage, the possibility that eotaxin stimulation may trigger the production of IL-5, IL-3, and GM-CSF or other endogenous cytokines relevant for inducing eosinophil differentiation from progenitor cells cannot be ruled out. Our findings suggest an alternative IL-5-independent pathway of eosinophil differentiation, the existence of which was indeed suggested by the presence of eosinophils in mice with inactivated IL-5 gene (11).

We further compared the effects of eotaxin along with RANTES on the differentiation of cord blood cells. Our results show that RANTES, a CC chemokine that signals through CCR3, can also induce the differentiation of eosinophils. It is likely that the differentiation of eosinophils occurs through this receptor. It remains to be determined whether the concentrations of eotaxin and RANTES in the airways and the blood of asthmatic patients are similar to those that are effective in vitro. However, in allergic inflammatory diseases, the release of several eosinophils priming cytokines such as IL-5 is increased (59). Therefore, we tested the effects of IL-5 and eotaxin or RANTES combinations on the differentiation of eosinophils. We found that eotaxin and RANTES in combination with IL-5 in vitro induced a higher percentage of eosinophils, compared with chemokines or IL-5 alone, with morphological features characteristic of eosinophils (Fig. 8). There are several possibilities for this observation. Eotaxin and RANTES, acting through CCR3, could induce the secretion of IL-5 or other cytokines in culture that accelerate eosinophil differentiation from hemopoietic progenitor cells. Eotaxin could also induce the expression of IL-5R on CD34⁺ cells and prime these cells to respond to IL-5. Alternatively, IL-5 can prime CD34⁺ cells to respond to eotaxin or RANTES at even lower concentrations. This hypothesis is consistent with results in IL-5 transgenic mice where injection of eotaxin in the lungs stimulated a rapid and dramatic increase in the number of eosinophils in the bronchoalveolar lavage and airways (60). Thus, a potent and specific chemokine, such as eotaxin, could combine with specific and highly effective cytokines such as IL-5 to promote a selective CD34⁺ cells accumulation and differentiation in sites of allergic inflammation.

The existence of a CCR3-dependent pathway of eosinophil development calls for the identification of cells that produce eotaxin. Activated T cells represent an obvious candidate, a possibility that would be consistent with the T cell secretion of hemopoietic growth factors (including T cell-specific IL-5), the presence of T cells in the BM (61, 62, 63, 64, 65, 66), and the lack of eosinophilia observed in T cell-deficient animals (61). Recirculating CD34⁺ cells may also encounter epithelial cells in the airways, thereby contributing to local eosinophil differentiation within the respiratory mucosa. Recently, we and others have shown up-regulation of eotaxin in the bronchial mucosa and have localized its expression to lung epithelial cells in patients with atopic asthma (5). Eosinophil precursors (CD34/IL-5R⁺ cells) also have been identified within the lungs of asthmatics and following ex vivo stimulation with specific allergen or rhIL-5, human nasal mucosal tissue obtained from patients with allergic rhinitis exhibited fewer CD34/IL-5R⁺ cells but more major basic protein-immunoreactive and IL-5 mRNA⁺ cells (36). We speculate that eotaxin production by epithelial cells during allergic or inflammatory responses, such as asthma, may influence the differentiation and/or function of eosinophils at local sites of inflammation.

Taken together, our findings indicate that a novel eotaxin-dependent pathway exists for the differentiation of progenitor cells into eosinophils. This would provide an alternative and probably a necessarily redundant mechanism to satisfy the increased eosinophil demands that occur during the immune responses to parasitic infections or in allergic diseases. The eotaxin-CCR3 interaction may thus represent an important link between hemopoiesis, mobilization, and migration of eosinophils into the site of inflammation.

	Acknowledgments

 
We thank the nurses of Charles le Moyne Hospital for collecting the samples for this study. Special thanks are also addressed to all volunteers who, by their consent, allowed this study to be performed. Additionally, we thank S. Seguin and N. Tessier for facilitating the purification and the sorting of cord blood progenitor cells. We thank Dr. J. Martin for his advice, thoughtful comments, and review of this manuscript.

	Footnotes

 
¹ This work was supported by the Medical Research Council Grants MT14842, MT10397, and MOP38011.

² Address correspondence and reprint requests to Dr. Lamkhioued Bouchaib, Centre Hospitalier de l’Universite de Montreal Research Center, Notre Dame Hospital, Pavillon Mailloux M-4211K, 1560 Sherbrooke East, Montreal, H2L-4 M1, Quebec, Canada. E-mail address: b.Lamkhioued{at}umontreal.ca

³ Abbreviations used in this paper: SDF, stromal cell-derived factor; BM, bone marrow; CBC, cord blood mononuclear cell; DAPI, 4,6-diamidino-2-phenylindole; rh, recombinant human; EPO, eosinophil peroxidase; c, -chain.

Received for publication January 31, 2002. Accepted for publication October 11, 2002.

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