HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nagase, H. Articles by Hirai, K. Search for Related Content PubMed PubMed Citation Articles by Nagase, H. Articles by Hirai, K.

Expression of CXCR4 in Eosinophils: Functional Analyses and Cytokine-Mediated Regulation¹

Hiroyuki Nagase^*, Misato Miyamasu, Masao Yamaguchi, Takao Fujisawa^§, Ken Ohta^¶, Kazuhiko Yamamoto, Yutaka Morita^* and Koichi Hirai^2,

Departments of ^* Respiratory Medicine, Allergy and Rheumatology, and Bioregulatory Function, University of Tokyo Graduate School of Medicine, Tokyo, Japan; ^§ Department of Pediatrics, National Mie Hospital, Mie, Japan; and ^¶ Department of Internal Medicine, Teikyo University School of Medicine, Tokyo, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We examined the expression of transcripts of a panel of chemokine receptors in human eosinophils and found intense constitutive expression of CXCR4 mRNA. Although surface CXCR4 protein was hardly detectable in the peripheral blood or freshly isolated eosinophils, surface expression of CXCR4 became gradually apparent during incubation at 37°C. In contrast, the level of CCR3 expression was virtually unchanged during the incubation. Stromal cell-derived factor-1 (SDF-1), the natural ligand of CXCR4, elicited an apparent Ca²⁺ influx in these cells and induced a strong migratory response comparable to that by eotaxin. The surface expression of CXCR4 in eosinophils was up-regulated by IFN-, TNF-, and TGF-ß while it was down-regulated by IL-4 and eosinophil-directed hemopoietins such as IL-5. The CXCR4 expression did not always parallel the apoptotic changes in cytokine-treated eosinophils. In contrast to IL-4 and IFN-, IL-5 potently reduced the level of CXCR4 mRNA. It seems unlikely that CXCR4 is fundamentally involved in the pathogenesis of allergic disorders by inducing the migration of eosinophils toward inflammatory sites, because a Th2-dominant state down-regulates eosinophil CXCR4 expression. However, CXCR4 may affect the size of the mobilizable pool by holding eosinophils at noninflamed tissues. Th2-dominant state may favor the liberation of eosinophils by down-regulating CXCR4 expression. The interplay between CXCR4 and SDF-1 in eosinophils potentially plays an important role in the accumulation of these cells at the allergic inflammatory sites.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Massive accumulation of eosinophils is a characteristic aspect of inflammation associated with allergic diseases. Historically, eosinophils had long been assumed to play an antiinflammatory role in allergic disorders by virtue of their antagonizing effects on mast cell-derived mediators such as histamine. However, it has become apparent that the eosinophils involved in these conditions are highly destructive: eosinophil-derived mediators, especially various cationic proteins, contribute to the tissue damage associated with allergic diseases. Along with the progression of allergic reactions, eosinophils migrate from the blood compartment to inflamed tissues and function as allergic inflammatory cells (1). The processes involved in tissue eosinophilia consist of a complex interplay of various pathways and are not fully understood. However, several chemoattractants generated at inflammatory sites potentially play a pivotal role in the recruitment of eosinophils in humans as well as in animals (2).

Chemotactic cytokines, termed chemokines, are now recognized as essential participants in the sequence of events by which circulating leukocytes migrate toward inflammatory sites. Chemokines are divided into two major subfamilies based on the sequence of arranged cysteine groups: the CXC subfamily and the CC subfamily. Two minor subfamilies, i.e., the CX3C and the C chemokines, are also categorized. To date, 40 chemokines have been identified, and 15 chemokine receptors, i.e., five CXC chemokine receptors (CXCRs), eight CCRs, one CX3CR, and one XCR, have been cloned. It has been reported that eosinophils express CCR3, and ligands of CCR3 such as eotaxin induce strong migration of eosinophils (3, 4). Expression of CCR1 (5), and under certain circumstances expression of CXCR2 (6), have also been reported in eosinophils. The expression and function of these receptors have been extensively investigated. In contrast, there is little information regarding the expression and function of other chemokine receptors in eosinophils.

In this study, we have examined the expression of transcripts of a panel of chemokine receptors in human eosinophils and found intense constitutive expression of CXCR4 mRNA as well as CCR3 mRNA. Here, we demonstrate that surface expression of CXCR4 is inducible in eosinophils and that stromal cell-derived factor-1 (SDF-1)³ elicits strong eosinophil migration comparable to that induced by eotaxin. The effects of cytokines on eosinophil CXCR4 expression have also been investigated.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents and mAbs

The following reagents were purchased as indicated: recombinant human SDF-1, eotaxin, IL-4, and TGF-ß1 (PeproTech, London, U.K.); IFN- (Shionogi Pharmaceutical, Osaka, Japan); TNF- (Dainippon Pharmaceutical, Osaka, Japan); C5a, cytochalasin B, and cycloheximide (Sigma, St. Louis, MO); ionomycin (Seikagaku, Tokyo, Japan); pertussis toxin (PTX) (Calbiochem-Behring, La Jolla, CA); anti-Fas mAb (CH-11, subclass IgM) and mouse IgM1 with irrelevant specificity (Medical and Biological Laboratories, Nagoya, Japan and Organon Teknika, West Chester, PA, respectively). Anti-CCR3 mAb (7) was provided by Dr. H. Kawasaki (Institute of Medical Science, University of Tokyo, Tokyo, Japan). IL-3 and IL-5 were kindly donated by Kirin Brewery (Tokyo, Japan) and Suntory (Osaka, Japan), respectively.

Eosinophil separation and culture conditions

Eosinophils were separated from normal volunteers who had no history of allergy, as previously described (8). In brief, buffy coat cells were obtained from venous blood by dextran T500 sedimentation. Eosinophils were isolated by Percoll (1.088 g/ml; Pharmacia, Uppsala, Sweden) density centrifugation. Unless Percoll separation achieved purity of 90%, the eosinophils were further purified by negative selection using anti-CD16-bound micromagnetic beads (Miltenyi BioTech, Bergisch-Gladbach, Germany) and a magnetic-activated cell sorter column (Miltenyi BioTech) as the second step (9). After this negative selection, the mean eosinophil purity was consistently >99%, and the viability was consistently >95%. Eosinophils (0.5–1.0 x 10⁶) were cultured in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% FCS (Life Technologies) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) at 37°C in 5% CO₂ in a total volume of 500 µl in flat-bottom 48-well culture plates (Costar, Cambridge, MA) for indicated times.

RT-PCR analysis of chemokine receptors

RT-PCR analysis of eosinophil mRNA was performed as previously described (10). In brief, total RNA was extracted from 10⁶ eosinophils with a SNAP Total RNA Isolation kit (Invitrogen, Leek, The Netherlands). After precipitation with ethanol, the first-strand cDNA was reverse transcribed. Second-strand DNA synthesis and hot-start amplification were performed using a TaKaRa Thermal Cycler MP (Takara, Ohtsu, Japan) under oil-free conditions. Amplification of cDNA was performed as previously described using AmpliTaq Gold DNA polymerase (Perkin-Elmer, Branchburg, NJ). Chemokine receptor cDNA was amplified by PCR with primers specific for each receptor. The direct and reverse oligo primers used and the expected product sizes are shown in Table I. The PCR conditions included 9 min of preheating at 95°C followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, elongation at 72°C for 30 s, and final elongation at 72°C for 10 min. PCR products were electrophoresed through a 2% agarose gel and visualized with ethidium bromide. To semiquantify CXCR4 mRNA, visualized bands were quantified using BIO-PROFIL densitometry (Vilber Lourmat, Marne La Vallée, France), and the density of CXCR4 mRNA was compared with that of ß-actin mRNA.

Isolated eosinophils were washed in PBS supplemented with 3% FCS and 0.1% NaN₃, and then incubated with anti-CXCR4 mAb (12G5; PharMingen, San Diego, CA) at 10 µg/ml for 60 min at 4°C. An isotype-matched mouse IgG2a with irrelevant specificity (UPC 10; Sigma) was used as a negative control. After washing, the cells were stained with FITC-labeled goat F(ab')₂ against mouse IgG (Jackson ImmunoResearch, West Grove, PA) at 7 µg/ml for 30 min at 4°C. During flow cytometry procedures, contaminating neutrophils were discriminated on the basis of their different fluorescence properties.

Stained eosinophils were analyzed using EPICS XL System II (Coulter, Miami, FL). At least 3000 eosinophils were assessed to calculate the median value of fluorescence intensity. The median values of fluorescence intensity of human eosinophils were converted to the numbers of the molecules of equivalent soluble fluorochrome units (MESF) using Quantum 25 microbeads (Flow Cytometry Standards, San Juan, Puerto Rico) on each day of an experiment. Surface receptor levels expressed in MESF units were calculated using the following formula: (MESF of eosinophils stained by anti-CXCR4 mAb) - (MESF of eosinophils stained with isotype control IgG).

Flow cytometry of eosinophils in whole blood

FACS analysis of eosinophils in whole venous blood was performed as previously described (11). In brief, blood was anti-coagulated with EDTA, and an equal volume of FACS buffer (PBS with 3% FCS and 0.1% NaN₃) was added. Cells were stained with anti-CXCR4-FITC (12G5; R&D Systems, Minneapolis, MN) and anti-CD16-PE (Coulter) at 10 µg/ml for 45 min at 4°C. An isotype-matched mouse IgG2a-FITC (R&D Systems) and mouse IgG1-PE (Immunotech, Marseille, France) with irrelevant specificity were used as negative controls. Contaminating erythrocytes were eliminated with a lysis buffer (Ortho Diagnostic Systems, Tokyo, Japan). Granulocytes were discriminated on the basis of different forward/side scatter properties, and electronic gates were set on CD16-negative cells to identify eosinophils.

Measurement of intracellular calcium concentration

Purified eosinophils (purity, >99%) were resuspended in HBSS with Ca²⁺ and Mg²⁺ (Life Technologies) and 2% BSA at a cell density of 2.0 x 10⁶/ml. Fura-2 AM (Dojindo, Tokyo, Japan) was added at a final concentration of 2 µM. After incubation for 20 min, excess dye was removed by centrifugation, and the cells were resuspended in a buffer containing 119 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 0.03% human serum albumin, and 25 mM PIPES, pH 7.4, at a concentration of 1.6 x 10⁶ cell/ml. Ca²⁺ influx was measured using excitation at 340 and 380 nm on a Hitachi F-2500 fluorescence spectrometer (Hitachi, Tokyo, Japan). Calibration was performed using 0.1% Triton X-100 for total Ca²⁺ release and 10 mM EGTA to chelate free Ca²⁺.

Chemotaxis assay of eosinophils

Eosinophil migration was measured using a 96-well multiwell Boyden chamber (Neuroprobe, Bethesda, MD) and a 10-µm-thick polyvinylpyrrolidone-free polycarbonate membrane filter with pores of 5 µm in diameter (Neuroprobe), as previously described (8). Aliquots of 362 µl of triplicate samples were transferred into the lower wells, while 200 µl of a cell suspension that contained 1.5 x 10⁴ eosinophils was introduced into each well of the top compartment. After incubation for 90 min, the eosinophil peroxidase (EPO) activity of cells at the bottom of wells was determined with 200 µl of 0.05 M Tris-HCl, pH 8.0, containing 0.1% (v/v) Triton X-100, 0.1 mM o-phenylenediamine dihydrochloride (OPD; Sigma) and 50 mM hydrogen peroxide. The OD was read at 490/570 nm in an ELISA reader (Model 550; Bio-Rad, Hercules, CA). Data were analyzed with the Microplate Manager III program (Bio-Rad), and the numbers of migrated eosinophils were calculated based on a standard curve established with varying known numbers of eosinophils.

Eosinophil degranulation

Freshly isolated eosinophils (purity, >99%) were cultured for 24 h with or without IFN- (10 ng/ml). These cells were pretreated with 5 µg/ml of cytochalasin B for 5 min and then stimulated with SDF-1 (333 ng/ml) or C5a (5 x 10^-9 M) for 5 h. The level of eosinophil-derived neurotoxin (EDN) was measured with an EDN ELISA kit (Medical and Biological Laboratories).

Analysis of apoptotic cells

Differential analysis of apoptotic and necrotic cells was performed using a MEBCYTO apoptosis kit (Medical and Biological Laboratories). Apoptotic cells were quantitatively determined by their ability to bind annexin V and exclude propidium iodide. Cells stained with propidium iodide were considered to be necrotic cells. In some experiments, eosinophils were double-stained with anti-CXCR4 mAb (12G5, 10 µg/ml) followed by a second step reaction Ab (PE-conjugated goat F(ab')₂ against mouse IgG Fc; Beckman Coulter, Tokyo, Japan) and FITC-conjugated annexin V.

Statistics

Unless otherwise noted, all data are expressed as the mean ± SEM, and differences between values were compared by the paired t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of CXCR4 in eosinophils

Eosinophils were highly purified by means of Percoll density gradient centrifugation followed by negative selection using anti-CD16 microbeads to apparent homogeneity. Expression of transcripts of 15 chemokine receptors was investigated by RT-PCR, and Fig. 1 shows the results of a representative experiment from among four different donors. Although CXCR2 and CCR1 transcripts were weakly detected in some specimens, eosinophils from all of the donors strongly expressed both CXCR4 and CCR3 transcripts. Having observed the consistent expression of CXCR4 mRNA, we next studied the expression of the CXCR4 protein in eosinophils. Unexpectedly, surface expression of CXCR4 was hardly detectable when Percoll-separated eosinophils were immediately stained with anti-CXCR4 mAb. Although no significant expression of CXCR4 was induced when incubated at 4°C (Fig. 2A), these eosinophils started to express CXCR4 during incubation at 37°C. Apparent surface expression of CXCR4 was observed within 4 h of incubation at 37°C, and the level of expression increased linearly up to 24 h of incubation (Figs. 2, B and C). It should be mentioned that the level of CCR3 expression was virtually unchanged during the incubation at 37°C (Fig. 2D).

View larger version (32K): [in this window] [in a new window]   FIGURE 1. mRNA expression of chemokine receptors in human eosinophils. Eosinophils were purified by means of Percoll density gradient centrifugation followed by negative selection using anti-CD16 microbeads (purity, >99%), and RT-PCR was performed as described in Materials and Methods. The PCR products were electrophoresed and then visualized with ethidium bromide. A representative example of four separate experiments is shown, and the other experiments all showed similar results. BA; ß-actin.

View larger version (16K): [in this window] [in a new window]   FIGURE 2. Time course of surface CXCR4 expression in purified eosinophils. Eosinophils were purified by means of Percoll density gradient centrifugation followed by negative selection using anti-CD16 microbeads. Purified eosinophils (purity, >99%) were cultured at 4°C (A) or 37°C (B) for the indicated times in culture medium. The cells were then treated with anti-CXCR4 mAb or isotype control mouse IgG (shaded peak) for 60 min at 4°C, stained with FITC goat F(ab')2 against mouse IgG for 30 min at 4°C and analyzed by flow cytometry. A and B are representatives of three separate experiments, and the others showed similar results. C, The time course of surface CXCR4 expression is shown. All data are expressed as the mean ± SEM (n = 3) of MESF values calculated as described in Materials and Methods. **, p < 0.01 vs MESF values of eosinophils cultured at 37°C. D, Expression of CCR3 on freshly isolated (0 h) and 24-h-cultured eosinophils (a representative of three separate experiments) is shown.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Expression of CXCR4 on eosinophils in whole blood. Eosinophils in whole blood were double-stained with anti-CXCR4-FITC and anti-CD16-PE and analyzed by flow cytometry. Granulocytes were discriminated on the basis of different forward/side scatter properties (A) and electronic gates on CD16-negative cells were set to eliminate neutrophils (B). C, Surface CXCR4 expression on eosinophils in fresh whole blood and eosinophils in whole blood cultured for 24 h at 37°C. The shaded area shows the fluorescence of cells stained with isotype-matched mouse IgG2a-FITC. The data are representative of three independent experiments, all showing similar results.

In the next series of experiments, we investigated whether eosinophil CXCR4 is functionally activated by a specific ligand for CXCR4, SDF-1. As shown in Fig. 2, freshly isolated eosinophils did not express significant amounts of CXCR4 on their surface, but SDF-1 elicited a small but apparent Ca²⁺ influx in these cells (Fig. 4A). In cells incubated for 24 h at 37°C, much stronger influx of Ca²⁺ was observed (Fig. 4C). The magnitude of Ca²⁺ influx elicited by SDF-1 was comparable to that induced by eotaxin (Fig. 4D). Furthermore, SDF-1-induced Ca²⁺ influx was completely attenuated by treatment with PTX (Fig. 4E), indicating the involvement of G proteins of the G_i class in the signal transduction pathways. To determine whether CXCR4 expressed in eosinophils has functional relevance, we tested the migration-inducing ability of SDF-1. As shown in Fig. 5, SDF-1 was capable of inducing a migratory response in eosinophils: although SDF-1 did not induce significant migration in freshly isolated cells, eosinophils incubated for 24 h at 37°C exhibited a migratory response toward SDF-1 in a dose-dependent manner. Apparent migration was observed at 33 ng/ml of SDF-1, and much stronger migration was observed at 333 ng/ml of SDF-1. It should be noted that SDF-1 induced eosinophil chemotaxis as strongly as eotaxin, which is known as the most potent eosinophil chemoattractant. Furthermore, the migration induced by SDF-1 was chemotactic rather than chemokinetic: when the same concentration of SDF-1 was added to both the upper and lower wells, the migration was significantly reduced (data not shown). No significant decrease in granulation was noted in cells that had migrated in response to SDF-1 compared with freshly isolated or 24-h-cultured eosinophils (Fig. 6). In fact, in vitro experiments showed that CXCR4 was not involved in the process leading to degranulation: SDF-1 failed to induce significant release of EDN from eosinophils cultured for 24 h with or without IFN- or from freshly isolated cells (Fig. 7).

View larger version (27K): [in this window] [in a new window]   FIGURE 4. SDF-1 caused calcium influx to eosinophils. Calcium influx in highly purified eosinophils (purity, >99%) just after purification (A and B) and eosinophils cultured for 24 h at 37°C without (C and D) or with (E) further treatment with PTX. E, Cultured cells were treated with PTX at 100 nM for 2 h at 37°C, and then stimulated sequentially with SDF-1 and ionomycin. The data shown are representative of two independent analyses from different donors, each showing similar results. The concentration of chemokines and ionomycin were 400 ng/ml and 1 µM, respectively.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. SDF-1 induced migration of eosinophils. The migration-inducing activity of SDF-1 and eotaxin was analyzed using eosinophils just after purification (purity, 96.3 ± 1.3%) and eosinophils cultured for 24 h at 37°C. The data are expressed as the percentage of total cells introduced into each of the upper wells (mean ± SEM, n = 4). The effects of the concentration of chemokines were analyzed by the two-way ANOVA test. When this test indicated a significant differences between concentrations (indicated as ++, p < 0.01), Fisher’s protected least significant difference (PLSD) test was used to compare individual groups. **, p < 0.01 vs values of eosinophil migration in control buffer at the same time point.

View larger version (50K): [in this window] [in a new window]   FIGURE 6. Photomicrographs of eosinophils that were freshly isolated (A), 24-h-cultured (B), and migrated in response to SDF-1 (C). The photos are representatives of two different experiments and another showed similar results. Magnification, x600.

View larger version (20K): [in this window] [in a new window]   FIGURE 7. Effect of SDF-1 on EDN release from eosinophils. Freshly isolated eosinophils (purity, >99%) were cultured for 24 h with or without IFN- (10 ng/ml). Degranulation experiments were performed as described in Materials and Methods. Percent release of EDN was calculated as the ratio of EDN content in the supernatant to total EDN in the lysate. Results are expressed as the mean ± SEM of the percentage of EDN release in four duplicate experiments. *, p < 0.05 vs values of EDN release in control buffer at the same time point.

In the next series of experiments, we examined the effects of cytokines on CXCR4 expression by eosinophils during 24 h of incubation. As shown in Table II, TGF-ß1, IFN-, and TNF- up-regulated the expression of CXCR4. In contrast, eosinophil-directed hemopoietins, i.e., IL-3, IL-5, and GM-CSF, almost completely attenuated the surface expression of CXCR4. In addition, IL-4 also drastically down-regulated the CXCR4 expression (Table II and Fig. 8A). As shown in Fig. 8B, as small as a femtomolar level of IL-5 was sufficient to inhibit the CXCR4 expression. Half-maximal inhibition was observed at a concentration of 100 fM of IL-5 with dose-dependent inhibition seen between 0.1 fM and 10 pM. For IL-4, half-maximal inhibition was observed at a concentration of 1 pM with dose-dependent inhibition seen between 10 fM and 10 pM. On a molar basis, IL-5 was 10-fold more potent than IL-4. Furthermore, delayed addition of IL-5 or IL-4 effectively down-regulated CXCR4 expression. When eosinophils were cultured for 24 h without addition of any factors and then treated with IL-5 or IL-4, the level of CXCR4 expression in the eosinophils was decreased at 6 and 24 h after the addition of each cytokines (data not shown).

View larger version (16K): [in this window] [in a new window]   FIGURE 8. Effect of IL-4 or IL-5 on eosinophil CXCR4 expression. A, Expressions of CXCR4 on 24-h-cultured eosinophils (purity, >99%) with (+) or without (-) the indicated cytokine (10 ng/ml) are shown. Shaded peak shows the fluorescence of cells stained by control mouse IgG. The data are representative of three separate experiments, and the others showed similar results. B, Dose-depending effect of IL-4 or IL-5 on surface CXCR4 expression in eosinophils: eosinophils (purity, >99%) were cultured with IL-4 or IL-5 at the indicated concentrations for 24 h at 37°C, and the surface CXCR4 expression was then analyzed by flow cytometry. The data are expressed as a percentage of the calculated MESF values of eosinophils cultured in medium alone without cytokine (mean ± SEM, n = 3). *, p < 0.05; **, p < 0.01 vs control (=100%).

View larger version (20K): [in this window] [in a new window]   FIGURE 9. Surface expression of CXCR4 in nonapoptotic eosinophils. A, Twenty-four-hour-cultured eosinophils (purity, >99%) were double-stained with anti-CXCR4 mAb or control mouse IgG and FITC-conjugated annexin V. B, Electronic gates were set on annexin V-negative cells, and CXCR4 expression was analyzed by flow cytometry (a representative of three separate experiments showing similar results). The shaded area indicates the fluorescence of cells stained with control mouse IgG.

The surface expression of CXCR4 by eosinophils was significantly but partially inhibited by treatment with a protein synthesis inhibitor, cycloheximide (MESF: 86.9 ± 3.0% and 59.5 ± 7.7% of inhibition for 4-h- and 24-h-cultured cells, respectively, n = 3), indicating that de novo protein synthesis is involved in the process leading to the surface expression of CXCR4. In the last series of experiments, we studied cytokine-mediated regulation of CXCR4 expression at the mRNA level. When we examined the expression of CXCR4 mRNA by RT-PCR amplification, we observed that treatment with IL-5, but not IL-4, apparently decreased the level of CXCR4 mRNA, although both cytokines suppressed surface protein expression of CXCR4. In addition, CXCR4 mRNA remained at the baseline level in eosinophils cultured with IFN-. In contrast, none of these cytokines had any effect on the level of CCR3 mRNA (Fig. 10).

View larger version (54K): [in this window] [in a new window]   FIGURE 10. Effect of cytokines on CXCR4 mRNA expression. Eosinophils (purity, >99%) were cultured for 4 h with cytokines (10 ng/ml). RT-PCR products were electrophoresed and visualized with ethidium bromide. A, The data shown are representatives of five (CXCR4) and three (CCR3) different experiments and others showed similar results. B, The density of bands was quantified by densitometry, and the intensity of CXCR4 mRNA and CCR3 mRNA was compared with that of ß-actin mRNA. Chemokine receptor mRNA expression (%) is calculated using the following formula: (receptor/ß-actin ratio of treated eosinophils)/(receptor/ß-actin ratio of freshly isolated eosinophils (0 h)) x 100. *, p < 0.05 vs control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Studies of time course and various conditions for CXCR4 expression raised a possibility that the expression of CXCR4 is related to apoptotic changes. IL-5, IL-3, and GM-CSF, all of which are known to rescue eosinophils from apoptosis (17, 18, 19), completely attenuated CXCR4 expression in eosinophils. Furthermore, the concentration of IL-5 necessary for the inhibition of CXCR4 induction matched the reported range required for inhibition of eosinophil apoptosis (20) (Fig. 8B). Consequently, it would be reasonable and even attractive to speculate that CXCR4 is preferentially induced in eosinophils undergoing apoptosis, and that these cells are then eliminated from the circulation by the interplay between CXCR4 and ubiquitously expressed SDF-1. However, the level of eosinophil CXCR4 expression did not always parallel the apoptotic changes (Table II). IL-4, which is devoid of anti-apoptotic effects, almost completely attenuated CXCR4 expression (Table II). On the contrary, IFN-, which is known to block eosinophil apoptosis (Ref. 20 and Table II), efficiently up-regulated CXCR4 expression. It might be possible that each cytokine may regulate eosinophil CXCR4 expression via different pathways or mechanisms. However, these discrepancies observed between apoptotic changes and CXCR4 expression in cytokine-treated eosinophils, together with the direct determination of CXCR4 expression in nonapoptotic populations (Fig. 9), strongly suggest that eosinophil CXCR4 expression cannot be explained merely as a consequence of apoptotic changes in these cells.

Spontaneous sustained increase in CXCR4 expression similar to our present findings has also been reported for monocytes and lymphocytes (21, 22). In these cells, surface expression of CXCR4 seems not to be regulated at the mRNA level but mainly at the protein level, such as receptor trafficking between the intracellular space and cell surface (23). In the present study, despite the constitutive expression of CXCR4 mRNA (Fig. 1), freshly isolated eosinophils did not express significant amounts of CXCR4 protein on their surface (Fig. 2). Furthermore, spontaneous sustained increase in CXCR4 expression was reversibly down-regulated by delayed addition of IL-4 and IL-5. These findings strongly suggest that surface CXCR4 expression in eosinophils is regulated, at least in part, at the level of receptor trafficking. However, the expression of surface CXCR4 dose not seem to be regulated merely at the level of posttranscription. The process leading to the expression of CXCR4 in eosinophils was partly inhibited by cycloheximide, indicating that the sustained induction of surface CXCR4 expression involves a cycloheximide-sensitive component. Although a direct relationship between the level of mRNA and the amount of surface protein was not always observed in cytokine-mediated alteration of CXCR4 expression, IL-5 apparently down-regulated the level of both mRNA and surface protein of CXCR4 (Table II and Fig. 10). Taken together, it seems plausible that surface CXCR4 expression in eosinophils is modulated not only at the level of posttranscription but also at least in part at the level of transcription.

The most striking findings of our study are that surface expression of CXCR4 in eosinophils has functional relevance, and that SDF-1 induced strong eosinophil migration comparable to that induced by eotaxin (Fig. 5). Eotaxin, a specific ligand for CCR3, has been known as the most potent chemokine for eosinophil chemotaxis (5), and an essential role of CCR3/eotaxin in selective accumulation of eosinophils has been established in vivo as well as in vitro. In contrast, the in vivo role of CXCR4/SDF-1-mediated chemotaxis is yet to be established and remains totally speculative, but it seems unlikely that CXCR4/SDF-1 is fundamentally involved in the pathogenesis of allergic disorders by inducing the migration of eosinophils toward inflammatory sites. Firstly, in contrast to CCR3, the expression of which is restricted only to eosinophils (4) and basophils (24, 25), CXCR4 is expressed in various white blood cells (reviewed in Ref. 26), indicating that CXCR4-mediated migration is not the primary mechanism of the "selective" accumulation of eosinophils observed in allergic inflammation. Secondly, transcription of eotaxin mRNA is induced by in vivo Ag challenge (27) and in vitro stimulation with certain cytokines (28). This inducible tissue-specific eotaxin expression is implicated in eosinophil influx observed during ongoing allergic reactions. However, SDF-1 is constitutively expressed in a variety of tissues (26, 29), and there has been little evidence demonstrating transcriptional regulation of SDF-1. Finally, allergic diseases exhibit polarized Th2 responses that are closely implicated in the onset and outcome of these disorders (30). However, the expression of CXCR4 in eosinophils is down-regulated by IL-4 and IL-5 and up-regulated by IFN-, indicating that a Th1-dominant state rather than a Th2-dominant state favors eosinophil CXCR4 expression. Thus, in view of Th1/Th2 governance in allergic inflammation, it seems unlikely that eosinophil CXCR4 mediates a major mechanism of migration of eosinophils in allergic diseases.

However, CXCR4 in eosinophils might be indirectly involved in the exacerbation of eosinophilic inflammation. A substantial body of evidence indicates that CXCR4 regulates myelopoiesis and lymphopoiesis in a stage-specific fashion: CXCR4 is preferentially expressed in immature progenitors in the bone marrow or thymus during the maturational stage (31, 32, 33, 34, 35), and SDF-1 retains these cells via CXCR4 to prevent their liberation from these tissues into the circulation. Thus, CXCR4 in immature leukocytes potentially serves as an anchor for these cells by inhibiting their movement rather than inducing their migration. A similar situation can be imagined for CXCR4 in mature eosinophils: eosinophil CXCR4 may affect the size of the mobilizable pool, and anchorage of mature eosinophils by SDF-1 may be crucial for retaining eosinophils in noninflamed tissues. Th2-dominant state may favor the liberation of eosinophils by down-regulating CXCR4 expression, which in turn would permit enhanced accumulation of eosinophils at allergic inflammatory sites by eosinophil-active chemokines such as eotaxin. The presence of IL-5 in the serum, which is often observed in patients with allergic and helminthic disorders, may increase the distribution of eosinophils to inflamed tissues.

In summary, we have demonstrated that functional expression of CXCR4 is inducible in eosinophils, and that SDF-1 elicits strong migration comparable to that induced by eotaxin. Th2 cytokines such as IL-4 and IL-5 drastically inhibited the expression of CXCR4. The interplay between CXCR4 and SDF-1 may affect the distribution and migration of eosinophils, thus indicating that CXCR4 in eosinophils might represent an important mechanism in diseases in which eosinophils play pathogenic roles.

	Acknowledgments

 
We thank Dr. H. Kawasaki for the donation of mouse lymphoma cells transfected with human CXCR4. Thanks are also extended to M. Imanishi and S. Takeyama for their excellent technical assistance and secretarial help, respectively.

	Footnotes

 
¹ This work was supported by a grant from the Manabe Medical Foundation, grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan (to M.M., M.Y., and K.H.), and grants-in-aid from the Ministry of Health and Welfare of Japan (to K.H.). M.M. is a Research Fellow of the Japan Society for the Promotion of Science.

² Address correspondence and reprint requests to Dr. Koichi Hirai, Department of Bioregulatory Function, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.

³ Abbreviations used in this paper: SDF-1, stromal cell-derived factor-1; PTX, pertussis toxin; MESF, molecules of equivalent soluble fluorochrome units; EPO, eosinophil peroxidase; OPD, o-phenylenediamine dihydrochloride; EDN, eosinophil-derived neurotoxin.

Received for publication October 29, 1999. Accepted for publication March 17, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Gleich, G. J., C. R. Adolphson, K. M. Leiferman. 1993. The biology of the eosinophilic leukocyte. Annu. Rev. Med. 44:85.[Medline]
2. Resnick, M. B., P. F. Weller. 1993. Mechanisms of eosinophil recruitment. Am. J. Respir. Cell Mol. Biol. 8:349.
3. Kitaura, M., T. Nakajima, T. Imai, S. Harada, C. Combadiere, H. L. Tiffany, P. M. Murphy, O. Yoshie. 1996. Molecular cloning of human eotaxin, an eosinophil-selective CC chemokine, and identification of a specific eosinophil eotaxin receptor, CC chemokine receptor 3. J. Biol. Chem. 271:7725.[Abstract/Free Full Text]
4. Ponath, P. D., S. Qin, D. J. Ringler, I. Clark-Lewis, J. Wang, N. Kassam, H. Smith, X. Shi, J. A. Gonzalo, W. Newman, J. C. Gutierrez-Ramos, C. R. Mackay. 1996. Cloning of the human eosinophil chemoattractant, eotaxin: expression, receptor binding, and functional properties suggest a mechanism for the selective recruitment of eosinophils. J. Clin. Invest. 97:604.[Medline]
5. Daugherty, B. L., S. J. Siciliano, J. A. DeMartino, L. Malkowitz, A. Sirotina, M. S. Springer. 1996. Cloning, expression, and characterization of the human eosinophil eotaxin receptor. J. Exp. Med. 183:2349.[Abstract/Free Full Text]
6. Schweizer, R. C., B. A. Welmers, J. A. Raaijmakers, P. Zanen, J. W. Lammers, L. Koenderman. 1994. RANTES- and interleukin-8-induced responses in normal human eosinophils: effects of priming with interleukin-5. Blood 83:3697.[Abstract/Free Full Text]
7. Sato, K., H. Kawasaki, H. Nagayama, R. Serizawa, J. Ikeda, C. Morimoto, K. Yasunaga, N. Yamaji, K. Tadokoro, T. Juji, T. A. Takahashi. 1999. CC chemokine receptors, CCR-1 and CCR-3, are potentially involved in antigen-presenting cell function of human peripheral blood monocyte- derived dendritic cells. Blood 93:34.[Abstract/Free Full Text]
8. Nagase, H., M. Yamaguchi, S. Jibiki, H. Yamada, K. Ohta, H. Kawasaki, O. Yoshie, K. Yamamoto, Y. Morita, K. Hirai. 1999. Eosinophil chemotaxis by chemokines: a study by a simple photometric assay. Allergy 54:944.[Medline]
9. Miyamasu, M., K. Hirai, Y. Takahashi, M. Iida, M. Yamaguchi, T. Koshino, T. Takaishi, Y. Morita, K. Ohta, T. Kasahara, et al 1995. Chemotactic agonists induce cytokine generation in eosinophils. J. Immunol. 154:1339.[Abstract]
10. Miyamasu, M., T. Nakajima, Y. Misaki, S. Izumi, N. Tsuno, T. Kasahara, K. Yamamoto, Y. Morita, K. Hirai. 1999. Dermal fibroblasts represent a potent major source of human eotaxin: in vitro production and cytokine-mediated regulation. Cytokine 11:751.[Medline]
11. Mawhorter, S. D., D. A. Stephany, E. A. Ottesen, T. B. Nutman. 1996. Identification of surface molecules associated with physiologic activation of eosinophils: application of whole-blood flow cytometry to eosinophils. J. Immunol. 156:4851.[Abstract]
12. Chelucci, C., I. Casella, M. Federico, U. Testa, G. Macioce, E. Pelosi, R. Guerriero, G. Mariani, A. Giampaolo, H. J. Hassan, C. Peschle. 1999. Lineage-specific expression of human immunodeficiency virus (HIV) receptor/coreceptors in differentiating hematopoietic precursors: correlation with susceptibility to T- and M-tropic HIV and chemokine- mediated HIV resistance. Blood 94:1590.[Abstract/Free Full Text]
13. Wang, J., A. Harada, S. Matsushita, S. Matsumi, Y. Zhang, T. Shioda, Y. Nagai, K. Matsushima. 1998. IL-4 and a glucocorticoid up-regulate CXCR4 expression on human CD4⁺ T lymphocytes and enhance HIV-1 replication. J. Leukocyte Biol. 64:642.[Abstract]
14. Jourdan, P., C. Abbal, N. Noraz, T. Hori, T. Uchiyama, J. P. Vendrell, J. Bousquet, N. Taylor, J. Pene, H. Yssel, N. Nora. 1998. IL-4 induces functional cell-surface expression of CXCR4 on human T cells. J. Immunol. 160:4153.[Abstract/Free Full Text]
15. Valentin, A., W. Lu, M. Rosati, R. Schneider, J. Albert, A. Karlsson, G. N. Pavlakis. 1998. Dual effect of interleukin 4 on HIV-1 expression: implications for viral phenotypic switch and disease progression. Proc. Natl. Acad. Sci. USA 95:8886.[Abstract/Free Full Text]
16. Annunziato, F., L. Cosmi, G. Galli, C. Beltrame, P. Romagnani, R. Manetti, S. Romagnani, E. Maggi. 1999. Assessment of chemokine receptor expression by human Th1 and Th2 cells in vitro and in vivo. J. Leukocyte Biol. 65:691.[Abstract]
17. Lopez, A. F., D. J. Williamson, J. R. Gamble, C. G. Begley, J. M. Harlan, S. J. Klebanoff, A. Waltersdorph, G. Wong, S. C. Clark, M. A. Vadas. 1986. Recombinant human granulocyte-macrophage colony-stimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression, and survival. J. Clin. Invest. 78:1220.
18. Sanderson, C. J.. 1992. Interleukin-5, eosinophils, and disease. Blood 79:3101.[Free Full Text]
19. Rothenberg, M. E., Jr W. F. Owen, D. S. Silberstein, J. Woods, R. J. Soberman, K. F. Austen, R. L. Stevens. 1988. Human eosinophils have prolonged survival, enhanced functional properties, and become hypodense when exposed to human interleukin 3. J. Clin. Invest. 81:1986.
20. Wallen, N., H. Kita, D. Weiler, G. J. Gleich. 1991. Glucocorticoids inhibit cytokine-mediated eosinophil survival. J. Immunol. 147:3490.[Abstract]
21. Förster, R., E. Kremmer, A. Schubel, D. Breitfeld, A. Kleinschmidt, C. Nerl, G. Bernhardt, M. Lipp. 1998. Intracellular and surface expression of the HIV-1 coreceptor CXCR4/fusin on various leukocyte subsets: rapid internalization and recycling upon activation. J. Immunol. 160:1522.[Abstract/Free Full Text]
22. Bermejo, M., J. Martin-Serrano, E. Oberlin, M. A. Pedraza, A. Serrano, B. Santiago, A. Caruz, P. Loetscher, M. Baggiolini, F. Arenzana-Seisdedos, J. Alcami. 1998. Activation of blood T lymphocytes down-regulates CXCR4 expression and interferes with propagation of X4 HIV strains. Eur. J. Immunol. 28:3192.[Medline]
23. Cole, S. W., B. D. Jamieson, J. A. Zack. 1999. cAMP up-regulates cell surface expression of lymphocyte CXCR4: implications for chemotaxis and HIV-1 infection. J. Immunol. 162:1392.[Abstract/Free Full Text]
24. Yamada, H., K. Hirai, M. Miyamasu, M. Iikura, Y. Misaki, S. Shoji, T. Takaishi, T. Kasahara, Y. Morita, K. Ito. 1997. Eotaxin is a potent chemotaxin for human basophils. Biochem. Biophys. Res. Commun. 231:365.[Medline]
25. Uguccioni, M., C. R. Mackay, B. Ochensberger, P. Loetscher, S. Rhis, G. J. LaRosa, P. Rao, P. D. Ponath, M. Baggiolini, C. A. Dahinden. 1997. High expression of the chemokine receptor CCR3 in human blood basophils. Role in activation by eotaxin, MCP-4, and other chemokines. J. Clin. Invest. 100:1137.[Medline]
26. Nagasawa, T., K. Tachibana, K. Kawabata. 1999. A CXC chemokine SDF-1/PBSF: a ligand for a HIV coreceptor, CXCR4. Adv. Immunol. 71:211.[Medline]
27. MacLean, J. A., R. Ownbey, A. D. Luster. 1996. T cell-dependent regulation of eotaxin in antigen-induced pulmonary eosinophilia. J. Exp. Med. 184:1461.[Abstract/Free Full Text]
28. Mochizuki, M., J. Bartels, A. I. Mallet, E. Christophers, J. M. Schröder. 1998. IL-4 induces eotaxin: a possible mechanism of selective eosinophil recruitment in helminth infection and atopy. J. Immunol. 160:60.[Abstract/Free Full Text]
29. Coulomb-L’Hermin, A., A. Amara, C. Schiff, I. Durand-Gasselin, A. Foussat, T. Delaunay, G. Chaouat, F. Capron, N. Ledee, P. Galanaud, et al 1999. Stromal cell-derived factor 1 (SDF-1) and antenatal human B cell lymphopoiesis: expression of SDF-1 by mesothelial cells and biliary ductal plate epithelial cells. Proc. Nat. Acad. Sci. USA 96:8585.[Abstract/Free Full Text]
30. Aebischer, I., B. M. Stadler. 1996. TH1-TH2 cells in allergic responses: at the limits of a concept. Adv. Immunol. 61:341.[Medline]
31. Kowalska, M. A., J. Ratajczak, J. Hoxie, L. F. Brass, A. Gewirtz, M. Poncz, M. Z. Ratajczak. 1999. Megakaryocyte precursors, megakaryocytes and platelets express the HIV co-receptor CXCR4 on their surface: determination of response to stromal-derived factor-1 by megakaryocytes and platelets. Br. J. Haematol. 104:220.[Medline]
32. Ma, Q., D. Jones, T. A. Springer. 1999. The chemokine receptor CXCR4 is required for the retention of B lineage and granulocytic precursors within the bone marrow microenvironment. Immunity 10:463.[Medline]
33. Nagasawa, T., S. Hirota, K. Tachibana, N. Takakura, S. Nishikawa, Y. Kitamura, N. Yoshida, H. Kikutani, T. Kishimoto. 1996. Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 382:635.[Medline]
34. Suzuki, G., Y. Nakata, Y. Dan, A. Uzawa, K. Nakagawa, T. Saito, K. Mita, T. Shirasawa. 1998. Loss of SDF-1 receptor expression during positive selection in the thymus. Int. Immunol. 10:1049.[Abstract/Free Full Text]
35. Berkowitz, R. D., K. P. Beckerman, T. J. Schall, J. M. McCune. 1998. CXCR4 and CCR5 expression delineates targets for HIV-1 disruption of T cell differentiation. J. Immunol. 161:3702.[Abstract/Free Full Text]
36. Neote, K., D. DiGregorio, J. Y. Mak, R. Horuk, T. J. Schall. 1993. Molecular cloning, functional expression, and signaling characteristics of a C-C chemokine receptor. Cell 72:415.[Medline]
37. Meucci, O., A. Fatatis, A. A. Simen, T. J. Bushell, P. W. Gray, R. J. Miller. 1998. Chemokines regulate hippocampal neuronal signaling and gp120 neurotoxicity. Proc. Natl. Acad. Sci. USA 95:14500.[Abstract/Free Full Text]
38. Samson, M., O. Labbe, C. Mollereau, G. Vassart, M. Parmentier. 1996. Molecular cloning and functional expression of a new human CC-chemokine receptor gene. Biochemistry 35:3362.[Medline]
39. Yanagihara, S., E. Komura, J. Nagafune, H. Watarai, Y. Yamaguchi. 1998. EBI1/CCR7 is a new member of dendritic cell chemokine receptor that is up-regulated upon maturation. J. Immunol. 161:3096.[Abstract/Free Full Text]
40. Wang, J. F., Z. Y. Liu, J. E. Groopman. 1998. The -chemokine receptor CXCR4 is expressed on the megakaryocytic lineage from progenitor to platelets and modulates migration and adhesion. Blood 92:756.[Abstract/Free Full Text]

This article has been cited by other articles:

				M. Watanabe, W. Matsuyama, Y. Shirahama, H. Mitsuyama, K.-i. Oonakahara, S. Noma, I. Higashimoto, M. Osame, and K. Arimura Dual Effect of AMD3100, a CXCR4 Antagonist, on Bleomycin-Induced Lung Inflammation J. Immunol., May 1, 2007; 178(9): 5888 - 5898. [Abstract] [Full Text] [PDF]

				S. M. A. Arraes, M. S. Freitas, S. V. da Silva, H. A. de Paula Neto, J. C. Alves-Filho, M. A. Martins, A. Basile-Filho, B. M. Tavares-Murta, C. Barja-Fidalgo, and F. Q. Cunha Impaired neutrophil chemotaxis in sepsis associates with GRK expression and inhibition of actin assembly and tyrosine phosphorylation Blood, November 1, 2006; 108(9): 2906 - 2913. [Abstract] [Full Text] [PDF]

				S. Basu and H. E. Broxmeyer Transforming growth factor-{beta}1 modulates responses of CD34+ cord blood cells to stromal cell-derived factor-1/CXCL12 Blood, July 15, 2005; 106(2): 485 - 493. [Abstract] [Full Text] [PDF]

				A. Katayama, T. Ogino, N. Bandoh, S. Nonaka, and Y. Harabuchi Expression of CXCR4 and Its Down-Regulation by IFN-{gamma} in Head and Neck Squamous Cell Carcinoma Clin. Cancer Res., April 15, 2005; 11(8): 2937 - 2946. [Abstract] [Full Text] [PDF]

				P. C. Fulkerson, N. Zimmermann, L. M. Hassman, F. D. Finkelman, and M. E. Rothenberg Pulmonary Chemokine Expression Is Coordinately Regulated by STAT1, STAT6, and IFN-{gamma} J. Immunol., December 15, 2004; 173(12): 7565 - 7574. [Abstract] [Full Text] [PDF]

				M. Iikura, M. Ebisawa, M. Yamaguchi, H. Tachimoto, K. Ohta, K. Yamamoto, and K. Hirai Transendothelial Migration of Human Basophils J. Immunol., October 15, 2004; 173(8): 5189 - 5195. [Abstract] [Full Text] [PDF]

				T. Nakayama, Y. Kato, K. Hieshima, D. Nagakubo, Y. Kunori, T. Fujisawa, and O. Yoshie Liver-Expressed Chemokine/CC Chemokine Ligand 16 Attracts Eosinophils by Interacting with Histamine H4 Receptor J. Immunol., August 1, 2004; 173(3): 2078 - 2083. [Abstract] [Full Text] [PDF]

				H. Nagase, S. Okugawa, Y. Ota, M. Yamaguchi, H. Tomizawa, K. Matsushima, K. Ohta, K. Yamamoto, and K. Hirai Expression and Function of Toll-Like Receptors in Eosinophils: Activation by Toll-Like Receptor 7 Ligand J. Immunol., October 15, 2003; 171(8): 3977 - 3982. [Abstract] [Full Text] [PDF]

				T. Jinquan, H. H. Jacobi, C. Jing, A. Millner, E. Sten, L. Hviid, L. Anting, L. P. Ryder, C. Glue, P. S. Skov, et al. CCR3 Expression Induced by IL-2 and IL-4 Functioning as a Death Receptor for B Cells J. Immunol., August 15, 2003; 171(4): 1722 - 1731. [Abstract] [Full Text] [PDF]

				H. Tachimoto, M. Kikuchi, S. A. Hudson, C. A. Bickel, R. G. Hamilton, and B. S. Bochner Eotaxin-2 Alters Eosinophil Integrin Function via Mitogen-Activated Protein Kinases Am. J. Respir. Cell Mol. Biol., June 1, 2002; 26(6): 645 - 649. [Abstract] [Full Text] [PDF]

				M. T. Borchers, T. Ansay, R. DeSalle, B. L. Daugherty, H. Shen, M. Metzger, N. A. Lee, and J. J. Lee In vitro assessment of chemokine receptor-ligand interactions mediating mouse eosinophil migration J. Leukoc. Biol., June 1, 2002; 71(6): 1033 - 1041. [Abstract] [Full Text] [PDF]