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(Journal of Leukocyte Biology. 2001;70:113-120.)
© 2001 by Society for Leukocyte Biology

Chemokine receptors in human basophils: inducible expression of functional CXCR4

Motoyasu Iikura*, Misato Miyamasu*, Masao Yamaguchi*, Hiroshi Kawasaki{dagger}, Kouji Matsushima{ddagger}, Motoji Kitaura§, Yutaka Morita||, Osamu Yoshie#, Kazuhiko Yamamoto* and Koichi Hirai**

* Department of Allergy and Rheumatology,
{ddagger} Department of Molecular Preventive Medicine and CREST,
|| Department of Respiratory Medicine, and
** Department of Bioregulatory Function, University of Tokyo Graduate School of Medicine, Tokyo, Japan;
{dagger} Department of Clinical Immunology and AIDS Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan;
§ Shionogi Institute for Medical Science, Osaka, Japan; and
# Department of Bacteriology, Kinki University, Osaka, Japan

Correspondence: Koichi Hirai, M.D., Ph.D., Department of Bioregulatory Function, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: hiraiko-tky{at}umin.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We examined the expression profile of chemokine receptors in human basophils and their regulation by cytokines. Basophils expressed transcripts of CC chemokine receptors (CCR)1, CCR2, CCR3, and CCR5 and CXC chemokine receptors (CXCR)1, CXCR2, and CXCR4. In contrast to the other receptors, surface-CXCR4 expression was not detected in fresh- and whole-blood basophils, but it became apparent gradually during incubation. Among 16 chemokines tested, eotaxin induced the most potent basophil migration. SDF-1 also induced a strong, migratory response comparable with that induced by eotaxin in 24-h, cultured basophils, but it failed to induce degranulation. IL-3 abrogated CXCR4 expression completely, and it only down-regulated CCR2 and CCR3 expression slightly. IL-5, GM-CSF, and IL-4 also down-regulated CXCR4 expression. Thus, expression of CXCR4 was the most strongly affected by cytokines, and this may represent an alternative mechanism for control of cell-specific, biological responses to SDF-1.

Key Words: SDF-1 • CCR5 • migration


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Since the first description by Paul Ehrlich, basophils have been recognized as unique, white-blood cells with metachromatic-staining properties. The outstanding characteristics of basophils, including expression of high-affinity receptors for immunoglobulin (Ig)E, histamine content, and metachromatic staining, are shared by tissue-dwelling mast cells [1 ]. However, it is evident that basophils and mast cells belong to completely different cell lineages and that eosinophils are the most closely related cells to basophils. Like eosinophils, basophils are motile cells; along with the progression of allergic reactions, basophils migrate from the blood compartment to inflamed tissues and function as allergic, inflammatory cells. Indeed, analyses of chemical mediators indicate that basophils contribute to the pathogenesis of late-phase, allergic reactions [1 ].

Chemokines are a family of secreted proteins of low molecular weight, and they possess chemotactic properties for cells, which mediate inflammation. Based on four conserved, cysteine residues, chemokines are categorized into two major subfamilies, namely CXC and CC chemokines. Two minor subfamilies, i.e., the CX3C and the C chemokines, are also categorized. To date, 5 CXC chemokine receptors (CXCRs), 10 CCRs, 1 CX3CR, and 1 XCR have been cloned. We and others [2 , 3 ] have shown the expression of CCR3 in human basophils at the mRNA and/or protein level. Eotaxin, a high-affinity and specific ligand for CCR3, induces the strongest migration of basophils [2 , 3 ]. Expression of CCR1, CCR2, CXCR1, and CXCR2 in basophils has also been demonstrated [3 4 5 ]. Basophil CCR2 mainly regulates degranulation; monocyte chemoattractant protein-1 (MCP-1), a high-affinity ligand for CCR2, induces the strongest histamine release from basophils [6 , 7 ]. A recent study by Jinquan et al. [8 ] also demonstrated constitutive expression of functional CXCR4 on human basophils. Furthermore, CCR4 was cloned from human leukemic, cell-line cells of basophil lineage [9 ].

In the present study, we profiled the expression and function of chemokine receptors in normal, human basophils at the mRNA and protein levels. Because modulation of chemokine-receptor expression is potentially important in turning the chemokine action, we also examined the expression of chemokine receptors in cytokine-stimulated basophils.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and monoclonal antibodies (mAbs)
The following reagents were purchased as indicated: murine anti-CCR1 mAb (IgG2b, clone 53504.111), anti-CCR2 mAb (IgG2b, clone 48607.121), anti-CXCR1 mAb (IgG2a, clone 42705.111), R-phycoerythrin (PE)-conjugated, anti-CCR2 mAb, carboxyfluorescein succinimidylester (CFS)-conjugated, anti-CXCR1 mAb, CFS-conjugated, anti-CXCR2 mAb (IgG2a, clone 48311.211), CFS-conjugated, mouse IgG2a, and PE-conjugated, mouse IgG2b (DAKO JAPAN, Kyoto, Japan); anti-CXCR2 mAb (IgG2a, clone 48311.211), PE-conjugated, anti-CCR1 mAb (IgG2b, clone 53504.111), and fluorescein isothiocyanate (FITC)-conjugated, mouse IgG2a (R&D Systems, Minneapolis, MN); FITC-conjugated or unconjugated, anti-CCR5 mAb (IgG2a, clone 2D7) and anti-CXCR4 (IgG2a, clone 12G5; PharMingen, San Diego, CA); biotin-conjugated, goat anti-human IgE mAb (Biosource, Camarillo, CA); and FITC-conjugated mouse IgG1 (Coulter Immunotech, Marseille, France). Anti-CCR3 mAb (IgG1, clone 444) [10 ] and anti-CCR4 mAb (IgG1, clone KM2160) [11 ] were prepared as described previously. Mouse IgG1 (MOPC-21) and IgG2a (UPC-10) with irrelevant specificity were purchased from Sigma (St. Louis, MO). Mouse IgG2b (MOPC141) was purchased from ICN Pharmaceutical (Aurora, OH).

Recombinant, human eotaxin was purchased from R&D Systems. Macrophage inflammatory protein (MIP)-1ß and thymus-expressed chemokine (TECK) were obtained from PeproTech (Rocky Hill, NJ). Other reagents used in the experiments were exactly the same as described previously [12 13 14 ].

Cell separation and culture conditions
Human basophils were isolated from venous blood obtained from consenting volunteers with no history of atopic diseases. Basophils were semi-purified by Percoll density-gradient centrifugation as described previously [12 ]. For most of the experiments, the basophils were purified further using a MACS Basophil Isolation Kit (Miltenyi Bio Tech, Bergisch-Gladbach, Germany), according to the manufacturer’s instructions. The purity of semi-purified and highly purified basophil preparations was approximately 16% and >98.5%, respectively. Their viability, measured by the trypan blue exclusion test, was consistently >95%. Mononuclear cells were isolated from venous blood by Ficoll (1.077 g/ml; Pharmacia, Uppsala, Sweden) density-gradient centrifugation. Eosinophils were purified as described previously [15 ]. In brief, crude eosinophils were obtained by dextran T500 sedimentation followed by Percoll (1.088 g/ml; Pharmacia) density centrifugation. Eosinophils were then purified further by negative selection using anti-CD16-bound micromagnetic beads (Miltenyi Bio Tech) and a magnetic cell-sorter column (Miltenyi Bio Tech) as the second step. The eosinophil purity was consistently >99%, and the viability was consistently >95%. Neutrophils were purified by two-step density centrifugation. In brief, buffy-coat cells were obtained from venous blood by dextran T500 sedimentation, and eosinophils were eliminated by Percoll (1.088 g/ml) density centrifugation. The buoyant fraction was collected and overlaid on Ficoll (1.077 g/ml) to eliminate mononuclear cells. The mean purity of neutrophils was consistently >98%, and the viability was consistently >95%.

Basophils were cultured in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (FCS; Gibco) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin; Gibco) at 37°C in 5% CO2 in flat-bottom, 48-well culture plates (Iwaki, Chiba, Japan) for the indicated times.

Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of chemokine receptors
Total RNA was extracted from highly purified basophils (~106 cells) using a QIAGEN Total RNA Isolation KitTM (Invitrogen, NV Leek, Netherlands) and treated with DNase I to remove contaminating DNA. RT for the first strand cDNA and hot-start PCR amplification (32 cycles) were performed as described previously [14 ]. Additionally, we constructed specific primers for the amplification of CCR9 mRNA: sense 5'-TAC TGG TAC TGC ACA AGA GTG-3' and anti-sense 5'-GTT TGG TGC TCT CAT CGC TAG-3', with an expected product size of 412 bp. The PCR products were electrophoresed through a 2% agarose gel and visualized with ethidium bromide.

Flow cytometric analysis of chemokine-receptor expression
For analysis of chemokine-receptor expression on basophils in whole blood, 200 µl uncoagulated blood was incubated with FITC- or PE- conjugated, mouse anti-chemokine-receptor mAb (10 µg/ml) and biotin-conjugated, goat anti-human IgE (10 µg/ml) for 60 min at 4°C. After washing in phosphate-buffered saline (PBS) supplemented with 3% FCS and 0.1% NaN3, red-blood cells were hemolyzed by hypotonic shock. After washing, the cells were incubated with PE-conjugated Extravidin (Sigma) or FITC-conjugated Extravidin (Sigma) at 10 µg/ml for 30 min at 4°C. Basophils were identified on the basis of strong staining with anti-IgE Ab.

For analysis of purified basophils, isolated basophils were washed in PBS supplemented with 3% FCS and 0.1% NaN3, preincubated with 3 mg/ml human IgG (Yoshitomi Pharmaceuticals, Osaka, Japan), and then incubated with anti-chemokine-receptor mAb at 10 µg/ml for 60 min at 4°C. An isotype-matched, murine mAb with irrelevant specificity was used as a negative control. After washing, the cells were stained with FITC-conjugated, goat F(ab')2 fragment, anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) at 7 µg/ml for 30 min at 4°C. For intracellular staining, cells were fixed with 4% paraformaldehyde in PBS for 30 min at 4°C and permeabilized with 0.1% Tween 20 in PBS for 30 min at 4°C.

For analysis of chemokine-receptor expression on lymphocytes or monocytes in mononuclear cells, these two cell types were discriminated on the basis of different forward-scatter and different side-scatter in flow cytometry [16 ].

Stained cells were analyzed using an EPICS XL SYSTEM II (Coulter, Miami, FL). At least 3000 cells were assessed to calculate the median value of fluorescence intensity. The median values of fluorescence intensity of cells were converted to the numbers of the molecules of equivalent, soluble fluorochrome units (MESF), as described previously [14 ]. Surface-receptor levels expressed in MESF units were calculated using the following formula: (MESF of cells stained with anti-receptor mAb) - (MESF of cells stained with isotype-matched, control mAb).

Measurement of intracellular calcium influx
Ca2+ influx was measured as described previously [14 ].

Basophil chemotaxis assay
Chemotaxicell (Kurabo, Osaka, Japan) with a 5-µm pore size was used for basophil chemotaxis assay. Percoll-separated basophils (3x104/100 µl) were added to the upper chambers, and samples to be tested (300 µl) were placed in the lower chambers. After incubation for 2 h at 37°C, the migrated cells in the lower chambers were collected and stained with FITC-conjugated, goat anti-human IgE (Biosource). The numbers of FITC-positive cells were counted by flow cytometry. Migration was expressed as a percentage of inoculated cells after subtracting the spontaneous migration (consistently <5%).

Histamine release from basophils
Basophil-histamine release was investigated as described previously [12 ]. In brief, Percoll-separated basophils were preincubated with or without IL-3 (5 ng/ml) for 15 min. The cells were then stimulated with a chemokine (100 nM) for 60 min at 37°C. The released histamine was measured using an automated, fluorometric technique. Histamine release was expressed as a percentage of the total cellular histamine after subtracting the spontaneous release.

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


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of chemokine-receptor mRNA in basophils
Basophils were purified to apparent homogeneity (>98.5%) by means of density centrifugation followed by negative selection, and the expression of 16 chemokine receptors was determined by RT-PCR analysis. Previous studies from our laboratory and others’ [2 3 4 5 , 8 ] demonstrated the expression of CCR1, CCR2, CCR3, CXCR1, CXCR2, and CXCR4 in human basophils. Consistent with those studies, we found strong CCR3 and CXCR4 transcript expression together with moderate CCR1 and CCR2 and weak CXCR1 and CXCR2 transcript expression in freshly isolated basophils. In addition to these chemokine receptors shown thus far, accumulation of mRNA of CCR5 was also detected (Fig. 1A ). Compared with resting basophils, no change in the basal-expression profile of chemokine receptors was noted in interleukin (IL)-3-stimulated basophils (Fig. 1B) . Conversely, no detectable expression of CCR4 mRNA was observed in freshly isolated or IL-3-stimulated basophils.



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  		Figure 1. mRNA expression of chemokine receptors in human basophils. Basophils were purified by means of Percoll density-gradient centrifugation followed by negative selection using micromagnetic beads (purity: >98.5%). RNA was extracted from freshly isolated basophils (A) and cells stimulated with IL-3 (10 ng/ml) for 4 h (B). RT-PCR was performed as described in Materials and Methods. A representative of three separate experiments is shown, and the other experiments showed similar results.

 
Expression of chemokine-receptor proteins in basophils
In the next series of experiments, we analyzed surface expression of chemokine receptors in whole-blood basophils. Whole-blood cells were stained with anti-receptor mAb and anti-IgE Ab, and the expression of CCR1, CCR2, CCR3, CCR4, CCR5, CXCR1, CXCR2, and CXCR4 was analyzed by flow cytometry (Fig. 2A and B ). Consistent with the mRNA results, blood basophils did not express CCR4 but clearly expressed CCR1, CCR2, CCR3, CCR5, CXCR1, and CXCR2 (Fig. 2C) . Conversely, despite the intense expression of mRNA, no significant surface expression of CXCR4 was observed in the whole-blood basophils at all.



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  		Figure 2. Surface, chemokine-receptor expression on whole-blood basophils. Whole-blood basophils were double-stained with anti-chemokine-receptor Ab and anti-IgE Ab and analyzed by flow cytometry. (A) Basophils and other peripheral blood mononuclear cells (PBMCs) were distinguished on the basis of different forward-/side-scatter properties (R1). (B) Electronic gates (R2) for anti-IgE-positive cells were set to eliminate contamination by lymphocytes and monocytes. (C) Surface, chemokine-receptor expression on basophils in fresh whole blood. The shaded area shows the fluorescence of cells stained with isotype-matched Ab. The data are representative of three independent experiments, and the others showed similar results.

 
We next purified basophils to apparent homogeneity and investigated the effects of cytokines on surface expression of CCR1, CCR2, CCR3, CCR4, CCR5, CXCR1, CXCR2, and CXCR4 in these cells. Freshly isolated basophils strongly expressed CCR3 at a level equivalent to eosinophils (Table 1A ). Basophils also expressed CCR2 and CXCR2 at levels similar to monocytes and neutrophils, respectively. The amount of CCR1 or CCR5 expression on basophils was smaller than that on monocytes and a subpopulation of lymphocytes, respectively. In contrast, freshly isolated basophils expressed clearly smaller amounts of CXCR1 compared with neutrophils (Table 1A) . The effects of cytokines are summarized in Table IB. IL-3 slightly inhibited surface expression of CCR2 and CCR3. Conversely, expression of CCR1, CCR5, CXCR1, and CXCR2 was not affected by any of the tested cytokines, albeit CCR1 and CXCR2 expression was almost completely diminished by 24-h incubation. Again, no significant expression of CCR4 was observed during 24 h of incubation in the presence or absence of various cytokines (Table 1B) .


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  		Table 1. Chemokine-Receptor Expression on Freshly Isolated Leukocytes (A) and Basophils Cultured with Cytokines (B)

 
In contrast to other chemokine receptors, the surface expression of CXCR4 on freshly isolated basophils was variable: Five donors expressed small amounts of CXCR4 on their surface, but six donors did not express CXCR4 at all. However, freshly isolated basophils possessed considerable amounts of intracellular CXCR4 protein, and the level of surface expression increased gradually up to 24 h of incubation (Fig. 3 ). SDF-1, the specific CXCR4 ligand, did not induce any calcium influx in freshly isolated basophils but caused strong influx in 24-h, cultured basophils, corresponding to the induction of surface-CXCR4 expression during culture (Fig. 4 ). As we demonstrated for eosinophils [14 ], expression of CXCR4 on basophils was highly regulated by several cytokines: The level of CXCR4 expression was markedly down-regulated by IL-3 and suppressed slightly by granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-5, and IL-4 (Table 1B) . However, interferon-{gamma} (IFN)-{gamma}, transforming growth factor-ß (TGF-ß), or tumor necrosis factor {alpha} (TNF-{alpha}) had no effect on basophil-CXCR4 expression in contrast to eosinophils. When the dose-dependent effects of IL-3 on surface expression of CXCR4 were studied, we found that a femtomolar order of IL-3 was sufficient to inhibit the expression of CXCR4 (unpublished results).



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  		Figure 3. Intracellular and surface expression of CXCR4 in basophils. (A) Intracellular and surface staining of CXCR4 in purified basophils. Shaded areas showed the fluorescence of cells stained with control mouse IgG2a. (B) Purified basophils were cultured at 37°C without exogenous factors, and surface-CXCR4 expression was analyzed after 4 and 24 h. Shaded areas showed the fluorescence of cells stained with control mouse IgG2a. Representatives of four separate experiments are presented, and the others showed similar results. (C) The time course of surface-CXCR4 expression is shown. All data are expressed as the mean ± SE (n=4) of MESF values calculated as described in Materials and Methods. *P < 0.05; ***P < 0.0005 versus MESF value at 0 h.

 


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  		Figure 4. SDF-1-induced Ca2+ influx in basophils. Freshly isolated basophils (purity: 99.8%; A) or 24-h, cultured basophils (B) were incubated with Fura-2 AM (2 µM) at 37°C for 20 min, and the Ca2+ influx induced by chemokines (50 nM) was measured with a fluorescence spectrometer. A separate experiment using the same donor basophils showed that surface expression was 93.4 and 87.6 (CCR2: x1000 MESF) and 0.1 and 87.6 (CXCR4: x1000 MESF) for freshly isolated basophils and 24-h, cultured basophils, respectively. The data shown are representative of two independent analyses from separate donors, each showing similar results.

 
Basophil activation in response to chemokines
Results of basophil migration by chemokines are shown in Table 2 . In freshly isolated basophils, eotaxin induced the strongest migration among a panel of chemokines (Table 2) . IL-8 and MCP-1 also exerted significant, migration-inducing ability, albeit to a lesser extent. Freshly isolated basophils failed to respond to SDF-1 at all, however in 24-h, cultured cells, SDF-1 elicited strong chemotaxis, comparable with that induced by eotaxin. Conversely, no significant migratory responses were observed in cells stimulated with other chemokines, including MIP-1{alpha}, MIP-1ß, or thymus and activation-regulated chemokine.


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  		Table 2. Chemokine-induced Basophil Migration

 
Table 3 summarized the results of basophil degranulation by chemokines. As demonstrated by others [6 7 ], MCP-1 was the most potent secretagogue: MCP-1 induced ca. 20% of histamine release from freshly isolated basophils, which was apparently amplified by priming with IL-3 (Table 3) . Freshly isolated basophils did not degranulate significantly in response to SDF-1, MIP-1{alpha}, or MIP-1ß. In contrast to migration, 24-h, incubated basophils also failed to degranulate in response to SDF-1, even after short-term priming with IL-3.


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  		Table 3. Chemokine-induced Basophil-Histamine Release

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous results from our laboratory and others’ [2 3 4 5 ] have revealed expression of CCR1, CCR2, CCR3, CXCR1, CXCR2, and CXCR4 in human basophils. In this study, we examined the expression of 15 chemokine receptors in human basophils at the mRNA and/or protein levels. We confirmed expression of CCR1, CCR2, CCR3, CXCR1, CXCR2, and CXCR4 and provided novel evidence of CCR5 expression in basophils. The CCR3 ligand, eotaxin, and the CXCR4 ligand, SDF-1, were able to induce strong migration in basophils, but degranulation was most strongly initiated by the CCR2 ligand, MCP-1. We also examined the effects of cytokines on chemokine-receptor expression and found that basophil-surface, CXCR4 expression was strictly regulated by several cytokines.

In contrast to the study by Jinquan et al. [8 ], who studied CXCR4 expression on isolated basophils, we failed to detect constitutive, surface-CXCR4 expression on basophils; lack of surface expression was confirmed by flow cytometric analyses of whole-blood cells (Fig. 2C) . However, basophils possess considerable amounts of intracellular CXCR4 store (Fig. 3A) , and the CXCR4-surface expression became apparent gradually during incubation (Fig. 3B and 3C) , analogous to our recent findings for eosinophils [14 ]. Because apparent surface-CXCR4 expression was observed after as short as 4 h of culture (Fig. 3B and 3C) , the conflicting observations concerning the expression of CXCR4 on resting basophils may be explained as a consequence of the separation conditions, especially the time-consuming purification process. In fact, in contrast to whole-blood basophils, we also observed small amounts of surface-CXCR4 expression on basophils just after purification in some experiments (5 out of 11).

The findings of the functional survey of various chemokines in this study confirmed the previous argument that CCR2 and CCR3 are responsible for degranulation and chemotaxis in resting basophils, respectively. Because ligands of both receptors are recognized as "inducible" chemokines up-regulated by inflammatory or immunological stimuli, CCR2 and CCR3 are potentially involved in disorderly conditions through activation and chemoattraction of basophils, respectively. Conversely, despite the strong, migration-inducing ability of SDF-1, a direct contribution of basophil CXCR4 to allergic inflammation is questionable. Because SDF-1 is expressed ubiquitously in almost all tissues, with little evidence of modulation of expression by inflammatory or immunological stimuli [17 ], CXCR4 may play a role in baseline trafficking of basophils into extravascular tissues rather than recruiting them to inflammatory sites.

In contrast to other chemokine receptors, surface-CXCR4 expression of basophils was strongly affected by cytokines. Previous studies from our laboratory and others’ have demonstrated that leukocyte CXCR4 expression is modulated in a stimulus- and cell-specific manner [14 18 19 ]. For example, IL-4 up-regulates, and IFN-{gamma} down-regulates CXCR4 expression in T cells [18 , 19 ], and both cytokines exerted completely opposite effects on eosinophil CXCR4 expression. Thus, modulation of surface-CXCR4 expression may represent an alternative mechanism for control of cell-specific, biological responses to SDF-1. In the present study, we observed that IL-3, the most potent basophil-directed hemopoietin, abrogated the expression of CXCR4 completely. CXCR4-surface expression was also down-regulated by IL-5 and GM-CSF, which share the receptor-common ß-chain with IL-3 [20 ]. These cytokines are assumed to play proinflammatory roles in allergic inflammation, because they can potently stimulate basophil, biological functions such as mediator release [21 , 22 ]. Our present finding is in line with the current concept for proinflammatory roles of these cytokines in allergic disorders. Because very low concentrations of IL-3 will down-regulate basophil CXCR4 expression theoretically, IL-3, and possibly IL-5 and GM-CSF, render circulating basophils less sensitive to tissue-derived SDF-1, thereby deaccelerating the exit of basophils from the circulation to noninflamed, extravascular tissues. This may work to accelerate local basophil accumulation at allergic, inflammatory sites. In this context, the expression profiles of chemokine receptors in basophils of allergic subjects and those at allergic inflammatory sites are intriguing questions meriting further study.

Basophils constitutively expressed considerable amounts of CCR5 on their surface. However, we found little functional significance in our assays (Table 1) . Furthermore, MIP-1{alpha} and MIP-1ß did not elicit any calcium influx at all (unpublished results). The expression of CCR5 on basophils was rather unexpected, because CCR5 is known to be expressed selectively in Th1 cells [23 ]. Basophils have been believed to exert effector functions in Th2-dominant states. Furthermore, studies of cytokine generation indicate that basophils produce Th2-type cytokines such as IL-4 [24 ] and IL-13 [25 ] but no Th1-type cytokines. Our findings indicate that basophils may also play a role under Th1-dominant, disease conditions.

An increasing body of evidence has identified basophils as important, allergic, inflammatory cells; along with progression of allergic responses, basophils migrate from the circulation to allergic, inflammatory sites, where they exert effector functions through the release of diverse, proinflammatory mediators. In fact, studies on experimentally induced, allergic reactions have revealed the influx of basophils to sites of inflammation several hours after antigen exposure [26 ]. Furthermore, analyses of chemical mediators at the sites of late-phase, allergic reactions have indicated that rather than mast cells, basophils are active participants in these reactions [26 ]. It is interesting that the functional, histochemical, and phenotypic properties of basophils show remarkable similarities with those of eosinophils (reviewed in ref. [27 ]). For example, growth, differentiation and activation of basophils and eosinophils are regulated by the exact same hemopoietins, i.e., IL-3, IL-5, and GM-CSF. Conversely, the expression and function of chemokine receptors are somewhat different between the two cell types. Eosinophils express CCR3 strongly and CCR1, CXCR1, and CXCR2 marginally on their surface (ref. [28 ] and unpublished results). Like basophils, CXCR4 was expressed on eosinophils upon culture. Unlike basophils, however, expression of CCR2 and CCR5 was not detected in eosinophils (unpublished results). Furthermore, degranulation was induced, via CCR3 in eosinophils [29 ] and via CCR2 in basophils [6 , 7 ]. Despite these notable differences, CCR3 and CXCR4 are able to induce the strongest chemotactic responses in basophils and eosinophils. Our results indicate the existence of overlapping mechanisms for locomotion of eosinophils and basophils. The in vivo distribution of both cell types may be regulated by a balance between homeostatic (CXCR4-mediated) and inducible (CCR3-mediated) mechanisms.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from the Manabe Medical Foundation, grants-in-aid from the Ministry of Health and Welfare of Japan (to K. H. and M. Y.), grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (to K. H. and M. Y.), and the Japan Society for the Promotion of Science Research Fellowships for Young Scientists (to M. I. and M. M.). We thank M. Imanishi and C. Tamura for their skilled, technical assistance. Thanks are also extended to S. Takeyama for her excellent secretarial help.

Received December 1, 2000; revised February 15, 2001; accepted February 19, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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