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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
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Department of Internal Medicine, Teikyo University School of Medicine, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 IgM
1 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.
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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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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, eosinophils in fresh
whole blood expressed no significant amount of CXCR4 on their surface.
However, as observed with Percoll-separated eosinophils, expression of
eosinophil CXCR4 in whole blood eosinophils became evident after
incubation for 24 h at 37°C (Fig. 3C). These results
indicate that the purification step did not modulate the expression of
CXCR4, and we therefore used Percoll-separated eosinophils in the
following experiments.
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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
Gi 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).
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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).
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20% for freshly
isolated and 24-h-cultured eosinophils, respectively). We determined
whether the surface expression of CXCR4 is related to apoptotic changes
of eosinophils. When 24-h-cultured eosinophils were double-stained with
anti-annexin V and anti-CXCR4, we observed the CXCR4 expression
in nonapoptotic, annexin V-negative eosinophils (Fig. 9). As shown in Table II, CXCR4
expression did not always parallel the apoptotic changes in
cytokine-treated eosinophils. IL-4 exerted no significant inhibitory
effect on apoptosis, but it almost completely suppressed CXCR4
expression. Conversely, IFN- significantly reduced the number of
apoptotic cells, while it failed to suppress and actually up-regulated
the eosinophil CXCR4 expression (Table II). Finally, treatment of
eosinophils with anti-Fas mAb slightly but significantly decreased
the number of living cells, whereas it failed to up-regulate CXCR4
expression (Table II).
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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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). While we were preparing this
manuscript, Chelucci et al. reported the surface expression of CXCR4 in
the eosinophil lineage during the culture of purified hemopoietic
progenitors (12), although they did not examine the
functional relevance of CXCR4 in these cells. In contrast to those
cultured eosinophils, we observed that surface CXCR4 protein was hardly
detected in freshly isolated eosinophils (Fig. 2). However, surface
CXCR4 expression gradually became apparent during incubation at 37°C.
Because surface CXCR4 in cultured eosinophils was widely variable, it
is of importance to clarify the control mechanisms of eosinophil CXCR4
expression. In this study, we observed that several cytokines exerted
reciprocal and divergent effects on eosinophil CXCR4 expression (Table II). The patterns of cytokine-mediated regulation of CXCR4 expression
in eosinophils seem to be distinct from those in other types of
leukocytes such as T lymphocytes. In T lymphocytes, IL-4 up-regulates
(13, 14, 15) and IFN- down-regulates (16)
CXCR4 expression, while both cytokines exerted completely opposite
effects on eosinophils.
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.
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Acknowledgments |
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Footnotes |
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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.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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]
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 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]
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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]
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 A. Valenzuela-Fernandez, T. Planchenault, F. Baleux, I. Staropoli, K. Le-Barillec, D. Leduc, T. Delaunay, F. Lazarini, J.-L. Virelizier, M. Chignard, et al. Leukocyte Elastase Negatively Regulates Stromal Cell-derived Factor-1 (SDF-1)/CXCR4 Binding and Functions by Amino-terminal Processing of SDF-1 and CXCR4 J. Biol. Chem., May 3, 2002; 277(18): 15677 - 15689. [Abstract] [Full Text] [PDF]
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H. Nagase, M. Miyamasu, M. Yamaguchi, M. Imanishi, N. H. Tsuno, K. Matsushima, K. Yamamoto, Y. Morita, and K. Hirai Cytokine-mediated regulation of CXCR4 expression in human neutrophils J. Leukoc. Biol., April 1, 2002; 71(4): 711 - 717. [Abstract] [Full Text] [PDF]
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 N. W. Lukacs, A. Berlin, D. Schols, R. T. Skerlj, and G. J. Bridger AMD3100, a CxCR4 Antagonist, Attenuates Allergic Lung Inflammation and Airway Hyperreactivity Am. J. Pathol., April 1, 2002; 160(4): 1353 - 1360. [Abstract] [Full Text] [PDF]
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Y. Dulkys, C. Kluthe, T. Buschermohle, I. Barg, S. Kno{beta}, A. Kapp, A. E. I. Proudfoot, and J. Elsner IL-3 Induces Down-Regulation of CCR3 Protein and mRNA in Human Eosinophils J. Immunol., September 15, 2001; 167(6): 3443 - 3453. [Abstract] [Full Text] [PDF]
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M. Iikura, M. Miyamasu, M. Yamaguchi, H. Kawasaki, K. Matsushima, M. Kitaura, Y. Morita, O. Yoshie, K. Yamamoto, and K. Hirai Chemokine receptors in human basophils: inducible expression of functional CXCR4 J. Leukoc. Biol., July 1, 2001; 70(1): 113 - 120. [Abstract] [Full Text] [PDF]
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M. J. Dobrzanski, J. B. Reome, and R. W. Dutton Role of Effector Cell-Derived IL-4, IL-5, and Perforin in Early and Late Stages of Type 2 CD8 Effector Cell-Mediated Tumor Rejection J. Immunol., July 1, 2001; 167(1): 424 - 434. [Abstract] [Full Text] [PDF]
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J.-W. Oh, K. Drabik, O. Kutsch, C. Choi, A. Tousson, and E. N. Benveniste CXC Chemokine Receptor 4 Expression and Function in Human Astroglioma Cells J. Immunol., February 15, 2001; 166(4): 2695 - 2704. [Abstract] [Full Text] [PDF]
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A. de Paulis, R. De Palma, L. Di Gioia, M. Carfora, N. Prevete, G. Tosi, R. S. Accolla, and G. Marone Tat Protein Is an HIV-1-Encoded {beta}-Chemokine Homolog That Promotes Migration and Up-Regulates CCR3 Expression on Human Fc{epsilon}RI+ Cells J. Immunol., December 15, 2000; 165(12): 7171 - 7179. [Abstract] [Full Text] [PDF]
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 G. Aust, C. Simchen, U. Heider, F. A. Hmeidan, V. Blumenauer, and K. Spanel-Borowski Eosinophils in the human corpus luteum: the role of RANTES and eotaxin in eosinophil attraction into periovulatory structures Mol. Hum. Reprod., December 1, 2000; 6(12): 1085 - 1091. [Abstract] [Full Text] [PDF]
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