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*
Department of Dermatology and Allergology, Hannover Medical University, Hannover, Germany; and
Serono Pharmaceutical Research Institute, Geneva, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain (binding
domain) and a common 
-chain (signaling domain), chemokines such as
eotaxin activate eosinophils via seven-transmembrane Gi
protein-coupled CCRs. Recent studies have demonstrated the importance
of CCR3 on human eosinophils that undergo receptor recycling after
chemokine activation, but the modulation of this receptor by cytokines
has not yet been addressed. In this study, we demonstrate that IL-3
induces a dose- and time-dependent down-regulation of CCR3 from the
surface of human eosinophils comparable to the CCR3-specific ligand
eotaxin, whereas IL-5, GM-CSF, IL-4, IL-10, IL-13, IFN-
, and TNF-
had no effect. Maximal down-regulation of CCR3 in response to IL-3 was
reached at 24 h. Reduction of CCR3 surface protein in response to
IL-3 could be prevented by an anti-IL-3 mAb and was neither due to
the release of CC chemokines nor to nonspecific binding of IL-3 to
CCR3. Moreover, down-regulation was prevented by phenylarsine
oxide, a nonspecific inhibitor of receptor internalization.
After 24 h, IL-3-induced decrease of CCR3 surface expression
correlated with diminished mRNA expression, suggesting a
transcriptional regulation mechanism. Since wortmannin partially
inhibited IL-3- but not eotaxin-induced CCR3 down-regulation, receptor
down-modulation seems to underlie different signaling events.
Therefore, these data suggest a novel role for the cytokine IL-3 in the
activation process of eosinophils and its predominant chemokine
receptor CCR3. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemokines are chemotactic proteins which play a central role in immune and inflammatory responses by the attraction and activation of leukocytes (7, 8). Chemokines differ from other cytokines in that they activate seven-transmembrane spanning G protein-coupled receptors (GPCR),3 and receptors for members of the two major subclasses (CC and CXC) of the chemokine family have been found to be expressed on eosinophils. The major chemokine receptor on eosinophils from normal patients is CCR3 (9, 10) but CCR1 is also expressed at a lower level, and there is evidence that eosinophils from atopic patients also express other CCRs (11).
Cytokines, in particular IL-3, IL-5, and GM-CSF, are not only
responsible for the generation of eosinophils from the bone marrow, but
also for their activation. Human eosinophils have prolonged survival,
enhanced functional properties, such as the release of toxic proteins
and reactive oxygen species, and become hypodense when exposed to these
cytokines (12, 13). The high-affinity receptors for IL-3,
IL-5, and GM-CSF are heterodimers consisting of a ligand-specific
-chain and a common -chain, with the three -chains being more
closely related to each other in primary sequence than to other members
of the cytokine family (14, 15). Although binding of IL-3,
IL-5, and GM-CSF to their respective receptor -chains is the first
step in receptor activation, it is the recruitment of the -chain
that allows high-affinity irreversible binding and leads to full
receptor activation and in consequence to signal transduction.
From in vitro experiments it has been established that eosinophil
functions, as well as expression of cell surface proteins, can be
induced or increased by several cytokines such as IL-2, IL-3, IL-4,
IL-5, GM-CSF, TNF-, or IFN- (2). More recently,
cytokines have been shown to be responsible for the up-regulation of
chemokines receptors on lymphocytes, mononuclear phagocytes, and
neutrophils (16, 17, 18). Only few reports exist about the
regulation of chemokine receptor expression regulated by cytokines on
eosinophils such as CXCR4 and CXCR3 (19, 20). However, to
date, there are no reports about the influence of cytokines on the
regulation of the dominant CCR on human eosinophils, CCR3.
The relationship between cytokines and chemokines in the activation
process is poorly understood. Down-regulation and recycling of
receptors, particularly chemokine receptors, during the migratory
response is very important for the adaptation and desensitization of
cells to the inflammatory environment (21). Recent studies
have demonstrated that chemokine activation of receptors does not
always result in the same trafficking pattern. RANTES induction of CCR5
down-regulation allows its recycling to the cell surface, whereas the
modified RANTES protein AOP-RANTES prevents the re-expression of cell
surface CCR5 (22). On the other hand, CCR1 does not
recycle after RANTES stimulation (23). CXCR2 has been
shown to be degraded in the lysosomal compartment (24)
whereas CCR3 appears to follow both fates; it is partially degraded in
the lysosomes, but 
70% of the receptors recycle (23, 25).
In this study, we report that a cytokine, IL-3, which is not known to
be a ligand for CCR3, very efficiently induces CCR3 down-regulation.
This activity was not shared by other cytokines, IL-5 and GM-CSF, which
signal through the common -chain of the receptor for these three
cytokines. The mechanism for this regulation of CCR3 expression remains
to be elucidated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eotaxin was obtained from PeproTech (London, U.K.). C5a was
obtained from Sigma (Deisenhofen, Germany). IL-3, IL-4, IL-5, GM-CSF,
IFN-, TNF-, and IL-13 were obtained from R&D Systems (Wiesbaden,
Germany). ELISA kits for eotaxin and RANTES were obtained from R&D
Systems.
Monoclonal Abs and protein inhibitors
The mAb against CCR3 (clone 7B11, mouse IgG2a) was a kind gift from Dr. Ponath. Since this Ab was no more obtainable, we used a rat mAb against human CCR3 (clone 61828.111; IgG2a) from R&D Systems which shows a similar binding pattern and inhibitory features than 7B11. The mouse and the rat IgG2a isotype control Ab were obtained from Immunotech (Hamburg, Germany). The PE-conjugated anti-CD16 mAb was obtained from Immunotech; anti-IL-3, anti-RANTES, and anti-eotaxin mAbs were obtained from R&D Systems. The protein inhibitors wortmaninn, staurosporin, and genistein were obtained from Calbiochem (San Diego, CA).
Eosinophil isolation
Human granulocytes were isolated from heparin-anticoagulated venous blood from normal nonatopic healthy or atopic donors using Ficoll (Pharmacia, Erlangen, Germany) density gradient centrifugation as described previously (23). For further purification, granulocytes were resuspended in HEPES-buffered HBSS (Life Technologies, Grand Island, N.Y.), pH 7.4, containing 1 mg/ml BSA (HBSS plus BSA). The granulocyte pellets were harvested and washed with PBS; the remaining erythrocytes were lysed using brief exposure to hypotonic solution (>0.85% NaCl); and the eosinophils were isolated using a MACS CD16 kit (Miltenyi Biotec, Auburn, CA) to remove neutrophils following the methods described by the manufacturer (26). The resulting eosinophil purity was 99% as determined by microscopic examination with Kimura staining and flow cytometric analysis (FACScan; BD Biosciences, Heidelberg, Germany) using anti-CD16. There were no differences in viability of the cells (<95%) between the fractions, as judged by trypan blue exclusion.
Immunofluorescence of eosinophils
Immunofluorescence of eosinophils was conducted as described previously (23). Aliquots (10 µl) containing 1 x 105 cells were incubated at 4°C for 30 min with the indicated mAb, stained in a second step by a FITC-conjugated goat anti-mouse (for clone 7B11) or goat anti-rat Ab (for clone 61828.111) (Immunotech) and analyzed by flow cytometry. Data are expressed as original plot (specific mAb vs isotype control), ratio of fluorescence intensity of specific mAb (median) and isotype control (median), or fluorescence intensity (percent) calculated as: (median channel fluorescence (stimulus) - median channel fluorescence (isotype control))/(median channel fluorescence (medium) - median channel fluorescence (isotype control)) x 100%.
Down-modulation and re-expression of chemokine receptors
These experiments were conducted as described in detail previously (23). Eosinophils were incubated in a total volume of 100 µl of RPMI 1640 for different time periods at 37°C in an incubator or on ice with the indicated cytokine, various concentrations of IL-3, IL-5, GM-CSF, and eotaxin. RPMI 1640 medium-cultured eosinophils were used as the control. Aliquots were taken at the times indicated and stained for receptor surface expression as described above. For re-expression experiments, cells were incubated for 24 h with IL-3 or eotaxin to induce maximal down-regulation of the receptor. Thereafter, eosinophils were washed three times with RPMI 1640 and further incubated in IL-3 and eotaxin-free medium (RPMI 1640) for the indicated time at 37°C. For data analysis, only cells were taken with a viability of <95%, as judged by trypan blue exclusion.
Inhibition of internalization
Eosinophils were treated for 5 min with 8 µM phenylarsine oxide (PAO; Sigma) at 37°C. Thereafter, cells were further incubated in the presence of PAO with IL-3 (5.7 x 10-9 M), eotaxin (1.25 x 10-7 M), or medium for the indicated time periods. After washing, immunofluorescence staining was conducted as described above. There were no differences in viability of the cells (<95%) between the fractions, as judged by trypan blue exclusion.
Inhibition of protein phosphorylation
Eosinophils were treated with the indicated inhibitor or medium in the presence or absence of eotaxin and IL-3 for 24 h at 37°C. Thereafter, cells were washed and immunofluorescence staining was conducted as described above. There were no differences in viability of the cells (<95%) between the fractions, as judged by trypan blue exclusion.
Real-time quantitative RT-PCR assay
RT-PCR analysis of eosinophil mRNA was performed with a
LightCycler Instrument (Roche Molecular Biochemicals, Mannheim,
Germany) which allows the quantification of mRNA as described
previously (27). In brief, poly(A)+
RNA was isolated from 106 eosinophils using a
mRNA isolation kit (Roche Molecular Biochemicals). The isolated mRNA
was used for cDNA synthesis with the first-strand cDNA synthesis kit
(Roche Molecular Biochemicals) following the instruction manual as
described previously (27). Specific PCR primers for human
mRNA of CCR3 and -actin were designed and conventional PCR was
performed under primer-specific conditions. The primers used for PCR
were CCR3 (sense, 5'-ATG CTG GTG ACA GAG GTG AT-3' and antisense,
5'-AGG TGA GTG TGG AAG GCT TA-3'; expected bp length 354), eotaxin
(sense, 5'-TGA AGC TTG GGC CAG CTT CTG TCC CAA CC-3' and antisense,
5'-TGG TCG ACT GGA GTT GGA GAT TTT TGG TC-3'), and -actin (sense,
5'- GAG CGG GAA ATC GTG CGT GAC ATT-3' and antisense, 5'- GAA GGT AGT
TTC GTG GAT GCC-3'; expected bp length 204) and were designed according
to the published sequences (GenBank accession nos.: CCR3,
AF026535, eotaxin, AJ223461, and -actin, AB004047). The standards
and unknown samples were added in a total volume of 20 µl/reaction as
described in detail previously (27). The standards used in
this experiment were obtained from a serial dilution of cDNA from
medium-stimulated eosinophils (24 h). The following experimental
protocol was adapted to use CCR3 and -actin: After an initial
denaturation step at 95°C for 30 s, 40 (CCR3) and 30 cycles
(-actin) of amplification were performed, respectively. Beginning
with a denaturation step at 95°C, followed by primer-specific
annealing (55°C for CCR3; 60°C for -actin), the elongation was
conducted at 72°C with an amplicon-depending incubation time (15 s
for CCR3; 9 s for -actin). Finally, the standard curve allowed
calculation of the template concentrations in the unknown samples using
LightCycler software. Agarose gel electrophoresis of the probes
obtained by the LightCycler revealed the predicted size for
CCR3.
Measurement of intracellular Ca2+ concentration ([Ca2+]i)
Eosinophils were incubated in a total volume of 100 µl of RPMI 1640 at 37°C for 24 h in an incubator with IL-3 and eotaxin to induce a down-regulation of CCR3. RPMI 1640 alone was used as the control. After 24 h, all samples were washed with PBS++/0.1% BSA and loaded for 30 min with 2 µM fura 2 (Molecular Probes, Eugene, OR). Thereafter, cells were washed three times with PBS++/0.1% BSA at room temperature and further incubated at 37°C. Aliquots were taken at the times indicated and [Ca2+]i was measured at 37°C in an Aminco Bowman series 2 spectrofluorometer (SLM-Aminco, Urbana, IL) as described previously (23). Data are expressed as the original plots or as the ratio of chemokine-stimulated maximal [Ca2+]i level and baseline [Ca2+]i level as described previously (28). The increase in magnitude of intracellular calcium basal level as well as C5a-stimulated maximal calcium concentration can be explained by a longer resting period of the cells in PBS++/0.1% BSA.
Lucigenin-dependent chemiluminescence
Lucigenin-dependent chemiluminescence was measured using a single-photon imaging system with a two-dimensional photon counting system which allows the simultaneous measurement and analysis of 96-well (MTP reader; Hamamatsu Photonics, Herrsching, Germany) as described previously (23). In brief, eosinophils were suspended at a density of 5 x 104 cells/ml in HBSS plus BSA containing 200 µM lucigenin (Sigma). Aliquots (100 µl) containing 5 x 103 eosinophils were placed into flat-bottom white microtiter plates (Microfluor; Dynatech, Denkendorf, Germany). Cells were preincubated in the presence and absence of the anti-CCR3 mAb 61828.111 and subsequently stimulated by IL-3 or medium as a control. Measurements were performed in triplicate at 37°C.
Statistical analysis
The number of experiments is stated in the legends of the figures as n and represents different donors. Unless otherwise stated, the data in the text and figures are expressed as mean + SEM as determined by SigmaStat (SPSS, Chicago, IL) analysis. Values of p > 0.05 were accepted as significant using Student’s t test: *, p > 0.05; **, p > 0.01; ***, p > 0.001.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the first set of experiments, eosinophils were incubated for
24 h with different cytokines at concentrations that have been
shown to modulate receptor expression on eosinophils (29).
From all cytokines tested (IL-3, IL-5, GM-CSF, IL-13, IFN-,
TNF-), only IL-3 was able to modulate CCR3 expression. Surprisingly,
it down-regulated CCR3 expression on the surface of human eosinophils
(Table I
). Therefore, we decided to
investigate the effect of IL-3 on CCR3 expression in comparison to the
CCR3-specific ligand eotaxin, which has recently been shown to induce
internalization of CCR3 on human eosinophils (23). Since
IL-3, IL-5, and GM-CSF are members of the same cytokine family sharing
a common receptor chain, we also examined CCR3 expression following
stimulation with these cytokines in different doses.
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However, CCR3 surface expression decreased to 40% (compared with
medium) following stimulation with IL-3 at a concentration of 5.7
x 10-11 M. Maximal down-regulation of CCR3 down
to 30% was reached at 5.7 x 10-9 M (Fig. 1). In comparison, maximal eotaxin-induced CCR3 down-regulation was
observed at 4 x 10-8 M. At optimal
concentrations, IL-3 was just as efficient as eotaxin to induce CCR3
down-regulation. However, at lower concentrations IL-3 was more
potent than eotaxin to induce CCR3 down-regulation (Fig. 1).
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To clarify whether the reduced cell surface expression in response to
IL-3 and eotaxin detected by binding of the anti-CCR3 mAb is
temperature dependent, eosinophils were incubated at 4°C. At this
temperature, the binding of the anti-CCR3 mAb was only slightly but
not significantly reduced after incubation with IL-3 (Fig. 2A). These data indicate that
IL-3 binding to IL-3R induced a temperature-dependent down-modulation
of CCR3.
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To investigate whether the IL-3-induced CCR3 down-regulation was
directly mediated by IL-3, eosinophils were incubated with a mixture of
IL-3 and a neutralizing anti-IL-3 mAb. Flow cytometric analysis
revealed that anti-IL-3 mAb prevented IL-3-induced down-regulation
of CCR3 (Fig. 2
B and Table I), further supporting the
receptor dependence of this action. To exclude the possibility that the
IL-3-induced down-regulation could be caused by the release of CCR3
ligands, such as eotaxin and RANTES, from eosinophils in response to
IL-3, down-regulation experiments were performed in the presence of the
neutralizing anti-eotaxin mAb 10C11. However, this mAb (at
concentrations of 1, 5, and 10 µg/ml) was not able to prevent the
IL-3-induced but could inhibit the eotaxin-induced CCR3
down-regulation of human eosinophils (Table I). No increased
protein expression for eotaxin and RANTES was detectable in
IL-3-treated eosinophils when compared with medium-treated cells as
determined by ELISA (Table I). Pretreatment of eosinophils with the
blocking anti-CCR3 mAb, 61828.111 did not prevent the release of
reactive oxygen species from eosinophils in response to IL-3 (data not
shown). Therefore, IL-3 induces a specific down-regulation of CCR3 on
human eosinophils which could not be explained by an IL-3-induced
release of eotaxin and RANTES from eosinophils or a direct receptor
interaction to CCR3.
Kinetics of CCR3 down-regulation and CCR3 re-expression
To further characterize the IL-3-induced down-modulation, we first
focused our interest on the time dependence of the process. As seen in
Fig. 3A, we could show that
IL-3-induced CCR3 down-regulation occurred significantly after 30 min
(p > 0.05) and 60 min
(p > 0.01). In contrast, at 10, 15, 120, and
180 min no significant down-regulation of IL-3-treated eosinophils
could be detected (Fig. 3A). The strongest down-regulation,
however, could be observed after 24 h (p
> 0.001; Fig. 3A). On the other hand, eotaxin, which is
well known to induce an internalization of CCR3 (23, 25),
induced a strong down-regulation of CCR3 after 10 min, with
significance after 15 min (p > 0.001; Fig. 3A).
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B).
Interestingly, flow cytometric analysis showed that the re-expression
of CCR3 on eotaxin- and IL-3-stimulated cells followed the same time
course (Fig. 3B). Re-expression of CCR3 slowly occurred
after 30 min of culturing in medium and even after 120 min in medium
only 40% of the receptors were detectable on the surface. After
24 h of reculturing in pure medium, the viability of human
eosinophils was too low to further investigate CCR3 re-expression. PAO prevented IL-3-induced CCR3 down-regulation
Previous studies revealed that eotaxin induces CCR3
internalization in eosinophils (23, 25). Therefore, we
next asked whether short-term IL-3 treatment induces internalization of
CCR3 and incubated eosinophils with IL-3 and eotaxin, respectively, in
the presence of PAO. PAO inhibits the protein tyrosine phosphatase and
has widely been used as a general inhibitor of receptor internalization
(30, 31). Treatment of human eosinophils with 8 µM PAO
prevented IL-3-induced as well as eotaxin-induced CCR3 down-regulation
as shown in Fig. 4 and Table II. In cells incubated for 15 min with
IL-3, CCR3 expression decreased down to 62.3%. Whereas in cells
additionally treated with PAO, down-regulation was almost completely
inhibited (98.2%; Fig. 4 and Table II). These data indicate that IL-3
induces a quick receptor-mediated endocytosis of CCR3 comparable to the
eotaxin-induced internalization of CCR3.
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To prove whether transcription is a regulative step leading to
reduced surface CCR3 expression, we examined the mRNA expression of
medium- and cytokine-stimulated eosinophils using real-time
quantitative RT-PCR. Therefore, eosinophils were stimulated for 24
h with cytokines or incubated with medium. After isolation of the mRNA,
real-time quantitative RT-PCR was performed. The melting curves in Fig. 5A indicate that mRNA for CCR3
was present in nonstimulated human eosinophils as well as in
eosinophils incubated for 24 h with different cytokines. Agarose
gel electrophoresis of the probes obtained by the LightCycler revealed
the predicted size for CCR3 (Fig. 5A). During amplification,
the fluorescence signal for CCR3 cDNA in IL-3-incubated eosinophils
increased later than for the other cytokines tested, indicating lower
CCR3 mRNA amounts in IL-3-treated cells (Fig. 5B). Comparing
the amplification of standard cDNA template (24-h medium-incubated
eosinophils) and cytokine-treated cDNA with the cDNA of a housekeeping
gene (-actin), we could detect a significant decrease in CCR3 cDNA.
Whereas after 60 min of stimulation, CCR3 mRNA was only lowered to
80%; after 24 h of incubation, CCR3 mRNA was dramatically
diminished (p > 0.001) down to 35% compared
with medium-cultured cells (Fig. 5C). The other cytokines
tested (IL-5, GM-CSF, and IFN-) had no significant effect on CCR3
mRNA expression. Therefore, the down-regulation of CCR3 surface protein
by IL-3 parallels with decreased CCR3 mRNA expression.
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The data presented in this study so far implicate that IL-3
induces CCR3 down-regulation via a pathway not directly mediated by
CCR3 signaling. Kinase inhibitors are a sensitive way to assess signal
transduction pathways and the method of choice when only low numbers of
cells are available, as in the present study. Pretreatment of
eosinophils with wortmannin was able to prevent in part the
IL-3-induced down-regulation of CCR3 in a dose-dependent manner (Fig. 6, A and B). On the
contrary, wortmannin was not able to prevent the eotaxin-induced CCR3
down-regulation (Fig. 6, A and C). In contrast to
wortmannin, other kinase inhibitors such as genistein, a tyrosine
kinase inhibitor, and staurosporin, a protein kinase C inhibitor, were
not able to prevent IL-3-induced down-regulation (data not shown).
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To address the question whether the CCR3 down-regulation parallels with eosinophil effector functions, [Ca2+]i assays were performed. Eosinophils were preincubated with IL-3 (5.7 x 10-8 M) or eotaxin (1.25 x 10-7 M) for 24 h to induce maximal CCR3 down-regulation and subsequently resuspended in fresh medium without IL-3 or eotaxin. After reincubation in ligand-free medium for various periods of time, eosinophils were exposed to different doses of eotaxin and [Ca2+]i was measured using spectrofluorometry.
Immediately after eotaxin-induced CCR3 down-regulation and washing
steps eosinophils did not show a
[Ca2+]i increase in
response to eotaxin (Fig. 7, 0 min). A
10-min incubation time in eotaxin-free medium was sufficient to induce
a small but detectable
[Ca2+]i rise following
stimulation with eotaxin. Prolonged incubation time (20, 30, or 60 min)
in eotaxin-free medium resulted in an enhanced
[Ca2+]i signal after
exposure to eotaxin (Fig. 7).
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, Control). However, IL-3 was not able
to block [Ca2+]i in
response to eotaxin, indicating that IL-3 did not bind to a region of
the CCR3 close to the binding side of the CCR3-specific ligand eotaxin
(Fig. 8, Control). Moreover, short-term IL-3 treatment did not
desensitize freshly eosinophils to eotaxin (Fig. 8, Control).
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, 0 min, and Fig. 9).
The insufficient resensitization to eotaxin at this early time period
(0 min) in IL-3-treated cells differed significantly
(p > 0.01) from that of medium-treated cells
and was dose and time dependent of eotaxin (Fig. 9). Longer
resuspension of eosinophils in IL-3-free medium resulted in a
resensitization to eotaxin and did not differ from that of
medium-pretreated cells (Figs. 8 and 9). Interestingly, whereas the
eotaxin-induced magnitude of
[Ca2+]i increased during
longer resuspension time in IL-3-free medium, the C5a-induced
[Ca2+]i magnitude was not
significantly affected by preincubation of eosinophils with IL-3 (Fig. 8). Taken together, we could clearly demonstrate that beside the
down-regulation of CCR3 surface protein and mRNA the effector functions
of eosinophils are markedly decreased in response to the CCR3 ligand
eotaxin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(16, 17) are able to
modulate chemokine receptor expression on lymphocytes, murine bone
marrow macrophages, monocytes, and neutrophils, to date only little
data exist on cytokine-induced chemokine receptor regulation on human
eosinophils such as CXCR4 and CXCR3, respectively (19, 20). Therefore, the aim of this study was to investigate the
role of cytokines in the regulation of CCR3 on human eosinophils, the
predominant chemokine receptor on these leukocytes.
In this study, we have found that a dose-dependent decrease in cell
surface CCR3 levels occurs in IL-3-treated eosinophils, while none of
the other cytokines tested (IL-5, GM-CSF, IL-1, IL-4, IL-10, IL-13,
IFN-, and TNF-) modified the CCR3 expression. This suggests a
central role of IL-3 in CCR3=mediated eosinophil activation. The
question that IL-3 binds directly to CCR3 could be raised. Although we
have not addressed this question by examining the ability of IL-3 to
bind to CCR3, for instance in CCR3-stable transfectants using iodinated
IL-3, we have investigated the heterologous desensitization of eotaxin
by IL-3 in freshly isolated eosinophils. IL-3 was not able to block the
calcium signal in response to eotaxin, indicating that IL-3 did not
functionally inactivate the response of CCR3 to eotaxin. A blocking
anti-CCR3 mAb did not inhibit the release of reactive oxygen
species from human eosinophils in response to IL-3. IL-3 was not able
to reduce the binding of a neutralizing anti-CCR3 mAb which blocks
eotaxin binding at 4°C, a temperature at which a receptor binding is
efficient, demonstrating that IL-3 does not impair eotaxin binding. We
therefore proposed that the IL-3-induced down-regulation of CCR3 could
be due to IL-3-induced release of CCR3 ligands such as eotaxin and
RANTES. However, no significant amount of eotaxin and RANTES could be
detected in the supernatant of IL-3-stimulated eosinophils. An
anti-IL-3 mAb but not an anti-eotaxin mAb prevented
IL-3-induced down-regulation of CCR3. Lastly, although Schroder
and Mochizuki (1) have demonstrated that eosinophils under
certain circumstances can release CC chemokines such as eotaxin and
RANTES, only picomolar amounts are released which are not sufficient to
induce CCR3 down-regulation. Thus, the possibility that IL-3-induced
down-regulation of CCR3 on human eosinophils could be due to a
nonspecific binding to CCR3 or due to IL-3-induced release of eotaxin
or RANTES from eosinophils can be excluded. Down-regulation of
chemokine receptors is induced by ligand activation which has been
shown for CXCR1/CXCR2 (34), CCR2 (35), CCR5
(22), and CCR3 (23, 25). However, the results
presented here show that a cytokine, IL-3, which is not a ligand for
the chemokine receptor CCR3, is able to induce down-regulation of this
receptor.
It is well known that eotaxin and other CC chemokines induce an internalization of CCR3 from the surface of human eosinophils (36, 37). Again, in this study eotaxin induced a strong internalization of CCR3 after 10 min. There exist several possibilities of cellular events leading to the modulation of receptor expression, e.g., endocytosis, exocytosis, shedding, modifications of the transcription of the gene, etc. (38). To find out the mechanism responsible for the marked reduction of CCR3 in response to IL-3, we conducted different approaches. In this report, we provide several lines of evidence that IL-3 has the capacity to activate the endocytosis of CCR3. First, the mechanism leading to the down-regulation of CCR3 after IL-3 treatment was temperature sensitive because IL-3-induced down-modulation did not occur at 4°C. Second, we could show that pharmacological treatment with PAO that blocks GPCR endocytosis prevented eotaxin- as well as IL-3-induced down-regulation of CCR3. Therefore, short-term exposure of human eosinophils to eotaxin as well as to IL-3 led to receptor down-regulation via the endosomal pathway.
Cytokines have been shown to modulate mRNA expression for certain
chemokine receptors. The first demonstration was that of Loetscher et
al. (18), who showed that IL-2 modulated the mRNA levels
of CCR1 and CCR2. It has also been shown that the expression of
chemokine receptors on particular leukocytes may be modified by
proinflammatory cytokines—neutrophils normally express the CXC
chemokine receptors CXCR1 and CXCR2 and respond to their ligands, but
after treatment with IFN-, there is induction of mRNA for the CCRs
CCR1 and CCR3 and the cells acquire responsiveness to the CC chemokines
that activate these receptors (16). Cytokine regulation of
receptors is not restricted to chemokine receptors; for example, the
mRNA for IL-5R is down-regulated in human eosinophils by the
cytokines IL-5, IL-3, and GM-CSF (39). More recently,
Nagase et al. (19) found intense expression of CXCR4
surface protein after 24-h culturing of human eosinophils which could
be prevented and down-regulated by several cytokines such as IL-3,
IL-4, IL-5, and GM-CSF. In another preliminary study from our
laboratory, we could confirm these results; however, there exists a
different regulation profile between CXCR4 and CCR3 in human
eosinophils. In contrast to CXCR4, CCR3 could only be down-regulated by
IL-3 and not by other cytokines, indicating a more specific interaction
between the IL-3R and CCR3.
To get more insight into the IL-3-induced CCR3 down-regulation,
real-time quantitative RT-PCR was conducted. Studying the mRNA level of
CCR3 after IL-3 stimulation, we could demonstrate that IL-3 diminished
the amount of CCR3 mRNA. Here, we could distinguish between the early
effect (60 min) and the late effect (24 h) of IL-3 on CCR3 mRNA
amounts. In contrast to IL-3, no changes in mRNA levels of IL-5-,
GM-CSF- and IFN--treated eosinophils could be detected. Therefore,
the down-regulation of CCR3 surface protein after long-term incubation
with IL-3 parallels with drastically diminished mRNA for CCR3. Changes
in the steady-state level of RNA expression of a gene result from
alterations in either the rate of transcription, the rate of
degradations of the message, or a combination of both
(40). Our data suggest that CCR3 mRNA expression in
eosinophils is dependent on continuos transcription of the CCR3 gene,
and, furthermore, that at least the late IL-3 effect is dependent upon
diminished transcriptional activity of the CCR3 gene.
Since IL-3 but not IL-5 and GM-CSF induced CCR3 down-regulation in human eosinophils raised the question as to whether a different signaling between these cytokines and a common cascade of signaling molecules between IL-3 and eotaxin may occur. Protein inhibitors are only indirect tools; however, they are suitable to investigate the signal transduction pathway particularly when the number of cells of interest is low (41). Here, we have demonstrated that neither genistein, a tyrosine kinase inhibitor, nor staurosporin, a protein kinase C inhibitor, were able to prevent IL-3-induced CCR3 down-regulation. In contrast, the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin prevented in part the IL-3-induced but not eotaxin-induced down-regulation of CCR3. Recently, Coffer et al. (42) reported that both cytokines such as IL-3 and chemoattractants such as RANTES and C5a induced PI3-kinase activity in human eosinophils. However, inhibition of IL-3-induced CCR3 down-regulation after wortmannin treatment is limited on micromolar doses of wortmannin. Other kinases such as myosin L chain kinase (43), phosphatidylinositol 4-kinase (43), and phospholipase A (44) are likely to be inhibited at these doses. To the best of our knowledge, up to now no data exist about an association between myosin L chain kinase or phosphatidylinositol 4-kinases or phospholipase A, respectively, and IL-3 signaling as detected by searching National Library of Medicine’s PubMed. Therefore, it appears that PI3-kinase may be partly responsible for the IL-3-induced down-regulation of CCR3. Furthermore, we showed that the protein tyrosine phosphatase inhibitor PAO almost completely prevented IL-3-induced CCR3 down-regulation. Tyrosine phosphorylation plays a major role in transmembrane signal transduction through most cell surface receptors. Binding of IL-3 to its cell surface receptor induces tyrosine phosphorylation of a set of protein substrates (45). Since PAO is also known as an inhibitor of PI3-kinase (46), our results indicate another hint for the involvement of PI3-kinase in IL-3-induced CCR3 down-regulation.
To more directly address the question as to whether the IL-3-induced CCR3 down-regulation parallels with eosinophil effector functions, [Ca2+]i assays were performed. Whereas short-term treatment of freshly isolated eosinophils with IL-3 did not desensitize cells to eotaxin, long-term IL-3 treatment for 24 h desensitized eosinophils to eotaxin. Resuspension of eosinophils in IL-3-free medium for 10 min resulted in a resensitization to eotaxin. This fast resensitization could be explained by quick mobilization of a few functional CCR3 from intracellular stores, since even 30 min after IL-3 removal only 30% of the receptors are re-expressed as shown by FACS analysis. The delayed CCR3 surface protein re-expression suggests a transcriptional control, which requires more time than the more "classical" resensitization due to the release of the receptor from intracellular stores. However, our data clearly demonstrate a loss of functional properties, concerning the major chemokine receptor CCR3, in eosinophils stimulated for a longer time with IL-3.
Cellular responses to GPCRs are usually rapidly attenuated. Mechanisms of signal attenuation include removal of agonists from the extracellular fluid, receptor desensitization, endocytosis, and down-regulation (21, 47). This process, termed desensitization, is the consequence of a combination of different mechanisms. These mechanism include the uncoupling of the receptor from heterotrimeric G proteins in response to receptor phosphorylation by intracellular kinases, the internalization of receptors to intracellular membranous compartments, and the down-regulation of the total cellular complement of receptors due to reduced receptor mRNA and protein synthesis (38). In this case, it is known that protein kinases not only phosphorylate agonist-activated, but also indiscriminately phosphorylate receptors that have not been exposed to agonists (48, 49). Therefore, it seems possible that IL-3 binding to its receptor activates kinases (e.g., PI3-kinase) which phosphorylate CCR3 and lead to receptor internalization. However, the underlying IL-3 (and not IL-5 and GM-CSF)-specific signal transduction pathway leading to CCR3 internalization, an altered transcription of the CCR3 gene and therewith to reduced surface protein expression, has to be clarified in further studies.
Although the effects of IL-3, IL-5, and GM-CSF on eosinophils are very similar in regard to stimulation of the production of eosinophils from the bone marrow and inhibition of apoptosis, this study reveals fundamental differences between these cytokines with respect to the recruitment of eosinophils. This suggests a novel role of the cytokine IL-3 in the activation process of eosinophils and its predominant chemokine receptor CCR3. Therefore, IL-3 may be responsible for a negative feedback mechanism of human eosinophils with respect to actions mediated via the CCR3 in Th2-associated inflammation.
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2 Address correspondence and reprint requests to Dr. Jörn Elsner, Department of Dermatology and Allergology, Hannover Medical University, Ricklinger Strasse 5, D-30449 Hannover, Germany. E-mail address: elsner{at}htp-tel.de
3 Abbreviations used in this paper: GPCR, G protein-coupled receptor; PAO, phenylarsine oxide; [Ca2+]i, intracellular Ca2+ concentration; PI3-kinase, phosphatidylinositol 3-kinase.
Received for publication March 13, 2001. Accepted for publication July 24, 2001.
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