| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Leukocyte Biology Section, Biomedical Sciences Division, Imperial College School of Medicine, London, United Kingdom;
Children’s Hospital, Harvard Medical School, Boston, MA 02115; and
LeukoSite Inc., Cambridge, MA 02142
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Eosinophils accumulate from the microcirculation in response to locally generated chemoattractants. Several types of chemoattractants are active on eosinophils in vitro (4, 5), but in allergic inflammation in vivo a central role is emerging for the C-C class of chemokines, in particular eotaxin (6, 7, 8, 9, 10, 11, 12). Eotaxin was first identified as the major eosinophil chemoattractant present in bronchoalveolar lavage fluid from a guinea pig model of allergic airway inflammation (6, 13), and using primers based initially on the guinea pig protein sequence, guinea pig (14, 15), murine (16), rat (17), and human eotaxin (8) have been cloned. In sensitized guinea pigs, the elevation in eotaxin concentrations in the lung tissue of allergen-challenged animals parallels the onset of eosinophil accumulation, and all the eosinophil chemoattractant activity in bronchoalveolar lavage fluid obtained 6 h after pulmonary allergen challenge is neutralized by an anti-eotaxin Ab (10).
Further studies have investigated in more detail the mechanisms of eosinophil accumulation in vivo, central to which is the generation of a blood eosinophilia (7). We have previously obtained evidence that effective eosinophil accumulation in response to local eotaxin generation in tissues is dependent upon the elevation of blood eosinophil levels, mediated by the actions of IL-5 to release a rapidly mobilizable bone marrow pool of eosinophils (7, 10). Recently, we have shown that eotaxin alone or acting in synergy with IL-5 can mobilize bone marrow eosinophils and their progenitors (18), supporting an important role for eotaxin in eosinophil recruitment acting through both local and remote pathways. Evidence is also accumulating for an important role for eotaxin in human disease. Eotaxin mRNA and protein are up-regulated in atopic asthma, and eotaxin contributes significantly to the eosinophil chemotactic activity present in bronchoalveolar lavage fluid from asthmatic patients (19, 20, 21). Eotaxin has also been implicated in eosinophil accumulation in other human diseases (22).
Chemoattractants, including chemokines, signal through seven-transmembrane, G protein-coupled receptors (23, 24, 25, 26). Guinea pig eotaxin was originally shown to be a potent stimulator of both guinea pig and human eosinophils (6). Binding and desensitization studies with eotaxin and human RANTES (hRANTES)3 on human eosinophils predicted a common receptor for these chemokines (8). The human eotaxin receptor, C-C chemokine receptor-3 (CCR3), was subsequently cloned from eosinophil cDNA using primers and probes based upon the human CCR1 and other chemokine receptor sequences (27, 28). Murine CCR3 has been cloned and characterized using similar strategies (29, 30).
Studies using an anti-human CCR3 mAb have confirmed CCR3 to be the
major eosinophil chemokine receptor in the majority of the human
population (12, 27), although in some individuals, eosinophils also
show potent responses to chemokines including hRANTES and MIP-1
via
another receptor (Ref. 31 and I. Sabroe et al., unpublished data).
Furthermore, eotaxin and its recently described functional homologue
eotaxin-2 (32, 33) are unusual among C-C chemokines in that they show
marked receptor selectivity, signaling only via CCR3 (8, 28, 32, 33).
In humans, CCR3 is also expressed on basophils (34) and some Th2 cells
(35, 36, 37, 38), supporting an important role for CCR3 signaling pathways in
allergic inflammation.
Blockade of CCR3 may therefore provide an effective therapy in the treatment of allergic diseases including asthma, and the development of CCR3 antagonists has been identified as a major therapeutic target (39). To understand better the role of eotaxin and its receptor in allergic inflammation, we have cloned and characterized the guinea pig eotaxin receptor, guinea pig CCR3. We have produced a blocking mAb to this receptor and demonstrate its ability to block the actions of eotaxin on eosinophils in vivo.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The Dunkin-Hartley guinea pig liver genomic DNA library in EMBL3A was purchased from Stratagene Cloning Systems (La Jolla, CA). 125I-labeled human monocyte chemotactic protein (hMCP)-3 and 125I-labeled hRANTES (each 2200 Ci/mmol) were from New England Nuclear (Boston, MA). Cell culture media and reagents including RPMI 1640, HEPES, FCS, sodium bicarbonate, sodium pyruvate, 2-ME, and geneticin (G418) were obtained from Life Technologies (Paisley, U.K.). Percoll, BSA, and general laboratory reagents were obtained from Sigma (Poole, U.K.). Restriction enzymes were purchased from Boehringer Mannheim (Indianapolis, IN). Human IL-8 was produced in Escherichia coli as described (40). Human eotaxin was purified from Sf-9 insect cell culture supernatants infected with a recombinant eotaxin baculovirus obtained using the Bac-to-Bac baculovirus expression system (Life Technologies). All other chemokines other than those below were from Peprotech EC, (London, U.K.). The following were generous gifts. Synthetic guinea pig eotaxin was from B. Guarino, G. Andrews, and H. Showell (Pfizer, Groton, CT). Recombinant human C5a (rhC5a) was from Dr. J. van Oostrum (Ciba-Geigy, Summit, NJ). ENA-2 anti-human E-selectin F(ab')2 fragments were from Dr. M. Robinson (Celltech Therapeutics, Slough, U.K.). The murine pre-B cell line L1.2 was from Dr. E. Butcher (Stanford University, Stanford, CA).
Isolation of guinea pig CCR3 genomic bacteriophage clones
Guinea pig CCR3 DNA clones were isolated by screening 5 x 105 plaque-forming units of the guinea pig genomic DNA library using 32P-labeled human (27) and mouse (29) CCR3 cDNA sequences as probes. Plaques showing hybridization to both probes under high stringency wash conditions were purified, and restriction fragments of phage DNA with homology to the CCR3 probes were subcloned into pBluescript SK+ (Stratagene) and sequenced (Sequenase; Amersham Life Science, Cleveland, OH). A 1.1-kb single-exon open reading frame was identified, subcloned by PCR into the expression vector pcDNA3 (Invitrogen Corp., San Diego, CA) incorporating a 5' BamHI and a 3' XbaI linker, and resequenced to confirm PCR fidelity. The PCR N- and C-terminal primers for this reaction were as follows: GPCKR3/1, 5'-CGG GAT CCC GCC AGT GGC ATG GCG C-3', and GPCKR3/2, 5'-GCT CTA GAG CCC TCG AGG TCG ACC-3'.
Generation of a guinea pig CCR3-expressing stable cell line
Twenty micrograms of the CCR3/pcDNA3 construct were linearized by digestion with Bsm1 (New England Biolabs, Beverly, MA) and used to transfect L1.2 cells as follows. Twenty five million cells were washed twice and resuspended in 0.8 ml of PBS. The cells were incubated for 10 min at room temperature with the linearized CCR3/pcDNA3 construct DNA and transferred to a 0.4-cm cuvette, and a single electroporation pulse was applied at 250 V, 960 µF. Electroporated cells were incubated for 10 min at room temperature and transferred to culture at 37°C in RPMI supplemented with 10% FCS. Geneticin (G418) was added to a final concentration of 800 µg/ml 48 h posttransfection, and the cells were plated in 96-well plates at 25,000 cells/well. After 2–3 wk under drug selection, geneticin-resistant cells were assessed for their ability to migrate to 100 nM guinea pig eotaxin in a Transwell (Costar, Cambridge MA) chemotaxis assay (8). Lines with robust migration were cloned by limiting dilution and rescreened for their ability to bind 125I-labeled hMCP-3 (described below) and their ability to migrate to guinea pig eotaxin.
Purification of guinea pig leukocytes
Guinea pigs (400–450 g) were humanely killed by the administration of an overdose of sodium pentobarbitone i.p. (May and Baker, Oxford, U.K.), and blood was taken by cardiac puncture into 10 mM EDTA. For some experiments, guinea pigs were pretreated with 1 µg/kg recombinant human IL-5 i.v. 90 min before euthanasia. The whole blood was spun at 260 x g for 20 min and the plasma discarded. Dextran (5 ml; 9% in normal saline) and 20 ml of normal saline were added per 15 ml of the buffy coat/erythrocyte cell pellet and gently mixed, and the erythrocytes were allowed to sediment over 30 min. The leukocyte-rich suspension was harvested, and the leukocytes were pelleted by centrifugation. The mononuclear cells and polymorphonuclear leukocytes (PMNLs, comprising eosinophils and neutrophils) were separated over a two-layer 70%/80% discontinuous PBS/Percoll gradient centrifuged at 400 x g for 25 min. Contaminating erythrocytes were removed by hypotonic shock lysis (41). The cells were washed in the appropriate assay buffer and counted using Kimura’s stain.
Chemotaxis assays
Chemotaxis experiments with L1.2 cells or L1.2 receptor transfectant cell lines were performed using Transwell tissue culture inserts (Costar) as described (8).
For guinea pig leukocyte chemotaxis, PMNLs were prepared from IL-5-treated animals as above, and the chemotaxis assay was performed as described (18). Briefly, aliquots of 5 x 105 PMNLs (containing 18% ± 3.2% (SD) eosinophils) were pretreated in 100 µl of buffer (RPMI 1640 containing 20 mM HEPES and 0.25% BSA) with or without Ab for 10 min at room temperature. The cells were transferred to Transwell inserts within 24-well tissue culture plates containing 400 µl of buffer, guinea pig eotaxin, or rhC5a per well. The plates were incubated for 1 h at 37°C, and the chemotactic responses were determined using FACS analysis to measure the eosinophil recruitment to the lower chamber, as described previously (18).
Radiolabeled ligand binding assays
Chemokine binding studies of transfected cells using 0.1 nM 125I-labeled hMCP-3 or 0.1 nM 125I-labeled hRANTES as the ligand were performed using a modified method previously reported (8). Briefly, cells were washed once in PBS and resuspended in buffer (1 x 107 cells/ml in PBS with 50 mM HEPES, pH 7.5; 1 mM CaCl2; 5 mM MgCl2; 0.5% BSA; and 0.05% azide). Aliquots of 50 µl (5 x 105 cells) were dispensed into 1.5-ml Eppendorf tubes, followed by the addition of cold competitor and radiolabeled chemokines as indicated. The final reaction volume was 200 µl. Nonspecific binding was determined in the presence of 500 nM unlabeled MCP-3. After a 60-min incubation at 37°C, the cells were washed twice with 200 µl of buffer containing 0.5 M NaCl. Cell pellets were then counted. The data are presented as the percentage of specific binding calculated according to the formula 100 x (S - B)/(T - B), where S is the radioactivity of the sample, B is background nonspecific binding, and T is total binding without competitors. Duplicates were used throughout the experiments, and the SDs were always less than 10% of the mean. All experiments were repeated at least three times. Curve fit was calculated by KaleidaGraph software (Synergy Software, Reading, PA). In some experiments, binding of 125I-labeled hMCP-3 and 125I-labeled hRANTES was performed in the RPMI-based buffer as used for chemotaxis assays (see above).
Binding assays using 125I-labeled guinea pig eotaxin were performed using a modified previously described method (41). Briefly, guinea pig eotaxin was iodinated using the Iodogen (Pierce & Warriner, Chester, U.K.) method as described (6), with a specific activity of 195 Ci/mmol. Transfectants were incubated with 0.3 nM 125I-labeled guinea pig eotaxin in RPMI 1640 + 20 mM HEPES + 0.5% BSA + 0.05% sodium azide (pH 7.4) and varying concentrations of unlabeled chemokines (in a final volume of 50 µl) at room temperature for 60 min. Fifty microliters of assay buffer containing NaCl was added to a final concentration of 0.5 M NaCl, and the samples were mixed and layered onto tubes containing 150 µl of silicone oil. The cells were pelleted through the oil by centrifugation (13,000 x g for 5 min at 15°C). The cell pellet and supernatant were counted separately in a Canberra Packard Cobra 5010 gamma counter (Canberra Packard, Pangebourne, U.K.). Data are presented without the subtraction of nonspecific binding. Curve fits and calculation of ligand Kd were performed using MacLigand software (NIH version 4.92).
Measurement of cytosolic calcium in purified guinea pig eosinophils
Guinea pig peritoneal eosinophils were elicited in response to horse serum and purified over discontinuous Percoll gradients as previously described (5). Eosinophils (1 x 107/ml) were loaded with 1 µM fura 2 as previously described (41), washed in PBS + 0.1% BSA, and resuspended at a final concentration of 1 x 106 cells/ml in PBS + 0.25% BSA + 10 mM HEPES + 10 mM glucose. Aliquots of cells (2 ml) were dispensed into quartz cuvettes and equilibrated with 1 mM calcium at 37°C for 10 min before use. Changes in fluorescence were measured in a Perkin-Elmer (Norwalk, CT) LS-50 fluorescence spectrophotometer as previously described (41). Calculation of intracellular free calcium was derived from the fluorescence spectra (excitation wavelengths, 340 and 380 nm; emission wavelength, 510 nm) in accordance with established methods (41).
Generation of anti-guinea pig CCR3 receptor mAbs and flow cytometry
mAbs reactive with guinea pig CCR3 were generated by immunizing C57BL6 mice with 1–2 x 107 receptor transfectants i.p. six to eight times at 2-wk intervals. The final immunization was given i.v. Three days later, the spleen was removed and the splenic lymphocytes were fused with the SP2/0 cell line as described (27). Candidate anti-guinea pig CCR3 mAbs were initially identified by flow cytometry. Hybridoma supernatants were incubated with guinea pig CCR3 receptor transfectants and untransfected L1.2 cells, and bound Ab were detected using FITC-conjugated F(ab')2 goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). mAbs showing selectivity for guinea pig CCR3 transfectants were cloned by limiting dilution and rescreened against a panel of L1.2 cell chemokine receptor transfectants and guinea pig leukocytes. For flow cytometry of guinea pig leukocytes, 5 x 105 cells were incubated with mAbs in 100 µl of buffer (PBS containing 10 mM HEPES, 10 mM glucose, and 0.1% BSA) for 60 min on ice, washed twice (220 x g for 7 min), and incubated for 30 min on ice with FITC-conjugated F(ab')2 goat anti-mouse IgG. The cells were washed once and resuspended in PBS before FACS analysis.
Generation of F(ab')2 fragments of mAb 2A8
For use in vivo, F(ab')2 fragments of mAb 2A8 were generated by TSD BioServices (Germantown, NY) as follows. The 2A8 (IgG2a) Ab was first purified from cell culture supernatant by passage over a protein A-Sepharose column (Pharmacia) and elution of Ab with 20 mM sodium acetate, pH 4.0. F(ab')2 fragments were prepared from the Ab by digestion with immobilized pepsin (Pierce and Warriner, Chester, U.K.) and purified from Fc by protein A chromatography. The final product contained <3.0 endotoxin units (EU) of endotoxin/mg.
Inhibition of 111In-labeled eosinophil accumulation by mAb pretreatment in vivo
Eosinophils from four donor animals were purified over discontinuous Percoll gradients, pooled, and labeled with 111InCl3 as previously described (5). The radiolabeled eosinophils were divided into two aliquots and pretreated with either a control F(ab')2 Ab (ENA2, anti-human E selectin; 100 µg/ml) or 100 µg/ml 2A8 F(ab')2 in buffer (HBSS + 0.25% BSA + 30 mM HEPES) for 10 min at room temperature. The cells were washed and injected i.v. via an ear vein into sedated recipient animals (2.5 x 106 cells/animal, with four animals receiving control F(ab')2 Ab-treated cells and four animals receiving anti-guinea pig CCR3 2A8 F(ab')2 Ab-treated cells). Five minutes later, test agents (in 100 µl) were injected intradermally into the shaved dorsal skin of the recipients in a randomized pattern. After 2 h, the recipients were humanely killed. Blood was taken by cardiac puncture for calculation of circulating eosinophil numbers, the back skin of the animals was removed, and the skin sites were punched out and counted in a Cobra 5010 gamma counter. Results are expressed as the calculated number of 111In-labeled eosinophils accumulating per site according to established protocols (5, 6).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Two clones from the guinea pig genomic library showing homology to
both human and murine CCR3 probe sequences were selected for further
investigation and were found to contain the same sequence; one clone
alone contained the complete open reading frame. Analysis of the open
reading frame showed it to encode a G protein-coupled
seven-transmembrane receptor with 67 and 69% homologies to human and
murine CCR3, respectively (Fig. 1
). The
predicted protein sequence contained the familiar amino acid motif,
DRYLAIV, a highly conserved chemokine receptor amino acid sequence
across both receptor types and animal species. Similarly to human CCR3,
and in contrast to many other human chemokine receptors (including
CCR1, CCR2, CCR4, CXCR1, and CXCR2), the N-terminus of the guinea pig
receptor lacks sites for N-linked glycosylation. In common with other
C-C and C-X-C chemokine receptors, the sequence contains a serine- and
a threonine-rich C terminus, sites that are likely to be important in
receptor regulation through phosphorylation by G protein receptor
kinases (41, 42, 43). RT-PCR using specific primers for the guinea pig CCR3
sequence amplified a band of predicted size from mRNA purified from
guinea pig peritoneal eosinophils (data not shown).
|
Stable guinea pig CCR3 transfectants were generated and assessed for their ability to migrate in response to guinea pig eotaxin. Two cell lines showing robust chemotaxis to guinea pig eotaxin (2A1 and 4B4) were further characterized for their ability to bind radiolabeled chemokines. Cell lines 2A1 and 4B4 showed identical ligand binding profiles, and therefore only data from the 4B4 line are shown here.
The 4B4 cells bound 125I-labeled guinea pig eotaxin (Fig. 2a), which was displaced by
unlabeled guinea pig eotaxin in a concentration-dependent fashion. In
previous studies using guinea pig eosinophils, binding of
125I-labeled guinea pig eotaxin was displaced both by
hMCP-3 and hRANTES as well as guinea pig eotaxin (44). In keeping with
these data, 4B4 cells, but not their untransfected parent cell line,
also bound 125I-labeled hMCP-3 (Fig. 2, a and
b) and 125I-labeled hRANTES (Fig. 2b). Scatchard analysis of the binding data indicated
dissociation constants (Kd) of 3.0 nM for hMCP-3
(Fig. 2a) and 4.5 nM for guinea pig eotaxin (mean of three
experiments; data not shown), with a mean receptor density of 47,000
receptors per cell (Fig. 2a).
|
b). hRANTES failed to compete for
125I-labeled guinea pig eotaxin or 125I-labeled
hMCP-3 binding in these experiments using the buffers described.
However, when 125I-labeled hMCP-3 binding studies were
performed in the RPMI buffer used in transfectant cell chemotaxis
assays, 100 nM hRANTES competed for 53% of 0.1 nM
125I-labeled hMCP-3 binding to 4B4 cells (data not shown).
To evaluate ligand specificity further, we tested the chemotactic
responses of the 4B4 cell line to the chemokines used in the
radiolabeled ligand binding assays. Human IL-8 was included as a
negative control. Untransfected L1.2 cells were unresponsive to all
chemokines tested (Ref. 27 and data not shown). Fig. 2c
shows that only guinea pig eotaxin was able to induce a chemotactic
response in the receptor transfectants, producing a characteristic
bell-shaped dose-response profile with a peak response at 100 nM. This
profile is similar to that found for human eotaxin acting on human CCR3
transfectants (27). Despite the similar Kds of
hMCP-3 and guinea pig eotaxin for guinea pig CCR3, hMCP-3 was unable to
induce a chemotactic signal at concentrations up to 500 nM.
Development of mAbs to guinea pig CCR3
Previous studies with a blocking mAb to human CCR3 have
demonstrated the importance of this receptor in chemokine-induced
chemotactic responses of eosinophils in vitro (12). This mAb does not
cross-react with guinea pig CCR3 on either transfectants or guinea pig
eosinophils, and therefore a panel of anti-guinea pig CCR3 mAbs was
generated to explore the in vivo relevance of CCR3 in well
characterized guinea pig models of allergic inflammation (6, 7, 10).
Following the first fusion, 1000 hybridomas were generated, and two
Abs, 1C3 and 2A8, were isolated. Both mAbs bound to guinea pig CCR3
transfectants and showed minor cross-reactivity with the human CCR3
transfectant cell line (data not shown). Neither Ab stained a murine
CCR3 transfectant or any other human CCR (1, 2, 4, 5) or CXCR
(1, 2, 3) transfectant tested (data not shown). The specificity of Abs 1C3
and 2A8 for guinea pig leukocytes was therefore investigated, and Fig. 3 shows that these Abs bound selectively
to guinea pig eosinophils in PMNL populations purified from blood.
There was no detectable binding of either 1C3 or 2A8 to guinea pig
blood neutrophils (Fig. 3), monocytes, or lymphocytes as determined by
flow cytometry (data not shown), in keeping with the previously
described expression patterns of human CCR3 (27). A second fusion and
round of hybridoma formation resulted in the identification of four
further anti-guinea pig CCR3 mAbs with binding characteristics
identical to those of 1C3 and 2A8 (data not shown).
|
To investigate the potential of the anti-guinea pig CCR3 mAbs
to block ligand binding, the 4B4 cell line stable guinea pig CCR3
transfectants were pretreated with mAb 1C3 or 2A8, and the subsequent
binding of 125I-labeled hMCP-3 was determined. Pretreatment
of transfectants with mAb 2A8 blocked 125I-labeled hMCP-3
binding to transfectants in a concentration-dependent manner; in
contrast, mAb 1C3 did not inhibit ligand binding at any concentration
tested (Fig. 4). The binding
characteristics of guinea pig CCR3 deduced from Fig. 2, a
and b, and Ref. 44 predict that guinea pig eotaxin and
hMCP-3 share a common binding site on guinea pig CCR3. Accordingly,
pretreatment with 2A8 also blocked chemotaxis of the guinea pig CCR3
transfectants to guinea pig eotaxin in a concentration-dependent
manner, at similar concentrations to those needed to inhibit
125I-labeled hMCP-3 binding (Fig. 4). Pretreatment of cells
with the mAb 1C3 did not inhibit chemotaxis.
|
mAb 2A8 bound specifically to both guinea pig CCR3 transfectants
and guinea pig eosinophils and blocked both ligand binding and
chemotactic responses of guinea pig CCR3 transfectants. Therefore, the
ability of 2A8 to block the in vitro responses of guinea pig
eosinophils to guinea pig eotaxin was investigated using assays of
chemotaxis and intracellular calcium flux. Pretreatment with 2A8 (10
µg/ml) of PMNLs purified from the blood of IL-5-treated guinea pigs
caused a potent inhibition of eosinophil chemotaxis in response to
guinea pig eotaxin but had no effect on the eosinophil chemotactic
response to rhC5a (Fig. 5). In the same
cell populations, guinea pig neutrophils showed a chemotactic response
to rhC5a, but not to guinea pig eotaxin, and this was not inhibited by
2A8 pretreatment (data not shown).
|
shows that mAb 2A8 (12
µg/ml) completely blocked the calcium signal in response to 3 nM
guinea pig eotaxin. In contrast, mAb 1C3 had no effect on the calcium
flux induced by guinea pig eotaxin at the same concentration. Blockade of guinea pig eosinophil CCR3 signaling by F(ab')2 fragments of mAb 2A8
In pilot experiments, i.v. administration of mAb 2A8 (1 mg/kg) to
guinea pigs caused a rapid clearance of eosinophils from the
circulation (data not shown), possibly mediated through nonspecific
interactions of the Fc portion of the mAb. Therefore, for the use of
2A8 as a blocking anti-guinea pig CCR3 Ab in vivo,
F(ab')2 fragments of 2A8 were prepared. Fig. 6 shows that 2A8 F(ab')2
fragments (30 µg/ml) bound to eosinophils similarly to whole Ab (10
µg/ml). 2A8 F(ab')2 fragments (30 µg/ml) retained their
ability to inhibit the eosinophil cytosolic calcium response to 3 nM
guinea pig eotaxin but did not inhibit responses to rhC5a, used as a
control to show that the eosinophil responses to other ligands were not
blocked (Fig. 6). Surprisingly, the results shown in Fig. 6 also
suggest that guinea pig eotaxin partially desensitized the calcium
response to rhC5a and that the blockade of eotaxin-induced responses by
2A8 pretreatment prevented this heterologous desensitization of the
rhC5a response. Previous studies have found little evidence of
heterologous desensitization of chemoattractant signaling in human
eosinophils (45). This is in contrast to the patterns of heterologous
desensitization observed in human neutrophils, in which C5a is a potent
desensitizer of responses to chemokines, but C5a responses are not
readily desensitized by chemokines (41, 45, 46).
|
In contrast to unfragmented 2A8 IgG, F(ab')2 2A8 did
not result in any clearance of circulating eosinophils when
administered i.v. to guinea pigs at a dose of 1 mg/kg (data not shown).
Eosinophils were labeled with 111In and pretreated with 100
µg/ml concentrations of either a control F(ab')2 mAb
(ENA-2, anti-human E selectin) or F(ab')2 2A8 as
described in Materials and Methods. The cells were washed to
remove unbound Ab and injected i.v. into recipient animals. Buffer,
guinea pig eotaxin, or leukotriene B4 (LTB4)
was then injected intradermally into the recipients, and the local
accumulation of circulating radiolabeled eosinophils was allowed to
proceed for 2 h. Fig. 7 shows that
pretreatment of eosinophils with F(ab')2 fragments of 2A8
significantly inhibited the dermal accumulation of eosinophils in
response to eotaxin, but not in response to LTB4. There was
no significant difference in circulating 111In-labeled
eosinophil numbers between control and 2A8-treated groups (14.7% ±
2.35% (SD) for ENA-2-pretreated cell recipients vs 10.1% ± 4.28%
for 2A8-pretreated cell recipients).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Simultaneous screening of a guinea pig genomic DNA library with human and murine CCR3 cDNA probes identified a clone with homology to both these sequences. The DNA contained a single exon open reading frame coding for a G protein-coupled receptor with highest homology to human and murine CCR3 (67 and 69%, respectively).
When stably expressed in L1.2 cells, the receptor bound 125I-labeled guinea pig eotaxin with high affinity (Kd 4.5 nM). The binding of radiolabeled ligand was effectively displaced both by unlabeled guinea pig eotaxin and hMCP-3. Human eotaxin was also able to displace 40% of the binding of 125I-labeled guinea pig eotaxin from the guinea pig CCR3 transfectants. The binding of 125I-labeled guinea pig eotaxin to guinea pig eosinophils has been shown to be competed by excess unlabeled guinea pig eotaxin, hMCP-3, and hRANTES (44). Indeed, hMCP-3 and hRANTES are antagonists of guinea pig eotaxin at this receptor in vitro, and hRANTES has been exploited as a prototypic receptor antagonist in vivo (44). In keeping with these data, the 4B4 cell stable guinea pig CCR3 transfectants also bound 125I-labeled hMCP-3 and 125I-labeled hRANTES. Binding of these ligands was effectively competed by an excess of unlabeled guinea pig eotaxin, indicating that they shared a common binding site on guinea pig CCR3.
125I-labeled hMCP-3 (available at a specific activity 11 times higher than that of 125I-labeled guinea pig eotaxin) was used to determine accurately guinea pig CCR3 expression levels on the transfectants and to verify the Kd. These studies demonstrated a mean receptor density on the 4B4 cells of 47,000 receptors per cell (Kd 3.0 nM), in keeping with expression levels previously observed for other chemokine receptors using this expression system (27).
The binding of ligands to human CCR3 is very susceptible to minor
variations in local buffer conditions (49). In our experiments,
competition of ligand binding to stable guinea pig CCR3 transfectants
(4B4 cells) was also partly influenced by the buffer (either a
PBS-based or RPMI-based buffer) in which the binding studies were
performed. In particular, competition of radiolabeled ligand binding to
guinea pig CCR3 transfectants by unlabeled hRANTES was more dependent
upon local conditions than the other ligands tested.
125I-labeled hRANTES bound to the 4B4 cells in both the
PBS-based and the RPMI-based buffers (Fig. 2b and data not
shown), and this binding was displaced by unlabeled guinea pig eotaxin,
hRANTES, and hMCP-3. However, displacement of 125I-labeled
guinea pig eotaxin and 125I-labeled hMCP-3 binding to the
4B4 cell line by unlabeled hRANTES was not observed (Fig. 2, a and b). When 125I-labeled hMCP-3
binding studies were then performed in the RPMI-based buffer (vs the
PBS-based buffer used in the initial experiments), 53% displacement of
125I-labeled hMCP-3 by hRANTES was demonstrated. These
variations in excess unlabeled hRANTES competition may also be partly
explained by the tendency of this chemokine to become extensively
aggregated at physiologic pH (50).
Of those ligands showing binding to guinea pig CCR3, only guinea pig eotaxin caused a functional response in the stably transfected 4B4 cell line. Chemotaxis, which was maximal in response to 100 nM guinea pig eotaxin, occurred over a concentration range similar to that seen with other L1.2 cell stable transfectants and their respective ligands (27). Guinea pig eotaxin is the only known native ligand for guinea pig CCR3. In particular, guinea pig RANTES has been cloned but has been shown to be active on guinea pig macrophages and not on guinea pig eosinophils (51).
To allow the investigation of the role of CCR3 and its ligands in vivo, we raised mAbs against guinea pig CCR3. From a panel of mAbs that bound to stable guinea pig CCR3 transfectants but not the parental L1.2 cell line, two (1C3 and 2A8) were chosen for further investigation. Both Abs bound selectively to guinea pig CCR3 transfectants; there was minimal cross-reactivity only with transfectants expressing human CCR3. These Abs also bound with high affinity to guinea pig eosinophils, but not monocytes, lymphocytes, or neutrophils, in keeping with the known expression patterns of CCR3 on human leukocytes (12). The mAb 2A8 inhibited both the binding of 125I-labeled hMCP-3 to stable transfectants and their chemotactic response to guinea pig eotaxin, and similarly inhibited the chemotactic and intracellular calcium responses of guinea pig eosinophils to guinea pig eotaxin.
In humans, there is evidence that T lymphocytes cultured in cytokines resulting in a Th2-type phenotype express CCR3 (35, 36, 37, 38). CCR3+ lymphocytes in the circulation occur in very small numbers (35) and, even under stimulated Th2-type culture conditions, lymphocyte CCR3 expression may be at low levels compared with receptors such as CCR4 (37). It is possible that the numbers of T lymphocytes expressing CCR3 in either blood or bronchoalveolar lavage may be up-regulated in sensitized animals after allergen challenge, and this is a subject of further studies.
In preliminary experiments, we found that 2A8 IgG when injected i.v. into guinea pigs caused clearance of eosinophils from the circulation. Therefore, before in vivo investigation of the role of CCR3 by Ab-mediated receptor blockade, we prepared F(ab')2 fragments of 2A8 to avoid possible Fc-mediated effects on eosinophil recruitment. These fragments retained their ability to block the guinea pig eosinophil responses to guinea pig eotaxin as determined in assays of intracellular calcium flux, with some reduction in potency, and had no effect on circulating eosinophil numbers.
To investigate the role of eosinophil CCR3 in the accumulation of eosinophils in response to chemokines, we pretreated 111In-labeled guinea pig eosinophils in vitro with control F(ab')2 fragments or F(ab')2 2A8 and examined their ability to accumulate in vivo in response to intradermal injections of guinea pig eotaxin and LTB4. Pretreatment of guinea pig eosinophils with F(ab')2 2A8 selectively inhibited their accumulation in response to eotaxin but not to LTB4. Inhibition of eosinophil recruitment in response to intradermal eotaxin by F(ab')2 2A8 was not complete. It is possible from the intradermal accumulation data alone to speculate the existence of a further functional guinea pig eotaxin receptor on guinea pig eosinophils that was not blocked by F(ab')2 2A8 pretreatment. Insufficient F(ab')2 2A8 was available to allow the coadministration of Ab i.v. with radiolabeled cells in sufficient concentration to block new or unblocked guinea pig CCR3 on circulating radiolabeled eosinophils and formally test this hypothesis. However, the existence of a second functional eotaxin receptor in the guinea pig would run counter to experience in all other species to date (52, 53, 54), and the in vitro data discussed above provide no support for the existence of a second functional eotaxin receptor on guinea pig eosinophils. This suggests that over the 2-h period during which the Ab-treated radiolabeled eosinophils circulated in vivo, there was either internalization of CCR3 with a subsequent reexpression of functional receptors not bound by 2A8 or some loss of F(ab')2 2A8 from the cell surface.
In summary, we have cloned and sequenced the guinea pig eosinophil eotaxin receptor, guinea pig CCR3. We have characterized the ligand binding and signaling of this receptor and raised mAbs against it, one of which is a functional blocker of the receptor on guinea pig eosinophils in vitro. We have shown that pretreatment of guinea pig eosinophils with F(ab')2 fragments of this Ab selectively inhibits eosinophil accumulation in response to guinea pig eotaxin in vivo, without affecting circulating eosinophil levels. Early evidence in vivo for the importance of chemoattractant receptors in leukocyte recruitment was provided by studies demonstrating the inhibition of neutrophil recruitment by pretreating cells with pertussis toxin (55). More recent studies have demonstrated that blockade of eosinophil CCR3 in the guinea pig using hRANTES can inhibit eosinophil accumulation in vivo (44). Similarly, methionine-RANTES has been shown to inhibit eosinophil accumulation in mice (56). The present study is the first to show inhibition of eosinophil accumulation in vivo by a specific Ab directed against a chemokine receptor. Abs to chemokine receptors will be valuable tools to analyze the component parts of different types of inflammatory reactions. Such Abs may prove to be efficacious in certain inflammatory diseases in humans and provide proof-of-principle data for the development of small-molecule chemokine receptor antagonists.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Ian Sabroe, Leukocyte Biology Section, Biomedical Sciences Division, Sir Alexander Fleming Building, Imperial College School of Medicine, South Kensington, London SW7 2A2, U.K.
3 Abbreviations used in this paper: h, human; CCR3, C-C chemokine receptor-3; MCP, monocyte chemotactic protein; rhC5a, recombinant human C5a; PMNL, polymorphonuclear leukocyte; LTB4, leukotriene B4.
Received for publication April 23, 1998. Accepted for publication August 7, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
induce the migration and activation of normal human eosinophil granulocytes. J. Exp. Med. 176:1489.This article has been cited by other articles:
|
|
 |
|
  A.H. Nissim Ben Efraim and F. Levi-Schaffer Review: Tissue remodeling and angiogenesis in asthma: the role of the eosinophil Therapeutic Advances in Respiratory Disease, June 1, 2008; 2(3): 163 - 171. [Abstract] [PDF]
|
|
|
|
 |
|
 A. Meiser, A. Mueller, E. L. Wise, E. M. McDonagh, S. J. Petit, N. Saran, P. C. Clark, T. J. Williams, and J. E. Pease The Chemokine Receptor CXCR3 Is Degraded following Internalization and Is Replenished at the Cell Surface by De Novo Synthesis of Receptor J. Immunol., May 15, 2008; 180(10): 6713 - 6724. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. M. Rosenkilde, T. Benned-Jensen, H. Andersen, P. J. Holst, T. N. Kledal, H. R. Luttichau, J. K. Larsen, J. P. Christensen, and T. W. Schwartz Molecular Pharmacological Phenotyping of EBI2: AN ORPHAN SEVEN-TRANSMEMBRANE RECEPTOR WITH CONSTITUTIVE ACTIVITY J. Biol. Chem., May 12, 2006; 281(19): 13199 - 13208. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Morokata, K. Suzuki, Y. Masunaga, K. Taguchi, K. Morihira, I. Sato, M. Fujii, S. Takizawa, Y. Torii, N. Yamamoto, et al. A Novel, Selective, and Orally Available Antagonist for CC Chemokine Receptor 3 J. Pharmacol. Exp. Ther., April 1, 2006; 317(1): 244 - 250. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. K. Fritz, C. Kerr, L. Tong, D. Smyth, and C. D. Richards Oncostatin-M Up-Regulates VCAM-1 and Synergizes with IL-4 in Eotaxin Expression: Involvement of STAT6 J. Immunol., April 1, 2006; 176(7): 4352 - 4360. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. W. Lukacs, A. L. Miller, and C. M. Hogaboam Chemokine Receptors in Asthma: Searching for the Correct Immune Targets J. Immunol., July 1, 2003; 171(1): 11 - 15. [Full Text] [PDF]
|
|
|
|
|
|
R. M. Phillips, V. E. L. Stubbs, M. R. Henson, T. J. Williams, J. E. Pease, and I. Sabroe Variations in Eosinophil Chemokine Responses: An Investigation of CCR1 and CCR3 Function, Expression in Atopy, and Identification of a Functional CCR1 Promoter J. Immunol., June 15, 2003; 170(12): 6190 - 6201. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Zhang, M. P. Soares, Y. Guan, S. Matheravidathu, R. Wnek, K. E. Johnson, A. Meisher, S. A. Iliff, J. S. Mudgett, M. S. Springer, et al. Functional Expression and Characterization of Macaque C-C Chemokine Receptor 3 (CCR3) and Generation of Potent Antagonistic Anti-macaque CCR3 Monoclonal Antibodies J. Biol. Chem., September 6, 2002; 277(37): 33799 - 33810. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Bryan, P. J. Jose, J. R. Topping, R. Wilhelm, C. Soderberg, D. Kertesz, P. J. Barnes, T. J. Williams, T. T. Hansel, and I. Sabroe Responses of Leukocytes to Chemokines in Whole Blood and Their Antagonism by Novel CC-Chemokine Receptor 3 Antagonists Am. J. Respir. Crit. Care Med., June 15, 2002; 165(12): 1602 - 1609. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Jopling, I. Sabroe, D. P. Andrew, T. J. Mitchell, Y. Li, M. R. Hodge, T. J. Williams, and J. E. Pease The Identification, Characterization, and Distribution of Guinea Pig CCR4 and Epitope Mapping of a Blocking Antibody J. Biol. Chem., February 22, 2002; 277(9): 6864 - 6873. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Penido, H. C. Castro-Faria-Neto, A. Vieira-de-Abreu, R. T. Figueiredo, A. Pelled, M. A. Martins, P. J. Jose, T. J. Williams, and P. T. Bozza LPS Induces Eosinophil Migration via CCR3 Signaling Through a Mechanism Independent of RANTES and Eotaxin Am. J. Respir. Cell Mol. Biol., December 1, 2001; 25(6): 707 - 716. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Martinelli, I. Sabroe, G. LaRosa, T. J. Williams, and J. E. Pease The CC Chemokine Eotaxin (CCL11) Is a Partial Agonist of CC Chemokine Receptor 2b J. Biol. Chem., November 9, 2001; 276(46): 42957 - 42964. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Qiu, K. A. Frait, F. Reich, E. Komuniecki, and S. W. Chensue Chemokine Expression Dynamics in Mycobacterial (Type-1) and Schistosomal (Type-2) Antigen-Elicited Pulmonary Granuloma Formation Am. J. Pathol., April 1, 2001; 158(4): 1503 - 1515. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. B. Nibbs, E. Kriehuber, P. D. Ponath, D. Parent, S. Qin, J. D. M. Campbell, A. Henderson, D. Kerjaschki, D. Maurer, G. J. Graham, et al. The {beta}-Chemokine Receptor D6 Is Expressed by Lymphatic Endothelium and a Subset of Vascular Tumors Am. J. Pathol., March 1, 2001; 158(3): 867 - 877. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Stellato, M. E. Brummet, J. R. Plitt, S. Shahabuddin, F. M. Baroody, M. C. Liu, P. D. Ponath, and L. A. Beck Cutting Edge: Expression of the C-C Chemokine Receptor CCR3 in Human Airway Epithelial Cells J. Immunol., February 1, 2001; 166(3): 1457 - 1461. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Heinemann, A. Hartnell, V. E. L. Stubbs, K. Murakami, D. Soler, G. LaRosa, P. W. Askenase, T. J. Williams, and I. Sabroe Basophil Responses to Chemokines Are Regulated by Both Sequential and Cooperative Receptor Signaling J. Immunol., December 15, 2000; 165(12): 7224 - 7233. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M A. El-Asrar, S. Struyf, S. A Al-Kharashi, L. Missotten, J. Van Damme, and K. Geboes Chemokines in the limbal form of vernal keratoconjunctivitis Br J Ophthalmol, December 1, 2000; 84(12): 1360 - 1366. [Abstract] [Full Text]
|
|
|
|
|
|
N. Matsushita, N. Nishi, M. Seki, R. Matsumoto, I. Kuwabara, F.-T. Liu, Y. Hata, T. Nakamura, and M. Hirashima Requirement of Divalent Galactoside-binding Activity of Ecalectin/Galectin-9 for Eosinophil Chemoattraction J. Biol. Chem., March 17, 2000; 275(12): 8355 - 8360. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. M. Murphy, M. Baggiolini, I. F. Charo, C. A. Hebert, R. Horuk, K. Matsushima, L. H. Miller, J. J. Oppenheim, and C. A. Power International Union of Pharmacology. XXII. Nomenclature for Chemokine Receptors Pharmacol. Rev., March 1, 2000; 52(1): 145 - 176. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Giembycz and M. A. Lindsay Pharmacology of the Eosinophil Pharmacol. Rev., June 1, 1999; 51(2): 213 - 340. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Sabroe, A. Hartnell, L. A. Jopling, S. Bel, P. D. Ponath, J. E. Pease, P. D. Collins, and T. J. Williams Differential Regulation of Eosinophil Chemokine Signaling Via CCR3 and Non-CCR3 Pathways J. Immunol., March 1, 1999; 162(5): 2946 - 2955. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Sabroe, M. J. Peck, B. J. Van Keulen, A. Jorritsma, G. Simmons, P. R. Clapham, T. J. Williams, and J. E. Pease A Small Molecule Antagonist of Chemokine Receptors CCR1 and CCR3. POTENT INHIBITION OF EOSINOPHIL FUNCTION AND CCR3-MEDIATED HIV-1 ENTRY J. Biol. Chem., August 18, 2000; 275(34): 25985 - 25992. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
