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J. Biol. Chem., Vol. 277, Issue 37, 33799-33810, September 13, 2002
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From the Departments of Immunology and Rheumatology and
Received for publication, June 3, 2002, and in revised form, July 3, 2002
Eosinophils are major effector cells implicated
in a number of chronic inflammatory diseases in humans, particularly
bronchial asthma and allergic rhinitis. The Bronchial asthma is a multifactorial disease characterized
clinically by reversible bronchoconstriction leading to shortness of
breath. In the pathophysiology of the disease, a chronic inflammatory condition persists in the airways of most patients, which involves a
complex interplay between blood leukocytes, airway epithelial cells,
and bronchial smooth muscle cells. One of the most striking aspects of
asthma is the selective accumulation and activation of distinct
subtypes of leukocytes into the airways, particularly eosinophils, and
it is these cells that are postulated to play a key role in the
pathophysiology of the disease (1, 2).
Inflammatory mediators, such as chemoattractants, generated at the
involved sites, promote the migration of eosinophils from the
vasculature into the tissue. Unlike eicosanoids and complement cleavage
fragments, which display activities on a wide variety of cells,
candidate molecules for the selective recruitment of eosinophils into
the airways are a class of proteins called chemotactic cytokines or
chemokines. Chemokines are a growing superfamily of >50 small
molecular mass proteins (~8-10 kDa) and are characterized by their
actions on distinct subtypes of leukocytes (3, 4). These proteins can
be classified into two major subfamilies based on the arrangement of
the first two conserved cysteines in the protein. In the Chemokines exert their effects by binding to members of the
G-protein-coupled receptor superfamily of receptors, which contain seven transmembrane domains. The discovery of a potent
eosinophil-specific CCR3-activating A prominent non-human primate model of asthma was established over a
decade ago in the cynomolgus macaque (30, 31), and its utility in the
preclinical evaluation of therapeutic agents in humans has proven to be
extremely valuable (31-40). To gain a better understanding of the role
of CCR3 and its ligands in this important animal model of asthma, we
have cloned and functionally expressed the macaque CCR3 in the murine
pre-B L1-2 cell line. We have also cloned the macaque eotaxin and
employed chemically synthesized protein to functionally characterize
the macaque CCR3. Moreover, we have generated murine monoclonal
antibodies directed against the macaque CCR3 using two different
immunizing antigens and have demonstrated that these mAbs are potent
functional antagonists of this receptor through a series of receptor
binding and signaling assays in vitro.
Southern Hybridization--
Genomic DNA was isolated from venous
blood of cynomolgus2 and
rhesus macaques (Macaca fascicularis and Macaca
mulatta, respectively; Merck Research Laboratories, in-house
colony) using the QIAamp DNA blood kit (Qiagen, Valencia, CA). Also,
cynomolgus macaque DNA was purchased from Therion (Troy, NY), and
rhesus macaque DNA was purchased from CLONTECH
(Palo Alto, CA). Southern blot hybridization was performed using
standard procedures (41). Briefly, genomic DNA (20 µg) was digested
with a set of restriction enzymes for 7 h, followed by
phenol/chloroform extraction, ethanol precipitation, and then
separation on a 0.7% agarose/Tris acetate-EDTA gel. The gel was
then saturated in 1.5 M NaCl and 0.5 M NaOH (to denature the DNA) for 45 min followed by neutralization in 1.5 M NaCl and 1 M Tris-Cl (pH 8.0) for 45 min. DNA
was transferred onto a Hybond-N+ membrane (Amersham
Biosciences) using 20× SSC as transfer buffer. Prehybridization was
carried out at 42 °C for 1 h in 6× SSC, 5× Denhardt's
solution, 0.5% SDS, 50% formamide, and 100 µg/ml salmon sperm DNA,
followed by hybridization for 16 h in the identical solution
containing 2 × 106 cpm/ml 32P-labeled
1.1-kb human CCR3 DNA fragment (Ready-to-Go DNA labeling kit; Amersham
Biosciences) comprising the open reading frame (7). The membrane was
washed twice in 2× SSC, 0.1% SDS at room temperature for 15 min;
twice in 0.1× SSC, 0.1% SDS at room temperature for 15 min; once at
55 °C for 15 min; and once at 65 °C for 15 min. The membrane was
then exposed onto Eastman Kodak Co. X-OMAT film and developed.
Cloning of Cynomolgus and Rhesus Macaque CCR3--
PCR was
performed using cynomolgus and rhesus macaque genomic DNA as template
with primers designed from the 5'- and 3'-untranslated regions of the
human CCR3 gene. The 5'-primer contained the sequence Cloning of Rhesus Macaque Eotaxin--
Poly(A)+
mRNA was isolated by oligo(dT)-cellulose chromatography from rhesus
macaque small intestine and used to generate first strand cDNA
using a tagged oligo(dT) primer (3'-RACE kit; Invitrogen). The
cDNA was then used as template for PCR performed with the following
two primers: 5'-primer (5'-AAC-CAC-CAC-CTC-TCA-CGC-C-3') and 3'-primer
(5'-CAG-GCT-CTG-GTT-TGG-TTT-CAA-3'). PCR was performed for 30 cycles:
94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min.
The resultant PCR product was subcloned into pCR2.1-TOPO (Invitrogen).
A consensus sequence for rhesus macaque eotaxin was identified by DNA
sequencing of six independent clones. The rhesus macaque eotaxin
protein was chemically synthesized (Gryphon Sciences, South San
Francisco, CA) based on the predicted amino acid sequence.
Expression of Macaque CCR3 in the Murine Pre-B Cell Line, L1-2,
and the Human Eosinophilic Cell Line, AML14.3D10--
The murine pre-B
L1-2 cell line was a generous gift from Dr. I. Weissman (Stanford
University). Transfection of the L1-2 and AML14.3D10 cell lines were
carried out as described (7, 8) with slight modifications. Briefly,
5 × 106 cells were washed in Hanks' balanced saline
solution, mixed with 20 µg of pcDEF3-rhesus CCR3 or pcDEF3-human CCR3
(for L1-2 transfection), and pBJ/Neo-cynomolgus CCR3 (for AML14.3D10
transfection) in a 0.4-cm electroporation cuvette, electroporated at
250 V and 960 microfarads, and then cultured in complete medium (RPMI
1640 with 10% fetal bovine serum). After 48 h, the cells were
placed in medium containing 0.8 mg/ml G418 and plated onto 96-well
plates at 18,000 cells/well. Clones were transferred into six-well
plates, and positive clones were selected by their ability to bind
125I-human eotaxin (Amersham Biosciences).
Chemokine Receptor Binding Assay--
125I-Human
eotaxin (2000 Ci/mmol) was obtained from PerkinElmer Life Sciences, and
125I-macaque eotaxin (2000 Ci/mmol) was
custom-radioiodinated at Amersham Biosciences. L1-2/macaque CCR3 cells
(30,000 cells/assay for 125I-macaque eotaxin and 200,000 cells/assay for 125I-human eotaxin) were mixed with 30 pM (~20,000 cpm) iodinated chemokine and varying
concentrations of Ligand-induced Ca2+ Mobilization--
Changes in
intracellular calcium level were measured on a fluorescence imaging
plate reader (FLIPR; excitation 488-nm argon laser line/emission 530-nm
bandpass interference filter; Molecular Devices, Inc., Sunnyvale, CA)
following the manufacturer's instructions with modifications.
L1-2/macaque CCR3 or L1-2/human CCR3 cells were washed in wash buffer
(Hanks' balanced saline solution containing 20 mM Hepes,
pH 7.2, 0.1% bovine serum albumin) and labeled with fluo-3 (Molecular
Probes, Inc., Eugene, OR) at 1.5 × 106 cells/ml in
labeling buffer at 37 °C for 1 h. Labeling buffer was prepared
as follows. 50 µg of fluo-3 was dissolved in 44 µl of 10% pluronic
F-127 in Me2SO and then added to 11 ml of wash buffer. Following labeling with fluo-3, cells were washed twice in wash
buffer. About 1.5 × 105 cells in 135 µl of wash
buffer were added to each well of a 96-well plate (black, clear bottom;
Corning Costar, Cambridge, MA) and then centrifuged for 5 min (no
brake). The plate was placed on FLIPR, 67.5-µl ligands (one-half of
the cell volume) at various concentrations were added, and fluorescence
changes were recorded. For blocking experiments, mAbs were preincubated
with cells for 20 min before 3 nM macaque eotaxin was
added. Results were calculated using KaleidaGraph.
Ligand-induced Chemotaxis--
L1-2/macaque CCR3 cells were
labeled with 5 µM calcein (Molecular Probes), washed, and
resuspended in chemotaxis buffer (RPMI 1640 plus 0.5% bovine serum
albumin) at 5 × 106 cells/ml. To the bottom chamber
of a 96-well Neuroprobe ChemoTx plate (5-µm pore size; NeuroProbe,
Inc., Gaithersburg, MD), increasing concentrations of chemokine were
added in a volume of 29 µl of chemotaxis buffer. To the top chamber,
30 µl of cells (1.5 × 105 total cells) were added,
and chemotaxis was allowed to proceed at 37 °C for 1 h.
Unmigrated cells were removed from membrane with a Kimwipe. The plate
was then analyzed in a CytoFluor II fluorometer (excitation, 485 nm;
emission, 530 nm; PerSeptive Biosystems, Framingham, MA). For blocking
experiments, 5 nM macaque eotaxin was added to the bottom
chamber. To the top chamber, 15 µl of cells (1.5 × 105 total) was mixed together with 15 µl of mAb at
various concentrations.
Rhesus Macaque Whole Blood Gated Autofluorescence Forward
Scatter (GAFS) Assay--
GAFS assays were performed as described
(44)3 with modifications for
whole blood. Briefly, 90 µl of fresh rhesus macaque blood was mixed
with varying concentrations of chemokines (in 10 µl) in 1.2-ml
polypropylene tubes, incubated at 37 °C for 10 min, and then
transferred onto ice. To preserve the shape change, the cells were
fixed in 250 µl of ice-cold optimized fixative solution containing
2.5% Cytofix (BD Biosciences, San Jose, CA), 22.5% H2O,
and 75% FACSflow (BD Biosciences) and left on ice for 2 min. The
mixture was then transferred into 1 ml of ice-cold lysis buffer (155 mM NH4Cl, 10 mM KHCO3)
in a Falcon tube (catalog no. 2052) and left on ice for another
20 min to achieve uniform red cell lysis. For blocking experiments,
aliquots of blood were preincubated with mAbs in a total volume of 90 µl for 20 min at room temperature before 10 µl of macaque eotaxin
(7.5 nM final concentration) was added. The samples were
run on a FACScan flow cytometer (BD Biosciences), and data from 30,000 cells from each sample were collected. Eosinophils were gated out based
on their high autofluorescence, and mean forward scatter was calculated by Consort 40/VAX software (Becton Dickinson, Immunocytometry Systems).
Typically, more than 4,500 eosinophils in each sample were analyzed.
The mean forward scatter of the eosinophils in each sample was
determined, and a dose-response curve for each chemokine or mAb was
generated accordingly.
Generation of Mouse Anti-macaque CCR3 mAb--
Two immunizing
antigens were used to generate monoclonal antibodies against the
macaque CCR3. The first approach used viable whole L1-2 cells stably
expressing the full-length macaque CCR3 sequence as immunogen,
essentially as described (27) with some modifications. About 1.5 × 107 L1-2/rhesus macaque CCR3 cells were injected into
C57b/6 mice. Intraperitoneal injections were performed four times at
2-week intervals (Cell Essentials, Boston, MA). Postimmune mouse sera were tested by flow cytometry and inhibition of
125I-human eotaxin binding on AML14.3D10-cynomolgus macaque
CCR3 transfectants versus untransfected AML14.3D10 cells.
The most potent serum blocked 125I-human eotaxin binding by
50% at 1:1000 serum dilution and blocked this binding completely at
1:125 dilution (data not shown). The mice whose sera were most potent
in flow cytometry and inhibition of 125I-human eotaxin
binding were chosen for fusion. The final injection of whole
L1-2/rhesus macaque CCR3 cells was performed intravenously. Three days
postinjection, mouse splenocytes were fused with SP2/0 myeloma cells.
Approximately 1600 hybridoma clones were obtained from two fusions
(Cell Essentials). To eliminate any clones that reacted to native L1-2
cell surface antigens, screening of hybridoma supernatants was
performed by flow cytometry on AML14.3D10/cynomolgus macaque CCR3
cells. In the second approach, a peptide (30-mer) was synthesized
(Gryphon Sciences), corresponding to the predicted NH2
terminus of the macaque CCR3 amino acid sequence
(TTS-LDT-VET-FGP-TSY-DDD-MGL-LCE-KAD-VGA-norleucinal-amide). The
peptide was conjugated to thyroglobulin and injected into BALB/c
mice at 3-week intervals. Mouse sera were tested by flow cytometry on
L1-2/macaque CCR3 cells versus L1-2 untransfected cells and
inhibition of 125I-human eotaxin binding to L1-2/macaque
CCR3 cells. Postimmune sera possessed similar potency as those using
whole L1-2/macaque CCR3 cells as immunogen. As above, the mouse that
produced the most potent serum was used for fusion. A total of about
600 hybridoma clones were obtained by this method from a single fusion.
Positive hybridoma clones were selected by enzyme-linked immunosorbent assay (see below) and tested by flow cytometry on L1-2/macaque CCR3
cells. Positive clones identified from both approaches tested positive
on L1-2/macaque CCR3 and negative on the parental L1-2 cell line by
flow cytometry. Hybridoma clones producing anti-macaque CCR3 mAbs were
subcloned to ensure purity and then injected into mice for ascites
production. The IgG fraction from the ascites fluid was purified by
protein A chromatography and dialyzed against PBS. Antibody
concentration was determined by the Bradford protein assay kit
(Bio-Rad). The isotype of the anti-macaque CCR3 mAbs was determined by
enzyme-linked immunosorbent assay.
Flow Cytometry--
About 5 × 105 cells
(L1.2/rhesus macaque CCR3, AML14.3D10/cynomolgus macaque CCR3, or
partially purified rhesus eosinophils or PBMCs) were washed in staining
buffer (PBS, 5% fetal bovine serum); subsequently, normal goat IgG (10 µg/ml final concentration, ICN Pharmaceuticals, Costa Mesa, CA) was
added and incubated at room temperature for 10 min. Hybridoma
supernatant (50 µl), purified anti-macaque CCR3 mAbs, mouse IgG (ICN)
(negative control), or mouse anti-human CD11b (BD Biosciences; clone
ICRF44), all at 3 µg/ml, was then added to the cells incubated at
room temperature for 30 min. Cells were washed, and then fluorescein
isothiocyanate-labeled goat F(ab')2 anti-mouse IgG (5 µg/ml; ICN) was added to the cells and incubated at room temperature
for 30 min. For the rhesus macaque PBMCs, R-phycoerythrin-conjugated
mouse anti-human CD3 mAb (clone SP34; BD Biosciences) was incubated as
above. The cells were washed and resuspended at 106/ml in
staining buffer containing 1 µg/ml propidium iodide. Fluorescein isothiocyanate fluorescence was analyzed on a FACScan flow cytometer or
FACSCalibur (BD Biosciences).
Enzyme-linked Immunosorbent Assay--
Bovine serum
albumin-conjugated macaque CCR3 NH2-terminal peptide (4 µg/ml in PBS) was loaded onto 96-well plates (100 µl/well) and
incubated at 4 °C for 18 h. The plates were then washed three times with PBS. Blocking solution (3% fish gel in PBS, 300 µl/well) was added, and the plates were incubated at room temperature for 2 h. The plates were then washed, and hybridoma supernatant was added to
each well and incubated for 1 h. The supernatant was removed, and
the plates were washed in PBS containing 0.05% (v/v) Nonidet P-40.
Horseradish peroxidase enzyme-conjugated secondary antibody was added
to each well, and the plates were incubated for 30 min. The plates were
washed again in PBS containing 0.05% Nonidet P-40. ABTS-100 substrate
solution was then added and incubated for 30 min for color development.
Clones that gave a signal/noise ratio of greater than 3 were picked for
further analysis.
Partial Purification of Rhesus Macaque Leukocyte
Subtypes--
Rhesus macaque eosinophils were prepared by the
following method. In a 50-ml conical tube, 7 ml of NIM2a and 7 ml of
NIM2b solutions (Cardinal Associates, Santa Fe, NM) were prewarmed to room temperature and carefully layered sequentially. Ten ml of rhesus
macaque whole blood was slowly layered on top and centrifuged at
1000 × g in a swinging bucket rotor at room
temperature for 40 min (brake off). The top portion containing plasma
and NIM solution was removed, and the granulocyte cell layer was then transferred to another tube. The granulocyte layer was washed once in
wash buffer (Hanks' balanced saline solution containing 0.1% bovine
serum albumin) and incubated in lysis buffer (155 mM
NH4Cl, 10 mM KHCO3) for 15 min to
lyse the red blood cells. The remaining cells were then washed twice in
wash buffer. The final granulocyte cell preparation contained ~25%
eosinophils by differential staining with the remaining cells mostly
consisting of neutrophils. For the preparation of rhesus macaque PBMCs,
the following method was employed. In a 50-ml conical tube, 6.8 ml of
Lympholyte-Mammal solution (Cedarlane Laboratories, Hornby, Ontario,
Canada) was added. Subsequently, 9 ml of diluted blood (1:1 with PBS)
was added and centrifuged at room temperature for 20 min. The PBMCs
were carefully removed from the interface with a Pasteur pipette,
diluted with PBS, and centrifuged as above. The PBMCs were then washed
twice in PBS. The final rhesus macaque PBMC preparation contained
~52% T-lymphocytes by anti-CD3 staining followed by flow cytometry.
Cloning of CCR3 from Macaque
To determine the complexity of the CCR3 genes from different
non-human primate species, a Southern blot analysis was performed on
both cynomolgus and rhesus macaque genomic DNAs. Southern blots hybridized with the 32P-labeled human CCR3 cDNA probe
surprisingly revealed that the hybridization banding pattern was
identical for both of these macaque species when the genomic DNAs were
digested with BamHI, BglII, EcoRI, and
HindIII (Fig. 1). Although
this banding pattern was different from that of human genomic DNA
(8),4 a simple hybridization
pattern was observed, indicating that CCR3 is encoded by a single copy
gene in both the cynomolgus and rhesus macaque.
Given that human CCR3 lacked intervening sequences within the coding
region (45), it was postulated that the coding region of the rhesus and
cynomolgus macaque CCR3 genes would be intronless as well. Using PCR
based on primers designed from the 5'- and 3'-untranslated regions of
the human CCR3 gene, we independently cloned the rhesus and cynomolgus
macaque CCR3 genes from genomic DNA, and the sequences we obtained were
genetically distinct from those previously reported (46-48). The
sequences of the cynomolgus and rhesus CCR3 genes contained open
reading frames of 1065 nucleotides in length, which encoded
G-protein-coupled receptor proteins of 355 amino acids in length.
Genomic DNA from six cynomolgus monkeys were used separately as
templates in PCR to amplify the CCR3 gene. For each individual, the PCR
products from at least four separate reactions were pooled for cloning,
and six clones were sequenced to obtain a consensus sequence. Analysis
of the cynomolgus macaque CCR3 sequences revealed a nucleotide
polymorphism of either G or A at the first position of codon 250, encoding either a valine or isoleucine at that position, respectively
(one animal was heterozygous, containing both A and G at that position,
whereas four animals contained only an A and one animal contained only
a G). To clone the rhesus macaque CCR3, genomic DNA from three rhesus
monkeys were used as templates in PCR. As with cloning the cynomolgus CCR3, four PCR products from each of the rhesus template genomic DNAs
were pooled and subcloned, and six clones were sequenced for each.
Consensus sequences of clones obtained from these three animals
contained only an A at the first position of codon 250, encoding only
isoleucine. All other nucleotide polymorphisms in either the cynomolgus
or rhesus macaque CCR3 sequences were silent (see GenBankTM
entries). Throughout this report, we will refer to the sequence containing isoleucine at position 250 as that of macaque CCR3. Despite
the fact that multiple clones were sequenced from at least two sources
each of rhesus macaque and cynomolgus macaque DNAs, we were unable to
confirm the amino acid changes previously reported for rhesus macaque
CCR3 (Lys176 (46); Lys176, Lys180,
and Lys202 (47)) and cynomolgus macaque CCR3
(Val152, Ile267, Pro271,
Val285, Trp295, and Pro324 (48)).
These differences probably reflect polymorphisms in the various rhesus
and cynomolgus macaque CCR3 genes present in these colonies.
The predicted amino acid sequence of macaque CCR3 is shown as a
serpentine diagram (Fig. 2A).
The macaque CCR3 sequence is 92 and 94% identical to the human CCR3
sequence at the amino acid and nucleotide level, respectively.
Specifically, there are 28 amino acid differences between the human and
macaque CCR3 proteins. Interestingly, of the 17 nonconservative changes
between human and macaque CCR3, 13 of these residues are located in the
extracellular domains of the molecule and evenly distributed along the
NH2 terminus and the first, second, and third
extracellular loops (Fig. 2A), regions important for
chemokine binding and activation of the receptor (49). Fig.
2B represents an amino acid lineup of the macaque CCR3 to
all of the known CCR3 sequences to date, which include human, African
green monkey (96% identical at the amino acid level), sheep (72%
identity), guinea pig (68% identity), rat (67% identity), and mouse
(69% identity for both SV/129 and Balb/c strains). Some important
characteristics of the macaque CCR3 sequence are worth noting. Like
human CCR3, macaque CCR3 appears to lack any triplet amino acid
consensus sites for N-linked glycosylation
(N-X-S/T; where X is any amino acid), motifs that are present in nearly all
Functional Expression and Characterization of Macaque C-C
Chemokine Receptor 3 (CCR3) and Generation of Potent Antagonistic
Anti-macaque CCR3 Monoclonal Antibodies*
, Comparative Medicine, Merck Research Laboratories,
Rahway, New Jersey 07065
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chemokine receptor C-C
chemokine receptor 3 (CCR3) provides a mechanism for the
selective recruitment of eosinophils into tissue and thus has recently
become an attractive biological target for therapeutic intervention. In
order to develop in vivo models of inflammatory diseases,
it is essential to identify and characterize the homologues of human
eotaxin (C-C chemokine ligand 11) and CCR3 from other species,
such as non-human primates. Accordingly, we cloned the macaque eotaxin
and CCR3 genes and revealed that they were 91 and 92% identical at the
amino acid level to their human homologues, respectively. Macaque CCR3
expressed in the murine pre-B L1-2 cell line bound macaque eotaxin with high affinity (Kd = 0.1 nM) and
exhibited a robust eotaxin-induced Ca2+ flux and
chemotaxis. Characterization of -chemokines on native macaque CCR3
on eosinophils was performed by means of eotaxin-induced shape change
in whole blood using a novel signaling assay known as gated
autofluorescence forward scatter. Additionally, mAbs were raised
against macaque CCR3 using two different immunogens: a 30-amino acid
synthetic peptide derived from the predicted NH2 terminus
of macaque CCR3 and intact macaque CCR3-transfected cells. These
anti-macaque CCR3 monoclonal antibodies exhibited potent antagonist
activity in receptor binding and functional assays. The
characterization of the macaque eotaxin/CCR3 axis and development of
antagonistic anti-macaque CCR3 monoclonal antibodies will facilitate the development of CCR3 small molecule antagonists with the hope of
ameliorating chronic inflammatory diseases in humans.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or
C-X-C family, these two cysteines are separated by
any amino acid, whereas in the or C-C family, these two cysteines are adjacent to one another. Some members of this latter family have
been discovered to possess strong eosinophil migratory and activating properties.
-chemokine, eotaxin, isolated from the
bronchoalveolar lavage (BAL)1
fluid from ovalbumin-challenged guinea pigs (5), led to the identification of CCR3 (6-8), the third -chemokine receptor characterized in an expanding family that numbers close to 20 to date
(3). Although its expression was first thought to be limited to
eosinophils, CCR3 is now known to be more widely expressed on cells
involved in allergic inflammation, such as basophils (9), mast cells
(10), airway epithelial cells (11), and potentially TH2
T-lymphocytes (12). Chemokines that are selective for CCR3 include
eotaxin, eotaxin-2, and eotaxin-3, whereas the potent
eosinophil-activating -chemokines RANTES, MCP-3, and MCP-4 bind to
other chemokine receptors in addition to CCR3 (13).
-chemokines are elevated in human asthmatics
(14-16) and are markedly up-regulated upon allergen challenge in
animal models (5, 17-21) and in humans (22-26). The relatively high
expression of CCR3 on eosinophils, along with expression of specific
CCR3-activating -chemokines in the involved tissue affords a potent
mechanism for the selective recruitment, activation, and retention of
this cell type into tissue sites during allergic inflammation. It has
been shown with a blocking monoclonal antibody directed against human
CCR3 that the actions of multiple -chemokines on eosinophils are
mediated through CCR3 (27), indicating that CCR3 is the major
-chemokine receptor expressed on these cells. For these reasons,
CCR3 has recently emerged as a significant anti-inflammatory
pharmacological target, and CCR3 antagonists are currently being
developed for the treatment of asthma and other allergic disorders (28,
29).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
23 to 3 bp
upstream of the ATG initiation codon and a HindIII site
(5'-GGC-TTA-AGC-TTC-TAT-CAC-AGG-GAG-AAG-TG-3'). The 3'-primer contained
the sequence 11-29 bp downstream of the TAG termination codon and a
NotI site
(5'-CTT-CAT-CTC-CTT-GCG-GCC-GCT-CCT-CTT-TAG-GCA-ATT-TTC-3'). PCR was
performed for 30 cycles: 94 °C for 60 s, 55 °C for 60 s, and 72 °C for 2 min in a PerkinElmer Life Sciences model 9600 DNA
thermal cycler. The resultant PCR products from the cynomolgus and
rhesus DNA were subcloned into expression vector pBJ-Neo (7) and pcDEF3
(42) (a generous gift from Dr. J. Langer, University of Medicine and
Dentistry of New Jersey), respectively.
-chemokines (PeproTech, Rocky Hill, NJ) in a
binding buffer consisting of 50 mM Hepes, pH 7.2, 1 mM CaCl2, 5 mM MgCl2,
0.1% gelatin and incubated at 30 °C for 60 min essentially as
described (7). Radioactivity retained on polyethyleneimine-treated
Whatman GFC membranes after washing in binding buffer (using a
PerkinElmer Life Sciences harvester) containing 0.5 M NaCl was counted in a TopCount (PerkinElmer Life Sciences). Binding results were analyzed using KaleidaGraph
(Synergy Software, Reading, PA) and LIGAND (43).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Southern blot analysis of the macaque CCR3
genes. Rhesus macaque (lanes 1-4) and
cynomolgus macaque (lanes 5-8) genomic DNAs were
digested with restriction enzymes, and Southern blot was performed as
described under "Experimental Procedures." Lanes
1 and 5, BamHI; lanes
2 and 6, BglII; lanes
3 and 7, EcoRI; lanes
4 and 8, HindIII. DNA molecular size
markers in kilobase pairs (kb) are designated by
arrows.
- and -chemokine receptors to date. The
four cysteine residues postulated to form two disulfide bonds, one in
each of the four extracellular domains, are located at positions 24, 106, 183, and 273. The macaque CCR3 also contains the amino acid motif,
DRYLAIVHA, distal to TMIII in intracellular loop 2 (i2), which is
strikingly conserved in all species of CCR3 sequences to date (Fig.
2B) and is critical for G-protein coupling and signal
transduction upon ligand binding to G-protein-coupled receptors (50).
The cytoplasmic tail of macaque CCR3 also contains eight
serine/threonine residues at positions 333, 339, 340, 341, 343, 345, 346, and 352. Serine- and threonine-containing cytoplasmic tails are a
common feature of chemokine receptors that may become phosphorylated by
G-protein coupled receptor kinases, resulting in desensitization
(51).
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Fig. 2.
A, serpentine diagram of the macaque
-chemokine receptor CCR3. The four extracellular segments including
the NH2 terminus (OUT), and the four
intracellular segments including the COOH-terminal tail (IN)
are shown. The seven transmembrane-spanning domains are shown between
the two horizontal lines. The four cysteines, one
in each of the four extracellular segments, postulated to form two
disulfide bridges, are indicated with a thick black line.
Nonconservative amino acid changes between the macaque and the human
CCR3 sequence are indicated in white letters.
Conservative amino acid changes are encircled within a
thick black line. B, amino
acid alignment of CCR3 amino acid sequences from various species. The
figure shows the predicted CCR3 sequences for macaque
(rhesus), human (7), African green monkey (AGM) (46), sheep
(accession number AF266468), guinea pig (GP) (56), rat (83),
murine SV/129 (84), and murine Balb/c. The positions of the seven
putative transmembrane-spanning regions are designated with
overlines. A minimum of four identical residues is indicated
in the shaded region. The complete nucleotide
sequence of the rhesus CCR3 (accession numbers AF405535-AF405537),
cynomolgus CCR3 (accession numbers AF405533 and AF405534), and murine
Balb/c CCR3 (accession number AY049018) are available from
EMBL/GenBankTM/DDBJ.
Cloning of Eotaxin from Macaque
Since it has been previously shown that eotaxin is highly
expressed in the gastrointestinal tract in humans (52), the rhesus macaque small intestine was chosen as a source of mRNA to clone the
eotaxin cDNA by reverse transcriptase-PCR from this species. The
two-loop structure of the predicted amino acid sequence of rhesus
macaque eotaxin is shown (Fig.
3A) (hereafter referred to as
macaque eotaxin throughout this report). The macaque eotaxin is 88 and
94% identical to the human eotaxin sequence at the amino acid and
nucleotide level, respectively. As shown, there are only seven amino
acid differences between the mature human and macaque eotaxins (six
nonconservative, one conservative), with six of the differences
occurring within the NH2 terminus-proximal 30 amino acids
of the protein. Fig. 3B represents an amino acid lineup of
the macaque eotaxin to all of the known eotaxin sequences to date,
which include human, horse (66% identical at the amino acid level),
bovine (59% identity), guinea pig (58% identity), rat (59%
identity), and mouse (56% identity).
|
Expression of Macaque CCR3 in Murine Pre-B L1-2 Cells
Both the human and macaque CCR3 were subcloned into expression vector pcDEF3 (42), which uses the human elongation factor 1a promoter and has been proven to drive high level expression of proteins in heterologous cell lines. Given that the murine pre-B L1-2 cell line was successfully used for the heterologous expression of human CCR3 (8), we also chose to use this cell line for functional expression of macaque CCR3. Positive clones were picked by their ability to bind 125I-human eotaxin, and one, designated clone 19, was chosen for further analysis.
Competition Binding of Various -Chemokines on
L1-2/Macaque CCR3 Cells
Competition binding studies were performed with
125I-macaque eotaxin on clone 19 in order to characterize
the pharmacological properties of macaque CCR3. As shown in Fig.
4A and Table
I, unlabeled macaque eotaxin competed
with a Kd of 0.1 nM. Scatchard analysis
demonstrated that macaque eotaxin bound with a single affinity and that
clone 19 expressed 3 × 105 receptors/cell (Fig.
4A, inset). These results were similar to those
obtained when human CCR3 was expressed in AML14.3D10 cells when
125I-human eotaxin was used as the radiolabeled ligand (7).
The rank order of potency of the various -chemokines on
macaque CCR3 upon competition with 125I-macaque
eotaxin is macaque eotaxin > murine eotaxin ~ human eotaxin > human MCP-4 > human eotaxin-2 > human
eotaxin-3 > human MCP-3 > human RANTES (Fig. 4B
and Table I).
|
|
The pharmacological properties of macaque CCR3 were compared with that
of human CCR3 by competition binding of the various -chemokines
against 125I-human eotaxin on recombinant L1-2 cells
expressing these receptors (Table I). Most of the ligands have
comparable binding affinity on the human and macaque CCR3, with the
exception of human eotaxin-3 and RANTES. Human eotaxin-3 has high
affinity on human CCR3 (Kd = 0.9 nM) yet
binds to macaque CCR3 with >5-fold lower affinity (Kd = 5.2 nM). Likewise, human RANTES
binds human CCR3 with 6-fold higher affinity (Kd = 1.5 nM) than macaque CCR3 (Kd = 9.2 nM).
Functional Coupling of Macaque CCR3 in L1-2 Cells
Ligand-induced Ca2+ Mobilization--
To investigate
the ability of -chemokines to activate signal transduction in
L1-2/macaque CCR3 cells, agonist-induced calcium mobilization was
measured (Table II). The amplitude at
which intracellular calcium reaches its highest level in L1-2 cells
occurs within 10 s of ligand addition. The rank order of potency
obtained by binding affinity via competition of -chemokines on
macaque CCR3 with 125I-macaque eotaxin was comparable with
the agonist potency obtained by calcium mobilization (see Tables I and
II); however, the functional EC50 values were ~10-fold
lower than the binding Kd values for most of the
chemokine ligands. Interestingly, macaque eotaxin was shown to be
equipotent as an agonist on the human and macaque CCR3. Furthermore,
human RANTES was unable to trigger a calcium flux even at a
concentration of 100 nM.
|
Ligand-induced Chemotaxis--
Since heterologous chemokine
receptors are functionally coupled to the chemotaxis pathway in murine
pre-B L1-2 cells, chemotaxis assays were carried out with various
-chemokines on L1-2/macaque CCR3 cells. The rank order of potency of
the various -chemokine ligands was as follows: macaque eotaxin ~ human eotaxin > human MCP-4 > human eotaxin-2 ~ human MCP-3 > human eotaxin-3 > human RANTES (Fig.
5, b and d; Table
III). Similar to human CCR3, no response
was observed with human MIP-1. This rank order of potency was
generally consistent with radiolabeled binding and calcium flux data,
except for human MCP-3, which induces chemotaxis with a higher relative
potency than it does in agonist-induced calcium flux. Unlike the other
chemotactic -chemokines that are active on L1-2/macaque CCR3, human
eotaxin-2 is only a partial agonist, exhibited by a chemotactic index
of less than 50% of the amplitude observed with the other
eotaxins.
|
|
Activation of Native Macaque CCR3: Ligand-induced Shape Change of Rhesus Macaque Eosinophils
Activation of native macaque CCR3 was assessed by
-chemokine-induced shape change of rhesus macaque eosinophils in
whole blood by GAFS assay (44). Eosinophils, like all
leukocytes, undergo cytoskeletal rearrangement (via actin
polymerization) upon chemokine receptor activation, resulting in a
change of cellular morphology. These shape changes can be readily
monitored via increases in forward scatter by flow cytometry. Moreover,
eosinophils are highly autofluorescent and therefore can be gated out
in whole blood using flow cytometry without separation of the different leukocytes. For the GAFS assay, blood from a rhesus macaque that contained a high circulating level of eosinophils (>15%) was used. The rank order of potency for the various -chemokine ligands in the
rhesus eosinophil GAFS assay was as follows: macaque eotaxin > human eotaxin ~ human MCP-4 > human MCP-3 > human
eotaxin-2 > human eotaxin-3 > human RANTES (Fig. 5,
a and c; Table III). This rank order was very
similar, but not identical, to that from the chemotaxis assay. Most
notably, macaque eotaxin was more potent than human eotaxin, and human
eotaxin-2 did not appear to be acting as a partial agonist in the GAFS
assay. Unlike human eosinophils, which express CCR1 (7, 44), the rhesus
macaque eosinophils did not respond to human MIP-1 in the GAFS
assay, most likely reflecting the absence of CCR1 on these cells or
species differences for activity of human MIP-1 between human and
macaque CCR1.
Identification of Monoclonal Antibodies against Macaque CCR3
Two approaches were taken to produce mouse mAbs against macaque
CCR3 (see "Experimental Procedures"). The first approach used the
L1-2/macaque CCR3 cell line as the immunizing antigen, and one clone,
designated mAb 5B9 (IgG2a/
), was identified by flow cytometry (Fig.
6a). The second approach used
a 30-amino acid NH2-terminal macaque CCR3 synthetic peptide
as the immunizing antigen, and six positive clones were identified by
enzyme-linked immunosorbent assay. Only two of these clones, designated
mAb 51 (IgG1/) and mAb 52 (IgG2b/), were positive by flow
cytometry on L1-2/macaque CCR3 cells (Fig. 6, b and
c). All three of these anti-macaque CCR3 mAbs were negative
on the parental L1-2 cell line by flow cytometry (Fig. 6,
a-c), indicating specificity for macaque CCR3. Only mAb 52 exhibited some specificity for human CCR3 by flow cytometry, whereas
none of these three anti-macaque CCR3 mAbs were found to be positive
for murine CCR3 (data not shown).
|
Anti-macaque CCR3 mAbs Recognize Native CCR3 on Rhesus Macaque Eosinophils
The anti-macaque CCR3 mAbs were assessed by flow cytometry on primary rhesus eosinophils to determine binding to native CCR3. Partial purification of the rhesus eosinophils yielded a final mixture that contained 24% eosinophils, 1% lymphocytes, and 75% neutrophils by differential staining. Of the three anti-macaque CCR3 mAbs, only two of these (mAb 5B9 and mAb 51) bound to a subset of the rhesus leukocyte cell suspension by flow cytometry (~22%), the exact proportion by differential staining representative of eosinophils (Fig. 6, e and f), whereas mAb 52 did not react with this subset at all (Fig. 6g). An anti-integrin mAb (anti-CD11b) was used as a positive control, which stained the entire population of cells in the rhesus leukocyte mixture (Fig. 6h). In contrast to data observed with human T-lymphocytes (12), rhesus macaque T-lymphocytes were not shown to express CCR3 via double staining the rhesus macaque PBMC mixture with anti-CD3 and anti-macaque CCR3 (5B9) mAbs, followed by flow cytometry (data not shown).
Anti-macaque CCR3 mAbs Block Eotaxin Binding to L1-2/Macaque CCR3 Cells
The anti-macaque CCR3 mAbs were next tested for inhibition of
eotaxin binding. As shown in Fig.
7a, mAbs 5B9 and 51 exhibited highly potent inhibition of 125I-human eotaxin binding to
L1-2/macaque CCR3 cells with IC50 values of 0.027 and 0.051 µg/ml, respectively. Also shown in Fig. 7a, mAb 52 or
mouse IgG control did not demonstrate any inhibition of eotaxin binding
at concentrations up to 100 µg/ml. None of the three anti-macaque
CCR3 mAbs inhibited 125I-human eotaxin binding to
L1-2/human CCR3 (data not shown).
|
Anti-macaque CCR3 mAbs Display Potent Functional Antagonism
The anti-macaque CCR3 mAbs were analyzed in a series of functional
assays to determine antagonist activity. As shown in Fig. 7b, pretreatment of L1-2/macaque CCR3 cells with mAbs 5B9
and 51 inhibited macaque eotaxin-induced calcium flux with
IC50 values of 0.5 and 1.6 µg/ml, respectively.
Additionally, none of the anti-macaque CCR3 mAbs blocked human
eotaxin-induced calcium flux in L1-2/human CCR3 cells (data not shown).
Moreover, the mAbs were also evaluated in both the rhesus macaque whole
blood GAFS assay and in chemotaxis on L1-2/macaque CCR3. As shown in
Fig. 7c, mAbs 5B9 and 51 antagonized macaque eotaxin-induced
shape change of rhesus eosinophils with IC50 values of
0.088 µg/ml and 2.1 µg/ml, respectively. Finally, mAbs 5B9 and 51 blocked macaque eotaxin-induced chemotaxis of the L1-2/macaque CCR3
cell line with IC50 values of 0.05 and 0.3 µg/ml,
respectively (Fig. 7d). These two anti-macaque CCR3
antagonist mAbs also inhibited human MCP-4-induced chemotaxis of
L1-2/macaque CCR3 cells with a potency comparable with that of macaque
eotaxin (data not shown). Consistent with the binding studies, mAb 52 or control mouse IgG did not exhibit any inhibition in either the
calcium flux assay (at concentrations up to 1 mg/ml) or the GAFS and
chemotaxis assays (at concentrations up to 100 µg/ml for both) (data
not shown).
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
In this paper, we describe the molecular cloning and functional
expression of the macaque CCR3 -chemokine receptor. Previous studies
investigating macaque CCR3 have principally focused on AIDS research
exploring the mechanisms of human immunodeficiency virus type 1 and 2 co-receptor usage (46) and AIDS-associated neuropathogenesis in the
macaque (53-55). In addition, two sequence notes have previously been
reported (47, 48). This is the first report describing full
pharmacological characterization of macaque CCR3 with a variety of
-chemokine ligands in a series of radiolabeled binding and signal
transduction assays. The macaque CCR3 sequences described in this
report are genetically distinct from those previously reported, most
likely reflecting genetic polymorphisms in the macaque CCR3 gene. We
have also cloned the macaque -chemokine eotaxin and were able to
employ chemically synthesized protein in the biochemical and functional
assays described in this report.
In addition, we describe the generation of monoclonal antibodies directed against the macaque CCR3 and have demonstrated that these mAbs are functional antagonists in a series of in vitro assays. The in vitro potency of the anti-macaque CCR3 mAbs reported here compares favorably with other antagonistic murine anti-CCR3 mAbs raised against CCR3 from other species, which include human CCR3 (mAb 7B11) (27) and guinea pig CCR3 (mAb 2A8) (56). In addition, in vivo studies demonstrated that the anti-guinea pig CCR3 mAb 2A8 was able to block eotaxin-induced eosinophil recruitment into the guinea pig skin. Furthermore, a rat mAb was generated against murine CCR3 (57). Although this antibody was nonneutralizing, it was very effective at depleting eosinophils in the blood, lung, and BAL fluid in a Nippostrongylus brasiliensis-infected model of eosinophil accumulation in mice. This is the first report describing the development of blocking mAbs specifically raised against a macaque chemokine receptor.
The anti-macaque CCR3 mAbs were generated by two different immunizing antigens, intact macaque CCR3-transfected cells, and a macaque CCR3 NH2-terminal synthetic peptide of 30 amino acids in length. The method utilizing heterologous stably transfected cell lines as immunogens has proven most successful at producing antagonist mAbs against chemokine receptors (27, 56, 58-65). We were able to produce two antagonist mAbs against macaque CCR3 (5B9 and 51), one from each of the two methods; however, the more potent mAb was the one employing intact macaque CCR3-transfected L1-2 cells as the immunogen (mAb 5B9). This mAb (5B9) was 2-fold more potent in blocking radiolabeled eotaxin binding and, in functional assays, 3-, 24-, and 6-fold more potent in eotaxin-induced calcium flux, shape change, and chemotaxis, respectively, than mAb 51 (see Fig. 7).
Antagonistic monoclonal antibodies raised against chemokine receptors have been invaluable tools for deciphering ligand binding domains on these receptors. These mAbs essentially fall into two main categories, those that bind to the NH2-terminal region and those that recognize the second extracellular loop, although some mAbs generated against human CCR5 have been mapped to multidomain conformational epitopes (63). Besides the anti-macaque CCR3 mAb 51 described in this report, neutralizing mAbs that have been shown to bind to the NH2 terminus of chemokine receptors include those that were raised against human CXCR1 and CXCR2 (58, 59, 61), human CXCR3 (66), human CCR4 (67), and human CCR5 (63). Neutralizing mAbs that recognize the second extracellular loop of chemokine receptors include human CCR2 (68), CCR5 (62, 63), and CXCR4 (69). Although beyond the scope of this report, it would be interesting to compare the neutralizing epitopes on CCR3 of the anti-macaque CCR3 mAb 5B9 as described here with that of the anti-human CCR3 mAb 7B11 (27), given the close homology between these two sequences and that both of these mAbs were generated with identical strategies using intact L1-2 transfectants as immunogens in mice.
Whereas we were able to generate a total of three mAbs that reacted with recombinant macaque CCR3 expressed on either L1-2 or AML14.3D10 cells, only two of these were able to recognize native CCR3 on primary rhesus macaque eosinophils by flow cytometry (mAbs 5B9 and 51; see Fig. 6). The inability of mAb 52 to recognize macaque CCR3 on the rhesus eosinophils has several plausible explanations. The epitope recognized by this mAb is masked by physical association with some other molecular entity on the cell surface (i.e. endogenous surface proteins, sugar moieties, etc.). Alternatively, this antigenic determinant on macaque CCR3 expressed on recombinant cell lines is in a conformation that is distinct from that on native rhesus eosinophils, resulting in the lack of reactivity with mAb 52. In support of this concept, the principle human immunodeficiency virus type 1 co-receptors CCR5 and CXCR4 have been shown to exist in multiple conformational states on the cell surface using a panel of specific neutralizing mAbs (63, 70, 71). Interestingly, mAb 52 was the only one of the three anti-macaque CCR3 mAbs that recognized recombinant human CCR3.
There have been numerous reports describing the functionality of the
-chemokine RANTES as a very potent activator of human eosinophils.
These biological effects include calcium mobilization, chemotaxis,
integrin up-regulation, degranulation, and transendothelial migration
in vitro (72-77). In addition, intradermal injection of
human RANTES into allergic human subjects was shown to cause a rich
eosinophil recruitment into the skin in vivo (78). In another human study, intranasal administration of RANTES into allergic
rhinitis patients resulted in an inflammatory infiltrate into the nasal
mucosa that was rich in eosinophils (79). Furthermore, we and others
have demonstrated that this -chemokine binds to CCR3 on human
eosinophils and stimulates signal transduction through CCR3 with high
potency (7, 8, 27). In contrast, human RANTES competed with low potency
against radiolabeled macaque eotaxin (Kd = 80 nM, Table I; Fig. 4) and was ineffective at stimulating a
calcium flux in macaque CCR3-transfected L1-2 cells (Table II). RANTES
was able only at very high concentrations to trigger a shape change in
rhesus macaque eosinophils and chemotaxis in L1-2/macaque CCR3 cells
(Table III; Fig. 5). Additional evidence for the lack of human RANTES
to activate macaque CCR3 was attained from studies in which human
RANTES was injected intradermally into cynomolgus macaques in
vivo. In these studies, a complete absence of eosinophil
recruitment was observed, whereas the other human CCR3-active
-chemokines eotaxin, MCP-4, and MCP-3 stimulated a robust eosinophil
accumulation into the skin of this species (80). Taken together, these
results would indicate that human RANTES is not an efficacious ligand
for macaque CCR3 due to species differences. It is unknown whether
native macaque RANTES is a functional agonist for macaque CCR3.
We have been able to assess functionality of native macaque CCR3
on rhesus eosinophils through a novel signaling assay known as GAFS
(44). This assay proved to be straightforward, efficient, and extremely
rapid. The GAFS assay is based on the fact that leukocytes undergo a
rapid shape change through actin polymerization upon activation by
chemokine receptor agonists, and this signal transduction pathway is
necessary for directional migration. This increase in shape change can
readily be measured by a shift in forward scatter using flow cytometry.
The GAFS assay was initially developed on partially purified human
leukocytes (PBMCs versus granulocytes). We were able to
adapt this assay to rhesus leukocytes, and the ability of the flow
cytometer to gate out eosinophils in rhesus whole blood based on their
high autofluorescence was extremely effective. Agonist properties using
GAFS of an assortment of -chemokines on rhesus eosinophils in whole
blood were very analogous to those in chemotaxis assays using the
transfected macaque CCR3 in the murine pre-B L1-2 cell line.
Furthermore, the GAFS assay readily determined antagonistic properties
of the anti-macaque CCR3 mAbs on eosinophils in rhesus whole blood, an assay condition not suitable for agonist-induced calcium flux and chemotaxis.
The most well established model of asthma in the non-human primate is the cynomolgus macaque (M. fascicularis) model (30, 31). These animals demonstrate a naturally occurring and reproducible airway sensitivity to Ascaris suum extract via inhalation. In the model, animals are exposed to single or multiple challenges of A. suum antigen, resulting in a prominent BAL eosinophilia. Indeed, eotaxin has been observed in the BAL rapidly within 6 h of after allergen challenge and remained high for 24 h in this cynomolgus macaque asthma model (17). Moreover, these non-human primates exhibit an early and late phase bronchoconstriction as well as a prominent airway hyperresponsiveness (AHR). The degree of AHR has been shown to be directly correlated with the level of eosinophil-derived major basic protein recovered from the BAL fluid, implying that eosinophil activation products may mediate the development and maintenance of AHR (30). Further support of eosinophil involvement in this primate asthma model has resulted from studies in which direct instillation of purified major basic protein into the airways caused the AHR (81) observed in these animals.
The use of the cynomolgus macaque model has proven crucial in the
preclinical evaluation of therapeutic agents for the treatment of
asthma in humans. Inhibition of airway eosinophilia and AHR in this
non-human primate model was observed with several classes of compounds
with disparate mechanisms of action. They include Rolipram, a specific
inhibitor of phosphodiesterase IV (32), the corticosteroid
dexamethasone (31, 33), the leukotriene D4 receptor
antagonist, ICI 198,615 (34), and mAbs directed against intercellular
adhesion molecule-1 (35) and interleukin-5 (TRFK-5) (36). Other
therapeutic agents that have shown efficacy in AHR include the long
acting 2-agonist, Salmeterol (33), and the leukotriene
B4 antagonist, CP-105,696 (37). Furthermore, agents that
have shown efficacy in late phase bronchoconstriction include the
platelet-activating factor receptor antagonist WEB 2170 (38), the
5-lipoxygenase inhibitor, BI-L-239 (39), and a monoclonal antibody
directed against endothelial leukocyte adhesion molecule-1 (40),
although it is unknown what effect these compounds have on eosinophil
recruitment into the airways. In a related non-human primate asthma
model, the leukotriene D4 receptor antagonist Montelukast
sodium (Singulair), also known as MK-0476 (82), inhibited early and
late phase bronchoconstriction in an A. suum challenge model
in squirrel monkeys; however, the effect on BAL eosinophils in this
model are unknown.
In summary, we have cloned and pharmacologically characterized the
recombinant macaque CCR3 expressed in the murine pre-B cell line L1-2.
We have also generated blocking monoclonal antibodies against macaque
CCR3 using two different immunogens in mice. The mAbs were very potent
in the inhibition of radiolabeled eotaxin binding to macaque CCR3, in
addition to being potent CCR3 receptor antagonists in a variety of
in vitro functional assays. These signaling assays include
eotaxin-induced calcium mobilization and chemotaxis on recombinant
macaque CCR3 and shape change on rhesus macaque eosinophils in whole
blood. It is hoped that the blocking anti-macaque CCR3 mAbs reported
here will be used in the macaque in proof-of-principle experiments to
validate the CCR3 hypothesis preclinically as a potential therapeutic
for asthma and other allergic diseases in humans.
| |
ACKNOWLEDGEMENTS |
|---|
We express our sincere gratitude to Drs. Trevor Hansel and Shannon Bryan for valuable discussions regarding the whole blood GAFS assay, to Paul Fisher and Dr. Dutch Boltz for valuable discussions regarding flow cytometry, to Alison Kulick for obtaining whole blood from the rhesus macaques, and to Charlotte Trainor for performing the leukocyte differentials on the rhesus macaque blood samples.
| |
FOOTNOTES |
|---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Atherosclerosis and Endocrinology, Merck Research Laboratories, Mail Code: RY80W-107, 126 E. Lincoln Ave., Rahway, NJ 07065. Tel.: 732-594-5681; Fax: 732-594-7926; E-mail: bruce_daugherty@merck.com.
Published, JBC Papers in Press, July 5, 2002, DOI 10.1074/jbc.M205488200
2 All animal protocols were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with applicable local and federal animal welfare regulations.
3 T. Hansel, personal communication.
4 B. L. Daugherty, unpublished data.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: BAL, bronchoalveolar lavage; CCR, C-C chemokine receptor; CXCR, C-X-C chemokine receptor; RANTES, regulated upon activation, normal T-cell expressed and secreted; MCP, monocyte chemotactic protein; GAFS, gated autofluorescence forward scatter; mAb, monoclonal antibody; AHR, airway hyperreactivity; PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
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)
(A) and human eotaxin (
), human eotaxin-2 (
), human
MCP-4 (
), and human RANTES (
) (B) were used to compete
against a fixed concentration of 125I-rhesus macaque
eotaxin (Rh-Eotaxin). All values are the averages of
triplicate determinations. Typically, 3000-4000 cpm of iodinated
ligand was bound in the absence of competitor, with signal/noise
ratios exceeding 10. Results are representative single experiments. A
Scatchard plot of the binding isotherm in A is shown
(inset).
), human eotaxin-3 (
), human RANTES (
).
