Cloning and characterization of the guinea pig eosinophil eotaxin receptor, C-C chemokine receptor-3: blockade using a monoclonal antibody in vivo.
Certain C-C chemokines, signaling via the eotaxin receptor C-C chemokine receptor-3 (CCR3), are thought to be central mediators of eosinophil accumulation in allergic inflammation. To investigate the role of CCR3 in vivo, we cloned the guinea pig eotaxin receptor (guinea pig CCR3) from a genomic DNA library. We isolated a single-exon open reading frame coding for a 358-amino acid chemokine receptor protein with 67 and 69% homology to human and murine CCR3, respectively. When expressed in stable transfectants, this receptor bound 125I-labeled guinea pig eotaxin, 125I-labeled human monocyte chemotactic protein-3, and 125I-labeled human RANTES. In chemotaxis assays, guinea pig CCR3 transfectants responded only to guinea pig eotaxin, with a maximal effect at 100 nM. mAbs were raised that bound selectively to both guinea pig CCR3 transfectants and guinea pig eosinophils. One of these mAbs, 2A8, blocked both ligand binding to transfectants and their chemotaxis in response to eotaxin. The Ab also inhibited chemotaxis and the elevation of cytosolic calcium in guinea pig eosinophils in response to eotaxin. F(ab')2 fragments of 2A8 were prepared that retained the ability to inhibit eosinophil calcium responses to eotaxin. Pretreatment of (111)In-labeled eosinophils in vitro with F(ab')2 2A8 selectively inhibited their accumulation in response to eotaxin in vivo. These data demonstrate that functional blockade of eosinophil chemokine receptors can be achieved in vivo and provide further support for the development of novel anti-inflammatory drugs targeting eosinophil recruitment through chemokine receptor antagonism.
Sabroe
I
I
Biomedical Sciences Division, Imperial College School of Medicine, London, United Kingdom.
Conroy
D M
DM
Gerard
N P
NP
Li
Y
Y
Collins
P D
PD
Post
T W
TW
Jose
P J
PJ
Williams
T J
TJ
Gerard
C J
CJ
Ponath
P D
PD
D000595:Amino Acid Sequence; D000818:Animals; D019138:Antibodies, Blocking / Q000008:administration & dosage* / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000008:administration & dosage*; D054413:Chemokine CCL11; D019742:Chemokines, CC; D002631:Chemotactic Factors, Eosinophil / Q000378:metabolism; D002634:Chemotaxis, Leukocyte / Q000276:immunology; D003001:Cloning, Molecular; D016207:Cytokines / Q000378:metabolism*; D004804:Eosinophils / Q000378:metabolism* / Q000637:transplantation; D006168:Guinea Pigs; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000008:administration & dosage / Q000494:pharmacology; D007205:Indium Radioisotopes / Q000378:metabolism; D007275:Injections, Intravenous; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000276:immunology; D054397:Receptors, CCR3; D019707:Receptors, Chemokine / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D015398:Signal Transduction / Q000276:immunology; D014162:Transfection
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; C515456:CCL11 protein, human; C515350:CCR3 protein, human; C515457:Ccl11 protein, mouse; C515351:Ccr3 protein, mouse; D054413:Chemokine CCL11; D019742:Chemokines, CC; D002631:Chemotactic Factors, Eosinophil; D016207:Cytokines; D007140:Immunoglobulin Fab Fragments; D007205:Indium Radioisotopes; D008024:Ligands; D054397:Receptors, CCR3; D019707:Receptors, Chemokine