Functional Expression and Characterization of Macaque C-C Chemokine Receptor 3 (CCR3) and Generation of Potent Antagonistic Anti-macaque CCR3 Monoclonal Antibodies

Eosinophils are major effector cells implicated in a number of chronic inflammatory diseases in humans, particularly bronchial asthma and allergic rhinitis. The β-chemokine receptor C-C chemokine receptor 3 (CCR3) provides a mechanism for the selective recruitment of eosinophils into tissue and thus...

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Bibliographic Details
Published in:The Journal of biological chemistry 2002-09, Vol.277 (37), p.33799-33810
Main Authors: Zhang, Liwen, Soares, Marco P., Guan, Yanfen, Matheravidathu, Stephen, Wnek, Richard, Johnson, Kristine E., Meisher, Anna, Iliff, Susan A., Mudgett, John S., Springer, Martin S., Daugherty, Bruce L.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Antibodies, Monoclonal - biosynthesis
Antibodies, Monoclonal - therapeutic use
Binding, Competitive
Calcium - metabolism
Cell Line
Chemokine CCL11
Chemokine CCL5 - physiology
Chemokines, CC - genetics
Chemokines, CC - metabolism
Chemotaxis
Cloning, Molecular
Eosinophils - physiology
Humans
Macaca mulatta
Mice
Mice, Inbred BALB C
Molecular Sequence Data
Receptors, CCR3
Receptors, Chemokine - antagonists & inhibitors
Receptors, Chemokine - chemistry
Receptors, Chemokine - genetics
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Summary:Eosinophils are major effector cells implicated in a number of chronic inflammatory diseases in humans, particularly bronchial asthma and allergic rhinitis. The β-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 β-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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M205488200