CDB15:0000185 CCL11 — CCR3

ABSTRACT: The specificity of leukocyte trafficking in inflammation is controlled by the interactions of chemokines with chemokine receptors. Reliable structure-function studies of chemokine-receptor interactions would benefit from cell lines that express consistent high levels of chemokine receptors. We describe herein two new Chinese hamster ovary (CHO) cell lines in which the genes for chemokine receptors CCR2 and CCR3 have been incorporated into identical positions in the host genome. CCR2 is the primary receptor for the chemokine monocyte chemoattractant protein-1 (MCP-1) whereas CCR3 is the primary receptor for the chemokines eotaxin-1, eotaxin-2 and eotaxin-3. Both receptors are expressed at >5,000,000 copies per cell, substantially higher levels than in previous cell lines, and both are competent for binding and activation by the cognate chemokines for these receptors. Using these cell lines we confirm that eotaxin-1 and eotaxin-3 can act as an agonist and an antagonist, respectively, of CCR2. In addition, we show that eotaxin-2 is an antagonist of CCR2 and MCP-1 is an agonist of CCR3. Comparison of the chemokine sequences reveals several positions that are identical in MCP-1 and eotaxin-1 but different in eotaxin-2 and eotaxin-3, suggesting that these amino acids play a role in CCR2 activation.

ABSTRACT: CC chemokine ligand 11 (CCL11/eotaxin) and other CC chemokine receptor 3 (CCR3) ligands (CCL24/eotaxin-2, CCL26/eotaxin-3, CCL13/monocyte chemotactic protein-4, etc.) play important roles in the chemotaxis and activation of eosinophils and other CCR3-expressing cells (basophils, mast cells, and CD4(+) T helper 2 cells) in allergic inflammation incidents, including asthma and rhinitis. A newly synthesized compound, N-{(3R)-1-[(6-fluoro-2-naphthyl)methyl]pyrrolidin-3-yl}-2-{1-[(5-hydroxy-3-methylpyridin-2-yl)carbonyl]piperidin-4-ylidene}-acetamide hemifumarate (YM-355179), inhibited the binding of CCL11 and CCL5/regulated on activation normal T cell expressed and secreted to CCR3-expressing B300-19 cells with IC(50) values of 7.6 and 24 nM, respectively. In contrast, YM-355179 did not affect the binding of CCL5 to CCR1 or CCR5. In functional assays, YM-355179 inhibited CCL11-induced, intracellular Ca(2+) influx, chemotaxis, and eosinophil degranulation with IC(50) values of 8.0, 24, and 29 nM, respectively. YM-355179 did not, however, affect any CC chemokine receptor (CCR1, CCR2, CCR4, or CCR5)-mediated Ca(2+) influx signals. Furthermore, oral administration of YM-355179 (1 mg/kg) inhibited CCL11-induced shape change of whole blood eosinophils in cynomolgus monkeys. Intravenous injection of YM-355179 (1 mg/kg) also inhibited eosinophil infiltration into airways of cynomolgus monkeys after segmental bronchoprovocation with CCL11. These results indicate that YM-355179 is a novel, selective, and orally available CCR3 antagonist with therapeutic potential for treating eosinophil-related allergic inflammatory diseases.

ABSTRACT: The CC chemokine eotaxin, identified in guinea pigs and also recently in mice, may be a key element for the selective recruitment of eosinophils to certain inflamed tissues. Using a partial mouse eotaxin CDNA probe, the human eotaxin gene was cloned and found to be 61.8 and 63.2% identical at the amino acid level to guinea pig and mouse eotaxin. Human eotaxin protein was a strong and specific eosinophil chemoattractant in vitro and was an effective eosinophil chemoattractant when injected into the skin of a rhesus monkey. Radiolabeled eotaxin was used to identify a high affinity receptor on eosinophils (0.52 nM Kd), expressed at 4.8 x 10(4) sites per cell. This receptor also bound RANTES and monocyte chemotactic protein-3 with lower affinity, but not macrophage inflammatory protein-1 alpha. Eotaxin could desensitize calcium responses of eosinophils to RANTES and monocyte chemotactic protein-3, although RANTES was able to only partially desensitize eosinophil calcium responses to eotaxin. Immunohistochemistry on human nasal polyp with antieotaxin mAbs showed that certain leukocytes as well as respiratory epithelium were intensely immunoreactive, and eosinophil infiltration occurred at sites of eotaxin upregulation. Thus eotaxin in humans is a potent and selective eosinophil chemoattractant that is expressed by a variety cell types in certain inflammatory conditions.

ABSTRACT: Although there is a mounting body of evidence that eosinophils are recruited to sites of allergic inflammation by a number of beta-chemokines, particularly eotaxin and RANTES, the receptor that mediates these actions has not been identified. We have now cloned a G protein-coupled receptor, CC CKR3, from human eosinophils which, when stably expressed in AML14.3D10 cells bound eotaxin, MCP-3 and RANTES with Kds of 0.1, 2.7 and 3.1 nM, respectively. CC CKR3 also bound MCP-1 with lower affinity, but did not bind MIP-1 alpha or MIP-1 beta. Eotaxin, RANTES, and to a lessor extent MCP-3, but not the other chemokines, activated CC CKR3 as determined by their ability to stimulate a Ca(2+) -flux. Competition binding studies on primary eosinophils gave binding affinities for the different chemokines which were indistinguishable from those measured with CC CKR3. Since CC CKR3 is prominently expressed in eosinophils we conclude that CC CKR3 is the eosinophil eotaxin receptor. Eosinophils also express a much lower level of a second chemokine receptor, CC CKR1, which appears to be responsible for the effects of MIP-1 alpha.

ABSTRACT: The chemokine eotaxin is unusual in that it appears to be a highly specific chemoattractant for eosinophils. Ligand-binding studies with radiolabeled eotaxin demonstrated a receptor on eosinophils distinct from the known chemokine receptors CKR-1 and -2. The distinct eotaxin binding site on human eosinophils also bound RANTES (regulated on activation T expressed and secreted) and monocyte chemotactic protein (MCP)3. We have now isolated a cDNA from eosinophils, termed CKR-3, with significant sequence similarity to other well characterized chemokine receptors. Cells transfected with CKR-3 cDNA bound radiolabeled eotaxin specifically and with high affinity, comparable to the binding affinity observed with eosinophils. This receptor also bound RANTES and MCP-3 with high affinity, but not other CC or CXC chemokines. Furthermore, receptor transfectants generated in a murine B cell lymphoma cell line migrated in transwell chemotaxis assays to eotaxin, RANTES, and MCP-3, but not to any other chemokines. A monoclonal antibody recognizing CKR-3 was used to show that eosinophils, but not other leukocyte types, expressed this receptor. This pattern of expression was confirmed by Northern blot with RNA from highly purified leukocyte subsets. The restricted expression of CKR-3 on eosinophils and the fidelity of eotaxin binding to CKR-3, provides a potential mechanism for the selective recruitment and migration of eosinophils within tissues.

ABSTRACT: Eosinophil leukocytes express high numbers of the chemokine receptor CCR3 which binds eotaxin, monocyte chemotactic protein (MCP)-4, and some other CC chemokines. In this paper we show that CCR3 is also highly expressed on human blood basophils, as indicated by Northern blotting and flow cytometry, and mediates mainly chemotaxis. Eotaxin and MCP-4 elicited basophil migration in vitro with similar efficacy as regulated upon activation normal T cells expressed and secreted (RANTES) and MCP-3. They also induced the release of histamine and leukotrienes in IL-3-primed basophils, but their efficacy was lower than that of MCP-1 and MCP-3, which were the most potent stimuli of exocytosis. Pretreatment of the basophils with a CCR3-blocking antibody abrogated the migration induced by eotaxin, RANTES, and by low to optimal concentrations of MCP-4, but decreased only minimally the response to MCP-3. The CCR3-blocking antibody also affected exocytosis: it abrogated histamine and leukotriene release induced by eotaxin, and partially inhibited the response to RANTES and MCP-4. In contrast, the antibody did not affect the responses induced by MCP-1, MCP-3, and macrophage inflammatory protein-1alpha, which may depend on CCR1 and CCR2, two additional receptors detected by Northern blotting with basophil RNA. This study demonstrates that CCR3 is the major receptor for eotaxin, RANTES, and MCP-4 in human basophils, and suggests that basophils and eosinophils, which are the characteristic effector cells of allergic inflammation, depend largely on CCR3 for migration towards different chemokines into inflamed tissues.

ABSTRACT: The solution structure of the CCR3-specific chemokine, eotaxin, has been determined by NMR spectroscopy. The quaternary structure of eotaxin was investigated by ultracentrifugation and NMR, and it was found to be in equilibrium between monomer and dimer under a wide range of conditions. At pH