(126). It is also expressed on basophils, Th2 lymphocytes, and mast cells (127-129). Based on its presence in a large array of cell types that are crucial for the induction and maintenance of inflammation in asthma and other allergic diseases, there is a significant effort to discover CCR3 receptor antagonists. In 1993, eotaxin (CCL11) was identified as an eosinophil-specific CC chemokine (130, 131). Subsequently, CCR3 was found to be activated by several other CC chemo-kines including MCP-3 (CCL7), MCP-4 (CCL13), RANTES (CCL5), eotaxin 2 (CCL24), and eotaxm 3 (CCL26) ^126, 132t 133).
In human asthma, increased mRNA and protein expression of eotaxin have been demonstrated, as well as CCR3 expression on local infiltrating eosinophils, as determined by biopsies (134). Results obtained from mice genetically deficient for the eotaxin gene (135), as well as studies using eotaxin-blocking antibodies (117), have demonstrated a significant effect on the course of a lung allergic reaction. By contrast, another report describing the response cf eotaxin-deficient mice, in a similar model of allergic lung inflammation, showed no significant difference from that of wild-type
(136).The reason for this discrepancy is not understood. CCR3~/_ mice have shown that this receptor plays an important role in eosinophil migration into the lung and skin. In intraperitoneally (i.p.) sensitized CCR3_/_ mice, eosinophil recruitment to bronchoalveo-lar lavage fluid and lung is decreased compared to that of wild-type after aerosol challenge. However, airway hyperreactivity (AHR) is enhanced under the same conditions
(137). If the mice are sensitized epicutane-ously with the same antigen, a diminished response and eosinophil recruitment is observed (138). In the i.p. sensitized mice | there is a significant mobilization of mast cells to the airway epithelium, but not into the skin. These results suggest that the mecha-| nism of induction of AHR might be different, depending on the route of sensitization.
6.3.1 CCR3 Receptor Structure. Chimera studies of CCR3 and CCR1 receptors show that the N-terminal segments of CCR3 appear to be important for eotaxin binding (77). How-
ver, eotaxin remained an effective agonist at this chimeric receptor in either calcium flux or chemotaxis assays. These data are consistent with a multisite model for chemokine-chemo-ne receptor interaction.
6.3.2 CCR3 Antibodies. A specific CCR3 monoclonal antibody, 7B11, blocks binding of eotaxin, MCP-3, and RANTES to the CCR3 receptor, and also blocks chemokine-induced emotaxis and calcium flux in human eosin-ophils (126). Because the CCR3 receptor is resent on basophils, it is not surprising that 7B11 blocks chemotaxis of these cells in response to CCR3 ligands (127). In these same studies, 7Bll was shown to inhibit histamine and leukotriene release mediated by eotaxin. This mAb has also been shown to inhibit the release of reactive oxygen species (ROS) after stimulation with eotaxin-1 or eotaxin-2 (139), and blocks the chemotactic response of eosin-ophils on HUVEC cells stimulated with TNFa and IFNy (140). Because CCR3 has been implicated in having a role in HIV-1 pathology, 7Bll has also been used to define the involvement of CCR3 in contributing to HIV-1-spe-cific pathology (141).
CCR3 monoclonal antibodies have also been evaluated in vivo. A rat mAb specific for mouse CCR3 receptor depletes blood eosinophil levels in Nippostrongyos brasiliensis-m-fected mice, and reduced eosinophil levels in lung tissue and bronchoalveolar lavage fluid (BAL) fluid after repeated treatment (142). Surprisingly, this antibody had no effect on CCR3-regulated Ca2+ flux. In another report, a guinea pig-specific CCR3 mAb, 2A8, was also evaluated both in vitro and in vivo (143).2A8 blocks the binding of guinea pig eotaxin to guinea pig eosinophils and guinea pig CCR3 transfectants, and shows functional antagonism of guinea pig eosinophils, as measured by Ca2+ flux and chemotaxis. In animals pre-treated with inIn-labeled eosinophils, 2AB inhibited accumulation of these eosinophils in response to eotaxin.
6.3.3 CCR3 Peptide Antagonists. As demonstrated for other chemokines, modification of the N-terminal region has been employed as a successful strategy to identify receptor antagonists. Truncation of MCP-3 (MCP3110-761) provides a ligand that retains potent receptor binding, yet is unable to induce chemotaxis, enzyme release, or Ca2+ flux (144). Dipeptidyl peptidase is able to truncate RANTES[1_68] to RANTES[3-68] and eotaxinL1-74] to eotaxm[3"74], and both have demonstrated functional antagonism in vitro (145-147).
RANTES, which can signal through CCR1, CCR3, or CCR5, has been modified to MetRANTES, as described earlier. Met-RANTES acts as a functional antagonist and blocks human eosinophil chemotaxis, Ca2+ flux, actin polymerization, and release of ROS after stimulation with RANTES, MCP-3, and eotaxin (148). Met-RANTES apparently antagonizes the response of eosinophils through CCRl at low concentrations and through CCR3 at higher concentrations.
Met-chemokine J37 (Ckj87) is an N-termi-nal-modified form of macrophage inflammatory protein (MIP4), wherein an alanine is replaced with methionine at the extreme N-terminal residue (149). MIP4 [also known as pulmonary and activation-regulated chemokine (PARC) (150); alternative macrophage activation-associated CC chemokine (AMAC) (151); or dendritic cell-derived chemokine (DCCK) (152,153)] itself shows some degree of CCR3 antagonistic activity. However, Met-Ckj37 is significantly more potent as a CCR3 antagonist than MIP4, Met-RANTES, or AOP-RANTES, and blocks eotaxin or MCP4-induced eosinophil chemotaxis at concentrations as low as 1 nAT. Direct binding of Ckj37 to CCR3 is evidenced by its ability to displace radioiodinated eotaxin or MCP4 from CCR3.
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