Pharmacological characterization of the chemokine receptor, hCCR1 in a stable transfectant and differentiated HL-60 cells:: antagonism of hCCR1 activation by MIP-1β

1 C-C chemokine receptor-1 (CCR1) has been implicated in mediating a variety of inflammatory conditions including multiple sclerosis and organ rejection. Although originally referred to as the MIP-1alpha/RANTES receptor, CCR1 is quite promiscuous and can be activated by numerous chemokines. 2 We used radioligand binding and [S-35]-GTPgammaS exchange assays in membranes from a cell line transfected to express CCR1 (Ba/F3-hCCR1) to characterize a panel of chemokines (HCC-1, MIP-1alpha, MIP-1beta, MIP-1delta, MPIF-1, MCP-2, MCP-3, and RANTES) as CCR1 ligands. In this recombinant model, these chemokines displaced I-125-MIP-1alpha with a wide range of potencies and, with the exception of MCP-2, acted as full agonists in stimulating [S-35]-GTPgammaS exchange. 3 We then assessed the utility of HL-60 cells cultured with known differentiating agents (PMA, DMSO, dibutyryl-cAMP or retinoic acid) for investigating CCR1 pharmacology. In [S-35]-GTPgammaS exchange assays, membranes from cells cultured with retinoic acid (4-6 days) were the most responsive to activation by MIP-1alpha and MPIF-1. FACS analysis and comparative pharmacology confirmed that these activities were mediated by CCR1. 4 Using [S-35]-GTPgammaS exchange assays, intracellular calcium flux and/or whole cell chemotaxis assays in HL-60(Rx) cells, we validated that MIP-1alpha was the most potent CCR1 ligand (MIP-1alpha MPIF-1 > RANTES greater than or equal to MIP-1beta) although the ligands differed in their efficacy as agonists. MPIF-1 was the more efficacious (MPIF-1 > RANTES = MIP-1alpha >> MIP-1beta). I-125-MIP-1beta binding in Ba/F3-hCCR1 and HL-60(Rx) membranes was competitively displaced by MIP-1alpha, MPIF-l and MIP-1beta. The binding K-i for these chemokines with I-125-MIP-1beta were essentially identical in the two membrane systems. 5 Lastly, MIP-1beta antagonized [S-35]-GTPgammaS exchange, Ca2+ flux and chemotaxis in HL-60(Rx) cells in response to robust agonists such as MIP-1alpha, RANTES and MPIF-1. Based on our results, we propose that MIP-1beta could function as an endogenous inhibitor of CCR1 function.