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-1α/RANTES receptor, CCR1 is quite promiscuous and can be activated by numerous chemokines. 2. We used radioligand binding and [(35)S]-GTPγS exchange assays in membranes from a cell line transfected to express CCR1 (Ba/F3-hCCR1) to characterize a panel of chemokines (HCC-1, MIP-1α, MIP-1β, MIP-1δ, MPIF-1, MCP-2, MCP-3, and RANTES) as CCR1 ligands. In this recombinant model, these chemokines displaced (125)I-MIP-1α with a wide range of potencies and, with the exception of MCP-2, acted as full agonists in stimulating [(35)S]-GTPγS 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 [(35)S]-GTPγS exchange assays, membranes from cells cultured with retinoic acid (4–6 days) were the most responsive to activation by MIP-1α and MPIF-1. FACS analysis and comparative pharmacology confirmed that these activities were mediated by CCR1. 4. Using [(35)S]-GTPγS exchange assays, intracellular calcium flux and/or whole cell chemotaxis assays in HL-60(Rx) cells, we validated that MIP-1α was the most potent CCR1 ligand (MIP-1α>MPIF-1>RANTES⩾MIP-1β) although the ligands differed in their efficacy as agonists. MPIF-1 was the more efficacious (MPIF-1>RANTES=MIP-1α>>MIP-1β). (125)I-MIP-1β binding in Ba/F3-hCCR1 and HL-60(Rx) membranes was competitively displaced by MIP-1α, MPIF-1 and MIP-1β. The binding K(i) for these chemokines with (125)I-MIP-1β were essentially identical in the two membrane systems. 5. Lastly, MIP-1β antagonized [(35)S]-GTPγS exchange, Ca(2+) flux and chemotaxis in HL-60(Rx) cells in response to robust agonists such as MIP-1α, RANTES and MPIF-1. Based on our results, we propose that MIP-1β could function as an endogenous inhibitor of CCR1 function