CCR2 and CXCR4 regulate peripheral blood monocyte pharmacodynamics and link to efficacy in experimental autoimmune encephalomyelitis

<p>Abstract</p> <p>Background</p> <p>CCR2 plays a key role in regulating monocyte trafficking to sites of inflammation and therefore has been the focus of much interest as a target for inflammatory disease.</p> <p>Methods</p> <p>Here we examined the effects of CCR2 blockade with a potent small molecule antagonist to determine the pharmacodynamic consequences on the peripheral blood monocyte compartment in the context of acute and chronic inflammatory processes.</p> <p>Results</p> <p>We demonstrate that CCR2 antagonism <it>in vivo </it>led to a rapid decrease in the number of circulating Ly6C^himonocytes and that this decrease was largely due to the CXCR4-dependent sequestration of these cells in the bone marrow, providing pharmacological evidence for a mechanism by which monocyte dynamics are regulated <it>in vivo</it>. CCR2 antagonism led to an accumulation of circulating CCL2 and CCL7 levels in the blood, indicating a role for CCR2 in regulating the levels of its ligands under homeostatic conditions. Finally, we show that the pharmacodynamic changes due to CCR2 antagonism were apparent after chronic dosing in mouse experimental autoimmune encephalomyelitis, a model in which CCR2 blockade demonstrated a dramatic reduction in disease severity, manifest in a reduced accumulation of monocytes and other cells in the CNS.</p> <p>Conclusion</p> <p>CCR2 antagonism <it>in vivo </it>has tractable pharmacodynamic effects that can be used to align target engagement with biologic effects on disease activity.</p

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