Pharmacological characterization of the chemokine receptor, hCCR1 in a stable transfectant and differentiated HL-60 cells: antagonism of hCCR1 activation by MIP-1beta.
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. We used radioligand binding and [35S]-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 125I-MIP-1alpha with a wide range of potencies and, with the exception of MCP-2, acted as full agonists in stimulating [35S]-GTPgammaS exchange. 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 [35S]-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. Using [35S]-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>or=MIP-1beta) although the ligands differed in their efficacy as agonists. MPIF-1 was the more efficacious (MPIF-1>RANTES=MIP-1alpha>>MIP-1beta). 125I-MIP-1beta binding in Ba/F3-hCCR1 and HL-60(Rx) membranes was competitively displaced by MIP-1alpha, MPIF-1 and MIP-1beta. The binding K(i) for these chemokines with 125I-MIP-1beta were essentially identical in the two membrane systems. Lastly, MIP-1beta antagonized [35S]-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.
Chou
Chuan-Chu
CC
Department of Immunology, Schering-Plough Research Institute, Kenilworth, New Jersey, NJ 07033, USA.
Fine
Jay S
JS
Pugliese-Sivo
Catherine
C
Gonsiorek
Waldemar
W
Davies
Liza
L
Deno
Gregory
G
Petro
Mary
M
Schwarz
Martin
M
Zavodny
Paul J
PJ
Hipkin
R William
RW
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D018925:Chemokines / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D018922:HL-60 Cells / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D019402:Macrophage Inflammatory Proteins / Q000494:pharmacology*; D051379:Mice; D054389:Receptors, CCR1; D019707:Receptors, Chemokine / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods
C515300:CCR1 protein, human; C515301:Ccr1 protein, mouse; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D018925:Chemokines; D019402:Macrophage Inflammatory Proteins; D054389:Receptors, CCR1; D019707:Receptors, Chemokine