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More MS news articles for October 2002

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

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12381680&dopt=Abstract

Br J Pharmacol 2002 Nov 5;137(5):663-675
Chou CC, Fine JS, Pugliese-Sivo C, Gonsiorek W, Davies L, Deno G, Petro M, Schwarz M, Zavodny PJ, Hipkin RW.
Department of Immunology, Schering-Plough Research Institute, Kenilworth, New Jersey, NJ 07033, U.S.A.

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 [(35)S]-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 (125)I-MIP-1alpha with a wide range of potencies and, with the exception of MCP-2, acted as full agonists in stimulating [(35)S]-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 [(35)S]-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 [(35)S]-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>/=MIP-1beta) although the ligands differed in their efficacy as agonists.

MPIF-1 was the more efficacious (MPIF-1>RANTES=MIP-1alpha>>MIP-1beta).

(125)I-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 (125)I-MIP-1beta were essentially identical in the two membrane systems.

5. Lastly, MIP-1beta antagonized [(35)S]-GTPgammaS exchange, Ca(2+) 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.