The CC family of chemokine receptors couple to the Gi/o family of G-proteins, leading to the regulation of a number of intracellular signalling molecules, such as cAMP and Ca²⁺ (Arai and Charo, 1996). However, the chemokines to which this family of receptors bind show surprising promiscuity. At least six known chemokines have been shown to bind the CC chemokine receptor, CCR5 (Blanpain et al., 1999), but each chemokine shows a unique pharmacological profile.

The present study therefore set out to investigate the effects of several CC chemokines (MIP-1, MIP-1ß, RANTES, MCP-2 and MCP-4) for their ability to inhibit forskolin stimulated cAMP production in a stable CHO cell line expressing human CCR5 (CHO.CCR5) (Mueller et al., 2002). The [FP²] cAMP kit (Perkin Elmer) was used to evaluate the inhibition of cAMP production in whole cells. Briefly, 2x10⁴ CHO.CCR5 cells were stimulated with forskolin (10µM) and a range of chemokine concentrations for 30 minutes in the presence of anti-cAMP antibody, before cells were lysed and exposed to fluorescein-labelled cAMP (fluo-cAMP). Following 40 minutes incubation at room temperature, fluorescence polarisation was measured using a Tecan Ultra 384 plate reader. Data for each chemokine were converted to the percentage of the maximum inhibition induced by MIP-1, measured in a parallel assay.

MIP-1, MIP-1ß, RANTES and MCP-2 all inhibited forskolinstimulated cAMP production in CHO.CCR5 cells (Table 1). MIP-1 and RANTES displayed the highest potency in this assay (IC₅₀ 4.7 nM), whilst MIP-1ß and MCP-2 showed at least a ten fold lower potency (IC₅₀ 36.1 and 123.4 nM, respectively). Although MCP-4 was unable to elicit a response, MIP-1 and MIP-1ß were full agonists, whereas RANTES and MCP-2 were partial agonists in the cAMP assay. The rank order of potency seen in the cAMP assay is similar to that seen for chemokine stimulation of [³⁵S]GTPS binding at CCR5 (Mueller et. al, 2002), although most of the chemokines were less potent at inhibiting cAMP production. This reduction in potency may reflect a number of processes, such as desensitisation of CCR5, which could only occur in whole cells.

	pIC50 (IC50[nM])	Emax (% MIP-1response)
MIP-1	8.33 ± 0.1 (4.7)	100
MIP-1ß	7.44 ± 0.1 (36.1)	95 ± 11
RANTES	8.33 ± 0.1 (4.7)	70 ± 9*
MCP-2	6.91 ± 0.1 (123.4)	81 ± 4*

Table 1 Mean±s.e. mean pIC₅₀ and E_max values for inhibition of cAMP production from at least three separate experiments (*p<0.05 unpaired Student's t-test relative to MIP-1)

A. Mueller et al. (2002). Br J Pharmacol, 135, 1033-43.
C. Blainpain et al. (1999). Blood, 96, 1638-45H.
Arai and I.F. Charo, (1996). J Biol Chem, 271, 21814-9.

We thank Novartis and the BBSRC for sponsorship of the project.