Characterization of Functional Chemokine Receptors (CCR1 and CCR2) on EoL-3 Cells: A Model System to Examine the Role of Chemokines in Cell Function

A growing family of proteins, known as the chemokines, play an important role in the recruitment and activation of inflammatory cells. The purpose of these studies was to characterize the chemokine receptors present on human sodium butyrate differentiated EoL-3 cells (dEoL-3 cells). Using a combination of 3′ rapid amplification of cDNA ends and nested polymerase chain reaction, we detected mRNA for CC chemokine receptor (CCR)1, CCR2, CCR3 and low level of CCR5. Radioligand binding studies demonstrated high-affinity saturable binding for both 125 I-macrophage inflammatory protein (MIP)-1α and 125 I-regulated upon activation normal T cell expressed and secreted (RANTES) with K d values of 1.4 and 7 nM, respectively. Competition binding with chemokines demonstrated exactly the same rank order of potency for displacement of both ligands: MIP-1α ∼ monocyte chemoattractant protein (MCP)-3 ∼ RANTES > MIP-1β >> MCP-1 >>> IL-8. RANTES, MCP-3 and MIP-1α all produced concentration-dependent transient increases in intracellular calcium concentrations in dEoL-3 cells. Desensitization studies indicated that RANTES, MIP-1α and MCP-3 interacted at the same receptor, which is identical in characterization to the cloned CCR1. 125 I-MCP-1 also demonstrated high-affinity satuable binding to dEoL-3 cells with a K d value of 0.4 nM. Competition studies showed that MCP-3 was slightly more potent than MCP-1 and MCP-2. MIP-1α, MIP-1β and RANTES were unable to displace 125 I-MCP-1. Addition of either MCP-1 or MCP-3 produced a concentration-dependent elevation of intracellular calcium with a maximun response 2-fold higher than that seen with RANTES or MIP-1α. Desensitization studies indicated that MCP-1 and MCP-3 function through CCR2 on these cells. Thus binding and functional studies indicate that dEoL-3 cells express functional CCR1 and CCR2 and that these cells may serve as an important system with which to study the regulation and role of these receptors.