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Vol. 283, Issue 1, 411-418, 1997
Departments of
Pulmonary Pharmacology, Gene Expression Sciences
(M.E.B.) and
Molecular Immunology (J.R.W.), SmithKline Beecham
Pharmaceuticals
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 125I-macrophage inflammatory protein
(MIP)-1
and 125I-regulated upon activation normal T cell
expressed and secreted (RANTES) with Kd 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-1 ~ monocyte chemoattractant protein
(MCP)-3 ~ RANTES > MIP-1
>> MCP-1
>>> IL-8. RANTES, MCP-3 and MIP-1 all produced
concentration-dependent transient increases in intracellular calcium
concentrations in dEoL-3 cells. Desensitization studies indicated that
RANTES, MIP-1 and MCP-3 interacted at the same receptor, which is
identical in characterization to the cloned CCR1.
125I-MCP-1 also demonstrated high-affinity satuable binding
to dEoL-3 cells with a Kd value of 0.4 nM.
Competition studies showed that MCP-3 was slightly more potent than
MCP-1 and MCP-2. MIP-1, MIP-1 and RANTES were unable to displace
125I-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-1.
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.
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