Sarau H M, Rush J A, Foley J J, Brawner M E, Schmidt D B, White J R, Barnette M S
Department of Pulmonary Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406-0939, USA.
J Pharmacol Exp Ther. 1997 Oct;283(1):411-8.
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)-1alpha 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-1alpha approximately monocyte chemoattractant protein (MCP)-3 approximately RANTES > MIP-1beta >> MCP-1 >>> IL-8. RANTES, MCP-3 and MIP-1alpha all produced concentration-dependent transient increases in intracellular calcium concentrations in dEoL-3 cells. Desensitization studies indicated that RANTES, MIP-1alpha 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-1alpha, MIP-1beta 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-1alpha. 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.
一类不断增多的蛋白质，即趋化因子，在炎症细胞的募集和激活过程中发挥着重要作用。这些研究的目的是鉴定人丁酸钠分化的EoL-3细胞（dEoL-3细胞）上存在的趋化因子受体。通过结合3' cDNA末端快速扩增和巢式聚合酶链反应，我们检测到了CC趋化因子受体（CCR）1、CCR2、CCR3的mRNA以及低水平的CCR5。放射性配体结合研究表明，125I-巨噬细胞炎性蛋白（MIP）-1α和125I-活化后正常T细胞表达和分泌调节因子（RANTES）均有高亲和力的饱和结合，其解离常数（Kd）值分别为1.4 nM和7 nM。与趋化因子的竞争结合显示，两种配体置换的效力顺序完全相同：MIP-1α≈单核细胞趋化蛋白（MCP）-3≈RANTES＞MIP-1β＞＞MCP-1＞＞＞白细胞介素（IL）-8。RANTES、MCP-3和MIP-1α均可使dEoL-3细胞内钙浓度产生浓度依赖性的瞬时升高。脱敏研究表明，RANTES、MIP-1α和MCP-3作用于同一受体，其特性与克隆的CCR1相同。125I-MCP-1与dEoL-3细胞也表现出高亲和力的饱和结合，Kd值为0.4 nM。竞争研究显示，MCP-3的效力略强于MCP-1和MCP-2。MIP-1α、MIP-1β和RANTES无法置换125I-MCP-1。添加MCP-1或MCP-3均可使细胞内钙浓度产生浓度依赖性升高，最大反应比RANTES或MIP-1α高2倍。脱敏研究表明，MCP-1和MCP-3在这些细胞上通过CCR2发挥作用。因此，结合和功能研究表明，dEoL-3细胞表达功能性CCR1和CCR2，这些细胞可能是研究这些受体的调节和作用的重要系统。
