The T Cell-directed CC Chemokine TARC Is a Highly Specific Biological Ligand for CC Chemokine Receptor 4

Toshio Imai,Masataka Baba,Miyuki Nishimura,Mayumi Kakizaki,Shin Takagi,Osamu Yoshie

doi:10.1074/jbc.272.23.15036

Abstract

Thymus and activation-regulated chemokine (TARC) is a recently identified CC chemokine that is expressed constitutively in thymus and transiently in stimulated peripheral blood mononuclear cells. TARC functions as a selective chemoattractant for T cells that express a class of receptors binding TARC with high affinity and specificity. To identify the receptor for TARC, we produced TARC as a fusion protein with secreted alkaline phosphatase (SEAP) and used it for specific binding. By stably transfecting five orphan receptors and five known CC chemokine receptors (CCR1 to -5) into K562 cells, we found that TARC-SEAP bound selectively to cells expressing CCR4. TARC-SEAP also bound to K562 cells stably expressing CCR4 with a high affinity (Kd = 0.5 nM). Only TARC and not five other CC chemokines (MCP-1 (monocyte chemoattractant protein-1), RANTES (regulated upon activation, normal T cells expressed and secreted), MIP-1alpha (macrophage inflammatory protein-1alpha), MIP-1beta, and LARC (liver and activation-regulated chemokine)) competed with TARC-SEAP for binding to CCR4. TARC but not RANTES or MIP-1alpha induced migration and calcium mobilization in 293/EBNA-1 cells stably expressing CCR4. K562 cells stably expressing CCR4 also responded to TARC in a calcium mobilization assay. Northern blot analysis revealed that CCR4 mRNA was expressed strongly in human T cell lines and peripheral blood T cells but not in B cells, natural killer cells, monocytes, or granulocytes. Taken together, TARC is a specific functional ligand for CCR4, and CCR4 is the specific receptor for TARC selectively expressed on T cells.

Highlights

Chemokines are small secreted polypeptides that play important roles in a wide range of inflammatory and immunological processes by recruiting selected subsets of leukocytes [1, 2]
CCR1 is a receptor for MIP1␣, RANTES, and MCP-3 [32,33,34,35]; CCR2 is a receptor for MCP-1 and MCP-3 [35, 36]; CCR3 is a receptor for eotaxin, RANTES, and MCP-3 [14, 37, 38]; CCR4 is a receptor for MIP-1␣, RANTES, and MCP-1 [39]; and CCR5 is a receptor for MIP-1␣, MIP-1␤, and RANTES (40 – 42)
RANTES and LARC, partially (Ͻ30%) inhibited Thymus and activation-regulated chemokine (TARC)-secreted alkaline phosphatase (SEAP)(His)6 binding. Such partial inhibition by heterologous chemokines was not, seen with Jurkat using either 125I-labeled TARC [17] or TARC-SEAP(His)6. These results may indicate that Hut78 expresses at least two types of TARC receptors, one highly specific for TARC and another shared by RANTES and LARC

Summary

Introduction

Chemokines are small secreted polypeptides that play important roles in a wide range of inflammatory and immunological processes by recruiting selected subsets of leukocytes [1, 2]. We have demonstrated that CCR4 is the specific high affinity functional receptor for TARC that is expressed selectively on T cells. Cells were incubated for 1 h at 16 °C with 0.1 nM of 125I-RANTES, 125I-MCP-1, 125I-MIP-1␣, or TARC-SEAP(His)6 without or with 200 nM of unlabeled chemokines in 200 ␮l of low salt binding buffer (50 mM Hepes, pH 7.5, 1 mM CaCl2, 5 mM MgCl2, 0.5% BSA, and 0.05% sodium azide).

Results

Conclusion