Leukocyte trafficking at the endothelium requires both cellular adhesion molecules and chemotactic factors. Fractalkine, a novel transmembrane molecule with a CX3C-motif chemokine domain atop a mucin stalk, induces both adhesion and migration of leukocytes. Here we identify a seven-transmembrane high-affinity receptor for fractalkine and show that it mediates both the adhesive and migratory functions of fractalkine. The receptor, now termed CX3CR1, requires pertussis toxin-sensitive G protein signaling to induce migration but not to support adhesion, which also occurs without other adhesion molecules but requires the architecture of a chemokine domain atop the mucin stalk. Natural killer cells predominantly express CX3CR1 and respond to fractalkine in both migration and adhesion. Thus, fractalkine and CX3CR1 represent new types of leukocyte trafficking regulators, performing both adhesive and chemotactic functions.

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). The known chemokines are divided into two major subfamilies based on the spacing of the first two cysteines in the conserved motif. The CXC chemokine subfamily, which includes IL-8 1and IP-10 (4), is characterized by the presence of a single amino acid separating the first two cysteines. The two cysteines are adjacent in the CC chemokine subfamily, which includes RAN-TES (5), MCP-1 (6, 7), MCP-2 (8), MCP-3 (9), MCP-4 (10), MIP-1␣ (11), MIP-1␤ (12), I-309 (13), eotaxin (14, 15), HCC-1 (16), TARC (17), and LARC (18). The CXC chemokines preferentially attract and activate neutrophils, whereas the CC chemokines usually attract and activate monocytes and also basophils, eosinophils, or lymphocytes with variable selectivity (19). Recently, lymphotactin/single C motif 1 that carries only the second and the fourth of the four cysteine residues conserved in other chemokines has been identified, suggesting the existence of the C type chemokine subfamily (20, 21). The human genes for the CXC, CC, and C chemokines are clustered on human chromosomes 4, 17, and 1, respectively (1, 22, 23). Recent studies indicate that genes for certain chemokines are present outside these clusters. For example, a CXC chemokine SDF-1/PBSF has been mapped to human chromosome 10 (24), and CC chemokines TARC and LARC have been mapped to human chromosomes 16 and 2, respectively (18, 25). In addition to chemotactic activity, some chemokines have a regulatory activity on hematopoiesis and angiogenesis (26 -28). Recently, it has been shown that three CC chemokines, MIP-1␣, MIP-1␤, and RANTES, block infection of macrophage-tropic strains of human immunodeficiency virus type 1, while a CXC chemokine, SDF-1/PBSF, blocks infection of T cell line-tropic human immunodeficiency virus type 1 strains (29, 30). The specific effects of chemokines on target cells are mediated by seven-transmembrane G-protein-coupled receptors (31). To date, at least five human CC chemokine receptors have been defined for ligand specificity. CCR1 is a receptor for MIP-1␣, RANTES,; CCR2 is a receptor for 36); CCR3 is a receptor for eotaxin, RANTES,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). The specific ligands for CCR1, CCR2, CCR3, and CCR5 were demonstrated by specific binding and functional assays such as chemotaxis and calcium flux using cDNA-transfected mammalian cells. In the case of CCR4, however, only marginal levels of binding of MIP-1␣ and RANTES were shown with HL-60 cells transfected with CCR4 (43), while a chloride current induction in response to MIP-1␣, RANTES, and MCP-1 was demonstrated in CCR4 cRNA-injected oocytes (39). Except for CCR3 that is almost exclusively expressed on eosinophils (38, 44), other receptors were reported to be expressed on monocytes and lymphocytes. Notably, CCR4 that was originall...

By searching the expressed sequence tag (EST) data base, we identified partial cDNA sequences encoding a novel human CC chemokine. We determined the complete cDNA sequence that encodes a highly basic polypeptide of a total 98 amino acids with 20 to 30% identity to other human CC chemokines. We termed this novel chemokine from EBI1-Ligand Chemokine as ELC (see below). The ELC mRNA was most strongly expressed in the thymus and lymph nodes. Recombinant ELC protein was expressed as a fusion protein with the Flag tag (ELC-Flag). For receptor-binding assays, recombinant ELC protein fused with the secreted form of alkaline phosphatase (SEAP) was used. By stably expressing five CC chemokine receptors (CCR1 to 5) and five orphan receptors, ELC-SEAP was found to bind specifically to an orphan receptor EBI1. Only ELC-Flag, but not MCP-1, MCP-2, MCP-3, eotaxin, MIP-1␣, MIP-1␤, RANTES (regulated on activation normal T cell expressed and secreted), thymus and activation-regulated chemokine (TARC), or liver and activation-regulated chemokine (LARC), competed with ELC-SEAP for EBI1. ELC-Flag-induced transient calcium mobilization and chemotactic responses in EBI1-transfected cells. ELCFlag also induced chemotaxis in HUT78 cells expressing endogenous EBI1 at high levels. By somatic hybrid and radiation hybrid analyses, the gene for ELC (SCYA19) was mapped to chromosome 9p13 instead of chromosome 17q11.2 where the genes for CC chemokines are clustered. Taken together, ELC is a highly specific ligand for EBI1, which is known to be expressed in activated B and T lymphocytes and strongly up-regulated in B cells infected with Epstein-Barr virus and T cells infected with herpesvirus 6 or 7. ELC and EBI1 may thus play roles in migration and homing of normal lymphocytes, as well as in pathophysiology of lymphocytes infected with these herpesviruses. We propose EBI1 to be designated as CCR7.

؉ and CD8 ؉ T cells was strongly up-regulated by IL-2. Taken together, GPR-CY4 is the specific receptor for LARC expressed selectively on lymphocytes, and LARC is a unique functional ligand for GPR-CY4. We propose GPR-CY4 to be designated as CCR6.

Precursors of most secreted and cell surface molecules carry signal sequences at their amino termini. Here we describe an efficient signal sequence trap method and isolation of a novel CC chemokine. An expression library was constructed by inserting 5' portion-enriched cDNAs from phytohemagglutinin-stimulated peripheral blood mononuclear cells into upstream of signal sequence-deleted CD4 cDNA in an Epstein-Barr virus shuttle vector. After electroporation into Raji cells, CD4 antigen-positive cells were enriched by repeated cell sorting and plasmids were recovered in Escherichia coli. Out of 100 plasmid clones examined, 42 clones directed expression of CD4 antigen on the cell surface. Among them were signal sequences of CD6, beta2-microglobulin, MGC-24, and T cell receptor epsilon-chain, and at least four novel potential signal sequences. A cDNA clone encoding a novel CC chemokine was isolated by using one of the trapped fragments. The gene designated as TARC from Thymus and Activation-Regulated Chemokine was expressed transiently in phytohemagglutinin-stimulated peripheral blood mononuclear cells and constitutively in thymus. Radiolabeled recombinant TARC specifically bound to T cell lines and peripheral T cells but not to monocytes or granulocytes. The binding of radiolabeled TARC to the high-affinity receptor (Kd, 2.1 nM) on Jurkat was displaced by TARC but not by interleukin-8, MIP-1alpha, RANTES, or MCP-1. TARC also bound to the promiscuous chemokine receptor on erythrocytes (Kd, 17 nM). TARC induced chemotaxis in T cell lines Hut78 and Hut102. Pretreatment of Hut78 with pertussis toxin abolished the TARC-induced cell migration. Collectively, T cells express a highly selective receptor for TARC that is coupled to pertussis toxin-sensitive G-protein. TARC may a factor playing important roles in T cell development in thymus as well as in trafficking and activation of mature T cells.

Thymus and activation-regulated chemokine (TARC) is a recently identified lymphocyte-directed CC chemokine which specifically chemoattracts T helper type 2 CD4 ϩ T cells in human. To establish the pathophysiological roles of TARC in vivo, we investigated whether a monoclonal antibody (mAb) against TARC could inhibit the induction of hepatic lesions in murine model using Propionibacterium acnes and lipopolysaccharide (LPS). P . acnes -induced intrahepatic granuloma formation in the priming phase is essential to the subsequent liver injury elicited by a low dose of LPS.The priming phase appears to be dominated by Th1 type immune responses determined by the profile of chemokine and chemokine receptor expression. TARC was selectively produced by granuloma-forming cells, and CC chemokine receptor 4 (CCR4)-expressing CD4 ϩ T cells migrated into the liver after LPS administration. In vivo injection of anti-TARC mAb just before LPS administration protected the mice from acute lethal liver damage, which was accompanied by a significant reduction of both CCR4 mRNA expression and IL-4 production by liver-infiltrating CD4 ϩ T cells. Moreover, both TNF-␣ and Fas ligand expressions in the liver were decreased by anti-TARC treatment. These results suggest that recruitment of IL-4-producing CCR4 ϩ CD4ϩ T cells by granuloma-derived TARC into the liver parenchyma may be a key cause of massive liver injury after systemic LPS administration. ( J. Clin. Invest. 1998Invest. . 102: 1933Invest. -1941

The liver and activation-regulated chemokine (LARC), also termed MIP-3 § and Exodus, is a novel human CC chemokine with a selective chemotactic activity for lymphocytes and den-dritic cells. Here we describe genomic and cDNA clones encoding the murine orthologue of LARC (mLARC). The gene consists of four exons and three introns. The 5'-noncoding region of about 400 bp contains typical TATA and CAAT boxes but no other potential regulatory elements so far described. The cDNA encodes a CC chemokine of 97 amino acid residues with the highest homology to human LARC (64 % amino acid identity). The 3'-noncoding region contains as many as five potential mRNA destabilization signals. mLARC was strongly and transiently induced in the murine monocytoid cell line J774 by lipopolysaccharide (LPS) but not by cytokines such as TNF-§ , IFN-+ , IL-1 g or IL-4. In normal mice, mLARC mRNA was expressed selectively in intestinal tissues such as small intestine and colon. Upon treatment with LPS, mLARC expression was enhanced in intestinal tissues and induced in some lym-phoid tissues such as lymph nodes. Because of alternative splicing, there are two types of transcripts encoding mLARC and its variant mLARCvar with and without an N-terminal ala-nine in the mature protein, respectively. Both types of transcripts appeared to be expressed in various mouse tissues. In situ hybridization revealed that epithelial cells of intestinal tissues , especially those lining lymphoid follicles, expressed mLARC. Localization of LARC mRNA in epithelial cells was also demonstrated in a human appendix. Furthermore, mLARC was efficiently chemotactic for cells such as + ˇ type T cells in intestinal epithelium and naive B cells in Peyer's patches. Thus, in both humans and mice, LARC may be physiologically involved in formation and function of the mucosal lymphoid tissues by attracting lympho-cytes and dendritic cells toward epithelial cells.

Secondary lymphoid tissue chemokine (SLC) is a CC chemokine expressed mainly in lymph nodes, appendix and spleen, and specifically chemotactic for lymphocytes (Nagira et al., J. Biol. Chem. 1997. 272: 19518-19524). Here, we carried out transendothelial migration assays to determine the classes and subsets of lymphocytes migrating toward SLC. SLC attracted freshly isolated B cells with high efficiency and T cells modestly. Thus, SLC is the first CC chemokine with a strong chemotactic activity on fresh B cells. Among T cell types and subsets, SLC broadly attracted CD4+ and CD8+ cells, CD45RO- (naive) and CD45RO+ (memory) cells, and CD26high (activated) and CD26low- (resting) cells. SLC also attracted both L-selectin+ and L-selectin- subpopulations of various T cell subsets and B cells. Furthermore, mitogenic stimulation strongly enhanced migratory responses of T cells and B cells toward SLC. By in situ hybridization, SLC mRNA was detected in the cortical parafollicular regions (the T cell areas) of a lymph node and an appendix. Collectively, SLC may be a basic chemokine supporting homeostatic migration of a broad spectrum of lymphocytes into the secondary lymphoid tissues. SLC may also be involved in immune responses by inducing highly efficient migration of T and B cells following antigenic stimulation.