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.

Helper T cells are classified into Th1 and Th2 subsets based on their profiles of cytokine production. Th1 cells are involved in cell-mediated immunity, whereas Th2 cells induce humoral responses. Selective recruitment of these two subsets depends on specific adhesion molecules and specific chemoattractants. Here, we demonstrate that the T cell-directed CC chemokine thymus and activation-regulated chemokine (TARC) was abundantly produced by monocytes treated with granulocyte macrophage colony stimulating factor (GM-CSF) or IL-3, especially in the presence of IL-4 and by dendritic cells derived from monocytes cultured with GM-CSF + IL-4. The receptor for TARC and another macrophage/dendritic cell-derived CC chemokine macrophage-derived chemokine (MDC) is CCR4, a G protein-coupled receptor. CCR4 was found to be expressed on approximately 20% of adult peripheral blood effector/memory CD4+ T cells. T cells attracted by TARC and MDC generated cell lines predominantly producing Th2-type cytokines, IL-4 and IL-5. Fractionated CCR4+ cells but not CCR4- cells also selectively gave rise to Th2-type cell lines. When naive CD4+ T cells from adult peripheral blood were polarized in vitro, Th2-type cells selectively expressed CCR4 and vigorously migrated toward TARC and MDC. Taken together, CCR4 is selectively expressed on Th2-type T cells and antigen-presenting cells may recruit Th2 cells expressing CCR4 by producing TARC and MDC in Th2-dominant conditions.

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...

We have isolated a novel cell-cell adhesion system localized at cadherin-based adherens junctions (AJs). This system consists of at least nectin, a Ca 2؉ -independent immunoglobulin-like adhesion molecule, and afadin, an actin filament-binding protein, that connects nectin to the actin cytoskeleton. Nectin constitutes a family consisting of two members, nectin-1 and -2. We have isolated here a third member of the nectin family and named it nectin-3. Nectin-3 has three splicing variants, nectin-3␣ (biggest), -3␤ (middle), and -3␥ (smallest). Like nectin-1 and -2, nectin-3␣ consists of three extracellular immunoglobulin-like domains, a transmembrane segment, and a cytoplasmic region with the C-terminal consensus motif for binding to the PDZ domain. Nectin-3␣ formed a cis-homo-dimer and showed Ca 2؉ -independent trans-homo-interaction to cause homophilic cell-cell adhesion. Nectin-3␣ furthermore showed trans-hetero-interaction with nectin-1 or -2 but did not form a cis-hetero-dimer with nectin-1 or -2. Nectin-1 did not show trans-heterointeraction with nectin-2. The affinity of trans-heterointeraction of nectin-3␣ with nectin-1 or -2 was higher than that of trans-homo-interaction of nectin-1, -2, or -3␣. Nectin-2 and -3 were ubiquitously expressed, whereas nectin-1 was abundantly expressed in brain. Nectin-3␣ was colocalized with nectin-2 at cadherinbased AJs and interacted with afadin. These results indicate that the nectin family consists of at least three members, nectin-1, -2, and -3, all of which show homophilic and heterophilic cell-cell adhesion activities and are localized at cadherin-based AJs.

Directed differentiation and purification of mesencephalic dopaminergic (mesDA) neurons from stem cells are crucial issues for realizing safe and efficient cell transplantation therapies for Parkinson's disease. Although recent studies have identified the factors that regulate mesDA neuron development, the mechanisms underlying mesDA neuron specification are not fully understood. Recently, it has been suggested that mesencephalic floor plate (FP) cells acquire neural progenitor characteristics to generate mesDA neurons. Here, we directly examined this in a fate mapping experiment using fluorescence-activated cell sorting (FACS) with an FP cell-specific surface marker, and demonstrate that mesencephalic FP cells have neurogenic activity and generate mesDA neurons in vitro. By contrast, sorted caudal FP cells have no neurogenic potential, as previously thought. Analysis of dreher mutant mice carrying a mutation in the Lmx1a locus and transgenic mice ectopically expressing Otx2 in caudal FP cells demonstrated that Otx2 determines anterior identity that confers neurogenic activity to FP cells and specifies a mesDA fate, at least in part through the induction of Lmx1a. We further show that FACS can isolate mesDA progenitors, a suitable transplantation material, from embryonic stem cell-derived neural cells. Our data provide insights into the mechanisms of specification and generation of mesDA neurons, and illustrate a useful cell replacement approach for Parkinson's disease.

Macrophage-derived chemokine (MDC) is a recently identified member of the CC chemokine family. MDC is not closely related to other chemokines, sharing most similarity with thymus-and activation-regulated chemokine (TARC), which contains 37% identical amino acids. Both chemokines are highly expressed in the thymus, with little expression seen in other tissues. In addition, the genes for MDC and TARC are encoded by human chromosome 16. To explore this relationship in greater detail, we have more precisely localized the MDC gene to chromosome 16q13, the same position reported for the TARC gene. We have also examined the interaction of MDC with CC chemokine receptor 4 (CCR4), recently shown to be a receptor for TARC. Using a fusion protein of MDC with secreted alkaline phosphatase, we observed high affinity binding of MDC-secreted alkaline phosphatase to CCR4-transfected L1.2 cells (K d ‫؍‬ 0.18 nM). MDC and TARC competed for binding to CCR4, while no binding competition was observed for six other chemokines (MCP-1, MCP-3, MCP-4, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein-1␣, macrophage inflammatory protein-1␤). MDC was tested for calcium mobilization in L1.2 cells tranfected with seven different CC chemokine receptors. MDC induced a calcium flux in CCR4-transfected cells, but other receptors did not respond to MDC. TARC, which also induced calcium mobilization in CCR4 transfectants, was unable to desensitize the response to MDC. In contrast, MDC fully desensitized a subsequent response to TARC. Both MDC and TARC functioned as chemoattractants for CCR4 transfectants, confirming that MDC is also a functional ligand for CCR4. Since MDC and TARC are both expressed in the thymus, one role for these chemokines may be to attract CCR4-bearing thymocytes in the process of T cell education and differentiation.Chemokines are small secreted proteins that mediate recruitment of leukocytes to sites of inflammation (1, 2). The complexity and functions of the chemokine family have become increasingly diverse as more members have been identified and characterized. There are four subfamilies of chemokines based on the relative position of conserved cysteine residues (1, 3, 4).The largest subfamily consists of the CC chemokines, which generally induce migration of monocytes, T lymphocytes, and in some cases eosinophils, basophils, or mast cells. All chemokines mediate their activities through G protein-coupled receptors, which have a characteristic seven-transmembrane structure. These receptors are very selective and bind specific ligands with high affinity. Eight different CC chemokine receptors have been characterized to date. CCR1, 1 CCR2, CCR3, and CCR5 each has ligand specificity for at least three CC chemokines (5-12), while single high affinity ligands have been identified that recognize CCR4, CCR6, CCR7, and CCR8 (13-17).Macrophage-derived chemokine (MDC) is a novel CC chemokine synthesized by macrophages and dendritic cells (18). MDC shares only limited homology wit...

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.

Fractalkine/CX3C ligand 1 and its receptor CX3CR1 are known to mediate both cell adhesion and cell migration. Here we show that CX3CR1 defines peripheral blood cytotoxic effector lymphocytes commonly armed with intracellular perforin and granzyme B, which include NK cells, γδ T cells, and terminally differentiated CD8+ T cells. In addition, soluble fractalkine preferentially induced migration of cytotoxic effector lymphocytes. Furthermore, interaction of cytotoxic effector lymphocytes with membrane-bound fractalkine promoted subsequent migration to the secondary chemokines, such as macrophage inflammatory protein-1β/CC ligand 4 or IL-8/CXC ligand 8. Thus, fractalkine expressed on inflamed endothelium may function as a vascular regulator for cytotoxic effector lymphocytes, regardless of their lineage and mode of target cell recognition, through its ability to capture them from blood flow and to promote their emigration in response to other chemokines.