Miyuki Nishimura

Identification and Molecular Characterization of Fractalkine Receptor CX3CR1, which Mediates Both Leukocyte Migration and Adhesion

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.

Macrophage-derived Chemokine Is a Functional Ligand for the CC Chemokine Receptor 4

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-1α, macrophage inflammatory protein-1β). 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.

Molecular Cloning of a Novel Human CC Chemokine Secondary Lymphoid-Tissue Chemokine That Is a Potent Chemoattractant for Lymphocytes and Mapped to Chromosome 9p13

By searching the Expressed Sequence Tag (EST) data base, we identified partial cDNA sequences potentially encoding a novel human CC chemokine. We determined the entire cDNA sequence which encodes a highly basic polypeptide of 134 amino acids total with a putative signal peptide of 23 amino acids. The predicted mature protein of 111 amino acids has the four canonical cysteine residues and shows 21–33% identity to other human CC chemokines, but has a unique carboxyl-terminal extension of about 30 amino acids which contains two extra cysteine residues. The mRNA was expressed strongly in tissues such as the lymph nodes, Appendix, and spleen. The recombinant protein, which was produced by the baculovirus system and purified to homogeneity, was a highly efficient chemoattractant for certain human T cell lines and a highly potent one for freshly isolated peripheral blood lymphocytes and cultured normal T cells expanded by phytohemagglutinin and interleukin 2. Unlike most other CC chemokines, however, this novel chemokine was not chemotactic for monocytes or neutrophils, suggesting that it is specific for lymphocytes. From these results, we designated this novel CC chemokine as SLC fromsecondary lymphoid-tissuechemokine. SLC fused with the secreted form of alkaline phosphatase (SLC-SEAP) was used to characterize the SLC receptor. Binding of SLC-SEAP to freshly isolated lymphocytes was blocked by SLC (IC50, 0.12 nm) but not by any other CC chemokine so far tested, suggesting that resting lymphocytes express a class of receptors highly specific for SLC. By using somatic cell hybrids, radiation hybrids, and selected yeast and bacterial artificial chromosome clones, we mapped the SLC gene (SCYA21) at chromosome 9p13 and between chromosomal markers, D9S1978(WI-8765) and AFM326vd1, where the gene for another novel CC chemokine termed ELC from EBI1-ligandchemokine (SCYA19) also exists. Collectively, SLC is a novel CC chemokine specific for lymphocytes and, together with ELC, constitutes a new group of chemokines localized at chromosome 9p13.

Differences in neurogenic potential in floor plate cells along an anteroposterior location: midbrain dopaminergic neurons originate from mesencephalic floor plate cells

Directed differentiation and purification of mesencephalic dopaminergic (mesDA) neurons from stem cells are crucial issues for realizing safe and efficient cell transplantation therapies for Parkinsons 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 Parkinsons disease.

Dual Functions of Fractalkine/CX3C Ligand 1 in Trafficking of Perforin + /Granzyme B + Cytotoxic Effector Lymphocytes That Are Defined by CX3CR1 Expression

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.

Identification of Single C Motif-1/Lymphotactin Receptor XCR1

Single C motif-1 (SCM-1)/lymphotactin is a member of the chemokine superfamily, but retains only the 2nd and 4th of the four cysteine residues conserved in other chemokines. In humans, there are two highly homologous SCM-1 genes encoding SCM-1α and SCM-1β with two amino acid substitutions. To identify a specific receptor for SCM-1 proteins, we produced recombinant SCM-1α and SCM-1β by the baculovirus expression system and tested them on murine L1.2 cells stably expressing eight known chemokine receptors and three orphan receptors. Both proteins specifically induced migration in cells expressing an orphan receptor, GPR5. The migration was chemotactic and suppressed by pertussis toxin, indicating coupling to a Gα type of G protein. Both proteins also induced intracellular calcium mobilization in GPR5-expressing L1.2 cells with efficient mutual cross desensitization. SCM-1α bound specifically to GPR5-expressing L1.2 cells with a K d of 10 nm. By Northern blot analysis, GPR5 mRNA of about 5 kilobases was detected strongly in placenta and weakly in spleen and thymus among various human tissues. Identification of a specific receptor for SCM-1 would facilitate our investigation on its biological function. Following the set rule for the chemokine receptor nomenclature, we propose to designate GPR5 as XCR1 from XC chemokine receptor-1.

Inhibition of Fractalkine Ameliorates Murine Collagen-Induced Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with massive infiltration of inflammatory cells in the synovium of multiple joints. We and others have shown that fractalkine (FKN/CX3CL1), a chemokine expressed on fibroblast-like synoviocytes and endothelial cells in RA synovium, may contribute to the accumulation of T cells, macrophages, and dendritic cells, which express CX3CR1, the receptor for FKN. This interaction might be involved in adhesion of the inflammatory cells to endothelial cells, migration into the synovium, and cytokine production. In this study, we examined the effect of FKN inhibition on murine collagen-induced arthritis. Anti-FKN mAb significantly lowered clinical arthritis score compared with control Ab, and reduced infiltration of inflammatory cells and bone erosion in the synovium. However, anti-FKN mAb did not affect the production of either serum anti-collagen type II (CII) IgG or IFN-γ by CII-stimulated splenic T cells. Furthermore, treatment with anti-FKN mAb inhibited migration of adoptively transferred splenic macrophages into the inflamed synovium. Our results suggest that anti-FKN mAb ameliorates arthritis by inhibiting infiltration of inflammatory cells into the synovium. Thus, FKN can be a new target molecule for the treatment of RA.

Molecular cloning of a novel C or γ type chemokine, SCM-1

From human PBMC stimulated with PHA, we have isolated cDNA clones encoding a novel cytokine named SCM‐1, which is significantly related to the CC and the CXC chemokines but has only the 2nd and the 4th of the four cysteines conserved in these proteins. Its gene is also distinctly mapped to human chromosome 1. SCM‐1 is strongly induced in human PBMC and Jurkat T cells by PHA stimulation. Among various human tissues, SCM‐1 is expressed most strongly in spleen. SCM‐1 is found to be 60.5% identical to lymphotactin, a recently described murine lymphocyte‐specific chemokine, which also retains only two cysteines. SCM‐1 and lymphotactin may thus represent the human and murine prototypes of a novel C or γ type chemokine family.

Similar and differential behaviour between the nectin‐afadin‐ponsin and cadherin‐catenin systems during the formation and disruption of the polarized junctional alignment in epithelial cells

We have recently identified a novel cell‐cell adhesion system, named NAP system, which is localized at cadherin‐based cell‐cell adherens junctions (AJs). The NAP system is composed of at least nectin, afadin and ponsin. Nectin is an immunoglobulin‐like cell adhesion molecule. Afadin is an actin filament‐binding protein which associates nectin with the actin cytoskeleton. Ponsin is an afadin‐binding protein which furthermore binds to vinculin and provides a possible linkage of nectin‐afadin to cadherin‐catenin through vinculin. We compared here the behaviour of the NAP and cadherin‐catenin systems during the formation and disruption of the polarized junctional alignment in epithelial cells.

Chemokines as Novel Therapeutic Targets for Inflammatory Bowel Disease

The inflammatory bowel diseases (IBD) such as Crohns disease (CD) and ulcerative colitis (UC) are illness characterized by a chronic clinical course of relapse and remission associated with self‐destructive inflammation of the gastrointestinal tract. In both UC and CD, leukocyte infiltration into the intestine is fundamental event in disease development and progression where the chemokines and their receptors are orchestrating the tissue‐specific and the cell type–selective trafficking of leukocytes. In this review, we will discuss the homeostatic and inflammatory roles of the chemokines and their receptors with their potentials and promise as molecular targets for therapeutic interventions in human IBD, focusing on the recently identified role of the CX3CL1–CX3CR1 axis, as well as the CCL20–CCR6, CCL25–CCR9, and CXCL10–CXCR3 pathways.