Human thymocytes express CCR-3 and are activated by eotaxin.

Eotaxin has been characterized as a chemokine involved in eosinophil activation; however, mRNA for this C-C chemokine has been shown to be constitutively expressed in thymus. Immunohistochemical analysis showed a punctate distribution pattern, with eotaxin expression localized mainly in the medulla and in Hassle's corpuscles. Moreover, the receptor for eotaxin, CCR-3, was detected on thymocytes, with the highest level of expression being on the CD8 single-positive population. Equilibrium binding analyses on unfractionated thymocytes demonstrated specific 125I-eotaxin binding profiles comparable with CCR-3 transfectants. Eotaxin induced cell migration and mobilization of intracellular calcium in all thymocytes except the immature CD4(-)/CD8(-) population. Eotaxin also induced the secretion of the chemokines interleukin-8, RANTES, and macrophage inflammatory protein-1beta from thymocyte cultures in vitro. These results suggest that eotaxin-induced thymocyte activation may have important physiological implications for lymphocyte mobilization within and from this lymphoid organ.

Macrophage inflammatory protein-1beta induces migration and activation of human thymocytes.

The CC chemokine macrophage inflammatory protein 1beta (MIP-1beta), has been shown to be a chemoattractant preferentially activating CD4(+) CD45RA+ T lymphocytes. Further analysis of chemokine action on lymphocytic cells has shown the potent migration-promoting capacity of MIP-1beta on human thymocytes. The responding cells were the CD4(+) and CD8(+) single-positive (SP), as well as the CD4(+) CD8(+) double-positive (DP) populations, with little if any migratory activity on the double-negative (DN) population. The activation of thymocytes by MIP-1beta appeared to be a direct, receptor-mediated event as evidenced by the rapid mobilization of intracellular calcium, increase in proteins phosphorylated on tyrosine, and activation of the mitogen-activated protein kinase (MAPK) pathway. Radioligand binding analyses showed specific and displaceable binding of MIP-1beta to thymocytes with a Kd of approximately 1 nmol/L, a profile that was comparable with MIP-1beta binding to CCR-5-transfected NIH 3T3 cells. In addition, CCR-5 mRNA was detected in total thymocyte populations indicating that activation of thymocytes by MIP-1beta may occur through binding to CCR-5. Further dissection of the subpopulations showed that only the DP and CD8(+) SP populations expressed CCR-5 and expression data on these two populations was confirmed using anti-CCR-5 monoclonal antibody. These data may be suggestive of a role for MIP-1beta in human thymocyte activation, and show a potential route for HIV infectivity in the developing immune system.

CCR6, a CC chemokine receptor that interacts with macrophage inflammatory protein 3alpha and is highly expressed in human dendritic cells.

Dendritic cells initiate immune responses by ferrying antigen from the tissues to the lymphoid organs for presentation to lymphocytes. Little is known about the molecular mechanisms underlying this migratory behavior. We have identified a chemokine receptor which appears to be selectively expressed in human dendritic cells derived from CD34+ cord blood precursors, but not in dendritic cells derived from peripheral blood monocytes. When stably expressed as a recombinant protein in a variety of host cell backgrounds, the receptor shows a strong interaction with only one chemokine among 25 tested: the recently reported CC chemokine macrophage inflammatory protein 3alpha. Thus, we have designated this receptor as the CC chemokine receptor 6. The cloning and characterization of a dendritic cell CC chemokine receptor suggests a role for chemokines in the control of the migration of dendritic cells and the regulation of dendritic cell function in immunity and infection.

HNMP-1: a novel hematopoietic and neural membrane protein differentially regulated in neural development and injury.

The hnmp-1 (hematopoietic neural membrane protein) gene encodes a protein with striking similarity to the tetra-transmembrane-spanning protein encoded by pmp22. hnmp-1 was cloned from an elutriated human monocyte library and is expressed in various human hematopoietic and lymphoid lineages as well as adult mouse spleen and thymus. In the mouse nervous system, HNMP-1 mRNA is temporally expressed by Schwann cells during sciatic nerve myelination. Dorsal root ganglia sensory and spinal cord alpha-motoneurons acquire HNMP-1 protein selectively throughout development. In the fiber tracts of the spinal cord and in sciatic nerve, HNMP-1 protein is axon-associated. Additionally a rapid and sustained level of HNMP-1 expression is observed in response to acute PNS injury. HNMP-1 is constituitively induced in sciatic nerve of Trembler J mice, which are mutant for pmp22 and have a demyelinating/hypomyelinating phenotype. The expression pattern of HNMP-1 suggests a possible role for this molecule during active myelination.

Identification through bioinformatics of two new macrophage proinflammatory human chemokines: MIP-3alpha and MIP-3beta.

An increasing number of proinflammatory peptides, known as chemokines, are constantly being described and characterized. Because of their proven biologic functions in allergy, AIDS and, in general, inflammatory processes, these proteins have recently gained more attention. In this study we report the identification through bioinformatics of two new human chemokines: MIP-3alpha and MIP-3beta. Both of them belong to the beta- or CC chemokine family. Expression studies indicate that MIP-3alpha is predominantly expressed in lymph nodes, appendix, PBL, fetal liver, fetal lung and several cell lines. However, MIP-3beta expression is restricted to lymph nodes, thymus and appendix. Interestingly enough, both chemokines manifested a pattern of expression strongly regulated by IL-10. In contrast with other CC chemokines, MIP-3beta maps to chromosome 9. Here we show the importance of bioinformatics to discover new molecules with possible therapeutic effects and regulatory functions.

Biochemical and genetic characterization of multiple splice variants of the Flt3 ligand.

We have performed a comprehensive analysis of cell lines and tissues to compare and contrast the expression patterns of Flt3 ligand (FL), c-Kit ligand (KL), and macrophage colony-stimulating factor as well as their receptors, Flt3, c-Kit, and c-Fms. The message for FL is unusually ubiquitous, whereas that of its receptor is quite restricted, apparently limiting the function of the ligand to fetal development and early hematopoiesis. We have also sequenced a mouse FL genomic clone, revealing how the three splice variant FL mRNAs that we have isolated arise. The chromosomal location of the FL gene has been mapped, by in situ hybridization, to chromosome 7 in mouse and chromosome 19 in human. Natural FL protein has been purified from a stromal cell line and shown to be a 65 kD nondisulfide-linked homodimeric glycoprotein comprised of 30 kD subunits, each containing 12 kD of N- and O-linked sugars. Pulse-chase experiments show that one of the splice variants (T110) is responsible for producing the bulk of soluble FL, but only after it has first been expressed at the cell surface as a membrane-bound form. The other splice-variant forms produce molecules that are either obligatorily soluble (T169) or membrane-bound but released only very slowly (T118). Finally, even though most cell lines express some amount of FL mRNA, we found that very little FL protein is actually made, with T cells and stromal cells being the major producers. The data suggests that FL plays its roles over very short distances, perhaps requiring cell-cell contact.

DNAM-1, a novel adhesion molecule involved in the cytolytic function of T lymphocytes.

Intercellular adhesion molecules play an important role in the generation of T lymphocyte-mediated immune responses. Here, we describe a novel accessory molecule, DNAX accessory molecule-1 (DNAM-1), that is constitutively expressed on the majority of peripheral blood T lymphocytes. DNAM-1 is a 65 kDa transmembrane glycoprotein consisting of 318 aa including two immunoglobulin-like domains. Anti-DNAM-1 monoclonal antibody (MAb) inhibits T and NK cell-mediated cytotoxicity against a variety of tumor cell targets and blocks cytokine production by alloantigen-specific T cells. In addition, DNAM-1 is a tyrosine-phosphorylated signal-transducing molecule that participates in primary adhesion during cytotoxic T lymphocyte (CTL)-mediated cytotoxicity.

Molecular cloning of NKB1. A natural killer cell receptor for HLA-B allotypes.

The expression of certain MHC class I allotypes by potential target cells can inhibit NK cell-mediated cytotoxicity. We recently identified the NKB1 surface Ag, expressed on T and NK cell subsets, as a putative inhibitory receptor for HLA-B class I molecules possessing the Bw4 serologic epitope. NKB1 is a 70-kDa glycoprotein that after deglycosylation migrates as a 50-kDa protein as determined by SDS-PAGE. A cDNA encoding the NKB1 receptor was cloned from a NKB1+T cell cDNA library by expression in COS-7 cells using the anti-NKB1 mAb DX9. NKB1 is a member of the lg superfamily containing three lg-like domains in the extracellular region and is related to the recently identified family (p58/NKAT) of human NK and T cell surface molecules that appear to function as inhibitory receptors for HLA class I.

Motor deficit and impairment of synaptic plasticity in mice lacking mGluR1.

Metabotropic glutamate receptor 1 (mGluR1) is a member of a large family of G-protein-coupled glutamate receptors, the physiological functions of which are largely unknown. Mice deficient in mGluR1 have severe motor coordination and spatial learning deficits. They have no gross anatomical or basic electrophysiological abnormalities in either the cerebellum or hippocampus, but they show impaired cerebellar long-term depression and hippocampal mossy fibre long-term potentiation. mGluR1-deficient mice should therefore be valuable models for studying synaptic plasticity.