No abstract

Abstract. To protect the body efficiently from infectious organisms, leukocytes circulate as nonadherent cells in the blood and lymph, and migrate as adherent cells into tissues. Circulating leukocytes in the blood have first to adhere to and then to cross the endothelial lining. CD31/PECAM-1 is an adhesion molecule expressed by vascular endothelial cells, platelets, monocytes, neutrophils, and naive T lymphocytes. It is a transmembrane glycoprotein of the immunoglobulin gene superfamily (IgSF), with six Ig-like homology units mediating leukocyte-endothelial interactions. The adhesive interactions mediated by CD31 are complex and include homophilic (CD31-CD31) or heterophilic (CD31-X) contacts. Soluble, recombinant forms of CD31 allowed us to study the heterophilic interactions in leukocyte adhesion assays. We show that the adhesion molecule otv[33 integrin is a ligand for CD31. The leukocytes revealed adhesion mediated by the second Ig-like domain of CD31, and this binding was inhibited by av[33 integrin-specific antibodies. Moreover Otv~ 3 was precipitated by recombinant CD31 from cell lysates. These data establish a third IgSF-integrin pair of adhesion molecules, CD31-otvI33 in addition to VCAM-1, MadCAM-1/ot4 integrins, and ICAM/B2 integrins, which are major components mediating leukocyteendothelial adhesion. Identification of a further versatile adhesion pair broadens our current understanding of leukocyte-endothelial interactions and may provide the basis for the treatment of inflammatory disorders and metastasis formation.

The melanoma cell adhesion molecule (MCAM)/CD146 is expressed as two isoforms differing by their cytoplasmic domain (MCAM long (MCAM-l) and MCAM short (MCAM-s)). MCAM being expressed by endothelial cells and activated T cells, we analyzed its involvement in lymphocyte trafficking. The NK cell line NKL1 was transfected by MCAM isoforms and submitted to adhesion on both the endothelial cell monolayer and recombinant molecules under shear stress. MCAM-l transfection reduced rolling velocity and increased NKL1 adhesion on the endothelial cell monolayer and VCAM-1. Scanning electron microscopy revealed that MCAM-l induced microvilli formation and extension. In contrast, MCAM short or mock transfection had no effect on adhesion of NKL1 cells and microvilli formation. As shown by mutagenesis, serine 32 of the MCAM-l cytoplasmic tail, belonging to a putative protein kinase C phosphorylation site, was necessary for MCAM-l-actin cytoskeleton interaction and microvilli induction. Accordingly, chelerythrine chloride, a protein kinase C inhibitor, abolished MCAM-l-induced microvilli and rolling of MCAM-l-transfected NKL1 cells. Inhibition of adhesion under shear stress by anti-MCAM Abs suggested that both lymphoid MCAM-l and endothelial MCAM were also directly involved in lymphocyte endothelium interaction. MCAM-l-transfected NKL1 and activated CD4 T cells adhered to rMCAM under shear stress whereas anti-MCAM Ab treatment inhibited this process. Taken together, these data establish that MCAM is involved in the initial steps of lymphocyte endothelium interaction. By promoting the rolling on the inflammation marker VCAM-1 via microvilli induction and displaying adhesion receptor activity involving possible homophilic MCAM-l-MCAM-l interactions, MCAM might be involved in the recruitment of activated T cells to inflammation sites.

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Abstract. We have characterized the adhesion molecule HEMCAM, which is expressed by hemopoietic progenitors of embryonic bone marrow. HEMCAM belongs to the immunoglobulin superfamily and consists of the V-V-C2-C2-C2 Ig domains. There are three mRNA splice variants. One has a short cytoplasmic tail; another has a long tail; while the third seems to lack transmembrane and cytoplasmic regions. Except for the NH2-terminal sequence, HEMCAM is identical to gicerin, a molecule involved in neurite outgrowth and Wilm's kidney tumor progression in the chicken and it is significantly homologous with MUC18 a molecule involved in melanoma progression and metastasis in human beings.In the bone marrow the HEMCAM ÷ cell population contains c-kit + subsets. HEMCAM ÷ cells coexpressing the receptor tyrosine kinase c-kit give rise to T cells at a frequency of 0.17 when injected intrathymically in congenic animals. As HEMCAM ÷, c-kit + cells differentiate into myeloid and erythroid CFU's the double-positive cell population seems to contain precursors for multiple lineages. HEMCAM promotes cell-cell adhesion of transfected cells. Cross-linking of murine HEM-CAM leads to cell spreading of T-lymphocyte progenitors adhering to the vascular adhesion molecules, PECAM-1 and VCAM-1. Thus, HEMCAM is likely to be involved in cellular adhesion and homing processes. large proportion of immunoglobulin supeffamily (IgSF) 1 molecules serve as adhesion receptors on the cell surface. In the recent literature there has emerged a prominent subgroup with a characteristic V-V-C2-C2-C2 Ig domain structure. Important representative members of this subgroup of molecules are B-CAM (human), Lutheran blood group antigen (human), BEN/DM-GRASP/SC1 (chicken), ALCAM (human), KG-CAM (rat), irreC-rst (drosophila), Neurolin (goldfish), Gicerin (chicken), and MUC18 (human) (3-5, 26, 28, 38, 41, 45, 47, 57, 60). Although most of these molecules are involved in adhesion and migration processes of neural cells, several of them are also found directly or indirectly associated with Please address all correspondence to O. Vainio,

The embryonic thymus is colonized by the influx of hemopoietic progenitors in waves. To characterize the T cell progeny of the initial colonization waves, we used intravenous adoptive transfer of bone marrow progenitors into congenic embryos. The experiments were performed in birds because intravenous cell infusions can be performed more efficiently in avian than in mammalian embryos. Progenitor cells, which entered the vascularized thymus via interlobular venules in the capsular region and capillaries located at the corticomedullary junction, homed to the outer cortex to begin thymocyte differentiation. The kinetics of differentiation and emigration of the T cell progeny were analyzed for the first three waves of progenitors. Each progenitor wave gave rise to γ/δ T cells 3 d earlier than α/β T cells. Although the flow of T cell migration from the thymus was uninterrupted, distinct colonization and differentiation kinetics defined three successive waves of γ/δ and α/β T cells that depart sequentially the thymus en route to the periphery. Each wave of precursors rearranged all three TCR Vγ gene families, but displayed a variable repertoire. The data indicate a complex pattern of repertoire diversification by the progeny of founder thymocyte progenitors.

In birds and mammals T cells develop along two discrete pathways characterized by expression of either the ␣␤ or the ␥␦ T-cell antigen receptors (TCRs). To gain further insight into the evolutionary significance of the ␥␦ T-cell lineage, the present studies sought to define the chicken TCR␥ locus. A splenic cDNA library was screened with two polymerase chain reaction products obtained from genomic DNA using primers for highly conserved regions of TCR and immunoglobulin genes. This strategy yielded cDNA clones with characteristics of mammalian TCR ␥ chains, including canonical residues considered important for proper folding and stability. Northern blot analysis with the TCR␥ cDNA probe revealed 1.9-kb transcripts in the thymus, spleen, and a ␥␦ T-cell line, but not in B or ␣␤ T-cell lines. Three multimember V␥ subfamilies, three J␥ gene segments, and a single constant region C␥ gene were identified in the avian TCR␥ locus. Members of each of the three V␥ subfamilies were found to undergo rearrangement in parallel during the first wave of thymocyte development. TCR␥ repertoire diversification was initiated on embryonic day 10 by an apparently random pattern of V-J␥ recombination, nuclease activity, and P-and N-nucleotide additions to generate a diverse repertoire of avian TCR␥ genes early in ontogeny.Studies in the chicken, Gallus gallus domesticus, suggest that avian T-cell differentiation and function are similar to those described in mammals (1, 2). Divergent pathways of T-cell development are characterized in both phyla by expression of either an ␣␤ or a ␥␦ T-cell antigen receptor (TCR). The genes encoding the chicken TCR ␣-and TCR ␤-chains, and their mode of repertoire diversification, resemble their mammalian counterparts (2-4). However, the chicken TCR ␣ and ␤ loci are relatively simple in that each contains only two V subfamilies (3-5) versus the 20-30 subfamilies of V␣ and V␤ genes found in mice and humans (6, 7). Interestingly, ␣␤ T cells that express the prototypic V␤1 genes migrate preferentially to the chicken intestine, where they provide help to mucosal B cells for IgA antibody production (8).Avian T cells bearing a ␥␦ TCR are the first to be generated during ontogeny (9) and they comprise up to 50% of the recirculating T-cell pool in mature birds (9,10). This relative abundance of ␥␦ versus ␣␤ T cells and the experimental accessibility of avian embryos make the chicken an attractive model in which to explore unresolved issues in ␥␦ T-cell development and function. However, more information on the TCR ␥␦ genes is needed to exploit this avian model. A chicken TCR␥ gene candidate has been isolated by PCR using short, minimally degenerate oligonucleotide primers complementing conserved V region segments to amplify TCR-like products from genomic DNA (11,12). The present studies refine the definition of this candidate TCR␥ gene, outline the composition of the chicken TCR␥ locus, determine the embryonic pattern of TCR␥ gene expression, and examine the initial TCR␥ repertoire diversification in th...

T cell precursors enter the chick thymus in three waves during embryonic life. Each wave of thymocyte precursors colonizing the thymus gave rise to a similar TCR V beta repertoire in thymus, spleen and intestine both in terms of V beta 1 and J beta usage as well as in the length of V beta‐D beta‐J beta junctions. Seventeen V beta 1s were utilized, and a new J beta segment was found. In the progeny of the third wave, more nucleotides were deleted at the 5′ end of the J beta segment, but the overall size of the CDR3 was conserved by a concomitant increase of N nucleotide addition at the V beta‐D beta‐J beta junctions during rearrangement. This CDR3 modification was observed in the spleen but not in the intestine, implying that progeny of the third wave migrate preferentially to the spleen, a possibility that was confirmed by adoptive cell transfers into congenic chickens. Very low frequencies of non‐productive rearrangements in the intestine suggested that negative selection may occur in this organ. The present analysis indicates that V beta 1+ T cells in spleen and intestine are primarily of thymic origin, this colonization of both organs occurs in waves and is not characterized by preselection of the TCR V beta 1 repertoire.