Smith is a fellow of the European Community Human Capital and Mobility program
Smith is a fellow of the European Community Human Capital and Mobility program. Abbreviations used in this paper ECvascular endothelial cellsecto-5NTCD73/ecto-5-nucleotidaseGPIglycosyl-phosphatidylinositolHUVEChuman umbilical vein endothelial cellsIFimmunofluorescenceMFImean fluorescence intensityPBLperipheral blood lymphocytesPI-PLCphosphatidylinositol-specific phospholipase C Footnotes Please address all correspondence to Laura Airas, MediCity Research Laboratory, Tykist?katu 6A, 20520 Turku, Finland. whereas triggering of lymphocyte CD73 can induce tyrosine phosphorylation. Despite the functional differences, CD73 molecules on lymphocytes and EC were practically identical structurally, when studied at the protein, mRNA, and cDNA level. Thus, CD73 is an interesting example of a molecule which lacks structural variants but yet has a wide diversity of biological functions. We suggest that the ligand- induced shedding of lymphocyte CD73 represents an important and novel means of controlling NIC3 lymphocyteC EC interactions. Lymphocyte recirculation is known to be finely regulated by different adhesion molecules expressed on lymphocytes and endothelial cells (EC)1 and by chemokines, controlling the activation status of the cells (30). Lymphocytes make the initial contact with EC by rolling along the vessel wall in the high endothelial venules of lymphoid tissues or along the flat-walled endothelium at sites of inflammation. Lymphocyte rolling is a well characterized phenomenon, and it is known to be mediated by selectins, situated on the tips of microvilli both on the lymphocyte and EC surface (42). More stable adhesion is achieved through binding of activated integrins to their EC counterparts, and lymphocytes eventually migrate through the vessel wall using integrins and integrin ligands of the Ig superfamily (8, 30). CD73/ecto-5-nucleotidase (ecto-5-NT), is a 70-kD glycosyl-phosphatidylinositol (GPI)Clinked molecule which can be detected in several different mammalian tissues and cell types (46). Ecto-5-NT enzyme activity catalyzes the extracellular dephosphorylation of nucleoside monophosphates to their corresponding nucleosides. This enables the uptake of adenosine, inosine, and guanosine into the cell and their subsequent reconversion into ATP and GTP in the purine salvage pathway (36). The physiological role of ecto-5-NT, however, probably differs in various organisms and tissues, and it most likely extends beyond its enzymatic activity (46). Plasma membraneCbound ecto5-NT (CD73) has been shown to be involved in controlling lymphocyteCEC interactions, as binding of lymphocytes to cultured EC can be inhibited by an anti-CD73 mAb (2, 3). Ecto-5-NT has also been NIC3 implicated in cellC matrix interactions in chicken fibroblasts (33) and as a signal transducing molecule in the human immune system (9, 24, 37). In particular, its role as a costimulatory molecule in T cell activation has been well established (14, 24). Transient expression of CD73 on neuronal cells has been described during developmental processes and, on lymphocytes, CD73 serves as a maturation marker, being absent from the surface of both immature B and NIC3 T cells (15, 37). A subpopulation of peripheral blood lymphocytes (PBL) expresses CD73 on the majority of B cells and CD8+ T cells but on only about 10% of CD4+ T cells (10, 38, 40). CD73/ecto-5-NT has also been detected in nervous tissue: on venules in various tissues and on follicular dendritic cells in the secondary lymphoid tissue (2, 10, 40). The subcellular expression of ecto-5-NT has been studied intensively in rat liver tissue where the molecule is expressed both intracellularly and on the surface of hepatocytes. A similar localization has been observed in rat fibroblasts, guinea pig neutrophils, and capillary EC (18, 23, 28, 45, 46). Continuous recycling of ecto-5-NT between the cell surface and the intracellular pools has been described in hepatocytes, fibroblasts, and rat hepatoma cells (31, 41, 45). The natural ligand(s) of CD73 are not known at present. As the diversity of ecto-5-NTCexpressing cells and tissues is substantial and the molecule NIC3 has several putative roles, it is important to clarify its functional and structural properties on different cell types. Thus, we studied properties of CD73 expressed on both cell types having importance in lymphocyte homing, PBL and vascular EC. Significant differences were found in the function and cell surface modulation of CD73 on these cell types. Materials and Methods Cells, Cell Lines, and Antibodies HUVECs (human umbilical vein endothelial cells) were isolated as described earlier (2). They were cultured on gelatin-coated flasks in EC basal medium (Clonetics Corp., San Diego, CA) supplemented with AOM 10% human AB-serum (Finnish Red Cross, Helsinki, Finland), 50 g/ml EC growth factor (Intl., Buckinghamshire, UK) using a dot blot apparatus (The Convertible, Filtration Manifold System; Intl.), following the manufacturer’s instructions. Light emission was detected using Hyperfilm MP (Intl.). Labeling and Immunoprecipitation of Cells Freshly isolated PBL and HECs were iodinated with 125I by the lactoperoxidase method (17)..