Preferential homing of naive lymphocytes to secondary lymphoid organs is thought to involve the action of chemokines, yet no chemokine has been shown to have either the expression pattern or the activities required to mediate this process. Here we show that a chemokine represented in the EST database, secondary lymphoid-tissue chemokine (SLC), is expressed in the high endothelial venules of lymph nodes and Peyer's patches, in the T cell areas of spleen, lymph nodes, and Peyer's patches, and in the lymphatic endothelium of multiple organs. SLC is a highly efficacious chemoattractant for lymphocytes with preferential activity toward naive T cells. Moreover, SLC induces firm adhesion of naive T lymphocytes via ␤2 integrin binding to the counter receptor, intercellular adhesion molecule-1, a necessary step for lymphocyte recruitment. SLC is the first chemokine demonstrated to have the characteristics required to mediate homing of lymphocytes to secondary lymphoid organs. In addition, the expression of SLC in lymphatic endothelium suggests that the migration of lymphocytes from tissues into efferent lymphatics may be an active process mediated by this molecule.

The leukocyte adhesion molecule, L-selectin, mediates the recruitment of lymphocytes to secondary lymphoid organs via interactions with specific ligands presented on high endothelial venules (HEV). Although the HEV-derived ligands for L-selectin are still incompletely defined, they share a common sialomucin-like structure which is thought to present clustered oligosaccharides to the lectin domain of L-selectin. Podocalyxin-like protein (PCLP) is a transmembrane sialomucin that is similar in structure to the well-characterized L-selectin ligand CD34. PCLP has been shown previously to be expressed on the foot processes of podocytes in the kidney glomerulus as well as on vascular endothelium at some sites. We have determined that PCLP is present on HEV, where it binds to both recombinant L-selectin and the HEV-specific monoclonal antibody MECA-79. Furthermore, purified HEV-derived PCLP is able to support the tethering and rolling of lymphocytes under physiological flow conditions in vitro. These results suggest a novel function for PCLP as an adhesion molecule and allow the definition of conserved structural features in PCLP and CD34, which may be important for L-selectin ligand function.

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Integrin activation, ligand binding, and integrin clustering were analyzed using alphaIIb beta3 reconstituted into phospholipid vesicles and into supported planar lipid bilayers. Strong and specific binding of fibrinogen and the gamma-chain dodecapeptide of fibrinogen to alphaIIb beta3 indicated that the integrin is in an activated state after membrane reconstitution. Cryoelectron and fluorescence microscopy suggested a nonclustered state of the protein in the vesicle membrane. Supported planar lipid membranes were generated by fusion of vesicles in which approximately equal fractions of integrins were pointing inside-out and outside-in. This distribution led to an immobilization of about 40% of the integrin in supported bilayers due to attachment of the large extracellular domains to the quartz support. Fluorescence recovery after photobleaching indicated a diffusion coefficient of D = (0.70 +/- 0.06) x 10(-8) cm2/s, consistent with a nonclustered state of the mobile integrin. Upon fibrinogen binding, the integrins became immobile, and fluorescence micrographs showed a patchy distribution of fibrinogen-integrin complexes consisting of approximately 250 molecules. In addition to the expected dimer formation by bivalent fibrinogen, additionally induced fibrinogen clustering may account for the large size of the complexes. In contrast, binding of monovalent GRGDS pentapeptide or the gamma-chain dodecapeptide of fibrinogen altered neither the mobile fraction nor the association state of alphaIIb beta3. Our data indicate that integrin alphaIIbb3 is activated while monodisperse, and became clustered upon fibrinogen binding, leading to an irreversibly bound state.