Abstract. Leukocyte interactions with vascular endothelium at sites of inflammation can be dynamically regulated by activation-dependent adhesion molecules. Current models, primarily based on studies with polymorphonuclear leukocytes, suggest the involvement of multiple members of the selectin, integrin, and immunoglobulin gene families, sequentially, in the process of initial attachment (rolling), stable adhesion (arrest), spreading and ultimate diapedesis. In the current study, IL-4-activated human umbilical vein endothelium, which selectively expresses VCAM-1 and an L-selectin ligand but not E-selectin, and appropriate function blocking monoclonal antibodies, were used to study monocyte-endothelial interactions in an in vitro model that mimics microcirculatory flow conditions. In this system, L-selectin mediates monocyte rolling and also facilitates ouBl-integdn-dependent arrest, whereas/~-integrins are required for spreading of firmly attached monocytes on the endothelial cell surface but not their arrest. These findings provide the first in vitro evidence for human monocyte rolling on cytokine-activated endothelium, and suggest a sequential requirement for both/~1-and/~2-integfin-dependent adhesive mechanisms in monocyte-endothelial interactions.p ERIPHERAL blood monocytes interact with the vascular endothelial lining as an initial step in a wide range of pathological processes including acute and chronic inflanunation, immune reactions, and atherosclerosis (12,13,45). As a consequence of their transendothelial migration, monocytes are recruited into tissues, organs and body cavities, undergo maturation to macrophages, and participate in defending the host against invading pathogens and regulating the behavior of vascular and non-vascular cells through the secretion of cytokines and other chemical mediators.Early in vitro studies of monocyte adhesion to cultured endothelial cells were typically performed under static conditions and indicated that basal adhesion of purified blood monocytes was relatively high

Abstract. L-selectin expressed by granulocytes, lymphocytes, and monocytes is responsible for initial leukocyte attachment to inflamed endothelium and high endothelial venules of peripheral lymph nodes. After leukocyte activation in vitro, L-selectin is rapidly shed from the cell surface. In this study, shed L-selectin (sL-selectin) from both lymphocytes and neutrophils was demonstrated to be present in high levels in human plasma by Western blot analysis and using a quantitative ELISA. In serum from normal human blood donors, a mean sL-selectin level of 1.6 ___ 0.8 #g/ml (n = 63) was found by ELISA. In addition, semipurified sL-selectin from plasma inhibited L-selectinspecific attachment of lymphocytes to cytokineactivated endothelium in a dose-dependent manner. L-selectin-dependent leukocyte attachment was completely inhibited at sL-selectin concentrations of 8-15 /zg/ml, while physiological concentrations of sLselectin caused a small but consistent inhibition of lymphocyte attachment, sL-selectin in plasma also inhibited anti-L-selectin mAb (2-5 #g/ml) binding to the surface of leukocytes. Interestingly, one epitope present within the EGF-like domain of L-selectin was lost in sL-selectin, suggesting a conformational change in the structure of the receptor after shedding. The presence of serum sL-selectin with functional activity indicates a potential role for sL-selectin in the regulation of leukocyte attachment to endothelium.T HE ability of leukocytes to leave the circulation and to migrate into tissues is a critical feature of the immune response. Several adhesion molecules are involved in the process of adhesion and transmigration of leukocytes through vascular endothelium at sites of inflammation (45). One molecule responsible for the initial attachment of leukocytes to endothelium is L-selectin (Leukocyte Adhesion Molecule-1 [LAM-1] t MEL-14) (24,30,43,44). L-selectin is a member of the selectin family of adhesion molecules (3,11,27,36,37,49) that includes E-selectin (Endothelial-Leukocyte Adhesion Molecule-1 [ELAM-1]) (1, 2, 31, 32) and CD62 (P-selectin, PADGEM, GMP-140) (13,18,25,26). The selectins are derived from evolutionarily related genes (7,9,18,33,52), and are characterized by a NH2-terminal, Ca-dependent lectin domain, an epidermal growth factor (EGF)-like domain followed by multiple short consensus repeat (SCR) domains, a transmembrane region, and a cytoplasmic tail.L-selectin is expressed on the surface of most leukocytes,

Abstract. Vascular cell adhesion molecule 1 (VCAM-1), a member of the Ig superfamily originally identified on activated endothelium, binds to the integrin very late antigen-4 (VLA-4), also known as ot4~l or CD49d/CD29, to support cell-cell adhesion. Studies based on cell adhesion to two alternatively spliced forms of VCAM-1 or to chimeric molecules generated from them and intercellular adhesion molecule-1 (ICAM-1) have demonstrated two VLA-4 binding sites on the predominate form of VCAM-1. Here, we studied VLA-4-dependent adhesion of the lymphoid tumor cell line Ramos to cells expressing wild type and mutant forms of VCAM-1. Results based on domain deletion mutants demonstrated the existence and independence of two VLA-4-binding sites located in the first and fourth domains of VCAM-1. Results based on amino acid substitution mutants demonstrated that residues within a linear sequence of six amino acids found in both domain 1 and 4 were required for VLA-4 binding to either domain. Five of these amino acids represent a conserved motif also found in ICAM domains. We propose that integrin binding to these Ig-like domains depends on residues within this conserved motif. Specificity of integrin binding to Ig-like domains may be regulated by a set of nonconserved residues distinct from the conserved motif.