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,

Reactive hemophagocytic syndrome (RHS) is a disease of overwhelming macrophage activity triggered by infection, malignancy or autoimmune disorders. Currently used laboratory markers for the quantitative assessment of monocyte/macrophage activation lack lineage-restricted expression patterns and thus specificity. Serum levels of the macrophage specific scavenger receptor CD163 were determined by enzyme-linked immunosorbent assay (ELISA) and were found to be highly increased in patients with RHS (median 39.0 mg/L). Significantly lower levels were determined in patients with sepsis (median 9.1 mg/L), acute mononucleosis (median 8.2 mg/L), Leishmania infection (median 6.7 mg/L) and healthy controls (median 1.8 mg/L). Follow-up of patients with a relapsing course of the disease revealed close correlations of sCD163 with clinical disease activity, serum ferritin and other markers of macrophage activity. Large sinusoidal accumulations of CD163 expressing macrophages actively engaged in phagocytosis of blood cells were detected in spleen sections of RHS patients. Our data suggests sCD163 to be a macrophage-specific marker in patients with disorders of inappropriate macrophage activation.

In an attempt to understand the regulatory mechanisms governing passage of neutrophils from the vascular bed to the interstitial tissue, we analyzed the effect of the pleiotropic monokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) on transendothelial neutrophil traffic. Short-time preincubation of human umbilical vein endothelial cell (HUVE) monolayers with IL-1 and TNF led to an impressive time-and dose-dependent increase of endothelial cell-associated neutrophils when working in a full plasma system on petri dishes. Electron microscopic analysis revealed junctional penetration of monolayers by neutrophils. More quantitatively, when using a monolayer-on-filter-system, priming led to a severalfold increase in complete layer passage occurring in the absence of an external chemotactic gradient. Direct comparison with an upside-down modification of the system together with data demonstrating the vectorial behavior of such migration revealed that IL-i-stimulated transendothelial neutrophil traffic is polarized. The described enhancement of neutrophil transendothelial passage was found to be a unique feature of IL-1/TNF-activated HUVE since HUVE-dependent transmigration potentiation was not observed as a consequence of mere neutrophil attachment to endothelial cells (e.g., induced by Fcmediated adherence of PMN to HUVE). IL-1 acts selectively on endothelial cells as demonstrated by total inhibition of its effect by actinomycin D. Moreover, IL-1 does not induce HUVE monolayers to secrete a chemotaxin, and the neutrophil passage guiding principle is removable from the HUVE surface by short trypsin exposure. Congruent results were obtained with human adult arterial as well as saphenous vein endothelial cells. As shown by blockade of neutrophil migration with pertussis toxin, IL-1-and TNF-inducible transendothelial migration can be dissected into an initial anchoring step, which is succeeded by active neutrophil migration, possibly along a putative endothelial membrane-bound gradient.

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