Of the several families of adhesion receptors involved in leukocyte-endothelial cell interactions, only the selectins have been shown to initiate leukocyte interaction under physiologic shear; indeed, beta 2 (CD18) intergrins responsible for neutrophil arrest are unable to engage without prior selectin-mediated rolling. In contrast, alpha 4 (CD49d) integrins are shown here to initiate lymphocyte contract ("tethering") in vitro under shear and in the absence of a selectin contribution. The alpha 4 integrin ligands MAdCAM-1 and VCAM-1 support loose reversible interactions including rolling, as well as rapid sticking and arrest that is favored following integrin activation. Moreover, alpha 4 beta 7 mediates L-selectin (CD62L)-independent attachment of blood-borne lymphocytes to lamina propria venules in situ. Scanning electron microscopy of alpha 4 beta 7hi lymphoid cells reveals that, like L-selectin, alpha 4 beta 7 is highly concentrated on microvillous sites of initial cellular contact, whereas the beta 2 integrin LFA-1 is excluded from villi. Thus, alpha 4 but not beta 2 integrins can initiate leukocyte adhesion under flow, a capacity that may be in part a function of topographic presentation on microvilli.

Leukocyte adhesion to endothelium requires specialized mechanisms for contact initiation under flow. L-selectin (CD62L), an efficient initiator of adhesion, is clustered on the tips of leukocyte microvilli. To test whether microvillous presentation is critical for contact formation ("tethering"), we transfected lymphoid cells with chimeras of L-selectin and CD44, an adhesion molecule that is excluded from microvilli. CD44 transmembrane and intracellular (TM-IC) domains targeted the L-selectin ectodomain to the planar body, whereas L-selectin TM-IC segments conferred CD44 ectodomain clustering on microvilli. Wild-type and chimeric transfectants bound similarly to anti-ectodomain MAbs in static assays, but MAb binding under flow was much more efficient in the context of microvillous presentation. Similarly, wild-type and chimeric L-selectin possessed equivalent lectin activity, but microvillous presentation dramatically enhanced contact initiation on a native ligand. These findings demonstrate a critical role for receptor topography in leukocyte adhesion and suggest a novel regulatory mechanism of leukocyte trafficking.

Oxidized LDL (oxLDL) has been identified as a potent stimulus of leukocyte adhesion to endothelium, a hallmark of early atherogenesis. A cytofluorometric study was performed to further characterize the mechanisms by which oxLDL stimulates the rapid adhesion of leukocytes to endothelium in vitro and in vivo. Incubation (30 minutes at 37 C) of whole blood (diluted with buffered saline to 1 x 10(6) leukocytes/ml) with oxLDL (0.85 mg LDL cholesterol/ml; oxidized by 7.5 mumol/L Cu2+ for 18 hours) but not native LDL stimulated the upregulation of CD11b/CD18 adhesion receptors on neutrophils (anti-leu-15 binding: 178 +/- 16% of baseline, P < 0.01, means +/- SD of n = 10 experiments) and on monocytes (169 +/- 34% of baseline, P < 0.01). This phenomenon was almost entirely inhibited by n-butanol or the vasoactive drug pentoxifylline (PTX), which also significantly reduced oxLDL-induced leukocyte adhesion to venular and arteriolar endothelium, as assessed by intravital microscopy on the dorsal skinfold chamber in hamsters (venules: 49 +/- 19 versus 120 +/- 34 cells/mm2, P < 0.05; arterioles: 9 +/- 4 versus 52 +/- 7 cells/mm2, P < 0.01) 30 minutes after intravenous injection of oxLDL (4 mg/kg body weight; means +/- SD of n = 7 hamsters per group). Butanol and PTX also significantly reduced the upregulation of CD11b/CD18 by f-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) but not by phorbol myristate acetate (PMA). Whereas fMLP and PAF stimulate leukocytes via binding to specific cell surface receptors and triggering complex signal transduction pathways, PMA bypasses these pathways and directly activates intracellular protein kinase C. By analogy, we propose that oxLDL upregulates CD11b/CD18 through its previously documented ability to stimulate the generation of second messengers. The effect of n-butanol and PTX on receptor presentation cannot be explained by changes in plasma membrane fluidity, as both agents failed to reverse the decrease in plasma membrane fluidity of neutrophils after stimulation with oxLDL, as assessed by fluorescence anisotropy measurement of the membrane marker diphenylhexatriene. Incubation of isolated neutrophils but not of whole blood with oxLDL resulted in a significant loss of L-selectin from the neutrophil surface (anti-TQ-1 binding: 40 +/- 13% of baseline, P < 0.01). A significant loss of this adhesion receptor on neutrophils and monocytes was also observed after stimulation of isolated neutrophils and whole blood with fMLP, PAF, and PMA.(ABSTRACT TRUNCATED AT 400 WORDS)

We have examined the topographical distribution of L-selectin on surface membrane domains of human lymphocytes and murine L1-2 cells transfected to express human L-selectin. L-selectin was immunolocalized using murine monoclonal DREG 200 Fab antibody and a 12 nm colloidal gold-conjugated secondary antibody. Cell surface morphology and surface distribution of gold-labelled L-selectin were visualized using backscatter electron images obtained by high-resolution, field emission scanning electron microscopy. The topographical morphologies of lymphocytes of both types were complex. The surface of human lymphocytes was composed of both microvilli and ruffles; that of the murine cells was composed of long microvilli and few, if any, ruffles. L-selectin on human lymphocytes was observed primarily as focal clusters on the apical surfaces of ruffles and microvilli. Similarly, on the transfected murine cells, L-selectin was detected predominantly on the apical surface of microvilli. We conclude that L-selectin has a common spatial distribution and clustered organization on all leukocytes examined to-date, and that these features of receptor expression likely facilitate rolling of circulating leukocytes on the endothelial surface.