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Jens V. Stein^*, Guiying Cheng^*, Britt M. Stockton, Brian P. Fors^*, Eugene C. Butcher, and Ulrich H. von Andrian^*

From the ^* Center for Blood Research and the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115; and Department of Pathology, Tufts University, Boston, Massachusetts 02111; and the Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University Medical School, Stanford, California 94305; and The Veterans Affairs Palo Alto Health Care Systems, Palo Alto, California 94304

Adhesion receptors that are known to initiate contact (tethering) between blood-borne leukocytes and their endothelial counterreceptors are frequently concentrated on the microvilli of leukocytes. Other adhesion molecules are displayed either randomly or preferentially on the planar cell body. To determine whether ultrastructural distribution plays a role during tethering in vivo, we used pre-B cell transfectants expressing L- or E-selectin ectodomains linked to transmembrane/intracellular domains that mediated different surface distribution patterns. We analyzed the frequency and velocity of transfectant rolling in high endothelial venules of peripheral lymph nodes using an intravital microscopy model. Ectodomains on microvilli conferred a higher efficiency at initiating rolling than random distribution which, in turn, was more efficient than preferential expression on the cell body. The role of microvillous presentation was less accentuated in venules below 20 µm in diameter than in larger venules. In the narrow venules, tethering of cells with cell body expression may have been aided by forced margination through collision with erythrocytes. L-selectin transfected cells rolled 10-fold faster than E-selectin transfectants. Interestingly, rolling velocity histograms of cell lines expressing equivalent copy numbers of the same ectodomain were always similar, irrespective of the topographic distribution. Our data indicate that the distribution of adhesion receptors has a dramatic impact on contact initiation between leukocytes and endothelial cells, but does not play a role once rolling has been established.

Key Words: selectins • lymphocyte homing • lymph nodes • microvilli • intravital microscopy

This study complies with NIH guidelines and was approved by the Institutional Review Committees on Animals of both Harvard Medical School and CBR. Animals were under complete surgical anesthesia throughout all experimental procedures.

J.V. Stein and G. Cheng contributed equally to this work.

Abbreviations used: BCECF, 2',7',-bis-(2-carboxyethyl)- 5(and-6) carboxyfluorescein; EC, endothelial cells; ED, ectodomain; HEV, high endothelial venules; LN, lymph node; MFI, mean fluorescence intensity; PLN, peripheral lymph nodes; PNAd, peripheral node addressin; PSGL-1, P-selectin glycoprotein ligand-1; TM/IC, transmembrane and intracellular.

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