Leukocyte Rolling in Rat Mesentery Venules: Distribution of Adhesion Bonds and the Effects of Cytoactive Agents YIHUA ZHAO, 1 SHU CHIEN, 1, RICHARD SKALAK, 1 and HERBERT H. LIPOWSKY 2 1 The Whitaker Institute of Biomedical Engineering and Department of Bioengineering, University of California, San Diego, La Jolla, CA and 2 Department of Bioengineering, Pennsylvania State University, University Park, PA (Received 11 February 2000; accepted 20 February 2001) Abstract—A new method of analyzing in vivo measurements of leukocyte WBC rolling along venular endothelium EC has been developed to extract insightful information on the dynam- ics of WBC–EC bond formation and disruption. The rolling velocity of WBCs was obtained by intravital microscopy of rat mesenteric venules. For the ‘‘spontaneous’’ rolling observed following exteriorization of the mesentery, we estimated that the average distance between clusters of adhesion bonds which tether a rolling cell to the venular wall was about 2 m, and that the average lifetime of a bond cluster at the trailing edge of the rolling cell, from its exposure to the tensile force to its release, was on the order of 0.05 s. Both the inter-cluster distance and the lifetime were significantly reduced by treat- ments with the chemoattractant N-formyl-methionyl-leucyl- phenylalanine and the cytokine interleukin-1, while the average lifetime of the stretched bond clusters was not significantly changed by treatment with the cytoskeleton-modifying agents cytochalasin B and colchicine. Each of the four treatments significantly reduced the heterogeneity in the cell rolling veloc- ity, presumably by the selective recruitment of WBC subsets from the circulating WBC population or by a reduction in the heterogeneity of endothelial adhesiveness. These results were analyzed in the context of in vitro data in the literature on molecular bonds of cell adhesion. The findings suggest that, in the case of ‘‘spontaneous’’ rolling, there are on average ap- proximately 2–3 clusters of adhesion bonds between a rolling cell and the vessel wall, and approximately five bonds in each cluster. © 2001 Biomedical Engineering Society. DOI: 10.1114/1.1366676 Keywords—Cell adhesion, Selectin, Integrin, In vivo, Intravi- tal microscopy, Cytokine, Heterogeneity, Leukocyte recruit- ment. INTRODUCTION The recruitment of leukocytes WBCs to sites of in- flammation involves a series of adhesive interactions with the vascular endothelial cell EC. Intravital micro- scopic observations have shown that, after their initial tethering, the WBCs first roll along the wall of postcap- illary venules, then firmly adhere to and spread on the vessel wall, and eventually emigrate from the microcir- culation into the surrounding tissue. The initial tethering and rolling adhesion are mediated mainly by selectins, whereas the firm adhesion is mediated by integrins. There are three known selectins. L-selectin is consti- tutively expressed by most circulating WBCs on their surfaces, and P- and E-selectins are expressed on stimu- lated ECs. Each of these selectins is capable of mediat- ing WBC rolling in vitro under flow conditions mimick- ing physiological states. 2,15,18,17 In vivo, the rolling adhesion may involve several adhesion pathways that work in a synergetic and overlapping manner. For ex- ample, trauma-induced WBC rolling in mesenteric venules is largely mediated by P- and L-selectins, with P-selectin playing a predominant role in the early phase and L-selectin at the later phase. 20 This type of in vivo WBC rolling is the animal model used in the control group of the present study. It has been shown that opti- mal rolling in vivo requires intercellular adhesion molecule-1 ICAM-1, 32 an EC ligand for WBC integrins LFA-1 lymphocyte function-associated molecule-1, or CD11a/CD18 and Mac-1 CD11b/CD18. In vitro stud- ies have shown that ICAM-1 alone cannot support neu- trophil rolling. 17 Two high-affinity ligands for the EC selectins have been identified: P-selectin glycoprotein ligand-1 PSGL-1 is expressed on most WBCs, can bind to both P- and E-selectins, and accounts for all or most of the P-selectin-dependent rolling of myeloid cells in vitro. 23 E-selectin ligand-1 ESL-1 is expressed by myeloid cells and some lymphoid cells, and is a major ligand for E-selectin. The EC selectins may also interact with the carbohydrate ligands presented by L-selectin. 18,25 How- ever, L-selectin can mediate WBC rolling in inflamma- tion in vivo independent of P- and E-selectins, indicating that the ECs of postcapillary venules must be able to present other inducible ligands for L-selectin. 19 Address correspondence to Shu Chien, The Whitaker Institute of Biomedical Engineering and Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093-0427. Electronic mail: schien@bioeng.ucsd.edu Richard Skalak is deceased. Annals of Biomedical Engineering, Vol. 29, pp. 360–372, 2001 0090-6964/2001/295/360/13/$15.00 Printed in the USA. All rights reserved. Copyright © 2001 Biomedical Engineering Society 360