Differential Requirements for Core2 Glucosaminyltransferase for Endothelial L-Selectin Ligand Function In Vivo 1 Markus Sperandio,* S. Bradley Forlow,* Jayant Thatte,* Lesley G. Ellies, ‡ Jamey D. Marth, ‡ and Klaus Ley 2 * † L-selectin is a calcium-dependent lectin on leukocytes mediating leukocyte rolling in high endothelial venules and inflamed microvessels. Many selectin ligands require modification of glycoproteins by leukocyte core2 1,6-N-acetylglucosaminyltrans- ferase (Core2GlcNAcT-I). To test the role of Core2GlcNAcT-I for L-selectin ligand biosynthesis, we investigated leukocyte rolling in venules of untreated and TNF--treated cremaster muscles and in Peyer’s patch high endothelial venules (HEV) of Core2GlcNAcT-I null (core2 / ) mice. In the presence of blocking mAbs against P- and E-selectin, L-selectin-mediated leukocyte rolling was almost completely abolished in cremaster muscle venules of core2 / mice, but not littermate control mice. By contrast, leukocyte rolling in Peyer’s patch HEV was not significantly different between core2 / and control mice. To probe L-selectin ligands more directly, we injected L-selectin-coated beads. These beads showed no rolling in cremaster muscle venules of core2 / mice, but significant rolling in controls. In Peyer’s patch HEV, beads coated with a low concentration of L-selectin showed reduced rolling in core2 / mice. Beads coated with a 10-fold higher concentration of L-selectin rolled equivalently in core2 / and control mice. Our data show that endothelial L-selectin ligands relevant for rolling in inflamed microvessels of the cremaster muscle are completely Core2GlcNAcT-I dependent. In contrast, L-selectin ligands in Peyer’s patch HEV are only marginally affected by the absence of Core2GlcNAcT-I, but are sufficiently functional to support L-selectin-dependent leukocyte rolling in Core2GlcNAcT- I-deficient mice. The Journal of Immunology, 2001, 167: 2268 –2274. L eukocytes are migratory cells characterized by their abil- ity to leave the vascular system and transmigrate into sites of inflammation or into lymphoid tissue during lympho- cyte homing. The recruitment of leukocytes requires a multistep adhesion cascade that begins with leukocyte capture and rolling and leads to firm adhesion and transmigration (1–3). The early steps of leukocyte recruitment, capture, and rolling are largely me- diated by E-, P-, and L-selectin (4). L-selectin (CD62L) is consti- tutively expressed on most leukocytes, including granulocytes, monocytes, and naive and some memory lymphocytes. Observation of inflamed venules in vivo has shown that leuko- cytes (5, 6) and L-selectin transfectants (7) can roll by binding to unidentified endothelial L-selectin ligands. Usually, L-selectin co- operates with P- and E-selectin to mediate rolling, but L-selectin- dependent rolling can be isolated in E/P -/- mice (8) or in mice in which P- and E-selectin are blocked by mAbs (9). The physiolog- ically relevant L-selectin ligands in inflamed venules are unknown. L-selectin ligand activity can be induced by cytokine treatment of cultured endothelial cells (10), but the relevant glycoprotein bear- ing the ligand has not been identified. L-selectin ligands on in- flamed endothelial cells express fucosylation-dependent epitopes such as HECA452 (11). In addition, sensitivity to chlorate treat- ment (12) and data from two newly identified carbohydrate sulfo- transferases, CHST1 and CHST2 (13), suggest that sulfotrans- ferase activity is required for L-selectin ligand expressed by inflamed venules. In high endothelial venules (HEV) 3 of secondary lymphoid or- gans, L-selectin is the predominant selectin mediating leukocyte rolling (14). The importance of L-selectin for lymphocyte traffick- ing is evident in L-selectin-deficient mice. These mice show small, lymphocyte-depleted peripheral lymph nodes with reduced bind- ing of lymphocytes to HEV using the Stamper-Woodruff assay (15). L-selectin is also required for most leukocyte rolling in Pey- er’s patch HEV (16), but  4  7 integrin mediates sufficient rolling in L-selectin-deficient mice to ensure normal size and cellularity of Peyer’s patches. L-selectin ligands on HEV of secondary lymphoid organs require carbohydrate-based post-translational modifications for recognition. Similar to ligands for P- and E-selectin, L-selectin ligands are thought to carry sialylated and fucosylated sequences on core2 1,6-N-acetylglucosaminyltransferase (Core2GlcNAcT- I)-modified O-linked glycans (17, 18). L-selectin ligands on HEV also require sulfation on C-6 of N-acetylglucosamine and/or C-6 of galactose of sialyl Lewis x (sLe x ) (19, 20). The L-selectin ligand function of HEV can be blocked by the carbohydrate-binding mAb MECA-79, which helped to identify candidate L-selectin ligands on HEV, including glycosylation-dependent cell adhesion mole- cule-1 and CD34 (21). It is thought that L-selectin ligands on HEV are distinct from those expressed on inflamed venules. Because of the transient nature of the bond formed between L-selectin and its ligands, the direct demonstration of functional *Department of Biomedical Engineering, and † Cardiovascular Research Center, Uni- versity of Virginia, Charlottesville, VA 22908; and ‡ Howard Hughes Medical Insti- tute, University of California, La Jolla, CA 92119 Received for publication March 13, 2001. Accepted for publication June 6, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grants HL64381 and HL54136 (to K.L.) and National Institutes of Health Grant DK48247 (to J.D.M.). M.S. is supported by a stipend from the German Research Foundation (SP 621/1-1). L.G.E. is supported by National Cancer Institute Award K08-CA88035. 2 Address correspondence and reprint requests to Dr. Klaus Ley, Department of Bio- medical Engineering, University of Virginia Health Sciences Center, Box 377, Char- lottesville, VA 22908. E-mail address: kfl3@virginia.edu 3 Abbreviations used in this paper: HEV, high endothelial venule; FucT, fucosyl- transferase; Core2GlcNAcT-I, core2 1,6-N-acetylglucosaminyltransferase; core2 -/- , Core2GlcNAcT-I deficient. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00