alpha-Neup5Ac-(2--3)-beta-D-Galp-(1--4)-[alpha-L-Fucp-(1--3)]-D-GlcpNAc and Dermatitis--Contact

Sialyl Lewis X (sLe(x)) antigen functions as a common carbohydrate determinant recognized by all three members of the selectin family. However, its expression and function in mice remain undefined due to the poor reactivity of conventional anti-sLe(x) monoclonal antibodies (mAbs) with mouse tissues. Here, we developed novel anti-sLe(x) mAbs, termed F1 and F2, which react well with both human and mouse sLe(x), by immunizing fucosyltransferase (FucT)-IV and FucT-VII doubly deficient mice with 6-sulfo-sLe(x)-expressing cells transiently transfected with an expression vector encoding CMP-N-acetylneuraminic acid hydroxylase. F1 and F2 specifically bound both the N-acetyl and the N-glycolyl forms of sLe(x) as well as 6-sulfo-sLe(x), a major ligand for L-selectin expressed in high endothelial venules, and efficiently blocked physiological lymphocyte homing to lymph nodes in mice. Importantly, both of the mAbs inhibited contact hypersensitivity responses not only when administered in the L-selectin-dependent sensitization phase but also when administered in the elicitation phase in mice. When administered in the latter phase, F1 and F2 efficiently blocked rolling of mouse leukocytes along blood vessels expressing P- and E-selectin in the auricular skin in vivo. Consistent with these findings, the mAbs blocked P- and E-selectin-dependent leukocyte rolling in a flow chamber assay. Taken together, these results indicate that novel anti-sLe(x) mAbs reactive with both human and mouse tissues, with the blocking ability against leukocyte trafficking mediated by all three selectins, have been established. These mAbs should be useful in determining the role of sLe(x) antigen under physiological and pathological conditions.

Topics: Animals; Antibodies; Base Sequence; Dermatitis, Contact; DNA Primers; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Fluorescent Antibody Technique; Humans; Leukocyte Rolling; Leukocytes; Mice; Mice, Inbred C57BL; Oligosaccharides; Polymerase Chain Reaction; Sialyl Lewis X Antigen

Lymphocyte homing is mediated by specific interaction between L-selectin on lymphocytes and the carbohydrate ligand 6-sulfo sialyl Lewis X on high endothelial venules. Here we generated mice lacking both core 1 extension and core 2 branching enzymes to assess the functions of O-glycan-borne L-selectin ligands in vivo. Mutant mice maintained robust lymphocyte homing, yet they lacked O-glycan L-selectin ligands. Biochemical analyses identified a class of N-glycans bearing the 6-sulfo sialyl Lewis X L-selectin ligand in high endothelial venules. These N-glycans supported the binding of L-selectin to high endothelial venules in vitro and contributed in vivo to O-glycan-independent lymphocyte homing in wild-type and mutant mice. Our results demonstrate the critical function of N-glycan-linked 6-sulfo sialyl Lewis X in L-selectin-dependent lymphocyte homing and recruitment.

Topics: Animals; Antigens, Surface; Cell Adhesion; Dermatitis, Contact; Endothelium, Lymphatic; L-Selectin; Lewis X Antigen; Lymph Nodes; Lymphocytes; Membrane Proteins; Mice; Mice, Knockout; Microscopy, Fluorescence; Oligosaccharides; Polysaccharides; Sialyl Lewis X Antigen

Lymphocyte homing is mediated by specific interactions between L-selectin on lymphocytes and sulfated carbohydrates restricted to high endothelial venules in lymph nodes. Here we generated mice deficient in both N-acetylglucosamine-6-O-sulfotransferase 1 (GlcNAc6ST-1) and GlcNAc6ST-2 and found that mutant mice had approximately 75% less homing of lymphocytes to the peripheral lymph nodes than did wild-type mice. Consequently, these mice had lower contact hypersensitivity responses than those of wild-type mice. Carbohydrate structural analysis showed that 6-sulfo sialyl Lewis X, a dominant ligand for L-selectin, was almost completely absent from the high endothelial venules of these mutant mice, whereas the amount of unsulfated sialyl Lewis X was much greater. These results demonstrate the essential function of GlcNAc6ST-1 and GlcNAc6ST-2 in L-selectin ligand biosynthesis in high endothelial venules and their importance in immune surveillance.

Topics: Animals; Carbohydrate Conformation; Carbohydrate Sequence; Carbohydrate Sulfotransferases; Cell Movement; Dermatitis, Contact; Endothelium, Lymphatic; L-Selectin; Leukocyte Rolling; Lewis X Antigen; Ligands; Lymph Nodes; Lymphocytes; Mice; Mice, Knockout; Mucins; Mutation; Oligosaccharides; Sialyl Lewis X Antigen; Substrate Specificity; Sulfotransferases

Inhibition of leukocyte migration into target organs has long been an attractive, though challenging, basis for anti-inflammatory strategies. However, to date, the manipulation of leukocyte rolling along blood vessels has not yielded successful new therapies. An important study may now open new avenues in this exciting field of anti-inflammatory therapies by introducing a putative inhibitor of poly-N-acetyllactosamine biosynthesis that affects selectin ligand activity and shows efficacy in a rodent skin inflammation model.

Topics: Acetylglucosamine; Animals; Antigens, Differentiation, T-Lymphocyte; Antigens, Neoplasm; Cell Movement; Dermatitis, Contact; E-Selectin; Endothelium, Vascular; Membrane Glycoproteins; Mice; Oligosaccharides; Sialyl Lewis X Antigen; Skin; T-Lymphocytes