Sulfation of L-selectin ligands: An in vivo, biochemical, and biophysical analysis

Lymphocytes from the blood home to secondary lymphoid tissues through a process of tethering, rolling, firm adhesion, and transmigration. Specialized high endothelial cells (HEC) which line the histologically distinct high endothelial venules (HEV) of lymphoid organs mediate this recruitment process. Tethering and rolling of lymphocytes is mediated by the interaction of L-selectin on lymphocytes with ligands expressed by HEC. These glycoprotein ligands require post-translational carbohydrate sulfation in order to be recognized by L-selectin. HEC-GlcNAc6ST is a sulfotransferase highly restricted to HEV. Mice genetically deficient in HEC-GlcNAc6ST have lost MECA79 reactivity and L-selectin ligand activity from the luminal aspect of HEV. Lymphocytes roll significantly faster and have reduced sticking in subcortical HEV of HEC-G1cNAc6ST-/- mice. These observations are consistent with decreased lymphocyte homing and reduced lymphocyte numbers in peripheral lymph nodes of HECGlcNAc6ST-/- mice. Whereas MECA79 dramatically inhibited lymphocyte rolling and homing in wild-type mice, MECA79 had no effect in HEC-GlcNAc6ST deficient mice. L-selectin ligands from HEC-GlcNAc6ST-/- mice incorporated significantly less sulfate and have diminished MECA79 reactivity compared to ligands from wild-type mice. However, lymphocyte rolling on purified GlyCAM-1 from BEC-GlcNAc6ST-/- mice was blocked by MECA79. These results suggest that alternative, non-MECA79 reactive L-selectin ligands are present in peripheral lymph nodes in the absence of HEC-GlcNAc6ST. A phenotypic analysis of resident lymphocytes in peripheral lymphoid tissues of HEC-GlcNAc6ST-/- mice was remarkable only for a relative deficiency of B-cells in peripheral lymph nodes. The leukocyte rolling flux fraction in HEV of Peyer's patches of HEC-GlcNAc6ST-/- mice was increased, whereas the leukocyte rolling flux fraction in TNFalpha-treated cremaster muscle was decreased.; The ability to maintain the phenotype of human tonsillar HEC in culture would aid in the analysis of the enzymes relevant to L-selectin ligand modification in human. The morphology of human HEC changes over time in culture and expression of the MECA79 epitope decreases with cellular proliferation. The addition of afferent human lymph to cultured BEC had variable effects on MECA79 expression. 35S-labeling of L-selectin ligands derived from human HEC in culture revealed that GlcNAc-6-SO4 is the dominant sulfated saccharide in human PNAd.