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J. Cell Biol.,
Volume 140, Number 3, February 9, 1998 721-731
The Center for Blood Research and Harvard Medical School, Department of Pathology, Boston, Massachusetts 02115
During the process of lymphocyte recirculation, lymphocytes bind via L-selectin to sulfated sialyl-Lewisx (sLex)-containing carbohydrate ligands expressed on the surface of high endothelial venules
(HEV). We have examined the expression of sLex on
HEV using a panel of mAbs specific for sLex and sLex-related structures, and have examined the function of
different sLex-bearing structures using an in vitro assay
of lymphocyte rolling on HEV. We report that three
sLex mAbs, 2F3, 2H5, and CSLEX-1, previously noted
to bind with high affinity to glycolipid-linked sLex, vary
in their ability to stain HEV in different lymphoid tissues and bind differentially to O-linked versus N-linked
sLex on glycoproteins. Treatment of tissue sections with
neuraminidase abolished staining with all three mAbs
but slightly increased staining with MECA-79, a mAb
to a sulfation-dependent HEV-associated carbohydrate
determinant. Treatment of tissue sections with O-sialoglycoprotease under conditions that removed the vast
majority of MECA-79 staining, only partially reduced
staining with the 2F3 and 2H5 mAbs. Using a novel
rolling assay in which cells bind under flow to HEV of frozen tissue sections, we demonstrate that a pool of
O-sialoglycoprotease-resistant molecules is present on
HEV that is sufficient for attachment and rolling of
lymphocytes via L-selectin. This interaction is not inhibited by the mAb MECA-79. Furthermore, MECA-79 mAb blocks binding to untreated sections by only
30%, whereas the sLex mAb 2H5 blocks binding by
~60% and a combination of MECA-79 and 2H5 mAb
blocks binding by 75%. We conclude that a pool of
O-glycoprotease-resistant sLex-like L-selectin ligands
exist on human HEV that is distinct from the mucin-associated moieties recognized by MECA-79 mAb. We
postulate that these ligands may participate in lymphocyte binding to HEV.
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