Hyaluronan anchoring and regulation on the surface of vascular endothelialcells is mediated through the functionally active form of CD44

CD44 on lymphocytes binding to its carbohydrate ligand hyaluronan can media te primary adhesion (rolling interactions) of lymphocytes on vascular endot helial cells. This adhesion pathway is utilized in the extravasation of act ivated T cells from the blood into sites of inflammation and therefore infl uences patterns of lymphocyte homing and inflammation. Hyaluronan is a glyc osaminoglycan found in the extracellular matrix and is involved in a number of biological processes. We have shown that the expression of hyaluronan o n the surface of endothelial cells is inducible by proinflammatory cytokine s. However, the manner through which hyaluronan is anchored to the endothel ial cell surface so that it can resist shear forces and the mechanism of th e regulation of the level of hyaluronan on the cell surface has not been in vestigated. In order to characterize potential hyaluronan receptors on endo thelial cells, we performed analyses of cell surface staining by flow cytom etry on intact endothelial cells and ligand blotting assays using membrane fractions, Hyaluronan binding activity was detected as a major species corr esponding to the size of CD44, and this was confirmed to be the same by Wes tern blotting and immunoprecipitation. Moreover, alterations in the surface level of hyaluronan after tumor necrosis factor-cu stimulation is regulate d primarily by changes in the cell surface levels of the hyaluronan-binding form of CD44. In laminar flow assays, lymphoid cells specifically rob on h yaluronan anchored by purified CD44 coated on glass tubes, indicating that the avidity of the endothelial CD44/hyaluronan interaction is sufficient to support rolling adhesions under conditions mimicking physiologic shear for ces. Together these studies show that CD44 serves to anchor hyaluronan on e ndothelial cell surfaces, that activation of CD44 is a major regulator of e ndothelial surface hyaluronan expression, and that the non-covalent interac tion between CD44 and hyaluronan is sufficient to provide resistance to she ar under physiologic conditions and thereby support the initial steps of ly mphocyte extravasation.