Effects of glycosylation inhibitors on human CLA+ T-cell rolling on E-, P-, and L-selectins.
To assess the effects of glycosylation inhibitors on the de novo synthesis of selectin ligands expressed by CLA+ T cells, cells were treated with neuraminidase to cleave all preformed selectin ligand on the cell surface. Cells were then recultured for 30 hours in PBS (diluent control), tunicamycin (0.02 mM), swainsonine (0.23 mM), 4-F-GlcNAc (0.05 mM), BAG (2.0 mM), and GlcNAc (5.0 mM; negative drug control). Cell rolling assessments were made at 2.0 dyne/cm2 from the midpoint of the chamber viewing field (4 fields per selectin spot; 3 different experiments). When compared with recovery in PBS, tunicamycin, 4-F-GlcNAc, and BAG significantly inhibited the re-expression of E- and P-selectin ligand activity (panels A and B), whereas 4-F-GlcNAc and BAG also inhibited L-selectin ligand activity (panel C) (*P < .01; Student ttest). Inhibition of P- and L-selectin–mediated T-cell rolling after bromelain treatment confirmed the role of PSGL-1 and revealed that a possible glycolipid component might be contributing to residual E-selectin ligand activity. # indicates that 0.02 mM tunicamycin inhibited protein synthesis and PSGL-1 expression as determined by flow cytometry and autoradiography.
