Adhesion of artificial leukocytes to P-selectin: Study and application

Adhesion of leukocytes to endothelium is a critical step in the inflammatory response. P-selectin, a primary mediator of leukocyte adhesion, is expressed on vascular endothelium, in response to injury or inflammatory signals. A physiological ligand for P-selectin was previously identified (P-selectin glycoprotein ligand-1, PSGL-1), though biochemical requirements for rolling adhesion on P-selectin have not been established. We used a reconstituted, cell-free system to identify the minimal recognition unit for rolling adhesion on P-selectin. In contrast to previously published findings, we established that the tetrasaccharide sialyl Lewis X (sLeX) represents the minimal functional element for rolling adhesion on P-selectin. Using a panel of biochemically well-characterized PGSL-1 peptides, we further showed that the presence of sulfotyrosine, in addition to sLeX, enhances the strength of adhesion on P-selectin. We show that PSGL-1 containing sulfotyrosine groups in the absence of sLeX can also support rolling adhesion on P-selectin. Using techniques established for the investigation of P-selectin-mediated adhesion, we examined how rolling adhesion is influenced by particle size. A panel of differently sized beads having identical surface chemistry was used to determine how particle size modulates rolling adhesion. We found that the smallest beads studied roll with the slowest velocities, but that the largest bead studied (10.9 μm) roll with a lower velocity than beads that are 7.75 μm in diameter. Our observations appear to be explained by differences in the gap width between the particle and substrate for the different sizes of beads. Finally, the cell-free system was used as a model for the development of biodegradable microspheres to selectively bind selectin-coated surfaces (model endothelium). An oil-in-water emulsion/solvent evaporation technique was used to fabricate micropheres from poly-lactic-co-glycolic acid (PLGA), a biodegradable polyester. PLGA microspheres were shown to be able to encapsulate fluorescent dyes (model drugs), and their surface was modified to express carbohydrate ligands for the selectins. As a result, specific adhesion to a selectin-coated surfaces was demonstrated