P-SELECTIN (P-SEL) SUPPORT OF NEONATAL NEUTROPHIL (N-PMN) ROLLING: CONTRIBTUION OF NEUTROPHIL P-SELECTIN-GYLCOLIGAND-1 (PSGL-1) † 1351

Marginating PMNs roll along the post-capillary wall prior to stopping and emigrating. This process is mediated in part by P-SEL on the endothelial cell and its ligand, PSGL-1, and requires the presence of sialyl Lewis^x and sulfation of one or more tyrosine in N-terminal sequence. We have previously shown that while P-SEL supports adult PMN (A-PMN) rolling, N-PMNs demonstrate an impairment in their ability to roll, and require CD11a/CD18 to resist detachment to increased shear stress (Ped Res 1996;39:302A). We hypothesize that these findings are in part due to differences in neonatal PSGL-1. Here we investigate N-PMN rolling on a confluent cell line expressing human P-SEL under two conditions: 1)attachment under continuous flow at a wall shear stress of 2 dyn/cm²; 2)detachment with step-wise increases in shear stress (0.6-22 dyn/cm²) in the presence of an anti-CD11a MoAb after a two minute stationary contact between PMNs and monolayer. We developed and purified a polyclonal antibody directed against the N-terminal sequence of PSGL-1 (anti-PSGL Ig) containing the putative sulfation sites. Using flow cytometry we could detect no statistical differences in the amount of PSGL-1 on N-PMNs compared to A-PMNs (173±43 and 210±60, respectively). In the attachment assay under continuous flow, anti-PSGL Ig inhibited the number of rolling N-PMNs (109±33) and A-PMNs (125±94) significantly compared to control Ig (N-PMN: 599±108; A-PMN: 651±57, p<0.01). In the detachment assay, anti-PSGL Ig decreased the number of both N-PMNs and A-PMNs at 0.6 dyn/cm² compared to control Ig by 80% and 60% respectively. With anti-PSGL Ig treatment the number of A-PMN rolling was not statistically different than N-PMNs at each shear stress. However, A-PMN and N-PMN rolling after stationary adhesion could be discriminated by two conditions: 1) the number of control Ig treated N-PMNs significantly decreased at shear stress of 12 and 22 dyn/cm²; 2) with anti-PSGL Ig treatment the number of N-PMNs significantly decreased at the highest shear stress. Adult PMN adhesion was not affected. PSGL-1 supports the rolling of both N- and A-PMNs on P-SEL. Nonetheless, there appear to be functional differences in either neonatal PSGL-1 or other ligands for P-SEL which become manifested at increased shear stress.