Activation of human leukocytes reduces surface P-selectin glycoprotein ligand-1 (PSGL-1, CD162) and adhesion to P-selectin in vitro

P-selectin glycoprotein ligand-l (PSGL-1), the primary ligand for P-selecti n, is constitutively expressed on the surface of circulating leukocytes. Th e objective of this study was to examine the effect of leukocyte activation on PSGL-1 expression and PSGL-1-mediated leukocyte adhesion to P-selectin, PSGL-1 expression was examined via indirect immunofluorescence and flow cy tometry before and after leukocyte stimulation with platelet activating fac tor (PAF) and PMA. Human neutrophils, monocytes, and eosinophils were all d emonstrated to have significant surface expression of PSGL-1 at baseline, w hich decreased within minutes of exposure to PAF or PMA. PSGL-1 was detecte d in the supernatants of PAF-activated neutrophils by immunoprecipitation. Along with the expression data, this suggests removal of PSGL-1 from the ce ll surface. Soluble PSGL-1 was also detected in human bronchoalveolar lavag e fluids. Down-regulation of PSGL-1 was inhibited by EDTA, However, inhibit ors of L-selectin shedding and other sheddase inhibitors did not affect PSG L-1 release, suggesting that PSGL-1 may be shed by an as Set unidentified s heddase or removed by some other mechanism. Functionally, PSGL-1 down-regul ation was associated with decreased neutrophil adhesion to immobilized P-se lectin under both static and flow conditions, with the most profound effect s seen under flow conditions. Together, these data indicate that PSGL-1 can be removed from the surface of activated leukocyte, and that this decrease in PSGL-1 expression has profound effects on leukocyte binding to P-select in, especially under conditions of flow.