Related Experiment Videos
Expression and function of P-selectin glycoprotein ligand 1 (CD162) on human basophils
M L Taylor1, M E Brummet, S A Hudson
1Department of Medicine, Division of Clinical Immunology, The Johns Hopkins University School of Medicine, Johns Hopkins Asthma and Allergy Center, Baltimore, MD 21224-6801, USA.
Background:
The endothelial cell adhesion molecule P-selectin may contribute to selective leukocyte migration in allergic diseases by binding to its ligand, P-selectin glycoprotein ligand 1 (PSGL-1), on eosinophils and other leukocytes. Although expression of PSGL-1 on basophils has been detected in leukocyte typing workshops, its function on basophils has not been explored.
Objective:
We sought to characterize the expression and function of PSGL-1 on human basophils and a basophil-like cell line (KU812) and to compare these characteristics with those for PSGL-1 on eosinophils and neutrophils.
Methods:
Basophils, eosinophils, and neutrophils were enriched from peripheral blood by using density gradient centrifugation and immunomagnetic negative selection. KU812 cells were cultured by using standard techniques. Indirect immunofluorescence and flow cytometry were used to determine surface PSGL-1 expression under various conditions, and Western blotting was used to analyze the molecular forms of PSGL-1 on each cell type. Static adhesion assays were performed by using immobilized recombinant P-selectin and relevant blocking antibodies. Histamine release assays were done by using adherent and nonadherent basophils to determine whether adhesion by means of PSGL-1 altered basophil releasability.
Results:
The expression of PSGL-1 on basophils was similar to that on neutrophils but was approximately 30% less bright than levels on eosinophils. Levels on basophils were 10-fold higher than on KU812 cells. Basophil activation by means of IgE cross-linking resulted in reductions in surface expression of PSGL-1 and L-selectin, as well as increased CD11b expression. Western blot analysis of PSGL-1 revealed that the molecular weights of the bands for neutrophils and basophils were similar, whereas those for eosinophils were of greater molecular weights. Static adhesion assays demonstrated that basophils bound well to P-selectin, whereas KU812 cells bound poorly. Adhesion of basophils to P-selectin was completely blocked by antibodies to either P-selectin or PSGL-1. Finally, adhesion to P-selectin did not alter the magnitude or kinetics of anti-IgE-induced histamine release.
Conclusion:
Expression of PSGL-1 on basophils is more similar to that on neutrophils than that on eosinophils. KU812 cells express much lower levels of this molecule but, like basophils and other cells, bind to P-selectin by means of PSGL-1. P-selectin expression at sites of allergic inflammation is likely to play an important role in human basophil recruitment, but adhesion by means of PSGL-1 does not alter IgE-dependent basophil histamine release.