Direct real-time observation of E- and P-selectin-mediated rolling on cutaneous lymphocyte-associated antigen immobilized on Western blots.
Human memory T cells associated with cutaneous inflammatory responses are characterized by their expression of cutaneous lymphocyte-associated Ag (CLA), a carbohydrate determinant differentially expressed on P-selectin glycoprotein ligand-1 (PSGL-1). Although expression of the CLA epitope on PSGL-1 (CLA(+) PSGL-1) by memory T cells is associated with acquisition of E-selectin ligand activity, it is not known whether CLA(+) PSGL-1, itself, is a ligand for E-selectin on human T cells or whether other glycoproteins, with or without CLA modification, support E-selectin-dependent rolling in shear flow. To address this issue, we developed a method for real-time analysis of functional adhesive interactions between selectin-bearing cells in shear flow with leukocyte ligands resolved by SDS-PAGE and immobilized on standard Western blots. The results of these studies provide direct evidence that CLA(+) PSGL-1 is a functional ligand for both E- and P-selectin, confirm that the P-selectin ligand activity of PSGL-1 is independent of CLA modification, and identify a distinct, non-PSGL-1 E-selectin ligand on CLA-positive human memory T cells.
Fuhlbrigge
Robert C
RC
Department of Dermatology, Brigham and Women's Hospital, Boston, MA 02115, USA.
King
Sandra L
SL
Dimitroff
Charles J
CJ
Kupper
Thomas S
TS
Sackstein
Robert
R
D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte; D000951:Antigens, Neoplasm; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D019040:E-Selectin / Q000502:physiology*; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism* / Q000502:physiology*; D019007:P-Selectin / Q000502:physiology*
D000945:Antigens, Differentiation, T-Lymphocyte; D000951:Antigens, Neoplasm; C063419:CTAGE1 protein, human; D019040:E-Selectin; D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein