The Studies On PSGL-1-mediated Signaling During Leukocytes' Rolling Adhesion

The recruitment of leukocytes from the flowing bloodstream is a very rapid process that requires special mechanisms for the establishment of leukocyte-endothelium contacts. The selectins represent a class of cell adhesion molecules that is specialized for this purpose. Selectins and their ligands mediate the capture of leukocytes from the bloodstream and rolling of leukocytes along the endothelial cell surface. This initial contact is named rolling adhesion, which initiates not only the recruitment of leukocytes to inflamed tissue but also the homing and recycling of lymphocytes, the homing of hemopoietic progenitor to the marrow, thrombopoiesis, atherosclerosis, wound healing and tumor metastasis as well.Selectins, a new-found family of adhesion molecules, are Ca2+-dependent zoo-agglutinins expressed within vascular system and consist of P-, L- and E-selectin. Like all zoo-agglutinins, the tetrasaccharide sialyl Lewis* and its isomer sialyl Lewis are binding determinants for three selectins. Three selectins all show their high affinity to some macromolecules carrying sialyl-Lewis oligosaccharides (glycolipid or glycoprotein).PSGL-1 (P-selectin glycoprotein ligand 1) is a mucin expressed on microvilli of almost all types of leukocytes. Accumulating in vivo and in vitro studies have shown that PSGL-1 is a high-affinity ligand of three selectins. In addition to its direct role in capture of leukocytes from the bloodstream, PSGL-1 also has been demonstrated to function as a signal-transducing receptor and initiate a series of intracellular signal events during the activation of leukocytes, including activation of P2-integrin, tyrosine-protein phosphorylation, secretion of cytokines and transcriptional activation as well.The dynamics of F-actin-based cytoskeleton plays a important role in the process of leukocytes activation. Cytoskeleton dynamics is the basis of cell morphological changes, and leukocytes require drastic morphological changesduring homing and inflammation to enable themselves adhere and spread on the endothelial cells and transmigrate out of the endothelial barrier. Signals initiated from many leukocyte adhesion molecules (LFA-1, CD28, CD31 and L-selectin) can induce the alteration of the F-actin-based cytoskeleton. In order to investigate if the engagement of PSGL-1 and its ligand can also induce the alteration of the F-actin-based cytoskeleton in leukocytes during rolling adhesion, we isolated and prepared human peripheral blood neutrophils and crosslinked PSGL-1 with mAb. The results showed that PSGL-1 associated with cytoskeleton and became a detergent-insoluble component after antibody crosslinking. In addition, F-actin filaments assembled and redistributed, and along with this alteration of F-actin, PSGL-1 polarized to the end of cell where F-actin located. Treatment of cells with cytochalasin B eliminated the polarization of PSGL-1. Taken together, the results suggested that signals transduced by PSGL-1 can activate neutrophils and induce the assembly and redistribution of F-actin, and the polarization of PSGL-1 is under the direction of F-actin-based cytoskeleton.Except for the alteration of F-actin, we detected a new transcriptional-level signal event by the technique of transient transfection, luciferase assay and RT-PCR, that is the engagement of PSGL-1 promoted the transcriptional activity of gene CSF-1.Recently, the participation of non-receptor tyrosine kinase within the leukocyte signal pathway is an area of intense research. To determinate if there are non-receptor tyrosine kinase involved in the signal events observed in our study (the alteration of F-actin and the transcriptional activation of CSF-1), we emphatically investigated the role of non-receptor tyrosine kinase c-Abl and Syk respectively in the two signal pathways.First, we showed that c-Abl was redistributed and re-localized to the periphery or an end of the neutrophil where F-actin concentrated after antibody crosslinking of PSGL-1. Furthermore, STI571, a special inhibitor for c-Abl, obviously blocked the alteration of F-acti...