No abstract

P-selectin glycoprotein ligand-1 (PSGL-1) mediates the initial tethering of leukocytes to activated platelets and endothelium. We report molecular cloning and characterization of the rat PSGL-1 gene. A neutralizing Ab was generated, and its binding epitope was mapped to the N-terminal binding region of rat PSGL-1. We examined the effects of early PSGL-1 blockade in rat liver models of cold ischemia, followed by ex vivo reperfusion or transplantation (orthotopic liver transplantation (OLT)) using an anti-PSGL-1 Ab with diminished Fc-mediated effector function. In the ex vivo hepatic cold ischemia and reperfusion model, pretreatment with anti-PSGL-1 Ab improved portal venous flow, increased bile production, and decreased hepatocellular damage. Rat pretreatment with anti-PSGL-1 Ab prevented hepatic insult in a model of cold ischemia, followed by OLT, as assessed by 1) decreased hepatocellular damage (serum glutamic oxaloacetic transaminase/glutamic-pyruvic transaminase levels), and ameliorated histological features of ischemia/reperfusion injury, consistent with extended OLT survival; 2) reduced intrahepatic leukocyte infiltration, as evidenced by decreased expression of P-selectin, ED-1, CD3, and OX-62 cells; 3) inhibited expression of proinflammatory cytokine genes (TNF-α, IL-1β, IL-6, IFN-γ, and IL-2); and 4) prevented hepatic apoptosis accompanied by up-regulation of antiapoptotic Bcl-2/Bcl-xL protective genes. Thus, targeting PSGL-1 with a blocking Ab that has diminished Fc-mediated effector function is a simple and effective strategy that provides the rationale for novel therapeutic approaches to maximize the organ donor pool through the safer use of liver transplants despite prolonged periods of cold ischemia.

P-Selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like glycoprotein expressed on the surface of leukocytes that serves as the major ligand for the selectin family of adhesion molecules and functions in leukocyte tethering and rolling on activated endothelium and platelets. Previous studies have implicated the highly conserved cytoplasmic domain of PSGL-1 in regulating outside-in signaling of integrin activation. However, molecules that physically and functionally interact with this domain are not completely defined. Using a yeast two-hybrid screen with the cytoplasmic domain of PSGL-1 as bait, a novel protein designated selectin ligand interactor cytoplasmic-1 (SLIC-1) was isolated. Computer-based homology search revealed that SLIC-1 was the human orthologue for the previously identified mouse sorting nexin 20. Direct interaction between SLIC-1 and PSGL-1 was specific as indicated by co-immunoprecipitation and motif mapping. Colocalization experiments demonstrated that SLIC-1 contains a Phox homology domain that binds phosphoinositides and targets the PSGL-1/SLIC-1 complex to endosomes. Deficiency in the murine homologue of SLIC-1 did not modulate PSGL-1-dependent signaling nor alter neutrophil adhesion through PSGL-1. We conclude that SLIC-1 serves as a sorting molecule that cycles PSGL-1 into endosomes with no impact on leukocyte recruitment.Supporting Information for this article is available at http://www.wiley-vch.de/contents/jc_2040/2008/37777_s.pdf IntroductionThe initial tethering and rolling of leukocytes on activated endothelium and platelets is a key biological event mediated by P-selectin glycoprotein ligand-1 (PSGL-1), a mucin-like glycoprotein that functions as a unique high-affinity ligand for the selectin family of adhesion molecules (for reviews, see [1][2][3][4]). Structurally, PSGL-1 is a type I integral membrane protein consisting of an N-terminal selectin-binding domain followed by a region of multiple decameric repeats bearing O-linked glycans, a short transmembrane domain, and lastly a cytoplasmic tail [5][6][7][8][9][10]. Comparison of human and murine PSGL-1 reveals that the 69-amino acid intracellular domain of PSGL-1 is highly conserved. While overall identity between human and Abbreviations: EEA-1: early endosomal antigen-1 Á EGFP: enhanced green fluorescent protein Á ERM: ezrin/radixin/ moesin Á L-E/I: L cell monolayer expressing E-selectin and ICAM-1 Á PI: phosphatidylinositol Á PI 3-kinase: phosphatidylinositol 3-kinase Á PSGL-1: P-selectin glycoprotein ligand-1 Á PtdIns(3)P: phosphatidylinositol 3-phosphate Á PtdIns(3,4)P 2 : phosphatidylinositol 3,4-bisphosphate Á PtdIns(3,4,5)P 3 : phosphatidylinositol 3,4,5-trisphosphate Á PX domain: phox homology domain Á SLIC-1: selectin ligand interactor cytoplasmic-1 Á SNX20: sorting nexin 20 Á TM: transmembrane Ulrich Y. Schaff et al.

No abstract

No abstract

No abstract