Molecular Characterization of Rat Leukocyte P-Selectin Glycoprotein Ligand-1 and Effect of Its Blockade: Protection from Ischemia-Reperfusion Injury in Liver Transplantation

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.

FK330, a Novel Inducible Nitric Oxide Synthase Inhibitor, Prevents Ischemia and Reperfusion Injury in Rat Liver Transplantation

“…Snap frozen liver samples were processed, as described (7,8). Primary antibodies (Abs) against P-selectin, (CD62P; Pharmingen, San Diego, CA, USA), intracellular adhesion molecule 1 (ICAM-1; Pharmingen), ED-1 (macrophage/monocyte; CHEMICON International, Inc., Temecula, CA, USA), CD3 (T cell; Pharmingen) were added at optimal dilutions.…”

“…However, FK330 treatment effectively reduced these cytokine, which otherwise up-regulate the expression of adhesion molecules favoring leukocyte-sinusoidal EC interactions and triggering cytokine cascades. Leukocyte-EC interactions are well established as playing a role in the pathophysiology of hepatic IRI (7,8). Initial leukocyte tethering in sinusoidal EC requires expression of CD62 selectins.…”

“…It causes up to 10% of early liver transplant failure, and can lead to the higher incidence of acute and chronic rejection (1). The mechanism of IRI involves microcirculatory flow disturbances caused by endothelial cell (EC) adhesion, leukocyte tethering with subsequent sequestration in tissue, activation of Kupffer cells leading to the release of pro-inflammatory cytokines and chemokines, as well as the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), resulting in sinusoidal EC death and ultimately in hepatocyte damage (1)(2)(3)(4)(5)(6)(7)(8).…”

Antibody immunosuppressive therapy in solid organ transplant

“…With infusion of PSLG-1 antibody prior to reperfusion, the study showed a reduction in hepatic injury and inflammation. 18 Although the specific pathophysiology of IRI has been elucidated, recipient that was successfully treated with eculizumab in conjunction with plasmapheresis, IVIg and rituximab. The authors concluded that eculizumab's benefit resulted from the prevention of antibody-induced damage while plasmapheresis and IVIg cleared donor-specific antibodies.…”

Platelet-mediated adhesion facilitates leukocyte sequestration in hypoxia-reoxygenated microvessels

“…However, the treatment of the monoclonal antibody against the leukocyte adhesion molecule alleviates I-R injury and reduces the no-reflow incidence (Duilio et al, 2001). The interventions against adhesion molecules on endothelial cells (ECs) also exhibit beneficial effects (Dulkanchainun et al, 1998;Garcia-Criado et al, 1995;Tsuchihashi et al, 2006;Wong et al, 1997). These results suggest that leukocyte adhesion to the endothelium is the initial power behind leukocyte TEM or sequestration.…”