Human Polymorphonuclear Neutrophils are Recruited by Porcine Chemokines Acting on CXC Chemokine Receptor 2, and Platelet-Activating Factor

Abstract

Pig-to-human xenotransplantation is hampered by strong humoral and cellular immune responses, including acute vascular rejection (AVR). Infiltration of vascular xenografts by recipient polymorphonuclear neutrophils (PMN) is an early feature of AVR. Since little is known about the initiation of PMN recruitment, the present study investigated whether activated porcine endothelial cells (EC) release factors that induce human PMN recruitment. Primary and immortalized porcine aortic EC cultures were stimulated with phorbol-myristate acetate/ionomycin, lipopolysaccharide, tumor-necrosis factor-alpha, or interferon-gamma. The interleukin (IL)-8 concentration of porcine EC supernatants was tested by ELISA. Human and porcine PMN were isolated from peripheral blood by Ficoll sedimentation and centrifugation, characterized by morphology and flow cytometry, and analyzed for chemotaxis using Boyden chambers or Transwells. PMN chemokine receptor desensitization was determined by intracellular calcium-flux measurements. Porcine EC supernatants contained significant amounts of porcine IL-8 and triggered chemotaxis in both human and porcine PMN. Chemotaxis of porcine, but not human, PMN was inhibited by anti-porcine IL-8 antibodies and recombinant porcine IL-8 induced strong chemotaxis only in porcine PMN. Porcine EC supernatants desensitized human PMN CXC-chemokine receptor (CXCR) 2, but not CXCR1, a receptor for human IL-8. Human PMN chemotaxis induced by porcine EC supernatants was significantly inhibited by blocking CXCR2 and platelet-activating factor (PAF). Both chemokines acting via CXCR2 and PAF are released by porcine EC inducing efficient chemotaxis of human PMN. These mechanisms responsible for the recruitment of human PMN to porcine endothelium during cell-mediated rejection of xenografts represent potential targets for preventive strategies.