Cytoprotection of endothelial cells (EC) is important in EC biology and pathophysiology, including graft rejection. Using porcine aortic EC and human complement as an in vitro model of xenotransplantation, we have reported that ligation of EC Gal alpha (1-3)Gal epitopes (alpha Gal) with antibodies or lectins BS-I and IB4 induces EC resistance to injury by complement. However, before the protective response is observed, alpha Gal ligation induces an early, proinflammatory response. Using a similar model, we now investigated whether the early inflammatory response, as well as NF-kappa B activation, is required for induction of cytoprotection. Despite up-regulation of EC mRNA for many inflammatory cytokines rapidly after BS-I stimulation, recombinant cytokines or conditioned media from EC incubated with BS-I failed to induce protection when used to stimulate EC. While the lectin-induced inflammatory response was markedly reduced by inhibition of NF-kappa B, the protection from complement and apoptosis was unaffected. The lectins caused up-regulation of mRNA for protective genes A20, porcine inhibitor of apoptosis protein and hemoxygenase-1, which was not modified by NF-kappa B inhibition. These findings suggest that induction of cytoprotection in porcine EC by alpha Gal ligation results from activation of pathways that are largely independent of those that elicit NF-kappaB activation and the inflammatory response.