Interleukin-8 mediates neutrophil-endothelial interactions in pig-to-human xenogeneic models.

Abstract

Human neutrophils are sequestered by pig lung xenografts within minutes during ex vivo perfusion. This phenomenon is not prevented by pig genetic modifications that remove xeno-antigens or added human regulatory molecules intended to down-regulate activation of complement and coagulation pathways. This study investigated whether recipient and donor interleukin-8 (IL-8), a chemokine known to attract and activate neutrophils during inflammation, is elaborated in the context of xenogeneic injury, and whether human or pig IL-8 promote the adhesion of human neutrophils in in vitro xenograft models. Plasma levels of pig, human or non-human primate (NHP) IL-8 from ex vivo pig lung perfusion experiments (n=10) and in vivo pig-to-baboon lung transplantation in baboons (n=22) were analysed by ELISA or Luminex. Human neutrophils stimulated with human or pig IL-8 were analysed for CD11b expression, CD18 activation, oxidative burst and adhesion to resting or TNF-activated endothelial cells (EC) evaluated under static and flow (Bioflux) conditions. For some experiments, human neutrophils were incubated with Reparixin (IL-8/CXCL8 receptor blocker) and then analysed as in the in vitro experiments mentioned above. Plasma levels of pig IL-8 (~6113pg/mL) increased more than human (~1235pg/mL) between one and four hours after initiation of ex vivo lung perfusion. However, pig IL-8 levels remained consistently low (<60pg/mL) and NHP IL-8 plasma levels increased by ~2000pg/mL after four hours in a pig-to-baboon lung xenotransplantation. In vitro, human neutrophils' CD11b expression, CD18 activation and oxidative burst all increased in a dose-dependent manner following exposure to either pig or human IL-8, which also were associated with increased adhesion to EC in both static and flow conditions. Reparixin inhibited human neutrophil activation by both pig and human IL-8 in a dose-dependent fashion. At 0.1mg/mL, Reparixin inhibited the adhesion of IL-8-activated human neutrophils to pAECs by 84±2.5%. Pig IL-8 increased in an ex vivo model of pig-to-human lung xenotransplantation but is not detected in vivo, whereas human or NHP IL-8 is elevated to a similar degree in both models. Both pig and human IL-8 activate human neutrophils and increase their adhesion to pig aortic ECs, a process significantly inhibited by the addition of Reparixin to human neutrophils. This work implicates IL-8, whether of pig or human origin, as a possible factor mediating in lung xenograft inflammation and injury and supports the evaluation of therapeutic targeting of this pathway in the context of xenotransplantation.