Abstract
Introduction: In solid organ xenotransplantation the first interactions between the recipient immune system and the graft occur at the endothelium. Reactive oxygen species activate the endothelium, leading to modulation of endothelial phenotype and permeability. This process may be inhibited by free radical scavengers, such as the low toxic nitrone S-PBN (2-sulfophenyl-N-tertbutyl-nitrone). We previously showed that S-PBN significantly prolongs 15-deoxyspergualin-induced survival of mouse heart xenografts in rats. To study the mechanism by which S-PBN interferes with the early recipient immune responses to a graft, we established an in vitro model to study the influence of S-PBN on human peripheral blood mononuclear cell (PBMC) activation and adhesion on porcine endothelial cells (pEC). Methods: Human PBMC or pEC derived from Gala1,3Gal-KO minipigs were preincubated with S-PBN and cocultured in adhesion assays under shear stress for 20 min. Adhesion of T, B, and NK cells and monocytes was quantified by cell counting and staining for cell-lineage markers of adherent PBMC by flow cytometry. Cells were analyzed for the expression of adhesion and activation markers after S-PBN preincubation and after the adhesion assay. Influence of S-PBN on chemotactic activity in PBMC induced by pEC-derived soluble factors was analyzed in chemotaxis assays. Toxic effects of S-PBN on pEC and PBMC were analyzed by ECIS, microscopy, and propidium iodide staining. Results: Preincubation of human PBMC, but not pEC, with S-PBN decreased the PBMC adhesion in a dose-response dependent manner. Significant reduction of monocyte and T-cell adhesion was induced by 0.2 mM S-PBN or more and of NK and B cells at 1 mM S-PBN or more. The most pronounced inhibition of adhesion was detected in the monocyte population, reaching 30%, 44%, and 60% with 0.2, 1, and 5 mM S-PBN, respectively. S-PBN treatment induced a significant downregulation of the adhesion receptor L-selectin (CD62L) on PBMC but had no significant effect on the adhesion receptor expression on pEC. Further, induction of chemotactic activity in PBMC by pEC-derived soluble factors was reduced by S-PBN. No toxic effect of S-PBN on PBMC or pEC was detected at concentrations up to 25 mM, whereas 125 mM S-PBN was toxic for pEC. Conclusion: Non-toxic doses of S-PBN significantly reduce the adhesion of human PBMC on pEC, with the most pronounced effect on the monocyte subpopulation. The reduction may in part be due to a decreased adhesion receptor expression and inhibition of activation in PBMC. Thus, S-PBN may inhibit xenograft infiltration by attenuating human leukocyte interactions with the porcine endothelium.
Published Version
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