IgM‐dependent activation of the lectin pathway of complement in xenotransplantation

Abstract

Background: The classical pathway is the dominant initiator of complement activation in xenotransplantation. By amplification of C3b generation, the alternative pathway is also critical. However, little attention has been paid up to date to the involvement of the lectin pathway in xenograft rejection. Natural IgM, containing anti‐Gal, is a major initiator of classical pathway complement activation, but recently it has been shown that during ischemia/reperfusion injury, IgM also induces lectin pathway activation. Thus, the present study was focused on lectin pathway as well as interaction of IgM and MBL in a pig‐to‐human in vitro xenotransplantation model.Methods: Cell ELISA using porcine aortic endothelial cells (PAEC) and normal human serum (NHS) was used to assess activation of the different complement pathways. To confirm activation of the lectin pathway and to analyze the role of natural IgM in it's activation, co‐localized deposition of MBL/MASP2 with C3b/c, C4b/c & C6 and IgM with MBL & MASP2 was investigated by immunofluorescence (IF)/confocal microscopy on PAEC. Influence of IgM presence on MBL binding to PAEC was tested using IgM depleted/repleted and anti‐Gal immunoabsorbed NHS. Finally, tissue samples from ex vivo xenoperfusion of pig limbs with whole human blood were tested for IgM mediated lectin pathway activation by IF staining.Results: Activation of all the three pathways of complement system was observed in vitro as indicated by IgM, C1q, MBL and Factor Bb binding on PAEC. MBL deposition was co‐localized with MASP2, C3b/c, C4b/c and C6, suggesting a predominant role of the lectin pathway in xenograft rejection. IgM co‐localization with MBL and MASP2 as well as dose‐dependently increased deposition of MBL on PAEC in the presence of human polyclonal IgM, further supports the idea that upon deposition of IgM a binding site for MBL is exposed. In addition, co‐localized deposition of MBL with IgM, C4b/c and C6 was also observed on ex vivo xenoperfusion samples.Conclusion: The lectin pathway of complement activation was shown to be involved in xenotransplantation. Co‐localization of MBL / MASP2 with IgM and complement proteins indicate that lectin pathway activation in xenotransplantation is dependent on antigen recognition by naturalIgM. These findings suggest that, similar to ischemia/reperfusion injury, the lectin pathway has a functional role in endothelial damage in xenotransplantation.