Activation of the Lectin Pathway of Complement in Pig-to-Human Xenotransplantation Models

Background. Natural IgM containing anti-Gal antibodies initiates
classic pathway complement activation in xenotransplantation.
However, in ischemia-reperfusion injury, IgM also induces lectin
pathway activation. The present study was therefore focused on
lectin pathway as well as interaction of IgM and mannose-binding
lectin (MBL) in pig-to-human xenotransplantation models. Methods.
Activation of the different complement pathways was assessed by
cell enzyme-linked immunosorbent assay using human serum on
wild-type (WT) and alpha-galactosyl transferase knockout
(GalTKO)/hCD46-transgenic porcine aortic endothelial cells (PAEC).
Colocalization of MBL/MASP2 with IgM, C3b/c, C4b/c, and C6 was
investigated by immunofluorescence in vitro on PAEC and ex vivo in
pig leg xenoperfusion with human blood. Influence of IgM on MBL
binding to PAEC was tested using IgM depleted/repleted and anti-Gal
immunoabsorbed serum. Results. Activation of all the three
complement pathways was observed in vitro as indicated by IgM, C1q,
MBL, and factor Bb deposition on WT PAEC. MBL deposition
colocalized with MASP2 (Manders' coefficient 3D] r(2) = 0.93),
C3b/c (r(2) = 0.84), C4b/c (r(2) = 0.86), and C6 (r(2) = 0.80). IgM
colocalized with MBL (r(2) = 0.87) and MASP2 (r(2) = 0.83). Human
IgM led to dose-dependently increased deposition of MBL, C3b/c, and
C6 on WT PAEC. Colocalization of MBL with IgM (Pearson's
coefficient 2D] r(p)(2) = 0.88), C3b/c (r(p)(2) = 0.82), C4b/c
(r(p)(2) = 0.63), and C6 (r(p)(2) = 0.81) was also seen in ex vivo
xenoperfusion. Significantly reduced MBL deposition and complement
activation was observed on GalTKO/hCD46-PAEC. Conclusion.
Colocalization of MBL/MASP2 with IgM and complement suggests that
the lectin pathway is activated by human anti-Gal IgM and may play
a pathophysiologic role in pig-to-human xenotransplantation.