Immunohistologic evaluation of mechanisms mediating hyperacute lung rejection, and the effect of treatment with K76-COOH, FUT-175, and anti-Gal column immune adsorption

Although most investigators agree that lung dysfunction occurs rapidly in v arious pig-to-primate hyperacute lung rejection (HALR) models, the basic me chanisms mediating this phenomenon remain in question. Here we describe an immunohistochemical method for assessment of mechanisms driving HALR. Using an established model wherein piglet lungs are perfused ex vivo with human blood, six experimental groups (K76 COOH; FUT-175; K76 with FUT; anti-cc-Ga l column adsorption; column with FUT; and column with K76) and two control groups (unmodified human blood; autologous pig blood) were studied. Each lu ng was biopsied serially during perfusion, and assessed using an immunohist ochemical technique, with vWF staining as an internal control to quantitate binding of human IgM, IgG, C3, C5b-9, properdin, and Clq. The effect of ea ch treatment and subsequent lung perfusion on IgG and IgM anti-a-Gal titers (by ELISA) and on pig endothelial cell cytotoxicity were correlated with hi stologic findings. We found that [1] the classical complement activation pa thway was activated, as has been shown for other pig organs in primate or h uman blood environments [2]; alternative complement pathway activation is a lso seen, which has not been described for other organs in pig-to-primate m odels, but only in the context of classical pathway activation; and [3] ant i-Gal column absorption, pharmacologic inhibition of complement, or combina tion therapy each was associated with histologic evidence of partial protec tion, consistent with what would be predicted for each intervention. Furthe r, immunohistologic differences correlated with physiologic outcomes [8] an d with antibody assay results, and revealed that treatments used were incom pletely effective. Our data suggest that more complete inhibition of antibo dy- and complement-driven pathways than was achieved in these experiments w ill be necessary to prevent the antibody and complement-mediated facets of hyperacute lung rejection. This immunohistologic technique may also help us identify additional pathogenic mechanisms important to eventual clinical a pplication of pig-to-human lung xenografts.