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

Abstract

Although most investigators agree that lung dysfunction occurs rapidly in various pig-to-primate hyperacute lung rejection (HALR) models, the basic mechanisms 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-alpha-Gal column adsorption; column with FUT; and column with K76) and two control groups (unmodified human blood; autologous pig blood) were studied. Each lung was biopsied serially during perfusion, and assessed using an immunohistochemical technique, with vWF staining as an internal control to quantitate binding of human IgM, IgG, C3, C5b-9, properdin, and C1q. The effect of each treatment and subsequent lung perfusion on IgG and IgM anti-alpha-Gal titers(by ELISA) and on pig endothelial cell cytotoxicity were correlated with histologic findings. We found that [1] the classical complement activation pathway was activated, as has been shown for other pig organs in primate or human blood environments [2]; alternative complement pathway activation is also seen, which has not been described for other organs in pig-to-primate models, but only in the context of classical pathway activation; and [3] anti-Gal column absorption, pharmacologic inhibition of complement, or combination therapy each was associated with histologic evidence of partial protection, consistent with what would be predicted for each intervention. Further, immunohistologic differences correlated with physiologic outcomes [8] and with antibody assay results, and revealed that treatments used were incompletely effective. Our data suggest that more complete inhibition of antibody- and complement-driven pathways than was achieved in these experiments will 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 application of pig-to-human lung xenografts.

Beyond Antibody-Mediated Rejection: Hyperacute Lung Rejection as a Paradigm for Dysregulated Inflammation

“…Nevertheless, unlike for other organs, where efficient antibody absorption reliably prevents hyperacute rejection, the results using analogous approaches have been disappointing in the lungs [12,17,19,24]. Although Gal specific antibody columns remove the majority of antispecies antibody, HALR is not reliably prevented [13,19,25]. Similarly, conventional plasmapheresis, removing all immunoglobulins as well as other plasma proteins, or columns which remove all immunoglobulins showed no significant advantages for the lung [24], in contrast to findings in other organs [26].…”

Hyperacute lung rejection in the pig-to-human model. III. platelet receptor inhibitors synergistically modulate complement activation and lung injury

“…Acetone-fixed 6-m sections were subsequently stained for immunohistologic analysis as described previously (18,19). Briefly, monoclonal antibodies specific for human immunoglobulin (Ig)M, C3b, C5b-9, and CD41 were used to assess XNA, complement, and platelet deposition in the lung tissue.…”

Hyperacute rejection of mouse lung by human blood: characterization of the model and the role of complement1

“…The lung tissue was divided for formalin fixation or snap-frozen with optimum cutting temperature in liquid nitrogen and stored at Ϫ70°C. Six-micron sections were prepared, fixed in acetone, and subsequently stained for immunohistologic analysis as described previously (15). Monoclonal antibodies anti-immunoglobulin (Ig)G (Pharmingen, San Diego, CA), anti-IgM (Immunotech, Westbrook, ME), and C3 (Quidel, San Diego, CA) (diluted at 1:50, 1:50, and 1:100, respectively) were used to assess antibody and complement deposition in the graft.…”

“…In addition, two soluble-phase complement inhibitors yield mechanistically similar results in both models. When our current findings are considered in the context of previous work using pig lungs (2,10,15) or other organs (16,17), we find that the characteristics of HALR are similar in detail (edema, hemorrhage, thrombosis, Igs, and complement tissue deposits) in the mouse and pig models: (A) Survival for organs perfused with unmodified blood is approximately 10 min; (B) PVR increases rapidly, and high PVR is the usual mode of graft failure in absence of complement inhibition; (C) IgG and IgM, including anti-␣Gal antibodies, are deposited on endothelium; (D) complement is activated and (E) deposited on endothelium, a process (F) refractory to regulation with soluble-phase complement inhibitors (10,11), and (G) intravascular platelet thrombi and neutrophil sequestration are prominent.…”