[Show abstract][Hide abstract] ABSTRACT: Transplants from alpha1,3-galactosyltransferase (Gal) gene-knockout pigs to nonhuman primates are largely protected from hyperacute but not acute humoral xenograft rejection. The present study investigates the role of Gal in cytokine responses using a novel pig-to-human whole blood in vitro model, developed for species-specific analysis of porcine and human cytokines. Porcine (n = 7) and human (n = 27) cytokines were measured using ELISA or multiplex technology, respectively. Porcine aortic endothelial cells from control (Gal(+/+)) and Gal-deficient (Gal(-/-)) pigs were incubated with human lepirudin anticoagulated whole blood from healthy donors. E-selectin expression was measured by flow cytometry. The C3 inhibitor compstatin and a C5aR antagonist were used to study the role of complement. Cytokine species specificity was documented, enabling detection of 2 of 7 porcine cytokines and 13 of 27 human cytokines in one single sample. Gal(+/+) porcine aortic endothelial cells incubated with human whole blood showed a marked complement C5b-9 dependent up-regulation of E-selectin and secretion of porcine IL-6 and IL-8. In contrast, Gal(-/-) cells responded with E-selectin and cytokine expression which was so weak that the role of complement could not be determined. Human IL-6, IL-8, IFN-gamma, MIP-1alpha, MIP-1beta, eotaxin, and RANTES were detected in the Gal(+/+) system, but virtually no responses were seen in the Gal(-/-) system (p = 0.03). The increase in human cytokine release was largely complement dependent and, in contrast to the porcine response, mediated through C5a. Species-specific analysis of cytokine release revealed a marked, complement-dependent response when Gal(+/+) pig cells were incubated with human whole blood, compared with Gal(-/-) cells which induced virtually no cytokine release.
The Journal of Immunology 06/2008; 180(9):6346-53. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The generation of Galalpha1-3Gal (Gal) transferase deficient pigs has increased the interest in non-Gal antigens potentially representing important targets for xenoreactive antibody binding leading to xenograft rejection. The present study addressed the levels and immunoglobulin isotypes of preformed human anti-non-Gal antibodies and their potential to activate porcine endothelial cells.
Porcine endothelial cells lacking the Gal epitope (Gal-/-) were used to measure immunoglobulin (Ig) M and IgG subclass anti-non-Gal antibodies, using sera from 80 blood donors and pooled human AB serum. Antibodies specific for the non-Gal Hanganutziu-Deicher (HD) xenoantigen were measured by enzyme-linked immunosorbent assay. Activation of Gal-/- and Gal+/+ endothelial cells by human serum was measured, in the presence or absence of complement inhibitors, by E-selectin cell-surface expression using flow cytometry.
Anti-non-Gal antibody levels varied considerably among individual sera and comprised approximately 10% of total anti-porcine antibodies without sex or age differences. Among the IgG subclasses only IgG1 and IgG2 were detected. Human serum-induced E-selectin expression on Gal-/- cells was less than 20% compared with Gal+/+ cells, correlated with anti-HD IgM and IgG antibody levels (P=0.027 and 0.032, respectively), and was largely complement-independent in accordance with the lack of IgG3 anti-non-Gal antibodies. In contrast, E-selectin upregulation on Gal+/+ cells was reduced in complement blocking experiments.
Preformed anti-non-Gal antibodies, in particular anti-HD antibodies, were present in all human sera samples, activated porcine endothelial cells, and may therefore play a role in xenograft rejection using organs from GalT-/- pigs.
[Show abstract][Hide abstract] ABSTRACT: Complement-activating naturally occurring anti-porcine endothelial cell antibodies (Abs) are responsible for hyperacute rejection in porcine-to-primate transplantation, whereas the role of complement in acute vascular rejection, characterized by type II endothelial cell activation, is less well understood. We previously demonstrated a correlation between porcine type II endothelial cell activation, as detected by E-selectin expression, and human immunoglobulin (Ig)G3 anti-Gal alpha1-3Gal (Gal) Abs, which was not seen for IgG1, IgG2 or IgG4. The present study was undertaken to investigate whether there is a causal relationship between human anti-porcine IgG3 Abs and porcine endothelial cell activation.
IgG3 was isolated employing a Protein A column to 98.3% purity. Porcine endothelial cells were incubated with isolated human IgG3 or the combination of IgG1, IgG2 and IgG4. E-selectin expression and complement activation were investigated by flow cytometry and Western blotting, respectively.
Purified IgG3, in contrast to the other IgG subclasses, induced a substantial increase in E-selectin expression. This activation was accompanied by complement activation as detected by C3 cleavage, and was abolished by heat inactivation or by adding the complement inhibitor FUT-175. Depletion of anti-Gal Abs reduced E-selectin expression by 60%, consistent with the presence of complement-activating anti-porcine non-Gal Abs of the IgG3 subclass.
Collectively, these data strengthen the hypothesis that human anti-porcine endothelial cell Abs of the IgG3 subclass are essential for endothelial cell activation in porcine-to-human species grafts and demonstrate such activation to be partly independent of Gal epitopes.
[Show abstract][Hide abstract] ABSTRACT: Naturally occurring anti-Galalpha1-3Gal (anti-Gal) antibodies and complement induce hyperacute rejection (HAR) of porcine organs transplanted to primates. If the hyperacute reaction is prevented, an acute vascular rejection (AVR) occurs within hours to few days. Antibodies are important for the development of AVR, whereas the role of complement is still not clarified. AVR is characterized by protein synthesis-dependent endothelial cell (EC) activation. In the present study we investigated the relation between EC activation as measured by E-selectin expression, and the concentrations of anti-Gal antibodies of IgM, IgG and IgG subclasses in sera from 80 healthy blood donors selected on the basis of sex and age. There was a significant correlation between E-selectin expression and the concentration of IgG3 anti-Gal (r=0.39; P=0.019), which was not seen for the other IgG subclasses or for total IgG anti-Gal. A modest, but significant correlation was found between the concentration of IgM anti-Gal and E-selectin expression (r=0.38; P=0.040), but not between IgM and IgG3 anti-Gal. There was a large interindividual variation in anti-Gal antibodies, 50-fold for IgM and 70-fold for IgG. Females had significantly higher concentrations of IgM anti-Gal than males (P=0.0006), which was explained by a substantial increase in IgM anti-Gal concentration in younger women. The concentration of IgG anti-Gal and the degree of E-selectin expression did not differ between sex or age groups. In conclusion, the close correlation between anti-Gal antibodies of the potent complement activating IgG3 subclass and porcine EC activation, may imply that these antibodies play a role in EC activation characteristic of AVR.