Pathogenesis Of Hyperacute Rejection Research Articles

Coagulation cascade activation triggers early failure of pig hearts expressing human complement regulatory genes

Hyperacute rejection (HAR) and early graft failure (EGF) have been described in a minority of pig-to-baboon heart transplants using organs transgenic for human complement regulatory proteins (hCRP). Here we investigate the role of coagulation cascade activation in the pathogenesis of HAR and EGF in a consecutive series where a high incidence of these outcomes was observed. Twenty-eight naïve wild-caught Papio anubis baboons received heterotopic heart transplants from pigs transgenic for hDAF (n = 23) or hMCP (n = 5). Immunosuppression consisted of cyclosporine A, cyclophosphamide and MMF (n = 18) or anti-CD154 mAb (IDEC-131) and ATG (n = 10). Eleven received anti-Gal carbohydrates (GAS914, n = 8, or NEX1285, n = 3), of which four also underwent extracorporeal immunoadsorption (EIA), and 12 also received pharmacologic complement inhibitors (C1 INH, n = 9, or APT070, n = 3). Excluding one technical failure, 14 of 27 transplants (11 hDAF, 3 hMCP) exhibited either HAR (n = 10) or EGF (n = 4). Surprisingly, neither complement inhibition (with C1 INH or APT070) nor anti-Gal antibody depletion with GAS914, NEX1285, or additional EIA consistently prevented HAR or EGF despite low or undetectable complement deposition. Strikingly, most grafts with HAR/EGF exhibited prominent fibrinogen and platelet deposition associated with systemic coagulation cascade activation, consistent with non-physiologic intravascular coagulation, in many instances despite little evidence for antibody-mediated complement activation. We conclude that dysregulated coagulation correlates closely with and probably causes primary failure of pig hearts transgenic for hCRP. These data support efforts to define effective strategies to prevent dysregulated coagulation in pig organ xenografts.

Understanding the pathogenesis and the pathology of hyperacute cardiac rejection

The terminology of hyperacute rejection (HAR) has become outmoded and confusing due both to advances that have been made in delaying its onset and due to a proliferation of synonyms for the same pathologic process. Until such time as antibody-mediated xenograft rejection can be classified by the type of causative antibody, it is recommended that the term hyperacute rejection be applied to antibody-mediated rejection with classical HAR occurring within 24 h. Delayed HAR is the same pathologic process encountered after 24 h. Recognition of the key role that venous thrombosis plays in the pathogenesis of HAR allows the microscopist to intelligently interpret biopsies from various portions of a transplanted organ according to the pathologic effects of the obstructed venous drainage of the organ. Particularly in the heart, HAR often shows different pathologic features in the inner compared to the outer myocardium. Once xenografting becomes feasible, it will be possible to apply a grading system of HAR in clinical practice.

Both IgG and IgM anti-pig antibodies induce complement activation and cytotoxicity.

Hyperacute rejection of pig xenografts transplanted in humans is caused by endothelial cell binding of pre-formed xenoreactive antibodies (XAb) and activation of the classical pathway of complement. Human XAb mainly consist of anti-Galalpha1 3Gal antibodies, which occur in IgM, IgG and IgA classes. Whereas IgM anti-Galalpha1 3Gal antibodies have an established role in hyperacute rejection, the potential role of IgG XAb in this process is still controversial. The aim of the present study was to assess the specificity and functional properties of IgG and IgM XAb. Both classes were present in all human plasma samples tested, with a high inter-individual variability. Levels of IgG XAb did not correlate with levels of IgM XAb. Binding to Galalpha1 3Gal is strongly correlated with binding to the pig cell line PK15, both for IgG and for IgM, pointing to Galalpha1 3Gal as the major antigen recognized. Both purified IgM and IgG induced C3 deposition on PK15 cells and complement-dependent cytotoxicity in a dose-dependent way. The combination of IgG and IgM XAb resulted in an additive effect on cytotoxicity. Affinity-purified IgG anti-Galalpha1 3Gal antibodies were 22 times less potent than IgM in induction of cytotoxicity. These results indicate a quantitative, but not a qualitative, difference between IgM and IgG anti-pig antibodies concerning their complement-activating properties. Therefore, both classes of XAb are of importance in the pathogenesis of hyperacute rejection, and the relative importance of each class may differ considerably between individual patients, depending on the ratio of IgG and IgM XAb present in serum.

Both IgG and IgM anti-pig antibodies induce complement activation and cytotoxicity

Hyperacute rejection of pig xenografts transplanted in humans is caused by endothelial cell binding of pre-formed xenoreactive antibodies (XAb) and activation of the classical pathway of complement. Human XAb mainly consist of anti-Galalpha1 3Gal antibodies, which occur in IgM, IgG and IgA classes. Whereas IgM anti-Galalpha1 3Gal antibodies have an established role in hyperacute rejection, the potential role of IgG XAb in this process is still controversial. The aim of the present study was to assess the specificity and functional properties of IgG and IgM XAb. Both classes were present in all human plasma samples tested, with a high inter-individual variability. Levels of IgG XAb did not correlate with levels of IgM XAb. Binding to Galalpha1 3Gal is strongly correlated with binding to the pig cell line PK15, both for IgG and for IgM, pointing to Galalpha1 3Gal as the major antigen recognized. Both purified IgM and IgG induced C3 deposition on PK15 cells and complement-dependent cytotoxicity in a dose-dependent way. The combination of IgG and IgM XAb resulted in an additive effect on cytotoxicity. Affinity-purified IgG anti-Galalpha1 3Gal antibodies were 22 times less potent than IgM in induction of cytotoxicity. These results indicate a quantitative, but not a qualitative, difference between IgM and IgG anti-pig antibodies concerning their complement-activating properties. Therefore, both classes of XAb are of importance in the pathogenesis of hyperacute rejection, and the relative importance of each class may differ considerably between individual patients, depending on the ratio of IgG and IgM XAb present in serum.

VALIDATION OF THE MOUSE-TO-RABBIT COMBINATION AS A MODEL FOR PIG-TO-PRIMATE XENOTRANSPLANTATION

110 Hyperacute rejection (HAR) of porcine organs transplanted into primates is initiated by binding of preformed natural anti-porcine antibodies to the vascular endothelium of the graft and activation of the classical pathway of complement (C). Yet, in the small animal models currently employed, HAR is initiated mainly by activation of the alternative C pathway. Therefore it would be useful to develop a small animal model of more clinical relevance that reproduced the rejection process that takes place in pig-to-primate xenotransplantation. With this aim, we performed mouse-to-rabbit heterotopic cardiac transplantation to study graft survival time, histology and immunopathology of the rejected hearts. We then investigated, in vitro, the activation mechanisms that are triggered when mouse red cells are incubated with rabbit serum as a source of antibody and C. We found a mean graft survival of 37 ± 9.6 min (n=10) for mouse-to-weanling rabbit transplants and 40 ± 11.1 min (n=5) for mouse-to-adult rabbit transplants. Histology of rejected grafts showed diffuse interstitial hemorrhage, endothelial cell damage and myocyte necrosis consistent with HAR. Immunofluorescence showed moderate diffuse deposition of IgG and dense deposition of IgM and C3 on the vascular endothelium of the graft. To determine whether HAR in this combination is C-mediated, we performed 2 mouse-to-C6-deficient rabbit transplants. The first graft was rejected in 21 hr and the second recipient died with a functioning graft after 6.5 hr. Histology of the first graft showed severe interstitial hemorrhage, cellular necrosis and a moderate cellular infiltrate of neutrophils and some macrophages, while that of the second graft showed normal myocytes and vasculature, with minimal spotty interstitial hemorrhage. This markedly delayed rejection in C6-deficient rabbits implies that the membrane attack complex of C plays a major role in the pathogenesis of HAR in this model. To establish the role of natural antibodies and the pathway of C activation, we showed that rabbit serum 1) contains high titers of cytotoxic antibodies against mouse red cells (62 ± 11.7 units), 2) has strong classical pathway activity when tested with antibody-sensitized mouse red cells (49.6 ± 19.3 units, similar to that of human serum), and 3) has no significant alternative pathway activity against mouse red cells (in contrast to human serum which had 23.7 ± 8.5 units). Thus the cytotoxicity of rabbit serum for mouse red cells is mediated by natural antibodies and activation of the classical C pathway. In conclusion, these studies suggest that HAR in the mouse-to-rabbit combination is mediated by natural antibodies that trigger activation of the classical C pathway. Thus, the pathogenesis of HAR in this model is similar to that seen in pig-to-primate combinations. This model has potential for performing clinically relevant studies at low cost with animals that are readily accessible and easy to maintain.

Potential of Anticomplement Therapy in Clinical Transplantation

The activation of complement contributes to tissue damage in many ways. Prevention of complement activation in organ transplantation has largely centred on studies in xenotransplantation, where complement plays a key role in the pathogenesis of hyperacute rejection. Transgenic porcine organs expressing human regulators of complement activation in combination with soluble inhibitors as well as appropriate immunosuppression may be sufficient to alleviate the major immunological barriers that currently prevent the use of xenogeneic organs for human transplantation.

Xenoreactive natural antibodies in the world of natural antibodies: typical or unique?

Xenoreactive natural antibodies are thought to be responsible for initiating the hyperacute rejection of porcine organs transplanted into primates. Progress has been made in recently years in the characterization of the specificity and functions of these antibodies. Xenoreactive natural antibodies recognize Galα1-3Gal, a carbohydrate related to the blood group A and blood group B antigens. The presence of Galα1-3Gal may not be sufficient to allow the binding of xenoreactive natural antibodies under physiological conditions; rather, the clustering of Galα3-3Gal determinants may dictate the extent to which xenoreactive natural antibodies attach to surfaces on which Galα1-3Gal is expressed. The predominant role of xenoreactive natural antibodies in the pathogenesis of hyperacute rejection in porcine-to-primate xenotransplantation involves the activation of complement. Complement activation is mediated by IgM, not IgG xenoreactive natural antibodies. Based on functional avidity, thermal binding optima, thermal liability and homogeneity of binding interactions, xenoreactive natural IgM appear to be members of a family of natural antibodies which includes isohaemagglutinins. The classification of xenoreactive antibodies and isochaemagglutinins into a family of antibodies may provide further insight into the nature and physiological function of xenoreactive natural antibodies.

Cardiac xenografts between primate species provide evidence for the importance of the alpha-galactosyl determinant in hyperacute rejection.

Transplants performed between phylogenetically disparate species are subject to hyperacute rejection initiated by binding of xenoreactive natural Abs to endothelium in the donor organ. Binding of these Abs activates complement, leading to tissue injury and destruction of the graft. Human xenoreactive natural Abs recognize Gal alpha 1-3Gal beta 1-4GlcNAc (galactose alpha 1-3galactose beta 1-4-N-acetylglucosame); however, the relative importance of this Ag in graft rejection has not been proved. The present study was conducted to test the potential importance of alpha-galactosyl (alpha-Gal) determinants in the pathogenesis of hyperacute rejection. To this end, hearts (n = 3) from New World monkeys (Saimiri scureus, squirrel monkey), which can synthesize Gal alpha 1-3Gal, were transplanted heterotopically into Old World monkeys (Papio species, baboon), which do not synthesize Gal alpha 1-3Gal determinants and which have circulating anti-alpha Gal Abs. The xenografts were rejected in 51 to 56 min (mean +/- SD = 53.3 +/- 2.5), results similar to those observed in porcine grafts transplanted into baboons. Histologic analysis of the hearts revealed thrombosis and intraparenchymal hemorrhage and immune deposits consisting of IgM, Clq, C3, C4, C5b, and the membrane attack complex, but not properdin or factor B of the recipient deposited on graft endothelium. Sera obtained from baboons after perfusion of squirrel monkey kidneys revealed depletion of alpha-Gal-specific Abs and anti-pig endothelial cell Abs. These findings provide strong evidence that the Abs that accumulate in New World monkey organs during perfusion with baboon blood are the same Abs that would accumulate in a porcine organ transplanted into a primate and suggest that hyperacute rejection is not necessarily a reflection of phylogenetic distance but that the expression of terminal alpha-Gal residues provides an adequate target to initiate that process.

Depletion of IgM Xenoreactive Natural Antibodies by Injection of anti‐μ Monoclonal Antibodies

It is believed that IgM xenoreactive natural antibodies (XNA) and activation of complement are the two main effectors involved in the hyperacute rejection (HAR) of discordant xenografts, such as pig-to-primate kidney, liver or heart transplants. We have hypothesized that long-term depletion of circulating IgM XNA might be able to overcome HAR and induce the "accommodation" of pig-to-primate vascular discordant xenografts. Several techniques have been described to eliminate circulating XNA in primates but, up to now, none has been able to totally deplete these antibodies for a sufficiently long period of time in order to test the hypothesis of discordant xenograft "accommodation". Previous reports from our laboratory have shown that, in rodents, B-cell immunosuppression could be achieved by neonatal administration of anti-mu antibodies. Recently we have shown that administration of an anti-mu mAb, in adult rats, was able to totally deplete circulating IgM and IgM XNA, without immune complex disease. Furthermore, we have used different methods such as splenectomy, plasma exchange and an anti-B cell immunosuppressive agent mycophenylate mophetil (RS61443, Syntex, Palo Alto, USA) to pre-deplete circulating IgM before administration of anti-mu mAb (MARM-7) and showed that the effectiveness of anti-mu mAb to deplete circulating IgM was increased by 100-fold. Depletion of circulating IgM in adult rats by anti-mu mAb (MARM-7) was used as an experimental model to study the role of IgM XNA in the pathogenesis of HAR in guinea pig-to-rat cardiac xenografts. Our data show that IgM XNA play a major role in HAR, even if in this discordant combination direct activation of complement, probably through the alternative pathway, seems to be the main effector involved in HAR. We have analyzed the mechanisms of anti-mu depletion of circulating IgM in adult animals and shown that, besides anti-mu/IgM immune complex formation, depletion of circulating IgM results from the very significant inhibition of B-cell differentiation and secretion of IgM following in vivo crosslinking and internalization of surface IgM on B cells. As well, we provide evidence demonstrating that anti-mu mAb blocks B cells at an early stage of maturation, probably in the bone marrow. Furthermore, we have developed several rat anti-human and anti-baboon IgM mAb and tested their ability to deplete circulating IgM and IgM XNA in baboons, after splenectomy or splenectomy and plasma exchange.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanisms of tissue injury in hyperacute xenograft rejection.

Vascularized organs transplanted between phylogenetically distant species are subject to hyperacute rejection. The pathologic features of hyperacute rejection appear to be caused by profound dysfunction of small blood vessels in the graft. The author describes a model for the initiation and pathogenesis of hyperacute rejection. The model focuses on the endothelial cell as a target of the recipient immune reaction. Rejection is initiated by the binding to donor endothelial cells of recipient xenoreactive natural antibodies, which, in turn, triggers the complement cascade. These events lead to activation of graft endothelial cells, which promotes the thrombus formation and loss of vascular integrity characteristic of hyperacute rejection.

THE ROLE OF NATURAL ANTIBODIES IN THE ACTIVATION OF XENOGENIC ENDOTHELIAL CELLS

Hyperacute rejection of organ xenografts is thought to be mediated by the reaction of naturally occurring antibodies and complement of the recipient with blood vessels in the donor organ. We have suggested previously that the pathogenesis of hyperacute rejection might involve the activation of endothelial cells in the graft. To evaluate the potential role of natural antibodies and complement in hyperacute xenograft rejection, sixteen human sera were tested for variation in the ability to activate porcine endothelial cells as manifested by the release of biosynthetically labeled heparan sulfate from the cells. It was then asked to which extent such variation might reflect differences in natural antibody titer and/or complement activity. The sera mediated release of 3.6-57% of endothelial cell-associated heparan sulfate. Heparan sulfate release correlated significantly with the titer, in the sera, of IgM antibodies that bound to cultured endothelial cells (P = 0.0008) or to a triad of glycoproteins believed to represent the major targets of natural antibodies in porcine to primate xenografts (P = 0.001); correlation was also observed with the total concentration of IgM (P = 0.0046). The release of heparan sulfate did not correlate with corresponding properties of serum IgG, with anti-swine hemagglutination or with isohemagglutination titers. Heparan sulfate release correlated with deposition on endothelial cells of iC3b (P = 0.0095), but not with serum complement activity. Our findings indicate that in the reaction between human serum and xenogeneic endothelial cells, it is the concentration of xenoreactive IgM and not differences in complement activity that limits the ensuing pathophysiologic events.

Discordant xenografting: challenges and controversies

The transplantation of organs between individuals of disparate species is being considered as a potential solution to a serious shortage of donor organs for clinical transplantation. We summarize recent papers concerning the immunologic barrier to xenotransplantation, the pathogenesis of hyperacute rejection and reports on efforts made to prevent or mitigate hyperacute xenograft rejection.

Neutrophil adhesion to xenogeneic endothelium via iC3b.

Neutrophils are thought to play an important role in the pathogenesis of hyperacute rejection, a dramatic form of tissue injury caused by the reaction of antigraft antibodies with endothelial cells of an organ allograft or xenograft. We asked whether the interactions of human polymorphonuclear leucocytes (PMN) with xenogeneic endothelium might be promoted by the binding of natural anti-endothelial antibodies and complement by using porcine aortic endothelial cells (PAEC), human serum, and human PMN in an in vitro model of hyperacute rejection. Pretreatment of PAEC with 10% human serum followed by washing markedly increased PMN adhesion from 15.7 +/- 1.8% to 62.5 +/- 3.6% (p less than 0.001). Complement and anti-endothelial antibodies were necessary for the increase, because heat-inactivated serum or serum depleted of IgM did not significantly increase PMN adhesion to treated endothelium. The induction of increased PMN adhesion to PAEC by human serum was observed within 1 min. The essential role of complement was defined using complement-depleted serum. Ten percent C2-deficient serum did not increase PMN adhesion whereas 10% C5-depleted or 10% C8-depleted serum caused the same increase in PMN adhesion as observed with normal human serum. These results suggested that C3 might play a critical role in enhanced neutrophil adhesion. In fact, PAEC treated with 10% human serum for 15 min and incubated with an F(ab')2 antihuman C3 for 10 min completely abolished the enhanced adhesion. PAEC treated with 10% human serum or C5-depleted serum displayed fluorescence of iC3b whereas monolayers treated with heat-inactivated serum or C2-deficient serum were non-reactive. The enhanced PMN adhesion to serum-treated PAEC was mediated through neutrophil receptors binding iC3b because mAb directed against CD11b/CD18 inhibited the serum-enhanced adhesion of PMN. We conclude that PMN adhesion to endothelium can be significantly enhanced by the endothelial deposition of iC3b.

Hyperacute renal allograft rejection in the rabbit. Vasoconstriction demonstrated by microangiography.

A quantitative microangiographic study of renal allografts in rabbits was carried out to determine the pathogenesis of hyperacute rejection. A group of 10 rabbits, presensitized with multiple ear skin grafts from the kidney donor before receiving renal allografts, developed hyperacute rejection. Another 10 rabbits that were not presensitized served as a control group. The kidney grafts, excised at 2, 4, 8, 16 and 24 hr after renal transplantation, were examined histopathologically and microangiographically. In the presensitized group, striking vasoconstriction of afferent arterioles and interlobular arteries occurred within 2 hr after renal transplantation (p less than 0.01), but these vessels gradually recovered to a near-normal state after 24 hr. The number of glomeruli, however, began to decrease at 8 to 16 hours after renal transplantation (p less than 0.01), suggesting that the vasoconstriction produced thrombosis in the glomerular capillaries. These findings suggest that vasoconstriction at the level of afferent arterioles and interlobular arteries is the primary cause of hyperacute rejection.