Antibody-mediated activation of the classical complement pathway in xenograft rejection

Transplant rejection is a multifactorial process involving complex interactions between components of the innate and the acquired immune system. In view of the shortage of donor organs available for transplantation, xenotransplantation of pig organs into man has been considered as a potential solution. However, in comparison to allografts, xenografts are subject to extremely potent rejection processes that are currently incompletely defined. Consequently, an appropriate and safe treatment protocol ensuring long-term graft survival is not yet available. The first barrier that has to be taken for a xenograft is hyperacute rejection, a rapid process induced by the binding of pre-formed antibodies from the host to the graft endothelium, followed by activation of the classical complement pathway. The present review concentrates on the role of antibodies and complement in xenograft rejection as well as on the approaches for treatment that target these components. The first part focuses on porcine xenoantigens that are recognized by human xenoreactive antibodies and the different treatment strategies that aim on interference in antibody binding. The second part of the review deals with complement activation by xenoreactive antibodies, and summarizes the role of complement in the induction of endothelial cell damage and cell activation. Finally, various options that are currently under development for complement inhibition are discussed, with special reference to the specific inhibition of the classical complement pathway by soluble complement inhibitors.     

C1 inhibitor for prophylaxis of xenograft rejection after pig to cynomolgus monkey kidney transplantation

BACKGROUND: Early rejection of discordant porcine xenografts in primate recipients is initiated by the intragraft binding of either preformed (hyperacute xenograft rejection) or induced (acute vascular rejection) antiporcine recipient antibodies with subsequent complement activation via the classical pathway. We have investigated the efficacy of the supplemental administration of C1-inhibitor (C1-INH), a specific inhibitor of the classical complement activation pathway, for prophylaxis of xenograft rejection in a pig to primate kidney xenotransplantation setting. METHODS: Based on the results of pharmacokinetic studies performed in two nontransplanted monkeys, supplemental C1-INH therapy was administered daily to three Cynomolgus monkeys receiving a life-supporting porcine kidney transplant together with cyclophosphamide-induction/cyclosporine A/mycophenolat-mofetil/steroid immunosuppressive therapy. RESULTS: In the three monkeys receiving porcine kidney xenografts and continuous C1-INH treatment none of the grafts underwent hyperacute rejection; all xenografts showed initial function. Recipient survival was 13, 15, and 5 days. No graft was lost due to acute vascular rejection. All animals died with a functioning graft (latest creatinine 96, 112, and 96 micromol/liter) due to bacterial septicemia. CONCLUSION: We conclude that, in our model, supplemental C1-INH therapy together with a standard immunosuppressive regimen can be helpful for prevention of xenograft rejection in a pig to primate kidney xenotransplantation setting. The optimal dose and duration of C1-INH treatment, however, has yet to be determined.     

Preservation, reperfusion, and rejection in transgenic xenograft organs

The success of transplantation has resulted in increasing demand, despite a continuing fall, in donor organ supply. This widening gap encourages the argument for animals to act as a reservoir for donor organs (xenografts). Despite genetic manipulation, transgenic xenograft organs are at risk of vascular rejection in man (delayed xenograft rejection), a process in part involving endothelial cell activation. It appears that ischemia-reperfusion injury also involves endothelial cell activation. Evidence already exists to support the suggestion that ischemia-reperfusion injury may promote delayed xenograft rejection. The mechanisms of both these processes are briefly described and a case is made for optimum organ preservation of transgenic xenograft donor organs before clinical work is proposed.     

Acute vascular rejection is associated with systemic complement activation in a pig-to-primate kidney xenograft model

The introduction of h-DAF transgenic porcine organs into pre-clinical pig-to-primate discordant xenotransplantation has led to complete and reliable abrogation of hyperacute xenograft rejection (HAR). Despite additional heavy immunosuppression however, most xenografts are still lost due to acute vascular rejection (AVR), with current treatment protocols being of only limited value. In a life-supporting model of pig-to-primate kidney transplantation, unmodified (n=8) or h-DAF-transgenic (n=9) porcine kidneys were transplanted into cynomolgus monkeys under cyclophosphamide (CyP), cyclosporine and low-dose steroid immunosuppression. Longest recipient survival was 11 days in the control group and 68 days in the h-DAF transgenic group. Stable initial graft function with recipient survival >4 days was generated in eight animals (two controls and six transgenics). In these animals, plasma complement levels were analyzed during ongoing AVR. Compared with baseline levels, a two-fold increase in C3a levels and a four-fold increase in sC5b-9 levels were measured. In parallel to systemic complement activation, increased deposition of C3 and C5b-9 along with massive staining for recipient IgM immunoglobulins was detected in the xenografts on immunohistochemistry. We conclude that acute vascular xenograft rejection of porcine kidneys in cynomolgus monkeys is associated with classical pathway complement activation following binding of induced recipient anti-porcine antibodies. This complement activation can be observed despite membrane bound expression of human complement regulators in the porcine xenografts. Therefore, additional short-term fluid phase complement inhibition seems necessary for the future development of protocols designed for treatment of AVR in the pig-to-primate combination.     

Tolerance across discordant xenogeneic barriers

Abstract: In contrast to islet transplants, which appear to enjoy privileged survival when transplanted across some species barriers, organ xenografts are subject to vigorous immunologic rejection. Part of this rejection is immediate and is caused by the binding of natural antibodies to vascular endothelial cells, followed by activation of complement and coagulation factors, and hyperacute destruction of the graft. Such hyperacute rejection, in a pig-to-monkey kidney model that we are studying, can be avoided by removal of natural antibodies, which we have accomplished by absorption procedures. However, the cellular immune response to xenografts is also stronger than that to allografts. One might therefore expect the amount of nonspecific immunosuppression that would be required to avoid xenograft rejection to be so great that recipients would succumb to infectious complications. For this reason, we have pursued an approach to discordant xenografting involving the induction of tolerance though establishment of mixed lymphohematopoietic chimerism. We summarize here the present status of these experiments.     

B cell tolerance to xenoantigens

Abstract: Xenotransplantation of pig organs to humans is a possible solution to the shortage of donor organs for transplantation. Multiple immunologic barriers need to be overcome if pig-to-primate transplantation is to be successful. The presence, in humans, of natural antibodies (Abs) directed against Galα1–3Galβ1–4GlcNAc epitopes on pig vascular endothelium provides the major barrier, as antibody–antigen binding initiates the process of hyperacute rejection. Even if hyperacute rejection is prevented, acute vascular rejection develops. Acute vascular rejection is also mediated, in part, by xenoreactive Abs and may be complement-independent. Efforts being made to overcome antibody-mediated rejection include depletion of antibody by extracorporeal immunoadsorption, prevention of an induced Ab response by pharmacologic reagents, B-cell and/or plasma cell depletion, depletion or inhibition of complement, and the use of organs from pigs transgenic for human complement regulatory proteins. The ultimate solution would be the induction of B-cell tolerance to xenogeneic antigens, which is being explored by attempting to induce xenogeneic hematopoietic chimerism. Here, we review the properties of the B cell types responding to xenoantigens and the strategies for tolerizing those B cells.     

Immunopathology of hyperacute xenograft rejection in a swine-to-primate model

Hyperacute rejection is the inevitable consequence of the transplantation of vascularized organs between phylogenetically distant species. The nature of the incompatibility and the pathogenetic mechanisms that lead to hyperacute xenograft rejection are incompletely understood. We investigated these issues by the immunopathological analysis of tissues from swine renal and cardiac xenografts placed in rhesus monkeys. Hyperacute rejection was associated with deposition of recipient IgM and classic but not alternative complement pathway components along endothelial surfaces, the formation of platelet and fibrin thrombi, and the infiltration of neutrophils. In animals from which natural antibody was temporarily depleted by organ perfusion, rejection was observed at 3 days to 5 days posttransplant. The immunopathology of rejection in these tissues revealed focal vascular changes similar to those observed in hyperacute rejection. A xenograft functioning for a prolonged period in a recipient temporarily depleted of circulating natural antibody contained recipient IgM along endothelial surfaces but no evidence for significant deposition of complement, formation of platelet and fibrin thrombi, or infiltration of neutrophils. These results suggest that rhesus IgM contributes significantly to the development of hyperacute rejection in the swine to Rhesus model and that the fixation of complement is a critical factor in the recruitment of the coagulation cascade and platelet aggregation--and possibly in the adherence and infiltration of PMN.     

Histopathology of cardiac xenograft rejection in the pig-to-baboon model.

BACKGROUND: The use of pig organs transgenic for human decay accelerating factor (hDAF) has largely overcome the problems of hyperacute rejection. With improved immunosuppressive protocols, life supporting grafts are showing greater survival times bringing the possibility of clinical xenotransplantation closer. Examination of the histopathology of the rejection process provides insight into the underlying mechanism and may suggest ways in which new immunosuppressive strategies should be directed. METHODS: 44 baboons (Papio anubis) underwent heart transplants of which 39 were from transgenic donors. The transplanted organs were examined histologically and stained for evidence of immunoglobulin and complement deposition as well as cellular infiltrates. RESULTS: In the transgenic animals survival times were 2 to 99 days (mean 23.5) and the heterotopic group and 1 to 39 days (mean 11.7) in the orthotopic group. There were 3 cases of hyperacute rejection between the 2 groups. Rejected organs showed areas of old and recent myocardial infarction associated with vascular thrombosis. There was widespread deposition within vessels of immunoglobulins IgM and IgG together with complement fractions C3 and C5b to 9 in those organs that were rejected. The amount of complement positive in the longer surviving organs was less than those rejecting early. Cellular infiltate was predominantly macrophage with some later appearing T or natural killer cells. CONCLUSIONS: The histopathological changes support the importance of immunoglobulin and complement in delayed xenograft or acute vascular rejection. With time there is an increase in cellular infiltrate predominantly macrophages and these findings suggest an increasingly important role for the cells and the rejection process. The presence of areas of infarction and underlying vascular thrombosis is in keeping with endothelial activation and the establishment of procoagulant phenotype which may be due to immunoglobulin, complement, secreted cytokines and direct cellular effects.     

Mouse-to-rabbit xenotransplantation: a new small animal model of hyperacute rejection mediated by the classical complement pathway

BACKGROUND: Hyperacute rejection of porcine organs transplanted into primate recipients is initiated by the binding of preformed xenoreactive natural antibodies to the vascular endothelium of the graft and activation of the classical complement pathway. Several small animal models are currently employed to study various aspects of xenograft rejection; however, none has been shown to manifest hyperacute rejection mediated by the classical pathway of complement activation. METHODS: We performed heterotopic mouse heart transplants into weanling rabbits, adult rabbits, and C6-deficient rabbits. The recipients received no immunosuppression. Rejected grafts were subjected to histologic analysis and immunofluorescence staining for rabbit IgG, IgM, and C3. Levels of preexisting cytotoxic antibodies as well as classical and alternative complement pathway activities were determined in rabbit serum using mouse red cells as targets. RESULTS: Mean graft survival was 37+/-9.6 min for mouse-to-weanling rabbit transplants (n=10), and 40+/-11.1 min for mouse-to-adult rabbit transplants (n=5). Rejected grafts showed diffuse interstitial hemorrhage, endothelial cell damage, myocyte necrosis, moderate diffuse deposition of rabbit IgG, and dense deposition of rabbit IgM and C3 on the vascular endothelium of the graft, consistent with hyperacute rejection. One mouse-to-C6-deficient rabbit transplant was rejected at 21 hr with severe interstitial hemorrhage, cellular necrosis and a moderate cellular infiltrate consisting primarily of neutrophils and some mononuclear cells. A second transplant in a C6-deficient rabbit was functioning when the recipient died at 6.5 hr as a result of complications of surgery; the graft had normal myocytes and vasculature with minimal spotty interstitial hemorrhage. Both weanling and adult rabbit serum were found to have high titers of cytotoxic IgM anti-mouse antibodies and strong classical complement pathway activity with minimal alternative pathway activity towards mouse red cells. CONCLUSIONS: The mouse-to-rabbit species combination manifests hyperacute xenograft rejection. In vitro studies suggest that this process is mediated by IgM anti-mouse natural antibodies and activation of the classical pathway of complement.     

Discordant xenograft rejection in an antibody-free model.

Newborn pigs prevented from suckling colostrum were shown to have less than 0.05 micrograms/ml total immunoglobulin present in their serum. Rabbit heart xenografts transplanted heterotopically into the neck of such pigs were hyperacutely rejected, with a mean survival time of 92 +/- 14 min (mean +/- SD). Pigs which had been allowed to suckle and whose serum contained 10-15 mg/ml maternal immunoglobulin hyperacutely rejected rabbit heart xenografts in 109 +/- 62 min. Histological studies showed no Ig binding but complement component 3 (C3) binding to rabbit hearts placed in immunoglobulin-negative pigs. Prolongation of rabbit heart xenograft survival was achieved by administering cobra venom factor (1 mg/kg) to the pigs pretransplant. These data show hyperacute xenograft rejection in the absence of antibody and suggest that its cause is activation of complement by the alternative pathway.     

The Role of Macrophages in Xenograft Rejection

Safe and effective xenotransplantation would provide a valuable answer to many of the limitations of allogenic transplantation. Such limitations include scarcity of organ supply and morbidity to donors in cases of living-related donor transplantation. The main hurdle to the efficacious application of xenotransplantation in clinical medicine is the fierce host immune response to xenografts. This immune response is embodied in 3 different types of xenograft rejection. Both hyperacute rejection and delayed xenograft rejection are mediated by natural antibodies and are concerned primarily with whole organ rejection. Cellular xenograft rejection (CXR), on the other hand, is concerned with both whole organ and CXR and is mediated by innate immunity rather than natural antibodies. Macrophages, which are cells of the innate immune system, play a role in all 3 types of xenograft rejection (not just CXR). They impart their effects both directly and through T-cell activation.     

Pathology of xenograft rejection: a commentary

Abstract: Trends in solid organ xenograft pathology are presented, with the focus on pig-to-nonhuman primate models. A simplified classification of rejection is followed, including hyperacute rejection (HAR), acute humoral xenograft rejection (AHXR), and acute cellular xenograft rejection (ACXR). The main components in HAR are natural xenoreactive antibodies in combination with complement activation. This is evident from the prevention of HAR in recipients in whom either antibodies or complement activation is depleted or inhibited. However, these strategies generally fail to prevent AHXR, which occurs later. AHXR is a multifactorial process in which natural and elicited antibodies may play roles, possibly in conjunction with complement, coagulation factors, and white blood cells. A main target appears to be the microvasculature which, in kidney grafts, is associated with a glomerular thrombotic microangiopathy. It is not clear to what extent species-specific physiologic disparities in complement and coagulation processes may play a role, separate from antibody-initiated processes. As rejection of solid organ xenografts is currently from AHXR, ACXR has not yet received close attention. In addition to intragraft rejection events, systemic complications following host–graft interactions have emerged, including (often fatal) consumptive coagulopathy and immune complex disease. It is anticipated that rejection processes will change when pigs with new genetic modifications become available. For instance, the precise role of natural antibodies to Galα1,3Gal will be able to be distinguished from other factors when pigs that lack the target antigen are available, and their organs can be evaluated in large animal xenotransplantation models.     

''Homologous restriction'' in complement lysis: roles of membrane complement regulators

The complement system is a powerful bactericidal immune defence with the potential to damage self cells. Protection of self is provided by expression on cells of a battery of membrane regulators that inhibit activation of complement. Roles of complement in the rejection of transplanted organs have long been recognized, and are particularly relevant in xenotransplantation, where hyperacute rejection is complement-driven. Inhibiting complement was therefore considered early in the history of xenografting, and the use of membrane complement regulators to this end was proposed more than two decades ago. For each of the membrane regulators in humans, early studies implied a species-specificity of action, inhibiting human complement but not that from other species. The dogma of species-specificity dictated strategies for inhibiting complement in xenografts and drove the creation of donor transgenic pigs expressing human regulators. Here we critically evaluate the evidence for species-specificity in membrane complement regulators from humans and other animals. We challenge the dogma and show that there is considerable cross-species activity for each of the membrane regulators of complement. Acceptance of the fact that species selectivity is not a limitation will open new avenues for protection of the xenograft from complement damage.     

Xenotransplantation

BACKGROUND: The success of clinical transplantation has led to a large discrepancy between donor organ availability and demand; considerable pressure exists to develop an alternative source of organs. The use of animal organs for donation is a possible solution that is not yet clinically applicable. METHODS AND RESULTS: A literature review was performed based on a Medline search to find articles on xenotransplantation. Keywords included hyperacute, acute vascular, xenograft rejection combined with concordant and discordant. Additional references cited in these articles from journals not included in Medline were obtained from the British Library. Limited information on unpublished, preliminary work has been included from sources known to the authors, based on their research work in the field. One hundred and forty-six references and four personal communications have been included in this review article. CONCLUSION: A greater understanding of the pathogenesis of xenograft rejection is developing rapidly. Strategies to abrogate hyperacute rejection have proved successful, but control of antibody-driven acute vascular rejection has not yet been achieved. The safety and viability of xenotransplantation as a therapeutic modality are still unproven.     

Growing kidneys

The number of kidney transplantations performed per year is restricted by the limited availability of donor organs. One possible solution to this shortage is the use of renal xenografts. However, the transplantation of xenografts is complicated by hyperacute and acute rejection. A second possible solution is to 'grow a kidney' from a transplanted renal anlage. It has been postulated that the host immune response might be attenuated after the transplantation of such an anlage (metanephros) instead of a developed kidney. Transplanted metanephroi become chimeric organs in that their blood supply originates, at least partly, from the host. It is possible to transplant a developing metanephros, without the use of immunosuppression, from one rat to another. Transplanted metanephroi grow, differentiate, become vascularized, and function in host rats. 'Growing kidneys' via the transplantation of metanephroi may hold promise as a novel therapeutic approach to the treatment of chronic renal failure.     

Removal of xenoreactive antibodies by protein-A immunoadsorption: experience in 22 patients

Abstract: The presence of naturally occurring anti‐Galα1–3Gal (antiαGal) Ab in human serum is believed to be a major factor in the pathogenesis of hyperacute rejection of discordant organ xenografts such as the pig‐to‐human combination. Galα1–3Gal epitopes are expressed on pig tissues and the binding of anti‐Galα1–3Gal leads to endothelial cell activation and complement‐mediated hyperacute graft rejection. Several strategies have been suggested in donor animals or in the xenograft recipient to overcome the anti‐αGal barrier. Protein‐A immunoadsorption (PAIA) was developed for the in vivo removal of circulating Ab and it has been shown to be effective in cases where pathogenic auto or alloAb are present. The aim of our study was to analyze the effect of PAIA on total and xenoreactive serum anti‐αGal immunoglobulin levels in a group of patients treated with this technique for different diseases. After three consecutive sessions of PAIA, total and xenoreactive IgG and IgM immunoglobulin levels were decreased by more than 50% of pre‐treatment levels. So we conclude that PAIA is an effective method to significantly reduce circulating Ab, including xenogeneic IgM and IgG Ab. This mode of therapy might be considered as a tool to overcome hyperacute xenograft rejection. PAIA combined with other therapeutic approaches may well protect the xenograft.     

Transplantation of renal precursor cells: a new therapeutic approach

The number of kidney transplantations performed per year is limited due to availability of donor organs. One possible solution to the organ shortage is the use of renal xenografts. However, the transplantation of xenografts is complicated by hyperacute and acute rejection. It has been postulated that the host immune response might be attenuated following the transplantation of renal precursor cells or embryonic kidneys (metanephroi) instead of developed (adult) kidneys. Transplanted metanephroi become chimeric organs in that their blood supply originates, at least in part, from the host. It is possible to transplant a developing metanephros, without the use of immunosuppression, from one rat to another. Transplanted metanephroi grow, develop, become vascularized, and function in host rats. Transplantation of metanephroi may be a promising novel therapeutic approach for the treatment of chronic renal failure. Received: 29 December 1999 / Revised: 13 March 2000 / Accepted: 13 March 2000     

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.     

Genetic engineering for xenotransplantation

Xenotransplantation is being pursued vigorously to solve the shortage of allogeneic donor organs. Experimental studies of the major xenoantigen (Gal) and of complement regulation enable model xenografts to survive hyperacute rejection. When the Gal antigen is removed or reduced and complement activation is controlled, the major barriers to xenograft survival include unregulated coagulation within the graft and cellular reactions involving macrophages, neutrophils, natural killer (NK) cells, and T lymphocytes. Unlike allografts, where specific immune responses are the sole barrier to graft survival, molecular differences between xenograft and recipient that affect normal receptor-ligand interactions (largely active at the cell surface and which may not be immunogenic), are also involved in xenograft failure. Transgenic strategies provide the best options to control antigen expression, complement activation, and coagulation. Although the Gal antigen can be eliminated by gene knockout in mice, that outcome has only become a possibility in pigs due to the recent cloning of pigs after nuclear transfer. Instead, the use of transgenic glycosyl transferase enzymes and glycosidases, which generate alternative terminal carbohydrates on glycolipids and glycoproteins, has reduced antigen in experimental models. As a result, novel strategies are being tested to seek the most effective solution. Transgenic pigs expressing human complement-regulating proteins (DAF/CD55, MCP/CD46, or CD59) have revealed that disordered regulation of the coagulation system requires attention. There will undoubtedly be other molecular incompatibilities that need addressing. Xenotransplantation, however, offers hope as a therapeutic solution and provides much information about homeostatic mechanisms.