| Abstract|| |
Organ transplantation has been a major development in clinical medicine but its success has been marred by the immune system's capacity to respond to "non-self" cells and tissues. A full molecular understanding of this mechanism and the myriad triggers for immune rejection is yet to be elucidated. Consequently, immunosuppressive drugs remain the mainstay of post-transplant management; however, these interventions have side effects such as increased incidence of cancer, post-transplant lymphoproliferative disorders, susceptibility to infection if not managed appropriately and the inconvenience to the patient of lifelong treatment. Novel therapeutic approaches based on molecular understanding of immunological processes are thus needed in this field. The notion that factors influencing successful transplants might be of use as therapeutic approaches is both scientifically and medically appealing. Recent developments in the understanding of successful transplants are expected to provide new opportunities for safer transplantation. This article reviews the present understanding of the molecular basis of rejection and the role of complement in this process as well as the possibility of generating "intelligent" therapy that better target crucial components of hyperacute rejections.
Keywords: Organ Transplantation, Complement, Hyper acute Rejection, Xenogeneic, Allogeneic, Isotypes, Epitopes
|How to cite this article:|
Al-Rabia MW. Complement and hyper acute rejection. Saudi J Kidney Dis Transpl 2009;20:362-9
| Introduction|| |
The success of clinical allergenic transplantation has been limited by the problem of demand exceeding the supply of donor tissue. As long ago as the early 1960s, the possibility of using tissues from non-human primates for transplantation into humans, xeno-transplantation, was explored with a view to ameriolating this problem. The potential supply of xenogeneic tissue would be almost inexhaustible, and thus several experiments were conducted using renal xenografts in severely ill patients.  However, the survival of these transplants was very short.  Also, there was concern about the risk of transmitting diseases from such primates to the human recipients. 
These and other factors, including the problems of breeding non-human primates for experimental work, led to a waning of interest in xenogeneic transplantation. However, over the past 15 years, the potential of this untapped resource has been reinvestigated. There are strong concerns, not least the evolutionary proximity of non-human primates to man, and thus the increased risk of transfer of infection, that have been raised against the use of non-human primates in xenotransplantation.  These issues would make it impossible to apply primate xenografting in the clinical setting. Thus, the focus now has changed to the use of non-primates as donors, in particular, the pig, since it can be bred easily, has organs of similar anatomy and physiology to humans, and, as it is more phylogenetically distant than nonhuman primates, there is a lower risk of transmitting disease.
The unsolved problem that remains is graft rejection. This is much more rapid in xenogeneic than in allogeneic transplantation. Studies have shown that the exchange of organs between phylogenetically closely related species (e.g. rats and mice) are less rapidly rejected than transplants between more distantly related species (e.g. humans and pigs). In 1970, Calne described the former as "concordant" and the latter as "discordant" combinations.  He proposed that concordant xenograft rejection, being less vigorous, was similar to first set allograft rejection, and that discordant combinations led to hyperacute rejection (HAR). However, phylogenetic differences were not the only factors that determined HAR, e.g. grafts transplanted across blood group barriers may also be rejected in this manner.  Hyperacute xenograft rejection is a well defined barrier to clinical pig to human xenotransplantation, and intense research in this area has identified potential solutions. 
| Hyperacute Rejection|| |
This form of rejection occurs in a very short time span, within minutes to hours after transplantation of vascularized grafts. The morphological changes in kidney xenotransplants begin immediately upon restitution of the circulation following anastomosis of graft vasculature to that of the recipient, and the kidney becomes blue. Histologically, the lesions are characteristic of the Arthus reaction, the graft showing edema, vascular thrombosis, hemorrhage, and infarction, all leading to total tissue destruction.
It results from preformed "natural" antibodies activating complement and graft endothelial cell "activation". This causes pro-coagulatory changes which result in extensive hemostasis, eventually resulting in destruction of the graft. The unsolvable barrier of HAR requires persistence and ingenuity. Numerous approaches have been suggested to circumvent this problem, including the depletion of these antibodies by techniques such as ex vivo porcine perfusion, plasmapheresis, immunoadsorption and complement inhibition. The introduction of accommodation and molecular chimerism has further improved the knowledge of this newly conceived field. Gal-deficient pigs have recently been developed by eliminating the alpha 1, 3 galactosyltransferase gene. These discoveries together with better immunosuppression raise hope for the yet unrealized promise of xenotranplantation. 
| Endothelial Cells|| |
Endothelial cells normally form a barrier to the cellular and protein components of blood. However, upon appropriate stimulation, these cells undergo phenotypic (i.e. structural and metabolic) changes and become "activated". This occurs as a result of either direct stimulation, e.g. by C5a  or indirectly, e.g. by cytokines such as tumor necrosis factor (TNF)  and interleukin-1 (IL-1).
Some of the changes occurring in activated cells happen very quickly, and include the upregulation (and de novo expression) of cell surface adhesion molecules, such as P-selectin, E-selectin, VCAM and ICAM-1. These molecules aid leukocyte tethering and their migration out of blood into the underlying tissue, through the endothelial cell layer. Activated cells also secrete cytokines that recruit other cells to the site; e.g. IL-8, which is a chemotactic factor for neutrophils and T lymphocytes.
Upon activation, endothelial cells change shape, and lose the integrity of the cell layer thereby promoting vascular permeability and effusion of blood constituents. Other metabolic changes result in the expression of a plethora of proteins important in the activation of the kinin and clotting cascades. These include expression of tissue factor or thromboplastin; synthesis and secretion of von Willebarand factor (vWF); and secretion of platelet activation factor (PAF), causing platelet aggregation and release of active compounds such as histamine.
Clearly, endothelial cells have a very critical role in normal health. However, their response to the insult of "natural" antibody binding and subsequent complement activation appears to be the critical event in HAR, as first proposed by Platt and colleagues.  They associated the pathology of tissue destruction in HAR to donor endothelial cell activation and consequently suggested that they are central to the development of HAR. A result of such endothelial cell activation is the loss of heparin sulphate from the surface of endothelial cells. This molecule is normally responsible for binding anticoagulatory molecules to the endothelial surface and its loss may be important to the procoagulatory changes seen in HAR.  However, Auchincloss  suggested that the role of natural antibodies in activating the classical complement cascade is as important, if not more so, to the occurrence of HAR.
Despite the association between C4d and immune complex deposition, immunoglobulin has not been detected in PTC where C4d is detectable. The reason for this is unclear and remains an important question. As humoral rejection probably results from direct antibody attack on the target endothelial cell, modulation from the surface may make antibody detection difficult. Endothelial cells dislodge surface antibody through "capping", "shedding", or "internalization".  C4d resists modulation from the surface due to its covalent binding to tissue structures.  Ischemia and reperfusion injury leading to endothelial damage has also been considered a possible non-alloimmune stimulus for C4d deposition. A prospective analysis of renal allograft biopsies including 35 pairs of pre- and one hour post-perfusion intraoperative biopsies demonstrated a lack of C4d in the pre-transplant biopsies and presence of C4d in only two of 82 perioperative biopsies.  The two allografts demonstrating C4d in the perioperative samples were from sensitized individuals who later had biopsy evidence of antibody mediated rejection (AMR). Thus, it was determined that C4d deposition could be evident within one hour of transplantation, and its presence was a marker of AMR and not of ischemic injury. 
| Natural Antibodies|| |
Preformed "natural" antibodies are found to constitute a large proportion (> 1%) of the circulating antibody repertoire. It has been suggested that these antibodies are induced by, and provide defense against, gastrointestinal bacteria since they recognize antigens similar to those found on these bacteria.  The binding of these antibodies to epitopes on the surface of the endothelium of vascularized grafts activates complement and is thought to cause HAR. The evidence for the involvement of these antibodies came from a number of sources. Antibody mediated rejection frequently causes refractory graft dysfunction. A randomized controlled trial, which was designed to evaluate whether immunoadsorption is effective in the treatment of severe AMR, suggests that this treatment is indeed effective in reversing severe AMR. 
One study demonstrated that recipient antibodies can be found deposited on rejected donor tissue by immunofluorescence.  Another study showed that removal of these antibodies, e.g. by plasmapheresis,  delays HAR. Also, the pathology of xenogeneic HAR is similar to allogeneic HAR, seen when allografts are transplanted across the ABO blood group barrier and, this response has been attributed to naturally occurring antibodies against ABO antigens. , Another study was performed on eight consecutive renal transplant patients presenting with acute humoral rejection to assess the efficacy of monoclonal anti-B cell antibodies. All eight patients had elevated serum creatinine, the appearance of donor specific alloantibodies, and the presence of C4d in the allograft biopsy.  The diagnosis of AMR was presumptive; historically, based on the lack of response to standard treatment regimens and the occasional presence of severe histologic findings. The presence of severe vascular rejection, categorized as Banff grade III or Cooperative Clinical Trials in Transplantation (CCTT) type III rejection, was felt to represent an antibody mediated event. These refractory cases were associated with a high incidence of graft loss. 
| Isotypes|| |
The exact isotypes that are involved in HAR in xenogeneic transplantation is still under investigation, but progress has been aided by studies which showed deposition of IgM on graft endothelium.  The same group then suggested that IgM (but not IgG) was the isotype responsible for HAR, since IgM titers in test sera appeared to have an effect on endothelial cell activation, as represented by heparin sulphate release from porcine endothelial cells.  Other groups showed deposition of both IgM and IgG on porcine endothelium perfused with human blood. , The difficulties of identifying which antibodies are involved in HAR may be due to the array of detection methods used and so, an ELISA was developed.  However, although both IgM and IgG isotypes can be detected by this method, only the IgM antibodies were found to activate complement.  Other isotypes have also been implicated in xeno-recognition, e.g. IgA, in the context of species combinations other than man and pig. 
Because of the strong implications of the involvement of complement in xeno-rejection,  it is most probable that the antibody isotypes of importance in HAR are those that are best at fixing complement. However, natural antibodies might also act independently of complement either by directly activating the endothelial cells, or by involving other cellular members of the immune system, such as NK cells and granulocytes, as shown in complement free models. 
| Epitopes|| |
Xenoreactive antibodies were originally studied by hemagglutination assays,  and this led to ambiguity in identifying the epitopes recognized by these antibodies, i.e. the epitopes recognized in xenogeneic HAR were unlikely to be blood group antigens, since these would not be expressed in non-primates. Endothelial cell membrane components from pigs have been isolated and analysis suggested that a triad of glycoproteins (named "gp115/135") possessed the epitopes recognized by human natural antibodies.  An alternative approach involved eluting the antibodies from donor grafts and showed that most of these reacted against synthetic carbohydrates containing unfucosylated terminal non-reducing disaccharides of the a galactose linear-B epitope α Gal1 → 3(3 Gal-R,  also known as galα (1-3) epitope. This sugar residue can be carried on a variety of protein backbones,  provided the enzymes necessary for its synthesis are present. Galili showed that this epitope is recognized by human anti-α Gal antibodies and xenoreactive antibodies.  However, it is still unclear that all xenoreactive antibodies are accounted for by naturally occurring anti-a Gal antibodies. Studies have shown that the HAR occurring within minutes after transplantation is mediated by binding of natural antibodies to the Galalpha (l-3) Gal epitope on the endothelial cells with subsequent complement activation. Whereas inhibition of complement activation protects against HAR, the role of complement in the later rejection phases is less clarified.  The disaccharide galactose (alpha)1,3 galactose (the alphaGal epitope) is the major xenoantigen responsible for the hyperacute vascular rejection occurring in pig-to-primates organ transplantation. The synthesis of the alphaGal epitope is catalyzed by the enzyme alpha1,3 galactosyltransferase (alpha1,3GalT). 
Upon binding to the endothelial cell surface epitopes, xenoreactive antibodies can fix complement via the classical pathway, leading to production of molecules that are known to activate endothelial cells.
| Complement|| |
The major biological role of complement is to provide effector functions to humoral immunity. There are two complement activation pathways which lead to the terminal lytic pathway to form the membrane attack complex (MAC) and this is represented in [Figure 1]. However, MAC is not the sole final active component since various fragments are created en route, some with potent biologically active properties.
The complement system is an important part of the innate immune system, mediating several major effector functions and modulating adaptive immune responses. Three complement activation pathways exist: the classical pathway (CP), the alternative pathway (AP), and the lectin pathway (LP). The LP is the most recently discovered and least characterized. The CP and the LP are generally viewed as working through the generation of the C3 convertase, C4bC2b, and are here referred to as the "standard" pathways. In addition to the standard CP and LP, so called bypass pathways have also been reported, allowing C3 activation in the absence of components otherwise believed critical [Figure 1]. The classical bypass pathways are dependent on C1 and components of the AP. A recent study has shown the existence also of a lectin bypass pathway dependent on mannan-binding lectin (MBL) and AP components. The emerging picture of the complement system is more that of a small "scale free" network, where C3 acts as the main hub, than that of three linear pathways converging in a common terminal pathway. 
| The Classical Pathway|| |
In man, single molecules of IgM or aggregates of IgG1 or IgG3 (immune complexes) can activate the complement cascade by interaction with the early complement components in the classical pathway. C3 is degraded and the rise in C3b levels can bring the alternative pathway into play by opening up the amplification loop.
The first component that interacts with antibody is C1, of the classical pathway. This consists of a single C1q molecule associated with dimers of C1r and C1s. C1q is a hexamer made up of 18 polypeptides, six each of A, B and C types, that are encoded on chromosome 1 (this is also an important chromosome in complement regulation as it is the site of RCA gene complex). The six globular head regions of C1q can bind to the CH2 domain of IgG and the CH3 domain of IgM, and activation of C1 occurs when two or more heads are bound to antibody, ,, and so free antibody cannot activate C1. C1r and C1s are serine proteases, normally existing in an inactive pro-enzyme form, but, in the presence of Ca 2+ ions, are activated.  C1r is thought to be cleaved in some autocatalytic manner and once both C1r molecules are activated they act on the C1s dimer, which in turn cleaves C4 and then C2. The C4 cleavage products are the small fragment, C4a (a weak anaphylatoxin) and the larger fragment, C4b (an opsonin). The latter molecule may be bound to complement 'activator' surfaces, and some may be degraded by regulatory factors in the plasma, but the remainder is bound to C2b to form the classical pathway C3 convertase. 
Detection of the complement split product C4d in renal allograft biopsies is an important adjunctive tool to help understand the alloimmune response and, in particular, to diagnose antibody mediated rejection. C4d is a degradation product of the classic complement pathway. After an antigen antibody complex fixes complement, a cascade of events follows with activation of several complement proteins. The complement protein C4 is split into C4a and C4b. C4b is then converted to C4d. A unique feature of C4d is that it binds covalently to the endothelial and collagen basement membranes, thereby avoiding removal and raising the possibility of serving as an immunologic footprint of complement activation and antibody activity. 
| Conclusion|| |
In the setting of acute antibody mediated rejection we currently use a protocol of plasmapheresis, steroids, and intravenous immunoglobulin with or without antilymphocyte therapy. We also convert to the more potent immunosuppressive combination, in the setting of either acute or chronic antibody mediated rejection.
If severe cell mediated rejection (Cooperative Clinical Trials in Transplantation type II rejection) is detected concurrently with antibody mediated rejection, anti-lymphocyte therapy with steroids may provide substantial benefit in combination with other antibody mediated rejection guided treatments. If cell mediated rejection is mild or not detected, then a pulse of steroids is likely warranted in addition to other treatment for antibody mediated rejection.
| Recommendations|| |
Staining for C4d, a marker of antibody mediated injury should be incorporated into all biopsies obtained for allograft dysfunction. This complement component may be detected in either acute or chronic allograft dysfunction, and the presence of C4d staining of peritubular capillaries strongly suggests antibody mediated rejection. The presence of donor specific antibody should also be assessed anytime C4d is positive or whenever there is concern for antibody mediated rejection.
Recommendations differ in part based on whether the patient with suspected antibody mediated rejection has or has not undergone desensitization protocols prior to the transplant.
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Mohammed WO Al-Rabia
Department of Hematology and Immunology, College of Medicine, Umm Al-Qura University, Makkah