CONTRIBUTION OF ENDOGENOUS PIG COMPLEMENT REGULATORY MOLECULES

A shortage of donor organs is the major obstacle in clinical transplantation. Successful xenotransplantation using pigs as a source of organs and tissues may quickly become a biomedical reality. Xenotransplantation is a new and rapidly expanding field which may save thousand of human lives each year. Clinical application of xenotransplantation is limited by a severe immunological reaction of the recipient against the graft, known as hyperacute rejection. This destruction of the grafted organ occurs within minutes of implantation and is mediated by natural antibodies and complement. One of the strategies being tested to reduce complement activation is the breeding of pigs expressing human complement regulators, in the expectation that organs from these pigs will be better protected from hyperacute rejection. However, the potential roles of the endogenous pig complement inhibitors have received little attention. This paper reviews the current knowledge of the endogenous pig complement inhibitors and their relevance in the context of current efforts to express human complement regulators in the pig. 1. Xenotransplantation Human beings live longer than most other mammals, but sometimes our organs begin to expire before we ourselves are ready to go. Organ transplantation has recently become a relatively routine solution to this dilemma. However, when a person needs an organ transplant, the immune system suddenly became a deadly force, attacking and destroying the implant. Immune system suppressing drugs, such as cyclosporin, have made human organ transplantation possible. Organs such as hearts, lungs, livers and kidneys can now be transplanted successfully. Yet this has led to a new problem: a supply shortage. There simply are not enough donor organs to be transplanted and it is estimated that almost 4,000 people die each year waiting for a human donor. In order to overcome this lack of organs, scientist recently considered the possibility of using animals as potential donor for humans [1]. Xenotransplantation describes the process where organs, cell or tissues from one organism are transferred to an organism of a different species. 2. The pig as the animal of choice The choice of a source animal will be of prime importance for xenotransplantation. Ideally, the donor animal would show immunological, anatomical and physiological similarities to human, possess an organ of similar size to humans, would breed fast and produce large litters under farmed conditions and would show minimal risk of transmitting infectious agents to humans. Two groups of animals have been considered as donor: non-human primates and pigs. For several reasons, pigs rather than non-human primates are currently viewed as the most promising xenograft donors [2, 3]. First, pigs are available in large numbers and in appropriate sizes while non-human primates are relatively scarce and generally much smaller than adult humans. Second, the use of pigs does not raise such severe ethical considerations as the use of species more closely related to human. Third, there is a possibility of genetically engineering pigs using technologies, which for practical reasons, cannot be applied to non human primates. Fourth, the risk of xenozoonoses (transmission of infectious agents between species) is increased with close species relationship. Therefore monkeys are unlikely to provide an adequate supply of clean tissues for xenotransplantation 3. Hyperacute rejection The major barrier to the use of pigs as a source of organs for clinical transplantation is that the organs are subject to severe and irreversible rejection, a phenomenon known as hyperacute rejection [4]. This involves the destruction of the vascular endothelium of the donor organ within minutes of exposure to the recipient’s blood. Hyperacute rejection cannot be prevented with standard anti-rejection medication, such as immunosuppressive drugs. The lysis of endothelial cells occurs following the binding of naturally occurring antibodies to carbohydrate antigens on the glycolipids and glycoproteins of the donor cells [5]. Most of the preexisting antibodies react with the carbohydrate Gal (α1,3)Gal [6]. The Galα1,3Gal carbohydrate is expressed in many mammals, but not in humans. Humans and Old World primates lack the enzyme glycosyltransferase responsible for the synthesis of Galα1,3Gal [6]. Binding of natural antibodies to the graft activates an important set of human serum proteins, known as complement [7]. The activation of the complement system results in an intense infiltration of neutrophils into the grafted tissue. The ensuing inflammatory reaction causes massive blood clots preventing vascularization of the graft and leading to the organ’s death [8, 9]. Figure 1. The complement system. Membrane–immobilized C3b, produced by either classical, alternative or lectin pathway, triggers a further cascade of reactions that leads to the assembly of membrane attack complexes causing the cell lysis. The sites of action of the three membrane regulatory proteins are shown. 4. The complement system The complement system, consist of nearly 30 serum and membrane proteins. Complement is present in the plasma of all vertebrates from fish to mammals. These proteins work together in a highly regulated cascade, to generate reaction products that facilitate antigen clearance and generation of an inflammatory response. There are three pathways of complement activation: the classical pathway, which depends on antibodies, the alternative pathway and the lectin pathway. Activation of the complement system via each route can result in the same end-stage reaction, that is, the generation of a macromolecular membrane-attack complex (MAC) (Figure 1). Complement performs its task in self-defence against invading microorganisms by binding C3b and/or MACs to the surface of cells, bacteria and viruses. These binding events do not depend on specific receptor interactions. Therefore, the complement system is non specific and able to attack both host cells and invading microorganisms. Elaborate mechamisms are required in order to confine the reactions to designated targets. One of these mechanisms is the expression of a group of complement inhibitor proteins on the host cell surfaces. Control of complement activation is focussed at two stages in the pathway, the formation of the C3/C5 convertase and the assembly of the MAC [10]. 5. Regulation of complement activation Numerous investigators have sought to reduce complement activation in hyperacute rejection by removing the natural antibodies and/or by inhibiting complement [11]. In human, three membrane proteins are important in host protection from complement. Decayaccelerating factor (DAF or CD55), membrane cofactor protein (MCP or CD46) and CD59. DAF and MCP both inhibit the formation of the C3 convertase, the major amplification step in the complement activation. This family of C3 convertase regulatory proteins are related structurally by the presence of short 60 amino acid repeating sequences or motifs, termed short concensus repeats (SCRs). CD59 does not belong to this family. These proteins are also related genetically, as they are encoded at a single chromosomal location known as the regulators of complement activation (RCA) gene cluster. In human, the RCA gene cluster is located in the long arm of chromosome 1. The regulatory protein CD59 operates at the level of the membrane attack complex. The ability of the C5b67 complex to be released and then bind to nearby cells must be regulated to prevent the lysis of healthy cells. The membrane bound molecule CD59 protects cells from non-specific complement mediated lysis. Endogenous membrane-bound complement inhibitors in the donor organ were suggested to be ineffective against complement of the recipient, especially at the high levels of attack seen during rejection. To overcome this proposed deficit, several groups have introduced human complement-regulatory proteins into pig organs or cells to obtain high levels of expression in the expectation that organs from these pigs will escape hyperacute rejection [12]. Experimental data indicate that this strategy is so far promising. Porcine aortic endothelial cells transfected with human complement regulatory proteins were effectively protected from lysis by human serum [13, 14]. Furthermore, pigs transgenic for human CD59 and /or DAF have been developed and organs obtained from these donors showed enhanced survival in ex-vivo models of xenotransplantation [15, 16] and when transplanted into primates [17, 18]. However, as a necessary preliminary to these studies, it is essential to understand the properties of the endogenous pig complement regulators, particularly with respect to their capacity to inhibit human complement. We here review the current knowledge about three important membrane complement inhibitors in the pig: CD59, MCP and DAF. 6. Pig CD59 Human CD59, the major inhibitor of the complement membrane attack complex, is an 18 to 20 kDa glycoprotein, linked to the membrane via a glycosylphosphatidylinositol (GPI) anchor [19]. It is expressed on a wide range of cell types of both hemopoetic and nonhemopoetic origins [20]. The complement inhibitory activity of CD59 lies in its ability to bind to the α chain of C8 in the C5b-8 complex, and also to the b domain of C9 [21]. CD59 binding to these two molecules prevents C9 insertion through the lipid membrane, and also prevents C9 polymerization thus inhibiting the formation of the pore structure of the membrane attack complex (Figure 2). The gene for Figure 2. Inhibition of MAC assembly by CD59. CD59 binds to the C5b-8 complex, formed in the membrane by the C5 convertase, preventing C9 incorporation and polymerization. human CD59 is localised to the short arm of chromosome 11, p14-p13 [22] The pig analogue of CD59 was identified in 1994, when CD59 was isolated from red blood cells by preparative electrophoresis [23]. Identificatio