The reaction mechanism of beta-1C-globulin (C'3) in immune hemolysis.

During immune hemolysis by human complement, C'3 (beta(1C)-globulin) becomes physically attached to the erythrocyte membrane. Binding of C'3 was found to be mediated by cell-bound, activated C'2 and to have the characteristics of an enzymatic reaction. A single C'4,2a site on the cell surface effected the binding of several hundred molecules of C'3 if the latter was provided in excess. The accumulation of hemolytically inactive, physicochemically altered C'3 in the fluid phase was found to be an inherent feature of the process of C'3 binding. It is postulated that C'4,2a activates C'3 for its subsequent reaction with cell membrane receptors. Antierythrocyte antibody did not play an essential role in C'3 uptake; C'3 could be bound to erythrocyte-C'4,2a complexes which were entirely devoid of antibody. Cell-bound C'3 proved hemolytically active, the degree of hemolysis being proportional to the number of C'3 molecules per cell. Binding of a large number of C'3 molecules per cell was found to be a prerequisite for the production of the immune adherence phenomenon and for immune hemolysis.

Release of heparan sulfate from endothelial cells. Implications for pathogenesis of hyperacute rejection.

Heparan sulfate proteoglycan associated with endothelial cells in normal blood vessels inhibits intravascular coagulation and egress of blood cells and plasma proteins, key features of hyperacute rejection. It was shown herein that exposure of cultured porcine endothelium to human serum as a source of natural antibodies and complement caused cleavage and release of 5% of endothelial cell proteoglycans within 4 min and greater than 50% within 1 h. Proteoglycan release depended on activation of the classical complement pathway and preceded irreversible cell injury. These findings suggest that loss of endothelial cell proteoglycan may be a critical step in the pathogenesis of hyperacute rejection and in diseases involving humoral injury to endothelial cells.

Interleukin-4 induces lipogenesis in porcine endothelial cells, which in turn is critical for induction of protection against complement-mediated injury.

Interleukin (IL)-4 has been shown to induce protection in porcine vascular endothelial cells (ECs) from killing by human complement. This protection is dependent on the PI3K/Akt signaling pathway. In this study, we investigated mechanisms downstream of Akt and found that activation of the lipid biosynthesis pathway is required for protection from complement in ECs treated with IL-4. Cells incubated with IL-4 for 48 hours contained increased fatty acids and phospholipids but cholesterol was not increased when compared with medium-treated controls. The transcription factor SREBP-1, which regulates fatty acid synthesis, was found to be activated in extracts of ECs incubated with IL-4 for 6 hours. Finally, induction of protection from complement killing with IL-4 was fully prevented by the presence of the SREBP inhibitor 25-OH cholesterol. This study showed that IL-4 induces lipid biosynthesis in porcine ECs through activation of SREBP-1 and that the activation of this pathway is critical for IL-4 to induce protection of porcine ECs from killing by human complement. Further study of these mechanisms may provide new strategies for the prevention of complement-mediated vascular injury as it occurs in xenograft rejection.

Neoantigen of the polymerized ninth component of complement. Characterization of a monoclonal antibody and immunohistochemical localization in renal disease.

A monoclonal antibody to a neoantigen of the C9 portion of the membrane attack complex (MAC) of human complement has been developed and characterized. The distribution of this neoantigen was assessed by indirect immunofluorescence microscopy in nephritic and nonnephritic renal diseases. The antibody (Poly C9-MA) reacted on enzyme-linked immunosorbent assay (ELISA) with a determinant in complement-activated serum that was undetectable in normal human serum (NHS). Zymosan particles incubated in NHS had positive immunofluorescent staining with Poly C9-MA; however, binding of Poly C9-MA was not observed with zymosan particles incubated in sera deficient in individual complement components C3, C5, C6, C7, C8, or C9. Reconstitution of C9-deficient sera with purified C9 restored the fluorescence with Poly C9-MA. Poly C9-MA reacted positively by ELISA in a dose-dependent manner with purified MC5b-9 solubilized from membranes of antibody-coated sheep erythrocytes treated with NHS but not with intermediate complement complexes. Poly C9-MA also reacted in a dose-dependent manner on ELISA and in a radioimmunoassay with polymerized C9 (37 degrees C, 64 h) (poly C9) but not with monomeric C9. Increasing amounts of either unlabeled poly C9 or purified MC5b-9 inhibited the 125I-poly C9 RIA in an identical manner. These studies demonstrate that Poly C9-MA recognizes a neoantigen of C9 common to both the MAC and to poly C9. By immunofluorescence, Poly C9-MA reacted minimally with normal kidney tissue in juxtaglomerular loci, the mesangial stalk, and vessel walls. Poly C9-MA stained kidney tissue from patients with glomerulonephritis in a pattern similar to that seen with polyclonal anti-human C3. In tissue from patients with nonnephritic renal disease--diabetes, hypertension, and obstructive uropathy--Poly C9-MA was strongly reactive in the mesangial stalk and juxtaglomerular regions, tubular basement membranes, and vascular walls. Poly C9-MA binding was especially prominent in areas of advanced tissue injury. Poly C9-MA frequently stained loci where C3 was either minimally present or absent. These studies provide strong evidence for complement activation not only in nephritic but also in nonnephritic renal diseases.

Killing of Streptococcus pneumoniae by capsular polysaccharide-specific polymeric IgA, complement, and phagocytes.

The role of IgA in the control of invasive mucosal pathogens such as Streptococcus pneumoniae is poorly understood. We demonstrate that human pneumococcal capsular polysaccharide-specific IgA initiated dose-dependent killing of S. pneumoniae with complement and phagocytes. The majority of specific IgA in serum was of the polymeric form (pIgA), and the efficiency of pIgA-initiated killing exceeded that of monomeric IgA-initiated killing. In the absence of complement, specific IgA induced minimal bacterial adherence, uptake, and killing. Killing of S. pneumoniae by resting phagocytes with immune IgA required complement, predominantly via the C2-independent alternative pathway, which requires factor B, but not calcium. Both S. pneumoniae-bound IgA and complement were involved, as demonstrated by a 50% decrease in killing with blocking of Fcalpha receptor (CD89) and CR1/CR3 (CD35/CD11b). However, IgA-mediated killing by phagocytes could be reproduced in the absence of opsonic complement by pre-activating phagocytes with the inflammatory products C5a and TNF-alpha. Thus, S. pneumoniae capsule-specific IgA may show distinct roles in effecting clearance of S. pneumoniae in the presence or absence of inflammation. These data suggest mechanisms whereby pIgA may serve to control pneumococcal infections locally and upon the pathogen's entry into the bloodstream.

Hemodialysis leukopenia. Pulmonary vascular leukostasis resulting from complement activation by dialyzer cellophane membranes.

Acute leukopenia occurs in all patients during the first hour of hemodialysis with cellophanemembrane equipment. This transient cytopenia specifically involves granulocytes and monocytes, cells which share plasma membrane reactivity towards activated complement components. The present studies document that complement is activated during exposure of plasma to dialyzer cellophane, and that upon reinfusion of this plasma into the venous circulation, granulocyte and monocyte entrapment in the pulmonary vasculature is induced. During early dialysis, conversion of both C3 and factor B can be demonstrated in plasma as it leaves the dialyzer. Moreover, simple incubation of human plasma with dialyzer cellophane causes conversion of C3 and factor B, accompanied by depletion of total hemolytic complement and C3 but sparing of hemolytic C1. Reinfusion of autologous, cellophane-incubated plasma into rabbits produces selective granulocytopenia and monocytopenia identical to that seen in dialyzed patients. Lungs from such animals reveal striking pulmonary vessel engorgement with granulocytes. The activated complement component(s) responsible for leukostasis has an approximate molecular weight of 7,000-20,000 daltons. Since it is generated in C2-deficient plasma and is associated with factor B conversion, it is suggested that activation of complement by dialysis is predominantly through the altermative pathway.

Complement and HLA. Further definition of high-risk haplotypes in insulin-dependent diabetes.

The families of 41 probands with type I (insulin-dependent) diabetes mellitus (IDDM) were typed for HLA-A, HLA-B, and HLA-DR antigens in addition to the complement polymorphisms C2, C4A, C4B, and Bf. All of these loci are encoded on the short arm of human chromosome 6 in a narrow region. Alleles at HLA-B (8, 15, 18, and 40), HLA-DR (3 and 4), and Bf (F1) have been associated with increased relative risk (RR) for IDDM, while HLA-B7 and HLA-DR2 have been associated with decreased RR for IDDM. This study confirms those significant risks in addition to confirming increased risk for the null (silent) allele for C4A (C4AQ0) and a rare C4B variant (C4B2.9). The significantly associated antigens (alleles) and risks were: HLA-B8 (RR = 3.1), HLA-DR3 (RR = 5.2), HLA-DR4 (RR = 4.3), and BfF1 (RR = 7.1), in addition to C4AQ0 (RR = 2.8) and C4B2.9 (RR = 12.6). Significantly low risk was associated only with HLA-DR2 (RR = 0.1). In a recent study, we defined five high-risk haplotypes that were determined solely by HLA-B, Bf, and HLA-DR (B8-BfS-DR3, B8-BfS-DR4, B15-BfS-DR4, B18-BfF1-DR3, and B40-BfS-DR4). By inclusion of information from the complement polymorphism, we have defined in greater detail three of these five high-risk haplotypes. One previously identified haplotype (B40-BfS-DR4) showed no complement clustering, while the rare high-risk haplotype (B8-BfS-DR4) was seen only once in this smaller sample.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of complement-mediated activation of xenogeneic endothelial cells in an in vitro model of xenograft hyperacute rejection by C1 inhibitor.

The complement system plays a major role in hyperacute rejection of discordant xenografts. In an immediately vascularized xenograft of a porcine organ to a primate, natural antibodies bind to the vascular endothelium of the graft, triggering activation of complement via the classical pathway. One consequence of antibody binding and complement activation is the activation of endothelial cells leading to the loss from the cells of heparan sulfate. We explored to what extent the classical pathway regulatory protein C1 inhibitor (C1 inh) would inhibit complement-mediated cytotoxicity and activation of endothelial cells. Cultured porcine aortic endothelial cells were used as a model for a xenogeneic organ and human serum as a source of xenoreactive natural antibody and complement. Addition of purified human C1 inh to human serum inhibited deposition of C4b and iC3b and cytotoxicity after the serum was reacted with the cultured cells. C1 inh prevented, in a dose-dependent manner, activation of the endothelial cells, as manifested by release of heparan sulfate. These observations demonstrate that C1 inh added in sufficient amounts to human serum can effectively inhibit C1 activation in an antigen-antibody system. These studies extend our previous findings consistent with the concept that complement activation occurs via the classical pathway in models of hyperacute rejection in which porcine vascular endothelial cells are in contact with human serum containing xenogeneic natural antibodies against the endothelial cells. Thus, our results suggest a potential clinical use of C1 inh in conjunction with other therapies to prevent hyperacute rejection in xenogeneic combinations mediated by complement activation via the classical pathway.

Selective IgM depletion prolongs organ survival in an ex vivo model of pig-to-human xenotransplantation.

In the pig-to-primate model, xenograft hyperacute rejection (HAR) is mediated by antibody and complement. Previous studies have implicated xenoreactive IgM natural antibody (nAb) as the predominant immunoglobulin involved in HAR. To further evaluate the role of IgM, we selectively reduced IgM levels in human blood, without changing IgG and IgA levels, and then used this blood to perfuse porcine hearts ex vivo. Specific IgM depletion was accomplished with an immunoabsorption column containing sheep anti-human IgM (mu-chain specific) conjugated to Sepharose beads. Human blood was separated into plasma and cellular components. For control experiments, those components were unmodified and recombined in the perfusion system. For experiments with IgM reduced blood, the plasma was passed through the IgM column. Immunoabsorption resulted in approximately 90% reduction in xenoreactive IgM levels, as measured by ELISA. Porcine hearts perfused with unmodified human blood survived 25 +/- 5.6 min (n=5). Porcine hearts perfused with human blood containing reduced levels of IgM survived 229 +/- 45.2 min (n=4; P<0.01). Organ survival was negatively associated with xenoreactive IgM nAb levels measured immediately before perfusion (r=-0.83; P=0.01), and not with IgG nAb levels (r=-0.21; P=0.62). The ability of plasma from IgM-depleted blood to elicit complement activation, measured by iC3b binding to porcine aortic endothelial cells in vitro, was also strongly associated with IgM xenoreactive nAb levels (r=0.92; P<0.0001). Control hearts perfused with unmodified human blood showed typical widespread histologic features of HAR, while porcine hearts perfused with IgM-reduced blood demonstrated milder and less uniform changes. Immunopathological analysis of heart tissues obtained at the completion of each study showed similar deposition of IgG between groups but markedly less IgM, C3, C4, and C9 in the IgM reduction group. These results suggest that selective IgM reduction delays HAR with prolongation of survival and that xenoreactive IgM may be the predominant immunoglobulin involved in HAR in the pig-to-human combination.

Evidence that rat xenoreactive antibodies recognize multiple protein antigens on guinea pig endothelial cells and platelets.

Xenoreactive antibodies are an integral part of the natural immune barrier to successful xenotransplantation between phylogenetically disparate species. Studies in primates suggest that the critical targets involved in hyperacute rejection of pig organs are glycoproteins expressed on endothelial cells and platelets. However, there is little information regarding the targets of xenoreactive antibodies and their cellular distribution in other experimental models of hyperacute xenograft rejection, including the commonly used guinea pig-to-rat model. The aim of this study was to characterize the target antigens on guinea pig platelets and endothelial cells that are recognized by rat anti-guinea pig antibodies. Using guinea pig platelet membrane proteins or intact guinea pig endothelial cells as antigens in an ELISA, we demonstrated that rat serum contains IgG* and IgM anti-guinea pig antibodies; levels of antiplatelet antibodies correlated with those against endothelial cells. Serum from naive or complement-depleted rats transplanted with a guinea pig heart was investigated by immunoblotting against membrane proteins extracted from guinea pig platelets and endothelial cells. Normal rat serum revealed numerous bands on either platelet or endothelial cell immunoblots, without obvious similarities in banding pattern under reduced or nonreduced conditions. Absorption of rat serum with intact guinea pig endothelial cells reduced reactivity against numerous bands on endothelial cell membrane blots, but only partially reduced reactivity with three platelet bands (141, 155, and 210 kDa). Absorption of rat serum with intact guinea pig platelets resulted in reduction in reactivity against both platelet and endothelial cell immunoblots. Antibodies eluted from those intact guinea pig endothelial cells and platelets used to absorb rat sera were found to yield patterns of membrane blot reactivity similar to those with unabsorbed sera, suggesting that the proteins recognized are expressed on the cell surface. Lectin affinity blotting demonstrated many of the guinea pig endothelial cell and platelet membrane proteins to be glycosylated. However, digestion of endothelial cell and platelet immunoblots with a mixture of glycosidases failed to change the xenoreactivity with naive rat serum. Immunoblot analysis of serum samples taken daily from complement-depleted rats carrying a functioning guinea pig heart for 3-4 days showed an increase in intensity for specific protein bands on platelets (38, 48, 56, 141, and 155 kDa) and endothelial cells (30, 210, and 270 kDa); no new protein bands were observed. Increased reactivity at several bands correlated with highly elevated rat serum anti-guinea pig antibody titers, as determined by ELISA.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of complement-mediated endothelial cell cytotoxicity by decay-accelerating factor. Potential for prevention of xenograft hyperacute rejection.

Complement plays a major role in hyperacute rejection of discordant xenografts. In immediately vascularized xenografts, such as porcine organs to humans, C activation contributes to triggering of endothelial cell activation and adhesion of leukocytes and platelets to the endothelial cells, which is followed by thrombosis and tissue necrosis. We investigated the potential utility of the membrane-associated inhibitor of C, decay accelerating factor (DAF), in the prevention of C-mediated tissue injury. We used an in vitro model of xenotransplantation consisting of porcine aortic endothelial cells incubated with human serum as the source of xenogeneic natural antibodies and C. Because C inhibitors such as DAF may be relatively species-specific, we tested whether human DAF would incorporate into porcine endothelial cells and function to inhibit cytotoxicity of such cells by human C. We found that purified radiolabeled human DAF incorporated into porcine endothelial cells in a dose-dependent manner and that human DAF very significantly protected the endothelial cells from the cytotoxic effect of human C.

Alpha-galactosyl epitope-mediated activation of porcine aortic endothelial cells: type I activation.

BACKGROUND: The galactose alpha(1-3)galactose (alpha-gal) epitope associated with membrane glycoproteins and glycolipids represents a major determinant recognized on porcine cells by human xenoreactive natural antibodies (XNA). Together, bound XNA and complement rapidly induce porcine aortic endothelial cell (PAEC) activation; this process is associated with cellular shape changes, transient development of intercellular gaps, and loss of ATDPase and thrombomodulin, with release of heparan sulfate. The aim of this study was to evaluate patterns of type I endothelial cell activation (i.e., activation that does not require protein synthesis) following ligation of alpha-gal epitopes with anti-Gal antibodies and alpha-gal-specific lectins. METHODS AND RESULTS: PAEC incubated in the presence of the alpha-gal binding, Bandeiraea simplicifolia lectin (BS-I) underwent cellular shape changes associated with the formation of intercellular gaps. PAEC exposure to BS-I was also associated with the tyrosine phosphorylation of a protein (apparent molecular mass of approximately 130 kDa), not observed following lipopolysaccharide, tumor necrosis factor, or XNA stimulation. This lectin-induced tyrosine phosphorylation was not affected by cytochalasin D (inhibitor of actin filament polymerization), by genistein (inhibitor of tyrosine kinases), or by staurosporine (inhibitor of tyrosine phosphorylation and protein kinase C). In addition, incubation of PAEC with BS-I and monoclonal anti-Gal IgM induced p42/44 map kinase and activated the transcription factor NF-kappaB. CONCLUSIONS: Agonist binding of alpha-gal can evoke endothelial cell activation independently of complement activation. These observations have implications for the survival of xenografts.

Alpha-galactosyl epitope-mediated activation of porcine aortic endothelial cells: type II activation.

BACKGROUND: Xenoreactive natural antibodies (XNAs) and complement mediate hyperacute rejection of discordant xenografts. Inhibition of complement alone results in some prolongation of graft survival, but delayed xenograft rejection still precludes long-term graft survival. In vitro data provide evidence for the direct proinflammatory activation of endothelial cells (ECs) by XNAs. These antibodies are primarily directed against galactose alpha(1-3)-galactose (alpha-gal), the major xenoantigen in the pig to primate xenotransplant model. Previous studies have shown EC activation by XNAs but failed to address the question of whether alpha-gal-specific ligands can induce EC activation. The aim of this study was to investigate whether agonist binding to the alpha-gal epitope by alpha-gal-specific lectins as compared with XNAs or elicited xenoreactive antibodies can directly elicit type II porcine aortic EC (PAEC) activation (i.e., activation that requires protein synthesis). METHODS AND RESULTS: The tetravalent, alpha-gal-binding Bandeiraea simplicifolia lectin I (BS-I), the wholly alpha-gal-specific BS-I isolectin B4, and elicited primate anti-pig xenoreactive antibodies (decomplemented cynomolgus monkey anti-porcine serum) induced E-selectin protein expression in PAECs. This induction was alpha-gal-specific, as preincubation with synthetic alpha-gal carbohydrate or adsorption of lectin or serum to rabbit, but not human, red blood cells removed the activating component. E-selectin expression, induced by BS-I, was inhibited in the presence of genistein, a tyrosine kinase inhibitor, and by mepacrine, an inhibitor of phospholipase A2. Human and primate XNAs lacked this activity when tested at relevant concentrations; however, stimulation of PAECs with affinity-purified human XNA (IgM and IgG) resulted in slightly increased interleukin-8 and P-selectin mRNA levels but had no apparent effects on E-selectin transcription. BS-I strongly induced E-selectin, P-selectin, intercellular adhesion molecule-1, and interleukin-8 mRNA in an NF-kappaB-dependent manner. CONCLUSIONS: Several agonists that specifically bind to alpha-gal can evoke type II EC activation. Hence, anti-Gal antibodies may contribute directly to xenograft rejection in the absence of complement activation.

Activation of intragraft endothelial and mononuclear cells during discordant xenograft rejection.

Most studies of discordant xenograft rejection have focused on the roles of recipient xenoreactive antibody and complement as mediators of hyperacute rejection; there are essentially no data from in vivo studies as to the contribution of endothelial cell responses to the pathobiology of xenograft rejection. We hypothesized that the mechanism by which xenoreactive natural antibodies and complement of the recipient are involved in rejection of a discordant, immediately vascularized xenograft involves donor organ endothelial cell activation, with the consequences of such activation contributing significantly to the rejection process. We performed a kinetic analysis of rejection of guinea pig hearts by untreated Lewis rats or recipients depleted of complement activity that underwent delayed xenograft rejection. We report that in both hyperacute rejection and delayed xenograft rejection there is widespread evidence of endothelial cell activation, including expression of P-selectin and E-selectin, upregulation of tissue factor, and downregulation of thrombomodulin and antithrombin III expression. Many of these changes occur very early posttransplantation in grafts that are not completely rejected until approximately 3 days. In delayed xenograft rejection, an intense cellular infiltrate is seen that results from progressive accumulation of activated macrophages and natural killer cells. T cell receptor alpha/beta+T cells are present only at relatively low levels. This cellular infiltrate is associated with dense expression of pro-inflammatory cytokines, including interferon gamma, interleukin 1, and tumor necrosis factor-alpha. We conclude that both endothelial cell activation and infiltration by activated macrophages and natural killer cells may play an important role in xenograft rejection. These newly described features of the xenogeneic rejection response may require targeting by future therapeutic regimens aimed at prolonging xenograft survival.

A recombinant soluble chimeric complement inhibitor composed of human CD46 and CD55 reduces acute cardiac tissue injury in models of pig-to-human heart transplantation.

BACKGROUND: Inasmuch as complement plays a critical role in many pathological processes and in xenograft rejection, efficient complement inhibitors are of great interest. Because the membrane-associated complement inhibitors are very effective, recombinant soluble molecules have been generated. METHODS: We tested the efficacy of complement activation blocker-2 (CAB-2), a recombinant soluble chimeric protein derived from human decay accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), in two models of pig-to-human xenotransplantation in which tissue injury is complement mediated. The in vitro model consisted of porcine aortic endothelial cells and human serum, and the ex vivo model consisted of a porcine heart perfused with human blood. RESULTS: In vitro, addition of CAB-2 to serum inhibited cytotoxicity and the deposition of C4b and iC3b on the endothelial cells. Ex vivo, addition of CAB-2 to human blood prolonged organ survival from 17.3 +/- 6.4 min in controls to 108 +/- 55.6 min with 910 nM (100 microg/ml) CAB-2 and 219.8 +/- 62.7 min with 1820 nM (200 microg/ml) CAB-2. CAB-2 also retarded the onset of increased coronary vascular resistance. The complement activity of the perfusate was reduced by CAB-2, as was the generation of C3a and SC5b-9. The myocardial tissues had similar deposition of IgG, IgM, and Clq; however, CAB-2 reduced the deposition of C3, C4, and C9. Hearts surviving >240 min demonstrated trace to no deposition of C9 and normal histologic architecture. CONCLUSION: These results indicate that CAB-2 can function as an inhibitor of complement activation and markedly reduce tissue injury in models of pig-to-human xenotransplantation and thus may represent a useful therapeutic agent for xenotransplantation and other complement-mediated conditions.

Assessment of hyperacute rejection in a rat-to-primate cardiac xenograft model.

We studied a rat-to-cynomolgous monkey model for xenotransplantation of vascularized organs and found that a rat heart was rejected in 5.5 +/- 1.4 min (n = 10). This hyperacute rejection (HAR) was consistent with kinetic experiments in vitro that showed damage to rat endothelial cells (ECs) after 3 min of incubation with primate serum. Histopathology and ultrastructural analysis of rejected hearts showed marked EC damage and early adherence of platelets and polymorphonuclear leukocytes to the endothelium. Immunohistochemical analysis revealed deposition along endothelial surfaces of IgG, IgM, and complement (C) components of the classical but not the alternative pathway, suggesting that, as in the pig-to-primate model, HAR is mediated by the binding of recipient xenogeneic natural antibodies and C activation. The effect of C depletion on xenograft survival was evaluated in two recipients that were treated with cobra venom factor (CVF). CVF caused complete C inactivation, demonstrated by lack of serum hemolytic activity and C-dependent EC cytotoxicity at engraftment and until the animals died. The rat cardiac transplants survived for at least 9 hr and 77 hr. Histology showed massive interstitial hemorrhage, edema, and cellular infiltration with scanty fibrin deposits. These results in CVF-treated recipient monkeys indicate that C activation mediates the development of HAR in this rat-to-primate model. We suggest that the model may be of interest as an alternative to the more expensive and time-consuming pig-to-primate model for testing the efficacy of transgenic modification of donor organs to prolong xenograft survival and for studying mechanisms of discordant xenograft rejection.

Expression of human CD59 in transgenic pig organs enhances organ survival in an ex vivo xenogeneic perfusion model.

The serious shortage of available donor organs for patients with end stage organ failure who are in need of solid organ transplantation has led to a heightened interest in xenotransplantation. The major barrier to successful discordant xenotransplantation is hyperacute rejection. Hyperacute rejection results from the deposition of preformed antibodies that activate complement on the luminal surface of the vascular endothelium, leading to vessel occlusion and graft failure within minutes to hours. Endogenous membrane-associated complement inhibitors normally protect endothelial cells from autologous complement -- however, these molecules are species-restricted and therefore are ineffective at inhibiting activated xenogeneic complement. To address the pathogenesis of hyperacute rejection in the pig-to-human combination, F1 offspring were generated from a transgenic founder animal that was engineered to express the human terminal complement inhibitor hCD59. High-level cell surface expression of hCD59 was detected in the hearts and kidneys of these transgenic F1 animals, similar to expression levels in human kidney tissue. The hCD59 was expressed on both large vessel and capillary endothelium. Ex vivo perfusion experiments, using human blood as the perfusate, were performed with transgenic porcine hearts and kidneys to evaluate the ability of hCD59 to inhibit hyperacute rejection. These experiments demonstrated that transgenic organs expressing hCD69 resisted hyperacute rejection, as measured by increased organ function for both the hearts and the kidneys, as compared with control pig organs. Hearts from hCD59-expressing animals demonstrated a five-fold prolongation in function compared with controls, 109.8 +/- 20.7 min versus 21.2 +/- 2.9 min (P = 0.164). The hCD59-expressing kidneys also demonstrated significantly prolonged function at 157.8 +/- 27.0 min compared with 60.0 +/- 6.1 min for controls (P = 0.0174). Deposition of C9 neoantigen In the vasculature of porcine organs perfused with human blood was markedly reduced in organs expressing hCD59. These studies demonstrate that C5b-9 plays an important role in hyperacute rejection of a porcine organ perfused with human blood and suggest that donor pigs transgenic for hCD59 may be an integral component of successful clinical xenotransplantation.

Inhibition of platelet integrin GPIIbIIIa prolongs survival of discordant cardiac xenografts.

The integrin GPIIbIIIa is known to be crucial to the formation of platelet aggregates and potentiates adhesion to subendothelial matrices via fibrin(ogen), von Willebrand factor, and vitronectin. Given the demonstration by us and others of widespread platelet aggregation during xenograft rejection, we hypothesized that platelet thrombi might contribute to graft dysfunction during development of hyperacute rejection (HAR), as well as during what we have termed delayed xenograft rejection (DXR), e.g., as seen in complement-depleted rat recipients of guinea pig cardiac xenografts. We therefore tested the effects of a specific GPIIbIIIa antagonist (SDZ GPI 562) during xenograft rejection. Lewis rats received heterotopic guinea pig cardiac xenografts and were treated with GPI 562 alone (HAR model) or in combination with cobra venom factor (CVF) (DXR model). A high (0.5 mg/kg) or a low dose (0.1 mg/kg) of GPI 562 was administered perioperatively and then given twice daily in the same dose until rejection. CVF was given daily until rejection. Plasma drawn after the first dose of GPI 562 and at the time of rejection was tested for the ability to inhibit ADP-stimulated platelet aggregation in vitro. Rejected grafts were analyzed by immunohistology. Plasma from animals in the high-dose group completely inhibited platelet aggregation in vitro, whereas plasma from the low-dose group resulted in only partial inhibition. Similarly, whereas low-dose GPI 562 failed to prolong graft survival, high-dose GPI 562 showed a statistically significant increase in graft survival in both HAR and DXR groups. Immunohistologic studies of HAR showed little effect of GPI 562 on platelet aggregation or activation and no effect on fibrin deposition. However, the combination of high-dose GPI 562 and CVF resulted in a significant decrease in intragraft platelet aggregation, P-selectin expression, and leukocyte infiltration compared with CVF alone. In conclusion, GPIIbIIIa antagonist therapy can inhibit platelet aggregation in vitro and prolong xenograft survival. The diminution of intragraft platelet microthrombi formation and leukocyte infiltration suggests an important role for platelet-dependent mechanisms in leukocyte recruitment during DXR.

The immunopathology of cardiac xenograft rejection in the guinea pig-to-rat model.

The mechanisms underlying rejection by rats of vascularized guinea pig xenografts have been controversial. The aim of this study was to define, using sequential immunopathologic analysis, the contributions of xenoreactive antibody, complement, and effector cells to the rejection of guinea pig cardiac xenografts by Lewis rats. In untreated recipients, hyperacute rejection of guinea pig cardiac xenografts occurred in 20 +/- 10.2 min and was characterized by focal endothelial deposition of IgM and by diffuse deposition of C3. IgG was not localized to endothelial surfaces, but was present in the same locations as albumin, suggesting that the accumulation of IgG might reflect nonspecific leakage of plasma proteins from blood vessels. No polymorphonuclear or monocytic infiltrate was observed. Depletion from rats of xenoreactive antibody to undetectable levels prolonged the survival of guinea pig cardiac xenografts, but did not prevent hyperacute rejection; the rejected xenografts contained deposits of C3 along the microvasculature but no deposits of IgM or IgG. No cellular infiltrate was observed. Depletion of complement with cobra venom factor prolonged the survival of xenografts up to 96 hr. Xenograft tissues from complement-depleted animals had diffuse deposits of IgM along the microvasculature, but no detectable deposits of C3 or IgG were noted. Graft tissues obtained at various times after transplantation into complement-depleted animals revealed cellular infiltrates consisting of granulocytes, monocytes, and lymphocytes, but few cells bearing an NK cell phenotype. Our findings are consistent with the concept that complement activation is essential for the hyperacute rejection of discordant xenografts, and that in this particular model complement activation can proceed without the involvement of antibody. However, our findings also suggest that xenoreactive antibody contributes to hyperacute rejection and, along with effector cells, contributes to the later rejection of a xenograft when hyperacute rejection has been averted. Finally, we show that when hyperacute rejection is avoided, a form of vascular rejection occurs in which certain of the pathologic features--i.e., interstitial hemorrhage, interstitial edema, and thrombosis--are very similar to those observed in hyperacute rejection. Whether this form of rejection is a delayed form of the process that leads to hyperacute rejection or a novel pathologic process of graft rejection has yet to be determined.

Prolongation of cardiac xenograft survival by depletion of complement.

Complement (C) activation is thought to be critical for the hyperacute rejection of xenografts. We investigated the role of C in the rejection of discordant cardiac xenografts by studying outcome in recipients depleted of C, using a highly purified form of cobra venom factor (CVF) in both a small (guinea pig [GP]-to-rat) and large (pig-to-baboon) animal model. A single dose of 30 or 60 units CVF given i.v. to rats completely abrogated hemolytic C activity for up to 72 hr. The lack of hemolytic C activity correlated with nearly undetectable serum levels of C3. Doses of 30 U/kg daily or 60 U/kg every other day over a 7-day period sustained C depletion without morbidity or mortality. Rats receiving GP cardiac xenografts during CVF therapy had significantly prolonged xenograft survival (88 +/- 10 hr in CVF-treated rats vs. 18.6 +/- 7.2 min in control rats, P < 0.001). Rats that rejected GP xenografts at 4 days posttransplant had higher levels of anti-GP antibodies than control rats, without hemolytic C activity at rejection. This rise in xenoreactive Ig reflected an increase in circulating IgG and IgM against GP antigens recognized before transplantation. Histologic analysis of GP cardiac xenografts taken from CVF-treated rats revealed leukocyte and monocyte margination along blood vessels, beginning at 12 hr posttransplant. Progressive cell infiltration, interstitial hemorrhage, and necrosis were observed over the next 72 hr. Rejected GP xenografts showed diffuse deposition of IgM and fibrin within blood vessels but no evidence of C3 deposition. A nonspecific pattern of IgG deposition was noted. CVF was tested in baboons. Complete C depletion was achieved with a dose of 60 U/kg, and was not associated with any morbidity or mortality. Xenotransplantation of a pig heart was performed in one baboon receiving CVF, 60 U/kg/day, for 2 consecutive days. Xenograft survival was prolonged to 68 hr, compared with 90 +/- 30 min in control baboons. Lack of hemolytic activity was noted during engraftment and at rejection. Histology showed evidence of vascular rejection. Immunopathology showed diffuse deposition of IgM, fibrin, and C4, and absence of C3 or membrane attack complex. We conclude that highly purified CVF can achieve marked C depletion with minimal morbidity and no associated fatalities. CVF alone can significantly prolong discordant cardiac xenograft survival. In the GP-to-rat model, the improvement in graft survival achieved with CVF was better than with conventional immunosuppression or isolated acute antibody depletion.(ABSTRACT TRUNCATED AT 400 WORDS)