Heterologous Complement Research Articles

GhTLP1, a thaumatin-like protein 1, improves Verticillium wilt resistance in cotton via JA, ABA and MAPK signaling pathway-plant pathways

Verticillium wilt of cotton is a very serious soil-borne disease and there is no effective control method. The mechanism of Gossypium hirsutum thaumatin-like protein 1(GhTLP1) in upland cotton regulating Verticillium wilt resistance has been an uncovered research approach. GhTLP1 is mainly localized in the cell wall. Overexpression of GhTLP1 significantly enhanced Arabidopsis plants resistance to Verticillium dahliae, while its homologous mutant tlp1 in Arabidopsis was more susceptible to the pathogen, and the heterologous complement line (EC) recovered resistance to V. dahliae. GhTLP1 responds to jasmonate acid (JA) and abscisic acid (ABA) hormones and regulates mitogen-activated protein kinase (MAPK) signaling pathway-plant pathway to enhance Arabidopsis plants resistance to V. dahliae. Silencing GhTLP1 resulted decrease in cotton plants resistance to V. dahliae. Moreover, the mutation of GhTLP1 at site Tyr97 and Tyr199 with the phosphorylation also decreased plant resistance to V. dahliae. Therefore, GhTLP1 phosphorylation was observed important in cotton plants against V. dahliae. Further analysis demonstrated that GhTLP1 interacted with gossypium hirsutum laccase 14 (GhLAC14) to enhance plants resistance to V. dahliae. Silencing GhLAC14 resulted decrease in cotton plants resistance to V. dahliae. Here, we propose that GhTLP1 is a potential molecular target for improving resistance to Verticillium wilt in cotton.

Neutrophils express CD52 and exhibit complement-mediated lysis in the presence of alemtuzumab

Neutropenia is a recognized adverse event in patients treated with the humanized anti-CD52 monoclonal antibody alemtuzumab. However, as it is widely believed that neutrophils do not express CD52, the etiology of alemtuzumab-associated neutropenia is unclear. We have found that neutrophils express both mRNA coding for CD52 and the protein itself on the cell surface. We confirmed cell-surface expression using 3 different anti-CD52 antibodies, and note that neutrophils express lower levels of CD52 than lymphocytes and eosinophils. Further, incubation of alemtuzumab with neutrophils results in dose-dependent, complement-mediated lysis in the presence of both heterologous and autologous complement. These data offer an explanation for the etiology of alemtuzumab-associated neutropenia. In a climate of increased use of alemtuzumab in leukemia and other disease states, as well as in transplantation, these data highlight the need for increased vigilance of emerging neutropenia in patients treated with alemtuzumab.

Thyroid follicular cell function after non-lethal complement membrane attack

Terminal complement complexes have been identified around thyroid follicles in Graves' disease and Hashimoto's thyroiditis, and the concentrations of such complexes are increased in the sera of these patients, suggesting a role for complement activation and membrane attack complexes (MAC) in autoimmune thyroiditis. This has been investigated further using cultured human and rat thyroid cells. Thyrocytes were resistant to lysis by homologous complement, in contrast to the effects of heterologous (rabbit) complement. The formation of non-lethal amounts of MAC, using reactive lysis or classical pathway activation, significantly reduced cAMP production by these cells in response to thyroid-stimulating hormone (TSH) (P less than 0.01); similar effects were seen with thyroid-stimulating antibodies. Thyroid cells were able to recover rapidly from complement attack after washing and incubation for 30 min. Non-lethal MAC formation also resulted in reactive oxygen metabolite production, detected by luminol-dependent chemiluminescence in three out of five thyroid cell preparations tested. Ionomycin, but not TSH, also stimulated reactive oxygen metabolite production. These results suggest that repeated or continuous sub-lethal complement attack on thyroid cells may exacerbate hypothyroidism in Hashimoto's thyroiditis, or partially counter the effects of thyroid-stimulating antibodies in Graves' disease. Furthermore, the production of reactive oxygen metabolites in these circumstances could increase the intra-thyroidal inflammatory response; oxygen radical scavenging by anti-thyroid drugs (which are concentrated by thyrocytes) may account in part for the amelioration of thyroiditis observed with such treatment.

Human DAF on pig cells protects against human and non-human primate sera cytotoxicity mediated by exogenous or endogenous complement, as determined by flow cytometry

Expression of human complement regulatory proteins (CRP) in pig cells through transgenesis was proposed to prevent complement activation and the ensuing rejection of pig tissues and organs following pig-to-primate transplantation. Transplantation in non-human primates of organs from transgenic pigs for human decay accelerating factor (hDAF) did not undergo hyperacute rejection, but hDAF could not prevent humoral xenograft rejection (AHXR). A possible explanation for the lack of efficacy of the expression of human complement regulatory proteins in pig cells to prevent AHXR may be interspecies differences between human and non-human complement regulatory system. We assayed the efficacy of transgenic hDAF expressed on porcine cells to inhibit the in vitro complement activity of primate sera. The individual cytotoxicity of sera from seven untreated baboons and of pools of normal human and baboon sera was assayed with endogenous and exogenous complement using a flow-cytometry complement-mediated cytotoxicity assay (FCCA) against peripheral blood lymphocytes (PBL) from hDAF and non-transgenic pigs. We also analyzed the anti-Galα1-3Gal (αGal) antibody titre of the baboon sera by ELISA and the expression of hDAF on the PBL surface by immunofluorescence. Transgenic hDAF expression was capable of protecting pig cells against injury produced by both baboon and human serum. Cellular expression of hDAF reduced cytotoxicity mediated by endogenous and exogenous complement, although the former was slightly higher. Humoral cytotoxicity was not related to a particular antibody but was inversely related to hDAF expression. The presence of hDAF protected pig cells against lysis by NHS more effectively than against NBS. These results confirm in vitro the protective role of hDAF in pig cells to heterologous complement mediated damage, but they also suggest that the extent of hDAF protection decreases, however, if cells express low levels of hDAF.

Pseudorabies virus (PRV) is protected from complement attack by cellular factors and glycoprotein C (gC)

Swine kidney derived CPK cells were resistant to swine complement attack in vitro while rabbit kidney derived RK13 cells were destroyed by swine complement. To rabbit complement, RK13 cells were resistant but CPK cells were sensitive. This phenomenon was known as homologous restriction (Proc. Natl. Acad. Sci. USA 78 (1981) 5118). The gC deletion mutant of pseudorabies virus (PRVdlgC) grown in CPK cells was resistant to swine complement while the same virus grown in RK13 cells was neutralized by swine complement. PRVdlgC grown in RK13 cells was more resistant to rabbit complement than the virus grown in CPK cells. Hence, the sensitivity of PRVdlgC to swine or rabbit complement was similar to that of the cells in which the virus was grown. It would appear that cell derived factors were present on the virion and they were protective against homologous complement but not against heterologous complement. The expression of gC rendered PRV more resistant to swine or rabbit complement, but the protective effect of gC was much less than that of cell derived factors. The best protection against complement was obtained when gC and cell derived factors were coexistent on the virion.

Human and rodent decay-accelerating factors (CD55) are not species restricted in their complement-inhibiting activities.

Homologous complement activation is restricted on cells by the complement regulators, decay-accelerating factor (DAF), membrane cofactor protein (MCP) and CD59. These proteins act in concert with other membrane structures to protect cells from homologous complement attack. In contrast, cells are usually sensitive to heterologous complement attack. It has been suggested that species-specific restriction of complement activation can be attributed to the inability of regulators to inhibit across species. We have investigated the capacities of human, rat and mouse analogues of DAF to regulate homologous and heterologous complement. Cells transfected with cDNA encoding these analogues were protected from heterologous complement attack. C3b-deposition experiments indicated that whilst cells were best protected by DAF from the same species, all three analogues inhibited human, rat and mouse complement. Comparable results were obtained in haemolysis assays using soluble, recombinant forms of the proteins. Inhibition of the classical pathway (CP) was best achieved with homologous DAF, although human DAF also inhibited rat complement, rat DAF also inhibited human complement and mouse DAF inhibited complement from all species. Human DAF was the best inhibitor of alternative pathway (AP)-mediated attack, inhibiting complement from all species. Mouse DAF inhibited mouse and rat AP, whilst rat DAF inhibited only rat AP. These data indicate that human and rodent analogues of DAF are not species restricted and highlights interesting differences in the capacity to regulate AP and CP. This has implications in broader fields of research, such as xenotransplantation, where cross-species regulation of complement is of paramount importance.

THE AVOIDANCE OF FORSSMAN-LIKE REACTION IN HAMSTERS RECEIVING RAT ANTISERUM IS DUE TO ACCOMMODATION TO HETEROLOGOUS COMPLEMENT

532 Forssman reaction can be induced in guinea pigs by injecting anti-Forssman Ab, but this reaction can be avoided if the same is given in progressively increasing doses. Shedding of Forssman antigen-antibody complexes from endothelium has been postulated as the mechanism of desensitization from this reaction and it has been hypothesized that organ xenografts might become less vulnerable to hyperacute rejection, if exposed to tolerable levels of xenoantibodies prior to transplantation (Tx). A Forssman-like reaction can be produced in hamsters (Forssman +) by injecting into them a lethal dose of rat (Forssman −) antihamster Ab. We examined whether desensitizing hamsters to this reaction could render their hearts less susceptible to humoral rejection when transplanted into rats. Anti-hamster Ab was prepared by implanting hamster hearts into rats and collecting the sera 7 days after Tx. The titer of the anti-hamster Ab was 1/4000 and 0.2 cc of this unmodified antiserum was determined to be the lethal dose (LD) required to induce fatal Forssman-like reaction. Hamsters were placed in 3 groups according to these treatments: (1) a single LD of anti-hamster Ab (n=5); (2) desensitization by injecting hamsters (i.v.) with unmodified anti-hamster antiserum in progressively increasing doses over a period of 48 h for a total of 25 LD (n=5); and (3) a single injection of 10 LD of antiserum that had been heat-inactivated (56°C for 30 min) to deplete complement (n=5). The hearts from the desensitized hamsters in Group 2 were harvested and transplanted into rats. Samples from lungs, liver and heart xenografts were taken at death or sacrifice for H&E and staining for rat IgM and C3. All hamsters in Group 1 died of lung edema within 5 min of Ab infusion. Histology revealed severe congestion of the lungs, perivascular edema and hemorrhage. Moreover, strong binding of rat IgM and C3 in the lungs were observed. On the contrary, hamsters that had received 25 LD in Group 2 appeared unharmed. However, the hearts taken from these "adapted" hamsters suffered hyperacute rejection in 2-9 min when transplanted into rats (normal rejection time: 3 d). Adapted hamsters showed moderate lung congestion and arteritis, the liver was infarcted with evidence of multifocal hepatocyte necrosis. Rat IgM was bound to the lungs, heart and kidney, indicating that the xenoantigen was still present. However, there was no evidence of C3 deposits in the lungs. Hamsters in Group 3 who received a single injection of 10 LD of decomplemented antiserum did not undergo the Forssman-like reaction and all survived. At sacrifice, their lungs and livers were mildly congested with strong binding of IgM but little or no C3. In conclusion, adaptation of lung endothelium to heterologous complement seems to be the main mechanism underlying this phenomenon and not the antigenic modulation on endothelial cells. Moreover, this adaptation is not conferred to other organs in the same animal.

Identification of a novel complement-dependent serum-elicited inward current in the Xenopus oocyte provoking Ca 2+ influx and subsequent activation of Cl − channels

The membrane spanning complement channel is assumed to be a nonselective ion ‘pore’, although little evidence is available to support this hypothesis. In this paper we provide evidence that Ca 2+ entry and Cl − exit occur rapidly after complement activation and precede the development of a long-lasting complement-dependent inward current. Addition of rabbit serum (a source of heterologous complement) and mouse anti-human insulin receptor antibody to a single Xenopus oocyte expressing human insulin receptor was shown to stimulate an initial hyperpolarising current followed by a sustained depolarising current. On voltage clamping the oocyte, a novel long-lasting inward current generated by serum addition was detected. Complement classical pathway-stimulated calcium influx into the oocyte was directly demonstrated using 45Ca influx measurements. In addition, we found that Ca 2+ influx was required for the stimulation of the complement alternative pathway-dependent inward current. The novel conductance elicited by the classical pathway was outwardly rectifying, had a reversal potential of −35 ± 8 mV (or −52 ± 7 mV in the presence of chloride channel inhibitors), was inhibited by nifedipine, and was observed in the presence but not in the absence of the pore-forming complement component C9. As overactivation of complement does play a role in many inflammatory or autoimmune diseases, inhibition of early complement-mediated ion flux might restrict tissue damage and aid recovery from such diseases.

Role of decay-accelerating factor in regulating complement activation on the erythrocyte surface as revealed by gene targeting.

Decay-accelerating factor (DAF) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that inhibits both the classical and the alternative pathways of complement activation. DAF has been studied extensively in humans under two clinical settings: when absent from the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) patients, who suffer from complement-mediated hemolytic anemia, and in transgenic pigs expressing human DAF, which have been developed to help overcome complement-mediated hyperacute rejection in xenotransplantation. Nevertheless, the exact role of DAF in regulating complement activation in vivo on the cell surface and the species specificity of this molecule remain to be fully characterized. To address these issues, we have used gene targeting to produce mice lacking GPI-anchored DAF. We found that erythrocytes from mice deficient in GPI-anchored DAF showed no increase in spontaneous complement activation in vivo but exhibited impaired regulation of zymosan-initiated bystander and antibody-triggered classical pathway complement activation in vitro, resulting in enhanced complement deposition. Despite a high level of C3 fixation, no homologous hemolysis occurred. It is noteworthy that GPI-linked DAF knockout erythrocytes, when tested with human and guinea pig sera, were more susceptible to heterologous complement lysis than were normal erythrocytes. These results suggest that DAF is capable of regulating homologous as well as heterologous complement activation via the alternative or the classical pathway. They also indicate that DAF deficiency alone is not sufficient to cause homologous hemolysis. In contrast, when the assembly of the membrane-attack complex is not properly regulated, as in the case of heterologous complement activation or in PNH patients, impaired erythrocyte DAF activity and enhanced C3 deposition could lead to increased hemolytic reaction.

Targeted disruption of the murine decay-accelerating factor (DAF) gene: Studies of erythrocytes reveal activity of DAF in regulating both autologous and heterologous complement activation

Targeted disruption of the murine decay-accelerating factor (DAF) gene: Studies of erythrocytes reveal activity of DAF in regulating both autologous and heterologous complement activation

Complement-mediated pulmonary xenograft injury: studies in swine-to-primate orthotopic single lung transplant models.

The pathogenesis of acute pulmonary xenograft injury has not yet been determined. The present study evaluates the role of complement in mediating pulmonary xenograft dysfunction by using cobra venom factor (CVF) to deplete recipient complement and transgenic swine, which express human regulators of complement activation (human decay-accelerating factor [hDAF] and hCD59). Fifteen orthotopic lung transplants were performed as follows: group I, swine-to-swine (n=5); group II, unmodified swine-to-baboon (n=3); group III, unmodified swine-to-(CVF treated) baboon (n=3); and group IV, hCD59/hDAF swine-to-baboon (n=4). Left pulmonary artery flow and pulmonary vascular resistance were measured at 30-min intervals. Serial lung biopsies were examined by light microscopy and immunofluorescence. The activation of complement was quantified by measurement of baboon plasma CH50 and C4 functional activity. Group II xenotransplants ceased functioning within 30 min of reperfusion. Histopathologic ab normalities included erythrocyte/platelet aggregates and hemorrhagic pulmonary edema. Groups I and IV showed excellent function throughout. hDAF/hCD59 lungs (group IV) showed trace venular fibrin plugs and moderate loss of alveolar architecture. Pretreatment with CVF (group III) was ineffective in preventing xenograft injury. These results characterize the fundamental features of discordant pulmonary xenotransplantation. Correction of the known defects in the regulation of heterologous complement activation was partially effective in preventing pulmonary xenograft dysfunction, suggesting that complement mediates, in part, some of the features of acute lung injury after discordant lung xenotransplantation.

Neuronal protection of oligodendrocytes from antibody-independent complement lysis.

Cultured rat oligodendrocytes are lysed by complement via antibody-independent activation of the classical pathway. This susceptibility to complement lysis has been demonstrated to be due to lack of CD59, a complement regulatory protein which inhibits assembly of the membrane attack complex. In this study the effects of homologous and heterologous complement were examined in a co-culture system of rat oligodendrocytes and peripheral neurones, where axonal ensheathment was observed as early as 4 days after the addition of glial progenitors to the neurones. Following exposure to complement, ensheathing oligodendrocytes were markedly less sensitive to antibody-independent but not antibody-dependent complement lysis than were cells grown without neurones. Immunocytochemical data revealed that co-cultured oligodendrocytes remained CD59 negative, but in contrast to oligodendrocytes cultured alone, were negative for C3b when incubated with C7-deficient serum. Taken together these data indicate that the decreased sensitivity of co-cultured oligodendrocytes to complement lysis is not attributed to the increased expression of CD59, but rather in a failure to activate complement. Incubation of oligodendrocytes with neurone-conditioned medium afforded significant protection (68%), against antibody-independent complement attack, suggesting that soluble neuronal factors can protect oligodendrocytes from complement-mediated lysis.

Mapping the regions of the complement inhibitor CD59 responsible for its species selective activity.

CD59 is a widely distributed membrane-bound glycoprotein that inhibits the formation of the cytolytic membrane attack complex (MAC) of complement on host cells. CD59 from different species varies in its capacity to inhibit heterologous complement, and this species selective function of CD59 contributes to the phenomenon of homologous restriction. Here, we demonstrate that human CD59 is not an effective inhibitor of rat complement, although rat CD59 inhibits rat and human complement equally well. By constructing human-rat CD59 chimeric proteins, we have mapped the residues important in conferring human CD59 species selectivity to two regions; 40-47 and 47-66 in the primary structure. Analysis of a model of the molecular surface of human CD59 revealed that residues 40-66 mapped to a region in the three-dimensional structure that surrounds residues previously identified as important for CD59 function.

Production of the Rat Complement Regulator, Crry, as an Active Soluble Protein inPichia pastoris

In this report, we describe the use of the methylotrophic yeastPichia pastorisfor the production of the rat complement regulator, Crry. Crry normally exists as an intrinsic membrane protein containing six to seven short consensus repeats (SCRs), a transmembrane region, and a cytoplasmic tail. To produce Crry as a soluble recombinant protein, nucleotides encoding the five N-terminal SCRs from the rat Crry cDNA were amplified by PCR, and cloned into theP. pastorisexpression vector, pPIC9. This vector contains the yeast α-factor signal sequence, thereby leading to secretion of recombinant protein. This construct was subsequently integrated intoP. pastorisstrain GS115 genomic DNA. Secreted soluble Crry was produced by induction of theAOX1promoter with methanol. Recombinant Crry protein was purified to homogeneity by sequential Mono Q and Mono P chromatography. The protein was highly active toward the alternative and classical pathways of complement, inhibiting the latter by ≈90% at a concentration of 15 nm. TheP. pastorissystem offers an efficient method for the production of soluble recombinant Crry. Production of active rat Crry offers opportunities to study long-term models of disease in rats, which has not been possible with available heterologous complement inhibitors.

In Vivo Immunological Suppression of Spinal Cord Myelin Development

The onset of myelination in the embryonic chick spinal cord begins on embryonic day (E) 12 or E13 of the 21 day in ovo developmental period. This event coincides with a loss of functional axonal regeneration following complete transection of the thoracic spinal cord. In this study, we have characterised an immunological method for delaying the developmental onset of myelination in vivo until later stages of development (developmental myelin-suppression). A single injection of heterologous or homologous serum complement proteins plus myelin-specific, complement-binding antibodies into the spinal cord prior to E13 delayed the onset of myelination until E17. The state of spinal cord myelin was assessed with immunohistochemical, histological and ultrastructural techniques. Northern blot analysis indicated that myelin basic protein mRNA was not downregulated in myelin-suppressed spinal cords, which suggests that oligodendrocytes survived developmental myelin-suppression. Glial fibrillary acidic protein immunostaining of normal and treated tissue indicated that myelin-suppression did not alter the resident astrocyte population of the spinal cord or elicit astrogliosis. Immunostaining with microtubule-associated protein-2 and thionine staining of normal and myelin-suppressed tissue further indicated that the neuronal architecture was unaffected by the immunological protocol.

CD59 homologue regulates complement-dependent cytolysis of rat Schwann cells

<h2>Abstract</h2> Antibody (Ab) sensitized sciatic nerve Schwann cells (SchC) of 2-day-old rats (SchC/2d) were significantly more susceptible to cytolysis by both heterologous, guinea pig (GP), and homologous rat serum complement (40 ± 3.8% and 21.2 ± 3.1%, respectively) than SchC of 6-day-old rats (SchC/6d) (7.9 ± 5.9% and 2.6 ± 3.1%, respectively). To determine if resistance to complement (C)-mediated cytolysis correlated with expression of membrane proteins which regulate C activation, we used Western blot and FACS analysis. Binding of specific polyclonal Ab demonstrated similar concentrations of Crry, a regulator of C3 convertase formation, on plasma membranes of SchC 2d and 6d. During C activation, both C3b deposition and iC3b formation were greater on SchC/6d than on SchC/2d and the C3b deposition did not correlate with enhanced cytolysis. In contrast, 2.1-fold more rat CD59, a regulator of C8 and C9 incorporation into C5b-9, detected with Western blot on SchC/6d compared with SchC/2d was confirmed by FACS. Further, both rat and GP C8/C9 lysed SchC/2d expressing human C5b-7 (20.1 ± 3.7 and 21.6 ± 4.7%, respectively), while only GP C8/C9 caused cytolysis of 10.7 ± 4.3% SchC/6d expressing hu C5b-7 and rat C8/C9 did not (0.5 ± 0.5%). Preincubation of SchC/6d with an F(ab)₂ fragment of an mAb to rCD59 with blocking capacity, increased cytolysis mediated by rat serum C more than 6-fold to 16.7 ± 3.0% but only 1.7-fold (maximum cytolysis 37.4 ± 11.2%) in SchC/2d. Our data suggest that expression of rat CD59 on SchC increased almost two-fold between postnatal days 2 and 6, and this increased expression on more terminally differentiated SchC is a significant factor in regulating terminal complement complex formation and limiting cytolysis of rat SchC by homologous serum complement.

CD59 homologue regulates complement-dependent cytolysis of rat Schwann cells

Antibody (Ab) sensitized sciatic nerve Schwann cells (SchC) of 2-day-old rats (SchC/2d) were significantly more susceptible to cytolysis by both heterologous, guinea pig (GP), and homologous rat serum complement (40 ± 3.8% and 21.2 ± 3.1%, respectively) than SchC of 6-day-old rats (SchC/6d) (7.9 ± 5.9% and 2.6 ± 3.1%, respectively). To determine if resistance to complement (C)-mediated cytolysis correlated with expression of membrane proteins which regulate C activation, we used Western blot and FACS analysis. Binding of specific polyclonal Ab demonstrated similar concentrations of Crry, a regulator of C3 convertase formation, on plasma membranes of SchC 2d and 6d. During C activation, both C3b deposition and iC3b formation were greater on SchC/6d than on SchC/2d and the C3b deposition did not correlate with enhanced cytolysis. In contrast, 2.1-fold more rat CD59, a regulator of C8 and C9 incorporation into C5b-9, detected with Western blot on SchC/6d compared with SchC/2d was confirmed by FACS. Further, both rat and GP C8/C9 lysed SchC/2d expressing human C5b-7 (20.1 ± 3.7 and 21.6 ± 4.7%, respectively), while only GP C8/C9 caused cytolysis of 10.7 ± 4.3% SchC/6d expressing hu C5b-7 and rat C8/C9 did not (0.5 ± 0.5%). Preincubation of SchC/6d with an F(ab) 2 fragment of an mAb to rCD59 with blocking capacity, increased cytolysis mediated by rat serum C more than 6-fold to 16.7 ± 3.0% but only 1.7-fold (maximum cytolysis 37.4 ± 11.2%) in SchC/2d. Our data suggest that expression of rat CD59 on SchC increased almost two-fold between postnatal days 2 and 6, and this increased expression on more terminally differentiated SchC is a significant factor in regulating terminal complement complex formation and limiting cytolysis of rat SchC by homologous serum complement.

Human CD59 expressed in transgenic mouse hearts inhibits the activation of complement

Porcine-to-human xenotransplantation offers a potential solution to the critical shortage of human organs. The major immunological barrier to xenotransplantation between these species is a rapid rejection process mediated by preformed natural antibodies and complement. Xenogeneic organ grafts are especially susceptible to complement mediated injury because complement regulatory proteins, which ordinarily protect cells from inadvertent injury during the activation of complement, function poorly in regulating activation of heterologous complement. Removal of xenoreactive antibodies or systemic inhibition of complement activity has been shown to prolong graft survival. As an alternative to the systemic inhibition of complement activity, we have established a model system using transgenic animals to test whether the expression of human membrane bound complement regulatory proteins on mouse endothelial cells can inhibit the activation of human complement. CD59, which acts at the terminal stage of complement activation by inhibiting the formation of the membrane attack complex, was used as a paradigm for this model. A CD59 construct containing the putative CD59 gene promoter linked to the CD59 coding region was used to demonstrate expression of the human CD59 protein in various tissues of transgenic mice, including endothelial cells in the heart. In addition, we show that the transgenic CD59 protein is biologically active as determined by the ability to inhibit the formation of membrane attack complex in transgenic mouse hearts perfused ex vivo with human plasma. These results demonstrate that expression of membrane bound complement regulatory proteins can achieve complement inhibition in a xenogeneic organ and suggest that this approach may be useful for successful xenotransplantation between discordant species.

Active Heymann nephritis in complement component C6 deficient rats

The mechanisms of renal injury that result in proteinuria in active Heymann nephritis (AHN) remain unclear, though data suggest that in analogy of the passive form of the disease the membrane attack complex C5b-9 may be involved. AHN was induced in an inbred strain of PVG/c-rats that are totally deficient in the C6 component of complement and are unable to form the lytic C5b-9 complex, as well as in non-complement deficient PVG/c+ rats that are immunologic identical to the deficient strain. In both groups of animals comparably high titers of anti-Fx1A autoantibodies were found after three weeks and persisted at 40 weeks. Proteinuria was also similar in both groups, and was first evident at six weeks. High levels of urinary protein, ranging from 200 mg/24 hr to 500 mg/24 hr, were found after 10 weeks and persisted up to one year. Renal biopsy findings at various times post-immunization were identical in both groups, including immunofluorescence staining for Ig and C3 deposits, and also EM findings of subepithelial electron-dense deposits were not different. The injection of heterologous rabbit complement, that partially and temporarily restored the CH50 activity in PVG/c- rats did not alter or hasten the disease. Long-term follow-up showed that all rats in both groups continued to have severe proteinuria and that most animals died between 8 to 12 months after disease induction, without renal impairment. EM findings in serial biopsies demonstrated that the growth of the subepithelial deposits as measured by surface area occurred between weeks 4 and 12. A positive correlation (r = 0.94) between the size of the deposits and the level of proteinuria was found. These studies demonstrate that the membrane attack complex of complement does not play a major role in AHN. The relationship of the size of the immune deposits to the level of proteinuria suggests that the growth of the immune deposits on itself initiate secondary mechanisms that damage the permselective characteristics of the glomerular membrane.

Human complement regulatory proteins protect swine-to-primate cardiac xenografts from humoral injury

The susceptibility of xenografts to hyperacute rejection is postulated to reflect in part failure of complement regulatory proteins (CRPs) to control activation of heterologous complement on graft endothelium. To test this concept, transgenic swine expressing the human CRP decay accelerating factor and CD59 were developed using a novel expression system involving transfer of the proteins from erythrocytes to endothelial cells. Hearts from transgenic swine transplanted into baboons had markedly less vascular injury and functioned for prolonged periods compared to hearts from nontransgenic swine. These results indicate that expression of human CRPs in xenogeneic organs may contribute to successful xenografting and suggest that intercellular protein transfer might be a useful approach for expression of heterologous proteins in endothelial cells.