Formation of high affinity C5 convertase of the classical pathway of complement.

C3/C5 convertase is a serine protease that cleaves C3 and C5. In the present study we examined the C5 cleaving properties of classical pathway C3/C5 convertase either bound to the surface of sheep erythrocytes or in its free soluble form. Kinetic parameters revealed that the soluble form of the enzyme (C4b,C2a) cleaved C5 at a catalytic rate similar to that of the surface-bound form (EAC1,C4b,C2a). However, both forms of the enzyme exhibited a poor affinity for the substrate, C5, as indicated by a high Km (6-9 microM). Increasing the density of C4b on the cell surface from 8,000 to 172,000 C4b/cell did not influence the Km. Very high affinity C5 convertases were generated only when the low affinity C3/C5 convertases (EAC1,C4b,C2a) were allowed to deposit C3b by cleaving native C3. These C3b-containing C3/C5 convertases exhibited Km (0.0051 microM) well below the normal concentration of C5 in blood (0.37 microM). The data suggest that C3/C5 convertase assembled with either monomeric C4b or C4b-C4b complexes are inefficient in capturing C5 but cleave C3 opsonizing the cell surface with C3b for phagocytosis. Deposition of C3b converts the enzymes to high affinity C5 convertases, which cleave C5 in blood at catalytic rates approaching Vmax, thereby switching from C3 to C5 cleavage. Comparison of the kinetic parameters with those of the alternative pathway convertase indicates that the 6-9-fold greater catalytic rate of the classical pathway C5 convertase may compensate for the fewer numbers of C5 convertase sites generated upon activation of this pathway.

Membrane attack complex contributes to destruction of vascular integrity in acute lung allograft rejection.

The lung is known to be particularly susceptible to complement-mediated injury. Both C5a and the membrane attack complex (MAC), which is formed by the terminal components of complement (C5b-C9), can cause acute pulmonary distress in nontransplanted lungs. We used C6-deficient rats to investigate whether MAC causes injury to lung allografts. PVG.R8 lungs were transplanted orthotopically to MHC class I-incompatible PVG.1U recipients. Allografts from C6-sufficient (C6(+)) donors to C6(+) recipients were rejected with an intense vascular infiltration and diffuse alveolar hemorrhage 7 days after transplantation (n = 5). Ab and complement (C3d) deposition was accompanied by extensive vascular endothelial injury and intravascular release of von Willebrand factor. In contrast, lung allografts from C6-deficient (C6(-)) donors to C6(-) recipients survived 13-17 days (n = 5). In the absence of C6, perivascular mononuclear infiltrates of ED1(+) macrophages and CD8(+) T lymphocytes were present 7 days after transplantation, but vascular endothelial cells were quiescent, with minimal von Willebrand factor release and no evidence of alveolar hemorrhage or edema. Lung allografts were performed from C6(-) donors to C6(+) recipients (n = 5) and from C6(+) donors to C6(-) recipients (n = 5) to separate the effects of systemic and local C6 production. Lungs transplanted from C6(+) donors to C6(-) recipients had increased alveolar macrophages and capillary injury. C6 production by lung allografts was demonstrated at the mRNA and protein levels. These results demonstrate that MAC causes vascular injury in lung allografts and that the location of injury is dependent on the source of C6.

C6 produced by macrophages contributes to cardiac allograft rejection.

The terminal components of complement C5b-C9 can cause significant injury to cardiac allografts. Using C6-deficient rats, we have found that the rejection of major histocompatibility (MHC) class I-incompatible PVG.R8 (RT1.A(a)B(u)) cardiac allografts by PVG.1U (RT1.A(u)B(u)) recipients is particularly dependent on C6. This model was selected to determine whether tissue injury results from C6 produced by macrophages, which are a conspicuous component of infiltrates in rejecting transplants. We demonstrated that high levels of C6 mRNA are expressed in isolated populations of macrophages. The relevance of macrophage-produced C6 to cardiac allograft injury was investigated by transplanting hearts from PVG. R8 (C6-) donors to PVG.1U (C6-) rats which had been reconstituted with bone marrow from PVG.1U (C6+) rats as the sole source of C6. Hearts grafted to hosts after C6 reconstitution by bone marrow transplantation underwent rejection characterized by deposition of IgG and complement on the vascular endothelium together with extensive intravascular aggregates of P-selectin-positive platelets. At the time of acute rejection, the cardiac allografts contained extensive perivascular and interstitial macrophage infiltrates. RT-PCR and in situ hybridization demonstrated high levels of C6 mRNA in the macrophage-laden transplants. C6 protein levels were also increased in the circulation during rejection. To determine the relative contribution to cardiac allograft rejection of the low levels of circulating C6 produced systemically by macrophages, C6 containing serum was passively transferred to PVG.1U (C6-) recipients of PVG.R8 (C6-) hearts. This reconstituted the C6 levels to about 3 to 6% of normal values, but failed to induce allograft rejection. In control PVG.1U (C6-) recipients that were reconstituted with bone marrow from PVG.1U (C6-) donors, C6 levels remained undetectable and PVG.R8 cardiac allografts were not rejected. These results indicate that C6 produced by macrophages can cause significant tissue damage.

Complement C6 and C2 biosynthesis in syngeneic PVG/c- and PVG/c+ rat strains.

A PVG rat with total deficiency of C6 and partial deficiency of C2 (PVG/c-), and a syngeneic control strain (PVG/c+), were used to study the production of extrahepatically synthesized complement. Livers of complement deficient rats were transplanted in sufficient rats (Tx-L). The C6 and C2 levels in Tx-L rats declined within 2 days to 25% and 30%, respectively, and remained stable for more than 6 weeks. To investigate the contribution of C6 synthesis by the liver, C6 sufficient livers were grafted in deficient rats (Tx + 1). After an initial increase, with maximum C6 levels of 119% at 10 days following transplantation, the C6 levels decreased gradually and C6 was no longer detectable 28 days after transplantation. This decline in C6 levels was dependent on antibody production against C6. No significant change in the C3, C4, factor H and factor B levels was observed. Expression of C6 mRNA in the grafted PVG/c+ sufficient liver was comparable to the expression of C6 mRNA in control PVG/c+ livers while C6 mRNA expression in the transplanted PVG/ c- liver and the control PVG/c- liver was lower. In conclusion, it was demonstrated in vivo that not only C6 but also C2 is synthesized extrahepatically in PVG/c rats.

Human polymorphonuclear leukocytes store large amounts of terminal complement components C7 and C6, which may be released on stimulation.

Secretion of the C factors C7, C6, and C3 by human polymorphonuclear leukocytes (PMNs) and PBMCs was studied by ELISA and immunoblot. The release of C7 and C6 by PMNs during 24 h of culture was 16-fold and 6-fold higher, respectively, than the C3 release, with median concentrations of 50.2 ng/ml, 18.3 ng/ml, and 3.1 ng/ml, respectively. In PBMC cultures, C release was considerably lower, and there was a different secretory pattern with a 6-fold higher release of C3 compared with C7 and C6. Stimulation with PMA led to a more rapid and complete secretion of the components to the culture media, whereas treatment with unopsonized Candida species did not affect the release. PMN release of C factors was not dependent on protein biosynthesis, and there was no indication of a selective uptake of C7 from serum as demonstrated by incubating PMNs from a subject with allotype C7 N in C7 M serum. Thus, the C components were probably produced by the PMNs or their bone marrow precursors before ex vivo culture. In cell lysates of freshly isolated cells, median C7, C6, and C3 contents of 1 x 10(7) PMNs were 149.7, 60.1, and 10.4 ng/ml, respectively, whereas the corresponding values for 1 x 10(7) PBMCs were 3.2, 2.6, and 14.6 ng/ml, respectively. The C6 and C7 were shown to incorporate into the terminal complement complex, and their molecular integrity was supported by identical m.w. to C6 and C7 present in normal serum. PMNs may represent a major source of C7 and C6 and may be more important than monocytes or macrophages in contributing terminal C components at a site of inflammation. This suggests a new role for the PMN as a C membrane attack modulator.

Extrahepatic synthesis of C6 in the rat is sufficient for complement-mediated hyperacute rejection of a guinea pig cardiac xenograft.

The liver is the major source of complement (C) components, but extrahepatic sources of C, such as macrophages and endothelial cells, have been hypothesized to contribute to inflammation. Our experiments demonstrate that extrahepatically produced C6 can contribute to hyperacute rejection. PVG (RT1c) rats with normal C activity (PVG (C+)) reject guinea pig cardiac xenografts in 0.5 +/- 0.2 hr, but fully C6-deficient PVG (RT1c) rats (PVG (C-)) reject guinea pig cardiac xenografts in 45 +/- 9 hr. PVG (C+) rats, which received liver transplants from PVG (C-) rats and retained all extrahepatic sources of C6, rejected guinea pig cardiac xenografts in 0.6 +/- 0.03 hr (n = 3). PVG (C-) rats, which received bone marrow transplants from PVG (C+) rats, had C6 levels restored to 10% of that of the donor and rejected guinea pig cardiac xenografts in 9 +/- 3.2 hr (n = 5). Thus, extrahepatic sources of C6 can contribute to xenograft rejection.

Hepatic and extrahepatic biosynthesis of complement factor C6 in the rat.

The relative contributions of liver and bone marrow (BM) constituents to systemic C6 production were compared in a rat model. Liver grafts were transplanted from C6-sufficient PVG (RT1c) rats (PVG (C+)) to profoundly C6-deficient PVG rats (PVG (C-)). C6 levels were restored to 32% within 24 h and reached more than 80% of that of the PVG (C+) donor within 7 days post-grafting, which indicates that the liver is a primary source of systemic C6 production. When livers were transplanted from PVG (C-) to PVG (C+) rats (n = 3), levels of C6 dropped to 42% of pretransplant levels within 24 h and remained between 30 and 40% for more than 100 days after grafting. To determine the source of extrahepatic C6 production, BM was transplanted from PVG (C+) to PVG (C-) rats after total body irradiation. Levels of C6 increased from undetectable levels to 5% of C6 levels of the donor PVG (C+) rat within 60 days. Replenishing PVG (C-) recipients with BM after treatment of recipients with busulfan, which preferentially allows reconstitution with donor myelomonocyte stem cells, resulted in restoration of C activity. Treatment with cyclophosphamide before BM transplantation, which preferentially allows reconstitution of lymphoid stem cells, did not restore hemolytic C activity in PVG (C-) rats. These results were confirmed directly by the successful restoration of C activity with BM depleted of lymphocytes by counterflow centrifugal elutriation from PVG (C+) rats. These in vivo experiments demonstrate that the liver is a primary, but not the sole, source of C6 in the rat and that extrahepatic sources, such as myelomonocytes, and not lymphoid cells in the BM produce a significant amount of systemic C6 in the rat.

C6 epitope expression by an unrelated antisense cDNA clone: an inadvertent surface-simulation mimotope.

A cDNA clone which directs the expression of a fusion protein reacting with anti-C6 antibodies has been isolated and sequenced. A synthetic peptide corresponding to the 14 C-terminal residues of the expressed protein elicited the production of antibodies which are specific for native C6, confirming the presence of a C6 epitope on the expressed protein. However, analysis of the intron-exon boundaries of a corresponding genomic clone revealed that the expression clone is in antisense orientation, and is therefore not C6 cDNA. Comparison of the sequences of the expression clone and expressed protein with those for C6 have not demonstrated any significant sequence homology. It is therefore apparent that what has been cloned is a mimotope for C6 which includes in its continuous sequence an epitope that is conformational in C6 and not represented as a continuous sequence in the C6 molecule. Although this was not the purpose of the investigation, these results confirm that screening random expression libraries with antibodies may be an alternative to the synthetic peptide approach to obtain mimotopes reacting with particular antibodies.

Human umbilical vein endothelial cells synthesize functional C3, C5, C6, C8 and C9 in vitro.

Human endothelial cells (EC), cultured serum-free, synthesize de novo protein which increasingly bind to agarose beads (an alternative pathway activator), until a plateau phase is reached after 24-48 h. EC synthesize functional C3, C5, C6, C8 and C9, which were detected on co-cultured agarose beads, using relevant polyclonal anti-complement antibodies. Two monoclonal anti-C9 neoepitope antibodies (aE11, poly C9-MA) bound to the co-cultured beads, showing that the terminal complement complex (TCC) (C5b-9) was assembled on the beads. This also suggests that C7 is synthesized. There seems to be a positive correlation between the amount of agarose-bound labelled protein and agarose-bound complement. The results indicate that EC produce and secrete the components for the functional alternative and terminal pathways of complement.

Secretion of the terminal complement proteins, C5-C9, by human platelets.

The terminal complement components, C8 and C9, and to a lesser extent C5, C6, and C7, but minimal amounts of C3, were shown to be associated with washed human platelets. In unactivated platelets, the complement components were detected in the platelet pellet by hemolytic assays after centrifugation and disruption of the platelets by freeze-thawing. However, after platelets had been activated by collagen, thrombin, or aggregated IgG to induce aggregation, the complement components were released into the supernatant. The rank order of hemolytic activity of C9, C8, C7, C6, and C5 detected in the supernatants of activated platelets was quite different from that found in serum from the same donors, in the same assays. In particular, the serum C7 hemolytic titer was more than twice the serum C9 hemolytic titer, whereas the activity of C9 detected from platelets was more than twice that of C7. This argues against a purely nonspecific uptake of these proteins by platelets from plasma. The functional role of terminal complement components released from platelets during activation is unknown, but it is tempting to speculate that these proteins may have a role in platelet-dependent immunological tissue injury. Because the C5b-9 membrane attack complex activates platelets, it is possible that release of terminal complement proteins serves to amplify platelet activation and may also play a role in diseases in which complement membrane attack complexes have been implicated.

Human peritoneal macrophages. Production in vitro of the active terminal complement components C5 to C9 and a functional alternative pathway of complement. Brief report.

Endotoxin-stimulated human peritoneal macrophages were cultured in serum-free medium with agarose beads. Monospecific antibodies to human C3c, C3g, C5, C6, C7, C8, C9 and to C9-neoantigen bound to the beads. This shows that activated C3 and the terminal complement complex (TCC), made from complement components C5 to C9, were generated on the beads. De novo synthesis was confirmed by agarose binding of tritium-labelled protein. Moreover, C3-derivatives and C9-neoantigen were detected on normal serum-treated agarose beads but not on beads kept in factor B-depleted or heat-inactivated sera, implying that an intact alternative complement pathway was required for our findings. The macrophages thus synthesize the active complement components of the alternative and terminal pathways in vitro.

Human alveolar macrophages synthesize active complement components C6, C7, and C8 in vitro.

We investigated whether serum-free human alveolar macrophage cultures synthesize active C6, C7, and C8. There was a significant binding of polyclonal anti-human C6 antibodies to agarose beads incubated with unstimulated macrophages for 24 or 48 h. Endotoxin stimulation of the macrophages was necessary for significant binding of polyclonal anti-C7 and anti-C8 antibodies to agarose beads co-cultured for 48 or 96 h. Two monoclonal antibodies (poly C9-MA and MCaE11) specific for a neoantigen of polymerized C9 in the terminal complement complex (TCC), bound to beads mainly incubated with endotoxin stimulated macrophages. The MCaE11 was more sensitive than the poly C9-MA in detecting the C9 neoantigen on beads incubated with the macrophages or human serum diluted 1:16. We thus conclude that human alveolar macrophages synthesize active C6, C7, and C9 that together with C5 and C9, assemble as the TCC on co-cultured agarose beads. Activation of the alternative pathway on the agarose with generation of fixed C3 and C5 convertases is a prerequisite for the subsequent generation of the TCC.

Functional active complement components secreted by guinea pig peritoneal macrophages.

In our studies on complement secretion functional C1, C4, C2, C3, P, D and B were clearly identified in the same cultures. Functional assays did not allow the detection of C5 to C9. Spontaneous C3 activation occurred at a very low level in culture supernatants. The responsible enzyme was identified as a metallo-enzyme. Upon addition of antibody-coated sheep erythrocytes (EA) to culture supernatant it was possible to induce C3 activation as indicated by the apparent formation of EAC1423. Zymosan was also able to activate C3 in culture supernatant after addition of purified functional factor B indicating efficient cooperation of factors of the alternative pathway. Thus in this in vitro system macrophages not only provide C3 but also all factors for spontaneous and induced C3 activation. If these secretory functions reflect in vivo properties of macrophages, our results may indicate that C3 and its activating systems are most relevant for local cooperation between macrophages and the complement system in inflammation and antimicrobial defense. Therefore availability of these essential factors at any time is secured by local production.

C6: synthesis by the liver in vivo.

The allotype of the sixth component of complement was determined in a patient before and after liver transplantation. The C6 phenotype changed from A before transplantation to B (the donor phenotype) within 10 days of the transplant and remained wholly of the donor phenotype at 17 wk. This demonstrates that the liver is the exclusive or predominant site of C6 synthesis in vivo in man.

Human platelet-initiated formation and uptake of the C5-9 complex of human complement.

We have studied the interaction of radiolabeled complement components with normal human platelets, platelets from a patient with paroxysmal nocturnal hemoglobinuria, and rabbit platelets in the absence of known complement activators or in the presence of cobra venom factor (CVF). When unwashed platelets in platelet-rich plasma, or washed platelets suspended in serum or autologous plasma, were incubated for 30 min, C3 and terminal components (C5, C8, and C9) were found to bind to them. The terminal components were shown to be bound as the C5-9 complex, rather than as individual proteins, by eluting them from the platelet membrane and examining their behavior on ultracentrifugation. They cosedimented at a rate characteristic of the stable C5-9 complex (22S). As many as 370-1,380 C5-9 complexes/platelet were calculated to have been bound during the incubation period. The complex so formed did not differ by ultracentrifugational criteria from that binding to rabbit platelets after CVF activation of complement. Though C3 was not included in the complex, it did not appear to be bound by nonspecific absorption. It could not be removed by washing but rather was eluted by the freeze-thaw technique used to elute the C5-9 complex. Incubation of radiolabeled components in platelet-free plasma did not result in C5-9 complex formation, indicating an initiating role for platelets in this reaction. In contrast to platelets, erythrocytes incubated in analogous plasma did not induce detectable C5-9 formation. Neither EDTA, phenylmethylsulfonylfluoride, nor epsilon-amino-N-caproic acid prevented platelet-initiated formation of C5-9, suggesting that the reaction may involve mechanisms of complement activation not previously described.

Effects of anesthesia, surgery and inflammation upon host defense mechanisms. I. Effects upon the complement system.

Complement protein levels and C7 hemolytic activity were measured in four individuals following anesthesia and surgery, and in a group of 20 patients with inflammatory diseases. Seven of the eight complement components studied characteristically were elevated, most dramatically C1s and C3PA. Elevation of C1s often was greater than elevation of C1q, displaying an independent variation of C1s and C1q in both postoperative and inflammatory disease patient groupds. The major increases of C components were seen subsequent to the peak C-reactive protein response, as was the occurrence of the 'reactor state', a propensity to formation of C56 which surprisingly was associated with increased levels of C7. Levels of properdin frequently were reduced postoperatively. It is concluded that multiple complement components, with the notable exception of properdin, respond as acute phase reactants which are elevated and changed in proportion postoperatively and during inflammatory disease.