Synthesis of C3, C5, C6, C7, C8, and C9 by Human Fibroblasts

We investigated the ability of human fibroblasts to produce the components of the final common pathway (C3-C9) of complement in vitro by co-culturing an alternative complement activator (agarose beads) with the cells. The test system involved incubation of beads with anti-complement antibodies followed by radioactive-labelled anti-Ig detection antibodies. Subsequently, the beads were examined in a radioimmunoassay. Our results indicate that human fibroblasts produce C3, C5, C6, C7, C8, and C9. A neoepitope selectively expressed on activated C9 was detected, indicating assembly of the terminal complement complex and thus formation of a functional terminal complement pathway by the fibroblasts.     

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 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.     

Synthesis of Complement Components C5, C6, C7, C8 and C9 in Vitro by Human Monocytes and Assembly of the Terminal Complement Complex

Monocytes cultured under serum-free conditions secreted protein which bound covalently and non-covalently to agarose beads, an activator of the alternative pathway of complement. There was a significantly binding of monoclonal anti-C3c antibodies, polyclonal anti-C5, anti-C6, anti-C7, anti-C8, and anti-C9 antibodies, and of a monoclonal antibody against a neoantigen of polymerized C9 to agarose beads incubated with the monocytes for 24, 48, 72 or 96 h. From these results, we conclude that monocytes produce C5, C6, C7, C8 and C9 that assemble as the terminal complement complex on the surface of the agarose beads. Activation by agarose of the alternative pathway with generation of particle bound C3 and C5 convertases is a prerequisite for the subsequent formation of the terminal complement complex. Whether SC5b-9 or the membrane attack of complement (C5b-9) is formed on the beads will be examined.     

Expression of terminal complement components by human keratinocytes

Human keratinocytes are important constituents of the skin immune system. They produce several cytokines, chemokines as well as some complement proteins. As regards soluble complement proteins, so far keratinocytes have been shown to synthesize only C3, factor B, factor H and factor I. Synthesis and regulation of synthesis of other complement proteins has not yet been studied. Here we studied the synthesis of terminal complement components, C5-C9 by human keratinocytes. We also studied the regulation of terminal complement synthesis in keratinocytes by several cytokines, namely, IL-1alpha, IL-2, IL-6, TGF-beta1, TNF-alpha, and IFN-gamma. Human keratinocytes constitutively expressed C5, C7, C8gamma and C9 mRNA but not C6, C8alpha and C8beta mRNA. They released C7 and C9, but not C5, C6 and C8. None of the cytokines tested had any influence on the synthesis of terminal components except TNF-alpha, which strongly upregulated C9 production. In conclusion, we demonstrate that keratinocytes are capable of synthesizing some of the terminal complement components and that the synthesis of C9 is regulated by TNF-alpha.     

Human Alveolar Macrophages Synthesize the Functional Alternative Pathway of Complement and Active C5 and C9 in Vitro

Attachment of protein to agarose beads cultured with macrophages in protein-free medium containing 3H-leucine, shows that de novo synthesis of protein with affinity to the beads takes place. We also found that monoclonal antibodies against human C3c, C3g, and a C9-neoantigen as well as polyclonal antibodies against human C5 and C9, bound to agarose beads that had been kept with the macrophage cultures. Demonstration of C3 derivatives on the agarose beads shows that the essential complement factors of the alternative pathway are synthesized and have been activated by the beads. Deposition of C5 and the detection of a neoantigen of C9 on the beads, indicates that the whole terminal complement pathway has been formed and activated. We conclude that human alveolar macrophages form in vitro the functional alternative pathway of complement, C5 and C9, and we have indirect evidence for synthesis of C6, C7, and C8.     

Membrane signaling by complement C5b-9, the membrane attack complex

The terminal complement complexes C5b-7, C5b-8 and C5b-9 are able to generate nonlethal cell signals. One universal consequence of a cell being targeted by C5b-8 or C5b-9 is an influx of Ca²⁺. In addition, other second messengers, including cAMP, inositol phosphate intermediates and arachidonate metabolites, are generated by the terminal complement complexes in specific cell types. In vivo, terminal complement complexes have been found in a wide variety of inflammatory processes in humans and in experimental animal models. Some of these models of inflammation putatively induced by terminal complement complexes have been tested in complement-deficient animals, and indeed no inflammation results, which supports the critical role of the terminal complement complexes in the pathogenesis of the lesion.     

Quantification of the Terminal Complement Complex in Human Plasma by an Enzyme-Linked Immunosorbent Assay Based on Monoclonal Antibodies against a Neoantigen of the Complex

The fluid-phase terminal complement complex (TCC), consisting of the components C5b, C6, C7, C8, C9, and the S-protein, has recently been detected in normal human plasma by using antibodies against native terminal complement components. Increased amounts of TCC were then found in several patients with in vivo activation of complement. We now describe a sensitive, specific, and reliable enzyme-linked immunosorbent assay for quantification of the TCC, based on monoclonal antibodies against a neoantigen of the complex. The results indicate that the TCC is present in normal human plasma and in increased amounts in patients with complement activation in vivo, thus confirming previously obtained results. The assay is easy to perform and can be used for examination of large numbers of plasma samples.     

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.     

The complement membrane attack complex stimulates the prostanoid production of cultured glomerular epithelial cells

Incubation of cultured rat glomerular epithelial cells (GEC) with sublytic amounts of the purified complement components C5b6, C7, C8 and C9 greatly stimulated the release of the prostanoids prostaglandin E (PGE) and thromboxane B2. Incubation of GEC with C5b-8 was also stimulatory, whereas omission of C7 abolished the enhanced prostanoid production. These effects were dose-dependent. The increased release of PGE was biphasic with peaks at 5 min and 24 h of incubation. The second peak could be prevented by treatment with cycloheximide, suggesting its dependence on protein synthesis. The observations on cultured GEC provide evidence that terminal complement components alter the metabolism of glomerular cells, resulting in increased production of prostanoids. The results are consistent with the concept that deposition of nonlytic amounts of complement in the glomerular capillary wall may affect the GEC in vivo and may indirectly contribute to abnormalities of the glomerular filter as it is seen in glomerular disease.     

Electrophoresis of guinea-pig complement components (C3, C5, C6, C7, C8 and C9) on polyacrylamide gel

The electrophoretic mobilities of C3, C5, C6, C7, C8 and C9 of guinea-pig complement were studied using polyacrylamide (PAA) gel as a supporting medium. The haemolytic activity of each complement component was readily revealed as a discrete haemolytic band in blood agar containing intermediate red blood cells and appropriate reagents which was overlayed on the PAA gel plate after electrophoresis. By this method, the relative mobilities of C3, C5, C6, C7, C8 and C9 were determined as 0.19, 0.40, 0.17, 0.27, 0.31 and 0.57 respectively. An additional, distinct, haemolytic C5 band was detected when native guinea-pig serum was run. The relative mobility of the haemolytic activity which was not detected when purified C5 was electrophoresed, was 0.25.     

Formation of the Terminal Complement Complex on Agarose Beads: Further Evidence that Vitronectin (Complement S-Protein) Inhibits C9 Polymerization

Vitronectin occupies the metastable binding site of C5b-7, which is unable to insert membranes as part of the complement lytic attack. Some evidence has been presented that vitronectin inhibits also membrane-associated pore formation by inhibiting C9 polymerization in the terminal complement complex (TCC). The authors wished to add to this background by studying the effect of vitronectin on formation of TCC on a carbohydrate surface like agarose beads, an alternative complement pathway activator. Bound TCC was detected by monoclonal and polyclonal antibodies to C9-neoepitopes. Soluble SC5b-7 and TCC (SC5b-9) did not bind to the agarose beads. Using serum or isolated complement factors for the alternative and terminal pathways, the authors found that vitronectin reduced the density of C9-neoepitopes on the beads. As there was no convincing evidence for association of vitronectin with the factors C5b-8 of the agarose-bound TCC, it was concluded that vitronectin bound directly to C9 in TCC and inhibited C9 polymerization within the complex. The authors have shown that TCC can bind to a carbohydrate surface like agarose (an alternating polymer of galactose moieties) in the absence of lipid. These results suggest that vitronectin can limit the lytic effect of membrane-bound TCC by inhibiting C9 polymerization.     

Vitronectin-mediated inhibition of complement: evidence for different binding sites for C5b-7 and C9.

In the activated complement system, vitronectin (complement S-protein) occupies the metastable membrane binding site of the nascent precursor complex C5b-7, so that the newly formed SC5b-7 is unable to insert into cell membranes. Some evidence also indicates that vitronectin limits on-going membrane-associated pore formation by inhibiting C9 polymerization. It has been assumed that these two stages of terminal complement complex (TCC) inhibition take place through charge interactions between the heparin-binding region of vitronectin and homologous cysteine-rich sequences of the late complement proteins C6, C7, C8 and C9. We examined SC5b-7 formation and inhibition of C9 binding in the TCC using separate haemolytic assays. The mode of action of vitronectin in these assays was compared with two 15mer peptides which span residues 348-379 of the heparin-binding region, and a heparin-affinity polypeptide, protamine sulphate. The results showed that vitronectin acts predominantly through SC5b-7 production with a lesser effect on the inhibition of C9 lytic pore formation. In contrast, protamine sulphate did not prevent C5b-7 membrane attachment, but was a potent inhibitor of C9-mediated lysis. The peptides did not inhibit C5b-7 membrane insertion and only one affected C9 binding. These data suggest that the two stages of TCC inhibition involve separate binding sites on the vitronectin molecule. The site for association with nascent C5b-7 is unknown, whereas inhibition of C9 binding and pore formation takes place through the heparin-binding region.     

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.     

A natural auto-inhibitory factor of the terminal complement pathway in serum of Ctenodactylus gondi

The serum of Ctenodactylus gondi, a Tunisian rodent, contains a unique inhibitor of the terminal complement pathway. The auto-inhibitor has been partially characterized as a heat-stable euglobulin that is slightly retarded on a DEAE-ion exchange column at pH 7 and elutes as a symmetrical peak on Sephacryl S-300 in the mol. wt region of approximately 200,000. The inhibitor acts by preventing attachment of cytolytic C5b-9 complexes to natural target cells. It does not appear to affect formation and function of C3-convertase, does not exert inhibitory effects at stages later than C5b-7 formation, and also does not prevent formation of SC5b-9 in serum. That the factor prevents attachment of C5b-7/C5b-9 to cells has been demonstrated in hemolysis model systems using sheep EA + human serum, and in the C3-independent reactive lysis system with the use of ELISA methods and quantitative assays with radioiodinated C8. Addition of partially purified inhibitory factor to human sera or to sera of other animal species abolishes the hemolytic activities of these sera. The inhibitory factor of Gondi serum is the first inhibitor of the terminal pathway which has been shown to be capable of preventing cytolysis of cells undergoing complement attack under physiological conditions. The presence of this factor is probably partially responsible for the remarkable susceptibility of C. gondi towards bacterial and parasitic infections.     

Terminal complement complex (TCC) and S-protein (vitronectin) on follicular dendritic cells in human lymphoid tissues.

Follicular dendritic cells (FDC) present in germinal centres trap considerable amounts of C3-containing immune complexes (IC). Activation of the terminal pathway of complement (C) on biological membranes normally generates C5b-9(m), which is the membranolytic form of the terminal complement complex (TCC). By contrast, when C activation takes place in the extracellular fluid phase, S-protein binds to C5b-7 and the non-lytic soluble SC5b-9 is formed. In this study deposits of C3d, C5, C9 TCC neoepitope and S-protein were demonstrated by immunohistochemistry on FDC in human tonsils, lymph nodes, spleens, appendices and colonic mucosae. TCC and S-protein were likewise observed on FDC in imprints from tonsils. The identical spatial distribution revealed by paired staining suggested that TCC had been generated in situ. The staining intensity for TCC and S-protein varied in parallel, which suggested that the S-protein may be incorporated in the membrane-bound TCC--perhaps rendering it non-lytic. However, the actual mechanism and function of the observed TCC and associated S-protein deposition needs further elucidation.     

Immunohistochemical Localization of C9 Neoantigen and the Terminal Complement Inhibitory Protein CD59 in Human Endometrium

PROBLEM : Human endometrium expresses complement components, receptors, and regulatory proteins, many of which appear to be expressed in a hormone-dependent manner. Whether terminal complement components are also present in the endometrium is unknown. CD59, a broadly expressed protein that blocks association of C9 with C8 in the membrane attack complex, is localized in reproductive tissue to human spermatozoa, seminal plasma, amniotic fluid, and placenta. The present study examines human endometrium for the presence of CD59 and terminal complement proteins. METHOD : Endometrial biopsies were obtained from six normal women from various phases of the menstrual cycle and analyzed by immunohistochemistry, using MEM-43 anti-human CD59 and anti-human SC5b-9 murine monoclonal antibodies and the immunoperoxidase technique. RESULTS : Both CD59 protein and SC5b-9 (C9 neoantigen) were demonstrated to be present in endometrial glandular epithelium throughout the menstrual cycle. No specific staining was demonstrated in the stromal compartment. CONCLUSION : CD59 protein and terminal complement proteins are expressed in glandular epithelial cells of normal human endometrium, in both proliferative and luteal phases, suggesting that expression is not hormonally dependent. These analyses further support the presence of a functionally active complement system in normal human endometrium.     

Cleavage of the fifth component of complement and generation of a functionally active C5b6-like complex by human leukocyte elastase

Activation by complement C3/C5 convertases of the fifth component of human complement, C5, leads to two active cleavage products: C5a, a chemotactic peptide, and C5b, the activated form of C5. Human leukocyte elastase (HLE) has long been known to also release from C5 a chemotactic, C5a-like fragment. This, however, cannot be identical with C5a, since HLE does not cleave peptide bonds at the carboxyl group of arginine, the cleavage site that separates C5a and C5b after the exposure to the complement convertases. Therefore, the question arose whether HLE is capable of releasing a functionally C5b-like product from C5. The results show that this is, indeed, so. Treatment of human C5 with HLE in the presence of C6 leads to the formation on an active C5b6-like complex that lyses non-sensitized guinea pig red cells upon addition of the terminal components C7, C8, and C9. However, since C6 is highly sensitive to the hydrolytic action of HLE, the yield of the activation complex is rather low. The results offer a third possibility for the activation of C5: 1) classical cleavage at Arg74 by complement convertases, 2) oxidation of methionine residues without cleavage, and, as shown here, 3) cleavage by elastase at (a) site(s) distal from Arg74. The three procedures may modulate the relative yield of the two activities generated from C5, C5a-like and C5b-like effects.     

C3-independent immune haemolysis: haemolysis of EAC14oxy2 cells by C5-C9 without participation of C3.

C3-independent immune haemolysis was studied using EAC14oxy2 cells and purified C5, C6, C7, C8 and C9. We have found that EAC14oxy2 cells were lysed by C5-C9 and that haemolysis occurs, even after pretreatment of the cells and the C5-C9 preparation with anti-C3. This indicates that EAC14oxy2 can be lysed by C5-C9 without any participation of C3. In contrast, EAC1 and EAC14 cells are not lysed by C5-C9, suggesting that our C5-C9 preparation lacks activated complement components, such as C3bBb, C5b6 or C(56)a. Based on our study of the haemolysis of EAC14oxy2, we have determined that: (i) EAC14oxy2 cells are lysed by a preparation of C5, C6, C7, C8 and C9, but no lysis occurs when any one of these complement is absent, (ii) for significant haemolysis of EAC14oxy2, a higher concentration of C5 is necessary as compared to the C5 requirement when haemolysis occurs in the presence of C3, (iii) the degree of haemolysis is linearly related to the concentration of C5 and does not reach a plateau, despite the addition of as much as 3,200 U of C5, and (iv) the degree of haemolysis is linearly related to the concentration of cell bound C42. These observations suggest that, in the absence of C3, the C3 convertase C42 can activate C5 directly, resulting in the formation of the membrane attack complex, C5b-9.     

Complement component C5 modulates the systemic tumor necrosis factor response in murine endotoxic shock.

Patients with disseminated Neisseria meningitidis infections (meningococcemia) suffer from a fulminant shock syndrome that is accompanied by extraordinarily high concentrations in serum of tumor necrosis factor (TNF). People with homozygous deficiencies of late complement components (C5, C6, C7, and C8) experience a high incidence of disseminated neisserial infections yet suffer from an attenuated form of the disease. The mechanisms that account for this disparity in host response are unclear, but they may in part be related to differences in the systemic TNF response that are modulated by terminal complement components (C5 to C9). The role of C5 in the modulation of the systemic endotoxin-induced TNF response was studied with matched strains of C5-deficient (B10 D2/Osn) and complement-sufficient (B10 D2/Nsn) mice. Following lipopolysaccharide (LPS) administration, complement-sufficient mice exhibited more rapid increases in pulmonary and hepatic vascular permeabilities than did C5-deficient controls. Complement-sufficient mice developed acute passive hepatic congestion, they appeared more ill than C5-deficient mice, and they exhibited a twofold greater rise in serum TNF activity compared with that by C5-deficient mice. C5-deficient mice reconstituted with normal serum before an LPS injection exhibited pulmonary and hepatic vascular permeability increases and serum TNF levels approaching those observed in complement-sufficient mice. Alveolar and peritoneal macrophages isolated from complement-sufficient and C5-deficient mice and incubated in heat-inactivated serum did not exhibit differences in TNF mRNA expression or secreted TNF activity following stimulation with LPS. However, incubation of macrophages in complement-sufficient mouse serum (before LPS stimulation) resulted in increased TNF mRNA expression and TNF activity compared with those in cells incubated in C5-deficient serum. In vitro studies employing human complement components and peripheral blood monocytes revealed that recombinant C5a, in the presence or absence of LPS, can induce increased concentrations of TNF and that C5b to C9 had no additional modulatory effect on the TNF response. These data suggest that C5 modulates the endotoxin-triggered TNF response. The role of complement components distal to C5 (i.e., C5b to C9) in the endotoxin-triggered TNF response remains unclear.