Mode of action of human C9: adsorption of multiple C9 molecules to cell-bound C8.
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Adsorption↗
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Interaction between components of the human classical complement pathway and immobilized Cibacron Blue F3GA.
The interaction between the complement components in human serum and the dye, Cibacron Blue F3GA, immobilized on cross-linked agarose (Affi-Gel Blue) has been studied. All nine components of the classical complement pathway bound to the dye and could be recovered using a linear salt gradient. With the exception of C5 and C8, all the components were eluted over a narrow NaCl concentration range, with the following yields: C1, 17%; C2, 69%; C3, 92%; C4, 87%; C6, 105%; C7, 109%; C9, 128%. C5 and C8 eluted throughout the NaCl gradient with yields of 103% and 14%, respectively. Since all components could be eluted without substantial contamination by albumin or IgG, this procedure may prove valuable as an initial step in the purification of complement components. In addition, the ability of immobilized Cibacron Blue F3GA to physicallly remove complement components may prove useful for both the decomplementation of serum and in elucidating the role of complement in immunological reactions.
Anthracenes↗
[Determination of complements in serum and middle ear effusion of the patients with secretory otitis media].
Complements in serum and middle ear effusion were determined in 20 patients with secretory otitis media, and compared with those in the normal controls. The C5 and C1-INH in serum of the patients were significantly higher. On the contrary, C9 and B factor (Bf) were significantly lower, and the circulatory immunocomplex was also higher. In patients with secretory otitis media, the middle ear effusion levels of C3, C4 and C5 were significantly lower, and Bf and immunocomplex were significantly higher than those in serum. The results suggest that the ability of complements in clearing immunocomplex is low. Therefore, the immunocomplex may deposit in the mucosa of the middle ear. Thus the permeability of capillary will be increased, and the middle ear effusion occurs.
Adolescent↗
Preconditioning reduces myocardial complement gene expression in vivo.
This investigation examined the effect of preconditioning in an in vivo model of ischemia-reperfusion injury. Anesthetized New Zealand White rabbits underwent 30 min of regional myocardial ischemia followed by 2 h of reperfusion. Hearts preconditioned with two cycles of 5 min ischemia-10 min reperfusion (IPC) or with the ATP-sensitive K (K(ATP)) channel opener, diazoxide (10 mg/kg), exhibited significantly (P < 0.05) smaller infarcts compared with control. These treatments also significantly (P < 0.001 to P < 0.05) reduced C1q, C1r, C3, C8, and C9 mRNA in the areas at risk (AAR). The K(ATP) channel blocker 5-hydroxydecanoate (5-HD; 10 mg/kg) attenuated infarct size reduction elicited by IPC and diazoxide treatment. 5-HD partially reversed the decrease in complement expression caused by IPC but not diazoxide. There were no significant differences in complement gene expression in the nonrisk regions and livers of all groups. Western blot analysis revealed that IPC also reduced membrane attack complex expression in the AAR. The data demonstrate that preconditioning significantly decreases reperfusion-induced myocardial complement expression in vivo.
Animals↗
The bovine complement system.
Methods were developed for titrating bovine C3b-inactivator, C2, C3 and C4 by non-hemolytic means, and for assaying by hemolysis all the components of the bovine classical complement system except C2. All components were detected at serum dilutions above 1:1000, and some at dilutions above 1:100,000. C1, C4, C5, C7 and C9 titers were very high in adult bovine serum, and C2 and C8 were relatively low. C1, C2, and C8 were quite heat-labile at 56 degrees C, and C7 was moderately so, while C3 amd C6 titers increased after heating due to inactivation of a heat-labile inhibitor. Fetal bovine serum contained approximately 1-3% of adult levels of conglutinin, C1 and C6, and 5-50% of adult levels of the remaining components except C3. C3 antigen was found, but C3 functional activity was undetectable in most fetal bovine sera, though present at low levels in a few.
Animals↗
Mechanism of complement activation in the hyperacute rejection of porcine organs transplanted into primate recipients.
The authors investigated the importance of natural antibody and complement in the pathogenesis of hyperacute xenograft rejection using in vivo and in vitro pig to primate models. Studies were carried out in rhesus monkeys transplanted with a pig heart or kidney in which hyperacute rejection was observed within a few hours. The rejected organs showed deposits of IgM, C3, C4, C5, and C9 neoantigen along small blood vessels, but few deposits of factors B and P. Removal of anti-endothelial cell "natural" antibodies by plasmapheresis, immunoabsorption, and immunosuppression techniques resulted in marked prolongation of the survival of a subsequently transplanted heart, even when complement levels were within the normal range. Thus, complement, in the absence of natural antibodies, did not initiate hyperacute rejection in this species combination. The requirements for complement activation in human serum to cause cytotoxicity of porcine endothelial cells were then evaluated. Cytotoxicity was abrogated by depleting human serum of IgM, C2, or C5, but not of factor B. Restoration of the effect of serum on endothelial cells was achieved by reconstitution of the respective depleted sera with purified IgM or with the corresponding complement proteins, indicating that IgM and the classical, but not the alternative, pathway of complement, were involved. Identical conclusions were drawn from experiments to ascertain the requirements for complement activation in human serum to mediate binding of iC3b to porcine endothelial cells. The authors conclude that in a pig to primate xenograft complement does not directly initiate injury to the graft but rather requires activation by bound xenoreactive natural antibodies; IgM antibodies directed against endothelial cells activate the classical complement pathway, which then contributes to endothelial cell activation and subsequent events characteristic of hyperacute rejection.
Animals↗
Distinct restriction of complement- and cell-mediated lysis.
Complement- and cell-mediated killing utilize related effector proteins (C8/C9 and perforin, respectively), suggesting that proteins which protect cells against complement- and cell-mediated attack may also be similar. In homologous complement-mediated killing two protective proteins, which are anchored to the cell membrane by phosphatidylinositol glycan (PIG) tails, are known. To study whether similar PIG-tailed proteins protect against lymphocyte-mediated killing, nucleated cell lines with a mutation in the biosynthesis of the PIG anchor were used. It was found that PIG-tailed membrane proteins restrict homologous complement-mediated lysis but not three different types of cell-mediated killing or lysis by purified perforin. Furthermore, E from patients with an acquired defect in PIG tail biosynthesis did not differ from normal E in sensitivity to antibody-dependent cell-mediated cytotoxicity, in spite of their increased sensitivity to human C8 and C9.
Antibody-Dependent Cell Cytotoxicity↗
Inhibition by epsilon-aminocaproic acid of the activation of the first component of the complement system.
The influence of EACA on C1 in whole human serum and on C1 and C (see article) as isolated molecules was assessed hemolytically. There was selective inhibition of C1 without effect on the levels of C4, C2, C3, and C9 in whole human serum that was reversed by dialysis. EACA was found to inhibit the intrinsic activation of C1 without inhibiting the already active molecule. This was confirmed by the capacity of trypsin to uncover C1 activity in cellular intermediates formed by C1 treated with EACA that did not evolve in the absence of this extrinsic activating mechanism. Inasmuch as the trypsin-dependent recovery of C1 was incomplete, an effect on binding cannot be excluded.
Aminocaproates↗
Decomplementation antigen, a possible determinant of staphylococcal pathogenicity.
We report the existence of an extracellular staphylococcal product, designated staphylococcal decomplementation antigen (DA), that causes rapid consumption of early-reacting complement components up to and including C5 in human serum. Complement activation occurs as a consequence of immune complex formation between DA and specific human immunoglobulin G antibodies and proceeds primarily via the classical pathway. The terminal components C7, C8, and C9 are not consumed during the process. Levels of DA production do not correlate with the expression of classical pathogenic factors, such as coagulase, clumping factor, protein A, or alpha-toxin. DA is a nondialyzable macromolecule eluting in a molecular-weight region of 70,000 to 120,000 on Sephacryl S-300 and displaying an apparent sedimentation coefficient of 3 to 4 S on sucrose density gradients. The molecule is remarkably stable and resists destruction upon boiling for 30 min or by treatment with pronase, lysostaphin, DNase, or RNase. We anticipate that DA protects staphylococci from complement attack through induction of abortive, complement-consuming reactions in the fluid phase.
Antigens, Bacterial↗
Regulatory control of complement on blood platelets. Modulation of platelet procoagulant responses by a membrane inhibitor of the C5b-9 complex.
Antibody against a membrane inhibitor of the C5b-9 complex has been used to investigate regulatory control of the terminal complement proteins on blood platelets. Monospecific rabbit antibody (alpha-P18) was raised against the purified 18-kDa erythrocyte membrane inhibitor of C5b-9 (Sugita, Y., Nakano, Y., and Tomita, M. (1988) J. Biochem. (Tokyo) 104, 633-637). In addition to its interaction with erythrocytes, this antibody (and its Fab) bound specifically to platelet membranes. In immunoblots of cell membrane proteins prepared under non-reducing conditions, alpha-P18 bound specifically to an 18-kDa erythrocyte membrane protein and to a 37-kDa platelet membrane protein. Absorption of this antibody by platelet membranes competed its binding to the purified 18-kDa erythrocyte protein, suggesting that epitopes expressed by the erythrocyte 18-kDa C5b-9 inhibitor are common to the platelet. When bound to the platelet surface, the Fab of alpha-P18 increased C9 activation by membrane C5b-8, monitored by exposure of a complex-dependent C9 neo-epitope. Although alpha-P18 caused little increase in the cytolysis of platelets treated with C5b-9 (total release of lactate dehydrogenase less than 5%), it markedly increased the cell stimulatory responses induced by these complement proteins, including, secretion from platelet alpha- and dense granules, conformational activation of cell surface GP IIb-IIIa, release of membrane microparticles from the platelet surface, and exposure of new membrane binding sites for components of the prothrombinase enzyme complex. Prior incubation of C5b67 platelets with 100 micrograms/ml alpha-P18 (Fab) lowered by approximately 10-fold the half-maximal concentration of C8 required to elicit each of these responses (in the presence of excess C9). Incubation with alpha-P18 (Fab) alone did not activate platelets, nor did incubation with this antibody potentiate the stimulatory responses of platelets exposed to other agonists. These data indicate that a membrane inhibitor of the C5b-9 complex normally serves to attenuate the procoagulant responses of blood platelets exposed to activated complement proteins, and suggest the mechanism by which a deletion or inactivation of this cell surface component would increase the risk of vascular thrombosis.
Blood Platelets↗
mRNA expression of complement components and regulators in rat arterial smooth muscle cells.
The presence of C5b-9 complexes, some complement regulators, and abundant cytokines in atherosclerotic lesions has been reported. However, it is unclear whether these complement-associated proteins are produced by vascular smooth muscle cells (SMCs) and how they are influenced by the cytokines. In the present study, we demonstrated, by the reverse transcription-polymerase chain reaction method, the mRNA expression of complement components (C3, C4, and C5) and membrane regulators (decay-accelerating factor, membrane cofactor protein, Crry, and CD59) in cultured SMCs derived from the rat carotid artery. The expression of C9 mRNA was also induced upon stimulation by interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and/or lipopolysaccharide (LPS). Northern blot analysis showed that the mRNA expression of C3, C4, DAF and Crry was up-regulated, but that of CD59 was down-regulated by IFN-gamma, TNF-alpha and/or LPS alone or by synergy. The increase of C3 mRNA by TNF-alpha or LPS and that of C4 mRNA by IFN-gamma was induced in a dose-dependent manner. The results indicate that the arterial SMCs of rat have the ability to produce complement components and regulators, which is affected by cytokines and/or LPS. Since atherosclerosis is characterized by the intimal proliferation of SMCs, the complement system including its regulators may be involved in the pathogenesis of the disease.
Animals↗
Letter: Elevation of serum C9 level associated with Behçet disease.
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Adult↗
Serum complement pattern in essential mixed cryoglobulinaemia.
In twenty-six patients affected by essential cryoglobulinaemia, 188 determinations of serum complement components (SCC) were made. A peculiar pattern was observed which was characterized by: (a) low levels of early components (Clq, Cls and C4), (b) normal levels of C3 and high concentrations of late components (C5, C9) and (c) CH50 values significantly lower than normal. No relationship could be observed between early SCC and C3 levels. Thirty-three crossed immunoelectrophoreses were performed in thirteen patient's blood samples. The C3c peaks were not different from normal. Follow-up data (156 serum samples from twenty-four patients) during a 6-40 month period showed a non-homogeneous SCC behaviour. However, no relationship was found between the complement concentrations and clinical score. These findings suggest that SCC abnormalities are related to a complement hyposynthesis, which could be caused by a reduced C2 production or by a negative feedback effect of active components or their fragments.
Complement C3↗
Lysis of paroxysmal nocturnal hemoglobinuria erythrocytes by acid-activated serum.
Erythrocytes from paroxysmal nocturnal hemoglobinuria patients (PNH-E) are much more susceptible to lysis by acid-activated human serum than normal human erythrocytes. Acidification of normal human serum to pH 6.4 in the absence of erythrocytes generates this lytic activity independently of the alternative pathway of complement activation. A shift of pH of a mixture of purified human C5 and C6 to 6.4 at 0 degrees C generates a similar activity C(56)a that lyses PNH-E together with C7-C9 much more efficiently than normal erythrocytes. Since acid-activation of normal human serum occurs in the absence of C3, the acid-activated C56 appears to be the lytic principle in acidified human serum.
Animals↗
Insights into the human CD59 complement binding interface toward engineering new therapeutics.
CD59 is a 77-amino acid membrane glycoprotein that plays an important role in regulating the terminal pathway of complement by inhibiting formation of the cytolytic membrane attack complex (MAC or C5b-9). The MAC is formed by the self assembly of C5b, C6, C7, C8, and multiple C9 molecules, with CD59 functioning by binding C5b-8 and C5b-9 in the assembling complex. We performed a scanning alanine mutagenesis screen of residues 16-57, a region previously identified to contain the C8/C9 binding interface. We have also created an improved NMR model from previously published data for structural understanding of CD59. Based on the scanning mutagenesis data, refined models, and additional site-specific mutations, we identified a binding interface that is much broader than previously thought. In addition to identifying substitutions that decreased CD59 activity, a surprising number of substitutions significantly enhanced CD59 activity. Because CD59 has significant therapeutic potential for the treatment of various inflammatory conditions, we investigated further the ability to enhance CD59 activity by additional mutagenesis studies. Based on the enhanced activity of membrane-bound mutant CD59 molecules, clinically relevant soluble mutant CD59-based proteins were prepared and shown to have up to a 3-fold increase in complement inhibitory activity.
Alanine↗
Lysis of erythrocytes by complement in the absence of antibody.
A new pathway of complement-mediated hemolysis has been described. It is independent of antibody and does not require binding of the first four complement components to the target-cell surface. The actual attack of the target cell begins with the attachment of C5, C6, and C7. The binding reaction is catalyzed by C4, 2, 3, an enzyme which may be formed in cell-free solution. C4, 2, 3 may effect binding of C5, 6, 7 by acting from the fluid phase or from the surface of another cell to which it is specifically bound (EAC 4, 2, 3). In either case, the resulting product is EC5, 6, 7 which is susceptible to lysis by C8 and C9. Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) were particularly susceptible to lysis by the above described mechanism. PNH cells, but not normal human erythrocytes, could also be lysed through activation of complement by cobra factor. These observations allow the operational distinction of an activation and an attack mechanism of complement.
Animals↗
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.
Animals↗
Mapping the active site of CD59.
CD59 is a widely distributed membrane-bound inhibitor of the cytolytic membrane attack complex (MAC) of complement. This small (77 amino acid) glycoprotein is a member of the Ly6 superfamily of proteins and is important in protecting host cells from the lytic and proinflammatory activity of the MAC. CD59 functions by binding to C8 and/or C9 in the nascent MAC and interfering with C9 membrane insertion and polymerization. We present data obtained from a combination of molecular modeling and mutagenesis techniques, which together indicate that the active site of CD59 is located in the vicinity of a hydrophobic groove on the face of the molecule opposite to a "hydrophobic strip" suggested earlier. In addition, removal of the single N-linked glycosylation site at Asn18 of CD59 resulted in an enhancement of complement inhibitory activity.
Amino Acid Sequence↗