Studies of complement complex C5b,6 eluted from--EAC-6: reaction of C5b,6 with EAC4b,3b and evidence on the role of C2a and C3b in the activation of C5.
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Animals↗
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Results for “Complement C5b”
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Thromboxane B2 synthesis in human platelets induced by the late complement components C5b-9.
Sublytic doses of purified C5b6, C7, C8 and C9 induce the release of thromboxane B2 (TXB2) in human platelets. In the present study, we attempted to analyze the signal by which C5b-9 triggers the prostanoid synthesis in platelets. TXB2 release was accompanied by liberation of Ca++ from intracellular stores. Influx of Ca++ was not observed, indicating that C5b-9 did not form a channel large enough to permit passage of Ca++ ions. Therefore, channel formation is apparently not required for cell stimulation.
Arachidonic Acid↗
Arachidonic acid mobilization and phosphoinositide turnover by the terminal complement complex, C5b-9, in rat oligodendrocyte x C6 glioma cell hybrids.
Previously, we have shown that rat oligodendrocytes release phospholipid and generate arachidonic acid (AA) and leukotriene B4 in response to sublytic C5b-9 formation. In the present study, we investigated the biochemical pathways by which C5b-9 generates AA from clone ROC-1, a fusion product of rat oligodendrocytes and C6 glioma. Cells were incubated for 24 h in the presence of [3H]AA or [3H]myoinositol. They were then sensitized with antibody against hybrid cell stroma and treated for 1 h with C9-depleted human serum (C9D-HS) or C9D-HS reconstituted with C9. Alternatively, cells were treated with C8,C9D-HS or C8,C9D-HS reconstituted with C8 or C8 plus C9 for 1 h. Qualitative and quantitative analysis of the released [3H]AA and [3H]myoinositol radiolabeled products were performed by thin layer chromatography/autoradiography and anion exchange chromatography, respectively. The major [3H]AA radiolabeled products after C5b-9 stimulation comigrated with intact phospholipid and AA standards, and the major [3H]myoinositol radiolabeled product was inositol-1-phosphate. Treatment of cells with phospholipase A2 inhibitors, mepacrine and bromophenacyl bromide, abolished AA release by C5b-9. In the absence of extracellular Ca2+, C5b-9 also failed to induce the release of AA. Interestingly, 1-(5-isoquinolinsulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinases, inhibited AA release by C5b-9, whereas AA release stimulated by the calcium ionophore A23187 was not blocked by H-7. The results suggest that AA generation by C5b-9 from the ROC-1 clone involves activation of Ca2+-dependent phospholipase A2 which is regulated by protein kinase-dependent mechanisms.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Complete primary structure and functional characterization of the sixth component of the human complement system. Identification of the C5b-binding domain in complement C6.
Complement C6 is one of five plasma proteins that are incorporated into the lytic terminal complement complex on lipid membranes (C5b-9m) upon activation of the complement cascade. Oligonucleotide probes derived from partial amino acid sequences of purified C6 were used to isolate cDNA clones from a human liver cDNA library. The complete polypeptide structure of mature C6 deduced from the cDNA sequence consists of 913 amino acid residues preceded by a typical 21-residue signal peptide. C6 is most similar in structure to complement C7, sharing 33.5% identical residues with C7 including all 56 cysteine residues. The low density lipoprotein receptor class A and B modules, the thrombospondin type I module at the carboxyl terminus, and the two short consensus repeat modules are arranged in the same way as in C7. In contrast to C7 and other terminal complement proteins, the thrombospondin type I module at the amino terminus occurs as a tandem repeat in C6. The last tandem repeat at the carboxyl terminus of C6 and C7 has been identified as a new distinct module (factor I module), which is closely related to a segment in the heavy chain of complement control factor I. Binding studies with filter-bound C6 fragments generated by proteolysis showed that the C5b-binding domain of C6 was located in the 34-kDa carboxyl terminal fragment consisting of two short consensus repeats and two factor I modules.
Amino Acid Sequence↗
[Immunoenzyme determination of the C5b-9 complex of the complement system].
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Aorta↗
The C5b-9 complex: subunit composition of the classical and alternative pathway-generated complex.
The membrane attack complex of complement (C5b-9) is identical in composition regardless of which pathway of activation was instrumental in its formation. Band V protein was consistently a subunit of the soluble complex. Since band V protein is not required for complement-dependent cytolysis, it probably represents a membrane site equivalent in serum of the nascent C5b-9 complex.
Centrifugation, Density Gradient↗
Increased ion permeability of planar lipid bilayer membranes after treatment with the C5b-9 cytolytic attack mechanism of complement.
The ion permeability of planar lipid bilayers, as measured electrically, was found to increase modestly upon treatment with purified complement complex C5b,6 and complement components C7 and C8. The subsequent addition C9 greatly amplified this change. No permeability changes occurred when components were added individually to the membrane, or when they were used in paired combinations, or when C5b, C7, C8, and C9 were admixed prior to addition. Thus, there is a significant parallel between the permeability changes induced in the model membrane and damage produced in biological membranes by the C5b-9 complement attack sequence. The efficiency of membrane action by C5b-9 was critically dependent on the order in whcih components were added to the membrane. There were also differences in the electrical properties of membranes treated with C5b-8 and C5b-9, though in both cases the enhanced bilayer permeability is best attributed to the formation of trans-membrane channels. Collectively, the data are consistent with the hypothesis that the mechanism of membrane action by complement involves the production of a stable channel across the lipid bilayer, resulting in cell death by colloid-osmotic lysis.
Cell Membrane Permeability↗
Elevated urinary excretion of the C5b-9 complex in membranous nephropathy.
In experimental membranous nephropathy, antibody binding to glomerular epithelial cell membrane antigens results in complement activation and formation of complement C5b-9 membrane attack complexes in glomeruli. During active disease, the C5b-9 complexes are shed into the urine. To test the hypothesis that a similar mechanism might be operative in human membranous nephropathy, we measured urinary excretion of C5b-9 and C5 in 146 proteinuric patients with biopsy-proven glomerular diseases or diabetes mellitus. Urinary excretion of C5b-9 relative to C5 excretion was higher in 40 patients with membranous nephropathy than in 106 patients with proteinuria due to non-membranous glomerulonephritis when analyzed by covariance analysis (P less than 0.0002). Urinary C5b-9 excretion was higher in membranous nephropathy than in membranoproliferative glomerulonephritis (N = 13, P less than 0.05), minimal change-focal sclerosis (N = 33, P less than 0.001), mesangial proliferative glomerulonephritis (N = 9, P less than 0.02) and IgA nephropathy (N = 7, P less than 0.025). Urinary C5b-9 excretion was also higher in patients with lupus nephritis (N = 18, P less than 0.02) compared to those with non-membranous glomerulonephritis. The lupus patients with the highest excretion had clinical or pathological features of membranous nephropathy. Nine patients with membranous nephropathy and elevated urinary C5b-9 excretion had a shorter duration of disease (P less than 0.05), lower serum creatinine levels (P less than 0.05) and more proteinuria (P less than 0.02) than the 31 membranous nephropathy patients with normal values.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
Bactericidal but not nonbactericidal C5b-9 is associated with distinctive outer membrane proteins in Neisseria gonorrhoeae.
In this study, we examined the bacterial constituents associated with the complement C5b-9 complex in detergent extracts from serum-treated Neisseria gonorrhoeae (GC). 125I surface-labeled GC were incubated in 10% serum, were washed, and were solubilized in the zwitterionic sulfobetaine detergent SB12. Immunoprecipitation of 125I-GC from the extract with anti-C5 Sepharose was followed by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography of immunoprecipitated material. Polyacrylamide gel analysis of surface-labeled 125I-GC showed prominent bands for proteins I and III for both serum-resistant GC strain 6305 and serum-sensitive GC strain 7189. These same bands were visible with similar intensity in the SB12 extracts from presensitized and non-presensitized 6305 and 7189 after serum incubation. For those organisms bearing bactericidal C5b-9 (6305 + IgG and 7189 +/- IgG), additional distinctive bands immunoprecipitated with antibody to C5 Sepharose. These components were of 93,000, 44,000 40,000, and 15,000 daltons for 6305 + IgG, and were of 90,000, 50,000, 44,000, and 19,000 daltons for 7189 +/- IgG. Nonbactericidal C5b-9 extracted from the surface of 6305 incubated in serum, but not sensitized with antibody, was not associated with these distinctive proteins. However, this nonbactericidal C5b-9 did have a different pattern of associated bacterial surface constituents from that observed in control samples incubated with antibody to human serum albumin, which were similar to those with nonserum-incubated organisms. These studies support our earlier experiments which demonstrated that C5b-9 is in a different molecular configuration on the surface of serum-resistant GC from that on the surface of serum-sensitive GC or resistant GC rendered sensitive with bactericidal antibody.
Animals↗
Formation of the membrane attack complex of complement (MAC) on erythrocytes from monocyte-produced terminal complement components.
By using antibodies against C5, C6, C7, C8, and C9, we found that terminal complement components were deposited on IgM-coated sheep erythrocytes (EIgM) kept in serum-free endotoxin-stimulated monocyte cultures for 24 or 48 h. Monoclonal antibodies revealed C9 neoantigens on the EIgM. There was no specific binding of an anti-S protein antibody, which reacts with the SC5b-9 complex, to the EIgM. Controls were native sheep erythrocytes (E) treated similarly which, in contrast to EIgM, do not activate the classical pathway of complement. Cycloheximide (1.0 microgram/ml) in the cell cultures resulted in no specific binding of the anti-C9 antibodies to EIgM. A fraction of the EIgM was lysed during incubation with the monocytes. We conclude that the monocytes secrete C5, C6, C7, C8, and C9, which form the membrane attack complex of complement (C5b-9) on the EIgM.
Animals↗
Localization of S protein and its relationship to the membrane attack complex of complement in renal tissue.
The S protein (S) binds to the attack complex of complement (C5b-9) in plasma preventing cytolysis. Using immunofluorescence microscopy, the authors determined the distribution of S in human renal tissue and its relationship to C5b-9, immunoglobulins, C3, albumin, and fibronectin. They examined normal and diseased human kidney tissue from patients with several forms of glomerulonephritis, diabetic nephropathy, and arterionephrosclerosis. S and C5b-9 were found in all diseased tissues; their amounts and distribution directly correlated with severity and location of injury. S and C5b-9 were colocalized in all immune deposits and in all injured glomeruli, tubular basement membranes, and vessel walls. Other than within immune deposits, S and C5b-9 were usually not colocalized with C3. This study demonstrates that S is deposited in areas of tissue injury and thus may participate in the pathogenesis of renal damage. Because in tissue S and C5b-9 are always associated, the attack complex in tissue must either be derived from the circulation as SC5b-9 or it must be capable of binding S after the formation in situ of C5b-9.
Anions↗
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.
Antibodies, Monoclonal↗
Detection of the membrane inhibitor of reactive lysis (CD59) in diseased neurons of Alzheimer brain.
The membrane inhibitor of reactive lysis (MIRL) protects host cells from complement-mediated lysis. It was detected immunohistochemically in tangled neurons and dystrophic neurites of Alzheimer disease (AD) tissue in a pattern highly similar to that observed for the membrane attack complex of complement, C5b-9. MIRL was also detected in cultured IMR-32 neuroblastoma cells. The mRNA for MIRL was detected in RNA extracts of both AD and normal brain. These data provide the first evidence of brain neuronal expression of MIRL and its upregulation in neurons exposed to complement attack. They are consistent with the previously advanced hypothesis that complement-mediated neuronal injury may play a role in AD.
Alzheimer Disease↗
Inhibition of acute passive transfer experimental autoimmune myasthenia gravis with Fab antibody to complement C6.
The role of the lytic complement C5b-9 membrane attack complex (MAC) in acute passive transfer experimental autoimmune myasthenia gravis (EAMG) produced in rats was investigated by in vivo inhibition of MAC formation with anti-C6 Fab. Anti-C6 Fab totally inhibited in vitro serum hemolytic activity, but did not consume or inhibit early complement pathways. Injection of rats with 0.12 mg/ml anti-C6 Fab reduced serum C6 to 8% and inhibited the muscle weakness, electrophysiologic abnormalities and loss of acetylcholine receptor (AChR) associated with acute EAMG. This level of C6 inhibition reduced the total serum complement hemolytic activity to 29% of normal but did not reduce the serum levels of complement components C3, C5, or C7. Treatment of rats with lower amounts of anti-C6 Fab (0.08 mg/ml) also inhibited clinical and electrophysiologic signs of EAMG, however, the lower amount of anti-C6 did not prevent the loss of muscle AChR. Both the higher and the lower amount of anti-C6 Fab inhibited the accumulation of macrophages at muscle motor end-plates. The inhibition by anti-C6 indicates that muscle weakness and electrophysiologic abnormalities associated with EAMG are dependent on the complement MAC, and that muscle weakness results from tissue injury in addition to loss of muscle membrane and AChR.
Animals↗
The membrane attack complex of complement: lipid insertion of tubular and nontubular polymerized C9.
The membrane-restricted photoactivatable carbene generator 3-(trifluoromethyl)-3-(m-[125I]-iodophenyl)diazirine [Brunner, J., & Semenza, G. (1981) Biochemistry 20, 7174-7182] was used to label the subunits of the membrane attack complex of complement (C5b-9). C5b-9 complexes either were assembled from serum on erythrocyte membranes or were reconstituted from purified components on liposomes. After irradiation, most of the probe is bound to C9 independent of the membrane system used, indicating that the wall of the transmembrane channel is predominantly composed of C9. No difference was observed whether polymerized C9 was in the tubular or nontubular form [Podack, E. R., & Tschopp, J. (1983) J. Biol. Chem. 257, 15204-15212], showing that tubule closure is not essential for successful lipid insertion. The same label distribution between the two forms of polymerized C9 was obtained by analyzing zinc-polymerized C9 in the absence of C5b-8. Since the photoreactive probe reacted with at least two distinct polypeptide segments within C9, lipid interaction does not occur via a single segment of hydrophobic amino acids.
Animals↗
The C8-binding protein of human erythrocytes: interaction with the components of the complement-attack phase.
C8-binding protein is an intrinsic membrane protein of the human erythrocyte. It inhibits the complement (C5b-9)-mediated lysis in a species-restricting manner. In the present study we incorporated C8bp, isolated from human erythrocytes, into sheep erythrocytes (SRBC). SRBC, normally sensitive to lysis by human C5b-9, became insensitive to lysis. Furthermore, we found that C8bp is incorporated into the membrane-attack complex C5b-9, most probably by interacting with C8, since C8bp has an affinity for C8, particularly for the C8 alpha-gamma-subunit. Antibodies to C8bp react with the C8 alpha-subunits and with C9, pointing to the possibility of a partial homology between these proteins.
Animals↗
Activated terminal complement in cerebrospinal fluid in Guillain-Barré syndrome and multiple sclerosis.
A quantitative enzyme-linked immunosorbent assay was used to measure the concentration of fluid-phase complement C5b-9 complexes (SC5b-9) in the cerebrospinal fluid (CSF) of 14 patients with acute monophasic Guillain-Barré Syndrome (GBS), 21 patients with multiple sclerosis (MS), and 11 patients with noninflammatory central nervous system (CNS) diseases. SC5b-9 complexes were detected in the CSF of 13 of 14 patients with acute GBS (mean, 3.08 micrograms/ml; range, 0 to 7.1 micrograms/ml) and 16 of 21 patients with MS (mean, 1.83 micrograms/ml; range, 0 to 7.5 micrograms/ml). In the control group of patients with noninflammatory CNS diseases, SC5b-9 was not detected in eight of 11 and was present in low concentrations in the remaining three patients (mean, 0.28 micrograms/ml; range, 0 to 1.7 micrograms/ml). The finding of SC5b-9 complexes in the CSF of patients with GBS and MS suggests that terminal complement components may participate in the tissue-damaging processes in these diseases.
Complement Activation↗
Absence of homologous restriction factor does not affect CTL-mediated cytolysis.
Homologous restriction factor (HRF) is a membrane protein of erythrocytes and leukocytes that inhibits the complement (C5b-9)-mediated lysis in a species-restricting manner. HRF has also been reported to inhibit perforin-mediated cytolysis and postulated to play a role in cytotoxic T lymphocyte (CTL) self-protection. We show that paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes, lacking HRF, are no more sensitive to Ca2+-dependent human CTL-mediated lysis than normal erythrocytes. Furthermore, mouse and normal human erythrocytes, as well as PNH erythrocytes, are similarly lysed by isolated murine perforin-containing granules. We conclude that HRF does not inhibit perforin-mediated lysis and therefore is not likely to play a role in CTL self-protection.
Blood Proteins↗