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PubMed1988

The terminal complement complex, C5b-9, a marker of disease activity in patients with systemic lupus erythematosus.

Concentrations of the terminal complement complex (TCC), C5b-9, were examined in 120 serum samples from 28 patients with systemic lupus erythematosus. Eleven patients with various manifestations of the disease were followed longitudinally for a 2-year period during active and inactive phases of the disease. In 9 of the 11 patients, elevations in TCC concentrations correlated with disease exacerbations. In many of these patients, C3 and C4 levels remained normal during the study and were less sensitive indicators of disease activity than were TCC concentrations. We believe that measurements of TCC are useful in monitoring patients with rheumatic diseases in which complement activation is a component.

Adult↗
PubMed1988

Complement proteins C5b-9 initiate secretion of platelet storage granules without increased binding of fibrinogen or von Willebrand factor to newly expressed cell surface GPIIb-IIIa.

The functional and conformational activation of cell surface glycoproteins IIb-IIIa (GPIIb-IIIa) was probed in platelets stimulated to secrete by complement proteins C5b-9. Gel-filtered human platelets exposed to the purified human C5b-9 proteins exhibited non-lytic secretory release of both alpha- and dense granule storage pools with only a small increase in total binding of 125I-fibrinogen (less than 3000 molecules/cell) to the cell surface. By contrast to ADP- or thrombin-activated platelets, increased 125I-fibrinogen bound to C5b-9 platelets was not inhibited by Arg-Gly-Asp-containing peptides, suggesting that the high affinity membrane receptor for fibrinogen is not expressed under these conditions. C5b-9-stimulated platelets also failed to bind 125I-von Willebrand factor (less than 1 ng/10(8) platelets), confirming that the adhesive protein receptor function of cell surface GPIIb-IIIa is not expressed in these cells. Although specific binding of 125I-fibrinogen or 125I-von Willebrand factor did not significantly increase after C5b-9 assembly, these proteins elicited de novo expression of the GPIIb-IIIa activation-associated epitope recognized by monoclonal antibody PAC-1, and binding of this antibody to C5b-9 platelets was fully competed by Arg-Gly-Asp-containing peptides. These data suggest that the metabolic events which trigger granule secretion after C5b-9 insertion into the plasma membrane cause cell surface GPIIb-IIIa to be expressed in an activation-associated but functionally incompetent conformation.

Adenosine Diphosphate↗
PubMed1985

Membrane damage by channel-forming proteins: staphylococcal alpha-toxin, streptolysin-O and the C5b-9 complement complex.

One mechanism through which cells can be damaged involves insertion of alien proteins into the membrane bilayer and the formation of hydrophilic transmembrane pores. Three examples for this process are discussed, namely membrane damage by staphylococcal alpha-toxin, streptolysin-O, and by the terminal C5b-9 complement complex. Common to all is the principle of a transition of the proteins from a water-soluble state to an amphiphilic state, occurring through the appearance or exposure of apolar surfaces during oligomerization of the protein molecules into supramolecular aggregates. The resulting complexes or protein oligomers insert spontaneously into the target lipid bilayer and assume properties akin to those of integral membrane proteins. The protein channels can be isolated from membranes after their solubilization by mild detergents and characterized on a bio-immunochemical and ultrastructural level.

Animals↗
PubMed1989

Complement proteins C5b-9 induce secretion of high molecular weight multimers of endothelial von Willebrand factor and translocation of granule membrane protein GMP-140 to the cell surface.

The effect of immune activation of the serum complement system on the secretory response of human endothelial cells was examined. Exposure of antibody sensitized cultured umbilical vein endothelial cells to human serum resulted in secretion of very high molecular weight multimers of von Willebrand factor which coincided with new surface expression of the intracellular granule membrane protein GMP-140. This response required complement activation through deposition of C5b-9 and was not observed with cells exposed to antibody plus C8-deficient serum or to membrane C5b-8 (in the absence of C9). This C5b-9-induced secretion was observed with minimal cell lysis, as assessed by the release of lactic dehydrogenase. Delayed addition of C8 and C9 to cells exposed to antibody plus C8-deficient serum revealed a rapid decay of membrane C8 binding sites accompanied by loss of the secretory response, suggesting a process of removal or inactivation of nascent C5b67 complexes deposited on the endothelial surface. Membrane assembly of C5b-9 complexes caused an increase in endothelial cytosolic [Ca2+], due to influx across the plasma membrane. This C5b-9-dependent increase in cytosolic [Ca2+] and concomitant von Willebrand factor secretion were both abolished by removal of external calcium. In addition to being linked to the level of external Ca2+, the C5b-9-induced secretory response was partially inhibited by the protein kinase inhibitor, sphingosine. The capacity of the C5b-9 proteins to stimulate endothelial cells to secrete a platelet adhesive protein provides one mechanism for increased platelet deposition at sites of inflammation, and suggests the potential for other functional changes in endothelium exposed to C5b-9 during intravascular complement activation.

Antibodies↗
PubMed1981

Permeability characteristics of complement-damaged membranes: evaluation of the membrane leak generated by the complement proteins C5b-9.

Permeability characteristics of the membrane lesion generated by the terminal complement proteins are considered in light of recent observations that the measured diffusion of solute across complement-damaged membranes does not conform to the "doughnut hole" model of a discrete transmembrane pore formed by the inserted C5b-9 complex. By using the measured kinetics of steady-state tracer isotope diffusion of nonelectrolytes across resealed erythrocyte ghost membranes treated with C5b-9, a new transport model is developed. This model considers the apparent membrane lesion strictly in terms of the operational criteria of a functional conducting pathway for the observed diffusing solute, independent of a priori assumptions about the geometry or molecular properties of the membrane lesion. With this definition of the unit membrane lesion and the assumption that the exclusion size of the conducting pathway varies directly with the multiplicity of bound C5b-9 (as suggested by previous measurements under conditions of varying input of C5b-9), numerical estimates of te apparent permeability of the complement-damaged membrane to four diffusing nonelectrolytes are derived. These results suggest that the pathway for a particle diffusing across the complement lesion cannot be a pore and is functionally equivalent to an aqueous leak pathway, free of pore constraints. Implications of these results are discussed in terms of current molecular models for the mechanism of membrane damage by the complement proteins.

Biological Transport↗
PubMed1995

Release of cytokines, polymorphonuclear elastase and terminal C5b-9 complement complex by infusion of wound drainage blood.

25 patients undergoing total hip replacement surgery were studied in an investigation of release of cytokines (interleukin-1 beta, IL-1 beta; interleukin-6, IL-6; interleukin-8, IL-8; and tumor necrosis factor-alpha, TNF-alpha), PMN elastase and terminal C5b-9 complement complexes (TCC) at the time of collection and transfusion of autologous blood. 15 patients received wound blood that was washed and centrifuged before being transfused as an erythrocyte suspension. In this blood there were no elevations in the concentrations of cytokines, TNF-alpha, PMN elastase or TCC, and there was no increase in these variables in plasma after transfusion of wound blood. 10 patients received postoperatively-collected drainage blood. There were high amounts of cytokines, PMN elastase and TCC in this blood, and filtration of the collected drainage blood did not reduce the concentrations of these factors, except those of TCC. When the collected drainage blood was infused, elevated plasma concentrations of IL-6, IL-8 and PMN elastase were observed 1 and 60 minutes after completing the transfusion. No differences regarding blood pressure, oxygen saturation (SpO2), and hemoglobin concentration between the groups were recorded.

Aged↗
PubMed1985

Cyanine dye fluorescence used to measure membrane potential changes due to the assembly of complement proteins C5b-9.

The fluorescent potentiometric indicator diS-C3-(5) has been used to investigate changes in membrane potential due to assembly of the C5b-9 membrane attack complex of the complement system. EAC1-7 human red blood cells and resealed erythrocyte ghosts--bearing membrane-assembled C5b67 complexes--were generated by immune activation in C8-deficient human serum. Studies performed with these cellular intermediates revealed that the membrane potential of EAC1-7 red cells and ghosts is unchanged from control red cells (-7 mV) and ghosts (O mV), respectively. Addition of complement proteins C8 and C9 to EAC1-7 red cells results in a dose-dependent depolarization of membrane potential which precedes hemolysis. This prelytic depolarization of membrane potential--and the consequent onset of hemolysis--is accelerated by raising external [K+], suggesting that the diffusional equilibration of transmembrane cation gradients is rate limiting to the cytolytic event. In the case of EAC1-7 resealed ghosts suspended at either high external [K+] or [Na+], no change in membrane potential (from O mV) could be detected after C8/C9 additions. When the membrane potential of the EAC1-7 ghost was displaced from O mV by selectively increasing the K+ conductance with valinomycin, a dose-dependent depolarization of the membrane was observed upon addition of C8 and C9. In these experiments, lytic breakdown of the ghost membranes was less than 5%. Conclusions derived from this study include: (i) measured prelytic depolarization of the red cell Donnan potential directly confirms the colloid-osmotic theory of immune cytolysis. (ii) The diffusional transmembrane equilibration of Na+ and K+ through the C5b-9 pore results in a dose-dependent depolarization of the membrane potential (Em) which appears to be rate-limiting to cytolytic rupture of the target erythrocyte. (iii) Enhanced immune hemolysis observed in high K+ media cannot be attributed to cation-selective conductance across the C5b-9 pore, and is probably related to the near-equilibrium condition of potassium-containing red cells when suspended at high external K+. These experiments demonstrate that carbocyanine dye fluorescent indicators can be used to monitor electrochemical changes arising from immune damage to the plasma membrane under both cytolytic and noncytolytic conditions. Potential application of this method to the detection of sublytic pathophysiological changes in the plasma membrane of complement-damaged cells are discussed.

Complement Membrane Attack Complex↗
PubMed1993

Complement activation in septic baboons detected by neoepitope-specific assays for C3b/iC3b/C3c, C5a and the terminal C5b-9 complement complex (TCC).

We have investigated the cross-reactivity of various species in neoepitope-specific methods for quantification of human complement activation products. In contrast to most other species examined, baboon showed a substantial cross-reactivity supporting a high degree of homology between human and baboon complement. An assay for C3b, iC3b and C3c (MoAb bH6) showed moderately good reactivity, in contrast to a C3a assay which did not cross-react. Excellent reactivity was found for C5a using MoAbs C17/5 and G25/2. The reactivity of an established TCC assay (MoAb aE11 to a C9 neoepitope and polyclonal antibody to C5) was improved substantially by replacing the anti-C5 antibody with a new MoAb to C6 particularly selected on the basis of baboon cross-reactivity. Plasma samples from baboons receiving 2.5 x 10(9) and 1.0 x 10(10) live Escherichia coli bacteria/kg were examined with the assays described. In vivo complement activation with the lowest dose was moderate and kept under control, in contrast to the highest dose, where an uncontrolled increase in all activation products continued throughout the infusion period. These results support the hypothesis that sufficiently high amounts of endotoxin lead to uncontrolled activation of complement as seen in irreversible septic shock. The results are discussed with particular emphasis on activation of the terminal complement pathway.

Animals↗
PubMed2004

C5b-9 terminal complement complex assembly on apoptotic cells in human arterial wall with atherosclerosis.

Apoptosis plays an important role in atherosclerosis. The factors regulating this process are not well defined. We examined the relation of apoptotic cells with the terminal complement complex C5b-9 in human atherosclerotic lesions. The extent of apoptosis was determined using TdT dUTP nick-end labeling (TUNEL) and immunohistochemistry of apoptosis regulators caspase-3, caspase-9, Bax, and Bcl-2. C5b-9 was localized by immunohistochemistry and immunoelectron microscopy. The apoptotic index was higher in fibrous plaques when compared with intimal fatty streaks and intimal thickenings. Bax expression was present in TUNEL+ apoptotic cells, and Bcl-2 was rarely present in the atherosclerotic wall. Active caspase 9 and caspase 3 deposits were present in the same areas, suggesting an involvement of the mitochondrial pathway. C5b-9 deposits colocalized with TUNEL+ cells, and the percent of double-positive cells was 2% in fatty streaks, 12% in intimal thickenings, and 35% in fibrous plaques. Colocalization of apoptotic cells with C5b-9 was also confirmed by immunoelectron microscopy. In conclusion, some apoptotic cells carry C5b-9 deposits, suggesting that complement might be activated by apoptotic cells and involved in the promotion of apoptosis, contributing to the progression of atherosclerotic lesions.

Aorta↗
PubMed1996

Interaction between complement proteins C5b-7 and erythrocyte membrane sialic acid.

The initial phase of membrane attack by complement is the interaction between C5b6, C7, and the cell membrane that leads to the insertion of C5b-7. Here we investigate the role of sialic acid residues in the assembly of C5b-7 intermediates on erythrocyte cell membranes. We find that C5b6 binds to glycophorin, whereas C5 or C6 does not bind, and desialylation of the glycophorin abolishes C5b6 binding. Complement lysis is inhibited by either masking glycophorin sialic acid with F(ab) fragments of an mAb, or by removal of the sialylated region of glycophorin by mild trypsinization. Gangliosides inhibit C5b-7 deposition when added to the aqueous phase. Asialogangliosides and synthetic gangliosides lacking the carboxylic acid residue have no inhibitory activity. We conclude that C5b6 binds to sialylated molecules on the erythrocyte surface. We propose a new model of membrane attack in which C5b6 initially binds to membranes via ionic forces. C7 then binds to C5b6, disrupting the ionic interaction and leading to the exposure of hydrophobic domains. Sialic acid is known to inhibit complement activation. Thus, these findings reveal a paradoxical role for sialic acid in complement attack; the presence of sialic acid inhibits the generation of C5b6, but once the membrane attack pathway is initiated, sialic acid enhances complement lysis.

Animals↗
PubMed1993

Interaction of the terminal complement components C5b-9 with synovial fibroblasts: binding to the membrane surface leads to increased levels in collagenase-specific mRNA.

The late complement components, apart from their lytic function, are known to trigger the release of various proinflammatory substances from different types of nucleated cells. In the present study, the interaction of C5b-9 with synovial fibroblast cells (SFC) was examined. It was found that incubation of SFC with activated complement components resulted in binding of C5b-9 to the cell membrane; subsequently an increase in abundance of collagenase-specific mRNA was seen, as assessed by Northern blotting. When C8-deficient serum was used as source of complement neither binding of C5b-9 nor an increase in collagenase-specific mRNA could be detected. These findings suggest that C5b-9, which might be generated during rheumatoid inflammation, may contribute to chronic joint destruction by triggering collagenolytic activity.

Arthritis, Rheumatoid↗
PubMed1993

Terminal complement complex C5b-9 stimulates mitogenesis in 3T3 cells.

The membrane attack complex of complement (MAC) can induce reversible changes in cell membrane permeability resulting in significant but transient intracellular ionic changes in the absence of cell lysis. Because ion fluxes and cytosolic ionic changes are integral steps in the signaling cascade initiated when growth factors bind to their receptors, we hypothesized that the MAC-induced reversible changes in membrane permeability could stimulate cell proliferation. Using purified terminal complement components we have documented a mitogenic effect of the MAC for quiescent murine 3T3 cells. The MAC enhances the mitogenic effects of serum and PDGF, and also stimulates cell proliferation in the absence of other exogenous growth factors. MAC-induced mitogenesis represents a novel effect of the terminal complement complex that could contribute to focal tissue repair or pathological cell proliferation locally at sites of complement activation.

3T3 Cells↗
PubMed1986

Single-channel analysis of the conductance fluctuations induced in lipid bilayer membranes by complement proteins C5b-9.

Single-channel analysis of electrical fluctuations induced in planar bilayer membranes by the purified human complement proteins C5b6, C7, C8, and C9 have been analyzed. Reconstitution experiments with lipid bilayer membranes showed that the C5b-9 proteins formed pores only if all proteins were present at one side of the membrane. The complement pores had an average single-channel conductance of 3.1 nS at 0.15 M KCl. The histogram of the complement pores suggested a substantial variation of the size of the single channel. The linear relationship between single-channel conductance at fixed ionic strength and the aqueous mobility of the ions in the bulk aqueous phase indicated that the ions move inside the complement pore in a manner similar to the way they move in the aqueous phase. The minimum diameter of the pores as judged from the conductance data is approximately 3 nm. The complement channels showed no apparent voltage control or regulation up to transmembrane potentials of 100 mV. At neutral pH the pore is three to four times more permeable for alkali ions than for chloride, which may be explained by the existence of fixed negatively charged groups in or near the pore. The significance of these observations to current molecular models of the membrane lesion formed by these cytolytic serum proteins is considered.

Complement Membrane Attack Complex↗
PubMed1989

Complement proteins C5b-9 induce transbilayer migration of membrane phospholipids.

Transbilayer migration of membrane phospholipid arising from membrane insertion of the terminal human complement proteins has been investigated. Asymmetric vesicles containing pyrene-labeled phosphatidylcholine (pyrenePC) concentrated in the inner monolayer were prepared by outer monolayer exchange between pyrenePC-containing large unilamellar vesicles and excess (unlabeled) small unilamellar vesicles, using bovine liver phosphatidylcholine-specific exchange protein. After depletion of pyrenePC from the outer monolayer, the asymmetric large unilamellar vesicles were isolated by gel filtration and exposed to the purified C5b-9 proteins at 37 degrees C. Transbilayer exchange of phospholipid between inner and outer monolayers during C5b-9 assembly was monitored by changes in pyrene excimer and monomer fluorescence. Membrane deposition of the C5b67 complex (by incubation with C5b6 + C7) caused no change in pyrenePC fluorescence. Addition of C8 to the C5b67 vesicles resulted in a dose-dependent decrease in the excimer/monomer ratio. This change was observed both in the presence and absence of complement C9. No change in fluorescence was observed for control vesicles exposed to C8 (in the absence of membrane C5b67), or upon C5b-9 addition to vesicles containing pyrenePC symmetrically distributed between inner and outer monolayers. These data suggest that a transbilayer exchange of phospholipid between inner and outer monolayers is initiated upon C8 binding to C5b67. The fluorescence data were analyzed according to a "random walk" model for excimer formation developed for the case where pyrenePC is asymmetrically distributed between lipid bilayers. Based on this analysis, we estimate that a net transbilayer migration of approximately 1% of total membrane phospholipid is initiated upon C8 binding to C5b67. The potential significance of this transbilayer exchange of membrane phospholipid to the biological activity of the terminal complement proteins is considered.

Androgen-Binding Protein↗
PubMed1980

Re-incorporation of the terminal C5b-9 complement complex into lipid bilayers: formation and stability of reconstituted liposomes.

The formation and stability of lecithin liposomes carrying re-incorporated C5b-9(m) complexes prepared through a detergent-dialysis procedure was studied. Confluent aggregates of phospholipid and protein formed at low initial lipid-protein ratios (1:1, w/w), higher lipid-protein ratios (e.g. 5:1, w/w) were required for formation of lipid vesicles with recognizable bilayer structure. The unfractionated preparations comprised a heterogeneous population of vesicles that sedimented according to their lipid-protein content to varying positions upon centrifugation in CsCl density gradients. The vesicles were stable and C5b-9(m) complexes did not detach from the membranes during centrifugation through CsCl. They also resisted elution from the bilayer by treatment with salt solutions of low or high ionic strength, or of high pH. The results indicate anchorage of C5b-9(m) in the membrane through apolar interactions, akin to that of an integral membrane protein, and are compatible with the channel concept of complement lysis.

Centrifugation, Density Gradient↗
PubMedDate not supplied

Immunohistochemical localization of the terminal C5b-9 complement complexes associated to S-protein and macrophage immunoreactive deposits in human damaged myocardial areas.

The indirect and double-labelling immunoperoxidase techniques were used to localize the C5b-9 neoantigens, S-protein and macrophage immunoreactive deposits in myocardial areas with necrosis and sclerosis. Granular masses of C5b-9 neoantigens, diffuse areas of S-protein and nucleated and anucleated immunoreactive deposits of macrophage origin were localized in damaged myocardial areas but not in areas free of lesion. The presence of C5b-9 neoantigens associated to S-protein and macrophage deposits is suggestive for a local complement activation in the complex heterolytic events of myocardial injury.

Complement Membrane Attack Complex↗
PubMed1992

Inhibition of the complement membrane attack complex by the galactose-specific adhesion of Entamoeba histolytica.

The human complement system is an important early host defense against infection. Entamoeba histolytica activates the complement system but is resistant to killing by complement C5b-9 complexes deposited on the membrane surface. Our aim was to identify components of the amebic plasma membrane that mediate resistance to human complement C5b-9 by screening for neutralizing monoclonal antibodies. A monoclonal antibody was identified that abrogated amebic resistance to C5b-9, and the mAb was shown to recognize the parasite's galactose-specific adhesin. The purified adhesin bound to C8 and C9 and conferred C5b-9 resistance to sensitive ameba upon reconstitution; these activities of the adhesin were inhibited by the antiadhesin mAb. The E. histolytica adhesin shared sequence similarities and antigenic cross-reactivity with CD59, a membrane inhibitor of C5b-9 in human blood cells, suggesting both molecular mimicry and shared complement-inhibitory functions.

Amino Acid Sequence↗
PubMed1989

Cells carrying C5b-9 complement complexes in human atherosclerotic wall.

Fibrous plaques and intimal thickenings of 5 femoral and 5 iliac human arteries obtained at surgery were processed for indirect and double-labeling immunoelectron microscopy using an affinity purified rabbit IgG anti-C5b-9 neoantigen and the EBM 11 monoclonal antibody anti-human macrophages. The C5b-9 complexes were localized in intact cells, disintegrated cells and cell debris enmeshed in the connective tissue matrix. Some of the cell debris bearing C5b-9 deposits was found to be of macrophage origin. Endocyted or exocyted pieces of membrane with pore-forming C5b-9 complexes were also identified. Damage of cells by complement in atherosclerotic lesions may contribute to atherogenesis.

Antibodies, Monoclonal↗