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PubMed1987

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↗

PubMed1986

The pore-forming protein (perforin) of cytolytic T lymphocytes is immunologically related to the components of membrane attack complex of complement through cysteine-rich domains.

Structural, functional and immunological similarities between the ninth component of complement (C9) and the lymphocyte pore-forming protein (PFP, perforin) have recently been described (8-10). PFP is shown here to be immunologically related to all other components of the membrane attack complex (MAC) of human complement, namely, C5b-6, C7, C8, and C9. Polyclonal antibodies raised against purified human C5b-6, C7, C8, or C9 react with other components of the MAC and with mouse lymphocyte PFP. The antigenic epitopes shared by human complement proteins and mouse lymphocyte PFP are limited to cysteine-rich domains. Only complement proteins that have been reduced and alkylated elicit the production of crossreactive antibodies when used as immunogens. The nonreduced forms of complement components or lymphocyte PFP neither react with these antibodies nor give rise to crossreactive antibodies. The homologous domains of complement proteins and lymphocyte PFP may play related functions in their attachment to lipid membranes and assembly of membrane lesions.

Animals↗

PubMed1990

Regulation of the membrane attack complex of complement. Evidence that C8 gamma is not the target of homologous restriction factors.

Inability of the membrane attack complex of C (C5b-9) to efficiently lyse E from the same species has been attributed to one or more membrane-associated proteins that are collectively called homologous restriction factors. These include a 65,000 Mr protein referred to as the C8 binding protein or homologous restriction factor and a 20,000 Mr protein referred to as P-18, HRF20, CD59 Ag, or MIRL. Both are found on nucleated cells as well as E and both protect against complement-mediated lysis by interfering with C8 and/or C9 function within C5b-9. The exact mechanism by which these factors restrict activity is unknown but studies with purified C8 binding protein suggest they may interact specifically with the gamma subunit of C8. To determine directly if gamma is the target of restriction factors, a derivative of human C8 lacking this subunit was evaluated for its potential to lyse homologous cells. This derivative (C8') was previously shown to be functionally equivalent to normal C8 in a heterologous sheep E system. Here, it is compared to normal C8 by using human E as target cells. Results indicate no difference between the ability of C8 and C8' to incorporate into HuEAC1-7, to mediate subsequent C9 binding and to promote hemolysis. Thus, the presence or absence of gamma has no effect on homologous restriction of C5b-9, therefore gamma cannot be the primary target of homologous restriction factors.

Blood Proteins↗

PubMed1997

Sublytic concentrations of the membrane attack complex of complement induce endothelial interleukin-8 and monocyte chemoattractant protein-1 through nuclear factor-kappa B activation.

Activation of the complement cascade and subsequent assembly of the membrane attack complex (MAC) occur in a number of pathophysiological settings. When formed on the surface of endothelial cells in sublytic concentrations, the MAC can induce a number of proinflammatory activities, including the secretion of soluble mediators (eg, interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1) and the up-regulation of cell surface adhesion molecules. Available data indicate that MAC-induced cell activation may occur through several complex signal transduction pathways, but little is known about the intranuclear mechanisms by which complement-derived products promote the up-regulation of inflammatory mediators. Using purified distal complement proteins (C5-9) to assemble functional MAC on early-passage human umbilical vein endothelial cells (HUVECs), we examined mechanisms of MCP-1 and IL-8 induction. Formation of sublytic concentrations of MAC promoted an increase in nuclear factor (NF)-kappa B DNA binding activity within 60 minutes as determined by serial electrophoretic mobility shift assay. Cytosolic to nuclear translocation of NF-kappa B was confirmed by Western immunoblot and immunocytochemical analyses. Formation of the C5b-8 complex also promoted NF-kappa B translocation but to a lesser degree than observed in HUVECs containing complete MAC. No cytosolic to nuclear translocation of the p65 NF-kappa B subunit was observed in unstimulated HUVECs or in cells incubated with the MAC components devoid of C7. Preincubation of HUVECs with pyrrolidine dithiocarbamate prevented MAC-induced increases in IL-8 and MCP-1 mRNA concentrations and protein secretion. A direct cause and effect linkage between MAC assembly and NF-kappa B activation was established through examination of the pharmacological effect of the peptide SN50 on IL-8 and MCP-1 expression. SN50 is a recently engineered 26-amino-acid peptide that contains a lipophilic cell-membrane-permeable motif and a nuclear localization sequence that specifically competes with the nuclear localization sequence of the NF-kappa B p50 subunit. This study provides direct in vitro evidence that the distal complement system (MAC) can promote proinflammatory endothelial cell activation, specifically, increases in IL-8 and MCP-1 mRNA concentrations and protein secretion, and that cytosolic to nuclear translocation of NF-kappa B is necessary for this response.

Blotting, Northern↗

PubMed1982

Polymerization of the ninth component of complement (C9): formation of poly(C9) with a tubular ultrastructure resembling the membrane attack complex of complement.

The ninth component of complement (C9) has a marked propensity to polymerize. C9 polymers [poly(C9)] formed spontaneously in Veronal-buffered saline upon incubation of purified C9 for 64 hr at 37 degrees C or within 2 hr at 46--56 degrees C. Poly(C9) formed at 37 degrees C was visualized by electron microscopy as a tubular structure with an internal diameter of 110 A and a length of 160 A. Its ultrastructure suggested a dodecameric composition and resembled that of the membrane attack complex of complement. The wider end of the tubular structure was formed by an approximately 30-A-thick torus with inner and outer diameters of 110 A and 220 A, respectively. Because the dimensions of C9 within poly(C9) were 160 x 55 A (maximal) and 20 A (minimal) and because monomeric C9 has dimensions of approximately 80 x 55 A, it is proposed that monomeric C9 unfolds during polymerization into tubules. Polymerization also occurred upon treatment of C9 for 1 hr at 37 degrees C with 0.6 M guanidine . HCl, 0.1 M octyl glucoside, or 1.5% sodium deoxycholate. Guanidine . HCl-induced C9 polymers consisted of elongated highly curved strands 55--80 A wide, suggesting that these polymers were formed by globular C9 that had not unfolded.

Cell Membrane Permeability↗

PubMed1990

Involvement of membrane attack complex of complement in UV-B-induced acantholysis in pemphigus.

The initial acantholytic process induced by irradiation of UV-B on uninvolved skin of patients with pemphigus was immunohistologically studied using monoclonal antibodies that bind to antigenic determinants present in the C5b9 complex. Membrane attack complex of complement-related antigens in suprabasal intercellular sites could be detected after 5 hours of irradiation, at which time there was no acantholysis. At 24 hours, the staining intensity markedly increased and suprabasal clefts with acantholytic epidermal cells could be seen. In contrast, acantholysis did not develop in the absence of membrane attack complex formation at intercellular sites even 24 hours following UV-B irradiation. These findings suggest that UV-B can induce membrane attack complex assembly in the epidermis, which may itself be involved in the process of acantholysis and provide additional evidence for involvement of the complement system in pemphigus.

Acantholysis↗

PubMedDate not supplied

Inhibition of neutrophil adhesion and the membrane attack complex of complement synergistically prolongs cardiac xenograft survival.

BACKGROUND: Hyperacute xenograft rejection is affected by activation of the complement cascade. Split products of early complement components influence the localization, activation, and effector function of platelets, granulocytes, and lymphocytes, whereas the formation of the membrane attack complex (C5b-9) leads to direct cellular injury. In a unique strain of PVG rats deficient in the C6 component of complement, the terminal membrane attack complex is not formed. However, production of the chemotactic and vasoactive components C3a and C5a proceeds normally. Guinea pig cardiac xenografts in these C6-deficient rats have prolonged survival, and at the time of rejection the inflammatory infiltrate is composed primarily of neutrophils. NPC 15669, a member of a class of antiinflammatory agents called leumedins, is known to inhibit neutrophil adhesion. The purpose of this study was to determine whether inhibition of neutrophil recruitment in animals incapable of membrane attack complex formation would prolong cardiac xenograft survival. METHODS: Cardiac xenografts from male Hartley guinea pigs were heterotopically grafted into PVG (C-) and PVG (C+) male rats. Experimental animals received 20 mg/kg of NPC 15669 i.v. before cross-clamp release and 10 mg/kg of NPC 15669 intravenously on postoperative day 1. Control animals received intravenous saline solution only. RESULTS: Complement sufficient PVG (C+) rats rejected cardiac xenografts hyperacutely despite mode of treatment: PVG (C+) rats which received saline solution (n = 5) rejected their xenografts at 10.8 +/- 2.6 minutes, and those receiving NPC 15669 (n = 5) rejected at 13.9 +/- 5.3 minutes. Histologic examination showed edema, platelet aggregation, and hemorrhage but no cellular inflammatory infiltrate. As expected, complement-deficient PVG (C-) rats had markedly longer xenograft survival in the saline solution-treated group (n = 5) with graft function being sustained 14.7 +/- 6.1 hours. NPC 15669 treatment (n = 4) further prolonged graft function to 61.0 +/- 4.7 hours. In addition to edema, platelet aggregation, and hemorrhage, histologic analysis of these grafts at the time of rejection was characterized by an infiltration of neutrophils. CONCLUSIONS: We conclude that neutrophils play a critical role in cardiac xenograft rejection when complement activation is restricted. Combined inhibition of complement and neutrophil adhesion prolongs xenograft survival longer than inhibition of either component alone.

Animals↗

PubMed1987

The membrane attack complex of Xenopus laevis complement.

Rabbit erythrocyte membranes lyzed by Xenopus laevis serum exhibited a typical ultrastructural complement lesion with an inner diameter of 80 +/- 9 A. The protein pattern associated with lyzed membrane is compared to a similar human preparation.

Animals↗

PubMed1993

The cerebrospinal-fluid soluble form of Alzheimer's amyloid beta is complexed to SP-40,40 (apolipoprotein J), an inhibitor of the complement membrane-attack complex.

The amyloid fibrils deposited in Alzheimer's neuritic plaque cores and cerebral blood vessels are mainly composed of aggregated forms of a unique peptide, 39-42 amino acids long, named amyloid beta (A beta). A similar, although soluble, A beta ('sA beta') has been identified in cerebrospinal fluid, plasma and cell supernatants, indicating that it is normally produced by proteolytic processing of its precursor protein, amyloid precursor protein (APP). Using direct binding experiments we have isolated and characterized an 80 kDa circulating protein that specifically interacts with a synthetic peptide identical with A beta. The protein was unmistakably identified as SP-40,40 or ApoJ, a cytolytic inhibitor and lipid carrier, by means of amino acid sequence and immunoreactivity with specific antibodies. Immunoprecipitation with anti-SP-40,40 retrieved soluble A beta from cerebrospinal fluid, indicating that the interaction occurs in vivo.

Amino Acid Sequence↗

PubMed1979

Proteolytic transformation of SC5b-9 into an amphiphilic macromolecule resembling the C5b-9 membrane attack complex of complement.

Proteolysis of fluid-phase SC5b-9 left a major part of the macromolecule intact and caused transition of the molecule from a hydrophilic to an amphiphilic state. The transformed complex exhibited neoantigens characteristic of the C5b-9 membrane attack complex of the complement. It yielded an SDS gel electrophoresis pattern that was similar, but not identical to that of the proteolysed, membrane attack complex. The proteolytically altered SC5b-9 complex bound lipid and incorporated into artificial lipid vesicles to yield a membrane-bound structure resembling the C5b-9 complement lesion.

Antigens↗

PubMed1985

Monoclonal antibodies which react with lymphocyte-lysed target cells and which cross-react with complement-lysed ghosts.

Monoclonal antibodies were raised against a human natural killing system and screened against targets lysed either by human lymphocytes in antibody-dependent cellular cytotoxicity (ADCC), or by human complement. Two monoclonals were identified which bound specifically to both types of killed targets. More detailed studies with one antibody showed that it inhibited ADCC, both as intact antibody and as the F(ab')2 fragment. Intact antibody enhanced natural (NK) killing, although the F(ab')2 fragment inhibited NK killing. The data support the hypothesis that lymphocyte-mediated killing involves a complex analogous in nature to the complement membrane attack complex. In addition, the antibodies provide evidence to suggest that this complex has antigenic determinants in common with the complement membrane attack complex, and indicate the possibility that the two systems are derived from a common ancestor.

Animals↗

PubMed1995

Induction of cellular procoagulant activity by the membrane attack complex of complement.

In addition to their well-recognized role in immune defense, there is a growing recognition that the proteins of the complement system impact directly on vascular homeostatic mechanisms, evoking cellular responses that serve to both promote adherence of blood cells to the walls of blood vessels, and the formation of fibrin through the enzyme mechanisms of the coagulation system. This clot-promoting or 'procoagulant' activity initiated through the complement system entails both receptor-mediated as well as receptor-independent pathways of cell activation. In this review, we will focus specifically upon the role that is now thought to be played by the membrane attack complex of the complement system (MAC) in the induction of the procoagulant properties of human platelets and endothelium.

Blood Coagulation↗

PubMed1993

Pathogenesis of glomerulonephritis.

This review summarizes current understanding of the mechanisms which mediate immune glomerular injury in glomerulonephritis. Non-inflammatory lesions resembling minimal change disease and membranous nephropathy, respectively, are induced by non-complement fixing antibodies to glomerular epithelial cell (GEC) membrane antigens (minimal change disease) and GEC membrane insertion of the C5b-9 membrane attack complex of complement (membranous nephropathy). The cellular mechanisms of these effects are unclear but may involve GEC activation and release of local mediators, such as proteases or oxidants, or GEC detachment from underlying basement membrane. Inflammatory types of glomerular lesions are mediated by circulating inflammatory cells (neutrophils, platelets, macrophages) or by resident glomerular cells (mesangial cells) or both. Neutrophil-mediated injury involves local release of GBM degrading proteases or GBM halogenation induced by the interaction of neutrophil-derived myeloperoxidase with H2O2 and a halide. Neutrophil induced glomerular injury is augmented by platelets. Recent evidence establishes that mesangial cell proliferation in glomerulonephritis is mediated by complement and platelets. Mesangial cell proliferation is accompanied by increased expression of PDGF and PDGF receptor proteins and the genes for these proteins resulting in an autocrine mechanism of cell proliferation. Mesangial cell proliferation is also accompanied by increased release of neutral proteinase in sites of basement membrane damage. Mesangial cell proliferation precedes up-regulation of genes for extracellular matrix components in several models and may be important in the pathogenesis of glomerulosclerosis.

Animals↗

PubMed1995

The membrane attack complex of complement mediates peripheral nervous system demyelination in vitro.

The present study used cocultures of rat dorsal root ganglia (DRG) and peritoneal macrophages to define the role of activated complement components during demyelination. The complement cascade was activated in vitro by treatment of the cultures with natural rat serum and lipopolysaccharides. Complement activation was examined by detection of the membrane attack complex of complement (MAC) with an antibody directed against rat C5-9. Detection of MAC in vitro by immunoelectron microscopy was associated with morphological changes of the myelin sheath. The sheath's regular structure was disrupted. Myelin lamellae were split and showed signs of decompaction. These changes were followed by a selective macrophage attack on myelin sheaths resulting in demyelination. Schwann cell viability was not affected by complement activation. Axons and sensory ganglion cells also survived this attack. The specificity of the complement effect was tested in experiments using treatment regimens with natural rat serum or lipopolysaccharides alone. In these experiments, no morphological changes of the myelin sheath were observed as well as no macrophage attack on myelin.

Animals↗

PubMed1986

Assembly of the membrane attack complex of complement on small unilamellar phospholipid vesicles.

Light-scattering intensity was shown to be a reliable, direct, and quantitative technique for monitoring the assembly of the membrane attack complex of complement (proteins C5b-6, C7, C8, and C9) on small unilamellar phosphatidylcholine vesicles. The assembly on vesicles occurred in a simple fashion; complexes of C5b-7 bound noncooperatively to the vesicles, and final assembly of C5b-9 did not induce vesicle aggregation or fragmentation. When C5b-6 and C7 were mixed in the presence of vesicles but at molar protein/vesicle ratios of less than 1, there was quantitative binding of C5b-7 to the vesicles with no concomitant aggregation of C5b-7. If C7 was added at a slower rate, quantitative binding was obtained at molar C5b-7/vesicle ratios of up to 5. The latter observations (a) were consistent with the proposal that C5b-7 aggregation and membrane binding were competitive events and (b) defined conditions under which light-scattering intensity measurements could monitor C5b-9 assembly on vesicles without contribution from the fluid-phase assembly. The C8/C5b-7 ratio in the phospholipid-C5b-8 complex was 0.97 +/- 0.12, and the maximum ratio of C9/C5b-8 in the final complex was 16.2 +/- 2.0. One C9 molecule associated rapidly with each phospholipid-C5b-8, followed by slower incorporation of the remaining C9 molecules. The initial velocity of the slow phase of C9 addition was easily saturated with C9 and gave an activation energy of 37 kcal/mol. This was identical with the value measured for the analogous process in the fluid-phase assembly.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement C7↗

PubMed1980

Membrane attack complex of complement: a structural analysis of its assembly.

This study was conducted to gain insight into the process of assembly of the membrane attack complex (MAC) of complement through structural analysis. Four intermediate complexes and the MAC were examined by electron microscopy and by sucrose density-gradient ultracentrifugation. The C5b-6 complex has a sedimentation rate of 11S, an elongated, slightly curved shape and dimensions of 160 x 60 x 60 A. At protein concentrattions greater than 1 mg/ml, and physiologic ionic strength and pH, the complex forms paracrystals that have the appearance of parallel strands. Equimolar quantities of C5b-6 and C7 mixed in the absence of lipids or detergents give rise to C5b-7 protein micelles which are soluble in aqueous media and have a sedimentation rate of 36S, suggesting a tetrameric composition. Ultrastructurally, C5b-7 protein micelles consist of four half-rings, each measuring 200 x 50 A, which are connected to one another by short stalks extending from the convex side of the half-rings. C5b-7 bound to dioleoyl lecithin (DOL) vesicles has a similar ultrastructural appearance. After extraction with deoxycholate (DOC), C5b-7 has a sedimentation velocity of 36S which further suggests the occurrence of C5b-7 in the form of tetrameric protein micelles. Attachment of C8 to vesicle-bound C5b-7 results in dissociation of the protein micelles. An individual C5b-8 complex appears as a half-ring attached to the DOL-vesicle via a 100-A-long and 30-A-wide stalk. After extraction from the DOL-vesicles with DOC, C5b-8 has a sedimentation velocity of approximately 18S. Binding of C9 to DOL-vesicle bound C5b-8 induces the formation of the typical ultrastructural complement lesions. C5b-9 extracted from the vesicles with DOC has a sedimentation rate of 33S, which is characteristic of the C5b-9 dimer. It is concluded that dimerization is a function of C9. C5b-9 monomers are visualized when a single C5b-9 complex or an odd number of complexes were bound per DOL-vesicle. The C5b-9 monomer has an ultrastructural appearance that is theoretically expected of a half-dimer: a 200- x 50-A half-ring which is attached to the DOL-vesicle by a 100- x 80-A appendage. Extracted with DOC, the C5b-9 monomer has a sedimentation rate of 23S. At a higher multiplicity of MAC per DOL-vesicle, large structural defects in the lipid bilayer are seen which are attributed to direct physical destruction of membranes by the known lipid-binding capacity of the MAC. It is proposed that protein micelle formation at the C5b-7 stage of MAC assembly and dissociation of these micelles upon binding of C8 are events that facilitate dimerization of C5b-9 and thus MAC formation.

Binding Sites↗

PubMed1994

Blocking antibodies specific for human albumin interfere with the hemolytic activity of the membrane attack complex of complement.

Earlier, we showed that antibodies specific for human serum albumin are able to bind to erythrocyte-associated albumin and inhibit complement mediated hemolysis. In the present study we determine if inhibition is occurring at the membrane attack phase of complement activation or at an earlier step. We show that although cell-bound anti-albumin antibodies do not inhibit binding and activation of C3 or uptake of C9, they do appear to cause cells to become refractory to lysis by the membrane attack complex as they inhibit both the kinetics and the extent of hemolysis in a reactive lysis system which employs preformed C5b6 plus C7, C8, and C9. We believe that this is the first report of inhibition of the hemolytic activity of the membrane attack complex by antibodies bound to an erythrocyte surface antigen.

Animals↗

PubMed1989

Increased urinary excretion of C5b-9 distinguishes passive Heymann nephritis in the rat.

Increased urinary excretion of C5b-9 distinguishes passive Heymann nephritis from other forms of experimental glomerulonephritis in the rat. In the passive Heymann nephritis (PHN) model of membranous nephropathy (MN) subepithelial deposits form from anti-Fx1A antibody reacting with antigen expressed on the glomerular epithelial cell membrane followed by membrane patching and shedding of immune complexes. Immune complex deposits are accompanied by deposits of C5b-9 which is required for the mediation of proteinuria. We tested the hypothesis that C5b-9 assembly on the epithelial cell membrane might result in C5b-9 excretion in the urine, which would distinguish this autoimmune mechanism of MN from other processes that result in subepithelial immune complex deposits. Using monoclonal antibodies developed to rat C6 and a rat C5b-9 neoantigen, in a sensitive ELISA assay, elevated urinary excretion of rat C5b-9 was documented in PHN associated with on-going glomerular immune deposit formation. No urinary C5b-9 was detectable in MN induced by an exogenous antigen (cationized IgG) despite equivalent glomerular C5b-9 deposits, or in models of nephrotoxic nephritis, subendothelial immune complex nephritis, anti-mesangial cell membrane antibody-induced nephritis or two non-immune nephropathies. Infusion of preformed C5b-9 in proteinuric animals excluded glomerular filtration of C5b-9 as a contributing mechanism to urinary C5b-9 excretion. We conclude that in the rat, increased urinary excretion of C5b-9 is a marker of MN induced by antibody to a glomerular epithelial cell antigen. Urine C5b-9 excretion reflects active glomerular immune deposit formation and distinguishes MN induced by this mechanism from other forms of MN as well as from other glomerular diseases with equivalent glomerular C5b-9 deposits.

Animals↗