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PubMed1988

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↗
PubMed1986

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↗
PubMed1989

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↗
PubMed1987

Trypanosoma lewisi: restriction of alternative complement pathway C3/C5 convertase activity.

The rat parasite Trypanosoma lewisi was incubated in vitro with rat or human serum, washed, and extracted in detergent. Extracts were fractionated by electrophoresis in denaturing gels, transferred to nitrocellulose, allowed to renature, then immunoblotted with polyclonal antibodies to rat complement component C3 and human complement components C3, C5, and factor B. Molecules that reacted with these antibodies were detected in the extracts. Fragments of rat C3 were detected in extracts of parasites that had not been exposed to serum in vitro. Additional complement deposition occurred during in vitro incubations; human complement components deposited in vitro could be distinguished from rat components deposited in vivo. Complement deposition in vitro required magnesium ions and did not occur when heat inactivated serum was used. Components reacting with antibodies to human C3 included a group of bands with molecular weights higher than C3 alpha or beta chains. Blotting with affinity purified, chain specific antibodies demonstrated that a 68 kDa component on parasites is C3 beta and that a 44 kDa molecule is derived from C3 alpha. A 73 kDa component that was difficult to resolve from C3 beta is probably also a C3 alpha fragment. This suggests that an inactive iC3b-like molecule is present on parasites. Kinetic studies showed that cleavage of C3 alpha is rapid and that the amount of C3 alpha fragments and C3 beta on intact parasites reached a steady state after 15 min. When parasites were trypsinized prior to incubation in C5 or C6 deficient serum, the rate and extent of C3 and C5 deposition increased. Unprocessed C3 alpha' and C5 alpha' chains were detected. Trypsinized parasites were lysed by the alternative complement pathway in normal serum. Intact parasites could be lysed by complement in the presence of antibody. The data support our previous suggestion that trypsin sensitive surface proteins on intact T. lewisi limit alternative pathway activity by restricting C3/C5 convertase activity.

Animals↗
PubMed1988

Rapidly progressive glomerulonephritis: classification, pathogenetic mechanisms, and therapy.

Immunopathologic studies over the past two decades have demonstrated that rapidly progressive glomerulonephritis (RPGN) can result from glomerular deposition of anti-GBM antibody, immune complexes, or from some as yet undefined mechanism that does not involve glomerular antibody deposition. The latter process may be cell mediated and resembles a small vessel vasculitis. Most cases of idiopathic RPGN are not accompanied by pathogenic glomerular immunoglobulin deposition. Recent experimental studies of immune mechanisms of glomerular injury have identified several new processes that can induce damage to the capillary wall sufficient to result in crescentic glomerulonephritis (GN). These include direct effects of anti-GBM antibody alone and of the complement C5b-9 (membrane attack) complex, nephritogenic effects of inflammatory effector cells that involve reactive oxygen species and glomerular halogenation, and injury mediated by sensitized lymphocytes independently of antibody deposition. Macrophages have been shown to participate in both intracapillary and extracapillary fibrin deposition and crescent formation as well as to mediate capillary wall damage. The role of resident glomerular cells and cell-cell interactions in glomerulonephritis is still under active investigation. Despite these several advances in understanding immune injury to the glomerulus, therapy for RPGN remains largely empiric. Although the prognosis in RPGN has clearly improved over time, no form of disease-specific therapy has been clearly shown yet to be beneficial in a controlled study. Interpretation of the existing literature on therapy is complicated by the availability of only historical rather than concurrent controls, lack of attention to several variables known to affect disease outcome, and uncertainty regarding bias in favor of reporting positive results. Available data suggests that optimal outcomes may be achieved in anti-GBM nephritis by treatment with steroids, immunosuppression and plasma exchange, particularly when therapy is directed at patients with mild but rapidly progressive disease before oliguria or severe azotemia develop. Pulse steroids are probably the most cost-effective therapy for the idiopathic form of RPGN, but treatment with cytotoxic agents should be considered if clinical or histologic evidence of vasculitis is present.

Glomerulonephritis↗
PubMed1989

Activation-dependent epitopes in the terminal complement pathway.

Assembly of the terminal complement complex (TCC) during complement activation is associated with considerable antigenic changes in the individual C5, C6, C7, C8 and C9 components. Numerous neoepitopes specific for the TCC are exposed. Similarly, native-restricted epitopes specific for the nonactivated components are concealed in the TCC. The present paper reviews these antigenic changes with special reference to available monoclonal antibodies and their utilization in assays to detect and quantify the TCC. Application of such assays in clinical medicine is discussed. Finally, a recommended terminology for the terminal complement pathway is put forward.

Antibodies, Monoclonal↗
PubMedDate not supplied

[The humoral and cellular factors of glomerular damage].

The present review summarizes current concepts on glomerular non-inflammatory and inflammatory immune injury. Non-inflammatory glomerular injury can be ascribed to mechanisms involving antibody alone, terminal component of complement (C5b-9) or cytokines having as primary target the glomerular epithelial cells. These mechanisms induce changes in cell cytoskeleton and in surface distribution of adhesion molecules leading to functional alterations which may account for loss of glomerular permselectivity. Inflammatory glomerular injury is mediated by neutrophils, macrophages, platelets and lymphocytes as well as by resident glomerular cells. Cell derived mediators include proteases, reactive oxygen species, cationic proteins, cytokines and growth factors that may variably affect recruitment of inflammatory cells, proliferation of resident glomerular cells and production of extracellular matrix. These alterations may determine proteinuria, impairment of renal function and eventually progression of glomerular injury to sclerosis. The inflammatory type of glomerular injury is mainly initiated by subendothelial-mesangial immune deposits and/or activation of cell mediated immunity. The role of factors such as the nature, intensity and persistence of initiating mechanisms, the types of inflammatory cells involved and the response of intrinsic glomerular cells, in expression and progression of glomerular injury are discussed.

Antibody Formation↗
PubMed1992

[Detection of ischemic myocardial damage].

Cases of cardiac death (myocardial infarction, coronary artery thrombosis, coronary artery sclerosis) were investigated with histochemical and immunohistochemical methods. Histochemical staining methods (HE, LFB, CAB) were used, with immunohistochemical methods were detected the proteins Desmin, Myoglobin, Fibrinogen and Complement C5b-9. Macroscopically visible myocardial infarctions were detected with both methods clearly. The damage pattern was associated to the type of destruction. At myocardial infarction the changes were focal at the other groups more disseminated.

Coronary Artery Disease↗
PubMed1985

Monoclonal antibodies against neoantigens of the terminal C5b-9 complex of human complement.

Assembly of the terminal C5b-C9 complement components into the cytolytic C5b-9 complex is accompanied by exposure of characteristic neoantigens on the macromolecule. We report the production and characterization of mouse monoclonal antibodies to C9-dependent neoantigens of human C5b-9. Binding-inhibition assays with EDTA-human plasma and micro-ELISA assays with purified C9 showed that the antibodies did not react with native complement components and thus confirmed the specificity of the antibodies for the neoantigens. The monoclonal antibodies did, however, cross-react with cytolytically inactive, fluid-phase C5b-9 complexes. Thus, expression of the neoantigenic determinants was not dependent on the formation of high molecular weight C9 polymers with the complex, since these are absent in fluid-phase C5b-9. Radioiodinated antibodies could be utilized in immunoradiometric assays for the detection and quantitation of C5b-9 on cell membranes. Cross-reactivities of the antibodies with C9-dependent neoantigens of several other animal species were examined and antibody clones cross-reacting with rabbit (clones 3B1, 3D8, and 2F3), sheep (clones 3D8 and 2F3) and guinea-pig (clone 3D8) neoantigens were identified. Three of four tested clones (3D8, 2F3, 1A12) precipitated C5b-9 complexes in double-diffusion assays, probably due to their interaction with multiple and repeating C9-epitopes on the terminal complexes. The monoclonal antibodies will be of value for definitive identification and quantitation of C5b-9 on cell membranes and in tissues, and for establishing immunoassays for detection and quantitation of terminal fluid-phase C5b-9 complexes in plasma.

Animals↗
PubMed1987

Generation of complement anaphylatoxins and C5b-9 by crystalline cholesterol oxidation derivatives depends on hydroxyl group number and position.

Cholesterol crystals activate the human alternative complement pathway. Loss of Factor B hemolytic activity in C2-deficient serum was comparable to that in normal human serum after incubation with cholesterol crystals. Consumption of Factor B hemolytic activity in normal serum incubated with cholesterol occurred in a time- and dose-dependent manner. The reduced capacity of crystals-absorbed serum to activate C2, but not Factor B, on fresh crystals, indicated that cholesterol mediates antibody-dependent classical pathway activation in addition to alternative pathway activation in whole serum. Cholestane triol, an oxidation derivative of cholesterol which bears three hydroxyl groups, cleaved 5-fold more C3 than cholesterol in normal human serum. Three cholesterol derivatives, each bearing two hydroxyl groups, were intermediate activators between cholesterol and cholestane triol. The compounds differed, however, in their complement-activating ability, indicating that hydroxyl position as well as number exerts an influence on complement activation. Measurements of C3adesArg and C5adesArg antigens in cholesterol crystal treated serum revealed that approx. 10% of total serum C3 was cleaved and that, on a molar basis, only 3% C5 cleavage occurred relative to C3 cleavage. For 1 mole of C5a generated, 0.1 moles of fluid-phase C5b-9 was detected. Although the extent of C3 cleavage varied with each cholesterol derivative depending on the position and number of hydroxyl groups, the relative coupling efficiency of C3 and C5 cleavage and C5a and C5b-9 generation was similar for all compounds. The ability of cholesterol and its oxidation products to generate anaphylatoxins and C5b-9 complexes may be of importance in mediating inflammatory processes involved in atherogenesis.

Anaphylatoxins↗
PubMed1987

Nucleated cell killing by complement: effects of C5b-9 channel size and extracellular Ca2+ on the lytic process.

For C5b-9 channels to mediate cytolysis of a nucleated cell, a sufficient number of channels must be formed in the plasma membrane to override the compensatory mechanisms that nucleated cells might employ to survive. It is well known that nucleated cells are relatively resistant to lysis by complement in comparison to erythrocytes, and it is now evident that this resistance is due, in part, to the ability of nucleated cells to rapidly eliminate C5b-9 from the cell surface. The ability of nucleated cells to eliminate complement complexes is related to physiochemical properties of the complex, such as channel diameter, which in turn affect Ca2+ fluxes that stimulate metabolic processes involved in the elimination process. Paradoxically, these same channel properties that stimulate the defense response may also be responsible for the lethal effects of complement. To further study the role of channel size on cytolysis of nucleated cells by C5b-9, we examined the lytic efficiency of larger C5b-9 channels containing several C9 molecules in comparison with smaller C5b-9 channels containing fewer C9. We have obtained data to indicate that although the larger channels were more cytolytically potent, the channel size had little influence on the rate of cell death. In contrast, the rate of lysis of erythrocytes was substantially slower when smaller C5b-9 channels were present. In evaluating the effect of the extracellular Ca2+ concentration, [Ca2+]o, on nucleated cell lysis in the presence of a lytic number of C5b-9 complexes, it was observed that when the [Ca2+]o was increased the rate of cell death also increased. These findings suggest that lysis of nucleated cells by C5b-9, unlike erythrocytes, may not be entirely due to colloid osmotic deregulation.

Animals↗
PubMed1989

The pathobiology of the terminal complement complexes.

C5b and the other late-acting complement components can assemble the two terminal complexes (TCC) C5b-9 and SC5b-9. In addition to the lytic effects of C5b-9 it has been demonstrated that sublytic amounts of C5b-8 or C5b-9 can stimulate several important cellular activities. These effects may be important to explain the role of C5b-9 in the production and progression of several pathological conditions that has been demonstrated in experimental models of disease. With the help of antibodies that specifically recognize C9 neoantigens, deposits of TCC have been identified in human tissues, not only in immunological diseases but also in certain nonimmunological diseases. In the latter it has been shown that often there is no concordance between deposits of TCC and those of immunoglobulins and C3. Methods for measuring SC5b-9 in biological fluids have also been developed. Normal plasma was found to have low levels of SC5b-9. Increased plasma levels of SC5b-9 have been observed during the active phase of SLE nephritis, in certain infections and during cardiopulmonary bypass. Increased levels were also found in the cerebrospinal fluid of patients with inflammatory diseases of the central nervous system and in the synovial fluid of patients with rheumatoid arthritis. Autoantibodies to C9 neoantigens in plasma of certain patients with autoimmune, infectious or neoplastic diseases have recently been recognized. Additional work is needed to better delineate the potential usefulness of these findings for diagnosis and evaluation of disease activity.

Adult↗
PubMed1978

Complement lysis: evidence for an amphiphilic nature of the terminal membrane C5b-9 complex of human complement.

The terminal, membrane-derived C5b-9 complex of human complement (C) is an apparently hollow, cylindrical macromolecule vertically oriented on the target membrane. In the present study, an antiserum to the complex has been used to probe its immunobiochemical properties. "Neoantigenic" determinants characteristic of the complex have been detected, which are absent on native C5-C9 molecules. Evidence that the C5b-9 complex is an amphiphilic molecule that possesses apolar, detergent-binding surfaces has been obtained by using charge-shift crossed immunoelectrophoresis, and by direct demonstration of Triton X-100 binding to the complex in quantitative immunoelectrophoresis. By the same criteria, serum C5, C6, and C9 are hydrophilic molecules. The results indicate that assembly of C5-C9 into the terminal membrane C5b-9 complex is accompanied by conformational changes in the individual C components that lead to the exposure of apolar molecular regions in the complex. It is proposed that this constitutes the basis for the lipid-binding properties of the macromolecule, which enable it to become inserted into biologic and artificial lipid membranes with apparent generation of a transmembrane pore.

Animals↗
PubMed1986

Immunohistochemical study of the C5b-9 complex of complement in human kidneys.

The presence and localization of the C5b-9 neoantigens of the terminal complement sequence, of antigens expressed by cleavage fragments of C3, and of Factor H antigens have been studied by immunohistochemical techniques in morphologically normal adult human kidneys and in biopsy specimens from patients with a wide range of renal diseases with and without immune deposits. In morphologically normal kidneys, C5b-9 neoantigens were observed within all connective matrices (arteriolar media, glomerular basement membrane (GBM), mesangial matrix and tubular basement membrane). The C3d and C3g antigens of the C3dg, and C3bi cleavage fragments of C3 and Factor H antigens were found in similar locations. None of the matrices stained for immunoglobulins. Immunoelectron microscopy demonstrated that C3d, C3g, H antigens and the C5b-9 neoantigens were localized on membranous and vesicular structures embedded in the connective matrices. These structures represent cell membranes shed from adjacent cells as evidenced by their ultrastructural appearance and by the fact that those which were in close vicinity to pedicles within the GBM expressed the C3b receptor antigen, a specific marker for podocyte membranes. Formation of C5b-9 complexes in the shielded environment of connective matrices may explain their persistence over long periods of time in the absence of apparent immunopathological consequences. Biopsies from pathological kidneys were classified into three groups based on the pattern of glomerular staining with anti-C5b-9 antibodies. In the first group, a sparse mesangial labeling was seen, similar to that observed in normal kidneys. In the second group, abundant clusters of C5b-9 were seen in the same location as immune deposits. Activation of the complement system to completion could be documented in the absence of detectable C3 (C3c) antigen in glomeruli. Immunoelectron microscopy demonstrated that C5b-9 neoantigens were present on cell remnants in connective matrices in all specimens that were studied. Labeled cell remnants were present in large amounts in sclerotic matrices. C5b-9 neoantigens were constantly found on old and large immune deposits, and absent or occasionally present on recent and small immune deposits. In membranous nephropathy stage I, proteinuria appeared to be independent of the presence or absence of detectable C5b-9 neoantigens on immune deposits. Thus, the presence of C5b-9 neoantigens in pathological renal tissue does not have an univocal significance, and requires analysis of the localization of the antigens and appropriate controls in order to assess the potential role of C5b-9 in tissue damage.

Adult↗
PubMed1989

Non-enzymic activation of the fifth component of human complement, by oxygen radicals. Some properties of the activation product, C5b-like C5.

Purified human C5 was converted non-enzymically to an activated form as defined by its ability to participate in reactive lysis. This conversion occurred following exposure to systems that generate oxygen radicals, namely addition of H2O2 in the presence of ascorbic acid and iron or the addition of xanthine oxidase, acetaldehyde and iron. The conversion of C5 to a functionally active species was iron-dependent and inhibited by hydroxyl radical scavengers such as DMSO. The findings suggest that OH. is the active oxygen species that converts C5. The conversion product of C5, termed C5(H2O2), is C5b-like due to its ability to bind C6 and cause reactive lysis. C5(H2O2) is much more stable than C5b obtained by complement convertases. Although C5(H2O2) has lost the binding site of native C5 for C3b it can be cleaved by complement-derived convertases; the cleavage is, however, less efficient than in the case of native C5. The resulting cleavage product, which is C5a-like, is chemotactic although C5(H2O2) is not chemotactic. C5(H2O2) serves as a better substrate for plasma kallikrein than native C5, resulting in the generation of a C5a-like chemotactic product. These data indicate that oxygen radicals can bring about a conformational change in C5, causing it to behave as a functionally activated molecule of the complement system. This may have implications for the role of complement and its activation in the inflammatory response.

Complement Activation↗
PubMed1989

Terminal complement complex and endothelial cells.

The effects of the terminal complement sequence on two endothelial cell functions (prostacyclin (PGI2) generation and permeability of an endothelial cell monolayer) were examined in antibody-sensitized pulmonary artery endothelial cells. Whereas C5b-7 complement complexes induced on PGI2 formation and no enhanced endothelial permeability, addition of purified complement component C8 resulted in a time- and dose-dependent burst of PGI2 release and in a substantially increased permeability of an endothelial cell monolayer in the absence of overt cell damage. Formation of the complete terminal complement complex C5b-9 enhanced PGI2 release but was accompanied by cytolysis. Extracellular Ca2+ was required for C5b-8 dependent PGI2 formation. Three different blockers of physiological calcium channels failed to suppress the observed stimulatory effect. One minute after addition of C8 to endothelial cells carrying C5b-7 complexes, a six to sevenfold enhanced passive influx of 45Ca2+ into the cells was noted. An enhanced passive influx was also observed for 51CrO4(2-), 3H-aminobutyric acid, and 3H-sucrose, but not for 3H-inulin and 3H-dextran. These data together suggest that complement C5b-8 complexes may serve as Ca2+-bypass gates in endothelial cells, the ensuing influx of Ca2+ leading to subsequent activation of the arachidonic acid pathway and the actin-myosin system.

Arachidonic Acid↗
PubMed1991

Complement activation in relation to development of preeclampsia.

Six hundred eighty-five primigravidas followed as a series had complement activation evaluated by the formation of anaphylatoxins (C3a and C5a) and terminal C5b-9 complement complexes in venous blood. Samples for complement determinations were obtained four times during pregnancy, in pregnancy weeks 12-16, 20-24, 28-32, and 34-36. Seven of the women developed preeclampsia and one of them the syndrome of hemolysis, elevated liver enzymes, and low platelet count (HELLP syndrome). Eleven others with uncomplicated pregnancies were selected as a control group. Plasma samples were taken from these 18 women at delivery and 1 and 7 days after delivery. At delivery, plasma C5a levels were significantly greater in the preeclamptics than in controls, and four of the seven preeclamptics had elevated plasma C3a values compared with controls. One week after delivery, these plasma anaphylatoxins had returned to normal. Elevations of the anaphylatoxins could not be detected before the women developed clinical signs of preeclampsia. No alterations in terminal C5b-9 complement complexes could be observed in the women with preeclampsia. However, the women who developed HELLP syndrome had elevated plasma concentrations of C3a, C5a, and terminal C5b-9 complement complex at delivery. These values returned to the normal range 1 week after delivery. We conclude that complement activation in the systemic circulation does not occur early in pregnancy and that plasma concentrations of C3a, C5a, or terminal C5b-9 complement complex cannot be used as predictors of preeclampsia.

Adult↗
PubMed1975

On the mechanism of cytolysis by complement: evidence on insertion of C5b and C7 subunits of the C5b,6,7 complex into phospholipid bilayers of erythrocyte membranes.

The doughnut hypothesis of cytolysis by complement [Mayer, M. M. (1972) Proc. Nat. Acad. Sci. USA 69, 2954-2958] describes an annular structure made up of C5b-9 (complement factors C5b, C6, C7, C8, and C9) which becomes inserted in the lipid bilayer of the cell membrane, thus creating a hole. We now present initial explorations of this hypothesis. EAC1-6 and EAC1-7 (sheep erythrocytes carrying rabbit antibody and complement factors C1 through C6 or C1 through C7, respectively), prepared with either 125I-C3 or 125I-C5 were incubated with trypsin and the release of bound 125I was measured. In the case of 125I-C3, all of the radioactivity was released by trypsin from both intermediates. With 125I-C5, trypsin released all of the 125I from EAC1-6, but only 40-55% from EAC1-7. Possible reasons for resistance of the C5b subunit in EAC1-7 to tryptic digestion are discussed; in terms of the doughnut hypothesis it would be due to shielding by lipid molecules as a consequence of insertion into the lipid bilayer. In accord with this interpretation we have also found that C5b in EAC1-7, but not in EAC1-6, resists elution by 0.3 M NaC1. Similarly, we have found that 125I-C7 in EAC1-7 resists stripping by trypsin. Hence, we now propose the hypothesis that hydrophobic polypeptide chains from the C5b and the C7 subunits of C5b,6,7 complex become inserted in the phospholipid bilayer and that subsequent reactions with C8 and C9 open a channel across the membrane.

Animals↗