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PubMed1987

Heymann antibodies induce complement-dependent injury of rat glomerular visceral epithelial cells.

The aim of this study was to investigate the in vitro role of the complement membrane attack complex (MAC) in the injury induced by nephritogenic anti-brush border vesicle (Fx1A) antibodies on rat glomerular visceral epithelial cells (GEC). Both sheep and rabbit anti-rat brush border vesicle IgG-induced complement-dependent lysis of cultured GEC. Fab fragments of sheep anti-rat brush border vesicles and polyclonal or monoclonal gp330 IgG were devoid of lytic activity. Shedding of cell-surface antigens induced by sheep or rabbit anti-rat brush border vesicle IgG protected GEC from subsequent exposure to lytic antibodies and complement, an effect that was not obtained with Fab fragments. When GEC were incubated with sheep or rabbit anti-rat brush border vesicle IgG in capping conditions, the C3 component was co-redistributed with Heymann immune complexes; in contrast, the MAC remained diffusely bound to the cell surface, indicating that it was not associated with the antigen-antibody complexes. The MAC was demonstrated on the surface of GEC by immunofluorescence staining with anti-MAC neoantigen and by electron microscopy of negatively stained membranes showing focal clusters of 110 A MAC lesions. When GEC were treated with sheep IgG or rabbit IgG plus C6-deficient sera, the cells were not lysed and MAC was not demonstrable on the surface; however, lytic activity was restored when C6-deficient sera were reconstituted with purified C6. The results are consistent with the interpretation that injury induced by Heymann antibodies on GEC is MAC-dependent.

Animals↗
PubMed2000

Evaluation of the membrane attack complex of complement for the detection of a recent myocardial infarction in man.

The diagnosis of an acute myocardial infarction (MI) can be cumbersome for pathologists. Even with a positive nitroblue tetrazolium (NBT) reaction, haematoxylin and eosin (H&E) evaluation of the myocardial tissue can remain inconclusive. Early signs presumed diagnostic for myocardial infarction, such as hypereosinophilia, waviness, and contraction band necrosis, have to be considered non-specific and are probably reversible signs of ischaemia. Several studies implicate the complement system, and especially complement factor C9, as part of the membrane attack factor (MAC), in cardiomyocyte damage during MI. In a post-mortem study on well-documented cardiological autopsies, we evaluated the use of complement factor C9 immunostaining as a marker for the detection of very recent MI. Forty-three tissue samples from 40 patients were obtained from the left ventricular free wall only, a region that can be specifically attributed to one corresponding coronary artery. As some patients presented with MIs of various stages in that perfusion area, in total 57 observations were possible. C9 immunostaining specifically detected irreversibly damaged (=infarcted) cardiomyocytes, as is implied by the lytic activity of C9/MAC binding to cell membranes. Most interesting was the group of clinically suspected, NBT-positive MIs resulting from very recent myocardial ischaemia. In this population, where H&E evaluation by (cardio-) experienced pathologists was not conclusive, C9 immunostaining clearly pointed towards myocardial infarction in 47% of the cases. In conclusion, C9 immunostaining, routinely practicable in the pathology laboratory, has an additional value in discriminating between reversible ischaemia and infarcted cardiomyocytes in very early MIs.

Adult↗
PubMed1979

Quantitation of the membrane attack complex of complement in an air-driven ultracentrifuge.

A sensitive assay of complement (C) activation via either the classical or alternative pathway was developed by evaluating assembly of the terminal complexes (C5b-9)2 or SC5b-9. Activation of serum containing [125I]C7 resulted in the formation of a stable, radiolabeled complex which was separable from its precursors by sedimentation in an air-driven ultracentrifuge. The radioactivity in the sediment was directly proportional to the amount of complex formed and assembly of the complex could be detected after C activation by aggregated IgG in concentrations as low as 10 micrograms/ml. Mild detergents such as Triton X-100 could be included in the reaction mixture, because they affected neither the assembly nor the integrity of the complexes. The assay, which detects both assembly of the membrane attack complex (MAC or (C5b-9)2) on target membranes and formation of SC5b-9 in fluid phase, measures the potential of certain substances to trigger the cytolytic phase of C regardless of whether the classical or alternative pathway was activated. However, by using serum depleted of either factor B or C1q, activation of either pathway can be assessed individually.

Cell Membrane↗
PubMed1987

Complement membrane attack (MAC) in idiopathic IgA-glomerulonephritis.

Antigens of the membrane attack complex of complement (MAC), such as C5, C6, C9 and MAC-related neoantigen(s), were demonstrated in the mesangium of 23 cases with IgA-glomerulonephritis (IgA-GN) and two cases with Henoch-Schönlein purpura nephritis (HSP). High specificity of the polyclonal antibodies was verified by dot-blot analysis. Control specimens lacking immunoglobulin deposits were negative for MAC-related antigens. Markers of classical pathway activation (Clq and C4) were observed only in two of 24 and one of 23 cases of IgA-GN and HSP, respectively. Glomerular distribution patterns (mesangial vs. mesangio-peripheral) of immunoglobulin or complement deposits were correlated for IgA and C3b/iC3b (P less than 0.002), for IgA and properdin (P less than 0.002) and for IgA and MAC neoantigens (P less than 0.01). Double immunostaining experiments revealed co-localization of IgA and MAC neoantigens at identical mesangial and capillary sites. Glomerular distribution of the less pronounced IgG or IgM deposits did not correlate with that of any complement-derived antigen. The pattern of MAC-related antigens was found to be uniformly either mesangial or mesangio-peripheral. Staining for MAC-related antigens was less intense in IgA-GN cases with minimal glomerular lesions than in cases with more advanced non-sclerosing lesions. IgA, C3d, and MAC localized in corresponding glomerular sites. This is consistent with complete local activation of complement by glomerular IgA deposits via the alternative pathway. The possibility exists that MAC plays a pathogenetic role, such as by irritation of bystander cells, in IGA-GN and HSP.

Adolescent↗
PubMed1999

Targeting of functional antibody-CD59 fusion proteins to a cell surface.

Complement is involved in the pathogenesis of many diseases, and there is great interest in developing inhibitors of complement for therapeutic application. CD59 is a natural membrane-bound inhibitor of the cytolytic complement membrane attack complex (MAC). In this study, the preparation and characterization of antibody-CD59 (IgG-CD59) chimeric fusion proteins are described. Constructs were composed of soluble CD59 fused to an antibody-combining site at the end of CH1, after the hinge (H), and after CH3 Ig regions. The antigen specificity of each construct was for the hapten 5-dimethylamino-naphthalene-1-sulfonyl (dansyl). Correct folding of each IgG-CD59 fusion partner was indicated by recognition with anti-CD59 antibodies specific for conformational determinants and by IgG-CD59 binding to dansyl. The IgG-CD59 fusion proteins all bound specifically to dansyl-labeled Chinese hamster ovary cells and provided targeted cells, but not untargeted cells, with effective protection from complement-mediated lysis. Data indicate that CD59 must be positioned in close proximity to the site of MAC formation for effective function, and that modes of membrane attachment other than glycophosphatidylinositol linkage can affect CD59 functional activity.

Animals↗
PubMed1999

Hindlimb ischemia-reperfusion increases complement deposition and glycolysis.

BACKGROUND: Hindlimb ischemia-reperfusion (HIR) impairs cellular energy metabolism and causes local muscle injury possibly through free radical or complement-mediated mechanisms. MATERIALS AND METHODS: To determine the relationship among myocellular energetics, histopathological injury, and mediator activity, male Wistar rats underwent 4 h of Sham (n = 8), Unilateral (n = 8), or Bilateral (n = 8) hindlimb ischemia followed by 4 h of reperfusion. All rats underwent 31P magnetic resonance spectroscopy of their right gastrocnemius muscle to determine various high-energy phosphate ratios including ATP to Pi (ATP/Pi, a measure of energy status) and phosphocreatine to Pi (PCr/Pi, a measure of thermodynamic capacity). Gastrocnemius muscles were then harvested to determine muscle damage and complement membrane attack complex (MAC) deposition by immunohistochemical staining [grade 0 (none) to 3 (very severe)] and to measure glutathione (GSH), DNA, and enzyme activities: beta-hydroxyacyl-CoA dehydrogenase, phosphofructokinase, and citrate synthetase. RESULTS: HIR was associated with significant declines in ATP/Pi and PCr/Pi (P < 0.001). Progressively more severe HIR (Sham, Unilateral, Bilateral) was associated with greater MAC deposition (0. 0 +/- 0.0, 1.0 +/- 0.3, 1.5 +/- 0.4, P = 0.06, mean +/- SEM) and histological damage (0.0 +/- 0.0, 0.9 +/- 0.3, 1.3 +/- 0.4, P < 0. 05). GSH levels, beta-hydroxyacyl-CoA dehydrogenase, and citrate synthetase activities were not affected by HIR, but phosphofructokinase activity increased (24.09 +/- 2.42, 35.16 +/- 5. 26, 59.29 +/- 9.82 mmol/mg of DNA/min, P < 0.05). Although GSH levels were not significantly altered, complement deposition was closely associated with skeletal muscle injury and compensatory changes in glycolysis. Alterations in myocellular bioenergetics after HIR closely paralleled complement deposition rather than GSH depletion. CONCLUSIONS: Therapeutic strategies aimed at controlling complement activity and assessment techniques based on bioenergetics may allow more precise determinations of the effects of HIR injury.

Adenosine Triphosphate↗
PubMed1985

The membrane attack complex of complement: C5b-8 complex as accelerator of C9 polymerization.

Polymerization of C9 occurs spontaneously or can be induced by the tetramolecular complex C5b-8. Spontaneous C9 (0.15 mg/ml) polymerization required more than 3 days at 37 degrees C. In the presence of C5b-8, C9 polymerization was complete within 10 min. The molar C9:C5b-8 ratio determined the extent of tubular poly C9 formation by C5b-8-bearing phospholipid vesicles. When this ratio was 9:1 or 12:1, 72% of complex-bound C9 was present as SDS resistant tubular poly C9 (Mr = 1.1 X 10(6]. At lower C9:C5b-8 ratios, poly C9 was bound primarily in nontubular form. Tubular poly C9, as part of C5b-9, could also be generated on rabbit erythrocytes by using whole human serum as a complement source. At limiting serum concentration (molar C9 to C8 ratio approximately 2), no SDS-resistant tubular poly C9 was detected. At high serum concentration or when using serum that was supplemented with C9, up to 40% of the C9 was SDS-resistant tubular poly C9, and the rest was poly C9, which was incompletely polymerized. It is suggested that the C5b-8 complex acts as an accelerator of C9 polymerization, and that its relative concentration to C9 determines the ultrastructure of the C5b-9 complex.

Animals↗
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↗
PubMed1986

C5b-9 assembly: average binding of one C9 molecule to C5b-8 without poly-C9 formation generates a stable transmembrane pore.

Membrane attack by serum complement normally results in the formation of C5b-9 complexes that are heterogeneous with respect to their C9 content. We here report that an apparently homogeneous population of C5b-9 complexes can be generated through treatment of C5b-7-laden sheep erythrocytes with C8 and C9 for 60 min at 0 degree C. Experiments performed by using radioiodinated C8 and C9 components have indicated that binding of C8 to these target cells is essentially temperature independent. In contrast, when a surplus of C9 molecules is offered to C5b-8 cells, an approximately fourfold to 4.5-fold higher number of C9 molecules become cell bound at 37 degrees C as opposed to 0 degree C. C5b-9 complexes isolated from target membranes treated with C9 at 0 degree C contain no polymerized C9 and do not exhibit the ring structure characteristic of the classical complement lesion. Nevertheless, these complexes generate stable transmembrane channels and cause hemolysis at 37 degrees C. The pores have been sized to 1 to 3 nm effective diameter by osmotic protection experiments. SDS-PAGE of the isolated complexes indicates an average stoichiometry of only one molecule C9 bound per C5b-8 complex. The results show that oligomerization of C9 with formation of ring lesions is not a basic requirement for the generation of stable transmembrane complement pores in sheep erythrocytes. They indirectly support the contention that terminal complement components other than C9 contribute to the intramembrane domains of C5b-9 pores.

Animals↗
PubMed2000

Hepatitis C virus infection and myositis: a polymerase chain reaction study.

Muscle biopsy tissue from a patient with chronic hepatitis, who was hepatitis C virus (HCV) positive and showed slight weakness of the right arm and leg associated with increased serum creatine kinase levels, was studied using immunocytochemical and polymerase chain reaction (PCR) techniques. Muscle biopsy showed changes compatible with an inflammatory myopathy. Immunohistochemical studies included the use of monoclonal antibodies against human T lymphocytes, macrophages, immunoglobulins, major histocompatibility complex class I molecules (MHC-I), and the neoantigens of the terminal C5b-9 complement membrane attack complex (MAC). In addition to confirming the potential importance of cytotoxic T cells and MHC-I antigen expression in inducing muscle pathology, we demonstrated MAC deposition and the presence of HCV-RNA in the muscle of our patient, suggesting that direct involvement of the virus leading to complement activation might be important in inducing muscle damage.

Adult↗
PubMedDate not supplied

Novel mechanisms of immune evasion by Schistosoma mansoni.

The interaction of Schistosoma mansoni with its host's immune system is largely affected by multiple specific and non-specific evasion mechanisms employed by the parasite to reduce the host's immune reactivity. Only little is known about these mechanisms on the molecular level. The four molecules described below are intrinsic parasitic proteins recently identified and studied in our laboratory. 1. m28--A 28kDa membrane serine protease. m28 cleaves iC3b and can thus restrict attack by effector cells utilizing complement receptors (especially CR3). Treatment with protease inhibitors potentiates killing of schistosomula by complement plus neutrophils. 2. Smpi56--A 56kDa serine protease inhibitor. Smpi56 binds covalently to m28 and to neutrophil's elastase and blocks their proteolytic activity. 3. P70--A 70kDa C3b binding protein. The postulated activity of P70 includes binding to C3b and blocking of complement activation of the C3 step. 4. SCIP-1--A 94kDa schistosome complement inhibitor. SCIP-1 shows antigenic and functional similarities to the human 18kDa complement inhibitor CD59. Like CD59, SCIP-1 binds to C8 and C9 and blocks formation of the complement membrane attack complex. Antibodies directed to human CD59 bind to schistosomula and potentiate their killing by complement. The structure and function of these four proteins as well as their capacity to induce protection from infection with S. mansoni are under investigation.

Animals↗
PubMed1992

Sub-minimal inhibitory concentrations of cefmetazole enhance serum bactericidal activity in vitro by amplifying poly-C9 deposition.

Serum-resistant organisms grown in sub-minimal inhibitory concentrations (subMICs) of antibiotics in vitro may be rendered sensitive to complement-mediated, serum bactericidal activity. We measured 125I-C3 and 125I-C9 deposition on genetically serum resistant Salmonella montevideo SH5770 (SH5770) that was rendered serum sensitive by growth in sub-MICs of cefmetazole (CMZ), a parenteral, second generation, cephamycin-group antibiotic. Three times as much C3 and over six times as much C9 bound to SH5770 grown in one-fourth the MIC of CMZ compared to broth-grown bacteria. SDS-PAGE analysis and autoradiography showed that neither the ratio of C3b:iC3b (approximately 1:2.5) nor the nature of the C3-bacterial bond was changed by growing the organisms in CMZ. Large amounts of complement membrane attack complexes containing poly-C9 were seen only on CMZ-grown SH5770 by SDS-PAGE and autoradiography. Poly-C9 was also detected only on CMZ-grown bacteria by indirect immunofluorescence and ELISA using a murine monoclonal antibody directed against a neoantigen of poly-C9. Bacterial hydrophobicity increased after growth in CMZ, and transmission electron micrographs of CMZ-grown SH5770 showed cell wall disruption and blebbing. These results indicate that growth in subMICs of CMZ increases bacterial hydrophobic domains available for interacting with the membrane attack complex, C5b-9, allowing formation and stable insertion of bactericidal complexes containing poly-C9.

Blood Bactericidal Activity↗
PubMed1993

Regulation of CD59 expression on the human endothelial cell line EA.hy 926.

CD59 (protectin) is an 18-20-kDa inhibitor of the membrane attack complex of complement. It protects homologous cells from complement-mediated damage and has been shown to be present on the endothelial cell membranes both in vitro and in vivo. In this study we observed that the surface expression of CD59 on the cultured EA.hy 926 endothelial cell line can be up-regulated to an approximately threefold higher level after a 72-h stimulation by the protein kinase C inducers phorbol-12-myristate-13 acetate (PMA; 10 nM) and calcium ionophore, A23187 (100 nM). Similarly, an increase in the level of CD59 expression was seen by the protein kinase A inducer dibutyryl-cyclic adenosine monophosphate. In Northern blot analysis increases were observed in CD59 mRNA expression, particularly in the level of the longest 1.9-kb, 2.1-kb and 5.8-kb transcripts. A functional significance for the increased CD59 expression was implied by an observed increased resistance of the PMA-stimulated EA.hy 926 cells to complement-mediated cell lysis.

Antigens, CD↗
PubMed1996

Terminal components of carp complement constituting a membrane attack complex.

The membrane attack complex (MAC) of carp complement was extracted with deoxycholate from rabbit erythrocytes lysed by carp serum and purified by a two-step chromatographic procedure. On two-dimensional SDS-PAGE of carp MAC, eight bands were detected. The band of M(r) 91,000 was identified as carp C9 by western blotting using anti-carp C9, and two bands of M(r) 62,000 and one band of M(r) 22,000 were confirmed as those of carp C8 alpha, C8 beta and C8 gamma, respectively, by their N-terminal amino acid sequences. The bands of M(r) 102,000 and 73,000, which generated from a 180,000 band under reducing conditions, were those corresponding to human C5b alpha and C5b beta, respectively. The remaining bands of M(r) 115,000 and 106,000 were identified as those corresponding to human C6 and C7, as determined by their molecular size, single-chain structures and similarities in N-terminal amino acid sequences to their mammalian counterparts. Densitometric scan of the gels showed the molar ratio of C5b, C6, C7, C8 and C9 in carp MAC to be 1:1:1:1:4. Based on these results, it appears that, as with mammals, the cytolytic pathway of bony fish complement is composed of five terminal components from C5 to C9.

Amino Acid Sequence↗
PubMed1987

Activation of terminal components of complement in patients with Guillain-Barré syndrome and other demyelinating neuropathies.

In the present study, the role of antiperipheral nerve myelin antibody (anti-PNM Ab) in demyelination by generating the terminal attack complex (C5b-9) of complement was explored in patients with Guillain-Barré syndrome (GBS) and other demyelinating neuropathies. The presence in serum of SC5b-9, an inactive C5b-9 containing S protein, was assessed quantitatively by enzyme-linked immunosorbent assay using an antibody (Ab) to neoantigens expressed on C9 when complexed with C5b-8 or after tubular polymerization. SC5b-9 was detected in all 19 GBS, four patients with paraprotein-associated neuropathy and five of six patients with chronic recurrent polyneuritis. No SC5b-9 was detected in 10 normal controls. Kinetic studies from six GBS patients showed the highest values of SC5b-9 on the 3rd to 5th d of admission; in contrast, the anti-PNM Ab were highest on the day of admission. Anti-PNM Ab fell rapidly to very low levels by the 15th to 20th d. SC5b-9 declined with similar kinetics to undetectable levels by the 30th d. Levels of Ab and SC5b-9 did not quantitatively correlate with soluble immune complexes in these patients' serum. Membrane-bound C5b-9 was also detected by immunohistochemistry in the peripheral nerves from a GBS patient. These results, which show a relationship between levels of complement-fixing anti-PNM Ab and the tissue-damaging C5b-9 complex, suggest that peripheral nerve myelin may serve as the target for Ab-mediated complement attack.

Antigen-Antibody Complex↗
PubMed1994

Plasmid-mediated virulence genes in non-typhoid Salmonella serovars.

Specific non-typhoid Salmonella serovars carry large virulence plasmids that promote sustained extra-intestinal infections. These plasmids all share a highly conserved 8-kb region containing the spv operon, consisting of the regulatory spvR locus and the four structural spvABCD genes. The SpvR protein belongs to the LysR/MetR family of transcriptional activators, and induces spvABCD expression in the stationary phase in response to nutrient limitation. spv expression also depends on the chromosomal stationary phase sigma factor RpoS (KatF), and is markedly induced when salmonellae enter eukaryotic cells. Additional plasmid genes encode complement resistance including the rck locus which is homologous to ail from Yersinia. Rck blocks formation of the complement membrane attack complex on the bacterial surface. Several loci involved in plasmid replication and stable maintenance have also been identified.

Complement Membrane Attack Complex↗
PubMed1991

Membrane vesiculation protects erythrocytes from destruction by complement.

Nucleated cells can resist attack by C by exocytosis or endocytosis of the terminal C components C5b-9 (membrane attack complex) (MAC), but it is generally accepted that formation of a single MAC channel on E leads to lysis (one-hit theory). We find that human and guinea pig E, but not SRBC, can eliminate the MAC from the membrane in the form of microvesicles and escape destruction. When guinea pig or human E are incubated with C5b-9, vesiculation proceeds without a lag and is detected at nonlytic doses of C9. Continuous Ca2+ influx is required for vesiculation. The amount of released vesicles is in direct relation to Ca2+ concentration, and the increase in vesiculation is associated with a parallel decrease in lysis. SRBC, which do not vesiculate when Ca2+ loaded, are lysed by C5b-9 with the same efficiency in the presence or absence of Ca2+. Vesicles released from guinea pig RBC under C5b-9 attack are enriched in C9 by a factor of 10, compared with the unlysed cells, and by a factor of 3 to 4, compared with ghosts. We conclude that E are protected from lysis not only by CD59 and C8bp/HRF, which prevent MAC assembly, but also by selective elimination of the MAC.

Animals↗
PubMed1996

IgM natural antibody against an asialo-oligosaccharide, gangliotetraose (Gg4), sensitizes HIV-I infected cells for cytolysis by homologous complement.

Although cells are usually resistant to homologous complement, the IgM antibody against gangliotetraose (Gg4), an asialo-oligosaccharide of GM1, was found to cause cytolysis of HIV-1 infected cells by homologous complement. Due to its size, IgM might enable the initiation of the complement cascade away from membrane complement inhibitors such as decay accelerating factor and membrane cofactor protein. Furthermore, HRF20 (CD59), which restricts formation of membrane attack complexes (MAC) of complement on homologous cell membranes, was significantly decreased on HIV-infected cells and therefore formation of MAC on cell membranes would be facilitated. IgM antibodies reactive with HIV-infected cells could result in the elimination of infected cells via complement-mediated cytolysis in HIV-infected patients, since all tested sera from HIV-positive hemophilia patients who have survived for >12 years contained IgM antibody activity against HIV-infected MOLT4 cells in a preliminary experiment. Therefore, administration of IgM antibodies reactive with HIV-infected cells may be effective in the treatment of HIV carriers.

CD4-Positive T-Lymphocytes↗