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PubMed1987

Presence of C5b-9 complement complex and S-protein in human myocardial areas with necrosis and sclerosis.

Myocardial fragments with acute infarction (10 cases), scars after chronic infarction (6 cases), areas with focal sclerosis and necrosis (8 cases) compared with normal myocardial areas (8 cases), were processed for indirect and double-labelling immunoperoxidase techniques to localize C5b-9 neoantigens, S-protein, C3d and apolipoprotein B. Granular masses of C5b-9 and C3d and diffuse areas of S-protein and apolipoprotein B were localized in the acute or chronically damaged areas but not in areas free of lesion. Double-labelling data revealed similarly damaged areas of localization for C5b-9 and S-protein, and for C3d and apolipoprotein B, respectively, on rather different than usual tissue structures. C5b-9 determination by ELISA from myocardial eluates revealed lower levels of neoantigens in normal areas (2.3 +/- 0.3 micrograms/g dried tissue), higher levels in areas with sclerosis (7.9 +/- 0.7 micrograms/g dried tissue) and the highest amounts in areas with acute infarction (11.1 +/- 1.2 micrograms/g dried tissue). The presence of C5b-9 neoantigens in damaged myocardial areas with a different localization than S-protein is suggestive of local complement activation.

Complement Membrane Attack Complex↗
PubMed1984

Interaction of human complement proteins with serum-sensitive and serum-resistant strains of Escherichia coli.

Exposure of serum-susceptible Escherichia coli strains to lethal concns of lysozyme-free human serum resulted in stable binding of complement components to the outer membrane (OM), but not to the cytoplasmic membrane (CM). The short prekilling phase of the reaction was accompanied by binding of C3b; loss of viability was immediately preceeded by stable deposition onto the OM of the component proteins of the membrane attack complex. During the early stages of the active killing phase, bound monomeric C9 could be resolved into two distinct bands on SDS-polyacrylamide gels. Serum exposure lead to a progressive loss of CM recoverability, which appeared to result from partial degradation of CM phospholipids. In contrast, exposure of a resistant E, coli strain to human serum resulted in little change in the membrane profile and very little stable deposition of terminal complement components onto the OM.

Blood Bactericidal Activity↗
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↗
PubMed1994

Antibodies to glycolipids activate complement and promote proteinuria in passive Heymann nephritis.

Passive Heymann nephritis is an experimental rat model of human membranous nephropathy induced by injection of antisera against crude renal cortical fractions such as Fx1A or rat tubular microvilli. This results in the formation of subepithelial immune deposits, the activation of the C5b-9 membrane attack complex of complement, and severe proteinuria. While the formation of immune deposits is attributed to in situ immune complex formation with antibodies specific for the gp330-Heymann nephritis antigenic complex (HNAC), activation of complement and proteinuria appear to be caused by at least one additional antibody species present in anti-Fx1A sera. We have separated by affinity absorption polyspecific antisera against Fx1A and rat microvilli into one IgG fraction directed specifically against microvillar proteins (anti-Fx1A-prot) and another IgG fraction specific for glycolipids (ant-Fx1A-lip) of tubular microvilli. When injected into rats, the anti-Fx1A-prot fraction induced immune deposits but failed to activate complement or produce proteinuria, similar to results obtained with affinity-purified anti-gp330 IgG. When the antibodies of the anti-Fx1A-lip fraction were injected alone they did not bind to glomeruli. By contrast, when the IgGs specific for the Fx1A-prot fraction (or for gp330-HNAC) were combined with those directed against the Fx1A-lip glycolipid preparation, immune deposits were formed, in situ complement activation was observed, and also proteinuria was induced. It is concluded that within anti-Fx1A and anti-microvillar sera there are at least two IgG fractions of relevance for the development of PHN: one directed against the gp330-HNAC complex which is responsible for the development of immune deposits, and a second specific for glycolipid antigen(s) which activate(s) the complement cascade.

Animals↗
PubMed1986

Complement activation in synovial fluid and tissue from patients with juvenile rheumatoid arthritis.

Synovial fluid (SF) and synovial tissue from 10 patients with juvenile rheumatoid arthritis were examined. The SFs were heterogeneous with respect to the degree of complement activation. Quantification of C3dg and the terminal complement complex revealed a positive correlation between activation of the early and the late parts of the cascade in all patients. The amount of C-reactive protein and the number of white blood cells in the SF correlated significantly with the degree of complement activation. Weak deposits of C3, C3dg, or terminal complement complex were observed in a few vessels in the synovial tissue from 5 of the patients. There was no correlation between complement activity in SF and in the corresponding tissue. Furthermore, there was no correlation between clinical activity in the joints and the degree of complement activation. It is concluded that there is a discrepancy between synovial tissue and synovial fluid with respect to complement activation. C-reactive protein may, to some extent, be responsible for activation in SF, and the accumulation of white blood cells may be due to complement activation products.

Adolescent↗
PubMed2002

Local production of complement proteins in rheumatoid arthritis synovium.

OBJECTIVE: Complement has been repeatedly implicated in the pathogenesis of rheumatoid arthritis (RA) based on studies showing reduced levels of native complement components and increased levels of complement metabolites in plasma, synovial fluid (SF), and synovial tissue (ST) of RA patients. However, there is limited information on local production and activation of key factors of the complement cascade in RA synovium and their potential modulation by novel anticytokine therapies. This study was undertaken to characterize the expression of complement proteins and receptors in RA SF and ST. METHODS: Using in situ hybridization, immunohistochemistry, and Western blot techniques, we assessed the presence of complement proteins C3, factor B (FB), and C5b-9, as well as the expression of complement receptors C3aR and C5aR in rheumatoid synovium. C3 and FB levels in SF were determined by enzyme-linked immunosorbent assay. Functional assessment was performed by examining the effects of soluble tumor necrosis factor receptor (sTNFR) p55 gene transfer in the SCID mouse model of RA. RESULTS: Complement proteins and receptors could be localized in all RA synovial specimens, whereas in osteoarthritis (OA) synovium, only a few, single cells expressed complement proteins and receptors. No differences were noted in the concentration of C3 between RA and OA in SF; however, FB levels were markedly reduced in RA versus OA SF. In RA synovium, in contrast to OA synovium, local expression of complement factor and complement receptor messenger RNA was found throughout the various ST compartments, suggesting that activation of the complement cascade occurs in all parts of the rheumatoid synovium. Moreover, C5aR expression was up-regulated following overexpression of sTNFR p55 by adenovirus-based gene transfer. CONCLUSION: In summary, local complement production and activation may play an important role in RA, and specific modulation and inhibition of local complement production could be an attractive therapeutic target for RA.

Aged↗
PubMed1983

Consequences of cell membrane attack by complement: release of arachidonate and formation of inflammatory derivatives.

Treatment of [3H]arachidonic acid [( 3H]C20:4)-labeled and antibody-sensitized Ehrlich ascites tumor cells with guinea pig or rabbit serum complement (C) released up to about 20 or 25% of the incorporated [3H]C20:4 into the aqueous phase as a consequence of C-induced hydrolysis of cellular phospholipid. The dose-response curve of release of [3H]C20:4 from Ehrlich ascites tumor cells, with respect to C, was approximately in the same range as the cytolytic response. In the case of [3H]C20:4-labeled and antibody-sensitized peritoneal mouse macrophages, treatment with C induced release of about 11% of the incorporated 3H as C20:4 and about 6% as prostaglandins, thromboxane B2, and hydroxyicosatetraenoic acids. C6- and C8-deficient rabbit and human sera, respectively, induced release of small amounts of [3H]C20:4 from Ehrlich ascites tumor cells and macrophages; these deficient sera also released traces of oxygenated derivatives from macrophages. Addition of purified C6 or C8 effectively restored release from both cell types, indicating that the terminal C proteins, up to and including C8, are required for the major part of the release. Our results do not rule out a possible requirement for C9.

Animals↗
PubMed1995

On the pathogenesis of atherosclerosis: enzymatic transformation of human low density lipoprotein to an atherogenic moiety.

Combined treatment with trypsin, cholesterol esterase, and neuraminidase transforms LDL, but not HDL or VLDL, to particles with properties akin to those of lipid extracted from atherosclerotic lesions. Single or double enzyme modifications, or treatment with phospholipase C, or simple vortexing are ineffective. Triple enzyme treatment disrupts the ordered and uniform structure of LDL particles, and gives rise to the formation of inhomogeneous lipid droplets 10-200 nm in diameter with a pronounced net negative charge, but lacking significant amounts of oxidized lipid. Enzymatically modified LDL (E-LDL), but not oxidatively modified LDL (ox-LDL), is endowed with potent complement-activating capacity. As previously found for lipid isolated from atherosclerotic lesions, complement activation occurs to completion via the alternative pathway and is independent of antibody. E-LDL is rapidly taken up by human macrophages to an extent exceeding the uptake of acetylated LDL (ac-LDL) or oxidatively modified LDL. After 16 h, cholesteryl oleate ester formation induced by E-LDL (50 micrograms/ml cholesterol) was in the range of 6-10 nmol/mg protein compared with 3-6 nmol/mg induced by an equivalent amount of acetylated LDL. At this concentration, E-LDL was essentially devoid of direct cytotoxic effects. Competition experiments indicated that uptake of E-LDL was mediated in part by ox-LDL receptor(s). Thus, approximately 90% of 125I-ox-LDL degradation was inhibited by a 2-fold excess of unlabeled E-LDL. Uptake of 125I-LDL was not inhibited by E-LDL. We hypothesize that extracellular enzymatic modification may represent an important step linking subendothelial deposition of LDL to the initiation of atherosclerosis.

Arteriosclerosis↗
PubMed1985

Cell damage by viruses, toxins and complement: common features of pore-formation and its inhibition by Ca2+.

Haemolytic paramyxoviruses interact with cells in the following way: a potentially leaky viral envelope fuses with the plasma membrane, creating a hydrophilic pore of approximately 1 nm in diameter; this allows ions and low molecular weight compounds, but not proteins, to leak into and out of cells. Other viruses act similarly if the pH is reduced to 5. Leakage (measured by collapse of membrane potential, by movement of monovalent cations and by loss of phosphorylated intermediates from cells) is prevented by extracellular Ca2+. Ca2+ does not affect binding or fusion of virus to cells. It inhibits leakage as well as preventing it, and it aids in the recovery (i.e. the restoration of non-leakiness) of cells. Certain 'anti-Ca2+' drugs have an opposite effect. Experiments with the bee venom protein melittin, with the alpha-toxin of Staphylococcus aureus and with activated complement, show that the lesions produced by these agents, too, are sensitive to extracellular Ca2+ and to 'anti-Ca2+' drugs. The mechanisms of these effects are discussed.

Animals↗
PubMed1998

Characterization in vitro and in vivo of the pig analogue of human CD59 using new monoclonal antibodies.

CD59 is the sole characterized regulator of the complement membrane attack complex in humans. It is very widely and abundantly distributed, being present on all circulating cells, endothelia and epithelia, and in most tissues. CD59 analogues in rodents are distributed similarly. Interest in complement regulation in the pig has developed out of the current enthusiasm to exploit this species as a donor in xenotransplantation of organs to humans. We have recently isolated and cloned the pig analogue of human CD59. We here report the development and characterization of monoclonal antibodies against pig CD59. We have used these antibodies to develop efficient methods for the purification of pig CD59 to homogeneity from erythrocyte membranes and have obtained new information on the structure and function of the purified protein. The antibodies were found to function well in immunohistochemistry and have been used to perform a comprehensive survey of the expression and distribution of pig CD59 on cells and in organs of normal pigs. Pig CD59, like human CD59, is broadly expressed but there are some striking differences in tissue distribution, notably the apparent lack of pig CD59 on circulating platelets and on a subset of leucocytes in blood and lymphoid organs. The reported findings have important implications for the current approaches to avoiding complement-mediated hyperacute rejection in pig-to-human xenografts.

Animals↗
PubMed1992

Clusterin in renal tissue: preferential localization with the terminal complement complex and immunoglobulin deposits in glomeruli.

The membrane attack complex (MAC) of complement is activated by immune and non-immune mechanisms in the kidney. MAC has been found associated with glomerular immune deposits, but also to cell remnants, particularly along tubules and in vessel walls. Clusterin and S-protein (vitronectin) bind to MAC, rendering it cytolytically inactive. Both have been found associated with MAC in renal tissue. Here we analysed the deposition of clusterin and S-protein in 118 renal biopsies relative to the localization of the MAC using MoAbs. Statistical analysis was performed comparing no or little versus evident or strong staining by immunofluorescence (IF). In glomeruli, out of the 92 biopsies where both MAC and immunoglobulins were evaluated, deposits of MAC were found in the presence (32 out of 41) but also in the absence of immunoglobulins (20/51). Clusterin and S-protein deposits were seen, respectively, in 25 out of 61 and 36 out of 61 biopsies containing glomerular MAC, and almost never in its absence (one out of 50 for both). The association of the two inhibitors with MAC was observed mainly in glomeruli containing immunoglobulin deposits (respectively, 21 out of 32 and 25 out of 32), but not when immunoglobulins were absent (three out of 20 and seven out of 20) (coefficient of concordance, K = 0.47 and 0.43). The localization of MAC along tubules and in vessels was easily identified in most biopsies (93 out of 118) and was accompanied by S-protein in most cases (tubules, 86 out of 93; vessels, 82 out of 93) (K = 0.58 and 0.57 respectively) but not by clusterin (28 out of 93 and 24 out of 93). These results suggest that clusterin does not co-localize with MAC whenever there is formation and fixation of the MAC. It seems that clusterin has a particular affinity for MAC which is associated with immunoglobulin. This observation should help to distinguish between the different forms of MAC, and might indicate that MAC associated with immunoglobulin is essentially in its cytolytically inactive form.

Biopsy↗
PubMed1984

Detection and quantification of the terminal C5b-9 complex of human complement by a sensitive enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay for detection and quantification of the terminal complexes (SC5b-9 and membrane attack complex) of human complement is described. We separate the complex from the native complement components, to use antibodies against the native components in a 'double-antibody sandwich' technique. It is thereby possible to detect the terminal complement complex in solution without the requirement of specific antibodies against the neoantigens. The results show that the assay is both sensitive and specific. Evidence is presented that a terminal complement complex occurs in a normal plasma pool. The terminal complement complex may be valuable for evaluating both the physiology and pathophysiology of the complement system in vivo.

Antibody Specificity↗
PubMed1990

Incorporation of SP-40,40 into the soluble membrane attack complex (SMAC, SC5b-9) of complement.

When SC5b-7 was prepared from the C8-depleted serum activated with inulin, it contained SP-40,40 as well as S-protein. From the densitometry of each component in SC5b-9 after SDS-PAGE, it was estimated that SC5b-9 was constituted of one molecule each of C5b, C6, C7, C8, S-protein, and SP-40,40 and two molecules of C9. SP-40,40 was depleted from normal serum with an affinity column using mouse monoclonal anti-SP-40,40 antibody. When the resulting SP-40,40-depleted serum was activated with inulin, SC5b-9 lacking SP-40,40 could be formed. S-Protein-depleted serum was also prepared with an affinity column using mouse monoclonal anti-S-protein antibody. Similarly, SC5b-9 lacking S-protein could be formed by the inulin activation of the S-protein-depleted serum. These results indicate that either SP-40,40 or S-protein should be able to form a soluble C5b-9 complex.

Blood Proteins↗
PubMed1989

Biotinylation: a simple method for labelling complement component C8 with preservation of functional activity.

Biotinylation of human C8 with the water-soluble biotin derivative biotinylamidohexanoic acid, N-hydroxysulfosuccinimide ester is an excellent method for labelling this terminal complement component with preservation of its functional activity. The biotinylated product can be detected both in native form and also following its incorporation into the terminal complement complexes. Detection assays include Western blotting, crossed immunoblotting, ELISA, and immunocytochemistry. Biotinylation is an attractive alternative method for labelling C8 and may be used for detecting and quantifying C8 and C5b-9 complexes in their soluble and membrane-bound forms.

Biotin↗