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PubMed1986

Isolation of a human erythrocyte membrane protein capable of inhibiting expression of homologous complement transmembrane channels.

Erythrocytes are poorly lysed by homologous complement, whereas they are readily lysed by heterologous complement. This phenomenon had been attributed to an interference by the cell surface with the action of complement components C8 and C9. To isolate the responsible membrane constituent, detergent-solubilized human erythrocyte (EH) membranes were subjected to affinity chromatography by using human C9-Sepharose. The isolated protein had a mass of 38 kDa and, incorporated into liposomes, was highly effective in inhibiting complement-mediated channel expression, including the C5b-8, membrane attack complex, and tubular polymer of C9 channels. Antibody produced to the 38-kDa protein caused a 20-fold increase in reactive lysis of EH by isolated C5b6, C7, C8, and C9. The antibody did not enhance C5b-7 uptake, but it affected C9 binding to the target cell membrane. Antibody to human decay-accelerating factor, used as a control, had no effect on reactive lysis of EH. Anti-38-kDa protein did not enhance the action on EH of C8 and C9 from other species, indicating that the action of this regulatory protein is species specific. It was therefore termed homologous restriction factor (HRF). Blood cells other than erythrocytes, such as polymorphonuclear leukocytes, also exhibited cell-surface HRF activity. In immunoblots of freshly isolated EH membranes, anti-38-kDa HRF detected primarily a 65-kDa protein, suggesting that the 38-kDa protein constitutes an active fragment of membrane HRF. Because of the specific binding reaction observed between HRF and C8 or C9, HRF was tested with anti-human C8 and anti-human C9. A limited immunochemical relationship of HRF to C8 and C9 could be established and solid-phase anti-C9 proved an efficient tool for the isolation of HRF from solubilized EH membranes.

Blood Proteins↗
PubMed1989

Vesicular removal by oligodendrocytes of membrane attack complexes formed by activated complement.

Oligodendrocytes synthesize myelin in the central nervous system and maintain it in lamellar sheaths around axons. Techniques for studying oligodendrocyte development in vitro can be used, indirectly, to investigate the myelin injury that occurs in human and experimental demyelinating disease. Cell-mediated immune mechanisms are necessary but not sufficient to induce myelin damage in vivo; more recently complement has also been implicated in the pathogenesis both of multiple sclerosis and experimental allergic encephalomyelitis. Previously we have demonstrated that antibody-independent complement activation occurs in vitro at the oligodendrocyte surface. Here we show that the ensuing oligodendrocyte injury is reversible, and that recovery involves the release of membrane-attack complex-enriched vesicles from the surface of viable cells. The demonstration of morphologically and immunochemically identical vesicles in the cerebrospinal fluid of patients with multiple sclerosis suggests that reversible complement-mediated injury contributes to myelin damage in vivo.

Animals↗
PubMed1994

Expression and function of multiple regulators of complement activation in autoimmune thyroid disease.

Membrane attack complexes of complement occur around thyroid follicles in Graves' disease and Hashimoto's thyroiditis. The lytic potential of such complexes is controlled by membrane-bound and fluid phase regulators and we have investigated the role of these in autoimmune thyroid disease. By immunohistochemical staining, clusterin and S-protein were found in all nine thyroid specimens from patients with Graves' disease and S-protein was found in one of two Hashimoto glands. CD46, CD55 and CD59 were found on thyroid cells in all specimens. CD46 and CD55 expression occurred on thyroid cells cultured in vitro and was increased significantly by culture with interleukin-1 (IL-1) and interferon-gamma (IFN-gamma), which are known to be released by the lymphocytic infiltrate in these conditions. Blocking CD55 had a weak and inconsistent effect on complement-mediated thyroid cell killing in vitro but, in four of five experiments, blocking CD46 enhanced killing. However, the effect of blocking CD59 was greater in all cases than blocking CD46 or CD55. Expression of these fluid phase and membrane-bound proteins may be important in determining the severity of thyroid damage produced by complement fixation in Graves' disease and Hashimoto's thyroiditis.

Antigens, CD↗
PubMed1987

A role for thromboxane in complement-mediated glomerular injury.

The membrane attack complex (MAC) of complement (C) has been shown to stimulate prostaglandin (PG) and thromboxane (Tx) synthesis in nucleated cells. Because glomerular epithelial cell injury and altered permeability in rat membranous nephropathy are mediated by the MAC, the authors examined whether MAC-induced proteinuria is linked to glomerular prostanoid synthesis. In kidneys containing non-nephritogenic, non-C-fixing gamma 2 sheep anti-Fx1A (planted antigen) that were perfused in vitro with C-fixing guinea pig anti-sheep IgG and a C source (fresh human plasma, 50% vol/vol in buffered bovine albumin), heavy proteinuria developed, reaching 4.27 +/- 1.20 mg/min/g at 100-120 minutes (n = 8). Cyclooxygenase blockade with 10(-4) M indomethacin (n = 6) inhibited urinary PGE2 excretion (569 +/- 47 to 124 +/- 18 pg/min/g, P less than 0.001) and lowered proteinuria (1.06 +/- 0.42 mg/min/g, P less than 0.001). Reduced protein excretion (0.88 +/- 0.12 mg/min/g, n = 6, P less than 0.001) also occurred with inhibition of Tx synthetase by OKY-046, 10(-4) M, a dose that was shown in separate perfusions to inhibit urinary TxB2 excretion by greater than 85%. Control kidneys, without planted antigen and perfused with anti-sheep IgG and plasma, excreted 0.30 +/- 0.05 mg protein/min/g (n = 6). Because inulin clearance was reduced by indomethacin, renal hemodynamic factors may have contributed to the reduction in proteinuria observed with this drug. However, insulin clearance was not significantly affected by OKY-046, implying that inhibition of Tx synthetase reduced proteinuria independently of changes in renal hemodynamics. Thus, proteinuria in rat membranous nephropathy is due to MAC-dependent glomerular epithelial injury and is mediated, in part, by Tx.

Animals↗
PubMed2003

Calpain inhibitors protect against axonal degeneration in a model of anti-ganglioside antibody-mediated motor nerve terminal injury.

Miller Fisher syndrome-associated anti-GQ1b ganglioside antibodies produce an acute complement-dependent neuroexocytic effect at the mouse neuromuscular junction (NMJ) that closely resembles the effect of alpha-latrotoxin (LTx). This pathophysiological effect is accompanied by morphological disruption of the nerve terminal involving the loss of major cytoskeletal components, including neurofilament. Both LTx and the membrane attack complex of complement form membrane pores that allow free ionic movement and we have previously hypothesized that Ca2+ ingress and the subsequent activation of Ca2+-dependent proteases, calpains, may lead to substrate degradation resulting in structural disorganization of the terminal. Here, we treated mouse NMJs in hemidiaphragm preparations with anti-GQ1b antibodies and complement, or with LTx in the presence and absence of extracellular Ca2+, and studied possible neuroprotective effects of the calpain inhibitors calpeptin and calpain inhibitor V. Both Ca2+ depletion and calpain inhibition protected the cytoskeleton from degradation, as assessed by immunohistological and ultrastructural analysis. Calpain inhibitors may therefore be useful therapeutically in limiting nerve terminal and axonal injury in autoimmune peripheral neuropathy and in human latrodectism.

Animals↗
PubMed2004

Anti-disialoside antibodies kill perisynaptic Schwann cells and damage motor nerve terminals via membrane attack complex in a murine model of neuropathy.

Anti-disialoside antibodies (Abs) that bind NeuAc(alpha2-8) NeuAc epitopes on GQ1b and related gangliosides are found in human autoimmune neuropathy sera and are considered to be pathogenic. In a model system in mice, one mechanism by which anti-disialoside Abs have been demonstrated to induce paralysis is through a complement dependent blocking effect on transmitter release at the neuromuscular junction, similar to the effects of alpha-latrotoxin. Although direct targeting of presynaptic neuronal membranes occurs in this model, concomitant injury to perisynaptic Schwann cells (pSC) could indirectly contribute to this paralytic effect by influencing nerve terminal function and survival. To examine this possibility and the specific complement components that might mediate these effects, we exposed neuromuscular junctions in vivo and in vitro to an anti-disialoside Ab in conjunction with intact and selectively deficient complement sources. Using immuno-electron microscopy, we observed Ab deposits equally distributed on both neuronal and pSC membranes, and ultrastructural evidence of injury at both sites. Presynaptic neuronal injury was demonstrated functionally with microelectrode recordings and histologically as neurofilament loss. As hypothesized, concomitant pSC injury occurred, as indicated by abnormal uptake of ethidium dimer into pSC nuclei. The pSC and nerve terminal damage indicators correlated well with deposition of the pore-forming terminal complement component, membrane attack complex (MAC) in pSC and nerve terminal membranes. Furthermore, both neuronal and pSC injury were exacerbated in tissues from mice lacking the inhibitory complement regulator, CD59, where MAC formation is increased. These data demonstrate that both presynaptic neuronal membranes and pSCs are targets for anti-disialoside Abs, and that the injury to both sites is mediated by MAC and further regulated by CD59. This is the first demonstration that complement mediated pSC injury occurs in a model of autoimmune neuropathy and provides a rationale for investigating the possibility of pSC injury in equivalent conditions in man.

Animals↗
PubMed1997

Antiphospholipid antibody-dependent C5b-9 formation.

The relationship between the presence of antiphospholipid antibodies (APA) and the production of the terminal membrane attack complex (MAC) of complement (C5b-9) was studied. Serum samples from known high positive APA patients induced platelet activation and destruction which was inhibited by heat-inactivation of the sera. The response was restored if the heat-inactivated APA-positive sera were supplemented with normal sera. Adsorption of the APA-positive sera with phospholipid (PL)-coated polystyrene beads inhibited platelet destruction. Addition of monoclonal antibody (mAb) to C5b-9 (aE11) also inhibited platelet destruction, suggesting that the APA-dependent platelet destruction might be complement-mediated. Purified APA, in the presence of normal serum, induced C5b-9 formation and binding to PL-coated beads in a dose-dependent manner as detected by flow cytometry. Prospective analysis of 200 serum samples for C5b-9 production showed that all sera testing negative for the presence of APA also tested negative for C5b-9 production. All sera with high levels of IgG binding to PL (GPL) showed evidence of C5b-9 production. Sera with low or moderate GPL values showed varying levels of C5b-9 production. These data suggest that complement may play a key role in APA-dependent platelet activation, in vivo.

Adenosine Triphosphate↗
PubMed1993

Immunolocalization and characterization of the rat analogue of human CD59 in kidney and glomerular cells.

CD59, a potent inhibitor of the human membrane attack complex (MAC) of complement, is present on many different tissues throughout the body. Recently we identified and characterized the rat analogue of CD59 and produced a number of monoclonal antibodies (mAb). We have now used these antibodies to demonstrate, by immunofluorescence microscopy, that rat CD59 was widely expressed in the rat kidney. Staining of renal sections revealed the presence of rat CD59 in abundance in the glomerulus, collecting ducts and distal tubules. Staining was abolished by treatment of sections with phosphatidylinositol-specific phospholipase C (PIPLC). Rat mesangial cells also stained strongly for rat CD59, giving an intensely granular staining pattern. In complement attack experiments the cultured mesangial cells were rendered more susceptible to lysis by rat or human complement after preincubation with the anti-inhibitor mAb. The results demonstrate that the rat analogue of CD59 is present and functionally active in rat renal tissue. The protective effect of CD59 against MAC-mediated tissue injury can now be examined in rat models of human renal disease.

Animals↗
PubMed1994

Affinity of the C9 molecule for the C5b-8 complex compared with that for the complex containing C9 molecules.

Gram-negative bacterial cells exposed to a complement source may carry membrane attack complexes containing variable numbers of C9 molecules per C5b-8 site. In order to investigate the assembly of this complex, the ability of C9 molecules to bind to C5b-8 complexes was compared with the binding characteristics of C9 for C5b-8 complexes containing variable numbers of bound C9 molecules. The apparent dissociation constant (Kd) of the C9 molecule for the C5b-8 site on a complement-sensitive strain of Escherichia coli was 1.2 (+/- 0.15) nM at 0 degree C. These conditions allow the binding of one C9 molecule per C5b-8 site. The C5b-8 site containing one C9 molecule bound a second C9 molecule at 0 degree C only after incubation at 37 degrees C. The binding of C9 to a C5b-8 site containing one C9 molecule was found to be 1.3 (+/- 0.2) nM. Therefore, the presence of a C9 molecule did not significantly alter the binding capacity of the C5b-8 site for additional C9 molecules. A similar result was obtained by using rabbit erythrocytes bearing either C5b-8 sites or C5b-8 sites containing one molecule of C9 per complex at 0 degree C. The similarity of binding characteristics for the first and second C9 molecules argues that the initial C9 molecule in the complex does not affect the binding of subsequent C9 molecules. This suggests that a unique C9 binding site that does not involve previously bound C9 molecules may exist on the forming membrane attack complex.

Animals↗
PubMed2005

Impact of inverse plasma filtration on complement activation in porcine blood.

During extracorporeal circulation, activation of the complement system as a bioincompatibility reaction upon contact with artificial surfaces is well known. This may induce adverse effects for the patient as well as for the bioreactor when using a hybrid liver support system. At present, the achievable clearance rates in hybrid liver support systems are restricted by the limitations of blood flow through the capillaries of the plasma filter. In order to enhance clearance rates it is desirable to develop a high-flow plasma separation unit in which the activation of the complement system is as low as possible. Conventional plasma filters operating in normal and in inverse (blood flow around the capillaries and the capillary lumen serving as plasma compartment) modes were investigated applying an in vitro circuit (n = 4 in each group). For this purpose heparinized porcine blood was used. Complement activation was measured as generation of the soluble membrane attack complex, SC5b-9. With a normal flow mode the maximum achievable filtration rates varied from 25 to 40 mL/min, whereas the maximal filtration rates with an inverse flow mode varied from 40 to 100 mL/min. While these values were significantly higher than those with regularly operating plasma filters, there was no significant difference between normal and inverse mode in the production of SC5b-9. We conclude that the outer surface of the investigated conventional plasma filtration membranes is suitable for an inverse mode and does not lead to an excessive activation of the complement system upon dynamic blood contact. Thus, the inversion of the blood and the plasma compartments may be a possible way forward in the development of a high-flow plasma separation filter.

Animals↗
PubMedDate not supplied

Binding of vitronectin and clusterin by coagulase-negative staphylococci interfering with complement function.

Coagulase-negative staphylococci (CoNS) are commonly associated with infections of prosthetic devices mediated by adsorbed host factors on biomaterial surfaces. Complement activation is known to occur and induce unspecific inflammation around the biomaterials. Human vitronectin (Vn) and clusterin (Clu), two potent inhibitors of complement, can be bound by CoNS. With a hypothesis whether binding of Vn or Clu influences complement activation, two measurements were determined. For Vn, complement activation was measured with a mouse anti-activated human C9 antibody. In the presence of Vn-binding strain, Staphylococcus hemolyticus SM13I, complement activation on a surface pre-coated with Vn occurred as it did in the absence of Vn pre-coating. For S. epidermidis 3380, which does not express binding of Vn, complement activation on a Vn-presented surface was significantly decreased. For Clu, erythrocytes lysis was measured to reflect the end product of complement activation (membrane attack complex). The complement-induced hemolysis increased when human serum was pre-incubated with Clu-binding strains, S. epidermidis J9P. The enhancement of hemolysis by J9P decreased when serum was supplemented by exogenous Clu. The data imply that interaction between CoNS and Vn or Clu interferes with one of their physiological functions, complement inhibition.

Journal Article↗
PubMed1990

Protection of human amniotic epithelial cells (HAEC) from complement-mediated lysis: expression on the cells of three complement inhibitory membrane proteins.

Cultured human amniotic epithelial cells (HAEC) were found by immunofluorescence microscopy to express three complement inhibitory membrane proteins, CD59 antigen, decay-accelerating factor (DAF) and membrane attack complex (MAC) inhibitory protein (MIP), on their surfaces. The effects of incubation with Fab2 fragments of monoclonal antibodies (mAb) raised against these proteins on susceptibility of sensitized cells to lysis by homologous complement was examined. Percentage cell lysis was markedly increased in the presence of Fab2 anti-CD59 and to a lesser, but significant, extent in the presence of Fab2 anti-DAF. Fab2 anti-MIP did not alter the sensitivity of the cells to lysis by complement.

Amnion↗
PubMed1994

Aberrant regulation of complement by the erythrocytes of hereditary erythroblastic multinuclearity with a positive acidified serum lysis test (HEMPAS).

Susceptibility to hemolysis in acidified serum is a pathognomonic feature of hereditary erythroblastic multinuclearity with a positive acidified serum lysis test (HEMPAS, congenital dyserythropoietic anemia type II). The purpose of the studies reported herein was to determine if aberrant regulation of complement contributes to the susceptibility of HEMPAS erythrocytes to acidified serum lysis. The results of these experiments have demonstrated that regulation of both the C3 convertase of the alternative pathway and the membrane attack complex of complement by HEMPAS erythrocytes is aberrant. However, these abnormalities are not a consequence of quantitative or functional deficiencies of the erythrocyte complement-regulatory proteins, decay accelerating factor (DAF, CD55), or membrane inhibitor of reactive lysis (MIRL, CD59). Our recent studies have shown that glycophorin A (GPA), the major erythrocyte sialoglycoprotein is a complement regulatory protein. Analysis by radioimmunoprecipitation suggested that GPA on HEMPAS erythrocytes is abnormally glycosylated. Further analysis indicated that the abnormality involves the O-linked oligosaccharide moiety. Together, these studies show that complement regulation by HEMPAS erythrocytes is abnormal and that constituents other than DAF and MIRL participate in controlling complement activation on the erythrocyte membrane. Additionally, these studies suggest that the glycosylation defect that is characteristic of HEMPAS involves GPA.

Anemia, Dyserythropoietic, Congenital↗
PubMed1988

Complement fixation by pemphigus antibody. V. Assembly of the membrane attack complex on cultured human keratinocytes.

Previous studies have shown that pemphigus vulgaris (PV) IgG will fix early complement components (C1q, C4, and C3) to cultured murine epidermal cell surfaces and that PV IgG and complement alter epidermal cell membrane integrity. The present study was undertaken to determine if assembly of terminal complement components (C5, C6, C7, C8, and C9) and expression of C5b-9 neoantigens occur when PV IgG interacts with human keratinocyte (HuK) cell surface antigens in the presence of a source of complement. Monoclonal antibodies specific for C5, C6, C7, C8, C9, and C5b-9 neoantigens were screened for reactivity to the individual complement components in an assembled complex of human C5b-9 on rabbit red blood cell ghosts. Monoclonal antibodies (tissue culture supernatants) that bound to antigenic determinants accessible in the C5b-9 complex were selected for this study using immunofluorescence methods. HuK treated with PV IgG fixed C5, C6, C7, C8, C9, and C5b-9 neoantigens in a characteristic speckled pattern, while normal IgG did not. Heat inactivation or EDTA treatment of the complement source, or substitution of C2-depleted serum abolished C5, C6, C7, C8, C9, and C5b-9 neoantigen staining. PV IgG and complement also resulted in significant cytotoxicity to cell membranes as assessed using an ethidium bromide-fluorescein diacetate assay. These results suggest that PV IgG will activate the membrane attack complex of the complement system on HuK cell surfaces, resulting in cytotoxicity to cell membranes, further implicating complement in the pathogenesis of pemphigus.

Autoantigens↗
PubMed1992

Sublytic complement attack protects tumor cells from lytic doses of antibody and complement.

Sublytic doses of the membrane attack complex (MAC) of complement are known to exert multiple stimulatory effects on metabolically active cells. Results presented herewith demonstrate that pretreatment of the human leukemic cells K562 and HL-60 with sublytic doses of antibody and normal human serum protects them from lytic complement concentrations, a phenomenon proposed to be called "complement-induced protection". C7- and C8-deficient human sera are ineffective in inducing resistance unless they are reconstituted with purified human C7 and C8, respectively. The complement-induced protection is inhibitable by actinomycin D and cycloheximide indicating that the increased complement resistance depends on RNA and protein synthesis triggered by the sublytic complement doses. Free extracellular Ca2+ is also required to achieve maximal protection, indicating a role for Ca2+ ions in the cell stimulatory events which culminate in increased complement resistance. Quantitative analysis of bound complement components indicated that similar amounts of C3 and C9 molecules are deposited on "protected" and control cells during complement activation. The "protected" K562 and HL-60 cells regain sensitivity to lytic MAC doses after about 8 or 3 h, respectively, of culture in growth medium, in the absence or presence of actinomycin D and cycloheximide. The "induced protection" is not species restricted and protection from human complement can be induced in K562 cells by treatment with sublytic doses of antibody and rabbit or guinea pig sera.

Animals↗
PubMed1995

[Activation of complement during hemodialysis].

In hemodialysis using 3 types of dialysis membrane materials [regenerated cellulose (RC), cellulose triacetate (CTA), and polysulfone (PS)], activation of the complement, reduction of white blood cells, and variation of vitronectin (VN) were observed. RC membrane caused a significant reduction of white blood cells and elevations of Bb and soluble membrane attack complex (S-MAC), indicating a strong activation of the alternative complement pathway. Especially, the increase of S-MAC persisted for a long time during hemodialysis. Because reduction of VN was transient, it was assumed that the S-MAC escaping removal by VN receptors might have persisted in the circulation. These findings suggested that S-MAC would become useful as an index for evaluating biocompatibility of various artificial organs including dialysis membranes.

Aged↗