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PubMed1991

C6 depletion reduces proteinuria in experimental nephropathy induced by a nonglomerular antigen.

The role of the complement membrane attack complex, C5b-9, in mediating glomerular injury has been well defined in models of membranous nephropathy induced by antibody to endogenous glomerular epithelial cell membrane antigens. The effect of selective C6 depletion (to prevent C5b-9 formation) on morphologic characteristics and proteinuria in a model of in situ subepithelial immune complex nephritis induced by an exogenous cationized antigen (human immunoglobulin G (IgG)) followed by rabbit antibody to human IgG was studied. Selective C6 depletion was achieved by repeated administration of a goat antibody to rat C6. Other groups were treated with cobra venom factor to induce generalized complement depletion and with sublethal irradiation to deplete circulating leukocytes. In C6-depleted rats, C6 levels were reduced to less than 3% of baseline throughout the 2 days of the study compared with over 100% in controls. At 4 h after disease induction, glomerular deposition of antigen and antibody were similar in C6D and control groups by immunofluorescence and by direct measurement of glomerular deposition of radiolabeled antigen and antibody (cationized 131I human IgG, 9.1 +/- 0.1 micrograms/38,000 glomeruli in C6D versus 9.8 +/- 0.9 in controls; P = was not significant; rabbit 125I-labeled anti-human IgG, 104 +/- 10 ng in C6D versus 80 +/- 9 ng in controls; P = was not significant). Circulating C3 levels and glomerular C3 deposition were also similar in C6D and control groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Regulatory control of complement on blood platelets. Modulation of platelet procoagulant responses by a membrane inhibitor of the C5b-9 complex.

Antibody against a membrane inhibitor of the C5b-9 complex has been used to investigate regulatory control of the terminal complement proteins on blood platelets. Monospecific rabbit antibody (alpha-P18) was raised against the purified 18-kDa erythrocyte membrane inhibitor of C5b-9 (Sugita, Y., Nakano, Y., and Tomita, M. (1988) J. Biochem. (Tokyo) 104, 633-637). In addition to its interaction with erythrocytes, this antibody (and its Fab) bound specifically to platelet membranes. In immunoblots of cell membrane proteins prepared under non-reducing conditions, alpha-P18 bound specifically to an 18-kDa erythrocyte membrane protein and to a 37-kDa platelet membrane protein. Absorption of this antibody by platelet membranes competed its binding to the purified 18-kDa erythrocyte protein, suggesting that epitopes expressed by the erythrocyte 18-kDa C5b-9 inhibitor are common to the platelet. When bound to the platelet surface, the Fab of alpha-P18 increased C9 activation by membrane C5b-8, monitored by exposure of a complex-dependent C9 neo-epitope. Although alpha-P18 caused little increase in the cytolysis of platelets treated with C5b-9 (total release of lactate dehydrogenase less than 5%), it markedly increased the cell stimulatory responses induced by these complement proteins, including, secretion from platelet alpha- and dense granules, conformational activation of cell surface GP IIb-IIIa, release of membrane microparticles from the platelet surface, and exposure of new membrane binding sites for components of the prothrombinase enzyme complex. Prior incubation of C5b67 platelets with 100 micrograms/ml alpha-P18 (Fab) lowered by approximately 10-fold the half-maximal concentration of C8 required to elicit each of these responses (in the presence of excess C9). Incubation with alpha-P18 (Fab) alone did not activate platelets, nor did incubation with this antibody potentiate the stimulatory responses of platelets exposed to other agonists. These data indicate that a membrane inhibitor of the C5b-9 complex normally serves to attenuate the procoagulant responses of blood platelets exposed to activated complement proteins, and suggest the mechanism by which a deletion or inactivation of this cell surface component would increase the risk of vascular thrombosis.

Blood Platelets↗
PubMed1988

Deposition of terminal C5b-9 complement complexes on erythrocytes and leukocytes during cardiopulmonary bypass.

Hemolysis, leukopenia, a hemostatic deficit, and nonspecific systemic reactions collectively known as the postperfusion syndrome develop in patients who undergo cardiopulmonary bypass. We now report that terminal C5b-9 complement complexes are deposited on erythrocytes and polymorphonuclear neutrophilic leukocytes during cardiopulmonary bypass. Plasma samples taken from 48 unselected patients during and at the end of cardiopulmonary bypass contained raised levels of fluid-phase SC5b-9 complement complexes, indicating that the complement sequence had been activated to completion. Various degrees of overt intravascular hemolysis were observed in all the patients, and lysed erythrocyte membranes were recovered from the blood samples. Immunoassays performed with use of antibodies to C5b-9 neoantigens demonstrated the presence of C5b-9 on red-cell ghosts but not on intact erythrocytes. The appearance of ghosts carrying C5b-9 always coincided with hemolysis. Furthermore, granulocytes isolated from 20 patients during bypass were all found to carry C5b-9 complexes, whereas cells isolated before or 24 hours after surgery carried no C5b-9. The neoantigen-positive material present in detergent extracts of granulocytes sedimented in a broad peak (25 to 40 sedimentation coefficient [S]) in sucrose-density gradients, exactly as did pore-forming C5b-9 complexes. Deposition of C5b-9 on blood cells during cardiopulmonary bypass may be partly responsible for the hemolysis and may augment granulocyte activation by the stimulation of arachidonate metabolism in those cells.

Autoantibodies↗
PubMed1987

Haemolytic 'efficiency' of C5b-9 complexes in drug-induced immune haemolysis: role of cellular C5b-9 distribution.

We report on quantitative analyses of C5b-9 binding to target red blood cells (RBC) lysed through the action of drug (nomifensine)-dependent antibodies (ddab) and anti-RBC antibodies (warm plus cold agglutinins). Immunoradiometric assays showed that, at any given degree of haemolysis, more C5b-9 was bound to cells sensitized with anti-RBC antibodies compared to cells lysed via ddab. Thus, C5b-9 complexes deposited through the action of ddab appeared to be haemolytically more 'efficient' than those deposited via anti-RBC antibodies. However, data obtained from flow-cytometric assays and immunoelectron microscopy demonstrated a major role for the distribution (total number) of C5b-9 lesions among the cells within a given cell population. Enhanced haemolysis with a given total amount of C5b-9 complexes appeared to be derived from a more even distribution of these complexes among the cells rather than from higher lytic efficiency of individual complexes. Since ddab bind with low affinity to cells, we suggest that these antibodies diffuse from cell to cell and cause extensive haemolysis through the deposition of relatively few C5b-9 complexes at each site. In contrast, high affinity antibodies remain largely bound to relatively restricted sites causing deposition of larger amounts of C5b-9 on, and hence lysis of, numerically fewer cells. With respect to the net intravascular haemolytic effect, our findings may explain why complement-activating antibodies of low affinity are often more detrimental than high affinity antibodies.

Antibody Affinity↗
PubMed1986

Repolarization of the membrane potential of blood platelets after complement damage: evidence for a Ca++ -dependent exocytotic elimination of C5b-9 pores.

Gel-filtered blood platelets exposed to complement proteins C5b-9 have previously been shown to undergo a reversible depolarization of membrane potential (Em) in the absence of lytic plasma membrane rupture. In this paper, we examine the mechanism by which C5b-9 damaged platelets restore their basal electrochemical state, despite increased ion conductance due to membrane insertion of these cytolytic serum proteins. Repolarization of Em after formation of the C5b-9 membrane pore is shown to be accompanied by a Ca++-dependent vesiculation of the platelet surface, which results in the release of these proteins from the plasma membrane and a restoration of the membrane's functional integrity. This exocytotic elimination of C5b-9 complexes from the plasma membrane is accompanied by a ouabain-inhibitable repolarization of Em, which presumably reflects restoration of transmembrane cation gradients by the plasma membrane Na/K ATPase. The role of external Ca++ in the platelet's response to membrane-insertion of the C5b-9 proteins is discussed both in the context of the known cellular effects of this ion and in the context of recent observations suggesting sublytic changes in platelet function after complement-mediated plasma membrane damage.

Adult↗
PubMed1997

The cytolytically inactive terminal complement complex activates endothelial cells to express adhesion molecules and tissue factor procoagulant activity.

The membrane attack complex of complement (C) in sublytic concentrations stimulates endothelial cells (EC) to express adhesion molecules and to release biologically active products. We have examined the ability of a cytolytically inactive form of this complex, which is incapable of inserting into the cell membrane, to upregulate the expression of adhesion molecules and of tissue factor (TF) procoagulant activity. The inactive terminal C complex (iTCC) was prepared by mixing C5b6, C7, C8, and C9 and was purified by fast protein liquid chromatography on a Superose 12 column. Binding of this complex to EC was found to be dose dependent and was inhibited by anti-C9 antibodies, as assessed both by ELISA using an mAb anti-C9 neoantigen and by measuring cell-bound 125I-labeled iTCC. Exposure of EC to iTCC resulted in a dose- and time-dependent expression of endothelial leukocyte adhesion molecule 1, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 accompanied by increased levels of the corresponding mRNA, but not in the rapid expression of P-selectin. Inactive TCC also induced increased TF activity evaluated by a chromogenic assay that measures the formation of factor Xa. These effects were inhibited by anti-C9 antibodies. The data support the conclusion that iTCC may induce proinflammatory and procoagulant activities on EC.

Cell Adhesion Molecules↗
PubMed1987

Immunohistochemical detection of the membrane and fluid-phase terminal complement complexes C5b-9(m) and SC5b-9. Consequences for interpretation and terminology.

Activation of the terminal pathway of complement on a membrane results in the generation of the membrane-damaging terminal C5b-9(m) complement complex, whereas the non-lytic water-soluble SC5b-9 complex is formed when complement is activated in the fluid phase. Both forms of the terminal complement complex (TCC) can be immunohistochemically detected, but not distinguished, by antibodies recognizing neoantigens in the complexes. By means of monoclonal antibodies against C9 neoantigens and against the S-protein, it was demonstrated that deposits of the TCC in tissue sections may be either in the form of C5b-9(m) or SC5b-9. The consequences of this for the interpretation of the histochemical data and the terminology of the two complexes are discussed.

Adult↗
PubMed1981

Permeability characteristics of complement-damaged membranes: evaluation of the membrane leak generated by the complement proteins C5b-9.

Permeability characteristics of the membrane lesion generated by the terminal complement proteins are considered in light of recent observations that the measured diffusion of solute across complement-damaged membranes does not conform to the "doughnut hole" model of a discrete transmembrane pore formed by the inserted C5b-9 complex. By using the measured kinetics of steady-state tracer isotope diffusion of nonelectrolytes across resealed erythrocyte ghost membranes treated with C5b-9, a new transport model is developed. This model considers the apparent membrane lesion strictly in terms of the operational criteria of a functional conducting pathway for the observed diffusing solute, independent of a priori assumptions about the geometry or molecular properties of the membrane lesion. With this definition of the unit membrane lesion and the assumption that the exclusion size of the conducting pathway varies directly with the multiplicity of bound C5b-9 (as suggested by previous measurements under conditions of varying input of C5b-9), numerical estimates of te apparent permeability of the complement-damaged membrane to four diffusing nonelectrolytes are derived. These results suggest that the pathway for a particle diffusing across the complement lesion cannot be a pore and is functionally equivalent to an aqueous leak pathway, free of pore constraints. Implications of these results are discussed in terms of current molecular models for the mechanism of membrane damage by the complement proteins.

Biological Transport↗
PubMed1983

Size distribution and stability of the trans-membrane channels formed by complement complex C5b-9.

We have performed double marker sieving experiments with molecules ranging from ca. 0.5-3 nm dia. in order to evaluate the size distribution of the channels formed by complement in resealed sheep erythrocyte ghosts. Evidence is presented that marker release through the channels reached equilibrium between the ghosts and the extracellular fluid in a period of 3 hr and that the channels are stable at 37 degrees C for this period of time. Under these experimental conditions we have observed a differential in the endpoint release of inositol and sucrose, which indicates that some of the ghosts carried channels measuring between 0.7 and 0.9 nm dia. No differential was observed between release of sucrose and raffinose (0.9 and 1.1 nm mol. dia., respectively). Comparisons between sucrose and inulin (0.9 and 3 nm mol. dia, respectively) showed a difference in marker release. Also, there was substantial release of inulin, indicating the presence of channels above 3 nm in dia. Hence, the present data indicate formation of channels in three size ranges, namely, 0.7-0.9 nm, 0.9-3 nm and greater than 3 nm.

Animals↗
PubMed1999

Increased susceptibility to erythrocyte C5b-9 deposition and complement-mediated lysis in chronic renal failure.

BACKGROUND: Decreased red blood cell survival contributes to the anemia of chronic renal failure patients. Because patients on chronic dialysis therapy are frequently exposed to excessive complement activation, we investigated the susceptibility of this patient population to erythrocyte C5b-9 deposition, complement-mediated lysis, and ghost formation. METHODS: We developed a flow cytometric assay using antibodies to both glycophorin and the C5b-9 complex to detect C5b-9 deposition on intact erythrocytes and erythrocyte ghosts. Serum C5b-9 levels and C5b-9 deposition on erythrocyte ghosts were measured by enzyme-linked immunosorbent assay. RESULTS: A significant increase in C5b-9 deposition on intact erythrocytes was demonstrated in patients with advanced chronic renal failure (2.2 +/- 0.5%) and in patients on chronic maintenance hemodialysis (2.3 +/- 0.4%) compared with normal volunteers (0.9 +/- 0.1%, P = 0.005 vs. chronic renal failure, P < 0.001 vs. chronic hemodialysis patients). There was also a significantly higher percentage of C5b-9-positive erythrocyte ghosts in patients with advanced chronic renal failure (20.6 +/- 5%) and in chronic hemodialysis patients (15.5 +/- 3.1%) compared with normal controls (2.6 +/- 0.9%, P < or = 0.001 vs. advanced chronic renal failure and chronic hemodialysis patients). Treatment of erythrocyte preparations with cobra venom factor, which activates the complement cascade, resulted in dramatic increases in the percentages of C5b-9-positive erythrocyte ghosts in patients with chronic renal failure (49.9 +/- 6.9%) and in chronic hemodialysis patients (45.0 +/- 4.2%) compared with normal volunteers (22.3 +/- 2.7%, P < 0.001 vs. chronic renal failure and chronic hemodialysis patients). Erythrocyte membrane expression of the complement regulatory proteins CD59 and CD55 did not significantly differ between normal controls and hemodialysis patients. Plasma C5b-9 levels after cobra venom factor stimulation were higher in chronic renal failure patients (538 micrograms/ml) compared with normal controls (345 micrograms/ml, P < 0.001). CONCLUSIONS: Patients with chronic renal failure and on hemodialysis therapy are susceptible to erythrocyte C5b-9 deposition with subsequent lysis and ghost formation. Susceptibility to complement-mediated erythrocyte injury may contribute to the anemia of chronic renal disease.

Cell Survival↗
PubMed1982

Cutaneous localization of the membrane attack complex in discoid and systemic lupus erythematosus.

Biopsy specimens of skin lesions from three patients with discoid lupus erythematosus and six patients with systemic lupus erythematosus contained the membrane attack complex, which comprises C5b through C9, as well as immune complexes at the dermal-epidermal junction. The basilar epithelium in these areas was vacuolated and edematous, and the dermis contained an inflammatory infiltrate. In contrast, 19 of 29 specimens of normal-appearing skin from patients with discoid or systemic lupus erythematosus showed only immune complexes at the dermal-epidermal junction, without the membrane attack complex. The other 10 specimens, all from patients without cutaneous involvement, showed neither immune complexes nor membrane attack complexes. These data suggest that immune complexes within skin lesions selectively generate the assembly of the membrane attack complex, which mediates membrane injury. A synergistic interaction of immune complexes and cofactors may be required to activate complement in areas of skin that are predisposed to tissue injury.

Adult↗
PubMed1985

Formation of soluble immune complexes by complement in sera of patients with various hypocomplementemic states. Difference between inhibition of immune precipitation and solubilization.

To examine whether the ability of complement to form soluble immune complexes plays a role in preventing immune complex-mediated diseases, we analyzed the capacity of complement to inhibit immune precipitation (IIP) and to solubilize preformed immune aggregates (SOL) in 23 sera of patients with various hypocomplementemic states, and we correlated the results of these studies with the clinical syndromes found in the various patients. In sera with deficiency or depletion of early classical pathway components, IIP was profoundly altered, whereas SOL was delayed but in the normal range. In contrast, in sera with C3 depletion but intact classical pathway and in properdin-deficient serum, IIP was initially preserved, whereas SOL was abolished. Since the incidence of immune complex diseases in various hypocomplementemic states correlates with the severity of IIP defects, but not with reduced SOL, it is suggested that IIP is an essential biological function of complement that prevents the rapid formation of insoluble immune complexes in vivo.

Antigen-Antibody Complex↗
PubMed1987

Paroxysmal nocturnal hemoglobinuria type III. Lack of an erythrocyte membrane protein restricting the lysis by C5b-9.

The complement-mediated lysis is inefficient when complement and target cells are homologous with regard to the species. In erythrocytes from patients suffering from paroxysmal nocturnal hemoglobinuria (PNH), the species restriction is lost: PNH-erythrocytes (PNH-E) are susceptible to lysis by human complement. In human erythrocytes (huE) the species restriction is ascribed to an integral membrane protein, designated C8-binding protein (C8bp). In the present study, we tested membranes of PNH-E type III for the presence of C8bp. A protein with C8-binding capacity could not be detected. C8bp, which was isolated from the membrane of huE, inhibited the lysis of PNH-E by C5b-9 as well as the C9 polymerization. Thus, addition of C8bp restored the species restriction in PNH-E. In conclusion, we propose that lack of C8bp might represent the defect in PNH-E type III membranes, which is responsible for their enhanced lytic susceptibility towards lysis by the late complement components.

Complement C8↗
PubMed1988

Paroxysmal nocturnal hemoglobinuria. Enhanced stimulation of platelets by the terminal complement components is related to the lack of C8bp in the membrane.

Recently, a protein isolated from the membrane of human E, the so-called C8 binding protein (C8bp), has been described. C8bp is characterized as a 65-kDa protein that binds to C8 and inhibits the C5b-9-mediated lysis in a homologous system. In the present study, membranes of peripheral blood cells were tested for the presence of C8bp by SDS-PAGE and immunoblotting. In all cells a protein band reacting with anti-C8bp was seen, the Mr, however, was only about 50 kDa. To further analyze the 50-kDa protein, we isolated the protein by phenol-water extraction and isoelectric focusing from papain-treated platelets. The isolated protein behaved similar to the E-derived C8bp: it inhibited the lysis of model target cells by C5b-9. To examine the function of C8bp in platelets, we tested platelets from patients suffering from paroxysmal nocturnal hemoglobinuria (PNH). These platelets were deficient in C8bp, being in accordance with their higher lytic susceptibility in vitro. In response to sublytic C5b-9 doses, the PNH platelets released considerably more serotonin and thromboxane B2 than normal platelets. By addition of purified C8bp, the thromboxane B2 release was suppressed, indicating that C8bp not only restricts the lytic complement attack, but also regulates the C5b-9-mediated stimulation of target cells. Thus, lack of C8bp might not only result in enhanced hemolysis, but also in enhanced stimulation of platelets, which in turn might contribute to the thrombotic complications seen in some PNH-type III patients.

Blood Platelets↗
PubMed1995

[Comparison of complement activation between tuberculous and malignant pleuritis].

Complement activation products in pleural effusions were studied to clarify the causes of tuberculous and malignant pleuritis. Pleural SC5b-9, an activation product of complement common pathway, was significantly higher in tuberculous effusions than in malignant ones. In the tuberculous effusions, the levels of SC5b-9 and LDH (a marker of tissue damage) were significantly correlated, but in the malignant effusions these two values were not correlated. In the tuberculous effusions, SC5b-9 and Bb values were significantly correlated, but SC5b-9 and C4d were not, and nor were SC5b-9 and immune complex. In the malignant effusions, SC5b-9 and Bb values were low and were not significantly correlated. These results suggest that complement activation plays a significant role in tuberculous pleuritis, but not in malignant pleuritis.

Antigen-Antibody Complex↗
PubMed1994

Glycosylation of IgA is required for optimal activation of the alternative complement pathway by immune complexes.

To investigate the effect of carbohydrate on activation of the alternative pathway of complement by IgA immune complexes, aglycosylated monoclonal IgA was made biosynthetically in the presence of tunicamycin. When immune complexes were incubated with normal human serum (NHS), the aglycosylated IgA immune complexes caused less depletion of the alternative pathway activity of the serum. They also bound less C3 and produced less terminal complement complexes. The binding of C3 to both immune complexes was mainly through hydroxylamine sensitive ester bonds. C3 did not bind to free IgA.

Antigen-Antibody Complex↗
PubMed1996

The transient pore formed by homologous terminal complement complexes functions as a bidirectional route for the transport of autocrine and paracrine signals across human cell membranes.

BACKGROUND: We have previously shown that the membrane attack complex (MAC) of complement stimulates cell proliferation and that insertion of homologous MAC into the membranes of endothelial cells results in the release of potent mitogens, including basic fibroblast growth factor (bFGF). The mechanism of secretion of bFGF and other polypeptides devoid of signal peptides, such as interleukin 1 (IL-1) is still an open problem in cell biology. We have hypothesized that the homologous MAC pore itself could constitute a transient route for the diffusion of biologically active macromolecules in and out of the target cells. MATERIALS AND METHODS: Human red blood cell ghosts and artificial lipid vesicles were loaded with labeled growth factors, cytokines and IgG, and exposed to homologous MAC. The release of the 125I-macromolecules was followed as a function of time. The incorporation of labeled polypeptides and fluorescent dextran (MW: 10,000) was measured in MAC-impacted human red blood cells and human umbilical endothelial cells (HUVEC), respectively. RESULTS: Homologous MAC insertion into HUVEC resulted in the massive uptake of 10-kD dextran and induced the release of bFGF, in the absence of any measurable lysis. Red blood cell ghosts preloaded with bFGF, IL-1 beta, and the alpha-chain of interferon-gamma (IFN-gamma) released the polypeptides upon MAC insertion, but they did not release preloaded IgG. MAC-impacted ghosts took up radioactive IFN-gamma from the extracellular medium. Vesicles loaded with IL-I released the polypeptide when exposed to MAC. CONCLUSIONS: The homologous MAC pore in its nonlytic form allows for the export of cytosolic proteins devoid of signal peptides that are not secreted through the classical endoplasmic reticulum/Golgi exocytotic pathways. Our results suggest that the release, and perhaps the uptake, of biologically active macromolecules through the homologous MAC pore is a novel biological function of the complement system in mammals.

Biological Transport↗