Mechanism of complement-dependent haemolysis via the lectin pathway: role of the complement regulatory proteins.
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Mannan-binding lectin (MBL) is an acute phase protein which activates the classical complement pathway at the level of C4 and C2 via two novel serine proteases homologous to C1r and C1s. We recently reported that haemolysis via this lectin pathway requires alternative pathway amplification. The present experiments sought to establish the basis for this requirement, and hence focused on the activity and regulation of the C3 convertases. Complement activation was normalized between the lectin and classical pathways such that identical amounts of bound C4 and of haemolytically active C4,2 sites were present on the indicator cells. Under these conditions, there was markedly less haemolysis, associated with markedly less C3 and C5 deposited, via the lectin pathway than via the classical pathway, particularly when alternative pathway recruitment was blocked by depletion of factor D. Lectin pathway activation was associated with enhanced binding in the presence of MBL of complement control proteins C4bp and factor H to C4b and C3b, respectively, with decreased stability of the C3-converting enzyme C4b,2a attributable to C4bp. Immunodepletion of C4bp and/or factor H increased lectin pathway haemolysis and allowed lysis to occur in absence of the alternative pathway. Thus, the lectin pathway of humans is particularly susceptible to the regulatory effects of C4bp and factor H, due at least in part to MBL enhancement of C4bp binding to C4b and factor H binding to C3b. (+info)
Glomerular complement regulation is overwhelmed in passive Heymann nephritis.
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BACKGROUND: An injection of anti-Fx1A antibodies in rats leads to passive Heymann nephritis (PHN), a model of membranous nephropathy. Fx1A is a crude extract of renal cortex that contains megalin as a principal component. However, when rats are given anti-megalin antibodies, abnormal proteinuria does not occur. Because of the established complement dependence of PHN, we hypothesized that antibodies neutralizing complement regulatory proteins in the rat glomerulus also were required to induce PHN. Two likely targets are Crry and CD59, proteins abundant on the rat podocyte and contained within Fx1A that inhibit the C3 convertase and C5b-9 assembly, respectively. METHODS: Rats were injected with anti-megalin monoclonal antibodies, followed by anti-Crry and/or anti-CD59 F(ab')(2) antibodies five days later. In a second group of experiments, rats were injected with anti-Fx1A or anti-Fx1A immunodepleted of reactivity against Crry and/or CD59. RESULTS: In the setting of podocyte-associated anti-megalin monoclonal antibodies, simultaneous neutralization of Crry and CD59 function led to the development of significant proteinuria (11.0 +/- 2.1 mg/day, P < 0.001 vs. all other groups). In contrast, animals that had neither or only one of these complement regulators inhibited had normal urinary protein excretion (< or =6 mg/day). In animals given anti-Fx1A depleted of anti-Crry and/or anti-CD59, all groups developed typical PHN, characterized by heavy proteinuria and extensive glomerular deposition of C3 and C5b-9. CONCLUSION: Crry and CD59 play an important role in restraining complement-mediated injury following subepithelial immune complex deposition; however, in PHN, their regulatory capacity is overwhelmed. (+info)
Role of membrane cofactor protein (CD46) in regulation of C4b and C3b deposited on cells.
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C4b and C3b deposited on host cells undergo limited proteolytic cleavage by regulatory proteins. Membrane cofactor protein (MCP; CD46), factor H, and C4b binding protein mediate this reaction, known as cofactor activity, that also requires the plasma serine protease factor I. To explore the roles of the fluid phase regulators vs those expressed on host cells, a model system was used examining complement fragments deposited on cells transfected with human MCP as assessed by FACS and Western blotting. Following incubation with Ab and complement on MCP(+) cells, C4b was progressively cleaved over the first hour to C4d and C4c. There was no detectable cleavage of C4b on MCP(-) cells, indicating that MCP (and not C4BP in the serum) primarily mediates this cofactor activity. C3b deposition was not blocked on MCP(+) cells because classical pathway activation occurred before substantial C4b cleavage. Cleavage, though, of deposited C3b was rapid (<5 min) and iC3b was the dominant fragment on MCP(-) and MCP(+) cells. Studies using a function-blocking mAb further established factor H as the responsible cofactor. If the level of Ab sensitization was reduced 8-fold or if Mg(2+)-EGTA was used to block the classical pathway, MCP efficiently inhibited C3b deposition mediated by the alternative pathway. Thus, for the classical pathway, MCP is the cofactor for C4b cleavage and factor H for C3b cleavage. However, if the alternative pathway mediates C3b deposition, then MCP's cofactor activity is sufficient to restrict complement activation. (+info)
Regulation of glycoprotein IIb-IIIa receptor function studied with platelets permeabilized by the pore-forming complement proteins C5b-9.
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Recent evidence suggests that the cytoplasmic domains of platelet glycoprotein (GP) IIb-IIIa are involved in the agonist-initiated transformation of this integrin into a receptor for fibrinogen. To identify intracellular reactions that regulate the receptor function of GP IIb-IIIa, membrane-impermeable agonists and antagonists were introduced into the platelet by permeabilizing the plasma membrane with the pore-forming complement proteins C5b-9. Platelet responses were then analyzed by flow cytometry. Non-lytic concentrations of C5b-9 caused permeabilization of the platelet plasma membrane, as determined by uptake of a water-soluble fluorescent tracer dye. The complement pores were large enough to permit the entry of fluorescein isothiocyanate (FITC)-labeled oligopeptides in a size-dependent manner. Under conditions of low external Ca2+, C5b-9 treatment per se did not activate GP IIb-IIIa, as measured by binding of the activation-dependent antibody FITC-PAC1. However, FITC-PAC1 binding to C5b-9-permeabilized platelets was stimulated by a thrombin receptor agonist acting at the cell surface and by guanosine 5'-O-(thiotriphosphate), a membrane-impermeable activator of G proteins. Permeabilization also permitted the entry of cyclic AMP and the peptide, RFARKGALRQKNV, a pseudo-substrate inhibitor of protein kinase C. Each of these inhibited agonist-induced FITC-PAC1 binding to permeabilized platelets but not to intact platelets. Agonist-induced GP IIb-IIIa activation in permeabilized platelets was also inhibited by tyrphostin-23, a protein tyrosine kinase inhibitor. Thus, C5b-9 can be used to permeabilize the plasma membrane to permit the selective entry of small peptides and other bioactive compounds into permeabilized platelets. Results obtained with these platelets indicate that GP IIb-IIIa receptor function is regulated by a network of signaling reactions involving G proteins, serine/threonine kinases, and tyrosine kinases. (+info)
Persistent complement activation on tumor cells in breast cancer.
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The neoantigens of the C5b-9 complement complex, IgG, C3, C4, S-protein/vitronectin, fibronectin, and macrophages were localized on 17 samples of breast cancer and on 6 samples of benign breast tumors using polyclonal or monoclonal antibodies and the streptavidin-biotin-peroxidase technique. All the tissue samples with carcinoma in each the TNM stages presented C5b-9 deposits on the membranes of tumor cells, thin granules on cell remnants, and diffuse deposits in the necrotic areas. When chemotherapy and radiation therapy preceded surgery, C5b-9 deposits were more intense and extended. The C5b-9 deposits were absent in all the samples with benign lesions. S-protein/vitronectin was present as fibrillar deposits in the connective tissue matrix and as diffuse deposits around the tumor cells, less intense and extended than fibronectin. IgG, C3, and C4 deposits were present only in carcinoma samples. The presence of C5b-9 deposits is indicative of complement activation and its subsequent pathogenetic effects in breast cancer. (+info)
The quantitative role of alternative pathway amplification in classical pathway induced terminal complement activation.
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Complement activation with formation of biologically potent mediators like C5a and the terminal C5b-9 complex (TCC) contributes essentially to development of inflammation and tissue damage in a number of autoimmune and inflammatory conditions. A particular role for complement in the ischaemia/reperfusion injury of the heart, skeletal muscle, central nervous system, intestine and kidney has been suggested from animal studies. Previous experiments in C3 and C4 knockout mice suggested an important role of the classical or lectin pathway in initiation of complement activation during intestinal ischaemia/reperfusion injury while later use of factor D knockout mice showed the alternative pathway to be critically involved. We hypothesized that alternative pathway amplification might play a more critical role in classical pathway-induced C5 activation than previously recognized and used pathway-selective inhibitory mAbs to further elucidate the role of the alternative pathway. Here we demonstrate that selective blockade of the alternative pathway by neutralizing factor D in human serum diluted 1 : 2 with mAb 166-32 inhibited more than 80% of C5a and TCC formation induced by solid phase IgM and solid- and fluid-phase human aggregated IgG via the classical pathway. The findings emphasize the influence of alternative pathway amplification on the effect of initial classical pathway activation and the therapeutic potential of inhibiting the alternative pathway in clinical conditions with excessive and uncontrolled complement activation. (+info)
Recombinant C345C and factor I modules of complement components C5 and C7 inhibit C7 incorporation into the complement membrane attack complex.
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Complement component C5 binds to components C6 and C7 in reversible reactions that are distinct from the essentially nonreversible associations that form during assembly of the complement membrane attack complex (MAC). We previously reported that the approximately 150-aa residue C345C domain (also known as NTR) of C5 mediates these reversible reactions, and that the corresponding recombinant module (rC5-C345C) binds directly to the tandem pair of approximately 75-residue factor I modules from C7 (C7-FIMs). We suggested from these and other observations that binding of the C345C module of C5 to the FIMs of C7, but not C6, is also essential for MAC assembly itself. The present report describes a novel method for assembling a complex that appears to closely resemble the MAC on the sensor chip of a surface plasmon resonance instrument using the complement-reactive lysis mechanism. This method provides the ability to monitor individually the incorporation of C7, C8, and C9 into the complex. Using this method, we found that C7 binds to surface-bound C5b,6 with a K(d) of approximately 3 pM, and that micromolar concentrations of either rC5-C345C or rC7-FIMs inhibit this early step in MAC formation. We also found that similar concentrations of either module inhibited complement-mediated erythrocyte lysis by both the reactive lysis and classical pathway mechanisms. These results demonstrate that the interaction between the C345C domain of C5 and the FIMs of C7, which mediates reversible binding of C5 to C7 in solution, also plays an essential role in MAC formation and complement lytic activity. (+info)
Regulatory control of the terminal complement proteins at the surface of human endothelial cells: neutralization of a C5b-9 inhibitor by antibody to CD59.
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Functionally inhibitory antibody to the plasma membrane complement inhibitor CD59 has been used to investigate control of the terminal complement proteins at the endothelial cell surface. Antibodies against purified human erythrocyte CD59 (polyclonal anti-CD59 and monoclonal antibodies [MoAbs] 1F1 and 1F5) were found to bind specifically to monolayers of cultured human umbilical vein endothelial cells, and by Western blotting to recognize an 18- to 21-Kd endothelial protein. When bound to the endothelial monolayer, anti-CD59 (immunoglobulin G or Fab fragment) potentiated membrane pore formation induced upon C9 binding to C5b-8, and augmented the C5b-9-induced cellular responses, including stimulated secretion of von Willebrand factor and expression of catalytic surface for the prothrombinase enzyme complex. Although potentiating endothelial responses to the terminal complement proteins, anti-CD59 had no effect on the response of these cells to stimulation by histamine. Taken together, these data suggest that human endothelial cells express the CD59 cell surface inhibitor of the terminal complement proteins, which serves to protect these cells from pore-forming and cell-stimulatory effects of the C5b-9 complex. These data also suggest that the inactivation or deletion of this cell surface regulatory molecule would increase the likelihood for procoagulant changes in endothelium exposed to complement activation in plasma. (+info)