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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↗
PubMed1998

Therapeutic inhibition of the complement system.

The use of powerful methodologies in molecular biology, biochemistry, and physiology in the last 2 decades had led to impressive progress in our understanding of the mechanisms of complement activation and its role as either a protective or a pathogenic factor in human disease. With respect to disease pathogenesis, the complexity of the complement cascade provides opportunities for several different therapeutic targets within the complement pathways. More than a century after complement was first described, we are about to witness in the near future the availability of a variety of complement inhibitors for specific therapies. Progress in the area of xenotransplantation has been substantial, but formidable obstacles remain to selective inhibition of the factors that block successful clinical xenotransplantation. Bispecific antibodies, designed to enhance rather than inhibit existing complement pathways, hold strong promise for the clearance of viral and bacterial pathogens from the circulation.

Animals↗
PubMed1992

Enhanced expression of the complement-regulatory factor C8 binding protein (C8bp) on U937 cells after stimulation with IL-1 beta, endotoxin, IFN-gamma, or phorbol ester.

C8 binding protein (C8bp) is a 65-kDa membrane glycoprotein that inhibits complement-mediated lysis by homologous C5b-9. C8bp was first identified on human erythrocytes, but could also be detected on peripheral blood cells, platelets, glomerular cells and synovial fibroblasts. Lack of C8bp as seen in patients with paroxysmal nocturnal hemoglobinuria type III results in enhanced susceptibility of the cells toward C5b-9. We studied C8bp expression on the promonocytic cell line U937. In addition to the membrane-bound C8bp, a cytoplasmic form of C8bp could also be identified by immunofluorescence, blotting, and precipitation. Stimulation of the cells with IL-1 beta, endotoxin, IFN-gamma, or phorbol ester increased C8bp surface expression. Because cycloheximide did not inhibit enhanced surface expression, it was most probably mobilized from cytoplasmic reservoirs. Thus, resistance of nuclear cells to complement attack seems to be based on two events: 1) the removal of the C5b-9 complex from the membrane; and 2) expression of regulatory surface proteins such as C8bp, which inhibit C5b-9-mediated lysis. We propose that the C8bp mobilization by cytokines might provide an additional protection against complement attack by its known interference with the C5b-9 assembly.

Blood Proteins↗
PubMed1991

Inhibitors of membrane lipid metabolism enhance complement-mediated nucleated cell killing through distinct mechanisms.

The ability of nucleated cells to survive limited complement attack has been attributed to metabolic processes unique to these cells, such as rapid elimination of terminal complement complexes (TCC) from their surfaces. The biochemical processes activated by complement channels responsible for cell defense remain poorly defined. Metabolic inhibitors affecting membrane lipid turnover have been shown to increase the complement-mediated cell death. Whether these metabolic inhibitors increase lytic susceptibility of target cells by reducing the rate of TCC elimination has not been previously evaluated. In the present study, inhibitors of membrane lipid transmethylation and lysolecithin reacylation were evaluated in view of the observations that TCC concurrently increase lipid transmethylation and inhibit lysolecithin reacylation, and the inhibition of lipid transmethylation correlates with increased complement-mediated cell death. We have measured the formation as well as the elimination of C5b-9 on the target membrane that affect the outcome of cell death. Our results in the present communication indicated that inhibitors of transmethylation and lysolecithin reacylation increased TCC-mediated cell death through distinct pathways, the former by allowing more efficient deposition of TCC, and the latter by impairing TCC elimination.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1991

Detection of the terminal fluid-phase complement complex, SC5b-9, in the plasma of patients with insulin-dependent (type I) diabetes mellitus. Relation to increased urinary albumin excretion and plasma von Willebrand factor.

An ELISA was used to measure the fluid-phase complement complex in the plasma of 54 patients with insulin-dependent (type I) diabetes mellitus. Sixty-seven per cent of the diabetic patients had increased levels of SC5b-9. In individual diabetic patients, increased SC5b-9 was found to be significantly associated with the occurrence of anti-heparan sulphate cross-reactive anti-ssDNA antibodies and in some cases with circulating immune complexes. There was a significant correlation between levels of SC5b-9 and those of urinary albumin excretion rate (AER) (r = 0.39, P less than 0.01). Levels of AER were 8.4 +/- 2.26 micrograms/min and 2.04 +/- 0.35 micrograms/min in the SC5b-9 positive and negative patients, respectively (P less than 0.01). A relationship was also found between SC5b-9 and plasma von Willebrand Factor (r = 0.45, P less than 0.02), von Willebrand factor was 189.2 +/- 19.3% and 132.3 +/- 19.6% in SC5b-9 positive and negative patients, respectively (P less than 0.05). It may be that the abnormalities found in this study play a role in the pathogenesis of the late diabetic vascular complications.

Adolescent↗
PubMed1986

Membrane factors responsible for homologous species restriction of complement-mediated lysis: evidence for a factor other than DAF operating at the stage of C8 and C9.

Species-restricted lysis of complement refers to the relative inefficiency of complement to lyse cells from the homologous species. Restriction occurs at least at the steps involving C3/C5 convertase formation and the C9 insertion phase of the complement cascade, and is presumed to be mediated by inhibitory factors in the target cell membrane. In this study, we have examined whether decay accelerating factor (DAF), a membrane protein known to modulate C3/C5 convertase activities on cell surfaces, acts as a regulatory protein in species-restricted lysis of human erythrocyte (E). The role of DAF was assessed in homologous lysis by the classic pathway, in reactive lysis, and in lytic steps requiring C8 and C9. The results indicated that DAF participated in regulating C3/C5 deposition on the surface of homologous E, but had no effect on homologous restriction in reactive lysis and in the reaction of C8 and C9 with antibody-sensitized E C1-7. Treatment of E with pronase or with dithiothreitol (DTT) abolished the restricting effect of homologous C8/C9, indicating that species-restricted lysis by C5b-9 involves membrane factor(s) sensitive to pronase and DTT.

Animals↗
PubMed1985

Effect of complement proteins C5b-9 on blood platelets. Evidence for reversible depolarization of membrane potential.

The carbocyanine dye 3,3'-dipropylthiodicarbocyanine iodide has been used to investigate changes in membrane potential (Em) which occur upon binding of complement proteins C5b-9 to the plasma membrane of blood platelets. Gel-filtered platelets exposed to C5b6 and C7 in serum-free medium show no change in Em from that of controls, as indicated by either 3,3,'-dipropylthiodicarbocyanine iodide fluorescence or by the distribution of [14C]tetraphenylphosphonium bromide. Addition of complement proteins C8 and C9 to the C5b67 platelets results in partial depolarization of Em, which spontaneously repolarizes to basal levels within 15-20 min at 37 degrees C. Under these conditions, C5b-9-treated platelets show no increase in lysis over complement-free controls. Isotonic replacement of external sodium by either potassium or choline alters both the rate and extent of membrane depolarization and inhibits the platelets' capacity to repolarize after C5b-9 assembly. Repolarization of Em to basal levels is also completely blocked by addition of ouabain, confirming that this recovery is mediated by the plasma membrane Na+/K+ pump. These results demonstrate that membrane binding of the C5b-9 proteins can induce a transient change in Em when bound to the plasma membrane at a sublytic concentration, providing a mechanism for target cell activation by these potentially cytolytic proteins.

Adult↗
PubMed1984

Stable insertion of C5b-9 complement complexes into the outer membrane of serum treated, susceptible Escherichia coli cells as a prerequisite for killing.

Escherichia coli 17, a K12 derivative, was rapidly killed by human serum following a short lag period of 10 min. Stable binding of terminal C5b-9 complement complexes was investigated in time course experiments. Serum treated E. coli cells were lysed osmotically and the resulting outer and cytoplasmic membrane vesicles separated by sucrose gradient centrifugation. Exposure of E. coli 17 to serum rapidly reduced the degree of recoverability of cytoplasmic membrane vesicles. Electron microscopy revealed no interaction of C5b-9 complexes with CM vesicles. In contrast there was a clear time-dependent deposition of terminal complement complexes onto OM-vesicles. Very few complexes were detected during the prekilling phase of the reaction; initiation of the active killing phase was accompanied by a large increase in complement lesions. In contrast, no C5b-9 complexes could be visualised on outer or cytoplasmic membrane vesicles of a smooth, serum-resistant E. coli strain. We conclude that complement-mediated killing is a consequence of stable binding of C5b-9 complexes to the outer membrane of susceptible strains.

Blood Bactericidal Activity↗
PubMed1985

Cyanine dye fluorescence used to measure membrane potential changes due to the assembly of complement proteins C5b-9.

The fluorescent potentiometric indicator diS-C3-(5) has been used to investigate changes in membrane potential due to assembly of the C5b-9 membrane attack complex of the complement system. EAC1-7 human red blood cells and resealed erythrocyte ghosts--bearing membrane-assembled C5b67 complexes--were generated by immune activation in C8-deficient human serum. Studies performed with these cellular intermediates revealed that the membrane potential of EAC1-7 red cells and ghosts is unchanged from control red cells (-7 mV) and ghosts (O mV), respectively. Addition of complement proteins C8 and C9 to EAC1-7 red cells results in a dose-dependent depolarization of membrane potential which precedes hemolysis. This prelytic depolarization of membrane potential--and the consequent onset of hemolysis--is accelerated by raising external [K+], suggesting that the diffusional equilibration of transmembrane cation gradients is rate limiting to the cytolytic event. In the case of EAC1-7 resealed ghosts suspended at either high external [K+] or [Na+], no change in membrane potential (from O mV) could be detected after C8/C9 additions. When the membrane potential of the EAC1-7 ghost was displaced from O mV by selectively increasing the K+ conductance with valinomycin, a dose-dependent depolarization of the membrane was observed upon addition of C8 and C9. In these experiments, lytic breakdown of the ghost membranes was less than 5%. Conclusions derived from this study include: (i) measured prelytic depolarization of the red cell Donnan potential directly confirms the colloid-osmotic theory of immune cytolysis. (ii) The diffusional transmembrane equilibration of Na+ and K+ through the C5b-9 pore results in a dose-dependent depolarization of the membrane potential (Em) which appears to be rate-limiting to cytolytic rupture of the target erythrocyte. (iii) Enhanced immune hemolysis observed in high K+ media cannot be attributed to cation-selective conductance across the C5b-9 pore, and is probably related to the near-equilibrium condition of potassium-containing red cells when suspended at high external K+. These experiments demonstrate that carbocyanine dye fluorescent indicators can be used to monitor electrochemical changes arising from immune damage to the plasma membrane under both cytolytic and noncytolytic conditions. Potential application of this method to the detection of sublytic pathophysiological changes in the plasma membrane of complement-damaged cells are discussed.

Complement Membrane Attack Complex↗
PubMed1989

Effect of complement on the lateral mobility of erythrocyte membrane proteins. Evidence for terminal complex interaction with cytoskeletal components.

The lateral mobilities of erythrocyte membrane proteins and terminal complement complexes (TCC) were measured on C-treated erythrocyte ghosts by the technique of fluorescence redistribution after photobleaching. Results showed that the lateral diffusion coefficient of the bulk membrane proteins decreased with the assembly of TCC on the membrane at low C dose and was significantly reduced with assembly of the full membrane attack complex (C5b-9), even in the absence of cell lysis. At high serum doses, the mobility of the membrane proteins increased slightly above that of the control cells. The diffusion coefficients of the TCC on the erythrocyte membrane range from 1.18 to 4.37 x 10(-11) cm2/s, values characteristic of anchored membrane proteins. Spectrin-depletion of the C-lysed erythrocytes results in 25- and 45-fold increases in the diffusion coefficients of the membrane proteins and the C5b-9 complex, respectively. Conversely, oxidative cross-linking of spectrin by diamide reduced the diffusion coefficients of both membrane and C proteins. These studies indicate that the deposition of TCC on an erythrocyte can result in a substantial change in the physical and structural properties of the target membrane, aside from the creation of functional lesions. The low mobilities of the terminal complexes on the target membrane suggest possible interactions with cytoskeletal elements or with anchored membrane proteins.

Animals↗
PubMed1982

Photolabeling of a hydrophobic domain of the ninth component of human complement.

Recent experiments with membrane-restricted, photoactivatable probes indicated a preferential labeling of C9 within the assembled membrane attack complex (MAC) of complement, suggesting a direct role for C9 in the interaction of the MAC with membrane lipids. To further characterize the lipid-binding sites on C9, we have now used C9 that has been cleaved by alpha-thrombin. This enzyme cleaves C9 at one site but the newly generated peptides, C9a and C9b, respectively, remain noncovalently associated and the cleaved protein suffers no loss in hemolytic activity. When cleaved C9 was incorporated into the MAC during assembly on phospholipid vesicles and photolabeled, subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and fluorography revealed that only the larger fragment C9b, but not the smaller fragment C9a, became labeled. C9 attached alone to vesicles through heat aggregation in the absence of the precursor complex C5b-8 is also accessible to the hydrophobic photolabel. When cleaved C9 is used in the heat-induced assembly on vesicles and the polymerized C9 is photolabeled, the label associates again predominantly with C9b and not C9a. These results not only show that, within C9 polymers or within the assembled MAC, C9 possesses a two-domain structure, but also lend considerable support to the structure proposed for C9 by Biesecker et al. (Biesecker, G., Gerard, C., and Hugli, T. E. (1982) J. Biol. Chem. 257, 2584-2590) who classified C9a as hydrophilic and C9b as hydrophobic.

Azides↗