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At least 235 records · Page 13Linked to original sources
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↗
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↗
PubMed2003

Expression and characterization of the C345C/NTR domains of complement components C3 and C5.

Complement components C3, C4, and C5 are members of the thioester-containing alpha-macroglobulin protein superfamily. Within this superfamily, a unique feature of the complement proteins is a 150-residue-long C-terminal extension of their alpha-subunits that harbors three internal disulfide bonds. Previous reports have suggested that this is an independent structural module, homologous to modules found in other proteins, including netrins and tissue inhibitors of metalloproteinases. Because of its distribution, this putative module has been named both C345C and NTR. To assess the structures of these segments of the complement proteins, their relationships with other domains, and activities as independent structures, we expressed C345C from C3 and C5 in a bacterial strain that permits cytoplasmic disulfide bond formation. Affinity purification directly from cell lysates yielded recombinant C3- and C5-C345C with properties consistent with multiple intramolecular disulfide bonds and high beta-sheet contents. rC5-, but not rC3-C345C inhibited complement hemolytic activity, and surface plasmon resonance studies revealed that rC5-C345C binds to complement components C6 and C7 with dissociation constants of 10 and 3 nM, respectively. Our results provide strong evidence that this binding corresponds to the previously described reversible binding of C5 to C6 and C7, and taken together with earlier work, indicate that the C5-C345C module interacts directly with the factor I modules in C6 and C7. The high binding affinities suggest that complexes composed of C5 bound to C6 or C7 exist in plasma before activation and may facilitate assembly of the complement membrane attack complex.

Amino Acid Motifs↗
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↗