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PubMed1984

Protective effects of various compounds on lysis of antibody-coated sheep erythrocytes by hypotonic shock or complement.

The combination of the two well known in vitro methods of hypotonic shock and immune hemolysis employing sheep erythrocytes (E) were used to evaluate membrane stabilization. Direct drug-induced hemolysis of antibody-coated E (EA), protection against hypotonic shock and complement (C)-mediated hemolysis can be recorded from a consecutive preparative procedure to give information to irreversible drug effects. The most active probable working mechanisms are interference with binding sites of the complement membrane attack complex (MAC) and unspecific membrane stabilization. Protection against C-mediated erythrolysis appeared more sensitive than against hypotonic shock.

Animals↗
PubMed2005

Human colon cancer cells lacking Bax resist curcumin-induced apoptosis and Bax requirement is dispensable with ectopic expression of Smac or downregulation of Bcl-XL.

Multiple apoptotic stimuli induce conformational changes in Bax, a proapoptotic protein from the Bcl-2 family and its deficiency is a frequent cause of chemoresistance in colon adenocarcinomas. Curcumin, a dietary compound from turmeric, is known to induce apoptosis in a variety of cancer cells. To understand the role of Bax in curcumin-induced apoptosis we used HCT116 human colon cancer cells with one allele of Bax gene (Bax+/-) and Bax knockout HCT116 (Bax-/-) cells in which Bax gene is inactivated by homologous recombination. Cell viability decreased in a concentration-dependent manner in Bax+/- cells treated with curcumin (0-50 microM) whereas only minimal changes in viability were observed in Bax-/- cells upon curcumin treatment. In Bax-/- cells curcumin-induced activation of caspases 9 and 3 was blocked and that of caspase 8 remained unaltered. Curcumin-induced release of cytochrome c, Second mitochondria derived activator of caspase (Smac) and apoptosis inducing factor (AIF) was also blocked in Bax-/- cells and reintroduction of Bax, downregulation of the antiapoptotic protein Bcl-XL by antisense DNA as well as the overexpression of Smac, highly sensitized the Bax-/- cells toward curcumin-induced apoptosis. There was no considerable difference in the percentage of apoptotic cells in Bak RNAi transfected Bax+/- or Bax-/- cells treated with curcumin when compared with their corresponding vector transfected cells treated with curcumin. The present study demonstrates the role of Bax but not Bak as a critical regulator of curcumin-induced apoptosis and implies the potential of targeting antiapoptotic proteins like Bcl-XL or overexpression of proapoptotic proteins like Smac as interventional approaches to deal with Bax-deficient chemo-resistant cancers for curcumin-based therapy.

Antineoplastic Agents↗
PubMed1990

Bacterial killing and inhibition of inner membrane activity by C5b-9 complexes as a function of the sequential addition of C9 to C5b-8 sites.

The assembly of the C5b-9 complex on the outer membrane of C-sensitive cells of Escherichia coli results in a rapid inhibition of inner membrane function and ultimately a loss of cell viability. Cells bearing C5b-8 sites suffer no deleterious effects; however, the addition of C9 results in a rapid inhibition of inner membrane function and cell death. An attempt was made to examine the relationship between the toxic effects of the C5b-9 complex and the number of C9 molecules per C5b-8 site. Cells bearing C5b-8 sites were exposed to excess C9 at 0 degrees C and washed three times at 4 degrees C. The number of C9 molecules bound to each cell was equivalent to the number of C5b-8 sites present on each cell, and no additional C9 molecules could be bound when the cells were maintained at 4 degrees C. These cells were then incubated at 37 degrees C for 3 min and returned to 0 degrees C, a technique which exposed additional C9-binding sites equivalent to the number of C9 molecules previously bound to the cells. This technique was repeated and demonstrated that the sequential build-up of a C5b-9 site with two C9 molecules per C5b-8 site was capable of inhibiting both inner membrane function (respiration and amino acid transport) and cell viability. Three C9 molecules per complex had effects that approached the inhibitory effects of complexes formed in the presence of excess C9.

Bacterial Physiological Phenomena↗
PubMed2000

Deficiency of complement defense protein CD59 may contribute to neurodegeneration in Alzheimer's disease.

Complement defense 59 (CD59) is a cell surface glycophosphoinositol (GPI)-anchored protein that prevents complement membrane attack complex (MAC) assembly. Here, we present evidence from ELISA assays that CD59 protein levels are significantly decreased in the frontal cortex and hippocampus of Alzheimer's disease (AD) compared with nondemented elderly (ND) patients, whereas complement component 9, a final component to form MAC, is significantly increased. To further confirm the CD59 deficit, PI-specific phospholipase C (PIPLC) was used to cleave the CD59 GPI anchor at the cell surface in intact slices from AD and ND cortex. CD59 released by PIPLC cleavage was significantly reduced in AD compared with ND samples. By the use of a ribonuclease protection technique, amyloid beta-peptide was found to downregulate CD59 expression at the mRNA level, suggesting a partial explanation of CD59 deficits in the AD brain. To evaluate the pathophysiological significance of CD59 alterations in neurons, we exposed cultured NT2 cells, which normally underexpress CD59, and NT2 cells transfected to overexpress CD59 to homologous human serum. Lactic acid dehydrogenase assays revealed significant complement-induced cell lysis in CD59-underexpressing NT2 cells and significant protection from such lysis in CD59-overexpressing NT2 cells. Moreover, cells expressing normal levels of CD59 showed no evidence of MAC assembly or damage after exposure to homologous serum, whereas pretreatment of these cells with a CD59-neutralizing antibody resulted in MAC assembly at the cell surface and morphological damage. Taken together, these data suggest that CD59 deficits may play a role in the neuritic losses characteristic of AD.

Aged↗
PubMed1995

Human polymorphonuclear leukocytes store large amounts of terminal complement components C7 and C6, which may be released on stimulation.

Secretion of the C factors C7, C6, and C3 by human polymorphonuclear leukocytes (PMNs) and PBMCs was studied by ELISA and immunoblot. The release of C7 and C6 by PMNs during 24 h of culture was 16-fold and 6-fold higher, respectively, than the C3 release, with median concentrations of 50.2 ng/ml, 18.3 ng/ml, and 3.1 ng/ml, respectively. In PBMC cultures, C release was considerably lower, and there was a different secretory pattern with a 6-fold higher release of C3 compared with C7 and C6. Stimulation with PMA led to a more rapid and complete secretion of the components to the culture media, whereas treatment with unopsonized Candida species did not affect the release. PMN release of C factors was not dependent on protein biosynthesis, and there was no indication of a selective uptake of C7 from serum as demonstrated by incubating PMNs from a subject with allotype C7 N in C7 M serum. Thus, the C components were probably produced by the PMNs or their bone marrow precursors before ex vivo culture. In cell lysates of freshly isolated cells, median C7, C6, and C3 contents of 1 x 10(7) PMNs were 149.7, 60.1, and 10.4 ng/ml, respectively, whereas the corresponding values for 1 x 10(7) PBMCs were 3.2, 2.6, and 14.6 ng/ml, respectively. The C6 and C7 were shown to incorporate into the terminal complement complex, and their molecular integrity was supported by identical m.w. to C6 and C7 present in normal serum. PMNs may represent a major source of C7 and C6 and may be more important than monocytes or macrophages in contributing terminal C components at a site of inflammation. This suggests a new role for the PMN as a C membrane attack modulator.

Candida↗
PubMed1995

Synergistic inhibition of human cell-mediated cytotoxicity by complement component antisera indicates that target cell lysis may result from an enzymatic cascade involving granzymes and perforin.

A widely accepted theory of lymphocyte-mediated cytotoxicity (CMC) proposes that upon effector cell (EC) and target cell (TC) interaction, release of perforin, serine proteases and other lytic moieties contained within cytoplasmic granules results in TC lysis. Complement activation and the activation of the various enzymatic activities associated with cytotoxic granules have strikingly similar modes of action and both lead to pore formation in their respective targets. We report here that by using antisera to early and late complement components we were able to inhibit CTL, NK and ADCC cytotoxicity up to 100%, even though binding of EC to TC was unaffected. Furthermore, we showed that addition of C1q or C1s (two serine proteases) antisera to C9 antisera, at titers too low to inhibit separately, resulted in synergistic inhibition of CMC. Anti-C1s together with anti-C1q (or anti-C8 with anti-C9) did not result in synergy. This finding supports a cascade model of activation for lytic molecules released from EC. In addition, we demonstrated that anti-C1q and anti-C1s bind to proteins in the 30-kD region and anti-C9 binds to proteins in the 70-kD region, coinciding with published molecular weights of granzymes and perforin, respectively. Finally, lytic ability of purified granules was also inhibited by complement antisera, further suggesting that activation occurs outside of TC. Taken as a whole, these data indicate that TC lysis may be the result of a cascade of events involving granzymes and perforin, analogous to that seen with the complement system.

Antibodies, Monoclonal↗
PubMed1987

A unique epitope exposed in native complement component C9 and hidden in the terminal SC5b-9 complex enables selective detection and quantification of non-activated C9.

Recently, monoclonal antibodies recognizing epitopes exposed in activation products of complement but hidden in the native components have been characterized and used for selective quantification of the activated protein. We now demonstrate that an epitope in the native component C9 is hidden in the terminal SC5b-9 complex. A monoclonal antibody against this epitope enabled selective detection of C9 without influence of the amount of SC5b-9 present. This antibody recognizing native soluble C9 was used to construct a quantitative double-antibody ELISA with unique sensitivity and specificity. Combination of this assay with an assay previously described for selective quantification of the SC5b-9 complex provides an important tool for evaluating terminal pathway activation of complement.

Animals↗
PubMed1984

Complement protein C9 labeled with fluorescein isothiocyanate can be used to monitor C9 polymerization and formation of the cytolytic membrane lesion.

Human complement protein C9 was covalently labeled with the fluorescent chromophore fluorescein isothiocyanate (FITC) with only a small reduction in the cytolytic activity of the protein. Polymerization of the labeled protein--either by incubating with lipid vesicles treated with complement proteins C5b-8 (activating the C5b-9 membrane lesion) or by heating the protein [Tschopp, J., Muller-Eberhard, H.J., & Podack, E.R. (1982) Nature (London) 298, 534]--resulted in a 40-60% decrease in the fluorescence emission from FITC. The decrease in total fluorescence was accompanied by an increase in the steady-state anisotropy following activation and polymerization of FITC-C9 by C5b-8 membranes, while heat-induced aggregation of the protein resulted in a dramatic depolarization of fluorescence. Only small changes in either the absorbance spectrum or fluorescence lifetime of the chromophore were detected upon FITC-C9 polymerization. Evidence is presented that the measured changes in FITC fluorescence upon C9 activation are due to self energy transfer between closely apposed fluorescein chromophores which occur in the polymerized form of the protein. The significance of these observations to the molecular structure of the assembled C5b-9 complex is discussed, as are the potential applications of this fluorescent derivative of C9.

Animals↗
PubMed1985

Elimination of terminal complement intermediates from the plasma membrane of nucleated cells: the rate of disappearance differs for cells carrying C5b-7 or C5b-8 or a mixture of C5b-8 with a limited number of C5b-9.

We have previously shown that multiple complement (C) channels are required for lysis of a nucleated cell in contrast to the single channel requirement for erythrocytes. To further investigate this multichannel requirement for nucleated cells, we examined the stability of terminal C complexes in the plasma membrane of Ehrlich ascites tumor cells. Ehrlich cells bearing C5b-7 or C5b-8 with or without C9 were incubated at 37 degrees C or 0 degree C for various time intervals before converting the remaining complexes to lytic C5b-9 channels. C5b-7, C5b-8, and C5b-8 in the presence of a limited number of C5b-9 complexes disappeared functionally from the plasma membrane at 37 degrees C, with initial half-lives of 31, 20, and 10 min, respectively. Disappearance of these complexes did not occur at 0 degree C, nor did disappearance occur at 37 degrees C when formed on sheep erythrocytes. The fate of C5b-8 complexes on the surface of Ehrlich cells was traced with colloidal gold particles bound to C5 determinants on C5b-8 with the use of immunoelectron microscopy. Colloidal gold could be seen on the cell surface after specific binding to cells carrying C5b-8 sites at 0 degree C. After incubating these cells at 37 degrees C, gold particles were internalized into the cell continuously via endocytic vesicles. It is postulated that terminal C complexes may stimulate or accelerate the removal of these complexes from the cell surface.

Animals↗
PubMed1988

Fluorescence analysis of size distribution and mode of dye release from carboxyfluorescein-loaded vesicles: application to the study of complement-membrane interactions.

We wish to report a novel method for visualizing large unilamellar vesicles loaded with a fluorescent dye and for monitoring changes in the size distribution as well as state of aggregation of such dye-loaded liposomes. In addition, we demonstrate that this method can be used to distinguish between all-or-none release of dye and graded release of dye from individual vesicles. Using this technique, we have characterized complement-mediated release of carboxyfluorescein from large unilamellar vesicles and have found that C5-8 complexes mediate a graded release of dye while C5-9 complexes cause an all-or-none release. Furthermore, complement appears to preferentially attack the medium to larger-sized vesicles in our population of large unilamellar vesicles while smaller vesicles appear to be selectively spared.

Cholesterol↗
PubMed1986

Complement lysis of U937, a nucleated mammalian cell line in the absence of C9: effect of C9 on C5b-8 mediated cell lysis.

Previous studies have demonstrated that in general, nucleated cells are more resistant to killing by serum complement than are erythrocytes. During studies aimed at defining the mechanisms of nucleated cell resistance, we found that the human histiocytic cell line U937 was easily lysed by homologous serum. U937 cells were also killed by serum depleted of C9, but not by serum depleted of C8, implying that the C5b-8 complex was sufficient to cause lysis of these cells. Enumeration of complexes on the cell surface demonstrated that approximately 40-fold more complexes were required to lyse U937 cells in the absence of C9 than in the presence of an excess of C9. Examination of the effects of small amounts of C9 on lysis of U937 cells by the C5b-8 complex demonstrated that at very low doses, C9 inhibited C5b-8 mediated lysis. The use of radiolabeled anti-C8 antibody showed that C5b-8 complexes were eliminated from the surface of U937 cells at 37 degrees C, and C9 at the dose causing inhibition of lysis accelerated the elimination of complexes. These results suggest that the increased lytic potential resulting from binding of small amounts of C9 to C5b-8 complexes is outweighed by enhanced elimination of complexes resulting in decreased cell death.

Cell Line↗
PubMed1981

Fluid-phase SC5b-8 complex of human complement: generation and isolation from serum.

A rapid, simple method for isolating SC5b-9 generated in human serum, and SC5b-8 generated in C9-depleted serum is described. The procedure leads to approximately 30% recovery of either complex and consists of polyethyleneglycol precipitation followed by 1 DEAE ion exchange chromatography and a sucrose density gradient ultracentrifugation. The SC5b-8 complex is a water-soluble macromolecular of alpha-electrophoretic mobility. Apart from the absence of C9, it exhibits an identical SDS-gel electrophoresis polypeptide pattern as SC5b-9. A sedimentation coefficient of 19 to 20S and an effective molecular radius of approximately 10 nm were determined by sucrose density gradient centrifugation and gel filtration, respectively. From these values, a m.w. of 800 to 850,000 is tentatively assigned to the SC5b-8 complex. The S-protein was not dissociable from either SC5b-8 or SC5b-9 through treatment with desoxycholate and no alterations in the hydrodynamic properties of either complex were discernible after action of this detergent.

Alpha-Globulins↗
PubMed1994

Effect of growth temperature on complement-mediated killing of mesophilic Aeromonas spp. serotype O:34.

Mesophilic Aeromonas spp. strains (serotype O:34) showed sensitivity to complement-mediated killing when they were cultivated at 37 degrees C (serum-sensitive) but not when they were cultivated at 20 degrees C (serum-resistant). These strains produced smooth lipopolysaccharide when they were grown at 20 degrees C and rough lipolysaccharide when cultivated at 37 degrees C. The reason for the resistance to complement-mediated killing could be that C3b is rapidly degraded (possibly because it is bound far from the cell membrane), consequently the lytic complex (C5b-9) is not formed.

Aeromonas↗
PubMed1988

Assembly of the membrane attack complex promotes decay of the alternative pathway C3 convertase on Neisseria gonorrhoeae.

C3, C4, factor B, properdin, and C2 binding to serum-sensitive and serum-resistant gonococci was quantitated in C8-deficient and normal human serum by using fluorescein-conjugated antibodies and 3H-labeled components. Organism and serum-specific differences were noted, the most striking of which involved factor B and properdin binding to the serum-sensitive strains in the different sera. C3 binding to these organisms was quantitatively and kinetically equivalent in C8-deficient and normal human serum. In contrast, factor B and properdin binding reached a plateau after 5 min in C8-deficient serum but peaked and fell to control values in normal human serum. Identical results were obtained with normal human serum immunochemically depleted of C8. Between 7 and 15% of the bound C3 participated in formation of the alternative pathway convertase C3bBb/P. Reconstitution of the C cascade by adding purified C8 to C8-deficient serum led to the loss of factor B previously bound to the organisms. Factor B loss occurred coincident with bacterial killing and membrane disruption as observed by electron microscopy. Prevention of membrane disruption by depleting normal human serum of lysozyme had no effect on killing and failed to prevent factor B loss. Stabilization of the C3bBb complex with Ni2+ prevented factor B loss as well as gross membrane disruption but not bacterial killing. C2 (the classical pathway analog of factor B) binding to gonococci was equivalent in C8-deficient and normal human serum peaking within 2.5 min and falling to control values in both sera thereafter. We conclude that the assembly of the membrane attack complex promotes decay of C3bBb/P with release of factor B and properdin but not C3 from the organism surface. Membrane disruption does not appear to be required for this effect. This activity may represent a mechanism to limit continued C consumption.

Blood Bactericidal Activity↗
PubMed2001

Expression of complement regulatory proteins on islets of Langerhans: a comparison between human islets and islets isolated from normal and hDAF transgenic pigs.

BACKGROUND: The expression of regulators of complement activity (RCAs) on islet cells may be of great importance for protecting them against complement-mediated lysis in the immediate posttransplant period after intraportal islet transplantation. We examined porcine and human islet cells for expression of RCA. We also examined to what extent human decay accelerating factor (hDAF) is expressed on adult and fetal islet cells isolated from hDAF transgenic (TG) pigs having a high transgene expression on endothelial cells. Moreover, the susceptibility of the various types of cells to lysis in human serum and blood was investigated. METHODS: Adult human islets (n=5), normal adult and fetal porcine islets (n=9 and n=8, respectively), and islets from adult and fetal hDAF TG pigs (n=5 and n=6, respectively) were examined. With islet single-cell suspensions and flow cytometry, adult human islet cells were examined for expression of hDAF (CD55), hCD59, and human membrane cofactor protein (hMCP; CD46), while porcine islet cells were examined for expression of pCD59 and pMCP. Islet cells from hDAF TG pigs were also examined for hDAF expression. Porcine peripheral blood lymphocytes, normal and hDAF TG porcine endothelial cell lines, a human endothelial cell line, and the human cell line U937 served as controls. Islet cytotoxicity was assayed after incubation of the islet cells with fresh human serum. Furthermore, adult islets from normal control pigs and hDAF TG pigs were exposed to fresh human blood in vitro for 60 min, and the inflammatory reaction elicited was compared between the different types of islets. RESULTS: All human islet cell preparations expressed hCD59, two of five expressed hMCP, but none expressed hDAF. Porcine islet cells expressed both pCD59 and pMCP. Normal adult porcine islet cells exposed to fresh human serum resulted in 74+/-5.4% cell lysis (mean+/-SEM, n=16). In comparison, only 1.3+/-2.8% (n=20, P<0.001) of human islet cells were lysed in the human serum. One islet cell preparation from an hDAF TG pig expressed small amounts of hDAF. This preparation from hDAF TG pigs bound significantly less C3c than did normal control islets (mean fluorescence ratio 16+/-2.2 and 58+/-4.3, respectively; P=0.046) and were partially protected from cell lysis in fresh human serum (47+/-10% and 78+/-18% cell lysis, respectively; P=0.046). The other four preparations from hDAF TG pigs were negative for hDAF and were equally susceptible to lysis as normal control islets. All fetal pancreatic islet cells from hDAF TG pigs analyzed were negative for hDAF expression. When exposed to fresh human blood in vitro, adult and fetal islets from hDAF TG pigs elicited equally strong inflammatory changes as did the normal control islets. The inflammatory changes were characterized by activation of the complement and coagulation systems, resulting in islet damage with "dumping" of insulin into the blood. CONCLUSIONS: Porcine and human islet cells express species-restricted complement regulatory proteins, with the human islet cells expressing mainly hCD59. A low expression of hDAF was detected on islet cells from one of five hDAF TG pigs. These islet cells displayed reduced islet cell cytotoxicity in fresh human serum. We conclude that protection from complement-mediated lysis will be important in the context of intraportal pig-to-human islet transplantation, and expression of a human RCA on islet cells should be beneficial in this context.

Animals↗
PubMed1986

Immunohistochemical detection of the terminal C5b-9 complement complex in children with glomerular diseases.

Kidney biopsies were obtained in 28 children with glomerular diseases and studied using indirect immunofluorescence and immunoperoxidase for IgG, IgA, IgM, C1q, C3c, C4, fibrinogen and the neoantigens of the terminal C5b-9 complement complex. C5b-9 deposits were detected in glomeruli of 14, in tubules of eight and in vessels of 12 cases. Patients with C5b-9 deposits fared worse than those without such deposits, even with the same histopathological type of glomerulonephritis. The C5b-9 complex suggests an in situ complement activation.

Adolescent↗