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PubMed1983

Complement pores in erythrocyte membranes. Analysis of C8/C9 binding required for functional membrane damage.

The number of membrane-bound terminal complement proteins (C5b-9) required to generate a functional pore in the human erythrocyte membrane ghost has been determined. Resealed erythrocyte ghost membranes (ghosts) were treated with human complement proteins C5b6, C7, 131I-C8, and 125I-C9 under non-lytic conditions. Following C5b-9 assembly, sucrose-permeant ghosts were separated from C5b-9 ghosts that remained impermeant to sucrose by centrifugation over density barriers formed of 43% (w/v) sucrose. Analysis of 131I-C8 and 125I-C9 bound to sucrose-permeant and sucrose-impermeant subpopulations of C5b-9 ghosts revealed: 1. Sucrose-permeant C5b-9 ghosts show increased uptake of both 131I-C8 and 125I-C9 as compared to ghosts that remain impermeant to sucrose. Ghosts with less than 300 molecules 131I-C8 bound remain impermeant to sucrose, irrespective of the total C9 input, or, the multiplicity of C9 uptake by membrane C5b-8. 2. In the presence of excess 125I-C9, the ratio of 125I-C9/131I-C8 bound to membrane C5b67 is 3.2 +/- 0.8 (mean +/- 2 S.D.), suggesting an average stoichiometry of 3 C9 per C5b-8. Under these conditions, the ratio of 125I-C9/131I-C8 bound to sucrose-permeant ghosts (3.3 +/- 0.7) does not significantly differ from the ratio bound to sucrose-impermeant ghosts (2.9 +/- 0.6). 3. With limiting C9 input, the threshold of total C5b-8 uptake required for sucrose permeability increases significantly above 300 per cell when the ratio of bound 125I-C9/131I-C8 is decreased below unity. In the complete absence of C9, 11 700 C5b-8 complexes are bound to sucrose-permeant ghosts. It is concluded that more than 300 C5b-9 complexes must bind to the human erythrocyte to form a sucrose-permeant lesion. Although the binding of one C9 per C5b-8 is critical to the pore-forming activity of these proteins, the binding of additional molecules of C9 to each complex (C9/C8 greater than 1) does not significantly alter the threshold of total C5b-9 uptake required for lesion formation.

Complement C8↗
PubMed1981

Synthesis of two components of human complement, beta 1H and C3bINA, during fetal life.

The levels of beta 1H and C3bINA were estimated in sera from human fetuses, cord and maternal samples. Both components of complement were detected in fetuses more than 12 weeks old. The serum concentrations tended to increase with the gestational age. The mean levels of beta 1H and C3bINA in cord sera were near 54 and 61% of the mean values in sera from normal adults. Elevated levels of beta 1H were observed in maternal sera at the end of the gestational period. When the levels of beta 1H and C3bINA were compared with those of two other components of complement, it was confirmed that the mean levels of C9 were low in fetal and newborn sera, while the serum concentrations of both C9 and Factor B were elevated in maternal samples. Newly synthesised beta 1H was detected in the culture fluids of fetal liver and peritoneal cells, as judged by the incorporation of labelled aminoacids and the autoradiography of he specific immunoprecipitates in agar gel.

Complement C3b Inactivator Proteins↗
PubMed1976

Lysis of tumor cells by antibody and complement. VII. Complement-dependent 86Rb release--a nonlethal event?

The guinea pig hepatoma (line-1) treated with anti-Forssman antibody (TA) and GPC sequentially released 86Rb, 14C from 14C aminoidobutyric acid and failed to exclude trypan blue. Incubation of TA with fluid phase GPC for 1 min caused maximal 86Rb release; however, if the GPC was removed at this time, the cells were not subsequently killed. Using a number of naturally occurring human sera deficient in a complement component we have shown 86Rb release requires the binding of the complement components 1 through 8, but there was no absolute requirement for C9. Irreversible damage to the cell as measured by 14C AIB release or uptake of trypan blue required the complete sequence of complete sequence of complement components. These observations indicate that 86Rb release is not a relible indicator cytotoxicity.

Animals↗
PubMed1985

Quantitation of activation of the human terminal complement pathway by ELISA.

We have devised an enzyme-linked immunosorbent assay (ELISA) to quantitate fluid phase terminal complement pathway activation. Upon activation to form C5b-9, terminal complement components express neoantigens not present in the unassembled individual components. Expression of one of these neoantigens occurs at the step of C9 activation. C9 neoantigen is present in fluid phase SC5b-9 complexes, membrane-bound MC5b-9 complexes, and in in vitro polymerized C9. Under physiologic conditions, the presence of C9 neoantigen indicates that the terminal complement pathway is activated through the terminal component C9. In our assay for C9 neoantigen, we used rabbit antiserum to polymerized C9 rendered specific for C9 neoantigenic determinants by serial absorption with human serum, human C9, and other terminal complement components bound to Sepharose. Using the IgG from this antiserum, we devised a sandwich ELISA to bind SC5b-9 from solution onto polystyrene plates. The ELISA plates were developed with the use of goat antiserum to native C9 epitopes followed by a swine anti-goat IgG-alkaline phosphatase conjugate. Quantitation of SC5b-9 in solution was performed by comparing sample OD to a standard curve generated with human SC5b-9 that was purified from zymosan-activated serum. The assay was sensitive to as little as 100 ng of SC5b-9/ml and should be useful for screening plasma, serum, cerebrospinal fluid, or other biological fluids for the presence of terminal complement pathway activation.

Antibody Specificity↗
PubMed1992

Mechanism of resistance to complement-mediated killing of bacteria encoded by the Salmonella typhimurium virulence plasmid gene rck.

We find that pADEO16, a recombinant cosmid carrying the rck gene of the Salmonella typhimurium virulence plasmid, when cloned into either rough or smooth Escherichia coli and Salmonella strains, confers high level resistance to the bactericidal activity of pooled normal human serum. The rck gene encodes a 17-kD outer membrane protein that is homologous to a family of virulence-associated outer membrane proteins, including pagC and Ail. Complement depletion, C3 and C5 binding, and membrane-bound C3 cleavage products are similar in strains with and without rck. Although a large difference in C9 binding was not seen, trypsin cleaved 55.7% of bound 125I-C9 counts from rough S. typhimurium with pADEO16, whereas only 26.4% were released from S. typhimurium with K2011, containing a mutation in rck. The majority of C9 extracted from rck strain membranes sediments at a lower molecular weight than in strains without rck, suggesting less C9 polymerization. Furthermore, SDS-PAGE analysis of gradient peak fractions indicated that the slower sedimenting C9-containing complexes in rck strains did not contain polymerized C9 typical of the tubular membrane attack complex. These results indicate that complement resistance mediated by Rck is associated with a failure to form fully polymerized tubular membrane attack complexes.

Antibodies, Monoclonal↗
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↗
PubMed1986

Number of hits necessary for complement-mediated hemolysis.

The number of hits necessary for the C8 and C9 steps of immune hemolysis was reexamined with a previously unemployed experimental design, in which various numbers of EAC1-7, excess of the supplementary component and a constant amount of the component tested were incubated in a constant volume (Inoue et al. 1976. Infect. Immun. 13: 337). Our results were consistent with previous findings; the steps of guinea pig C8 and C9, the human C8 each followed a one-hit mechanism, while that of human C9 showed ka multi-hit response. When lysis of sensitized erythrocytes (EA) by normal human serum was analysed in a similar way, one-hit curves were obtained. This result, taken together with the above results, suggests that immune hemolysis occurs by a single lesion including a single C8 and multiple C9 in the case of human complement and that normal human serum contains sufficient excess of C9. On the other hand, when C9-deficient human serum was used for lysis of EA, multiple-hit curves were obtained. The mechanism of lysis by C5b-8 may differ from that by C5b-9.

Animals↗
PubMed1975

Inhibition of the terminal stage of complement-mediated lysis (reactive lysis) by zinc and copper ions.

The effect of various metal ions, Fe++, Fe+++, Cu++, Zn++, Co++, on the terminal stage of reactive lysis (a form of complement-mediated hemolysis in which only late-acting components of complement are required) was studied. Only Cu++ and Zn++ exhibited an inhibitory effect on the lysis of EC5678 (sheep erythrocytes reacted with C56, C7 and C8) induced by C9. The mode of action of these metal ions was further explored. Both Cu++ and Zn++ inhibited the formation of hemolytically active EC56789 from EC5678 and C9. Their effect appeared to be primarily due to the inhibition of C9 binding to EC5678 through their reversible interaction with C9. Furthermore, Cu++ is shown to inactivate irreversibly the hemolytic activity of EC5678. EC5678 pretreated with Cu++ was capable of binding C9, but the resulting EC56789 was hemolytically inactive. Besides their effect on complement, both metal ions were shown to affect directly the erythrocyte membrane, since the mechanical lysis of hemolytic intermediate cells (E, EC567, EC5678) was suppressed by Cu++ and Zn++. The lysis of EC56789, a process of internal activation, was also inhibited by both Cu++ and Zn++.

Animals↗
PubMed1987

Release of leukotriene B4 from sublethally injured oligodendrocytes by terminal complement complexes.

In the present study, the interaction of the terminal complement complexes with oligodendrocytes was investigated for observation of its effect on membrane lipid hydrolysis. [14C]Arachidonic acid was incorporated into the membrane lipids of cultured oligodendrocytes before sensitization with anti-galactocerebroside antiserum. Cells were then exposed to excess C6-deficient rabbit serum reconstituted with limiting doses of C6 to form various numbers of C5b-9 complexes. Qualitative analysis of the supernatants by HPLC revealed the presence of compounds that coeluted with arachidonic acid and its oxygenated derivatives, prostaglandin E2, leukotrienes E4 and B4, and 15-hydroxyeicosatetraenoic acid. The kinetics of leukotriene B4 release by excess C5b-8 was quantitated by radioimmunoassay. Leukotriene B4 release approached a maximum around 30 min, and C6 dose-response studies performed at 1 h showed that maximal levels of leukotriene B4 were detected over a range of sublytic C5b-9 attack. Maximal release of leukotriene B4 was also achieved by C5b-8 without further enhancement by addition of lytic doses of C9. Results indicate that sublytic attack of oligodendrocytes by complement induces release of lipid-derived inflammatory mediators.

Animals↗
PubMed1996

Identification of complement activation sites in human immunodeficiency virus type-1 glycoprotein gp120.

Recombinant glycoprotein 120 (rgp120) of human immunodeficiency virus type-1 (HIV-1) activates the human complement system in the absence of anti-gp120 antibodies. HIV-1 glycoprotein gp120 can dissociate from the viral envelope either spontaneously or after binding of HIV-1 to the CD4 molecule. As a consequence, gp120 can circulate in the patient's serum and attach to the surface of uninfected CD4+ T cells. Complement activation by cell-bound HIV-1 glycoprotein gp120 with subsequent opsonization may represent a mechanism for the elimination of uninfected CD4+ cells by the reticuloendothelial system, thereby enhancing the progression of HIV disease. In the current study, the complement proteins C4,C3,C5,C9, and properdin were found to bind to a synthetic peptide covering positions 233-251 of the gp120BRU sequence on incubation with normal human serum. Complement activation by the peptide was comparable with that induced by aggregated IgG, complete rgp120, and the previously described complement-activating gp41-peptide 609-623. Activation occurred via the classical pathway and was abrogated in the presence of EDTA, Mg2+/EGTA, or C4-deficient human serum. Peptides partly overlapping the sequence 233-251 activated complement to a lesser extent. The complement-activating capacity of the gp120 sequence 233-251 was not restricted to the HIV-1BRU isolate, because a peptide from the corresponding sequence of the HIV-1MN strain was also capable of activating complement. An additional strong complement-activating site was identified in the gp120 sequence 321-360 of the HIV-1MN strain. These data indicate that distinct sites in gp120 are able to activate human serum complement via the classical pathway in the absence of anti-gp120 and independent of glycosylation.

Amino Acid Sequence↗
PubMed1986

Evasion of the alternative complement pathway by metacyclic trypomastigotes of Trypanosoma cruzi: dependence on the developmentally regulated synthesis of surface protein and N-linked carbohydrate.

Epimastigotes (EPI) of Trypanosoma cruzi are highly sensitive to lysis in fresh normal human serum by the alternative complement pathway (ACP). In contrast, metacyclic trypomastigotes (CMT) derived from EPI in stationary culture fail to activate the ACP and are thus resistant to serum-mediated lysis. To investigate the nature of the parasitic surface molecules which enable infective metacyclic trypomastigotes to evade the ACP, CMT were treated with a variety of different proteolytic and glycosidic enzymes, and their sensitivity to ACP-dependent lysis was tested. Pretreatment with pronase was found to cause a near complete reversal in the resistance of CMT to serum lysis, whereas trypsin or chymotrypsin induced smaller increases in complement sensitivity. Similarly, pretreatment with N-glycanase or neuraminidase also partially abrogated the resistance of CMT to ACP-dependent lysis. The effect of these enzymes on susceptibility to complement-mediated lysis was paralleled in increased C3 and C9 deposition on the organism. In addition, electrophoretic analysis of parasite-bound C3 indicated that the hemolytically inactive fragment, iC3b, was the major form of the molecule on CMT, while the hemolytically active fragment, C3b, predominated on pronase-treated CMT. Furthermore, when C3 was deposited on the parasite surface by means of purified ACP components, 80% of C3b on pronase-pretreated CMT but only 14% of the C3b on CMT bound the amplification protein factor B with high affinity, a prerequisite for efficient ACP activation. When cultured at 37 degrees C after pronase treatment, CMT gradually regained their resistance to ACP-mediated lysis. This process was blocked if puromycin, cycloheximide, or tunicamycin were included in the culture medium. The above findings suggest that evasion of the ACP by CMT is dependent on the developmentally regulated synthesis of protein as well as N-linked carbohydrate chains. A stage-specific 90,000 to 115,000 m.w. glycoprotein doublet present on the surface of CMT was shown to be uniquely sensitive to pronase digestion. Thus, this complex, which is also recognized by a CMT-specific monoclonal antibody, may be the glycoprotein component responsible for control of ACP activation

Animals↗
PubMed1987

Formation of the membrane attack complex of complement (MAC) on erythrocytes from monocyte-produced terminal complement components.

By using antibodies against C5, C6, C7, C8, and C9, we found that terminal complement components were deposited on IgM-coated sheep erythrocytes (EIgM) kept in serum-free endotoxin-stimulated monocyte cultures for 24 or 48 h. Monoclonal antibodies revealed C9 neoantigens on the EIgM. There was no specific binding of an anti-S protein antibody, which reacts with the SC5b-9 complex, to the EIgM. Controls were native sheep erythrocytes (E) treated similarly which, in contrast to EIgM, do not activate the classical pathway of complement. Cycloheximide (1.0 microgram/ml) in the cell cultures resulted in no specific binding of the anti-C9 antibodies to EIgM. A fraction of the EIgM was lysed during incubation with the monocytes. We conclude that the monocytes secrete C5, C6, C7, C8, and C9, which form the membrane attack complex of complement (C5b-9) on the EIgM.

Animals↗
PubMed1994

Affinity of the C9 molecule for the C5b-8 complex compared with that for the complex containing C9 molecules.

Gram-negative bacterial cells exposed to a complement source may carry membrane attack complexes containing variable numbers of C9 molecules per C5b-8 site. In order to investigate the assembly of this complex, the ability of C9 molecules to bind to C5b-8 complexes was compared with the binding characteristics of C9 for C5b-8 complexes containing variable numbers of bound C9 molecules. The apparent dissociation constant (Kd) of the C9 molecule for the C5b-8 site on a complement-sensitive strain of Escherichia coli was 1.2 (+/- 0.15) nM at 0 degree C. These conditions allow the binding of one C9 molecule per C5b-8 site. The C5b-8 site containing one C9 molecule bound a second C9 molecule at 0 degree C only after incubation at 37 degrees C. The binding of C9 to a C5b-8 site containing one C9 molecule was found to be 1.3 (+/- 0.2) nM. Therefore, the presence of a C9 molecule did not significantly alter the binding capacity of the C5b-8 site for additional C9 molecules. A similar result was obtained by using rabbit erythrocytes bearing either C5b-8 sites or C5b-8 sites containing one molecule of C9 per complex at 0 degree C. The similarity of binding characteristics for the first and second C9 molecules argues that the initial C9 molecule in the complex does not affect the binding of subsequent C9 molecules. This suggests that a unique C9 binding site that does not involve previously bound C9 molecules may exist on the forming membrane attack complex.

Animals↗
PubMed1977

Hereditary deficiency of the third component of complement in a child with fever, skin rash, and arthralgias: response to transfusion of whole blood.

A previously well 34-month-old male presenting with fever, skin rash, and arthralgias was found to lack C3 by immunochemical (undetectable) and hemolytic (1% normal) assays. No infectious agent could be demonstrated. Protein levels of Clq. C4, C5, properdin, and C3b-INA and hemolytic activities of complement components C1 to C9 except C3 were normal or elevated; total hemolytic complement activity was 13% of normal and was reconstituted by purified C3. Properdin factor B was 702 (normal 175 to 275) mug/ml, and was not cleaver upon addition of zymosan or cobra venom factor. The serum had normal immune adherence activity, but was deficient in ability to opsonize Candida albicans for uptake and Escherichia coli for killing by neurophils, generate neutrophil chemotactic factors and inhibit the growth of E. coli; these activities were restored by purified C3. A transfusion of 320 ml 1-hour-old normal whole blood on the fifty-second day resulted in transitory elevation of the C3 level to 25 mg/dl with a fall-off (approximately 2 1/2% per hour) to undetectable levels by 69 hours; it was followed by disappearance of the skin rash and arthralgias and return to normal of the previously elevated temperature and CRP levels. C3 levels in family members (seven of 24 half-normal), lack of anti-C3 activity, normal C3b-INA levels and a normal rate of catabolism of transfused C3 indicated that the deficiency was inherited with autosomal codominance and involved decreased synthesis of C3. Thus, this child is a unique individual with inherited C3 deficiency presenting with absence of repeated infections, whose symptoms of fever, skin rash, and arthralgia were abated by whole blood transfusion.

Blood Transfusion↗
PubMed1984

An immunochemical method for assessing the function of the alternative complement pathway.

Activation of the alternative pathway of human complement (C) by soluble C activators resulted in a decrease of C9 antigen, measured by single radial immunodiffusion, concomitant with a decrease of C9 hemolytic activity. In the presence of ethylene glycol bis-(beta-aminoethylether)-tetraacetic acid (EGTA) and Mg2+, this decrease of C9 antigen in the presence of soluble activators such as dinitrophenylated bovine serum albumin depends mainly on the activation of the alternative C pathway. Therefore, an assay system to express the total activity of the alternative C pathway in human serum - the C9 depletion test (C9DT) -, was devised. C9DT showed significantly lower values for patients with systemic lupus erythematosus and rheumatoid arthritis than for control patients.

Antigens↗
PubMed1994

Identification of a putative pore-forming hemolysin active at acid pH in Leishmania amazonensis.

Several organisms, including the protozoa Entamoeba histolytica and Trypanosoma cruzi, have been shown to contain pore-forming proteins (PFP) thought to play a role in the pathogenesis of the diseases they generate. In the present report, we show that promastigotes of Leishmania amazonensis express a hemolysin that appears to cause colloid-osmotic lysis, typical of pore formation. This hemolysin affects red blood cells of different species at 37 degrees C, but not at 0 degrees C, with maximum activity at pH 5.0. The hemolytic activity is heat-labile, but lysis is not affected by protease inhibitors. These results suggest the involvement of a protein with no proteolytic or detergent activity. Hemolysis is inhibited by polyethyleneglycol, suggesting its colloid-osmotic nature. Hemolytic extracts of the parasite contain a polypeptide that reacts with antibodies to perforin from mouse cytotoxic T lymphocytes or to C9 from human complement. In addition, genomic DNA of L. amazonensis contains a fragment that hybridizes to a perforin cDNA probe. The circumstantial evidence suggests that the L. amazonensis hemolytic activity may be mediated by a PFP homologous to perforin and C9.

Animals↗
PubMed1980

Immunological studies of human placentae: complement components in immature and mature chorionic villi.

The localization and distribution of complement components in term and pre-term normal human placentae have been studied by using haemadsorption and immunofluorescence experiments. The components Clq, C4, C5, C6 and C9 were identified in characteristic locations. Receptors for C3 and C4 were not found. Complement was associated with certain stromal cells, areas of fibrinoid necrosis within the trophoblastic mantle, and in the walls and endothelia of foetal stem vessels. Activation of the complement system on trophoblastic basement membranes (TBM) did not appear to involve the early reacting components of the classical pathway of complement activation, because C1q, C4 and C2 could not be identified on TBM. The C6 component was identified within cytoplasmic granules of foetal stem vessel endothelia, suggesting that it may be synthesized by these cells. These findings put forward the possibility that complement may play an immunobiological role in the materno-foetal relationship during normal human pregnancy.

Basement Membrane↗