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PubMed1999

Peritoneal mesothelial cells produce complement factors and express CD59 that inhibits C5b-9-mediated cell lysis.

The CD59 membrane protein confers protection from C5b-9-mediated cell lysis. Because evidence exists for complement (C) activation and generation of C5b-9 in the peritoneal cavity during chronic peritoneal dialysis (CPD), we investigated, on mesothelial cell (MC) lines, the expression of CD59 and the production of C components. Four MC lines were obtained from children on CPD, and two from non uremic children. CD59 expression on MCs was investigated with anti-CD59 monoclonal antibody (mAb) and polyclonal goat immunoglobulin G (IgG). MC lines were positive for staining with anti-CD59 mAb. Western blotting analysis of MC membrane demonstrated a band with the same molecular weight as CD59. Incubation of MC with anti-CD59 mAb abrogated the protective effect of CD59 (100% cytotoxicity). C3, C4, and C6 were detected in the supernatants of MC; in non uremic MC supernatants, C5, C7, C8, and C9 were also detectable, and C4 concentration was tenfold higher. CD59 expression confers to MCs protection from C5b-9-mediated lysis. MCs produce C factors. These findings suggest that production of complement components and expression of CD59 on MCs could play a role both in peritoneal cavity infection (decreased complement production) and in peritoneal membrane damage (decreased CD59 expression and reduced remesothelialization owing to MC lysis).

CD59 Antigens↗
PubMed1999

Complement regulators C1 inhibitor and CD59 do not significantly inhibit complement activation in Alzheimer disease.

Proteins characteristic of activated complement are associated with Alzheimer disease (AD) lesions. The classical complement pathway can be activated only when the influence of such endogenous regulators as C1-inhibitor (C1-inh) and CD59 are overcome. We used the techniques of reverse transcriptase-polymerase chain reaction and Western blotting to assess the mRNA and protein levels of C1-inh and CD59 in AD and control brains in comparison with levels of the complement components with which they interact. The inhibitors were only slightly upregulated and then only in heavily affected areas of AD brain such as the entorhinal cortex, hippocampus, midtemporal gyrus and midfrontal gyrus. The ratio of AD to control mRNAs in these four areas was 1.17 for C1-inh and 1.12 for CD59, compared to 3.06 for C1r, 2.67 for C1s, 2.35 for C5, 2.56 for C6, 2.42 for C7, 5. 08 for C8 and 16.3 for C9. Peripheral organ expression of C1-inh and CD59 mRNAs was no different in AD than controls but was slightly upregulated in infarcted heart tissue. Again, the increase was small compared with that of the competitive complement components. These data indicate that the forces which upregulate and activate complement in AD and myocardial infarction are not effectively suppressed by the endogenous regulators, C1-inh and CD59.

Adult↗
PubMed1979

Studies on the terminal stages of immune hemolysis. VI. Osmotic blockers of differing Stokes' radii detect complement-induced transmembrane channels of differing size.

We have previously shown that 0.1 M EDTA could be used to distinguish functionally different transmembrane channels produced during complement-(C) mediated hemolysis of E. In this paper we have studied the ability of sugars of varying Stokes' radii to prevent hemoglobin release from E intermediates whose lysis was inhibitable or not inhibitable by EDTA. On the basis of these experiments we propose that the inhibition of E transformation by high molarity EDTA occurs by virtue of the size of the EDTA molecule in solution. Studies on the effect of EDTA on red cell lysis induced by polyene antibiotics that form transmembrane channels of a defined size support this conclusion. The results of these experiments were interpreted to mean: 1) The EDTA inhibitable lesion of E has a smaller effective radius than the noninhibitable lesion; 2) the effective radius of the smallest lesion that yields a lytic site was less than 3.6 A; 3) the lesions produced in the red cell membrane by C are not uniform but vary in size depending on the C9 to SACl-8 ratio used to produce E.

Animals↗
PubMed1989

Complement proteins C5b-9 induce secretion of high molecular weight multimers of endothelial von Willebrand factor and translocation of granule membrane protein GMP-140 to the cell surface.

The effect of immune activation of the serum complement system on the secretory response of human endothelial cells was examined. Exposure of antibody sensitized cultured umbilical vein endothelial cells to human serum resulted in secretion of very high molecular weight multimers of von Willebrand factor which coincided with new surface expression of the intracellular granule membrane protein GMP-140. This response required complement activation through deposition of C5b-9 and was not observed with cells exposed to antibody plus C8-deficient serum or to membrane C5b-8 (in the absence of C9). This C5b-9-induced secretion was observed with minimal cell lysis, as assessed by the release of lactic dehydrogenase. Delayed addition of C8 and C9 to cells exposed to antibody plus C8-deficient serum revealed a rapid decay of membrane C8 binding sites accompanied by loss of the secretory response, suggesting a process of removal or inactivation of nascent C5b67 complexes deposited on the endothelial surface. Membrane assembly of C5b-9 complexes caused an increase in endothelial cytosolic [Ca2+], due to influx across the plasma membrane. This C5b-9-dependent increase in cytosolic [Ca2+] and concomitant von Willebrand factor secretion were both abolished by removal of external calcium. In addition to being linked to the level of external Ca2+, the C5b-9-induced secretory response was partially inhibited by the protein kinase inhibitor, sphingosine. The capacity of the C5b-9 proteins to stimulate endothelial cells to secrete a platelet adhesive protein provides one mechanism for increased platelet deposition at sites of inflammation, and suggests the potential for other functional changes in endothelium exposed to C5b-9 during intravascular complement activation.

Antibodies↗
PubMed1987

Nucleated cell killing by complement: effects of C5b-9 channel size and extracellular Ca2+ on the lytic process.

For C5b-9 channels to mediate cytolysis of a nucleated cell, a sufficient number of channels must be formed in the plasma membrane to override the compensatory mechanisms that nucleated cells might employ to survive. It is well known that nucleated cells are relatively resistant to lysis by complement in comparison to erythrocytes, and it is now evident that this resistance is due, in part, to the ability of nucleated cells to rapidly eliminate C5b-9 from the cell surface. The ability of nucleated cells to eliminate complement complexes is related to physiochemical properties of the complex, such as channel diameter, which in turn affect Ca2+ fluxes that stimulate metabolic processes involved in the elimination process. Paradoxically, these same channel properties that stimulate the defense response may also be responsible for the lethal effects of complement. To further study the role of channel size on cytolysis of nucleated cells by C5b-9, we examined the lytic efficiency of larger C5b-9 channels containing several C9 molecules in comparison with smaller C5b-9 channels containing fewer C9. We have obtained data to indicate that although the larger channels were more cytolytically potent, the channel size had little influence on the rate of cell death. In contrast, the rate of lysis of erythrocytes was substantially slower when smaller C5b-9 channels were present. In evaluating the effect of the extracellular Ca2+ concentration, [Ca2+]o, on nucleated cell lysis in the presence of a lytic number of C5b-9 complexes, it was observed that when the [Ca2+]o was increased the rate of cell death also increased. These findings suggest that lysis of nucleated cells by C5b-9, unlike erythrocytes, may not be entirely due to colloid osmotic deregulation.

Animals↗
PubMed1971

Production of ultrastructural membrane lesions by the fifth component of complement.

A direct quantitative relationship has been demonstrated between the number of cell bound C4,2 complexes or C5 molecules and the number of ultrastructural lesions visualized on the cell membrane subsequent to immune hemolysis. When bound C4,2 complexes exceeded bound C5 molecules, the number of ultrastructural lesions seen corresponded to the number of C5 molecules. However, in the reverse situation, with bound C5 molecules in excess of bound C4,2 complexes, the latter determined the number of lesions. During the complement-reaction sequence, the lesions first became visible in the nonlytic intermediate complex EAC1,4,2,3,5 and their number was unaffected when lysis was induced by C6-C9. Since the lesions were also demonstrable on the intermediate complex EC5,6,7, it is concluded that the protein C5 is responsible for their production. Once formed, the physical presence of the C5 molecule is no longer required for the manifestation of the lesions as indicated by persistence of lesions after removal of C5 protein by trypsin. The C5-dependent ultra-structural phenomenon has therefore been interpreted to represent a true structural change of the membrane which, however, is not accompanied by a permeability defect.

Animals↗
PubMed1985

Characterization of human S protein, an inhibitor of the membrane attack complex of complement. Demonstration of a free reactive thiol group.

S protein, an inhibitor to the membrane attack complex of complement, was purified from human plasma. The procedure involved barium citrate adsorption and fractionation by poly(ethylene glycol) 4000 precipitation, followed by chromatography on DEAE-Sephacel, Blue Sepharose, Sephacryl S-200, and finally anti-albumin-Sepharose. Reduced glutathione was added throughout to inhibit spontaneous formation of disulfide-linked S-protein dimers. The recovery was 7%, resulting in approximately 10 mg of pure S protein from 1 L of starting plasma. S protein is a single-chain molecule; sedimentation equilibrium ultracentrifugation yielded a molecular weight of 83 000; the s020,W value was estimated to be 4.0 S. The purified protein contained a free, reactive thiol group causing spontaneous formation of disulfide-linked S-protein dimers. Alkylated and nonalkylated S proteins were equally active in inhibiting C9 polymerization, catalyzed by the C5b-8 complex. In parallel with the inhibition of C9 polymerization, nonalkylated S protein catalyzed the formation of disulfide-linked C9 dimers, presumably through disulfide interchanges.

Amino Acid Sequence↗
PubMed1988

C5 gene influences the development of murine myasthenia gravis.

The influence of the C5 gene and C5 deficiency on murine experimental autoimmune myasthenia gravis (EAMG) susceptibility was evaluated. Two co-isogenic strains, B10.D2/nSn (C5 sufficient) and B10.D2/oSn (C5 deficient), which are genetically identical except for the C5 gene locus, were immunized with acetylcholine receptors (AChR) in CFA to induce myasthenia gravis. Both strains had equivalent concentration of serum autoantibodies to muscle AChR and antibodies bound to muscle AChR. C5-sufficient B10.D2/nSn, but not C5-deficient B10.D2/oSn, demonstrated increased incidence of clinical disease and death and lost significant amounts of muscle AChR. Therefore, C5 deficiency in B10.D2/oSn prevented EAMG. C5 gene, which codes for C component C5, may influence EAMG pathogenesis through activation of the terminal lytic C sequence (C5 to C9) required for muscle AChR destruction, which is the primary pathology.

Animals↗
PubMed1981

Host modification of Sindbis virus sialic acid content influences alternative complement pathway activation and virus clearance.

Previous studies have shown that Sindbis virus, an enveloped alphavirus of the togavirus group, activates the alternative complement pathway in the absence of detectable antiviral immunoglobulin. The present studies examined the role of the host-determined sialic acid content of Sindbis virus on activation of the alternative complement pathway. Purified Sindbis virus grown in baby hamster kidney (BHK-SV) and in mosquito (MOSQ-SV) cells yielded virus with 10.2 and less than 2.0 nmol sialic acid/mg viral protein, respectively. Sindbis virus deficient in sialic acid (2.0 nmol sialic/mg) was also produced by treating the BHK-SV with neuraminidase (NANase-SV). When MOSQ-SV or NANase-SV was incubated in either C4DGPS or C2DHS, each consumed significantly more C3 than did BHK-SV, indicating that the ability of Sindbis virus to activate the alternative pathway is inversely related to its sialic acid content. Studies in vivo showed that virus deficient in sialic acid (MOSQ-SV) was cleared from the blood of mice much more efficiently than was virus rich in sialic acid (BHK-SV), after i.v. inoculation. Furthermore, when animals were depleted of C3 through C9 by cobra venom factor (CoVF) treatment, no differences in the clearance of high and low sialic acid-containing viruses were observed. Thus both the activation in vitro and complement-dependent clearance in vivo are significantly affected by the host-determined sialic acid content of Sindbis virus.

Animals↗
PubMed1987

Persistently circulating C3 nephritic factor (C3 NeF)-stabilized alternative pathway C3 convertase (C3 CoF) in serum of an 11-year-old girl with meningococcal septicemia--simultaneous occurrence with free C3 NeF.

Hemolytic complement was found to be absent in the serum of an 11-yr-old girl (R.N.) with meningococcal septicemia. C1, C4, and C2 were slightly decreased, C3 was absent, C5-C9 within the normal range. B levels immunochemically and electrophoretic mobility of B were normal. C3d was greater than 1000% of a pooled EDTA-plasma standard indicating hypercatabolism of C3. On incubation of the patient's serum with normal human serum activation of C3 occurred even in the presence of 0.04 M EDTA. The amount of C3b generated was, however, greater without any chelating agent or in Mg-EGTA. On gel filtration of the serum two protein containing peaks were found to be responsible for activation of C3: the IgG containing peak was able to activate C3 in normal human serum without chelating agents and in Mg-EGTA but not in the presence of EDTA. The IgM-containing peak activated the third component of complement even in the presence of EDTA. The factor responsible for this phenomenon was termed C3 converting factor (C3 CoF). The IgG fraction of the patients serum caused activation of C3 in Mg-EGTA. However, in the presence of EDTA no activation of C3 could be induced even if physiological concentrations of the patients IgG were added to normal human EDTA-plasma. Thus the activity of the patient's IgG did not differ from typical C3 nephritic factor. The decay of C2 in EAC42 intermediates in the presence of the patient's IgG was uninfluenced indicating that it did not carry autoantibody activity against the classical pathway convertase C4b,2a, an activity recently termed NFc.(ABSTRACT TRUNCATED AT 250 WORDS)

Child↗
PubMed1986

The interaction of Escherichia coli with normal human serum: the kinetics of serum-mediated lipopolysaccharide release and its dissociation from bacterial killing.

We have examined the killing of E. coli and kinetics of lipopolysaccharide (LPS) release after the exposure of the bacteria to normal human serum (NHS) and sera deficient in complement components, or with inactivated complement components. LPS of the galactose epimerase-deficient strain E. coli J5 were specifically radiolabeled by growing the bacteria in a medium containing [3H]galactose. Exposure of the washed bacteria to NHS resulted in a significant reduction (greater than 99%) in viability within 15 min and the concomitant release of radiolabeled LPS. However, maximal release of LPS was consistently 30% of the total radiolabel incorporated into the LPS molecules. The amount of tritium-labeled LPS released was shown to be directly proportional to the concentration of bacteria exposed to NHS, suggesting that release of LPS was not limited by the availability of some critical serum component(s). The consumption of complement in NHS by incubation with E. coli was demonstrated by decreased alternative and classical pathway-specific hemolytic activity. The use of Factor D-depleted and VEM-treated human sera demonstrated that, with these bacteria, both the alternative and classical pathways of complement contribute to bacterial killing and release of LPS. It is noteworthy that, in VEM-treated and Factor D-depleted sera, the rate of killing and the kinetics of LPS release were somewhat slower as compared to control serum. Bacterial killing in C7-depleted and C9-deficient human sera was minimal. Neither killing nor LPS release occurred in heat-inactivated (56 degrees C, 30 min) human serum. The amount of [3H]LPS released by C9-deficient serum was qualitatively similar to the amount released by the action of NHS. Tritium-labeled LPS was not released in C7-depleted serum. These data indicate that bacterial killing can be dissociated from LPS release, and suggest that, whereas LPS release may be necessary for the bactericidal effects of serum complement, it is probably not sufficient to effect killing. Furthermore, a significant fraction of LPS can be removed from the outer membrane of the bacteria without an apparent affect on viability.

Adult↗
PubMed2000

Identification of mutations in rat CD59 that increase the complement regulatory activity.

Formation of the membrane attack complex (MAC) of complement on host cells is inhibited by the glycosylphosphatidylinositol- (GPI-) anchored glycoprotein CD59. Published data on the active site of human CD59 are confusing. To clarify these data, we set out to elucidate the active site of a nonprimate CD59 molecule by site-directed mutagenesis. We also undertook to investigate a region of potential species selectivity, and to this end rat CD59 was chosen for all mutations. Our investigations confirmed the proposal that the active site of CD59 is the major hydrophobic groove, with mutations Y36A, W40A, and L54A ablating complement inhibitory function of CD59. Other mutations reducing the function of rat CD59 were I56E, D24A, and D24R. Importantly, mutations at one residue increased the function of rat CD59. The K48E mutation significantly increased function against human rat or rabbit serum, whereas the K48A mutation increased function against human serum alone. A similar mutation in human CD59 (N48E) had no effect on activity against human or rat serum but completely abolished all activity against rabbit serum. These findings suggest that the alpha-helix of human CD59, adjacent to the hydrophobic groove, influences the interaction between human CD59 and rabbit C8, C9, or both.

Amino Acid Substitution↗
PubMed1973

Formation of a hemolytically active cellular intermediate by the interaction between properdin factors B and D and the activated third component of complement.

The present studies demonstrate that the factor B-dependent C3 convertase can be affixed to an erythrocyte by use of an intermediate bearing C3b and that this convertase brings the hemolytic reaction to completion with an efficiency comparable to that of classical convertase. The evidence that the EAC43 intermediate was lysed by a new pathway includes requirements for factors B and D and cell-bound C3b for subsequent lysis by the terminal components, C3-C9. The linear stoichiometry of the effective molecule titrations of C3b and factor B, and the first-order kinetics displayed by the generation and decay of the factor B-dependent hemolytic site are characteristics consistent with the one-hit theory as initially developed for the classical complement system. The use of hemolytically active cellular intermediates to examine the reactions occurring with C3b and factors B and D has allowed extension of the one-hit theory to this molecular sequence, development of effective molecule titrations, recognition of the analogies to the functional characteristics of the classical C3 convertase, and discrimination of the probable mechanism of terminal complement activation from reactive lysis.

Complement System Proteins↗
PubMed1997

Presence of immunoglobulins, C3 and cytolytic C5b-9 complement components on the surface of erythrocytes from patients with beta-thalassaemia/HbE disease.

The occurrence of IgG, IgM, IgA, C3 and C5b-9 complement complexes on erythrocytes from 43 patients with beta-thalassaemia HbE disease was investigated. Indirect immunoradiometric assays using radioiodinated protein A were employed to quantify the individual components. We confirmed that circulating erythrocytes from thalassaemic patients contained elevated amounts of IgG, and small but significant amounts of C3. In addition, small but significant amounts of C5b-9 were detected. Levels of cell-bound IgG, C3 and C5b-9 were higher in splenectomized versus non-splenectomized patients. The presence of C5b-9 on circulating cells from five splenectomized patients was confirmed by an ELISA employing a monoclonal antibody specific for a C5b-9 neoantigen. When C5b-9 positive cells from two patients were solubilized with detergent and subjected to sucrose density gradient centrifugation, the terminal complexes sedimented as 25-40S macromolecules, thus behaving as membrane C5b-9 complexes. The presence of C8 and C9 in these high molecular weight fractions was directly demonstrated by Western blotting. These results constitute the first demonstration that circulating diseased erythrocytes may carry low numbers of potentially cytolytic C5b-9 complement complexes which may be partly responsible for the known ionic disturbances found in thalassaemic cells. Both bound C3 and C5b-9 could promote removal of diseased cells in the reticuloendothelial system.

Adult↗
PubMed1993

Complement activation in septic baboons detected by neoepitope-specific assays for C3b/iC3b/C3c, C5a and the terminal C5b-9 complement complex (TCC).

We have investigated the cross-reactivity of various species in neoepitope-specific methods for quantification of human complement activation products. In contrast to most other species examined, baboon showed a substantial cross-reactivity supporting a high degree of homology between human and baboon complement. An assay for C3b, iC3b and C3c (MoAb bH6) showed moderately good reactivity, in contrast to a C3a assay which did not cross-react. Excellent reactivity was found for C5a using MoAbs C17/5 and G25/2. The reactivity of an established TCC assay (MoAb aE11 to a C9 neoepitope and polyclonal antibody to C5) was improved substantially by replacing the anti-C5 antibody with a new MoAb to C6 particularly selected on the basis of baboon cross-reactivity. Plasma samples from baboons receiving 2.5 x 10(9) and 1.0 x 10(10) live Escherichia coli bacteria/kg were examined with the assays described. In vivo complement activation with the lowest dose was moderate and kept under control, in contrast to the highest dose, where an uncontrolled increase in all activation products continued throughout the infusion period. These results support the hypothesis that sufficiently high amounts of endotoxin lead to uncontrolled activation of complement as seen in irreversible septic shock. The results are discussed with particular emphasis on activation of the terminal complement pathway.

Animals↗
PubMed1984

Deposition of the membrane attack complex of complement in bullous pemphigoid.

Bullous pemphigoid is associated with deposition of IgG and C3 at the dermal-epidermal junction. In order to see whether complement activation in bullous pemphigoid resulted in deposition of membrane attack complex (MAC) at the basement membrane zone, skin biopsies from patients with bullous pemphigoid were examined using a direct immunofluorescence technique. By employing a monoclonal antibody to a neoantigen of C9, the MAC was demonstrated in linear pattern at the basement membrane zone. These deposits were seen in both involved and uninvolved skin but the amount of MAC was greater in involved skin as judged by intensity of staining. Stippled deposits of MAC were also present in or around epidermal basal cells. The MAC could be generated in vitro by reaction of normal plasma with antibasement membrane antibody bound to sections of monkey esophagus. The IgG antibody activated complement and this complement activation proceeded all the way to the terminal step.

Basement Membrane↗
PubMed1984

Molecular composition of the tubular structure of the membrane attack complex of complement.

The composition of the tubular structure of the membrane attack complex of complement (MAC) which migrates as a high molecular weight band (Mr approximately 1.2- 1.3 X 10(6) upon sodium dodecyl sulfate, polyacrylamide gel electrophoresis under reducing conditions was analyzed and compared to the high molecular weight band (Mr approximately 1.1 X 10(6] of tubular poly(C9). The sodium dodecyl sulfate-resistant band of the MAC, designated MAC-poly(C9), is composed of C6, C7, C8 alpha-gamma, and poly(C9), in approximate molar ratios of the protomers of 1:1:1:10-18. This conclusion is based 1) on the results of the incorporation of labeled proteins into MAC-poly(C9); 2) on the immunostaining of MAC-poly(C9) with anti-C6, anti-C7, anti-C8 alpha-gamma, and anti-C9 and its lack of immunostaining with anti-C5 and anti-C8 beta; and 3) on the dissociation of MAC-poly(C9) to 1 mol of C6, C7, C8 alpha-gamma and 10 to 18 mol of C9 upon treatment with 8 M guanidine isothiocyanate. Ultrastructurally the sodium dodecyl sulfate-resistant poly(C9) tubule and MAC-poly(C9) tubule are indistinguishable, suggesting a similar ultrastructure of the C6, C7, C8 alpha-gamma, and C9 subunits in the MAC-poly(C9) tubule. Further analogies among these four proteins are their tendency to form disulfide-linked dimers. It is concluded that the transmembrane channel of the MAC is formed by a tubule in which C6, C7, C8 alpha-gamma are copolymerized with poly(C9), whereas the C5b and C8 beta subunits are not part of the tubule structure and may form the 170-A long appendage of the MAC. This appendage is dissociated upon boiling in sodium dodecyl sulfate whereas the tubule remains stable.

Complement Membrane Attack Complex↗
PubMed1977

Activation of complement by opportunist pathogens and chemotypes of Salmonella minnesota.

Washed cells of Staphylococcus aureus, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, and Salmonella minnesota chemotypes (S, Rb, and Re) were tested for their ability to activate the alternative complement pathway (ACP). Parameters of ACP activation were (i) conversion of C3 in 10 mM ethyleneglycol-bis-(beta-aminoethyl ether)-N,N1-tetraacetic acid-treated human serum supplemented with 2.5 mM MgCl2, (ii) lysis of glutathione-treated human erythrocytes in the presence of human serum, and (iii) C3 to C9 consumption in C4-deficient guinea pig serum. With the exception of S. minnesota Re and S. aureus, all of the strains were highly active in the test systems when compared with inulin. S. minnesota Re and S. aureus initiated C3 conversion in untreated human serum, suggesting that these microorganisms were capable of activating complement by a mechanism other than the ACP. These results provide direct evidence for ACP activation by opportunist gram-negative bacilli and refute the hypothesis that the lipid A moiety of the lipopolysaccharide cell wall is responsible for ACP activation.

Adult↗