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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↗
PubMed1997

Heterogeneity in the complement-dependent bacteriolysis within the species of Borrelia burgdorferi.

Sixteen Borrelia burgdorferi strains, including all three species, were compared in a colorimetric bactericidal assay for their ability to escape the complement-dependent bacteriolysis on incubation in normal human serum free of specific antibodies (NHS). The species B. afzelii was found to be serum resistant (EB1, EB3, FEM1, FEM2, Pko), whereas strains of the species B. garinii were found to be serum sensitive (1/B29, G1, G2, PSth, PBr, PTrob). Six strains, mainly B. burgdorferi sensu stricto, were only partially sensitive (Z25, 297, B31, PKa-I, PBi). All strains activated the complement cascade in NHS, whereas only four strains (G1, G2, PBr, PSth) could activate complement in the presence of EGTA-Mg. After complement activation, covalently bound C3 fragments (C3b, iC3b) were detected on serum-sensitive as well as serum-resistant borrelial strains. Heterogeneity, however, was observed between serum-resistant and serum-sensitive strains with respect to deposition of C6 and C9. Whereas serum-sensitive strains were strongly positive for C6 and C9 and were, therefore, killed by the terminal complement complex (TCC), serum-resistant strains were devoid of C6 and C9 on their cell surface. The serum resistance may, therefore, be due to an absent or only transient formation of TCC on the bacterial surface.

Bacteriolysis↗
PubMed1994

Oxidants generated by the myeloperoxidase-halide system activate the fifth component of human complement, C5.

Hypochlorite and taurine chloramine (T-NCI) convert native fifth component of human complement (C5) to an activated state. This is evident from loss of functional properties of native C5 and acquisition of a binding site for C6 which is characteristic of C5b, the physiological activation fragment of C5. The complex of activated C5 with C6 is capable of combining with the components C7, C8, and C9 forming the cytotoxic terminal complement complex C5-9. The activation of C5 and its assembly with the late reacting complement components has been detected by reactive lysis, i.e. hemolysis of unsensitized red cells upon incubation with the activated C5 and the reacting complement components C6-C9. T-NCl does, however, not cleave any peptide bond in C5 as happens in the physiological activation process but converts the intact protein to the activated form. The conversion is accompanied and probably caused by oxidation of methionine residues in the C5 protein to methionine sulfoxide. Since hypochlorite and T-NCl are biological products generated by the myeloperoxidase-halide system of stimulated leukocytes, the activation of C5 by these agents may be one way to complement activation during inflammation and tissue injury.

Complement Activation↗
PubMed2000

Molecular basis for a link between complement and the vascular complications of diabetes.

Activated terminal complement proteins C5b to C9 form the membrane attack complex (MAC) pore. Insertion of the MAC into endothelial cell membranes causes the release of growth factors that stimulate tissue growth and proliferation. The complement regulatory membrane protein CD59 restricts MAC formation. Because increased cell proliferation characterizes the major chronic vascular complications of human diabetes and because increased glucose levels in diabetes cause protein glycation and impairment of protein function, we investigated whether glycation could inhibit CD59. Glycation-inactivation of CD59 would cause increased MAC deposition and MAC-stimulated cell proliferation. Here, we report that (i) human CD59 is glycated in vivo, (ii) glycated human CD59 loses its MAC-inhibitory function, and (iii) inactivation of CD59 increases MAC-induced growth factor release from endothelial cells. We demonstrate by site-directed mutagenesis that residues K41 and H44 form a preferential glycation motif in human CD59. The presence of this glycation motif in human CD59, but not in CD59 of other species, may help explain the distinct propensity of humans to develop vascular proliferative complications of diabetes.

Amino Acid Sequence↗
PubMed2003

Serum-resistant strains of Borrelia burgdorferi evade complement-mediated killing by expressing a CD59-like complement inhibitory molecule.

Borrelia burgdorferi, the etiological agent of Lyme disease, comprises three genospecies, Borrelia garinii, afzelii, and burgdorferi sensu strictu, that exhibit different pathogenicity and differ in the susceptibility to C-mediated killing. We examined C-sensitive and C-resistant strains of B. burgdorferi for deposition of C3 and late C components by fluorescence microscope and flow cytometry. Despite comparable deposition of C3 on the two strains, the resistant strain exhibited reduced staining for C6 and C7, barely detectable C9, and undetectable poly C9. Based on these findings, we searched for a protein that inhibits assembly of C membrane attack complex and documented an anti-human CD59-reactive molecule on the surface of C-resistant spirochetes by flow cytometry and electron microscopy. A molecule of 80 kDa recognized by polyclonal and monoclonal anti-CD59 Abs was identified in the membrane extract of C-resistant strains by SDS-PAGE and Western blot analysis. The molecule was released from the bacterial wall using deoxycholate and trypsin, suggesting its insertion into the bacterial membrane. The CD59-like molecule acts as C inhibitor on Borrelia because incubation with F(ab')(2) anti-CD59 renders the serum-resistant strain exquisitely susceptible to C-mediated killing and guinea pig erythrocytes bearing C5b-8, unlike the RBC coated with C5b-7, are protected from reactive lysis by the bacterial extract. Western blot analysis revealed preferential binding of the C inhibitory molecule to C9 and weak interaction with C8 beta.

Antibodies, Blocking↗
PubMed1983

[Complement components, whole complement activity, and circulating immune complexes in neoplastic diseases].

Serum levels of complement components(cc), whole complement activity (CH 50), and circulating immune complexes (IC) were measured in 41 patients with neoplastic diseases. The level of cc was higher than in healthy controls; the levels of C1q, C1INA, C4, C3c, C3ACT, C5, and C9 were statistically higher. In patients with lung cancer, the levels of cc were correlated with the clinical stage as well as the performance status. Both the IC serum level and the incidence of high serum IC levels in lung cancer were higher in stage III and IV than in stage I and II. Serum CH 50 was higher than in healthy controls, but not correlated with the clinical stage.

Aged↗
PubMed1996

Evidence for the involvement of complement proteins in platelet aggregation by Streptococcus sanguis NCTC 7863.

We investigated the mechanisms of platelet aggregation by the type strain of Streptococcus sanguis (NCTC 7863). This species is one of the major aetiological agents of infective endocarditis. S. sanguis NCTC 7863 caused aggregation of normal human platelets in vitro following a lag period that varied between donors (7-19 min). Platelet aggregation was dependent on one or more plasma constituents and all the necessary factors gradually became bound to the bacterial surface during the lag period. The length of the lag period was determined by the plasma of the donor and not by a feature of their platelets. Platelet aggregation by S. sanguis NCTC 7863 could be inhibited by heating plasma at 56 degrees C, by treating plasma with cobra venom factor, or by incubating with soluble Complement Receptor 1, all of which inhibit or deplete complement. Complement activation required Mg2+, but not Ca2+ ions and the the cleavage fragment, Ba, of factor B was produced, indicating that the alternative pathway was operative. Zymosan- and S. sanguis-induced aggregation showed similarities, including the same variability in lag times among donors, and absorption of plasma with zymosan prevented the plasma from supporting platelet aggregation by S. sanguis, C3, C9 and vitronectin were found to bind to S. sanguis NCTC 7863, but the latter two were present at very low levels on a non-aggregating strain of S. sanguis, SK96. The rate of assembly of the C5b-9 complex on the NCTC 7863 bacterial surface correlated with the lag time. These data suggest a role for the complement pathway in platelet aggregation by the type strain of S. sanguis.

Complement C1↗
PubMed1996

Activation of the complement cascade and increase of clusterin in the brain following a cortical contusion in the adult rat.

The aim of the present study was to examine the glial cell response and the possible involvement of the complement cascade following a cerebral cortical contusion. The lesion was produced using a standardized weight-drop technique in adult rats. The blood-brain barrier was damaged, as demonstrated by a decrease of immunoreactivity for a tight junction protein normally expressed by endothelial cells of small vessels in the central nervous system. Increased immunoreactivity for microglial (OX42) and astroglial cells (glial fibrillary acidic protein), as well as macrophages expressing ED1-immunoreactivity (IR) were found in the vicinity of the lesion at all postoperative survival times (2-14 days). In the present study complement factor C3d- and C9-IR was found around the lesion, indicating that activation of the complement cascade had taken place. Furthermore, immunoreactivity for the putative complement inhibitor clusterin (sulfated glycoprotein-2) was found in some of the injured neurons. The contralateral hemisphere showed no evidence of the reaction found in the ipsilateral hemisphere. The balance between complement activation and complement inhibitors may have an impact on the degenerative components in the brain following traumatic injury and in particular on the events leading to nerve cell death.

Animals↗
PubMed2007

Conformationally altered hyaluronan restricts complement classical pathway activation by binding to C1q, C1r, C1s, C2, C5 and C9, and suppresses WOX1 expression in prostate DU145 cells.

Linear non-sulfated hyaluronan (HA) does not bind complement proteins yet inhibits their hemolytic function. We have previously induced the complement inhibitory function of HA by heat treatment. However, heated HA readily loses its anti-complementary activity probably due to instantaneous interchain re-association. Here, HA solutions were heated and then freeze-dried. Compared to native HA, heated/freeze-dried HA stably restricted serum complement-mediated hemolysis via the classical pathway, in which serum C1 hemolytic function and C3 activation were blocked. Also, treated HA had a significantly increased binding of component C1q, C1r, C1s, C2, C5, C9, P, D and H. Further, when HA was gel-fractionated by electrophoresis and then freeze-dried, its anti-complementary activity was stably induced. Both native and heated/freeze-dried HA stimulated ERK phosphorylation in prostate DU145 cells. However, treated HA suppressed the expression of tumor suppressors WOX1 and WOX2. Together, HA with an altered conformation stabilizes its inhibition and binding of complement proteins. It may recognize cell surface receptors differently from native HA, thereby differentially regulating the expression of cellular proteins.

Cell Line, Tumor↗
PubMed1977

C567-initiated cytolysis of lymphoid cells: description of the phenomenon and studies on its control by C567 inhibitors.

Cells of the Raji human lymphoblastoid line, when pretreated with the metabolic inhibitor puromycin were found to be susceptible to killing by the isolated proteins of the complement attack mechanism (C5-9). Incubation of 51Cr-labeled lymphoblastoid cells with purified C56 and C7 resulted in the formation of a lymphoblast-C567 (LC567) intermediate, and the addition of purified human C8 and C9 resulted in release of 51Cr from these cells. Serum C567 inhibitors (C567-INH), purified human lipoproteins, and dextran sulfate, each previously shown to inhibit the attachment of the C567 trimolecular complex to erythrocytes, also inhibited the formation of LC567, and as a consequence, C56-initiated cytotoxicity; the polycation protamine sulfate counteracted the inhibitory effect of dextran sulfate. Thus, the potential for damage of bystander nucleated cells exists when C56 is generated in solution, and is influenced by agents known to modulate the hemolytic activity of C567. It is suggested that these mechanisms may be involved in the control of the function as well as the viability of various nucleated cells.

Cell Line↗
PubMed1985

Inside-out vesicles prepared from complement lysed sheep red blood cells.

Antibody sensitized sheep red blood cells were lysed to 68% by diluted human serum. The ghosts were shown to undergo vesiculation induced by low ionic strength buffer and gentle shearing forces like osmotically lysed ghosts. About 45% of the vesicles derived from complement lysed ghosts (as referred to vesicle protein) remained floating on top of a linear 3-18% dextran T110 gradient during ultracentrifugation. Using antibodies as probes for sideness about 90% of the floating vesicles displayed an inside-out orientation. Floating inside-out vesicles bear a slightly reduced but significant number of C9 molecules on their inverted extracellular side of the membrane as compared to the (leaky or collapsed) vesicles banding in the gradient. This finding suggests that the floating is not due to a lack of C5b-9 (under the assumption that all C9 was bound to C5b-9). It is concluded that lysis with diluted human serum may result partly in ineffective C5b-9 complexes and/or in lesions with limited permeability which do not impair vesicle floating on top of polymer gradients.

Animals↗
PubMed1989

Toxoplasma gondii: mechanism of resistance to complement-mediated killing.

Tachyzoites of the obligate intracellular protozoan Toxoplasma gondii are resistant to lysis in non-immune human serum. We have examined the mechanism of this serum resistance in RH and P strain organisms, which differ markedly in virulence, but are equally resistant to serum killing. Rapid, but limited, activation of the alternative complement pathway occurred in non-immune human serum, with deposition of equivalent amounts of C3 on the two strains. C component C3 bound covalently to parasite acceptor molecules via an ester linkage. The predominant form of C3 was iC3b which cannot participate in formation of a lytic C5b-9 complex. Multiple membrane constituents of the tachyzoite of T. gondii may serve as acceptors for the limited amount of C3 deposited during incubation in non-immune serum. When tachyzoites were presensitized with the lytic anti-p30 mAb 7B8, new amide-linked C3-acceptor complexes formed. Nearly equivalent C3 binding but a threefold enhancement of 125I-C9 binding occurred when mAb 7B8 pre-sensitized tachyzoites were compared to native organisms. These results indicate that tachyzoites of T. gondii are serum resistant because of failure to activate C efficiently. Presensitization with a lytic mAb alters the site of complement deposition and augments C5b-9 formation.

Animals↗
PubMed1987

Human alveolar macrophages synthesize active complement components C6, C7, and C8 in vitro.

We investigated whether serum-free human alveolar macrophage cultures synthesize active C6, C7, and C8. There was a significant binding of polyclonal anti-human C6 antibodies to agarose beads incubated with unstimulated macrophages for 24 or 48 h. Endotoxin stimulation of the macrophages was necessary for significant binding of polyclonal anti-C7 and anti-C8 antibodies to agarose beads co-cultured for 48 or 96 h. Two monoclonal antibodies (poly C9-MA and MCaE11) specific for a neoantigen of polymerized C9 in the terminal complement complex (TCC), bound to beads mainly incubated with endotoxin stimulated macrophages. The MCaE11 was more sensitive than the poly C9-MA in detecting the C9 neoantigen on beads incubated with the macrophages or human serum diluted 1:16. We thus conclude that human alveolar macrophages synthesize active C6, C7, and C9 that together with C5 and C9, assemble as the TCC on co-cultured agarose beads. Activation of the alternative pathway on the agarose with generation of fixed C3 and C5 convertases is a prerequisite for the subsequent generation of the TCC.

Adult↗
PubMed1990

Role of the terminal complement pathway in accelerated autologous anti-glomerular basement membrane nephritis.

The terminal complement pathway (C5b to C9) has been demonstrated to have an important role in the mediation of glomerular immune injury in various models of experimental glomerulonephritis. In the present studies, the role of the terminal complement pathway in the accelerated autologous phase of anti-glomerular basement membrane (GBM) nephritis in the rabbit was investigated. Normocomplementemic rabbits and rabbits deficient in C6 (C6D) who are therefore unable to form the terminal complement pathway were immunized with sheep immunoglobulin G (IgG) before being injected with a subnephrotoxic dose of the gamma 2 fraction of sheep anti-rabbit GBM. C6D animals had a delay in the onset of the glomerular injury, as manifested by proteinuria. At 72 hours, controls had a greater degree of proteinuria (15.2 +/- 8.8 mg protein/mg creatinine vs. 2.6 +/- 2.1, P = 0.197), but at 120 hours there were no differences in proteinuria between C6D and control animals (11.1 +/- 3.6 mg protein/mg creatinine vs. 12.2 +/- 6.2, P = 0.89). Light microscopy demonstrated more severe glomerular injury in C6D animals with marked cellular proliferation and large areas of glomerular necrosis. Interestingly, C6D animals had significantly higher levels of sheep IgG remaining in their glomeruli at 120 hours (0.95 +/- 0.12 micrograms sheep IgG/1 x 10(4) glomeruli, N = 11, vs. 0.57 +/- 0.07, N = 11, P = 0.014) and 72 hours (1.22 +/- 0.25 micrograms, N = 3, vs. 0.60 +/- 0.15, N = 3, P = 0.104) compared with 24 hours when there was no difference (1.25 +/- 0.22 micrograms, N = 7, vs. 1.08 +/- 0.14, N = 7, P = 0.53). C6D rabbits had a greater rise in serum creatinine at 120 hours (2.3 +/- 0.5 mg/dl vs. 1.3 +/- 6.4, P = 0.132). We conclude that in C6D animals, the persistence of glomerular immune deposits is responsible for more severe renal injury and renal failure.

Animals↗
PubMed1986

Does complement kill E. coli by producing transmural pores?

Three lines of evidence are presented to indicate that C5b-9 kills serum-sensitive E. coli K 12 cells by generating functional pores across the outer and inner bacterial membrane. First, viable cells carrying C5b-8 complexes are impermeable to o-nitrophenyl-beta-D-galactoside (ONPG), but lose viability and become permeable to this marker upon post-treatment with purified C9 in the absence of lysozyme. Cells killed with colicin E1 or gentamicin are also impermeable to ONPG but take up the marker if they are post-treated with lysozyme-free serum. Second, killing by C5b-9 is highly effective, deposition of only a small number of complexes being lethal. This has been demonstrated in experiments where viable cells carrying 2000-4000 C5b-7 complexes per CFU were permitted to multiply in broth culture, and the daughter generations subsequently treated with purified C8 and C9. Fifty percent killing was observed in the fifth to sixth generation, corresponding to a dilution of C5b-7 complexes to 50-100 molecules/CFU. In the presence of 2 mM EDTA, further dilution of C5b-7 down to 8-30 complexes/CFU still caused 50% killing of daughter cells. Third, treatment of C5b-7 cells with purified CC8 and C9 results in the release of intracellular K+, which commences immediately after addition of C8/C9. This was shown in experiments where C5b-7 cells were packed to high density in saline, post-treated with C8 + C9, and K+ directly measured in the cell supernatants. Based on these results, we propose that C5b-9 pores deposited in the outer bacterial membrane periodically fuse with the inner membrane, the transmural pores thus generated permitting rapid K+ efflux, with cell death ensuing through the collapse of membrane potential.

Bacteriolysis↗
PubMed1982

Studies on the mechanism of bacterial resistance to complement-mediated killing. I. Terminal complement components are deposited and released from Salmonella minnesota S218 without causing bacterial death.

The mechanism of resistance of gram-negative bacteria to killing by complement was investigated. Complement consumption and uptake of purified, radiolabeled complement components on bacteria was studied using a serum- sensitive and a serum-resistant strain of Salmonella minnesota. Twice as many molecules of (125)I C3 were bound per colony-forming unit (CFU) of the smooth, serum-resistant S. minnesota S218 as were bound per CFU of the rough, serum-sensitive S. minnesota Re595 in 10 percent pooled normal human serum (PNHS), although 75-80 percent of C3 was consumed by both organisms. Hemolytic titrations documented total consumption of C9 by 5 min and more than 95 percent consumption of C5 and C7 by 15 min in the reaction with S218 with 10 percent PNHS. In contrast, negligible C5 depletion, 10 percent C7 consumption, and only a 26 percent decrease in C9 titer occurred with the serum-sensitive Re595. Binding of (125)I C5, (125)I C7, and (125)I C9 to S218 and Re595 was measured in 10 percent PNHS. A total of 6,600 molecules C5/CFU, 5,200 molecules C7/CFU, and 3,100 molecules C9/CFU bound to S218 after 5-10 min of incubation at 37 degrees C, but 50-70 percent of the C5, C7, and C9 bound to S218 was released from the organism during incubation at 37 degrees C for 60 min. Binding of 2,000 molecules C5/CFU, 1,900 molecules C7/CFU, and 9,000 molecules C9/CFU to Re595 was achieved by 20 min and was stable. The ratio of bound C9 molecules to bound C7 molecules, measured using (131)I C9 and (125)I C7, was constant for both organisms after 15 min and was 4.3:1 on Re595 and 0.65:1 on S218 in 10 percent PNHS. With addition of increasing amounts of purified, unlabeled (29 to 10 percent PNHS, there was no change in the C9:C7 ratio on Re595. However, with S218 there was a linear increase of the C9:C7 ratio, which approached the ratio on Re595. There was no (14)C release from S218 incubated in PNHS, nor was there evidence by electron microscopy of outer membrane damage to S218. Therefore, S. minnesota S218 is resistant to killing by PNHS, despite the fact that the organism consumes terminal complement components efficiently and that terminal components are deposited on the surface in significant amounts. The C5b-9 complex is released from the surface of S218 without causing lethal outer membrane damage.

Adsorption↗
PubMed1995

Calcium-loaded erythrocytes have a defect in complement regulation distinct from that resulting from exposure to 2-aminoethylisothiouronium bromide.

Calcium-loaded red blood cells (RBCs) previously have been shown to have an increased sensitivity to complement-mediated hemolysis and particularly to lysis mediated by the C5b-9 membrane attack complex (MAC) of complement. Because RBCs exposed to 2-aminoethylisothiouronium bromide (AET) also have been shown to be particularly sensitive to the MAC, a direct comparison of calcium-loaded and AET-treated RBCs was performed. Calcium-loaded and AET-treated RBCs shared a marked increase in sensitivity to lysis by the MAC in two different assays. However, measurements of C5b-7 and C9 binding suggested that different mechanisms were responsible. AET-treated RBCs showed an increase in C9 binding and an increased C9/C7 ratio consistent with functional loss of CD59/membrane inhibitor of reactive lysis (MIRL). In contrast, calcium-loaded RBCs had minimally increased C9 binding that resulted in C9/C7 ratios that were less than those for untreated RBCs, suggesting that CD59/MIRL inactivation had not occurred. When RBCs were incubated in acidified serum, AET-treated cells demonstrated a marked increase in C3b binding and hemolysis that was observed in neither control nor calcium-loaded RBCs. These results suggest that the underlying lesions responsible for an increase in susceptibility to complement-mediated hemolysis are different for calcium-loaded and AET-treated RBCs.

Adult↗
PubMed1979

Complement alterations in inflammatory bowel disease.

A prospective evaluation of the activity of the complement system was undertaken in 32 patients at the time of diagnosis of inflammatory bowel disease, before the onset of therapy. Serum classical pathway components and function were normal, while significant abnormalities of the alternative pathway were found. Depressions of serum properdin and properdin convertase were noted in association with diminished consumption of C3--C9 after reaction with cobra venom. These abnormalities of alternative pathway integrity were most significant in regional enteritis and in ulcerative colitis with extraintestinal complications. Sequential studies extending into clinical remission revealed resolution of all significant abnormalities.

Adolescent↗