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PubMed1993

Antibody response to meningococcal polysaccharides A and C in patients with complement defects.

Patients with defects of terminal complement components are particularly exposed to the risk of developing neisserial infections and seem to respond poorly to meningococcal capsular polysaccharide (PS) C via natural immunization. The sole meningococcal PSC is, on the other hand, an excellent immunogen in normal people. Considering the great importance of vaccine prophylaxis for the prevention of meningococcal infections in patients with complement defects, it is crucial to study the antibody response to the sole meningococcal PS in these patients. We therefore analysed the levels of anti-PSA and PSC antibodies in the members of four families including patients with homozygous and heterozygous defects of C7, C8 or factor H, before and after vaccination with the sole PSA + C. Surprisingly, we found the highest levels of antibodies before vaccination in homozygous subjects, followed by heterozygous and normal controls, whereas, after vaccination, homozygous subjects showed the lowest increase of specific antibodies, indicating their relative incapability to respond to sole meningococcal PS. In conclusion, this study demonstrates (1) the capacity to respond to meningococcal PS via natural immunization by patients with total complement defects, and (2) the low responsiveness to meningococcal PS via vaccine immunization by the same patients. We propose that vaccination should be given to patients lacking specific antibodies and their serological response should be assessed. In addition this study confirms previous observations on a likely lower immunogenic power of meningococcal serogroup C via natural immunization compared with the better immunogenicity of the sole PSC.

Adolescent↗

PubMed1986

Fluid-phase assembly of the membrane attack complex of complement.

The dynamics and protein stoichiometry of the fluid-phase assembly of the membrane attack complex of complement were characterized by using light-scattering intensity measurements. The assembly proceeded in an ordered manner with generation of stable and highly reproducible intermediates. In the absence of phospholipid or C8, mixtures of C5b-6 and C7 self-associated to fluid phase-C5b-7 which had a weight-average molecular weight of (4.1 +/- 0.2) X 10(6). This corresponded to an average of nine C5b-7 complexes per particle. The particles appeared heterodisperse on sucrose gradients with S20,W values ranging from 21 to 39 S. Addition of C8 and C9 caused no further aggregation or disassembly of the particles. When excess C8 was added to the aggregated C5b-7, the ratio of C8 incorporated per C5b-7 moiety was 0.98 +/- 0.03. At saturating levels of C9, the C9/C5b-8 ratio in the particles was 7.2 +/- 0.6. Incorporation of C8 caused a small increase in the Z-averaged particle diffusion coefficient [(9.9-10.3) X 10(-8) cm2/s], indicating that it added in a manner that "filled in the gaps" in the C5b-7 particles. C9 caused only small decreases in the particle diffusion coefficient and substantially decreased the f/fmin ratio. The time course for C9 incorporation into fluid phase-C5b-8 indicated an initial rapid phase followed by a slow phase. The rapid phase corresponded to the incorporation of about one C9 for every two C5b-8 complexes. This suggested that one C9 binding site was accessible on about half of the C5b-8 complexes. This may imply that only about half of the C5b-8 complexes were capable of C9 polymerization so that the ratio of C9 incorporated per functional C5b-8 was (14 +/- 2)/1. The initial velocity of the slow phase of C9 addition gave an activation energy of 37 kcal/mol. The activation energy for C5b-8-independent polymerization of C9 had a similar value of 41 kcal/mol. Light-scattering intensity measurements seemed to be a highly reliable method for quantitative characterization of the fluid-phase assembly.

Complement C7↗

PubMed1990

Purification of 1F5 antigen that prevents complement attack on homologous cell membranes.

A mAb, 1F5, has the ability to cause hemolysis by human serum of human E treated with neuraminidase via the alternative C pathway. By Western blotting, this mAb reacts with a glycoprotein having a molecular mass of 20 kDa (1F5Ag). 1F5Ag was isolated from human E by affinity chromatography with mAb-coupled Sepharose. Purified 1F5Ag was then adsorbed to guinea pig E rendering them resistant to human C attack by both the classical and the alternative pathways. Furthermore, experiments with isolated C components revealed that 1F5Ag interferes with both homologous human C8 and C9 in the terminal stage of the C reaction, whereas it has little effect on hemolysis by rabbit C8 and C9. Therefore, 1F5Ag can be called HRF20, which stands for homologous restriction factor of 20 kDa.

Amino Acid Sequence↗

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↗

PubMed1988

Restriction of cell lysis by homologous complement: I. An analysis of membrane attack complex formation on target membranes.

The hemolytic efficiency and binding of C9 to homologous and heterologous erythrocytes was evaluated by using a standardized passive sensitization procedure to prepare antigen- and antibody-coated erythrocytes (EA) and human serum for lysis. Heterologous bovine EA were readily lysed by human serum, whereas human EA were quite resistant to lysis. Human EA bound as many C8 and C9 molecules per cell as bovine EA when incubated under identical conditions, but four times as much bound C9 was required to lyse an equal number of human EA compared with bovine EA. The susceptibility of human erythrocytes did not increase when increased volumes of undiluted human serum were used although C9 binding increased to as much as 100,000 molecules per cell. Sodium dodecyl sulfate-resistant polymerized C9 (poly(C9)) was detected on both lysed ghosts and unlysed EA bearing complement proteins C1 through C9 (EAC1-9) after incubation with undiluted human serum; however, the ratio of poly(C9) to monomeric C9 was higher on unlysed cells than on ghosts. Although bovine and human EA bound equal amounts of human C9 at the end point, the rate of lysis and C9 uptake was slower on homologous cells. The rate-limiting step occurred before C9 binding and lysis because the rates of lysis and C9 binding were equal on homologous and heterologous EAC1-8 targets, but the extent of lysis of homologous cells was still lower than lysis of heterologous cells. Human erythrocytes lose restriction against homologous hemolysis during storage in autologous plasma or in isotonic buffers.

Animals↗

PubMed1994

Blocking antibodies specific for human albumin interfere with the hemolytic activity of the membrane attack complex of complement.

Earlier, we showed that antibodies specific for human serum albumin are able to bind to erythrocyte-associated albumin and inhibit complement mediated hemolysis. In the present study we determine if inhibition is occurring at the membrane attack phase of complement activation or at an earlier step. We show that although cell-bound anti-albumin antibodies do not inhibit binding and activation of C3 or uptake of C9, they do appear to cause cells to become refractory to lysis by the membrane attack complex as they inhibit both the kinetics and the extent of hemolysis in a reactive lysis system which employs preformed C5b6 plus C7, C8, and C9. We believe that this is the first report of inhibition of the hemolytic activity of the membrane attack complex by antibodies bound to an erythrocyte surface antigen.

Animals↗

PubMed1986

Single-channel analysis of the conductance fluctuations induced in lipid bilayer membranes by complement proteins C5b-9.

Single-channel analysis of electrical fluctuations induced in planar bilayer membranes by the purified human complement proteins C5b6, C7, C8, and C9 have been analyzed. Reconstitution experiments with lipid bilayer membranes showed that the C5b-9 proteins formed pores only if all proteins were present at one side of the membrane. The complement pores had an average single-channel conductance of 3.1 nS at 0.15 M KCl. The histogram of the complement pores suggested a substantial variation of the size of the single channel. The linear relationship between single-channel conductance at fixed ionic strength and the aqueous mobility of the ions in the bulk aqueous phase indicated that the ions move inside the complement pore in a manner similar to the way they move in the aqueous phase. The minimum diameter of the pores as judged from the conductance data is approximately 3 nm. The complement channels showed no apparent voltage control or regulation up to transmembrane potentials of 100 mV. At neutral pH the pore is three to four times more permeable for alkali ions than for chloride, which may be explained by the existence of fixed negatively charged groups in or near the pore. The significance of these observations to current molecular models of the membrane lesion formed by these cytolytic serum proteins is considered.

Complement Membrane Attack Complex↗

PubMed1996

The mechanism of platelet aggregation induced by HLA-related antibodies.

Immune-mediated platelet activation is emerging as an important pathogenic mechanism of thrombosis. In vitro studies have suggested two distinct pathways for immune-mediated platelet activation; one involving clustering of platelet Fc gamma RIIa, the other involving platelet-associated complement activation. HLA-related antibodies have been shown to cause platelet aggregation, but the mechanism has not been clarified. We evaluated the mechanism of platelet aggregation induced by HLA-related antibodies from nine patients. Antibody to platelet Fc gamma RIIa failed to block platelet aggregation with 8/9 samples, indicating that engagement of platelet Fc gamma RIIa is not necessary for the platelet aggregation induced by HLA-related antibodies. In contrast, platelet aggregation was blocked by antibodies to human C8 (5/7) or C9 (7/7). F(ab')2 fragments of patient IgG failed to induce platelet activation although they bound to HLA antigen on platelets. Intact patient IgG failed to aggregate washed platelets unless aged serum was added. The activating IgG could be adsorbed by incubation with lymphocytes and eluted from the lymphocytes. These results indicate that complement activation is involved in the aggregation response to HLA-related antibodies. This is the first demonstration of complement-mediated platelet aggregation by clinical samples. Five of the patients developed thrombocytopenia in relationship to blood transfusion and two patients developed acute thromboembolic disease, suggesting that these antibodies and the complement-dependent pathway of platelet aggregation may be of clinical significance.

Adult↗

PubMed1988

Complement induces a transient increase in membrane permeability in unlysed erythrocytes.

The effects of low concentrations of human serum on antibody-sensitized sheep erythrocytes (EA) were studied. We report that exposure to low concentrations of serum induced a large but transient increase in the membrane permeability of those EA that do not lyse. This change in the permeability of the erythrocyte membrane resulted in net uptake of Na+ and decrease in cell K+, without affecting the total internal cation content. Although exposure to serum also allowed for net uptake of larger molecules like L-glucose, it did not lead to cell swelling. Experiments with sera genetically deficient in one of the terminal complement components showed that C8, but not C9, was required to produce the observed change in membrane permeability. Therefore, we propose that the C5b-8 complex can mediate the transient increase in permeability observed in unlysed erythrocytes during complement activation by whole serum.

Animals↗

PubMed1990

Functional properties of the asialo-fifth component of human complement.

Removal of exposed, terminal sialic acid (SA) from carbohydrate chains N-glycosidically linked to asparagine residues of highly pure human C5 with bacterial sialidase increased C-mediated hemolysis of antibody-sensitized sheep E maximally 2.77-fold. Sialidase-treated C5 used as a reagent for the titration of C6, C7, C8, and C9 resulted in increased titers of all these components compared to buffer-treated C5. As determined by a fluorometric method, ca. 65% of the SA was enzymically hydrolyzed under optimal conditions. Endoglycosidase F incubated with C5 followed by monosaccharide analyses by anion exchange chromatography with pulsed amperometric detection revealed both high mannose and complex (terminate in SA) oligosaccharides were hydrolyzed; no effect was found on the functional activity of C5. Approximately 4% of the complex oligosaccharides were hydrolyzed from C5. Comparison of sialidase- and buffer-treated C5 decay rates from EAC1gp(4b,oxy2a,3b)hu resulted in two linear components of the decay curve with sialidase-treated C5, but one linear component with buffer-treated C5. Of the sialidase-treated 125I-C5 15% was bound to EAC1gp(4b,oxy2a,3b)hu compared to 9.3% of buffer-treated 125I-C5. Furthermore, 27% of sialidase-treated 125I-C5 was bound to EAC1gp,4bhu compared to 16.6% of buffer-treated 125I-C5, but no lysis occurred after the addition of C6-C9. The mechanism of increased hemolytic activity after removal of SA from C5 is: the Tmax is prolonged at 30 degrees C (ca. 15 min vs 9 min), and a higher percentage of C5 binds to cellular intermediates compared to buffer-treated C5.

Asialoglycoproteins↗

PubMed1979

Membrane attack complex of complement: generation of high-affinity phospholipid binding sites by fusion of five hydrophilic plasma proteins.

The molecular basis of the membranolytic activity of the membrane attack complex (MAC) of complement was investigated. By using density gradient equilibrium ultracentrifugation, the binding of egg yolk lecithin to the isolated MAC and to its intermediate complexes and precursor proteins was measured. No stable phospholipid--protein complexes were formed with the MAC precursor components C5b--6, C7, C8, and C9. Stable complexes of phospholipid and protein were formed by C5b--7, C5b--8, C5b--9, and the MAC (C5b--9 dimer) and they exhibited densities of 1.2164, 1.184, 1.2055, and 1.2275 g/ml, respectively. The molar phospholipid/protein ratios for the four complexes were determined to be: C5b--7, 399:1, C5b--5, 841:1; C5b--9, 918:1; and C5b--9 dimer, 1460:1. Electron microscopy of the isolated phospholipid--protein complexes revealed no lipid bilayer structures. The magnitude of the phospholipid binding capacity of the MAC is consistent with the interpretation that the MAC forms phospholipid--protein mixed in micelles in lipid bilayers and biological membranes and thus causes formation of hydrophilic lipid channels.

Binding Sites↗

PubMed1989

Erythrocyte membrane protein deficiencies in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder characterized by intermittent hemolytic anemia. Membrane abnormalities of blood cells from patients with PNH are the reason for the unusual sensitivity to lysis by autologous plasma complement. A patient with typical clinical disease consistent with PNH is described together with a few strategies and pitfalls for treatment. Commonly used in vitro assays are discussed that document the complement-mediated lysis of aberrant PNH erythrocytes. Membrane-associated proteins that are abnormal in PNH cells, the characteristics of these proteins, and their mechanism(s) of action are described; these include the decay accelerating factor that inhibits the C3/C5 convertases of both complement pathways on cell surfaces, the C8 binding protein that modulates a step in terminal complement lysis, and other proteins that regulate complement-mediated lysis at early or late steps of the complement cascade.

Adult↗

PubMed1985

Enhanced reactive lysis of paroxysmal nocturnal hemoglobinuria erythrocytes by C5b-9 does not involve increased C7 binding or cell-bound C3b.

The most complement (C)-sensitive type of erythrocytes (E) occurring in paroxysmal nocturnal hemoglobinuria (type III PNH E) have previously been found to exhibit approximately twofold to fourfold greater lysis than normal human E when exposed to isolated human C5b6, C7, C8, and C9 (reactive lysis), in the absence of a known source of C3- or C5-convertases or fluid-phase C3. In further studies on the mechanism of this phenomenon, we now report that C5b6-dependent binding of 125I-C7 to two samples of PNH E (greater than 95% type III) is equal to that found with normal human E at each of several C5b6 inputs tested. Lysis developed by excess C8 and C9, however, was consistently greater for the PNH E. Thus, the exaggerated sensitivity of type III PNH E to reactive lysis cannot be explained by abnormally high uptake of C5b6 or C7 from the fluid phase. Rather, the data indicate that cell-bound C5b67 sites are converted to effective hemolytic sites with greater efficiency on type III PNH E than on normal human E, assuming that the distribution of cell-bound C7 throughout both cell populations is similar. In related studies we have addressed the proposal by other investigators that C3b putatively bound to PNH E in vivo might account for their increased sensitivity to reactive lysis in vitro, by analogy to prior observations on C3b-potentiated reactive lysis of sheep E. The latter hypothesis was made more appealing by the recent discovery that type III PNH E lack an integral membrane protein, decay-accelerating factor (DAF), which in normal E accelerates the decay of membrane-bound C3 convertases. Against this hypothesis, however, is our present finding that preincubation of PNH E with four different goat or rabbit polyclonal antibodies to human C3 failed to inhibit the subsequent reactive lysis of these cells. Under these same conditions, the C3b-dependent increment in reactive lysis of sheep EAC4b3b was abrogated by pretreatment with similar dilutions of these anti-C3 antibodies, generally in association with agglutination. Furthermore, sheep EAC4b3b displayed increased 125I-C7 binding in proportion to augmented lysis, in contrast to the findings with PNH E. Therefore, deficiency of DAF in type III PNH E does not adequately explain their supranormal sensitivity to reactive lysis unless DAF can modulate the terminal lytic steps by a mechanism distinct from its effect on C3 convertase decay. Alternatively, type III PNH E could have a more general abnormality in which DAF deficiency is one manifestation and increased sensitivity to reactive lysis is another.

Animals↗

PubMed1977

Hereditary C5 deficiency in man. III. Studies of hemostasis and platelet responses to zymosan.

Platelet-rich-plasma from two hemostatically normal individuals, genetically lacking the fifth component of complement (C5), failed to exhibit normal platelet aggregation, or serotonin release, in the presence of zymosan. This abnormality was found to reside in the C5D plasma rather than in the platelets as demonstrated by the inability of the deficient plasma to activate zymosan for the aggregation of washed normal platelets. The defect could be corrected by the addition of normal plasma, normal serlm, or highly purified human C5. A plasma abnormality similar to that found in the C5D individuals was also noted in plasmas deficient in C3, C6, and C7; whereas C8 plasma D behaved normally. These data suggest that this platelet reaction requires late acting C components, perhaps as the C567 complex, bound to the zymosan particles.

Blood Platelets↗

PubMed1991

Formation of ion-conducting channels by the membrane attack complex proteins of complement.

The effects of sequential additions of purified human complement proteins C5b-6, C7, C8, and C9 to assemble the C5b-9 membrane attack complex (MAC) of complement on electrical properties of planar lipid bilayers have been analyzed. The high resistance state of such membranes was impaired after assembly of large numbers of C5b-8 complexes as indicated by the appearance of rapidly fluctuating membrane currents. The C5b-8 induced conductance was voltage dependent and rectifying at higher voltages. Addition of C9 to membranes with very few C5b-8 complexes caused appearance of few discrete single channels of low conductance (5-25 pS) but after some time very large (greater than 0.5 nS) jumps in conductance could be monitored. This high macroscopic conductance state was dominated by 125-pS channels having a lifetime of approximately 1 s. The high conductance state was not stable and declined again after a period of 1-3 h. Incorporation of MAC extracted from complement-lysed erythrocytes into liposomes and subsequent transformation of such complexes into planar bilayers via an intermediate monolayer state resulted in channels with characteristics similar to the ones produced by sequential assembly of C5b-9. Comparison of the high-conductance C5b-9 channel characteristics (lifetime, ion preference, ionic-strength dependence) with those produced by poly(C9) (the circular or tubular aggregation product of C9) as published by Young, J.D.-E., Z.A. Cohn, and E.R. Podack. (1986. Science [Wash. DC]. 233:184-190.) indicates that the two are significantly different.

Animals↗

PubMed1987

A natural auto-inhibitory factor of the terminal complement pathway in serum of Ctenodactylus gondi.

The serum of Ctenodactylus gondi, a Tunisian rodent, contains a unique inhibitor of the terminal complement pathway. The auto-inhibitor has been partially characterized as a heat-stable euglobulin that is slightly retarded on a DEAE-ion exchange column at pH 7 and elutes as a symmetrical peak on Sephacryl S-300 in the mol. wt region of approximately 200,000. The inhibitor acts by preventing attachment of cytolytic C5b-9 complexes to natural target cells. It does not appear to affect formation and function of C3-convertase, does not exert inhibitory effects at stages later than C5b-7 formation, and also does not prevent formation of SC5b-9 in serum. That the factor prevents attachment of C5b-7/C5b-9 to cells has been demonstrated in hemolysis model systems using sheep EA + human serum, and in the C3-independent reactive lysis system with the use of ELISA methods and quantitative assays with radioiodinated C8. Addition of partially purified inhibitory factor to human sera or to sera of other animal species abolishes the hemolytic activities of these sera. The inhibitory factor of Gondi serum is the first inhibitor of the terminal pathway which has been shown to be capable of preventing cytolysis of cells undergoing complement attack under physiological conditions. The presence of this factor is probably partially responsible for the remarkable susceptibility of C. gondi towards bacterial and parasitic infections.

Animals↗

PubMed1982

Cooperative interaction of factor B and other complement components with mononuclear cells in the antibody-independent lysis of xenogeneic erythrocytes.

Synergistic cytotoxicity is a term used to describe a cytotoxic system in which xenogeneic erythrocyte target cells are lysed in the presence of nonimmune human mononuclear effector cells and antibody-depleted normal human serum. Neither the mononuclear cells nor the serum alone are cytolytic to the target erythrocytes. Previous studies have shown that the serum activity is not immunoglobulin and is heat-labile, suggesting a similarity to serum complement. In this report, sera deficient in various complement components as well as highly purified single complement components were tested with whole mononuclear cell populations and purified monocytes and lymphocytes to further characterize this cytotoxicity system. Whole mononuclear cell populations failed to mediate target cell lysis in sera deficient in C5 or factor B. However, C3-deficient serum, even in the presence of anti-C3 antibody, supported synergistic cytotoxicity normally. Purified lymphocytes were also normally cytotoxic in C3-deficient serum but failed to lyse targets in sera deficient in C5, C7, C8, or depleted of factor B. Purified monocytes failed to lyse the target cells only in factor B-depleted serum and could lyse the target cells in serum-free medium when purified factor B alone was added. Monocyte-mediated cytotoxicity induced by factor B was inhibited 73-100% by adding lymphocytes back to the purified monocytes. Thus, both lymphocytes and monocytes can serve as effector cells in this form of cytotoxicity but require cooperative interaction with different sets of complement components. In addition, lymphocytes can modulate the monocyte-mediated form of target cell lysis associated with factor B.

Animals↗

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↗