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PubMed1987

Inactivation of influenza B virus by normal guinea-pig serum.

Normal guinea-pig serum (GPS) lacking detectable antiviral antibody efficiently neutralized the infectivity of influenza B virus grown in chick embryos or MDCK cells. The inhibitor was heat-labile and sensitive to trypsin digestion. This beta-like inhibitor required Ca2+ and the complement components C1 and C4 for its activity. In contrast, GPS did not inactivate influenza A virus. Influenza B virus from which the neuraminidase activity of the spikes on the viral envelope had been eliminated by trypsin digestion was also inactivated to a level comparable to untreated virus. Complement component C1 alone bound directly to influenza B virus and inhibited its haemagglutinin activity. We suggest that the beta-like inhibitor in GPS is a component of the classical complement pathway which is triggered by the protein moiety of influenza B virus haemagglutinin, leading to virus neutralization.

Animals↗
PubMed1991

The role of lipopolysaccharide in complement-killing of Aeromonas hydrophila strains of serotype O:34.

The role of lipopolysaccharide (LPS) in the susceptibility of Aeromonas hydrophila strains of serotype O:34 to non-immune human serum was investigated using isogenic mutants (serum-sensitive), previously obtained on the basis of phage resistance, and characterized for their surface components. The classical complement pathway was found to be principally involved in the serum-killing of these sensitive strains. LPS preparations from serum-resistant or serum-sensitive strains, or purified core oligosaccharides (low-molecular-mass LPS) inactivated both bactericidal and complement activity of whole serum, while the O-antigen molecules (high-molecular-mass LPS) did not. The results indicate that LPS core oligosaccharide composition contributes to complement resistance of A. hydrophila strains from serotype O:34 with moderate virulence.

Aeromonas hydrophila↗
PubMed1979

Effects of zinc chloride on guinea pig complement component activity in vitro: concentration-dependent inhibition and enhancement.

We have studied the in vitro effects of zinc chloride on the hemolytic activity of each component of the guinea pig classical complement pathway over a wide range (25 to 500 muM) of zinc concentrations. At high concentrations (>200 muM) the activity of all components was strongly inhibited by this metal. Concentrations of 500 muM inhibited C1 and C5 by 80 and 65%, respectively, whereas all other components were inhibited by more than 94%. Zinc chloride at 25 muM produced more varied effects, with C2, C3, and C6 inhibited by 36, 35, and 55%. C7 and C8 were inhibited by approximately 25%, whereas C1, C4, and C9 were not appreciably affected. The activity of the fifth component, on the other hand, was strongly enhanced by the presence of zinc. Concentrations of 25, 50, and 100 muM zinc chloride produced increases of 92, 44, and 18%, respectively, in C5 titers when present during the activation-binding step of this component. Further studies indicated that the activities of cell-bound complement components were unaffected by zinc treatment after activation and/or binding to the sheep erythrocyte surface had occurred. In addition, zinc did not appear to inhibit by causing irreversible denaturation of either total complement proteins or its various components. Rather, it appears that zinc must be present as a reactant during the activation and/or binding step of each component for inhibition or enhancement to occur.

Animals↗
PubMed1985

Immune immobilization of Treponema pallidum: antibody and complement interactions revisited.

The Treponema pallidum immobilization test was designed for serodiagnosis of syphilis and is dependent upon specific antibody and a heat labile component of normal serum. Investigators have shown the component to be dependent upon divalent cations and it is presumed to be complement. Experiments were performed to reevaluate the interactions of antibody and complement and the mechanism of immobilization. The loss of treponemal motility was correlated to the loss of complement activity in the reaction mixture. When motility of treponemes incubated with immune serum IgG and complement had dropped to 50% (3.4 h), 72% of the available complement had been consumed. At the same time, treponemes incubated with normal serum IgG and complement were 82% motile and only 51% of the complement had been consumed. C6 deficient rabbit serum and C4 deficient guinea pig serum were used in conjunction with immune serum IgG to determine which components of the complement cascade were necessary for immobilization. Treponemes were not immobilized by either sera. Results suggest that the heat labile factor in normal sera is complement, that both early and late components of the complement cascade are necessary, and that the reaction proceeds via the classical complement pathway. Although T. pallidum is susceptible to the actions of antibody and complement, the organisms must interact with these components for at least 2 h before immobilization will result.

Animals↗
PubMed1984

Membrane-bound hemagglutinin mediates antibody and complement-dependent lysis of influenza virus-treated human platelets in autologous serum.

Influenza A virus-treated human platelets were lyzed in autologous serum. Lysis required the presence of antibody and occurred predominantly through activation of the classical complement pathway. Binding of the virus followed by its elution at 37 degrees C resulted in a dose-dependent desialation of the cells with a maximal release of 45% of total platelet sialic acid. In contrast, platelets that had been treated with Vibrio cholerae neuraminidase and from which 55% of total sialic acid had been removed were not lyzed in autologous serum and did not bind C3 as shown in binding assays using radiolabeled monoclonal anti-C3 antibody. Thus, the immune-mediated lysis of virus-treated platelets in autologous serum did not involve neoantigens expressed by desialated cells. To assess the effect of viruses on the platelet surface, treated platelets were incubated with galactose oxidase and sodium [3H]borohydride prior to separation and analysis of the labeled glycoproteins by SDS-PAGE. Viral treatment resulted in a desialation of each of the surface glycoproteins. At the same time, a labeled component of Mr 72,000 (nonreduced) and Mr 55,000 (reduced) was observed that was not present when V. cholerae-desialated platelets were examined in the same way. Immunoblotting experiments performed using antiwhole virus and anti-hemagglutinin antibodies demonstrated this component to be viral hemagglutinin. Involvement of membrane-bound hemagglutinin in antibody and in complement-mediated lysis of virus-treated platelets in autologous serum was supported by the increased lytic activity of a postvaccinal serum containing an elevated titer of complement fixing anti-hemagglutinin antibodies. Binding of a viral protein to the platelet surface provides a model for immune thrombocytopenias occurring during acute viral infections at the time of the specific immune response.

Adult↗
PubMed1983

The role of complement in the opsonization of mucoid and non-mucoid strains of Pseudomonas aeruginosa.

Requirements for complement and/or antibody for opsonization were assessed for 34 strains of Pseudomonas aeruginosa. Included were mucoid strains from patients with cystic fibrosis (CF), non-mucoid derivatives of these strains, and non-CF strains with classical morphology. Non-CF strains are known to vary as to opsonic requirements, but this study shows that mucoid strains are also diverse. Among the 14 mucoid strains, five could not be opsonized and completely resisted phagocytosis. All non-mucoid strains can be opsonized. When the bacteria were incubated in fresh human serum and stained with fluorescein, conjugated anti-C3 non-opsonizable strains did not bind C3 on the surface whereas five of six mucoid strains, which could be opsonized by complement alone, stained with anti-C3. In mucoid strains, surface characteristics correlate with differences in functional requirements for opsonization. In non-CF strains this specificity was not seen. Most mucoid strains required an intact classical complement pathway for opsonization. A number of mucoid strains could not be opsonized in the absence of a functional alternative complement pathway whereas in contrast, non-CF strains were not greatly affected by inactivation of the alternative pathway.

Antibodies, Bacterial↗
PubMed1990

Evidence of IgG-mediated enhancement of the antibody response in vivo without complement activation via the classical pathway.

The complement (C) dependency of IgG-mediated enhancement of the antibody response was investigated by immunizing mice with trinitrophenyl-coupled keyhole limpet hemocyanin (TNP-KLH) and either a C-activating TNP-specific monoclonal IgG2a antibody (Hy-1.2) or a mutant, non-C-activating variant of Hy-1.2 (M12). Hy-1.2 as well as M12 efficiently enhanced the anti-KLH response, although Hy-1.2 was more active. In addition, also a naturally non-C-activating TNP-specific IgG1 antibody enhanced the response to TNP-coupled bovine serum albumin. Moreover, C-activating IgG could enhance the antibody response in mice depleted of C3 by treatment with cobra venom factor. These findings suggest that the classical pathway of C activation is not required for IgG-mediated enhancement.

Animals↗
PubMed1990

A mechanism of activation of the alternative complement pathway by the classical pathway: protection of C3b from inactivation by covalent attachment to C4b.

In this work we studied the role of the classical pathway complement component C4b in the activation of the alternative pathway. It was found that nascent C3b attaches with high efficiency to C4b and that C3b in C4bC3b complexes is protected from inactivation by factors H and I. Activation of C3 by factors B and D in the presence of Mg2+ ions and excess C4b led to 35% incorporation of nascent C3b into C4bC3b complexes in the fluid phase. In comparison, when human IgG was tested as an acceptor under similar conditions, only 12% of generated C3b was incorporated into IgGC3b complexes. The half-life time of dissociation of C3b from purified C4bC3b complexes was approximately 2.3 h at 37 degrees C. C4b in these complexes protected C3b from inactivation as effectively as any known alternative pathway activator. Thus, C3b bound to C4b was tenfold more stable than free C3b or C3b bound to a nonactivating surface. In comparison, the protection provided by attachment to human IgG was only 67% of that of C4b. The results provide an explanation for observations of alternative pathway recruitment following classical pathway activation and for the stability of the classical pathway C5 convertase on surfaces which do not provide protection for C3b from factors H and I.

Complement C3-C5 Convertases↗
PubMed1986

Effect of the nephrotic syndrome on the concentration of serum complement components.

The concentration of 12 component and four control proteins of the complement system was measured in serum from 43 children with a nephrotic syndrome, which subsequently proved to be steroid-responsive, and from 13 children with focal glomerulosclerosis (FGS) and was compared with values from 197 normal subjects. Of classical pathway complement components, 40% of patients had low C1q levels and 20%, low C2 levels. Mean serum levels of C1s, C4, C1INH, and C4bp were elevated. Of alternative pathway components, factors B and I were low in one third, while levels of C3 and H were commonly elevated. Of the terminal components, only C8 and C9 were low. In five patients with FGS with hypoalbuminemia without edema, all component levels were normal. With the exception of C1q, C1s, and C8, high molecular weight (mol wt) components were in high concentration and low mol wt components in low concentration. The three exceptions may be explained by the subunit structure of C1 and C8. From a practical standpoint, the study indicates that edematous patients with a nephrotic syndrome may have low serum levels of C1q and C2, simulating classical pathway complement activation such as commonly occurs in glomerulonephritis. However, low levels of C4, and possibly C1s, can be used as indicators of classical pathway activation since their levels are not reduced by a nephrotic syndrome.

Adolescent↗
PubMed1983

Neutralization of influenza virus by normal human sera: mechanisms involving antibody and complement.

All normal human sera examined neutralized WS/33 H1N1 influenza virus efficiently by one of two antibody-dependent mechanisms. A minority of the sera contained moderate levels of IgG antibody directed against the viral hemagglutinin that had the ability to directly neutralize the virus. The majority of sera tested contained very low levels of IgG anti-hemagglutinin antibody, which was detectable with a specific ELISA but not by conventional HAI assays. Such IgG antibody was unable to directly neutralize the virus. Studies with agammaglobulinemic serum and with sera depleted of and reconstituted with complement components established essential roles for IgG and the components of the classical complement pathway through C3 for neutralization. The components of the alternative and membrane attack pathways were not needed for neutralization. As anticipated from the requirement for IgG and exclusive mediation of neutralization by the classical pathway, the virus-IgG immune complex activated purified C1. Binding of C3 and C4 to the virus was demonstrated, as was classical pathway-mediated triggering of the alternative pathway, with recruitment of properdin. In addition, the H1N1 influenza virus also directly activated the alternative complement pathway in human serum, leading to C3 and properdin deposition on the viral envelope. Such direct alternative pathway activation also required immunoglobulin. However, the alternative pathway alone was unable to neutralize the virus. Thus, most normal sera examined contain low levels of IgG anti-hemagglutinin antibody, which activate the classical pathway of the complement system and neutralize WS/33 influenza virus by deposition of C3 and C4 on the viral envelope.

Agammaglobulinemia↗
PubMed1987

The classical pathway of complement prevents the formation of insoluble antigen-antibody complexes: biological implications.

There is now clear evidence for a complement dependent physiological system which is capable of processing immune complexes in and outside the vascular compartment so that they remain soluble, and transporting such complexes to the fixed macrophage system where they are safely eliminated. Defects in physiological immune complex disposal can occur at various stages described in this article, and it could well be that several of these stages could present subtle defects which are, however, additive so that under a given set of circumstances immune complexes end up in the wrong places, i.e. outside the fixed macrophage system.

Animals↗
PubMed1984

Simplified assays of hemolytic activity of the classical and alternative complement pathways.

Simplified hemolytic assays for the classical (CP) and alternative (AP) pathways of complement (C) were developed. The CP function was tested with sensitized sheep erythrocytes in a diluent containing Ca2+ and Mg2+, while AP was tested with unsensitized rabbit erythrocytes in a diluent containing Mg2+-EGTA. In contrast to the commonly used hemolytic titration (CH50) assays, the present techniques tested the activity in reaction mixtures containing C at final dilutions which would not affect its function. These ranges fell between 1/1 and approximately 1/20 for CP and between 1/1 and approximately 1/3 for AP. With the adopted assay techniques single aliquots of serum were tested at single final dilutions of 1/8 for CP and 1/2 for AP, in the presence of excess target cells. Hemolysis was allowed to take place at 37 degrees C for 20 min. The number of cells lysed by CP and AP under these conditions was directly proportional to the dose of serum and unaffected by the presence of a large excess of target cells. Each pathway was tested independently of the other. Serum C levels, measured as described, correlated strongly with those determined by standard hemolytic titration (CH50) assays. The modified assays should offer less laborious alternatives for the functional assay of C than current routine procedures.

Animals↗
PubMed1985

Suppression of mouse complement activity by contaminants of technical grade pentachlorophenol.

Pentachlorophenol (PCP) is an antimicrobial agent used chiefly for the preservation of wood. Subchronic oral exposure (14 days) to Technical Grade PCP significantly inhibited the functional activity of female B6C3F1 mouse complement when measured in a microtiter hemolytic assay. When evaluated one day following the final exposure the highest administered dose (100 mg/kg) significantly suppressed the Classical complement pathway, the Spontaneous C1 autoactivation pathway, the Alternate pathway and the level of complement component, C3. Reconstitution studies using C5-deficient serum also demonstrated deleterious effects on this complement component. The Classical pathway was the most sensitive to Technical Grade PCP effects. Animals treated with 100 mg/kg Technical Grade PCP had CH 50 levels 30% of vehicle controls. Animals treated for 14 days and allowed a 15 day recovery period had CH 50 values 36% of control and animals which recovered for 30 days had only 52% of the complement activity of control animals. C3 recovery studies also demonstrated continued suppression on days 15 and 30 post-final exposure. Doses of 10 and 30 mg/kg did not produce the marked effects observed with the highest dose; however, a dose-dependent trend was observed for all responses. Animals treated with 100 mg/kg of EC-7, a PCP preparation with reduced amounts of contaminating dioxins and dibenzofurans, did not demonstrate detrimental effects on the complement system.

Animals↗
PubMed1986

Development of a rapid kinetic assay for the function of the classical pathway of the complement system and for C2 and C4.

A simple functional complement assay (FCA) is described which is based on kinetic turbidimetry. 25 microliter of sample are combined with 250 microliter of a suspension of antibody coated ovine erythrocytes in a microprocessor controlled photometer. After a lag phase the activation of complement leads to a rapid lysis of the cells which is measured photometrically at 578 nm. The time for a decrease of absorbance of 0.1 is defined as the endpoint of the reaction. Normal citrated plasma which is the preferred specimen for the assay shows a reaction time of about 42 sec whereas pathological or diluted samples show a prolongation. With specific antibodies it was demonstrated that the assay was sensitive for a deficiency of factors of the classical pathway. Factors of the alternative pathway do not influence the assay. In combination with deficient sera a modified form of the assay allows also the determination of C2 and C4. Because of its simplicity, speed and accuracy these assays may be well suited to perform functional complement assays in a routine laboratory.

Animals↗
PubMed1986

The activation of C5 in the fluid phase and in the absence of C3 through the classical pathway of the complement system.

Unsensitized guinea-pig erythrocytes (Egp) were lysed by a combination of eight isolated, human-derived complement components, Cls, C4, C2, C5, C6, C7, C8 and C9 (Cls-C9exC3), even in the presence of anti-C3. It was determined that a factor was generated in the reaction mixture of Cls, C4, C2, C5 and C6, which had a lytic activity against Egp when C7, C8 and C9 were added. The lytic factor was similar to C56 in the following properties: the activity of the lytic factor decreased when incubated with C7 prior to its reaction with Egp, the lytic factor did not bind to Egp by itself but it did bind in the presence of C7, EDTA did not have any inhibitory effect on the lytic factor, and the activity of the lytic factor was lost by treatment with anti-C5 or anti-C6 but not by treatment with anti-C4. Furthermore, C5a, a cleavage product of C5, was clearly detected in the reaction mixture of Cls, C4, C2 and C5. These findings indicate that C5 can be activated proteolytically into C5a and C5b in the fluid phase solely by the classical pathway C3 convertase, C42, without any participation of C3.

Animals↗
PubMed1988

Pulmonary alveolar type II epithelial cells synthesize and secrete proteins of the classical and alternative complement pathways.

The serum complement system is a major mediator of inflammation reactions. Two of the complement proteins, the third (C3) and fifth (C5) components, are precursors of potent phlogistic molecules, C3a and C5a. C5a has potent chemotactic activity and plays an active role in pulmonary inflammation. We present evidence suggesting that several complement proteins, including C5, are synthesized locally in the lung in alveolar type II epithelial cells. Lung tissue from normal mice synthesized and secreted C5 protein similar to the C5 protein in mouse serum, whereas lung tissue from C5-deficient mice did not. Lung tissues from both normal and C5-deficient mice synthesized C3. Rat lung tissue synthesized and secreted C5, as well as C2, C4, C3, and factor B. Cultures of type II cells (95% type II cells, 5% macrophages) regularly synthesized all these proteins. In contrast, cultures of macrophages alone synthesized large amounts of C2 and factor B, and in some experiments C3 and C4, but never C5. The C5 synthesized by the rat cells was slightly larger than serum C5 (200 kD compared with 180 kD) and was not processed to the two-chain molecule seen in serum. Rat lung tissue and purified type II cells contained C5 mRNA with the same molecular mass as the C5 mRNA in rat liver and in mouse lung and liver. Human type II cells also synthesized C5, as well as C2, C4, C3, and factor B. Human pulmonary macrophages synthesized only C2, factor B, and, in some experiments, C3. Synthesis of complement proteins in cells that line the alveolar wall may provide a local source of these proteins for inflammatory responses in the lung. Local synthesis of complement proteins could be regulated independently of the synthesis in the liver.

Animals↗
PubMed1990

C4b-binding protein, a regulatory component of the classical pathway of complement, is an acute-phase protein and is elevated in systemic lupus erythematosus.

A radioimmunoassay using monoclonal and polyclonal antihuman C4b-binding protein (C4BP) antibody was developed to quantitate C4BP in serum. Using the assay, the levels of C4BP in healthy individuals, in patients with systemic lupus erythematosus (SLE), and in acute-phase individuals were determined. The levels of C4BP are significantly elevated in individuals with SLE (186%; p = 0.0001) and are even higher in individuals during the acute phase (286%; p = 0.0001). To confirm whether or not individuals were in the acute-phase response, serum C-reactive protein (CRP) levels were assessed. In the acute-phase response, CRP levels were 100-fold elevated over normals, but did not correlate with increases in C4BP (r = -0.031; p = 0.899). In SLE patients, the CRP levels were significantly, but moderately, elevated (5-fold; p = 0.028). The data indicate that C4BP is an acute-phase reactant and is differentially regulated from CRP during the acute-phase response.

Acute-Phase Proteins↗