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PubMed1993

New procedure for the detection of complement deficiency by ELISA. Analysis of activation pathways and circumvention of rheumatoid factor influence.

A procedure using enzyme-linked immunosorbent assays for the assessment of complement function has been evaluated. The sera investigated were incubated in microtiter plates with solid-phase complement activators. Human polyclonal IgG or monoclonal IgM were used for classical activation pathway assays and Salmonella typhosa lipopolysaccharide (LPS) for alternative activation pathway assays. The analysis focussed on deposition of C9 and properdin as detected with enzyme-conjugated antibodies. In an attempt to avoid spurious results due to rheumatoid factors in patient sera, monoclonal mouse and chicken antibodies were unsuccessfully tested as indicator reagents in the assay with solid-phase IgG. However, the use of solid-phase IgM as an activator completely circumvented the influence of rheumatoid factors. With solid-phase IgG or IgM, properdin deposition occurred in the absence of factor D. A combination of assays is suggested for diagnostic purposes: IgM-coated plates with detection of bound C9 and properdin for the classical pathway and LPS-coated plates with detection of bound properdin for the alternative pathway. The procedure distinguished between defects of the classical activation pathway (C1, C4, C2), the alternative activation pathway (C3, factor B, factor D, properdin) and the terminal components (C5-C9). This analytical approach may be useful for detection of inherited complement deficiency and the assessment of complement function in acquired complement deficiency states.

Complement Fixation Tests↗
PubMed1982

Effects of decomplementation on mercuric chloride-induced glomerulonephritis in Brown-Norway rats.

The course of mercuric chloride-induced immune glomerulonephritis is characterized by complement activation, intensive proteinuria, linear and then granular IgG and C3 deposits in the glomeruli. To assess the role of complement activation in the occurrence of the disease, decomplementation was achieved by intravenous injections of cobra venom factor in rats injected with mercuric chloride. In these animals, proteinuria still appeared while rats were decomplemented by cobra venom factor through the alternative pathway. These rats exhibited linear IgG deposits without detectable C3 deposits. In the rats injected with cobra venom factor alone, no proteinuria, no classical pathway complement activation and no renal IgG or C3 deposits were observed. Therefore, in Brown-Norway rats intoxicated with mercuric chloride, proteinuria appears to be at least in part complement independent.

Animals↗
PubMed2002

Heat shock protein 70 is a potent activator of the human complement system.

According to new hypotheses, extracellular heat shock proteins (Hsps) may represent an ancestral danger signal of cellular death or lysis-activating innate immunity. Recent studies demonstrating a dual role for Hsp70 as both a chaperone and cytokine, inducing potent proinflammatory response in human monocytes, provided support for the hypothesis that extracellular Hsp is a messenger of stress. Our previous work focused on the complement-activating ability of human Hsp60. We demonstrated that Hsp60 complexed with specific antibodies induces a strong classical pathway (CP) activation. Here, we show that another chaperone molecule also possesses complement-activating ability. Solid-phase enzyme-linked immunosorbent assay was applied for the experiments. Human Hsp70 activated the CP independently of antibodies. No complement activation was found in the case of human Hsp90. Our data further support the hypothesis that chaperones may messenger stress to other cells. Complement-like molecules and primitive immune cells appeared together early in evolution. A joint action of these arms of innate immunity in response to free chaperones, the most abundant cellular proteins displaying a stress signal, may further strengthen the effectiveness of immune reactions.

Complement Pathway, Classical↗
PubMed2003

Evasion of phagocytosis through cooperation between two ligand-binding regions in Streptococcus pyogenes M protein.

The M protein of Streptococcus pyogenes is a major bacterial virulence factor that confers resistance to phagocytosis. To analyze how M protein allows evasion of phagocytosis, we used the M22 protein, which has features typical of many M proteins and has two well-characterized regions binding human plasma proteins: the hypervariable NH2-terminal region binds C4b-binding protein (C4BP), which inhibits the classical pathway of complement activation; and an adjacent semivariable region binds IgA-Fc. Characterization of chromosomal S. pyogenes mutants demonstrated that each of the ligand-binding regions contributed to phagocytosis resistance, which could be fully explained as cooperation between the two regions. Deposition of complement on S. pyogenes occurred almost exclusively via the classical pathway, even under nonimmune conditions, but was down-regulated by bacteria-bound C4BP, providing an explanation for the ability of bound C4BP to inhibit phagocytosis. Different opsonizing antisera shared the ability to block binding of both C4BP and IgA, suggesting that the two regions in M22 play important roles also under immune conditions, as targets for protective antibodies. These data indicate that M22 and similar M proteins confer resistance to phagocytosis through ability to bind two components of the human immune system.

Amino Acid Sequence↗
PubMed1998

High dose intravenous immunoglobulin does not affect complement-bacteria interactions.

Pooled IgG preparations for i.v. use (IVIg) have been shown to possess anticomplementary activity in autoimmune and systemic inflammatory diseases. Both in vitro and in vivo, IVIg is a preferential acceptor of activated C4 and C3, thus diverting complement activation from the target surface. We explored the effect of IVIg on complement-bacteria interactions in an attempt both to determine the safety of IVIg preparations in relation to natural immunity to bacteria and to extend our knowledge of the physiologic mechanism of action of IVIg. Using both complement-sensitive and complement-resistant bacterial strains, we investigated the effect of IVIg on C3 binding to bacterial surfaces. In all cases, whether complement could be directly activated by bacteria through the classical or the alternative pathway, IVIg had no effect on the amount of C3 bound to bacteria. In addition, IVIg did not inhibit complement-dependent bacterial lysis. Interestingly, increasing concentrations of IVIg induced an increase in C1q binding, suggesting the presence of low affinity complement-fixing antibacterial Abs in certain preparations. Using serum samples from patients treated with IVIg, complement binding to and lysis of complement-sensitive bacterial strains were not modified as compared with normal controls and pretreatment samples, although a decrease in C3 binding to sensitized human erythrocytes was observed. Our data suggest that IVIg does not affect direct complement-bacteria interactions, although it is a potent agent to use for diversion of complement activation on sensitized target surfaces.

Animals↗
PubMed1996

Distinct characteristics of initiation of the classical and alternative complement pathways by Candida albicans.

Candida albicans is a potent activator of the complement system. The objective of this study was to characterize factors that influence the kinetics for activation of C3 and binding of C3 fragments to C. albicans. Factors that were examined included the surface properties of the yeast and contributions of the classical and alternative complement pathways. The results showed that incubation of hydrophobic, hydrophilic, or germinating yeast cells in normal human serum (NHS) containing radiolabeled C3 led to immediate accumulation of C3 on all three cell types, although the rate of accumulation of C3 on germinating cells was lower. An examination of the sites for early C3 binding showed that classical pathway initiation led to immediate, synchronous binding over the entire cell surface. A blockade of the classical pathway by absorption of putative classical pathway initiators or by chelation of calcium limited activation to the alternative pathway. Binding of C3 solely via the alternative pathway was characterized by a significant lag in the initial binding kinetics. In the absence of classical pathway initiation, the early cellular sites for C3 binding appeared as random, asynchronous foci of C3 that appeared to expand with time. The factor(s) mediating rapid deposition of C3 that was characteristic of the classical pathway initiation was reciprocally cross-absorbed by hydrophilic and hydrophobic C. albicans but was not removed by absorption of NHS with Saccharomyces cerevisiae, encapsulated Cryptococcus neoformans, or nonencapsulated C. neoformans. Delayed binding of C3 produced by absorption of serum was largely reversed by addition to the absorbed serum of immunoglobulin G isolated from NHS, indicating a significant role for a naturally occurring anti-C. albicans immunoglobulin C. in classical pathway initiation.

Antibodies, Fungal↗
PubMed1992

Cold target competition analysis of the classical activation pathway of complement-mediated cytotoxicity: a non-interaction model for competing lysis.

A mathematical analysis of cold target competition experiments of complement-mediated lysis is presented, aimed at developing a minimal model of lysis where no interaction between the competing populations of sensitized blood group A and B erythrocytes is presumed. The model is able to predict the extent of lysis from the input values with remarkable accuracy suggesting that under the conditions used no stimulation and/or inhibition of the lysis of the sensitized erythrocytes occurs. The distribution of complement between the competing A and B erythrocyte populations is approximated by the model and found to be proportional to the 5th and 4th power of the ratios of the antibody and target cell concentrations, respectively. In accordance with earlier observations, suggesting that the interaction between the antibody and the C1q molecules is based on polar electrostatic charges, we propose that the sensitizing antibody provides an electrostatic field around the erythrocytes which attracts C1q molecules towards their membranes.

Antibodies, Monoclonal↗
PubMed1985

Molecular cloning and characterization of the cDNA coding for C4b-binding protein, a regulatory protein of the classical pathway of the human complement system.

By using synthetic oligonucleotides as probes, plasmid clones containing portions of cDNA coding for human C4b-binding protein were isolated from a liver cDNA library. The entire amino acid sequence of the C4b-binding protein can be predicted from this study of the cloned cDNA when allied to a previous sequence study at the protein level [Chung, Gagnon & Reid (1985) Mol. Immunol. 22, 427-435], in which over 55% of the amino acid sequence, including the N-terminal 62 residues, was obtained. The plasmid clones isolated allowed the unambiguous determination of 1717 nucleotides of cDNA sequence between the codon for the 32nd amino acid in the sequence of C4b-binding protein and the 164th nucleotide in the 3' non-translated region. The sequence studies show that the secreted form of C4b-binding protein, found in plasma, is composed of chains of apparent Mr 70 000 that contains 549 amino acid residues. Examination of the protein and cDNA sequence results show that there are at least two polymorphic sites in the molecule. One is at position 44, which can be glutamine or threonine, and the other is at position 309, which can be tyrosine or histidine. Northern-blot analysis indicated that the mRNA for C4b-binding protein is approx. 2.5 kilobases long. The N-terminal 491 amino acids of C4b-binding protein can be divided into eight internal homologous regions, each approx. 60 amino acids long, which can be aligned by the presence in each region of four half-cystine, one tryptophan and several other conserved residues. These regions in C4b-binding protein are homologous with the three internal-homology regions that have been reported to be present within the Ba region of the complement enzyme factor B and also to the internal-homology regions found in the non-complement beta 2-glycoprotein I.

Amino Acid Sequence↗
PubMed1991

Complement-mediated inactivation of interferon-gamma in ELISA systems.

The recovery of a predetermined amount of interferon-gamma (IFN-gamma) added to normal serum was studied in two independent sandwich ELISA systems specific for rat and human IFN-gamma. In both assays the ELISA activity was rapidly lost in fresh but not in heat-inactivated (30', 56 degrees C) serum. Ninety percent of the initial activity had disappeared within 30 minutes upon incubation at 37 degrees C. Serum-mediated inhibition was not species-specific as the ELISA activity of rat IFN-gamma diminished equally well in rat and human sera. Inhibition was critically dependent on the isotype of the solid-phase monoclonal antibody (mAb) used in the ELISA systems. IgG1 and IgG2a mAbs efficiently inhibited the ELISA activity of IFN-gamma, whereas an IgA mAb was ineffective. The inhibition was not influenced by a wide variety of anti-proteolytic agents but was effectively blocked by anti-complementary substances or treatments directed to the first (C1) and third (C3) component of complement. Our results indicate that activation of the classical pathway of complement (CPC) and the concomitant covalent binding of C3 to the IFN-gamma molecule play a major role in the inhibitory process. It is concluded that reduction of the ELISA activity is attributable to diminished accessibility of the detector antibody for the IFN-gamma protein as a consequence of C3 binding.

Binding Sites↗
PubMed1981

Screening for deficiencies in the classical and alternative pathways of complement by hemolysis in gel.

Two assays based on hemolysis in gel were assessed for screening complement (C) component deficiencies. In one assay sensitized sheep erythrocyte (EA) were incorporated in agarose gel containing Ca2+ and Mg2+, in the other guinea pig erythrocytes (GpE) were used in the presence of Mg2+and EGTA. With few exceptions, fresh samples from healthy individuals produced homogeneous areas of complete hemolysis in both assays. Clearly aberrant patterns were observed in approximately 4% of healthy blood donors. Sera from patients having complete deficiencies of Clq, C2 or C4 produced clear lysis of GpE only, whereas in sera lacking C3 or C8 lysis was grossly impaired in both assays. Properdin deficient serum produced very slight lysis of GpE but normal lysis of EA. Reconstitution of these C-deficient sera gave normal lysis. Together, the two assays supplement immunochemical C3 and C4 determinations for screening out C disorders.

Animals↗
PubMed2001

The complement system in central nervous system diseases.

The activation of complement system is an important factor participating in inflammatory, neurodegenerative, and cerebrovascular diseases. Astrocytes and neurons are able to synthesize complement components. Myelin and oligodendrocyte (OLG) activate the classical pathway of complement in vitro in the absence of antibodies. Sublytic C5b-9 in the absence of cell death induces proto-oncogenes, activates cell cycle, and enhances cell survival in OLG. In addition, C5b-9 reverses the differentiation phenotype in OLG and enhances cell survival. beta amyloid protein is an activator of the complement system and neurons are susceptible to bystander complement mediated damage. These findings indicate that complement activation and membrane assembly of C5b-9 play an important role in pathogenesis of central nervous system (CNS) disorders.

Animals↗
PubMed1986

Opsonic requirements for hepatic clearance of pneumococci.

An in situ isolated, perfused rat liver system was used to evaluate various opsonins for hepatic trapping and killing of encapsulated, virulent, type 3 pneumococci. Pneumococci were rapidly trapped in the liver in the presence of all potential opsonins including Hanks balanced salt solution with added colloid. However, with some of the potential opsonins the organisms remained viable and could be recovered from the liver. With others there was killing of pneumococci. The combination of rat antibody and complement was effective as an opsonin and produced about the same degree of killing as the same human components. When tested separately killing was not promoted by rat complement, human complement, or human antibody. However, when ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-Mg2+ was added to block the classical pathway and preserve the alternative pathway there was significant pneumococcal killing which may have been complement mediated or due to an adverse effect of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-Mg2+ on the organisms. Fibronectin and C-reactive protein reduced the number of surviving microorganisms, although the change was not significant. Immunoglobulin G (IgG)-IgM, IgG, or IgM with complement, and antibody + the classical complement pathway, significantly reduced survival of the organisms. The best killing occurred with antibody plus complement plus fibronectin, which reduced mean survival to 24% during the 1-h perfusion.

Animals↗
PubMed1976

The complement profile in babesiosis.

Rats have been infected with Babesia rodhaini, a protozoan agent that induces immune complex disease. Assays for complement have revealed depletion of C2, C3, C4, C5, and whole complement in the course of infection. No evidence of depletion in the alternative (properdin) pathway was found. These findings are consistent with the conclusion that the classical complement pathway is activated during the course of the protozoan infection.

Animals↗
PubMed1994

Complement activation by cellulosic dialysis membranes.

AIMS: To assess the effect of cellulosic dialysis membranes on the production of complement degradation products to determine to the role of the classical pathway. METHOD: Complement activation was studied in 33 patients during a single haemodialysis session using cellulosic membranes. Pre- and post-dialysis plasma EDTA valves of C3, C4, C3dg, C4d and C reactive protein (CRP) were measured. Statistical analysis was done using the Wilcoxon signed rank test. RESULTS: Post-dialysis C4 (p = 0.0003), C3dg (p < 0.0001), and C4d (p = 0.003) concentrations were increased compared with pre-dialysis values. There was no significant change in C3 (p = 0.095) and CRP (p = 0.13) values. Post-dialysis C3dg and C4d concentrations correlated significantly (p = 0.007). IgG, an undialysed molecule, was quantified and post-dialysis valves were significantly higher than those before dialysis (p = 0.0002), indicating a degree of haemoconcentration. To remove this effect, the C3:IgG, C4:IgG, C3dg:IgG, C4d:IgG and CRP:IgG ratios were calculated. Compared with pre-dialysis values, post-dialysis C3dg:IgG and C4d:IgG ratios were increased and C3:IgG decreased significantly. No change was observed in C4:IgG and CRP:IgG ratios. CONCLUSION: This study confirms that significant complement activation takes place following dialysis with cellulosic membranes. This is denoted by an increase in C3dg. This was paralleled by a rise in C4d, implying a contributory role for the classical pathway. Concomitant post-dialysis increases in IgG and C4 indicate a degree of haemoconcentration; but removal of this effect shows that C3dg and C4d are increased following dialysis--suggesting classical, in addition to alternative, pathway activation.

Adult↗
PubMed1986

Immunomodulators and the complement system.

The possible role of immunomodulators in the host-defense mechanism against neoplasm is discussed from the standpoint of the complement system. Serum complement is activated by the majority of immunomodulators in vitro via either the classical or the alternative pathway, and this activation is sometimes observed following systemic administration of immunomodulators. Besides activating the complement, systemic administration of immunomodulators elevates serum complement levels, and in some cases binds complements to tumor cells. Activated serum complement by an immunomodulator induced an accumulation of PMNs in ascites with tumor cell destruction when intraperitoneally injected, indicating that complement-derived chemotactic factors C3a and C5a generated by an immunomodulator had an antitumor effect. This evidence strongly supports the concept that complement system plays an important role in the host-defense mechanism against neoplasm.

Adjuvants, Immunologic↗
PubMed1982

Complement mediated lysis in chickens.

Rat erythrocytes, when either injected i.v. into chickens, or incubated in vitro with chicken serum, will be lysed within minutes. This lysis is complement dependent but does not involve specific antibodies since Ig-free bursectomized chickens behave in exactly the same way as normal chickens with high levels of haemagglutinating antibodies. The lysis is mediated by activation of complement component C3 via the alternative complement pathway and various observations lead us to doubt the functional existence in this lytic reaction of the classical complement pathway.

Animals↗
PubMed1985

Properties of glycans that activate the human alternative complement pathway and interact with the human monocyte beta-glucan receptor.

The glycans used in an earlier study to define the ligand specificity of the human monocyte phagocytic receptor for unopsonized particulate activators were assessed for their capacities to activate the proteins of the human alternative complement pathway. Normal human serum was preincubated with glycans under conditions of chelation to prevent activation of the classical complement pathway, and the activation-depletion of the alternative complement pathway was determined by the subsequent capacity of the serum to lyse rabbit erythrocytes (Er). When serum was preincubated at a 1/2 dilution in 8 mM EGTA/2 mM Mg with increasing numbers of yeast glucan or zymosan particles, and was evaluated at final serum dilutions of 1/8, its capacity to lyse Er was found to be reduced by 50% with 1.9 X 10(6)/ml yeast glucan particles and 1.4 X 10(6)/ml zymosan particles. At 2 mg/ml of serum diluted 1/2 in 8 mM EGTA/2 mM Mg, nonturbid preparations of mannan, laminarin, or pyrogen-free inulin and turbid suspensions of cellulose, Sephadex, agarose, or purified inulin failed to activate the alternative complement pathway. In contrast, activation-depletion of the alternative pathway was induced by turbid preparations of crude inulin, nigeran, pachyman, barley beta-glucan, and pustulan, which at 700 micrograms/ml, 500 micrograms/ml, 350 micrograms/ml, 60 micrograms/ml, and 27 micrograms/ml, respectively, effected 50% reductions in the subsequent lysis of Er. After centrifugation of 2 mg/ml suspensions of barley beta-glucan at 1100 X G for 5 min and at 15,000 X G for 15 min, the supernatants contained 90 to 92% and 65% of the barley beta-glucan, respectively, as determined by the anthrone method. On a weight basis, the 1100 X G supernatant exhibited the same capacity to activate the alternative pathway as the corresponding original suspension, whereas the 15,000 X G supernatants had less than 3% of the original anti-complementary activity. Preincubation of adherent human monocytes with increasing concentrations of barley beta-glucan suspensions, 100,000 X G supernatants containing 64% of the original beta-glucan, and laminarin all decreased subsequent ingestion of 1.25 X 10(6) zymosan particles in a dose-related fashion. The numbers of monocytes from three different donors phagocytosing zymosan were reduced by 50% after pretreatment with 30 to 65 micrograms/ml, 25 to 48 micrograms/ml, and 12 to 15 micrograms/ml of barley beta-glucan suspensions, 100,000 X G supernatants of barley beta-glucan, and laminarin, respectively, even though the latter two preparations were fully soluble and had no capacity to activate the alternative pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Complement Activation↗
PubMed1987

Antibody and complement-binding activity of viable and nonviable human spermatozoa.

Antibody and complement fixation by viable and nonviable spermatozoa were studied by means of immunofluorescence and a hemolytic assay (CH-50). Spermatozoa were incubated in sera from fertile male and female donors and in peritoneal fluid from fertile women undergoing laparoscopy. Nonviable spermatozoa were able to bind antibody or complement from sera and peritoneal fluid. There was no evidence of antibody or complement fixation by viable spermatozoa. The antibodies present in the serum that bind to spermatozoa belong to the IgG and IgM class; in peritoneal fluid, only IgG could be found. Complement fixation occurred via the classical (antibody-mediated) and alternative pathway. Viable spermatozoa possess antigenic properties different from nonviable spermatozoa. The lack of immunological reaction of women to viable spermatozoa and a possible mechanism for immunological infertility is considered.

Antibodies↗