Complement Activation Fragments Research Articles

Local neutrophil influx following lateral fluid-percussion brain injury in rats is associated with accumulation of complement activation fragments of the third component (C3) of the complement system

Traumatic brain injury can lead to locally destructive secondary events mediated by several inflammatory components. Following lateral fluid-percussion (FP) brain injury in rats, we examined cortical and hippocampal sections for neutrophil infiltration and accumulation of complement component C3. Neutrophil influx into the brain after injury was detected by an improved myeloperoxidase (MPO) microassay and manual cell counting, while C3 accumulation was detected using immunocytochemistry. MPO levels were elevated in the injured cortical tissue, whereas C3 immunoreactivity was increased in both injured cortical and ipsilateral hippocampal sections. These results show that the FP model of head injury leads to an intense local inflammatory reaction and subsequent tissue destruction.

The effect of the antithyroid drug propylthiouracil on the alternative pathway of complement in rats

The effect of propylthiouracil (PTU) on the lytic activity of complement in rat serum was investigated in vivo. Rats (180±10 g) were treated daily by gavage with PTU doses of 1–50 mg/200 g body weight for time intervals ranging from 1 to 30 days. Serum classical pathway (CP) and alternative pathway (AP) activities were determined 24 h after the last dose. A single dose of 50 mg/200 g body weight was administered to additional groups and the animals were sacrificed after periods of 1–48 h. The results showed a relatively small reduction (∼30%) in CP activity, evident only in animals treated with 50 mg of PTU for three weeks. However, a clear and opposite effect of PTU, an increase in lytic activity reaching values up to 180% of controls, was observed on AP activity. This effect was seen at all PTU doses used, and occurred within 4 days of treatment with the highest dose. Maximum activity was observed at intermediate intervals, depending on the PTU dose, with a return to control levels occurring after the longer periods of treatment. The lytic activity of serum from animals treated with a single PTU dose of 50 mg/200 g body weight and sacrificed 1–48 h after dosing did not differ from controls. Serum levels of thyroid hormone (triiodo l-thyronine, T3, and thyroxine, T4) were determined in representative groups of treated animals (injected with 5 mg of PTU/200 g body weight/day). These were either undetectable or considerably lower than those of controls. The serum PTU levels of these rats increased for up to 22 days, reaching values of 2–4 μg/ml. PTU is described in the literature as a modulator of both cellular immune responses and antibody production. Upon complement activation fragments of complement components bind to immune complexes and to specific receptors on cells of the immune system. Thus, alteration in AP activity caused by PTU treatment suggests a possible mechanism by which the drug exerts its modulatory effect. Increased complement AP activity might affect events as antigen presentation and hence the onset and course of the immune response.

INHIBITION OF COMPLEMENT-MEDIATED XENOGENEIC INJURY BY THE MEMBRANE ATTACK COMPLEX IS SUFFICIENT TO ALLOW INDUCTION OF ACCOMMODATION IN THE HAMSTER-TO-RAT MODEL

55 Accommodation is long-term graft survival in the presence of xenoreactive antibodies and complement. Accommodation can be obtained in the hamster-to-rat cardiac model by a combination of complement inactivation with cobra venom factor (CVF) and daily administration of cyclosporin A (CSA). Since CVF inactivates both C3 and subsequent components following C3, experiments with CVF suggest that several complement activation fragments, in addition to the membrane attack complex (MAC), may play a role in preventing accommodation in this model. To define which complement-mediated process needs to be inhibited to allow induction of accommodation, we investigated accommodation of a hamster heart xenograft in C6-deficient (C6D) PVG rats and in normocomplementemic PVG rats treated with antibodies against C6. Golden Syrian hamster hearts were transplanted heterotopically into normal or C6D rats. C6D rats had complete deficiency of C6 hemolytic activity; addition of purified C6 to the serum fully restored CH50 total complement activity, confirming that other complement components were intact. Graft survival in untreated rats was 3-4 days (n=4) and in untreated C6D rats was 5 days (n=5). Experimental groups received either CVF (60 U/Kg at day-1 pre-tx. and 20U/day × 9 days) or polyclonal goat anti-C6 antiserum (Cappel) (140 mg IgG/kg/day × 5 days) with or without CSA. The table shows the number of grafts performed in each group and grafts surviving >100 days. C6D rats bearing hamster hearts had no reconstitution of hemolytically active C6. Histopathologic examination was performed on rejected hearts (<100 day survivors) or after sacrifice (>100 day survivors).TableC6D rats treated with CSA or CSA and CVF had 90% long-term graft survival. Normal rats treated with CSA and CVF, or CSA and anti-C6 antibodies had >60% long-term graft survival. Histologically, rejected hearts had myofiber necrosis, interstitial hemorrhage, acute inflammation and vascular damage. In contrast, hearts from >100 day survivors lacked these findings and had preservation of normal architecture; however, a mononuclear cell infiltrate was present. These features are characteristic of accommodated hearts in the hamster-to-rat model. In conclusion, the finding that C6D rats and normal rats treated with anti-C6 antibodies develop accommodation without CVF treatment to inactivate C3 and C5 demonstrates that xenogeneic injury due to the MAC is responsible for preventing the development of accommodation in the hamster-to-rat model. Accommodation occurred in the face of unabated formation of fragments derived from C3 and C5.

Complement-dependent proinflammatory properties of the Alzheimer's disease beta-peptide.

Large numbers of neuritic plaques (NP), largely composed of a fibrillar insoluble form of the beta-amyloid peptide (Abeta), are found in the hippocampus and neocortex of Alzheimer's disease (AD) patients in association with damaged neuronal processes, increased numbers of activated astrocytes and microglia, and several proteins including the components of the proinflammatory complement system. These studies address the hypothesis that the activated complement system mediates the cellular changes that surround fibrillar Abeta deposits in NP. We report that Abeta peptides directly and independently activate the alternative complement pathway as well as the classical complement pathway; trigger the formation of covalent, ester-linked complexes of Abeta with activation products of the third complement component (C3); generate the cytokine-like C5a complement-activation fragment; and mediate formation of the proinflammatory C5b-9 membrane attack complex, in functionally active form able to insert into and permeabilize the membrane of neuronal precursor cells. These findings provide inflammation-based mechanisms to account for the presence of complement components in NP in association with damaged neurons and increased numbers of activated glial cells, and they have potential implications for the therapy of AD.

Immunoblot analysis of proteins associated with HEMA-MMA microcapsules: Human serum proteins in vitro and rat proteins following implantation

Human serum proteins and their fragments, associated with hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) copolymer microcapsules, were characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. Capsules were incubated with serum for 1 week in vitro and then dissolved in ethanol to also precipitate the adsorbed protein. The precipitate was dissolved in 2% (w/v) SDS (the ‘capsule eluate’) to be assayed by electrophoresis. The majority of proteins probed for in the immunoblots were detected in the capsule eluates. These included fibronectin, plasminogen, IgG, vitronectin, Factor B, Factor H, Factor I, C3, but not β-lipoprotein, fibrinogen, HMWK, or IgM. Complement activation fragments were detected in both the immunoblots of the capsule eluates and the medium containing serum without capsules. Thus, the adsorption of these fragments, formed independent of capsule presence, may be partially or completely responsible for the complement fragments associated with capsules. The prevention of complement activation by the addition of 5.8 mM EDTA, at the begining of the week-long incubation, resulted in fewer low-molecular-weight C3 fragments associated with capsules. Rat proteins were also detected in immunoblots of the eluate of ‘free-floating’ capsules from the rat peritoneal cavity following implantation for 1 day using anti-human antibodies. Detected proteins included HMWK, fibrinogen, antithrombin III, transferrin, α 1-antitrypsin, fibronectin, albumin, α 2-macroglobulin, vitronectin, β 2-microglobulin, Factor B and Factor I. Rat fibrinogen, IgG, and complement C3 fragments were also detected in these immunoblots, but with monoclonal antibodies against the rat proteins.

Inhibition of interleukin-6 synthesis in an animal model of septic shock by anti-C5a monoclonal antibodies.

The complement activation fragment C5a was recently shown to induce interleukin (IL)-6 synthesis by peripheral blood mononuclear cells. To understand better the role of C5a in cytokine regulation in vivo, we investigated the effects of complement depletion by cobra venom factor (CVF) or of anti-C5a monoclonal antibodies (mAb) on IL-6 generation in an animal model of septic shock. Complement-depleted pigs which were subsequently challenged with Escherichia coli generated significantly (p < 0.05) less IL-6 during the 6-h observation period than complement-sufficient controls. To address specifically the role of C5a in IL-6 regulation, we produced a C5a(57-74) peptide-specific mAb (T13/9) which neutralizes the bioactivity of porcine C5a. The mAb T13/9 does not cross-react with the precursor protein C5. The pretreatment of pigs with anti-C5a mAb T13/9 prior to the induction of sepsis resulted in a decrease of over 75% in serum IL-6 bioactivity compared to control animals (p < 0.0001). These results indicate a role for C5a in the modulation of IL-6 synthesis in Gram-negative bacteremia.

Isolation of the porcine complement activation fragments C3c and C3d and their use in the preparation of monospecific antibodies

The hemolytic test to date has been the sole analytic technique applied to study the complement reaction in the swine. To improve the analytical possibilities for this species we have developed polyclonal antibody reagents with specificities for the C3c and C3d activation fragments of swine C3. Access to these reagents, by which activation products can be analysed in tissues and biological fluids, will offer new possibilities for a more precise analysis of the complement reaction.

Rheumatoid Factor Expression and Complement Activation in Children Congenitally Infected with Human Immunodeficiency Virus

Rheumatic disease manifestations and autoimmune phenomena are common in adults infected with human immunodeficiency virus (HIV). On the other hand, rheumatic disease manifestations are rare in children congenitally infected with HIV. We studied the relationship between autoimmune phenomena and rheumatic diseases by examining HIV-infected children for the presence of rheumatoid factors (RFs), immune complexes, and complement activation fragments. RFs (principally IgA) were detected in 12 of the 24 HIV-infected patients (50%) and in none of the uninfected (HIV-exposed, seroreverted) controls ( n = 22). Mean levels of complement activation fragments C3a and Bb were elevated in the HIV-infected children compared with the controls. A correlation was seen between the presence of IgA RF and alternative complement pathway activation as measured by plasma levels of Bb. None of the children had clinical or laboratory evidence of rheumatic disease.

The apolipoprotein(a) moiety of lipoprotein(a) interacts with the complement activation fragment iC3b but does not functionally affect C3 activation or degradation

A previous study has shown that complement component C3 binds to recombinant apolipoprotein(a) (r-apo(a)). In the present report we have investigated the interactions between lipoprotein(a) (Lp(a)), r-apo(a) and C3 in relation to complement activation and degradation. Neither Lp(a) nor r-apo(a) affected complement activation as indicated by sheep and rabbit red blood cell hemolytic assays, and by assessment of the amount of C3a generated in zymosan-activated human serum in the presence or absence of Lp(a). Crossed immunoelectrophoretic analyses indicated that Lp(a) retarded the migration of iC3b in complement-activated serum but had no effects on C3, C3b, C3c or C3dg. Recombinant apo(a) exhibited the same properties as intact Lp(a) indicating that it is the apo(a) portion of Lp(a) that mediates this effect and not the lipid moiety. Low density lipoprotein had no effect on the migration of C3 cleavage fragments. Treatment of Lp(a) or apo(a) with neuraminidase abolished their capacity to alter iC3b migration. SDS-PAGE immunoblotting analysis of C3 activation fragments generated in the presence of Lp(a) demonstrated the usual physiologic C3 cleavage fragments. Rocket intermediate gel immunoelectrophoresis of complement-activated serum demonstrated that Lp(a) did not hinder or accelerate the generation of C3c and C3dg breakdown fragments of iC3b. The results indicate that the apo(a) moiety of Lp(a) alters the migration of iC3b in an electric field but does not affect complement activation or degradation of activated C3. The sialic acid residues on apo(a) are necessary for the apo(a)-iC3b interaction.

Generation of iC3 at the Interface between Blood and Gas

Earlier studies have shown that C3 can be denatured when blood comes in contact with a polystyrene surface. This study was undertaken to see if similar denaturation of C3 occurs at the gas-plasma interface which is found in all kinds of oxygenator used during cardio-pulmonary operations. An in vitro system consisting of gas bubbling through human blood, serum or plasma was used. The generation of C3a, as an indicator of complement activation, and iC3 and iC3 fragments were monitored. Both C3a and iC3/iC3 fragments levels were increased during bubbling. In contrast to the C3a level, no reduction in iC3/iC3 fragments formation was seen in the presence of EDTA, indicating that it was independent of complement activation. The rate of iC3/iC3 fragments generation was unaffected by the composition of the gas (pure oxygen, pure nitrogen or air), suggesting that the denaturation of C3 indeed occurred at the serum-gas interface. C3 and iC3/iC3 fragments were isolated from bubbled EDTA-chelated serum by PEG precipitation and chromatography on FPLC, using a Mono S column and detected by two ELISAs, specific for native C3 and iC3/iC3 fragments. After 240 min approximately 20% of the total amount of C3 consisted of intact iC3 and it was confirmed that this population bound to human erythrocytes.

The Complement System in HIV Disease

Different aspects of the relationship between the HIV infection and the complement system were studied. 1. No significant differences were found between seronegative controls, asymptomatic, and symptomatic (ARC, AIDS) HIV-seropositive patients in the plasma levels of complement components C4, Bf, and C3. 2. Using sensitive ELISA assays, a significant increase was observed in the levels of protein-protein complexes which are formed at the activation of the classical (C1r-C1s-C1-INH) and alternative (C3b-Bb-P) pathways, indicating that both complement pathways are activated in the HIV disease. No significant differences were found, however, in the levels of these complexes between the groups of asymptomatic and symptomatic HIV-infected patients. 3. Artificial immune complexes of synthetic peptides representing some immunodominant epitopes of HIV envelope (gp120, and gp41) proteins, and human polyclonal anti-HIV IgG were found to weakly activate both the classical and alternative complement pathways. 4. An elevated percentage of the lymphocytes carrying a complement activation fragment, C3d, was detected in the blood of HIV seropositive patients as compared to the seronegative controls. No significant positive correlation was found between the percentage of these cells and that of any T cell subsets tested.

Complement activation in psoriasis

Plasma from 16 patients with psoriasis and 12 healthy control subjects were measured for iC3b, C4d, and Bb fragments generated by complement activation. Plasma concentrations for iC3b, C4d, and Bb fragments were found to be significantly increased in the patients with psoriasis. The highest concentrations of these complement activation fragments were seen in patients with erythrodermic pustular psoriasis, psoriatic arthritis, and Reiter's syndrome. The serum concentrations of complement components and regulatory proteins were normal or elevated in almost all samples.

Normal polymorphic variations and transcription of the decay accelerating factor gene in paroxysmal nocturnal hemoglobinuria cells.

In paroxysmal nocturnal hemoglobinuria (PNH), an acquired hemolytic anemia, deficiency of decay accelerating factor (DAF) renders blood cells susceptible to increased deposition of autologous complement activation fragments (C3b) and complemented-mediated injury. To investigate the mechanism of the DAF defect, DNA and mRNA from normal and PNH leukocytes were compared in blot hybridization assays by using DAF cDNA and oligonucleotide probes. Southern analyses of DNA from normal cells revealed a single gene spanning approximately equal to 35 kilobases of DNA. Six HindIII banding patterns were distinguishable among normal individuals. In family studies, the patterns segregated as three homozygous and three heterozygous genotypes deriving from three haplotypes: A, B, and C with frequencies of 0.47, 0.36, and 0.17, respectively. Oligonucleotide mapping localized the polymorphic HindIII sites to two noncoding regions in the vicinity of exons encoding (i) the protein oligosaccharide-rich domain and (ii) the mRNA 3'-untranslated region. Analyses of DNA from DAF-negative leukocytes of eight PNH patients demonstrated restriction fragment profiles identical to those of normal individuals for all enzymes studied. Three patients had the BC (normals = 3/32), three patients had the AA (normals = 6/32), and two patients had the AC (normals = 8/32) HindIII genotype. Of the three PNH patients exhibiting the BC genotype, family studies of two demonstrated the expected inheritance patterns, and RNA gel blot analyses of two showed mRNA transcripts indistinguishable from those in normal cells. The absence of DAF gene or mRNA alterations in affected PNH cells that lack other glycolipid-anchored proteins as well as DAF argues that the lesion underlying PNH cells resides in the glycolipid-anchor pathway.

Inhibitory effect of Zn2+ ions on the degradation of the complement activation fragment C3b

Conference Article| February 01 1986 Inhibitory effect of Zn2+ ions on the degradation of the complement activation fragment C3b ANTHONY J. DAY; ANTHONY J. DAY 1M.R.C. Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K. Search for other works by this author on: This Site PubMed Google Scholar ROBERT B. SIM ROBERT B. SIM 1M.R.C. Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1986) 14 (1): 73–74. https://doi.org/10.1042/bst0140073 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation ANTHONY J. DAY, ROBERT B. SIM; Inhibitory effect of Zn2+ ions on the degradation of the complement activation fragment C3b. Biochem Soc Trans 1 February 1986; 14 (1): 73–74. doi: https://doi.org/10.1042/bst0140073 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: CRI, complement receptor type I, C4bp, C4b-binding protein, SDS, sodium dodecyl sulphate This content is only available as a PDF. © 1986 Biochemical Society1986 Article PDF first page preview Close Modal You do not currently have access to this content.

Complement activation in Type 1 human diabetes

Complement activation was quantitated in serum and plasma of diabetic and normal subjects by sensitive competitive equilibrium radioimmunoassays (RIA) for C3a, C4a, C5a, Factor B, and a newly described C5 neoantigen (termed C5 activation antigen, and abreviated C5-AA) in a stable 54-kDa fragment of C5. Plasma C3a levels were significantly elevated in 8 of 16 patients with newly diagnosed Type 1 diabetes ( P < 0.0005) with the mean C3a concentration for these patients being more than 10-times greater than the mean value of normal controls. C4a levels were also elevated in 2 of these patients ( P < 0.02), but C5a levels, although higher than normal, were not significantly increased. In contrast, the levels of C5-AA in the serum of all patients ( 11 11 ) with chronic Type 1 diabetes were significantly higher than in control Type 2 patients (non-insulin-dependent diabetes) ( P < 0.0005) and 4 of 7 patients with new onset insulin-dependent diabetes mellitus also had significantly higher levels of C5-AA than the Type 2 patients ( P < 0.01). The levels of Factor B in the serum of 5 of 9 patients with new onset diabetes were significantly higher than normal ( P < 0.0025). Five recent onset Type 1 diabetes patients were evaluated longitudinally for C3a, C4a, and C5a: in 3 the levels of C3a were elevated during new onset disease decreasing into the normal range during remission; in 2 of these patients C4a was also significantly elevated and the levels decreased during remission; and in 3 patients the levels of C5a were not significantly elevated but they decreased during remission. Purified human complement proteins and complement hemolytic assays were used to measure complement activation in serum during incubation with rat pancreatic islet cells. With diluted normal human serum, less than 20% of C3 or Factor B were consumed during 30 min at 37°C, while with new onset Type 1 diabetic patient sera up to 90% of C3 and Factor B were consumed in 5 6 sera and 4 6 sera, respectively. These findings suggest (a) that complement activation fragments C3a, C4a, and C5a are generated in vivo in new onset Type 1 diabetes; (b) that both the classical and the alternative complement pathways may be activated; and (c) that this may result in a measurable activation of C5 generating biologically and immunologically active C5a and other C5 activation fragments. Elevated levels of C3a, C4a, Factor B, and C5-AA in plasma or serum (respectively) appear correlated with clinical disease activity in new onset Type 1 diabetes.

Primary structure of bovine complement activation fragment C4a, the third anaphylatoxin. Purification and complete amino acid sequence.

Purification of C4a from heat-activated bovine plasma by elution from CM-Sephadex C-50 at pH 7.4 and gel filtration on Sephadex G-50 gives a 20% yield of pure C4a. The complete amino acid sequence of bovine C4a has been determined by automatic sequencer degradation of CNBr and enzymic fragments, and by carboxypeptidase digestion. The 77-residue bovine sequence shows 12 differences from the human sequence with five of these differences occurring in the C-terminal 11 residues. The sequence of C4a confirms earlier suggestions of homology with C3a and C5a: the three sequences show an almost equal number of identities with each other. The six cysteine residues of the 'disulphide knot' are conserved as well as seven other residues including the C-terminal arginine.

Characterization of human C4a anaphylatoxin.

Human C4a anaphylatoxin was isolated from a Cls digest of the fourth component of complement. Isolation required a two-step procedure involving ion-exchange chromatography on CM-Sephadex C-50 and gel filtration on Sephadex G-50. Characterization of C4a indicated it is a highly cationic polypeptide (pI = 9.0-9.5) containing 77 residues with Mr = 8,759. C4a is devoid of tryptophan, histidine, and carbohydrate. Judged by the shape and magnitude of its circular dichroism spectrum, 54% of the polypeptide backbone of C4a assumes an alpha-helical conformation. Partial NH2-terminal sequence determination of C4a revealed a sequence identical with that published by Bolotin et al. (Bolotin, C., Morris, S., Tack, B., and Prahl, J. (1977) Biochemistry 16, 2008-2015) for the NH2 terminus of the alpha-subunit of human C4. Comparison of the NH2-terminal sequence of C4a with the sequences of complement activation fragments C3a (Hugli, T.E. (1975) J. Biol. Chem. 250, 8293-8301) and C5a (Fernandez, H.N., and Hugli, T.E. (1978) J. Biol. Chem, 253-6955-6962) showed that of the first 24 NH2-terminal residues of C4a, 6 were identical with those of C3a (25% homology) and 8 were identical with those of C5a (33% homology). These data represent the first chemical evidence for the existence of an evolutionary relationship among anaphylatoxins C3a, C4a, and C5a, and imply that a similar relationship exists among their precursor proteins.

Studies on the mechanism of the complement-mediated inhibition of the Fc and C3 receptors of B lymphocytes

The mechansim of the complement-mediated inhibition of the Fc receptors on human peripheral lymphocytes incubated with normal serum was studied. The pretreatment of lymphocytes with a purified C1-esterase inhibitor preparation almost completely blocked the inhibitory effect of the serum on the binding of FITC-conjugated IgG molecules. Twenty to twenty-five percent of the cells in a lymphocyte suspension incubated in normal human serum became immune adherance positive. When normal lymphocytes were treated with a C3 preparation, no inhibition of the IgG aggregate binding occurred. However, if the C3b fragments were cleaved from the C3 molecules in the presence of lymphocytes a strong inhibitory effect on the Fc receptors was observed. The same inhibition by C3b was demonstrated after the digestion of the C3b receptors from the surface of lymphocytes. Furthermore, the EAC rosette formation was inhibited on lymphocytes incubated in normal serum. Pretreatment of the cells by C1-esterase inhibitor or elimination of C1 by EDTA also strongly reduced this effect of the serum. It was concluded from these findings that C1 present on the surface of lymphocytes triggers the classical pathway activation in the serum. During the complement activation, C4b and C3b fragments are cleaved from the C4b and C3 molecules. Part of these fragments fixes to the C3b receptors of the B lymphocytes and inhibits the ability of these cells to form EAC rosettes. The second part of the C4 and C3b fragments binds to the membrane of lymphocytes through their labile binding site outside the C3b receptors. The possible relationship of the portion of lymphocyte membrane which fixes the labile binding sites of the complement-derived fragments and the Fc receptors is discussed.