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PubMed1986

Alternative complement pathway activation by C4b deposited during classical pathway activation.

Sheep erythrocytes (E) sensitized with anti-E antibody (A) were reacted with guinea pig C1 (C1gp) and human C4 (C4hu) or guinea pig C4 (C4gp) to prepare EAC1, 4b. Treatment of the EAC1, 4b with a buffer containing EDTA removes C1rgp and C1sgp, resulting in the formation of EAC4b. EAC4b prepared in this way were found to be lysed by human or guinea pig serum in a gelatin Veronal-buffered saline containing 2 mM MgCl2 and 8 mM EGTA (Mg-EGTA-GVB). In the hemolytic sensitivity of EAC4bhu, essentially no difference was noted whether IgG or IgM antibodies were used for preparation of EAC4bhu. The extent of the hemolysis of EAC4bhu was dependent on the dose of C4bhu. Because EAC4bhu were lysed even by C2-deficient human serum, C3 convertase of the classical complement pathway would not be involved in the hemolysis of EAC4bhu. Furthermore, the reactivity of EAC4bhu with serum in Mg-EGTA-GVB remained even after treatment of the intermediate cells with 1 mM PMSF, indicating that any remaining C1gp was not responsible for the hemolysis. Therefore, the hemolysis of EAC4b by sera in Mg-EGTA-GVB was considered to be mediated via activation of the alternative complement pathway (ACP). Pretreatment of EAC4bhu with anti-C4hu antibody or C4-binding protein suppressed the hemolysis of EAC4bhu via the ACP activation. Furthermore, EAC4bhu were more sensitive to hemolysis by the reaction with a mixture of C3, B, D, and H followed by rat serum in EDTA-GVB than EAC1qgp were. These results indicate that C4b molecules on the cell membrane participate in the activation of ACP.

Animals↗
PubMed1995

Animal models for complement deficiencies.

The complement system plays a key role in host defense and in the development of autoimmunity. Three types of animal models of complement-mediated disease have traditionally been used: they involve normal animals, animals with spontaneously arising genetic deficiency, and animals treated with complement-inactivating agents. All of these approaches have had partial success in our attempts to understand complement mechanisms. Most animal models of genetic deficiency have been studied relatively little, as the availability of such animals is limited. C4, C2, and partial C3 deficiency in the guinea pig are well characterized, although only C4 deficiency in the guinea pig has been exclusively studied. C3 deficiency in the dog and C6 deficiency in the rabbit are well described, although studies are limited in number. C6 deficiency in the rat has been described recently and C5 deficiency in inbred mice strains has been studied fairly extensively. Factor H deficiency in the Yorkshire pig has also been described. Relatively few agents that inhibit complement are in use. Most widely used in animal studies is cobra venom factor. This inactivates the alternative complement pathway in the fluid phase and thereby depletes complement protein levels. The antigenicity of this protein, purified from the venom of cobras, limits its duration of use in most animal models. Complement-inhibiting agents are rare and, as yet, not widely used. We recently described the use of intravenous immune globulin for inhibiting complement in animal studies and present data on its use in animals, including discordant xenograft rejection, and its potential use in human disease. New developments in molecular biology provide the potential for a vast new array of deficiency models. A limited number of laboratories are actively engaged in the production of animals with inactivated genes. For example, gene knockout mice with no C3, and with no factor B, have been generated. Several complement control proteins have been prepared by genetic molecular biological techniques. Most promising among these is CR1, which limits complement damage in several animal models. Transgenic animals, which complement regulatory proteins expressed on their cells, have been prepared. As complement control proteins tend to be more efficient at regulating complement of the same species type as the regulatory protein, these animals may be useful in such areas as xenograft transplantation. The various animal models are reviewed and their potential application to understanding of human disease is emphasized.

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↗
PubMed1990

Two types of C3 nephritic factor: properdin-dependent C3NeF and properdin-independent C3NeF.

The IgG fraction of serum from patients with membranoproliferative glomerulonephritis (MPGN) types I and II or partial lipodystrophy (PLD) was found to contain C3 nephritic factor (C3NeF) which reacts with the alternative pathway C3 convertase C3bBb and stabilizes it. Two types of C3NeF were detected, of which one was heat sensitive (56 degrees C for 30 min) and properdin dependent (C3NeF:P) but the other was heat stable and properdin independent (C3NeF:nP). C3NeF:P was found in sera from patients with MPGN types I and II and it displayed the properties of properdin and IgG. C3NeF:P was observed in patients with reduced serum concentrations of C3 and terminal complement components (TCC), and the generation of SC5b-9 complex was increased in mixtures with normal human serum. C3NeF:nP was found in sera from patients with MPGN type II and PLD, whose sera revealed a selective decrease in C3 concentrations.

Autoantibodies↗
PubMed1990

Proteolytic elimination of decay-accelerating factor (DAF): lytic abnormality coincides with removal of DAF in papain-treated human erythrocytes.

Erythrocytes (E) from patients with paroxysmal nocturnal hemoglobinuria (PNH) lack decay-accelerating factor (DAF) and this partly causes increasing susceptibility of the E to complement. Several reagents have been used to convert normal E to the complement-sensitive (PNH-like) cells. The relationship between DAF amounts and complement susceptibility of these PNH-like cels has been examined. Of the reported reagents for preparation of PNH-like cells, 2-amino-ethylisothiouronium bromide (AET), papain, and periodate efficiently converted normal E to the complement-sensitive cells, but only papain reduced the quantity of DAF on the cells. Further, of the proteases we tested only papain cleaved DAF to liberate its major fragment from the cells. The papain-treated cells lysed in a similar fashion to PNH cells as the serum concentration increased. The major papain-digested product of DAF had Mr, 55,000, lacked hydrophobicity, and retained the ability to inhibit the C3 convertases. These findings suggest that papain allows liberation from cells of functional domains as well as most of the antigenic epitopes of DAF to generate a PNH-like cell.

Blood Proteins↗
PubMedDate not supplied

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 beta-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 beginning 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, alpha1-antitrypsin, fibronectin, albumin, alpha2-macroglobulin, vitronectin, beta2-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.

Animals↗
PubMed2000

Control of the classical and the MBL pathway of complement activation.

The activation of complement via the mannan-binding lectin (MBL) pathway is initiated by the MBL complex consisting of the carbohydrate binding molecule, MBL, two associated serine proteases, MASP-1 and MASP-2, and a third protein, MAp19. In the present report we used an assay of complement activation specifically reflecting the physiological activity of the MBL complex to identify biological and synthetic inhibitors. Inhibitor activity towards the MBL complex was compared to the inhibition of the classical pathway C1 complex and to a complex of MBL and recombinant MASP-2. A number of synthetic inhibitors were found to differ in their activities towards complement activation via the MBL pathway and the classical pathway. C1 inhibitor inhibited both pathways whereas alpha2-macroglobulin (alpha2M) inhibited neither. C1 inhibitor and alpha2M were found to be associated with the MBL complex. Upon incubation at 37 degrees C in physiological buffer, the associated inhibitors as well as MASP-1, MASP-2, and MAp19 dissociated from MBL, whereas only little dissociation of the complex occurred in buffer with high ionic strength (1 M NaCl). The difference in sensitivity to various inhibitors and the influence of high ionic strength on the complexes indicate that the activation and control of the MBL pathway differ from that of the classical pathway. MBL deficiency is linked to various clinical manifestations such as recurrent infections, severe diarrhoea, and recurrent miscarriage. On the other hand, impaired control of complement activation may lead to severe and often chronically disabling diseases. The results in the present report suggests the possibility of specifically inhibiting of the MBL pathway of complement activation.

Animals↗
PubMedDate not supplied

Correlation between inhibited alternative complement activity and the protective effect induced by Nocardia lysozyme digest (NLD) during Klebsiella pneumoniae infection in mice.

Nocardia lysozyme digest (NLD) was administered intraperitoneally (i.p.) at a dose of 500 micrograms/kg to normal and immunosuppressed mice for 3 consecutive days prior to inoculation with Klebsiella pneumoniae. A protective effect was observed when the pathogen was injected subcutaneously and intravenously, as opposed to an aggravating effect obtained in the case of intraperitoneal inoculation The i.p. administration of NLD partially restored the immunosuppression caused by cyclophosphamide but did not change cobra venom-induced deterioration of the infection. The results obtained could be regarded as a consequence of the lowered alternative pathway serum complement activity and the crucial role of the diminished level of complement in the peritoneal cavity.

Adjuvants, Immunologic↗
PubMed1988

Vaccinia virus encodes a secretory polypeptide structurally related to complement control proteins.

Several polypeptides are secreted into the medium of cells infected with vaccinia virus, a cytoplasmic DNA virus belonging to the poxvirus family. One of these, a polypeptide of relative molecular mass 19,000 is structurally related to epidermal growth factor and binds to epidermal growth factor receptor stimulating proliferation of uninfected cells in vitro and in vivo. Here, we show that a second, and much more abundant secretory polypeptide, is also encoded by vaccinia virus and is structurally related to the superfamily of complement control proteins. Members of this family can block complement-mediated induction of the inflammatory response, and engulfment, killing and lysis of bacteria and viruses.

Amino Acid Sequence↗
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↗
PubMed2000

Conservation of plasma regulatory proteins of the complement system in evolution: humans and fish.

The complement system is an important defense system of innate immunity. The recent identification of structurally and functionally related complement regulatory proteins in the teleost, barred sand bass (Paralabrax nebulifer), and humans, two species which are separated in evolution by 100 million years, indicates a high level of conservation and the early presence of this defense system in evolution. The complement regulatory protein of barred sand bass, SBP1, is related to both the human alternative pathway regulator factor H, and to the classical pathway regulator C4bp, and displays regulatory activities in both human pathways. In addition, molecules with homology to the recently identified human factor-H-related proteins are also present in the sand bass genome. Here, we summarize the structural and functional aspects of these homologies and discuss the consequences for the evolution of the complement system.

Animals↗
PubMed1989

Relationship between the membrane inhibitor of reactive lysis and the erythrocyte phenotypes of paroxysmal nocturnal hemoglobinuria.

Susceptibility to hemolysis initiated by activated cobra venom factor (CoF) complexes is a characteristic that distinguishes the most complement-sensitive type III erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) from the intermediately sensitive type II and the normally sensitive type I cells. Recently we isolated a membrane constituent from normal erythrocytes that inhibits CoFBb-initiated hemolysis, and this protein was designated membrane inhibitor of reactive lysis (MIRL). To investigate the molecular basis of the variability in complement sensitivity among PNH erythrocytes, the surface expression of MIRL and decay accelerating factor (DAF) on the three phenotypes of PNH was quantified immunochemically. Both complement regulatory proteins were markedly deficient on the erythrocytes from a patient with predominately type III cells. The erythrocytes from patients with a majority of either type II or I cells were also significantly deficient in both MIRL and DAF. While cytofluorometric analysis confirmed the quantitative deficiencies, segregation of erythrocytes into discrete subpopulations that expressed either no MIRL or normal amounts of MIRL was not observed. The results of immunoprecipitation studies were consistent with quantitative, but not qualitative abnormalities of MIRL and DAF. Selective removal of the sensitive erythrocytes indicated that approximately 20% of the normal amount of MIRL is sufficient to protect cells from CoF-initiated lysis. These studies suggest that relatively subtle quantitative differences in membrane complement regulatory proteins underlie the variability in complement sensitivity of PNH erythrocytes.

Blood Proteins↗
PubMed2000

Complement-dependent acute-phase expression of C-reactive protein and serum amyloid P-component.

The acute-phase response (APR) is regulated by TNF-alpha, IL-1beta, and IL-6 acting alone, in combination, or in concert with hormones. The anaphylotoxin C5a, generated during complement activation, induces in vitro the synthesis of these cytokines by leukocytes and of acute-phase proteins by HepG2 cells. However, there is no clear evidence for a role of C5a or any other complement activation product in regulation of the APR in vivo. In this study, using human C-reactive protein (CRP) transgenic mice deficient in C3 or C5, we investigated whether complement activation contributes to induction of the acute-phase proteins CRP and serum amyloid P-component (SAP). Absence of C3 or C5 resulted in decreased LPS-induced up-regulation of the CRP transgene and the mouse SAP gene. Also, LPS induced both the IL-1beta and IL-6 genes in normocomplementemic mice, but in complement-deficient mice it significantly induced only IL-6. Like LPS injection, activation of complement by cobra venom factor led to significant elevation of serum CRP and SAP in normocomplementemic mice but not in complement-deficient mice. Injection of recombinant human C5a into human CRP transgenic mice induced the IL-1beta gene and caused significant elevation of both serum CRP and SAP. However, in human CRP transgenic IL-6-deficient mice, recombinant human C5a did not induce the CRP nor the SAP gene. Based on these data, we conclude that during the APR, C5a generated as a consequence of complement activation acts in concert with IL-6 and/or IL-1beta to promote up-regulation of the CRP and SAP genes.

Acute-Phase Reaction↗