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PubMed1992

The complement system of Calomys callosus, Rengger, 1830 (Rodentia, Cricetidae).

The complement system (C) of Calomys callosus, Rengger, 1830 (Rodentia, Cricetidae), a wild reservoir for several infectious agents in Latin America, was characterized. Sera from normal adult animals lysed sheep erythrocytes (Es) previously sensitized with rabbit serum anti-Es (Ar) in the presence of veronal-buffered saline containing 0.15 mM CaCl2 and 0.5 mM MgCl2, pH 7.4, or unsensitized rabbit erythrocytes (Er) in the presence of one-half isotonic strength veronal-buffered-saline containing 2.5% glucose, 2 mM MgCl2 and 10 mM EGTA, pH 7.4. Both hemolytic curves were sigmoidal in shape, with CH50 values of 30-40 for females and 20-30 for males. C5, determined hemolytically using the intermediate cells EsArClm4m2m3m, was approximately 4.5 x 10(8)/ml and 4.0 x 10(8)/ml for females and males, respectively. Immunochemical serum analyses by double immunodiffusion or by immunoblotting using polyclonal antisera against human C1s, C1q, C2, C3, C4, C5, C8 and factors B, I and H indicated that C. callosus C components factor B, C4 and C3 cross-reacted with the corresponding human C components. Thus, C. callosus was found to contain effective classical and alternative pathways (CP, AP) and common pathways, reasonable amounts of C5 and common epitopes in the key C components, factor B, C4 and C3, which were preserved during evolution.

Animals↗
PubMed1998

A further link between innate and adaptive immunity: C3 deposition on antigen-presenting cells enhances the proliferation of antigen-specific T cells.

Murine cells of the B lymphoblastoid line A20 and concanavalin A-elicited peritoneal macrophages are shown to activate and fix C3 fragments covalently when incubated in fresh, autologous serum under conditions allowing the initiation of the alternative complement pathway. For the detection of cell-bound C3, cytofluorimetry was performed using FITC-labeled F(ab')2 fragments of anti-mouse C3. Cell-bound C3 fragments are not internalized or shed by the cells under culture conditions for at least two hours. When the antigen-presenting capacity of serum-treated cells was tested using various antigens and experimental systems, augmentation of the proliferation of antigen-specific T cells was found. This enhancing effect was particularly pronounced at suboptimal antigen doses. The elevation of T cell proliferation induced by C3-opsonized antigen-presenting cells (APC) could be abrogated by F(ab')2 fragments of goat anti-mouse C3, suggesting the involvement of C3 receptors expressed by T cells in the process. Using the 7G6 mAb recognizing murine CR1/CR2, the presence of these complement receptors on activated T cells is demonstrated by cytofluorimetry and immunoprecipitation, as well. These results point to the role of C3 bound to acceptor sites on APC in the facilitation of antigen presentation, providing a further link between innate and adaptive immunity.

Animals↗
PubMed1997

Activation, binding, and processing of complement component 3 (C3) by Blastomyces dermatitidis.

Complement plays a key role in phagocyte recognition and killing of Blastomyces dermatitidis, but little is known about how complement components interact with the yeast. We report the characteristics of activation, binding, and processing of C3 by B. dermatitidis. In pooled normal human serum (NHS), deposition of C3 on the yeast was detectable within 2 min, whereas in NHS containing MgEGTA, deposition was delayed by 6 min, indicating that the yeast activates C3 by both classical and alternative pathways. When both pathways were operative, maximal binding of 4.5 x 10(6) C3 molecules/cell was achieved in less than 30 min. In the absence of the classical pathway, yeast cells bound 80% of the maximum C3, indicating that the yeast intrinsically activates the alternative pathway. Delayed deposition of C3 in NHS-MgEGTA was similar to that in NHS preabsorbed by the yeast or by immobilized protein A/G to remove serum immunoglobulin. Purified immunoglobulin restored C3 binding to NHS preabsorbed by the yeast, suggesting that antibody in nonimmune serum initiates the classical pathway. beta-Glucan absorption of NHS abolished the classical pathway, suggesting that cell wall beta-glucan is a target of initiating antibodies. Hydroxylamine treatment of NHS-opsonized yeast cells showed that 76% of C3 was bound to yeast cells by ester linkage, supporting a role for hydroxyl groups on cell wall polysaccharides. Hydroxylamine-cleaved fragments were chiefly C3b and iC3b; 70% of hydroxylamine-sensitive C3b was converted to iC3b within 1 min of opsonization, and the ratio was stable over 1 h. Our data predict that C3b and iC3b on opsonized yeast cells direct binding to CR1 and CR3 receptors on human phagocytes, which, in turn, may influence the fate of this host-pathogen interaction.

Blastomyces↗
PubMed1988

The cytolytic C5b-9 complement complex: feedback inhibition of complement activation.

We describe a regulatory function of the terminal cytolytic C5b-9 complex [C5b-9(m)] of human complement. Purified C5b-9(m) complexes isolated from target membranes, whether in solution or bound to liposomes, inhibited lysis of sensitized sheep erythrocytes by whole human serum in a dose-dependent manner. C9 was not required for the inhibitory function since C5b-7 and C5b-8 complexes isolated from membranes were also effective. No effect was found with the cytolytically inactive, fluid-phase SC5b-9 complex. However, tryptic modification of SC5b-9 conferred an inhibitory capacity to the complex, due probably to partial removal of the S protein. Experiments using purified components demonstrated that C5b-9(m) exerts a regulatory effect on the formation of the classical- and alternative-pathway C3 convertases and on the utilization of C5 by cell-bound C5 convertases. C5b-9(m) complexes were unable to inhibit the lysis of cells bearing C5b-7(m) by C8 and C9. Addition of C5b-9(m) to whole human serum abolished its bactericidal effect on the serum-sensitive Escherichia coli K-12 strain W 3110 and suppressed its hemolytic function on antibody-sensitized, autologous erythrocytes. Feedback inhibition by C5b-9(m) represents a biologically relevant mechanism through which complement may autoregulate its effector functions.

Complement Activation↗
PubMed1979

An anticomplementary agent, K-76 monocarboxylic acid: its site and mechanism of inhibition of the complement activation cascade.

A monocarboxylic acid derivative (K-76 COOH) of K-76, purified from the culture filtrate of Stachybotrys complement I nov. sp. K-76, inhibits complement (C) activity. Its inhibitory action is mainly on C5 step. It strongly inhibits the generation of EAC1,4b,2a,3b,5b from C5 and EAC1,4b,2a,3b, and accelerates the decay of EAC1,4b,2a,3b,5b. It also causes some inhibition of the reactions of the reactions of C2,C3,C6,C7 and C9 with their respective preceding intermediate cells. It has no effect on the generation of EAC1,4b from C4 and EAC1, or of EAC-8 from C8 and EAC-7, and apparently increases the generation of EAC1,4b from C1 and EAC4b probably by inhibiting transfer or turnover of C1. It does not affect the rate of decay of EAC1,4b,2a or the T max of generation of EAC1,4b,2a, and it inhibits immune adherence only at high concentration. K-76 COOH also strongly inhibits hemolysis through the alternative pathway of C activation by cobra venom factor, but it does not seem to inhibit the early steps of the alternative pathway, because it has little affect on the consumption of C3 or the conversion of beta 1C to beta 1A on treatment of C serum with zymosan. K-76 COOH probably combines with C5 molecules, forming the inactive complexes, or it causes the structural alteration of C5.

Animals↗
PubMed1993

Extracts of wheat gluten activate complement via the alternative pathway.

We studied the ability of wheat gluten and its subfractions to activate complement directly. A sensitive sandwich ELISA employing a monoclonal antibody (MoAb) to a C9 neoepitope exposed in the terminal complement complex (TCC), a functional haemolytic assay for C5b6 generation, and Laurell's electrophoretic method of estimating C3 conversion to C3bi were used. On a weight-for-weight basis, enzyme solubilized Frazer's fraction three of gluten (FIII) produced approximately 75% of the complement activation seen with the potent activator zymosan. By contrast, activation with whole insoluble undigested gluten was very weak and similar to that seen with ovalbumin or beta-lactoglobulin. The results were the same using normal human serum or sera from patients with coeliac disease, dermatitis herpetiformis, or hypogammaglobulinaemia as the complement source. Activation by both zymosan and FIII was blocked in 0.01 M EDTA, but not in 0.01 M EGTA with 0.0025 M magnesium chloride. Zymosan and FIII activated complement in a serum from a patient with an intact alternative pathway but classical pathway haemolytic activity (CH50) of zero. Preferential heat inactivation of the alternative pathway inhibited both zymosan- and FIII-induced activation. Our results confirm that FIII is a strong activator of the alternative pathway. We discuss how gluten enteropathy might be initiated by complement.

Adult↗
PubMed1984

The influence of classical pathway components during alternative pathway--modulated immune complex aggregation: the role of C1 INH.

Alternative pathway (AP)-triggered reactions as well as classical pathway (CP)-mediated ones, were investigated turbidimetrically and/or immune electrophoretically, either in the presence or in the absence of in situ-generated immune complexes (ICs; tetanus toxoid/human anti-tetanus toxoid-IgG; ICs of equivalence) during the early stages of reaction. Monospecific Fab'- or Fab-fragments (rabbit) were used to block the complement function in normal human serum (NHS). C1q, functionally available following the addition of ethylene-glycol-bis-(beta-aminoethyl ether), N,N'-tetraacetic acid to NHS (EGTA-NHS), was found to increase the IC aggregation, thereby producing a biological surface upon which AP-dependent proteins were deposited. The functional inhibition of C1INH caused a C1s-mediated C3 conversion irrespective of the fact whether C1s was incorporated within macromolecular C1 (NHS) or dissociated from it (EGTA-NHS), thus, in the latter case inhibiting the AP-dependent portion of turbidity. It seemed probable that C3 conversion was effected by a fluid-phase CP C3 convertase. This process, normally counteracted by C1INH, worked more efficiently in EGTA-NHS than in NHS, indicating that the C1s-mediated reactions, initiated by presently unknown mechanisms, were less extensively regulated outside of the Ca2+-dependent C1 complex. The study demonstrates that in EGTA-NHS, too, where AP-triggered reactions have usually been investigated, sections of CP activation may play an important role, especially in situations where the function of C1INH is restricted.

Antigen-Antibody Complex↗
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↗
PubMed2006

Primary glomerulonephritis with isolated C3 deposits: a new entity which shares common genetic risk factors with haemolytic uraemic syndrome.

INTRODUCTION: Abnormal control of the complement alternative pathway (CAP) (factor H, factor I and membrane cofactor protein (MCP) deficiencies) is a well established risk factor for the occurrence of haemolytic uraemic syndrome (HUS). In some instances, HUS may be associated with an unusual glomerulonephritis with isolated C3 deposits (glomerulonephritis C3). We determined whether HUS and glomerulonephritis C3 share common genetic susceptibility factors. METHODS: We identified 19 patients with glomerulonephritis C3. We measured levels of circulating complement components, performed assays for the detection of C3 nephritic factor (C3NeF) and screened factor H, factor I and MCP coding genes for the presence of mutations. RESULTS: Patients were divided in two groups based on renal pathology findings: group I (n = 13) had typical features of type I membranoproliferative glomerulonephritis (glomerulonephritis C3 with membranoproliferative glomerulonephritis (MPGN)) and group II (n = 6) was characterised by mesangial and epimembranous C3 deposits in the absence of mesangial proliferation (glomerulonephritis C3 without MPGN). Mutations in complement regulatory genes were detected in 4/6 patients with glomerulonephritis C3 without MPGN (heterozygous mutations in factor H gene (two patients) with low factor H antigenic level in one case, heterozygous mutations in factor I gene (two patients)) and in only 2/13 patients with glomerulonephritis C3 with MPGN (heterozygous mutations in factor H gene (one patient) and double heterozygous mutation in CD 46 gene (one patient)). In contrast, C3NeF was present in 5/13 patients with glomerulonephritis C3 with MPGN and in 2/6 patients with glomerulonephritis C3 without MPGN, one of whom had a factor H mutation. CONCLUSION: HUS and glomerulonephritis C3 without MPGN share common genetic risk factors. Constitutional or acquired dysregulation of the CAP is probably associated with a wide spectrum of diseases, ranging from HUS to glomerulonephritis C3 with MPGN.

Adolescent↗
PubMed1979

Activation of the classical complement pathway by nephritic factor bound to the alternative pathway C3/C5 convertase.

Nephritic Factor (NF), the potent alternative pathway activator, which is occasionally found in association with certain types of nephritis has recently been identified as an IgG class autoantibody specific for the C3 convertase (C3bB) of the alternative pathway. In these studies we have examined the possibility that the cell-bound NF-stabilized C3 convertase (EC3 bBNF) binds and activates the first component of the classical pathway of complement. EC3bBNF bound C1q, and the extent of binding was dependent upon the number of NF molecules bound per cell and decreased parallel to the dissociation and release of NF from the cells. Interaction of C1 with bound NF resulted in its activation as shown by the proteolytic conversion of proenzyme C1s to its activated form C1s. As was the case with C1q binding, C1 activation was dependent on the number of NF molecules bound per cell. Thus the NF-stabilized C3 convertase binds and activates C1.

Binding Sites↗
PubMed1985

[Mechanisms of the mediated neutrophil reactivity to Escherichia coli peptidoglycan].

The opsonic properties of normal serum with respect to E. coli peptidoglycan was studied under the actual conditions of the oxygen-dependent metabolism of neutrophils. In the course of the differentiated study of the influence of antibodies, the classical and the alternative cascades of complement the serum was heated, treated with ethylenediaminetetraacetate and ethylene glycol tetraacetate, exhausted in the cold. In serial experiments the opsonic activity of purified fibronectin was studied. The indirect reactions were shown to be the leading mechanisms of the neutrophil-stimulated activity of E. coli peptidoglycan. IgG was found to be in the center of the opsonic cooperation and thus to determine the quantitative manifestation of the total phenomenon. Complement proved to be of lesser importance; depending on the conditions of the experiment, the activation of complement occurred by the alternative way (after the removal of antibodies) or the classical way (whole serum). The actual contribution of IgG-independent and complement-independent opsonins was insignificant. Fibronectin in physiological concentrations showed no opsonic activity.

Antibodies, Bacterial↗
PubMed1987

Complement effects of the infectivity of Plasmodium gallinaceum to Aedes aegypti mosquitoes. II. Changes in sensitivity to complement-like factors during zygote development.

During transformation into ookinetes, the zygotes of Plasmodium gallinaceum are initially resistant to lysis by heat-labile and EDTA-sensitive factors in the serum of their natural host, the chicken. Between 6 and 8 hr postgametogenesis, zygotes cultured in vitro lose their resistance to these factors. Loss of resistance to these factors in vitro is reflected by loss of infectivity of the zygotes to Aedes aegypti mosquitoes in the presence of native chicken serum. These factors are probably components of the alternative pathway of complement (APC) of chicken serum. Gametocytes of P. gallinaceum in chicken blood are able to infect A. aegypti mosquitoes apparently due to inactivation of the APC in a blood meal within 3-4 hr after ingestion, i.e., several hours before the zygotes lose their resistance to chicken APC. In addition to the heat-labile factors (APC) in chicken serum, the zygotes are transiently sensitive to other factor(s) in the mosquito blood meal. These factor(s) are not destroyed by prior heating of the chicken serum given in a blood meal and therefore cannot be complement components. The antiparasitic effects of the factors are neutralized by addition of EDTA to the blood meal and could be due to an EDTA-sensitive metalloprotease present in the mosquito midgut.

Aedes↗
PubMedDate not supplied

[Assessment of total complement activity of blood plasma (serum) using reader for immunoenzyme analysis].

A method for instrumental assessment of the total complementary activity of human blood plasma (serum) by the classical and alternative activation is proposed. The test is carried out by the micromethod in round-bottom 96-well plates for immunological tests. The results are read by automated analyzer for enzyme immunoassay recording at 405 nm wavelength.

Complement Activation↗
PubMed2001

A novel chicken membrane-associated complement regulatory protein: molecular cloning and functional characterization.

A cDNA encoding a membrane-associated complement (C) regulatory protein was identified here for the first time in an oviparous vertebrate, chicken. This protein, named Cremp, possessed five short consensus repeats (SCRs) and one SCR-like domain followed by a transmembrane domain and a cytoplasmic tail. SCR1/SCR2 of Cremp were 43.6% identical with SCR2/SCR3 of human decay-accelerating factor (CD55), and SCR3/SCR4 were 45.3% identical with those of human membrane cofactor protein (CD46). Cremp is likely to be an ancestral hybrid protein of human decay-accelerating factor and membrane cofactor protein rather than a homolog of rodent C receptor 1-related protein y, which structurally resembles human CR1 (CD35). Chinese hamster ovary cells transfected with Cremp were efficiently protected from chicken C but not from human or rabbit C in both classical and alternative pathways. Thus, chicken Cremp is a membrane C regulator for cell protection against homologous C. Cremp mRNA was seen as a doublet comprised of a faint band of 2.2 kb and a thick band of 3.0 kb on RNA blotting analysis. An Ab against chicken Cremp recognized a single band of 46.8 kDa on immunoblotting. mRNA and protein of Cremp were ubiquitously expressed in all chicken organs tested. Minute amounts of dimer were present in some tissues. Surface expression of Cremp was confirmed by flow cytometry and immunofluorescence analysis. These results suggested that even in nonmammals a C regulatory membrane protein with ubiquitous tissue distribution should be a prerequisite for protection of host cells from homologous C attack.

Amino Acid Sequence↗
PubMed1996

Mouse complement component C4 is devoid of classical pathway C5 convertase subunit activity.

It has long been known that mouse C4 has unusually low hemolytic activity relative to the C4 of other mammalian species (e.g. human and guinea pig), the measurements being done in most cases using a C4-deficient guinea pig serum reagent in a one-step assay with EA. This low activity for mouse C4 previously had been attributed to "technical" difficulties such as lability of the protein during blood collection and partial species incompatibilities with guinea pig components. Recently, we presented evidence for the involvement of human C4 beta-chain residues 455-469, a putatively exposed hydrophilic segment, in contributing to a C5 binding site in the C4b subunit of the classical pathway C5 convertase, C4b3b2a. Given that there were five sequence differences between the human and mouse protein within this segment, we hypothesized that these substitutions may have compromised the C5 convertase subunit activity of mouse C4, thereby resulting in its low hemolytic activity. Using a multi-step hemolytic assay which was totally dependent upon C5 cleavage by the classical pathway, we found that mouse C4 was completely devoid of classical pathway C5 convertase subunit activity. We have been able to rule out the most obvious potential species incompatibilities (e.g. between C4mo and C5gp) as being responsible for this lack of activity. Moreover, we found that the low level of hemolytic activity of mouse C4 measured in the one-step assay can be ascribed totally to C5 cleavage, and subsequent terminal component assembly, by the alternative pathway C5 convertase, (C3b)2Bb. However, the assembly of the latter enzyme complex is dependent upon the presence of C3b molecules deposited initially via the classical pathway C3 convertase in which mouse C4b is a subunit. Finally, whereas conversion of human residues 458RP to the mouse-like sequence PL was sufficient to abrogate classical pathway C5 convertase subunit activity in human C4, the five substitutions which "humanized" the 452-466 segment of mouse C4 (corresponding to human residues 455-469) were on their own insufficient to impart this activity to mouse C4. This implies that, in addition to the 455-469 beta-chain segment of human C4, there are other regions of the molecule contributing to C5 binding which are also non-conserved between human and mouse C4.

Amino Acid Sequence↗
PubMed1982

Nephritic factor (NeF) of alternate pathway (NeFA) and of classical pathway (NeFc).

NeFA and NeFc were studied in various cases (115 cases). 5 cases were followed up for a long time. NeFA assay was done by the "microtest plate method". We detected the NeF activity for the first time in the cases of Sjögren syndrome (SjS) and SjS+SLE+Hashimoto's disease (Hashimoto). In some cases NeF activity disappeared after therapy, and in one case NeFA and NeFc were positive at first, but then only NeFA activity became negative following the adequate therapy. As to the antibody nature of NeF, the possibility was suggested that NeFA might be anti C3b autoantibody and NeFc might be anti C4b and/or C4b2a auto-antibody.

Complement Activation↗
PubMed1993

Genetic, structural, and functional studies of a C3-like protein in the marsupial Setonix brachyurus.

A monospecific goat antiserum was prepared against a putative C3 protein (QuC3) from serum of the Western Australian macropod Setonix brachyurus (quokka) using the classical method previously used to produce antiserum against C3 from other mammalian species. Sodium dodecyl sulphate-polyacrylamide gel electrophoretic analysis of reduced immunoprecipitated QuC3 revealed two polypeptide chains with an estimated M(r) of 128,000 and 82,000, respectively--presumably reflecting subunit molecules similar to the C3 alpha and beta subunits found in other mammalian species. No variation in the size of either of the QuC3 alpha or beta subunits was found in different quokka serum samples. Furthermore, no variation in the electrophoretic mobility of QuC3 was detected amongst the same samples using a standard immunofixation electrophoretic technique. The addition of zymosan or immune complexes to fresh quokka serum resulted in activation of QuC3. Immune complex mediated activation of QuC3 was inhibited by the addition of EDTA and Mg(2+)-EGTA. By contrast, EDTA only inhibited activation of QuC3 following incubation of fresh quokka serum with zymosan, suggesting that the quokka has a classical and an alternative C3 activation pathway, similar to those found in eutherian mammals. This study has shown for the first time that one of the macropod marsupials (S. brachyurus) has a C3-like protein with similar structural and functional characteristics to C3 found in eutherian mammals. However, unlike C3 in other mammalian species, genetic variability of the structure of the quokka C3-like protein may be restricted.

Animals↗