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PubMed1995

Mutation of residues in the C3dg region of human complement component C3 corresponding to a proposed binding site for complement receptor type 2 (CR2, CD21) does not abolish binding of iC3b or C3dg to CR2.

Most evidence points toward there being a shared binding site in complement receptor type 2 (CR2, CD21) for the complement ligand C3dg and the EBV surface envelope glycoprotein gp350/220. Indeed, synthetic peptide studies have suggested that the CR2-binding sites in human C3dg and EBV gp350/220 share a similar sequence motif. The proposed CR2-binding sequence in C3dg is EDPGKQLYNVEA (residues 1199-1210 of mature C3), whereas that in EBV gp350/220 is EDPGFFNVEI (residues identical to C3dg are underlined). To further examine the role of amino acids 1199-1210 in the binding of the C3 fragments iC3b and C3dg to CR2, the following alanine-substitution variants of human C3 were tested in two independent CR2-binding assays: ED1199,1200AA; KQ1203,1204AA; L1205A; Y1206A; NV1207,1208AA; E1209A; and ED-KQ-NV1199,1200-1203,1204-1207,1208AA-AA-AA. Also engineered and tested was a chimeric C3 molecule in which the 1199-1210 sequence (PVPGGYQLTLEA) from the non-CR2-binding trout C3 molecule was grafted onto a human C3 background. Recombinant C3 proteins were expressed transiently in COS-1 cells, deposited as C3b on C3 convertase-bearing sheep erythrocytes and finally converted to cell-bound iC3b or C3dg using factors H and I. Binding of EAC423bi and EAC423dg to CR2 on Raji cells or EAC423dg to soluble CR2 was assessed. In most cases, the substitutions had little effect on CR2-binding activity and even in the case of the most highly substituted variants, the decrease in CR2-binding activity was less than twofold. Thus, contrary to the results anticipated from synthetic peptide studies, the single and multiple substitutions to the C3 sequence tested failed to corroborate a role for the 1199-1210 sequence in the C3dg-CR2 interaction.

Amino Acid Sequence↗

PubMed1993

Expression of the complement classical pathway by human glioma in culture. A model for complement expression by nerve cells.

In this paper we demonstrate the synthesis of the components of the classical complement pathway, namely C1q, C1r, C1s, C1-Inh, C2, C4, and C5, by human glioma cell lines (U118MG, T193, and T98G). All these components were structurally, antigenically, and functionally similar to their serum counterparts as determined by biosynthetic labeling experiments, Western blot analysis, and hemolytic assays. Northern blot analysis of mRNA demonstrated that, for each of these components, their specific mRNA had the same size as the equivalent mRNA from hepatic tissue. We could not detect the synthesis of C4bp by these cell lines, and the secretion of C1q was only detected after stimulation by interferon-gamma. All these syntheses were up-regulated by interferon-gamma and tumor necrosis factor. Interleukin-1 beta only increased C2 expression and reproducibly down-regulated C5 secretion when used at high doses. Glioma cell lines appear to be an efficient and convenient model for the analysis of complement expression in human astrocytic cells.

Astrocytes↗

PubMed1983

Studies of the mechanism of bacterial resistance to complement-mediated killing. V. IgG and F(ab')2 mediate killing of E. coli 0111B4 by the alternative complement pathway without increasing C5b-9 deposition.

The mechanism of antibody-dependent complement-(C) mediated killing of Escherichia coli 0111B4, strain 12015 (12015), was examined. 12015 was resistant to serum killing when incubated in hypogammaglobulinemic serum (H gamma S) or pooled normal human serum (NHS) that had been previously adsorbed to remove specific antibody (Abs NHS). Presensitization with immune rabbit serum or purified immune rabbit IgG resulted in 1 to 3 log killing when 5 X 10(8) colony forming units (CFU)/ml were incubated in 10 to 40% Abs NHS. Binding of 125I-C3 and 131I-C9 to the bacterial surface of the presensitized and the nonpresensitized strain was quantitated when these organisms were incubated in 10, 20, and 40% Abs NHS. Stable binding of up to 3.0 X 10(5) molecules of C3 and 8.0 X 10(4) molecules of C9 to presensitized and nonpresensitized isolates occurred in the highest concentration of serum, but there was no killing without presensitization. Similar results were found when Abs NHS was chelated with ethylene bis glycoltetraacetic acid containing 2 mM MgCl2 (Mg EGTA) to block classical pathway activation, indicating that antibody mediated the bactericidal reaction through the alternative pathway. Deposition of C3 and C9 and killing of 120 15 in 10% Abs NHS or 10% H gamma S was measured after presensitization with increasing amounts of IgG, F(ab')2, or Fab'. There was a dose-dependent increase in C3 deposition and killing, but only minimal change in C9 binding when 1.0 X 10(3) to 3.2 X 10(4) IgG or F(ab')2/CFU were bound to the bacterial surface. In contrast, there was no increase in C3 or C9 binding and no bacterial killing when 1 X 10(3) to 3.4 X 10(4) molecules Fab'/CFU were bound to the bacterial surface. These experiments show that immune IgG and F(ab')2 can mediate killing of E. Coli 0111B4 by the alternative pathway without changing the extent of terminal C component attachment to the bacterial surface.

Animals↗

PubMed1977

Role of complement in thrombogenesis. I. Complement-dependent coagulation in a model system.

Chondroitin sulphate was used to isolate from plasma a system that clotted with Russell's viper venom, brain extract and activated contact factors. Clotting appeared to depend on concomitant change in C4, C3 and C1s in the system. Brain extract additionally reacted with C9. Reconstitution of specifically defective plasmas suggested a specific role for each of these complement components in clotting.

Antigen-Antibody Reactions↗

PubMed1988

The significance of complement activation in the pathogenesis of hypersensitivity pneumonitis: sequential changes of complement components and chemotactic activities in bronchoalveolar lavage fluids.

Hypersensitivity pneumonitis (HP) is believed to be induced by immunological mechanisms, the details of which remain to be clarified. While a role for cellular immunity is accepted in the pathogenesis of HP, several clinical observations also suggest a role for immune-complex-mediated lung injury. We have previously demonstrated the presence of chemotactic factors for polymorphonuclear cells (PMNs) in bronchoalveolar lavage (BAL) fluids of acutely ill patients with the summer type of HP found in Japan. The present study correlated chemotactic factors for PMNs with the level of C5a des Arg in BAL fluids obtained from patients with summer type HP. Furthermore, this study demonstrated that PMNs were increased in BAL fluids obtained after 2 days of avoidance of exposure to the presumptive causative agent. The percentage of PMNs in the BAL increased in proportion to the activity of the chemotactic factors. Finally, leukotriene B4 was not detected in concentrated BAL or supernatant fluids of cultured macrophages. These results suggest that complement activation in the respiratory tract may occur as the early event in the pathogenesis of HP.

Alveolitis, Extrinsic Allergic↗

PubMed1985

Regulatory proteins for the activated third and fourth components of complement (C3b and C4b) in mice. II. Identification and properties of complement receptor type 1 (CR1).

We identified on the membrane of mouse spleen cells a polypeptide of Mr 190,000 (S190), with binding affinity for the mouse third component of the complement system (C3). S190, purified by affinity chromatography on C3-Sepharose, has properties resembling those of the human C3 receptor type 1 (CR1). Thus, S190, like CR1, served as a cofactor for the C3b inactivator (I)-mediated cleavage of fluid-phase C3b into iC3b, and had cofactor activity comparable to that of serum factor H (H). S190 also acted as a cofactor for the cleavages of membrane-bound C3b or membrane-bound iC3b into C3c (Mr 140,000) and C3dg (Mr 40,000) by serum factor I. As is the case with CR1, the specific activity of S190 for the cleavages leading to C3c-C3dg formation was approximately 100-fold greater than that of H. We therefore conclude that S190 and CR1 are analogous proteins.

Animals↗

PubMed1980

The effect of aging on complement activity (CH50) and complement protein levels.

Total complement activity (CH50), and the protein levels of C1q, C4, C3, C5, C9, factor B and C1 inactivator were measured in the 1,130 sera obtained from healthy subjects aged between 20 and 79 years old. Increase in the levels of CH50, C1q, C4, C3, C5 and C9 and decrease of factor B were found in the older age group. The level of C1 inactivator showed little variation with aging.

Adult↗

PubMed1986

Complement C3b receptors on erythrocytes, circulating immune complexes, and complement C3 split products in patients with primary Sjögren's syndrome.

Twenty-one patients with primary Sjögren's syndrome (SS) and 22 age- and sex-matched healthy controls were examined for levels of C3b receptors (CR1) on erythrocytes (by an enzyme-linked immunosorbent assay), levels of circulating immune complexes (IC) (by a polyethylene glycol precipitation complement consumption method), and C3 split products, C3d and C3c (by intermediate gel rocket immunoelectrophoresis). CR1 varied within a wide range (20-124%), and a preponderance of low numbers was found among patients with primary SS (P less than 0.05). The levels of circulating IC and C3d were elevated in primary SS patients (P less than 0.01) and were mutually correlated (P less than 0.01; tau = 0.5), but levels of circulating IC and C3d did not correlate with CR1 values. C3c concentrations were the same in patients and controls. It is possible that the CR1 level serves as a predictor for the development of IC-induced manifestations, and the C3d concentration serves as an objective parameter for disease activity in primary SS.

Adult↗

PubMed1980

Analysis of the different types of leukocyte membrane complement receptors and their interaction with the complement system.

The specificity, distribution, and structure of 8 different types of leukocytes membrane complement (C) receptors (CR1, CR2, CR3, and receptors for C1q, beta 1H, C3e, C3a, and C5a) are discussed. Recent data are reviewed on the synthesis of C components by macrophages and B lymphocytes, and how these components may function in the activation of these two cell type by the C system. Commonly used C receptor assay procedures are evaluated in terms of both specificity and sensitivity. Specific assay procedures are recommended for measuring CR1 (C4b-C3b receptor), CR2 (C3d receptor), CR3 (C3bi receptor), and the beta 1H receptor. Assays include both rosette and fluorescence procedures for detection of C receptors on either mouse or human leukocytes. Primary systems have been selected for optimal sensitivity and specificity, and where possible, acceptable alternative systems that are less sensitive or specific are suggested for laboratories lacking facilities for C purification.

Animals↗

PubMed1984

Measurement of the ratio of the eighth and ninth components of human complement on complement-lysed membranes.

The mole ratio of the eighth (C8) and ninth (C9) components of human complement on membranes carrying the cytolytic C5b-9 complex was measured by direct binding assays. Erythrocytes from two different species were used as the membrane system. Antibody-treated sheep erythrocytes carrying a relatively small number of precursive membrane-bound C5b-7 complexes were prepared by exposure to human C8-depleted serum. These complexes were subsequently converted to C5b-8 by addition of saturating amounts of C8. Parallel binding assays using 125I-C8 were used to determine the exact amount bound and thus the number of C5b-8 complexes per cell. These cells were subsequently incubated with excess 125I-C9 and the amount bound relative to C8 on the membrane was measured. Results indicated the C8:C9 ratio remained constant at approximately 1:4 as the number of complexes varied from 40 to 310 per cell. Similar results were obtained regardless of whether C8 and C9 were added sequentially or simultaneously to cells bearing C5b-7. For comparison, experiments were also performed using membranes that contained a high number of complexes. Here, rabbit erythrocytes which carried approximately 25 000 C5b-7 per cell were incubated with limited amounts of C8 to form C5b-8 complexes on the membrane surface, the exact number of which was measured by 125I-C8 binding assays. When erythrocytes prepared in this manner were incubated with excess 125I-C9, the ratio of C8:C9 on the membrane was found to be essentially constant at approximately 1:3 as the number of these complexes varied from 50 to 4000 per cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1979

Relationships between the haemolytic activities of the human complement system and complement components.

The relationships between the haemolytic activities of complement and its components were studied. The activities studied included CH50 (classical pathway), AP50 (alternative pathway), CV50 (early part of alternative pathway) and C(3--9)H50 ((the late part of both pathways). The components included C3, C4, C5, C9, B and D. There was a good correlation between CH50 and AP50. AP50 had a good correlation with B and CV50. There was no correlation between AP50 and C(3--9)H50, and none between C(3--9)H50 and C5 or C9. AP50 may primarily represent changes in the early part of the alternative pathway. C(3--9)H50 is not influenced by respective changes in the amounts of C5 or C9. Since cell lesion is now considered to be caused by a unit of C5b to C9, a change in each component of C5 to C9 may not influence haemolytic activity.

Adult↗

PubMed1991

Human IgG subclass pattern of inducing complement-mediated cytolysis depends on antigen concentration and to a lesser extent on epitope patchiness, antibody affinity and complement concentration.

The relative complement-mediated lytic capability of the IgG subclass isotypes was studied using a matched set of mouse-human chimeric anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies. The subclass pattern was shown to be highly dependent on variations in antigen concentration and to lesser extent on variation in epitope patchiness, antibody binding affinity and complement concentration. In general, the IgG3 subclass was most effective in inducing cytolysis at the different conditions used and only at high antigen concentration did the IgG1 subclass mediated more efficient cytolysis than IgG3. The IgG2 isotype required a relative high antigen concentration to be cytolytic while the IgG4 isotype was not cytolytic at any of the conditions tested. These individual characters of each of the IgG subclasses makes it conceivable that a subtle system of immunoregulation exists among the subclasses.

Antibody Affinity↗

PubMed1997

Complementation analysis of fibroblasts from peroxisomal fatty acid oxidation deficient patients shows high frequency of bifunctional enzyme deficiency plus intragenic complementation: unequivocal evidence for differential defects in the same enzyme protein.

In the last few years many patients have been reported with a defect in peroxisomal fatty acid beta-oxidation of unknown origin. Using a combined approach based on direct activity measurements of straight-chain acyl-CoA oxidase and complementation analysis after somatic cell fusion of fibroblasts, we have now classified 13 patients into 4 distinct groups representing different gene defects. Remarkably, we found intragenic complementation in group 2 so that group 2 is in fact made up of 3 distinct subgroups. The underlying basis for this peculiar phenomenon probably has to do with the fact that bifunctional protein harbors two catalytic activities including enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase. In group 2A enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase are defective whereas in group 2B and 2C either the hydratase or 3-hydroxyacyl-CoA dehydrogenase component of the bifunctional protein is deficient.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1998

Nonsense mutation in exon 4 of human complement C9 gene is the major cause of Japanese complement C9 deficiency.

Deficiency of the ninth component of human complement (C9) is the most common complement deficiency in Japan but is rare in other countries. We studied the molecular basis of C9 deficiency in four Japanese C9-deficient patients who had suffered from meningococcal meningitis. Direct sequencing of amplified C9 cDNA and DNA revealed a nonsense substitution (CGA-->TGA) at codon 95 in exon 4 in the four C9-deficient individuals. An allele-specific polymerase chain reaction system designed to detect exclusively only one of the normal and mutant alleles indicated that all the four patients were homozygous for the mutation in exon 4 and that the parents of patient 2 were heterozygous. The common mutation at codon 95 in exon 4 might be responsible for most Japanese C9 deficiency.

Adolescent↗

PubMed1978

The effect of limited fragmentation of porcine 19 S IgM on agglutination and complement fixation. Evidence that at least two intact subunits are required for complement fixation by IgM antibody.antigen complexes.

Limited fragmentation of pig IgM anti-Salmonella with pepsin gave a 12 S product which agglutinated Salmonella almost as efficiently as the original IgM but the complement fixing ability of the complexes was substantially lowered. The 12 S product was shown to consist of molecular entities having intact Fc regions with an average of 3 or 4 intact Fab arms still attached. Our results appeared to show that removal of Fab arms by pepsin was a random process and on this basis a mathematical model was set up. From this model it was possible to calculate the frequencies of molecular entities which would give rise to the observed average of 3 or 4 Fab arms. The most likely explanation of our results is that complexes need to be formed by molecules having at least two intact subunits before efficient complement fixation takes place.

Animals↗

PubMed1989

Methylprednisolone inhibits granulocytopenia induced by infusion of complement-activated serum but not of complement-activated plasma in rabbits.

The effect of methylprednisolone was examined on complement-induced granulocytopenia in vivo following infusion of zymosan-activated autologous plasma or serum into rabbits. Methylprednisolone only inhibited granulocytopenia and thrombocytopenia when zymosan-activated serum or fibrinogen-depleted plasma were infused. It was ineffective at preventing granulocytopenia in animals infused with zymosan-activated plasma. Only zymosan-activated plasma contained fibrin monomers which could directly cause granulocytopenia independently of the generation of C5a. Hence, during complement activation in whole blood in vivo, little or no effect of methylprednisolone should be expected in preventing granulocytopenia.

Agranulocytosis↗

PubMed1986

Treatment of cancer ascites by intraperitoneal administration of a streptococcal preparation OK-432 with fresh human complement--role of complement-derived chemotactic factor to neutrophils.

The role of complement in the polymorphonuclear leukocyte (PMN)-mediated tumor cell destruction in cancer ascites was investigated in relation to a streptococcal preparation OK-432, a so-called biological response modifier. Incubation of OK-432 with fresh human serum at 37 degrees C for 60 min resulted in the generation of C3a and C5a chemotactic factors. Intraperitoneal (i.p.) injection of the mixture to a patient with cancer ascites revealed an accumulation of PMNs in the ascitic fluid for a longer period with a rapid reduction of the ascitic fluid, than an intraperitoneal injection of OK-432 alone examined in the same patient. PMNs were found to invade clusters of the tumor cells and then form rosettes followed by the destruction of tumor cells. These findings induced by OK-432 continued over 10 days in the presence of fresh serum, while diminished within 3-4 days when OK-432 alone was injected. When fresh human plasma or fresh frozen plasma was used instead of serum and i.p. injected with OK-432 avoiding preincubation, the same cytological and clinical changes were observed in other patients. These data strongly indicate that OK-432 activates human complement either in vitro or in the peritoneal cavity, and induces PMNs to accumulate in the ascitic fluid. Although the mechanism of killing of tumor cells by PMNs is obscure, addition of human serum or plasma to i.p. use of OK-432 seems to be valuable for the management of patients with malignant ascites.

Ascites↗

PubMed1999

Telomerase activity in hybrids between telomerase-negative and telomerase-positive immortal human cells is repressed in the different complementation groups but not in the same complementation group of immortality.

The expression of telomerase is essential for cells to be immortalized, and most immortal cell lines possessed telomerase activity. Using the cell fusion technique, it has been shown that mortal and telomerase-negative phenotypes of normal cells are dominant over immortal and telomerase-positive phenotypes, suggesting that the normal cells possessed dominant repressor-type activity for telomerase expression. Several telomerase-negative immortal human cell lines were reported, in which telomerase-independent mechanisms was supposed to maintain telomere length. We aimed at seeing whether the telomerase-negative phenotype of these immortal cells is dominant over telomerase-positive phenotype of other immortal cells in correlation with cellular mortality. Results showed that, when telomerase-positive and -negative immortal parental cell lines belonging to the different complementation groups were fused, telomerase-negative mortal hybrid clones arose, i.e. telomerase-negative phenotype was dominant as well as mortal phenotype. However, when immortal hybrid cells arose from telomerase-positive and -negative immortal parents belonging to either the same or different complementation groups, they were all telomerase-positive, i.e. telomerase-negative phenotype appeared to be recessive. Telomerase-negative immortal hybrid was never established from any combinations between telomerase-negative and -positive immortal parental cells.

Base Sequence↗