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PubMed1999

Up-regulated production and activation of the complement system in Alzheimer's disease brain.

We used reverse transcriptase-polymerase chain reaction and Western blotting techniques to measure the levels of complement mRNAs and their protein products in Alzheimer's disease (AD) brain compared with non-AD brain. mRNAs for C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9 were detected in the 11 regions of brain that were investigated. The mRNA levels were markedly up-regulated in affected areas of AD brain. In the entorhinal cortex, hippocampus, and midtemporal gyrus, which had dense accumulations of plaques and tangles, C1q mRNA was increased 11- to 80-fold over control levels, and C9 mRNA 10- to 27-fold. These levels were substantially higher than in the livers of the same cases. Western blot analysis of AD hippocampus established the presence of all of the native complement proteins as well as their activation products C4d, C3d, and the membrane attack complex. These data indicate that high levels of complement are being produced in affected areas of AD brain, that full activation of the classical complement pathway is continuously taking place, and that this activation may be contributing significantly to AD pathology.

Adult↗
PubMed1997

Characterization of neuronal cell death induced by complement activation.

The complement system plays an important role in human immune defense mechanism. Its activation via either the classical or the alternative pathway can lead to the formation of membrane attack complex (MAC) and subsequently kills target cells. Activation of the classical pathway can be initiated with binding of C1q which is first factor of complement cascade to the Fc (fragment crystalline) region of immunoglobulin. This triggers a cascade of proteolytic events resulting in the activation of C5 convertase which cleaves C5 into C5b and C5a. The C5b then binds C6, C7, C8 to form a C5b-8 complex. Binding of C9 molecules to C5b-8 forms C5b-9, the MAC, which pore size increases as the number of C9 in the complex increases. If this membrane lesion persists and results in uncontrolled ion fluxes, the cells swell and eventually lyse. To restrict the activity of the complement system, endogenous complement inhibitors are available to regulate complement-mediated cytolysis. This enables the complement system to distinguish "self" from "foreign" and protect the host from inadvertent complement attack. Activation of the classical complement cascade has been reported in Alzheimer's disease and other neurodegenerative disorders. Recently, we demonstrated that complement activation causes neuronal cell death in vitro, and this neurodegenerative process is regulated by homologous restriction. In this article, we describe the use of two cell lines as in vitro models to evaluate cell injury/cell death induced by complement activation.

Animals↗
PubMedDate not supplied

[Membrane proteins as regulators of the complement system].

The data on structure, biochemical properties and functions of membrane proteins, performing cell defence against complement lysis, are summarized. Proteins DAE (decay accelerating factor) and CR1 (complement receptor of type 1) reduce the stability of complement convertases and cause their dissociation. MCP (membrane cofactor protein) and CR1 act as cofactors. In factor I mediated proteolysis of the convertase fragments C3b and C4b. The proteins C8bp-(C8-binding protein) and protectin affect membrane attack complex assembly. In contrast to MCP and CR1, which are integrative proteins, DAF, C8bp and protectin are bound to membranes with their glycophospholypid anchors. Tissue distribution of the proteins and the ways of their solubilization into biological fluids are reviewed.

Cell Membrane↗
PubMed2005

Insights into the human CD59 complement binding interface toward engineering new therapeutics.

CD59 is a 77-amino acid membrane glycoprotein that plays an important role in regulating the terminal pathway of complement by inhibiting formation of the cytolytic membrane attack complex (MAC or C5b-9). The MAC is formed by the self assembly of C5b, C6, C7, C8, and multiple C9 molecules, with CD59 functioning by binding C5b-8 and C5b-9 in the assembling complex. We performed a scanning alanine mutagenesis screen of residues 16-57, a region previously identified to contain the C8/C9 binding interface. We have also created an improved NMR model from previously published data for structural understanding of CD59. Based on the scanning mutagenesis data, refined models, and additional site-specific mutations, we identified a binding interface that is much broader than previously thought. In addition to identifying substitutions that decreased CD59 activity, a surprising number of substitutions significantly enhanced CD59 activity. Because CD59 has significant therapeutic potential for the treatment of various inflammatory conditions, we investigated further the ability to enhance CD59 activity by additional mutagenesis studies. Based on the enhanced activity of membrane-bound mutant CD59 molecules, clinically relevant soluble mutant CD59-based proteins were prepared and shown to have up to a 3-fold increase in complement inhibitory activity.

Alanine↗
PubMed1985

Decomplementation antigen, a possible determinant of staphylococcal pathogenicity.

We report the existence of an extracellular staphylococcal product, designated staphylococcal decomplementation antigen (DA), that causes rapid consumption of early-reacting complement components up to and including C5 in human serum. Complement activation occurs as a consequence of immune complex formation between DA and specific human immunoglobulin G antibodies and proceeds primarily via the classical pathway. The terminal components C7, C8, and C9 are not consumed during the process. Levels of DA production do not correlate with the expression of classical pathogenic factors, such as coagulase, clumping factor, protein A, or alpha-toxin. DA is a nondialyzable macromolecule eluting in a molecular-weight region of 70,000 to 120,000 on Sephacryl S-300 and displaying an apparent sedimentation coefficient of 3 to 4 S on sucrose density gradients. The molecule is remarkably stable and resists destruction upon boiling for 30 min or by treatment with pronase, lysostaphin, DNase, or RNase. We anticipate that DA protects staphylococci from complement attack through induction of abortive, complement-consuming reactions in the fluid phase.

Antigens, Bacterial↗
PubMed1981

Complement levels in cigarette smokers: elevation of serum concentrations of C5, C9, and C1-inhibitor.

Serum levels of 13 individual complement component and control proteins were measured in 25 male cigarette smokers and 25 male non-smokers. A significant elevation of the mean serum levels of C5, C9 and C1-inhibitor was demonstrated for the smokers while levels of C1q, C2, C4, C3, C6, C8, Factor B, properdin, C3b inactivator and beta 1H did not differ significantly between the two groups. Serum level of C9 in individual smokers correlated significantly with both the present amount of consumption and cumulative consumption, while C5 level correlated with present consumption. The complement profile in the cigarette smoker is similar to that observed in certain inflammatory conditions. The elevation of C5, C9 and C1-inhibitor levels may reflect the presence of a low grade inflammatory process in the cigarette smoker.

Adolescent↗
PubMed1992

The 4-nitroquinoline 1-oxide mutational spectrum in single stranded DNA is characterized by guanine to pyrimidine transversions.

4-Nitroquinoline-1-oxide is a potent mutagen and carcinogen which induces two main guanine adducts at positions C8 and N2. In ds or ss damaged DNA the ratio C8/N2 adducts is 1:2 and 8-10:1, respectively. In bacteria and yeast 4NQO has been shown to be a base substitution mutagen acting at G residues inducing mainly G to A transitions. We determined the mutational spectrum induced by the 4NQO metabolite, acetoxy-4-aminoquinoline 1-oxide, in the M13lacZ'/E. coli lacZ delta M15 alpha complementation assay using ssDNA. Among 68 Ac-4HAQO induced mutants, G to Pyr transversion was the most frequent base substitution observed. By comparison with dsDNA based systems, our data suggest that dGuo-C8-AQO induces G to Pyr transversions. A mechanism to explain how this lesion may induce transversions is proposed.

4-Nitroquinoline-1-oxide↗
PubMed1984

Solubilization of immune complexes in complement factor deficient sera and the influence of temperature, ionic strength and divalent cations on the solubilization reaction.

The complement-mediated solubilization (CMS) of immune complexes (IC) and the initial kinetics (IKS) of this reaction in human sera depleted of or deficient in C2, C3, C8, factors B, P and I were investigated. Sera depleted of B or P and those lacking native C3 or factor I showed virtually no CMS whereas the IKS and CMS capacity of a C8-deficient serum were within the reference range. The IKS of a C2-deficient serum was markedly retarded while the CMS capacity was normal. Addition of C2 normalized the kinetics of the reaction. There was a good correlation between the kinetics of CMS determined by a radioassay and kinetic data for the binding of C3b to preformed immune complexes. The CMS capacity reached maximum at 39-41 degrees C and at an ionic strength of approximately 0.20 mu. Selective chelation of Mg2+ completely abolished the CMS of IC. Maximal CMS was observed at Mg2+ concentration of about 2mM. Chelation of Ca2+ in serum by Mg2+-ethylene glycol tetraacetic acid reduced the CMS capacity by up to 50% and the IKS was markedly retarded. Varying the Zn2+ or Mn2+ ion concentrations in serum influenced neither the IKS nor the CMS capacity.

Antigen-Antibody Complex↗
PubMed2000

Preconditioning reduces myocardial complement gene expression in vivo.

This investigation examined the effect of preconditioning in an in vivo model of ischemia-reperfusion injury. Anesthetized New Zealand White rabbits underwent 30 min of regional myocardial ischemia followed by 2 h of reperfusion. Hearts preconditioned with two cycles of 5 min ischemia-10 min reperfusion (IPC) or with the ATP-sensitive K (K(ATP)) channel opener, diazoxide (10 mg/kg), exhibited significantly (P < 0.05) smaller infarcts compared with control. These treatments also significantly (P < 0.001 to P < 0.05) reduced C1q, C1r, C3, C8, and C9 mRNA in the areas at risk (AAR). The K(ATP) channel blocker 5-hydroxydecanoate (5-HD; 10 mg/kg) attenuated infarct size reduction elicited by IPC and diazoxide treatment. 5-HD partially reversed the decrease in complement expression caused by IPC but not diazoxide. There were no significant differences in complement gene expression in the nonrisk regions and livers of all groups. Western blot analysis revealed that IPC also reduced membrane attack complex expression in the AAR. The data demonstrate that preconditioning significantly decreases reperfusion-induced myocardial complement expression in vivo.

Animals↗
PubMed1987

Recovery of human neutrophils from complement attack: removal of the membrane attack complex by endocytosis and exocytosis.

Nucleated cells can resist lysis by and recover from complement attack even after formation of the potentially cytolytic membrane attack complex on the cell surface. We have found that human neutrophils resist complement lysis by the physical removal of membrane attack complexes by both endocytic and exocytic process. The latter mechanism predominates, vesiculation being detectable within 60 sec of initiating the complement cascade. Sixty-five percent of the formed complexes are removed on plasma membrane vesicles, although only 2% of the cell surface is lost. Ultrastructural examination revealed that these vesicles were covered with ring-like "classical" complement lesions. Analysis of these vesicles by gel electrophoresis indicated that C9 was present exclusively in the form of a sodium dodecyl sulfate-resistant, high m.w. complex. In contrast, the 35% of C9 that remained associated with the cells was found to be inaccessible to a C9-specific monoclonal antibody, and was partly degraded, suggesting internalization of the membrane attack complex and proteolysis of some C9 molecules. The molar ratio of C9 to C8 was 12 to 1 on shed vesicles and on recovered cells.

Cell Membrane↗
PubMed1989

Distinct restriction of complement- and cell-mediated lysis.

Complement- and cell-mediated killing utilize related effector proteins (C8/C9 and perforin, respectively), suggesting that proteins which protect cells against complement- and cell-mediated attack may also be similar. In homologous complement-mediated killing two protective proteins, which are anchored to the cell membrane by phosphatidylinositol glycan (PIG) tails, are known. To study whether similar PIG-tailed proteins protect against lymphocyte-mediated killing, nucleated cell lines with a mutation in the biosynthesis of the PIG anchor were used. It was found that PIG-tailed membrane proteins restrict homologous complement-mediated lysis but not three different types of cell-mediated killing or lysis by purified perforin. Furthermore, E from patients with an acquired defect in PIG tail biosynthesis did not differ from normal E in sensitivity to antibody-dependent cell-mediated cytotoxicity, in spite of their increased sensitivity to human C8 and C9.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1993

The complement system and systemic sclerosis.

Serum concentrations of the various complement components including the classical and the alternative pathways were determined in 58 control healthy subjects and 80 patients with systemic sclerosis (SSC). The mean concentrations of C1q, C2, C5, C6, C7, C9, and factor B were significantly increased in the SSC patients in comparison to controls, while the increases were not significant for C3 and C8. C4 was an exception in that the mean levels were found to be decreased, with 18 patients having levels < 65% of the mean normal value. Properdin was also found to be decreased, but not significantly. We found a similarity between the pattern of serum complement component concentrations in SSC patients and patients with primary biliary cirrhosis, two disorders frequently associated in the same patients. The significance of complement component patterns in these diseases is discussed.

Complement Activation↗
PubMed1993

Solid-phase synthesis of oligonucleotides containing site-specific N-(2'-deoxyguanosin-8-yl)-2-(acetylamino)fluorene adducts using 9-fluorenylmethoxycarbonyl as the base-protecting group.

Eight oligodeoxyribonucleotides containing a site-specific N-(2'-deoxyguanosin-8-yl)-2-(acetyl-amino)fluorene (dG-C8-AAF) adduct were prepared successfully by solid-phase DNA synthesis using the 2-cyanoethyl N,N-diisopropylphosphoramidites of dA, dC, dG, dT, and dG-C8-AAF, with 9-fluorenyl-methoxycarbonyl (Fmoc) as the base-protecting group. The oligonucleotides were deprotected and released from the support by 1:9 piperidine/MeOH at room temperature for 22-36 h or by 1:1 diisopropylamine in MeOH at 55 degrees C for 15 h, purified by HPLC, and fully characterized. About 6 mg of HPLC-purified d[GTGGCG(C8-AAF)CCAAGT] and 7 mg of d[GTGATG(C8-AAF)ATAAGT] were obtained from the 10-mumol-scale synthesis, and their 1D 1H NMR spectra were consistent with the presence of a dG-C8-AAF adduct. The dG-C8-AAF oligonucleotides were also deacetylated to afford the corresponding dG-C8-AF oligonucleotides. d[GTGGCG(C8-AAF)CCAAGT] formed stable 1:1 duplexes with both the fully complementary 12-mer and a GC-deleted (across the adduct) 10-mer complement, and identical melting temperatures were observed for both duplexes. The multidimensional NMR study of these duplexes is presently under investigation.

2-Acetylaminofluorene↗
PubMed1997

Mapping the active site of CD59.

CD59 is a widely distributed membrane-bound inhibitor of the cytolytic membrane attack complex (MAC) of complement. This small (77 amino acid) glycoprotein is a member of the Ly6 superfamily of proteins and is important in protecting host cells from the lytic and proinflammatory activity of the MAC. CD59 functions by binding to C8 and/or C9 in the nascent MAC and interfering with C9 membrane insertion and polymerization. We present data obtained from a combination of molecular modeling and mutagenesis techniques, which together indicate that the active site of CD59 is located in the vicinity of a hydrophobic groove on the face of the molecule opposite to a "hydrophobic strip" suggested earlier. In addition, removal of the single N-linked glycosylation site at Asn18 of CD59 resulted in an enhancement of complement inhibitory activity.

Amino Acid Sequence↗
PubMed2000

Cleavage of the fifth component of complement and generation of a functionally active C5b6-like complex by human leukocyte elastase.

Activation by complement C3/C5 convertases of the fifth component of human complement, C5, leads to two active cleavage products: C5a, a chemotactic peptide, and C5b, the activated form of C5. Human leukocyte elastase (HLE) has long been known to also release from C5 a chemotactic, C5a-like fragment. This, however, cannot be identical with C5a, since HLE does not cleave peptide bonds at the carboxyl group of arginine, the cleavage site that separates C5a and C5b after the exposure to the complement convertases. Therefore, the question arose whether HLE is capable of releasing a functionally C5b-like product from C5. The results show that this is, indeed, so. Treatment of human C5 with HLE in the presence of C6 leads to the formation on an active C5b6-like complex that lyses non-sensitized guinea pig red cells upon addition of the terminal components C7, C8, and C9. However, since C6 is highly sensitive to the hydrolytic action of HLE, the yield of the activation complex is rather low. The results offer a third possibility for the activation of C5: 1) classical cleavage at Arg74 by complement convertases, 2) oxidation of methionine residues without cleavage, and, as shown here, 3) cleavage by elastase at (a) site(s) distal from Arg74. The three procedures may modulate the relative yield of the two activities generated from C5, C5a-like and C5b-like effects.

Animals↗
PubMed1992

Complement profile in primary biliary cirrhosis.

PBC is a chronic progressive liver disease of unknown etiology. Several abnormalities found in PBC support the hypothesis that it may be considered an autoimmune disease. Despite the complex and interesting relationship that exists between autoimmune disorders and the complement system, very few reports on the level of the serum complement component in PBC have been published, and most of these comprised only a few patients or analyzed only a scant number of the complement components. In the present study, sera of 73 PBC patients were analyzed for the levels of 10 complement components. It was found that the levels of most of the serum complement components, including C1q, C2, C3, C5, C7, properdin and factor B were significantly elevated in patients with PBC in comparison to healthy controls. The level of C4 was slightly lower than that of the normal controls (p = 0.019), while the levels of C6 and C8 were within the normal range. The number of PBC patients with serum levels of C4 and C6 < 60% of normal pooled serum was higher than in the respective control groups (6/69 compared with 0/26 and 4/71 compared with 0/27, respectively). However, the difference was not statistically significant. Thus, our study shows alterations in the levels of most complement components in PBC, the reasons for which are discussed.

Autoimmune Diseases↗
PubMed1970

Lysis of erythrocytes by complement in the absence of antibody.

A new pathway of complement-mediated hemolysis has been described. It is independent of antibody and does not require binding of the first four complement components to the target-cell surface. The actual attack of the target cell begins with the attachment of C5, C6, and C7. The binding reaction is catalyzed by C4, 2, 3, an enzyme which may be formed in cell-free solution. C4, 2, 3 may effect binding of C5, 6, 7 by acting from the fluid phase or from the surface of another cell to which it is specifically bound (EAC 4, 2, 3). In either case, the resulting product is EC5, 6, 7 which is susceptible to lysis by C8 and C9. Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) were particularly susceptible to lysis by the above described mechanism. PNH cells, but not normal human erythrocytes, could also be lysed through activation of complement by cobra factor. These observations allow the operational distinction of an activation and an attack mechanism of complement.

Animals↗