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PubMed1986

House dust allergen activates the classical complement pathway in mouse serum.

A house dust fraction was tested for complement activation in mouse serum using a microtitre complement fixation assay. It was observed that the preparation was a potent activator of the classical, but not of the alternative pathway suggesting an analogy with the complement activation in human serum. The activation showed similarity with that by classical complement activators such as aggregated IgG, DNA, lipopolysaccharide (LPS), but some discrepancy with mite allergen was observed. The contamination of the preparation with LPS was negligible and could not account for the anticomplementary effect. The role of DNA fragments in the activation of mouse complement by the house dust fraction is discussed. Our results suggest that the mouse is suited to study the role of complement activation by house dust constituents in the induction of the IgE response.

Allergens↗
PubMed1986

Interaction of purified lipoteichoic acid with the classical complement pathway.

Glycerophosphate-containing lipoteichoic acids (LTAs) interact with the first component of the classical complement pathway (C1). This resulted in the activation of the classical complement pathway in serum, shown by the consumption of C1, C2, and C4. The dose-dependent interaction of LTAs with purified C1 and C1q was dependent on the negative charges of the phosphate groups of LTA. It was reduced by charge compensation through D-alanine ester substituents and by sterical hindrance through di- and trihexosyl residues linked to position 2 of the glycerol moieties. The charge density of LTA may also play a role: poly(digalactosylglycerophosphate) LTAs, in which the phosphate groups are in a greater distance from each other, were less effective, and the loss of micellar organization by deacylation of LTA drastically reduced the complement activation capacity.

Complement Activating Enzymes↗
PubMed1992

Covalent binding of C3b to C4b within the classical complement pathway C5 convertase. Determination of amino acid residues involved in ester linkage formation.

C5 convertase of the classical complement pathway is a protein complex consisting of C4b, C2a, and C3b. Within this complex C3b binds to C4b via an ester linkage. We now present evidence that the covalent C3b-binding site on human C4b is Ser at position 1217 of C4. We also show that formation of the covalently linked C4b.C3b complex occurs in the mouse complement system and that the C3b-binding site on mouse C4b is Ser at position 1213 which is homologous to Ser-1217 of human C4. Therefore, covalent binding of C3b to a single specific site on C4b within the classical pathway C5 convertase is likely a common phenomenon in the mammalian complement system. Specific noncovalent association of metastable C3b with C4b would occur first, leading to reaction of the thioester with a specific hydroxy group. This is supported by two lines of experimental evidence, one which shows that a mutant C4 that does not make a covalent linkage with C3b is still capable of forming C5 convertase and a second in which the C4b.C3b complex has been demonstrated by cross-linking erythrocytes bearing this C5 convertase.

Amino Acid Sequence↗
PubMed1976

C4 synthesis in C4-deficient guinea pig radiation chimeras: restoration of the classic complement pathway.

Bone marrow transplants from normal Albany strain guinea pigs established a functional classical pathway of complement (C) in C4-deficient (C4D) guinea pigs. Seventeen days after transplant the Albany leads to C4D chimeras had detectable C4 and total hemolytic C activities. Maximum C4 levels (2 to 8% of normal were reachered by day 73 and restored total C to 40% of normal. Classical pathway function persisted for about 150 days and, thereafter, declined to undetectable levels by day 385. In contrast, Albany guinea pigs transplanted with C4D marrow maintained normal C4 levels throughout the experiment, suggesting that the C4-producing cells are radioresistant and long-lived. Unlike unmanipulated C4D animals, Albany leads to C4D chimeras were unable to produce antibodies to guinea pig C4 when immunized with normal guinea pig serum. These experiments suggest that bone marrow cell progeny produce C4 in vivo.

Animals↗
PubMed1989

Reflection of disease activity in rheumatoid arthritis by indices of activation of the classical complement pathway.

Levels of C4d, a fragment of C4 generated during activation of the classical complement pathway, were measured in the plasma of 77 patients with rheumatoid arthritis and 30 healthy subjects. Disease activity was judged according to Ritchie's articular index to be mildly active in 31 (group 1), moderately active in 29 (group 2), and severely active in 17 patients (group 3). Plasma levels of C3d, a fragment of C3, and serum levels of C4, C3, and immune complexes were also measured. The ratios C4d/C4 and C3d/C3 were calculated. The C4d/C4 and C3d/C3 ratios and the levels of circulating immune complexes correlated with the degree of disease activity without significantly departing from linear trend and discriminated between patients with different grades of disease activity. C4d, C3d, C4, and C3 also correlated with disease activity but in a non-linear relationship. A significant correlation was found between C4d and C3d, and between C4d/C4 and C3d/C3. C4d and C4d/C4 also correlated with circulating immune complexes. These results indicate that indices of C4 and C3 activation, in particular the ratios C3d/C3 and C4d/C4, provide a sensitive assessment of disease activity in rheumatoid arthritis, and confirm the major part played by the classical complement pathway in the pathogenesis of this disease.

Adult↗
PubMed1991

Human immunodeficiency virus (HIV)-infected cells and free virus directly activate the classical complement pathway in rabbit, mouse and guinea-pig sera; activation results in virus neutralization by virolysis.

Since animal models of human immunodeficiency virus (HIV) infection are being used increasingly in determining various aspects of virus/host interaction and as models for virus expression, it will be important to assess any significant differences in anti-viral immune responses between animals and humans. Previous studies have shown that incubation of HIV with non-immune sera from several animal species results in virus neutralization, and that rabbit serum can lyse HIV-infected cells. The objectives of the current study were to evaluate the animal complement pathway(s) activated by HIV and HIV-infected cells and determine the mechanism by which complement could mediate viral neutralization. Incubation of HIV-infected cells with mouse, guinea-pig or rabbit sera resulted in cell-surface deposition of C3 fragments. Deposition of C3 fragments did not occur either in the presence of C4-deficient guinea-pig serum or in the absence of Ca2+, indicating that activation by infected cells occurred via the classical pathway. Neutralization of free virus was also mediated by the classical pathway since C4-deficient guinea-pig serum and Ca(2+)-chelated sera lacked activity. Serum treatment of virus resulted in release of HIV reverse transcriptase (RT), suggesting that neutralization occurred by C5b-9-mediated virolysis. RT was also released from simian immunodeficiency virus by animal complement. Antibodies in animal sera were not responsible for the classical pathway activation by free virus or HIV-infected cells. These results define several substantial differences between animal and human complement reactivity with HIV which could significantly affect the ability of HIV to replicate in animals, and which need to be considered in the assessment of animal models of HIV infection.

Animals↗
PubMed1986

Surface-bound capsular polysaccharide of type Ia group B Streptococcus mediates C1 binding and activation of the classic complement pathway.

The role of surface-bound type Ia group B Streptococcus (GBS) capsular polysaccharide in antibody-independent binding of C1 and activation of the classic complement pathway was investigated. In a radiolabeled bacterial-polymorphonuclear leukocyte (PMN) association assay, a measure of bacterial opsonization, preincubation of 3H-type Ia GBS with purified F(ab')2 to the organism blocked the association of the bacteria with PMN', and the inhibitory effect was dose dependent. The specificity of F(ab')2 blocking was shown after adsorption of F(ab')2 with type Ia polysaccharide-sensitized erythrocytes. Polysaccharide-adsorbed F(ab')2 had a 70% decrease in ability to block the association of bacteria with PMN. Evidence for the requirement of the capsular polysaccharide in classic complement pathway activation came from a C1 transfer assay with the use of neuraminidase-digested type Ia GBS. Neuraminidase digestion removed 80% of the terminal sialic acid residues from the native polysaccharide. These neuraminidase-digested organisms had a 72% decrease in binding and transfer of purified C1 compared with non-enzyme-treated organisms. Type Ia capsular polysaccharide bound to sheep erythrocytes promoted classic complement pathway-mediated hemolysis of the cells. The role of C1 inhibitor (INH) in modulation of C1 activation by the organisms was investigated. The possibility existed that the C1 INH could be bound by the bacteria, allowing C1 activation to occur in the fluid phase. The inhibitor was purified from human serum, and its activity was measured before and after incubation with type Ia GBS. The organisms had no effect on C1 INH activity. Thus surface-bound capsular polysaccharide of type Ia GBS mediates C1 binding and classic pathway activation, and this does not involve the C1 INH.

Adsorption↗
PubMed1987

Activation of the classical complement pathway by BioRex-70.

The cation exchange resin BioRex-70 was able to activate the classical complement pathway in human serum at 37 degrees C over the resin concentration range 0-5% (v/v). Using zymosan-treated human serum, it was found that the activation proceeded as far as complement protein C3.

Cation Exchange Resins↗
PubMed1989

Immune complex mediated activation of the classical complement pathway.

The activation of classical C pathway by immune complexes depends on the binding and activation of C1, the first component of C. The Ig in the complex must be of the right class and in the right configuration to accomplish the conversion of precursor (zymogen) C1s to C1s, the active enzyme, whose substrates are C4 and C2. The primary question discussed in this paper is evidence that indicates that epitope distribution and density is a major factor in controlling the configuration of antibodies which in turn controls the activation of bound C1. This evidence confirms earlier findings that binding of C1 is a necessary but not sufficient condition for activating C1.

Animals↗
PubMed1989

Studies on activation and levels of haemolytic complement of buffalo (Bubalus bubalis). 1. Classical complement pathway.

Optimum conditions for haemolytic complement (HC) assay in buffalo serum were standardized. In all, 11 indicator systems of red blood cells (RBC) and haemolysins were investigated. Maximum HC CH50 titre was obtained with rabbit RBC sensitized with goat haemolysin. The effect of pH, Ca2+ and Mg2+ concentration, ionic strength, time and temperature were studied. Of all the variables, ionic strength influenced the HC activity most significantly. The standard system for titrating the HC consisted of rabbit RBC sensitized with goat haemolysin, sucrose-veronal buffer with pH 7.5, ionic strength 0.023 M and Ca2+ and Mg2+ concentrations 6 x 10(-4) and 2 x 10(-3) M, respectively. Incubation at 37 degrees C for 2 h gave highest haemolytic activity. With this protocol 5-7-fold higher HC activity was recorded than with prestandardized conditions. Levels of HC were determined in the sera of 98 buffaloes aged from 1 month to 12 years. The lowest mean CH50 units of 401 +/- 0.35 per ml were recorded in buffalo calves below 3 months of age. The mean HC levels increased with age, reaching peak values of 2349 +/- 62.25 CH50 units/ml in 2-3-year-old buffalo. Animals in the age group 5-12 years had significantly decreased (P less than 0.05) mean HC levels of 1545 +/- 68.94.

Animals↗
PubMed1989

Isolation and characterization of a novel plasma protein which binds to activated C4 of the classical complement pathway.

We report here the isolation and partial characterization of a previously unrecognized protease-sensitive plasma protein identified during the development of a novel protocol for the purification of the second component of human complement (C2). This new protein is physicochemically similar to C2. It coprecipitates with C2 on polyethylene glycol fractionation and specifically binds, like C2, to Sepharose-bound iC4/C4b. Binding occurs both in the presence and absence of C2. The purified protein has a chain structure similar to C2 as determined by sodium dodecyl sulfate-gel electrophoresis in the presence or absence of reducing agent and has a molecular mass of 120 kDa, only somewhat greater than C2 at 95 kDa. Both proteins radioiodinate under similar conditions to the same specific activities with each of two different methods that yield 10-fold disparate results. Quantitative Mancini analysis identifies 300 micrograms/ml of the 120-kDa protein in plasma and serum. The protein is present at normal concentrations in serum from individuals genetically deficient in C2, has no C2 functional activity, and is not cleaved as is C2 when serum complement is activated. Potent monospecific polyclonal anti-serum to each do not cross-immunoprecipitate using standard gel techniques. However, these anti-sera identify epitopes in common by Western blotting. The data presented indicate that the 120-kDa protein is a distinct plasma component and suggest that the protein is not an "immature" form of C2. Initial experiments to delineate a functional role for the 120-kDa protein have demonstrated a consistent inhibition of C1 site generation on EAC4b which is dose-dependent and reversible. Thus, this protein appears to be a new complement regulatory factor.

Blood Proteins↗
PubMed1989

Activation of the classical complement pathway in brain tissue of Alzheimer patients.

Positive immunohistochemical staining of Alzheimer brain tissue was obtained with antibodies to proteins associated with classical, but not the alternative, complement pathway. Clq, C3d, C4d are fractions of complement proteins that bind to tissue when the classical complement pathway is activated. Antibodies to these fractions stained senile plaques, dystrophic neurites and some neurofibrillary tangles. C5b-9 is the membrane attack complex which promotes cell lysis when assembled on the plasma membrane. An antibody to a neoantigenic site on this complex stained dystrophic neurites and many neurofibrillary tangles, but not extracellular amyloid. Properdin and fraction Bb of factor B, two proteins that bind to tissue when the alternative complement pathway is activated, were not detected immunohistochemically.

Aged↗
PubMed1992

Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease.

Serum mannose-binding protein (MBP) is a C-type lectin that binds to terminal mannose and N-acetylglucosamine moieties present on surfaces of certain pathogens and activates the classical complement pathway. In the present study, we describe the mechanism underlying the activation triggered by MBP. The human serum MBP fraction was obtained by sequential affinity chromatography on mannan-Sepharose, anti-IgM-Sepharose and anti-MBP-Sepharose in the presence of calcium ions. This fraction contained a C1s-like serine protease as assessed by C4 consumption. The C1s-like serine protease, designated MBP-associated serine protease (MASP), was separated from MBP by rechromatography on anti-MBP-Sepharose in the presence of ethylenediaminetetraacetic acid. MASP exhibited both C4- and C2-consuming activities. The molecular mass of MASP was estimated to be 83 kD with two polypeptides of heavy (66 kD) and light (L) (31 kD) chains linked by disulfide bonds. The serine residue responsible for protease activity is located on the L chain. Reconstitution experiments using MASP and MBP revealed that combination of the two components restores C4- and C2-activating capacity on mannan. Based on analyses of molecular size, antigenicity, and 11 NH2-terminal amino acid sequences of the L chain, we conclude that MASP is a novel protein different from C1r or C1s. Our findings are not in accord with a proposed mechanism by which MBP utilizes the C1r2-C1s2 complex to initiate the classical complement pathway.

Amino Acid Sequence↗
PubMed1991

High doses of intravenous immunoglobulin do not affect the recognition phase of the classical complement pathway.

We have recently found that intravenous immunoglobulin (IVIg) prevents deposition of C3 and C4 fragments onto antibody sensitized erythrocytes. To find out if such an effect results from the blockade of the recognition phase of the classical complement cascade, we investigated the ability of human serum containing high concentrations of IVIg to deposit the recognition subunit of the first complement component (C1q) onto targets. Normal human serum supplemented in vitro with IVIg did not demonstrate reduced C1q binding to targets as determined by radiolabeled antihuman C1q antibody uptake. Similarly, methylamine-treated normal human serum to which IVIg was added was equally effective in terms of C1q binding as the same serum without IVIg. At increasing doses of sensitizing antibody, C1q uptake decreased proportionally; however, at all antibody dilution points C1q uptake was not significantly different in the serum with IVIg in comparison with normal serum. Serum from a patient treated with IVIg did not differ in its capacity to deposit C1q from the same patient's serum before therapy. Our data suggest that IVIg does not interfere with the recognition step of classical complement pathway. This is a US government work. There are no restrictions on its use.

Complement C1↗