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At least 19 recordsLinked to original sources
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↗
PubMed2006

Classical pathway complement activation on human endothelial cells.

Endothelial cells regulate vascular integrity and express complement binding proteins including gC1qR/p33 (gC1qR), which recognize C1q, a subunit of the first component of the classical complement pathway. Experiments were performed to investigate classical complement pathway activation on resting endothelial cells and endothelial cells exposed to shear stress. C1q deposition and C4 activation (C4d) were demonstrated by solid phase ELISA and flow cytometry on human microvascular and umbilical vein endothelial cells after exposure to serum or plasma. C4d deposition was accompanied by downstream complement activation including C3b and C5b-9 deposition. C4 activation failed to occur in C1q depleted serum, but was not affected by Factor B depleted serum, confirming classical complement pathway activation. Moreover, C4 activation occurred following exposure of endothelial cells to purified C1 and C4, in the absence of other plasma proteins, and in the absence of detectable cell surface IgG and IgM. Shear stress (18 dynes/cm2) increased C1q (n=9, p<0.05) and C4d (n=9, p<0.05) deposition approximately two-fold, and enhanced endothelial cell gC1qR expression (n=7, p<0.05). Treatment of endothelial cells with anti gC1qR monoclonal antibody F(ab')2 fragments reduced C4d deposition by approximately 20% (n=5, p<0.05). These data demonstrate direct classical complement pathway activation on endothelial cells. gC1qR appears to play a minor but definable role, whereas cell surface IgG or IgM are not required.

Animals↗
PubMed1997

Neurons express proteins of the classical complement pathway in Alzheimer disease.

Occurrence of the classical pathway complement proteins C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8 and C9 was studied in human hippocampus and temporal cortex by immunohistochemistry and Western blotting. In Alzheimer disease (AD) cases, positive staining for all of these proteins was observed in pyramidal neurons and senile plaques. In control cases, weaker pyramidal neuron staining was observed except for C1q and C1s which were not detected. On Western blots of AD hippocampal extracts, bands corresponding to those detected in normal serum were found for each of the complement proteins. Comparable bands were also detected in normal hippocampal extracts with the exception of C1s which was not observed. The intensity of the bands was generally stronger in AD than in normal extracts, but, in the latter, there was considerable variability between cases and between bands in a single case. These data suggest that pyramidal neurons may be a source of the complement components known to be associated with Alzheimer lesions.

Aged↗
PubMed2001

Regulation of complement classical pathway by association of C4b-binding protein to the surfaces of SK-OV-3 and Caov-3 ovarian adenocarcinoma cells.

The role of fluid-phase regulators of complement is to inhibit excessive complement activation and maintain homeostasis in blood. By binding to and inactivating complement components on cell surfaces, they can also protect autologous cells from complement-mediated cytotoxicity and phagocytosis. In this study, we wanted to find out whether C4b-binding protein (C4bp), a fluid-phase regulator of the classical complement pathway, could directly bind to cell surfaces in a functionally active form. After screening several malignant cell lines, we observed that the ovarian adenocarcinoma cell lines SK-OV-3, Caov-3, and SW626 were capable of binding C4bp. Binding tests with recombinant deletion mutants suggested that the primary binding site on C4bp is located on the alpha-chain complement control protein 4 domain. Functional tests showed that tumor cell-bound C4bp retained its cofactor activity for factor I-mediated inactivation of C4b, thus increasing the control of classical complement pathway activation on the surfaces of these cells. These results demonstrate a novel mechanism of complement regulation on cell surfaces, particularly on those of malignant ovarian tumor cells.

Adenocarcinoma↗
PubMed1997

Changes in classical pathway complement activity in dromedary camels experimentally infected with Trypanosoma evansi.

The complement system is known to have important effector functions in immune responses. However, its role in camel trypanosomosis has not been determined. The present study was undertaken to evaluate haemolytic complement activity in Trypanosoma evansi-infected and uninfected camels. Five dromedary camels were experimentally infected with T. evansi and classical pathway haemolytic complement activity was assayed. Parasitaemia and packed cell volume were also monitored. Following infection, classical pathway haemolytic complement showed a slight initial increase (7%) in all the camels. The amounts later dropped as the infection progressed and correlated negatively with parasitaemia. Haemolytic complement recovered following elimination of trypanosomes by treatment with melarsomine. Treatment of uninfected camels had no effect on complement. This study has demonstrated that complement concentration increases in the initial phase of infection followed by a drop as the infection progresses towards chronicity. In addition, the study has shown that activation of the classical complement pathway occurs in camels infected with T. evansi. Complement could therefore be involved in the in vivo control of parasitaemia in dromedary camels infected with T. evansi. Decreased complement levels in this species could lead to immunosuppression, widely reported in animal trypanosomosis.

Animals↗
PubMed2004

Immunoglobulin M-enriched intravenous immunoglobulin inhibits classical pathway complement activation, but not bactericidal activity of human serum.

Acute or even hyperacute humoral graft rejection, mediated by classical pathway complement activation, occurs in allo- and xenotransplantation due to preformed anti-graft antibodies. Intravenous immunoglobulin (IVIg) preparations can prevent complement-mediated tissue injury and delay hyperacute xenograft rejection. It is known that IgM-enriched IVIg (IVIgM) has a higher capacity to block complement than IVIgG. Different IVIgs were therefore tested for specificity of complement inhibition and effect on anti-bacterial activity of human serum. IVIgM-I (Pentaglobin), 12% IgM), IVIgM-II (IgM-fraction of IVIgM-I, 60% IgM), and three different IVIgG (all >95% IgG) were used. The known complement inhibitor dextran sulfate was used as control. Hemolytic assays were performed to analyze pathway-specificity of complement inhibition. Effects of IVIg on complement deposition on pig cells and Escherichia coli were assessed by flow cytometry and cytotoxicity as well as bactericidal assays. Complement inhibition by IVIgM was specific for the classical pathway, with IC50 values of 0.8 mg/ml for IVIgM-II and 1.7 mg/ml for IVIgM-I in the CH50 assay. Only minimal inhibition of the lectin pathway was seen with IVIgM-II (IC50 15.5 mg/ml); no alternative pathway inhibition was observed. IVIgG did not inhibit complement in any hemolytic assay. Classical pathway complement inhibition by IVIgM was confirmed in an in vitro xenotransplantation model with PK15 cells. In contrast, IVIgM did not inhibit (mainly alternative pathway mediated) killing of E. coli by human serum. In conclusion, IgM-enriched IVIg is a specific inhibitor of the classical complement pathway, leaving the alternative pathway intact, which is an important natural anti-bacterial defense, especially for immunosuppressed patients.

Animals↗
PubMed1988

Activation of classical pathway complement in chronic inflammation. Elevated levels of circulating C3d and C4d split products in rheumatoid arthritis and Crohn's disease.

Split products of complement component 3 (C3) and complement component 4 (C4) derived from activation of the alternative and classical complement pathways were measured in untreated outpatients, 20 with Crohn's disease and 19 with rheumatoid arthritis. Elevated levels of the d split product of C4 (C4d) were observed in 12 of 19 patients with rheumatoid arthritis and in 9 of 20 patients with Crohn's disease. Levels of the d split product of C3 (C3d) were increased in 14 of 19 patients with rheumatoid arthritis and in 6 of 20 Crohn's disease patients. The median values of C4d and C3d were significantly increased in both groups of patients. C3d concentrations correlated positively with C4d levels (rs = 0.51-0.56, p less than 0.005). The complement activation was not reflected in reduced plasma levels of native C3 and C4. The data indicate activation of the classical complement pathway in both rheumatoid arthritis and Crohn's disease.

Adolescent↗
PubMed2005

Classical pathway complement activity in schizophrenia.

There is considerable evidence to suggest a role for complement in the pathogenesis of schizophrenia, but the data related to the classical pathway complement activity in patients with schizophrenia are conflicting. In the present study, the total hemolytic activity of the complement and the activities of individual complement components, C1, C2, C3 and C4, were determined in the blood serum of schizophrenic patients with positive family history of the disease and healthy subjects. In comparison to the healthy subjects, the mean values of the hemolytic activities of the C1, C3 and C4 complement components in the serum of the schizophrenic patients were significantly higher, and the mean value of the hemolytic activity of the C2 complement component was significantly lower. However, no significant difference was found between the mean values of the total hemolytic activity of complement in schizophrenic patients and healthy subjects. The C3 hemolytic activity was 2.17 times higher in medicated patients than in drug-free patients. Within each group examined no significant difference was found between smokers and non-smokers or between males and females. The results of this study suggest that the pathogenesis of schizophrenia is associated with alterations in activities of complement classical pathway components.

Adult↗
PubMed1990

Localization of the covalent C3b-binding site on C4b within the complement classical pathway C5 convertase, C4b2a3b.

C5 convertase of the classical complement pathway is a trimolecular protein complex consisting of C4b, C2a, and C3b. In the complex there is an ester bond between C3b and C4b. We analyzed the C5 convertase formed on erythrocytes and localized the covalent binding site of C3b to a small region on C4b. The covalently linked C4b.C3b complex was purified from a detergent extract of the erythrocytes and digested with lysyl endopeptidase. An Mr 17,000 fragment containing the ester linkage between C4b and C3b was purified and its amino-terminal sequence was examined. Two amino acids were obtained at each cycle and identified with those in the sequences of C3 and C4. The sequence derived from C3 corresponded to the thioester region. The sequence derived from C4 started at Ala-1186. Alkali treatment of the fragment yielded an Mr 7,000 peptide derived from C4, which thus appeared to span the region of C4 from Ala-1186 to Lys-1259. Therefore, the covalent C3b-binding site on C4b is located within a 74-residue region of the primary structure. This finding supports the notion that after cleavage of C3 by the C4b2a complex, the covalent binding of metastable C3b to C4b is a specific reaction to form a trimolecular complex with a defined quaternary structure.

Amino Acid Sequence↗
PubMed1982

Activation of the classical complement pathway by a polysaccharide from sugar cane.

The effects of an immunostimulating polysaccharide, Bo, from sugar cane, on the complement system have been investigated. Bo, a glucan of about 10,000 mol wt, was found to activate complement in whole human and guinea pig serum in vitro by the classical pathway. Complement consumption was also demonstrated in guinea pigs upon intravenous injection. Specifically, C1 is activated, and C4 and C2, as well as C3, are consumed. The activation is prevented when Ca++ ions are chelated by ethyleneglycoltetraacetic acid, and when C1q is lacking. Hence, it does not rest on direct activation of C1s. Supplementation of C1q-deficient human serum with purified C1q restores the ability to be activated by Bo. The alternative pathway of complement is little if at all affected by the polysaccharide. The activation of C1 seems to be mediated by immune complex formation between Bo and naturally occurring immunoglobulins. Complement in sera from two severely hypogammaglobulinemic patients was not activated by Bo, but was made reactive by addition of purified human immunoglobulin G.

Adjuvants, Immunologic↗
PubMed2000

Serum amyloid P component bound to gram-negative bacteria prevents lipopolysaccharide-mediated classical pathway complement activation.

Although serum amyloid P component (SAP) is known to bind many ligands, its biological function is not yet clear. Recently, it was demonstrated that SAP binds to lipopolysaccharide (LPS). In the present study, SAP was shown to bind to gram-negative bacteria expressing short types of LPS or lipo-oligosaccharide (LOS), such as Salmonella enterica serovar Copenhagen Re and Escherichia coli J5, and also to clinical isolates of Haemophilus influenzae. It was hypothesized that SAP binds to the bacteria via the lipid A part of LPS or LOS, since the htrB mutant of the nontypeable H. influenzae strain NTHi 2019-B29-3, which expresses a nonacetylated lipid A, did not bind SAP. This was in contrast to the parental strain NTHi 2019. The binding of SAP resulted in a clear inhibition of the deposition of complement component C3 on the bacteria. SAP inhibited only the activation of the classical complement pathway; the alternative route remained unaffected. In the classical route, SAP prevented the deposition of the first complement component, Clq, probably by interfering with the binding of Clq to LPS. Since antibody-mediated Clq activation was not inhibited by SAP, SAP seems to inhibit only the LPS-induced classical complement pathway activation. The SAP-induced inhibition of C3 deposition strongly diminished the complement-mediated lysis as well as the phagocytosis of the bacteria. The binding of SAP to gram-negative bacteria, therefore, might influence the pathophysiology of an infection with such bacteria.

Animals↗
PubMed1982

Antibody-independent complement activation by myelin via the classical complement pathway.

Murine or rabbit whole brain homogenates were shown to activate human complement via the classical pathway by an antibody-independent reaction. This activity required Ca++ ions. Anticomplementary activity in fractionated murine brain was found to reside in the myelin fraction and in purified myelin. It was absent, however, both from highly purified myelin basic protein (MBP) and from the MBP-free residue. Because purified MBP is a monomer and this protein exists in brain tissue largely as a dimer, the ability of the cross-linked form of MBP to activate complement was investigated. MBP, dimerized with difluorodinitrobenzene, was highly anticomplementary. The murine brain, inactive when taken from the newborn mouse, was shown to first acquire the capacity to activate complement at 7 d after birth. This finding is consistent with the report that the synthesis of myelin protein has been shown to be initiated in murine brain 8 d after birth. Complement activation by MBP could play an important role in the pathological changes observed in neurological disorders.

Animals↗
PubMed1985

Opsonization of Legionella pneumophila in human serum: key roles for specific antibodies and the classical complement pathway.

Legionella pneumophila has previously been shown to require serum factors for efficient uptake by phagocytic cells. In this investigation, the roles of specific antibody and complement in phagocytosis of L. pneumophila by human polymorphonuclear leucocytes (PMN) and tissue macrophages were determined. Opsonization was assessed by quantitating the uptake of [3H]-labelled Legionellae. Compared to other Gram-negative and to Gram-positive bacterial species, L. pneumophila was highly resistant to the opsonic activity of normal pooled human serum (PHS). Of 12 donor sera tested, only four promoted significant L. pneumophila uptake when used at full strength. Experiments with immune antibody, and with human sera deficient in immunoglobulins, or the complement components C2, C3, or C5, revealed that L. pneumophila opsonization was dependent on antibody-mediated activation of the classical complement pathway; activation of the alternative pathway could not be detected. At high concentrations, immune antibody alone could adequately opsonize L. pneumophila. Human alveolar and peritoneal macrophages required very similar amounts and types of opsonins for L. pneumophila phagocytosis as did human PMN. Heating L. pneumophila to temperatures greater than or equal to 80 degrees abolished its resistance to opsonization by diluted PHS; however, activation of complement via the alternative pathway or via other antibody-independent routes remained undetectable. These studies show that, in addition to immune antibody, the classical pathway of complement plays an important role in the opsonization of L. pneumophila. The limited ability of these bacteria to interact with human complement provides a likely explanation for their resistance to opsonization and may be partly based on heat-sensitive structures on the surface of L. pneumophila.

Antibodies, Bacterial↗
PubMed1999

Comparison of three different methods for measuring classical pathway complement activity.

The complement system plays an important role in host defense against infection and in most inflammatory processes. The standard 50% hemolytic complement (CH50) assay is the most commonly used method of screening patient sera for functional activity of the classical complement pathway. Our objective in this study was to compare two newer methods (the enzyme immunoassay and the liposome immunoassay) to a commercial CH50 assay for measuring total classical complement activity. We conclude that both newer methods compare well with a CH50 assay and are equally sensitive in screening routine clinical sera.

Adult↗
PubMed1979

Interaction between components of the human classical complement pathway and immobilized Cibacron Blue F3GA.

The interaction between the complement components in human serum and the dye, Cibacron Blue F3GA, immobilized on cross-linked agarose (Affi-Gel Blue) has been studied. All nine components of the classical complement pathway bound to the dye and could be recovered using a linear salt gradient. With the exception of C5 and C8, all the components were eluted over a narrow NaCl concentration range, with the following yields: C1, 17%; C2, 69%; C3, 92%; C4, 87%; C6, 105%; C7, 109%; C9, 128%. C5 and C8 eluted throughout the NaCl gradient with yields of 103% and 14%, respectively. Since all components could be eluted without substantial contamination by albumin or IgG, this procedure may prove valuable as an initial step in the purification of complement components. In addition, the ability of immobilized Cibacron Blue F3GA to physicallly remove complement components may prove useful for both the decomplementation of serum and in elucidating the role of complement in immunological reactions.

Anthracenes↗
PubMed1985

Activation of the human classical complement pathway by a mouse monoclonal hybrid IgG1-2a monovalent anti-TNP antibody bound to TNP-conjugated cells.

A mouse hybridoma selected and cloned for anti-TNP specificity produced three distinct monoclonal antibody species that were separated on protein A-Sepharose by stepwise acid elution. The IgG1 kappa product of the parental myeloma was eluted at pH 6.0. An IgG2a kappa bivalent anti-TNP antibody was eluted at pH 4.5, whereas elution at pH 5.0 yielded a hybrid IgG1-2a kappa monovalent anti-TNP antibody. The IgG2a molecules agglutinated TNP-conjugated sheep erythrocytes (TNP-ES) and lysed TNP-ES in the presence of normal human serum (NHS). Hybrid IgG1-2a antibody was also capable of lysing the cells in NHS, although it did not agglutinate TNP-ES. A threshold in monovalent antibody input was necessary for the lysis of TNP-ES, indicating a requirement for a minimal density of bound monovalent IgG to trigger complement activation. Lysis occurred in NHS-VBS++ but not in NHS-MgEGTA, and it was associated with a dose-dependent consumption of C1, C4, and C2 hemolytic activities. Quantitation of the antibody bound to TNP-ES when using radiolabeled rabbit anti-mouse Fab antibody demonstrated that for similar inputs, 5.4 times as much bivalent as monovalent antibody bound to TNP-ES. When similar amounts of antibody were effectively bound to TNP-ES, monovalent hybrid IgG1-2a was five times less efficient than bivalent IgG2a to yield 50% cell lysis in the presence of NHS. These results indicate that neither bivalent binding nor the presence of two identical heavy chains are necessary requirements for antibody-dependent activation of the classical complement pathway.

Agglutination↗
PubMed2005

Complexes between C1q and C3 or C4: novel and specific markers for classical complement pathway activation.

Classical pathway activation is often assessed by measuring circulating levels of activated C4. However, this parameter does not discriminate between activation through the classical or the lectin pathway. We hypothesized that during classical pathway activation, complexes are formed between C1q and activated C4 or C3. Using ELISA, we investigated whether such complexes constitute specific markers for classical pathway activation. In vitro, C1q-C3d/C4d complexes were generated upon incubation of normal recalcified plasma with aggregated IgG or an anti-C1q mAb that activates C1 (mAb anti-C1q-130). In contrast, during incubation with C1s or trypsin, C1q-C3d/C4d complexes were not generated, which excludes an innocent bystander effect. Additionally, C1q-C3d/C4d complexes were not generated during activation of the alternative or the lectin pathway. Repeated freezing and thawing did not influence levels of C1q-C3d/C4d complexes in recalcified plasma. To measure C1q-complement complexes in plasma samples, we separated unbound complement proteins from C1q-C3d/C4d complexes in the samples prior to testing with ELISA. In samples from patients undergoing cardiopulmonary bypass surgery or suffering from rheumatoid arthritis, we found higher levels of C1q-C4 complexes than in samples from healthy individuals. We conclude that complexes between C1q and C4 or C3 are specific markers of classical complement pathway activation.

Arthritis, Rheumatoid↗