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Characterization of the macrophage receptro for complement and demonstration of its functional independence from the receptor for the Fc portion of immunoglobulin G.

The complement receptor of the macrophage membrane recognizes particle-bound C3b but does not recognize particle-bound C3d. C3-b-coated sheep erythrocytes were bound to macrophages via their C3b receptors, and the preparations were then incubated with either latex particles or opsonized pneumococci (test particles). Macrophages ingested the test particles, but erythrocytes were not ingested; they remained bound to C3b receptors of the macrophage plasma membrane. Thus, a signal initiating ingestion via one type of receptor is not transmitted to all receptors which have the potential to mediate phagocytosis.

Animals↗

Immune complexes and complement catabolism in ankylosing spondylitis.

In serum samples from 37 patients with ankylosing spondylitis (AS), immune complexes were quantitated by the 125I-Clq binding test; in paired plasma samples the C3 breakdown product C3d was measured by an immunochemical method. Compared to results in 30 blood donors, the Clq binding activity was significantly (greater than 2 SD), although discretely, increased in 5 of 8 patients with seropositive AS, but not in other AS patients. All C3d levels were within the normal range. In a parallel investigation, increased Clq binding activity and C3d levels were found in 87% and 90%, respectively, of patients with seropositive rheumatoid arthritis.

Antigen-Antibody Complex↗

Haemodialysis-induced activation of complement. Effects of different membranes.

The ability of cellulose-based (Cuprophan, saponified cellulose ester) and synthetic (polyacrylonitrile, polycarbonate, polymethylmethacrylate) haemodialysis membranes to activate complement during treatment was compared, using functional, immunochemical, radioimmunoassays, and fast centrifugal analysis assay techniques. Cellulosic and synthetic membranes show a striking similarity in their complement activation when measured by immunochemical assays. Functional haemolytic assays for alternate pathway and CH50 demonstrate no significant differences from predialysis values. C3d levels were also unable to demonstrate differences between the membranes. C3a levels, on the other hand, demonstrated significant differences between cuprophan, polyacrylonitrile, and polymethylmethacrylate but not between Cuprophan and polycarbonate membranes. Since comparable alternate-pathway activity of both cellulosic and synthetic membranes was demonstrated, but their C3a release differed, it is possible that certain surfaces that activate complement also possess the ability to absorb components of the alternate pathway.

Acrylic Resins↗

Demonstration and characterization of C3 receptors on rat glomerular epithelial cells.

Receptors for C3 have been demonstrated on the glomerular podocyte in humans. There is conflicting evidence regarding the presence of C3 receptors on rat glomerular cells. Even when shown to be present, the ligand specificity of the receptor has not been determined. Decapsulated rat glomeruli obtained from male Sprague-Dawley rats weighing 50 to 100 g were placed in enriched culture media. On days four to eight, cells of epithelial morphology were observed growing out of glomeruli. Receptors for C3 were detected by rosette formation of sheep erythrocytes (E) coated with antibody (A) and complement (EAC) around the glomerular epithelial cells in culture. The EACs were prepared by incubating antibody-coated sheep erythrocytes with C5-deficient mouse serum or with individual components of complement. Results indicate the presence of two types of C3 receptors on glomerular epithelial cells--CR1 for C3b and CR2 for C3d. The functional roles of these receptors remain to be elucidated.

Animals↗

Cryoprecipitate of patients with cryoglobulinemic glomerulonephritis contains molecules of the lectin complement pathway.

Serological and histological studies were carried out to explore the role of the lectin complement pathway in the pathogenesis of cryoglobulinemic glomerulonephritis. Sixteen patients with mixed cryoglobulinemia type II with glomerulonephritis (GN) were enrolled. All cases had hepatitis C virus (HCV) infection. The serum concentration of mannose-binding lectin (MBL) was significantly higher in the GN patients than in the normal controls according to ELISA (P < 0.01). IgG, IgM, C1q, C4d, HCV envelope antigen, MBL, and MBL-associated serine protease-1 (MASP-1) could be visualized in the cryoprecipitate of the 16 patients by Dot blot assay. Renal biopsy specimens obtained from 3 patients were examined by immunohistochemistry, and the glomeruli strongly stained for IgG, IgM, MBL, MASP-1, C4d, C3c, and C3d in a fringe-like pattern. The pattern of HCV constituent deposition was partially fringe-like. The complement profiles of the 16 cases were distinctive; briefly, the serum levels of C1q, C2, and C3 were reduced, although the levels of circulating regulatory proteins (C1-inhibitor, factor H, and factor I) were in the normal range. The serum C4 level was significantly reduced. These results indicate that immune complex formation involves molecules of the lectin pathway and leads to organ damage in cryoglobulinemic glomerulonephritis.

Adolescent↗

Effect of chorioamnionitis on the levels of serum proteins in the cord blood of premature infants.

OBJECTIVE: To determine the relationships among complements, other serum proteins (including acute-phase reactant), and the cytokine interleukin-6. DESIGN: Prospective observational study. SETTING: A perinatal center, children's hospital, and research institute in Osaka, Japan. PATIENTS: Two hundred fifteen newborn infants ranging from 17 to 42 weeks in gestational age. MAIN OUTCOME MEASURES: We measured the serum levels of several proteins and complements in the cord blood of neonates with and without chorioamnionitis by immunological assays. RESULTS: The levels of C3d, haptoglobin, interleukin-6, IgM, C-reactive protein, and IgA were not influenced by gestational age, and levels of C5, C1q, C2, albumin, C9, and IgG were not influenced by chorioamnionitis status. The levels of C3, C4, CH50, factor B, and orosomucoid were influenced by both factors. CONCLUSIONS: Our investigation of the mutual relation of the data suggests that the increase of cytokine interleukin-6 affects each other component. We suggest that, compared with the serum levels of proteins in neonates with chorioamnionitis, levels in neonates without chorioamnionitis are more similar to those in the normal fetus.

Blood Proteins↗

Surface markers of complement receptor lymphocytes.

Normal blood lymphocytes bearing complement receptors (CRL) were divided into two populations, one expressing both CR1 (C4b-C3b receptor) and CR2 (C3d receptor) and a second expressing only CR1. Nearly all of the population that expressed both CR1 and CR2 also bore membrane surface immunoglobulins (Ig) and Ia antigens. The majority of cells that had only CR1 lacked detectable surface Ig. These Ig- CR1+ CR2- cells could be distinguished from the majority of monocytes and immature granulocytes, in that the latter ingested latex particles and expressed CR2 as well as CR1. The Ig- CR1+ cells were further subdivided into an Ia-bearing subpopulation and another that lacked Ia. Among the Ig- Ia- CR1+ cells, one third formed spontaneous rosettes with sheep erythrocytes while all of the remaining CRL were erythrocyte-rosette negative. Essentially all CRL in normal blood had IgG Fc receptors, but a qualitative heterogeneity in the Fc receptors of Ia+ CRL vs. Ia- CRL was observed in their binding of different immune complex systems.

Antigens, Surface↗

An analysis of the levels of complement components in the synovial fluid in rheumatic diseases.

A linear relationship between the synovial fluid to serum concentration ratios and log molecular weight was found for six plasma proteins, which are largely synthesized by the liver. Production or utilization of a given protein in the joint can, therefore, be determined by its deviation from the calculated diffusion line. Based on this diffusion model the role of the complement system was investigated in the joint effusions of 48 patients with rheumatoid arthritis (RA), 6 patients with osteoarthritis (OA) and 7 patients with meniscus lesions (ML). Among these three groups quantitative differences were found in the metabolism or utilization of several complement components, based on the fact that the ratios were lower than expected for diffusion of proteins of similar molecular weight. The ratios for the RA group were the lowest. In the three patient groups, results showed increased consumption mainly of C3 and C4 locally in the joint. The existence of a real complement activation in the joints of the three different patient groups was further proved by the elevated levels of C3 breakdown products (C3d). Overall this kind of calculation provides us with a method for studying the role of other proteins which may be important in the inflammatory process of the joint.

Arthritis, Rheumatoid↗

Cell surface heterogeneity of human blood neutrophils and monocytes.

Until recently, human blood neutrophils (PMN) and monocytes have been considered to be homogeneous cell populations. However, much evidence has accumulated on their functional heterogeneity. This functional heterogeneity suggests the existence of different subsets of myeloid cells analogous to T and B subsets of lymphoid cells. The goal of this study was to investigate this question of myeloid subsets by examining myeloid cells for cell surface reactivity for IgG and complement (C). Normal PMN and monocytes were examined from 60 subjects for the presence of two types of IgG-Fc receptors and two activated C components, C3b and C3d. Most PMN and monocytes showed Fc receptor activity for rabbit IgG (Fc-R). In addition, the majority of monocytes but very few PMN reacted with human IgG (anti-Rh0) coated Rh-positive erythrocytes (Fc-H). Most PMN and monocytes showed C receptor reactivity for C3b, but only a minor subpopulation of both myeloid cells had C3d receptors. These data provide evidence that human blood myeloid cells may be composed of subsets with different membrane marker reactivities.

Complement C3↗

The kinetics of [3H]-dsDNA/anti-DNA immune complex formation, binding by red blood cells, and release into serum: effect of DNA molecular weight and conditions of antibody excess.

[3H]dsDNA/anti-DNA immune complexes (IC) formed, fixed complement, and bound rapidly to red blood cells (RBC) in whole blood (less than 5 min), but were released from the cells more slowly. The rate of release was dependent on both the antibody:DNA ratio and the m.w. of the DNA in the complex. For example, complexes formed with high m.w. DNA (6 X 10(6) daltons) were released more slowly (t1/2 = 60 min) than complexes formed with lower m.w. DNA (2 to 6 X 10(5) daltons, t1/2 = 15 to 20 min). The [3H]dsDNA/anti-DNA complexes, which were released from the cells as intact antigen/antibody/complement complexes, did not rebind to RBC, but did bind to Raji cells and could be precipitated by monoclonal antibody to C3d. When these released IC (RIC) containing high m.w. DNA were incubated with additional anti-DNA antibody and fresh complement, they rebound to RBC. However, RIC containing lower m.w. DNA (5 X 10(5) daltons) did not rebind to RBC under the same conditions. These data suggest that IC containing high m.w. DNA bind to and remain bound to RBC more effectively than IC containing lower m.w. DNA, and thus may be more easily cleared from the circulation by the RBC IC clearance mechanism. Thus, the size of the DNA in the IC may be a significant factor in the pathogenicity of DNA/anti-DNA complexes in SLE.

Antibodies, Antinuclear↗

Acute hepatitis: significance of changes in complement components.

Serial measurements of complement components were performed in fifty-nine patients with acute, uncomplicated hepatitis and twelve with alcoholic cirrhosis. Thirty-one of the former group had detectable hepatitis B antigen. Abnormal complement profiles were observed in nine patients with hepatitis B and seven with antigen-negative hepatitis. Low levels of C4, C3 and factor B were common in the subjects with cirrhosis and confined to those cases with severe reduction in serum albumin and/or prothrombin index. By contrast, the complement changes in the patients with hepatitis occurred without significant alteration in these parameters; certain subjects also had reduction in C1q and C5 and a significant number had C3d detectable in fresh plasma. The pattern of abnormality suggests predominant involvement of the classical pathway and it is concluded that this results, at least in part, from an immune process evident only in the early clinical phase of hepatitis. Such gross changes in complement are likely to reflect immune-complex activity and it is proposed that these complexes may be important in the clearance of virus material. The data supports a previous suggestion that recovery from acute hepatitis is primarily dependent on host immune competence rather than viral cytotoxicity or generation of immune complexes.

Acute Disease↗

Clearance kinetics and organ uptake of complement-solubilized immune complexes in mice.

C3-bearing immune complexes were prepared by in vitro solubilization of BSA-anti-BSA complexes at equivalence. Sucrose density gradient analyses showed a size-heterogeneous population of solubilized complexes with a range of 7S to greater than 29S and a peak at around 19S. The presence of C3bi was demonstrated by precipitation with antibodies to C3c and to C3d and by binding to conglutinin. Immune complexes solubilized in two and three times antigen excess were selected as controls due to their size similarities with complement-solubilized complexes. Blood clearance curves were very similar for C3-bearing complexes and controls. At 1 hr, the percentage of injected material remaining in the circulation for complement-solubilized and two and three times antigen excess complexes were 29.5 +/- 1.3, 30.9 +/- 1.7 and 26.1 +/- 2.7, respectively. Uptake by liver accounted for the majority of complement- and antigen-solubilized immune complexes removed from circulation. Although the uptake by the spleen was no more than one-tenth of the liver uptake, more complement-solubilized complexes than antigen-solubilized complexes were removed by this organ. The present data indicate that soluble immune complexes bearing C3 components and soluble immune complexes without C3 components, but of comparable size, are cleared from the circulation of mice at comparable rates. The mechanisms of clearance of these two populations of complexes, however, may differ.

Animals↗

Complement activation and HLA-B27.

The efficiency of complement activation was studied in sera from HLA-B27 positive and negative subjects (27 with previous yersinia arthritis and 35 controls). Activation of complement with zymosan induced higher mean levels of the anaphylatoxin C3a in HLA-B27 positive sera (mean (SD) 7.40 (1.66) mg/l) than in HLA-B27 negative sera (6.41 (1.79) mg/l). Similarly, higher levels of C3d,g, another C3 breakdown fragment, were obtained in HLA-B27 positive sera after Escherichia coli 0111:B4 lipopolysaccharide treatment (17.6 (3.7)% v 15.0 (3.8)%). The differences occurred irrespective of previous arthritis, complement C4 or Bf phenotype, or variation in background complement levels. The findings suggest that an increased responsiveness to complement activators may contribute to the pathogenesis of HLA-B27 associated inflammatory diseases.

Adult↗

Complement components, complement activation, and acute phase response in systemic lupus erythematosus.

The investigation concerned 33 systemic lupus erythematosus (SLE) patients assigned to three groups representing mild SLE, more severe extra renal SLE, and SLE with significant renal involvement. In patients with extrarenal disease, the inflammatory plasma protein response was often pronounced during exacerbation, as evidenced by markedly increased concentrations of C-reactive protein (CRP), alpha 1-antichymotrypsin, alpha 1-antitrypsin, and orosomucoid. CRP responses were rare in patients with renal involvement, despite the increased concentrations of other acute-phase reactants in some of these patients. Superimposed bacterial infections were not clearly distinguished by raised CRP concentrations. The classical pathway of complement was activated in all patients during exacerbation, as indicated by increased concentrations of C1r-C1s-C1 inactivator complexes and C2a fragments. C1, C2, and probably also C3 activation varied according to the amounts of circulating C1q-binding immune complexes, as measured by solid-phase assay. Manifest hypocomplementemia was usually associated with glomerulonephritis. Participation of complement components in the inflammatory plasma protein response apparently counteracted the development of hypocomplementemia in many patients with extrarenal SLE. Circulating C3d was detected in all patients during exacerbation of renal disease and in most patients with severe extrarenal manifestations. Inverse relationships were found between immunochemical C2 concentrations and the percentage of cleaved C2 and between C3 and C3d. There was no appreciable consumption of factors B and D and properdin of the alternative pathway in the patients. High concentrations of factor D, a low molecular weight protein, were exclusively found in patients with renal involvement and could be ascribed to retention due to reduced glomerular filtration.

Antigen-Antibody Complex↗

Evidence for an important interaction between a complement-derived CD21 ligand on follicular dendritic cells and CD21 on B cells in the initiation of IgG responses.

The addition of Ags to mononuclear leukocyte cultures typically elicits modest Ab responses, implying that cosignals beyond those provided by T cells and macrophages may be needed. Recently, we reported that Ab responses could be dramatically enhanced (10-1000-fold) by the addition of follicular dendritic cells (FDC), suggesting that FDC may provide an important costimulatory signal. This result prompted a study of molecules involved in FDC-mediated enhancement of Ab responses stimulated by specific Ag with memory T and B cells or nonspecifically by the addition of LPS. In this study, we report evidence supporting the concept that FDC bear a ligand that engages complement receptor II (CR2 or CD21) on B cells and provides a critical cosignal for both Ag-specific and polyclonal responses. A blockade of the CR2 ligand on FDC by the use of soluble CR2 or a blockade of CR2 on B cells by use of CR2 knockout mice (or B cells with CR2 blocked) reduced Ab responses from the microg/ml to the ng/ml range (10-1000-fold reductions). FDC from C3 knockout mice, which cannot generate the CR2-binding fragments (iC3b, C3d, and C3dg), were unable to provide costimulatory activity, suggesting the CR2 ligand on FDC consists of C3 fragments. FDC trap complement-activating Ag-Ab complexes, and it appears that FDC present B cells with both specific Ag to engage B cell receptors and a CR2 ligand to engage B cell-CR2. In short, optimal induction of specific Ab responses appears to require the combination of specific Ag and costimulatory molecules from both T cells and FDC.

Animals↗

Heterogeneity of complement receptor expression on surface immunoglobulin-bearing cells from neonatal and adult mice.

The ontogeny of acquisition of complement receptors (CR) on splenic B lymphocytes from mice of varying ages was examined. Reagents used for the identification of CR included antibody- and complement-coated erythrocytes (EAC), bacteria (BAC) and bacteria treated with complement alone (BC). When whole mouse serum used as the source of complement both EAC and BAC failed to bind to human erythrocytes (C3b receptor-negative), showed binding to Raji and Daudi cells (C3d receptor) and formed rosettes with human neutrophils (C3bi receptor). Therefore these reagents bore C3d and C3bi, but not intact C3b. Although EAc and BAC detected nearly equal percentages of CR lymphocytes in adult mice, BAC bound to a higher percentage of neonatal lymphocytes (8-12%) than did EAC (1-3%). The ability of BAC to detect EAC-negative lymphocytes among neonatal spleen lymphocytes appeared to be due to the increased sensitivity of this bacterial reagent for binding to CR. Further evidence of this enhanced sensitivity was that BAC were less inhibitable than EAC by soluble antigen-antibody-complement complexes.

Animals↗

Mucosal type mast cells express complement receptor type 2 (CD21).

Fragments of complement component C3 generated upon activation of the cascade play an important role in the induction and regulation of immune responses. Receptors interacting with various fragments of this versatile complement protein are expressed on a wide variety of cell types, including lymphocytes, macrophages, dendritic cells, follicular dendritic cells, granulocytes, erythrocytes and consequently, C3-products may influence several biological functions at different sites of the body, where complement activation occurs. Regarding the expression of various C3-receptors on mast cells, mainly rodent serosal type mastocytes have been investigated so far. It has been known for a long time that C3a triggers the release of mediators of immediate type hypersensitivity via binding to serosal-type cells. Complement receptor type 1 (CR1/CD35) and type 2 (CR2/CD21) interacting with the larger activation products, such as C3b and C3d, have so far been shown on serosal type mast cells only. In this study, the expression of CR1/2 on mucosal type mast cells is demonstrated. Using mouse CR1/2 specific single chain antibodies and the natural ligand C3d in cytofluorimetric measurements, we show that the rat mucosal mast cell line RBL-2H3 and mouse bone marrow-derived mast cells (BMMC) express CD21. RT-PCR experiments carried out with mouse CR1 and CR2 specific primers show CD21, but not CD35 specific products in BMMC. It is also demonstrated that, in contrast to serosal type mast cells, mucosal mastocytes do not express CD19. In an attempt to reveal the possible function of CR2 on mucosal type mast cells, the effect of receptor-clustering was tested regarding degranulation, Ca-response and IL-6 production, but no CR2-mediated change was detected in any of these processes.

Animals↗

Interaction between C3 and IL-2; inhibition of C3b binding to CR1 by IL-2.

We previously reported that C3 has a role in the enhancement of the IL-2 dependent proliferation of helper T cells. Because the IL-2R has a structural homology with the complement proteins, such as CR1 and CR2, we studied the possible ligand crossreactions on CR1 and IL-2-receptor, and the direct interaction between C3 and IL-2. While C3 has an enhancing effect on the IL-2 dependent proliferation of HT-2, a CR1-positive mouse T-cell line, the growth of the CTLL-16 line (CR1-negative) is not affected by C3. It has been proven that neither the insolubilized C3 nor the soluble C3b-like C3 react with the IL-2 binding epitope of the IL-2 receptor. However, using human RBC we have demonstrated that the binding of aggregated C3 to CR1 is inhibited by rIL-2, in a dose-dependent manner. When RBC were incubated with rIL-2 and FITC-labelled Fab-anti-CR1 simultaneously, there was no inhibition in the fluorescence intensity. As detected by ELISA, rIL-2 was bound to the same extent by insolubilized C3, C3b, and C3c, while C3d coat had lower binding capacity. The receptor-binding epitope of IL-2 is intact in the complex of complement proteins and rIL-2, as demonstrated by the binding of DMS1, a monoclonal antibody reacting with the receptor site of IL-2. It is strongly suggested that C3b may play a role in the growth of CR1 positive T cells.

Cell Line↗