[Anaphylactoid purpura and Streptococcus sanguis--culture medium of Streptococcus sanguis induce C3c binding activity on vascular endothelial cells].
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Adolescent↗
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Activation of the lectin complement pathway in Henoch-Schönlein purpura nephritis.
BACKGROUND: We previously reported the existence of complement activation through the alternative and lectin pathways in patients with immunoglobulin A (IgA) glomerulonephritis (GN). The current study aims to elucidate the correlation between each complement pathway and clinicopathologic findings in patients with Henoch-Schonlein purpura nephritis (HSPN). METHODS: Immunohistologic staining was performed on renal specimens obtained from 31 patients with HSPN and 20 controls as non-IgA GN by using antibodies against IgG, IgA, IgA1, IgA2, IgM, C1q, C3c, C4, fibrinogen, factor B, C4-binding protein (C4-bp), C5b-9, CD59, mannose-binding lectin (MBL), and MBL-associated serine protease-1 (MASP-1). RESULTS: No control showed deposition of any antibody. In 16 patients with mesangial IgA1/IgA2 codeposits, mesangial deposits of C3c, C4, factor B, C4-bp, C5b-9, CD59, MBL, and MASP-1 were found. In the remaining 15 patients with mesangial IgA1 deposits alone, no mesangial deposits of C4 or MBL/MASP-1 were found, and mesangial deposits of C3c, factor B, C5b-9, and CD59 were evident in 11 patients. Glomerular deposits of fibrinogen were detected in 15 of 16 patients with IgA1/IgA2 codeposits and only 6 of 15 patients with IgA1 deposits. Severity of glomerular changes and degrees of hematuria and proteinuria at latest follow-up were greater in patients with IgA1/IgA2 codeposits than in those with IgA1 deposits. CONCLUSION: Complement activation through both the alternative and lectin pathways is found in patients with HSPN. Complement activation is promoted in situ in the glomerulus. MBL/MASP-1 may be associated with glomerular deposition of fibrinogen. Complement activation through the lectin pathway may contribute to the development of advanced glomerular injuries and prolonged urinary abnormalities in patients with HSPN.
Adolescent↗
Activation of human complement system Paracoccidioides brasiliensis and its deposition on the yeast form cell surface.
The yeast form of Paracoccidioides brasiliensis strain Pb18 was able to activate C3 of normal human serum diluted in phosphate-buffered saline or EGTA-MgCl2 in vitro. C3 convertase was also permissive when Pb18 cells were pre-treated with a pool of immune serum from patients with paracoccidioidomycosis and incubated in serum diluted in EDTA-CaCl2. The components C3, and fragments C3c, C3d, C3g, factor H, factor B, C4 and C5b-9 were demonstrated on the Pb18 cell surface by immunofluorescence although no effect was seen on fungal viability.
Complement Activation↗
Deposits of immunoglobulins and complement factor C3 in human dental periapical inflammatory lesions.
Thirty-five human dental periapical lesions were studied by immunofluorescence technique using polyclonal anti-IgG (F(ab)2), anti-IgM (F(ab)2), anti-IgA (F(ab)2), anti-IgE and anti-C3c as well as monoclonal anti-IgE. Prewashed ethanol-fixed specimens showed a great number of plasma cells staining for IgG. Cells containing IgA, IgM or IgE were also seen. Deposits of immunoglobulins and C3c suggestive of immune complexes were demonstrated using a double staining technique combining FITC-labeled immunoglobulins with TRITC-conjugated C3c. The complexes were located intracellularly in macrophages, as well as in vessel walls and in the basement membrane zone of proliferating epithelium. Immune complexes may be involved in the epithelial proliferation in those granulomas which lead to cyst formation. Monoclonal anti-IgE demonstrated plasma cells and mast cells, as well as intercellular IgE in epithelial strands of granulomas. The role of IgE in the epithelium is obscure. The study strongly indicates the involvement of hypersensitivity reactions type I and III in periapical inflammatory lesions.
Adult↗
Identification of the C3b receptor-binding domain in third component of complement.
We report here that complement receptor type one (CR1) binds to a region of C3b that is contained within the NH2 terminus of the alpha' chain. In an enzyme-linked immunosorbent assay, CR1 bound to C3b, iC3b, and C3c but not to C3d, and this binding was inhibited by soluble C3b and C3c. Further attempts to generate a small C3 fragment capable of binding CR1 were unsuccessful. However, elastase degradation of C3 generated four species of C3c (C3c I-IV), two of which bound CR1. NH2-terminal sequence analysis and sodium dodecyl sulfate-gel electrophoresis of the C3cs indicated that the beta chains and the 40,000-dalton COOH-terminal alpha' chain fragments were identical; the NH2-terminal alpha' chain fragments of C3c I-IV varied from 21,000 to 27,000 daltons and accounted for the differential binding to CR1. C3c-I and II, which do not bind CR1, were missing 8 and 9 residues from the NH2 terminus of the alpha' chain when compared with the intact alpha' chain of C3b. C3c-III and IV, which bind CR1, had NH2 termini identical to the intact NH2-terminal alpha' chain of C3b. Using iodinated concanavalin A and endoglycosidase H, we showed that the NH2-terminal alpha' chains of C3c-I and III were glycosylated, while C3c-II and IV were not. Therefore, these data indicated that the amino terminus of the NH2-terminal alpha' chain fragment of C3c was responsible for binding CR1 while the COOH terminus of this fragment was not involved since the presence or absence of this region in C3c did not affect CR1 binding to C3c. Subsequently, two peptides were synthesized from the NH2-terminal alpha' chain fragment of C3c: X42, 42 residues in length from the NH2 terminus and C30, 30 residues in length from the COOH terminus. X42 inhibited binding of CR1 to C3b, and this effect was also observed with antipeptide antibodies against the X42 peptide. The C30 and other C3-derived peptides and antipeptide antibodies had no effect on the binding of CR1 to C3b.
Amino Acid Sequence↗
Modulation of neutrophil expression of C3b receptors (CR1) by soluble monomeric human C3b.
Upon incubation at 37 degrees C with purified human C3b (500 micrograms/ml), polymorphonuclear neutrophils (PMN) were found to express up to 50% more C3b receptors (CR1) than PMN incubated with buffer alone. This up-regulation of CR1, assessed by the binding of radiolabeled CR1-specific monoclonal antibody, was dependent on the dose of C3b, occurred within 10-20 min and was stable for at least 90 min. PMN incubated with C3b also demonstrated enhanced CR1-dependent binding functions, such as EC3b rosette formation and phagocytosis of EIgGC3b particles. C3b at a concentration of 500 micrograms/ml induced up to 90% increase in the attachment or the phagocytic index. However, CR1 remained unable to promote phagocytosis of EC3b intermediates. Fc receptor-mediated functions were unaffected by the treatment with C3b. The active factor was characterized as monomeric C3b and, in particular, shown to be distinct from C5a. C3b purified by anion-exchange fast protein liquid chromatography on a Mono Q column or eluted from a monoclonal anti-C3b-Sepharose retained its modulating activity, while native C3 or C3 fragments such as iC3b, C3c or C3d,g were ineffective.
Antibodies, Monoclonal↗
Double-decker rocket immunoelectrophoresis for direct quantitation of complement C3 split products with C3d specificities in plasma.
A double-decker rocket immunoelectrophoresis (DD-RIE) method for direct quantitation of complement split products with C3d determinants in human plasma is described. The usefulness of the DD-RIE method for monitoring C3 activation has been assessed and compared with conventional crossed immunoelectrophoresis (CIE) for C3c determination in a patient with iatrogenic septic shock and patients with rheumatoid arthritis. In contrast with CIE the DD-RIE method is quantitative by reference to a standard curve based on an internal reference C3d preparation and its sensitivity and assay capacity are superior to CIE. All reagents and antibody preparation are commercially available and the production of standards is easy. No overlapping was observed between C3d values in plasma from healthy persons and patients with active classical rheumatoid arthritis. The DD-RIE is highly suitable for routine use in laboratories of clinical immunology.
Complement C3↗
Specificity of human lymphocyte complement receptors.
Erythrocytes, bone marrow-derived lymphocytes, monocytes, and granulocytes were shown to have a receptor activity for C4. Theis C4 receptor activity was studied in relation to the previously identified C3b and C3d receptors. By assay for inhibition of rosette formation by fluid-phase complement (C), only two different lymphocyte C receptors were demonstrated. The immune adherence receptor, the only one of the two shared in common with erythrocytes, was specific for C4 or the C3c region of C3b, but was unreactive with C3d. The other lymphocyte receptor, the C3d receptor, was specific for C3d fragments, but would also react to a lesser extent with the C3d region of uncleaved C3b. ThC3d receptor did not react with either C3c or C4. This specificity of the C3d receptor allowed certain cells which contained only C3d receptors to form rosettes with EAC1-3b and EAC1-3d, but not with EAC14. However, because C3d receptors bound EAC1-3d or C3d fragments more firmly than they did EAC1-3b or C3b fragments, many other types of cells containing only C3d receptors, formed rosettes with EAC1-3d but not with EAC1-3b. Erythrocytes and those lymphocytes which contained only immune adherence receptors, formed rosettes with EAC14 and EAC1-3D but not with EAC1-3d. A double-label assay was devised for the simultaneous detection of both types of C receptors on individual lymphocytes. This assay involved fluorescence labeling of one of the two C receptors with soluble C fragments in combination with the usual rosette method for labeling the other type of C receptor. With this double-label assay, it was observed that the two different lymphocyte C receptors capped independently and thus were located on different molecules which could each move through the fluid membrane matrix independently of the other.
Animals↗
Studies on the preservation of red blood cells. Attachment of the third component of human complement to erythrocytes during storage at 4 degrees C.
Utilizing an automated antiglobulin test, we have investigated the presence of the third and fourth components of human complement on normal red blood cells (RBCs). Only negligible amounts of the fourth component, C4, could be detected on either freshly collected or stored RBCs. The fragment C3d of the third component, C3, was detectable on both freshly collected and stored normal RBCs. A product derived from C3 and reacting with anti-C3c antibody was only barely detectable on freshly collected normal RBCs. During storage of blood at 4 degrees C, increasing quantities of this material were detected on the RBC membrane. Bromelin treatment rendered stored RBCs completely nonreactive with anit-C3c antibody, whereas only partial loss of reactivity was observed following incubation with heated plasma. In contrast, incubation of EC43 with heated plasma completely abolished their ability to react with anti-C3c antibody. We suggest that the presence of this C3 fragment on stored RBCs may contribute to the development of "preservation injury".
Agglutinins↗
Breakdown products of C3 and factor B in hemolytic-uremic syndrome.
Serum concentrations of Clq, C4, C3, and Factor B but not properdin were significantly decreased in patients with HUS compared to values in normal control subjects (p value less than 0.01). Sera from 13 HUS patients obtained early after the onset of the disease showed breakdown products of Factor B (Ba, Bb) by immunoelectrophoresis; 12 of these sera showed C3 breakdown products (C3c, C3d). Sera from seven patients studied 1 month to 3 years later no longer demonstrated any breakdown products of Factor B or C3. These data suggest that the complemented system is activate in HUS. The concept that immunological mechanisms play a major role in this disease is additionally supported by the occurrence of IgM, C3, and fibrin in glomeruli and renal vessels.
Adolescent↗
Effects of recombinant sCR1 on the immune inflammatory reaction in acute spinal cord injury tissue of rats.
OBJECTIVE: To determine the effects of recombinant soluble complement receptor type I (sCR1) on the immune inflammatory reaction in acute spinal cord injury tissue of rats and its protective effects. METHODS: SD rat models of acute spinal cord injury were prepared by modified Allen's method. The motor function of the rat lower extremities in sCR1 group and normal saline (NS) group was evaluated by the tiltboard experiment at 12 h, 1 d, 3 d, 7 d, and 14 d. The neutrophil infiltration and C3c positive expression were observed. The myeloperoxidase activity was assessed in the injury tissue at 12 h, 1 d, 3 d, 7 d, and 14 d after injury in the two groups. RESULTS: The motor function of rat in sCR1 group at 3 d, 7 d, and 14 d was obviously better than that in NS group (P<0.01, P<0.01, P<0.01). C3c positive expression in sCR1 group at each time point after injury was obviously less than that in NS group (P<0.01). The myeloperoxidase activity in sCR1 group at each time point after injury was obviously less than that in NS group (P<0.01). CONCLUSIONS: Recombinant soluble complement receptor type I (sCR1) can lessen the immune inflammatory reaction in acute spinal cord injury tissue and relieve secondary spinal cord injury by inhibiting the activation of the complement system.
Animals↗
Factor I deficiency and C3 nephritic factor: immunochemical findings and association with Neisseria meningitidis infection in two patients.
The complement system was examined in two patients with systemic Neisseria meningitidis infections, both of whom had reduced or nondetectable CH50 as analysed by both pathways. C3 measured by conventional technique revealed 19% anti-C3c-reactive protein in the plasma of patient 1 and 3% in patient 2. Patient 1 had circulating C3b but no detectable C3c, C3d, or C4d, whereas patient 2 had normal levels of C3c and C4d and strongly elevated levels of C3d. Factor B analysis revealed no demonstrable native factor B and small amounts of Bb in patient 1 and normal concentration of native factor B plus trace amounts of Bb in patient 2. The depletion of C3 in both patients was due to uncontrolled activation caused by complete factor I deficiency (patient 1) and circulating C3 nephritic factor (patient 2). Both parents of patient 1 had factor I concentrations below (mean-2 SD) that seen in normal healthy individuals (n = 20). Circulating immune complexes (IC) were demonstrated in patient 1 only, whereas serum from both patients had strongly reduced capacity to solubilize preformed IC.
Adolescent↗
C3d fragment of complement interacts with laminin and binds to basement membranes of glomerulus and trophoblast.
Two mouse monoclonal antibodies generated against human placental homogenate were found to react specifically with human complement component C3. In immunofluorescence of human tissues, these antibodies gave a bright linear staining outlining the glomerular basement membrane of the adult kidney and the trophoblast basement membrane of placenta. An identical staining pattern was observed with a rabbit C3d antiserum which also prevented binding of the monoclonal antibodies to tissue sections. Only negligible basement membrane staining was observed in the same tissues with antisera to human C3c, C5, IgG, IgA, or IgM. When interactions of C3 with basement membrane proteins were tested in enzyme immunoassays and column chromatography, C3(H2O) was found to bind efficiently to solid-phase laminin. Native C3 from fresh plasma did not bind to laminin but C3 from plasma treated with methylamine bound efficiently. When C3 was cleaved with trypsin, C3b and C3d but not C3c bound to laminin-Sepharose. The interaction of C3 and laminin was inhibited by soluble laminin and by high ionic strength. The results indicate that C3d, a biologically active breakdown product of C3, can be found in glomerular and placental basement membranes in the absence of signs for ongoing local complement activation or immune complex deposition. It is possible that binding affinities between C3 and basement membrane molecules, especially laminin, are involved in the retention of C3d at these sites. Such interactions between C3 and components of the glomerular basement membrane could play important roles in complement-related pathological processes of the glomerulus.
Antibodies, Monoclonal↗
Activated complement in the sputum from patients with cystic fibrosis.
14 cystic fibrosis (CF) patients chronically infected with mucoid P. aeruginosa and presenting multiple precipitins in serum against this bacterium (CF + P) and 13 CF patients without P. aeruginosa infection (CF-P) had their plasma and sputum sol phase examined for albumin, Clq. C3/C3c, C4 and C5 by means of electroimmunoassays. Their sputum sol phase was examined also for factor B by rocketimmunoelectrophoresis. C3c was demonstrated in the sputum sol phase but significantly more frequent (p less than 0.01) among the CF + P patients than among the CF-P patients. Factor B was also demonstrated in the sputum sol phase, but no significant difference in frequency could be demonstrated between the CF + P and the CF-P patients. None of the results indicated that a local pulmonary production of complement factors took place. Complement activation was significantly (p less than 0.01) associated with inflammation expressed as increased (formula: see text). The results show the importance of complement mediated inflammation in the pathogenesis of pulmonary tissue damage in patients with CF and support the concept of chronic P. aeruginosa lung infection as an immune complex disease in CF patients.
Adolescent↗
Three rat monoclonal antibodies to human C3.
Three monoclonal antibodies to human C3 have been obtained from a fusion of the rat myeloma line Y3 Ag 1.2.3. with spleen cells from rats immunized against C3. One, from clone 4, reacts with an antigenic determinant in C3c showing the expected reactivity of the 'C' antigen of C3. The specificity of the other two monoclonal antibodies correspond less clearly with known C3 antigens. By agglutination analysis of complement coated cells the determinant reacting with clone 3 is present in C3d while that for clone 9 appears as a neoantigen on C3bi. In both cases the co-precipitation results are anomalous and more direct studies are needed to define the exact specificity. The possibility that internal sequence duplications in C3 may explain some anomalies is discussed. None of the monoclonal antibodies significantly inhibit C3 functions. The monoclonal antibodies have been found to have unusual properties in co-precipitation assays being able to diffuse through a precipitation line with which they react to react with a further line. One antibody is also able to react strongly with the anodal half of what appears as a single line with a polyclonal antiserum.
Agglutination Tests↗
Specific inhibition of C3 to facilitate general complement inhibition on endotoxin affinity sorbents for apheresis applications.
Complement activation, as a result of human blood exposure to biomaterial surfaces, continues to be a concern in medical applications. The purpose of this study was to identify sorbent(s) and surface modification(s) that allow specific removal of endotoxin, while minimizing complement activation. Maleic anhydride (MA) modification of Sepharose CL-4B, cellulose, and Toyopearl HW-65F resulted in reduced generation of C3a, a marker of complement activation, by two orders of magnitude over unmodified surfaces. Surfaces modified with both MA and polymyxin B (PMB), utilized for binding endotoxin, reduced complement activation in a similar manner. Western Blot analysis of the larger C3 cleavage product C3dg showed a similar reduction, for all MA-modified sorbents, as observed for C3a by ELISA. C3alpha43 levels (constituent of iC3b and C3c) were also reduced, although only MA-Sepharose CL-4B levels were similar to C3a. Activation of C5, measured as the SC5b-9 complex, was also reduced by two orders of magnitude after MA modification of Sepharose CL-4B; the decrease was similar to all chemical modifications tested. PMB immobilized via CNBr on MA-modified cellulose maintained its endotoxin-binding capacity, while the latter was eliminated when PMB was immobilized via CNBr to MA-modified Sepharose CL-4B and Toyopearl HW-65F.
Adsorption↗
Solid-phase classical complement activation by C-reactive protein (CRP) is inhibited by fluid-phase CRP-C1q interaction.
C-reactive protein (CRP) interacts with phosphorylcholine (PC), Fcgamma receptors, complement factor C1q and cell nuclear constituents, yet its biological roles are insufficiently understood. The aim was to characterize CRP-induced complement activation by ellipsometry. PC conjugated with keyhole limpet hemocyanin (PC-KLH) was immobilized to cross-linked fibrinogen. A low-CRP serum with different amounts of added CRP was exposed to the PC-surfaces. The total serum protein deposition was quantified and deposition of IgG, C1q, C3c, C4, factor H, and CRP detected with polyclonal antibodies. The binding of serum CRP to PC-KLH dose-dependently triggered activation of the classical pathway. Unexpectedly, the activation was efficiently down-regulated at CRP levels > 150 mg/L. Using radial immunodiffusion, CRP-C1q interaction was observed in serum samples with high CRP concentrations. We propose that the underlying mechanism depends on fluid-phase interaction between C1q and CRP. This might constitute another level of complement regulation, which has implications for systemic lupus erythematosus where CRP is often low despite flare-ups.
C-Reactive Protein↗
Novel monoclonal antibodies against mouse C3 interfering with complement activation: description of fine specificity and applications to various immunoassays.
The role of complement proteins in various pathophysiological settings has been studied primarily using mouse models of disease. However, the specific contribution of C3-derived fragments to these biologic processes has not been addressed in a rigorous manner because of a lack of antibodies that can selectively recognize mouse C3 or any of its degradation fragments. Here we report the generation and characterization of a panel of rat monoclonal antibodies reacting with mouse C3 and its degradation products. We describe their performance in various immunological assays such as ELISA, Western blotting, flow cytometry and immunohistochemistry. Of all the antibodies generated, one selectively recognized the C3a anaphylatoxin, and all other reacted with C3c. Furthermore, two monoclonal antibodies preferentially reacted with the cleaved C3 fragments C3b/iC3b/C3c but not native C3. Except for the one recognizing C3a, all antibodies were suitable for detecting C3 deposited on cells and tissues, two effectively inhibited the hemolytic activity of mouse complement and one enhanced C3-deposition to the cell membrane. These novel monoclonal antibodies may serve as useful reagents for elucidating functions mediated by C3-derived fragments in various pathophysiological conditions.
Animals↗