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Six monoclonal antibodies raised against Escherichia coli O111 and against its rough mutant J5 (chemotype Rc) were studied. One IgG2A, one IgM anti-J5, and one IgG2A anti-O111 monoclonal antibody did not bind to lipopolysaccharides of the homologous strain, but cross-reacted with heterologous gram-negative rods in an enzyme-linked immunosorbent assay. These three monoclonal antibodies activated complement when incubated with homologous or heterologous strains, but were opsonic neither in the presence nor in the absence of complement. The other three monoclonal antibodies were directed against lipopolysaccharide of the homologous strain, but showed no cross-reactivity. The IgG3 and one IgM anti-J5 monoclonal antibodies activated complement and were opsonic only in the presence of complement. The IgM anti-O111 monoclonal antibody activated complement and was opsonic both in the presence and absence of complement. Thus, the outcome of the interaction between bacteria, antibodies, and complement is influenced primarily by whether antibodies are directed against lipopolysaccharides or against other cell wall components.
Eluates from one all-silicone and two different combined silicone-latex catheters and one latex and one vinyl procedure glove were mixed with normal human serum and tested in a C3 complement activation assay. Simultaneously the eluates were tested in a cellular assay to measure inhibition of cellular proliferation. The combined silicone-latex catheters and the latex procedure glove caused a pronounced complement activation and inhibited cell proliferation. However, the response ranging was opposite in the two assays, suggesting that different soluble factors were responsible for the observed reactions. No complement activation or inhibition of cellular proliferation was observed for the all-silicone catheter and the vinyl procedure glove. Our results demonstrate that different assays should be taken into account when biocompatibility is evaluated.
Serum levels of the complement proteins C3, C4, C1 inhibitor (C1 INH), factor I (C3b inactivator) and factor H (BIH) and plasma levels of cleavage products of C3 (C3c) and factor B were measured in 26 patients with acute pancreatitis. Breakdown of C3 occurred in 19 patients, as shown by a reduction in C3 level and the presence of C3c. C4 levels, however, did not fall and factor B breakdown products were not detected, thus suggesting that enzymatic cleavage of C3 occurred without significant involvement of either the early classical pathway or the alternative pathway. C1 INH and factor H both showed increases, presumably reflecting an acute phase response. Factor I showed an initial fall followed by a rise. There was no correlation between the presence or extent of C3 breakdown and the clinical condition of the patients. It is concluded that C3 cleavage in pancreatitis probably results from tryptic activity and that the measurement of complement components has no part to play in the management of the disease.
Activation of platelets during preparation and/or storage of platelet concentrates in plastic containers at room temperature has recently been recognized. Many different biologic causes of this activation have been postulated. Activated complement, as a multi-enzyme system, is one of the possible sources of molecules leading to platelet activation. To detect complement activation, functional complement activity and the generation of complement-derived ligands were investigated in platelet concentrate supernatant plasma during 5 days of storage at room temperature. Hemolytic tests for functional classical and alternative pathway activity were used, as was the kinetic test for complement-mediated inhibition of immune complex precipitation. The presence of C3 activation products (C3, C3c, C3dg) was investigated in plasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting procedures and on platelets by immunofluorescence. Activation of complement was evident during storage, and C3c and C3d fragments were clearly demonstrated in plasma. The amount of C3d fragments on platelets gradually rose during the first 3 days of storage. At the end of 5 days of storage, the platelets became C3d negative. There are two possible mechanisms of C3d disappearance--shedding and/or further degradation of C3d fragments. Those results indicated that complement activation and the generation of complement-dependent ligand-receptor interaction may be mechanisms for platelet activation in concentrates stored at room temperature.
BACKGROUND AND OBJECTIVES: The aim of this study was to investigate whether IgG antibodies to blood group A bind or activate complement after crossing the placenta. MATERIALS AND METHODS: IgG anti-A concentrations as well as C1-q-binding and the capacity of anti-A to form the C5b-9 membrane attack complex were measured in sera of 61 mother/child pairs. The enzyme-linked immunosorbent assay was used to quantitate anti-A or anti-B in maternal and neonatal serum samples. RESULTS: The concentrations of IgG anti-A in ABO-identical and ABO-compatible children born to type O or B mothers correlated significantly with maternal concentrations (p < 0.05), whereas type A or AB children had strikingly diminished concentrations of IgG anti-A. The IgG anti-A concentrations were analyzed in two different groups: first, 44 mother/child pairs with mothers and children group O or B (group I), and second, 17 pairs with mothers group O or B and children group A or AB (group II). In group I, identical or compatible pairs with regard to IgG anti-A, a significant correlation was found by Spearman's rank analysis, whereas the same analysis revealed no significant correlation in group II. The capacity of anti-A to activate complement was also more marked in newborns who were ABO-identical or ABO-compatible with their mothers, and this capacity was virtually absent in cord blood samples from incompatible babies. CONCLUSIONS: Tests for complement-dependent or complement-independent ABO blood group antibodies must be interpreted in the light of the blood group of the child.
In the present study, we demonstrate that a substituted soluble dextran derivative bearing 73% carboxylic groups and 15% benzylamide sulfonate groups, termed CMDBS25, inhibits complement activation and complement-mediated damage in an in vitro model of xenogeneic rejection. Incubation of porcine aortic endothelial cells with normal human serum resulted in time-dependent complement consumption as assessed by C3a generation in the fluid phase and deposition of activated complement fragments C3, C5 and of C5b-9 on target cells. The presence of C5b-9 membrane attack complex was associated with 51Cr release from prelabelled endothelial cells. The addition of 5-25 mg of CMDBS25/ml under the experimental conditions used, inhibited complement activation and C3a generation in a dose-dependent fashion. CMDBS25 (25 mg/ml) totally suppressed iC3b, C5 and C5b-9 cytolytic complex deposition on cells and inhibits by 42% lysis of target endothelial cells. Native dextran had no effect. Our observations document the anti-complementary properties of sulfonated dextran derivatives and their potential as therapeutic agents for the prevention of complement-dependent hyperacute xenograft rejection.
The aim of this study was to determine whether the biologically active complement peptides C3a and C5a are formed in pregnancy and whether amniotic fluid can activate complement. C3a and C5a are formed when complement is activated. They increase smooth-muscle contraction, vascular permeability, and histamine release from mast cells and basophils. Thirty pregnant women were studied, 16 with uncomplicated and 14 with preeclamptic pregnancies. The plasma C3a and C5a concentrations before delivery were significantly higher in the preeclamptic than in the normal group. The concentrations returned to normal within 1 week. Plasma, serum, and amniotic fluid from 12 pregnant women (eight uncomplicated and four preeclamptic pregnancies) were drawn in connection with delivery. Amniotic fluid was incubated in fresh autologous serum at 37C for 15 minutes. A dose-dependent formation of C3a and C5a was registered with increasing amounts of amniotic fluid.
Pulsed tunable dye laser (577 nm) (PTDL) therapy induces hemoglobin coagulation and tissue necrosis, which is mainly limited to blood vessels. To define whether this treatment activates complement in normal skin and senile hemangioma, we analyzed complement deposition in blood vessels by immunofluorescence. C3 fragments, C8, and C9 were detected with specific polyclonal antibodies. The membrane attack complex of complement (MAC) was demonstrated with a monoclonal antibody which reacts only with a neoantigen of MAC. Amplification of C3 deposition by the alternative pathway was determined on cryostat sections by indirect immunofluorescence with use of C4 deficient guinea pig (GP) serum. Normal skin and hemangiomas from three individuals were studied. In PTLD-irradiated normal skin, the main findings were as follows: 1) C3 fragments, C8, C9, and MAC were deposited in vessel walls; 2) these deposits were not due to denaturation of the proteins since they became apparent only 7 min after irradiation, contrary to immediate deposition of transferrin at the sites of erythrocyte coagulates; 3) the C3 deposits were shown to amplify complement activation by the alternative pathway, a reaction which was specific since tissue necrosis itself did not lead to such amplification; 4) these reactions preceded the local accumulation of polymorphonuclear leucocytes. Tissue necrosis was more pronounced in the hemangiomas. The larger angiomatous vessels in the center of the necrosis did not fix complement significantly. By contrast, complement deposition in the vessels situated at the periphery was similar to that observed in normal skin with one exception: C8, C9, and MAC were detected in some blood vessels immediately after laser treatment, a finding consistent with assembly of the MAC occurring directly without the formation of a C5 convertase. These results indicate that complement is activated in PTDL-induced vascular necrosis, and might be responsible for the ensuing inflammatory response.
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An increase in the occurrence of false-positive assays for C5a and C3 conversion, combined with hemolytic C assays discrepant between two laboratories, led to the suspicion that serum tubes with silicone separators might activate complement. Plasma incubated with some lots of such tubes showed evidence of complement activation via the alternative pathway, to an extent capable of influencing interpretation of results. The authors recommmend that such tubes not be used for C split-product assays, and be used only with caution for routine hemolytic C assays.
Alzheimer's disease (AD) brains contain large numbers of amyloid-beta peptide (Abeta) deposits associated with activated microglia, astrocytes and dystrophic neurites. Activated complement components and pro-inflammatory cytokines are also present, indicative of focal inflammation. However, neither Abeta, nor the chemokine-like mediator, C5a, which is generated by Abeta-mediated complement activation, significantly activates microglia, as assessed by pro-inflammatory cytokine release. We evaluated the possibility that both together would co-stimulate such release using the THP-1 human monocytic cell line as a microglial surrogate, and found this to be the case. These studies support the hypothesis that Abeta and C5a induce a chronic microglia-mediated focal inflammatory response in AD.
Binding of rituximab (RTX) to CD20+ B cells activates complement and promotes covalent deposition of C3b fragments on the cells. Previously, we reported that the deposited C3b is substantially co-localized with cell-bound RTX, and therefore C3b may block access of antibody probes specific for RTX. We examined the ability of several commercially available phycoerythrin (PE)-labeled anti-Mouse IgG antibodies to bind to B cells opsonized in milieu which allow or preclude complement activation. Even when large amounts of fluorescently labeled RTX are bound to the cells, binding of the anti-Mouse IgG probes is substantially inhibited if C3b is deposited on the cells. However, cell-bound RTX is still demonstrable on development with a monoclonal antibody (mAb) specific for the human Fc region of RTX. Our findings may provide an alternative explanation for data presented in recent reports suggesting that binding of RTX to cells in plasma leads to internalization of RTX and CD20.
Prolonged exposure to antigens may result in high IgG4 antibody titres as was shown in a previous paper (Aalberse et al., 1983b). In novice bee keepers, a shift in the IgG1/IgG4 ratio of the response against phospholipase-A (PLA; a major component of bee venom) occurred. This resulted in an IgG4-dominated response after approximately 2 years of bee-keeping experience. Subject of the present study was the influence of relatively high concentrations of IgG4 antibodies on the biological activity of immune complexes. In the PLA antigen model, it was demonstrated that IgG4-containing immune complexes do not activate complement and that IgG4 antibodies effectively inhibit immune precipitation and complement activation by IgG1 antibodies. Evidence is provided that IgG4 antibodies inhibit binding of C1q to IgG1 in mixed, IgG1- and IgG4-containing complexes. It is proposed that IgG4 antibodies protect against the biological effects of the complement-fixing IgG subclasses. For this reason, determination of the total IgG response or just determination of antibody activity in the complement-fixing isotypes is insufficient in immune-complex diseases. The modulating effect of the non-complement-fixing isotypes should be taken into account to predict the biological activity of the immune complexes.
There is evidence that activated complement fragments (C3b) can bind to human red cells (RBCs) serving as adsorbing surfaces, but not as antigens. This evidence prompted the present in vitro experiments. Using the standard antiglobulin test (AGT), the radioimmune antiglobulin test (RIAT), and the immune adherence test, we found that C3 can indeed be attached to "bystander" human RBCs if complement is activated either through the classical pathway (anti-A hemolysins plus blood group A1 RBCs) or the alternative pathway (activator surfaces, i.e., Escherichia coli bacteria, rabbit RBCs, or inulin). On Scatchard plot analysis, between 24,000 and 44,000 radiolabeled anti-C3d molecules were bound per one adjacent "bystander" RBC, while untreated control RBCs, or RBCs preincubated with fresh compatible serum, bound only 200 to 300, and 600 to 800 molecules, respectively. Despite strong coating with C3 fragments, only "bystander" paroxysmal nocturnal hemoglobinuria, but not normal, RBCs were hemolyzed by complement activation, i.e., through E. coli at pH 8.0. RBCs were not coated with C3 when complement was activated in the fluid phase by heat-aggregated IgG or a staphylococcal "decomplementation antigen." We conclude that the findings of our in vitro experiments accurately mimic some old, but as yet insufficiently understood, in vivo effects of complement activation on circulating blood cells.
Necrotic cells are generally considered to stimulate inflammation, whereas apoptotic cells should not. However, apoptotic cells have pro-inflammatory properties since they can activate complement. To what extent this activation compares to that by necrotic cells is not known. We compared complement activation by necrotic cells and apoptotic cells in plasma. Jurkat cells were made apoptotic or necrotic by incubation with etoposide or by heat shock, respectively. Cells incubated in recalcified plasma were tested for C3 and C4 fixation and fluid phase generation of complement activation products. Fixation of C3 and C4 to necrotic cells occurred mainly via the classical pathway, independent from the method of necrosis induction and the cell type. Depletion of IgM from plasma almost completely abrogated complement fixation by necrotic cells, which was restored by supplementation with purified IgM. Complement activation by late apoptotic cells was comparable to that by necrotic cells regarding the extent and dependence on IgM. Moreover, incubation of plasma with necrotic or late apoptotic cells led to the generation of comparable amounts of complement activation products. These results indicate that late apoptotic and necrotic cells employ similar complement activation mechanisms in the plasma environment.
Serum mannan-binding protein (S-MBP), a lectin specific for mannose and N-acetylglucosamine, was documented to activate complement through the classical pathway. In this study, we examined the mechanism that initiates this activation. By a passive hemolysis test using sheep erythrocytes coated with yeast mannan, the activation of complement by human S-MBP was shown to proceed in the absence of C1q. The following binding studies using 125I-labeled C1r2s2 and C1s indicated that the activated form of C1r2s2 bound to S-MBP located on the surface of the cells with high affinity. The binding of C1s to the cell-bound S-MBP require the presence of C1r, suggesting that C1r2s2 binds to S-MBP through C1r. The activation of C1s from a proenzyme to a protease was mediated by cell-bound S-MBP in the presence of C1r and the activated protease remained associated with the cells and was not released into the medium. The activation of complement with S-MBP was a solid phase event and did not proceed in a fluid phase. On the basis of these results, it was concluded that S-MBP is responsible for the initiation of carbohydrate-mediated complement activation as C1q does in immune complex-mediated complement activation.
Purified surface components from Staphylococcus aureus strain E 2371, which bind to human plasma proteins, were tested for their ability in vitro to activate complement present in normal human serum. Staphylococcal clumping factor as well as protein A both isolated from strain E 2371 were capable of activate complement, while the fibronectin-binding protein showed no activity. At identical molar concentrations clumping factor was more potent than protein A to induce complement consumption. Both clumping factor and protein A were able to activate the alternative complement pathway as demonstrated in serum chelated with ethylene-glycerol-tetra-acetic acid (10 mM) in the presence of MgCl2 (10 mM).