Opsonic potential of C3b-anti-band 3 complexes when generated on senescent and oxidatively stressed red cells or in fluid phase.
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Agglutinins↗
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Kinetic studies of neutrophil phagocytosis. V. studies on the co-operation between the Fc and C3b receptors.
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Complement C3b↗
Biosynthesis of the human C3b/C4b receptor during differentiation of the HL-60 cell line. Identification and characterization of a precursor molecule.
The C3b receptor (C3bR) of the human promyelocytic leukemia cell line (HL-60) was induced by incubating these cells with dimethylsulfoxide (DMSO) or retinoic acid. A majority of differentiated (DMSO- or retinoic acid-treated) but not undifferentiated cells formed rosettes with C3b-coated erythrocytes and were morphologically mature granulocytes. HL-60 cells were surface- or biosynthetically labeled and then solubilized in 1% Nonidet P-40 in the presence of multiple protease inhibitors. The C3bR was isolated either by immunoprecipitation with anti-C3bR antibodies or by affinity chromatography with hemolytically inactive components in which the internal thioester bond within the alpha-chain was cleaved (iC3)- or iC4-Sepharose. Autoradiographs of NaDodSO4-polyacrylamide gels indicated that the surface-labeled C3bR on the differentiated cells had an Mr of 210,000 (nonreduced) or 240,000 (reducing conditions). The bulk (approximately 85%) of the radiolabeled material that was isolated from biosynthetically labeled cells co-migrated with the surface-labeled band. A small fraction (approximately 15%) of the biosynthetically labeled material that was isolated by affinity chromatography or immunoprecipitation had an Mr of 188,000, which did not correspond to any surface-labeled band. This putative precursor molecule was characterized by pulse-chase experiments and by analysis of its carbohydrate. In pulse-chase (15-min pulse) studies of differentiated cells, only the 188,000-mol wt molecule was detected at 0 h. By 2 h, greater than 80% of counts had chased from the 188,000 to the 210,000-mol wt molecule. Treatment of these two molecules with endoglycosidases indicated that the 188,000-mol wt molecule possessed high mannose oligosaccharides, while the mature C3bR had complex oligosaccharides. We conclude from these data that the 188,000-mol wt molecule is a precursor of the C3b receptor of HL-60 cells. Other experiments indicated that the half-maximal time for newly synthesized receptor to attain an Mr of 210,000 was 45 min, and that the t1/2 for the disappearance of the receptor on the surface of differentiated HL-60 cells in tissue culture was approximately 10 h. The ability to observe the induction of the C3b receptor as the HL-60 cell line differentiates is an instructive model system to study the biosynthesis of a human integral membrane receptor glycoprotein.
Cell Differentiation↗
Role of complement in the pathogenesis of postpartum thyroiditis: relationship between complement activation and disease presentation and progression.
The aim of this study was to assess whether the presentation and progression of autoimmune postpartum thyroiditis (PPT) was related to the degree of thyroid peroxidase autoantibody (TPO-ab)-mediated activation of the complement cascade. One hundred and forty-eight thyroid autoantibody-positive women have been followed during their postpartum year. Seventy-five women remained euthyroid during this time whilst the remaining 73 showed one or more episodes of thyroid dysfunction. Fourteen women showed hyperthyroid PPT, 23 showed a biphasic PPT and the remaining 36 showed hypothyroid PPT. Hyperthyroid PPT was always transient but 29 of the 59 women with hypothyroidism remained hypothyroid or still required thyroxine replacement therapy at the conclusion of the study. Thyroid autoantibodies were measured by enzyme-linked immunosorbent assay (ELISA), free triiodothyronine and free thyroxine by the Amerlex M methods and thyrotrophin by the Amerlite TSH (monoclonal) assay. Complement component C3b, immobilized as a result of classical complement pathway activation in the presence of TPO/TPO-ab complexes in vitro, was measured by ELISA. Bioactive TPO-ab were calculated as the product of the C3 index and TPO-ab level. Basal levels of complement C3 activation were seen in the euthyroid TPO-ab-positive women (C3 index 0.06 at delivery rising to 0.36 at 12 months postpartum; bioactive TPO-ab activity 0.4 kIU/l at delivery rising to 10.4 kIU/l at 12 months' postpartum (N = 75). These parameters were elevated progressively as the severity of the clinical syndrome increased. In 14 hyperthyroid PPT women the C3 index was 0.47 at 8 months' postpartum (bioactive TPO-ab activity = 20 kIU/I; p vs euthyroid group, NS).(ABSTRACT TRUNCATED AT 250 WORDS)
Autoantibodies↗
Regulation of the release of alveolar macrophage-derived neutrophil chemotactic factor.
In order to further clarify the physiologic role of the neutrophil-directed chemotactic factor derived from alveolar macrophages, we evaluated those stimuli that possess the potential to regulate the quantity and kinetics of its release in vitro. In short-term culture, particulate stimuli (Staphylococcus albus, Micropolyspora faeni, zymosan, and Sepharose 4B) as well as IgG-immune complexes induced normal guinea pig alveolar macrophages to release significant quantities of this chemotactic factor. In addition, serum opsonization of particulate stimuli resulted in significant augmentation of release of the chemotactic factor from alveolar macrophages responding to these particles. This serum augmentation was associated with the fixation of C3b to the particle surface via the alternative complement pathway. Purified C3b, by itself, was also capable of inducing release of this macrophage-derived mediator. Partial characterization of this chemotactic factor revealed that it was a material of low molecular weight (400 to 600 daltons), and that it was antigenically and physically distinct from C5a. These studies suggested that the induction of chemotactic factor release from alveolar macrophages responding to microorganisms, noninfectious particulates, antigen-complexed IgG, and C3b may contribute to the pathophysiologic events observed in those lung diseases characterized by an influx of neutrophils into the pulmonary parenchyma.
Animals↗
A comparative evaluation of receptor reactivities for C3b, iC3b, and C3d on Raji lymphoblastoid cells.
Raji cells were described to carry receptors for iC3b, C3d, C3b-beta 1 H and beta 1 H. Controversial opinions, however, exist whether or not these cells carry also receptors for C3b. Using highly purified C3, definitely devoid of beta 1 H and C5, for preparation of C3b intermediates, it could be shown that Raji cells bound to C3b cells. Furthermore, Raji cells reacted with monoclonal antibodies that interfered with binding of C3b to human erythrocytes, lymphocytes and renal cells. The receptor for C3b on Raji cell, however, exhibited some special properties and, therefore, required some distinct experimental conditions for its detection: (1) The origin of the erythrocytes used for preparation of the C3b intermediates seemed to be important; this was not the case when iC3b and C3d receptor reactivity was assessed. (2) Rosettes already formed between Raji cells and EAC1423b showed the tendency to disintegrate within the first 30 min after the rosette formation assay. Again, this effect could not be observed with iC3b- and C3d-dependent rosette formation. (3) Incubation of the Raji cells at 37 degrees C as well as 4 degrees C before rosette formation resulted in a rhythmic loss and reappearance of C3b receptor reactivity. At room temperature (19-22 degrees C) this effect was much less expressed. There was no influence of preincubation at 4 and 37 degrees C, respectively, on the iC3b and C3d receptor reactivity of Raji cells. (4) Diisopropylfluorophosphate (DFP) present during rosette formation enhanced, within a certain range of concentration, the percentage of C3b-dependent rosette formation. iC3b and C3d receptor reactivity was not influenced. A similar reaction pattern was observed with pokeweed mitogen (PWM)-stimulated tonsil lymphocytes. In the concentrations tested, DFP showed no effect on the rosette formation between C3b, iC3b, and C3d cells, respectively, and unstimulated tonsil lymphocytes. The data presented suggest that C3b receptors on Raji cells undergo some special metabolism, possibly controlled by fluid phase or cell-bound proteases. This might be a common property of C3b receptors on blast-like and transformed cells, differing from that of unstimulated small lymphocytes.
Binding Sites, Antibody↗
Activation of rabbit C3: studies of the generation of cleavage products in vitro and of their metabolism in vivo.
The cleavage of purified, functionally active rabbit C3 by cobra venom factor and trypsin was analysed by reducing and non-reducing sodium dodecyl sulphate electrophoresis and autoradiography. The specific aim of the study was to compare these reactions to those that occur with human C3. Analysis showed that the pattern of breakdown was very similar to that for the human protein: while the beta-chain remained intact, there was step-wise degradation of the alpha-chain to form C3a, C3b, iC3b and C3c, all of which could be identified by gel analysis. The metabolic behaviour of three of these cleavage products, C3a, C3b and iC3b, was then examined in vivo using dual isotope techniques. Rabbits were studied simultaneously with 131I-C3 and 125I-labelled C3 breakdown products. Analysis of plasma and urine radioactivity for the subsequent 72 h showed that all three breakdown proteins had rapid rates of catabolism in vivo compared to the native molecule. Specifically, 93 and 98% of C3b and iC3b, respectively, were eliminated from the plasma compartment within 10 h of injection. C3a was completely eliminated within 10 h. By comparison, native C3 showed a half-life of 29 +/- 3 h (mean +/- SD) and a fractional catabolic rate of 4.30 +/- 0.75%/h. The data support the use of this species in studies of complement behaviour in models of human immune disease and further clarify the basis for changes in plasma C3 concentration that accompany active immune complex- and antibody-mediated activity, in vivo.
Animals↗
Potentiation of NK cytotoxicity by antibody-C3b/iC3b heteroconjugates.
The interaction of two Burkitt lymphoma lines, Raji and Rael, with human C and NK cells was analyzed. Raji cells activate the alternative C pathway (ACP) and then bind C3 fragments. Consequently, the cells become more sensitive to lysis by CR3-bearing NK cells but not to C lysis. In contrast, Rael cells are poor ACP activators, do not bind C3 fragments, and are therefore resistant to C-dependent NK lysis. As suggested earlier, the difference between Raji and Rael could be attributed to the presence or absence of CR2, respectively, on their surface. To potentiate C- and NK-dependent lysis of target cells, we generated heteroconjugates composed of a murine antitransferrin receptor mAb and of human C C3b or iC3b. Antibody-C3b conjugates induced C3 deposition on Rael cells and elevated C3 deposition on Raji cells in human serum. Both Raji and Rael cells coated with antibody-C3b conjugates were efficiently lyzed by the cytolytic ACP in human serum. This conjugate had a small enhancing effect on target cell lysis by NK cells which could be markedly increased by combined treatment of the target cell with antibody-C3b conjugate and C5-depleted human serum. On the other hand, antibody-iC3b conjugates efficiently potentiated lysis of target cells by NK cells in the absence of serum. The iC3b-directed cytotoxicity was mediated by CR3-bearing NK effector cells. Anti-C3 but not anti-mouse Ig antibodies abrogated the activity of the antibody-iC3b conjugate. These results further demonstrate that NK cytotoxicity may be potentiated by opsonizing the target cells with C3 fragments and suggest that antibody-C3b/iC3b conjugates could be potent tools for targeting and potentiation of the lytic action of both C and NK cells against tumor cells.
Antigen-Antibody Complex↗
Factors influencing the endocytosis of immune complexes.
The MPS plays an important role in the removal of both particulate and soluble immunogenic material from the circulation. E.IgG adhere to mononuclear phagocytes if the Fc portion of the IgG can interact with the phagocyte's Fc receptors (Fc gamma R). Simultaneous sensitization with IgG and C3b via complement activation enhances the effectiveness of binding and ingestion. E.IgM adhere mainly via C3b to C3b receptors (C3bR) of macrophages. Unstimulated macrophages do not ingest E.C3b. Stimulated macrophages, on the other hand, can ingest E.C3b. If soluble material cannot adhere to the surface of macrophages, it will be endocytosed in vitro via fluid-phase pinocytosis at the concentration that is present in the medium. If the material adheres to the cell's surface via its chemical properties or via specific receptors, it will be selectively concentrated at the cell's surface and endocytosed by adsorptive pinocytosis. Ingestion of IC via Fc gamma R and C3b depends on the ability of the antibodies to interact with Fc gamma R and their capacity to activate the complement system. IC-bound C3b enhances the adsorptive pinocytosis of IC. Soluble AIgG are also pinocytosed more effectively when C3b is bound to AIgG. The degree of endocytosis varies with the level of C3b sensitization. The highly effective C3b-mediated pinocytosis can be abolished by treating with trypsin to inactivate C3bR. This observation illustrates that C3b-mediated pinocytosis can replace Fc-mediated pinocytosis in unstimulated macrophages. When macrophages are stimulated in vivo, Fc-mediated pinocytosis increases significantly. Under these conditions, the binding of C3b no longer stimulates; instead, it sterically interferes with Fc-Fc gamma R interaction. In vivo, E.IgG are removed mainly by splenic macrophages. C4-deficient guinea pigs clear E.IgG less effectively than guinea pigs with an intact complement system. On the other hand, soluble IC and AIgG are removed from the circulation mainly by hepatic Kupffer cells. Complement depletion does not seem to influence the clearance rates of these soluble IC or AIgG. The different results obtained in vitro and in vivo and the finding that different effector organs are responsible for the removal of sensitized erythrocytes and soluble Ic in vivo, suggest that more reliable techniques have to be developed to measure IC clearance in patients with a supposedly deficient or saturated MPS.
Animals↗
Use of monoclonal anti-C3 antibodies to characterise the fragments of C3 that are found on erythrocytes.
Using monoclonal antibodies to C3 it has been shown that the red blood cells of patients with cold haemagglutinin disease carry on their cells C3d,g (alpha-2D-globulin) rather than C3d. C3d,g seems to be the final product of in vivo C3 activation in fluid phase and on red cells. The cleavage of C3dg to C3d and C3g does not appear to occur in vivo either in the fluid phase or on red cell bound C3bi. In vitro C3-coated red cells prepared by antibody or low ionic strength techniques produce cells with C3d and C3bi as the predominant C3 fragment, whereas the Fruitstone technique in which coating occurs by the alternative pathway has principally C3b. The activity of C3 cleaving enzymes in whole serum is strongly influenced by the ionic conditions of the serum.
Anemia, Hemolytic, Autoimmune↗
Inhibition by lysine and arginine of the conversion of C3 and B in the serum and a purified system.
When human serum was incubated at 45 degrees C for 30 min, C3 and B were converted to C3b and Bb. Molecular weights of purified C3 and B were shown not to change after incubation at 50 degrees C. Spectropolarimetry indicated that the secondary structures of C3 and B changed after incubation at higher temperature. The titration of SH groups in the C3 molecule showed the liberation of an SH group. These results show that the alternative complement pathway is activated at raised temperatures without known activators such as zymosan or lipopolysaccharide (LPS). This may be due to the accelerated interaction of conformationally changed components of the alternative pathway such as C3 and B. Using this system, effects of various substances on the interaction of C3 and B in serum and the purified system were investigated. The addition of arginine and lysine resulted in the inhibition of the conversion of C3 and B in the serum at elevated temperature. Other amino acids such as anionic amino-acids and NaCl did not influence the conversion. In the purified system, only arginine and lysine prevented the conversion of C3 and B, when C3, B and D were incubated in the presence of Mg++ and amino-acids. Since lysine and arginine did not inhibit the enzymatic activity of D, these data suggest that arginine and lysine prevent the interaction of C3 and B in the serum at elevated temperatures.
Animals↗
A single dose of endotoxin activates neutrophils without activating complement.
Both complement (C) and neutrophils (PMN) are activated in critically ill patients. To evaluate the role of endotoxin in this response, we studied C activation products and PMN cell surface receptors in seven normal subjects before and after endotoxin (USRef 20 U/kg) or saline solution administered on separate occasions. By 4 hours, with endotoxin only, all subjects had myalgia, headache, an increase in body temperature and heart rate, and leukocytosis that returned to normal by 24 hours. At the same time, PMN cell surface receptors for the complement opsonin C3b increased, as measured by indirect immunofluorescence, rising to 251 +/- 44% of baseline by 4 hours (p less than 0.01) and remaining elevated at 24 hours (237 +/- 16%, p less than 0.01). PMN receptors for iC3b increased to 308 +/- 49% of baseline by 4 hours (p less than 0.02) and returned to normal by 24 hours. There was no change in plasma of C3a desArg, C4a desArg, and C5a desArg (4 hours: mean C3a: 153.4 +/- 11.5 ng/ml versus 176.2 +/- 16.2 ng/ml for saline solution, p = ns; C4a: 159.6 +/- 32 ng/ml versus 151.4 +/- 21 ng/ml, p = ns; C5a: undetectable). To confirm the lack of C activation, we examined PMN chemotaxis (CTX) to C5a for any impairment caused by prior in vivo exposure to C5a. CTX to C5a was unaffected (4 hours: 109% +/- 22% of normal versus 114% +/- 10% for saline solution, p = ns). PMN CTX to formyl-methionyl-leucine-phenylalanine and PMN phagocytosis and killing of S. aureus were also unaffected by endotoxin. Thus, a single dose of endotoxin produced a subjective febrile illness and precipitated sustained PMN activation as indicated by increased PMN cell surface complement receptor number in the absence of C activation.
Adult↗
A C3 convertase assay for nephritic factor functional activity.
C3 nephritic factor (C3NeF) is an autoantibody against the C3 convertase which stabilizes this otherwise inherently labile neoenzyme and induces a continuous activation of the alternative pathway with C3 depletion. NeF is found in patients with membranoproliferative glomerulonephritis and/or partial lipodystrpohy. NeF activity is usually detected in plasma by hemolytic tests. In order to obtain reproducible data for the functional activity of purified C3NeF IgG a solid phase assay was developed. C3 convertase was generated on immobilized C3b by incubation with factors B and D in the presence of Ni(2+). Convertase sites were left to decay in the presence of normal IgG or NeF IgG. Residual convertase activity was measured by adding 125I-C3 and capturing nascent 125I-C3b on the plate surface via covalently coupled NH2-Glu-Tyr dipeptide. In the presence of factor H during C3 convertase decay, a dose dependent stabilizing activity was shown for NeF IgG including NeF IgG purified from urine. A second format of the assay was developed in which C3 convertase was assembled on C3b(2)-IgG complexes in the presence of Mg(2+). Since these complexes are more efficient as convertase precursors the signal was five-fold higher than with C3b. Convertase decay, on the other hand, was not influenced by the nature of the precursor and in both systems the stabilizing activity of NeF IgG was similar.
Adult↗
The interaction of C5 with C3b in free solution: a sufficient condition for cleavage by a fluid phase C3/C5 convertase.
We have measured the interactions of C3b with C5 in free solution under conditions that favor detecting weak binding interactions (high C3b and low C5 concentrations and low ionic strength). When a mixture of 125I-C5 (2 X 10(-8) M) and unlabeled C3b (3.8 x 10(-5) M) was ultracentrifuged in a sucrose gradient, virtually all of the C5 sedimented to the position of a 13 to 14S complex. In contrast, a sedimentation rate for C5 of 9S was obtained in the absence of C3b. The ability to bind C5 was observed to be a property of C3b since native C3 was unable to bind C5. It was also found that beta 1H by itself could inhibit the binding of C5 to cell-bound C3b. From inhibition studies, we estimate that the association constant for the C3b-C5 interaction is on the order of 2 x 10(6) M-1 in a low ionic strength buffer (mu = 0.06) and 5-fold weaker at physiologic ionic strength. C5 bound to C3b in free solution was cleaved by nephritic factor-stabilized fluid phase C3bB. C5 activation did not occur on omitting C3b. We conclude that the ability to bind C5 is a property of C3b molecules whether surface bound or in free solution and that when C5 is bound in either fashion, it can be cleaved by a fluid phase C3/C5 convertase.
Animals↗
Detection of a neoantigen on human C3bi and C3d by monoclonal antibody.
A neoantigen was detected on human C3bi and C3d by using the monoclonal antibody (MoAb) 130. The antibody bound to EC3bi and EC3d cells but not to EC3b. Although highly purified C3bi or C3d strongly inhibited the binding of the antibody to EC3d, highly purified C3c had no such effect. Native C3, C3b, or C3(H2O) inhibited this binding only weakly. The neoantigen was also detected in serum after activation with zymosan or heat-aggregated IgG, and it was found bound to the aggregated IgG and zymosan particles. Plasma samples from patients with immunologic disorders were tested for this neoantigen, and 25 out of 43 samples tested were found to have levels of neoantigen corresponding to 2 to 11.5% complement activation, whereas 13 out of 14 normal donor plasmas contained amounts of neoantigen indicating much less than 1% complement activation.
Antibodies, Monoclonal↗
Non-covalently bound C3 enhances lysis of rabbit erythrocytes through the alternative pathway.
Rabbit red blood cells (RaRBC, 3 x 10(7)/ml PBS) were incubated with different amounts of purified human C3 at 37 degrees for 30 min and washed twice in PBS. Different amounts of normal human serum containing 2 mM Mg2+ and 5 mM EGTA were added to the C3-treated and control RaRBC. The extent of lysis was measured after a further incubation at 37 degrees for 40 min. Enhanced lysis was observed with C3-treated RaRBC as compared to control cells. The enhancing effect was dependent on the dose of C3 used for the treatment of RaRBC. Investigation the kinetics of lysis, the lag phase was observed to be significantly shorter with the C3-treated than with the control RaRBC. No enhancement was found when RaRBC were pretreated with preformed C3b fragment. KSCN-treated C3 (C3b-like C3), however, had a lysis-enhancing effect. These results suggest that noncovalently bound C3 molecules may have a role in the initiation and/or maintenance of the alternative pathway activation on activator cells.
Animals↗
C3b-sensitized erythrocytes.
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Complement C3b↗
A study of C3b deposition on yeast surfaces by sera of known opsonic potential.
C3c fragments released by the actin of trypsin from C3b molecules bound to zymosan may be readily quantitated by conventional gel techniques to give a measurement of the efficiency of C3b opsonization. Using this approach, the rate of deposition of C3b molecules was found to be very rapid in sera known to opsonize yeast normally. In contrast, sera defective in yeast opsonization deposited C3b much more slowly. The C3b elution technique was found to correlate well with both a direct phagocytosis assay using baker's yeast (r = 0.87, P less than 0.001) and a recently described neutrophil iodide uptake assay (r = 0.88, P less than 0.001).
Adult↗