Deposition of C3b/iC3b leads to the concealment of antigens, immunoglobulins and bound C1q in complement-activating immune complexes.

Complement activation by bound IgG in serum at physiological concentrations is reflected in the deposition of C3b/iC3b in the absence of antigenic expression of the IgG or of any bound C1q on the target. The aim of this study was to investigate the functional requirements for this phenomenon and to establish its relationship to a release or concealment of the antigens. Microtiter wells coated with IgG by direct adsorption or by binding of IgG antibodies to pre-adsorbed homologous antigen were incubated with serum or serum reagents at 37 degrees C. The complement reaction was analyzed by ELISA to quantitate bound or released reaction products, and the release of IgG from the coated microtiter wells was gauged radiometrically. In the presence of serum, rapid binding of C1q and C3b occurred and was soon followed by a rapid loss of C1q expression; C3b binding remained high. Loss of IgG paralleled that of C1q. The functional requirement for the reaction was restricted to the activation and deposition of C3b/iC3b but was dependent of the combined function of the classical and alternative complement pathways. The loss of the IgG antigen was solely the result of antigen concealment, whereas the loss of C1q was only partly so. In biological terms, the concealment of bound IgG and C1q may reflect mechanisms by which complement down-regulates leukocyte responses stimulated by ligand-cell membrane receptor interactions.

Complement C3b interactions studied with surface plasmon resonance technique.

The surface plasmon resonance (SPR) phenomenon is utilized in a number of new real time biosensors. In this study, we have used this technique to study interactions between the central complement component C3b and its multiple ligands by using the Biacore equipment. The SPR technique is particularly suitable for analysis of the alternative complement pathway (AP) because the inherent nature of the latter is to amplify deposition of C3b on various surfaces. C3b was coupled onto the sensor surface and the coupling efficiency was compared under various conditions on both polystyrene and carboxymethylated dextran surfaces. After enzymatic C3b coupling or standard amine C3b coupling, we analyzed and compared the binding of four C3b ligands to the surface: factor B, factor H, C5 and the soluble complement receptor 1 (sCR1, CD35). Binding of each ligand to C3b was detected when C3b had been coupled either enzymatically or using the amine coupling, but the half-lives of the interactions were found to vary depending on the coupling procedure. Factor H binds to C3b via three interaction sites. The target sites are exposed on the C3b, C3c and C3d fragments of C3, respectively. Therefore, we also tested by using the Biacore whether factor B, C5 and sCR1 bind to C3c and/or C3d. It was found that factor B bound to C3d, but not to C3c. On the other hand, both C5 and sCR1 bound to C3c, but not to C3d. In conclusion, this study shows that SPR is a powerful tool in analyzing and mapping the interactions of C3b with its multiple ligands.

Defective elimination of C3b/iC3b-coated autologous erythrocytes in patients with primary biliary cirrhosis, alcoholic cirrhosis, and ulcerative colitis.

Delayed in vivo elimination of autologous erythrocytes coated with immunoglobulin G has been reported in autoimmune and inflammatory gastrointestinal diseases. Our aim was to elucidate whether impairment of the macrophages was restricted to the Fc receptors of the reticuloendothelial system or whether complement receptors were also affected. We studied elimination by complement receptors of autologous erythrocytes coated with fragments of C3 and C4 in patients with primary biliary cirrhosis, ulcerative colitis, and alcoholic cirrhosis. Impaired function was seen in all patient groups as compared with function in normal subjects, both concerning the mean half-life of the injected cells and the total number of eliminated erythrocytes. Neither of these parameters correlated with the levels of C3 fragments bound to the injected autologous erythrocytes. This is the first report of defective complement receptor function in ulcerative colitis and alcoholic cirrhosis. Immunoglobulin G-dependent elimination of erythrocytes was confirmed to be lowered in all patient groups. The results suggest severe macrophage functional aberrations involving both complement receptors and Fc receptors as the basis of phagocytic defects in autoimmune/inflammatory conditions. In contrast, a general loss of macrophages might cause the functional loss in alcoholic cirrhosis.

Inhibition of complement activation by soluble recombinant CR1 under conditions resembling those in a cardiopulmonary circuit: reduced up-regulation of CD11b and complete abrogation of binding of PMNs to the biomaterial surface.

The influence of soluble recombinant CR1 (sCR1) on complement activation, and its indirect effects on the coagulation system and cellular responses were assessed in two models for the study of blood/surface and blood/air interactions, as are encountered in e.g. cardiopulmonary bypass circuits. The concentrations of C3a and sC5b-9 and the amount of bound C3/C3 fragments were analyzed as indicators of complement activation. Thrombin-antithrombin complexes, the platelet count, surface-ATP, beta-thromboglobulin, and the expression of CD11b on leukocytes were the parameters analyzed to reflect coagulation and cellular responses. In addition, immunochemical analyses of the phenotypes of surface-bound leukocytes and platelets were performed. Recombinant sCR1, at doses ranging between 0.1-0.25 mg/ml, was found to completely inhibit the generation of sC5b-9, and of C3a by two thirds; the binding of C3 and/or C3 fragments to the surface was almost entirely abolished. As a result of the inhibition of complement activation, the expression of CD11b on PMNs, and the binding of these cells to the biomaterial surface was almost completely lost. In contrast, the thrombin-antithrombin complexes, the platelet count, and the adherence of platelets to the surface, as reflected by the ATP binding and the release of beta-thromboglobulin, were not affected. These data show that complement activation, in association with extra-corporeal treatment, causes activation and binding of PMNs to the biomaterial and that these effects can be completely abolished by the addition of soluble recombinant sCR1.

Reconstitution of the alternative pathway of complement by plasma infusions given to a patient with an SLE-like syndrome associated with a hereditary C3 dysfunction.

OBJECTIVE: To reconstitute a dysfunctional form of complement factor C3 in a patient with a systemic lupus erythematosus (SLE)-like syndrome. METHODS: The propositus was treated with plasma infusions during five sessions over a period of eight months. RESULTS: The alternative pathway was reconstituted to normal levels for approximately two to three days after each infusion. C3 fragments were incorporated into previously detected deposits of IgG and IgM at the dermal-epidermal junction and the immune complex levels gradually decreased during the whole treatment period. CONCLUSION: The reconstitution appears to result in the solubilisation of tissue immune complexes and a subsequent transportation to the fixed macrophage system.

Complement activation during cardiopulmonary bypass: effects of immobilized heparin.

The role of complement in biocompatibility reactions and the correlation between complement activation during cardiopulmonary bypass (CPB) and postperfusion syndrome have inspired attempts to improve the biocompatibility of extracorporeal blood oxygenation devices. Here we assessed the effect of immobilized heparin on the generation of C3a and terminal complement complexes during CPB. Thirty patients undergoing aortocoronary bypass were randomized to CPB with either heparin-coated (Duraflo II; Bentley, Irvine, CA) or noncoated control membrane oxygenators (Univox; Bentley). A standard dose of heparin (300 IU/kg) was given to the control group while the heparin dose was reduced to 30% (100 IU/kg) in the heparin-coated group. Significantly lower levels of terminal complement complexes were detected in the heparin-coated group by the end of CPB. From 28 +/- 5 AU/mL (heparin-coated group) and 26 +/- 3 AU/mL (control group, mean +/- standard error of the mean) the terminal complement complex levels increased to 391 +/- 35 AU/mL and 602 +/- 47 AU/mL, respectively (p < 0.002). This difference was still apparent 180 minutes after CPB. Although there was no difference in C3a levels between the two groups at the end of CPB, C3a levels were significantly lower in the heparin-coated group 30 minutes after CPB (194 +/- 18 ng/mL and 307 +/- 18 ng/mL in heparin-coated and control groups, respectively; p < 0.001). We conclude that the heparin-coated surface is more biocompatible with regard to complement activation than is the ordinary unmodified surface in extracorporeal circuits.

Increased in vivo activation of neutrophils and complement in sickle cell disease.

Eight patients with homozygous sickle cell anemia, 15 heterozygotes, and eight control individuals were investigated with respect to plasma concentrations of the inflammatory markers lysozyme and myeloperoxidase and the complement activation marker C3d. The patients showed significantly increased levels of myeloperoxidase and C3d, but not lysozyme, compared with the heterozygotes and the controls. The heterozygotes were also significantly different from the controls with regard to C3d concentration. The concentrations of myeloperoxidase and C3d in plasma showed a significant inverse correlation with the hemoglobin concentration. Myeloperoxidase and C3d showed a significant positive correlation. This suggests a role for the neutrophil and the complement system in the pathophysiology of sickle cell disease.

Isolation of the porcine complement activation fragments C3c and C3d and their use in the preparation of monospecific antibodies.

The hemolytic test to date has been the sole analytic technique applied to study the complement reaction in the swine. To improve the analytical possibilities for this species we have developed polyclonal antibody reagents with specificities for the C3c and C3d activation fragments of swine C3. Access to these reagents, by which activation products can be analysed in tissues and biological fluids, will offer new possibilities for a more precise analysis of the complement reaction.

Evidence for iC3 generation during cardiopulmonary bypass as the result of blood-gas interaction.

Earlier we have shown that iC3 is generated at the blood-gas interface in vitro and that the generation of this molecule is independent of complement activation and the composition of the gas. In order to investigate whether iC3 is also generated during cardiopulmonary bypass where blood comes into contact with oxygen bubbles, two bubble oxygenators were incubated at 37 degrees C with human heparinized blood. A continuous increase in the level of iC3 was shown in the oxygen-perfused bubble oxygenator (up to 100 nmol/l after 180 min) in contrast to the unbubbled control. Similarly, in plasma drawn from patients undergoing cardiopulmonary bypass using either bubble or membrane oxygenators, the levels of iC3 were shown to increase continuously during the operation. Furthermore, this form of C3 was found to be susceptible to cleavage by factor I. The formation of iC3 at the blood-gas interface in vivo could be a mechanism by which gas bubbles induce clinical manifestations associated with complement activation, e.g. during cardiopulmonary bypass, adult respiratory distress syndrome and decompression sickness.

Modification of the complement binding properties of polystyrene: effects of end-point heparin attachment.

In recent years, conjugation of heparin to biomaterials has been shown to improve its biocompatibility. The purpose of the present work was to compare complement activation and binding of C3 to unmodified and heparin-treated polystyrene surfaces of microtitre plates. When polystyrene was incubated with human serum, C3 was deposited on the surface by both adsorption and binding dependent on activation of the classical (CPW) and alternative (APW) pathways. After end-point attachment of heparin, significant C3 deposition, although at reduced levels, occurred only by CPW-mediated mechanisms, while adsorption and APW-mediated binding were strongly reduced. Generally, the modified surface bound lower amounts of protein, e.g. serum albumin and IgG, than the unmodified. By contrast, it had increased affinity for C1q which leads to binding of C1 and activation of complement via the CPW. Nevertheless, the net effect of the surface modification on the complement reaction was an overall reduction of C3 binding due to obliteration of APW. This can be related to an enhanced factor H/I-dependent down-regulation of C3b and to the lowered protein-adsorbing property of the surface, both of which have inhibitory effects on APW and on the C3 shunt-dependent activation of the complement system.

Conformational epitopes of C3 reflecting its mode of binding to an artificial polymer surface.

The aim of the study was to investigate the incompletely understood mechanisms of complement (C) activation and binding on artificial biomaterials. Polystyrene in the form of microtitre plates was used as target for C binding, detectable by ELISA using monoclonal anti-C3 antibodies specific for conformational epitopes expressed by bound C3 and C3 fragments. C3 binding in whole blood/plasma/serum is maximal at low dilutions and occurs predominantly by C activation. At higher dilutions, C3 binding occurs at approximately 1/3 of maximal levels and is solely an effect of adsorption. C3 adsorption in the lower serum dilution range, occurs at low but clearly detectable levels. Comparative epitope analysis between C3 fragments, actively bound to polystyrene in the presence of serum, and of iC3b bound to sheep erythrocytes, clearly indicates that C3 binding/activation on polystyrene takes place as a C3 convertase-mediated reaction, which in serum/plasma is followed by a secondary factor I-dependent degradation of the bound C3b into iC3b. The neo-epitope analysis of serum-contacting polystyrene revealed that the adsorbed C3, throughout the entire serum dilution range tested, deposits in a state closely similar to that observed for purified C3 at a high packing density. Polystyrene surfaces with adsorbed purified C3 expressing this epitope profile were found to mediate APW dependent deposition of C3b in pig serum, presumably by forming a hybrid convertase with porcine Bb. These data therefore suggest that adsorbed C3 on serum-contacting polystyrene surfaces may initiate complement activation via the APW.

Complement activation by polymethyl methacrylate minimized by end-point heparin attachment.

After intraocular lens implantation, despite good clinical results, many cataract patients develop a chronic uveitis, caused by an inflammatory response to the implant. One way to improve the biocompatibility of the intraocular lens is to modify the surface by end-point heparin attachment. This study shows that complement activation caused by poly(methyl methacrylate) can be diminished by end-point heparin attachment, as demonstrated by a significant reduction in the generation of C3a and fluid phase terminal complement complexes. It suggests that assessment of complement activation is a good indicator of the biocompatibility of intraocular lenses.

Detection and characterization of immunoconglutinins in patients with systemic lupus erythematosus (SLE): serial analysis in relation to disease course.

The levels of IgA, IgG and IgM immunoconglutinins (IK) were assessed in sera from 20 patients with SLE which were followed for 8-month periods. At the time of the exacerbation, IgG IKs were significantly increased to 226 +/- 90 arbitrary units (mean +/- s.e.m.) compared with both the minimum value of 75 +/- 28 in the SLE patients and with 31 +/- 2 in healthy controls (P < 0.05). There was no difference between SLE patients and controls in the levels of IgM and IgA IKs. Most of the SLE patients in this material showed maximal IgG IK levels before exacerbation, but there was no correlation between the clinical disease index and the levels of IgG IK. The specificity of IgG IKs showed a broad diversity for microtitre-fixed C3b, iC3b, C3c and C3dg. The antibodies were of IgG1, IgG3 and in two patients, IgG4 subclass. IgG IKs were correlated to the C3d/C3 ratio which suggested that the IK responses were secondary to C3 activation. In summary, unlike other conditions associated with complement activation where elevated IgM IKs are common, an increase in IgG IK levels was observed. It is possible that this diverging IK response contributes to the pathophysiology of the disease.

Purification and characterization of porcine C3. Studies of the biologically active protein and its split products.

Separation techniques for obtaining pure and biologically active swine C3 have been improved in this study. Using these procedures and through the further characterization of porcine C3, the possibilities for developing more specific techniques for the analysis of the complement system in swine have been improved. Plasma was initially treated with protease inhibitors, polyethylene glycol (PEG)-fractionation, plasminogen-depletion and a rapid chromatographic desalting step. The essential fractionation was carried out by DEAE-Sephacel chromatography. Contaminants were removed by size-exclusion (Sepharose CL-6B)- and hydroxylapatite-chromatography. The final recovery reached 56% with 73% retaining specific hemolytic activity. The amino acid composition (98.33%), the functional compatibility and the secondary structure of fragments and intact protein indicate a high degree of homology with human C3. In contrast with the findings of earlier studies was the considerable immunologic cross-reactivity observed with human C3, and the size difference between the human and the swine C3-beta subunit, which was found to be 10 kDa lighter than the human analogue. The finding that the swine C3b/iC3b/C3c fragments do not separate from C3 by agarose electrophoresis, unlike the human analogues, demonstrated that this commonly used simple parameter for the detection of complement activation cannot be used in the porcine model.

Lack of evidence for altered complement and immunoglobulin levels in patients with sickle cell anaemia.

Forty-three patients with homozygous sickle cell disease (haemoglobin SS), 24 heterozygous (AS) subjects and 24 controls (AA) from Sudan were investigated by haemolytic activation for complement function via the classical and the alternative pathways, respectively, and by determination of the plasma levels of C3, C4 and factor B as well as of immunoglobulins IgG, IgA and IgM. The study failed to reveal any direct involvement of the complement system in sickle cell disease and nor was any alteration of the immunoglobulin levels registered as a possible cause of increased susceptibility to infections in patients with homozygous sickle cell anaemia.

Hereditary dysfunction of the third component of complement associated with a systemic lupus erythematosus-like syndrome and meningococcal meningitis.

OBJECTIVE: We describe a dysfunction of C3 in a patient with a systemic lupus erythematosus (SLE)-like syndrome. Alternative pathway complement function was absent, but classical pathway complement function was partially intact. METHODS: We used functional, preparative, and immunochemical techniques in the study. RESULTS: The patient's C3 proved normally susceptible to trypsin proteolysis and partially resistant to classical pathway, but completely resistant to alternative pathway, convertase-dependent cleavage. CONCLUSION: The dysfunction, thus, was caused by a failure of C3 to interact with the C3 convertases, rather than by a lack of a proteinase-sensitive cleavage site in the deficient protein.

Conformational differences between surface-bound and fluid-phase complement-component-C3 fragments. Epitope mapping by cDNA expression.

In previous studies a subset of complement-component-C3 (C3) epitopes, C3(D), expressed in denatured and surface-bound C3 and C3 fragments, has been described. These epitopes were detected by antibodies raised against denatured C3. In the present study we used a cDNA expression strategy to localize epitopes recognized by monoclonal and polyclonal anti-C3(D) antibodies. First, DNAse I digestion of C3 cDNA was used to generate 200-300 bp fragments. These cDNA fragments were expressed as beta-galactosidase-C3 fusion proteins using the lambda gt11 vector. The fusion proteins were tested by Western-blot analysis for reactivity with monoclonal and polyclonal anti-C3 antibodies, and the location of the epitopes were determined by sequencing the cDNA fragments. Affinity-purified polyclonal anti-C3(D) antibodies specific for denatured C3 reacted strongly with the C3 fusion fragments corresponding to segments of the 40 kDa subunit of C3c (residues 1477-1510) and the C3d fragment (residues 1117-1155 and 1234-1294) of C3. Adsorption of the polyclonal antibodies with a mixture of EAC3b and EAC3bi (degradation fragments of C3 bound to sheep erythrocytes) abolished binding to fusion proteins spanning the C3d region, but not the 40 kDa fragment of C3c. No effect was seen with the corresponding soluble C3 fragments. The monoclonal anti-C3(D) antibodies (mAbs) 7D326.1 and 7D331.1, specific for EAC3b and EAC3bi, bound to a fusion protein corresponding to amino acid residues 1312-1404, whereas mAb 7D9.2, specific for EAC3d, reacted with a fusion protein spanning amino acid residues 1082-1118. mAbs 4SD11.1 and 4SD18.1, which did not bind to any physiological C3 fragment, detected a fusion protein covering residues 1477-1510. In summary, the segments of C3 represented by amino acid residues 1082-1118, 1117-1155, 1234-1294 and 1312-1404 accommodate C3(D) epitopes that are expressed by erythrocyte-bound C3 fragments, but not by the corresponding fluid-phase fragment, whereas the segments spanning residues 973-1026 and 1477-1510 contain C3(D) epitopes that are exposed exclusively in denatured C3 and therefore hidden in physiological fragments of the protein.

Generation of iC3 at the interface between blood and gas.

Earlier studies have shown that C3 can be denatured when blood comes in contact with a polystyrene surface. This study was undertaken to see if similar denaturation of C3 occurs at the gas-plasma interface which is found in all kinds of oxygenator used during cardio-pulmonary operations. An in vitro system consisting of gas bubbling through human blood, serum or plasma was used. The generation of C3a, as an indicator of complement activation, and iC3 and iC3 fragments were monitored. Both C3a and iC3/iC3 fragments levels were increased during bubbling. In contrast to the C3a level, no reduction in iC3/iC3 fragments formation was seen in the presence of EDTA, indicating that it was independent of complement activation. The rate of iC3/iC3 fragments generation was unaffected by the composition of the gas (pure oxygen, pure nitrogen or air), suggesting that the denaturation of C3 indeed occurred at the serum-gas interface. C3 and iC3/iC3 fragments were isolated from bubbled EDTA-chelated serum by PEG precipitation and chromatography on FPLC, using a Mono S column and detected by two ELISAs, specific for native C3 and iC3/iC3 fragments. After 240 min approximately 20% of the total amount of C3 consisted of intact iC3 and it was confirmed that this population bound to human erythrocytes.

RF-plasma-modified polystyrene surfaces for studying complement activation.

Five different plasma modified surfaces were made for studying different aspects of biocompatibility. These surfaces were: 1,2-diaminocyclohexane (DACH), acrylic acid (AA), Hydroxyethylmethacrylate (HEMA), methane and hexamethylene-disiloxane (HMDSO). In addition a polyethylene-glycol (PEG) was made by grafting aldehyde functional PEG to the DACH surface. PEG and HMDSO which are the most hydrophilic and the most hydrophobic surface shows the lowest amount of adsorbed protein of the three proteins studied here (albumin, IgG and C3). Methane, HMDSO and HEMA was found to activate via the classical (complement activation) pathway while the others activated via the alternative pathway.

Defective Fc receptor-mediated clearance in patients with primary biliary cirrhosis.

Fc receptor-mediated clearance of immunoglobulin G-coated autologous erythrocytes was studied in patients with primary biliary cirrhosis (n = 14), alcoholic liver cirrhosis (n = 5) and healthy reference individuals (n = 14). The mean half-life of the sensitized erythrocytes was significantly prolonged in patients with primary biliary cirrhosis (85 +/- 25 minutes; P less than 0.001) compared with the corresponding value in patients with alcoholic cirrhosis (16 +/- 2 minutes) and healthy reference individuals (20 +/- 5 minutes), respectively. No correlation between clearance rate and age, liver histopathology, or serum levels of bilirubin, aminotransferases, immunoglobulin G, immunoglobulin A, and Clq binding or C3-containing immune complexes was found. The results presented here indicate a profound disturbance of Fc receptor-mediated immune clearance function in patients with primary biliary cirrhosis.