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PubMed1984

Fc-dependent effector functions of idiotype-anti-idiotype immune complexes.

Some effector functions of antigen-antibody and antibody-antibody (idiotype-anti-idiotype) complexes were analyzed. As a model system a monoclonal IgM antibody specific for the hapten NP (antibody B1-8) was reacted either with hapten and hapten-carrier conjugates or with monoclonal anti-idiotope antibodies with specificity for B1-8 idiotopes. The precipitating, C1q-binding, complement-activating and Fc receptor binding properties of these complexes were compared. Binding of both hapten-carrier conjugates and anti-idiotope antibodies to B1-8 results in formation of complexes which depending on the B1-8:ligand ratio precipitate, activate complement, bind C1q and exhibit increased avidity for Fc mu and Fc gamma receptors of mouse spleen cells. In both types of complexes cross-linking of IgM molecules is essential for triggering these Fc-dependent functions, and a functional heterogeneity if idiotype-anti-idiotope complexes based on different idiotype-anti-idiotope ratios could also be observed. The functional similarity of B1-8-hapten-carrier and B1-8-anti-idiotope complexes suggests that regulatory functions so far assigned to antigen-antibody complexes could be carried out also by idiotype-anti-idiotope complexes.

Animals↗
PubMed1986

A molecular mechanism for the activation of the first component of complement by immune complexes.

The proposed activation mechanism is based upon several key concepts, including the "S"-structure for the folding of the C1r2C1s2 tetramer among the C1q arms [Poon, et al., J. molec. Biol. 168, 563-577 (1983)]; the locations of the catalytic domains on the tetramer and the resulting functional relevance of the "S"-structure [Colomb et al., Phil. Trans. R. Soc. B306, 282-292 (1984)]; the structure of C1-inhibitor [Odermatt et al., FEBS Lett. 131, 283-289 (1981)]; and the control of C1 activation by C1-inhibitor [Ziccardi, J. Immun. 128, 2505-2508 (1982)]. The proposed activation mechanism has four main features: steric exclusion of C1-inhibitor from C1 when it binds to an immune complex; signal generation through multivalent binding of the C1q heads to an irregularly-arranged cluster of antibody Fc regions, and signal transmission through the movement of the stiff C1q arms about their semi-flexible joints, causing distortion of the symmetrical cone of C1q arms; induction of rapid activation by a shift in equilibrium favoring the autocatalytic conformation of C1r2C1s2; and release of the activated C1s from the C1q arms, so that the ends of the tetramer are free for interaction with C4 and C2 and C1-inhibitor, and the C1q subcomponent becomes more flexible, allowing access of C1-inhibitor to C1r.

Antigen-Antibody Complex↗
PubMed1987

Regulation of the alternative pathway of human complement by C1q.

The interaction of C1q with C3b and its effect on C3b activities in the alternative pathway of complement (APC) have been studied. Purified C1q markedly inhibited C3b deposition on and lysis of rabbit erythrocytes by the isolated cytolytic APC. It also blocked formation of the C3 convertase, C3b, Bb as well as binding of Factors B and H to sheep erythrocytes (E) bearing C3b. The direct and specific binding of C1q to C3b was clearly demonstrated using the hemagglutination technique at low ionic strength (0.1 M NaCl). C1q concns of 2 micrograms/ml and higher agglutinated, in a dose-dependent fashion, EC3b but not E, EC3bi or EC3d. Addition of C1r and C1s to C1q and formation of C1 did not affect its capacity to agglutinate EC3b. The C1q-mediated agglutination of EC3b was inhibited by EDTA, MgEGTA, C3b and Factor B but not by native C3 or collagen. Heating C1q (56 degrees C) markedly potentiated its agglutinating activity whereas collagenase-treated C1q lost most of its activity. Taken together, these results suggest that C1q binds through its "heads" and in the presence of calcium ions to a site on C3b that is adjacent to the Factor B and Factor H binding sites. This interaction may down-regulate the activity of the alternative pathway of complement on surfaces which activate both the classical and alternative pathways of complement.

Animals↗
PubMed1987

Synergistic complement lysis by monoclonal antibodies to the human leukocyte common antigen requires both the classical and alternative pathways.

In a previous study we isolated a series of rat monoclonal antibodies to the human leukocyte common (LC) antigen and demonstrated that synergistic complement lysis was possible between IgG2b antibodies which recognised different epitopes. In this report we have examined the mechanisms that were involved in synergistic lysis. We found that the number of C1q binding sites increased from 30,000 to 40,000/cell using a single antibody to 90,000/cell when two IgG2b antibodies to different epitopes were used together. The affinity of C1q binding also increased approx. 3-fold for the synergistic pair, and there was a similar increase in the rate of C1 activation. Combinations of an IgG2b with IgG1 or IgG2a gave much smaller increases in the amount of C1q bound and in the rate of C1 activation. Despite the large number of C1q molecules bound with the optimal synergistic pair and the increased rate of C1 activation, lysis was inefficient in serum depleted of Factor D, suggesting a requirement for the alternative pathway. This is the first demonstration of the need for the alternative pathway in complement lysis by monoclonal antibodies.

Animals↗
PubMed1988

The distortive mechanism for the activation of complement component C1 supported by studies with a monoclonal antibody against the "arms" of C1q.

A mouse monoclonal antibody (IgG1 isotype) against human C1q (MAb 130) is presented that activates C1 in serum through its antigen-binding sites at an optimal molar ratio of 3 MAbs:1 C1q. The antibody does not inhibit binding of C1q to IgG. Experiments with pepsin- and collagenase-digested C1q showed that MAb 130 binds to the fibril-like strands (arms) of C1q, close to the globular heads. Bivalency of MAb 130 was a requirement for C1-activation, but not for binding to C1q. Increasing the segmental flexibility of the intact antibody by reduction and alkylation destroyed its capacity to activate C1. A MAb against the globular heads of C1q completely inhibited C1-activation by aggregated IgG (AHG), but did not prevent activation by MAb 130. C1, reconstituted by adding C1q-stalks that lack the globular heads to C1q-depleted serum was not activated by AHG, whereas activation by MAb 130 was not affected. Activation of serum-C1 by AHG and MAb 130 was inhibited by addition of excess purified C1-inhibitor in a comparable and dose-dependent manner. Sucrose-gradient analysis indicated a predominance of stable complexes of a single C1q-molecule with three MAbs at the optimal activating ratio. When isolated and added to C1q-depleted serum, these complexes activated C1 efficiently. A mechanism for activation by MAb 130 is proposed that supports the "distortive" model of C1-activation.

Antibodies, Monoclonal↗
PubMed1982

Activation of the classical complement pathway by a polysaccharide from sugar cane.

The effects of an immunostimulating polysaccharide, Bo, from sugar cane, on the complement system have been investigated. Bo, a glucan of about 10,000 mol wt, was found to activate complement in whole human and guinea pig serum in vitro by the classical pathway. Complement consumption was also demonstrated in guinea pigs upon intravenous injection. Specifically, C1 is activated, and C4 and C2, as well as C3, are consumed. The activation is prevented when Ca++ ions are chelated by ethyleneglycoltetraacetic acid, and when C1q is lacking. Hence, it does not rest on direct activation of C1s. Supplementation of C1q-deficient human serum with purified C1q restores the ability to be activated by Bo. The alternative pathway of complement is little if at all affected by the polysaccharide. The activation of C1 seems to be mediated by immune complex formation between Bo and naturally occurring immunoglobulins. Complement in sera from two severely hypogammaglobulinemic patients was not activated by Bo, but was made reactive by addition of purified human immunoglobulin G.

Adjuvants, Immunologic↗
PubMed1983

Polyions regulate the alternative amplification pathway of complement.

Polyanion has previously been shown to inhibit generation of amplification pathway convertase of complement and to inhibit H-mediated decay of this complex. Polycations, protamine sulfate, and poly-l-lysine (PLL) were examined in this study for direct effects on alternative amplification pathway mechanisms and for ability to regulate polyanion-induced inhibiton; they were found to inhibit generation of EAC4b,3b,Bb, and EAC4b,3b,Bb,P in a dose-related manner. The generation of EAC4b,3b,B was also inhibited by higher doses but was augmented by lower doses of polycation. Polycation interfered with the effective consumption of B in the fluid phase, bound minimally to EAC4b,3b, and did not cause accelerated decay of a preformed convertase. Polycation diminished the ability of polyanion (heparin) to inhibit generation of both cell-bound and fluid-phase amplification pathway convertase. In contrast, polycation enhanced the ability of polyanion to cause decay of preformed convertase even though polycation had no effect itself. These studies demonstrate that both polyionic substances have the capacity to regulate amplification pathway mechanisms directly.

Cations↗
PubMed1984

Factor D of the alternative pathway of bovine complement: isolation and characterization.

Factor D of the bovine alternative complement pathway has been purified by chromatography on CM-Sephadex C-50, Sephacryl S-200 and hydroxylapatite. The isolated factor D (0.25 mg from 1 litre of bovine serum) had an apparent molecular weight of 27,500 and a pI of 7.2. In whole bovine serum the pI of factor D was also 7.2. The isolated protein caused the Mg++-dependent cleavage of bovine factor B in the presence of cobra venom factor (CVF) to generate a haemolytically active C3-convertase as shown by SDS-polyacrylamide gel electrophoresis and haemolytic diffusion plate assays. Bovine factor D and human factor D were interchangeable in restoring the alternative pathway haemolytic activities of both bovine RD and human RD (factor D deficient sera). The haemolytic activity of bovine serum factor D was completely inhibited by 20 mM diisopropylfluorophosphate (DFP) but only 25% inhibited by 1 mM DFP. Serum heated at 56 degrees C for 10 min completely lost factor D activity but purified factor D was relatively more heat stable.

Animals↗
PubMed1988

A simple isolation procedure for functionally pure components of the bovine alternative complement pathway (ACP) C3 convertase and bovine conglutinin (K).

A simple multicomponent isolation procedure for bovine C3, factor B, factor D and conglutinin (K) from a single serum sample is described. The components of the alternative pathway C3 convertase were isolated in milligram quantities from 800 ml bovine serum and were found to be functionally pure with respect to each other and to factors H and I.

Animals↗
PubMed1987

Activation of the classical complement pathway by BioRex-70.

The cation exchange resin BioRex-70 was able to activate the classical complement pathway in human serum at 37 degrees C over the resin concentration range 0-5% (v/v). Using zymosan-treated human serum, it was found that the activation proceeded as far as complement protein C3.

Cation Exchange Resins↗
PubMed1987

C1 activation and dissociation in disease.

The composition of complexes containing C1 inactivator (C1 IA), C1r and C1s was investigated in normal serum after activation of C1 under various conditions. Analyses were performed with PAGE of eluates from Sepharose beads coated with F(ab')2 fragments of anti C1s followed by immunoblotting with anti C1 IA, anti C1s or anti C1r. Eluates obtained from serum treated with aggregated IgG (AGG) contained C1 IA in complex with C1r and C1s with both subcomponents in activated form. Eluates from serum incubated at 37 degrees C for 1, 2 or 3 days without activators showed C1 IA complexed with activated C1r and with C1s in proenzyme state associated to the complex. On analysis of serum, treated as mentioned above, by a variant of the electroimmunoassay using an intermediate gel containing anti-C1 IA and with anti-C1s in the anodal gel the two types of C1r--C1s--C1 IA complexes could be distinguished. Investigation of fresh sera and synovial fluids from patients with rheumatoid arthritis in this assay showed complexes containing C1 IA and C1r-C1s in activated form in the synovial fluids, while C1 IA-activated C1r-proenzyme C1s complexes were found in the corresponding sera.

Arthritis, Rheumatoid↗
PubMed1986

Binding of complement component C1q by spectrin.

125I-labelled human C1q was found to bind to human spectrin. Scatchard plots for the binding process were non-linear, indicating the possible presence of multiple classes of binding sites for C1q on spectrin. The binding was ionic-strength-dependent; the extent of binding decreased with increasing ionic strength. Chemical modification of arginine and histidine residues on C1q as well as pretreatment of C1q at pH 4.45 or at 56 degrees C reduced its spectrin binding activity. The amount of 125I-labelled C1q bound to immune complexes was reduced by the presence of spectrin. Spectrin was also able to deplete the complement haemolytic activity of human serum in a dose-dependent manner.

Complement Activating Enzymes↗
PubMed1988

Rosmarinic acid: a new inhibitor of complement C3-convertase with anti-inflammatory activity.

Rosmarinic acid (RA) is a naturally occurring compound, isolated from Rosmarinus officinalis or Melissa officinalis which inhibits the in vitro immunohaemolysis of antibody-coated sheep erythrocytes by guinea pig serum. In further experiments this reduced immunohaemolysis was found to be due to inhibition of the C3-convertase of the classical complement pathway. The threshold concentration for inhibition of C3-convertase was 10(-6) mol/l. RA with an optimal inhibitory concentration between 5 and 10 mumol/l., resulting in about 70% inhibition of haemolysis. However, higher concentrations of RA were less effective at inhibiting C3-convertase. The inhibition may not be specific for C3-convertase, since another serine protease, elastase, was also weakly inhibited by RA in vitro. RA also exhibited inhibitory activity in three in vivo models in which complement activation plays a role. Thus, RA (0.316-3.16 mg/kg i.m.) reduced paw oedema induced by cobra venom factor (CVF) in the rat, and at 1-100 mg/kg p.o. inhibited passive cutaneous anaphylaxis in the rat. In addition, at 10 mg/kg i.m. RA impaired in vivo activation by heat-killed Corynebacterium parvum (i.p.) of mouse macrophages, as measured by the decreased capacity of the activated macrophages to undergo the oxidative burst. RA (0.1-10 mg/kg i.m.) did not inhibit t-butyl hydroperoxide-induced paw oedema in the rat, indicating selectivity for complement-dependent processes.

Animals↗
PubMed1990

Autoantibody to complement neoantigens in membranoproliferative glomerulonephritis.

With the exception of C3 nephritic factor, autoantibody formation has not been commonly associated with membranoproliferative nephritis (MPGN). We measured autoantibodies (nephritic factors) to the C3 convertases C3bBb (NFa) and C3bBbP (NFt), which result in fast and slow C3 activation, respectively, and to a neoantigen on C1q fixed to a solid phase (spC1q) in sera from 29 patients with MPGN type I, 26 with type II, and 28 with type III. Autoantibody formation was common in all MPGN types. An autoantibody to a C3 convertase neoantigen was identified in more than 75% of the hypocomplementemic MPGN sera tested. Anti-C3bBb (NFa) was present in 81% of patients with MPGN type II but was rarely found in either type I or type III. Anti-C3bBbP (NFt) was common in both MPGN I and III. Anti-spC1q was present in 74% of patients with type I and in 38% and 48% of types II and III MPGN, respectively. Patients with MPGN types I, II, and III had one and two serum autoantibodies detected significantly more frequently than did a group of healthy subjects. The presence of any one autoantibody was not specifically associated with the presence of any other autoantibody. The results indicate that multiple autoantibody formation is common in all MPGN types. MPGN II, and possibly MPGN I, tend to form more specific autoantibodies.

Antigen-Antibody Complex↗
PubMed1990

Evidence that production of autoantibody to the alternative pathway C3 convertase is a normal physiologic event.

The origin of autoantibody production was studied with the use of antibody to the alternative pathway C3 convertase (C3 nephritic factor (C3NeF), as a model. Pokeweed mitogen stimulation of peripheral mononuclear cells from newborn infants, normal adults, and patients with membranoproliferative glomerulonephritis indicated that the ability to make C3NeF is apparently present in everyone from the time of birth. In addition, C3NeF appeared to express a single or very limited idiotope (21/21 isolates). The data also suggest that the elaboration of C3NeF may approximate an antibody response after immunization. Thus the C3NeF fraction of the total IgG or IgM produced in culture by pokeweed mitogen-stimulated mononuclear cells from normal neonates and adults, as well as from patients, was in the range of the production of specific antibody. Further, both IgG and IgM C3NeF produced by cells from these normal individuals, including newborn infants, had an affinity for antigen (10(8) to 10(9) L/mol) that was also in the range of specific antibody. Most of the autoantibody molecules (5/7) from serum were IgG3; two B cell clones producing C3NeF were CD5-negative. These experiments indicate that unmutated germline genes are used in the production of C3NeF and that a limited spectrum of antiidiotypic antibodies regulate its production.

Adult↗
PubMed1982

Serum hemolytic factor D values in children with steroid-responsive idiopathic nephrotic syndrome.

Serum hemolytic factor D activity, an important component of the ACP, was measured in patients with SR-INS. The mean serum factor D hemolytic activity of patients with SR-INS in relapse was significantly reduced compared to the mean of the control group. Twenty-one of 27 SR-INS patients in relapse (78%) had reduced serum factor D activity. In contrast, the mean serum factor D hemolytic activity of SR-INS patients in remission was not significantly different from that in the control group. Factor D hemolytic activity was also reduced in other types of renal disease with the nephrotic syndrome. Serum factor D values were highly correlated with the serum albumin concentration. Although hemolytic factor D activity could not be detected in the urine, the low molecular weight and the significant correlation with serum albumin concentration suggest that urinary loss is responsible for the low serum levels of factor D. Deficient serum values of factor D may contribute to the increased susceptibility of SR-INS patients in relapse to bacterial infections with organisms which activate the ACP.

Adolescent↗
PubMed1985

Atypical hypocomplementemic vasculitis syndrome in a child.

We report a patient who developed recurrent urticaria and angioedema at age 2 years, severe hypocomplementemic glomerulonephritis at 11 years, and end-stage renal disease at 14 years. His disease resembled the hypocomplementemic vasculitis syndrome but was atypical in its early age of presentation, severe hypocomplementemia, and progression to end-stage renal disease. Serum C1q levels were extremely low, and C4, C2, C3, and C5 levels were significantly reduced. Serum C1 inhibitor (C1INH) levels were slightly low, presumably from consumption. Circulating C1INH-C1r-C1s complexes were evidenced by reduced ratios of functional to antigenic C1INH and antigenic C1r to C1s. Family members had normal functional and antigenic levels of all complement components studied. The patient's serum, erythrocytes, platelets, and mononuclear cells did not activate complement when mixed with normal target serum. Absence of a circulating complement activator and the low serum C3 and C5 levels suggested the presence of a solid-phase complement activator, possibly related to renal or systemic vascular endothelium. As in patients with homozygous deficiencies of classical pathway components, a severe, prolonged, acquired C1q deficiency may have predisposed this patient to the development of glomerulonephritis.

Angioedema↗