Detection and analysis of inborn and acquired complement abnormalities.
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Animals↗
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Results for “Complement Activating Enzymes”
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Activation of the fifth component of human complement (C5) induced by monosodium urate crystals: C5 convertase assembly on the crystal surface.
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Arthritis↗
Quantitation of (C1INH)2 C1r-C1s complexes in glomerulonephritis as an indicator of C1 activation.
C1 activation was assessed in several forms of glomerulonephritis by radioimmunoassay quantitation of circulating (C1INH)2 C1r-C1s complexes (INC). Eight patients with active systemic lupus erythematosus (SLE) and nephritis had elevated serum INC (mean = 15.3 vs control = 5.8, P less than 0.01). Their INC levels were normal during remission. Serum INC had a weak inverse correlation with serum C1q greater than 3 mg/dl (r = 0.42, P = 0.02). In longitudinal studies, serum INC also had a weak inverse correlation with serum C3 and C4. Only 1 of 10 patients with type I and 1 of 15 with type III membrano-proliferative glomerulonephritis (MPGN) had elevated serum INC. No patient with type II MPGN had elevated levels. Two of 10 patients with poststreptococcal glomerulonephritis (P-SGN) had elevated serum INC, but all normalized with convalescence. Patients with IgA nephropathy had normal serum INC. The data demonstrate the importance of C1 activation in SLE and P-SGN. The mechanism of complement activation in types I and III MPGN remains unclear; the data suggest, but do not prove, that C1-independent complement activation may occur in these patients.
Antigen-Antibody Complex↗
Interaction of fibronectin with DNA/anti-DNA complexes from systemic lupus erythematosus: role of activated complement Cl in modulation of the interactions.
Fibronectin (Fn) is an integral constituent of the endothelial cell surface and the basement membrane. The mechanism for binding DNA/anti-DNA complexes to Fn was examined in a solid-phase assay. In physiological buffer, a low-affinity binding of DNA was observed with Fn and optimal binding was seen at pH 6.5 and in the absence of Ca2+. Further, the interaction of DNA to Fn was inhibited when DNA was complexed to anti-DNA antibody. However, complement Clq mediated the binding of complexes to Fn at pH 7.4 and it was proportional to the extent of the dissociation of Cl. Cl inactivator (Cl-In) appeared to play a modulating role; whereas at low concentrations (Cl:Cl-In::4: less than 1) it enhanced the binding of complexes to Fn, higher concentrations inhibited the binding. Further, sera from patients with active systemic lupus erythematosus reacted with Fn, which was shown to be dependent on the presence of Clq and was minimally affected by DNase treatment of sera, indicating a relatively minor role of DNA in the direct binding of DNA to Fn. These findings support "circulating immune complex" hypothesis in the pathogenesis of lupus glomerular immune complex deposition disease.
Antibodies, Antinuclear↗
Complement activation occurs through both classical and alternative pathways prior to onset and resolution of adult respiratory distress syndrome.
We have previously reported that plasma concentrations of the terminal complement (C) complex (TCC), C5b-9, increased significantly 2 days prior to onset of adult respiratory distress syndrome (ARDS) and also 1 day preceding its resolution. To determine the pathway of complement activation that preceded development and resolution of this acute inflammatory lung injury in septic patients, we quantified the C1rC1s-C1 inhibitor complex and the C3bP complex, which are generated following activation of classical and alternative complement pathways, respectively. Two days prior to diagnosis of ARDS, the plasma C1rC1s-C1 inhibitor complex and C3bP complex levels increased 22 and 14%, respectively. Furthermore, significant correlations were identified between concentrations of the TCC and C1rC1s-C1 inhibitor complex (r = 0.73, P = 0.003) and also with the levels of the TCC and C3bP complex (r = 0.81, P = 0.002) before onset of ARDS. Equally of interest, the C1rC1s-C1 inhibitor complex and C3bP complex concentrations increased 68 and 35%, respectively, 1 day before resolution of ARDS. Similarly, significant elevations of TCC concentrations preceding resolution of ARDS correlated with C1rC1s-C1 inhibitor complex (r = 0.66, P = 0.02) and also with C3bP complex (r = 0.72, P = 0.002) levels. Our results indicate that both the classical and alternative complement pathways are activated prior to onset of ARDS and also before its resolution in septic patients.
Complement Activating Enzymes↗
Plasma concentrations of complement-activation complexes correlate with disease activity in patients diagnosed with isolated central nervous system vasculitis.
Isolated central nervous system (CNS) vasculitis is rare medium- sized vessel disease limited to intracerebral vessels. The two most common symptoms of this inflammatory disorder observed at entry to a hospital are headaches and mild memory deficits. Further progression of this disease may result in focal neurologic alterations and seizures. Currently, the most common laboratory abnormality noted is an elevated erythrocyte sedimentation rate. The complement (C) system is known to play a role in many inflammatory processes; it may also be involved in CNS vasculitis. In this longitudinal study of patients with CNS vasculitis, we detected C activation by highly sensitive and specific assays that are capable of identifying breakdown products formed after C activation: C3a des arg, C4a des arg, C5a des arg, C1rC1s-C1-inhibitor complex, and terminal C complex (C5b-9). We present two cases of documented CNS vasculitis in which serial measurements of these C-activation products correlate with disease activity. Our results indicate that a temporal association exists between C activation and the clinical presentation of CNS vasculitis. We conclude that physicians should monitor C-activation by-products in plasma when they attempt to follow the clinical course of CNS vasculitis.
Adult↗
Interaction of bovine immunoglobulins with complement.
Whereas complement (C) in rabbit serum (CR) was bound by bovine antibodies in seven different IgG1 preparations, only two IgG1 preparations could bind the C in guinea pig serum (CGP). Addition of the Clq component of CR to CGP was alone sufficient to render the C-cascade in CGP activable in the presence of bovine erythrocytes sensitized with specific antisera, i.e. reagents. Normal bovine serum was also capable of restoring the haemolytic activity of CGP. However, the bovine serum was much more temperature sensitive than was CR and, as was observed in the sera from MZ twins, it showed considerable variation both in titre values and in prozones when added to CGP.
Agglutination Tests↗
Activation of Cl, the first component of complement, the generation of Clr-Cls and Cl- inactivator complexes in normal serum by heparin-affinity chromatography.
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Chromatography, Affinity↗
The binding and activation of the Clr-Cls subunit of the first component of human complement.
The value of the functional affinity constant between 125I-labelled Clq and the Clr-Cls tetramer (when free in solution) in the formation of Cl was found to be 3.6 X 10(7) M-1. When Clq was bound to activating immune complexes, the value of K was about 10-fold higher before initiation of activation and there was a further two to three-fold rise as activation proceeded. The addition of an excess of unlabelled Clq increased the rate of activation of 125I-labelled Cl, suggesting an interaction between Clr-Cls and two neighbouring Clq molecules. It is suggested that the tetramer Clr-Cls may bind bivalently to Clq when free in solution, but on binding to activating complexes, one of the Clr-Cls binding sites is detached from Clq and becomes bound to a site on the complex. The resultant spatial rearrangement of the Clr molecules within the tetramer may be optimal for autocatalytic activation of Clr.
Antigen-Antibody Complex↗
Interaction of C1q with DNA.
With the aim of clarifying the relationship between the activation of the first component of complement (C1) by immunoglobulin and by polyanions, the mode of interaction of C1q with DNA was investigated by structural and inhibition studies. DNA inhibits C1q binding to IgG immune complexes (ICs) through binding to C1q rather than to IgG, as seen from two lines of evidence. Firstly, DNA does not bind to ICs under conditions where full binding to C1q is observed. Secondly, at I - 0.15, DNA inhibits more strongly when mixed first with C1q rather than with ICs. The inhibition of C1q-IgG binding by DNA is subject to kinetic factors. Firstly, DNA is not an effective inhibitor if added after C1q has bound to ICs. This at least in part reflects a portion of the IgG-bound C1q that exchanges only very slowly with free C1q. Secondly, the relative rates of association of C1q to DNA and ICs at different ionic strengths are important in determining whether inhibition is observed. The existence of a kinetic effect in the inhibition by DNA means that inhibition experiments cannot be used to establish whether DNa binds to the same site on C1q as IgG. This question was therefore approached by structural studies. Precipitation of C1q with DNA was greatly diminished by heat or pH 4.45 denaturation of C1q, by pepsin digestion to remove the globular heads, and by limited modification with 1, 2-cyclohexanedione. In contrast, extensive modification with methyl acetimidate only had a limited effect. In these respects the structural requirements for C1q-DNA precipitation were similar to those for C1q-Igg binding, as would be consistent with binding of DNA and IgG to nearby or overlapping sites on C1q. In view of residual DNA-precipitating activity in the pepsin fragment preparation of C1q, there is the possibility that there are additional DNA sites on the collagenous tails.
Amino Acids↗
A reinterpretation of the evidence for an interaction between Clq and pairs of immunoglobulin molecules in an immune lattice.
An error in a previous article [Cohen S. (1968) J. Immun. 100, 407-413] was discovered which invalidates it as evidence that IgG immune complexes activate the classical complement pathway by an interaction involving pairs of antibody molecules, so-called doublet formation. The error concerns the correction to be made for the observed 8% residual activity of the modified antibody preparation used. When appropriate correction factors are applied, the data are found not to be consistent with the hypothesis of doublet formation. The analysis presented here has no bearing on the original article's experimental techniques, nor on its demonstration of co-operation between IgG molecules during complement fixation.
Antigen-Antibody Complex↗
Kinetics of activation of the first component of complement (C1) by IgG oligomers.
The rate of activation of the first component of complement, Cl, by IgG oligomers was investigated. The kinetics of Clr activation exhibited a pronounced lag phase at low IgG concentration and were followed by a rapid conversion of the proenzyme Cls to Cls. Activation of Cl resulted in a conformational change of this complex. Bases on these results, a model of Cl activation by IgG oligomers is proposed: The efficiency of Cl activation by IgG oligomers is in parallel to their binding affinity to Clq. Clr undergoes a slow rearrangement to a conformation which is not stable and is autoactivated, then Clr rapidly converts Cls to Cls.
Antigen-Antibody Complex↗
The interaction of immune serum globulin and immune globulin intravenous with complement.
The in vitro anticomplementary activity of untreated and heat-aggregated (63 degrees C, 10 min) immune serum globulin (ISG) and immune globulin intravenous (IGIV) prepared by partial reduction and alkylation have been evaluated by three assays, C3 activation, binding to C1q and enhancement of alternative pathway lysis of rabbit erythrocytes. Crossed immunoelectrophoresis was used to quantitatively measure the ability of ISG and IGIV to activate endogenous C3 in normal serum. Binding to C1q was determined according to the ability to inhibit binding of 125I-C1q to solid phase IgG. ISG and IGIV enhancement of lysis of rabbit erythrocytes by normal human serum adsorbed with rabbit erythrocytes in the presence of MgEGTA was used to determine activity in the alternative complement pathway. Unheated IGIV at 10 mg/ml only marginally activated endogenous C3 in normal serum, had about a 5-fold lower affinity for 125I-C1q (Ki = 138 to 356 microM vs Ki = 62.5 microM for ISG), but was very similar in ability to ISG on a weight basis in enhancing complement alternative pathway activity (RCH50 = 0.23 to 0.40 mg for IGIV vs 0.17 mg for ISG). Heat-aggregated IGIV at 5 mg/ml in normal human serum was about 2-fold less effective than heat-aggregated ISG in the activation of C3 in normal serum and had approximately 2- to 3-fold lower affinity in the C1q binding assay (Ki = 45 to 83 nM for heat-aggregated IGIV vs Ki = 14.6 nM for heat-aggregated ISG). These data suggest that IGIV prepared by chemical modification retains sufficient specific receptor activity to allow in vivo efficacy in complement-mediated amplification of host defense reactions, but is safe for intravenous use due to a lower capacity to initiate nonspecific complement activation.
Complement Activating Enzymes↗
Measurement of the association constants of the complexes formed between intact C1q or pepsin-treated C1q stalks and the unactivated or activated C1r2C1s2 tetramers.
The association constants between C1q and C1r2C1s2 and between C1q and C1r2C1s2 were measured in solution using a new technique which employs sucrose gradient ultracentrifugation to estimate thermodynamic association constants. In this technique, zones of dilute, radioiodine-labeled C1q were sedimented through uniform concentrations of either C1r2C1s2 or C1r2C1s2. The zones remained intact, indicating that the dynamic equilibrium was rapid compared with the time of centrifugation. The observed increases in the sedimentation coefficients of the C1q zones were assumed to be directly proportional to the fraction of C1q bound in the dynamic equilibrium. Binding curves were constructed by performing the measurements at many C1r2C1s2 and C1r2C1s2 concentrations. The association constants were estimated from the midpoints of the binding curves and found to be 6.7 X 10(7)M-1 for C1r2C1s2 binding to 125I-C1q. After activation of the C1r2C1s2 the association constant decreased 10-fold to 7.1 X 10(6)M-1. These association constants refer to solvent conditions of pH 7.35, 1 mM Tris, 5 mM Ca2+ and 150 mM NaC1, pH 7.35. Similar measurements were performed with the collagenous peptic fragment of C1q and both 125I-C1r2C1s2 and 125I-C1r2C1s2. The association constants were independent of the state of activation and both found to be about 2 X 10(7) M-1, suggesting that most if not all of the interactions between C1q and C1r2C1s2 were confined to the collagenous portion of C1q.
Binding Sites↗
The role of non-immune IgG in controlling IgG-mediated effector functions.
The majority of evidence supports the conclusion that IgG-dependent effectors respond to antibodies which have been polymerized artificially or by polyvalent antigens, but not to monomeric IgG antibodies. Effectors can distinguish polymerized IgG antibodies from monomeric IgG because they contain multiple receptor units and can interact multivalently with polymerized IgG. However, monomeric IgG is present at very high concns in plasma and interstitial fluids and will inhibit multivalent interactions in vivo between polymerized antibody and effectors. Such inhibition raises the question of how IgG-mediated effector responses could function in vivo. In this review we present a mathematical model which quantitatively predicts how polyvalent ligands interact multivalently with receptors in the presence of excess monovalent ligand. We then show that results from experiments in vitro using such diverse systems as the binding and endocytosis of immune complexes by macrophages, complement-mediated lysis of antibody-coated target cells, and ADCC can be explained qualitatively by the model. We conclude that monomeric IgG does not totally inhibit IgG-mediated effector functions but, rather, raises the threshold of antibody binding which is required to elicit a response. We then consider how non-immune IgG may serve as a homeostatic regulator of IgG-dependent responses, in vivo, perhaps for the purpose of inhibiting responses to low levels of cell-bound IgG autoantibodies.
Adult↗
Activation of the alternative complement pathway by the immune precipitate formed with F(ab')2 fragment of human IgG antibody.
The complement fixing ability of the F(ab')2 fragment of human IgG was studied using an immune precipitate (Ippt) formed between tetanus toxoid and the F(ab')2 of high-titer IgG antibody against tetanus toxin. A major subclass of the specific IgG antibody against tetanus toxin, which was separated by affinity column chromatography, was identified as IgG1. On incubation of normal human serum (NHS) with the Ippt formed at equivalence, a dose-dependent consumption of CH50, C3 and C5 activities was observed without significant loss of the early acting complement components. A similar consumption of CH50, C3 and C5 activities was found in NHS reacted with Ippt formed at any antigen/antibody ratio. The Ippt formed at antibody excess was more efficient in complement consumption than the Ippt formed at antigen excess. An apparent consumption of C3 and C5 activities was also noted in C4-deficient guinea pig serum treated with Ippt. When Ippt was incubated with Mg2+--EGTA-treated NHS, both C3 and C5 convertases of the alternative pathway were generated on the Ippt. From these results, it was concluded the the F(ab')2 of human IgG antibody, especially IgG1 antibody, when it formed an Ippt with antigen, could activate the alternative complement pathway.
Antibody Specificity↗
Interaction of Zn2+ with guinea-pig C5 convertase and guinea-pig C5.
A comparison of two methods of C5 activation, the standard method (EAC14 + C2, C3, C5, C6, and C7) and the washed-cell intermediate method (EAC1423 + C5 and washed), demonstrated that formation of hemolytically competent SAC14235 was reduced in the washed-cell method. Addition of Zn2+ in this method increased the formation of competent SAC14235 to the approximate level of the standard method. The optimum concn range of Zn2+ was 0.006-0.025 mM. In the standard method Zn2+ had no significant enhancing effect on the formation of competent SAC14235. Zn2+ had a greater affinity for EAC1423 than for fluid-phase C5 when reacted with each separately. Maximum enhancement, however, was obtained when Zn2+ was present during the reaction of C5 with EAC1423. The effect of Zn2+ on C5 activity was not related to the stabilizing property of C6 on cell-bound C5. In the washed-cell method there was an inverse relationship between the concn of C2 or C3 and the concn of C5 required to generate one competent SAC14235/cell. Over a 10-fold range of C2 or C3 concn there was an approximate four-fold increase in the number of competent SAC14235/cell formed in the presence of Zn2+. Zn2+ enhances formation of competent SAC14235 on cells that have a limited ability to activate C5 even though the C2 and C3 on the convertase are present in excess.
Animals↗
Effect of reduction and alkylation on structure and function of rabbit IgG antibody--II. Effects on classical pathway C3 convertase formation.
Anaerobic reduction of purified rabbit IgG antibody (Ab) with 1.5 moles of dithiothreitol per mole of Ab at pH 8.0, followed by alkylation, cleaves 39% of the inter-heavy-chain (H-H) disulfide (SS) bonds. This treatment has the following effects on the ability of the Ab to activate the classical pathway of complement. Compared to control Ab, reduced and alkylated (RA) Ab retained 4-5.6% of overall hemolytic activity and 55% of complement-fixing activity at 0 degrees C. Complexes of RA Ab and equivalent amounts of soluble Ag consumed C4, C2 and C3 at 37, 51 and 44%, respectively, of the rate at which these components were consumed by equal concns of complexes containing control Ab. Complexes made with RA Ab bound 18% as much C-1 as those made with native Ab. These data indicate that the principal, if not the only, effect of RA is on C-1 binding. Measurements of the ability of complexes of Ab with cell-bound Ag to bind C-1 showed at most a 20% loss of C-1 binding sites and a ca two-fold decrease in affinity for C-1. Similar results were obtained with purified (activated) C-1 and with native C1 in serum. No significant difference could be detected in the rate of activation of bound C1. Normal rabbit IgG which was reduced and alkylated under the same conditions retained 52% of its H-H SS bonds and 30% of its ability to bind C-1. This finding suggests that the impairment in C-1 binding results from an effect on the C1 binding site itself, rather than from an effect on the ability of the RA Ab to transmit a putative conformational "signal" from the Ag-binding site to the C1 binding site. Finally, our data show that the observed functional effect of reduction and alkylation depends strongly on the assay used to evaluate that effect.
Alkylation↗