The role of the classical pathway for the bactericidal effect of normal sera against gram-negative bacteria.
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Identification of complement regulatory domains in vaccinia virus complement control protein.
Vaccinia virus encodes a homolog of the human complement regulators named vaccinia virus complement control protein (VCP). It is composed of four contiguous complement control protein (CCP) domains. Previously, VCP has been shown to bind to C3b and C4b and to inactivate the classical and alternative pathway C3 convertases by accelerating the decay of the classical pathway C3 convertase and (to a limited extent) the alternative pathway C3 convertase, as well as by supporting the factor I-mediated inactivation of C3b and C4b (the subunits of C3 convertases). In this study, we have mapped the CCP domains of VCP important for its cofactor activities, decay-accelerating activities, and binding to the target proteins by utilizing a series of deletion mutants. Our data indicate the following. (i) CCPs 1 to 3 are essential for cofactor activity for C3b and C4b; however, CCP 4 also contributes to the optimal activity. (ii) CCPs 1 to 2 are enough to mediate the classical pathway decay-accelerating activity but show very minimal activity, and all the four CCPs are necessary for its efficient activity. (iii) CCPs 2 to 4 mediate the alternative pathway decay-accelerating activity. (iv) CCPs 1 to 3 are required for binding to C3b and C4b, but the presence of CCP 4 enhances the affinity for both the target proteins. These results together demonstrate that the entire length of the protein is required for VCP's various functional activities and suggests why the four-domain structure of viral CCP is conserved in poxviruses.
CD55 Antigens↗
Immune adherence and staphylococcus protein A binding of soluble immune complexes produced by complement activation.
Complement has been shown to affect the solubility of antigen-antibody complexes by two mechanisms: in the first, classical pathway dependent, complement inhibits the formation of the immune precipitate; in the second, alternative pathway dependent, complement reacts with a formed precipitate to bring about its solubilization. The biological properties of complement reacted immune complexes (IC) has been assessed by studying their binding to staphylococcus protein A (SPA) and to human erythrocytes. BSA-anti-BSA complement reacted IC bound to human erythrocytes and to SPA. Complexes generated by solubilization of immune precipitates showed greater immune adherence than complexes held in solution by complement, despite their similar size. Complexes held in solution in a factor D depleted human serum bound more efficiently to erythrocytes than complexes formed in normal serum. These experiments demonstrate that complement reacted IC cannot be regarded as biologically inert and that factors affecting complement function may have important effects on the properties of antigen-antibody complexes.
Antigen-Antibody Complex↗
Roles of antibodies and complement in phagocytic killing of enterococci.
The contributions of complement and antibodies to polymorphonuclear leukocyte (PMN)-mediated killing of enterococci were investigated with pooled normal human serum (PNHS) or immune human sera (IHS) from patients with serious enterococcal infections. Each IHS containing antienterococcal antibodies demonstrated by enzyme-linked immunosorbent assay and Western blotting (immunoblotting) was examined with the enterococcus strain isolated from the same patient. PNHS promoted PMN-mediated killing of enterococci similar to that for IHS. PMN-mediated killing was consistently abrogated after preopsonization with heat-inactivated PNHS, but some heat-inactivated IHS supported neutrophil bactericidal activity. Inhibition of the classical pathway of complement by chelation of either PNHS or IHS with Mg-EGTA [Mg-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] did not alter PMN-mediated killing, suggesting that activation of the alternative pathway of complement is sufficient to promote killing of enterococci by PMNs. PMN-mediated killing assays were also performed with normal rabbit serum and immune rabbit serum against enterococci. Preopsonization with heat-inactivated immune rabbit serum resulted in PMN-mediated killing of enterococci, which was ablated after adsorption of the serum with the same isolate used for immunization. The influence of different phenotypic enterococcal traits on neutrophil-mediated killing was also investigated. Similar kinetics of killing were observed for derivatives of Enterococcus faecalis strains regardless of resistance to antimicrobial agents or production of beta-lactamase, hemolysin, gelatinase, or surface proteins involved in the aggregative response to pheromones. In summary, PMN-mediated killing of enterococci appears to depend primarily on complement activation by either the classical or the alternative pathway. Human antienterococcal antibodies generated during infection variably promoted neutrophil bactericidal activity, while antibody raised in a rabbit supported PMN-mediated killing of the organism examined. Finally, the different phenotypic properties of E. faecalis examined did not influence the neutrophil-mediated killing of these organisms.
Adult↗
Activation of complement by myelin: identification of C1-binding proteins of human myelin from central nervous tissue.
Myelin isolated from central nervous tissue activates the classic pathway of complement by directly activating C1. Activation of C1 can proceed to form membrane attack complex, C5b-9, in the myelin. Such an interaction between myelin and complement may be important in diseases involving myelin damage, in view of the role of complement in membrane attack and inflammation. To identify the C1-activating protein, myelin was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The blots were incubated with C1 or with whole serum complement, followed by immunostaining for C1 or C3, respectively. A duplicate strip was stained with amido black or anti-myelin antibody to visualize the myelin proteins. The results showed that two major protein bands were capable of activating C1. An approximately 56-58-kilodalton band comigrated with the W2 protein and an approximately 45-47-kilodalton band migrated along with, but slightly behind, the W1 Wolfgram doublet.
Complement Activating Enzymes↗
Conformational changes in C1q after binding to immune complexes: detection of neoantigens with monoclonal antibodies.
The formation of neoantigens within the C1q molecule after the binding of C1r and C1s to C1q and the binding of C1q to immune complexes is described. The neoantigens were detected by different monoclonal anti-C1q antibodies. This immunochemical study supports the hypothesis drawn from functional studies that the activation of the classical C pathway results from conformational changes within the C1q molecule leading to the activation of C1r and subsequently C1s.
Animals↗
Deficiency of human complement factor I associated with lowered factor H.
Deficiencies of factor I and/or factor H result in an increased consumption of C3 and higher susceptibility to recurrent infections. Here we describe a case of human factor I deficiency and lowered factor H levels. C3 concentration was 50% lower than normal, the classical pathway-dependent hemolytic activity was reduced to almost 30% of normal, and alternative pathway-dependent activity was completely absent. The killing by peripheral leukocytes of Candida albicans treated with deficient serum and the production of complement-dependent chemotactic factors were reduced in the proband's serum when compared with normal serum. Finally, we observed that C3 antigen present in the proband's serum has a different electrophoretic mobility than native C3 (most likely C3b), confirming the deregulation of complement activation due to the lack of regulatory proteins factors I and H. The impaired complement system described in this case, the first of its kind described in a Chile, explains the higher susceptibility to infections found in the proband.
Animals↗
Amelioration of lytic abnormalities of paroxysmal nocturnal hemoglobinuria with decay-accelerating factor.
Purified decay-accelerating factor (DAF), from the stroma of normal human erythrocytes, was incorporated into the membranes of erythrocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH), and its effect on the complement sensitivity of the cells was investigated. Reconstitution with exogenous DAF restored the ability of the affected PNH cells to resist assembly of the homologous C3 convertase, C4b2a, on their surfaces, and decreased the susceptibility of the cells to lysis in acidified serum. Conversely, treatment of normal erythrocytes with monoclonal or polyclonal anti-DAF antibodies abrogated the capacity of the normal cells to circumvent C4b2a assembly and rendered the cells sensitive to acid lysis. These findings show that the previously reported association of DAF deficiency with PNH is causally related to the lytic abnormalities of the cells and clarify the molecular basis for restriction of autologous convertase formation on normal human erythrocytes.
Acids↗
Monomeric complement-activating IgG paraproteins.
Three patients presented a unique syndrome of recurrent panniculitis with an IgGkappa paraprotein and depletion of the early components of the classical pathway of complement. The IgGkappa paraproteins were monomers with a normal structure, and with no evidence for aggregation, as assessed by electron microscopy and ultracentrifugation. Both heavy and light chains were of normal molecular size (SDS-PAGE), and the paraproteins were not heavily glycosylated. However, the paraproteins from all three patients had unusual features that included abnormal behavior on gel filtration chromatography and a heavy chain of high pI. When analyzed by fast protein liquid chromatography (Superdex 200), elution of the paraproteins was retarded, particularly when the ionic strength was increased. This retardation was partially reversed in 20% alcohol, and fully reversed in 6 M guanidine-HCl. Neither anti-C1 inhibitor nor anti-C1q autoantibodies were found in any of the patients' sera. However, the paraproteins bound to the globular heads of C1q at normal ionic strength. They activated C4 in normal human serum, but not in C1q-deficient serum. Activation led to the formation of C1s-C1 inhibitor complexes. Taken together, the data suggest that the unusual paraproteins have the capacity to bind C1q, which then leads to activation of C1. The ability of these paraproteins to activate C1, in spite of their being soluble monomers, is likely to be related to their unique physicochemical features.
Chromatography, Gel↗
Complement: function and clinical relevance.
There is a remarkable array of proteins participating in the complement cascade, regulating the activation of the system, or recognizing a fragment of a component as a biologic signal. The classical pathway of complement activation depends on antigen-antibody interaction and is important as an effector arm of acquired humoral immunity to microorganisms. The alternative pathway functions as a form of innate humoral immunity by attacking membranes not having the characteristics of self-membrane. In addition, the alternative path provides amplification after triggering by either path. Absence or dysfunction of many of the components is associated with autoimmune or immunodeficiency disease. Absence of the inhibitor C1INH is associated with the unique syndrome of hereditary angioedema.
Complement Activation↗
Effect of fibronectin on the haemolytic activity of complement.
Sheep erythrocytes were coupled with trinitophenyl sulphonate, sensitized with anti-TNP (or-DNP) IgM monoclonal antibodies, and exposed to components of the classical pathway of complement activation. When human fibronectin (FN) was added after C1q, but before addition of C1r and C1s (subunits of the first complement component), inhibition of haemolytic activity was observed which was strictly dependent upon the dose of FN. When FN was added after addition of C1 (reconstituted from C1q, C1r and C1s), the haemolytic activity of complement was not affected by the presence of FN. These data suggest that FN binds on C1q by interfering with C1r and C1s fixation. In addition, FN was unable to displace the activated subcomponents (C1r and C1s) from their binding site on C1q. When using other systems (sheep erythrocytes sensitized with anti-Forssman IgM monoclonal antibodies), the quantity of FN required to inhibit complement haemolytic activity was greater than in the TNP system. In normal plasma, there is a 50-fold excess of FN compared to free C1q.
Animals↗
Activation of human complement by mouse and mouse/human chimeric monoclonal antibodies.
The complement (C)-activating capabilities in human serum of 32 mouse and 10 mouse/human chimeric MoAbs of different isotypes, and their fragments, were tested in vitro. Activation of C via the classical pathway (CP) was performed in 1% factor D-deficient serum in gelatin containing Veronal buffer in the presence of calcium and magnesium (GVB++), while activation of the alternative pathway of C (AP) was assessed in 10% C1q-depleted serum in the presence of 5 mM MgCl2 in GVB++. The C-activating ability of MoAbs was expressed relative to the degree of activation of complement by aggregated IgG for the CP and relative to mouse IgG1 for the AP. All of seven mouse IgG2a MoAbs were potent activators of the CP. The results of CP activation by IgG1, IgG2b and IgG3 isotypes were different for individual MoAbs. Only three (two IgG1 and one IgG3) of 32 mouse MoAbs were potent activators of the AP. IgG2a and IgG2b were relatively poor AP activators. There were a few MoAbs which activated both the AP and CP. Of 10 chimeric MoAbs, two IgG1, one IgG2 and one IgG4 were poor or non-activators of the CP. On the other hand, IgG2 and IgG4 were good AP activators. IgG3 was the most potent AP activator. Most of the F(ab')2 fragments were activators of the AP and displayed no activation of the CP. Fc fragments only activated the CP, whereas Fab' did not activate the CP or the AP. These studies suggest that the route of complement activation by class and subclass MoAbs can not always be predicted in advance and based only on their subclass identity.
Animals↗
Role of decay-accelerating factor in regulating complement activation on the erythrocyte surface as revealed by gene targeting.
Decay-accelerating factor (DAF) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that inhibits both the classical and the alternative pathways of complement activation. DAF has been studied extensively in humans under two clinical settings: when absent from the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) patients, who suffer from complement-mediated hemolytic anemia, and in transgenic pigs expressing human DAF, which have been developed to help overcome complement-mediated hyperacute rejection in xenotransplantation. Nevertheless, the exact role of DAF in regulating complement activation in vivo on the cell surface and the species specificity of this molecule remain to be fully characterized. To address these issues, we have used gene targeting to produce mice lacking GPI-anchored DAF. We found that erythrocytes from mice deficient in GPI-anchored DAF showed no increase in spontaneous complement activation in vivo but exhibited impaired regulation of zymosan-initiated bystander and antibody-triggered classical pathway complement activation in vitro, resulting in enhanced complement deposition. Despite a high level of C3 fixation, no homologous hemolysis occurred. It is noteworthy that GPI-linked DAF knockout erythrocytes, when tested with human and guinea pig sera, were more susceptible to heterologous complement lysis than were normal erythrocytes. These results suggest that DAF is capable of regulating homologous as well as heterologous complement activation via the alternative or the classical pathway. They also indicate that DAF deficiency alone is not sufficient to cause homologous hemolysis. In contrast, when the assembly of the membrane-attack complex is not properly regulated, as in the case of heterologous complement activation or in PNH patients, impaired erythrocyte DAF activity and enhanced C3 deposition could lead to increased hemolytic reaction.
Animals↗
C1q: its functions within the innate and adaptive immune responses and its role in lupus autoimmunity.
The complement cascade is a multi-faced effector component of the innate immune response. C1q is the recognition component of the classical pathway of complement activation. In addition, C1q has been recognized to serve a number of other biological functions including a modulating role on cellular functions within the adaptive immune response. The importance of C1q to normal immune regulation is reflected by the fact that greater than 90% of individuals who have complete congenital deficiency of C1q have been observed to develop early-onset photosensitive systemic lupus erythematosus (SLE). As a number of single nucleotide polymorphisms have been identified in three C1q genes, it is possible that more subtle variations in C1q expression could be a risk factor for cutaneous LE and SLE. Thus, a more comprehensive delineation of complotype could be of increasing clinical importance in the future.
Complement Activation↗
[Angioedema due to acquired complement-C1-inhibitor deficiency in a female patient with non-Hodgkin lymphoma and autoimmune hemolytic anemia].
A case of angioedema due to acquired deficiency of the regulatory protein C1-esterase-inhibitor (C1-INH) is reported. The edematous attack occurred 3 1/2 weeks after initiation of successful therapy for autoimmune-hemolytic anemia in the course of long-standing non-Hodgkin's lymphoma. At the time of acute edema the complement profile was typical: virtual absence of C1-INH function was associated with diminished concentrations of the components of the classical pathway of complement (C1q, C1r, C1s, C2, C4) and reduced complement hemolytic activity (CH50). Anti-C1-INH-autoantibodies were not detected. The angioedema lasted for about one week, and no further attacks occurred during the five-months follow-up period. Although there was only a minor adjustment to the therapy, the C1q, C2, C4 and CH50 values gradually increased to levels close to the lower limit of the normal range, while C1r and C1s showed normal values. In contrast to most other reports, this case was characterized by angioedema which was precipitated only after initiation of appropriate treatment for the underlying disease rather than before therapy or even diagnosis of the underlying disease.
Aged↗
Structural and functional differences between decay-accelerating factor and red cell acetylcholinesterase.
The abnormal erythrocytes in paroxysmal nocturnal hemoglobinuria, both PNH II (the moderately abnormal cells) and PNH III (the markedly abnormal cells), lack both acetylcholinesterase (AChE) activity and decay-accelerating factor (DAF) activity. Both of these activities are found on glycoprotein molecules with a molecular weight of about 70 Kd. To demonstrate that these two activities are in fact on different proteins, we have shown that binding to normal red cells of antibody to DAF does not inhibit the subsequent binding of monoclonal antibody to AChE nor AChE activity. Inhibition of DAF activity by polyclonal antibody increases the susceptibility of normal erythrocytes to lysis by complement but inhibition of AChE activity by antibody does not. The rate of decay of the C3 convertase complex of the classical pathway of complement activation was inhibited by DAF added in the fluid phase but not by AChE. When DAF was exhaustively immunoprecipitated from a solution of the erythrocyte membrane proteins, AChE remained and vice versa. These studies indicate that acetylcholinesterase and decay-accelerating factor are two different proteins, both of which are lacking on PNH II and PNH III erythrocytes.
Acetylcholinesterase↗
Paraproteins and complement depletion: pathogenesis and clinical syndromes.
Various clinical syndromes that associate paraproteinemia and complement depletion have been described in the last three decades. Among these, cryoglobulinemias, acquired Clq deficiency, and acquired deficiencies of the classical pathway of complement can be associated with B-cell lymphoproliferative disorders. Some specific symptoms should alert the clinician to suspect an underlying malignancy. In this report, we review the pathogenesis, symptomatology and therapeutic options of these clinical conditions.
Agammaglobulinemia↗
Complement activation by cross-linked B cell-membrane IgM.
The B cell membrane IgM (mIgM) occurs in a monomeric form incapable of activating C. However, when cross-linked by a polyvalent ligand, mIgM may activate C by assuming a polymeric structure like secreted IgM. This possibility was tested with CR2- lymphoma cells, which did not activate C spontaneously. When CR2-deficient mIgM(lambda)+ Ramos cells were treated with F(ab')2 goat anti-lambda, then exposed to human serum, a marked C3 deposition took place, as examined by the flow cytometry. Similarly, C3 deposition on mIgM(kappa)+, mIgD(kappa)+ P32 cells was induced by F(ab')2 of either anti-kappa or anti-mu. Anti-delta was without effect, but the C3 deposition resulting from anti-kappa was markedly enhanced after mIgD was modulated by anti-delta. The mIgM-cross-linked cells bound C1q, and C3 deposition on these cells was abrogated by depletion of C1q, but not Factor B nor D, from serum. The C1-binding step of the mIgM-mediated C activation was inhibited by monomeric Fab' of polyclonal anti-mu containing a blocking Ab to the hemolytic activity of human IgM Forssman Ab. A large proportion of C3 deposits on mIgM-cross-linked cells was found to be associated with mIgM in the form of C3dg or C3d. These results demonstrate that cross-linked mIgM indeed triggers the classical pathway of C.
B-Lymphocytes↗