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PubMed2002

The crystal structure of the zymogen catalytic domain of complement protease C1r reveals that a disruptive mechanical stress is required to trigger activation of the C1 complex.

C1r is the modular serine protease (SP) that mediates autolytic activation of C1, the macromolecular complex that triggers the classical pathway of complement. The crystal structure of a mutated, proenzyme form of the catalytic domain of human C1r, comprising the first and second complement control protein modules (CCP1, CCP2) and the SP domain has been solved and refined to 2.9 A resolution. The domain associates as a homodimer with an elongated head-to-tail structure featuring a central opening and involving interactions between the CCP1 module of one monomer and the SP domain of its counterpart. Consequently, the catalytic site of one monomer and the cleavage site of the other are located at opposite ends of the dimer. The structure reveals unusual features in the SP domain and provides strong support for the hypothesis that C1r activation in C1 is triggered by a mechanical stress caused by target recognition that disrupts the CCP1-SP interfaces and allows formation of transient states involving important conformational changes.

Amino Acid Sequence↗
PubMed2000

Studies on anti-complementary activity of extracts and isolated flavones from Ligustrum vulgare and Phillyrea latifolia leaves (Oleaceae).

Polar fractions and flavones isolated from methanolic extracts of the leaves of Ligustrum vulgare and Phillyrea latifolia (Oleaceae), whose popular use as an anti-inflammatory is well-known in Mediterranean historical medicine and ethnobotany, showed significant in vitro complement inhibiting effect on the classical pathway of the complement system. Among the isolated flavonoidic structures, apigenin-7-O-glucoside, apigenin-7-O-rutinoside, luteolin-4'-O-glucoside, luteolin-7-O-glucoside and ligustroflavone presented remarkable activity.

Apigenin↗
PubMed1979

Regulation of macrophage migration by products of the complement system.

Agents formerly shown to induce rapid macrophage spreading were examined for their ability to modify the migration of macrophages in the capillary tube assay. Products of the activation of the contact phase of blood coagulation as well as the purified component Bb, the large cleavage fragment of factor B of the alternative complement pathway produced a dose-dependent inhibition of migration. In addition, inflammatory macrophages elicited with either a lipopolysaccharide endotoxin or thioglycollate medium exhibited rapid spreading and inhibited migration, whereas resident cells did not. A close correlation existed, therefore, between enhanced spreading and inhibited migration under both in vitro induced and in vivo situations. Cleavage products of component C5 of the classical complement pathway enhanced macrophage migration and did not alter spreading. In mixtures of C5 cleavage products and Bb, the predominant peptide determined the outcome of the reaction. Factor B, a normal secretory product of macrophages, may represent a common substrate for several of the proteases that induce spreading, inhibit migration, and lead to the generation of the enzymatically active fragment Bb.

Ascitic Fluid↗
PubMed1996

Inhibition of the hemolytic activity of the first component of complement C1 by an Escherichia coli C1q binding protein.

Molecular mimicry is a well established mechanism via which bacteria protect themselves from complement-mediated killing. We have previously demonstrated that a number of human cells express receptors for C1q (C1qR) and that the soluble form of this receptor inhibits activation of the classical pathway of complement. We now investigated whether Escherichia coli possesses a C1qR-like protein that protects these bacteria from complement-mediated injury. By FACS analysis it was shown that approximately 60% of the bacteria bound C1q directly in the absence of Abs. With ELISA we confirmed that the bacterial cell envelope was able to bind C1q in a dose-dependent fashion. We isolated a cell envelope associated C1q binding protein (C1qBP) by C1q affinity chromatography, then by anion exchange chromatography and gel filtration chromatography. On SDS-PAGE, the m.w. of C1qBP appeared to be 57 kDa and 51 kDa under reducing and nonreducing conditions, respectively. It was demonstrated that C1qBP specifically binds C1q and inhibits the hemolytic activity of C1q in both a dose- and time-dependent fashion. The binding of C1qBP to C1q is inhibited by C1q itself and also by the collagen-like stalks and the globular heads of C1q. In this respect, bacterial C1qBP is different from human C1qR because the binding of C1q to C1qR is only inhibited by the collagen-like stalks of C1q and not by the globular heads of C1q. C1qBP, when bound to C1q, prevents the assembly with C1r and C1s to form a functional C1 complex. The occurrence of C1qBP is not limited to certain E. coli strains, but is also found on Staphylococcus aureus, Citrobacter freundii, and Pseudomonas aeruginosa. Also, the binding of 125(I)-labeled C1q to these bacteria is specific because the binding of C1q to these bacteria is inhibitable with isolated soluble C1qBP. These findings provide evidence for the existence of a C1qR-like protein on bacteria that might protect them from complement-mediated damage.

Animals↗
PubMed1988

C4-mediated inhibition of immune precipitation and differences in inhibitory action of genetic variants, C4A3 and C4B1.

The inhibition of immune precipitation is mediated by the classical complement pathway. We report here that the rate of precipitate formation depends on the genetic form of human C4 present during immune precipitation. C4A3 is more effective than C4B1 in its capacity to inhibit the rate of immune precipitate formation in serum and in serum-free reaction mixtures containing C1 and C4. Immune precipitates form within seconds after antigen is mixed with antibody, and the activation of the classical pathway is known to occur within seconds after C1 binds to antibody molecules. The covalent deposition of C4b on immune complexes is an essential step in the inhibition of immune precipitate formation, and if any of the reactions that lead to covalent C4b deposition become limiting, the rate of immune precipitation could exceed the complement system's inhibitory capacity. Hence, the inhibition of this rate may be an important function underlying the complement-mediated processing of immune complexes, and a decreased ability of the complement system to mediate this process in the presence of C4B1, in contrast to C4A3, could explain, at least in part, the association between the C4A-null phenotype and autoimmune diseases such as systemic lupus erythematosus.

Animals↗
PubMed1985

Responses of opsonic substances to major trauma and sepsis.

Adequate opsonic capability is vital for normal host defense against infection. The presence of complement fraction 3 (C3) indicates both classical and alternative pathways of complement activation, while complement fraction 4 (C4) indicates only classical pathway activation. C-reactive protein (CRP) functions as an opsonic substance. We measured serum levels of C3, C4, and CRP in 32 trauma patients. Six suffered minor injuries and 26 suffered major injuries. Nineteen of the major-injury patients developed post-trauma sepsis. Complement levels fell immediately after injury and remained low for approximately 8 days in all patients. CRP levels rose by 12 h, peaked at 48 h, and correlated with the severity of injury and the presence of sepsis. Elevated CRP levels after 4 days postinjury indicated the presence of sepsis in all cases. CRP elevations preceded clinical diagnosis by an average of 2.4 days. Serial measurements of CRP are valuable in monitoring the course of trauma patients and may indicate the presence of septic complications before clinical detection.

C-Reactive Protein↗
PubMed1992

[A study of the humoral immunity of mice injected with beryllium chloride].

We studied changes of humoral immunity, such as complement pathway activity, C3 contents and contents of immunoglobulin, in mice injected subcutaneously with BeCl2 or CuCl2 once a week for 12 weeks. Mean body weights of JCL: ICR female mice were approximately 30g in control mice (control group; n = 7), in mice injected with Be (Be group; n = 8) and in mice injected with Cu (Cu group; n = 8). Values of classical complement pathway activity (CH50) were 18.8 +/- 1.4 U per ml, 15.3 +/- 1.8 U per ml and 16.7 +/- 1.3 U per ml in the control group, Be group and Cu group, respectively. The CH50 values of Be and Cu groups were significantly lower than that of the control group (P < 0.01). In contrast, values of alternative complement pathway activity (ACH50) and contents of C3 were almost constant in the three groups. The immunoglobulin content in the Be group tended to increase. The activity of alanine aminotransferase in the Be group was markedly higher than that in the control group (P < 0.05), and the aspartate aminotransferase activity was also high. The CH50 value of mice injected with a small amount of Be once a week over a 12-week period decreased markedly, although either the ACH50 value or C3 content was the same as in the control group. The immunoglobulin content somewhat increased in the Be group. These results suggest the possibility that immune complex is induced by Be.

Animals↗
PubMed1984

Initiation of complement activation.

The complement system is an excellent one to look at as an example of a triggered enzyme system. It is found to use almost all the strategies of enzyme cascades and for initiation which were discussed in the introduction. The classical pathway is a fine example of an active zymogen form of activation and the alternative pathway is the canonical example of a tickover. Both may also be activated by exogenous enzymes from other systems. The strategies of enzymes waiting for substrates, of substrates waiting for enzymes, and of both waiting for modifying proteins are all seen in various stages of the reaction. There is an association of a linear cascade with the positive feedback amplification loop. This degree of evolutionary adaptation is not only aesthetically pleasing but must be taken to mean that the system is of considerable biological importance and has been over a long evolutionary time span.

Complement Activation↗
PubMed1998

Inhibition of liposome-induced complement activation by incorporated poly(ethylene glycol)-lipids.

Complement activation causes opsonization of foreign particles leading to particle elimination from the blood. Complement-mediated opsonization of charged and large liposomes presents a fundamental problem in their use to deliver therapeutic agents in vivo. To prolong the circulation half-lives of such liposomes, complement activation must be curtailed. The aim of this study was to assess the ability of poly(ethylene glycol)-lipids (PEG-lipids) to inhibit the in vitro activation of the classical pathway of complement in human serum by anionic liposomes. Incorporation of cholesterol-PEG600 (CH-PEG600), cholesterol-PEG1000 (CH-PEG1000), or phosphatidylethanolamine-PEG2000 (PE-PEG2000) resulted in dose-dependent inhibition of C1q binding and complement activation. The dose of PEG-lipid at which complement activation was blocked was inversely related to the PEG chain length. Complement activation was strongly inhibited when 15 mole% of CH-PEG600, 10 mole% CH-PEG1000, or 5 mole% PE-PEG2000 was incorporated into 100-nm anionic liposomes. PEG-lipid incorporation into larger liposomes (240 nm) was also successful in blocking C1q binding and complement activation. Radiolabeled cholesterol-PEG approximately 1400 was prepared and used to determine both the percentage of CH-PEG incorporated into the liposomes and the percentage maintained in the liposomes in the presence of 50% human serum at 37 degrees C for up to 24 h.

Cholesterol↗
PubMed1987

[Complement activation in extracorporeal circulation. Comparison of nylon versus polyester bubble oxygenators].

Complement activation during cardiopulmonary bypass is well known and may influence postoperative morbidity. As nylon can particularly induce complement activation, its influence was assessed by measuring total haemolytic complement and B, C3 and C4 factors, during cardiopulmonary bypass with bubble oxygenators for coronary surgery, comparing "nylon" circuits (20 patients, Bentley BOS 10) versus "polyester" circuits (19 patients, Shiley S 100 A). Complement activation began with induction of anaesthesia and surgical procedures, B, C3 and C4 levels falling significantly (respectively 15, 17 and 20% from baseline values). The alternative pathway was activated before the classical pathway. Complement activation continued during cardiopulmonary bypass, with no more consumption of complement factors (slight variations of about 0 to 3% of the levels found after anaesthetic induction and surgical procedures). No statistically significant difference appeared between the two groups. This suggested that nylon did not significantly increase complement activation during cardiopulmonary bypass. The bubble oxygenator material cannot therefore be considered as a criterion for choosing the type of equipment.

Complement Activation↗
PubMed2003

Oxidized low density lipoprotein and innate immune receptors.

PURPOSE OF REVIEW: Atherosclerosis is now recognized as a chronic inflammatory disease. This review discusses recent literature reporting that innate immune receptors bind oxidatively modified LDL and its many oxidized moieties and consequently modulate the atherogenic process. These innate pattern recognition receptors are known to play a central role in pro-inflammatory responses to bacteria by binding pathogen-associated molecular patterns. It is hypothesized that oxidized LDL exposes similar molecular patterns recognized by receptors of innate immunity. RECENT FINDINGS: Minimally modified LDL and its oxidized phospholipids have been found to bind to CD14 or activate Toll-like receptors on macrophages. In turn, various biological activities have been induced, including the stimulation of cytoskeletal rearrangements that alter phagocytic activity and the stimulation of cytokine secretion, such as IL-8. These findings link modified LDL with innate pattern recognition receptors, such as those involved in the lipopolysaccharide signaling pathway. Human epidemiological studies support the involvement of CD14 and TLR4 in cardiovascular diseases. Oxidized LDL has also been demonstrated to bind to C-reactive protein, an opsonic molecule activating classic complement pathway and Fcgamma receptor endocytosis. These data suggest that C-reactive protein may not only be a strong predictor of clinical disease, but may also play a role in atherogenesis. Recent data on other innate immune receptors are discussed in the context of their potential interactions with oxidized LDL and atherogenesis. SUMMARY: Recent findings suggest that oxidized forms of LDL interact with innate immune receptors. Further studies are needed to identify the role of these interactions in inflammation and atherosclerosis.

Animals↗
PubMed1997

A neoepitope-based enzyme immunoassay for quantification of C1-inhibitor in complex with C1r and C1s.

Monoclonal antibodies (MoAb) recognizing neoepitopes exposed on activation products of complement proteins but hidden in the native components have been used for quantification of activated complement. A previously produced and characterized mouse MoAb, recognizing a neoepitope on the human plasma protein C1-inhibitor complexed with its substrates, was used to design an enzyme immunoassay for detection of C1-inhibitor complexed with C1r and C1s. These complexes are indicators of early classical complement pathway activation. The standard was serum activated with heat aggregated IgG defined to contain 1000 arbitrary units (AU)/ml. The lower detection limit was approximately 0.05 AU/ml corresponding to 0.005% of fully activated serum. The reliability of the assay, including day-to-day variation, was tested. Intra-assay variation coefficients were 12% for low plasma control and 13% for high plasma control (n = 12 for both). Inter-assay variation coefficients were 12% for low control (n = 6), 19% for high control (n = 6) and 15% for the normal plasma control (n = 9). A 2.5-97.5 percentile reference range (normal blood donors) was 16-33 AU/ml. Two patients with systemic lupus erythematosus had considerably elevated plasma levels of the activation product (56 and 62 AU/ml), and six patients with hereditary angioedema had normal plasma levels despite considerably reduced C1-inhibitor concentration. We conclude that the present method is sensitive and reliable for detection of early classical pathway activation and superior to previously published methods by utilizing neoepitope specificity and non-radiolabelled reagents.

Animals↗
PubMed1984

In vitro cytodestruction of human leukemic cells using murine monoclonal antibodies and human complement.

We have investigated the ability of murine monoclonal antibodies (MoAb) to lyse human leukemic cells in vitro using human serum as a source of complement (C'). The human C'-fixing ability of five of seven MoAb is documented. Studies with two of these MoAb (BA-1 and BA-2) indicated that their human C'-fixing ability and subsequent lysis of leukemic cells was through activation of the classical pathway of C', was independent of donor serum source, and occurred with a number of different target cells. BA-1 and BA-2 could effectively lyse fresh leukemic cells in the presence of 100% human serum, and BA-1 plus human serum could effectively lyse leukemic cells in the presence of a 20-fold excess of normal human bone marrow cells. Our results have potential implications for immunotherapy trials utilizing murine MoAb.

Adult↗
PubMed1982

Activation of the alternative complement pathway of guinea-gip by liposomes incorporated with trinitrophenylated phosphatidylethanolamine.

By incorporation of trinitrophenylamino-caproyldipalmitoylphosphatidylethanolamine (TNP-Cap-DPPE) into liposomes composed of an equimolecular mixture of dimyristoylphosphatidylcholine (DMPC) and cholesterol (TNP-Cap-liposomes), liposomes became readily lysed by guinea-pig serum (GPS) in Mg++-EGTA-GVB (gelatin veronal buffered saline containing 2 mM MgCl2 and 10 mM ethyleneglycol-bis (beta-amino-ethyl ether)N-N'-tetraacetate) as well as in GVB++ (gelatin veronal buffered saline containing 0.15 mM CaCl2 and 0.5 mM MgCl2). Since the classical complement pathway (CCP) does not work in Mg++-EGTA-GVB, TNP-Cap-liposome lysis by GPS in Mg++-EGTA-GVB was thought to be mediated by the activation of the alternative complement pathway (ACP). This conclusion was supported by observations that heating of GPS at 50 degrees impaired its lytic activity while C4-deficient GPS was capable of lytic activity, no lysis occurred in EDTA, and there was noted consumption of complement in GPS treated with TNP-Cap-liposomes at 30 degrees. For TNP-Cap-liposome lysis by GPS in Mg++-EGTA-GVB, the epitope density of the TNP hapten was required to be 5% or more of the DMPC. Changing the acyl group of the phosphatidylcholine (PC) significantly influenced the ACP activating capacity of TNP-Cap-liposome. Dipalmitoyl-PC, DMPC and distearoyl-PC facilitated the ACP activating capacity of the TNP-Cap-liposome, while dilauroyl-PC, egg-PC and dioleoyl-PC did not. Furthermore, the length of spacer between TNP and dipalmitoylphosphatidylethanolamine (DPPE) also influenced the ACP activating capacity and maximum activation was noted when the spacer was aminocaproyl. These physicochemical characteristics which increase the ACP activating capacity coincided with those reported to increase the immunogenicity of hapten-sensitized liposomes.

Animals↗
PubMed2002

The biological functions of MBL-associated serine proteases (MASPs).

The Mannose-binding lectin-associated serine proteases (MASPs) have been the subject of intensive research particularly over the past 10 years. First one, then two, and currently 3 MASPs have been characterized. Initially it was thought likely that the MBL + MASPs system would resemble very closely the C1 complex of the complement classical pathway, and that MASP1 and MASP2 would have similar activities to their classical pathway homologues C1r and C1s. MASP2 does certainly have similar activities to C1s, but MASP1 does not have the activities of either C1r or C1s. MASP1 has been thought to act on the complement system by cleaving C3 directly, but work with recombinant and purified native MASP1 shows that direct C3 cleavage by this protease is very slow, and may not be biologically significant. MASP1 and MASP2 appear not to have such a narrow specificity as C1r and C1s, and may have significant substrates other than complement proteins. As an example, MASP1 does cleave fibrinogen, releasing fibrinopeptide B (a chemotactic factor) and also cleaves and activates plasma transglutaminase (Factor XIII). These reactions are also relevant to defence against microorganisms, and may represent a biologically significant action of MASP1.

Animals↗
PubMed1989

Complement system protein C4 and susceptibility to hydralazine-induced systemic lupus erythematosus.

21 patients with systemic lupus erythematosus induced by long-term treatment with hydralazine were investigated to see whether susceptibility to this syndrome was associated with deficiency of the classical pathway complement protein, C4. 16 of 21 (76%) patients had one or more C4 null (ie, non-productive) alleles compared with 35 of 82 normal subjects (43%). This difference was significant. The HLA-DR4 antigen, known to be in linkage disequilibrium with the C4B null allele, was also significantly more frequent in the patients (14 of 21 patients compared with 31 of 81 normal subjects). Susceptibility to hydralazine-induced lupus, as in idiopathic systemic lupus erythematosus, may depend partly upon genetically determined C4 levels.

Alleles↗
PubMed1995

Complement-activating properties of IgM rheumatoid factors reacting with IgG subclasses.

To estimate the complement-activating property (CAP) of IgM rheumatoid factor (RF), which was purified from synovial fluids of patients with rheumatoid arthritis, in a reaction with each IgG subclass, the activation and binding of C4 in the classical pathway of complement by IgM RF was measured in an enzyme-linked immunosorbent assay using biotinylated F(ab')2 antibody to human C4. The CAP of IgM RF reacting with IgG3 was significantly higher than that of IgM RFs bound to the other IgG subclasses (P < 0.01). These results suggest that IgM RF reacting with IgG3 in synovial fluid could induce a greater degree of complement-dependent inflammation in RA synovium than IgM RF reacting with other IgG subclasses.

Arthritis, Rheumatoid↗