Synthesis of classical pathway complement components by chondrocytes.

Using immunohistochemical studies, C1q, C1s, C4 and C2 were detected in chondrocytes in normal human articular cartilage and macroscopically normal articular cartilage from the inferior surfaces of hip joints of patients with osteoarthritis. Using reverse-transcribed polymerase chain reaction (RT-PCR), mRNA for C1q, C1s, C4 and C2 was also detected in RNA extracted from articular cartilage. C1r, C3, C1-inhibitor, C4-binding protein and factor I were not detected by either technique. Articular chondrocytes cultured in vitro synthesized C1r, C1s, C4, C2, C3 and C1-inhibitor but not C1q, C4-binding protein or factor I, as assessed by enzyme-linked immunosorbent assay (ELISA) and Northern blot analysis. Thus cultured articular chondrocytes have a complement profile that is similar to that of cultured human fibroblasts rather than that of articular chondrocytes in vivo. Complement synthesis in cultured chondrocytes was modulated by the cytokines interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), showing that cytokines can probably regulate complement synthesis in intact cartilage. The possible roles of local synthesis of complement components by chondrocytes in matrix turnover and the regulation chondrocyte function are discussed.     

Prevention of immune precipitation by purified classical pathway complement components

The role of the classical pathway of complement in the prevention of immune precipitation has been investigated using purified complement components and immune complexes (IC) consisting of rabbit anti-BSA and BSA. C1 reduced the rate of immune precipitation. As C1q, EDTA treated C1 or C1-inhibitor treated C1 were unable to retard the precipitation of IC, it was concluded that the intact C1 molecule was required for this function. Use of phenylmethylsulphonyl fluoride and benzamidine showed that the enzymatic site on C1 was not required for this activity. C4 and C2 did not affect immune precipitation significantly when C1 was present at the concentrations present in serum. When C3 was added to C1, C4 and C2 precipitation of IC did not occur. These data demonstrate that classical pathway activation alone is sufficient for the prevention of immune precipitation.     

Alzheimer's beta-amyloid peptides can activate the early components of complement classical pathway in a C1q-independent manner

beta-Amyloid (beta-A) accumulates in the brain of patients with Alzheimer's disease (AD) and is presumably involved in the pathogenesis of this disease, on account of its neurotoxicity and complement-activating ability. Although assembly of beta-A in particular aggregates seems to be crucial, soluble non-fibrillar beta-A may also be involved. Non-fibrillar beta-A does not bind C1q, so we investigated alternative mechanisms of beta-A-dependent complement activation in vitro. On incubation with normal human plasma, non-fibrillar beta-A 1-42, and truncated peptide 1-28, induced dose-dependent activation of C1s and C4, sparing C3, as assessed by densitometric analysis of immunostained membrane after SDS-PAGE and Western blotting. The mechanism of C4 activation was not dependent on C1q, because non-fibrillar beta-A can still activate C1s and C4 in plasma genetically deficient in C1q (C1qd). In Factor XII-deficient plasma (F.XIId) the amount of cleaved C4 was about 5-10% less that in C1qd and in normal EDTA plasma; the reconstitution of F.XIId plasma with physiologic concentrations of F.XII resulted in an increased (8-15%) beta-A-dependent cleavage of C4. Thus our results indicate that the C1q-independent activation of C1 and C4 can be partially mediated by the activation products of contact system. Since the activation of contact system and of C4 leads to generation of several humoral inflammatory peptides, non-fibrillar beta-A might play a role in initiating the early inflammatory reactions leading to a multistep cascade contributing to neuronal and clinical dysfunction of AD brain.     

The activation of the C3b feedback cycle with human complement components. I. Through the classical pathway

Reaction between the fourth, the oxidized second and the activated first components of human complement generated the stable enzyme C4oxy2 capable of cleaving the third component and depleting total complement in human serum. This enzyme was shown further to activate the C3b feedback cycle as shown by its ability to consume factor B in serum and the reduction in the extent of complement consumption in the presence of EDTA. OxyC2 on its own gave rise to C3 cleavage in normal human serum by a pathway needing classical pathway components. This unexpected finding suggests that there may be a 'C-1 tickover' in serum analogous to the 'C3b tickover'; the presence of oxyC2 allowing the 'capture' of the trivial amounts of C42 normally formed. In preliminary experiments in the rat, C4oxy2 was successfully formed in vivo, where it gave rise to cleavage of C3, consumption of C5, depletion of cobra venom factor cofactors and a biphasic change in the neutrophil count.     

Haemolytic assays in agarose plates for components of the classical complement pathway: interference by the alternative pathway.

It has been observed that when serum C6 is measured by the haemolytic radial diffusion technique a heat labile factor limits the size of the haemolytic rings. This reduction is haemolysis has been shown to be due to alternative pathway activation of C6 in the agarose plate; and that the heat labile factor is Factor B of the alternative pathway. This phenomenon is of practical importance when assaying for C6; however, it does not explain the observations of a C6 inactivator reported by Nelson & Biro (1968).     

Effect of concanavalin A on the hemolytic activity of the components of the classical complement pathway

The lectin concanavalin A (Con A) immobilized on Sepharose 4B was found to bind all the components of the guinea pig and human classical complement pathway except C6, C7 and C9. Fluid-phase Con A inhibited the hemolytic activity of guinea pig and human C2, C3, C5 and C8 and human C2 oxidized with iodine (C2^ox); however, with the exception of C3, the lectin did not inhibit the hemolytic activity of cell-bound complement components. This suggests that the primary effect of Con A was to hinder the binding of components to the cell surface and/or their activation. The degree of inhibition for a given quantity of a component was dependent on the concentration of Con A. The lectin had no effect on the extent or rate of decay of cell-bound C5 or unoxidized C2. These results suggest that several of the components of the human and guinea pig classical complement pathway contain exposed sugar moieties and that the interaction between these sugars and Con A can impair their functional activity.The lectin concanavalin A (Con A) has a high binding affinity for glucose and structurally-related sugars (Goldstein et al., 1965) and has been used extensively for the purification and identification of glycoproteins containing these sugars (Rapin & Burger, 1974; Nicholson, 1974). We have previously demonstrated that Con A can inhibit the hemolytic activity of fluid-phase guinea pig Cl and C2, but not C4 (Langone et al., 1977), suggesting that exposed sugar moieties may play a role in the functional activity of these components. In this study we have extended this approach to study the interaction between Con A and the later-acting guinea pig and human complement components.     

The classical pathway of complement prevents the formation of insoluble antigen-antibody complexes: biological implications

There is now clear evidence for a complement dependent physiological system which is capable of processing immune complexes in and outside the vascular compartment so that they remain soluble, and transporting such complexes to the fixed macrophage system where they are safely eliminated. Defects in physiological immune complex disposal can occur at various stages described in this article, and it could well be that several of these stages could present subtle defects which are, however, additive so that under a given set of circumstances immune complexes end up in the wrong places, i.e. outside the fixed macrophage system.     

补体经典激活途径C3转化酶的体外组装及活性观察

目的：体外组装包含人C4分子的补体经典激活途径C3转化酶，并对其转化酶活性及衰变特性进行观察。方法：利用豚鼠血清功能纯C1、C2及溶血中间体EAC4^hu体外组装经典途径C3转化酶，观察不同C1、C2用量及孵育温度对C3转化酶形成和自发性衰变的影响，以及人红细胞膜抽提蛋白对C3转化酶衰变化的影响。结果：高剂量和低剂量的C1均会影响C3转化酶的形成，增加C2用量可增加C3转化酶的形成数量，C3转化酶的自发性衰变随孵育温度的升高而加速，人红细胞膜抽提蛋白可抑制C3转化酶的自发性衰变过程，结论：C1、C2用量及孵育温度是影响C3转化酶形成和自发性衰变的主要因素，体外组装的补体经典激活途径C3转化酶可应用于相关补体调控蛋白的活性检测。     

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.     

On the site of C4 deposition upon complement activation via the mannan-binding lectin pathway or the classical pathway

The mannan-binding lectin (MBL) pathway and the classical pathway of complement activation are initiated by the binding of the recognition structure of the initiator complexes, MBL and C1q, respectively, to their ligands, i.e. carbohydrate structures or immune complexes. Proenzymes associated with MBL or C1q are then activated and generate C3 convertase through the activation of C4 and C2. The cleavage product of C4, C4b, attaches covalently to nearby hydroxyl or amino groups. The current picture is that C2 must then attach to C4b before being cleaved by the same associated proteases into the enzymatically active fragment, C2b. This suggests a stringent requirement for the deposition of C4b very close to the initiator complex, or indeed onto the initiator complex. We examined the possibility of C4b being bound to the initiator complex by a solid-phase assay, allowing for the selective elution of the initiator complexes, followed by quantification of the C4b being eluted and the C4b remaining on the solid phase. Also, we estimated the generation of complexes between the released initiator complex and C4b. More than 99% of deposited C4b was bound directly to the solid phase rather than to the initiator complex. Our approach cannot answer the question of the whereabouts of the C2 when it is cleaved.     

BrkA protein of Bordetella pertussis inhibits the classical pathway of complement after C1 deposition

Bordetella pertussis produces a 73-kDa protein, BrkA (Bordetella resistance to killing), which inhibits the bactericidal activity of complement. In this study we characterized the step in the complement cascade where BrkA acts, using three strains: a wild-type strain, a strain containing an insertional disruption of brkA, and a strain containing two copies of the brkA locus. Following incubation with 10% human serum, killing was greatest for the BrkA mutant, followed by that for the wild-type strain, while the strain with two copies of brkA was the most resistant. Complement activation was monitored by enzyme-linked immunosorbent assay (ELISA) or Western blotting. ELISAs for SC5b-9, the soluble membrane attack complex, showed that production of SC5b-9 was greatest with the brkA mutant, less with the wild type, and least with the strain containing two copies of brkA. Deposition of complement proteins on the bacteria was monitored by Western blotting. A decrease in deposition on the bacteria of C4, C3, and C9 corresponded with decreased complement sensitivity. Deposition of C1, however, was not affected by the presence of BrkA. These studies show that BrkA inhibits the classical pathway of complement activation and prevents accumulation of deposited C4.     

Inhibition effects of isolated compounds from Artemisia rubripes Nakai of the classical pathway on the complement system

The study evaluated the anticomplement activity from isolated compounds from Artemisia rubripes Nakai from South Korea on the classical pathway. In the previous works, Artemisia rubripes chloroform extracts showed inhibitory activity against complement system. The chromatographic separation of a chloroform chloride extract of Artemisia rubripes led to the isolation of three compounds. Their structures were characterized to be scopoletin (1), 11,(13)-triene-6,12-olide (2), and 1β,6α-dihydroxy-4(15)-eudesmene (3) by spectroscopic data. This is the first report of anticomplement activity of isolated compounds from Artemisia rubripes.     

Interaction between components of the human classical complement pathway and immobilized Cibacron Blue F3GA.

The interaction between the complement components in human serum and the dye, Cibacron Blue F3GA, immobilized on cross-linked agarose (Affi-Gel Blue) has been studied. All nine components of the classical complement pathway bound to the dye and could be recovered using a linear salt gradient. With the exception of C5 and C8, all the components were eluted over a narrow NaCl concentration range, with the following yields: C1, 17%; C2, 69%; C3, 92%; C4, 87%; C6, 105%; C7, 109%; C9, 128%. C5 and C8 eluted throughout the NaCl gradient with yields of 103% and 14%, respectively. Since all components could be eluted without substantial contamination by albumin or IgG, this procedure may prove valuable as an initial step in the purification of complement components. In addition, the ability of immobilized Cibacron Blue F3GA to physicallly remove complement components may prove useful for both the decomplementation of serum and in elucidating the role of complement in immunological reactions.     

Legionella pneumophila lipopolysaccharide activates the classical complement pathway.

Legionella pneumophila is a gram-negative bacterium capable of entering and growing in alveolar macrophages and monocytes. Complement and complement receptors are important in the uptake of L. pneumophila by human mononuclear phagocytes. The surface molecules of L. pneumophila that activate the complement system are unknown. To identify these factors, we investigated the effects of L. pneumophila lipopolysaccharide (LPS) on the classical and alternative complement pathways of normal human serum by functional hemolytic assays. Although incubation of LPS in normal human serum at 37 degrees C resulted in the activation of both pathways, complement activation proceeded primarily through the classical pathway. Activation of the classical pathway by LPS was dependent on natural antibodies of the immunoglobulin M class that were present in various quantities in sera from different normal individuals but were absent in an immunoglobulin-deficient serum obtained from an agammaglobulinemic patient. Additional studies using sheep erythrocytes coated with LPS suggested that the antibodies recognized antigenic sites in the carbohydrate portion of LPS. The ability of LPS to interact with the complement system suggests a role for LPS in the uptake of L. pneumophila by mononuclear phagocytes.     

Carbamylation of immunoglobulin abrogates activation of the classical complement pathway

Post‐translational modifications of proteins significantly affect their structure and function. The carbamylation of positively charged lysine residues to form neutral homoitrulline occurs primarily under inflammatory conditions through myeloperoxidase‐dependent cyanate (CNO^?) formation. We analyzed the pattern of human IgG₁ carbamylation under inflammatory conditions and the effects that this modification has on the ability of antibodies to trigger complement activation via the classical pathway. We found that the lysine residues of IgG₁ are rapidly modified after brief exposure to CNO^?. Interestingly, modifications were not random, but instead limited to only few lysines within the hinge area and the N‐terminal fragment of the CH2 domain. A complement activation assay combined with mass spectrometry analysis revealed a highly significant inverse correlation between carbamylation of several key lysine residues within the hinge region and N‐terminus of the CH2 domain and the proper binding of C1q to human IgG₁ followed by subsequent complement activation. This severely hindered complement‐dependent cytotoxicity of therapeutic IgG₁. The reaction can apparently occur in vivo, as we found carbamylated antibodies in synovial fluid from rheumatoid arthritis patients. Taken together, our data suggest that carbamylation has a profound impact on the complement‐activating ability of IgG₁ and reveals a pivotal role for previously uncharacterized lysine residues in this process.     

Anticomplementary activity of boswellic acids--an inhibitor of C3-convertase of the classical complement pathway.

Boswellic acids (BA), an anti-inflammatory and anti-arthritic principle/s of Boswellia serrata, were found to possess anticomplementary activity. It inhibits the in vitro immunohaemolysis of antibody-coated sheep erythrocytes by pooled guinea-pig serum. The reduced immunohaemolysis was found to be due to inhibition of C3-convertase of the classical complement pathway. The threshold concentration for inhibiting C3-convertase was found to be 100 micrograms. However, higher concentrations of BA showed constant inhibitory effects on immunohaemolysis. BA also exhibited weak inhibitory effects on individual components of the complement system. In vivo administration of BA also showed the inhibitory effect on guinea-pig serum.     

Staphylococcus aureus VraX specifically inhibits the classical pathway of complement by binding to C1q

VraX is a protein secreted by Staphylococcus aureus, an important human pathogen. A dramatic over expression of VraX is observed when S. aureus is exposed to several antimicrobial agents; however, its function remains unclear. Here, we aimed to reveal the function of this protein and the mechanism by which it affects the immune system to enhance the pathogenesis of the bacterium. Our results showed that VraX specifically inhibited the classical pathway of the complement system. In particular, VraX could bind to the C1q protein and block the formation of the C1 complex. Deletion of VraX decreased the pathogenesis of S. aureus. Our findings indicate that over expression of VraX might be a protective response for S. aureus survival.     

Control of the classical and the MBL pathway of complement activation

The activation of complement via the mannan-binding lectin (MBL) pathway is initiated by the MBL complex consisting of the carbohydrate binding molecule, MBL, two associated serine proteases, MASP-1 and MASP-2, and a third protein, MAp19. In the present report we used an assay of complement activation specifically reflecting the physiological activity of the MBL complex to identify biological and synthetic inhibitors. Inhibitor activity towards the MBL complex was compared to the inhibition of the classical pathway C1 complex and to a complex of MBL and recombinant MASP-2. A number of synthetic inhibitors were found to differ in their activities towards complement activation via the MBL pathway and the classical pathway. C1 inhibitor inhibited both pathways whereas alpha2-macroglobulin (alpha2M) inhibited neither. C1 inhibitor and alpha2M were found to be associated with the MBL complex. Upon incubation at 37 degrees C in physiological buffer, the associated inhibitors as well as MASP-1, MASP-2, and MAp19 dissociated from MBL, whereas only little dissociation of the complex occurred in buffer with high ionic strength (1 M NaCl). The difference in sensitivity to various inhibitors and the influence of high ionic strength on the complexes indicate that the activation and control of the MBL pathway differ from that of the classical pathway. MBL deficiency is linked to various clinical manifestations such as recurrent infections, severe diarrhoea, and recurrent miscarriage. On the other hand, impaired control of complement activation may lead to severe and often chronically disabling diseases. The results in the present report suggests the possibility of specifically inhibiting of the MBL pathway of complement activation.