Artesunate: A natural product-based immunomodulator involved in human complement.

Complement is an important innate immune defence machinery. Once dysregulated, it is often linked to pathogenesis of diverse autoimmune diseases. Artesunate (ART) is a well-known anti-malarial compound. Recently, ART has been highlighted by its potential therapeutic effects on certain complement-related autoimmune diseases. However, the underlying mechanisms are hitherto unknown. In the present study, we found that ART mediated complement interception as validated by analysis of complement haemolytic assay. In cell-based setup using dying Jurkat cells, ART-mediated complement interception was also confirmed. Further, we newly established an ELISA system selectively allowing complement activation via the classical pathway, the lectin pathway and the alternative pathway, respectively. ELISA analysis revealed that ART dose-dependently inhibited C4 activation, C3 activation and terminal complement complex assembly via the effector pathways. ART was found to blockade C1q, C3 and C5 with a lesser extent to properdin. The interaction of ART with C1q was determined to be mediated via C1q globular head region. FACS analysis using ART-conjugated mesoporous silica particles revealed that ART specifically bound the key therapeutic targets of C1q, C3 and C5 on microparticles. In conclusion, we for the first time report the anti-complement bioactivities of ART and suggest a potential therapeutic benefit of ART in the complement-related human diseases.

The Streptococcus agalactiae complement interfering protein combines multiple complement-inhibitory mechanisms by interacting with both C4 and C3 ligands.

Group B Streptococcus (GBS) colonizes the human lower intestinal and genital tracts and constitutes a major threat to neonates from pregnant carrier mothers and to adults with underlying morbidity. The pathogen expresses cell-surface virulence factors that enable cell adhesion and penetration and that counteract innate and adaptive immune responses. Among these, the complement interfering protein (CIP) was recently described for its capacity to interact with the human C4b ligand and to interfere with the classical- and lectin-complement pathways. In the present study, we provide evidence that CIP can also interact with C3, C3b, and C3d. Immunoassay-based competition experiments showed that binding of CIP to C3d interferes with the interaction between C3d and the complement receptor 2/cluster of differentiation 21 (CR2/CD21) receptor on B cells. By B-cell intracellular signaling assays, CIP was confirmed to down-regulate CR2/CD21-dependent B-cell activation. The CIP domain involved in C3d binding was mapped via hydrogen deuterium exchange-mass spectrometry. The data obtained reveal a new role for this GBS polypeptide at the interface between the innate and adaptive immune responses, adding a new member to the growing list of virulence factors secreted by gram-positive pathogens that incorporate multiple immunomodulatory functions.-Giussani, S., Pietrocola, G., Donnarumma, D., Norais, N., Speziale, P., Fabbrini, M., Margarit, I. The Streptococcus agalactiae complement interfering protein combines multiple complement-inhibitory mechanisms by interacting with both C4 and C3 ligands.

SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis.

Blood-feeding insects inject potent salivary components including complement inhibitors into their host's skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases.

Hereditary angioedema: a survey of UK emergency departments and recommendations for management.

Patients with hereditary angioedema may present to the emergency department (ED) with subcutaneous and submucosal swellings, the most important being the development of laryngeal oedema, which can rapidly obstruct the airways and cause death. The aim of this study was to establish whether local guidelines exist for the management of such patients and to determine where the C1 inhibitor concentrate (C1 INHC) was kept in the trusts. A questionnaire survey of the availability and use of C1 INHC was sent to 35 EDs across the UK with established immunology services within their trusts. A hundred percent response was received. Thirty-three trusts had a supply of the drug C1 INHC in varying quantities. Nineteen trusts had it in their ED. Only 17 departments had any guidance with regard to their use. There is a significant lack of guidance for C1 INHC use in the EDs surveyed. A guideline was developed as a result that can be used by EDs across Europe.

Transcriptional repression of C4 complement by hepatitis C virus proteins.

The fourth component of human complement (C4) plays an important role in innate immune function. C4 activity has been observed to be significantly lower in patients with chronic hepatitis C virus (HCV) infections, although the mechanism remains unknown. In this study, we have examined the mechanisms of C4 regulation by HCV. Liver biopsy specimens from patients with chronic HCV infections displayed significantly lower C4 mRNA levels than liver tissue samples from patients with unrelated liver disease. Further, C4 mRNA levels of the two isoforms (C4A and C4B) were significantly reduced in hepatocytes transfected with RNA from HCV genotype 1a or 2a. Subsequently, a significant C4 regulatory role of HCV core or NS5A upon C4 promoter activity was observed. HCV core or NS5A transgenic mice displayed a reduction in C4 mRNA. Gamma interferon (IFN-γ)-induced C4 promoter activation was also impaired in the presence of HCV proteins. We further demonstrated that HCV core reduced the expression of upstream stimulating factor 1 (USF-1), a transcription factor important for basal C4 expression. On the other hand, the expression of interferon regulatory factor 1 (IRF-1), which is important for IFN-γ-induced C4 expression, was inhibited by hepatocytes expressing HCV NS5A. These results underscore the roles of HCV proteins in innate immune regulation in establishing a chronic infection.

Antagonism of the complement component C4 by flavivirus nonstructural protein NS1.

The complement system plays an essential protective role in the initial defense against many microorganisms. Flavivirus NS1 is a secreted nonstructural glycoprotein that accumulates in blood, is displayed on the surface of infected cells, and has been hypothesized to have immune evasion functions. Herein, we demonstrate that dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV) NS1 attenuate classical and lectin pathway activation by directly interacting with C4. Binding of NS1 to C4 reduced C4b deposition and C3 convertase (C4b2a) activity. Although NS1 bound C4b, it lacked intrinsic cofactor activity to degrade C4b, and did not block C3 convertase formation or accelerate decay of the C3 and C5 convertases. Instead, NS1 enhanced C4 cleavage by recruiting and activating the complement-specific protease C1s. By binding C1s and C4 in a complex, NS1 promotes efficient degradation of C4 to C4b. Through this mechanism, NS1 protects DENV from complement-dependent neutralization in solution. These studies define a novel immune evasion mechanism for restricting complement control of microbial infection.

Complement activation by PEGylated single-walled carbon nanotubes is independent of C1q and alternative pathway turnover.

We have investigated the interaction between long circulating poly(ethylene glycol)-stabilized single-walled carbon nanotubes (SWNTs) and the complement system. Aminopoly(ethylene glycol)(5000)-distearoylphosphatidylethanolamine (aminoPEG(5000)-DSPE) and methoxyPEG(5000)-DSPE coated as-grown HIPco SWNTs activated complement in undiluted normal human serum as reflected in significant rises in C4d and SC5b-9 levels, but not the alternative pathway split-product Bb, thus indicating activation exclusively through C4 cleavage. Studies in C2-depleted serum confirmed that PEGylated nanotube-mediated elevation of SC5b-9 was C4b2a convertase-dependent. With the aid of monoclonal antibodies against C1s and human serum depleted from C1q, nanotube-mediated complement activation in C1q-depleted serum was also shown to be independent of classical pathway. Nanotube-mediated C4d elevation in C1q-depleted serum, however, was inhibited by N-acetylglucosamine, Futhan (a broad-spectrum serine protease inhibitor capable of preventing complement activation through all three pathways) and anti-MASP-2 antibodies; this strongly suggests a role for activation of MASP-2 in subsequent C4 cleavage and assembly of C4b2a covertases. Intravenous injection of PEGylated nanotubes in some rats was associated with a significant rise in plasma thromboxane B2 levels, indicative of in vivo nanotube-mediated complement activation. The clinical implications of these observations are discussed.

Biphasic effect of gingipains from Porphyromonas gingivalis on the human complement system.

Periodontitis is an inflammatory disease of the supporting structures of the teeth and is caused by, among other agents, Porphyromonas gingivalis. P. gingivalis is very resistant to killing by human complement, which is present in a gingival fluid at 70% of the serum concentration. We found that the incubation of human serum with purified cysteine proteases of P. gingivalis (gingipains) or P. gingivalis wild-type strains W83 and W50 resulted in a drastic decrease of the bactericidal activity of the serum. In contrast, serum treated with P. gingivalis mutants lacking gingipains (particularly strains without HRgpA) maintained significant bactericidal activity. To understand in detail the mechanism by which gingipains destroy the serum bactericidal activity, we investigated the effects of gingipains on the human complement system. We found that all three proteases degraded multiple complement components, with arginine-specific gingipains (HRgpA and RgpB) being more efficient than lysine-specific gingipain (Kgp). Interestingly, all three proteases at certain concentrations were able to activate the C1 complex in serum, which resulted in the deposition of C1q on inert surfaces and on bacteria themselves. It is therefore plausible that P. gingivalis activates complement when present at low numbers, resulting in a local inflammatory reaction and providing the bacteria with a colonization opportunity and nutrients. At later stages of infection the concentration of proteases is high enough to destroy complement factors and thus render the bacteria resistant to the bactericidal activity of complement.

Inhibition of complement components C3 and C4 by cadralazine and its active metabolite.

The effect of cadralazine and its active metabolite CGP 22639 on the covalent binding reaction of C4 and C3 has been studied. Trypsin-Sepharose was used to activate radio-labelled C3 and C4 and binding of the radio-labelled protein to the trypsin-Sepharose was measured. Cadralazine inhibited 50% of the binding of C3 and C4 at concentrations of 19 mmol/l and 15 mmol/l, respectively. Its active metabolite was more potent and inhibited 50% of the C3 and C4 binding at concentrations of 8 and 3.5 mmol/l, respectively. These concentrations are much higher than those found in plasma during therapy. This is consistent with the clinical observation that in patients with normal kidney function cadralazine is not an inducer of SLE.

Hereditary angioedema: therapeutic effect of danazol on C4 and C1 esterase inhibitors.

Hereditary angioedema (HAE) is an inherited deficiency of C1 esterase inhibitor (C1 inh). The two types of genetic C1 inh deficiency are type I, which is quantitative, and type II, which is functional. For the purpose of the present study, four HAE patients were selected. None of them had received any androgenic therapy. The group included three type I and one type II cases. All patients that entered the protocol received danazol, 400 mg/day for 14 days. The complement system was evaluated by monitoring C4, hemolytic complement 50% (CH50), circulating immune complexes (CIC), and antigenic and functional C1 inh during the study. The level of complement factors at the beginning and at the end of this period demonstrated a statistically significant increase in C4 and CH50 and the disappearance of CIC, while C1 inh remained unmodified. These results suggest that the therapeutic effect of danazol may have two mechanisms of action: (1) promotion of C4 synthesis by anabolic effect resulting in an improvement of the complement system with the disappearance of CIC and (2) a minor increase in C1 inh level primarily due to the lack of its consumption.

Inhibition of the covalent binding reaction of complement component C4 by penicillamine, an anti-rheumatic agent.

D(-)-Penicillamine [D(-)-beta beta-dimethylcysteine] is an anti-arthritic drug, but its use is limited by adverse side effects, which include problems in immune-complex clearance. Complement is important as a source of inflammatory mediators in rheumatoid arthritis and is also involved in immune-complex clearance. Thus inhibition of the complement cascade would be likely to contribute to both the therapeutic and the toxic effects of penicillamine. It is shown that penicillamine and cysteine are potent inhibitors of the covalent binding of activated complement component C4 to immune complexes. [35S]Cysteine itself becomes covalently bound to C4b through the thioester site. Penicillamine and cysteine are more reactive with the C4A isotype than with the C4B isotype of the HLA class III protein C4. The limited amino acid sequence differences between C4A and C4B include a cysteine/serine interchange, and it is suggested that the cysteine residue in C4A contributes to the increased rate of reaction of C4A with the alpha-amino-beta-thiol compounds.

[A factor from the venom of the Central Asiatic cobra Naja naja oxiana, which inactivates component C4 of human complement]. / Faktor iada sredneaziatskoi kobry Naja naja oxiana inaktiviruiushchii komponent C4 komplementa cheloveka.

An acid glycoprotein (mol. m. 60 kDa) containing 6 sialic acid residues and N-terminal Thr was isolated from the venom of the central asian cobra Naja naja oxiana. The protein has an anticomplementary activity selectively inactivating of the C4 component of the human complement. This factor (CFA-Ib) binds C4 with Ki = 0.27 +/- 0.13 microM and then irreversible inactivates it with a rate constant k = 0.75 +/- 0.25 min-1. Membrane bound C4b restores its ability of CFA-Ib binding. This binding hinders component C2 sorption on C4b and C3 convertase formation.

Metabolites of procainamide and practolol inhibit complement components C3 and C4.

Drug-induced systemic lupus erythematosus arises from toxic side-effects of administration of hydralazine, isoniazid, procainamide and practolol. Hydralazine and isoniazid are nucleophilic drugs and inhibit the covalent binding reaction of complement components, C3 and C4, an effect likely to lead to deposition of immune complexes (a feature of systemic lupus erythematosus). Procainamide and practolol do not themselves inhibit C3 and C4. A range of metabolites and putative metabolites of procainamide and practolol were synthesized, and tested for their ability to inhibit the covalent binding reactions of C3 and C4. The highly nucleophilic hydroxylamine metabolite of procainamide was strongly inhibitory in both tests, as was a putative hydroxylamine metabolite of practolol. These studies indicate a potential role for the hydroxylamine metabolites in mediating the toxic side-effects of procainamide and practolol, and emphasize the need for adequate measurements of hydroxylamine metabolites in human tissue.

Hydralazine binds covalently to complement component C4. Different reactivity of C4A and C4B gene products.

Long-term treatment with hydralazine is sometimes associated with deposition of immune complexes and development of systemic lupus erythematosus (SLE) as an adverse side-effect. Hydralazine inhibits the covalent binding reaction of the complement protein C4. We show that when hydralazine inhibits C4, it becomes covalently bound to the polypeptide chain containing the active site thiol ester. C4 is encoded at 2 adjacent polymorphic loci, C4A and C4B, within the major histocompatibility complex. We show that hydralazine binds more efficiently to the C4A than to the C4B gene product and suggest that C4 type may predispose patients to hydralazine-induced SLE.

Studies on polysaccharides from Angelica acutiloba--IV. Characterization of an anti-complementary arabinogalactan from the roots of Angelica acutiloba Kitagawa.

An anti-complementary polysaccharide, AR-arabinogalactan, was isolated from the roots of Angelica acutiloba Kitagawa (Japanese name = Yamato-Tohki), and purified by chromatography on DEAE-Sephadex A-50, Sephadex G-100, Sepharose CL-2B and concanavalin A-Sepharose. AR-arabinogalactan was composed of arabinose and galactose in a molar ratio of 1.2:1.0 and a small amount of galacturonic acid. The results of methylation and exo-alpha-L-arabinofuranosidase treatment showed that the polysaccharide was a branched arabinogalactan containing a backbone involving galactopyranosyl (1----6) linkages. Most of the arabinose was present as an alpha-L-furanosyl residue in the non-reducing terminals and side chains. The (1----3)-linked galactopyranosyl residue might be linked to the arabinosyl side chains. alpha-L-arabinofuranosidase digestion did not destroy the anti-complementary activity of the polysaccharide. After incubation of the serum with AR-arabinogalactan in the absence of Ca2+ ions, a cleavage of C3 in the serum was detected by immunoelectrophoresis as well as from the consumption of complement when rabbit erythrocytes were used in the assay system. A marked consumption of C4 was also observed to have occurred when serum was incubated with AR-arabinogalactan in the presence of Ca2+ ions. Collectively considered these results indicate that the mode of complement activation by AR-arabinogalactan is via both the alternative and the classical pathway.

Drugs that induce systemic lupus erythematosus inhibit complement component C4.

The syndrome resembling systemic lupus erythematosus (SLE) associated with long-term treatment with hydralazine and isoniazid seems to be due to the drugs themselves rather than their metabolites. This syndrome is associated with deposition of immune complexes; and in the complement system inhibition of C4 is likely to increase deposition of immune complexes. In vitro, hydralazine and isoniazid inhibited 50% of the binding of C4 at 840 mumol/l and 1.05 mmol/l, respectively--ie, within the concentration ranges that have been used in therapy. Acetylated metabolites were not inhibitory at the maximum concentrations tested; and iproniazid, which does not cause SLE, gave 50% inhibition only at a concentration far exceeding that used in therapy.

A comparison of the properties of two classes, C4A and C4B, of the human complement component C4.

A remarkable difference has been observed between the reactivity of the two forms of human complement component C4. C4B binds twice as effectively as C4A to antibody-coated red cells, but the reverse occurs with protein-antigen complexes. C4B reacts much more effectively with hydroxyl groups than C4A and this is reversed for reaction with amino groups in spite of the very small difference in amino acid sequence between the two forms of C4. No other differences in stability, activation or inactivation were observed. These findings emphasise the biological advantage of the duplication of the C4 gene in its reaction with a wide range of antigenic structures. The correlation of the presence of different forms of C4 with susceptibility to autoimmune diseases may be explicable by these big differences in binding reactivity.

Interactions of complement with an extract of airborne spring wheat dust.

The inhalation, by grain elevator workers, of airborne grain dusts can lead to pulmonary problems. Complement, which is present in human airways, can interact with various grain dusts, producing activation products that have been shown to participate in the inflammatory reaction. Because of this apparent connection between grain-dust inhalation, complement activation, and respiratory difficulties, we are studying the reaction of an aqueous extract of spring wheat dust (swd) with human complement. The swd extract activates both the classical and alternative pathways; it acts on purified C2 to inhibit it, and it reacts with undiluted serum to consume C4 with kinetics significantly different from those shown by a "typical" antigen-antibody complex (sensitized sheep erythrocytes). Enzyme susceptibility experiments suggest that the alternative and classical pathway activators of swd extract are neither protein nor nucleic acid; periodate oxidation indicates these substances are carbohydrate, and gel filtration suggests they are macromolecular. Enzyme vulnerability also indicates that the C2 inhibitor of swd extract is ribonucleic acid. Although endotoxin is present in swd extract, a gel-filtration experiment showed that a major fraction of the complement reactivity was not associated with this substance.

Complement receptor is an inhibitor of the complement cascade.

A glycoprotein from the membrane of human erythrocytes has been identified as a receptor for C3b (CR1). It promotes the dissociation of the alternative pathway C3 convertase C3b,Bb and the cleavage of C3b by C3b/C4b inactivator. We find that CR1 also inactivates the C3 and C5 convertases of the classical pathway. CR1 inhibits the consumption of C3 by C3 convertase EAC142 and enhances the decay of C4b,2a sites. On a weight basis, CR1 is approximately 5-10 times more active than C4 binding protein, a serum inhibitor of C4b,2a. The binding of 125I-CR1 to EAC14 cells is inhibited by C2. Therefore, it is likely that CR1 and C2 compete for a site on C4b. CR1 inhibited C5 convertase even more effectively, but had no effect on the assembly of the late complement components. At high concentrations, CR1 alone has no irreversible effects on cell-bound C4b. In the fluid phase, CR1 can function as a cofactor for the cleavage of the alpha' chain of C4b by C3b/C4b inactivator. A well-known function of CR1 is to promote adherence of microbes or immune complexes bearing C3b and C4b to cells. This interaction could result in a microenvironment damaging to the plasma membrane of the responding cell because the extrinsic C3b and C4b fragments can serve as additional sites of assembly of enzymes of the cascade. We therefore wish to propose that CR1 on the surface of cells supplies an increased local concentration of a strong inhibitor of the amplifying enzymes of the complement system and provides cells with a mechanism for circumventing damage when they bind C3b- and C4b-bearing substrates.