Classical Pathway Of Complement System Research Articles

Structural peculiarities of carrageenans from Far Eastern red seaweed Mazzaella parksii (Gigartinaceae)

The sulfated polysaccharides from cystocarpic plants of Mazzaella parksii were studied. Fractionation at a given KCl concentration allowed us to assume, and stepwise fractionation to prove, that these polysaccharides consisted of several carrageenans that differed in structure and molecular weight. As a result of stepwise fractionation with KCl, nine gelling (1–9) and one non-gelling (10) fractions were obtained. Using IR spectroscopy, it was shown that fractions 3, 4 and 5 were kappa/iota-, kappa- and kappa/beta-carrageenans, respectively. The structures of the main fractions 1, 2, 9 and 10 were investigated in more detail by methylation, NMR spectroscopy and mass spectrometry. Fractions 1 and 2 were hybrid kappa/iota-carrageenans with kappa:iota ratio 79:21 and 63:37, respectively. At the same time, fraction 9 contained kappa-, iota- and small amounts of nu-carrageenans. The fraction 10 had complex structure and was built from kappa-, iota-, beta-, mu- and nu-carrageenans and included agar-like structure, which explained the inability of this fraction to gel at 15 % KCl. It was shown that isolated polysaccharides activated the classical pathway of complement system, increasing the concentration of C1 inhibitor of serine protease by 50 % compared with the negative control.

Chemical characterization and complement modulating activities of an arabinogalactan-protein-rich fraction from an aqueous extract of avocado leaves

The aim of this study was to chemically characterize an arabinogalactan-protein-rich fraction (FRAGP) obtained from an aqueous extract of avocado leaves and investigate its effects on the classical pathway of the complement system. The FRAGP contained 4.6% ± 1.8%, 22.5% ± 4.9%, and 76.7% ± 8.8% of total protein, arabinogalactan-protein, and carbohydrates, respectively. Arabinose and galactose were the main monosaccharide constituents. FT-IR and NMR data, together with linkage analyses, indicated the presence of a structure that included a (1 → 3)-linked β-D-Galp main chain, mainly substituted at O-6 by Gal and Ara residues, which was characteristic of a type II arabinogalactan. The effect of FRAGP on the classical pathway of complement system was examined by a hemolytic fixation test and comparing with heparin, which was used as a control for inhibition. With pre-incubation, the IC50 of FRAGP was 1.90 ± 1.1 μg/mL, which was similar to that of heparin (IC50 = 2.90 ± 0.3 μg/mL). Without pre-incubation, the IC50 values were 18.6 ± 3.7 and 8.0 ± 4.1 μg/mL for FRAGP and heparin, respectively. Collectively, these results suggested that FRAGP has an inhibitory effect on the classical pathway of the complement system.

High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation.

High-mobility group box 1 (HMGB1), a well-known danger-associated molecular pattern molecule, acts as a pro-inflammatory molecule when secreted by activated immune cells or released after necrotic cell damage. HMGB1 binds to immunogenic bacterial components and augments septic inflammation. In this study, we show how HMGB1 mediates complement activation, promoting sterile inflammation. We show that HMGB1 activates the classical pathway of complement system in an antibody-independent manner after binding to C1q. The C3a complement activation product in human plasma and C5b-9 membrane attack complexes on cell membrane surface are detected after the addition of HMGB1. In an acetaminophen (APAP)-induced hepatotoxicity model, APAP injection reduced HMGB1 levels and elevated C3 levels in C1q-deficient mouse serum samples, compared to that in wild-type (WT) mice. APAP-induced C3 consumption was inhibited by sRAGE treatment in WT mice. Moreover, in a mouse model of brain ischemia–reperfusion injury based on middle cerebral arterial occlusion, C5b-9 complexes were deposited on vessels where HMGB1 was accumulated, an effect that was suppressed upon HMGB1 neutralization. We propose that the HMGB1 released after cell necrosis and in ischemic condition can trigger the classical pathway of complement activation to exacerbate sterile inflammation.

Autoimmune pancreatitis—a new evolving pancreatic disease?

Recent advances support the concept of autoimmune pancreatitis as a unique systemic disease because occasional extrapancreatic lesions such as sclerosing cholangitis, sclerosing sialoadenitis, and retroperitoneal fibrosis show similar pathological features with fibrosis and abundant infiltration of IgG4-positive plasma cells, and are steroid responsive. Based on these findings, several diagnostic criteria have been proposed. Although AIP is accepted worldwide as a unique clinical entity, pathogenetic mechanism still remains unclear. To clarify it, genetic background, humoral immunity, candidates of target antigens including self-antigens and molecular mimicry from microbes, cellular immunity including regulatory T cells, complement system, and experimental models are reviewed. Based on these findings, we have proposed a hypothesis for the pathogenesis of AIP in the biphasic mechanism of "induction" and "progression." In the early stage, initial response to self-antigens (LF, CA-II, CA-IV, PSTI, or α-fodrin) or molecular mimicry (Helicobacter pylori) is induced by decreased naive regulatory T cells (Tregs), and Th1 cells release proinflammatory cytokines (IFN-γ, IL-1b, IL-2, and TNF-α). In the chronic stage, progression is supported by increased memory Tregs and Th2 immune responses. The classical pathway of complement system may be activated by IgG1 immune complex. As Tregs seem to take important roles in progression as well as induction of the disease, further studies are necessary to clarify the pathogenesis.

9,10- seco-9,19-Cyclolanostane arabinosides from the roots of Actaea podocarpa

Seven 9,10- seco-9,19-cyclolanostane arabinosides, named podocarpasides A–G ( 1– 7), were isolated from the roots of Actaea podocarpa DC., a species closely related to black cohosh (a well known dietary supplement). Their structures were determined with the help of spectroscopic data including extensive 2D NMR spectroscopy. The isolates were found inactive, when tested for cytotoxic, estrogenic, and antioxidant activities in cell based assays. They were also tested for anticomplement activity against the classical pathway of complement system and only podocarpaside C ( 3) inhibited modest complement activity with an IC 50 value of 200 μM.

Oxiagin from the Naja oxiana cobra venom is the first reprolysin inhibiting the classical pathway of complement

A basic glycoprotein oxiagin with molecular mass of 49.8 kDa was isolated from the venom of Central Asian cobra Naja oxiana. Partial amino acid sequence determination has shown that oxiagin belongs to reprolysins, a subfamily of animal metalloproteinases possessing a characteristic multidomain structure. Oxiagin was found to inhibit the classical pathway of the complement system. A study of the oxiagin influence on the different stages of the classical pathway showed that it inhibited the formation of C3-convertase. To achieve it, oxiagin binds to IgG on the surface of sheep erythrocytes sensitized with rabbit antibodies, thus, preventing the interaction of component C2 (without its inactivation) with immobilized C4b. IC 50 for the inhibiton of classical pathway of complement system by oxiagin is 80 nM, while it does not affect the alternative pathway at concentrations up to 1.2 μM. Oxiagin possessed hemagglutinating activity towards sheep and rabbit erythrocytes, and this activity as well as the complement inhibition by oxiagin were suppressed by d-galactose. Oxiagin is the first representative of snake venom reprolysins that inhibits the complement system, utilizing non-proteolytic inhibiting strategy.

Flavonoids from the leaves ofLitsea japonica and their anti-complement activity

Four flavonoids, epicatechin (1), afzelin (2), quercitrin (3), and tiliroside (4), were isolated from the leaves of Litsea japonica (Thunb.) Jussieu (Lauraceae). The structures of compounds were identified by comparing their chemical and spectral data with those previously reported. The flavonoids (1-4) were tested for their anti-complement activity against classical pathway of complement system. Compounds 2-4 showed inhibitory activity against complement system with IC50 values of 258, 440, and 101 microm, respectively, whereas 1 was inactive. For the evaluation of the structure-activity relationship of 5,7-dihydroxyflavones, myricitrin (5) from Juglans mandshurica also tested for it's anti-complement activity and is inactive in this assay system. Furthermore, compounds 2, 3, and 5 were hydrolyzed with naringinase to give kaempferol (2a), quercetin (3a), and myricetin (5a), and these were also tested for their activity. Of the three aglycones, 2a exhibited anti-complement activity with an IC50 value of 730 microM, while 3a and 5a were inactive. The inhibitory potencies of 2, 2a, 3, 3a, 5, and 5a against complement activity increased in inverse proportion to number of free hydroxyls on B-ring of 5,7-dihydroxyflavone. Of the compounds tested, 4 showed the most potent inhibitory activity against the complement system.

Effects of triterpenoids from Pueraria lobata on immunohemolysis: beta-D-glucuronic acid plays an active role in anticomplementary activity in vitro.

The anticomplementary properties of kaikasaponin III (4) and soyasaponin I (8) from Pueraria lobata and their hydrolytic analogs were investigated in vitro. Diglycosidic saponins [kaikasaponin I (3), soyasaponin III (7)] showed most potent anticomplementary activities, followed by monoglycosidic saponins [soyasapogenol B monoglucuronide (6), sophoradiol monoglucuronide (2)] and triglycosidic saponins [soyasaponin I (8), kaikasaponin III (4)], whereas sophoradiol (1) and soyasapogenol B (5) showed enhancement of hemolysis under the presence of serum on the classical pathway of complement system. But all of them showed very weak or no anticomplementary activities on the alternative pathway of complement system. The anticomplementary activity of the saponins was influenced by the nature of glucuronic acid, where the free acid forms (-COOH) showed much more potent activity than the sodium salt forms (-COO-Na+) or methyl ester forms (-COOCH3), and the reduced forms (-CH2OH) decreased the activity significantly.

Complement System Is Not Activated in Primary Biliary Cirrhosis

There is controversial evidence suggesting that the classical pathway of complement system is chronically activated in primary biliary cirrhosis (PBC) and that complement activation may be important in development of bile duct injury. We have reevaluated this issue by measuring by-products of complement activation such as C4a, C3a, Bb, and terminal complement complexes (SC5b-9) in plasma of 44 PBC patients with sensitive methods not previously used to detect complement activation in this disease. Age-matched healthy women and patients with chronic hepatitis of different etiology were studied as controls. We found that PBC patients have normal C4a concentrations. This finding argues strongly against chronic classical pathway activation. Although a minor increase of C3a levels was observed in a minority of PBC patients, the C3a/C3 ratio, an index used to evaluate the extent of native protein conversion, was remarkably similar in all groups. Potentially lytic terminal complement complexes were not increased. PBC patients had normal Bb plasma levels, indicating that the alternative pathway is also not activated. C3 concentration was higher in PBC patients than in healthy subjects and in chronic hepatitis patients, particularly in the early stages of the disease. C3 and C4 concentrations became lower in PBC and chronic hepatitis with the progression of the disease. The increase of C3 concentration in PBC does not reflect liver inflammation, since serum levels of C-reactive protein are normal. We found high serum C3 levels in patients with rare chronic cholestatic syndromes without superimposed infections and observed that serum C3 levels paralleled those of bilirubin in a patient with benign recurrent intrahepatic cholestasis. In conclusion, our data indicate that complement is not activated in PBC and that the increase of serum C3 levels is related to cholestasis.

C1-Inhibitor substitution as ultima ratio therapy in septic shock: experience with 15 patients

Plasma C1-Inhibitor (C1-INH) is the major inhibitor of both the classical pathway of complement system and contact activation. A relative deficiency of C1-INH has been proposed as an important contributor to the development of shock and organ failure [1,2]. Among others, our working group has published two cases of successful C1-INH substitution in septic patients recently [3].

Purification and characterization of the C3 convertase of the classical pathway of human complement system by size exclusion high-performance liquid chromatography.

The C3 convertase of the classical pathway of the complement system is a liable complex, C4b,2a, and is activated by limited proteolysis of two components, C4 and C2, by C1s. By utilizing iodine-treated C2 and size exclusion high-performance liquid chromatography (HPLC), we have succeeded in isolating for the first time the classical pathway C3 convertase. Size exclusion HPLC demonstrated that the apparent molecular mass of the C3 convertase was 280K daltons. The C3 convertase decay-dissociates spontaneously into C4b and C2a. The decay-dissociation is a temperature-dependent reaction and the half-lives of the C3 convertase at 24, 30, and 37 degrees C were estimated to be 400, 180, and 60 min, respectively. The decay-dissociation was also dependent on pH and was accelerated by increasing pH. In addition, the decay-dissociation of the C3 convertase was accelerated by C2b. This result suggests that C2b acts as a feedback inhibitor on the activation of the classical pathway of complement system.

Human C4b-binding protein, C4bp: Chymotryptic cleavage and location of the 48 kDa active fragment within C4bp

C4bp, a regulator of the classical pathway of complement system, is composed of 6–8 disulfide-linked subunit chains of 75 kDa. Upon incubation with chymotrypsin, C4bp was rapidly cleaved into a nicked C4bp, composed of disulfide-linked 48 kDa and 27 kDa fragments. Subsequent slow cleavage on the 27 kDa fragment resulted in the liberation of the active site-containing 48 kDa fragment from the nicked C4bp. The N-terminal amino acid sequence of the 48 kDa fragment was identical to that of the parent subunit chain of C4bp, indicating that the 48 kDa active fragment was released from the N-terminal side of the parent subunit chain. Based on these results, a possible gross structure of C4bp is proposed.