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PubMed2000

Glycyrrhizin inhibits the lytic pathway of complement--possible mechanism of its anti-inflammatory effect on liver cells in viral hepatitis.

Glycyrrhizin, an aqueous extract of licorice root, has anti-inflammatory activity and has been used for the treatment of chronic viral hepatitis. In the present study we describe the mechanism by which glycyrrhizin inhibits complement. Glycyrrhizin inhibited the cytolytic activity of complement via the activation of both the classical and alternative pathways, while it had no effect on immune adherence, suggesting that it blocks C5 or a later stage of the complement cascade. Further analysis revealed that glycyrrhizin inhibits the lytic pathway in which the membrane attack complex (MAC) is formed. This mechanism suggests that glycyrrhizin may prevent tissue injury caused by MAC not only in chronic hepatitis but in many autoimmune and inflammatory diseases.

Animals↗
PubMed1988

Attachment of human polymorphonuclear leukocytes to herpes simplex virus-infected fibroblasts mediated by antibody-independent complement activation.

Herpes simplex virus (HSV)-infected cells can activate the human complement system without interference of specific anti-HSV antibodies. Analysis by flow cytometry showed that C3-like molecules were deposited on the membrane of the infected cell when incubated with human serum without specific antibodies. Depletion of calcium to block the classical pathway of the complement system had no effect on fluorescence intensity. The complement activation could be blocked by chelating both calcium and magnesium or by heating the serum. Furthermore, in the fluid phase C3 was converted to C3b by infected cells and not by uninfected cells. The antibody-independent activation did not lead to lysis of the virus-infected fibroblasts, indicating that the complement cascade is abrogated before formation of the membrane attack complex. This was also confirmed by measurement of the 50% hemolytic complement activities for total and alternative pathways. Polymorphonuclear leukocytes attached to infected fibroblasts after incubation of these fibroblasts with intact complement. This is most probably mediated by complement receptor binding of C3b and C3bi which is deposited on the membrane of the HSV-infected cell. Both type 1 and type 2 HSVs showed the same characteristics in complement activation and thereby mediated polymorphonuclear leukocyte adherence.

Calcium↗
PubMed2006

Suppression of complement regulatory proteins (CRPs) exacerbates experimental autoimmune anterior uveitis (EAAU).

This study was undertaken to explore the role of complement regulatory proteins (CRPs) in experimental autoimmune anterior uveitis (EAAU). We observed that the levels of CRPs, Crry and CD59, in the eyes of Lewis rats increased during EAAU and remained elevated when the disease resolved. The in vivo role of these CRPs in EAAU was explored using neutralizing mAbs, antisense oligodeoxynucleotides (AS-ODNs), and small interfering RNAs against rat Crry and CD59. Suppression of Crry in vivo at days 9, 14, or 19 by neutralizing mAb or AS-ODNs resulted in the early onset of disease, the exacerbation of intraocular inflammation, and delayed resolution. Suppression of CD59 was only effective when the Abs and ODNs were given before the onset of disease. The most profound effect on the disease was observed when a mixture of Crry and CD59 mAbs or AS-ODNs was administered. A similar effect was observed with a combination of Crry and CD59 small interfering RNA. There was no permanent histologic damage to ocular tissue after the inflammation cleared in these animals. Increased complement activation as determined by increased deposition of C3, C3 activation fragments, and membrane attack complex was observed in the eyes of Lewis rats when the function and/or expression of Crry and CD59 was suppressed. Thus, our results suggest that various ocular tissues up-regulate the expression of Crry and CD59 to avoid self-injury during autoimmune uveitis and that these CRPs play an active role in the resolution of EAAU by down-regulating complement activation in vivo.

Animals↗
PubMed2005

cDNA cloning and phylogenetic analysis of the sixth complement component in rainbow trout.

The sixth complement protein (C6) is an essential component of the membrane attack complex (MAC); the end product of the lytic pathway of complement activation. The MAC complex constitutes a supramolecular assembly containing the five precursor proteins C5b, C6, C7, C8, and C9. Once assembled on the target surface it forms transmembrane channels that cause membrane damage and cytolysis of complement-opsonized pathogens. Besides mediating direct pathogen elimination, exposure of cells to sublytic doses of MAC can trigger diverse cellular responses such as, cell activation, induction of apoptosis, cell cycle re-entry and proliferation in various biological settings. The terminal complement components (C6-C9) are structurally related proteins, differing in size and complexity. In order to study their evolution, we report here the cloning and molecular characterization of C6 component in rainbow trout. The deduced amino acid sequence of trout C6 exhibits 55 and 44% identity with zebra fish and human orthologs, respectively. The 'domain' architecture of trout C6 resembles that of mammalian counterparts, and the cysteine backbone is also conserved. Finally, trout C6 gene appears to exist as a single copy in the trout genome, and is expressed in a wide range of trout tissues.

Amino Acid Sequence↗
PubMed1997

Characterization of neuronal cell death induced by complement activation.

The complement system plays an important role in human immune defense mechanism. Its activation via either the classical or the alternative pathway can lead to the formation of membrane attack complex (MAC) and subsequently kills target cells. Activation of the classical pathway can be initiated with binding of C1q which is first factor of complement cascade to the Fc (fragment crystalline) region of immunoglobulin. This triggers a cascade of proteolytic events resulting in the activation of C5 convertase which cleaves C5 into C5b and C5a. The C5b then binds C6, C7, C8 to form a C5b-8 complex. Binding of C9 molecules to C5b-8 forms C5b-9, the MAC, which pore size increases as the number of C9 in the complex increases. If this membrane lesion persists and results in uncontrolled ion fluxes, the cells swell and eventually lyse. To restrict the activity of the complement system, endogenous complement inhibitors are available to regulate complement-mediated cytolysis. This enables the complement system to distinguish "self" from "foreign" and protect the host from inadvertent complement attack. Activation of the classical complement cascade has been reported in Alzheimer's disease and other neurodegenerative disorders. Recently, we demonstrated that complement activation causes neuronal cell death in vitro, and this neurodegenerative process is regulated by homologous restriction. In this article, we describe the use of two cell lines as in vitro models to evaluate cell injury/cell death induced by complement activation.

Animals↗
PubMed2006

Binding of the lipocalin C8gamma to human complement protein C8alpha is mediated by loops located at the entrance to the C8gamma ligand binding site.

Human C8 is one of five complement components (C5b, C6, C7, C8 and C9) that interact to form the membrane attack complex (MAC). C8 is composed of a disulfide-linked C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. C8alpha and C8beta are homologous to C6, C7 and C9, whereas C8gamma is the only lipocalin in the complement system. Lipocalins have a core beta-barrel structure forming a calyx with a binding site for a small molecule. In C8gamma, the calyx opening is surrounded by four loops that connect beta-strands. Loop 1 is the largest and contains Cys40 that links to Cys164 in C8alpha. To determine if these loops mediate binding of C8alpha prior to interchain disulfide bond formation in C8alpha-gamma, the loops were substituted separately and in combination for the corresponding loops in siderocalin (NGAL, Lcn2), a lipocalin that is structurally similar to C8gamma. The siderocalin-C8gamma chimeric constructs were expressed in E. coli, purified, and assayed for their ability to bind C8alpha. Results indicate at least three of the four loops surrounding the entrance to the C8gamma calyx are involved in binding C8alpha. Binding near the calyx entrance suggests C8alpha may restrict and possibly regulate access to the C8gamma ligand binding site.

Amino Acid Sequence↗
PubMed1992

Complement-mediated killing of susceptible gram-negative bacteria: an elusive mechanism.

Exposure of gram-negative bacteria to a source of antibody and complement frequently results in efficient cell killing; this effect follows the deposition of C5b-9 membrane attack complexes onto the bacterial surface. This review examines the molecular and physiological events culminating in C5b-9 deposition and cell death and advances an hypothesis that may account for cytoplasmic-membrane damage following complement-mediated perturbation of the outer membrane.

Animals↗
PubMed1995

Active Heymann nephritis in complement component C6 deficient rats.

The mechanisms of renal injury that result in proteinuria in active Heymann nephritis (AHN) remain unclear, though data suggest that in analogy of the passive form of the disease the membrane attack complex C5b-9 may be involved. AHN was induced in an inbred strain of PVG/c-rats that are totally deficient in the C6 component of complement and are unable to form the lytic C5b-9 complex, as well as in non-complement deficient PVG/c+ rats that are immunologic identical to the deficient strain. In both groups of animals comparably high titers of anti-Fx1A autoantibodies were found after three weeks and persisted at 40 weeks. Proteinuria was also similar in both groups, and was first evident at six weeks. High levels of urinary protein, ranging from 200 mg/24 hr to 500 mg/24 hr, were found after 10 weeks and persisted up to one year. Renal biopsy findings at various times post-immunization were identical in both groups, including immunofluorescence staining for Ig and C3 deposits, and also EM findings of subepithelial electron-dense deposits were not different. The injection of heterologous rabbit complement, that partially and temporarily restored the CH50 activity in PVG/c- rats did not alter or hasten the disease. Long-term follow-up showed that all rats in both groups continued to have severe proteinuria and that most animals died between 8 to 12 months after disease induction, without renal impairment. EM findings in serial biopsies demonstrated that the growth of the subepithelial deposits as measured by surface area occurred between weeks 4 and 12. A positive correlation (r = 0.94) between the size of the deposits and the level of proteinuria was found. These studies demonstrate that the membrane attack complex of complement does not play a major role in AHN. The relationship of the size of the immune deposits to the level of proteinuria suggests that the growth of the immune deposits on itself initiate secondary mechanisms that damage the permselective characteristics of the glomerular membrane.

Animals↗
PubMed2001

Clusterin is up-regulated in glomerular mesangial cells in complement-mediated injury.

BACKGROUND: Clusterin is a soluble complement regulatory protein that binds to C5b-7 and inhibits generation of membrane attack complex, C5b-9. Glomerular deposition of clusterin has been observed in human and experimental membranous nephropathy in association with C5b-9 and immune deposits. However, it is controversial as to whether clusterin observed in glomeruli is synthesized by the resident glomerular cells or is derived from the circulation. We examined whether clusterin is expressed by resident glomerular cells exposed to complement-mediated injury. METHODS: In vitro, cultured mesangial cells were exposed to antithymocyte serum immunoglobulin G and 5% normal rat serum as a complement source. In vivo, we induced anti-Thy1 nephritis in rats and examined the kidneys on days 8 and 29. RESULTS: We observed increased expression of clusterin in cultured rat glomerular mesangial cells stimulated by sublytic complement attack. We also demonstrated that in comparison with control rats, both a marked increase in clusterin mRNA in the glomeruli and marked deposition of clusterin protein in the mesangial area occurred in the OX-7-treated rats on day 8 in association with C5b-9 deposition and on day 29. CONCLUSION: Clusterin was induced in glomerular mesangial cells during the course of immune-mediated injuries. This up-regulation of clusterin may play a critical role in protecting mesangial cells from complement attack.

Animals↗
PubMed1988

Complement inhibitor S protein is associated with membranes of red blood cells from patients with paroxysmal nocturnal haemoglobinuria.

S protein is a plasma glycoprotein (Mr = 78,000) which binds to nascent C5b-7 complexes upon complement activation in the fluid phase in whole serum. It thereby protects innocent bystander cells from complement mediated lysis. It is unknown whether S protein also functions as complement inhibitor on cell surfaces. We here report that S protein is recognized on red blood cells (RBC) from patients with paroxysmal nocturnal haemoglobinuria (PNH), but not on normal RBC. RBC from eight PNH patients showed 12-48% haemolysis subsequent to complement activation in the fluid phase, while normal RBC did not respond. Preincubation of the PNH cells with affinity-purified antibodies against human S protein resulted in a three- to five-fold increase of haemolysis, while preincubation of these cells with S protein decreased haemolysis by 40%. In contrast, haemolysis remained unaffected by other unrelated antibodies, i.e. IgG anti-Rh(D) and anti-A. If PNH RBC, normal RBC pretreated with 2-amino-ethylisouronium bromide (AET), or untreated normal RBC, respectively, were incubated with purified S protein in vitro, the uptake of antibodies against S protein was significantly enhanced with PNH and with AET-treated, but not with untreated normal RBC. Additionally, while normal RBC did not respond to reactive lysis initiated by purified C5b-6 and C7, PNH as well as AET-RBC showed significant haemolysis that could be inhibited by S protein in a dose-dependent fashion. These findings strengthen the assumption that the increased sensitivity of PNH cells towards reactive complement lysis is either due to the lack of an inhibitor of the terminal complement sequence and/or enhanced insertion of the membrane attack complex. These defects of PNH RBC may partly be overcome by the fluid phase complement inhibitor S protein which binds to PNH RBC and may thereby suppress homologous cytolysis.

Complement Inactivator Proteins↗
PubMed2003

A small molecule C5a receptor antagonist protects kidneys from ischemia/reperfusion injury in rats.

BACKGROUND: C5a has been implicated in numerous pathophysiological conditions, including ischemia/reperfusion (I/R) injury of the kidney. We examined whether a novel and specific C5a receptor antagonist, the cyclic compound AcF-[OPdChaWR] could moderate I/R-induced renal injury in rats. METHODS: Female Wistar rats were subjected to renal ischemia (60 min) and reperfusion (5 h). Rats were treated with either 1 mg/kg IV in 5% ethanol/saline or 10 mg/kg PO in 25% ethanol/saline prior to ischemia. I/R injury was characterized by significant tissue hemorrhage with increased microvascular permeability, elevated renal tissue levels of tumor necrosis factor-alpha (TNF-alpha) and myeloperoxidase (MPO), increased serum levels of creatinine and aspartate aminotransferase (AST) and hematuria. RESULTS: Pre-ischemic treatment with the C5a receptor (C5aR) antagonist (1 mg/kg IV or 10 mg/kg PO) substantially inhibited or prevented I/R-induced hematuria, vascular leakage, tissue levels of TNF-alpha and MPO, and serum levels of AST and creatinine. Histological examination of kidneys from antagonist pretreated I/R animals showed a marked reduction in tissue damage compared to drug-free I/R rats. This antagonist, however, did not inhibit complement-mediated lysis of red blood cells, suggesting unimpaired formation of the membrane attack complex (MAC). CONCLUSIONS: The results demonstrate for the first time that a selective antagonist of both human and rat C5a receptors, given either intravenously or orally, significantly protects the kidney from I/R injury in the rat. We conclude that C5a is an important pathogenic agent in renal I/R injury, and that C5a receptor antagonists may be useful therapeutic agents for the pretreatment of anticipated renal reperfusion injury in humans.

Animals↗
PubMed1992

Studies on complement deposits in epidermolysis bullosa acquisita and bullous pemphigoid.

Epidermolysis bullosa acquisita (EBA) is an inflammatory subepidermal blistering disease characterized by circulating and tissue-bound autoantibodies specific for type VII collagen of the basement membrane zone. The antibodies consist of both complement- and noncomplement-binding populations and belong to all four subclasses of IgG. We investigated the presence of the membrane attack complex C3b, C5, and S protein in EBA and compared C3b and C5 in EBA and bullous pemphigoid. In 10 patients with EBA, these components were detected at the basement membrane zone as follows: membrane attack complex, 90%; S protein, 90%; direct C5, 90%; C3b, 100%; and C5 binding, 90%. In the patients with bullous pemphigoid, the results were as follows: direct C5, 58%; C3b, 33%; and C5 binding, 19%. These results provide additional evidence for complement activation at the basement membrane zone in EBA, show that complement activation in EBA proceeds to activation of terminal complement components, and suggest that EBA antibodies are more potent activators of C5 than are bullous pemphigoid antibodies.

Basement Membrane↗
PubMed2002

C6-like and C3-like molecules from the cephalochordate, amphioxus, suggest a cytolytic complement system in invertebrates.

The mammalian immune system has cytotoxic mechanisms, both cellular and humoral, that destroy the membrane integrity of target cells. The main effector molecules of these cytolytic mechanisms-perforin, used by killer lymphocytes, and the membrane attack complex (MAC) components of the complement system-share a unique module called the MAC/perforin module. Until now, both immunological cytotoxicity and the MAC/perforin module have been reported only in jawed vertebrates. Here, we report the identification of a protein containing the MAC/perforin module from the invertebrate cephalochordate, amphioxus ( Branchiostoma belcheri), using expressed sequence tag (EST) analysis of the notochord. The deduced amino acid sequence of this molecule is most similar to the primary structure of human complement component C6 and is designated AmphiC6. AmphiC6 shares a unique modular structure, including the MAC/perforin module, with human C6 and other MAC components. Another EST clone predicts the presence of a thioester-containing protein with the closest structural similarity to vertebrate C3 (therefore designated AmphiC3). AmphiC3 retains most of the functionally important residues of vertebrate C3 and is shown by phylogenetic analysis to be derived directly from the common ancestor of vertebrate C3, C4, and C5. Only opsonic activity has been assigned to the invertebrate complement system until now. Therefore, this is the first molecular evidence for complement-mediated immunological cytotoxicity in invertebrates.

Amino Acid Sequence↗
PubMed1975

The membrane attack mechanism of complement. Isolation and subunit composition of the C5b-9 complex.

Isolation of the C5b-9 complex from inulin-activated whole human serum was effected by molecular sieve column chromatography employing Biogel A-15 M, preparative Pevikon block electrophoresis, and removal of low density beta-lipoproteins by flotation in CsCl. The final product was homogeneous upon cellulose acetate strip electrophoresis and analytical ultracentrifugation. Ouchterlony analyses indicated that the complex reacted with antisera to C5, C6, C7, C8, and C9 to form a continuous, circular precipitin line without spurs. The C5b-9 complex was dissociated by sodium dodecyl sulfate (SDS) in the absence of reducing agents, and analytical SDS-polyacrylamide gel electrophoresis revealed seven protein bands after straining with Coomassie Blue. Bands 1, 2, 3, and 6 were identified as C5b, C7, C6, and C9, respectively. Bands 4 and 7 were identified as two noncovalently bound subunits of C8. Molar ratios among C5b, C6, C7, C8, and C9 dissociated from the complex by SDS were estimated to be 1:1:1:1:3. Band 5 protein, which had an estimated mol wt of 88,000 and was found to occur with a molar ratio of 3, has not yet been identified. Its nature and possible biological functions are discussed.

Animals↗
PubMed2000

Therapeutic potential of complement inhibitors in myocardial ischaemia.

Under normal conditions, the complement system functions to eradicate microbes and other membrane bound pathogens. In other situations, complement activation comprises a pivotal mechanism for mediating tissue demolition in inflammatory disorders, including ischaemia/reperfusion injury. Complement-mediated tissue damage has long been recognised as a significant contributor to myocardial reperfusion injury. However, clinical use of complement inhibitors to reduce the extent of irreversible tissue injury related to reperfusion, remains in the early stages of development. Activation of the complement system generates anaphylatoxins, opsonins and the lytic moiety known as the membrane attack complex (MAC). In addition, fragments of the complement cascade proteins (e.g., C3a and C5a) secondarily initiate processes deleterious to myocytes by recruiting and stimulating inflammatory cells, such as neutrophils and macrophages, within the area of reperfusion. Damaged tissue itself, is capable of upregulating the genes that encode the formation of complement proteins leading to assembly of the MAC, which in turn further advances tissue injury. All of these factors contribute to the development of myocardial infarction subsequent to ischaemia and reperfusion. This paper provides an overview of how the complement system operates and examines the various inhibitors, both endogenous and exogenous, that regulate the complement cascade. Activation and inhibition of the complement system will be discussed primarily in the context of myocardial ischaemia and reperfusion injury.

Animals↗
PubMed1998

Neuronal protection of oligodendrocytes from antibody-independent complement lysis.

Cultured rat oligodendrocytes are lysed by complement via antibody-independent activation of the classical pathway. This susceptibility to complement lysis has been demonstrated to be due to lack of CD59, a complement regulatory protein which inhibits assembly of the membrane attack complex. In this study the effects of homologous and heterologous complement were examined in a co-culture system of rat oligodendrocytes and peripheral neurones, where axonal ensheathment was observed as early as 4 days after the addition of glial progenitors to the neurones. Following exposure to complement, ensheathing oligodendrocytes were markedly less sensitive to antibody-independent but not antibody-dependent complement lysis than were cells grown without neurones. Immunocytochemical data revealed that co-cultured oligodendrocytes remained CD59 negative, but in contrast to oligodendrocytes cultured alone, were negative for C3b when incubated with C7-deficient serum. Taken together these data indicate that the decreased sensitivity of co-cultured oligodendrocytes to complement lysis is not attributed to the increased expression of CD59, but rather in a failure to activate complement. Incubation of oligodendrocytes with neurone-conditioned medium afforded significant protection (68%), against antibody-independent complement attack, suggesting that soluble neuronal factors can protect oligodendrocytes from complement-mediated lysis.

Animals↗
PubMedDate not supplied

C-type lectins and galectins mediate innate and adaptive immune functions: their roles in the complement activation pathway.

In recent years, a 'new' pathway for complement activation mediated by the mannose-binding lectin (MBL) has been described as a key mechanism for the mammalian acute phase response to infection. This complement activation pathway is initiated by a non-self recognition step: the binding of a humoral C-type lectin [mannose-binding lectin (MBL)] to microbial surfaces bearing 'foreign' carbohydrate determinants. The recognition factor, MBL, is associated with a serine protease [MBL-associated serine protease (MASP)] which, upon MBL binding to the microbial ligand, activates the complement component C3, leading to either (a) phagocytosis of the opsonized target via the complement receptor, or (b) humoral cell killing via assembly of the membrane attack complex. Galectins (formerly known as S-type lectins) modulate activity of the complement receptor 3 (CR3), the macrophage membrane receptor for complement components C3b and iC3b, downstream products of the MBL pathway which are covalently bound to 'target cells. Galectins also mediate macrophage- and dendrocyte-adhesion to lymphocytes activated by signaling through another C-type lectin, the L-selectin, leading to immunoglobulin-mediated responses. Thus, the functional interplay of MBL, galectins and L-selectin in the acute phase response neutralizes the microbial challenge, and lead to further adaptive immunity. Although the observation of various components of the lectin pathway in different invertebrate species demonstrates the high conservation and ancient roots of the components of innate immunity, there has previously been no evidence supporting the possibility that the integral lectin-mediated complement activation pathway is present in invertebrates. We now have evidence for the coexistence of homologs of all the pathway's key components (MBL, MASP, C3, and galectin) in the protochordate Clavelina picta, suggesting the lectin-mediated pathway of complement activation preceded the immunoglobulin pathway in evolution. Therefore, despite being 'new' to the textbooks, experimental evidence indicates that this pathway is ancient, and has been conserved intact throughout its evolution.

Amino Acid Sequence↗
PubMedDate not supplied

A rat model of pregnancy-induced sensitization to transplants.

Previous pregnancy is a known risk factor for alloantibody production and graft rejection in clinical transplantation. However, in previous rat models, immune responses to RT1.A antigens induced by allogeneic pregnancy resulted in prolonged survival of subsequent allografts. This study was designed to investigate the effects of a previous pregnancy on alloantibody response, complement activation, and allograft survival in a highly immunogenic rat strain combination. C6-sufficient and -deficient female PVG.1U (RT1.A(u)B(u)) rats were mated with allogeneic PVG.R8 (RT1.A(a)B(u)) males or control isogeneic PVG.1U (RT1.A(u)B(u)) males. Three weeks after parturition, experimental and control females received cardiac allografts from female PVG.R8 donors. A low dose of cyclosporine (CsA, 5 mg/kg on alternate days) was used for immunosuppression after transplantation. Allogeneic, but not control isogeneic, pregnancy elicited a weak, transient IgG alloantibody response that declined before transplantation. Experimental female recipients produced a rapid, vigorous IgM and IgG alloantibody response to the transplant despite CsA treatment. C6-sufficient recipients rejected their transplants at an accelerated rate (5 days, n = 6) compared with control animals (7 days, n = 5). In contrast, allografts to C6-deficient recipients functioned until sacrifice at 90 days in both the experimental group (n = 7) and control group (n = 4). Most experimental C6-deficient recipients continued to produce strong IgG alloantibodies for 90 days. Complement activation resulting from the alloantibody response was evidenced by the diffuse deposition of C3d on the vascular endothelium of the grafts. In summary, previous pregnancy leads to memory alloantibody responses that accelerate allograft rejection even with immunosuppression. Membrane attack complex is required for accelerated rejection induced by previous pregnancy.

Animals↗