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PubMed2004

Central role of complement membrane attack complex in monosodium urate crystal-induced neutrophilic rabbit knee synovitis.

OBJECTIVE: Monosodium urate monohydrate (MSU) crystals promote gouty inflammation that is critically mediated by neutrophil recruitment and activation. Interleukin-8 (IL-8) and closely related chemokines are major neutrophil chemotaxins in experimental gout. But MSU crystals also activate the classical and alternative pathways of complement, and MSU crystals directly cleave C5 on the crystal surface. Unlike IL-8, the roles in acute gout of individual complement-derived peptides and of the terminal C5b-9 complement components that comprise the membrane attack complex (MAC) are unclear. Hence, we studied rabbits deficient in the MAC component C6 to determine if MAC mediated urate crystal-induced arthritis. METHODS: We injected C6-deficient and C6-sufficient rabbit knee joints with 10 mg of pyrogen-free urate crystals and analyzed IL-8 levels, leukocyte influx, and joint inflammation 24 hours later. RESULTS: There was a significant decrease (>60%) in swelling in MSU crystal-injected knees of C6-deficient animals as compared with C6-sufficient animals (P < 0.05). An attenuated rise in MSU crystal-induced joint effusion levels of IL-8 also was observed, which was concordant with diminished numbers of neutrophils (P < 0.05) but not monocytes in MSU crystal-induced knee synovial fluid from C6-deficient animals. Synovial tissue analysis confirmed mononuclear leukocyte infiltration in response to MSU crystal injection in all animals, but substantial neutrophil infiltration only in C6-sufficient animals. CONCLUSION: MAC activation appears to play a major role in intraarticular IL-8 generation and in neutrophil recruitment in experimental acute gouty arthritis of the rabbit knee. C6 and MAC activation may represent novel therapeutic targets for suppression of neutrophil-mediated joint inflammation in gout.

Animals↗
PubMed2006

Complement mediators in ischemia-reperfusion injury.

BACKGROUND: Ischemia-reperfusion (I/R) injury occurs when a tissue is temporarily deprived of blood supply and the return of the blood supply triggers an intense inflammatory response. Pathologically, increased complement activity can cause substantial damage to blood vessels, tissues and also facilitate leukocyte activation and recruitment following I/R injury. Herein, previously published studies are reported and critically reviewed. METHODS: Medline and the World Wide Web were searched and the relevant literature was classified under the following categories: (1) Complement pathways; (2) The complement system and the inflammatory response; (3) Complement in ischemia-reperfusion injuries; and (4) Therapeutic approaches against complement in I/R injuries. RESULTS AND CONCLUSIONS: I/R injury is a common clinical event with the potential to seriously affect, and sometimes kill, the patient and is a potent inducer of complement activation that results in the production of a number of inflammatory mediators. Complement activation leads to the release of biologically active potent inflammatory complement substances including the anaphylatoxins (C3a and C5a) and the cytolytic terminal membrane attack complement complex C5b-9 (MAC). The use of specific complement inhibitors to block complement activation at various levels of the cascade has been shown to prevent or reduce local tissue injury after I/R. Several agents that inhibit all or part of the complement system, such as soluble complement receptor type 1 (sCR1), C1 inhibitor (C1-INH), C5a monoclonal antibodies, a C5a receptor antagonist and soluble CD59 (sCD59) have been shown to reduce I/R injury of various organs. The novel inhibitors of complement products may eventually find wide clinical application because there are no effective drug therapies currently available to treat I/R injuries.

Animals↗
PubMed2002

How safe is the port access technique in minimally invasive coronary artery bypass grafting?

BACKGROUND: This study compares conventional coronary artery bypass grafting (CABG) with port access CABG via a left anterior small thoracotomy in patients requiring surgical multivessel revascularization. Clinical, neuropsychological, and angiographic outcomes were studied, as well as parameters of myocardial and cerebral protection. Pathogenicity of cardiopulmonary bypass (CPB) was further evaluated by measuring parameters of peripheral limb ischemia and inflammatory whole-body response. METHODS: In a prospective randomized study, 40 patients who required multivessel CABG were assigned to either conventional CABG via complete median sternotomy (group A) or port access CABG via minithoracotomy (group B). Control angiograms were performed in group B only. In addition, patients underwent neuropsychological testing after the operation. CK, CK-MB, and Troponin T levels were documented. S-100B protein and neuron-specific enolase (NSE) served to quantify cerebral injury. The terminal complement complex (C5b-9) and myeloperoxidase concentrations were determined to analyze inflammatory whole-body response after CPB. RESULTS: There was no mortality. One patient suffered a retrograde aortic dissection immediately after onset of CPB, but had an uneventful postoperative course after surgical repair. Troponin T and CK-MB showed no difference between groups. CK and myoglobin were significantly higher in the minimally invasive cohort. Changes in complement activation (C5b-9) and myeloperoxidase during CPB markers of the whole-body inflammatory response were similar in both groups. S-100B concentrations in the port access group were significantly higher, whereas NSE levels were similar in both groups. Both groups did not display any significant difference in neuropsychological testing. CONCLUSIONS: Minimally invasive multivessel CABG via minithoracotomy using port access technology is feasible and safe. Though prolonged operating and CPB times with significantly higher S-100B concentrations were observed in group B, equivalent myocardial and cerebral protection and similar whole-body inflammatory response were documented.

Aged↗
PubMed1994

Immunohistochemical studies of vitronectin, C5b-9, and vitronectin receptor in membranous nephropathy.

To investigate the involvement of vitronectin, the terminal complement complex (C5b-9), and the vitronectin receptor in the pathogenesis of membranous nephropathy, the immunohistochemical localization of these antigens in the kidney was determined using the immunoperoxidase method and monoclonal antibodies: antivitronectin, anti-SC5b-9 (neoantigen), and antivitronectin receptor (specific for alpha v beta 3 and alpha v). The subjects were 6 patients with membranous nephropathy, and the controls were 2 patients with minimal-change nephrotic syndrome. In membranous nephropathy, vitronectin was localized in subepithelial deposits and in epithelial cell foot processes and was intensely positive in the foot processes adjacent to subepithelial deposits. C5b-9 showed a similar pattern of localization to vitronectin. Both alpha v beta 3 and alpha v were localized in the basal portions of the foot processes of visceral epithelial cells as well as along the borders of these cells adjacent to the urinary space. Deposition at the former site was heavier than at the latter, and localization was especially prominent adjacent to the subepithelial deposits. In addition, alpha v was localized around and within some of the electron-lucent subepithelial deposits in the basement membrane. In contrast, the deposition of vitronectin, C5b-9, alpha v beta 3, and alpha v was always less intense in minimal-change nephrotic syndrome than in membranous nephropathy. Vitronectin and C5b-9 were localized to small parts of mesangium and glomerular basement membrane, while alpha v beta 3 and alpha v deposits showed no difference in intensity between the basal portions of the foot processes and the urinary border of the visceral epithelial cells. Thus, membranous nephropathy featured increased localization of vitronectin, C5b-9, and vitronectin receptors both within and around the subepithelial deposits, suggesting that the mechanism of immune complex disposal via the vitronectin receptor and the vitronectin-C5b-9 complex, associated with complement activation due to subepithelial immune complex formation, may also be active.

Adult↗
PubMed1987

Generation of an activated form of human C5 (C5b-like C5) by oxygen radicals.

Treatment of purified human C5 with hydrogen peroxide leads to a reduction of functional activity in the classical immune haemolysis assay. The effect of H2O2 is enhanced by traces of iron-EDTA, and becomes even stronger when ascorbic acid is present in addition. The enhancement by iron and protective effects of various radical scavengers indicate that the conversion of C5 is brought about by hydroxyl radicals mainly, generated from H2O2 during its decomposition. By the conversion C5 acquires a new functional property: it becomes capable of binding C6, and the resulting complex C56 lyses non-sensitized red cells in cooperation with the late components C7, C8, and C9 (reactive lysis). In this respect, H2O2-treated C5 resembles the activation fragment of C5, C5b. However, unlike C5b, C5(H2O2) is not fragmented but comprises the whole molecule, according to polyacrylamide gel electrophoresis. In accordance with the lack of cleavage, no chemotactic activity indicating formation of C5a, became apparent after H2O2 treatment of C5. In whole human serum, however, the H2O2 system induces additional processes leading to the generation of C5a activity and indicating C5 cleavage. The pathways to this reaction are as yet not clear. The effect of oxygen radicals on C5 may be the basis of the enhancing effect of stimulated leukocytes on C5 activation in body fluids. Activated leukocytes are known to produce H2O2 and oxygen radicals.

Complement Activation↗
PubMed2005

Terminal complement complex in septic shock with capillary leakage: marker of complement activation?

BACKGROUND AND OBJECTIVE: The aim of this study was to evaluate the value of terminal complement complex (C5b-9) plasma levels as a marker for complement activation in septic shock with concomitant capillary leak syndrome. METHODS: In a prospective animal study 10 fasted, anaesthetized, mechanically ventilated and multi-catheterized pigs (20.6 +/- 1.3 kg) were investigated over a period of 8 h. Sepsis was induced by faecal peritonitis (1 g kg(-1) body weight faeces, n = 5) and compared to controls (n = 5). The animals received 6% hydroxyethyl starch 200/0.5 to maintain a central venous pressure of 12 mmHg. To quantify capillary leak syndrome, albumin escape rate was measured using 99mTc-labelled human serum albumin. Plasma levels of terminal complement complex were measured in a double antibody immunoassay (neoepitope-specific MoAb aE 11 as catching antibody). Immunohistological studies of renal specimens were performed to detect terminal complement complex deposition. RESULTS: Albumen escape rate increased in septic animals (+ 52%) compared to controls (+ 3%, P < 0.05). Plasma levels of terminal complement complex decreased during the study period in both groups. In septic animals this finding was accompanied by a significant deposition of terminal complement complex in renal specimens (P < 0.05). CONCLUSION: We found an activation of the complement system proven by marked deposition of terminal complement complex in renal specimen, while its plasma levels decreased during the study period in septic and control animals. These results suggest that in septic shock with capillary leak syndrome plasma level of terminal complement complex may not be a reliable marker of complement activation.

Animals↗
PubMed1977

Isolation and properties of complement-resistant strains of Escherichia coli K-12.

Several strains that were resistant to the bactericidal action of antibody and complement were isolated from Escherichia coli K-12 W3110/SM by selecting them through the medium containing antiserum and complement. They can be agglutinated by antiserum against the parent strain and showed similar immune adherence reactivity to the parent when sensitized with this antiserum. Few differences were found in the compositions of phospholipids and proteins between both inner and outer membranes of these strains and those of the parent. However, there were fewer short-chain and more long-chain fatty acids in these strains than in the parent. It was also found that unsaturated fatty acide decreased and saturated and cyclopropanoic acids increased in phosphatidylethanolamine and phosphatidylglycerol in both inner and outer membranes of one of these strains when compared with those from the parent. Therefore, the resistance of these strains to the complement-mediated bactericidal action was considered to be due to the rigidity of their membrane structures which might repel the insertion of membrane-attack complement complex C5b-9, although they could fix the earlier complement components up to the step of the formation of C4b,2a,3b complex enzyme.

Bacterial Proteins↗
PubMed1990

Channel fluctuations induced by membrane attack complex C5B-9.

The assembly of complement (C) components C5b-9 in membranes results in the formation of transmembrane lesions. The C9 component has been shown to be mainly responsible for formation of the ultrastructurally visible tubules associated with C5b-9 complexes. Several studies have disputed the role of C9 polymerization in C-mediated cytolysis on the grounds that C5b-9 lyses cells in the absence of tubular formation. Here, C5b-9 complexes were reconstituted into high-impedance planar lipid bilayers and shown to form channels which are heterogenous in size. The smallest channels had unitary conductances of 15 picoSiemens (pS) in 0.1 M NaCl. The closing of these channels showed voltage-dependence at membrane potentials exceeding 40 mV. These channels were more cation-selective, with K+ ions being favored over Na+. The 15-pS channels described here are much smaller than the channels attributed previously to either C5b-9 or polymerized C9 complexes but resemble channels formed by the C9b fragment, which does not polymerize into tubules. These results indicate that C5b-9 complexes are capable of damaging membranes by forming initially small ion channels which then aggregate in the membrane to form tubular lesions with much larger conductances. Like C5b-9, C5b-8 also increased membrane permeability. However, this increase in membrane conductance could not be resolved into single channels, suggesting that C5b-8 may induce membrane leakiness by perturbing the packing of membrane lipids, whereas addition of C9 results in authentic production of ion channels.

Cell Membrane Permeability↗
PubMed1986

Immunohistochemical analysis of C3 cleavage fragments, factor H, and the C5b-9 terminal complex of complement in de novo membranous glomerulonephritis occurring in patients with renal transplant.

Fifteen renal biopsies from 13 transplanted patients with de novo membranous nephropathy (DNMN) were investigated by immunofluorescence for the presence of C5b-9 neoantigens of the terminal sequence of complement and for antigens expressed by C3 cleavage fragments. DNMN lesions were classified as stage I, II or III upon light and electron microscopy examination. Seven biopsies were classified as stage I DNMN and 8 stage II-III. All patients were proteinuric. In six biopsies with stage I DNMN, staining for C5b-9 neoantigens was restricted to a fine granular labeling in mesangial areas which was analogous to that seen in normal kidneys in contrast with extensive parietal labeling for IgG, C3d and factor H antigens. In eight biopsies with stage II-III DNMN, the pattern of staining with anti-C5b-9 neoantigens antibodies was similar to that obtained with anti-IgG, anti-C3d and anti-factor H antibodies. These results suggest that in situ activation of the whole complement sequence throughout C5b-9 only occurs on large immune deposits (stage II-III DNMN).

Adolescent↗
PubMed2002

The membrane attack complex of complement induces caspase activation and apoptosis.

Activation of the terminal pathway of the complement system leads to insertion of terminal complement complexes (C5b-9) into the cell membrane, which may induce cytolysis. Recent data indicate that the terminal complement pathway can also result in apoptosis in vivo. To further define the cell death pathway induced by complement, we examined induction of apoptosis by complement in vitro. Rat mesangial cells opsonized with a complement-activating antibody and exposed to rat serum as a complement source underwent apoptotic cell death in a time- and dose-dependent fashion, as demonstrated by membrane exposure of phosphatidylserine and fragmentation of nuclei. No significant apoptosis was detected in either cultures treated with C6-deficient serum or in control cultures. The pan-caspase-inhibitor zVAD-fmk inhibited complement-induced apoptosis completely. In line with this observation, complement induced cleavage and activation of caspase 3. Importantly, cellular exposure to purified cytolytically inactive C5b-9, in the absence of antibody and early complement components, also resulted into caspase activation and apoptosis. Together, these results indicate that C5b-9 is involved in induction of apoptosis via a caspase-dependent pathway. Apoptosis as a consequence of complement-mediated cell damage may provide an explanation for the presence of apoptosis in inflammatory processes, for instance in hyperacute xenograft rejection.

Amino Acid Chloromethyl Ketones↗
PubMed1990

Human protectin (CD59), an 18,000-20,000 MW complement lysis restricting factor, inhibits C5b-8 catalysed insertion of C9 into lipid bilayers.

Human cells are relatively resistant to lysis by the homologous complement system. Here we describe the mechanism of action of a recently discovered and widely distributed 18,000-20,000 molecular weight (MW) membrane glycoprotein (CD59), which appears to act as a major protective element against complement-mediated lysis (hence called protectin). When incorporated into heterologous erythrocyte membranes, protectin efficiently prevented cell lysis by human serum. Neutralization with antibody of the naturally occurring protectin on human erythrocytes or on nucleated K562 cells increased their susceptibility to lysis by homologous complement. During complement activation, protectin became incorporated into the membrane attack complex (MAC). By interacting with newly exposed regions in the C5b-8 complex and in aggregating C9 it limited the number of C9 molecules associating with the C5b-8 complex to a C8:C9 ratio of 1:1.5 instead of a normal average of 1:3.5. The results demonstrate directly that protectin is a powerful inhibitor of complement cytolysis and acts by inhibiting the C5b-8 catalysed insertion of C9 into the lipid bilayer.

Antigens, Differentiation↗
PubMed2006

Candidate inhibitors of porcine complement.

Therapeutic complement inhibition is a promising strategy for treatment of a number of diseases as judged from rodent studies. The species distance from rodents to humans may limit the clinical relevance of these studies. The pig is an alternative animal for studies of human diseases like sepsis and ischemia/reperfusion injury. However, available complement inhibitors for use in pigs are scarce. The aim of the present study was to investigate and compare the efficacy of selected candidate inhibitors of porcine complement in vitro for possible future application in vivo. Sera from three different pigs were each incubated with three different activators of the complement system (zymosan, heat-aggregated immunoglobulin G (HAIGG) and Escherichia coli). Three groups of complement inhibitor candidates were tested: serine protease inhibitors (FUT-175 and C1-inhibitor), monoclonal antibodies (anti-factor B (fB) and anti-factor D (fD)) and a recombinant regulatory protein (vaccinia virus complement control protein (VCP)). Read-out was the terminal C5b-9 complement complex (TCC). The serine protease inhibitors FUT-175 and C1-inhibitor dose-dependently inhibited TCC formation in zymosan-, HAIGG- and E. coli-activated porcine sera, but with different efficacy. Complete inhibition of TCC was obtained using 0.2 mg/mL FUT-175, but required 16 mg/mL of C1-inhibitor. The monoclonal anti-fB and -fD antibodies both inhibited TCC formation dose-dependently, but in different ways. Anti-fB at high dose (1 mg/mL) completely inhibited TCC formation in sera activated with zymosan and virtually completely in sera activated with HAIGG, but not in sera activated with E. coli. Anti-fD inhibited all three activators at low dose (0.05 mg/mL), and approximately 50% TCC reduction was obtained. The recombinant complement regulatory protein VCP efficiently and dose-dependently inhibited TCC formation with a complete inhibition found at 0.05 mg/mL for all three activators. All candidates tested inhibited porcine complement activation, but in different ways and to different degrees. Of the complement-specific candidates, VCP inhibited all activators completely at low doses.

Animals↗
PubMed1975

Neoantigens of the membrane attack complex of human complement.

The membrane attack complex of complement is a fusion product of five complement proteins: C5b, C6, C7, C8, and C9. The complex causes complement-dependent cell membrane damage. It is assembled following complement activation both on the target cell surface and in the fluid phase. The isolated soluble complex, which has a molecular weight of one million, exhibited reduced expression of the antigenic determinants of the native precursor proteins. Antisera produced to the intact complex contained antibodies to neoantigens which were not detectable on the five precursor proteins. Antisera were rendered neoantigen-specific by adsorption with fresh human serum. Since the adsorbed antisera precipitated the complex, the complex must contain multiple neoantigenic sites. The complex-specific antibodies not only reacted with the soluble complex, but also with the target cell-bound membrane attack complex.

Antigens↗
PubMed1990

Quantitative measurement of SC5b-9 and C5b-9(m) in infarcted areas of human myocardium.

Previous immunohistochemical work has indicated that terminal C5b-9 complement complexes are selectively deposited in infarcted areas of human myocardium. In the present study, we sought to quantify C5b-9 levels in myocardial tissue, and to differentiate between the membrane-bound C5b-9 (m) and the cytolytically inactive SC5b-9 complex. Paired tissue specimens from infarcted and non-infarcted myocardium were obtained from 36 autopsies. The homogenized and washed tissues were extracted with n-octyl-beta-D-glucopyranoside (octylglucoside) detergent, and the concentrations of C5b-9 in the extracts were determined by ELISA. Membrane-derived C5b-9 (m) and SC5b-9 were differentiated from each other on the basis of their characteristic sedimentation behaviour in sucrose density gradients. It was found that infarcted myocardial tissue contained on average an approximately three-fold higher concentration of C5b-9, compared with non-infarcted tissue. This increase was due in part to an increase in levels of C5b-9 (m). The results corroborate previous immunohistochemical data and show that complement activation occurs to completion with the generation of potentially cytotoxic C5b-9 complexes in infarcted myocardial tissues.

Adult↗
PubMed1992

Isolated hyperthermic liver perfusion with cytostatic-containing perfusate activates the complement cascade.

Eight patients with advanced liver malignancy undergoing isolated hyperthermic liver perfusion with melphalan and cisplatin were studied with regard to complement activation and formation of anaphylatoxins (C3a and C5a) and terminal C5b-9 complement complexes (TCCs). Blood samples for complement variables (C1-INH, C3, C4, C5, C3a, C5a and TCCs) were taken before surgery, 1 min before the start of perfusion, 1, 2 and 3 h after the start of perfusion, and 24 h after operation. Samples were drawn from the perfusate 1 h after the start of perfusion. Activation of complement was observed during perfusion. Raised plasma concentrations of C3a and TCCs were recorded and high levels of C3a and TCCs were found in the perfusate. In vitro tests indicated that melphalan and cisplatin may activate complement. This activation occurred at 37 and 42 degrees C but was more pronounced at 42 degrees C.

Adult↗
PubMed1983

Role of the terminal complement pathway in experimental membranous nephropathy in the rabbit.

Our recent observations of a complement-mediated, cell-independent mechanism of altered glomerular permeability in rat membranous nephropathy suggested a possible role for the terminal complement pathway in the mediation of proteinuria in certain forms of glomerular disease. To directly determine whether the membranolytic terminal complement components (C5b-C9) are involved in glomerular injury, we studied the development of proteinuria in normal and C6-deficient (C6D) rabbits, in both of which a membranous nephropathy-like lesion develops early in the course of immunization with cationized bovine serum albumin (cBSA) (pI 8.9-9.2). C6 hemolytic activity of C6D was 0.01% that of control rabbits. After 1 wk of daily intravenous injections of cBSA, proteinuria developed in 71% of controls (median 154, range 1-3,010 mg/24 h, n = 24), whereas none of C6D were proteinuric (median 6, range 2-12 mg/24 h, n = 12, P less than 0.01). After 1 wk of cBSA, both groups had qualitatively identical glomerular deposits of BSA, rabbit IgG, and C3 on immunofluorescence microscopy, predominantly subepithelial electron-dense deposits on electron microscopy, and minimal glomerular inflammatory cell infiltration of glomeruli. Glomeruli were isolated from individual animals after 1 wk of cBSA and deposits of rabbit IgG antibody were quantitated by a standardized in vitro assay using anti-rabbit IgG-125I. Rabbit IgG deposits were found to be similar in control (29.8 +/- 13.2, range 12.7-48.6 micrograms anti-IgG/2,000 glomeruli, n = 6) and C6D rabbits (32.6 +/- 13.8, range 16.8-48.8 micrograms anti-IgG/2,000 glomeruli, n = 5, P greater than 0.05). After 2 wk, coincident with a prominent influx of mononuclear cells and neutrophils, proteinuria developed in C6D rabbits. These results document, for the first time, a requirement for a terminal complement component in the development of immunologic glomerular injury. Since the only known action of C6 is in the assembly of the membrane attack complex, these observations suggest that the membranolytic properties of complement may contribute to glomerular damage.

Animals↗
PubMed1993

Vitronectin-mediated inhibition of complement: evidence for different binding sites for C5b-7 and C9.

In the activated complement system, vitronectin (complement S-protein) occupies the metastable membrane binding site of the nascent precursor complex C5b-7, so that the newly formed SC5b-7 is unable to insert into cell membranes. Some evidence also indicates that vitronectin limits on-going membrane-associated pore formation by inhibiting C9 polymerization. It has been assumed that these two stages of terminal complement complex (TCC) inhibition take place through charge interactions between the heparin-binding region of vitronectin and homologous cysteine-rich sequences of the late complement proteins C6, C7, C8 and C9. We examined SC5b-7 formation and inhibition of C9 binding in the TCC using separate haemolytic assays. The mode of action of vitronectin in these assays was compared with two 15mer peptides which span residues 348-379 of the heparin-binding region, and a heparin-affinity polypeptide, protamine sulphate. The results showed that vitronectin acts predominantly through SC5b-7 production with a lesser effect on the inhibition of C9 lytic pore formation. In contrast, protamine sulphate did not prevent C5b-7 membrane attachment, but was a potent inhibitor of C9-mediated lysis. The peptides did not inhibit C5b-7 membrane insertion and only one affected C9 binding. These data suggest that the two stages of TCC inhibition involve separate binding sites on the vitronectin molecule. The site for association with nascent C5b-7 is unknown, whereas inhibition of C9 binding and pore formation takes place through the heparin-binding region.

Amino Acid Sequence↗