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PubMed1989

Deposits of terminal complement complex (TCC) in muscularis mucosae and submucosal vessels in ulcerative colitis and Crohn's disease of the colon.

Extensively washed, ethanol fixed and paraffin embedded colonic specimens from 15 patients with ulcerative colitis (UC) and nine patients with Crohn's disease (CD) of the colon, ileal specimens from six patients with CD of the ileum, and histologically normal control specimens obtained from 10 patients operated for colonic carcinoma, were examined by immunohistochemistry with a monoclonal antibody specific for a neoepitope in the C9 part of the terminal complement complex (TCC). The submucosal blood vessels in inflammatory bowel disease (IBD) showed significantly more TCC positivity than the controls, and vascular TCC deposition was statistically related (p less than 0.001) to degree of inflammation. Five of the six ileal CD specimens contained likewise vascular TCC deposits. In addition, five UC specimens and one colonic CD specimen contained TCC-positive fibrils in the muscularis mucosae or submucosa. There was no significant difference in vascular TCC deposits between UC and CD. The results suggested that terminal complement activation takes place in the intestinal lesions of IBD.

Adolescent↗
PubMed1998

Complement components and their activation products in pleural fluid.

STUDY OBJECTIVES: The aim of this study was to determine the role of complement components in pleural effusion measured with novel markers of complement activation, to assess which pathway of activation is predominant in different diseases, and to find out whether the analysis of complement components and their activation products could help in diagnostic procedure differentiating the etiologies of pleural effusion. PATIENTS: The study population consisted of 71 patients who had pleural effusion secondary to tuberculosis (n=23), rheumatic disease (n=10), or malignancy (n=38). MEASUREMENTS: Complement components and their activation products, including the soluble terminal complex SC5b-9, were measured in plasma and pleural fluid. RESULTS: In all patients with rheumatic pleurisy, pleural fluid SC5b-9 was higher than 2 AU/mL and in all patients with malignant pleural fluid it was lower than 2 AU/mL. The mean level of SC5b-9 in rheumatic pleural effusion was also significantly higher than in tuberculosis. In addition, the concentrations of pleural fluid C3 and C4 were significantly lower and the ratio C4d/C4 was significantly higher in rheumatic compared with tuberculous or malignant pleurisy. In plasma, both SC5b-9 and C1s-C1r-C1INH-complexes were significantly higher in rheumatic subjects than in other patients. In stepwise multinominal logistic regression analyses, the most significant predictors for rheumatic pleural fluid were high pleural fluid SC5b-9 and low C4. CONCLUSIONS: These observations indicate that the complement cascade is activated through both the classic and the alternative pathways in rheumatic pleurisy. Determinations of SC5b-9 and C4d/C4 in pleural fluid were the best variables differentiating rheumatic, tuberculous, and malignant effusions.

Adult↗
PubMed1987

Sensitive ELISA for quantitating the terminal membrane C5b-9 and fluid-phase SC5b-9 complex of human complement.

Activation of the complement system to completion results either in the generation of a pore-forming, cytolytic C5b-9(m) complex, or of a cytolytically inactive, fluid-phase SC5b-9 complex. In this paper, we describe a sensitive and reliable, sandwich ELISA for C5b-9(m) and SC5b-9, which is based on the use of a monoclonal antibody to a neoantigen of C5b-9 in combination with affinity-purified, polyclonal rabbit antibodies. The ELISA has been calibrated with purified C5b-9(m) and SC5b-9, and can detect 3 ng/ml C5b-9(m) and 20 ng/ml SC5b-9. We show that maximal conversion of C5-C9 in pooled human serum by insulin or zymosan activation generates 220 +/- 40 micrograms/ml SC5b-9. 65 of 100 normal human EDTA plasma samples analyzed in this study contained 100-600 ng/ml SC5b-9, corresponding to 0.04-0.24% of maximal conversion. Levels of circulating SC5b-9 in other donors were below the limit of detection. Incubation of serum at 37 degrees C always led to spontaneous generation of SC5b-9; concentrations ranged from 490-4725 ng/ml after 60 min, 37 degrees C, with a mean of 1848 +/- 1031 (SD) ng/ml amongst 25 donors studied. The terminal complement complex present in EDTA plasma was partially purified by PEG precipitation, DEAE-ion exchange chromatography and sucrose density gradient centrifugation, and was found to contain C8, C9 and S-protein as demonstrable by SDS-PAGE immunoblotting. Thus, the material most probably represented genuine SC5b-9. No significant age- or sex-dependent variations in SC5b-9 levels were noted. The present data call for a critical re-appraisal of several previously published methods for the determination of SC5b-9 levels in human plasma and serum.

Antibodies, Monoclonal↗
PubMed1988

The role of the C9b domain in the binding of C9 molecules to EAC1-8 defined by monoclonal antibodies to C9.

Three mAb to human C9, X195, X197, and P40 were used to analyze the roles of the C9a and C9b domains in the reaction of the C9 molecule with sensitized sheep E bearing C1 to C8 (EAC1-8). X195 bound to NH2-terminal (C9a) fragments, and X197 bound to COOH-terminal (C9b) fragments obtained by cleavage of C9 with alpha-thrombin or trypsin. P40 recognized the epitope on the C9b fragment obtained by alpha-thrombin cleavage but did not react with the NH2-terminal or COOH-terminal fragment obtained by trypsin cleavage. In this respect, P40 differed from mAb to C9 reported previously. P40 almost completely inhibited the hemolytic activity of C9. X195 and X197 also inhibited C9 activity, but less effectively than P40. C9 molecules bound to P40 could not bind to EAC1-8 cells. C9 bound to X197 could not bind rapidly to EAC1-8, but prolonged incubation of the C9-X197 complex with EAC1-8 caused considerable lysis of the cells. C9 molecules bound to X195 could bind rapidly to EAC1-8, but their lytic activity was partially inhibited by the bound antibody. From these results, it is concluded that the C9b but not C9a domain contributes to the binding of C9 to EAC1-8 and that the epitope recognized by P40 or a closely adjacent site may be the binding site of C9 molecule to EAC1-8.

Animals↗
PubMed1989

Complement activation and endotoxin levels in systemic meningococcal disease.

The activation state of the complement system in 39 consecutively admitted patients with systemic meningococcal disease was studied prospectively using two monoclonal antibodies reacting with neoepitopes exposed during complement activation. The fluid-phase C3 activation products and SC5b-9 (terminal complement complex) were strongly correlated to the levels of endotoxin (lipooligosaccharides, LOS) in plasma on admission (r = .79, P less than .0001 and r = .76, P less than .0001, respectively) and to fatality. Maximum complement activation in survivors occurred 7h (median; range 0-44 h) after initiation of antibiotic treatment. The most severely ill patients had the capacity to activate the whole complement cascade. In nonsurvivors, high-grade activation often continued until the patients died. The results suggest that LOS are important activators of complement in systemic meningococcal disease and that complement-activating products, in concert with other mediators, may contribute to the multiple organ failure and death occurring in the most severe cases.

Complement Activation↗
PubMedDate not supplied

Identification of a homologue of CD59 in a cyclostome: implications for the evolutionary development of the complement system.

We have employed a COS cell expression cloning procedure to isolate a full length cDNA clone encoding a hagfish leukocyte-associated membrane protein (HLMP1). The protein, which is identified by a monoclonal antibody (JB3) generated in our laboratory, is present on the majority of hagfish leukocytes and is also expressed on erythrocytes. The cDNA clone contained an open reading frame encoding a 120 residue polypeptide which exhibits 33% amino acid sequence identity with the precursor protein of human CD59, a leukocyte-associated membrane protein which regulates the action of the complement membrane attack complex on homologous cells. CD59 belongs to a family of structurally related glycoproteins which includes the Ly-6 proteins expressed on mouse lymphocytes. In addition to significant overall sequence homology HLMP1 shows conservation of 8 key cysteine residues with members of the CD59/Ly-6 family. Comparison of the hagfish sequence with that of the mature human CD59 protein suggested a processed protein consisting of 74 amino acids associated with the cell membrane via a GPI anchor. The latter was confirmed by immuno-flow cytometry following treatment of transfected COS cells with phospholipase. Phylogenetic analysis and tissue distribution of this protein in the hagfish are consistent with HLMP1 being a homologue of CD59. A three-dimensional model of HLMP1, constructed using the NMR-determined structure for human CD59 as a template, indicated conservation of a core structure of five strands of beta-sheet and a short helix stabilised by four disulfide bonds. These findings, when taken together with our previous identification of C5a-like chemotactic activity in LPS-activated serum, provide indirect evidence for the existence of the terminal lytic complement pathway (C5 to C9) in these primitive vertebrates.

Animals↗
PubMed1988

Detection of the terminal complement complex in patient plasma following acute myocardial infarction.

The mechanisms of inflammation responsible for the myocardial tissue damage seen after an acute myocardial infarction (AMI) have not been clearly identified. Recent lines of evidence, demonstrating depressed sera levels of individual complement components in patients after myocardial infarction, have suggested involvement of the complement (C) system in micro- and macrovascular injury subsequent to AMI. The present study assessed the role of complement as a mediator of myocardial inflammation by quantifying products of complement activation including, the terminal complement complex (TCC) the cytolytic component of the complement system, C1rC1s-C1 inhibitor complex and C3bBbP complex, formed following activation of the classical and alternative pathway, respectively, and anaphylatoxins C3a and C5a in 41 patients following AMI. Plasma TCC and C1rC1s-C1 inhibitor complex concentrations increased up to 32-fold (P less than 0.001) and 8-fold (P less than 0.001), respectively, while the C3bBbP complex, C3a des-Arg and C5a des-Arg each increased over 2-fold (P less than 0.001) 16 h after AMI, and were only minimally detectable during non-inflammatory myocardial conditions. Furthermore, TCC concentrations increased over 150% (P less than 0.001) one day after patients reinfarcted, subsequent to hospitalization for a primary AMI. These results demonstrate activation of complement after AMI and suggest that inflammatory mediators of the complement system may contribute to myocardial tissue damage during the infarction process.

Adult↗
PubMed1985

Localization of the terminal C5b-9 complement complex in the human aortic atherosclerotic wall.

The terminal C5b-9 complement complex was investigated in 15 aortic, 2 femoral fibrous plaques and 5 fatty streaks aortic intimae using indirect immunofluorescence and immunoperoxidase. All the fibrous plaques presented C5b-9 deposit-like threads in the fibrous cap and masses in the amorphous areas of the plaque. The deposits were frequently associated with other immune-related proteins such as: IgG, IgA, IgM, Clq, C3c and C4 which were simultaneously investigated. Fatty streaks intimae presented no C5b-9 and complement component deposits. Whereas the demonstration of the complement components could merely reflect a non-specific trapping, the presence of assembled C5b-9 in the damaged tissue is more indicative of the involvement of complement activation in the progression of atherosclerotic lesions.

Adult↗
PubMed1999

Inhibition of oligodendrocyte apoptosis by sublytic C5b-9 is associated with enhanced synthesis of bcl-2 and mediated by inhibition of caspase-3 activation.

We have previously shown that generation of sublytic C5b-9, the membrane attack complex of complement, induces oligodendrocytes to enter cell cycle and reduces apoptotic cell death in vitro. In the present study, the cellular factors involved in apoptosis of oligodendrocyte progenitor cells and oligodendrocytes, and the inhibitory effect of C5b-9 on apoptotic process were investigated. Oligodendrocyte progenitor cells identified by mAb A2B5 that were isolated from neonatal rat brains were differentiated into oligodendrocytes in serum-free defined medium. The differentiation, which occurs simultaneously with apoptotic cell death, was associated with a rapid loss of bcl-2 mRNA and increased expression of caspase-3 mRNA. Activation of caspase-3 in differentiating cells was demonstrated by the generation of 17- and 12-kDa fragments of caspase-3 proenzyme and by cleavage of poly(ADP-ribose) polymerase, a specific caspase-3 substrate. Cell death associated with differentiation was inhibited by the caspase-3 inhibitor DEVD-CHO in a dose-dependent manner. Assembly of sublytic C5b-9 resulted in inhibition of caspase-3 activation. In addition, synthesis of BCL-2 protein in oligodendrocytes was significantly increased by C5b-9. The TNF-alpha-induced apoptosis of oligodendrocytes was also inhibited by C5b-9. These results indicate that up-regulation of BCL-2 protein and inhibition of caspase-3 activation are potential mechanisms by which C5b-9 increases survival of oligodendrocyte in vitro and possibly in vivo during inflammation and immune-mediated demyelination affecting the CNS.

Animals↗
PubMed1995

Study of the in vitro activation of the complement alternative pathway by Echinococcus granulosus hydatid cyst fluid.

In the present study we have investigated the fluid phase activation of the complement (C) alternative pathway by Echinococcus granulosus sheep hydatid cyst fluid (SHCF) and its higher molecular weight fraction (SHCF-I) by quantitating the formation of both the terminal C intermediary C5b6 complex and the terminal C complex (TCC). Our results show that in vitro C activation progresses beyond the C5 step suggesting that potentially lytic complexes may be generated in vivo. In addition, SHCF and SHCF-I glucidic moieties are probably involved in C activation since 80% and 86% of SHCF and SHCF-I activity respectively was destroyed by periodate oxidation. Furthermore, partial deglycosylation with Peptide N-Glycosidase F of SHCF-I which had been digested with Pronase E, released an active fraction (MW < 14 KDa) which bound to Soybean agglutinin, suggesting that N-linked oligosaccharides containing alpha- or beta-linked N-acetyl galactosamine play a role in C activation by SHCF.

Acetylgalactosamine↗
PubMed2002

Extensive deposits of complement C3d and C5b-9 in the choriocapillaris of eyes of patients with diabetic retinopathy.

PURPOSE: To determine the presence of activated complement components in eyes affected by diabetic retinopathy. METHODS: Eyes of 50 deceased donors with diabetic retinopathy and of 10 nondiabetic subjects with uveal melanoma (n = 6) or phthisical eyes (n = 4), as well as eyes of 16 deceased donors without diabetic retinopathy were subjected to immunohistochemical studies, using a panel of antibodies directed against candidate markers of complement activation. RESULTS: Extensive deposits of complement C5b-9 complexes were detected in the choriocapillaris immediately underlying the Bruch membrane and densely surrounding the capillaries, in all 50 diabetic retinopathy specimens. Complement deposition was sometimes also observed around the larger choroidal vessels. Similarly intense staining for C5b-9 was absent in 25 of the 26 of the other donor eyes. Positive staining was observed in a case of systemic amyloidosis. Staining for C3d positively correlated with C5b-9 staining, corroborating the notion that complement activation had occurred in situ. Furthermore, positive staining was found for vitronectin, which forms stable complexes with extracellular C5b-9. When present, deposits under the pigment epithelium and drusen also stained positively for the activated complement components, independent of diabetic retinopathy. In contrast, there was no positive staining for C-reactive protein (CRP), mannose-binding lectin (MBL), C1q, or C4, indicating that complement activation did not occur through a C4-dependent pathway. CONCLUSIONS: The presence of C3d, C5b-9, and vitronectin indicates that complement activation occurs to completion, possibly through the alternative pathway in the choriocapillaris in eyes affected by diabetic retinopathy. Complement activation at this site may evoke a spectrum of pathologic sequelae that could contribute to ocular tissue disease and visual impairment.

Aged↗
PubMed1988

The terminal complement complex, C5b-9, a marker of disease activity in patients with systemic lupus erythematosus.

Concentrations of the terminal complement complex (TCC), C5b-9, were examined in 120 serum samples from 28 patients with systemic lupus erythematosus. Eleven patients with various manifestations of the disease were followed longitudinally for a 2-year period during active and inactive phases of the disease. In 9 of the 11 patients, elevations in TCC concentrations correlated with disease exacerbations. In many of these patients, C3 and C4 levels remained normal during the study and were less sensitive indicators of disease activity than were TCC concentrations. We believe that measurements of TCC are useful in monitoring patients with rheumatic diseases in which complement activation is a component.

Adult↗
PubMed1986

Does complement kill E. coli by producing transmural pores?

Three lines of evidence are presented to indicate that C5b-9 kills serum-sensitive E. coli K 12 cells by generating functional pores across the outer and inner bacterial membrane. First, viable cells carrying C5b-8 complexes are impermeable to o-nitrophenyl-beta-D-galactoside (ONPG), but lose viability and become permeable to this marker upon post-treatment with purified C9 in the absence of lysozyme. Cells killed with colicin E1 or gentamicin are also impermeable to ONPG but take up the marker if they are post-treated with lysozyme-free serum. Second, killing by C5b-9 is highly effective, deposition of only a small number of complexes being lethal. This has been demonstrated in experiments where viable cells carrying 2000-4000 C5b-7 complexes per CFU were permitted to multiply in broth culture, and the daughter generations subsequently treated with purified C8 and C9. Fifty percent killing was observed in the fifth to sixth generation, corresponding to a dilution of C5b-7 complexes to 50-100 molecules/CFU. In the presence of 2 mM EDTA, further dilution of C5b-7 down to 8-30 complexes/CFU still caused 50% killing of daughter cells. Third, treatment of C5b-7 cells with purified CC8 and C9 results in the release of intracellular K+, which commences immediately after addition of C8/C9. This was shown in experiments where C5b-7 cells were packed to high density in saline, post-treated with C8 + C9, and K+ directly measured in the cell supernatants. Based on these results, we propose that C5b-9 pores deposited in the outer bacterial membrane periodically fuse with the inner membrane, the transmural pores thus generated permitting rapid K+ efflux, with cell death ensuing through the collapse of membrane potential.

Bacteriolysis↗
PubMed1983

Deposition of the terminal C5b-9 complement complex on erythrocytes by human red cell autoantibodies.

A radioassay for the detection of complement activating autoantibodies (autohaemolysins) in patients with autoimmune haemolytic anaemia (AIHA) is described. The method is based on immunoradiometric quantitation of the cytolytic C5b-9 complement complex following its antibody-dependent deposition on red blood cells (RBC). The use of affinity-purified, radiolabelled antibodies directed against the neoantigens of the C5b-9 complex ensured specificity of the test which proved more sensitive than conventional haemolytic assays and was not subject to disturbances by haemolytic sera. The results obtained in 70 patients with various forms of AIHA (warm type N = 45); cold type (N = 22); Donath-Landsteiner type (N = 3] support the prevailing assumption that autohaemolysins can trigger the complement cascade to completion. Since intact RBC from patients with detectable autohaemolysins carried C3/C4 components but never C5b-9, it is inferred that regulatory mechanisms operate in vivo at the level of C4b/C3b inactivation to arrest the cascade and rescue the autologous cells.

Adolescent↗
PubMed1995

Membrane attack complex deposits in cutaneous lesions of dermatomyositis.

BACKGROUND AND DESIGN: The presence of membrane attack complex of complement (MAC) deposits in the intramuscular vasculature of biopsy specimens taken from patients with dermatomyositis (DM) has been implicated in the pathogenesis of this myopathy. The purpose of this study was to investigate the presence of MAC deposition in the skin lesions of patients with DM. Using immunohistochemical methods, we examined 22 biopsy specimens from lesional skin, six biopsy specimens from uninvolved skin, and 12 muscle biopsy specimens from patients with DM for the presence of MAC and vitronectin and CD59, two regulatory proteins of complement. RESULTS: The deposition of MAC was demonstrated in a large percentage of biopsy specimens obtained from the lesional skin of patients with DM. Deposits were found along the dermoepidermal junction in 19 (86%) of 22 biopsy specimens. Deposits on the vessel walls of the dermis were found in 17 (77%) of 22 biopsy specimens; but only in six of these biopsy specimens (27%) were deposits present in more than 10% of blood vessels. In contrast, deposits along the dermoepidermal junction and the vessel walls of the dermis were absent in specimens from uninvolved skin. In 12 muscle biopsy specimens obtained simultaneously from these patients, MAC deposits were found on the vessel walls in nine (75%), but only in six (50%) were deposits found in more than 10% of the intramuscular vessels. The pattern of vitronectin immunoreactivity in skin and muscle biopsy specimens obtained from patients with DM was similar to MAC deposits. The expression of CD59 was normal in all skin and muscle biopsy specimens. CONCLUSIONS: The deposition of MAC was found in a high percentage of biopsy specimens from the lesional skin of patients with DM; it was absent in uninvolved skin. These findings suggest that the complement system may be involved in the pathogenesis of the skin lesions of DM.

Adolescent↗
PubMed1989

Prelesional complement activation in experimental atherosclerosis. Terminal C5b-9 complement deposition coincides with cholesterol accumulation in the aortic intima of hypercholesterolemic rabbits.

Cholesterol accumulation in the tunica intima of large and medium sized arteries is a characteristic of atherosclerotic lesion development. Cholesterol activates the complement system in vitro generating C5b-9 complexes, and C5b-9 antigens have been observed in atherosclerotic lesions. We therefore investigated whether complement activation correlates temporally with accumulation of intimal cholesterol in vivo. Frozen sections of aortic tissue from rabbits fed a 0.3% cholesterol diet for 0, 2, 6, and 10 weeks were stained with antibodies directed against neoantigens of the C5b-9 complex. Lipids were detected with oil red O, filipin, and antibodies against apoprotein B. C5b-9 complexes were observed to colocalize with extracellular lipid in the subendothelial space beginning at 2 weeks on the diet and extending past 10 weeks. Quantitation of aorta extracted C5b-9 complexes by an enzyme-linked immunosorbent assay revealed a progressive increase in aortic C5b-9 content as a function of time on the cholesterol diet. An approximately 30-fold increase in aortic C5b-9 occurred between 0 and 10 weeks on the diet, whereas plasma C5b-9 remained at normal levels throughout this time. Aortic cholesterol and triglyceride levels approximately doubled between 0 and 10 weeks on the diet and plasma cholesterol increased nearly 50-fold. Intimal C5b-9 deposition preceded monocyte infiltration and foam cell development. The data indicates that early cholesterol accumulation in the aortic intima results in complement activation. Since complement activation products are chemotactic for monocytes and promote their adhesion to the endothelium, complement may provide a mechanism by which monocytes are attracted to arterial regions of lipid accumulation.

Animals↗
PubMed1987

Evidence that C5b recognizes and mediates C8 incorporation into the cytolytic complex of complement.

The aim of this study was to identify constituents of the intermediate C5b-7 complex of human complement that mediate binding of C8 and formation of C5b-8. Analysis of interactions between purified C8 and C5, C6, or C7 indicate that C5 and C8 associate to form a dimer in solution. This interaction is specific and involves a single C5 binding site located on the beta-subunit of C8. Simultaneous interaction of C8 with C5 and C9 in solution suggests that during assembly of the cytolytic C5b-9 complex on membranes, C8 binds to C5b-7 through association of beta with C5b, after which C9 associates through interaction with the previously identified C9-specific site on the alpha-subunit. Other evidence of interaction with C5b was provided by the fact that C8 can bind purified C5b6. Also, in situ cross-linking experiments showed that within C5b-8, the beta-subunit is in close proximity to C5b. These results indicate that C8 binding to C5b-7 is mediated by a specific C5b recognition site on beta, thus explaining the requirement for this subunit in C5b-8 formation. They also reveal that C5b contains a specific site for interaction with beta.

Binding Sites↗
PubMed1991

Evasion of Yersinia enterocolitica serotype 03 from complement-mediated killing.

Using isogenic strains of Y. enterocolitica serotype 03 (isolate 75) differing in LPS side chains (S and R), plasmid content (p+ and p-) or selective failure of YOP1 expression (YOP1-) we observed comparable C9 consumption via the alternative pathway by all strains. Differences became apparent in the bactericidal assays in which the 75S p+ strain was resistant whereas the plasmid-negative S and R strains were killed. Increased but submaximal resistance was observed with the 75R p+ and 75S YOP1- strains indicating that LPS side chains and plasmid-encoded factors other than YOP1 also contribute to serum resistance. Deposition of C9 and terminal complement complex (TCC) formation were greatly reduced on the resistant 75S p+ strain compared to the sensitive 75S p- strain. The 75S YOP1- variant behaved like the 75S p- strain with respect to C9 deposition and TCC formation suggesting that YOP1 prevents terminal C activation. A comparison of TCC deposited on resistant and sensitive Y. enterocolitica revealed no differences with respect to their salt- and protease-resistance and their size. We conclude from our experiments that serum resistance of 75S p+ depends on several surface components YOP1 being the most important. The YOP1 protein clearly interferes with C9 deposition and TCC formation and thereby contributes to serum resistance of plasmid-positive, YOP1 expressing Y. enterocolitica.

Centrifugation, Density Gradient↗