Stimulation of arachidonate metabolism by endotoxin and the terminal complement complex C5b-8.
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Animals↗
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Transfer of preformed terminal C5b-9 complement complexes into the outer membrane of viable gram-negative bacteria: effect on viability and integrity.
An efficient fusion system between Gram-negative bacteria and liposomes incorporating detergent-extracted C5b-9 complexes has been developed that allows delivery of preformed terminal complexes to the cell envelope (Tomlinson et al., 1989b). Fusion of Salmonella minnesota Re595 and Escherichia coli 17 with C5b-9-incorporated liposomes resulted in the transfer of 1900 C5b-9 complexes to each target bacterial cell. No loss in viability of bacteria was observed following fusion, even though the deposotion of 900 complexes onto the envelope following exposure to lysozyme-free serum effected a greater than 99% loss of viability. Increased sensitivity to antibiotics normally excluded from the cell by an integral outer membrane (OM), as well as the ability of the chromogenic substrate PADAC to gain access to periplasmically located beta-lactamase, indicated that transferred C5b-9 complexes functioned as water-filled channels through the OM. A similar conclusion was drawn from measurements demonstrating the uptake by cells of the lipophilic cation tetraphenylphosphonium (bromide), a result further indicating that the membrane potential across the cytoplasmic membrane was maintained following C5b-9 transfer to the OM. Examination of S. minnesota Re595 by electron microscopy revealed no obvious difference between cells exposed to lethal concentrations of lysozyme-free serum and cells following fusion with C5b-9-incorporated liposomes. These data suggest either that there are critical sites in the OM to which liposome-delivered C5b-9 complexes are unable to gain access or that bacterial cell death is related to events occurring during polymerization of C9 on the cell surface.
Cell Membrane↗
Size distribution and stability of the trans-membrane channels formed by complement complex C5b-9.
We have performed double marker sieving experiments with molecules ranging from ca. 0.5-3 nm dia. in order to evaluate the size distribution of the channels formed by complement in resealed sheep erythrocyte ghosts. Evidence is presented that marker release through the channels reached equilibrium between the ghosts and the extracellular fluid in a period of 3 hr and that the channels are stable at 37 degrees C for this period of time. Under these experimental conditions we have observed a differential in the endpoint release of inositol and sucrose, which indicates that some of the ghosts carried channels measuring between 0.7 and 0.9 nm dia. No differential was observed between release of sucrose and raffinose (0.9 and 1.1 nm mol. dia., respectively). Comparisons between sucrose and inulin (0.9 and 3 nm mol. dia, respectively) showed a difference in marker release. Also, there was substantial release of inulin, indicating the presence of channels above 3 nm in dia. Hence, the present data indicate formation of channels in three size ranges, namely, 0.7-0.9 nm, 0.9-3 nm and greater than 3 nm.
Animals↗
Presence of C5b-9 complement complex and S-protein in human myocardial areas with necrosis and sclerosis.
Myocardial fragments with acute infarction (10 cases), scars after chronic infarction (6 cases), areas with focal sclerosis and necrosis (8 cases) compared with normal myocardial areas (8 cases), were processed for indirect and double-labelling immunoperoxidase techniques to localize C5b-9 neoantigens, S-protein, C3d and apolipoprotein B. Granular masses of C5b-9 and C3d and diffuse areas of S-protein and apolipoprotein B were localized in the acute or chronically damaged areas but not in areas free of lesion. Double-labelling data revealed similarly damaged areas of localization for C5b-9 and S-protein, and for C3d and apolipoprotein B, respectively, on rather different than usual tissue structures. C5b-9 determination by ELISA from myocardial eluates revealed lower levels of neoantigens in normal areas (2.3 +/- 0.3 micrograms/g dried tissue), higher levels in areas with sclerosis (7.9 +/- 0.7 micrograms/g dried tissue) and the highest amounts in areas with acute infarction (11.1 +/- 1.2 micrograms/g dried tissue). The presence of C5b-9 neoantigens in damaged myocardial areas with a different localization than S-protein is suggestive of local complement activation.
Complement Membrane Attack Complex↗
Effect of the late complement components C5b-9 on human monocytes: release of prostanoids, oxygen radicals and of a factor inducing cell proliferation.
Recently, we reported stimulation of rat macrophages and human platelets by isolated C5b-9 to synthesize prostaglandin E (PGE) or thromboxane B2 (TXB2). In the present study, we tested whether besides prostanoids, C5b-9 also would induce the production of other mediators. We found that C5b-9 in sublytic concentrations stimulated human granulocytes (polymorphonuclear leukocytes) or monocytes to release oxygen radicals. Furthermore, monocytes release interleukin-1 in response to C5b-9. Thus, besides having a lytic capacity, C5b-9 also functions as a stimulator of various cells.
Complement Membrane Attack Complex↗
Tyrosine phosphorylation and activation of Janus kinase 1 and STAT3 by sublytic C5b-9 complement complex in aortic endothelial cells.
The pathway involving Janus kinase (JAK) and signal transducers and activators of transcription (STATs) plays an important role in differentiation and proliferation of cells initiated by receptor activation. In the present study we identified the JAK and STAT proteins activated by C5b-9 in human aortic endothelial cells (AEC). JAK1 but not JAK2 was tyrosine phosphorylated in response to sublytic C5b-9. STAT3 was rapidly tyrosine phosphorylated also by C5b-9. Pertussis toxin inhibited the C5b-9 induced JAK1 activation. However, phosphorylation of STAT3 was not inhibited by Pertussis toxin, although C5b-9 induced a time-dependent nuclear translocation of STAT3. These observations indicated that JAK1 is phosphorylated by C5b-9 through activation of trimeric G proteins of the Gi/Go family. Raf-1 and ERK1 were also activated by C5b-9 in human AEC in a G protein dependent manner. Therefore, JAK1 activity may be involved in activation of Raf-1 and ERK1 via G proteins activated by C5b-9. This study demonstrates the ability of membrane-inserted C5b-9 to activate JAK1 and STAT3 proteins, thus defining new signalling pathway by which C5b-9 may regulate gene activation.
Adult↗
Interaction between terminal complement proteins C5b-7 and anionic phospholipids.
We have recently shown that C5b-6 binds to the erythrocyte membrane via an ionic interaction with sialic acid before the addition of C7 and subsequent membrane insertion. In this study we assessed the role of anionic lipids in the binding of the terminal complement proteins to the membrane and the efficiency of subsequent hemolysis. Human erythrocytes were modified by insertion of dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylserine (DPPS), dipalmitoyl phosphatidylethanolamine (DPPE), or dipalmitoyl phosphatidic acid (DPPA). Lipid incorporation and the hemolytic assays were done in the presence of 100 micromol/L sodium orthovanadate to prevent enzymatic redistribution of lipid. We found that the neutral lipids, DPPC and DPPE, did not affect C5b-7 uptake or hemolysis by C5b-9. In contrast, the two acidic phospholipids, DPPS and DPPA, caused a dose-dependent increase in both lysis and C5b-7 uptake. We conclude that the presence of anionic lipids on the exterior face of the membrane increases C5b-7 uptake and subsequent hemolysis. It is known that sickle cell erythrocytes have increased exposure of phosphatidylserine on their external face and are abnormally sensitive to lysis by C5b-9. The data presented here provide a plausible mechanism for this increased sensitivity.
1,2-Dipalmitoylphosphatidylcholine↗
Terminal complement complex (C5b-9) in children with recurrent hemolytic uremic syndrome.
Recurrent hemolytic uremic syndrome (recHUS) is a heterogeneous group of disorders. The pathogenesis of recHUS is not fully understood. recHUS has a high risk of development of terminal renal insufficiency and other sequelae. Abnormalities in complement factor H or in membrane-bound complement inhibitors with consecutive complement activation can be found in approximately 30 to 50% of the patients. Starting in 2001, we evaluated 42 patients with recHUS from five European countries (Germany, Austria, Hungary, Switzerland, and the Czech Republic). We measured the terminal complement complex (TCC) by an enzyme-linked immunosorbent assay using a neoepitope-specific anti-C9 antibody in 17 patients in plasma (native complement activation), serum (after coagulation), and zymosan-activated serum (Z-serum; after stimulation of coagulation). We compared the results to those of 16 healthy persons. In patients with recHUS (eight males, nine females) with a median age of 10.8 years, the TCC values were higher in plasma (0.57 versus 0.48 microg/mL; P = 0.04) and serum (3.1 versus 2.2 microg/mL) than in those of the control group, with a median age of 28.6 years (six males, 10 females) The TCC values in patients with low C3 compared with patients with normal C3 levels were even higher in plasma and serum, and the ratio was much lower. Children with recHUS have higher concentrations of TCC in plasma and serum. The ratio of Z-serum to serum showed significantly lower values in children with recHUS (96.01 versus 150.3; P = 0.01). These findings indicate a higher grade of complement activation and consumption in recHUS, suggesting that TCC may mediate cell toxicity. This may play an important role in the inferior outcome of these patients. The isolated substitution of factor H, or other complement inhibitors to block TCC formation, may represent useful therapies for these patients.
Adolescent↗
Immunohistochemical alterations after muscle trauma.
The proteins fibrin, fibrinogen, fibronectin and complement C5b-9 were investigated in mechanically damaged skeletal muscle. An accumulation of fibrin, fibrinogen and fibronectin could be observed immediately after intra-vital trauma in damaged fibre zones, later an accumulation at the torn edges of the fibres. The accumulation of complement C5b-9 began one hour after trauma. After post mortem trauma no positive reactions could be observed for any of the proteins. The degree of expression of these proteins can therefore be used to differentiate between vital and postmortem muscle damage as well as the estimation of wound age in the early antemortem time period.
Animals↗
Complement proteins C5b-9 stimulate procoagulant activity through platelet prothrombinase.
The capacity of platelets treated with nonlytic concentrations of the C5b-9 proteins to catalyze prothrombin activation and thereby trigger clot formation has been investigated. When suspended in the presence of exogenous factors Xa and Va, gel-filtered platelets treated with purified C5b-9 proteins catalyzed prothrombin to thrombin conversion at rates up to tenfold above controls, and exceeded by up to fourfold the prothrombinase activity observed for thrombin-stimulated platelets. In the absence of added factor Va, C5b-9 assembly on the platelet surface significantly shortened the lag period before prothrombinase expression that was observed for untreated platelets and increased the maximum catalytic rate of thrombin formation. A comparison with other platelet stimuli revealed that the C5b-9-induced activation of platelet prothrombinase closely paralleled the effects mediated by calcium ionophore A23187. Our data suggest that the C5b-9 proteins promote the release of platelet factor V and the assembly of the prothrombinase complex, thereby potentiating the effects of thrombin on the activation of prothrombinase. Membrane assembly of the C5b-9 proteins was also observed to markedly accelerate the rate of platelet-catalyzed plasma clotting, suggesting a direct link between C5b-9-mediated prothrombinase activation and procoagulant activity accompanying immunologic damage to the platelet.
Blood Coagulation↗
Studies of complement complex C5b,6 eluted from--EAC-6: reaction of C5b,6 with EAC4b,3b and evidence on the role of C2a and C3b in the activation of C5.
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Animals↗
Analysis of solute diffusion across the C5b-9 membrane lesion of complement: evidence that individual C5b-9 complexes do not function as discrete, uniform pores.
We have investigated the diffusion of radiolabeled nonelectrolytes across the membranes of resealed erythrocyte ghosts that had been treated with the terminal complement components C5b-9 and incubated under nonlytic steady state conditions. For all solutes tested, we note that diffusion across the C5b-9 lesion is retarded to rates more than 2 orders of magnitude slower than can be anticipated for a transmembrane diffusional channel of the dimensions suggested by the ultrastructure of the C5b-9 membrane lesion. Furthermore, direct measurement of the relative selectivity of the C5b-9 membrane lesions to permeation by solutes of differing molecular radii suggests that individual membrane lesions are not of uniform functional size. Data are presented that suggest that the functional heterogeneity of the membrane lesion is due to the aggregation of individual membrane bound C5b-9 complexes into larger functional units and not due to variable stoichiometry of C9 within the individual C5b-9 complex.
Cell Membrane Permeability↗
Thromboxane B2 synthesis in human platelets induced by the late complement components C5b-9.
Sublytic doses of purified C5b6, C7, C8 and C9 induce the release of thromboxane B2 (TXB2) in human platelets. In the present study, we attempted to analyze the signal by which C5b-9 triggers the prostanoid synthesis in platelets. TXB2 release was accompanied by liberation of Ca++ from intracellular stores. Influx of Ca++ was not observed, indicating that C5b-9 did not form a channel large enough to permit passage of Ca++ ions. Therefore, channel formation is apparently not required for cell stimulation.
Arachidonic Acid↗
Arachidonic acid mobilization and phosphoinositide turnover by the terminal complement complex, C5b-9, in rat oligodendrocyte x C6 glioma cell hybrids.
Previously, we have shown that rat oligodendrocytes release phospholipid and generate arachidonic acid (AA) and leukotriene B4 in response to sublytic C5b-9 formation. In the present study, we investigated the biochemical pathways by which C5b-9 generates AA from clone ROC-1, a fusion product of rat oligodendrocytes and C6 glioma. Cells were incubated for 24 h in the presence of [3H]AA or [3H]myoinositol. They were then sensitized with antibody against hybrid cell stroma and treated for 1 h with C9-depleted human serum (C9D-HS) or C9D-HS reconstituted with C9. Alternatively, cells were treated with C8,C9D-HS or C8,C9D-HS reconstituted with C8 or C8 plus C9 for 1 h. Qualitative and quantitative analysis of the released [3H]AA and [3H]myoinositol radiolabeled products were performed by thin layer chromatography/autoradiography and anion exchange chromatography, respectively. The major [3H]AA radiolabeled products after C5b-9 stimulation comigrated with intact phospholipid and AA standards, and the major [3H]myoinositol radiolabeled product was inositol-1-phosphate. Treatment of cells with phospholipase A2 inhibitors, mepacrine and bromophenacyl bromide, abolished AA release by C5b-9. In the absence of extracellular Ca2+, C5b-9 also failed to induce the release of AA. Interestingly, 1-(5-isoquinolinsulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinases, inhibited AA release by C5b-9, whereas AA release stimulated by the calcium ionophore A23187 was not blocked by H-7. The results suggest that AA generation by C5b-9 from the ROC-1 clone involves activation of Ca2+-dependent phospholipase A2 which is regulated by protein kinase-dependent mechanisms.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
C5b-9 complement complex in autoimmune demyelination: dual role in neuroinflammation and neuroprotection.
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Animals↗
Participation of the blood platelet in immune reactions due to platelet-complement interaction.
OBJECTIVE: Review of different aspects of the primary interaction of complement with blood platelets in immunological reactions and the effect on platelet activation in healthy people and patients. DATA SOURCES AND SELECTION CRITERIA: Relevant original papers and review articles mainly of the English-written literature. RESULTS: Besides their major role in hemostasis and wound healing, blood platelets are involved in immunological reactions. They are not only able to interact with IgG through Fc receptors (FcR), they also react with complement components. This review summarizes interactions of complement with mainly human platelets. Such interactions may occur through complement receptors of the plasma membrane (e.g. C1q receptor, complement receptors 2 and 4), but also in a receptor-independent way including activation of the platelet by the membrane attack complex of complement C5b-9. In addition, activation of complement at the surface of the platelets may be induced after binding of anti-platelet antibodies to membrane glycoproteins (e.g. GpIIb/IIIa, GpIb/IX) or after binding of platelet-nonspecific immune complexes via FcR. Complement activation in turn may be regulated by various means including specific plasma or membrane proteins [e.g. decay-accelerating factor (DAF), membrane cofactor protein (MCP), membrane inhibitor of reactive lysis (MIRL), C8-binding protein (C8bp, homologous restriction factor hrf)]. As a further way of self-protection against complement attack, platelets may actively release C5b-9, deposited at the surface as C5b-9-enriched membrane vesicles. CONCLUSIONS: Two lines of interaction of platelet with complement can be distinguished. On the one hand, platelets are equipped with membrane proteins which protect them from complement attack against themselves. On the other hand, membrane receptors for activated complement components as well as for IgG are expressed on the surface, which enable the platelet to intervene in immunological reactions. This property varies between platelets of different species and needs further investigation also in view of the platelet as an intersection between immunology and hemostasis.
Antigen-Antibody Complex↗
The terminal membrane C5b-9 complex of human complement. Evidence for the existence of multiple protease-resistant polypeptides that form the trans-membrane complement channel.
C5b-9(m) complexes were incorporated into lecithin liposomes and subjected to proteolysis in the presence of DTT to remove the externally oriented annulus. Liposomes were recovered that selectively carried the membrane-bound, thin-walled cylindrical portion of the C5b-9(m) complex. The presence of DTT during proteolysis enhanced peptide bond cleavage in the C5b-9(m) complex. All C5-C9 components were degraded to lower m.w. fragments. A protease-resistant, but hydrophilic 85 to 86,000-dalton polypeptide derivative of C5, possibly representing the C5 beta-chain, was recovered in the fluid phase. This component is not intimately associated with the target lipid bilayer. Immunochemical analyses yielded evidence for the existence of minor C5-C9 antigenic determinants on the membrane-bound C5b-9(m) residue. SDS polyacrylamide gel electrophoreses of liposomes carrying the C5b-9(m) residues revealed the persistence of at least six major polypeptides of approximately m.w. 50,000, 45,000, 40,000, 38,000, 20,000, and 16,000. The data are interpreted to indicate that multiple protease-resistant polypeptide chains derived from several terminal C components participate in formation of the trans-membrane C channel.
Antigens↗
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↗