Quantitative measurement of C9 sites and their association to the ring-like "lesions" on complementlysed membranes: a morphometric immunoferritin study.
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Distinction between C8-mediated and C8/C9-mediated hemolysis on the basis of independent 86Rb and hemoglobin release.
The intermediate product EAC1-7 released hemoglobin when incubated with high concentrations of GPC8 in the absence of C9. The reaction failed to reach an end point within 8 hr at 37 degrees C, and analysis of the kinetics indicated that it did not conform to the one-hit theory of immune hemolysis, and was not, therefore, the result of C9 contamination of the C8 preparation. The release of 86Rb from labeled EAC1-7 incubated at 30 degrees C with limiting C8 and excess C9 was paralleled, within 5 to 10 min, by release of hemoglobin. In the absence of C9 and with higher concentrations of C8, 86Rb was released rapidly, but hemoglobin release was delayed for several hours. Addition of excess C9 to concentrations of C8, which did not alone cause 86Rb release, resulted in substantial release of the isotope. These observations indicate that C9 acts by producing a distinct lesion in the cell membrane rather than by accelerating the release of hemoglobin from the C8-initiated 86Rb-releasing lesions. It is concluded that 86Rb release cannot be used as a reliable indicator of cell lysis and that C8- and C8/C9-mediated hemolysis are the result of mechanistically different processes.
Animals↗
Bactericidal activity of C9-deficient human serum.
Escherichia coli B/SM, strain 1-1, was killed dose dependently by human hereditary C9-deficient serum (C9DHS), which was shown to contain no C9 Ag by an ELISA method. On the other hand, human hereditary C7-deficient serum did not kill the bacteria under similar conditions. The bactericidal activity of C9DHS was inhibited by rabbit anti-C5 antibody but not by murine anti-C9 mAb. The anti-C9 antibody decreased the bactericidal activity of normal human serum (NHS) to the level of that with C9DHS. Sheep anti-human lysozyme antibody did not affect the bactericidal activity of C9DHS or NHS even when added at more than twice the concentration required to block the serum lysozyme activity on Micrococcus luteus. After treatment with C9DHS and washing, surviving Escherichia coli were killed by C9, but not by lysozyme, transferrin, or both. Other strains of E. coli (K12 W3110, C600, and NIHJ) and Salmonella typhimurium (strain NCTC 74), all maintained in the laboratory, were also killed by C9DHS. However, pathogenic strains recently isolated from patients with traveler's diarrhea and some strains of S. typhimurium were resistant to both C9DHS and NHS, at least at the serum concentration tested. A concentration of 0.1 M Tris did not increase the susceptibility of serum-resistant strains of bacteria to C9DHS, but made one strain of S. typhimurium tested susceptible to NHS, but not to C9DHS. These results clearly showed that C9DHS kills bacteria that are sensitive to NHS through activation of C up to the step of C8 in the same way that C9-deficient C serum lyzed sensitized erythrocytes.
Blood Bactericidal Activity↗
Immunochemical quantitation of serum complement components in SFD and AFD infants.
Components of complement (protein concentrations of C1q, C3, C3-activator, C4, C5 and C9 and whole complement (hemolytic activity)) were measured in sera from full-term small-for-date (SFD) and appropriate-for-date (AFD) infants and their mothers. (1) Half the SFD infants showed lower C1q levels than the AFD infants. (2) SFD infants showed the same levels of C3 as AFD infants. (3) Although SFD infants showed slightly higher levels of C3-activator than AFD infants, there was no significant difference. (4) SFD infants and AFD infants showed the same level of C4. (5) With respect to C5, half the SFD infants showed lower levels than the AFD infants. (6) Levels of C9 in the SFD infants were essentially the same as those in the AFD infants. (7) The amount of protein in every complement component of an infant was smaller than that of the mother. In AFD infants, the infant-maternal ratios were 0.64 for C1q, 0.45 for C3, 0.29 for C3-activator, 0.43 for C4, 0.54 for C5 and 0.12 for C9. (8) The whole complement titers of umbilical cord sera from the AFD infants were approximately one half of those of the maternal sera. This is due to the smaller amount of complement protein itself in the infant sera. (9) The whole complement titers of umbilical cord serum agreeably correlate with gestational weeks and birth weight of the infants. (10) The whole complement titer in full-term SFD infants was lower than that in normal full-term AFD infants.
Complement C1↗
Isolation and characterization of the eighth component of the bovine complement system.
OBJECTIVE: To isolate and characterize the eighth component of the complement system (C8) in cattle. SAMPLE POPULATION: Fresh plasma obtained from beef cattle. PROCEDURES: Plasma samples were fractionated, using sequential precipitation and ion-exchange and gel-filtration chromatography, to yield C8. The protein was identified throughout the procedure on the basis of its hemolytic function. Electrophoresis in polyacrylamide gels was used to determine molecular weight and composition of polypeptide chains. Reconstitution of classical and alternative complement pathways was used to characterize the hemolytic function of bovine C8. RESULTS: The bovine C8 protein consisted of a disulfide-bonded alpha-gamma heterodimer that was noncovalently associated with a beta chain. Apparent molecular weight of the alpha, beta, and gamma chains under reducing conditions were 66, 61, and 23 kd, respectively. In the classical pathway of activation, bovine C8 and the ninth component of the complement system (C9) had species incompatibility with human C8 and C9 on sheep erythrocyte target cells. CONCLUSIONS: A simple 4-step fractionation procedure provided good yield of bovine C8 from plasma. The isolated protein was structurally comparable to C8 from other species. Purified bovine C8 may be useful in functional hemolytic assays to investigate the roles of complement-mediated lysis in the pathogenesis of inflammatory diseases and the killing of susceptible microorganisms.
Animals↗
Mechanisms and kinetics of the synthesis and release of platelet-activating factor (PAF) by polyacrylonitrile membranes.
Platelet-activating factor is a recognized mediator of anaphylaxis and bioincompatibility. Here, the mechanisms and the kinetics of the production of platelet-activating factor were studied in vivo during high-flux hemodialysis and in vitro in a recirculation model with polyacrylonitrile membranes, the AN-69 and the more recent SPAN, where the Na-metallilsulfonate group is partially substituted with the less polar methacrylate group. In in vivo studies, eleven patients were studied in cross over. Patients were randomly allocated to the AN-69 (5 patients) and to the SPAN membrane (6 patients) for two weeks. Measurements were made in the second week of use. After completion of the second week, the patients were switched to the other membrane for a further two weeks. Samples for leukocyte and platelet counts, PAF in whole blood or bound to platelets, the C3a des Arg and the C5b-C9 membrane attack complex as well as samples for clearances of urea, creatinine and phosphates were taken at different time intervals during treatment. PAF was detected by biological assay after methanol extraction of whole blood or of platelet pellets obtained by sequential centrifugation. C3a des Arg and the C5b-C9 fraction were detected by commercially available immunoassays. Results were analyzed by Minitab statistical package. PAF was detectable only during treatment with AN-69 but not with SPAN 1 min after start of the extracorporeal circulation in both whole blood (4.5 +/- 2.7 ng/ml) and on platelet surface (4.1 +/- 1.2 ng/ml). No statistical significant differences were observed between AN-69 and SPAN with regard to leukocyte and platelet counts, plasma C3a des Arg and C5b-C9 levels. The structure modification did not alter functional performances as indicated by the lack of statistically significant differences in clearance values between the two membranes. In in vitro experiments performed with normal washed and whole blood recirculated in a closed circuit demonstrated the presence of a plasma-dependent, complement-independent mechanisms responsible for the triggering of PAF synthesis and release with AN-69 but not SPAN membrane. PAF was extractable from the inner and outer side of both polyacrylonitrile membranes (AN-69: inner, 4.9 +/- 0.5 ng/ml; outer, 0.1 +/- 0.05 ng/ml; SPAN: inner, 5.5 +/- 0.6 ng/ml, outer: 3.3 +/- 0.7 ng/ml, SPAN vs. p < 0.001), suggesting that absorption may be relevant with both membranes.
Acrylic Resins↗
Neutralization of cytomegalovirus virions: the role of complement.
Complement provides a key immunologic defense against invading pathogens; thus, a clear understanding of the interactions between cytomegalovirus (CMV) and complement may permit the development of strategies to enhance CMV neutralization. In the presence of specific anti-CMV antibodies, complement enhanced the neutralizing ability of serum by 2- to 3-fold. However, in the absence of specific anti-CMV antibodies, complement was ineffective in neutralizing CMV virions by plaque assay. Although complement alone did not mediate any neutralizing effect, CMV consumed complement activity from seronegative serum, resulting in the deposition of C3 on the virion. However, only in the presence of specific anti-CMV antibody did complement activation continue to the deposition of C9 on the virions. These results strongly suggest complement regulation by CMV virions that is modulated by anti-CMV antibody; this regulation may be attributed to three host complement regulators on the virions: CD55, CD46, and CD59.
Antibodies, Viral↗
Computer imaging analysis of the correlation between intensities of glomerular immune-deposits and histopathology in patients with IgA nephropathy.
The relationship between the intensities of IgA, C3c, and C9 deposition in renal glomeruli and the severity of histopathologic injuries in patients with IgA nephropathy was examined using Microscope-Photometer 01K and a computer. Percentages of glomerular adhesion to Bowman's capsules, crescent formation, and glomerular sclerosis were calculated in the renal specimens. There was a significant correlation between the intensity of each C3c and C9 deposition in glomeruli and the degree of glomerular adhesion to Bowman's capsules and crescent formation in patients with IgA nephropathy. There was no significant correlation between the intensity of C3c or C9 deposition in glomeruli and the degree of glomerular sclerosis. No relationship was found between the intensity of IgA deposition in glomeruli and the degree of histopathologic injuries. The patients with negative or trace amounts of glomerular C3c deposits showed less severe glomerular injuries. Thus, the intensity of C3c and C9 deposition in glomeruli appears to be one of the critical factors responsible for the active progression of glomerular inflammatory process in patients with IgA nephropathy.
Complement C3c↗
Human serum complement requirements for bacterial killing and protoplast lysis of Escherichia coli ML308 225.
Normal human serum kills Escherichia coli ML308 225 and lyses protoplasts derived from this organism. Human serum which is depleted of complement component C9 or deficient in component C8 is not bactericidal, but C9-depleted serum will lyse protoplasts whereas C8-deficient serum will not. Bacterial lipopolysaccharide, which can protect bacteria from the serum bactericidal reaction, does not protect protoplasts from lysis by serum.
Bacteriolysis↗
Complement in acute and chronic arthritides: assessment of C3c, C9, and protectin (CD59) in synovial membrane.
OBJECTIVES: To investigate the role of complement cascade induced damage and protection against it in acute arthritides compared to rheumatoid arthritis and other chronic joint derangements. METHODS: C3c, C9, and protectin (CD59) were examined by avidin-biotin-peroxidase complex staining. RESULTS: Marked deposits of C3c and C9 were found in synovial vasculature and intercellular matrix of the lining in rheumatoid arthritis and in acute arthritides (including bacterial, reactive, and osteoarthritis flare up). Furthermore, protectin was not visible in synovial lining cells and was relatively weakly expressed in stromal and endothelial cells in rheumatoid arthritis; also in acute arthritides protectin expression was weak. In contrast, C3c and C9 deposits were not found in chronic conditions associated with degenerative diseases (osteoarthritis and osteochondritis dissecans) or mechanical causes (patellar luxation and a ruptured meniscus), in which also the protectin expression was prominent in synovial lining, endothelial and some stromal cells. CONCLUSIONS: Activation of the complement in rheumatoid arthritis and in acute arthritides seems to be associated with a decreased protection of synovial cells against cellular effects and lysis mediated by membrane attack complex.
Acute Disease↗
Molecular weight of the membrane C5b-9 complex of human complement: characterization of the terminal complex as a C5b-9 monomer.
The hydrodynamic properties of the detergent-solubilized, terminal membrane complex of serum complement components C5-C9 [C5b-9(m)] were studied to obtain an estimate of its molecular weight. In a solution of Triton X-100/deoxycholate, the protein complex binds 17% Triton X-100 and 11% deoxycholate by weight. The sedimentation coefficient of the protein-detergent complex is 26 S as determined by sucrose density gradient ultracentrifugation, and gel filtration indicated a molecular radius of 11 nm. It was ascertained by electron microscopy that these hydrodynamic parameters apply to mono-dispersed C5b-9(m) complexes, which were observed as nonaggregated, hollow protein cylinders and were identical to the complement "lesions" formed on target membranes. The calculated molecular weight of the protein-detergent complex is approximately 1,286,300 to which the protein moiety contributes approximately 1,000,000. The results indicate that the C5b-9(m) complex formed on biological membranes is a monomer entity of the C5-C9 complement components.
Complement Membrane Attack Complex↗
Small angle neutron scattering studies of C8 and C9 and their interactions in solution.
Small angle neutron scattering (SANS) results revealed that contrary to most reports C9 is not a globular protein. Its radius of gyration (Rg) at pH 8 and an ionic strength of 0.5 is 32.2 +/- 1.4 A increasing to 35 A at physiologic ionic strength. In contrast, C8, which has a 2.2-fold larger mass, has a similar Rg value [34.6 +/- 1.6 A]. Calibration plots of Rg vs. M(r) indicate that native C8 is a spherical protein whereas native C9 is elongated. From previous reports it was known that native C8 and C9 associate in solutions of low ionic strength. SANS results confirmed this observation but also demonstrated that C8-C9 heterodimers are already formed at physiologic ionic strength. The dimeric complex is globular [Rg = 40 +/- 0.8 A] indicating that the proteins associate side-by-side rather than end-to-end. In contrast, in presence of the drug Suramin, a potent inhibitor of the assembly of the C5b-9 complex, C9 forms a complex with twice the molecular mass that is still elongated (Rg = 48.8 +/- 0.8 A), suggesting that in this case the protein dimerizes end-to-end via a bridging Suramin molecule.
Biophysical Phenomena↗
Immunohistochemical detection of myocardial necrosis in stillbirth and neonatal death.
The aims of this study were to determine whether immunohistochemical staining for C9 can demonstrate myocardial necrosis in the fetus and neonate. Hearts from cases of stillbirth or neonatal death with confirmed myocardial necrosis (in neonates) or with ischemic lesions outside the heart (in neonates and stillborns) were stained immunohistochemically with antibodies to C9. All five cases with confirmed myocardial infarction showed positive immunohistochemical staining for C9, largely localized to the infarcted areas. The youngest subject was born at 24 weeks gestation and died at 4 days of age. One of two neonates without myocardial necrosis on H&E staining but with pathological evidence of ischemic lesions elsewhere showed staining of scattered fibers. Six out of ten hearts from macerated stillborn infants showed varying degrees of positive staining. Immunohistochemical staining for C9 detects myocardial necrosis in neonates of a gestational age of 24 weeks or more. C9 is also demonstrable immunohistochemically in macerated stillborns, and this is likely to represent myocardial necrosis. The method is of great potential value in the investigation of cardiac ischemia in the fetal and perinatal period.
Antibodies, Monoclonal↗
Purification of C8 and C9 from rat serum.
A procedure based on modifications of published methods for human proteins for the isolation of rat C8 and C9 from one batch of serum is described. The procedure allows the rapid, large-scale isolation of pure and haemolytically active proteins. Rat C9 had a slightly higher molecular weight than human C9 on SDS-PAGE and similar isoelectric point. Rat C8 differed from human C8 in the molecular weight of the gamma chain (23,000 and 21,000 kD respectively), and on isoelectric focusing (pI rat C8: 6.5-6.9; pI human C8: 7.4-7.9).
Animals↗
Role of complement in renal tubular damage.
Deposition of immunoglobulins, complement proteins C1q, C3c, C3d, C4, C5, C6, C7, C8, C9, and terminal complement complex neoantigens in the renal tubulointerstitium was studied in serial sections by immunofluorescence microscopy. Renal tissue from 45 cases with various glomerular diseases, including 8 controls, was studied. The patients were divided into groups; one with tubulointerstitial lesions (24 cases) and the other without (13 cases). The immunoproteins were deposited mainly in the tubular basement membrane and blood vessels. Compared with controls there was a significantly increased staining score for C5 to C9 in the tubular basement membrane in both disease groups. However, the increase in terminal complement complex neoantigens score was significant only in the disease group with tubulo interstitial lesions. The changes in C3d score were not significant. Serial sections showed consistent and heavy ribbon-like deposits of complement proteins C3d, C5 to C9, and terminal complement complex neoantigens in corresponding locations of the segments of tubular basement membrane, mainly in the disease group with tubulointerstitial lesions and especially in the damaged tubules. These findings suggest that in situ activation of the complement cascade leads to the deposition of terminal complement complex neoantigens. Complement activation in the basal area of the tubules may, therefore, be an important pathogenetic mechanism in tubulointerstitial damage.
Basement Membrane↗
Membrane factors responsible for homologous species restriction of complement-mediated lysis: evidence for a factor other than DAF operating at the stage of C8 and C9.
Species-restricted lysis of complement refers to the relative inefficiency of complement to lyse cells from the homologous species. Restriction occurs at least at the steps involving C3/C5 convertase formation and the C9 insertion phase of the complement cascade, and is presumed to be mediated by inhibitory factors in the target cell membrane. In this study, we have examined whether decay accelerating factor (DAF), a membrane protein known to modulate C3/C5 convertase activities on cell surfaces, acts as a regulatory protein in species-restricted lysis of human erythrocyte (E). The role of DAF was assessed in homologous lysis by the classic pathway, in reactive lysis, and in lytic steps requiring C8 and C9. The results indicated that DAF participated in regulating C3/C5 deposition on the surface of homologous E, but had no effect on homologous restriction in reactive lysis and in the reaction of C8 and C9 with antibody-sensitized E C1-7. Treatment of E with pronase or with dithiothreitol (DTT) abolished the restricting effect of homologous C8/C9, indicating that species-restricted lysis by C5b-9 involves membrane factor(s) sensitive to pronase and DTT.
Animals↗
Antibody to intermediate filaments of the cytoskeleton in patients with Behçet's disease.
Antibodies to 10-nm intermediate filaments (anti-IF) were determined in the sera of 30 patients with Behçet's disease (BD), in addition to C-reactive protein and C9, and an attempt has been made to determine whether the presence of anti-IF indicate disease activity. The vimentin type of anti-IF was found to be positive in 14 out of 30 patients with BD (47%), whereas it was positive in 35% of the patients with rheumatoid arthritis (20 cases), 16% of the patients with systemic lupus erythematosus (19 cases) and in only 9% of the normal controls. The anti-IF were predominantly IgG class and the titers in BD were significantly higher than those in normal controls. Out of the 14 patients with anti-IF, 10 showed significantly increased levels of serum C9 and 8 showed increased levels of CRP activity. Only one patient showed increased C9, but was negative for anti-IF and CRP. The presence of anti-IF in the patients' sera was found to be a more sensitive indicator, though not specific, for the clinical assessment of disease activity.
Antibody Specificity↗
[IgG, IgM, IgA, secretory IgA and the complement components C3, C4 and C9 in the large intestine in nonspecific ulcerative colitis and Crohn disease].
The immunohistological study of the large intestines excised from 20 patients with ulcerous colitis and Crohn's disease was made with the use of the indirect immunofluorescence test. A considerable increase in the number of IgG immunocytes has been noted in the lamina propria of the mucous membrane, especially in infiltrations around ulcers. In cases of a highly active inflammatory process accompanied by the pronounced destruction of the epithelium, IgG and C3, C4, C9 were detected in the epithelium of crypts and in blood vessels of the mucous membrane and the submucosa. One-third of the patients showed a decrease in the number of crypts with secretory and serum IgA. The authors suggest that immune complexes take part in maintaining the chronic inflammatory process.
Colitis, Ulcerative↗