Assay of complement components C1, C4, C2, C3 and C9 in whole rat serum.
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Assembly of the membrane attack complex of complement on small unilamellar phospholipid vesicles.
Light-scattering intensity was shown to be a reliable, direct, and quantitative technique for monitoring the assembly of the membrane attack complex of complement (proteins C5b-6, C7, C8, and C9) on small unilamellar phosphatidylcholine vesicles. The assembly on vesicles occurred in a simple fashion; complexes of C5b-7 bound noncooperatively to the vesicles, and final assembly of C5b-9 did not induce vesicle aggregation or fragmentation. When C5b-6 and C7 were mixed in the presence of vesicles but at molar protein/vesicle ratios of less than 1, there was quantitative binding of C5b-7 to the vesicles with no concomitant aggregation of C5b-7. If C7 was added at a slower rate, quantitative binding was obtained at molar C5b-7/vesicle ratios of up to 5. The latter observations (a) were consistent with the proposal that C5b-7 aggregation and membrane binding were competitive events and (b) defined conditions under which light-scattering intensity measurements could monitor C5b-9 assembly on vesicles without contribution from the fluid-phase assembly. The C8/C5b-7 ratio in the phospholipid-C5b-8 complex was 0.97 +/- 0.12, and the maximum ratio of C9/C5b-8 in the final complex was 16.2 +/- 2.0. One C9 molecule associated rapidly with each phospholipid-C5b-8, followed by slower incorporation of the remaining C9 molecules. The initial velocity of the slow phase of C9 addition was easily saturated with C9 and gave an activation energy of 37 kcal/mol. This was identical with the value measured for the analogous process in the fluid-phase assembly.(ABSTRACT TRUNCATED AT 250 WORDS)
Complement C7↗
Distributions and recoveries of complement components during cold ethanol fractionation of human plasma.
The distributions and recoveries of the hemolytic activities and quantities of protein of complement components during cold ethanol fractionation were studied. Most of the components were recovered in Cohn's fraction III, with slight activities in fractions I and IV. The hemolytic activities of C1 and C5 were recovered in high yields in fraction III. The recoveries of activities of late components of complement (C6-C9) in fraction II + III were also high. These results suggest that many complement components can be isolated from Cohn's ethanol fractions.
Cold Temperature↗
Platelet-associated complement in chronic ITP.
Chronic ITP is due to antibody-induced destruction of platelets by the reticuloendothelial (RE) system. The role of complement in this process is unclear. We measured platelet-associated complement (PAC) components C3, C3bi, C4 and C9 in 16 patients with chronic ITP, in two of these patients prior to and after splenectomy. Competitive solid-phase radioimmunoassays using monoclonal antibody (anti-C3d, anti-C3bi neoantigen or anti-C9) or affinity-purified heterologous antibody (anti-C4) were used. Mean values (+/- SD) of normal subjects (ng/10(7) plts) were: PAC3d 17.6 +/- 6.8; PAC3bi 11.6 +/- 2.3; PAC4 1.6 +/- 0.5; PAC9 9.9 +/- 2.6. Significantly elevated (greater than 2 SD) PAC3, PAC3bi, PAC4 and PAC9 levels occurred in 12/16, 11/14, 10/14 and 5/9 chronic ITP patients. The PAC3, PAC3bi and PAC9 values correlated inversely with the patients' platelet counts (P less than 0.001); PAC4 levels did not. A positive correlation was also noted between PAC3, PAC3bi and PAC9 while PAC4 values showed no correlation. Two patients with preoperative elevation of all four PAC proteins showed normalization of PAC3, PAC3bi and PAC9 values after a splenectomy-induced remission; PAC4 levels remained elevated for up to 5 months after surgery. We conclude that in vivo C activation occurs in most chronic ITP patients with binding of C3 and C9 to the platelet surface. This in vivo C activation may promote more efficient phagocytosis (C3b) and possibly platelet lysis (C5-9) in some ITP patients.
Adult↗
Increased ion permeability of planar lipid bilayer membranes after treatment with the C5b-9 cytolytic attack mechanism of complement.
The ion permeability of planar lipid bilayers, as measured electrically, was found to increase modestly upon treatment with purified complement complex C5b,6 and complement components C7 and C8. The subsequent addition C9 greatly amplified this change. No permeability changes occurred when components were added individually to the membrane, or when they were used in paired combinations, or when C5b, C7, C8, and C9 were admixed prior to addition. Thus, there is a significant parallel between the permeability changes induced in the model membrane and damage produced in biological membranes by the C5b-9 complement attack sequence. The efficiency of membrane action by C5b-9 was critically dependent on the order in whcih components were added to the membrane. There were also differences in the electrical properties of membranes treated with C5b-8 and C5b-9, though in both cases the enhanced bilayer permeability is best attributed to the formation of trans-membrane channels. Collectively, the data are consistent with the hypothesis that the mechanism of membrane action by complement involves the production of a stable channel across the lipid bilayer, resulting in cell death by colloid-osmotic lysis.
Cell Membrane Permeability↗
Protection of gingival epithelium against complement-mediated damage by strong expression of the membrane attack complex inhibitor protectin (CD59).
Adult periodontitis (AP) is a chronic inflammatory disease of the tooth-supporting apparatus. Activation products of the inflammation-inducing complement system have been detected in the gingival crevicular fluid at the site of gingival inflammation. In the present study, we examined whether evidence for ongoing complement activation in gingival tissues of patients with AP can be obtained. In light of the potential tissue-damaging effects of the complement system, we also examined how the gingival tissue is protected against the cytolytic activity of complement. Surgical and autopsy samples of AP (n = 18) and healthy (n = 11) gingiva were analyzed for the expression or deposition of the complement regulators protectin (CD59) and vitronectin (S-protein) and complement components C3d and C9 by indirect immunofluorescence microscopy with specific antibodies. In healthy gingiva, protection was strongly expressed on the membranes of epithelial cells and on the vascular endothelia of the underlying connective tissue. In AP, protectin was also strongly expressed by endothelial cells, but in the epithelia the expression was granular and weaker than in the healthy gingiva. Coarse granular deposits of complement components were seen in the subepithelial tissues of 61% (C3d), 39% (C9), and 33% (vitronectin) of AP patients, compared with 9% (one case in 11) in healthy controls. In addition, deposits of C3d, C9, and vitronectin were observed on the basement membranes of both pocket and oral epithelium of healthy and AP gingiva but not at sites of protectin expression. The results suggest an increased turnover of the complement system in the gingival tissues of AP patients. The gingival epithelium and connective tissue endothelia are well-protected against damage by the membrane attack complex of complement (MAC). Protection of the underlying connective tissue is insufficient, however, and may allow for deposition of MAC and autologous tissue damage in AP.
Adult↗
Enhancement of complement-mediated lysis of dithiothreitol-treated erythrocytes involves increased C9 insertion and polymerization.
Treatment of human erythrocytes with dithiothreitol (DTT) increases the sensitivity of normal cells to complement (C)-mediated lysis. We have investigated the mechanism through which DTT increases cell susceptibility to complement by comparing the interactions of complement proteins with DTT-treated erythrocytes and with normal cells. In addition, we have studied the effect of DTT on the physical state of the erythrocyte membrane. Results indicated that the DTT primarily affects the interactions of the late components of complement with the cell membrane. In particular, the insertion efficiency of C9 and its ability to form tubular poly-C9 are enhanced on DTT-treated cells. Electron spin resonance (ESR) spectroscopic analyses of the treated and untreated membranes showed essentially no correlation between bulk membrane fluidity and the DTT-induced change in lytic susceptibility, suggesting no gross disruption of the membrane lipid structure by DTT. In view of the fact that DTT-treated erythrocytes have been proposed as a possible model for the abnormally complement-sensitive erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) which are deficient in a 75,000 mol. wt membrane protein called decay accelerating factor (DAF), we explored the possibility that DAF might be affected by DTT. Studies with anti-DAF F(ab')2 antibodies indicated that DAF activity is protected from DTT-treatment. These results are reinforced by the observation that DTT-treatment of DAF-deficient Type III PNH-E also led to enhanced lysis of PNH-E, implying that DTT affects membrane structures other than DAF. Thus, we conclude: (1) that DTT increases the lytic susceptibility of human erythrocytes to late components of human complement by modifying membrane structures to facilitate C9 insertion and polymerization, and (2) that DTT-treated erythrocytes are not a suitable model for PNH erythrocytes.
Biopolymers↗
Complement gene expression by rabbit heart: upregulation by ischemia and reperfusion.
Activation of the complement system has been implicated in the pathogenesis of myocardial ischemia/reperfusion injury. It has always been assumed that liver is the primary source of complement components. In the present study, we used the reverse-transcriptase polymerase chain reaction technique to establish that the mRNAs for complement proteins C3 and C9 are expressed in rabbit heart. Rabbit liver, brain, spleen, and kidney were also shown to express C3 and C9 mRNAs. We used Western blotting to establish that these mRNAs in heart are translated into the corresponding proteins. We further established that dramatic upregulation of the mRNAs occurred in Langendorff-perfused isolated hearts subjected to ischemia and reperfusion. C3 mRNA was always expressed at higher levels than was C9 mRNA, but C9 mRNA showed greater upregulation under stress. Compared with levels in control hearts subjected to 5 minutes of normoxic perfusion, hearts subjected to 0.5 hours of ischemia followed by 1 hour of reperfusion had a 4.72-fold increase in C3 mRNA and a 19.5-fold increase in C9 mRNA. By contrast, C3 mRNA in hearts subjected to 3.5 hours of normoxic perfusion showed no change, and those subjected to 3.5 hours of ischemia showed only a 1.72-fold increase, whereas C9 mRNA levels increased by 5.17-fold after 3.5 hours of normoxic perfusion and 12.5-fold after 3.5 hours of ischemia. The results of this study demonstrate for the first time that heart tissue is capable of expressing genes and proteins of the complement system, although it is not yet known which cell types are responsible. They further demonstrate that ischemia and reperfusion of the heart promotes a rapid upregulation of the mRNAs encoding the complement proteins C3 and C9 and that these abnormal levels considerably exceed those of normal liver. These observations are consistent with the hypothesis that local production of complement proteins may contribute significantly to the degree of ischemic injury to the myocardium and that complement expression is augmented by reperfusion.
Animals↗
Restriction in the lytic efficiency of complement of different erythrocyte targets: a re-examination of the activities of horse C8 and C9.
Differences in the lytic efficiency of different complement sources have frequently been observed. This effect has been shown to be related to both the species of the target erythrocyte and the species composition of terminal complement components within the 5b-9 membrane attack complex. The majority of studies have indicated that the source of C9 is critical in controlling the range of erythrocyte species that can be lysed efficiently. One exception to this general finding was the report by Lachmann et al., 1973 (Immunology 24, 135-145), using horse serum as a complement source. In that study, horse C8 rather than C9 was implicated as the critical component. In this study, we have re-examined this observation and find that the restricted hemolytic potential of horse complement correlates absolutely with the presence of horse C9. The reason for the differences between our findings and those of the earlier study are discussed.
Animals↗
Further studies on C9 deficiency.
Further studies were carried out on the C9 deficiency (C9D). Her serum complement activity (CH50) was 15.7 units when assayed in high ionic strength buffer and 8.8 or less than 5.0 units when assayed in low ionic strength buffer containing glucose or sucrose, respectively. It was revealed that this buffer-dependent CH50 variation of C9D serum was due to the effect of the buffer on the spontaneous lysis of EAC1-8. The serum bactericidal activity of C9D was low, but the addition of specific antibody against bacteria increased the activity indicating an important role of antibody in the serum bactericidal activity. Neither C9 inactivator(s) nor antibody against C9 was detected in the serum, indicating that the case had a defect of C9 synthesis. However, the estimation of C9 levels in the sera of her family could not reveal the mode of inheritance of C9D.
Adult↗
Membrane attack complex of complement in dermatitis herpetiformis.
The assembly of membrane attack complex (MAC) of complement implies activation of complement to the attachment of C9 and the presence of MAC on tissue suggests a possible pathogenic role for complement in disease since MAC is able to damage membranes. We examined normal skin of five patients with dermatitis herpetiformis for the presence of MAC using a monoclonal antibody (poly C9-MA) that recognizes a neoantigen of C9 that is not present on monomeric C9 but is common to both isolated MAC and to polymerized C9. Granular deposits of polymerized C9 were found at the sites of IgA deposition in the dermal papillae of normal skin from all patients. The pathologic importance of this finding is uncertain.
Antibodies, Monoclonal↗
Purification and characterization of the ninth component of rabbit complement.
A method of purification of C9 from rabbit serum is described. The three-step procedure, consisting of anion exchange chromatography, gel-filtration and isoelectric focusing yielded a homogeneous, single band protein as judged by SDS-PAGE. With regard to its physicochemical properties, rabbit C9 resembled C9 purified from human or guinea-pig serum.
Animals↗
The human complement regulatory protein CD59 binds to the alpha-chain of C8 and to the "b"domain of C9.
The erythrocyte membrane inhibitor of the human terminal complement proteins, surface antigen CD59, has previously been shown to enter into a detergent-resistant complex with either the membrane-bound complex of C5b-8 or C5b-9 (Meri, S., Morgan, B. P., Davies, A., Daniels, R. H., Olavesen, M. G., Waldmann, H. and Lachmann, P. J. (1990) Immunology 71, 1-9; Rollins, S. A., Zhao, J., Ninomiya, H., and Sims, P. J. (1991) J. Immunol, 146, 2345-2351). In order to further define the interactions that underlie the complement-inhibitory function of CD59, we have examined the binding interactions between 125I-CD59 and the isolated components of human complement membrane attack complex, C5b6, C7, C8, and C9. By density gradient analysis, we were unable to detect interaction of 125I-CD59 with any of these isolated complement components in solution. Specific binding of 125I-CD59 to C8 and C9 was detected when these human complement proteins were adsorbed to either plastic or to nitrocellulose, suggesting that a conformational change that accompanies surface adsorption exposes a CD59-binding site that is normally buried in these serum proteins. The binding of 125I-CD59 to plastic-adsorbed C8 and C9 was saturable and competed by excess unlabeled CD59, with half-maximal binding observed at 125I-CD59 concentrations of 80 and 36 nM, respectively. No specific binding of 125I-CD59 was detected for surface-adsorbed human C5b6 or C7 nor was such binding observed for C8 or C9 isolated from rabbit serum. Binding of CD59 to human C8 and C9 was not mediated by the phospholipid moiety of CD59, implying association by protein-protein interaction. In order to further define the binding sites for CD59, ligand blotting with 125I-CD59 was performed after separation of C8 into its noncovalently associated subunits (C8 alpha-gamma and C8 beta) and after alpha-thrombin digestion of C9. These experiments revealed specific and saturable binding of 125I-CD59 to C8 alpha-gamma subunit (half-maximal binding at 75 nM), but not to C8 beta, and specific and saturable binding to the 37-kDa fragment (C9b) of thrombin-cleaved C9 (half-maximal binding at 35 nM), but not to the 25-kDa C9a fragment. Partial reduction of C8 alpha-gamma revealed that only C8 alpha polypeptide exhibited affinity for CD59, and no specific binding to the C8 gamma chain was detected.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Molecular mechanisms of cytotoxicity: comparison of complement and killer lymphocytes.
The membrane attack complex of complement is an amphiphilic fusion product of 5 glycoproteins, C5, C6, C7, C8 and C9. The membrane attack complex forms transmembrane channels that vary in size depending on the number of C9 molecules incorporated into the complex. The C5b-8 complex forms small channels and at high multiplicity can kill nucleated cells. At least 12 C9 molecules are required to form tubular poly C9 which evokes the ultrastructural image of the classical membrane lesion produced by complement. The membranes of erythrocytes and other blood cells contain a 70,000 dalton protein that can inhibit channel formation by the membrane attack complex. This protein is species specific and has been called homologous restriction factor. A cytotoxic protein immunochemically related to C9 was isolated from cytotoxic human large granular lymphocytes and from OKT3 activated human peripheral blood mononuclear cells. In the presence of Ca++, isolated C9 related protein (C9RP) formed circular structures that resembled poly C9. C9RP efficiently killed K562 cells, human melanoma cells, Raji cells and human large granular lymphocytes. The results suggest that the channel forming protein of cytotoxic lymphocytes and C9 of complement have a common evolutionary ancestry.
Complement C9↗
Regulation of the membrane attack complex of complement. Evidence that C8 gamma is not the target of homologous restriction factors.
Inability of the membrane attack complex of C (C5b-9) to efficiently lyse E from the same species has been attributed to one or more membrane-associated proteins that are collectively called homologous restriction factors. These include a 65,000 Mr protein referred to as the C8 binding protein or homologous restriction factor and a 20,000 Mr protein referred to as P-18, HRF20, CD59 Ag, or MIRL. Both are found on nucleated cells as well as E and both protect against complement-mediated lysis by interfering with C8 and/or C9 function within C5b-9. The exact mechanism by which these factors restrict activity is unknown but studies with purified C8 binding protein suggest they may interact specifically with the gamma subunit of C8. To determine directly if gamma is the target of restriction factors, a derivative of human C8 lacking this subunit was evaluated for its potential to lyse homologous cells. This derivative (C8') was previously shown to be functionally equivalent to normal C8 in a heterologous sheep E system. Here, it is compared to normal C8 by using human E as target cells. Results indicate no difference between the ability of C8 and C8' to incorporate into HuEAC1-7, to mediate subsequent C9 binding and to promote hemolysis. Thus, the presence or absence of gamma has no effect on homologous restriction of C5b-9, therefore gamma cannot be the primary target of homologous restriction factors.
Blood Proteins↗
Formation of transmural complement pores in serum-sensitive Escherichia coli.
The binding of C9 at 0 and 37 degrees C to viable Escherichia coli K-12 cells carrying C5b-8 complexes was quantified. At low temperature, limited average binding of only 1 to 1.4 molecules of C9 per C8 molecule occurred, whereas 6 to 8 C9 molecules were bound per C8 molecule at 37 degrees C. Despite incorporation of C9 into C5b-9 complexes at 0 degrees C, these terminal complexes caused no loss of bacterial viability even when present in very large numbers (1,000 to 1,500 per CFU) on the bacterial cells. In contrast, generation of 50 to 100 C5b-9 complexes carrying multiple C9 molecules per CFU caused loss of viability. The failure of C5b-81C91 complexes to generate transmural pores was confirmed by measurements of o-nitrophenyl-beta-D-galactoside influx into the cells. Whereas treatment of C5b-8-laden cells with C9 at 32 degrees C caused virtually instantaneous influx of the marker, almost no influx was registered in cells receiving C9 at 0 degrees C. When cells carrying C5b-7 were brought into the stationary phase and given C8 and C9 at 32 degrees C, a C9-dependent disruption of the outer membrane permeability barrier immediately occurred as demonstrated by cleavage of a chromogenic substrate by periplasmic beta-lactamase. In sharp contrast, o-nitrophenyl-beta-D-galactoside influx was markedly retarded over a prolonged period, with abrupt permeability increases of the inner membrane toward this molecule being noted just before bacterial cell division occurred. We conclude that killing of E. coli requires binding of C5b-9 complexes containing C9 oligomers to the outer membrane and suggest that formation of pores in the inner membrane occurs when these complexes are "hit" by transiently forming zones of bioadhesion. Formation of the latter may be a dynamic process that is accentuated during cell division and quiescent during the stationary phase.
Blood Bactericidal Activity↗
Self-protection of cytotoxic lymphocytes: a soluble form of homologous restriction factor in cytoplasmic granules.
A soluble form of homologous restriction factor (HRF) has been isolated from the cytoplasmic granules of human large granular lymphocytes that were cultured in the presence of recombinant interleukin 2 for 2-3 weeks. The granule-derived protein (approximately 65 kDa) is soluble in detergent-free solution and reacts with antibody produced to membrane HRF. HRF was first described as a 65-kDa membrane protein of human erythrocytes capable of inhibiting the formation of transmembrane channels by the membrane attack complex of complement. It has also been isolated from activated human lymphocytes and shown to confer upon these cells relative resistance to lysis by the membrane attack complex and by the complement component C9-related protein of human cytotoxic lymphocytes. The soluble HRF of lymphocyte granules inhibits reactive lysis of erythrocytes by the membrane attack complex of human complement. It was also found to be a potent inhibitor of (i) the cytolytic activity of the C9-related protein of human cytotoxic lymphocytes, (ii) human large granular lymphocyte cytotoxicity, and (iii) the cytotoxic activity of human CD8+ lymphocytes obtained by cell sorting from recombinant interleukin 2-activated peripheral blood mononuclear cells. It is proposed that granule-derived soluble HRF and cell surface-membrane-bound HRF are involved in the mechanism of self-protection of killer lymphocytes.
Blood Proteins↗
Cell membranes in cytotoxicity.
Silica particles are cytotoxic for macrophages because they damage the membranes around secondary lysosomes in which the particles are engulfed. Hydroxyl groups of silicic acid on the surface of the particles form hydrogen bonds with phosphate ester groups of phospholipids and disrupt a variety of natural and artificial membranes. Asbestos fibers induce secretion of hydrolytic enzymes from cultured macrophages. Magnesium hydroxide groups of chrysotile asbestos interact ionically with ionized sialic acid residues of membrane glycoproteins, increase passive cation flux and produce osmotic lysis. The terminal components of complement (C5b-C9) when inserted into the bilayer structure also increase passive cation flux and produce osmotic lysis. The small complement cleavage product C3a is lytic for several cell types, especially malignant cells. The mechanism by which specifically sensitized thymus-derived (T)-lymphocytes kill tumour cells is discussed. Plasma membranes from effector lymphocytes possess considerable cytolytic potential, which is dependent on the activity of a membrane-associated proteinase.
Animals↗