Molecular composition of the terminal membrane and fluid-phase C5b-9 complexes of rabbit complement. Absence of disulphide-bonded C9 dimers in the membrane complex

The terminal membrane C5b-9(m) and fluid-phase SC5b-9 complexes of rabbit complement were isolated from target sheep erythrocyte membranes and from inulin-activated rabbit serum respectively. In the electron microscope, rabbit C5b-9(m) was observed as a hollow protein cylinder, a structure identical with that of human C5b-9(m). Monodispersed rabbit C5b-9(m) exhibited an apparent sedimentation coefficient of 29 S in deoxycholate-containing sucrose density gradients, corresponding to a composite protein-detergent molecular-weight of approx. 1.4 X 10(6). Protein subunits corresponding to human C5b-C9 were found on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. By densitometry, there were consistently six molecules of monomeric C9 present for each monomeric C5b-8 complex. Fluid-phase rabbit SC5b-9 was a hydrophilic 23 S ma macromolecule that differed in subunit composition from its membrane counterpart in that it contained S-protein and only two to three molecules of C9 per monomer complex. The data are in accord with the previous report on human C5b-9 that C5b-9(m) contains more C9 molecules than SC5b-9 [Ware & Kolb (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6426-6430]. They corroborate the previous molecular-weight estimate of approx. 10(6) for C5b-9(m) and thus support the concept that the fully assembled, unit lesion of complement is a C5b-9 monomer [Bhakdi & Tranum-Jensen (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 1818-1822]. They also show that C9 dimer formation is not required for assembly of the rabbit C5b-9(m) protein cylinder, or for expression of its membrane-damaging function.     

Lipopolysaccharide-protein complexes of the outer membrane of gram-negative bacteria

The evidence for occurring lipopolysaccharide-protein complexes in the outer membrane of gram-negative bacteria has been summarized. The composition and supramolecular structure of these complexes as well as their functions in microbial envelope and substantial role in membrane organization have been discussed. The biological properties of the complexes as endotoxins and O-specific antigens have been considered.     

Complement lysis: evidence for an amphiphilic nature of the terminal membrane C5b-9 complex of human complement

The terminal, membrane-derived C5b-9 complex of human complement (C) is an apparently hollow, cylindrical macromolecule vertically oriented on the target membrane. In the present study, an antiserum to the complex has been used to probe its immunobiochemical properties. "Neoantigenic" determinants characteristic of the complex have been detected, which are absent on native C5-C9 molecules. Evidence that the C5b-9 complex is an amphiphilic molecule that possesses apolar, detergent-binding surfaces has been obtained by using charge-shift crossed immunoelectrophoresis, and by direct demonstration of Triton X-100 binding to the complex in quantitative immunoelectrophoresis. By the same criteria, serum C5, C6, and C9 are hydrophilic molecules. The results indicate that assembly of C5-C9 into the terminal membrane C5b-9 complex is accompanied by conformational changes in the individual C components that lead to the exposure of apolar molecular regions in the complex. It is proposed that this constitutes the basis for the lipid-binding properties of the macromolecule, which enable it to become inserted into biologic and artificial lipid membranes with apparent generation of a transmembrane pore.     

Monoclonal antibodies against neoantigens of the terminal C5b-9 complex of human complement

Assembly of the terminal C5b-C9 complement components into the cytolytic C5b-9 complex is accompanied by exposure of characteristic neoantigens on the macromolecule. We report the production and characterization of mouse monoclonal antibodies to C9-dependent neoantigens of human C5b-9. Binding-inhibition assays with EDTA-human plasma and micro-ELISA assays with purified C9 showed that the antibodies did not react with native complement components and thus confirmed the specificity of the antibodies for the neoantigens. The monoclonal antibodies did, however, cross-react with cytolyticaIly inactive, fluid-phase C5b-9 complexes, Thus, expression of the neoantigenic determinants was not dependent on the formation of high molecular weight C9 polymers with the complex, since these are absent in fluid-phase C5b-9. Radioiodinated antibodies could be utilized in immunoradiometric assays for the detection and quantitation of C5b-9 on cell membranes. Cross-reactivities of the antibodies with C9-dependent neoantigens of several other animal species were examined and antibody clones cross-reacting with rabbit (clones 3BI, 3Dg, and 2F3), sheep (clones 3Dg and 2F3) and guinea-pig (clone 3D8) neoantigens were identified . Three of four tested clones (3D8, 2F3, IA12) precipitated C5b-9 complexes in double-diffusion assays, probably due to their interaction with multiple and repeating C9-epitopes on the terminal complexes. The monoclonal antibodies will be of value for definitive identification and quantitation of C5b-9 on cell membranes and in tissues, and for establishing immunoassays for detection and quantitation of terminal fluid-phase C5b-9 complexes in plasma.     

Interaction of human beta-endorphin with the terminal SC5b-9 and "preterminal" SC5b-7 and SC5b-8 complexes of human complement

Human beta-endorphin (beta H-EP) is demonstrated to bind to the "preterminal" SC5b-7 and SC5b-8 complexes and to the terminal SC5b-9 complex of human complement. Detailed binding studies revealed saturability, reversibility and structural specificity of the beta H-EP interaction with high or low affinity non-opiate binding sites on SC5b-7 and SC5b-9 complexes. The high affinity binding sites seem to be located predominantly on C5b, C6 or C7 subunits of the complexes.     

Deposition of the terminal C5b-9 complement complex in infarcted areas of human myocardium

Poly- and monoclonal antibodies to neoantigens of the human C5b-9 complement complex, as well as polyclonal antibodies to C5, C8, and C9, were used to detect and identify C5b-9 deposits in human myocardial tissue. Immunocytochemical studies were performed on fresh-frozen autopsy material derived from patients with myocardial infarctions; in addition, in 17 of these patients, paraffin sections of formalin-fixed tissue were investigated. Sixteen autopsies from patients with noncardiac diseases were analyzed as controls. Without exception, C5b-9 positivity was registered selectively and exclusively on and in myocardial cells located within the zones of infarction. The selectivity of staining was confirmed by control reactions for succinic dehydrogenase activity performed in adjacent, respective double-stained sections. Most intensive staining with anti-neoantigen antibodies was observed in the peripheral areas of the infarctions. Weak staining for C3d, rather strong staining for C5 and C9, and intermediate staining with anti-C8 antibodies were observed in the same localizations. Stainings for C4 and IgA were negative, whereas immunocytochemical reactions for IgG and IgM revealed an irregular and very weak staining. Only very weak staining was also observed with a monoclonal antibody to complement S-protein, indicating that the terminal complement components were deposited mainly in the form of membrane-damaging C5b-9 complexes. Immunocytochemical staining for C5b-9 was found to represent a most sensitive tool for detection of ischemic myocardial lesions, permitting easy detection even of single cell necroses. As a working hypothesis, we suggest that initial ischemia may cause loss of the ability of the heart muscle cells to regulate complement turnover at the membrane level. The resulting deposition of C5b-9 on the cell membranes may contribute to functional disturbance and irreversible damage of myocardial cells during the infarction process.     

Elimination of terminal complement intermediates from the plasma membrane of nucleated cells: the rate of disappearance differs for cells carrying C5b-7 or C5b-8 or a mixture of C5b-8 with a limited number of C5b-9

We have previously shown that multiple complement (C) channels are required for lysis of a nucleated cell in contrast to the single channel requirement for erythrocytes. To further investigate this multichannel requirement for nucleated cells, we examined the stability of terminal C complexes in the plasma membrane of Ehrlich ascites tumor cells. Ehrlich cells bearing C5b-7 or C5b-8 with or without C9 were incubated at 37 degrees C or 0 degree C for various time intervals before converting the remaining complexes to lytic C5b-9 channels. C5b-7, C5b-8, and C5b-8 in the presence of a limited number of C5b-9 complexes disappeared functionally from the plasma membrane at 37 degrees C, with initial half-lives of 31, 20, and 10 min, respectively. Disappearance of these complexes did not occur at 0 degree C, nor did disappearance occur at 37 degrees C when formed on sheep erythrocytes. The fate of C5b-8 complexes on the surface of Ehrlich cells was traced with colloidal gold particles bound to C5 determinants on C5b-8 with the use of immunoelectron microscopy. Colloidal gold could be seen on the cell surface after specific binding to cells carrying C5b-8 sites at 0 degree C. After incubating these cells at 37 degrees C, gold particles were internalized into the cell continuously via endocytic vesicles. It is postulated that terminal C complexes may stimulate or accelerate the removal of these complexes from the cell surface.     

Killing of gram-negative bacteria by complement. Fractionation of cell membranes after complement C5b-9 deposition on to the surface of Salmonella minnesota Re595.

The effect of C5b-9 deposition on the envelope of target Gram-negative bacteria was studied. In order to understand the changes occurring after complement deposition on the bacterial surface, the preparation of Gram-negative bacterial membranes by different methods involving the osmotic lysis of spheroplasts was investigated. Subsequent fractionation of the outer membrane (OM) and cytoplasmic membrane (CM) by sucrose-density-gradient centrifugation showed differences in the membrane profiles obtained. The results indicate that optimum separation of OM and CM components requires effective digestion of DNA in the total membrane preparation before density-gradient fractionation. Salmonella minnesota Re595 carrying the intermediate complement complex C5b-7 (BC1-7) or C5b-8 (BC1-8) were efficiently killed upon incubation with purified C8 + C9 or C9 respectively. Human-alpha-thrombin-cleaved C9 (C9n), which is unable to form tubular poly(C9), was shown to be more effective at killing than native C9. By using an optimized system for the separation of OM and CM, it was found that, subsequent to lethal complement attack, the CM could not be recovered when C9 was used as the terminal complement component, but was recovered with reduced yield when C9n replaced C9. The results show that inability to recover the CM on sucrose density gradients after complement attack may not be a consequence of an essential membrane damage event required for complement-mediated killing of Gram-negative bacteria.     

The membrane attack complex of complement: C5b-8 complex as accelerator of C9 polymerization

Polymerization of C9 occurs spontaneously or can be induced by the tetramolecular complex C5b-8. Spontaneous C9 (0.15 mg/ml) polymerization required more than 3 days at 37 degrees C. In the presence of C5b-8, C9 polymerization was complete within 10 min. The molar C9:C5b-8 ratio determined the extent of tubular poly C9 formation by C5b-8-bearing phospholipid vesicles. When this ratio was 9:1 or 12:1, 72% of complex-bound C9 was present as SDS resistant tubular poly C9 (Mr = 1.1 X 10(6]. At lower C9:C5b-8 ratios, poly C9 was bound primarily in nontubular form. Tubular poly C9, as part of C5b-9, could also be generated on rabbit erythrocytes by using whole human serum as a complement source. At limiting serum concentration (molar C9 to C8 ratio approximately 2), no SDS-resistant tubular poly C9 was detected. At high serum concentration or when using serum that was supplemented with C9, up to 40% of the C9 was SDS-resistant tubular poly C9, and the rest was poly C9, which was incompletely polymerized. It is suggested that the C5b-8 complex acts as an accelerator of C9 polymerization, and that its relative concentration to C9 determines the ultrastructure of the C5b-9 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.     

Bacterial killing and inhibition of inner membrane activity by C5b-9 complexes as a function of the sequential addition of C9 to C5b-8 sites

The assembly of the C5b-9 complex on the outer membrane of C-sensitive cells of Escherichia coli results in a rapid inhibition of inner membrane function and ultimately a loss of cell viability. Cells bearing C5b-8 sites suffer no deleterious effects; however, the addition of C9 results in a rapid inhibition of inner membrane function and cell death. An attempt was made to examine the relationship between the toxic effects of the C5b-9 complex and the number of C9 molecules per C5b-8 site. Cells bearing C5b-8 sites were exposed to excess C9 at 0 degrees C and washed three times at 4 degrees C. The number of C9 molecules bound to each cell was equivalent to the number of C5b-8 sites present on each cell, and no additional C9 molecules could be bound when the cells were maintained at 4 degrees C. These cells were then incubated at 37 degrees C for 3 min and returned to 0 degrees C, a technique which exposed additional C9-binding sites equivalent to the number of C9 molecules previously bound to the cells. This technique was repeated and demonstrated that the sequential build-up of a C5b-9 site with two C9 molecules per C5b-8 site was capable of inhibiting both inner membrane function (respiration and amino acid transport) and cell viability. Three C9 molecules per complex had effects that approached the inhibitory effects of complexes formed in the presence of excess C9.     

Assembly of complement components C5b-8 and C5b-9 on lipid bilayer membranes: visualization by freeze-etch electron microscopy

We have visualized by freeze-etch electron microscopy the macromolecular complexes of complement, C5b-8 and C5b-9, respectively, assembled on synthetic phospholipid bilayers. These complexes were formed sequentially by using purified human complement components C5b-6 followed by C7, C8, and C9. Complexes of C5b-8 were observed on the external surface (ES) of vesicles as 12-nm particles that tended to form polydisperse aggregates. The aggregates were sometimes of a regular chainlike structure containing varying numbers of paired subunits. Etching of vesicles containing C5b-9 complexes revealed on the ES large rings of approximately 27-nm outer diameter. One or two knobs usually were attached to the perimeter of the rings. Splitting of the membrane resulted in partitioning of the C5b-9 with the outer leaflet. Thus, round holes of approximately 17-nm diameter were present in the protoplasmic face (PF), and raised circular stumps of a matching size were present on the exoplasmic face (EF) of C5b-9 vesicles. C5b-9 complexes were frequently localized in regions of the lowest lipid order. That is, in micrographs of the EF and ES, single C5b-9 complexes were located where the ripples of the P beta' phase bend or reach a dead end, and linear arrays of C5b-9 complexes outlined disclination-like structures in the lattice; the holes in the PF mirrored this distribution. The membrane immediately surrounding C5b-9 rings was often sunk inwardly over an area much larger than that of the ring itself.(ABSTRACT TRUNCATED AT 250 WORDS)     

Penetration of C8 and C9 in the C5b-9 complex across the erythrocyte membrane into the cytoplasmic space

We have developed a technique in which transglutaminase is used to measure the penetration of terminal complement proteins across the erythrocyte membrane into the cytoplasmic space. Penetration of a given terminal complement protein into the cytoplasmic space was assessed by labeling the protein of interest with radioactive iodine, forming the complement channel using the labeled protein, adding transglutaminase to only one side of the membrane, and allowing the enzyme to cross-link the susceptible proteins on that side of the membrane. Cross-linking was assessed by measuring the increase in molecular weight of the appropriate molecule on sodium dodecyl sulfate gels under reducing conditions. The results of these experiments indicate that C8 and C9 are rapidly cross-linked to high molecular weight from either the interior or the exterior of the membrane. In order to determine whether the cross-linking mediated by enzyme on the interior was occurring from within the ghosts and not via enzyme that had leaked into the extracellular medium, experiments were performed with dimethylcasein in the extracellular medium. In the presence of this protein, cross-linking of C8 and C9 from outside was negligible. Hence, if cross-linking occurs when transglutaminase is trapped inside the ghosts, it cannot be due to leakage of enzyme, but must be attributable to cross-linking from the inside. The results show that C9 definitely penetrated across the membrane into the intracellular space. With respect to C8, statistical evaluation indicates that C8 probably penetrated into the intracellular space.     

Multimeric C9 within C5b-9 is required for inner membrane damage to Escherichia coli J5 during complement killing

We have shown recently that an average of three or more C9 molecules must bind to C5b-8 on Escherichia coli strain J5 to cause direct complement killing in the absence of serum lysozyme. We initially confirmed and extended this observation by showing that deposition of a large number of C5b-9 complexes bearing 1C9 per C5b-8 was not bactericidal for J5. To identify the target site for bactericidal C5b-9 deposition, we measured release of periplasmic and cytoplasmic markers of different size from J5 as the C9:C5b-8 ratio was changed, because the diameter of the C5b-9 channel is known to increase as the C9:C5b-8 ratio increases. To facilitate measurement of release of the periplasmic marker beta-lactamase (BLA), J5 was transformed for high level constitutive TEM-1 BLA production (J5-Amp). Multimeric C9 within C5b-9 (C9:C5b-8 greater than 3) was required to release BLA (m.w. 28,900) from J5-Amp regardless of whether cells bore 310, 560, or 890 C5b-9/organism. Curves of both BLA release and killing vs C9:C5b-8 ratio were sigmoidal and nearly superimposable. Release of the small cytoplasmic marker 86Rb, a potassium analog, also required a minimum C9:C5b-8 ratio of 3:1; specific 86Rb release did not occur in the absence of killing. Release of the large cytoplasmic marker beta-galactosidase (m.w. 505,000) did not occur even at the highest achievable C9:C5b-8 ratio of 11:1, despite greater than 99.9% killing, indicating that there was no dissolution of the peptidoglycan layer due to incomplete removal of serum lysozyme. Complement-mediated killing of J5 requires sufficient damage to the outer membrane or formation of a sufficiently large C5b-9 channel to release the large periplasmic marker BLA. The requirement of multimeric C9 for 86Rb release suggests that at low C9:C5b-8 ratios, either C5b-9 does not have access to the cytoplasmic space or that the J5 K+ transport systems are able to compensate for putative C5b-9 channels.     

Effect of complement proteins C5b-9 on blood platelets. Evidence for reversible depolarization of membrane potential

The carbocyanine dye 3,3'-dipropylthiodicarbocyanine iodide has been used to investigate changes in membrane potential (Em) which occur upon binding of complement proteins C5b-9 to the plasma membrane of blood platelets. Gel-filtered platelets exposed to C5b6 and C7 in serum-free medium show no change in Em from that of controls, as indicated by either 3,3,'-dipropylthiodicarbocyanine iodide fluorescence or by the distribution of [14C]tetraphenylphosphonium bromide. Addition of complement proteins C8 and C9 to the C5b67 platelets results in partial depolarization of Em, which spontaneously repolarizes to basal levels within 15-20 min at 37 degrees C. Under these conditions, C5b-9-treated platelets show no increase in lysis over complement-free controls. Isotonic replacement of external sodium by either potassium or choline alters both the rate and extent of membrane depolarization and inhibits the platelets' capacity to repolarize after C5b-9 assembly. Repolarization of Em to basal levels is also completely blocked by addition of ouabain, confirming that this recovery is mediated by the plasma membrane Na+/K+ pump. These results demonstrate that membrane binding of the C5b-9 proteins can induce a transient change in Em when bound to the plasma membrane at a sublytic concentration, providing a mechanism for target cell activation by these potentially cytolytic proteins.     

Bactericidal but not nonbactericidal C5b-9 is associated with distinctive outer membrane proteins in Neisseria gonorrhoeae

In this study, we examined the bacterial constituents associated with the complement C5b-9 complex in detergent extracts from serum-treated Neisseria gonorrhoeae (GC). 125I surface-labeled GC were incubated in 10% serum, were washed, and were solubilized in the zwitterionic sulfobetaine detergent SB12. Immunoprecipitation of 125I-GC from the extract with anti-C5 Sepharose was followed by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography of immunoprecipitated material. Polyacrylamide gel analysis of surface-labeled 125I-GC showed prominent bands for proteins I and III for both serum-resistant GC strain 6305 and serum-sensitive GC strain 7189. These same bands were visible with similar intensity in the SB12 extracts from presensitized and non-presensitized 6305 and 7189 after serum incubation. For those organisms bearing bactericidal C5b-9 (6305 + IgG and 7189 +/- IgG), additional distinctive bands immunoprecipitated with antibody to C5 Sepharose. These components were of 93,000, 44,000 40,000, and 15,000 daltons for 6305 + IgG, and were of 90,000, 50,000, 44,000, and 19,000 daltons for 7189 +/- IgG. Nonbactericidal C5b-9 extracted from the surface of 6305 incubated in serum, but not sensitized with antibody, was not associated with these distinctive proteins. However, this nonbactericidal C5b-9 did have a different pattern of associated bacterial surface constituents from that observed in control samples incubated with antibody to human serum albumin, which were similar to those with nonserum-incubated organisms. These studies support our earlier experiments which demonstrated that C5b-9 is in a different molecular configuration on the surface of serum-resistant GC from that on the surface of serum-sensitive GC or resistant GC rendered sensitive with bactericidal antibody.     

Cyanine dye fluorescence used to measure membrane potential changes due to the assembly of complement proteins C5b-9

Summary The fluorescent potentiometric indicator diS–C₃-(5) has been used to investigate changes in membrane potential due to assembly of the C5b-9 membrane attack complex of the complement system. EAC1-7 human red blood cells and resealed erythrocyte ghosts—bearing membrane-assembled C5b67 complexes—were generated by immune activation in C8-deficient human serum. Studies performed with these cellular intermediates revealed that the membrane potential of EAC1-7 red cells and ghosts is unchanged from control red cells (–7 mV) and ghosts (0 mV), respectively. Addition of complement proteins C8 and C9 to EAC1-7 red cells results in a dose-dependent depolarization of membrane potential which precedes hemolysis. This prelytic depolarization of membrane potential—and the consequent onset of hemolysis—is accelerated by raising external [K⁺], suggesting that the diffusional equilibration of transmembrane cation gradients is rate limiting to the cytolytic event. In the case of EAC1-7 resealed ghosts suspended at either high external [K⁺] or [Na⁺], no change in membrane potential (from 0 mV) could be detected after C8/C9 additions. When the membrane potential of the EAC1-7 ghost was displaced from 0 mV by selectively increasing the K⁺ conductance with valinomycin, a dose-dependent depolarization of the membrane was observed upon addition of C8 and C9. In these experiments, lytic breakdown of the ghost membranes was <5%. Conclusions derived from this study include: (i) measured prelytic depolarization of the red cell Donnan potential directly confirms the colloid-osmotic theory of immune cytolysis. (ii) The diffusional transmenbrane equilibration of Na⁺ and K⁺ through the C5b-9 pore results in a dose-dependent depolarization of the membrane potential (E _m ) which appears to be rate-limiting to cytolytic rupture of the target erythrocyte. (iii) Enhanced immune hemolysis observed in high K⁺ media cannot be attributed to cation-selective conductance across the C5b-9 pore, and is probably related to the nearequilibrium condition of potassium-containing red cells when suspended at high external K⁺. These experiments demonstrate that carbocyanine dye fluorescent indicators can be used to monitor electrochemical changes arising from immune damage to the plasma membrane under both cytolytic and noncytolytic conditions. Potential application of this method to the detection of sublytic pathophysiological changes in the plasma membrane of complement-damaged cells are discussed.