Complex C5b-9 Research Articles

Monoclonal antibodies directed against gonococcal protein I vary in bactericidal activity.

Monoclonal antibodies (Mab) with specificity for protein I (PI) from Neisseria gonorrhoeae (GC) were examined for bactericidal activity. Mab 4G5 (gamma 3), ID3 (gamma 2a), and 1G6 (gamma 2a) bound to surface-exposed epitopes on PI of GC strain R11 (IA serotype) as assessed by co-agglutination and 125I protein A uptake. Mab 2H1 (gamma 3) that were directed against IB serotype strains and Mab 2E9 (gamma 2a) were negative in co-agglutination and protein A uptake assays and served as controls for some experiments. Only 4G5 and 1D3 were bactericidal for R11 when presensitized organisms were incubated in 10% absorbed, pooled normal human serum (PNHS) or 10% hypogammaglobulinemic serum (H gamma S) despite binding of nearly equivalent numbers of 4G5, 1D3, and 1G6 to R11 during presensitization, as assessed by 125I-protein A uptake. These Mab activated complement to a similar extent on GC R11, leading to deposition of 56.4 X 10(3), 61.9 X 1093), and 47.1 X 10(3) molecules of C3/organism during incubation in 10% C8-deficient serum. Deposition occurred almost exclusively via the classical complement pathway. Measurement of complement component C9 binding to R11 during incubation in H gamma S showed 35,700 molecules of C9/organism with 4G5, 32,600 C9/organism with 1D3, and surprisingly, 29,600 C9/organism with 1G6. Eight thousand four hundred molecules of C9/organism bound to 2E9-coated organisms, 6000 C9/organism to 2H1-coated bacteria, and 3600 C9/organism to nonpresensitized organisms. The C5b-9 complex deposited by 4G5 had a different sedimentation profile by sucrose density gradient analysis from the C5b-9 complex deposited by 1G6, consistent with a different molecular configuration of the bound complex. Mab 1G6 and 1D3, but not 2E9 or 2H1, were able to compete with 125I-4G5 for binding to GC R11. A Mab (2E6) directed against protein III of GC competed weakly with 125I-4G5 for binding to GC R11. Mab 1G6, but not 1D3, blocked 4G5-dependent killing in a dose-related fashion. Both 4G5 and IG6 reacted weakly with native PI of GC R11 by immunoblotting, but neither Mab recognized the 34,800 m.w. fragment of PI generated by trypsin and chymotrypsin treatment of outer membranes. In contrast, 2E9 reacted strongly by immunoblot with both native and cleaved PI of GC R11, suggesting binding to buried determinants of PI. These experiments show that Mab directed against identical or closely associated, surface-exposed epitopes on gonococcal PI differ markedly in bactericidal activity, despite leading to deposition of nearly equivalent numbers of C3 and C9 molecules per organism.

Characterization of human S protein, an inhibitor of the membrane attack complex of complement. Demonstration of a free reactive thiol group.

S protein, an inhibitor to the membrane attack complex of complement, was purified from human plasma. The procedure involved barium citrate adsorption and fractionation by poly(ethylene glycol) 4000 precipitation, followed by chromatography on DEAE-Sephacel, Blue Sepharose, Sephacryl S-200, and finally anti-albumin-Sepharose. Reduced glutathione was added throughout to inhibit spontaneous formation of disulfide-linked S-protein dimers. The recovery was 7%, resulting in approximately 10 mg of pure S protein from 1 L of starting plasma. S protein is a single-chain molecule; sedimentation equilibrium ultracentrifugation yielded a molecular weight of 83 000; the s020,W value was estimated to be 4.0 S. The purified protein contained a free, reactive thiol group causing spontaneous formation of disulfide-linked S-protein dimers. Alkylated and nonalkylated S proteins were equally active in inhibiting C9 polymerization, catalyzed by the C5b-8 complex. In parallel with the inhibition of C9 polymerization, nonalkylated S protein catalyzed the formation of disulfide-linked C9 dimers, presumably through disulfide interchanges.

The relationship between channel size and the number of C9 molecules in the C5b-9 complex.

Abstract We have recently shown by dose-response analyses with resealed erythrocyte ghosts that the channel formed by complement is a monomer of C5b-9 of the composition C5b61C71C81C9n, in which n = 1 for channels permitting passage of sucrose (0.9 nm molecular diameter) and n = 2 for channels allowing transit of inulin (3 nm molecular diameter) (1). We have now continued these experiments and expanded them by including ribonuclease A (molecular diameter, 3.8 nm) as a marker to assess whether additional C9 molecules enlarge the functional C5b-9 channel. Our results show that formation of C5b-9 channels displays one-hit characteristics with respect to C5b6 when tested by transmembrane passage of inulin or ribonuclease A. By contrast, analysis of dose-response curves of C9 indicate that n = 2-3 for channels allowing transit of inulin and n = 4 for channels allowing transit of ribonuclease A. We have also performed sieving experiments with ghosts carrying C5b-7 and containing two small markers, inositol and sucrose. Dose-response curves for C8 were performed in the presence of excess C9 to ensure conversion of all C5b-8 to C5b-9 channels. The results indicate that small channels (approximately 0.8 nm effective diameter) are not formed at high C9 multiplicity, thus confirming the results obtained with the larger markers, i.e., increase of C9 input leads to formation of larger channels.

Monoclonal antibodies to human plasma protein X alias complement S-protein.

Protein X alias complement S-protein was isolated by dissociation from purified XC5b-9 (fluid-phase terminal C5b-9) complexes with 250 mM deoxycholate and subsequent sucrose density gradient centrifugation and Sephacryl gel chromatography. Polyclonal rabbit and monoclonal mouse antibodies were used to preliminarily characterize the protein in human serum and plasma. In plasma, Protein X yielded a symmetrical immunoprecipitate of alpha 2-mobility in a crossed immunoelectrophoresis assay. However, a second immunoprecipitate of alpha 1-mobility was observed when serum was analysed; this precipitate represented Protein X in complex with antithrombin-III. The co-precipitation of Protein X with serum antithrombin-III was exploited for establishing a simple screening test for unequivocal identification of monoclonal anti-Protein X antibodies. SDS-PAGE immunoblotting with monoclonal antibodies showed that Protein X exhibits pronounced microheterogeneity, migrating as a diffuse moiety of approx. Mr 80-90 000. Additionally, a small amount of polymeric aggregates appear to be present in plasma. Reduction of disulfide bonds led to liberation of a polypeptide of approx. 15 K as discerned by two-dimensional SDS-PAGE immunoblotting. Protein X is not cleaved to lower molecular weight entities during the process of blood coagulation or during formation of fluid-phase terminal complement complexes. The plasma concentrations in healthy adults were in the range of 500-700 micrograms/ml. The availability of methods for isolating Protein X and raising monoclonal antibodies will facilitate further studies on the dual role of this protein in the terminal complement and coagulation cascades.

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.

The cobra complement system: II. The membrane attack complex

Rabbit erythrocytes are lysed by cobra plasma. The membranes of the lysed rabbit erythrocytes exhibited typical ultrastructural complement lesions with an inner diameter of 72 Å. Structures similar to human C5b-8 complexes were also visualized. The proteins found in membranes lysed by cobra plasma resembled closely the proteins of the human membrane attack complex in number and molecular weight. Cobra poly C9 that is resistant to reduction and boiling in SDS could also be demonstrated. These results indicate that the membrane attack complex of cobra complement is very similar to its mammalian counterpart.

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.

Possible involvement of terminal complement complex in active Heymann nephritis

We investigated whether the appearance of various complement components in renal deposits of immune complexes correlated with the development of proteinuria in rats with active Heymann nephritis. Sequential kidney biopsy specimens and serum samples were obtained from Lewis rats immunized with Fx1A in complete Freund's adjuvant. Circulating antibodies against purified auto-antigen renal tubular epithelial glycoprotein, as measured by ELISA, were found in the circulation together with a diffuse granular deposition of IgG1, IgG2a, and IgG2b in the glomeruli within 2 weeks after immunization. Biopsy specimens taken 4 weeks after immunization showed diffuse deposition of C4 and C3, which indicated that activation of complement by the classical pathway had occurred. The detection of the C5b-9 complex of complement in glomerular deposits coincided with the development of abnormal proteinuria indicating that the glomerular damage in this autoimmune disease may be caused by complement-mediated lesions in the glomerular capillary walls.

Effect of lethal doses of complement on the functional integrity of target enterobacteria.

Exposure of a wide variety of gram-negative bacteria to serum results in the activation of either the classical or alternative pathway of complement and the generation of macromolecular C5b-9 protein complexes. When formed on the surface of susceptible Escherichia coli (Bladen et al. 1966) or other gramnegative bacteria (Bladen et al. 1967; Swanson and Goldschneider 1969; Harriman et al. 1982), C5b-9 complexes form remarkably stable lesions embedded in the bacterial envelope where they behave essentially as integral membrane proteins (Joiner et al. 1983; Kroll et al. 1983). Complexes are formed from the fluid-phase proteins (C5b, C6, C7, C8, and C9) of the terminal complement membrane attack pathway by spontaneous association following enzymatic cleavage of C5 (Bhakdi 1980; Mayer 1981). Polymerization is accompanied by the appearance of terminal apolar regions on the cylindrical C5b-9 complex that facilitate insertion into hydrophobic domains of target membranes (Bhakdi and Tranum-Jensen 1978). There is a large body of evidence indicating that insertion of complexes into the bacterial envelope is directly responsible for initiating the sequence of events that results in the death of the target bacterial cell (Inoue et al. 1968 b; Schreiber et al. 1979; Joiner et al. 1982 a, b; Kroll et al. 1984). Conversely, serum-resistant strains appear to escape the potentially lethal action of complement because C5b-9 complexes fail to insert in a stable fashion into the target membrane (Joiner et al. 1982b; Kroll et al. 1983; Taylor and Kroll 1984).

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.

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.

High-density lipoprotein and its apolipoproteins inhibit cytolytic activity of complement. Studies on the nature of inhibitory moiety

Human high-density lipoprotein (HDL) and its apolipoproteins A-1 and A-II inhibit complement-mediated lysis of human and sheep erythrocytes. This inhibitory activity under study is exerted after C9 is bound to membrane-associated C5b-8 complexes but prior to completed assembly and insertion of the C5b-9 complex. In this paper, we define some structure-activity relationships of the inhibitory moiety. With the exception of weak lytic inhibitory activity found in LDL/VLDL pools and in some unconcentrated minor fractions of plasma obtained by hydrophobic chromatography, all inhibitor activity was found in fractions which contained either apolipoprotein A-I, apolipoprotein A-II, or both. Intact HDL has a high level of inhibitor activity but delipidation by chloroform-methanol extraction was associated with an increase in activity on a protein-weight basis. Purified apolipoprotein A-I and apolipoprotein A-II exhibited equal inhibitory activity, greater than that exhibited by intact HDL. Nevertheless, ultracentrifugal fractions in which no free apolipoproteins could be demonstrated still possessed inhibitory activity. These experiments suggest that delipidation of HDL is not necessary for expression of inhibitor activity, although we could not rule out the possibility that apolipoproteins in dynamic equilibrium with HDL are responsible for the inhibitor activity observed in whole serum and plasma and in HDL preparations. Limited proteinase digestion completely abolished the inhibitory activity of partially delipidated HDL. Phospholipase C had little or no effect on the inhibitory activity of delipidated HDL, apolipoprotein A-I or apolipoprotein A-II, but reduced the inhibitory activity of intact HDL. These data suggest that the phospholipid polar headgroups are not necessary for inhibitory activity. However, the loss of these headgroups is associated with decreased activity, possibly due to increased hydrophobicity of HDL, or increased association among HDL micelles, and subsequent decrease in effective molar concentration of the inhibitory moiety.

Increased plasma levels of the terminal complement complex in patients with evidence of complement activation.

The terminal C5b-9 complex of human complement has recently been described and quantified in normal human plasma by an enzyme-linked immunosorbent assay (ELISA). We collected EDTA plasma samples from 20 patients clinically suspected to have complement activation. The terminal complement complex (TCC) and C3d split products were measured. The TCC was increased in 8 patients, and 6 of these also had increased C3d values, whereas 4 patients had increased C3d and normal TCC values. Two different double-antibody assays were used to detect terminal pathway activation: the combination of anti-C6 and anti-C9 detecting only the whole complex, and the combination of anti-C6 and anti-C5 detecting intermediate complexes as well. There was a close correlation between the observations in these two assays, suggesting that in general the whole cascade including C9 is involved when the terminal pathway of complement is activated in vivo. Quantification of TCC in plasma is an important supplement to already established methods for the evaluation of complement activation in vivo.

Inside-out vesicles prepared from complement lysed sheep red blood cells.

Antibody sensitized sheep red blood cells were lysed to 68% by diluted human serum. The ghosts were shown to undergo vesiculation induced by low ionic strength buffer and gentle shearing forces like osmotically lysed ghosts. About 45% of the vesicles derived from complement lysed ghosts (as referred to vesicle protein) remained floating on top of a linear 3-18% dextran T110 gradient during ultracentrifugation. Using antibodies as probes for sideness about 90% of the floating vesicles displayed an inside-out orientation. Floating inside-out vesicles bear a slightly reduced but significant number of C9 molecules on their inverted extracellular side of the membrane as compared to the (leaky or collapsed) vesicles banding in the gradient. This finding suggests that the floating is not due to a lack of C5b-9 (under the assumption that all C9 was bound to C5b-9). It is concluded that lysis with diluted human serum may result partly in ineffective C5b-9 complexes and/or in lesions with limited permeability which do not impair vesicle floating on top of polymer gradients.

Stable insertion of C5b-9 complement complexes into the outer membrane of serum treated, susceptible Escherichia coli cells as a prerequisite for killing.

Escherichia coli 17, a K12 derivative, was rapidly killed by human serum following a short lag period of 10 min. Stable binding of terminal C5b-9 complement complexes was investigated in time course experiments. Serum treated E. coli cells were lysed osmotically and the resulting outer and cytoplasmic membrane vesicles separated by sucrose gradient centrifugation. Exposure of E. coli 17 to serum rapidly reduced the degree of recoverability of cytoplasmic membrane vesicles. Electron microscopy revealed no interaction of C5b-9 complexes with CM vesicles. In contrast there was a clear time-dependent deposition of terminal complement complexes onto OM-vesicles. Very few complexes were detected during the prekilling phase of the reaction; initiation of the active killing phase was accompanied by a large increase in complement lesions. In contrast, no C5b-9 complexes could be visualised on outer or cytoplasmic membrane vesicles of a smooth, serum-resistant E. coli strain. We conclude that complement-mediated killing is a consequence of stable binding of C5b-9 complexes to the outer membrane of susceptible strains.

Characterization of the interaction of human C4b-binding protein with physiological ligands.

The binding of C4b to C4b-binding protein (C4BP) was demonstrated at physiological ionic strength by analytical ultracentrifugation. The sedimentation rate of C4BP gradually increased from 9.4 S to a maximum of 18.5 S with increasing C4b concentration. The stoichiometry of different C4BP X C4b complexes was calculated from the sedimentation-velocity data. A linear relationship was established between the number of C4b bound per C4BP and the sedimentation rate of the complex. In order to define further the C4BP-C4b interaction, sucrose density gradient ultracentrifugation was also used. Trace amounts of 125I-C4BP were centrifuged through 12 sucrose density gradients, each of which contained a different concentration of C4b throughout the gradient. The sedimentation rate of the C4BP increased with increasing C4b input to a maximum of 19.5 S. These binding data, in conjunction with the stoichiometry measurements determined in the analytical ultracentrifuge, were analyzed by the methods of Scatchard and Hill. At physiological ionic strength, C4BP exhibited four binding sites for C4b, each having an association constant of 1.2 X 10(7) M-1. A Hill coefficient of 1.1 was calculated, indicating that the four binding sites were independent. At reduced ionic strength, two additional sites were detected. The sedimentation coefficient of C4BP(C4b)6 was 24 S. The hydrodynamic data suggest that after four C4b molecules have bound to C4BP, the binding of additional C4b is sterically hindered. This interpretation implies that all six binding sites on C4BP are identical. C4BP also bound C4(H2O) (the product resulting from spontaneous hydrolysis of the thiol ester bond in native C4) and weakly bound C4c, but had no measurable affinity for native C4 or C4d at physiological ionic strength. A low-affinity interaction between C3b and C4BP was also demonstrated in the analytical ultracentrifuge. The C4BP X C3b complex was specific because C4BP mediated the cleavage of C3b by Factor I to C3bi with concomitant dissociation of the complex.

Species-restricted target cell lysis by human complement: complement-lysed erythrocytes from heterologous and homologous species differ in their ratio of bound to inserted C9.

The cytolytic efficiency of the terminal complement complex (C5b-9) against erythrocytes of different species is, in part, dependent on the species of C9 origin. In the present study, we have examined the interaction of C9 with erythrocytes in terms of the binding, dimerization, and insertion of C9 into the membranes of sheep and human erythrocytes lysed by human complement (C). The membranes of these C-lysed erythrocytes were analyzed for bound, dimerized, and inserted C9 by a combination of photolabeling, SDS-PAGE, electroblotting, and immunostaining techniques. We found that neither binding nor dimerization of C9 could be correlated with the relative hemolytic efficiency of human C on these erythrocytes, but that C9 insertion into the membranes of these cells varied in direct relation to the extent of lysis. Interestingly, the binding of C3 to these cells under conditions of equivalent C1 fixation also correlated with lytic efficiency. These data indicate that the C9-related differences in the cytolytic efficiency of C against erythrocytes from different species is primarily due to the efficiency of C9 insertion into these cells. Moreover, these data emphasize that neither the binding of C9 to a target membrane nor the formation of C9 dimers necessarily leads to the insertion of C9 into the membrane, suggesting the presence of membrane-bound but inactive C5b-9 complexes. Because the extent of C3 binding also correlated with the relative degree of lysis of sheep vs human erythrocytes, the possibility exists that surface-bound C3 may regulate hemolysis by directing the insertion of C9 in terminal complexes into cells.

Mechanism of complement cytolysis and the concept of channel-forming proteins.

Complement damages membranes via the terminal reaction sequence that leads to the formation of membrane-bound, macromolecular C5b-9(m) protein complexes. These complexes represent C5b-8 monomers to which varying numbers of C9 molecules can be bound. Complexes carrying high numbers of C9 (ca. 6/8-12/16?) exhibit the morphology of hollow protein channels. Because they are embedded within the lipid bilayer, aqueous transmembrane pores are generated that represent the primary lesions caused by complement in the target cell membrane. Many other proteins damage membranes by forming channels in a manner analogous to the C5b-9(m) complex. Two prototypes of bacterial exotoxins, Staphylococcus aureus alpha-toxin and streptolysin-O, are discussed in this context, and attention is drawn to the numerous analogies existing among these protein systems. Common to all is the process of self-association of the native proteins to form supramolecular complexes. This event is in turn accompanied by a unique transition of the molecules from a hydrophilic to an amphiphilic state.

On the cause and nature of C9-related heterogeneity of terminal complement complexes generated on target erythrocytes through the action of whole serum.

The binding of C8 and C9 from human serum to target erythrocytes was quantified, and the molecular stoichiometries of C9:C8 within terminal C5b-9(m) complexes were determined. Low doses of serum generated terminal complexes with mean C9:C8 ratios of 2 to 3:1, whereas complexes generated by highest serum doses harbored an average of six to eight C9/C8 molecules. From the collective biochemical and ultrastructural data, we concluded that heterogeneous populations of terminal complexes regularly form on target membranes; those containing high numbers of C9 molecules (greater than or equal to six to eight) exhibit the structure of the classical "lesion", whereas those containing low numbers of C9 do not exhibit this typical structure, although they probably still function as small pores. A major cause for this heterogeneity of the lesions derives from shortage of C9, which is naturally present in a 2 to 1 molar ratio relative to C8 in serum. Generation of terminal complexes harboring high numbers of C9 on erythrocyte membranes is possible in spite of this natural shortage because SC5b-9 does not form in the fluid phase to compete for C9 binding. If interrupted, the process of C9-C9 oligomerization cannot be recontinued, and "incomplete" C5b-9 complexes are unable to bind additional C9 upon reincubation with this component. The demonstrated heterogeneity of terminal complexes with respect to their C9 content may explain the functional heterogeneity of complement lesions observed previously by other investigators.

Measurement of the ratio of the eighth and ninth components of human complement on complement-lysed membranes.

The mole ratio of the eighth (C8) and ninth (C9) components of human complement on membranes carrying the cytolytic C5b-9 complex was measured by direct binding assays. Erythrocytes from two different species were used as the membrane system. Antibody-treated sheep erythrocytes carrying a relatively small number of precursive membrane-bound C5b-7 complexes were prepared by exposure to human C8-depleted serum. These complexes were subsequently converted to C5b-8 by addition of saturating amounts of C8. Parallel binding assays using 125I-C8 were used to determine the exact amount bound and thus the number of C5b-8 complexes per cell. These cells were subsequently incubated with excess 125I-C9 and the amount bound relative to C8 on the membrane was measured. Results indicated the C8:C9 ratio remained constant at approximately 1:4 as the number of complexes varied from 40 to 310 per cell. Similar results were obtained regardless of whether C8 and C9 were added sequentially or simultaneously to cells bearing C5b-7. For comparison, experiments were also performed using membranes that contained a high number of complexes. Here, rabbit erythrocytes which carried approximately 25 000 C5b-7 per cell were incubated with limited amounts of C8 to form C5b-8 complexes on the membrane surface, the exact number of which was measured by 125I-C8 binding assays. When erythrocytes prepared in this manner were incubated with excess 125I-C9, the ratio of C8:C9 on the membrane was found to be essentially constant at approximately 1:3 as the number of these complexes varied from 50 to 4000 per cell.(ABSTRACT TRUNCATED AT 250 WORDS)