Fluid-phase assembly of the membrane attack complex of complement

The dynamics and protein stoichiometry of the fluid-phase assembly of the membrane attack complex of complement were characterized by using light-scattering intensity measurements. The assembly proceeded in an ordered manner with generation of stable and highly reproducible intermediates. In the absence of phospholipid or C8, mixtures of C5b-6 and C7 self-associated to fluid phase-C5b-7 which had a weight-average molecular weight of (4.1 +/- 0.2) X 10(6). This corresponded to an average of nine C5b-7 complexes per particle. The particles appeared heterodisperse on sucrose gradients with S20,W values ranging from 21 to 39 S. Addition of C8 and C9 caused no further aggregation or disassembly of the particles. When excess C8 was added to the aggregated C5b-7, the ratio of C8 incorporated per C5b-7 moiety was 0.98 +/- 0.03. At saturating levels of C9, the C9/C5b-8 ratio in the particles was 7.2 +/- 0.6. Incorporation of C8 caused a small increase in the Z-averaged particle diffusion coefficient [(9.9-10.3) X 10(-8) cm2/s], indicating that it added in a manner that "filled in the gaps" in the C5b-7 particles. C9 caused only small decreases in the particle diffusion coefficient and substantially decreased the f/fmin ratio. The time course for C9 incorporation into fluid phase-C5b-8 indicated an initial rapid phase followed by a slow phase. The rapid phase corresponded to the incorporation of about one C9 for every two C5b-8 complexes. This suggested that one C9 binding site was accessible on about half of the C5b-8 complexes. This may imply that only about half of the C5b-8 complexes were capable of C9 polymerization so that the ratio of C9 incorporated per functional C5b-8 was (14 +/- 2)/1. The initial velocity of the slow phase of C9 addition gave an activation energy of 37 kcal/mol. The activation energy for C5b-8-independent polymerization of C9 had a similar value of 41 kcal/mol. Light-scattering intensity measurements seemed to be a highly reliable method for quantitative characterization of the fluid-phase assembly.     

Activation of glomerular mesangial cells by the terminal membrane attack complex of complement

Treatment of cultured renal glomerular mesangial cells (MC) with nonlytic concentrations of the purified components (C5b-9) of the terminal membrane attack complex (MAC) of complement induced significant functional alterations characteristic of cellular activation. C5b-9-treated MC released large quantities of primarily vasodilatory prostaglandins. In addition, the secretion of an MC-derived auto-growth factor (MC interleukin 1) was greatly enhanced. Examination of the action of C5b-9 on MC phospholipid metabolism indicated that complement induced the activation of phospholipases, leading to quantitative changes in the fatty acid profile of MC membrane phospholipids. These findings demonstrate that cultured MC are highly responsive to nonlytic concentrations of the C5b-9 complex, and suggest that the mesangial deposition of the MAC in many forms of glomerular disease, with resultant cellular activation, may play a major role in the hemodynamic and cellular proliferative events characteristic of these disorders.     

Role of complement and complement membrane attack complex in laser-induced choroidal neovascularization

Choroidal neovascularization (CNV), or choroidal angiogenesis, is the hallmark of age-related macular degeneration and a leading cause of visual loss after age 55. The pathogenesis of new choroidal vessel formation is poorly understood. Although inflammation has been implicated in the development of CNV, the role of complement in CNV has not been explored experimentally. A reliable way to produce CNV in animals is to rupture Bruch's membrane with laser photocoagulation. A murine model of laser-induced CNV in C57BL/6 mice revealed the deposition of C3 and membrane attack complex (MAC) in the neovascular complex. CNV was inhibited by complement depletion using cobra venom factor and did not develop in C3(-/-) mice. Anti-murine C6 Abs in C57BL/6 mice inhibited MAC formation and also resulted in the inhibition of CNV. Vascular endothelial growth factor, TGF-beta2, and beta-fibroblast growth factor were elevated in C57BL/6 mice after laser-induced CNV; complement depletion resulted in a marked reduction in the level of these angiogenic factors. Thus, activation of complement, specifically the formation of MAC, is essential for the development of laser- induced choroidal angiogenesis in mice. It is possible that a similar mechanism may be involved in the pathophysiology of other angiogenesis essential diseases.     

PRELP protein inhibits the formation of the complement membrane attack complex

PRELP is a 58-kDa proteoglycan found in a variety of extracellular matrices, including cartilage and at several basement membranes. In rheumatoid arthritis (RA), the cartilage tissue is destroyed and fragmented molecules, including PRELP, are released into the synovial fluid where they may interact with components of the complement system. In a previous study, PRELP was found to interact with the complement inhibitor C4b-binding protein, which was suggested to locally down-regulate complement activation in joints during RA. Here we show that PRELP directly inhibits all pathways of complement by binding C9 and thereby prevents the formation of the membrane attack complex (MAC). PRELP does not interfere with the interaction between C9 and already formed C5b-8, but inhibits C9 polymerization thereby preventing formation of the lytic pore. The alternative pathway is moreover inhibited already at the level of C3-convertase formation due to an interaction between PRELP and C3. This suggests that PRELP may down-regulate complement attack at basement membranes and on damaged cartilage and therefore limit pathological complement activation in inflammatory disease such as RA. The net outcome of PRELP-mediated complement inhibition will highly depend on the local concentration of other complement modulating molecules as well as on the local concentration of available complement proteins.     

Recovery of human neutrophils from complement attack: removal of the membrane attack complex by endocytosis and exocytosis

Nucleated cells can resist lysis by and recover from complement attack even after formation of the potentially cytolytic membrane attack complex on the cell surface. We have found that human neutrophils resist complement lysis by the physical removal of membrane attack complexes by both endocytic and exocytic process. The latter mechanism predominates, vesiculation being detectable within 60 sec of initiating the complement cascade. Sixty-five percent of the formed complexes are removed on plasma membrane vesicles, although only 2% of the cell surface is lost. Ultrastructural examination revealed that these vesicles were covered with ring-like "classical" complement lesions. Analysis of these vesicles by gel electrophoresis indicated that C9 was present exclusively in the form of a sodium dodecyl sulfate-resistant, high m.w. complex. In contrast, the 35% of C9 that remained associated with the cells was found to be inaccessible to a C9-specific monoclonal antibody, and was partly degraded, suggesting internalization of the membrane attack complex and proteolysis of some C9 molecules. The molar ratio of C9 to C8 was 12 to 1 on shed vesicles and on recovered cells.     

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)     

Leukocyte complement: assembly of the membrane attack complex of complement by human peripheral blood leukocytes in the presence and absence of serum.

The specific neoantigenic determinants (neoAg) that are indicative of the assembled C5b-9 C complex are generated on the surface of peripheral blood leukocytes (PBL) during collection and processing of blood. Formation of neoAg on PBL could be prevented by collecting blood directly into 20 mM EDTA and, could be induced in vitro by adding autologous serum to isolated PBL that lacked neoAg. When neoAg was induced by the addition of serum containing 125I-labeled C8, the C8 was incorporated into a 23S complex which could be eluted from PBL. A mechanism for neoAg formation on PBL independent of exogenous serum factors was detected when PBL were placed into culture in serum-free medium. Results with metabolic inhibitors and 14C-leucine suggest that PBL can synthesize C5 and assemble the C5b-9 complex. The possible relevance of these findings to the understanding of mechanisms of cell-mediated cytotoxicity is discussed.     

The membrane attack complex of complement causes severe demyelination associated with acute axonal injury.

Complement is implicated in pathology in the human demyelinating disease multiple sclerosis and in animal models that mimic the demyelination seen in multiple sclerosis. However, the components of the complement system responsible for demyelination in vivo remain unidentified. In this study, we show that C6-deficient (C6-) PVG/c rats, unable to form the membrane attack complex (MAC), exhibit no demyelination and significantly reduced clinical score in the Ab-mediated experimental autoimmune encephalomyelitis model when compared with matched C6-sufficient (C6+) rats. In C6+ rats, perivenous demyelination appeared, accompanied by abundant mononuclear cell infiltration and axonal injury. Neither demyelination nor axonal damage was seen in C6- rats, whereas levels of mononuclear cell infiltration were equivalent to those seen in C6+ rats. Reconstitution of C6 to C6- rats yielded pathology and clinical disease indistinguishable from that in C6+ rats. We conclude that demyelination and axonal damage occur in the presence of Ab and require activation of the entire complement cascade, including MAC deposition. In the absence of MAC deposition, complement activation leading to opsonization and generation of the anaphylatoxins C5a and C3a is insufficient to initiate demyelination.     

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.     

Leukocyte complement: neoantigens of the membrane attack complex on the surface of human leukocytes prepared from defibrinated blood.

The neoantigenic determinants (neoAg) which have been identified in the human C5b-9 membranolytic C complex were detected here by the direct fluorescent antibody technique on the surface of 27 +/- 11% of viable peripheral blood leukocytes (PBL). The cells were prepared from defibrinated blood by sedimentation on Ficoll-Hypaque. Specificity of the antisera was established by quantitative inhibition of the fluorescent staining reaction, and of agglutination of EAC1-7, by highly purified C5b-9 complex. No inhibition was observed with fresh normal human serum. The majority of the PBL with surface neoAg was found in the B lymphocyte subpopulation that failed to form rosettes with sheep erythrocytes. NeoAg on B lymphocytes was removed to differing degrees by trypsin, papain, or pepsin treatment, and by maintaining the cells at 4 degree C for 20 hr in serum-free medium. The individual components, C5, C6, C7, C8, and C9, were also detected on the surface of PBL. With differential fluorescent stains, C5 and neoAg as well as C8 and neoAg could be detected on the same cells. The results indicate that viable B lymphocytes prepared from defibrinated blood, have the components of the membrane attack complex of C on their surface. The concomitant occurrence of the neoAg indicates that these proteins are present at least in part in the form of the assembled terminal complex.     

Membrane permeability to macromolecules mediated by the membrane attack complex

A simple and well-defined system of purified phospholipids and human complement proteins was used to study membrane permeability to macromolecules mediated by the membrane attack complex (MAC) of complement. Large unilamellar vesicles (LUVs) of phosphatidylcholine (PC) or phosphatidylserine (PS) containing trapped macromolecules [bovine pancreatic trypsin inhibitor (BPTI), thrombin, glucose-6-phosphate dehydrogenase (G6PD), and larger molecules] were used to monitor permeability. Membrane permeability to macromolecules was measured by thrombin inhibition by an external inhibitor or by separation of released molecules by gel filtration. Membrane-bound intermediates (C5b-8 or C5b-93) were stable for hours, and macromolecular permeability occurred without fragmentation, fusion, or aggregation of the vesicles. Quantitative membrane binding by C5b-7 as well as essentially quantitative release of thrombin was obtained for PS vesicles. MAC binding to PS-LUVs approximated the theoretical Poisson distribution curve for full release of vesicle contents by one complex per vesicle. Reactions with PC-LUVs occurred with some fluid-phase MAC assembly. Therefore, results from experiments with these vesicles were interpreted in a relative manner. However, the values obtained closely corroborated those obtained with PS-LUVs. At low C9/C5b-8 ratios, the size of the lesion was proportional to the C9 content of the MAC. Half-maximum release of BPTI, thrombin, and G6PD, by a single MAC per vesicle, required approximately 3,5, and 7 C9/C5b-8 (mol/mol), respectively. Larger molecules (greater than or equal to 118-A diameter) were not released from the vesicles. Release of G6PD (95.4-A diameter) required 45% of saturating C9. Therefore, it appeared that the last half of the bound C9 molecules did not increase pore size and the pore which released G6PD approached the diameter of the closed circular lesion measured (by others) in electron micrographs (approximately 100 A). The results were consistent with the formation of a stable membrane pore by a single complex per vesicle in which C9 molecules line only one side of the pore at low C9/C5b-8 ratios and maximum pore size is attained by incomplete, noncircular polymers of C9.     

The membrane attack complex of complement: relation of C7 to the metastable membrane binding site of the intermediate complex C5b-7

Isolated C7 (m.w. 120,000) in 1% deoxycholate (DOC) forms dimers with an apparent m.w. of 230,000 and a DOC-binding capacity of 82 mol per mol of dimer. Dimerization of C7 also occurs in the presence of DOC-phospholipid mixed micelles and eventuates in the insertion of C7 dimers into the lipid bilayer upon the removal of the detergent. C5b-7 complex formation in the fluid phase or on lipid vesicles likewise involves polymerization. C5b-7 sedimented with 17-40S, which suggests a dimeric to hexameric composition. In avidin-biotin binding experiments in which two differentially labeled forms of C5b,6 (biotinyl 125I-C5b,6, and 131I-C5b,6) were used in equimolar amounts to assemble C5b-7, more than 50% of the biotinyl 125I-C5b,6-containing complexes also contained 131I label; again suggesting that C5b-7 consisted of oligomers rather than monomers. The conformation of C7 in C5b-7 and in dimeric C7 appeared similar by the following criteria. On formation of C5b-7 from C5b,6 and C7, a 20% increase in beta-pleated sheet structure was observed by circular dichroism spectroscopy, and a similar change occurred on dimerization of isolated C7. Tryptic and thermolytic digests of C5b-7 and C7 dimers containing 125I-C7 were analyzed by autoradiography after SDS-polyacrylamide gel electrophoresis and were found to contain similar peptides that were distinct from those in the digests of monomeric C7. Direct evidence showing that the metastable membrane binding site of the C5b-7 complex resides in the C7 subunit was obtained by using the conjugates of C5b,6 and colloidal gold. Viewed in the electron microscope, these conjugates were aggregated upon the addition of isolated C7. In contrast, when conjugates of C7 and colloidal gold were treated with soluble C5b,6, no such aggregates occurred, but instead, individual C5b-7 complexes were observed arranged around single gold particles, resulting in star-like structures. The results strongly suggest that structures of C7 are responsible for the expression of the membrane binding site of metastable C5b-7.     

The inner mitochondrial membrane contains ion-conducting channels similar to those found in bacteria

Patch-clamp experiments were performed on rat liver mitochondria inner membranes. Application of voltage gradients of either polarity revealed the presence of several different conductances, ranging up to 1.3 nS in symmetrical 150 mM KCl. Evidence is presented that at least those higher than 0.3 nS are substates of the highest conductance channel. Increasing matrix-side-positive (unphysiological) transmembrane voltage gradients favored the switch of the 1.3 nS channel to operation in lower conductance states. The size of these conductances, the presence of substates and the channel behavior are strongly reminiscent on one hand of the observations on the membrane of protoplasts from the gram-positive bacterium Streptococcus faecalis, [Zoratti, M. and Petronilli, V. (1988) FEBS Lett. 240, 105-109], and on the other of some properties of previously described channels of mitochondrial origin.     

The membrane attack complex of complement: lipid insertion of tubular and nontubular polymerized C9

The membrane-restricted photoactivatable carbene generator 3-(trifluoromethyl)-3-(m-[125I]-iodophenyl)diazirine [Brunner, J., & Semenza, G. (1981) Biochemistry 20, 7174-7182] was used to label the subunits of the membrane attack complex of complement (C5b-9). C5b-9 complexes either were assembled from serum on erythrocyte membranes or were reconstituted from purified components on liposomes. After irradiation, most of the probe is bound to C9 independent of the membrane system used, indicating that the wall of the transmembrane channel is predominantly composed of C9. No difference was observed whether polymerized C9 was in the tubular or nontubular form [Podack, E. R., & Tschopp, J. (1983) J. Biol. Chem. 257, 15204-15212], showing that tubule closure is not essential for successful lipid insertion. The same label distribution between the two forms of polymerized C9 was obtained by analyzing zinc-polymerized C9 in the absence of C5b-8. Since the photoreactive probe reacted with at least two distinct polypeptide segments within C9, lipid interaction does not occur via a single segment of hydrophobic amino acids.     

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.     

Cutting edge: the membrane attack complex of complement is required for the development of murine experimental cerebral malaria

Cerebral malaria is the most severe complication of Plasmodium falciparum infection and accounts for a large number of malaria fatalities worldwide. Recent studies demonstrated that C5(-/-) mice are resistant to experimental cerebral malaria (ECM) and suggested that protection was due to loss of C5a-induced inflammation. Surprisingly, we observed that C5aR(-/-) mice were fully susceptible to disease, indicating that C5a is not required for ECM. C3aR(-/-) and C3aR(-/-) × C5aR(-/-) mice were equally susceptible to ECM as were wild-type mice, indicating that neither complement anaphylatoxin receptor is critical for ECM development. In contrast, C9 deposition in the brains of mice with ECM suggested an important role for the terminal complement pathway. Treatment with anti-C9 Ab significantly increased survival time and reduced mortality in ECM. Our data indicate that protection from ECM in C5(-/-) mice is mediated through inhibition of membrane attack complex formation and not through C5a-induced inflammation.     

Amplified gene expression in CD59-transfected Chinese hamster ovary cells confers protection against the membrane attack complex of human complement.

Protection against the pore-forming activity of the human C5b-9 proteins was conferred on a nonprimate cell by transfection with cDNA encoding the human complement regulatory protein CD59. CD59 was stably expressed in Chinese hamster ovary cells using the pFRSV mammalian expression vector. After cloning and selection, the transfected cells were maintained in media containing various concentrations of methotrexate, which induced surface expression of up to 4.2 x 10(6) molecules of CD59/cell. Phosphatidylinositol-specific phospholipase C removed greater than 95% of surface-expressed CD59 antigen, confirming that recombinant CD59 was tethered to the Chinese hamster ovary plasma membrane by a lipid anchor. The recombinant protein exhibited an apparent molecular mass of 21-24 kDa (versus 18-21 kDa for human erythrocyte CD59). After N-glycanase digestion, recombinant and erythrocyte CD59 comigrated with apparent molecular masses of 12-14 kDa, suggesting altered structure of asparagine-linked carbohydrate in recombinant versus erythrocyte CD59. The function of the recombinant protein was evaluated by changes in the sensitivity of the CD59 transfectants to the pore-forming activity of human C5b-9. Induction of cell-surface expression of CD59 antigen inhibited C5b-9 pore formation in a dose-dependent fashion. CD59 transfectants expressing greater than or equal to 1.2 x 10(6) molecules of CD59/cell were completely resistant to human serum complement. By contrast, CD59 transfectants remained sensitive to the pore-forming activity of guinea pig C8 and C9 (bound to human C5b67). Functionally blocking antibody against erythrocyte CD59 abolished the human complement resistance observed for the CD59-transfected Chinese hamster ovary cells. These results confirm that the C5b-9 inhibitory function of the human erythrocyte membrane is provided by CD59 and suggest that the gene for this protein can be expressed in xenotypic cells to confer protection against human serum complement.