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PubMed1985

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↗

PubMed1984

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↗

PubMed1992

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↗

PubMed1987

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↗

PubMed1990

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↗

PubMed1987

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↗

PubMed1988

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↗

PubMed1977

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↗

PubMed1993

Clusterin, the human apolipoprotein and complement inhibitor, binds to complement C7, C8 beta, and the b domain of C9.

Clusterin is a heterodimeric multifunctional protein expressed in a variety of tissues and cells. It forms high density lipid complexes in plasma and participates in the control of the lytic activity of the late complement complex (TCC, C5b-9). Together with vitronectin, clusterin binds to the nascent amphiphilic C5b-9 complex, rendering it water soluble and lytically inactive. To define the interactions that underlie the complement-inhibitory function of clusterin, we have examined the binding interactions between [125I]clusterin and the isolated components of the complex, C5b-6, C7, C8, and C9 and vitronectin. By using ligand blotting in the presence of Tween, specific binding of the labeled clusterin with C7, the beta-subunit of C8 and C9 was detected. Binding to C9 was competed by polymerized C9, but not by C8, C7, C6, and CD59, suggesting that the conformational change occurring during the hydrophilic-amphiphilic transition of C9 exposes the interaction site for clusterin. When thrombin-treated C9 was analyzed, clusterin was found to recognize the C9b fragment containing the hydrophobic membrane interaction segment. Both subunits of clusterin interact with C9 and are similarly potent in inhibiting C5b-9-mediated hemolysis and Zn+(+)-induced C9 polymerization. These results show that clusterin exerts its inhibitory effect by interacting with a structural motif common to C7, C8 alpha, and C9b.

Clusterin↗

PubMed1981

Effect of agents that produce membrane disorder on lysis of erythrocytes by complement.

To evaluate the effect of membrane lipid acyl-chain packing on the efficiency of cell lysis by complement, we have studied membrane modulation by 2-(2-methoxy)-ethoxyethyl-8-(cis-2-n-octylcyclopropyl)-octanoate (A2C) and by myristoleyl alcohol, the cis isomer of a C14:1 aliphatic alcohol. These substances are known to increase the membrane lipid disorder by virtue of the bend in their acyl chains, which is believed to loosen the phospholipid acyl-chain packing. We have found that both of these compounds markedly enhance the lysis of erythrocytes by the terminal complement proteins C5b-9. The enhancing effect by A2C is operative in the formation of erythrocytes carrying complement components C5b, C6, and C7, as well as in the subsequent reactions with complement components C8 and C9. We have also found that A2C-treated erythrocytes bind C5b6 to a measurable extent, whereas untreated erythrocytes do not. We attribute this to a shift in the partition equilibrium of C5b6 toward membrane association, which would improve lytic efficiency. The increase of membrane lipid disorder by these agents would also be expected to increase insertion of hydrophobic peptides from C7, C8, and C9, with consequent gain in lytic efficiency. Treatment of erythrocytes with sublytic doses of NaDodSO4, or Triton X-100 did not enhance lysis by C5b-9 appreciably, suggesting that enhancement of lysis by C5b-9 is not a general property of amphiphiles.

Complement C5↗

PubMed1983

Glycophorin A inhibits lysis by the complement attack phase.

A glycoprotein from human erythrocyte membranes has been found to inhibit lysis of target cells by the attack-phase components C5-C9 from human complement. The inhibiting molecule was purified and identified as glycophorin A. Thus, glycophorin A may have a regulatory function in the lytic complement attack on isologous cells.

Animals↗

PubMed1988

Restriction of cell lysis by homologous complement: II. Protection of erythrocytes against lysis by newly activated complement.

Our previous work revealed that homologous complement (C) was ineffective in lysing antibody-sensitized erythrocytes (EA) even at high concentrations. It was also shown that activation of complement on homologous EA resulted in the binding of C9 and the formation of EA bearing complement proteins C1 through C9 (EAC1-9), yet few hemolytic sites were formed. Instead, as shown here, the formation of homologous EAC1-9 caused the cells to become resistant to lysis even by heterologous complement during a second incubation. In contrast, when homologous EAC1-8 were produced by incubating EA with C9-depleted serum, such intermediates were not protected against lysis by heterologous complement during a second incubation. Furthermore, homologous C9 on EAC1-9 was able to reduce the hemolytic efficiency of heterologous complement without blocking C activation and the formation of new C5b-9 complexes. Protection was not modified when homologous EAC1-9 were produced in one step, by incubation of EA with serum, or sequentially by adding C9 to EAC1-8. The minimum number of 9-sites required to confer a protective effect on EAC1-9 was less than 200 per cell. Thus, in addition to its known effect in heterologous cell killing, homologous C9 is capable of protecting homologous cells against inadvertent complement lysis.

Ammonia↗

PubMed1984

Immunological studies of human placentae: complement components in pre-eclamptic chorionic villi.

Forty human placentae were studied by immunofluorescence for the presence of complement (C) components C1q, C4, C3d, C6 and C9 with the use of characterized antisera. The tissues were grouped as control placentae from 20 normal pregnancies and 20 from cases of pre-eclampsia (PE): the PE samples were sub-grouped as being obtained from patients with mild or severe PE. All of the C components studied were found in the same distribution for test and control samples, but statistical analysis of each pattern of distribution revealed that the deposition of C1q, C3d and C9 were increased in PE as compared to normal tissues. This impression was strengthened by the finding that the deposition of these C components was further increased when chorionic villus immunopathology was compared between mild and severe PE. These data indicate that immunological mechanisms are operating in PE chorionic villi, and they suggest that, among other mechanisms, immune processes may be operative in the pathophysiology of this clinical disease, and that more studies along these lines are in order to rule either in or out this possibility.

Adolescent↗

PubMed1998

Life-supporting pig-to-baboon heart xenotransplantation.

BACKGROUND: The aim of this study was to investigate the effect of the expression of human decay-accelerating factor in transgenic pigs on hyperacute rejection in a pig-to-baboon heterotopic heart transplantation model and to assess the ability of such transgenic pig hearts in supporting the life of a primate when transplanted orthotopically. METHODS: Hearts from pigs transgenic for human decay-accelerating factor were transplanted heterotopically (n = 3) and orthotopically (n = 5) into the baboon. All animals received cyclosporine, steroids, and cyclophosphamide. Blood was sampled regularly for total antipig antibody titers, trough cyclosporine levels, full blood count, electrolytes, and creatinine. Rejection of the heterotopic hearts was defined as the absence of palpable cardiac pulsation. Explanted hearts were examined histologically with hematoxylin and eosin and with immunochemistry for complement components C3, C4, C9, and immunoglobulin M. RESULTS: None of the hearts were hyperacutely rejected. In the heterotopic group one heart underwent acute vascular rejection on day 13, and the remaining two recipients with beating xenografts were killed on days 2 and 21. In the orthotopic group, one recipient with a life-supporting xenograft was killed on day 9 because of poor general condition. Histologic examination demonstrated no evidence of rejection. Two xenografts stopped beating on day 5, and histologic study showed acute vascular rejection in both. There were also two graft failures for technical reasons in this group. CONCLUSIONS: Hyperacute rejection is abrogated in pig-to-baboon heart xenotransplantation with the expression of the human decay-accelerating factor transgene. The human decay-accelerating factor transgenic pig heart is able to support primate life for a prolonged period.

Animals↗

PubMed2002

Indications for the immunological evaluation of patients with meningitis.

Although people with bacterial meningitis lack adequate protective antibody against the invading pathogen, most do not have an underlying immunodeficiency. Certain comorbid conditions increase the risk for development of bacterial sepsis and meningitis. In addition, certain congenital complement deficiencies, defects of antibody production, or asplenia may be first recognized by the occurrence of bacterial meningitis, particularly when it occurs in infants or young children. Deficiencies of the terminal components of complement (C5-C9) or properdin have been associated with recurrent or invasive neisserial infections, and asplenia, agammaglobulinemia, and deficiencies of the early components of complement (e.g., C1-C3) are associated with risks of infections caused by Streptococcus pneumoniae, Haemophilus influenzae, and meningococci. The presence of congenital or acquired immunodeficiencies should be considered in persons who present with bacterial meningitis on the basis of the etiology, clinical epidemiology, and presence of other risk factors.

Complement System Proteins↗

PubMed1985

Transmembrane channel-formation by five complement proteins.

Five serum proteins act in concert to form the membrane attack complex (MAC) of complement. The precursor proteins, C5, C6, C7, C8 and C9, are hydrophilic glycoproteins with molecular weights ranging from 70,000 to 180,000. When C5 is cleaved by the serine protease C5 convertase, nascent C5b is produced which forms together with C6 a soluble and stable bimolecular complex (C5b,6). Upon binding of C5b,6 to C7 a trimolecular complex (C5b-7) is formed, which expresses a metastable membrane binding site. Membrane-bound C5b-7 constitutes the receptor for C8 and the tetramolecular C5b-8 complex binds and polymerizes C9. During the assembly process the proteins undergo hydrophilic-amphiphilic transition and the end product consists of C5b-8 (Mr approx. 550,000) and of tubular poly C9 (Mr approx. 1,100,000). The functional channel size varies but its maximal diameter is approximately 10 nm. C9 polymerization appears to involve initial reversible associations of several C9 molecules, which leads to temperature dependent, constrained unfolding. Unfolded C9 monomers then associate laterally with each other and polymerization terminates with closure of the circular structure, which consists of 12-18 C9 monomers. Amino acid composition and sequence indicate that the N-terminal half of the single chain C9 molecule is hydrophilic and the C-terminal half rather hydrophobic. Phospholipid binding and insertion into membranes are functions of the C-terminal portion of the molecule.

Amino Acid Sequence↗

PubMed1986

Complement lysis of U937, a nucleated mammalian cell line in the absence of C9: effect of C9 on C5b-8 mediated cell lysis.

Previous studies have demonstrated that in general, nucleated cells are more resistant to killing by serum complement than are erythrocytes. During studies aimed at defining the mechanisms of nucleated cell resistance, we found that the human histiocytic cell line U937 was easily lysed by homologous serum. U937 cells were also killed by serum depleted of C9, but not by serum depleted of C8, implying that the C5b-8 complex was sufficient to cause lysis of these cells. Enumeration of complexes on the cell surface demonstrated that approximately 40-fold more complexes were required to lyse U937 cells in the absence of C9 than in the presence of an excess of C9. Examination of the effects of small amounts of C9 on lysis of U937 cells by the C5b-8 complex demonstrated that at very low doses, C9 inhibited C5b-8 mediated lysis. The use of radiolabeled anti-C8 antibody showed that C5b-8 complexes were eliminated from the surface of U937 cells at 37 degrees C, and C9 at the dose causing inhibition of lysis accelerated the elimination of complexes. These results suggest that the increased lytic potential resulting from binding of small amounts of C9 to C5b-8 complexes is outweighed by enhanced elimination of complexes resulting in decreased cell death.

Cell Line↗

PubMed2000

Comparison of two laboratory methods for the determination of serum resistance in Borrelia burgdorferi isolates.

A growth inhibition assay (GIA) and an immunofluorescence test detecting deposited complement components C6 and C9 were compared for their ability to classify Borrelia isolates with respect to their resistance to non-immune human serum (NHS). In both assays a total of 34 Borrelia isolates of all three human pathogenic genospecies were tested. Interestingly, 95% of the serum-sensitive or intermediate serum-sensitive isolates belonged to the genospecies B. burgdorferi s. s. and B. garinii, whereas most B. afzelii isolates (83%) proved serum-resistant. Consequently, a strong correlation between the assignment of the isolates to the different genospecies and their degree of serum sensitivity was seen. These findings were supported strongly by the quantitative analysis of the deposited complement components and the location of the terminal complement complex on the bacterial surface as detected by means of immunoelectron microscopy. The GIA displayed an obvious lack of sensitivity to slow growing isolates, whereas the IFA allowed classification of all Borrelia isolates. Discrimination between serum-sensitive and serum-resistant isolates in the IFA was the most specific provided that the detection of C6 and C9 was incorporated into the final classification of isolates. Accordingly, both assays, turned out to be effective and reliable tools for the investigation of borrelial serum sensitivity. The IFA, however, is regarded as superior to the GIA owing to the obvious ease of performance and its rapid capability for the classification of even very slow growing isolates.

Borrelia burgdorferi Group↗