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PubMed1982

Glutathione-catalyzed disulfide-linking of C9 in the membrane attack complex of complement.

The membrane attack complex of complement (the dimeric C5b-9 complex) is a multimolecular assemblage of five proteins (C5b, C6, C7, C8, and C9) which are held together by noncovalent forces. We found that C9 molecules in the complex can be covalently crosslinked (disulfide-linked) by glutathione. In this experiment, the tetramolecular C5b-8 complex bound to phospholipid vesicles was first prepared from purified C5b-6, incubated (37 degrees C, 20 min) with an excess of 131I-C9 in the presence of 1 mM glutathione; an average of 5.3 molecules of C9 per C5b-8 were bound and the C5b-9 complex formed was predominantly a dimeric C5b-9 complex. About one-third of C9 in this C5b-9 complex was found to be in a disulfide-linked dimeric form. The C5b-9 complex, having only an average of 0.9 molecules of C9 per C5b-8, was also prepared in the presence of glutathione; this C5b-9 preparation contained both monomeric and dimeric C5b-9 complexes, and about one-fifth of the C9 subunits was in a cross-linked dimeric form. By contrast, C9 in the absence of the C5b-8 complex was not significantly cross-linked by glutathione. These results indicate that C9 has a unique property to associate with itself upon reaction with the C5b-8 complex.

Centrifugation, Density Gradient↗
PubMed1986

C7 radiculopathy: importance of scapular winging in clinical diagnosis.

Lesions of the seventh cervical (C7) root are common and cause a readily recognised neurological syndrome. Recognition of this pattern is essential in differentiating C7 root lesions from lesions of the brachial plexus or peripheral nerves. Serratus anterior weakness is not generally included in this syndrome. We report six verified cases of C7 radiculopathy in which weakness of the serratus anterior was present in addition to the usual findings. This was manifest as winging of the scapula, when pushing forward against a wall, either with the hands at shoulder level or, in some cases, only when the hands were lowered to waist level. This latter method of testing places the muscle at a mechanical disadvantage and reveals partial paralysis. Analysis of this clinical finding complements anatomical evidence suggesting that the powerful lower digitations of the muscle may be primarily supplied by the C7 root in some cases. Scapular winging, apparent either in the usual position or the modified position described here, should be recognised as consistent with a diagnosis of C7 radiculopathy. When present, this sign serves to differentiate C7 radiculopathy from lesions of the brachial plexus or radial nerve.

Adult↗
PubMed1996

S-protein/vitronectin interaction with the C5b and the C8 of the complement membrane attack complex.

S-protein/vitronectin participates in the regulation of complement-mediated lysis by incorporating into the membrane attack complex C5b-9. Subsequently, soluble SC5b-7 and SC5b-9 macromolecules are not inserted into the membrane lipid bilayer nor induce lytic pore formation. Using a dot blot binding assay, it was determined that soluble S-protein/vitronectin interacted with immobilized C5b and C8 among the five separately immobilized components (C5b, C6, C7, C8, C9) of the membrane attack complex. Those interactions imply a new model of the formation of the complement membrane attack complex and its regulation by S-protein/vitronectin.

Animals↗
PubMedDate not supplied

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.

Alpha-Globulins↗
PubMed1994

Complement and antibody in neutrophil-mediated killing of type V group B streptococcus.

Serious infections caused by type V group B streptococci (GBS) are increasing. The requirements for antibody, complement, and neutrophil receptors in the killing of 12 clinical type V GBS isolates were investigated. When tested at concentrations of 33%, 5% and 1%, a human serum pool promoted neutrophil-mediated killing of the 12 isolates at a mean of 83% +/- 9%, 77% +/- 17%, and 14% +/- 28%, respectively. Addition of heated immune rabbit serum to the 5% or 1% pool increased killing significantly (97% +/- 2% or 80% +/- 15%, respectively, P < .01). With hypogammaglobulinemic serum, complement-mediated killing ranged from 74% +/- 2% for a strain designated resistant to 98% +/- 1% for a strain designated sensitive. Neutrophil-mediated killing was not altered by use of human sera deficient in C4 or C7 but was reduced significantly with C3-deficient serum (P < .05). Maximal inhibition of neutrophil-mediated killing was observed by monoclonal antibody blockade of complement receptor (CR) 3 alone or in combination with CR1 or Fc receptor III. Thus, C3 is required and specific antibody promotes neutrophil-mediated killing of type V GBS. Neutrophil CR3 and either CR1 or Fc receptor III optimize phagocytosis. A number of host responses function in concert to effect optimal neutrophil-mediated killing of clinical isolates of type V GBS.

Animals↗
PubMed1977

On the mechanism of cell membrane damage by complement: evidence on insertion of polypeptide chains from C8 and C9 into the lipid bilayer of erythrocytes.

The preceding paper (Hammer, C.H., A. Nicholson, and M. M. Mayer, 1975, Proc. Natl. Acad. Sci., 72:5076) presented evidence on insertion of polypeptide chains from the C5b and C7 subunits of C5b, 6, 7 complex into the phospholipid bilayer of erythrocyte membranes. In the present study, EAC1-8 and EAC1-9 (sheep erythrocytes carrying rabbit antibody and complement proteins C1 through C8 or C9, respectively), prepared with either 125I-C8 or 125I-C9, were incubated with trypsin or chymotrypsin and the release of 125I was measured. Only 9 to 19% of the specifically bound radioactivity was released. In addition, elution experiments were performed with 0.02 M EDTA-1.0 M NaCl. This solution did not elute C9 from EAC1-9. By contrast cellbound C9 was recovered from erythrocyte membranes with sodium dodecyl sulfate (SDS). Thus, enzymatic stripping and elution experiments indicate that cellbound C9 behaves like an integral membrane protein, presumably due to insertion into the lipid bilayer. EAC1-9 membranes that had been subjected to extended digestion with trypsin or chymotrypsin were extracted with SDS to recover the enzyme-resistant part of the C9 molecule from the membrane. Even though this domain of C9 carried 90% of the radioiodine associated with native C9, its m.w. was found to be only 18,000 daltons by analysis on SDS-PAGE. This represents one-quarter of the native C9 molecule.

Animals↗
PubMed1987

Terminal complement complex in plasma from patients with systemic lupus erythematosus and other glomerular diseases.

To evaluate terminal complement pathway activation in plasma from patients with systemic lupus erythematosus (SLE) and primary glomerular diseases, we developed an enzyme-linked immunosorbent assay (ELISA) for measuring the terminal complement complexes (TCC). The method is based on a sandwich technique using rabbit antibodies against native human C5, C7 and C9. To avoid interference by native components, we equilibrated plasma specimens with 5% polyethylene glycol buffer. The precipitates were measured by ELISA. TCC was detectable in all 14 normal controls (0.48 +/- 0.06 AU/ml; mean +/- s.e.m.). TCC levels were elevated in 18 of 54 patients with SLE (0.89 +/- 0.07 AU/ml; P less than 0.01) and in eight of 11 patients with membranoproliferative glomerulonephritis (MPGN) (3.15 +/- 0.62 AU/ml; P less than 0.01). However, only one of six patients with membranous nephropathy and none of 13 with mesangial proliferative glomerulonephritis showed high values. In SLE, TCC was correlated with circulating immune complexes and inversely with CH50, C3, C4, C5 and alternative complement pathway activity (AH50), and showed significantly high values even in normal CH50 cases (n = 34; P less than 0.01). In MPGN, TCC was inversely correlated with CH50, AH50, C3, C5 and C9. These results suggest that the classical pathway plays an important role for TCC generation in SLE and that the alternative pathway does in MPGN.

Adolescent↗
PubMed1989

Biosynthesis of the third and fifth complement components by isolated human lung cells.

Increasing research efforts have been directed at determining the contribution of locally synthesized (cell-derived) complement in host defense and inflammation. In the studies presented here, we determined the ability of a continuous cell line of type II pneumocytes (A549) and a cell line of human lung fibroblasts (WI-38) to produce complement components in vitro. Complement biosynthesis by A549 pneumocytes and WI-38 fibroblasts was demonstrated by incorporation of [35S]methionine into immunoprecipitable complement proteins. Using this technique, A549 pneumocytes were demonstrated to synthesize Clr, Cls, C4, C3, C5, C6, C7, C8, C9, Factor B, Factor H, Factor I, and C1s inactivator. In comparison, WI-38 fibroblasts were shown to synthesize Cls, C4, C3, C5, C6, C8, and C9. Because previous work has demonstrated the central role of C3, C5, and their activation products in regulating lung inflammation and tissue injury, we further investigated the production of C3 and C5 by both lung pneumocytes and fibroblasts using enzyme-linked immunospecific assays. A549 cells cultured in the presence of 15% fetal bovine serum (FBS) produced antigenic C3 (135 ng C3/ml/24 h) at a greater rate than did identical cells maintained in serum-free culture conditions (70 ng C3/ml/24 h). Similarly, antigenic C5 production by A549 pneumocytes was greatest in the presence of FBS when compared with cells maintained in serum-free culture conditions (245 ng C5/ml/24 h versus 155 ng C5/ml/24 h).(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗
PubMed1989

Effect of lipopolysaccharide on C3 and C5 production by human lung cells.

Although studies to date have demonstrated the ability of the monocyte/macrophage to produce C components in vitro, very few studies on C production by nonhepatic tissue cells have been reported. Recently, using 35S-methionine incorporation and immunoprecipitation techniques our laboratory has demonstrated the ability of tissue cells, i.e., the human lung type II pneumocyte (A549) and human lung fibroblast (WI-38), to synthesize and secrete a variety of early and terminal complement components, as well as several regulatory proteins in vitro, i.e., C1r, C1s, C4, C3, C5, C6, C7, C8, C9, factor B, factor H, factor I, and C1s inactivator. In our studies, we extended these observations by demonstrating the capability of LPS to modulate C3 production by A549 pneumocytes. Specifically, using a sensitive ELISA we demonstrated that A549 pneumocytes exposed to LPS induced an 80 to 180% increase in C3 levels when compared to untreated A549 cells. Interestingly, LPS had no effect on C5 production or total protein synthesis by A549 pneumocytes. In the case of the WI-38 fibroblast, LPS had no effect on 1) C3 production, 2) C5 production, or 3) total protein synthesis in vitro. These studies demonstrate that agents such as LPS have the potential to selectively regulate C production (i.e., C3) in individual lung cells in vitro, and suggests that in vivo LPS may alter the local tissue reservoir of C components during infection and lung injury, thus impacting on pulmonary inflammation and host defense.

Animals↗
PubMed1992

Human bactericidal/permeability-increasing protein and a recombinant NH2-terminal fragment cause killing of serum-resistant gram-negative bacteria in whole blood and inhibit tumor necrosis factor release induced by the bacteria.

The bactericidal/permeability-increasing protein (BPI) of neutrophils and BPI fragments neutralize the effects of isolated Gram-negative bacterial lipopolysaccharides both in vitro and in vivo. Since endotoxin most commonly enters the host as constituents of invading Gram-negative bacteria, we raised the question: Can BPI and its bioactive fragments also protect against whole bacteria? To determine whether the bactericidal and endotoxin-neutralizing activities of BPI/fragments are expressed when Gram-negative bacteria are introduced to the complex environment of whole blood we examined the effects of added BPI and proteolytically prepared and recombinant NH2-terminal fragments on: (a) the fate of serum-resistant encapsulated Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa that survive the antibacterial actions of whole blood and (b) the ability of these bacteria to trigger cytokine release. Added BPI in nanomolar concentrations killed each of three encapsulated strains of E. coli and in closely parallel fashion inhibited tumor necrosis factor (TNF) release. Holo-BPI and its NH2-terminal fragment were equipotent toward a rough LPS chemotype K1-encapsulated strain, but the fragment was substantially more potent than holo-BPI toward two encapsulated smooth LPS chemotype strains. TNF release induced by K. pneumoniae and P. aeruginosa was also inhibited by both holo-BPI and fragment but, at the protein concentrations tested, P. aeruginosa was killed only by the fragment and K. pneumoniae was not killed by either protein. The bactericidal action of BPI/fragment toward E. coli is inhibited by C7-depleted serum, but accelerated by normal serum, indicating that BPI, acting in synergy with late complement components, enhances extracellular killing of serum-resistant bacteria. Thus, BPI and an even more potent NH2-terminal fragment may protect against Gram-negative bacteria in the host by blocking bacterial proliferation as well as endotoxin-mediated effects, not only as components of the intracellular antibacterial arsenal of the neutrophil, but also as potentially therapeutic extracellular agents.

Antimicrobial Cationic Peptides↗
PubMed1994

Effect of cytokines on the secretion of the fifth and eighth complement components by HepG2 cells.

Liver cells can be induced by interleukin-1, tumor necrosis factor and interleukin-6 to secrete higher amounts of complement components. Information, so far available only for the early components, indicates that these cytokines exhibit different effects on various complement proteins. For instance, they promote the biosynthesis of C3 and B but have no effect on that of C4 and C2. These observations led us to evaluate the ability of interleukin-1, tumor necrosis factor and interleukin-6 to modulate the secretion of the late complement components by HepG2 cells, a human hepatoma-derived cell line known to produce several complement proteins. The amount of complement components in the culture supernatant was evaluated by a sensitive enzyme-linked immunosorbent assay revealing picogram levels of these proteins. The HepG2 cells were found to secrete a substantial amount of C3 (approximately 1 microgram/10(6) cells), easily detectable C5 (approximately 150 ng/10(6) cells) and C8 (approximately 10 ng/10(6) cells) and a low amount of C6 (approximately 0.5 ng/10(6) cells), whereas the levels of both C7 and C9 could not be measured. The addition of interleukin-1, tumor necrosis factor and interleukin-6 to the cell culture resulted in an enhanced secretion of C8, whereas that of C5 was only marginally increased. None of these cytokines had a clear effect on the secretion of C6 nor induced the production of C7 and C9.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma, Hepatocellular↗
PubMed1997

Neuronal expression of mRNAs for complement proteins of the classical pathway in Alzheimer brain.

To determine possible sources of complement proteins in the brain, we investigated by in situ hybridization expression of the mRNAs of C1q, C2, C3, C4, C5, C6, C7, C8 and C9 in postmortem Alzheimer disease (AD) and control brain tissue. We found detectable hybridization for all these components in the temporal cortex and hippocampus, with significantly higher levels being found in AD tissue. Hybridization signals were strongest over pyramidal neurons. Low or absent hybridization was seen in the visual cortex or cerebellum. These results suggest that the activated complement components found in association with AD lesions may be, in part, derived from neurons.

Aged↗
PubMed1989

CD59, an LY-6-like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells.

A novel cell surface antigen has been identified on a wide range of lymphoid cells and erythrocytes. A mAb YTH 53.1 (CD59) against this antigen enhanced the lysis of human red cells and lymphocytes by homologous complement. Studies of reactive lysis using different species of C56, and of whole serum used as a source of C7-9, indicated that the inhibitory activity of the CD59 antigen is directed towards the homologous membrane attack complex. CD59 antigen was purified from human urine and erythrocyte stroma by affinity chromatography using the mAb YTH 53.1 immobilized on Sepharose, and, following transient expression of a human T cell cDNA library in COS cells, the corresponding cDNA also identified using the antibody. It was found that the CD59 antigen is a small protein (approximately 20 kD as judged by SDS-PAGE, 11.5 kD predicted from the isolated cDNA) sometimes associated with larger components (45 and 80 kD) in urine. The sequence of CD59 antigen is unlike that of other complement components or regulatory proteins, but shows 26% identity with that of the murine LY-6 antigen. CD59 antigen was released from the surface of transfected COS cells by phosphatidylinositol-specific phospholipase C, demonstrating that it is attached to the cell membrane by means of a glycolipid anchor; it is therefore likely to be absent from the surface of affected erythrocytes in the disease paroxysmal nocturnal hemoglobinuria.

Antibodies, Monoclonal↗
PubMed1992

Role of TraT protein, an anticomplementary protein produced in Escherichia coli by R100 factor, in serum resistance.

Escherichia coli K12 strain W3110/SM bearing a plasmid containing the traT gene (traT+ strain) was more resistant to the bactericidal activity of guinea pig serum than the same strain bearing this plasmid without the traT gene (traT- strain). A murine mAb was generated against synthetic TraT peptide (86-99). This antibody reacted only with denatured TraT protein, but it was used for monitoring TraT protein by immunoblotting during purification of the protein. Six mAb were then generated against partially purified traT protein from the solubilized membrane fraction of the traT+ strain. These mAb reacted with the native protein even on living cells, and their F(ab) fragments were found to suppress the inhibitory effect of the TraT protein on the bactericidal activity of serum. TraT protein was purified from solubilized membranes of the traT+ strain by ion exchange and gel filtration chromatographies. The purified TraT protein inhibited the lysis of sensitized erythrocytes by serum complement. Its inhibitory action was mainly on the C6 step. It strongly inhibited the reaction of C6 with EAC14b2a3b and excess C5, C7, C8, and C9. TraT protein also inhibited the reaction of C7-deficient human serum with guinea pig erythrocytes when it was activated by cobra venom factor. It did not inhibit the reaction of preformed C5b6 complexes. However, TraT did not have any effect on the cleavage of 125I[C5] to 125I[C5b] in similar conditions. It also partially inhibited the reaction steps of C4, C5, and factor B and limited guinea pig complement serum in 0.1% gelatin veronal buffered saline, pH 7.4, containing 10 mM EDTA with their respective preceding intermediate cells. It had no effect on either the binding of C3 to EAC14b2a or the cleavage of C3b by factors H and I. TraT protein probably inhibits the formation of C5b6 complex or causes structural alteration of the complex to a nonfunctional form.

Amino Acid Sequence↗
PubMed2000

Expression of a complete and functional complement system by human neuronal cells in vitro.

We demonstrate in vitro expression of complement components, i.e. C3, factor H (FH), factor B (FB), C4, C1-inhibitor (C1-inh), C1q, C5, C6, C7 and C9, by four human neuroblastoma cell lines IMR32, SKNSH, SH-SY5Y and KELLY. Activating proteins C4, C9 and C1q, and regulatory proteins FH and C1-inh were produced constitutively by the four cell lines. C3, C6 and FB were mainly produced by SKNSH and SH-SY5Y. Western blot experiments showed that secreted proteins were structurally similar to their serum counterparts. An additional polypeptide of 43 kDa with FH immunoreactivity was detected, which could correspond to the N-terminal truncated form found in plasma. Regulation of complement expression by inflammatory cytokines, lipopolysaccharide and dexamethasone was tested in vitro. These factors had no significant effects on activating synthesis of components C3, FB and C4, but expression of regulating components C1-inh and FH was strongly increased particularly by IFN-gamma and tumor necrosis factor-alpha. The rate of synthesis of complement components was dependent on the differentiation of neuroblastoma cells. This effect of differentiation was also observed on normal rat neurons. Rat cerebellar granule cells constitutively expressed mRNA for C4 and C1q, but expression of C3 mRNA was induced by differentiation. This study shows that neurons could be another local source of complement in the brain, besides astrocytes and microglia. Human neuroblastoma cell lines can constitute an interesting model to analyze complement biosynthesis by human neurons. Local complement expression by neurons in vivo may be implicated in some physio-pathological processes.

Animals↗
PubMed1989

[Assembly of the membrane attack complex of complement in pemphigus vulgaris skin].

The time course of the deposition of the membrane attack complex of complement (MAC) in the skin of a case of pemphigus vulgaris was studied by immunofluorescence technique using monoclonal antibodies to human C5, C6, C7, C8, C9 and C5b-9 neoantigens. Biopsy specimens of skin lesions always contained the MAC-related antigens in the ICS areas. No MAC could be detected in the non-lesional skin. It was also noted that MAC could be generated in vitro on cryostat-sectioned normal human skin by the patient serum in the presence of complement. The titer of this complement-fixing antibody rose during the clinically active phase. Results of these studies suggest that complement activation, with subsequent assembly of MAC, may be related to acantholysis in the pemphigus skin.

Complement Membrane Attack Complex↗
PubMedDate not supplied

Structural/functional similarity between proteins involved in complement- and cytotoxic T-lymphocyte-mediated cytolysis.

Cytolysis mediated by complement or cytolytic lymphocytes results in the formation of morphology similar lesions in the target membrane. These lesions, formed by the polymerization of C9 or perforin respectively, contribute the major killing action by causing osmotic lysis of the target cell. Following the suggestion of Mayer that the mechanisms of humoral and cell-mediated cytotoxicity might be related, studies into the morphology of the membrane lesions formed, and the proteins responsible for causing the lesions, have shown several similarities. While the lesion caused by natural and T-killer cells is a little larger than that caused by complement, its overall shape is similar and in both cases the cylindrical pore is formed by polymerization of a monomeric subunit, C9 (relative molecular mass, Mr = 71,000) for complement, and perforin (Mr = 66,000) for cell-mediated cytotoxicity. C9 has an absolute requirement for a receptor in the target membrane formed by the earlier membrane attack complex components, C5b, C6, C7 and C8 (ref. 8). For perforin, polymerization in a target membrane requires no receptor, specificity being derived from the specific recognition between killer and target cell. Both proteins can be made to polymerize in vitro by the addition of divalent cations (Zn2+ for C9 (ref. 16) and Ca2+ for perforin) and the resultant complexes closely resemble their physiological counterparts. Antibodies raised against lymphocyte-killed targets have also been shown to cross-react with complement proteins, but the antigenically related proteins were not determined in these studies. We show here using purified proteins that perforin, C9 and complexes involving C7 and C8 share a common antigenic determinant which is probably involved in polymerization.

Amino Acid Sequence↗