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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↗
PubMed1989

Structural homology of complement protein C6 with other channel-forming proteins of complement.

The amino acid sequence of the amino-terminal half of the complement protein C6 has been found to show overall structural homology with the homologous regions of the channel-forming proteins C7, C8 alpha, C8 beta, and C9. In addition, two specific cysteine-rich segments common to the amino-terminal regions of C7, C8 alpha, C8 beta, and C9 also occur in their expected positions in C6, suggesting functional significance. Two cDNA clones encoding C6 were isolated from a human liver library in the bacteriophage vector lambda gt11. The predicted protein sequence contains an apparent initiation methionine and a putative signal peptide of 21 residues, as well as a site for N-glycosylation at residue 303. The sequence of the C6 protein reported here has 47-52% similarity with C7, C8 alpha, C8 beta, and C9, as well as 31-38% similarity with thrombospondin, thrombomodulin, and low density lipoprotein receptor. The sequence data have been interpreted by using computer algorithms for estimation of average hydrophobicity and secondary structure.

Amino Acid Sequence↗
PubMed1980

Identification of the activator system for antibody to Toxoplasma as the classical complement pathway.

In view of the many recent advances in our understanding of the composition and function of the complement system, it was decided to apply this newer knowledge to an investigation of the heat-labile activator required for the action of antibody to Toxoplasma gondii in the neutralization and dye tests. With use of antibody-coated toxoplasma trophozoites in a diluent of 0.2% gelatin in 0.85% NaCl and alkaline methylene blue as indicator, various component-deficient sera were added to activate the antibody on the protozoan membranes. It was determined that the classical complement system is required for antibody activity and that the properdin system plays no role in the reaction. Human sera genetically deficient in C5, C6, C7, and C8 were shown to be inactive as activators of antibody to Toxoplasma. The addition of specific missing components immediately restored full activity to the deficient sera.

Animals↗
PubMed1984

Formation of EAC142 and EAC1423 with macrophage culture supernatant containing the secreted complement components C1 to C3.

Culture supernatants of thioglycollate-elicited guinea pig peritoneal macrophages contained hemolytic C1, C4, C2 and C3, whereas hemolytic C5, C6, C7, C8 or C9 were not detected. Activity of C1, C2 and C3 increased up to a 48 h culture period, whereas C4 activity already declined in 2 day old cultures. After secretion, the hemolytic activity of C1 was least stable in culture supernatant. Sensitized sheep erythrocytes (EA) when incubated with culture supernatant initiated activation and functional cooperation of secreted C1 to C3 as indicated by formation of EAC142 and EA1423 intermediates. Decay and regeneration with purified C2 was shown for EAC142 and deposition of C3 fragments on EAC1423 was demonstrated with anti-C3. On an average, supernatants of 2 day old macrophage cultures were most suitable for formation of EAC142 and EAC1423 . The rate of EAC142 and EAC1423 formation, and also of C2 and C3 inactivation, during incubation of EA with culture supernatant was slow; addition of purified C1 to culture supernatant, however, greatly enhanced the same reactions of EA with supernatant which indicated that C1 was the rate limiting factor. Local secretion of hemolytic C1, C4, C2 and C3 by macrophages may have an important role in antimicrobial defense mechanisms due to the well-known functional cooperation between macrophages and activated C3.

Animals↗
PubMed1996

Identification of disulfide bonds in the ninth component (C9) of human complement.

C9 is the most abundant protein of the membrane attack complex of complement. By means of limited proteolysis, different chromatographic techniques, a thiol-specific fluorescence assay, amino acid analysis, and Edman degradation 9 out of 12 disulfide bridges are definitely assigned (Cys22-Cys57, Cys33-Cys36, Cys67-Cys73, Cys121-Cys160, Cys233- Cys234, Cys359-Cys384, Cys489-Cys505, Cys492-Cys507, Cys509-Cys518). Weaker evidence permits to reduce the number of possible configurations for the remaining 3 cystines (Cys80-Cys91, Cys86-Cys104, Cys98-Cys113, or Cys80-Cys91, Cys86-Cys113, Cys98-Cys104). These findings are discussed in comparison with the strongly related components C6, C7, C8alpha, and C8beta.

Amino Acid Sequence↗
PubMed1985

Depletion of complement by light and porphyrin does not depend on sequential activation.

The mechanism of complement depletion from human serum fortified with porphyrin and irradiated with light has been reinvestigated, with the conclusion that it did not depend on the normal sequence of complement activation. Thus, disappearance of the activities of C3, C4, and C5 was not dependent on divalent cations. Purified C3-C7 were labile to porphyrin/light treatment in the absence of other components. The depletion of C4 was not prevented by potent inhibitors of C1 and, unlike the depletion of C4 seen in response to aggregated gamma globulin, was insensitive to change in temperature. Electrophoresis showed an alteration of C3 unlike that caused by cobra venom factor and that light/porphyrin treatment nonspecifically altered many serum proteins.

Complement Activation↗
PubMed1987

Complementation between glucocorticoid receptor and lymphocytolysis in somatic cell hybrids of two glucocorticoid-resistant human leukemic clonal cell lines.

Somatic hybrids between two glucocorticoid-resistant clonal cell lines, CEM C1 and ICR-27, isolated independently from the CCRF-CEM human lymphoblastoid cell line, were constructed to investigate the complementation effect between the glucocorticoid receptor and the gene product(s) for inducing receptor-mediated lymphocytolysis. CEM C1 (r+ly-) has a normal amount of functional glucocorticoid receptor as compared to the steroid-sensitive clonal line CEM C7. Clone ICR-27 (r-ly?), which was originally isolated following mutagenesis of CEM C7 with the mutagen ICR 191, has few glucocorticoid receptors as determined by whole-cell receptor assay. The eight randomly selected CEM C1 X ICR-27 hybrid clones all showed sensitivity to 10(-6) M dexamethasone (ly+). The receptor site content of two near-tetraploid hybrids chosen for analysis was close to that of CEM C1 (r+). Hybrids constructed between CEM C1 and the receptor-bearing, steroid-sensitive clone, CEM C7 (r+ly+) also showed glucocorticoid sensitivity, and their receptor sites corresponded to the sum of those of CEM C1 and CEM C7 (r+r+ly+). These results indicate: that CEM C1 has no trans-active inhibitor of lysis; that CEM C1 has intact glucocorticoid receptor; and that ICR-27 and CEM C1 complement one another to restore lymphocytolysis. Therefore, CEM C1 cells can serve as a donor of human glucocorticoid receptors.

Cell Line↗
PubMed1986

The interaction of Escherichia coli with normal human serum: the kinetics of serum-mediated lipopolysaccharide release and its dissociation from bacterial killing.

We have examined the killing of E. coli and kinetics of lipopolysaccharide (LPS) release after the exposure of the bacteria to normal human serum (NHS) and sera deficient in complement components, or with inactivated complement components. LPS of the galactose epimerase-deficient strain E. coli J5 were specifically radiolabeled by growing the bacteria in a medium containing [3H]galactose. Exposure of the washed bacteria to NHS resulted in a significant reduction (greater than 99%) in viability within 15 min and the concomitant release of radiolabeled LPS. However, maximal release of LPS was consistently 30% of the total radiolabel incorporated into the LPS molecules. The amount of tritium-labeled LPS released was shown to be directly proportional to the concentration of bacteria exposed to NHS, suggesting that release of LPS was not limited by the availability of some critical serum component(s). The consumption of complement in NHS by incubation with E. coli was demonstrated by decreased alternative and classical pathway-specific hemolytic activity. The use of Factor D-depleted and VEM-treated human sera demonstrated that, with these bacteria, both the alternative and classical pathways of complement contribute to bacterial killing and release of LPS. It is noteworthy that, in VEM-treated and Factor D-depleted sera, the rate of killing and the kinetics of LPS release were somewhat slower as compared to control serum. Bacterial killing in C7-depleted and C9-deficient human sera was minimal. Neither killing nor LPS release occurred in heat-inactivated (56 degrees C, 30 min) human serum. The amount of [3H]LPS released by C9-deficient serum was qualitatively similar to the amount released by the action of NHS. Tritium-labeled LPS was not released in C7-depleted serum. These data indicate that bacterial killing can be dissociated from LPS release, and suggest that, whereas LPS release may be necessary for the bactericidal effects of serum complement, it is probably not sufficient to effect killing. Furthermore, a significant fraction of LPS can be removed from the outer membrane of the bacteria without an apparent affect on viability.

Adult↗