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Biomedical subjects

W Fiers

Publications and source records attributed to W Fiers.

At least 19 recordsLinked to original sources
PubMed1992

Tumor necrosis factor stimulates multiple serine/threonine protein kinases in Swiss 3T3 and L929 cells. Implication of casein kinase-2 and extracellular signal-regulated kinases in the tumor necrosis factor signal transduction pathway.

Incubation of Swiss 3T3 or L929 cells with tumor necrosis factor (TNF) leads to the rapid stimulation of several cytosolic Ser/Thr kinases active toward myelin basic protein, the S6 peptide (RRLSSLR), the G peptide (SPQPSRRGSESSEE), and Kemptide (LRRASLG). This confirms the hypothesis that kinases other than protein kinases A and C may be involved in the TNF signal transduction. Chromatography on Mono Q resolved multiple kinase peaks with each substrate tested and moreover revealed a TNF-mediated casein kinase-2 activation in both cell lines, measurable with the specific RRREEESEEE peptide or with the G peptide. The TNF-stimulated myelin basic protein kinases-1 and -2 were identified as extracellular signal-regulated kinases-2 and -1, respectively, based on their elution pattern on Mono Q chromatography, their inactivation by protein phosphatase action, their reaction with phosphothreonine and phosphotyrosine antibodies, and by their migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as 42- and 44-kDa proteins recognized by anti-extracellular signal-regulated kinase antibodies.

3T3 Cells↗
PubMed1992

Functional characterization of the human tumor necrosis factor receptor p75 in a transfected rat/mouse T cell hybridoma.

We investigated the biological role of the human tumor necrosis factor p75 (hTNF-R75), making use of the species specificity of TNF responses in murine (m) T cell lines. Several TNF-mediated activities on mouse T cells, such as cytokine induction or proliferation, showed a 100-500-fold difference in specific biological activity between mTNF and hTNF. After transfection of hTNF-R75 cDNA in a rat/mouse T cell hybridoma (PC60), however, the 100-fold lower specific biological activity of hTNF was converted to the same specific biological activity as mTNF. The TNF-mediated induction of granulocyte/macrophage colony-stimulating factor was strongly synergized by the addition of interleukin 1. In the presence of the latter cytokine, ligand-competing monoclonal antibodies against hTNF-R75 (utr-1, utr-2, utr-3) were agonistic on transfected PC60 cells. This agonistic activity was further enhanced by crosslinking with sheep anti-murine immunoglobulin antibodies. These data provide direct evidence for a functional role of TNF-R75, without ligand-dependent TNF-R55 involvement, in the induction of cytokine secretion in T cells.

Animals↗
PubMed1992

Molecular basis of the membrane-anchored and two soluble isoforms of the human interleukin 5 receptor alpha subunit.

By use of a 3' extension PCR strategy, cDNA clones were isolated spanning the transmembrane region and a complete cytoplasmic domain of the human interleukin 5 receptor alpha subunit (hIL5R alpha). These cDNAs differ from previously isolated clones encoding a soluble hIL5R alpha form by a sequence switch at position 1243. When expressed in COS-1 cells, only low-affinity binding of 125I-labeled human interleukin 5 was observed. Coexpression of the hIL5R beta chain led to a 2-fold increase in binding affinity. In addition, this same cloning strategy allowed us to identify a putative second soluble isoform of hIL5R alpha. Genomic data revealed that the two soluble variants arise from either a "normal" splicing event or from the absence of splicing, whereas synthesis of the membrane-anchored form requires alternative splicing.

Amino Acid Sequence↗
PubMed1992

TNF-mediated IL6 gene expression and cytotoxicity are co-inducible in TNF-resistant L929 cells.

Interleukin (IL)-6 gene induction was studied in the murine cell line, L929r2, which is resistant to the cytotoxic action of tumor necrosis factor (TNF). Increasing concentrations of TNF slightly elevated the background levels of IL6 expression as compared to non-induced cells. Under conditions where the resistant cells are sensitive to TNF by combined TNF/IFN-gamma treatment, the IL6 levels were strongly induced. This induction could be further enhanced by the addition of lithium chloride, or reduced by inhibitors of cytotoxicity, such as dexamethasone. These results confirm our earlier conclusions regarding the close relationship between TNF-mediated IL6 gene expression and the pathway leading to cytotoxicity.

Animals↗
PubMed1992

Expression of an exogenous tumor necrosis factor (TNF) gene in TNF-sensitive cell lines confers resistance to TNF-mediated cell lysis.

TNF, a cytokine with cytotoxic activity on a variety of tumor cells, is mainly produced by macrophages; however, some tumor cell types of non-macrophage origin, apparently resistant to TNF-mediated cell lysis, can also produce TNF. It is not clear whether these cells were TNF-resistant a priori or whether protective mechanisms against toxicity of autocrine TNF may be induced in TNF-producing cells. Murine L929sA fibrosarcoma cells, which are highly sensitive to TNF cytotoxicity, were transfected with the neomycin resistance (neor) gene, alone or in combination with the human (h) or the murine (m) TNF gene. All exogenous genes were under control of the constitutive SV40 early promoter. After cotransfection, the number of neor colonies was 10 to 100% as compared with the number of colonies upon transfection with the neor gene alone. An appreciable fraction of these colonies (50-100%) constitutively produced biologically active TNF. mTNF-producing L929 cells were fully TNF resistant, whereas hTNF-producing cells showed partial TNF resistance. Specific TNF binding could not be detected on mTNF-producing L929sA transfectants, whereas hTNF-producing cells showed reduced TNF binding. Apparently, TNF gene expression, even in a priori TNF-sensitive cells, can induce mechanisms to prevent toxicity by both autocrine and exogenous TNF. No TNF resistance was induced by expression of a gene sequence encoding the 9-kDa membrane-bound presequence part of the 26-kDa mTNF proform. Expression of a mutant 26-kDa TNF gene coding for a quasi-inactive mature mTNF induced only weak TNF resistance as compared with the complete resistance obtained after transfection with the wild-type gene. These findings show that the membrane-bound TNF presequence as such is not sufficient for induction of TNF resistance and imply that the active site of mature TNF is involved in modulation of TNF responsiveness upon autocrine TNF production.

Animals↗
PubMed1992

Production of functionally active murine and murine::human chimeric F(ab')2 fragments in COS-1 cells.

We report here the synthesis, in nonlymphoid cells, of two functionally active recombinant F(ab')2 fragments directed against the tumor marker, human placental alkaline phosphatase (hPLAP). The truncated heavy chain (HC) sequences, E6Hf2 and E6Hy3f2, of the murine F(ab')2 fragment, E6F2, and of the murine::human chimeric F(ab')2 fragment, E6(Hy3,kappa)F2, respectively, were engineered by introducing an in-phase stop codon within the second constant domain of the corresponding parental HC sequence. The antibody-encoding genes were placed under control of the simian virus 40 late promoter and each HC sequence, together with the light chain (LC) sequence, was transiently expressed in COS-1 cells. The truncated HCs were correctly synthesized, processed and assembled with the murine LC and subsequently secreted into the culture medium as functionally active entities with stable hinge region interactions. These results indicate that, under the conditions used, the hinge region was sufficient for the formation of divalent molecules. However, Western blotting revealed the presence of hPLAP-binding half-molecules of E6F2, which was not the case for E6(Hy3,kappa)F2. Since E6F2 and E6(Hy3,kappa)F2 mainly differ by the length of their hinge region (22 and 62 aa residues, respectively) and the number of inter-HC disulfide bridges (four and eleven, respectively), it may be concluded that F(ab')2 fragments with an extended hinge region and several inter-HC disulfide bridges are formed more efficiently.

Alkaline Phosphatase↗
PubMed1992

Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation.

Structural mitochondrial damage accompanies the cytotoxic effects of several drugs including tumor necrosis factor (TNF). Using various inhibitors of mitochondrial electron transport we have investigated the mechanism of TNF-mediated cytotoxicity in L929 and WEHI 164 clone 13 mouse fibrosarcoma cells. Inhibitors with different sites of action modulated TNF cytotoxicity, however, with contrasting effects on final cell viability. Inhibition of mitochondrial electron transport at complex III (cytochrome c reductase) by antimycin A resulted in a marked potentiation of TNF-mediated injury. In contrast, when the electron flow to ubiquinone was blocked, either at complex I (NADH-ubiquinone oxidoreductase) with amytal or at complex II (succinate-ubiquinone reductase) with thenoyltrifluoroacetone, cells were markedly protected against TNF cytotoxicity. Neither uncouplers nor inhibitors of oxidative phosphorylation nor complex IV (cytochrome c oxidase) inhibitors significantly interfered with TNF-mediated effects, ruling out the involvement of energy-coupled phenomena. In addition, the toxic effects of TNF were counteracted by the addition of antioxidants and iron chelators. Furthermore, we analyzed the direct effect of TNF on mitochondrial morphology and functions. Treatment of L929 cells with TNF led to an early degeneration of the mitochondrial ultrastructure without any pronounced damage of other cellular organelles. Analysis of the mitochondrial electron flow revealed that TNF treatment led to a rapid inhibition of the mitochondria to oxidize succinate and NADH-linked substrates. The inhibition of electron transport was dose-dependent and became readily detectable 60 min after the start of TNF treatment, thus preceding the onset of cell death by at least 3-6 h. In contrast, only minor effects were observed on complex IV activity. The different effects observed with the mitochondrial respiratory chain inhibitors provide suggestive evidence that mitochondrial production of oxygen radicals mainly generated at the ubisemiquinone site is a causal mechanism of TNF cytotoxicity. This conclusion is further supported by the protective effect of antioxidants as well as the selective pattern of damage of mitochondrial chain components and characteristic alterations of the mitochondrial ultrastructure.

Amobarbital↗
PubMed1992

Efficient synthesis of secreted murine interleukin-2 by Saccharomyces cerevisiae: influence of 3'-untranslated regions and codon usage.

Several expression vectors were compared which directed the synthesis of secreted murine interleukin-2 (mIL2) in the culture medium of Saccharomyces cerevisiae. We used the prepro-sequence of the alpha 1 mating-factor precursor as a secretion signal in S. cerevisiae in combination with different promoters. The yield of mature mIL2 was significantly improved by deleting the major part of the 3'-untranslated region (UTR). In Northern-blotting experiments we showed that a destabilizing sequence present in the 3' UTR might be responsible for rapid degradation of the mIL2 mRNA. The highest expression (about 10 micrograms/ml) was obtained under control of the GAL1 promoter in an S. cerevisiae strain where the regulatory GAL4 gene was overexpressed. No difference in expression level was observed in a construct wherein twelve consecutive codons were replaced by optimal codons for S. cerevisiae.

Animals↗
PubMed1992

The glucocorticoid antagonist RU38486 mimics interleukin-1 in its sensitization to the lethal and interleukin-6-inducing properties of tumor necrosis factor.

Although tumor necrosis factor (TNF) is undoubtedly a major mediator of the antitumor and shock-inducing activities of lipopolysaccharide (LPS), the outcome of a challenge with TNF is highly dependent on the presence or absence of other substances or conditions. We have previously shown that to obtain lethality in mice after TNF administration both TNF receptor (TNF-R) types have to be triggered. This is illustrated by the fact that recombinant human (rh) TNF, which is a selective murine (m) TNF-R55 agonist, is not lethal, whereas mTNF, which binds both mTNF-R55 and mTNF-R75, is lethal in mice. Triggering of TNF-R75 is, however, no longer needed when sensitizers such as galactosamine or low doses of LPS or interleukin (IL)-1 are also present. Here, we report that this selective species specificity of TNF is also reflected in patterns of induced IL-6: both rmTNF and rhTNF could induce considerable IL-6 peak levels in the plasma (up to 10 ng/ml) 2 to 3 h after TNF administration. However, only rmTNF was capable of inducing the same pattern of sustained IL-6 levels previously observed after lethal LPS doses, while rhTNF only caused induction of transient IL-6 levels, as found after nonlethal LPS doses. We also observed that the sensitizer IL-1 could complement rhTNF to induce such a sustained IL-6 induction. Since we were interested in sensitizers with a defined mechanism of action, we further investigated the effects of the glucocorticoid and progesterone inhibitor RU38486 on the lethal and IL-6-inducing properties of TNF. We observed that RU38486 closely mimicked IL-1: both had similar effects on IL-6 induction and sensitized mice to the lethal effects of TNF with comparable efficiency and kinetics. Using a monoclonal anti-IL-1R antibody, we finally observed that the effects of RU38486 were most probably not mediated by IL-1. These observations suggest that a glucocorticoid-antagonistic activity might be a key factor in the pathways leading to septic shock and that such activity could be a key target for the pharmacological manipulation of sepsis.

Animals↗
PubMed1992

Synergistic induction of interleukin-6 by tumor necrosis factor and lithium chloride in mice: possible role in the triggering and exacerbation of psoriasis by lithium treatment.

One of the side effects of treatment of manic depressive disease with lithium salts is the triggering or aggravation of psoriasis. In a murine model, subcutaneous (s.c.) injection of a combination of tumor necrosis factor (TNF) and lithium chloride (LiCl) induces a psoriasiform inflammatory reaction. Recent studies suggest that interleukin (IL)-6 and its inducer TNF may play an important role in the pathophysiology of psoriasis. To understand the mechanism involved in the exacerbation of psoriasis by lithium salts, the IL-1, IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels in murine skin injected with TNF in combination with LiCl were studied. IL-6 levels in skin extracts of mice treated s.c. with a combination of TNF and LiCl were considerably increased as compared to the levels found in skin extracts from mice treated with TNF or LiCl alone. In contrast, in the same skin extracts IL-1 levels were not changed and GM-CSF was even not detectable. Although less pronounced, increased IL-6 levels could also be found in the sera of mice treated s.c. with TNF and LiCl. Injection with IL-1, interferon-gamma, lipopolysaccharide, or phorbol 12-myristate 13-acetate also induced IL-6 in murine skin. However, these IL-6 levels were not enhanced by co-treatment with LiCl. Likewise, on inflammatory reaction could be seen in mice treated with these agents. These results suggest a role for endogenous TNF and IL-6 in the triggering or aggravation of psoriasis in lithium-treated patients.

Animals↗
PubMed1992

Limited involvement of interleukin-6 in the pathogenesis of lethal septic shock as revealed by the effect of monoclonal antibodies against interleukin-6 or its receptor in various murine models.

Several studies in human patients and in laboratory animals have revealed a correlation between serum interleukin (IL)-6 levels and outcome in clinical sepsis and in related animal models, respectively. In the present study, two monoclonal antibodies were used to investigate the contribution of IL-6 in the lethal action of tumor necrosis factor (TNF) and of lipopolysaccharide (LPS) in mice. We studied the potential protective properties of an anti-murine (m) IL-6 antibody and of an anti-mIL-6 receptor antibody. In controlled experiments, we observed that both monoclonal antibodies conferred a dose-dependent protection to a lethal dose of mTNF. Detailed studies with the monoclonal antibodies indicate, however, that protection was no longer observed when the mTNF dose was slightly higher than the lethal dose. Likewise, the anti-IL-6 monoclonal antibody protected against injections of LPS at a lethal-dose concentration, but here too failed to protect against higher doses of LPS. The anti-IL-6 monoclonal antibody was unable to protect against mTNF in mice sensitized by galactosamine, the corticoid receptor antagonist RU38486 or human (h) IL-1 beta. Protection did not correlate with the serum concentrations of IL-6. Finally, we demonstrate that hIL-6 injection did not change the sensitivity of mice towards mTNF. We conclude that, although IL-6 levels may be of value as a marker for the outcome in septic shock, this cytokine contributes only marginally in the pathogenesis leading to death. The small, but real, contribution of IL-6 in some situations might be due to its ability to up-regulate the level of TNF receptors.

Animals↗
PubMed1992

Cloning and expression of the canine interferon-gamma gene.

The canine interferon-gamma (cIFN-gamma) chromosomal gene was isolated from a recombinant library, containing the entire dog genome, by screening with a rat IFN-gamma genomic probe. The structural organization of the cIFN-gamma gene closely resembles that of the human, murine, and rat IFN-gamma genes. It contains three intervening sequences and encodes a signal sequence of 23 amino acids followed by a mature protein of 143 amino acids. Two potential N-glycosylation sites are located at positions 16 and 83 of the mature protein. Comparison of the cIFN-gamma protein sequence with that of the corresponding murine, rat, human, and bovine proteins revealed a homology of 40%, 42%, 65%, and 76%, respectively. The cIFN-gamma gene was expressed under control of the simian virus 40 early promoter in Chinese hamster ovary (CHO) cells, deficient in dihydrofolate reductase (DHFR), after cotransformation with a plasmid containing the murine dhfr gene. Initial transformants with dhfr+ phenotype produced cIFN-gamma titers ranging from 10 to 10,000 laboratory units (LU)/ml of culture medium. A cIFN-gamma cDNA sequence was identified in a cDNA library constructed with partially purified RNA from a cIFN-gamma-producing CHO cell line. In vitro transcription of the cDNA and translation of the mRNA in Xenopus laevis oocytes resulted in secreted IFN-gamma with an antiviral activity specific for canine cells.

Amino Acid Sequence↗
PubMed1992

Disulphide bridge formation in the periplasm of Escherichia coli: beta-lactamase:: human IgG3 hinge fusions as a model system.

We report the construction and the expression in Escherichia coli of three different fusion genes encoding the extended human IgG3 hinge region (Hi) fused in-phase to the C-terminal end of bacterial TEM1 beta-lactamase (Bla). In the first fusion gene blahi, TEM1 beta-lactamase (Bla). In the first fusion gene blahi, the hinge sequence was directly coupled to the 3' end of the beta-lactamase gene, whereas in the two other constructs, blal1hi and blal2hi, a linker encoding 14 and 10 amino acids, respectively, was inserted between the two subunits. After expression (24 h, 20 degrees C) under control of the constitutive kanamycin phosphoribosyl transferase promoter, the fusion proteins, BlaHi, BlaL1Hi and BlaL2Hi, respectively, were almost exclusively detected in the periplasmic fraction, and they conferred carbenicillin-resistance to the cells. These results indicate that beta-lactamase can efficiently direct the export of proteins fused to its C-terminus, and moreover, at least some of the exported fusion proteins must carry the beta-lactamase moiety in a properly folded form. Analysis of their assembly, however, revealed that only a minor fraction was recovered as the expected F(ab')2-like dimer. The presence in the periplasm of 'oxidized' monomers (with intrachain disulphide bonds) as well as of several high-molecular-mass proteins, probably resulting from the association between monomers and other cysteine-rich proteins, strongly suggests that the conditions in the bacterial periplasm are insufficient to allow proper assembly of multimeric proteins with several interchain disulphide bonds.

Bacterial Proteins↗
PubMed1992

Stimulation of lipolysis in cultured fat cells by tumor necrosis factor, interleukin-1, and the interferons is blocked by inhibition of prostaglandin synthesis.

Multiple cytokines induce a number of alterations in lipid metabolism which can produce hyperlipidemia. Recent studies have demonstrated that tumor necrosis factor (TNF) increases lipolysis, resulting in an increase in circulating FFA levels, which stimulates hepatic triglyceride production, thereby contributing to the hyperlipidemia induced by TNF. In the present investigation we have determined the effects of a variety of cytokines on lipolysis in cultured 3T3-F442A adipocytes. TNF increased lipolysis approximately 3-fold with a maximal effect at 100 ng/ml and a half-maximal increase at 5-10 ng/ml. This increase was first observed 8 h after incubation with TNF. Interleukin-1 (IL-1) and interferon-alpha (IFN), -beta, and -gamma also stimulated lipolysis in cultured adipocytes. The half-maximal increase in lipolysis occurred at approximately 10 ng/ml IL-1, 5 ng/ml IFN alpha, 10 ng/ml IFN beta, and 8 ng/ml of IFN gamma. Maximal lipolysis was observed at approximately 100 ng/ml for each of these cytokines, with the exception of IFN beta, for which maximal stimulation was observed at 1000 ng/ml. Neither platelet-activating factor nor IL-6 stimulated lipolysis; therefore, it is unlikely that these compounds mediate the increase in lipolysis induced by cytokines. However, indomethacin, a well known inhibitor of prostaglandin synthesis, prevented the increase in lipolysis induced by TNF, IL-1, IFN alpha, IFN beta, or IFN gamma. Indomethacin did not affect basal lipolysis or the acute stimulation of lipolysis induced by epinephrine. These results demonstrate that multiple cytokines can increase lipolysis and that this increase is mediated by cytokine-induced stimulation of prostaglandin synthesis.

3T3 Cells↗
PubMed1992

Selective species specificity of tumor necrosis factor for toxicity in the mouse.

The selective cytotoxic activity of tumor necrosis factor (TNF) on many transformed human or murine cell lines is (almost) not species-specific. There are, however, a limited number of biological assay systems such as gene induction in murine thymoma cells and the murine thymocyte proliferation assay, in which recombinant murine (rm) TNF, but not recombinant human (rh) TNF is active. We have now investigated the possible species specificity of the lethality-inducing properties of TNF in the mouse. When administered alone, only rmTNF, but not rhTNF caused lethality. This difference was not due to a different endotoxin contamination or to a different pharmacokinetic behavior. When a sensitizing agent, galactosamine, was added, the species specificity was abolished. We have shown previously that similar conclusions could be drawn for at least some of the antitumor mechanisms of TNF. We conclude that to mimic the two major phenomena caused by endotoxin administration, viz. lethality and antitumor effect, two distinct signals are needed. In mice, rmTNF can provide both, while rhTNF needs another agent (a sensitizer) to cause systemic toxicity. The results are discussed with respect to recent findings showing that, in the mouse, rhTNF can only bind to the TNF-R55 receptor type, while rmTNF can bind both to the TNF-R55 and TNF-R75 receptor types.

Animals↗
PubMed1991

Efficient KEX2-like processing of a glucoamylase-interleukin-6 fusion protein by Aspergillus nidulans and secretion of mature interleukin-6.

We have designed an expression vector for the secretion of human interleukin-6 (hIL-6) in which the mature protein is fused through a spacer peptide, containing a KEX-2 like protein processing signal, to the entire Aspergillus niger glucoamylase (glaA) gene. Transformation of Aspergillus nidulans with this vector results in fungal strains secreting equimolar amounts of the glucoamylase and IL-6 proteins. The KEX2-type processing signal, Lys-Arg, is recognized and cleaved efficiently by an enzyme present in A. nidulans resulting in the secretion of an authentic mature hIL-6 protein at levels of up to 5 mg/l.

Amino Acid Sequence↗