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Biomedical subjects

J Vilcek

Publications and source records attributed to J Vilcek.

At least 55 records · Page 3Linked to original sources
PubMed1993

TSG-6: a TNF-, IL-1-, and LPS-inducible secreted glycoprotein associated with arthritis.

TSG-6 (TNF-stimulated gene 6) was originally discovered by differential screening of a cDNA library prepared from TNF-stimulated human diploid FS-4 fibroblasts. We show that the 35-kDa protein encoded by TSG-6 was undetectable in the medium of untreated FS-4 cultures, whereas its production reached approximately 1400 and 700 ng/10(6) cells after 24-h treatment with IL-1 or TNF, respectively. Stimulation of TSG-6 protein and mRNA levels was also demonstrated in normal human mononuclear cells by treatment with TNF and, especially, by LPS. In view of the inducibility of TSG-6 by inflammatory cytokines and its earlier demonstrated affinity for hyaluronan, we examined the presence of TSG-6 protein in the synovial fluids from patients with various forms of arthritis. TSG-6 protein was undetectable in the joint fluids of persons with no known history of arthritis, but high levels of TSG-6 oere demonstrated in the synovial fluids of a majority of arthritis patients. TSG-6 protein was also detected in the sera of some of the arthritis patients, albeit at concentrations that were less than in the joint fluids. To investigate the source of TSG-6 in the synovial fluids, we examined the production of TSG-6 protein in cultures of synovial cells. Synoviocytes from rheumatoid arthritis patients produced TSG-6 protein constitutively, and this production was increased by treatment with TNF or IL-1, but not with TGF-beta. Steady-state levels of TSG-6 mRNA were also increased in synoviocytes after treatment with TNF or IL-1. The presence of high levels of TSG-6 protein in the synovial fluids of arthritis patients and its inducibility by inflammatory cytokines in fibroblasts, mononuclear cells, synoviocytes, and chondrocytes suggest a role for TSG-6 in arthritis and inflammation.

Adolescent↗
PubMed1993

Mice that lack the interferon-gamma receptor have profoundly altered responses to infection with Bacillus Calmette-Guérin and subsequent challenge with lipopolysaccharide.

Mice with a targeted disruption of the interferon gamma receptor gene (IFN-gamma R0/0 mice) and control wild-type mice were inoculated with the Bacillus Calmette-Guérin (BCG) strain of Mycobacterium bovis. BCG infection was not lethal for wild-type mice whereas all IFN-gamma R0/0 mice died approximately 7-9 wk after inoculation. Histological examination at 2 and 6 wk after BCG inoculation showed that livers of IFN-gamma R0/0 mice had higher numbers of acid-fast bacteria than wild-type mice, especially at 6 wk. In parallel, the livers of IFN-gamma R0/0 mice showed a reduction in the formation of characteristic granulomas at 2 wk after inoculation. Injection of lipopolysaccharide (LPS) 2 wk after BCG inoculation was significantly less lethal for IFN-gamma R0/0 mice than for wild-type mice. Reduced lethality of LPS correlated with a drastically reduced production of tumor necrosis factor alpha (TNF-alpha) in the IFN-gamma R0/0 mice. Interleukin 1 alpha (IL-1 alpha) and IL-6 levels in the serum were also significantly reduced in the IFN-gamma R0/0 mice after BCG infection and LPS challenge. The greatly reduced capacity of BCG-infected IFN-gamma R0/0 mice to produce TNF-alpha may be an important factor in their inability to resist BCG infection. These results show that the presence of a functional IFN-gamma receptor is essential for the recovery of mice from BCG infection, and that IFN-gamma is a key element in the complex process whereby BCG infection leads to the sensitization to endotoxin.

Animals↗
PubMed1993

Tumor necrosis factor-induced activation and increased tyrosine phosphorylation of mitogen-activated protein (MAP) kinase in human fibroblasts.

Tumor necrosis factor (TNF) is a pleiotropic cytokine whose many demonstrated actions include effects on cell growth and differentiation. TNF treatment of cells is known to lead to a rapid increase in serine/threonine phosphorylation of many cellular proteins, but the kinases responsible remain largely unidentified. We show that TNF treatment induces a rapid and transient increase in mitogen-activated protein kinase (MAPK) activity in the human diploid FS-4 cell line, for which TNF is known to be mitogenic. TNF-induced activation of MAPK was demonstrated by its enhanced ability to phosphorylate myelin basic protein in vitro and by a characteristic shift in the electrophoretic mobility of MAPK proteins. MAPK activation was accompanied by a significant increase of MAPK phosphorylation on tyrosine residues, which was demonstrated by 32P labeling of cells and isolation of the labeled proteins after immunoprecipitation with antibodies to phosphotyrosine, and by direct immunoblotting of SDS-polyacrylamide gel electrophoresis-fractionated unlabeled cell lysates with antibodies to phosphotyrosine. The pp42 and pp44 MAPK were the only proteins whose tyrosine phosphorylation was demonstrably increased in FS-4 cells after TNF treatment. MAPK activation is likely to represent an important component in the cascade of signals that link TNF receptors to various TNF-elicited cellular responses.

Blotting, Western↗
PubMed1993

Generation of nitric oxide and induction of major histocompatibility complex class II antigen in macrophages from mice lacking the interferon gamma receptor.

Availability of mice with a targeted disruption of the interferon gamma (IFN-gamma) receptor gene (IFN-gamma R0/0 mice) made it possible to examine parameters of macrophage activation in the absence of a functional IFN-gamma receptor. We asked to what extent other cytokines could replace IFN-gamma in the induction of nitric oxide or major histocompatibility complex class II antigen (Ia) expression in peritoneal macrophages. In thioglycollate-elicited macrophages from wild-type mice, tumor necrosis factor (TNF) alone was virtually ineffective in inducing release of NO2- (the endproduct of nitric oxide generation), but TNF enhanced NO2- release in the presence of IFN-gamma. In macrophages from IFN-gamma R0/0 mice, which were unresponsive to IFN-gamma, TNF completely failed to stimulate NO2- release. The stimulatory actions of IFN-alpha/beta on NO2- release were indistinguishable in wild-type and IFN-gamma R0/0 macrophages: IFN-alpha/beta was ineffective on its own, showed marginal stimulation of NO2- release in combination with TNF, and was moderately effective in the presence of lipopolysaccharide. The level of constitutive Ia antigen expression was not significantly different in peritoneal macrophages from wild-type and IFN-gamma R0/0 mice. An increased Ia expression was induced by IL-4 and granulocyte-macrophage colony-stimulating factor in both wild-type and IFN-gamma R0/0 macrophages, but the magnitude of this induction was less than with optimal concentrations of IFN-gamma in macrophages from wild-type mice. IFN-alpha/beta showed only a minor stimulatory effect on Ia expression in both wild-type and IFN-gamma R0/0 macrophages. Simultaneous treatment of wild-type macrophages with IFN-alpha/beta and IFN-gamma reduced the IFN-gamma-induced Ia expression in wild-type macrophages, but IFN-alpha/beta did not show an inhibitory effect on IL-4- or granulocyte-macrophage-colony-stimulating factor-induced Ia expression in either wild-type or IFN-gamma R0/0 macrophages. The important role of IFN-gamma in the regulation of the induced expression of major histocompatibility complex class II antigen was confirmed by showing that after systemic infection with the BCG strain of Mycobacterium bovis resident peritoneal macrophages from IFN-gamma R0/0 mice had a lower level of Ia expression than macrophages from wild-type mice. The inability of other cytokines to substitute fully for IFN-gamma in macrophage activation helps to explain the earlier observed decreased resistance of IFN-gamma R0/0 mice to some infections.

Animals↗
PubMed1993

Transcriptional regulation of TSG6, a tumor necrosis factor- and interleukin-1-inducible primary response gene coding for a secreted hyaluronan-binding protein.

TSG6 was originally identified as a tumor necrosis factor (TNF)-inducible gene in human fibroblasts. Earlier we showed that the secretory TSG6 protein is a member of a family of hyaluronan-binding proteins that includes cartilage link protein, proteoglycan core protein, and the adhesion receptor CD44. In the present study we have used Southern blot analysis to demonstrate that TSG6 is a single-copy gene in the human and murine species. With the aid of a somatic cell hybrid mapping panel, TSG6 was assigned to human chromosome 2. Nuclear run-on analysis revealed that TNF produced a rapid, primary transcriptional activation of the TSG6 gene in normal human FS-4 fibroblasts. In order to learn more about the regulation of TSG6 gene expression, we cloned the TSG6 gene from a genomic library of human white blood cells. Sequencing of a 1.3-kilobase fragment of the 5'-flanking region of the TSG6 gene identified TATA-like and CAAT sequences near the transcription start site. In addition, potential binding sites for NF-IL-6, AP-1, interferon regulatory factors (IRF)-1 and -2, and glucocorticoid response elements were identified in the 5'-flanking region. A single transcription start site was identified by primer extension. Deletion analysis of the 5'-flanking region of the TSG6 DNA linked to the chloramphenicol acetyltransferase reporter gene revealed that a construct containing TSG6 DNA from positions -165 to +78 could be transcriptionally activated by interleukin(IL)-1, and to a lesser extent by TNF, upon transfection into FS-4 fibroblasts. The region that imparts inducibility by IL-1 or TNF (positions -165 to -58) contains potential binding sites for IRF-1 and -2, AP-1, and NF-IL-6. A region mediating transcriptional silencing was localized further upstream (between positions -332 and -165). The results suggest that TSG6 gene expression is regulated by an interplay of positively and negatively acting transactivating factors.

Animals↗
PubMed1993

Immune response in mice that lack the interferon-gamma receptor.

Interferon-gamma (IFN-gamma) exerts pleiotropic effects, including antiviral activity, stimulation of macrophages and natural killer cells, and increased expression of major histocompatibility complex antigens. Mice without the IFN-gamma receptor had no overt anomalies, and their immune system appeared to develop normally. However, mutant mice had a defective natural resistance, they had increased susceptibility to infection by Listeria monocytogenes and vaccinia virus despite normal cytotoxic and T helper cell responses. Immunoglobulin isotype analysis revealed that IFN-gamma is necessary for a normal antigen-specific immunoglobulin G2a response. These mutant mice offer the possibility for the further elucidation of IFN-gamma-mediated functions by transgenic cell- or tissue-specific reconstitution of a functional receptor.

Animals↗
PubMed1993

TSG-14, a tumor necrosis factor- and IL-1-inducible protein, is a novel member of the pentaxin family of acute phase proteins.

TNF-stimulated gene (TSG)-14 was originally identified as a TNF-inducible gene in a differentially screened cDNA library derived from TNF-treated normal human FS-4 fibroblasts. Analysis of the TSG-14 cDNA sequence revealed a major open reading frame encoding a protein of 381 amino acids, including a hydrophobic signal peptide sequence. The predicted protein shows 23 to 27% sequence homology to C-reactive protein and serum amyloid P-component, members of the pentaxin family of acute phase proteins. In addition, TSG-14 protein contains a sequence motif common among the pentaxin proteins. The ability of the TSG-14 cDNA to encode a protein of the correct molecular size was confirmed in a cell-free transcription/translation system. In vitro translation in the presence of microsomes confirmed that the protein has a cleavable signal peptide sequence, and that it is glycosylated. TSG-14 mRNA is rapidly elevated from almost undetectable levels in untreated FS-4 cells to high levels in cells treated with TNF or IL-1. A moderate increase in TSG-14 mRNA was observed in FS-4 cells treated with the glucocorticoid dexamethasone. Nuclear run-on analysis indicated that TNF induces the expression of the TSG-14 gene at the transcriptional level, and that de novo protein synthesis is not required for induction of TSG-14 mRNA. Expression of TSG-14 mRNA was also detected after exposure to TNF in vascular endothelial cells; however, little or not expression of TSG-14 message was observed in cell lines derived from malignant tumors. Our data strongly suggest that TSG-14 is a novel member of the pentaxin family of acute phase proteins.

Acute-Phase Proteins↗
PubMed1993

Construction and initial characterization of a mouse-human chimeric anti-TNF antibody.

Tumor necrosis factor-alpha (TNF) has been implicated in the pathogenesis of a variety of human diseases including septic shock, cachexia, graft-versus-host disease and several autoimmune diseases. Monoclonal antibodies directed against TNF provide an attractive mode of therapeutic intervention in these diseases. We have generated a murine monoclonal antibody (A2) with high affinity and specificity for recombinant and natural human TNF. To increase its therapeutic usefulness, we used genetic engineering techniques to replace the murine constant regions with human counterparts while retaining the murine antigen binding regions. The resulting mouse-human chimeric antibody should have reduced immunogenicity and improved pharmacokinetics in humans. Molecular analysis of light chain genomic clones derived from the murine hybridoma suggests that two different alleles of the same variable region gene have rearranged independently and coexist in the same hybridoma cell. The chimeric A2 antibody (cA2) exhibits better binding and neutralizing characteristics than the murine A2 which was shown to contain a mixture of two kappa light chains. The properties of cA2 suggest that it will have advantages over existing murine anti-TNF antibodies for clinical use.

Animals↗
PubMed1992

Downregulation of interleukin 8 gene expression in human fibroblasts: unique mechanism of transcriptional inhibition by interferon.

The chemotactic cytokine interleukin 8 (IL-8) is produced upon stimulation by various agents in many cell types, including connective-tissue fibroblasts. Tumor necrosis factor (TNF) and IL-1 are potent inducers of IL-8 expression. Earlier we showed that TNF-induced stimulation of IL-8 mRNA accumulation in human FS-4 fibroblasts was inhibited by interferon beta (IFN-beta) or IFN-gamma. Here we show that this inhibition is not specific for TNF, since IFN-beta also reduced IL-8 mRNA accumulation induced by IL-1 or the double-stranded RNA poly (I-C). Treatment with IFN-beta also decreased TNF-induced IL-8 protein accumulation. Interestingly, the inhibitory effect was much less pronounced when IFN-beta was added greater than or equal to 1 hr before TNF. The inhibitory action of IFN-beta on IL-8 mRNA accumulation was undiminished in the presence of inhibitors of protein synthesis. Nuclear run-on assays demonstrated that IFN-beta caused a marked inhibition of TNF-induced IL-8 gene transcription; the transcriptional activation of several other TNF-induced genes was not inhibited by IFN-beta. The results suggest that the specific inhibition of the transcriptional activation of IL-8 by IFN is due either to a transient inactivation of a factor required for IL-8 transcription or to the activation of a selective inhibitory factor.

Cell Line↗
PubMed1992

Critical role of a common transcription factor, IRF-1, in the regulation of IFN-beta and IFN-inducible genes.

Interferon regulatory factor 1 (IRF-1) is a protein that binds to cis-elements within the promoter of interferon (IFN)-beta and some IFN-inducible genes. We used a human fibroblast line, GM-637, to generate stable transfectants constitutively expressing IRF-1 mRNA in either the sense or antisense orientation. Upon induction with poly-(I).poly(C) or Newcastle disease virus, cells expressing sense IRF-1 mRNA produced significantly higher levels of IFN-beta mRNA and protein than control cells, whereas cells expressing antisense IRF-1 mRNA produced little or no IFN-beta mRNA and protein. Furthermore, clear differences were seen among the transfectants in the level of expression of two IFN-induced genes (2'-5'-oligoadenylate synthetase and class I HLA). Our data show that IRF-1 is essential for the induced expression of the IFN-beta gene. The results also indicate an important role of IRF-1 in the expression of IFN-inducible genes and suggest a role for IRF-1 in many other cytokine actions.

2',5'-Oligoadenylate Synthetase↗
PubMed1992

Cytokine regulation of interleukin-6 gene expression in astrocytes involves activation of an NF-kappa B-like nuclear protein.

The cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) induce interleukin-6 (IL-6) gene expression in astrocytes. The molecular mechanism(s) by which these cytokines activate IL-6 expression was examined by transient transfection of the human IL-6 promoter linked to the reporter gene CAT (IL-6-CAT) in primary rat astrocytes. We show that both IL-1 beta and TNF-alpha exert their effects through the IL-6 promoter to increase CAT activity, indicating that the cytokines act at the transcriptional level. Use of deletion mutants revealed that the NF-kappa B-like binding site is required for cytokine induction of IL-6 promoter activity. The correlary effects of IL-1 beta and TNF-alpha on DNA-binding proteins specific for this element were examined. Treatment of astrocytes with either cytokine leads to a rapid activation (15 min) of a nuclear protein which specifically complexes with the NF-kappa B-like binding region in the IL-6 promoter. These results suggest that TNF-alpha and IL-1 beta activate IL-6 gene expression in astrocytes by a mechanism(s) involving activation of an NF-kappa B-like protein.

Animals↗
PubMed1992

Induction of HLA class I mRNA by cytokines in human fibroblasts: comparison of TNF, IL-1 and IFN-beta.

Expression of HLA class I antigens is known to be regulated by various cytokines at both the mRNA and protein levels. We have examined the induction of HLA-B7 by tumor necrosis factor alpha (TNF), interleukin 1 alpha (IL-1) and interferon beta (IFN-beta) in normal human diploid FS-4 fibroblasts. Optimal induction of HLA-B7 by TNF at 24 h was shown to require a continuous presence of TNF. Since TNF also induces IFN-beta in these cells and the latter cytokine itself has the capacity to upregulate HLA class I expression, we investigated the role of autocrine IFN-beta in the induction of HLA-B7 by TNF. Experiments with neutralizing polyclonal antibodies to recombinant IFN-beta showed that the induction of HLA-B7 mRNA by TNF was partially dependent on autocrine IFN-beta. However, TNF and IFN-beta induced HLA-B7 mRNA with similar kinetics and treatment with saturating concentrations of both TNF and IFN-beta resulted in an additive or possibly synergistic response. The latter findings support the idea that induction of HLA class I by TNF is not mediated solely by autocrine IFN-beta produced in response to TNF. In addition, experiments with the protein synthesis inhibitor cycloheximide suggested that the induction of mRNAs for both the heavy and light (beta 2-microglobulin) chains of the HLA class I antigen by TNF did not require de novo protein synthesis. IL-1 was also shown to increase steady-state mRNA levels of HLA-B7 with kinetics similar to those of TNF and IFN-beta in FS-4 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗
PubMed1992

A novel secretory tumor necrosis factor-inducible protein (TSG-6) is a member of the family of hyaluronate binding proteins, closely related to the adhesion receptor CD44.

TSG-6 cDNA was isolated by differential screening of a lambda cDNA library prepared from tumor necrosis factor (TNF)-treated human diploid FS-4 fibroblasts. We show that TSG-6 mRNA was not detectable in untreated cells, but became readily induced by TNF in normal human fibroblast lines and in peripheral blood mononuclear cells. In contrast, TSG-6 mRNA was undetectable in either control or TNF-treated human vascular endothelial cells and a variety of tumor-derived or virus-transformed cell lines. The sequence of full-length TSG-6 cDNA revealed one major open reading frame predicting a polypeptide of 277 amino acids, including a typical cleavable signal peptide. The NH2-terminal half of the predicted TSG-6 protein sequence shows a significant homology with a region implicated in hyaluronate binding, present in cartilage link protein, proteoglycan core proteins, and the adhesion receptor CD44. The most extensive sequence homology exists between the predicted TSG-6 protein and CD44. Western blot analysis with an antiserum raised against a TSG-6 fusion protein detected a 39-kD glycoprotein in the supernatants of TNF-treated FS-4 cells and of cells transfected with TSG-6 cDNA. Binding of the TSG-6 protein to hyaluronate was demonstrated by coprecipitation. Our data indicate that the inflammatory cytokine (TNF or IL-1)-inducible, secretory TSG-6 protein is a novel member of the family of hyaluronate binding proteins, possibly involved in cell-cell and cell-matrix interactions during inflammation and tumorigenesis.

Amino Acid Sequence↗
PubMed1992

Protodyne: an immunostimulatory protein component, prepared from gram-positive Bacillus subtilis.

A protein component derived from bacterial protoplasm, called Protodyne, increases the non-specific resistance to infections by bacteria and viruses. Here we show that Protodyne can be prepared not only from Gram-negative bacteria, but also from Gram-positive bacilli. Several preparations of Protodyne, prepared from Bacillus subtilis by phenol extraction or by ammonium sulfate precipitation, were evaluated for immunomodulatory activities in a variety of assays. Protodyne had a marked mitogenic activity on mouse spleen cells; it was a potent inducer of tumor necrosis factor (TNF) and stimulated production of interleukin-1 (IL-1) in human peripheral blood mononuclear cells; it increased the capacity of activated macrophages to undergo a respiratory burst, to produce intracellular killing of leishmanial parasite and extracellular lysis of mastocytoma cells; it also stimulated phagocytosis of latex particles, and prolonged survival of immunosuppressed mice infected with Pseudomonas aeruginosa. These activities were not inhibited by polymyxin B, indicating that the activity of Protodyne is not the result of contamination with exogenous lipopolysaccharide. It appears that Protodyne exerts its many immunomodulatory actions by inducing the release of soluble mediators, including TNF and IL-1.

Adjuvants, Immunologic↗
PubMed1992

Overexpression of metallothionein confers resistance to the cytotoxic effect of TNF with cadmium in MCF-7 breast carcinoma cells.

Experiments were designed to determine whether cells that overexpress metallothionein acquire resistance to the cytotoxic action of tumor necrosis factor (TNF). Human MCF-7 mammary carcinoma cells, which are sensitive to the cytotoxic action of TNF, were stably transfected with a vector in which the human metallothionein-IIA (hMT-IIA) gene was placed under the control of the constitutively active beta-actin promoter. MT-expressing clones displayed greater resistance to cadmium toxicity than control cell lines. Neither control-transfected nor MT-expressing cell lines were sensitive to the cytotoxic effect of TNF alone, even at concentrations as high as 200 ng/ml. However, treatment of control-transfected cells with TNF in the presence of CdCl2 produced a greater cytotoxic effect than CdCl2 alone. MT-expressing cell clones were protected from this synergistic cytotoxic effect of TNF and CdCl2. These results suggest that under certain conditions MT expression may protect tumor cells from the cytotoxic effects of TNF.

Cadmium↗