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J Content

Publications and source records attributed to J Content.

At least 37 records · Page 2Linked to original sources
PubMed1990

Inducible production of interleukin-6 by human polymorphonuclear neutrophils: role of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha.

The recent demonstration of the ability of human polymorphonuclear neutrophils (PMN) to secrete various cytokines in response to the granulocyte activator granulocyte-macrophage colony-stimulating factor (GM-CSF) but not to other cytokines, has led to the identification of PMN as biosynthetically active cells. In this study we have investigated the ability of PMN to secrete interleukin-6 (IL-6), a molecule known to be involved in inflammatory reactions. Using RNA blotting analysis and bioassays, we show that PMN could be induced to synthesize transcripts specific for IL-6, indistinguishable in size from IL-6 mRNA produced by activated human macrophages. Consequently, PMN released IL-6-like activity into their culture supernatants that could be neutralized by monospecific anti-IL-6 antibody. Interleukin-6 secretion by PMN, however, required previous stimulation with GM-CSF or tumor necrosis factor-alpha (TNF-alpha), whereas other cytokines, including interleukin-3 (IL-3), granulocyte-CSF (G-CSF), macrophage-CSF (M-CSF), interferon gamma (IFN-gamma), and lymphotoxin (LT), failed to induce IL-6 mRNA accumulation and protein secretion by PMN. Similar to GM-CSF and TNF-alpha, other compounds, including the inhibitor of protein synthesis cyclohexemide (CHX), endotoxin (Escherichia coli-derived lipopolysaccharide), and phorbol myristate acetate (PMA) (but not the chemoattractant N-formyl-methionyl-leucyl-phenylalanine [FMLP]), induced detectable levels of IL-6 transcripts in PMN.

Blotting, Northern↗
PubMed1990

Fate of interleukin-6 in the rat. Involvement of skin in its catabolism.

Iodinated recombinant human interleukin-6 (125I-rhIL-6) was intravenously injected into rats and its fate was studied during 24 h. Between 10-20 min after a single-dose injection, 125I-rhIL-6 accumulated in liver as previously reported [Castell et al. (1988) Eur. J. Biochem. 177, 357-361]. After 1 h, the radioactivity disappeared from the liver and accumulated in skin, reaching 35% of injected 125I-rhIL-6 5-8 h after injection. No comparable accumulation of radioactivity was found in skin when [125I]iodide or rat serum 125I-albumin was administered. Finally the radioactivity was detected as [125I]iodide in urine. Autoradiographic analysis of skin sections 5 h after 125I-rhIL-6 injection showed radioactivity in the interstitium. When the experiments were carried out with [35S]rhIL-6, essentially the same results were obtained: a decrease in radioactivity in the liver after 20 min, and a substantial increase in skin 7 h after injection. In vitro experiments showed that 125I-rhIL-6 is degraded by rat and human fibroblasts, whereas no degradation was observed with rat hepatoma cells (Fao) or human hepatocytes. These observations suggest the involvement of skin in the catabolism of IL-6.

Animals↗
PubMed1990

Excessive in vitro bacterial lipopolysaccharide-induced production of monokines in cirrhosis.

The objective of this study was to analyze monokine production by peripheral blood mononuclear cells from patients with alcoholic cirrhosis. The capacity of peripheral blood mononuclear cells and purified monocytes from these patients to produce tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6 was investigated. Spontaneous production of tumor necrosis factor alpha, interleukin 6 and interleukin 1 beta was similar in cirrhotic and healthy subjects, but serum levels of interleukin 6 (less than 2 U/ml vs. 9.5 +/- 3 U/ml) and tumor necrosis factor alpha (3.1 +/- 1.2 pg/ml vs. 12.0 +/- 1.2 pg/ml) were significantly higher in cirrhotic patients. However, peripheral blood mononuclear cells or purified monocytes from patients with alcoholic liver cirrhosis, stimulated in vitro with Escherichia coli lipopolysaccharide, displayed a marked increase of tumor necrosis factor alpha, interleukin 1 beta and interleukin 6 secretions compared with healthy controls. A striking feature of this overproduction was its reversibility as assessed by allowing cells to rest in vitro without lipopolysaccharide for 1 to 7 days before stimulation. In such conditions, tumor necrosis factor alpha and interleukin 6 secretions declined to levels present in healthy subjects in whom production remained stable, whereas interleukin 1 beta secretion markedly decreased in both groups to the point where no difference could be seen. This reversible oversecretion of cytokines after lipopolysaccharide stimulation, along with the lack of abnormality of spontaneous cytokine secretion, suggests that monocytes in these patients may have undergone an in vivo activation process analogous to a priming phenomenon. The in vitro activation with lipopolysaccharide may represent the correlate of in vivo endotoxemia observed during acute events such as sepsis.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1990

Glucocorticoid up-regulation of high-affinity interleukin 6 receptors on human epithelial cells.

Interleukin 6 (IL-6) is a potent pleiotropic cytokine, known, among others, to stimulate immunoglobulin production by B cells and to trigger acute-phase protein synthesis by hepatocytes. Similar to IL-1, it is produced by monocytes and macrophages following an inflammatory challenge. Analysis of IL-6 receptor (IL-6R) expression on different human cell lines indicates that dexamethasone could up-regulate the number of IL-6R on one epithelial cell line (UAC) and on two hepatoma cell lines (HepG2 and Hep3B). This effect was confirmed by Scatchard analysis of binding experiments, using [35S]methionine and [35S]cysteine metabolically labeled IL-6. It was confirmed at the level of mRNA expression by Northern blot analysis. These results provide evidence for a link between IL-6 and glucocorticoids. They could represent an example of a system in which one role of glucocorticoids is to define more accurately the target of cytokines, and they could explain, at least partly, the frequently observed synergy between IL-6 and glucocorticoids, notably in the case of hepatocytes.

Carcinoma, Hepatocellular↗
PubMed1990

The effects of various cytokines on interleukin-6 and interferon-alpha synthesis in human peripheral blood mononuclear cells.

The effect of various recombinant cytokines on the induction of interleukin-6 (IL-6) synthesis induced in adherent and nonadherent cells of human peripheral blood mononuclear cells (PBMNC) by bacterial lipopolysaccharide (LPS) or concanavalin A (CA) was studied. The results showed that human interferon-(HuIFN)-alpha, -beta, and gamma at a concentration of 100-10,000 IU/ml enhanced the LPS-induced IL-6 production in the adherent cell fraction of PBMNC. However, in nonadherent cells, treatment with HuIFN-alpha or -beta inhibited the CA-stimulated IL-6 production in a dose-dependent manner. Recombinant (r) IL-2 enhanced the IL-6 production of the adherent cells, while rIL-1 alone in the absence of other inducer induced IL-6 production in the nonadherent cell fraction. Other cytokines such as the recombinant tumor necrosis factor-alpha (rTNF-alpha) or rIL-6 itself did not modulate IL-6 production in human PBMNC. TNF and the interleukins studied did not affect the Sendai virus-induced IFN production in the adherent cells. In contrast, the different IFNs exerted a significant priming effect.

Cytokines↗
PubMed1990

Cloning and sequence analyses of cDNAs for interferon- and virus-induced human Mx proteins reveal that they contain putative guanine nucleotide-binding sites: functional study of the corresponding gene promoter.

The human protein p78 is induced and accumulated in cells treated with type I interferon or with some viruses. It is the human homolog of the mouse Mx protein involved in resistance to influenza virus. A full-length cDNA clone encoding the human p78 protein was cloned and sequenced. It contained an open reading frame of 662 amino acids, corresponding to a polypeptide with a predicted molecular weight of 75,500, in good agreement with the Mr of 78,000 determined on sodium dodecyl sulfate gels for the purified natural p78 protein. The cloned gene was expressed in vitro and corresponded in size, pI, antigenic determinant(s), and NH2 terminus sequence to the natural p78 protein. A second cDNA was cloned which encoded a 633-amino-acid protein sharing 63% homology with human p78. This p78-related protein was translated in reticulocyte lysates where it shared an antigenic determinant(s) with p78. A putative 5' regulatory region of 83 base pairs contained within the gene promoter region upstream of the presumed p78 mRNA cap site conferred human alpha interferon (IFN-alpha) inducibility to the cat reporter gene. The p78 protein accumulated to high levels in cells treated with IFN-alpha. In contrast, the p78-related protein was not expressed at detectable levels. The rate of decay of p78 levels in diploid cells after a 24-h treatment with IFN-alpha was much slower than the rate of decay of the antiviral state against influenza A virus and vesicular stomatitis virus, suggesting that the p78 protein is probably not involved in an antiviral mechanism. Furthermore, we showed that these proteins, as well as the homologous mouse Mx protein, possess three consensus elements in proper spacing, characteristic of GTP-binding proteins.

Amino Acid Sequence↗
PubMed1990

Lack of correlation in the interferon-induced small Mw 2-5A synthetase mRNA levels and sensitivity of two L cell sublines to the antiviral and priming effects of interferon.

MuIFN-alpha/beta at a concentration of 0.5 IU/ml exerted both antiviral and priming effects in the B, but not in the M subline of L929 cells. This concentration of IFN did not affect the small size 2-5A synthetase mRNA levels in either sublines, as determined by a Northern blot technique. Pretreatment with 5 or 50 IU/ml IFN resulted in more pronounced antiviral and priming effects in the L929B than in the L929M cells. However, the enhancements of the 2-5A synthetase mRNA levels due to the pretreating IFN dose were similar in both sublines.

Animals↗
PubMed1989

Different preparations of natural and recombinant human interleukin-6 (IFN-beta 2, BSF-2) similarly stimulate acute phase protein synthesis and uptake of alpha-aminoisobutyric acid by cultured rat hepatocytes.

1. Rat hepatocytes were cultured for 2 days in Williams E medium containing 1 microM insulin and dexamethasone. 2. Production of five plasma proteins was determined by electroimmunoassay in the media, and amino acid uptake was measured by [alpha-14C]aminoisobutyric acid accumulation in hepatocytes. 3. Supernatants from rat peritoneal macrophages and IL-6/IFN-beta 2/BSF-2 obtained from four different laboratories similarly stimulated synthesis of fibrinogen, alpha 1-cysteine proteinase inhibitor and alpha 2-macroglobulin, as well as [14C]-accumulation in cultured hepatocytes. 4. It is concluded that IL-6 is the principal hepatocyte stimulating factor responsible for typical features of the acute phase response of liver cells.

Acute-Phase Proteins↗
PubMed1989

Cloning, sequence determination, and expression of a 32-kilodalton-protein gene of Mycobacterium tuberculosis.

We describe the identification of the gene encoding an immunodominant 32-kilodalton (kDa) protein of Mycobacterium tuberculosis. The 32-kDa antigen is abundantly secreted into the culture supernatant of a variety of mycobacteria and appears to be a major stimulant of cellular and humoral immunity against mycobacteria. Recombinant clones expressing a 140- or 125-kDa beta-galactosidase fusion protein reactive with rabbit polyclonal anti-32 kDa protein serum were detected. The corresponding DNA sequence contains a 1,008-base-pair coding region. The deduced amino acid sequence corresponds to a 336-residue protein including the previously determined NH2-terminal sequence of the 32-kDa protein (J. De Bruyn, K. Huygen, R. Bosmans, M. Fauville, R. Lippens, J. P. Van Vooren, P. Falmagne, M. Weckx, H. G. Wiker, M. Harboe, and M. Turneer, Microb. Pathog. 2:351-366, 1987). Upstream of this NH2-terminal region, the gene codes for a signal peptide required for the secretion of a 294-amino-acid-long mature protein. A putative promoter sequence could be located upstream of the open reading frame. Comparison of the M. tuberculosis 32-kDa antigen with the Mycobacterium bovis BCG alpha-antigen (K. Matsuo, R. Yamaguchi, A. Yamazaki, H. Tasaka, and T. Yamada, J. Bacteriol. 170:3847-3854, 1988) revealed 73.8% homology between DNA sequences and 72.8% homology between amino acid sequences (signal and mature protein). Finally, the 140-kDa fusion protein could selectively be recognized by human tuberculous sera. This result confirms our previous finding that the 32-kDa antigen could be a valuable tool for the serological diagnosis of tuberculosis. Moreover, the availability of recombinant proteins opens perspectives for the localization of relevant B- and T-cell epitope regions on the 32-kDa antigen.

Amino Acid Sequence↗
PubMed1989

Regulation of the interferon-inducible IFI-78K gene, the human equivalent of the murine Mx gene, by interferons, double-stranded RNA, certain cytokines, and viruses.

The interferon-inducible gene (IFI-78K gene) that codes for a human protein, p78, of 78,000 Mr is the equivalent of the mouse Mx gene encoding Mx protein. The IFI-78K gene is located on chromosome 21 together with the alpha/beta interferon (IFN-alpha/beta) receptor. The p78 protein is important since it may be involved in resistance to influenza viruses. The regulation of the IFI-78K gene was studied in human diploid cells by using a cDNA probe to p78 mRNA and specific monoclonal antibodies to p78 protein. The IFI-78K gene, a normally quiescent gene, is transcriptionally regulated by IFN-alpha, and its induction does not require protein synthesis. The rate of transcription measured in a run-on assay increased rapidly but transiently. The level of p78 mRNA increased up to 8 h, declining slowly afterwards. The p78 protein, undetectable in untreated cells, accumulated up to 16 h, and its amount remained stable for at least 36 h after the addition of IFN-alpha. Cytokines such as tumor necrosis factor, interleukin-1 alpha, and interleukin-1 beta activated the IFI-78K gene at concentrations comparable to that of IFN-alpha. However, gene activation by these cytokines required protein synthesis. Poly(rI)-poly(rC) induced the IFI-78K gene directly at the transcriptional level without requirement for protein synthesis. Newcastle disease virus, influenza virus, and to a lesser extent vesicular stomatitis virus also induced the IFI-78K gene in the absence of any protein synthesis. Induction of transcription by viruses was markedly enhanced by pretreatment of cells with IFN-gamma (which by itself is a poor inducer of the IFI-78K gene), resulting in accumulation of p78 protein during the course of infection; this suggests that IFN-gamma programs cells to full antiviral activity upon virus infection.

Blotting, Northern↗
PubMed1989

High interleukin-6 serum levels and increased production by leucocytes in alcoholic liver cirrhosis. Correlation with IgA serum levels and lymphokines production.

Hypergammaglobulinaemia and enhanced serum IgA levels are common in alcoholic liver cirrhosis. Interleukin-6 (IL-6), which is identical to B cell differentiation factor BSF2 and is implicated in various autoimmune diseases, has been studied in patients with alcoholic liver cirrhosis. Increased serum levels and spontaneous or induced production of IL-6 by peripheral blood monoclonal cells have been found. IL-6 production correlates closely with IgA serum levels and negatively with impaired interleukin-2 and interferon gamma production. This abnormality could be related to overproduction of immunoglobulins and immune disturbances observed in this disease.

Female↗
PubMed1989

Paracrine rather than autocrine regulation of myeloma-cell growth and differentiation by interleukin-6.

To explore the mechanisms involved in the pathogenesis of human multiple myeloma (MM), we investigated the potential role of interleukin-6 (IL-6), a B-cell differentiation factor in humans, and a growth factor for rat/mouse heterohybridomas and murine plasmacytomas. Using a heterohybridoma assay, we found that two well-documented human myeloma cell lines, RPMI 8226 and U266, did not secrete IL-6 and did not express RNA messengers for IL-6. Neutralizing antibodies to IL-6 did not inhibit their proliferation, and recombinant IL-6 did not stimulate it. Taken together, these data show that IL-6 is not the autocrine growth factor of these human myeloma cell lines. A high production of IL-6 was found in the bone marrows of patients with fulminating MM, compared with patients with inactive or slightly active MM, or to healthy donors. This IL-6 production was assigned to adherent cells of the bone-marrow environment but not to myeloma cells. A spontaneous proliferation of myeloma cells freshly isolated from patients was observed in short-term cultures. Recombinant IL-6 was able to amplify it two- to threefold. The spontaneous proliferation of the myeloma cells was inhibited by anti-IL-6 antibodies and reinduced by recombinant IL-6. After 2 to 3 weeks of culture, the myeloma-cell proliferation progressively declined and no IL-6-dependent myeloma cell lines could be obtained despite repeated additions of fresh IL-6 and costimulation with other cytokines such as tumor necrosis factor (TNF)beta, or IL-1 beta. These data demonstrated a paracrine but not autocrine regulation of the growth and differentiation of myeloma cells by IL-6.

Aged↗
PubMed1988

Genes for IFN-beta-2 (IL-6), tumor necrosis factor, and IL-1 are expressed at high levels in the organs of normal individuals.

The gene of a cytokine designated IFN-beta-2, or IL-6, and recently identified as identical to the B cell-stimulatory factor 2, is transcribed at high levels in the spleen, liver, kidney, and peripheral blood leukocytes of normal individuals. The number of IFN-beta-2/IL-6 transcripts present endogenously in normal human tissues (0.6 to 16 copies/cell) is comparable to that present in normal cells induced in vitro with human rTNF. This is in marked contrast to the absence of detectable IFN-beta-1 transcripts (less than 0.0003 copy/cell) in the same samples of human tissue. The expression of the IFN-beta-2/IL-6 gene is closely associated with that of two other cytokines TNF, and IL-1. Thus, significant levels of IFN-beta-2/IL-6, TNF, IL-1 alpha, and IL-1 beta, mRNA were detected in all the samples of normal tissue tested and those samples which contained high levels of IFN-beta/IL-6 mRNA also contained high levels of TNF, and IL-1 beta mRNA. These results suggest that these cytokines may function in consort as regulators of cellular growth and function in normal tissues.

Gene Expression Regulation↗
PubMed1988

Recombinant interleukin 6 regulates the transcriptional activation of a set of human acute phase genes.

The phenomenon of acute phase (AP) response can be reproduced in vitro using cultured cells of hepatic origin by stimulation with the crude supernatant of activated monocytes (MoCM). Several monocyte-derived factors have been identified which might be responsible, alone or in combination, for the induction of AP response, but recently the attention has been focused on interleukin 6 (IL-6). We have previously shown that part of the AP response consists of the increase in the rate of transcription of the AP genes. Here we have treated the human hepatoma cell line Hep 3B with either crude MoCM or recombinant IL-6 and compared the effect of the two stimulants on the expression of both endogenous AP genes and recombinant plasmids introduced into the cells by transfection. The transfected plasmids contained the 5'-flanking region of AP genes fused to the coding region of the bacterial chloramphenicol acetyltransferase gene. We observe that the induction of mRNA accumulation of the endogenous genes corresponds to the transcriptional activation of the chloramphenicol acetyltransferase fusions. This is good evidence that the effect of IL-6 is totally or partially exerted at the level of transcription and that short segments of the 5'-flanking sequences of the inducible genes contain IL-6-responsive elements. Our results show that IL-6 is fully effective only on some of all the genes induced or repressed by MoCM, whereas others are only partially affected or totally nonresponsive.

Acetyltransferases↗
PubMed1988

Regulation of two interferon-inducible human genes by interferon, poly(rI).poly(rC) and viruses.

The IFI-56K and IFI-54K genes are transcriptionally stimulated when cells are treated by interferon. We have previously shown that the IFI-56K gene is in addition directly induced by poly(rI).poly(rC), and inducer of interferon-beta. Since the regulation of the IFI-56K and IFI-54K genes by interferon are very much alike, we tested whether the IFI-54K gene is also directly regulated by poly(rI).poly(rC). Treatment of various cell lines with poly(rI).poly(rC) leads to a clear accumulation of the IFI-54K mRNA to a level which sometimes even exceeds that obtained with high doses of interferon. Several interferon-resistant cell lines were investigated for the inducibility of both the IFI-56K and IFI-54K genes by interferons, poly(rI).poly(rC) and viruses (which are the natural inducers of interferon-alpha and -beta). Both genes appear to be coordinately regulated by these inducers. It was thus interesting to search for common regulatory element(s) in the control region of these two genes. The IFI-54K gene promoter region was isolated, from which a 520-base-pair segment was sequenced and compared with the promoter region of the IFI-56K gene that we had previously sequenced. The only homology was found is a well conserved 19-bp segment located just upstream of the TATA box of these genes; interestingly, this sequence is also homologous to the minimal region needed for the inducibility by poly(rI).poly(rC) of the interferon-beta gene. This conserved sequence might be responsible for the coordinate induction of the IFI-56K and IFI-54K genes by interferon, poly(rI).poly(rC) and viruses.

Base Sequence↗