Differential utilization of IFN-gamma-responsive elements in two maturationally distinct macrophage cell lines (original) (raw)
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Gene expression in IFN-gamma-activated murine macrophages
Brazilian Journal of Medical and Biological Research, 2004
Macrophages are critical for natural immunity and play a central role in specific acquired immunity. The IFN-γ activation of macrophages derived from A/J or BALB/c mice yielded two different patterns of antiviral state in murine hepatitis virus 3 infection, which were related to a down-regulation of the main virus receptor. Using cDNA hybridization to evaluate mRNA accumulation in the cells, we were able to identify several genes that are differently up-or down-regulated by IFN-γ in A/J (267 and 266 genes, respectively, up-and down-regulated) or BALB/c (297 and 58 genes, respectively, up-and downregulated) mouse macrophages. Macrophages from mice with different genetic backgrounds behave differently at the molecular level and comparison of the patterns of non-activated and IFN-γ-activated A/J or BALB/c mouse macrophages revealed, for instance, an up-regulation and a down-regulation of genes coding for biological functions such as enzymatic reactions, nucleic acid synthesis and transport, protein synthesis, transport and metabolism, cytoskeleton arrangement and extracellular matrix, phagocytosis, resistance and susceptibility to infection and tumors, inflammation, and cell differentiation or activation. The present data are reported in order to facilitate future correlation of proteomic/transcriptomic findings as well as of results obtained from a classical approach for the understanding of biological phenomena. The possible implication of the role of some of the gene products relevant to macrophage biology can now be further scrutinized. In this respect, a down-regulation of the main murine hepatitis virus 3 receptor gene was detected only in IFN-γ-activated macrophages of resistant mice.
Regulation of gene expression in RAW 264.7 macrophage cell line by interferon-gamma
Biochemical and biophysical research communications, 2006
Macrophages play an important role in immune responses and in inflammatory disease states such as atherosclerosis. Interferon-gamma (IFN-gamma) is a major cytokine involved in the activation of macrophages. To elucidate the primary response of various genes and biological pathways regulated by IFN-gamma in macrophage, we analyzed the gene expression profile in RAW 264.7 macrophage cells treated with IFN-gamma for 4h. Microarray analysis revealed that about 400 genes were differentially expressed, of which about 250 genes were up-regulated and 150 were down-regulated. Functional organization of the transcriptome revealed that induced genes are involved in antimicrobial and antiviral responses, antigen presentation, chemokine and cytokine signaling, and inhibition of cell growth. We also found that expression of genes involved in cell-cycle control, DNA repair, and lipid metabolism was suppressed by IFN-gamma. We also identified induction of multiple transcription factors by IFN-gamma...
Molecular and cellular biology, 1993
Gamma interferon activation factor (GAF) rapidly induces transcriptional activation of gamma interferon (IFN-gamma)-responsive genes. Conversion of the GAF from a latent cytoplasmic to an activated, DNA-binding form is an immediate step in the cellular response to IFN-gamma. The amount of IFN-gamma-activated GAF, measured by exonuclease III protection or gel shift assays, increased strongly upon monocytic differentiation of U937 cells. Activated GAF contained the IFN-responsive 91-kDa protein as its DNA-binding activity in gel shift or exonuclease III assays could be inhibited through direct addition of specific antiserum, and it was not present in p91-immunodepleted extracts. There was a differentiation-induced increase in the amount of nonphosphorylated (latent) p91. Transcription rate measurement demonstrated a strong induction of the p91 gene during monocytic differentiation of U937 cells. The amount of p91 which was rapidly phosphorylated in response to IFN-gamma was found to b...
Molecular and cellular biology, 1994
Rapid transcriptional induction of genes in response to gamma interferon (IFN-gamma) is mediated by the IFN-gamma activation site (GAS) and its cognate protein, the IFN-gamma activation factor (GAF). We describe a GAS-associated, differentiation-induced factor (DIF) as a potential molecular link between the activities of IFN-gamma and of growth and differentiation factors. DIF DNA binding was activated by colony-stimulating factor 1 in murine macrophages and also during tetradecanoyl phorbol acetate-induced differentiation or IFN-gamma treatment in myeloid U937 cells. IFN-gamma activation of DIF decreased significantly upon monocytic differentiation. DIF binding to DNA was inhibited by antiphosphotyrosine antibodies and could be induced by treatment of U937 cells with vanadate. Unlike GAF, DIF-DNA complexes did not contain the 91-kDa protein (p91) from ISGF-3. DIF bound with high affinity to GAS from the promoters of the IFP 53/tryptophanyl-tRNA synthetase and Fc gamma RI genes, int...
Regulation of gene expression in RAW 264.7 macrophage cell line by interferon-γ
Biochemical and Biophysical Research Communications, 2006
Macrophages play an important role in immune responses and in inflammatory disease states such as atherosclerosis. Interferon-c (IFN-c) is a major cytokine involved in the activation of macrophages. To elucidate the primary response of various genes and biological pathways regulated by IFN-c in macrophage, we analyzed the gene expression profile in RAW 264.7 macrophage cells treated with IFN-c for 4 h. Microarray analysis revealed that about 400 genes were differentially expressed, of which about 250 genes were up-regulated and 150 were down-regulated. Functional organization of the transcriptome revealed that induced genes are involved in antimicrobial and antiviral responses, antigen presentation, chemokine and cytokine signaling, and inhibition of cell growth. We also found that expression of genes involved in cell-cycle control, DNA repair, and lipid metabolism was suppressed by IFN-c. We also identified induction of multiple transcription factors by IFN-c in RAW 264.7 cells. Functional annotation of genes regulated by IFN-c in RAW 264.7 cells may provide novel insights into the role of macrophages in immunity and in inflammatory disease.
Potent activation of mouse macrophages by recombinant interferon-gamma
Cancer research, 1984
The ability of recombinant interferon-gamma (IFN-gamma) to activate mouse macrophages was investigated. The use of recombinant IFN-gamma has the advantage of being devoid of contaminating lymphokines. Two preparations of IFN-gamma were utilized, one which was not glycosylated and which was highly purified from Escherichia coli another which was glycosylated and which was from transfected COS-7 monkey cells. Both preparations of recombinant IFN-gamma activated murine macrophages to kill lymphoma and melanoma tumor targets, suggesting that glycosylation of the protein or the presence of other mammalian proteins is not essential for activation. Significant levels of cytolytic activity were induced from IFN-gamma (1 to 10 units/ml). This activity was undiminished by treatment of the IFN-gamma preparations with polymixin B at doses which neutralized endotoxin (50 micrograms/ml). Similarly, IFN-gamma, at low concentrations, induced an inhibition of migration by macrophages. Based on antiv...
Analysis of the IFN-g-Signaling Pathway in Macrophages at Different Stages of Maturation1
We previously demonstrated that the macrophage cell lines RAW 264.7 and WEHI-3 exhibit distinct patterns of gene expression in response to IFN-g. This difference is controlled at the transcriptional level and results from a specific inability of the less mature WEHI-3 cells to utilize either the IFN-stimulated response element or the g-activated sequence DNA regulatory element in response to stimulation with IFN-g, while other aspects of IFN-g gene induction remain intact. In the work described here, we examined the components of the IFN-g signal transduction pathway in RAW 264.7 and WEHI-3 cells to determine whether differences in pathway components or activity exist in WEHI-3 cells that could give rise to this difference in transcriptional response. Reverse transcriptase-PCR (RT-PCR) and flow cytometric analyses indicated that the levels of IFN- g receptor mRNA accumulation and protein expression are comparable for RAW 264.7 and WEHI-3 cells. RT-PCR and immunoblot analyses reveale...
Cellular Immunology, 1986
IFN-r can induce the expression of both class II histocompatibility antigens (Ia) and the lymphocyte function associated (LFA)-1 antigen on mutine peritoneal macrophages. We have examined the molecular changes which lead to altered expression of these two cell surface proteins to determine whether they are regulated by similar or independent mechanisms. While I-A antigen expression can be induced or enhanced by treatment of macrophages with either phorbol diesters and/or the Ca*' ionophore A23 187, these agents had no effect upon expression of LFA-1 under similar conditions. Macrophages from the A/J strain mouse exhibit a deficiency in their sensitivity to IF&y which is seen in our studies as an inability of RN-/ to elevate I-A antigen expression. However, expression of I-A could be modulated in these cells by treatment with either phorbol diesters or A23 187. In contrast, IFN-/ could induce LFA-1 antigen on A/J derived macrophages and this was not affected by either phorbol or A23 187. Thus these two antigens, despite coordinate expression in response to IFN-y in normal mouse strains, are clearly regulated independently. These results suggest that IFN-y generates at least two independent molecular events in macrophages which ultimately modulate the expression of cell surface proteins important to the performance of activated functions. 0 1986 Academic Press. Inc.
Analysis of the IFN-γ-Signaling Pathway in Macrophages at Different Stages of Maturation1
The Journal of Immunology, 1998
We previously demonstrated that the macrophage cell lines RAW 264.7 and WEHI-3 exhibit distinct patterns of gene expression in response to IFN-␥. This difference is controlled at the transcriptional level and results from a specific inability of the less mature WEHI-3 cells to utilize either the IFN-stimulated response element or the ␥-activated sequence DNA regulatory element in response to stimulation with IFN-␥, while other aspects of IFN-␥ gene induction remain intact. In the work described here, we examined the components of the IFN-␥ signal transduction pathway in RAW 264.7 and WEHI-3 cells to determine whether differences in pathway components or activity exist in WEHI-3 cells that could give rise to this difference in transcriptional response. Reverse transcriptase-PCR (RT-PCR) and flow cytometric analyses indicated that the levels of IFN-␥ receptor mRNA accumulation and protein expression are comparable for RAW 264.7 and WEHI-3 cells. RT-PCR and immunoblot analyses revealed that the principal components of this signaling pathway, including JAK1, JAK2, and STAT1, are present in both RAW 264.7 and WEHI-3 cells. However, analysis of STAT1 DNA-binding activity by electrophoretic mobility shift assay and of STAT1 phosphorylation by immunoblot revealed that this DNA-binding factor is active in RAW 264.7, but not in WEHI-3, cells after IFN-␥ stimulation. These results demonstrate that the components of the IFN-␥ signal transduction pathway are intact in WEHI-3 cells, but stimulation of these cells by IFN-␥ does not result in STAT1 activation.