The promoter region of the mouse gene for macrophage-inducible nitric oxide synthase (mac-NOS; EC 1.14.13.39) has been characterized. A putative TATA box is 30 base pairs upstream of the transcription start site. Computer analysis reveals numerous potential binding sites for transcription factors, many of them associated with stimuli that induce mac-NOS expression. To localize functionally important portions of the regulatory region, we constructed deletion mutants of the mac-NOS 5' flanking region and placed them upstream of a luciferase reporter gene. The macrophage cell line RAW 264.7, when transfected with a minimal promoter construct,
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Mouse macrophages can be stimulated by interferon (IFN)-␥ and bacterial lipopolysaccharide (LPS) to produce nitric oxide (NO) as the result of expression of the inducible NO synthase (iNOS; EC 1.14.13.39) gene. The iNOS gene promoter contains a candidate ␥-interferonactivated site (GAS). In transfection studies reported here, it was demonstrated that a luciferase reportergene construct, containing four synthetic copies of the iNOS GAS, was inducible when transfected macrophages were stimulated with either IFN-␥, LPS, or a combination of the two. Consistent with this finding were other transfection analyses, which showed that responsiveness of the intact iNOS promoter to these same agents was significantly reduced when two conserved nucleotide positions within the GAS were mutated. Oligonucleotide probes, which mimicked the iNOS GAS, formed a complex with proteins that appeared in the nuclei of IFN-␥ or IFN-␥ ؉ LPS-treated macrophages within 30 min of stimulation, as shown by electrophoretic mobility shift assay. LPS alone also caused the the appearance of a nuclear protein capable of binding the iNOS GAS-containing oligonucleotide; however, in contrast to binding induced by IFN-␥, approximately 2 h of stimulation with LPS were required. The protein bound to the iNOS GAS-containing oligonucleotide reacted specifically with an antibody raised against Stat1␣, regardless of the stimulus used. These data collectively support the conclusion that binding of Stat1␣ to the iNOS promoter's GAS is required for optimal induction of the iNOS gene by IFN-␥ and LPS.
Antiviral and macrophage-priming activities in the supernatant medium of a subelone of a concanavalin A-stimulated mouse T-cell hybridoma were investigated. The two activities were associated with a molecular weight of approximately 50,000 and could not be separated by various approaches. Both activities were eliminated by a highly specific neutralizing antibody against mouse interferon-v, but not by antibody against interferon-a and -(P. The ratio of-priming to antiviral activity in the hybridoma culture supernate was indistinguishable from the ratio obtained with mouse interferon-yprepared by recombinant DNA technology. It was concluded from these data that the priming activity in hybridoma culture supernates was attributable to interferon-and that this mediator is one form of the lymphokine macrophage-activating factor. Interferon-y was greater than 800 times more efficient at priming mouse macrophages for tumor cell killing than was a mixture of interferon-a and -(3. This finding contributes to growing awareness that type II interferon may have greater immunoregulatory potential than type I interferons.Macrophages can be activated to kill tumor cells in vitro by a nonspecific, extracellular mechanism that may be important in host defense against neoplastic cells in vivo. An important goal, therefore, is acquisition of better understanding of how the process of activation is regulated. Recently, it has been shown that under defined conditions the lymphokine macrophage-activating factor (MAF) does not render macrophages fully cytolytic (1-5). Instead, it heightens their responsiveness to a second signal(s) that then triggers the expression of killing-i.e., MAF "primes" macrophages to respond to a second, triggering stimulus.A cloned hybridoma (24/Gl) has been recognized that produces MAF, identified by its ability to prime mouse macrophages for tumor cell killing (6). The hybridoma-derived MAF was shown by various biochemical and functional criteria to be indistinguishable from conventionally prepared MAF (6). These supernates also contained antiviral activity attributed to interferon (IFN)-y.We report here that the antiviral and macrophage-priming activities produced by cells of a subclone of 24/Gl are not dissociable by various approaches. These results, together with data obtained by using recombinant mouse IFN-y, support a conclusion that priming activity produced by this hybridoma is attributable to IFN-y. We also report here that IFN-y is much more potent as a macrophage priming agent than type I interferon is. MATERIALS AND METHODSMice. Male C3H/HeN mice were obtained from Charles River Breeding Laboratories (Kingston, NY) and used at 6-9 weeks of age.
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