Sylvie Reveneau

Hyporesponsiveness to lipopolysaccharide alters the composition of NF-κB binding to the regulatory regions of inducible nitric oxide synthase gene

Repeated exposure to bacterial endotoxin causes a diminished response by the host to further exposure. One important feature of this hyporesponsiveness is a reduced macrophage production of nitric oxide (NO) via the inducible nitric oxide synthase (iNOS) pathway. Using a murine macrophage model, we observed that hyporesponsiveness was accompanied by a decrease in the levels of NO release (measured as nitrite), iNOS protein and iNOS gene transcription. The expression of the putative lipopolysaccharide (LPS) receptor, CD14, was not altered. In vivo genomic footprinting showed that the same binding sites are occupied in the iNOS promoter and enhancer of desensitized macrophages and of LPS‐responsive macrophages, yet the composition of NF‐κB in the nuclei of these cells was found to be altered. The transcriptionally inactive homodimer p50‐p50 represented the predominant binding activity in nuclei from LPS‐pretreated cells before and after stimulation. Nuclei from cells which had not been pretreated but were stimulated contained more of the transcriptionally active p50‐p65 heterodimer than their pretreated counterparts. Consistent with this, the cytosolic steady‐state level of an inhibitor of NF‐κB activity, I‐κBα, was decreased in normal cells but not in pretreated cells. We propose that the presence of an overwhelming excess of transcriptionally inactive p50 homodimers on their κB sites in the iNOS control region in pretreated cells may block κB site binding by p50‐p65, thereby reducing the activity of the protein complex governing iNOS transcription.

Expression of inducible nitric oxide synthase in tumors in relation with their regression induced by lipid A in rats

It is well documented that nitric oxide (NO) is an effector molecule of macrophage‐mediated tumor cell toxicity in vitro; however, little is known about the role of NO in the antitumor immune response in vivo. We have developed a treatment protocol using lipid A. We have investigated the effects of lipid A on inducible NO synthase (NOS II) expression and evolution inside tumors during the course of treatment. Lipid A (OM‐174) treatment induced tumor regression in rats bearing established colon tumors. Furthermore, NO was synthesized and secreted inside the tumors of lipid A‐treated rats, as demonstrated by the increase of NOS II mRNA and NOS II content in the tumors, as well as of NOS II activity and NO production. During treatment, NOS II was localized in tumor cells only. Lipid A had no direct effect on tumor cells in vitro, while the combination of interferon gamma (IFN‐γ) plus interleukin‐1 beta (IL‐1β) induced production of NO by tumor cells which was cytostatic. The content of IFN‐γ and IL‐1β in tumors was enhanced during lipid A treatment; this is in agreement with an indirect effect of lipid A in vivo via the IFN‐γ and IL‐1β pathways. Int. J. Cancer 81:755–760, 1999.

Heat shock enhances transcriptional activation of the murine inducible nitric oxide synthase gene

There is considerable interest in determining the conditions leading to enhanced inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) biosynthesis. Using in vivofootprinting, we demonstrate that heat shock of murine macrophages concurrent with lipopolysaccharide (LPS) treatment stimulated changes in guanine methylation sensitivity at ‐898/9, at a putative partial heat shock element (HSE) and at ‐893/4, a site bordering an E‐box, within the iNOS gene enhancer, suggesting inducible occupation by transcription factors at these regions. LPS treatment accompanied by heat shock provoked increased iNOS gene transcription, increased levels of iNOS protein, and increased production of NO compared with LPS treatment alone. Electrophoretic mobility shift analysis revealed low constitutive levels of specific binding to an E‐box and a partial HSE within the iNOS enhancer. Binding to the E‐box was increased by LPS treatment or by heat shock, achieving a greater increase by a combination of both treatments. The proteins occupying this site were identified as belonging to the USF family of transcription factors. Heat shock or LPS increased binding to the HSE, and the factor responsible for this interaction was identified as heat shock factor‐1 (HSF‐1). Mutations at the HSE revealed the importance of HSF‐1 in the induction of iNOS by LPS. Thus, our data reveal two novel regulatory sites in the murine iNOS gene, one of which is implicated in enhancing iNOS expression via LPS stimulation, and provide the first evidence that heat shock enhances transcription of the iNOS gene. These results could have implications in the host response mechanism to fever‐associated gram‐negative infection.

A repressor in the proximal human inducible nitric oxide synthase promoter modulates transcriptional activation

The human inducible nitric oxide synthase (iNOS or NOSII) gene is regulated through an extended and complex promoter. In this study, the transcriptional regulation of human NOSII is investigated in the human colon cell line HCT‐8R. Stimulation with a cytokine mix (interferon‐γ, interleukin 1‐β, and tumor necrosis factor α) induces NOSII mRNA accumulation, as well as promoter activity in these cells. Several random deletions were performed within the proximal 7 kb of the promoter, which led to the identification of a region, whose deletion provokes a marked increase in transcriptional activity upon cytokine stimulation. Furthermore, this region is shown to repress a viral‐driven luciferase construct, mainly at basal levels. An AP‐1‐like sequence present in this region that is specifically recognized by nuclear proteins is shown to be involved in the repressive effect. This element is capable of repressing a viral promoter, and its deletion augments cytokine‐stimulated transcription. These findings are confirmed in various cell lines and suggest a general mechanism for the control of basal levels of NOSII expression, to avoid unnecessary toxicity under normal conditions.

Oct-1 cooperates with the TATA binding initiation complex to control rapid transcription of human iNOS

Expression of the human inducible nitric oxide synthase (hiNOS) is generally undetectable in resting cells, but stimulation by a variety of signals including cytokines induces transcription in most cell types. The tight transcriptional regulation of the enzyme is a complex mechanism many aspects of which remain unknown. Here, we describe an octamer (Oct) element in hiNOS proximal promoter, located close to the TATA box. This site constitutively binds Oct-1 and its deletion abrogates cytokine-induced transcription, showing that it is indispensable though not sufficient for transcription. Increasing the distance between Oct and the TATA box by inserting inert DNA sequence inhibits transcription, and footprinting of this region shows no other protein binding in resting cells, suggesting an interaction between the two complexes. Chromatin immunoprecipitation assays detect the presence of Oct-1, RNA polymerase II and trimethyl K4 histone H3 on the proximal promoter in resting cells, confirming that the gene is primed for transcription before stimulation. RT-PCR of various fragments along the hiNOS gene shows that transcription is initiated in resting cells and this is inhibited by interference with Oct-1 binding to the proximal site of the promoter. We propose that, through interaction with the initiation complex, Oct-1 regulates hiNOS transcription by priming the gene for the rapid response required in an immune response.

Tumor-derived granzyme B-expressing neutrophils acquire antitumor potential after lipid A treatment

Neutrophils are known to possess both pro- and anti-tumor properties, a feature that could be related to the diversity and plasticity of these cells. Here we explored the hypothesis that under an appropriate environment and stimuli, neutrophils could induce an effective response against tumor cells. In a rat and mouse models, we show that a substantial amount of colon tumor associated-neutrophils (TAN) expressed the cytolytic enzyme granzyme B, which is absent in spleen or blood circulating neutrophils. This TAN population was also found into tumors of patients with colon cancer. Tumor neutrophil infiltration was correlated with an increase of chemokines known to attract neutrophils in both rat models and patients. These cells were involved in a Lipid A analog-mediated colon tumor regression. Mechanistically, treating the rats with the Lipid A analog triggered granzyme B release from neutrophils in tumor cell vicinity, which was correlated to tumor regression. Alteration of granzyme B function in tumor cells decreased the cytotoxic effect of Lipid A in rat and mouse models. Granzyme B expression in neutrophils could be induced by the lipid A analog but also by some of the cytokines that were detected in the tumor microenvironment. These results identify a subpopulation of neutrophils expressing granzyme B that can act as a key player of lipid A-mediated colon cancer regression in rat and mouse models and the molecular mechanisms involved may provide novel approaches for human therapeutic intervention.

Transcriptional Regulation of the Macrophage NOS2 Gene

Nitric oxide (NO), produced by macrophages, was first shown to be involved in the antitumor immune response in 1987, when the group of Hibbs reported on the in vitro cytotoxic activity of murine macrophage NO toward tumor cells (Hibbs et al., 1987). Using an in vivo rat colon cancer model, our laboratory has determined that during tumor growth, macrophages are responsible for NO production (Lejeune et al., 1994). These macrophages can be either splenic or tumor-associated, as has been shown by double-labeling flow cytometric analysis. This NO production can block the specific immune response by both inhibiting migration of specific T lymphocytes into the tumor, as well as by inhibiting their proliferation. Because of difficulties in determining what factors can stimulate human macrophages/monocytes to produce significant amounts of NO, most studies on the induction of NO production by macrophages have focused on control mechanisms in the murine macrophage model.