The human inducible nitric oxide synthase (iNOS) gene is overexpressed in a number of human inflammatory diseases. Previously, we observed that the human iNOS gene is transcriptionally regulated by cytokines and demonstrated that the cytokine-responsive regions are upstream of ؊3.8 kilobase pairs (kb). Therefore, the purpose of this study was to further localize the functional enhancer elements and to assess the role of the transcription factor NF-B in both human liver (AKN-1) and human lung (A549) epithelial cell lines. The addition of NF-B inhibitors significantly suppressed cytokinestimulated iNOS mRNA expression and NO synthesis, indicating that NF-B is involved in the induction of the human iNOS gene. Analysis of the first 4.7 kb of the 5-flanking region demonstrated basal promoter activity and failed to show any cytokine-inducible activity. However, promoter constructs extending to ؊5.8 and ؊7.2 kb revealed 2-3-fold and 4 -5-fold induction, respectively, in the presence of cytokines. DNA sequence analysis from ؊3.8 to ؊7.2 kb identified five putative NF-B cis-regulatory transcription factor binding sites upstream of ؊4.7 kb. Site-directed mutagenesis of these sites revealed that the NF-B motif at ؊5.8 kb is required for cytokine-induced promoter activity, while the sites at ؊5.2, ؊5.5, and ؊6.1 kb elicit a cooperative effect. Electromobility shift assays using a site-specific oligonucleotide and nuclear extracts from cells stimulated with cytokine-mixture, tumor necrosis factor-␣ or interleukin-1, but not interferon-␥, exhibited inducible DNA binding activity for NF-B. These data indicate that NF-B activation is required for cytokine induction of the human iNOS gene and identifies four NF-B enhancer elements upstream in the human iNOS promoter that confer inducibility to tumor necrosis factor-␣ and interleukin-1.
The human inducible nitric oxide synthase (hiNOS) gene is expressed in several disease states and is also important in the normal immune response. Previously, we described a cytokine-responsive enhancer between ؊5.2 and ؊6.1 kb in the 5-flanking hiNOS promoter DNA, which contains multiple nuclear factor  (NF-B) elements. Here, we describe the role of the IFN-Jak kinase-Stat (
Recombinant adenovirus (rAd) infection is one of the most effective and frequently employed methods to transduce dendritic cells (DC).Contradictory results have been reported recently concerning the influence of rAd on the differentiation and activation of DC. In this report, we show that, as a result of rAd infection, mouse bone marrow-derived immature DC upregulate expression of major histocompatibility complex class I and II antigens, costimulatory molecules (CD40, CD80, and CD86), and the adhesion molecule CD54 (ICAM-1). rAd-transduced DC exhibited increased allostimulatory capacity and levels of interleukin-6 (IL-6), IL-12p40, IL-15, gamma interferon, and tumor necrosis factor alpha mRNAs, without effects on other immunoregulatory cytokine transcripts such as IL-10 or IL-12p35. These effects were not related to specific transgenic sequences or to rAd genome transcription. The rAd effect correlated with a rapid increase (1 h) in the NF-B-DNA binding activity detected by electrophoretic mobility shift assays. rAd-induced DC maturation was blocked by the proteasome inhibitor N␣-p-tosyl-L-lysine chloromethyl ketone (TLCK) or by infection with rAd-IB, an rAd-encoding the dominant-negative form of IB. In vivo studies showed that after intravenous administration, rAds were rapidly entrapped in the spleen by marginal zone DC that mobilized to T-cell areas, a phenomenon suggesting that rAd also induced DC differentiation in vivo. These findings may explain the immunogenicity of rAd and the difficulties in inducing long-term antigen-specific T-cell hyporesponsiveness with rAd-transduced DC.As professional antigen-presenting cells (APC), dendritic cells (DC) exhibit the unique ability to stimulate both naive and memory T lymphocytes and play a critical role in central and peripheral T-cell tolerance (3,4,34,55,58). Their potential to determine the balance between immunity and tolerance makes DC targets for the therapeutic manipulation of immune responses against tumor cells or microorganisms or for the control of undesired immune reactions against allo-or autoantigens. In this respect, gene transfer approaches have been explored in an effort to potentiate the adjuvant (12, 29) or tolerogenic properties of DC (30,35,57). Recombinant adenovirus (rAd) has been demonstrated to be one of the most effective vehicles to deliver foreign DNA into DC (1,15,16,29,41,59,71). However, a fundamental problem with the use of replication-deficient rAd is that they generate the rapid development of natural killer (NK) cell and cytotoxic T-lymphocyte (CTL) responses that eliminate rAd-infected cells and induce neutralizing antibodies (Abs) that "limit" readministration of the same rAd serotype (65-67). The immunogenicity of rAd is a particular drawback when long-term transgene expression is required or when transduced DC are employed to generate antigen-specific tolerance for therapy of graft rejection or autoimmune diseases (26,30,35,(65)(66)(67). Although the mechanistic basis of rAd immunogenicity is unknown, evidence has accumulated...
The SWI/SNF complex is required for the enhancement of transcription by many transcriptional activators in yeast. Genetic and biochemical studies indicate that the complex facilitates activator function by antagonizing chromatin-mediated transcriptional repression. The absence of known DNA-binding motifs in several SWI/SNF subunits and the failure to identify SWI/SNF-dependent DNA-binding activities in crude yeast extracts have led to the belief that the complex does not bind DNA. Here we show that the SWI/SNF complex has a high affinity for DNA and that its DNA-binding properties are similar to those of proteins containing HMG-box domains. The complex interacts with the minor groove of the DNA helix, binds synthetic four-way junction DNA, and introduces positive supercoils into relaxed plasmid DNA. These properties are likely to be important in the remodelling of chromatin structure by the SWI/SNF complex
Aspirin is the most commonly used analgesic and antiinflammatory agent. In this study, at physiological concentrations, it profoundly inhibited CD40, CD80, CD86, and MHC class II expression on murine, GM-CSF + IL-4 stimulated, bone marrow-derived myeloid dendritic cells (DC). CD11c and MHC class I expression were unaffected. The inhibitory action was dose dependent and was evident at concentrations higher than those necessary to inhibit PG synthesis. Experiments with indomethacin revealed that the effects of aspirin on DC maturation were cyclooxygenase independent. Nuclear extracts of purified, aspirin-treated DC revealed a decreased NF-κB DNA-binding activity, whereas Ab supershift analysis indicated that aspirin targeted primarily NF-κB p50. Unexpectedly, aspirin promoted the generation of CD11c+ DC, due to apparent suppression of granulocyte development. The morphological and ultrastructural appearance of aspirin-treated cells was consistent with immaturity. Aspirin-treated DC were highly efficient at Ag capture, via both mannose receptor-mediated endocytosis and macropinocytosis. By contrast, they were poor stimulators of naive allogeneic T cell proliferation and induced lower levels of IL-2 in responding T cells. They also exhibited impaired IL-12 expression and did not produce IL-10 after LPS stimulation. Assessment of the in vivo function of aspirin-treated DC, pulsed with the hapten trinitrobenzenesulfonic acid, revealed an inability to induce normal cell-mediated contact hypersensitivity, despite the ability of the cells to migrate to T cell areas of draining lymphoid tissue. These data provide new insight into the immunopharmacology of aspirin and suggest a novel approach to the manipulation of DC for therapeutic application.
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