Raymond W. Ganster

Multiple NF-κB Enhancer Elements Regulate Cytokine Induction of the Human Inducible Nitric Oxide Synthase Gene

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-κB in both human liver (AKN-1) and human lung (A549) epithelial cell lines. The addition of NF-κB inhibitors significantly suppressed cytokine-stimulated iNOS mRNA expression and NO synthesis, indicating that NF-κB 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-κB cis-regulatory transcription factor binding sites upstream of −4.7 kb. Site-directed mutagenesis of these sites revealed that the NF-κB 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-α or interleukin-1β, but not interferon-γ, exhibited inducible DNA binding activity for NF-κB. These data indicate that NF-κB activation is required for cytokine induction of the human iNOS gene and identifies four NF-κB enhancer elements upstream in the human iNOS promoter that confer inducibility to tumor necrosis factor-α and interleukin-1β.

Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-κB

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 κβ (NF-κB) elements. Here, we describe the role of the IFN-Jak kinase-Stat (signal transducer and activator of transcription) 1 pathway for regulation of hiNOS gene transcription. In A549 human lung epithelial cells, a combination of cytokines tumor necrosis factor-α, interleukin-1β, and IFN-γ (TNF-α, IL-1β, and IFN-γ) function synergistically for induction of hiNOS transcription. Pharmacological inhibitors of Jak2 kinase inhibit cytokine-induced Stat 1 DNA-binding and hiNOS gene expression. Expression of a dominant-negative mutant Stat 1 inhibits cytokine-induced hiNOS reporter expression. Site-directed mutagenesis of a cis-acting DNA element at −5.8 kb in the hiNOS promoter identifies a bifunctional NF-κB/Stat 1 motif. In contrast, gel shift assays indicate that only Stat 1 binds to the DNA element at −5.2 kb in the hiNOS promoter. Interestingly, Stat 1 is repressive to basal and stimulated iNOS mRNA expression in 2fTGH human fibroblasts, which are refractory to iNOS induction. Overexpression of NF-κB activates hiNOS promoter–reporter expression in Stat 1 mutant fibroblasts, but not in the wild type, suggesting that Stat 1 inhibits NF-κB function in these cells. These results indicate that both Stat 1 and NF-κB are important in the regulation of hiNOS transcription by cytokines in a complex and cell type-specific manner.

Recombinant Adenovirus Induces Maturation of Dendritic Cells via an NF-κB-Dependent Pathway

ABSTRACT 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-κB–DNA binding activity detected by electrophoretic mobility shift assays. rAd-induced DC maturation was blocked by the proteasome inhibitorNα-p-tosyl-l-lysine chloromethyl ketone (TLCK) or by infection with rAd-IκB, an rAd-encoding the dominant-negative form of IκB. 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.

Aspirin Inhibits In Vitro Maturation and In Vivo Immunostimulatory Function of Murine Myeloid Dendritic Cells

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.

Cyclosporine A inhibits the expression of costimulatory molecules on in vitro-generated dendritic cells: association with reduced nuclear translocation of nuclear factor kappa B.

BACKGROUND The maturation of dendritic cells (DC) is influenced by various factors, in particular cytokine-mediated signaling events. These include modulation of the activation of nuclear factor kappa B (NF-kappaB), which controls the transcription of genes encoding major histocompatibility complex (MHC) antigens, and costimulatory/accessory molecules for T-cell activation. Here, we investigated the influence of cyclosporine A (CsA) on the in vitro maturation of DC, and on the nuclear translocation and DNA binding of NF-kappaB. METHODS DC progenitors were propagated from mouse bone marrow in granulocyte-macrophage colony-stimulating factor (GM-CSF) or in GM-CSF plus either transforming growth factor (TGF)-beta or interleukin (IL)-4, in the presence or absence of CsA (1 microg/ml). After 5 days of culture, cell surface expression of MHC class I/II, CD40, CD80, and CD86 was analyzed by flow cytometry, and nuclear NF-kappaB proteins by electrophoretic mobility shift, antibody supershift, and Western blot assays. The antigen-presenting function of DC was determined in one-way mixed leukocyte reactions. RESULTS Exposure of replicating DC progenitors propagated in GM-CSF or GM-CSF+TGF-beta to CsA reduced costimulatory molecule expression, without affecting MHC antigen expression. Nuclear extracts from the CsA-treated DC revealed a decrease in nuclear translocation of NF-kappaB (p50). Mixed leukocyte reaction data were consistent with the flow cytometry and gel shift assay results, and showed reduced allostimulatory ability of the CsA-treated cells compared with untreated controls. Addition of IL-4 from the start of DC cultures conferred resistance to CsA-induced inhibition of NF-kappaB nuclear translocation and DC maturation. CONCLUSIONS CsA differentially inhibits the expression of key cell surface costimulatory molecules by in vitro-generated DC. This effect can be overcome, at least in part, by IL-4 and augmented by TGF-beta. The inhibition is linked to a decrease in nuclear translocation/DNA binding of NF-kappaB. Thus, CsA can alter the antigen-presenting function of DC for T-cell activation.

Activation of the lipopolysaccharide signaling pathway in hepatic transplantation preservation injury.

Background. Endotoxin or lipopolysaccharide (LPS) initiates a cascade of complications of septic shock and multiple organ failure seen in Gram-negative bacterial infections. The first step of this pathway, which leads to activated nuclear factor (NF)-&kgr;B, activating protein (AP)-1, and other transcription factors, is the formation of the LPS receptor complex by LPS, LPS-binding protein (LBP), CD14, and toll-like receptor (TLR) 2 or 4. We examined whether the LPS signaling pathway is activated by hepatic ischemia/reperfusion injury in the transplant setting. Methods. Orthotopic syngeneic rat liver transplantation was performed with 0 to 18 hr of cold preservation in University of Wisconsin solution. Animals were killed 1 to 48 hr after reperfusion. Northern blot analysis for CD14, LBP, and TLR2 mRNA, immunohistochemistry for LBP, liver enzyme analysis, and gel shift assay for NF-&kgr;B and AP-1 were performed. Results. LPS levels were elevated early after reperfusion. Aspartate aminotransferase and alanine aminotransferase maximally increased 12 hr after transplantation. LBP mRNA and protein and CD14 mRNA were significantly up-regulated peaking at 6 to 12 hr after reperfusion. TLR2 mRNA was also increased. NF-&kgr;B activity showed a biphasic peak at 1 to 3 hr and 12 hr after reperfusion, whereas AP-1 activity showed a peak at 3 to 6 hr. The induction of CD14 mRNA correlated with the length of cold ischemia time. Conclusions. These data indicate that multiple components of the LPS signaling pathway are activated during ischemia/reperfusion injury after liver transplantation.

Inhibition of cytokine-induced nitric oxide synthase expression by gene transfer of adenoviral IκBα

Abstract Background: Nitric oxide is overexpressed in nearly every organ during sepsis and it has profound biologic effects. Previously, we showed that maximal inducible nitric oxide synthase (iNOS) expression is up-regulated by a combination of cytokines and that this effect is mediated by the transcription factor NF-κB. Therefore the purpose of this study was to establish whether gene transfer of the inhibitory molecule IκB would result in the abrogation of cytokine-induced iNOS expression. Methods: Cultured hepatocytes were infected with an adenoviral vector containing the IκBα gene (Ad5IκB) and after an 18-hour recovery period were stimulated with the cytokine mixture of tumor necrosis factor-α (500 U/mL) plus interleukin 1β (200 U/mL) plus interferon gamma (100 U/mL). Results: As expected, cytokine mixture induced significant hepatocyte nitrite (NO 2 – ) and iNOS messenger RNA production. Cells infected with the IκBα gene showed a dose-dependent decrease in NO 2 – and iNOS messenger RNA levels. Western blot analysis showed a marked decrease in iNOS protein levels in the presence of Ad5IκBα. Gel shift assays of nuclear extracts demonstrated that Ad5IκBα decreased the cytokine-induced DNA binding activity for NFκB. Conclusions: NFκB is an important regulator of cytokine-induced NO expression. These results identify a novel therapeutic approach where gene transfer of the inhibitory molecule IκBα can be used to down-regulate cytokine-induced iNOS expression as well as other NFκB-dependent genes that are up-regulated during the inflammatory response. (Surgery 1999;126:142-7.)

Induced nitric oxide inhibits IL-6-induced stat3 activation and type II acute phase mRNA expression.

Inducible nitric oxide synthase (iNOS) can be coexpressed with acute phase reactants in hepatocytes; however, it is unknown if NO can regulate the acute phase response. We tested the hypothesis that iNOS-derived nitric oxide (NO) attenuates the acute phase response by inhibiting IL-6-enhanced Stat3 DNA-binding activity and type II acute phase mRNA expression. iNOS was overexpressed in cultured rat hepatocytes via transduction with a replication defective adenovirus containing cDNA for human iNOS (AdiNOS), and Stat3 DNA-binding activity was determined by electrophoretic mobility shift assay (EMSA). EMSAs demonstrated that AdiNOS inhibits IL-6-induced Stat3 activation and that this inhibition is reversible in the presence of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMA). The induction of beta-fibrinogen mRNA by IL-6, a Stat3 dependent process, is attenuated in AdiNOS-transduced cells and partially reversed by L-NMA. Thus, iNOS overexpression suppresses IL-6-induced Stat3 activation and type II acute phase mRNA expression in cultured hepatocytes. This suppression may represent a mechanism by which NO down-regulates the acute phase response.

Chapter 8 – Molecular Regulation of Inducible Nitric Oxide Synthase

Publisher Summary This chapter summarizes the molecular mechanisms that govern inducible nitric oxide synthase (iNOS) gene expression, focusing on the regulation of iNOS transcription. The gene for iNOS is expressed by many species and in a large variety of cell types. Typically, the expression is repressed in resting cells, requiring activation by an expanding number of biological, chemical, or physical stimuli. The molecular regulation of iNOS expression is complex and occurs at multiple sites in the gene expression pathway, with both transcriptional and posttranscriptional control mechanisms. Induction of nitric oxide synthesis has been shown to elicit either beneficial or detrimental consequences depending on the physiological or pathophysiological condition. An understanding of the signaling pathways and molecular mechanisms that regulate iNOS expression is critical for the development of effective therapies to modulate its expression in disease states. The major differences between human and rodent gene regulation as well as the mechanisms that downregulate iNOS expression are highlighted.