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The role of NF-kappaB, the rapid-response transcription factor for multiple genes, in cold ischemia-reperfusion (I/R) injury was examined after syngeneic transplantation of liver grafts. Lewis rat recipients were killed 1-48 h after reperfusion of three different liver grafts: 1) uninfected control, 2) infected ex vivo with control adenoviral vector (AdEGFP), and 3) infected ex vivo with AdIkappaB. In uninfected control livers, NF-kappaB was activated biphasically at 1-3 and 12 h after reperfusion with aspartate transaminase (AST) levels of 4,244 +/- 691 IU/l. The first peak of NF-kappaB activation associated with an increase of mRNA for TNF-alpha, IL-1beta, and IL-10. AdEGFP transfection resulted in similar outcomes. Interestingly, AdIkappaB-transfected liver grafts suffered more severe I/R injury (AST9,000 IU/l). Transfected IkappaB was detected in transplanted livers as early as 6 h, and this correlated with the abrogation of the second, but not the first, peak of NF-kappaB activation at 12-48 h and increased apoptosis. Thus inhibition of the second wave of NF-kappaB activation in IkappaB-transfected livers resulted in an increase of liver injury, suggesting that NF-kappaB may have a dual role during liver I/R 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)-κB, 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-κB 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-κB 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.

Background. The inducible nitric oxide synthase (iNOS) is strongly expressed following inflammatory stimuli. Adenosine 3′,5′-cyclic monophosphate (cAMP) increases iNOS expression and activity in a number of cell types but decreases cytokine-stimulated iNOS expression in hepatocytes. The mechanisms for this effect are unknown. Methods. Rat hepatocytes were stimulated with cytokines to induce iNOS and cultured with cAMP agonists dibutyryl-cAMP (dbcAMP), 8-bromo-cAMP, and forskolin (FSK). Nitric oxide synthesis was assessed by supernatant nitrite levels and iNOS expression was measured by Northern and Western blot analyses. Nuclear factor κB binding was assessed by electromobility shift assay. Results. Cyclic AMP dose dependently decreased NO synthesis in response to a combination of proinflammatory cytokines or interleukin-1β (IL-1β) alone. The adenylate cyclase inhibitor SQ 22,536 increased cytokine- or IL-1β-stimulated NO synthesis. dbcAMP decreased iNOS mRNA expression and iNOS protein expression. Both dbcAMP and glucagon decreased iNOS promoter activity in rat hepatocytes transfected with the murine iNOS promoter and decreased DNA binding of the transcription factor NF-κB. Conclusion. These data suggest that cAMP is important in hepatocyte iNOS expression and agents that alter cAMP levels may profoundly alter the response of hepatocytes to inflammatory stimuli through effects onthe iNOS promoter region and NF-κB.

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.

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.)

Previously, we demonstrated that nuclear factor-kappaB (NF-kappaB) mediates cytokine-induced hepatic inducible nitric oxide synthase (iNOS) expression. NF-kappaB activation is regulated by kinases and phosphatases whose function is only beginning to be understood. Therefore, experiments were performed to determine the role of protein phosphatases (PPase) in cytokine-induced iNOS expression. Hepatocytes were stimulated with cytokines in the presence or absence of tyrosine phosphatase inhibitors (pervanadate [PV], phenylarsine oxide [PAO]) and a serine-threonine phosphatase inhibitor (okadaic acid [OA]). Cytokines induced hepatocyte iNOS mRNA, protein, and NO2- production that was substantially decreased by the addition of the tyrosine phosphatase inhibitors (PAO and PV). The serine-threonine phosphatase inhibitor (OA) decreased NO release and protein levels in a concentration-dependent fashion; however, iNOS mRNA levels were not significantly reduced. Nuclear run-on experiments demonstrated that protein tyrosine phosphatases (PTPases) are required for iNOS transcription, while the serine-threonine phosphatase inhibitor (OA) had no effect on iNOS transcription. Electromobility shift assays (EMSAs) revealed that the tyrosine-phosphatase inhibitors blocked cytokine-induced NF-kappaB activation, while OA did not have a significant effect on NF-kappaB DNA binding activity. Therefore, tyrosine phosphatases are involved in the regulation of cytokine-induced activation of NF-kappaB, while serine-threonine phosphatases posttranscriptionally regulate iNOS translation. These results identify the regulatory role of specific protein phosphatases (PPases) in hepatic iNOS expression.

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.

THE SWI/SNF complex is required for the enhancement of transcription by many transcriptional activators in yeast1,2. Genetic and biochemical studies indicate that the complex facilitates activator function by antagonizing chromatin-mediated transcriptional repression3–6. 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 DNA7,8. 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 domains9. 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.