Alvin Denenberg

Peroxisome Proliferator Activator Receptor-γ Ligands, 15-Deoxy-Δ12,14-Prostaglandin J2 and Ciglitazone, Reduce Systemic Inflammation in Polymicrobial Sepsis by Modulation of Signal Transduction Pathways

Peroxisome proliferator activator receptor-γ (PPARγ) is a nuclear receptor that controls the expression of several genes involved in metabolic homeostasis. We investigated the role of PPARγ during the inflammatory response in sepsis by the use of the PPARγ ligands, 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) and ciglitazone. Polymicrobial sepsis was induced by cecal ligation and puncture in rats and was associated with hypotension, multiple organ failure, and 50% mortality. PPARγ expression was markedly reduced in lung and thoracic aorta after sepsis. Immunohistochemistry showed positive staining for nitrotyrosine and poly(ADP-ribose) synthetase in thoracic aortas. Plasma levels of TNF-α, IL-6, and IL-10 were increased. Elevated activity of myeloperoxidase was found in lung, colon, and liver, indicating a massive infiltration of neutrophils. These events were preceded by degradation of inhibitor κBα (IκBα), activation of IκB kinase complex, and c-Jun NH2-terminal kinase and, subsequently, activation of NF-κB and AP-1 in the lung. In vivo treatment with ciglitazone or 15d-PGJ2 ameliorated hypotension and survival, blunted cytokine production, and reduced neutrophil infiltration in lung, colon, and liver. These beneficial effects of the PPARγ ligands were associated with the reduction of IκB kinase complex and c-Jun NH2-terminal kinase activation and the reduction of NF-κB and AP-1 DNA binding in the lung. Furthermore, treatment with ciglitazone or 15d-PGJ2 up-regulated the expression of PPARγ in lung and thoracic aorta and abolished nitrotyrosine formation and poly(ADP-ribose) expression in aorta. Our data suggest that PPARγ ligands attenuate the inflammatory response in sepsis through regulation of the NF-κB and AP-1 pathways.

Absence of inducible nitric oxide synthase modulates early reperfusion-induced NF-κB and AP-1 activation and enhances myocardial damage

The role of nitric oxide (NO) generated by the inducible NO synthase (iNOS) during myocardial ischemia and reperfusion is not understood. We investigated the role of iNOS during early reperfusion damage induced in genetically deficient iNOS (iNOS‐/‐) mice and wild‐type littermates. In wild‐type mice, ischemia (60 min) and reperfusion (60 min) induced an elevation in serum levels of creatine phosphokinase and myocardial injury characterized by the presence of scattered apoptotic myocytes and mild neutrophil infiltration. Northern blot analysis showed increased expression of iNOS, whose activity was markedly elevated after reperfusion. Immunohistochemistry showed staining for nitrotyrosine; Western blot analysis showed elevated expression of heat shock protein 70 (HSP70), a putative cardioprotective mediator. Plasma levels of nitrite and nitrate, tumor necrosis factor α (TNF‐α), interleukin 6 (IL‐6), and IL‐10 were also increased. These events were preceded by degradation of inhibitor κBα (IκBα), activation of IκB kinase complex (IKK) and c‐Jun‐NH2‐terminal kinase (JNK), and subsequently activation of nuclear factor‐κB (NF‐κB) and activator protein 1 (AP‐1) as early as 15 min after reperfusion. In contrast, iNOS‐/‐ mice experienced 35% mortality after reperfusion. The extensive myocardial injury was associated with marked apoptosis and infiltration of neutrophils whereas expression of HSP70 was less pronounced. Nitrotyrosine formation and plasma levels of nitrite and nitrate were undetectable. TNF‐α and IL‐6 were increased and IL‐10 was reduced in earlier stages of reperfusion. Activation of IKK and JNK and binding activity of NF‐κB and AP‐1 were significantly reduced. Thus, we conclude that iNOS plays a beneficial role in modulating the early defensive inflammatory response against reperfusion injury through regulation of signal transduction.—Zingarelli, B., Hake, P. W., Yang, Z., O’Connor, M., Denenberg, A., Wong, H. R. Absence of inducible nitric oxide synthase modulates early reperfusion‐induced NF‐κB and AP‐1 activation and enhances myocardial damage. FASEB J. 16, 327–342 (2002)

The Serine/Threonine Phosphatase, PP2A: Endogenous Regulator of Inflammatory Cell Signaling

We have investigated the regulation of kinases and phosphatases in early gene activation in monocytes because these cells are implicated in the pathogenesis of acute inflammatory states, such as sepsis and acute lung injury. One early gene up-regulated by endotoxin is c-Jun, a member of the activating protein (AP) family. C-Jun is phosphorylated by c-Jun N-terminal kinase (JNK) and associates with c-Fos to form the AP-1 transcriptional activation complex that can drive cytokine expression. Inhibition of the serine/threonine phosphatase, PP2-A, with okadaic acid resulted in a significant increase in JNK activity. This finding was associated with increased phosphorylation of c-Jun, AP-1 transcriptional activity, and IL-1β expression. Activation of PP2A inhibited JNK activity and JNK coprecipitated with the regulatory subunit, PP2A-Aα, supporting the conclusion that PP2A is a key regulator of JNK in the context of an inflammatory stimulus.

A green tea-derived polyphenol, epigallocatechin-3-gallate, inhibits IkappaB kinase activation and IL-8 gene expression in respiratory epithelium.

Interleukin-8 (IL-8) is a principle neutrophil chemoattractant and activator in humans. There is interest in developing novel pharmacological inhibitors of IL-8 gene expression as a means for modulating inflammation in disease states such as acute lung injury. Herein we determined the effects of epigallocatechin-3-gallate (EGCG), a green tea-derived polyphenol, on tumor necrosis factor-α (TNF-α)-mediated expression of the IL-8 gene in A549 cells. EGCG inhibited TNF-α-mediated IL-8 gene expression in a dose response manner, as measured by ELISA and Northern blot analysis. This effect appears to primarily involve inhibition of IL-8 transcription because EGCG inhibited TNF-α-mediated activation of the IL-8 promoter in cells transiently transfected with an IL-8 promoter-luciferase reporter plasmid. In addition, EGCG inhibited TNF-α-mediated activation of IκB kinase and subsequent activation of the IκBα/NF-κB pathway. We conclude that EGCG is a potent inhibitor of IL-8 gene expression in vitro. The proximal mechanism of this effect involves, in part, inhibition of IκB kinase activation.

Bacterial induction of inducible nitric oxide synthase in cultured human intestinal epithelial cells

BACKGROUND & AIMS Enterocytes play a major role in the mucosa as a source of proinflammatory cytokines and cytotoxins. We tested the hypothesis that bacteria induce expression of the inducible nitric oxide synthase (iNOS) in cultured human enterocytes. METHODS DLD-1 and Caco-2BBe cell monolayers exposed to Salmonella dublin were analyzed for iNOS up-regulation and nitric oxide production (NOx) in the presence of various proinflammatory cytokines. RESULTS S. dublin augmented NOx in interferon gamma (IFN-gamma)-primed cells but had no independent effect on iNOS expression. S. dublin-induced NOx was not mediated by endotoxin and was augmented by an enteroinvasive phenotype. In DLD-1 cells, S. dublin-mediated NOx was blocked by inhibitors of nuclear factor kappa B (NF-kappa B) and tyrosine kinase activation and was steroid resistant. Cis-acting elements in the human iNOS promoter responsive to endotoxin and S. dublin stimulation of IFN-gamma-treated DLD-1 cells were identified between 10.9 and 8.7 kilobases upstream of the transcription initiation site. CONCLUSIONS S. dublin alters the regulation of iNOS messenger RNA in IFN-gamma-treated intestinal epithelial cells via a steroid-resistant pathway involving NF-kappa B and tyrosine kinase activity. Because bacterial interaction with cytokine-primed epithelial cells induces the synthesis of NO, an endogenous antimicrobial agent, these findings may have implications for the regulation of mucosal immunity.

Poly(ADP-ribose) polymerase-1 regulates activation of activator protein-1 in murine fibroblasts.

Poly(ADP-ribose) polymerase (PARP)-1 is activated in response to DNA injury in the nucleus of eukaryotic cells and has been implicated in cell dysfunction in inflammation. We investigated the role of PARP-1 on the AP-1 pathway, which is involved in the signal transduction of the inflammatory process. In murine wild-type fibroblasts, oxidative challenge by peroxynitrite and hydrogen peroxide or immunological challenge by IL-1 and 20% FCS induced phosphorylation of the mitogen-activated protein kinase kinase-4, activation of c-Jun N-terminal kinase (JNK), and DNA binding of AP-1. In comparative experiments, peroxynitrite induced DNA binding of heat shock factor-1. Pretreatment of wild-type cells with 5-iodo-6-amino-1,2-benzopyrone, a PARP-1 inhibitor, inhibited JNK activation and DNA binding of AP-1. In parallel experiments in PARP-1-deficient fibroblasts, DNA binding of AP-1 was completely abolished. Activation of JNK was significantly elevated at basal condition, but it exhibited a lesser increase after oxidative or immunological challenge than in wild-type fibroblasts. Nuclear content of phosphorylated mitogen-activated protein kinase kinase-4 was observed in PARP-1-deficient cells after peroxynitrite challenge only. Western blotting analysis for AP-1 subunits indicated that c-Fos was similarly expressed in wild-type and PARP-1-deficient cells. Phosphorylated c-Jun was expressed after oxidative or immunological challenge, but not in basal condition, in wild-type cells; however, it was significantly elevated at basal condition and further enhanced after oxidative or immunological challenge in PARP-1-deficient cells. No DNA binding of heat shock factor-1 was observed in PARP-1-deficient cells. These data demonstrate that PARP-1 plays a pivotal role in the modulation of transcription.

Poly(ADP-ribose) synthetase activation mediates increased permeability induced by peroxynitrite in Caco-2BBe cells

BACKGROUND & AIMS Peroxynitrite induces cytotoxicity by generating DNA single-strand breaks and activating poly(ADP-ribose) synthetase (PARS), a nuclear enzyme that consumes oxidized nicotinamide adenine dinucleotide (NAD+) and depletes cellular adenosine triphosphate (ATP). The aim of this study was to examine this mechanism of injury in an intestinal epithelial cell model after exposure to exogenous peroxynitrite (ONOO-) and nitric oxide (NO). METHODS Caco-2BBe cell monolayers exposed to donors of peroxynitrite (3-morpholino-sydnonimine [SIN-1], 3 mmol/L) or NO (S-nitroso-N-acetyl penicillamine [SNAP]; 3 mmol/L) were analyzed for DNA strand breaks, [NAD+], [ATP], and transepithelial flux of fluorescein sulfonic acid. RESULTS SIN-1 but not SNAP induced DNA single-strand breakage. Both SIN-1 and SNAP reduced [ATP], but only SIN-1 reduced [NAD+]. Inhibition of PARS activity by the PARS inhibitors 5-iodo-6-amino 1,2-benzopyrone or 3-aminobenzamide prevented the SIN-1-induced reduction in [NAD+] and [ATP] but had no effect on the SNAP-induced reduction in [ATP]. PARS inhibition reduced SIN-1-but not SNAP-induced hyperpermeability. CONCLUSIONS Peroxynitrite but not NO increases transepithelial permeability by inducing DNA strand breaks that activate the PARS pathway and cause the depletion of intracellular energy stores. Inhibition of PARS activity may represent a novel strategy in ameliorating peroxynitrite-mediated epithelial injury during intestinal inflammation.

Increased expression of heat shock protein-70 protects A549 cells against hyperoxia

Acute and chronic lung injury secondary to hyperoxia remains an important complication in critically ill patients, and, consequently, there is interest in developing strategies to protect the lung against hyperoxia. Heat shock proteins (HSPs) confer protection against a broad array of cytotoxic agents. In this study, we tested the hypothesis that increased expression of the 70-kDa HSP (HSP70) would protect cultured human respiratory epithelium against hyperoxia. Recombinant A549 cells were generated in which human HSP70 was increased by stable transfection with a plasmid containing human HSP70 cDNA under control of the cytomegalovirus promoter (A549-HSP70 cells). A549-HSP70 cells exposed to hyperoxia had greater acute survival rates and clonogenic capacity compared with wild-type A549 cells and with control cells stably transfected with the empty expression plasmid. Hyperoxia-mediated lipid peroxidation and ATP depletion were also attenuated in A549-HSP70 cells exposed to hyperoxia. Increased expression of HSP70 did not detectably alter mRNA levels of the intracellular antioxidants manganese superoxide dismutase, catalase, and glutathione peroxidase. Collectively, these data demonstrate a specific in vitro protective role for HSP70 against hyperoxia and suggest that potential mechanisms of protection involve attenuation of hyperoxia-mediated lipid peroxidation and ATP depletion.

Contribution of MKP-1 regulation of p38 to endotoxin tolerance.

Endotoxin tolerance has been characterized as diminished TNF-α expression after a second LPS stimulus and is dependent on new protein synthesis. LPS-induced expression of TNF-α is partly regulated by the p38 mitogen-activated protein (MAP) kinase, which post-transcriptionally stabilizes TNF-α mRNA. The dual-specific phosphatase, MKP-1, has been shown to negatively regulate p38 via dephosphorylation. We hypothesized that MKP-1 expression induced during tolerance regulates TNF-α expression by inhibiting p38 activity. To test this hypothesis, tolerance was induced in THP-1 cells, and naive or tolerized cells were rechallenged 18 h later with LPS (1 μg/mL) and TNF-α production was measured. Under similar conditions, nuclear proteins were isolated after LPS stimulation and were analyzed for phospho-p38 and MKP-1 by Western blot. Transient overexpression of MKP-1 was achieved using an adenoviral expression strategy and infected cells subsequently treated with LPS for TNF-α production and p38 activation. Results showed that LPS tolerance was induced as reflected by decreased TNF-α. Induction of LPS hyporesponsiveness could be mimicked by overexpression of MKP-1 but not β-gal. MKP-1 expression was noted only in LPS-tolerized or Ad-MKP-1 infected cells. In the canonical and Ad-MKP-1-mediated tolerance models, decreased phospho-p38 activity was observed. MKP-1s role in mediating endotoxin tolerance was further confirmed by demonstrating the inability to fully tolerize peritoneal macrophages isolated from MKP-1 null mutant (vs. wild type) mice (24% vs. 72% reductions, respectively). These data demonstrate that the dual specific phosphatase MKP-1 is an important mediator of endotoxin tolerance via p38 regulation.

Circulating methemoglobin and nitrite/nitrate concentrations as indicators of nitric oxide overproduction in critically ill children with septic shock.

OBJECTIVES To examine the relationship between circulating methemoglobin and nitrite/nitrate concentrations and to compare these markers of nitric oxide overproduction with clinical variables in children diagnosed with septic shock. DESIGN Prospective, controlled, clinical study. SETTING Pediatric intensive care unit and outpatient clinic in a childrens hospital. PATIENTS Twenty-two children diagnosed with septic shock and ten age-matched healthy control patients. INTERVENTIONS Patients diagnosed with septic shock had blood specimens taken on study entry and every 6 hrs for 72 hrs for methemoglobin and nitrite/nitrate determinations. Single blood specimens were obtained from controls. MEASUREMENTS AND MAIN RESULTS Circulating methemoglobin and nitrite/nitrate concentrations were significantly higher in children diagnosed with septic shock in comparison with healthy control children (p = .01 and .05, respectively). Peak nitrite/nitrate concentrations correlated with serum creatinine (r2 = .19; p = .04) and were inversely correlated with arterial pH (r2 = .28; p = .01) and urine output (r2 = .21; p = .03) when analyzed by log-linear regression. There were no significant relationships between methemoglobin and nitrite/nitrate or between methemoglobin and any other clinical variable. CONCLUSIONS Circulating methemoglobin and nitrite/nitrate concentrations are increased in children diagnosed with septic shock. Plasma nitrite/nitrate values correlate with selected clinical variables in these children. Circulating methemoglobin measurements are not superior to plasma nitrite/nitrate concentrations as an indicator of endogenous overproduction of nitric oxide in children diagnosed with septic shock. A need remains to develop markers of endogenous nitric oxide activity that have greater accuracy and reliability.