Mitsuyoshi Utsugi

c-Jun N-terminal kinase negatively regulates lipopolysaccharide-induced IL-12 production in human macrophages: role of mitogen-activated protein kinase in glutathione redox regulation of IL-12 production.

Although c-Jun N-terminal kinase (JNK) plays an important role in cytokine expression, its function in IL-12 production is obscure. The present study uses human macrophages to examine whether the JNK pathway is required for LPS-induced IL-12 production and defines how JNK is involved in the regulation of IL-12 production by glutathione redox, which is the balance between intracellular reduced (GSH) and oxidized glutathione (GSSG). We found that LPS induced IL-12 p40 protein and mRNA in a time- and concentration-dependent manner in PMA-treated THP-1 macrophages, and that LPS activated JNK and p38 mitogen-activated protein (MAP) kinase, but not extracellular signal-regulated kinase, in PMA-treated THP-1 cells. Inhibition of p38 MAP kinase activation using SB203580 dose dependently repressed LPS-induced IL-12 p40 production, as described. Conversely, inhibition of JNK activation using SP600125 dose dependently enhanced both LPS-induced IL-12 p40 production from THP-1 cells and p70 production from human monocytes. Furthermore, JNK antisense oligonucleotides attenuated cellular levels of JNK protein and LPS-induced JNK activation, but augmented IL-12 p40 protein production and mRNA expression. Finally, the increase in the ratio of GSH/GSSG induced by glutathione reduced form ethyl ester (GSH-OEt) dose dependently enhanced LPS-induced IL-12 p40 production in PMA-treated THP-1 cells. GSH-OEt augmented p38 MAP kinase activation, but suppressed the JNK activation induced by LPS. Our findings indicate that JNK negatively affects LPS-induced IL-12 production from human macrophages, and that glutathione redox regulates LPS-induced IL-12 production through the opposite control of JNK and p38 MAP kinase activation.

Resolvin E1 dampens airway inflammation and hyperresponsiveness in a murine model of asthma.

Resolvin E1 (RvE1; 5S, 12R, 18R-trihydroxyeicosapentaenoic acid) is an anti-inflammatory lipid mediator derived from the omega-3 fatty acid eicosapentaenoic acid (EPA). It has been recently shown that RvE1 is involved in the resolution of inflammation. However, it is not known whether RvE1 is involved in the resolution of asthmatic inflammation. To investigate the anti-inflammatory effect of RvE1 in asthma, a murine model of asthma was studied. After RvE1 was administered to mice intraperitoneally, there were decreases in: airway eosinophil and lymphocyte recruitment, specific Th2 cytokine, IL-13, ovalbumin-specific IgE, and airway hyperresponsiveness (AHR) to inhaled methacholine. Moreover, RvE1-treated mice had significantly lower mucus scores compared to vehicle-treated mice based on the number of goblet cells stained with periodic acid-schiff (PAS). These findings provide evidence that RvE1 is a pivotal counterregulatory signal in allergic inflammation and offer novel multi-pronged therapeutic approaches for human asthma.

Regulation of LPS induced IL-12 production by IFN-γ and IL-4 through intracellular glutathione status in human alveolar macrophages

Interleukin‐12 (IL‐12) is secreted from monocytes and macrophages; it exerts pleiotropic effects on T cells and natural killer (NK) cells, and stimulates interferon‐γ (IFN‐γ) secretion. Glutathione tripeptide regulates the intracellular redox status and other aspects of cell physiology. We examined whether IFN‐γ and IL‐4 affect the balance between intracellular reduced glutathione (GSH) and oxidized (GSSG) glutathione, as this may affect IL‐12 production in human alveolar macrophages (AM). We used both AM from healthy non‐smokers obtained by bronchoalveolar lavage and the monocytic THP‐1 cell line in this study. Incubation of AM for 2 h with the GSH precursor N‐acetylcysteine (NAC) increased the intracellular GSH/GSSG ratio, and enhanced lipopolysaccharide (LPS)‐induced IL‐12 secretion by AM. In THP‐1 cells, NAC increased the GSH/GSSG ratio and the expression of LPS‐induced IL‐12 mRNA, whereas l‐buthionine‐[S,R]‐sulphoximine (BSO) decreased these. NAC and BSO offset their own effects on the intracellular GSH/GSSG ratio and the expression of LPS‐induced IL‐12 mRNA. Furthermore, exposure of AM to the helper T cell type 1 (Th1) cytokine IFN‐γ or the helper T cell type 2 (Th2) cytokine IL‐4 for 72 h increased and decreased the GSH/GSSG ratio, respectively. Lipopolysaccharide (LPS)‐induced secretion of IL‐12 in AM was enhanced by IFN‐γ but inhibited by IL‐4. These results suggest that IFN‐γ and IL‐4 oppositely affect the GSH/GSSG balance, which may regulate IL‐12 secretion from AM in response to LPS.

Glutathione redox regulates lipopolysaccharide-induced IL-12 production through p38 mitogen-activated protein kinase activation in human monocytes: role of glutathione redox in IFN-γ priming of IL-12 production

We examined whether changes in intracellular reduced (GSH) or oxidized (GSSG) glutathione of human monocytes regulate lipopolysaccharide (LPS)‐induced IL‐12 production and defined the molecular mechanism that underlies glutathione redox regulation. Monocytes exposed to glutathione reduced form ethyl ester (GSH‐OEt) or maleic acid diethyl ester (DEM) increased or decreased the intracellular GSH/GSSG ratio, respectively. LPS‐induced IL‐12 production and p38 mitogen‐activated protein (MAP) kinase activation were enhanced by GSH‐OEt but suppressed by DEM. Selective p38 inhibitors showed that p38 promoted GSH‐OEt‐enhanced IL‐12 production. Furthermore, IFN‐γ priming increased the GSH/GSSG ratio and enhanced IL‐12 production through p38, and DEM negated the priming effect of IFN‐γ on p38 activation and IL‐12 production as well as on the GSH/GSSG ratio. These findings reveal that glutathione redox regulates LPS‐induced IL‐12 production from monocytes through p38 MAP kinase activation and that the priming effect of IFN‐γ on IL‐12 production is partly a result of the glutathione redox balance.

Protective effect of resolvin E1 on the development of asthmatic airway inflammation.

Resolvin E1 (RvE1) is an anti-inflammatory lipid mediator derived from the omega-3 fatty acid eicosapentaenoic acid (EPA), and strongly acts in the resolution of inflammation. We previously reported that RvE1 dampens airway inflammation and hyperresponsiveness in a murine model of asthma. In the present study, to elucidate the effects of RvE1 on the development of asthmatic airway inflammation, we investigated whether RvE1 acts on different phases of an OVA-sensitized and -challenged mouse model of asthma. RvE1 treatments at the time of either OVA sensitization or at the time of OVA challenge were investigated and compared with RvE1 treatments at the time of both OVA sensitization and challenge. After RvE1 was administered to mice intraperitoneally at the time of both OVA sensitization and challenge, there were decreases in airway eosinophil and lymphocyte recruitment, as well as a reduction in Th2 cytokine and airway hyperresponsiveness. RvE1 treatment at the time of either OVA sensitization or challenge also improved AHR and airway inflammation. Our results suggest that RvE1 acts on several phases of asthmatic inflammation and may have anti-inflammatory effects on various cell types.

PI3K p110β Positively Regulates Lipopolysaccharide-Induced IL-12 Production in Human Macrophages and Dendritic Cells and JNK1 Plays a Novel Role

The PI3K family is thought to participate in TLR signaling, and it has been reported to be a negative regulator of TLR-mediated production of IL-12, a key inducer of Th1 responses. However, the role of individual PI3K subtypes in IL-12 production remains obscure. We defined the distinct regulation of LPS-mediated IL-12 production by p110α and p110β catalytic subunits of PI3K in human APCs. We observed that knockdown of PI3K p110β by small interfering RNA (siRNA) suppressed both LPS-induced IL-12 protein production and mRNA expression in monocyte-derived macrophages and dendritic cells (DCs). Knockdown of PI3K p110α by siRNA reduced LPS-induced IL-12 protein production in both cell types. Conversely, knockdown of PI3K p110α suppressed LPS-induced IL-12 mRNA expression in monocyte-derived macrophages but minimally affected monocyte-derived DCs. PI3K p110β siRNA inhibited JNK activation, but not p38 MAPK or ERK activation, stimulated by LPS, while PI3K p110α siRNA did not affect LPS-induced JNK, p38 MAPK, or ERK activation in both cell types. Transfection of siRNA against JNK1, JNK2, and both decreased LPS-induced IL-12 production. Furthermore, PI3K p110β siRNA attenuated LPS-induced JNK1 phosphorylation, while not affecting JNK2 phosphorylation. Our findings indicate that PI3K p110β positively controls LPS-induced IL-12 production through the JNK1-dependent pathway in human macrophages and DCs.

Rac1 Negatively Regulates Lipopolysaccharide-Induced IL-23 p19 Expression in Human Macrophages and Dendritic Cells and NF-κB p65 trans Activation Plays a Novel Role

IL-23 is a heterodimeric cytokine composed of a unique p19 subunit and of a p40 subunit that is also common to IL-12. We defined the distinct signaling mechanisms that regulate the LPS-mediated induction of IL-23 p19 and p40 in human macrophages and dendritic cells. We found that the overexpression of dominant-negative Rac1 (N17Rac1) enhanced LPS-induced IL-23 p19 expression but did not alter p40 expression or IL-12 p70 production in PMA-treated THP-1 macrophages and in human monocyte-derived dendritic cells. Although the inhibition of either p38 MAPK or JNK enhanced LPS-induced p19 expression, N17Rac1 did not influence either p38 MAPK or JNK activation. By contrast, N17Rac1 augmented both NF-κB gene expression and p65 trans activation stimulated by LPS without affecting the degradation of IκB-α or DNA binding to NF-κB. Furthermore, small interference RNA of NF-κB p65 attenuated cellular amounts of p65 and suppressed LPS-induced p19 expression but did not affect p40 expression. Our findings indicate that Rac1 negatively controls LPS-induced IL-23 p19 expression through an NF-κB p65 trans activation-dependent, IκB-independent pathway and that NF-κB p65 regulates LPS-induced IL-23 p19, but not p40, expression, which causes differences in the control of IL-23 p19 and p40 expression by Rac1.

A phase II study of amrubicin, a synthetic 9-aminoanthracycline, in patients with previously treated lung cancer.

PURPOSE This study was designed to confirm the efficacy and safety of amrubicin, a new anthracycline agent, in patients with previously treated non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). METHODS Eligible patients were required to have recurrent or refractory NSCLC and SCLC after one or two previous chemotherapy regimens. All patients received intravenous amrubicin 35 mg/m(2) on days 1-3 every 3 weeks. Overall response rate (ORR), progression-free survival (PFS), and overall survival (OS) were evaluated. RESULTS Sixty-six patients (37 NSCLC and 29 SCLC) were assessable for efficacy and safety evaluation. Grade 3 or 4 neutropenia was observed in 39.4% of all patients (NSCLC, 37.8%; SCLC, 41.4%). Nonhematological toxicities were mild. No treatment-related death was observed. The ORRs were 13.5% (95% CI, 4.5-28.8%) in NSCLC and 44.8% (95% CI, 26.4-64.3%) in SCLC. In SCLC, ORRs were 60.0% in the sensitive relapse and 36.8% in the refractory relapse (p=0.2332). In NSCLC, the PFS, OS, and 1-year survival were 3.3 months, 12.0 months, and 35.3%, respectively. In SCLC, the PFS, OS, and 1-year survival were 4.0 months, 12.0 months, and 46.7%, respectively. CONCLUSIONS Amrubicin is an active and well-tolerated regimen in patients with previously treated lung cancer. Amrubicin 35 mg/m(2) seems to achieve similar efficacy with less toxicity than amrubicin 40 mg/m(2) in this patient population. These results warrant further evaluation in previously treated lung cancer.

Proton Pump Inhibitor Improves Breath Marker in Moderate Asthma with Gastroesophageal Reflux Disease

Background: Gastroesophageal reflux disease (GERD) influences the symptoms of asthma with acid and oxidative stress. Objectives: The purpose of this study was to determine the usefulness of measurement of the acid stress marker pH and the oxidative stress marker 8-isoprostane by exhaled breath condensate in proton pump inhibitor (PPI) therapy effect on moderate asthma patients with GERD. Methods: The pH and the concentration of 8-isoprostane were measured in the exhaled breath condensate of patients with moderate asthma (n = 36) and healthy subjects (n = 26). Two months of PPI therapy (lansoprazole at 30 mg/day) were done in the asthma patients with (n = 13) or without (n = 13) GERD according to a questionnaire for the diagnosis of reflux disease, and exhaled markers were measured. Results: The pH was lower (7.3 ± 0.3) and the 8-isoprostane level was higher (27.7 ± 2.3) in the asthma patients than in the healthy control subjects (pH 7.5 ± 0.2 and 8-isoprostane 6.6 ± 1.2). Two months of PPI therapy improved the pH (from 7.2 ± 0.1 to 7.3 ± 0.1) and the 8-isoprostane concentration (from 32.7 ± 3.4 to 19.2 ± 3.4) in the asthma patients with GERD, along with improvement of GERD symptoms. However, these markers did not change in the asthma patients without GERD. Conclusions: Measurement of the pH and 8-isoprostane level of exhaled breath condensate may be useful to evaluate the influence of GERD on asthma, as well as to determine the timing of intermittent PPI therapy.

Lysophosphatidic Acid Inhibits CC Chemokine Ligand 5/RANTES Production by Blocking IRF-1–Mediated Gene Transcription in Human Bronchial Epithelial Cells

Lysophosphatidic acid (LPA) is a phospholipid mediator that exerts a variety of biological responses through specific G-protein–coupled receptors (LPA1–LPA5 and P2Y5). LPA is thought to be involved in airway inflammation by regulating the expression of anti-inflammatory and proinflammatory genes. Chemokines such as CCL5/RANTES are secreted from airway epithelium and play a key role in allergic airway inflammation. CCL5/RANTES is a chemoattractant for eosinophils, T lymphocytes, and monocytes and seems to exacerbate asthma. We stimulated CCL5/RANTES production in a human bronchial epithelial cell line, BEAS-2B, with IFN-γ and TNF-α. When LPA was added, CCL5/RANTES mRNA expression and protein secretion were inhibited, despite the presence of IFN-γ and TNF-α. The LPA effect was attenuated by Ki16425, a LPA1/LPA3 antagonist, but not by dioctylglycerol pyrophosphate 8:0, an LPA3 antagonist. Pertussis toxin, the inhibitors for PI3K and Akt also attenuated the inhibitory effect of LPA on CCL5/RANTES secretion. We also identify the transcription factor IFN regulatory factor-1 (IRF-1) as being essential for CCL5/RANTES production. Interestingly, LPA inhibited IFN-γ and TNF-α–induced IRF-1 activation by blocking the binding of IRF-1 to its DNA consensus sequence without changing IRF-1 induction and its nuclear translocation. Ki16425, pertussis toxin, and PI3K inhibitors attenuated the inhibitory effect of LPA on IRF-1 activation. Our results suggest that LPA inhibits IFN-γ– and TNF-α–induced CCL5/RANTES production in BEAS-2B cells by blocking the binding of IRF-1 to the CCL5/RANTES promoter. LPA1 coupled to Gi and activation of PI3K is required for this unique effect.