Shu Hashimoto

p38 Mitogen-Activated Protein Kinase and c-Jun-NH2-Terminal Kinase Regulate RANTES Production by Influenza Virus-Infected Human Bronchial Epithelial Cells

Airway epithelial cells which are the initial site of influenza virus (IV) infection are suggested to participate in airway inflammatory response by expressing various cytokines including RANTES; however, the intracellular signal that regulates RANTES expression has not been determined. In the present study, we examined the role of p38 mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase (Erk), and c-Jun-NH2-terminal kinase (JNK) in RANTES production by IV-infected human bronchial epithelial cells. The results showed that IV infection induced increases in p38 MAP kinase, and Erk and JNK phosphorylation and activity. SB 203580, PD 98059, and CEP-1347 attenuated IV-infection induced p38 MAP kinase activity, Erk activity, and JNK activity, respectively. SB 203580 and CEP-1347 attenuated RANTES production by 45.3% and 45.2%, respectively, but a combination of these inhibitors additively attenuated by 69.1%. In contrast, PD 98059 did not attenuate. Anti-IL-1α mAb, anti-IL-1β mAb, anti-TNF-α mAb, anti-IL-8 mAb, anti-IFN-β mAb, anti-RANTES mAb, and a combination of these mAbs did not affect IV infection-induced increases in p38 MAP kinase, Erk, and JNK phosphorylation, indicating that each cytokine neutralized by corresponding Ab was not involved in IV infection-induced phosphorylation of MAP kinases. N-acetylcysteine (NAC) did not affect IV infection-induced increases in MAP kinase phosphorylation, whereas NAC attenuated RANTES production by 18.2%, indicating that reactive oxygen species may act as a second messenger leading to RANTES production via p38 MAP kinase- and JNK-independent pathway. These results indicate that p38 MAP kinase and JNK, at least in part, regulate RANTES production by bronchial epithelial cells.

Mitophagy-dependent necroptosis contributes to the pathogenesis of COPD

The pathogenesis of chronic obstructive pulmonary disease (COPD) remains unclear, but involves loss of alveolar surface area (emphysema) and airway inflammation (bronchitis) as the consequence of cigarette smoke (CS) exposure. Previously, we demonstrated that autophagy proteins promote lung epithelial cell death, airway dysfunction, and emphysema in response to CS; however, the underlying mechanisms have yet to be elucidated. Here, using cultured pulmonary epithelial cells and murine models, we demonstrated that CS causes mitochondrial dysfunction that is associated with a reduction of mitochondrial membrane potential. CS induced mitophagy, the autophagy-dependent elimination of mitochondria, through stabilization of the mitophagy regulator PINK1. CS caused cell death, which was reduced by administration of necrosis or necroptosis inhibitors. Genetic deficiency of PINK1 and the mitochondrial division/mitophagy inhibitor Mdivi-1 protected against CS-induced cell death and mitochondrial dysfunction in vitro and reduced the phosphorylation of MLKL, a substrate for RIP3 in the necroptosis pathway. Moreover, Pink1(-/-) mice were protected against mitochondrial dysfunction, airspace enlargement, and mucociliary clearance (MCC) disruption during CS exposure. Mdivi-1 treatment also ameliorated CS-induced MCC disruption in CS-exposed mice. In human COPD, lung epithelial cells displayed increased expression of PINK1 and RIP3. These findings implicate mitophagy-dependent necroptosis in lung emphysematous changes in response to CS exposure, suggesting that this pathway is a therapeutic target for COPD.

N‐acetylcysteine attenuates TNF‐α‐induced p38 MAP kinase activation and p38 MAP kinase‐mediated IL‐8 production by human pulmonary vascular endothelial cells

We have previously shown that tumour necrosis factor‐α (TNF‐α) activates p38 mitogen‐activated protein (MAP) kinase to produce interleukin‐8 (IL‐8) by human pulmonary vascular endothelial cells. Reactive oxygen species (ROS) including H2O2 generated by TNF‐α can act as signalling intermediates for cytokine induction; therefore, scavenging ROS by anti‐oxidants is important for the regulation of cytokine production. However, the effect of N‐acetylcysteine (NAC), which acts as a precursor of glutathione (GSH) synthesis, on TNF‐α‐induced activation of p38 MAP kinase pathway and p38 MAP kinase‐mediated IL‐8 production by human pulmonary vascular endothelial cells has not been determined. To clarify these issues, we examined the effect of NAC on TNF‐α‐induced activation of p38 MAP kinase, MAP kinase kinase (MKK) 3 and MKK6 which are upstream regulators of p38 MAP kinase, and p38 MAP kinase‐mediated IL‐8 production. Human pulmonary vascular endothelial cells that had been preincubated with NAC were stimulated with TNF‐α and then the activation of p38 MAP kinase and MKK3/MKK6 in the cells and IL‐8 concentrations in the culture supernatants were determined. Intracellular GSH levels increased in NAC‐treated cells. NAC attenuated TNF‐α‐induced activation of p38 MAP kinase and MKK3/MKK6. NAC attenuated p38 MAP kinase‐mediated IL‐8 production by TNF‐α‐stimulated cells. These results indicate that the cellular reduction and oxidation (redox) regulated by intracellular GSH is critical for TNF‐α‐induced activation of p38 MAP kinase pathway and p38 MAP kinase‐mediated IL‐8 production by human pulmonary vascular endothelial cells, and we emphasize that anti‐oxidant therapy is an important strategy for the treatment of acute lung injury.

Proinflammatory cytokine-induced and chemical mediator-induced IL-8 expression in human bronchial epithelial cells through p38 mitogen-activated protein kinase-dependent pathway.

The p38 mitogen-activated protein (MAP) kinase is activated in various cells by proinflammatory cytokines and environmental stresses. However, little is known about the role of p38 MAP kinase in proinflammatory cytokine- and chemical mediator-induced cytokine expression in human bronchial epithelial cells (BECs). In this study we examined the role of p38 MAP kinase in IL-8 expression in BECs to clarify the signal transduction pathway regulating IL-8 expression in BECs stimulated with tumor necrosis factor-alpha (TNF-alpha), IL-1alpha, and platelet-activating factor (PAF). We used TNF-alpha, IL-1alpha, and PAF as inducers for the analysis of the signal transduction pathway and determined IL-8 expression in BECs because TNF-alpha, IL-1alpha, and PAF are known to induce cytokine expression in BECs, and these proinflammatory cytokines and PAF are described to have a role in the production of allergic inflammation. The results showed that TNF-alpha, IL-1alpha, and PAF induced tyrosine phosphorylation of p38 MAP kinase in a dose- and time-dependent manner. The specific p38 MAP kinase inhibitor, SB 203580, completely inhibited TNF-alpha-, IL-1alpha-, or PAF-induced IL-8 protein and mRNA expression in BECs. These results indicated that p38 MAP kinase plays an important role in TNF-alpha-, IL-1alpha-, or PAF-activated signaling pathway, which regulates IL-8 expression in BECs. In addition, these results provide new evidence on a strategy for treatment of airway inflammation with the specific p38 MAP kinase inhibitor.

Elevation of serum soluble intercellular adhesion molecule‐1 (sICAM‐1) and sE‐selectin levels in bronchial asthma

Adhesion molecules such as ICAM‐1 and E‐selection have been shown to play important roles in the production of allergic inflammation. In the present study, we measured serum soluble ICAM‐1 (sICAM‐1)and soluble E‐selection (sE‐selectin) levels by ELISA in 42 patients with bronchial asthma (22 atopic and 20 non‐atopic) during asthma attacks and in stable conditions in order to assess the state of ICAM‐1 and E‐selectin in allergic inflammation. Both serum sICAM‐1 levels and serum sE‐sclectin levels in sera obtained during bronchial asthma attacks were higher than those in sera obtained in stable conditions. These findings were observed regardless of atopic status. To examine the regulatory mechanism in the elevation of serum sICAM‐1 and sE‐selectin levels, serum tumour necrosis factor‐alpha (TNF‐α) levels were measured by ELISA. TNF‐α levels in sera obtained during bronchial asthma attacks were higher than those in sera obtained in stable conditions. There was a correlation between the nature of change in serum TNF‐α levels and the nature of change in serum slCAM‐1 levels or serum sE‐selectin levels, though serum TNF‐α levels did not correlate with serum sICAM‐1 levels or serum sE‐selectin levels. These results suggest that higher levels of sICAM‐l and sE‐selectin during asthma attacks may reflect the up‐regulation of ICAM‐1 and E‐selectin expression in allergic inflammation, and that the soluble form of these adhesion molecules may be useful markers for the presence of allergic inflammation. TNF‐α is shown to enhance the expression and release of ICAM‐1 and E‐selectin in vitro, however; the regulatory mechanism in the elevation of serum sICAM‐1 and sE‐selectin levels remains to be clarified.

Consensus statement for the diagnosis and treatment of drug-induced lung injuries.

Keishi Kubo, Arata Azuma, Minoru Kanazawa, Hideto Kameda, Masahiko Kusumoto, Akihiko Genma, Yasuo Saijo, Fumikazu Sakai, Yukihiko Sugiyama, Koichiro Tatsumi, Makoto Dohi, Hitoshi Tokuda, Shu Hashimoto, Noboru Hattori, Masayuki Hanaoka, Yuh Fukuda, the Japanese Respiratory Society Committee for formulation of Consensus statement for the diagnosis and treatment of drug-induced lung injuries Nagano Prefectural Hospital Organization, Japan Division of Pulmonary Medicine, Infections Diseases, and Oncology, Department of Internal Medicine, Nippon Medical School, Japan Department of Respiratory Medicine, Saitama Medical University, Japan Division of Rheumatology, Department of Internal Medicine, School of Medicine, Keio University, Japan Department of Diagnostic Radiology, National Cancer Center Hospital, Japan Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, Japan Department of Diagnostic Radiology, Saitama International Medical Center, Saitama Medical University, Japan Division of Pulmonary Medicine, Department of Internal Medicine, Jichi Medical University, Japan Department of Respirology, Chiba University Graduate School of Medicine, Japan Department of Allergy and Rheumatology, Tokyo University Graduate School of Medicine, Japan Department of Respiratory Medicine, Social Insurance Central General Hospital, Japan Division of Respiratory Medicine, Department of Internal Medicine, Nihon University School of Medicine, Japan Department of Molecular and Internal Medicine, Hiroshima University Graduate School of Biomedical & Health Sciences, Japan Department of Analytic Human Pathology, Graduate School of Medicine, Nippon Medical School, Japan

ASK1 regulates influenza virus infection-induced apoptotic cell death.

Apoptosis occurs in influenza virus (IV)-infected cells. There are a number of mechanisms for the regulation of apoptosis. However, the molecular mechanism of IV infection-induced apoptosis is still controversial. Apoptosis signal-regulating kinase1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the SEK1-c-Jun N-terminal kinase (JNK) and MKK3/MKK6-p38 MAPK signaling cascades. ASK1 has been implicated in cytokine- and stress-induced apoptosis. Here, we show the following: (1) IV infection activated ASK1 and concomitantly phosphorylated JNK and p38 MAPK in human bronchial epithelial cells; (2) the activation of JNK and p38 MAPK but not extracellular-regulated kinase (ERK) in embryonic fibroblasts (MEFs) derived from ASK1 knockout mice (ASK1(-/-) MEFs) was depressed compared to MEFs derived from wild type mice (ASK1(+/+) MEFs); and (3) ASK1(-/-) MEFs were defective in IV infection-induced caspase-3 activation and cell death. These results indicate that apoptosis in IV-infected BEC is mediated through ASK1-dependent cascades.

Curcumin inhibition of Dermatophagoides farinea-induced interleukin-5 (IL-5) and granulocyte macrophage-colony stimulating factor (GM-CSF) production by lymphocytes from bronchial asthmatics

Curcumin, a dietary pigment responsible for the yellow color of curry, has been used for the treatment of inflammatory diseases and exhibits a variety of pharmacological effects such as anti-inflammatory, anti-tumor, anti-oxidant, and anti-viral activity. However, it has not been determined whether the effect of curcumin on the production of cytokine affects eosinophil functions and IgE synthesis. In the present study, we examined the effect of curcumin on the production of interleukin (IL)-2, IL-5, granulocyte macrophage-colony stimulating factor (GM-CSF), and IL-4 by lymphocytes from atopic asthmatics in response to house dust mites (Dermatophagoides farinea: Df) in order to clarify a potential application for allergic diseases. Curcumin inhibited Df-induced lymphocyte proliferation and production of IL-2. Exogenous IL-2 reconstituted the proliferative responsiveness of lymphocytes to Df in the presence of curcumin. Furthermore, curcumin inhibited IL-5, GM-CSF, and IL-4 production in a concentration-dependent manner. These results indicate that curcumin may have a potential effect on controlling allergic diseases through inhibiting the production of cytokines affecting eosinophil function and IgE synthesis.

Role of mitogen-activated protein kinases in influenza virus induction of prostaglandin E2 from arachidonic acid in bronchial epithelial cells

Background Influenza virus (IV) infection causes airway inflammation; however, it has not been determined whether IV infection could catabolize arachidonic acid cascade in airway epithelial cells. In addition, the responsible intracellular signalling molecules that catabolize arachidonic acid cascade have not been determined.

Apoptosis signal-regulating kinase 1-mediated signaling pathway regulates hydrogen peroxide-induced apoptosis in human pulmonary vascular endothelial cells

ObjectiveReactive oxygen species initiate pulmonary vascular endothelial cell damage leading to an increase in endothelial permeability resulting in the production of pulmonary edema. Apoptosis signal-regulating kinase (ASK)-1 is a ubiquitously expressed mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the MKK3/MKK6-p38 MAPK and the SEK1-c-Jun N-terminal kinase (JNK) signaling cascade. ASK1 has been implicated in cytokine- and stress-induced apoptosis. However, little is known about the role of ASK1 in apoptosis in hydrogen peroxide (H2O2)-stimulated pulmonary vascular endothelial cells and how ASK1-mediated apoptosis is executed. To clarify this issue, we examined the role of ASK1-p38 MAPK/JNK cascade in apoptosis and caspase-3 activation in H2O2-stimulated pulmonary vascular endothelial cells. DesignExperimental laboratory study. SettingUniversity laboratory. SubjectsNormal human pulmonary artery endothelial cells. InterventionsWestern blot analysis and quantification of apoptosis in cells. Measurements and Main ResultsThe results showed that H2O2 induced ASK1 phosphorylation and concomitantly p38 MAPK and JNK phosphorylation as well as induced caspase-3 activation in pulmonary vascular endothelial cells. To further characterize the role of ASK1 cascade in H2O2-induced apoptosis of pulmonary vascular endothelial cells, the dominant negative form of ASK1-stably transfected porcine artery endothelial cells was used. p38 MAPK and JNK phosphorylation, caspase-3 activation, and apoptosis in the dominant negative form of ASK1-stably transfected porcine artery endothelial cells were depressed compared with those in the parental porcine artery endothelial cells. ConclusionASK1-p38 MAPK/JNK cascade regulates apoptosis of H2O2-stimulated human pulmonary vascular endothelial cells.