Tadayoshi Kawata

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.

Toll-like receptor 4 surface expression on human monocytes and B cells is modulated by IL-2 and IL-4

Human Toll-like receptor 4 (TLR4) has recently been identified, and it has been shown to be the main protein involved in recognizing gram-negative bacteria. We examined the regulation of TLR4 surface expression in human peripheral blood monocytes and B cells by interleukin-2 (IL-2) and IL-4. IL-2 up-regulated TLR4 surface expression on human peripheral blood monocytes, but did not change expression on human peripheral B cells. By contrast, IL-4 down-regulated TLR4 surface expression on human peripheral blood monocytes, but up-regulated TLR4 surface expression on human peripheral B cells. These results indicate that Th1 cytokine IL-2 enhances receptors involved in the response to gram-negative bacteria and that activation of cellular immunity may enhance defense against these pathogens through monocytes, but not B cells, whereas Th2 cytokine IL-4 modulates the receptor response to gram-negative bacteria and that activation of humoral immunity may enhance defense against these pathogens through B cells, but not monocytes.

Sensitized Mast Cells Migrate Toward the Agen: A Response Regulated by p38 Mitogen-Activated Protein Kinase and Rho-Associated Coiled-Coil-Forming Protein Kinase

Although mast cells accumulate within the mucosal epithelial layer of patients with allergic rhinitis and bronchial asthma, the responsible chemotactic factors are undefined. We investigated whether mast cells sensitized with Ag-specific IgE migrate toward the Ag. MC/9 mast cells sensitized with anti-DNP IgE migrated toward DNP-conjugated human serum albumin. This migration was directional, and the degree was stronger than that induced by stem cell factor. IL-3 and stem cell factor-dependent cultured mast cells derived from mouse bone marrow also migrated toward the Ag. Subsequent migration mediated by the FcεRI was significantly inhibited by incubating the cells with Y-27632, a Rho-associated coiled-coil-forming protein kinase inhibitor, or with SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor. Both p38 MAPK and MAPK-activated protein kinase (MAPKAPK)2 were activated following FcεRI aggregation, and activation of MAPKAPK2 was almost completely inhibited by 10μM SB203580. Wortmannin or a low concentration of SB203580 partially inhibited MAPKAPK2, but did not block mast cell migration. In contrast, Y-27632 did not affect the activation of MAPKAPK2. These results indicate that Ag works not only as a stimulant for allergic mediators from IgE-sensitized mast cells, but also as a chemotactic factor for mast cells. Both p38 MAPK activation and Rho-dependent activation of Rho-associated coiled-coil-forming protein kinase may be required for FcεRI-mediated cell migration.

Atrial natriuretic peptide inhibits tumor necrosis factor-alpha production by interferon-gamma-activated macrophages via suppression of p38 mitogen-activated protein kinase and nuclear factor-kappa B activation.

We investigated whether the atrial natriuretic peptide (ANP) might have an inhibitory effect on inflammatory cells. Treatment of RAW264.7 macrophages with interferon-gamma (IFN- gamma) caused a significant increase in tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) production. Activation of p38 mitogen-activated protein (MAP) kinase was observed 30 to 120 min after IFN-gamma, and transcription factor nuclear factor-kappa B (NF-kappaB) was activated about 7 to 9 times of the basal activity. Human ANP(99-126) and a specific p38 MAP kinase inhibitor SB203580 inhibited the IFN-gamma-induced TNF-alpha production in a dose-dependent manner without affecting NO production. ANP inhibited the IFN-gamma-induced p38 MAP kinase activation, and ANP and SB203580 inhibited NF-kappaB activation. To study the involvement of oxidative stress in this system, the effects of allopurinol and acetovanillone, inhibitors of xanthine oxidase and NADPH oxidase, respectively, were studied. Allopurinol or acetovanillone did not inhibit the IFN-gamma-induced production of TNF-alpha or NO, suggesting little involvement of oxidative stress in this system. This is the first evidence in vitro that ANP has an anti-inflammatory activity on IFN-gamma-activated macrophages by suppressing signal transduction pathway leading to p38 MAP kinase and NF-kappaB activation.

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.

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.

Glutathione redox regulates TGF-β-induced fibrogenic effects through Smad3 activation

Transforming growth factor‐β (TGF‐β) plays a pivotal role in the fibrogenic action involved in the induction of connective tissue growth factor (CTGF), extracellular matrix and fibroblast transformation. Smad3 mediates TGF‐β signaling related to the fibrotic response. In human lung fibroblasts or bronchial smooth muscle cells, we demonstrated that an increase in the intracellular glutathione level suppressed TGF‐β1‐induced phosphorylation of Smad3, while inhibiting TGF‐β1‐induced expressions of CTGF, collagen type1, fibronectin and transformation into myofibroblasts, which are characterized by the expression of α‐smooth muscle actin. These data indicate that the intracellular glutathione redox status regulates TGF‐β‐induced fibrogenic effects through Smad3 activation.

JNK1 and JNK2 differently regulate IL-12 production in THP-1 macrophage cells

Macrophages play a key role in initiating the innate responses to infection by secreting cytokines such as interleukin-12 (IL-12). This study defined the distinct regulation of lipopolysaccharide (LPS)-mediated IL-12 production by c-jun NH(2)-terminal kinase (JNK)1 and JNK2 isoforms in human macrophages. Knockdown of JNK1 and JNK2 by small interference RNA (siRNA) reduced and enhanced LPS-induced IL-12 p40 production in THP-1 macrophage cells, respectively. The simultaneous knockdown of JNK1 and JNK2 augmented LPS-induced IL-12 production as well as a specific JNK inhibitor. In addition, transfection of siRNA against phosphoinositide 3-kinase (PI3K) p110beta attenuated LPS-induced IL-12 production and JNK1 phosphorylation, while not affecting JNK2 phosphorylation. These findings indicate that JNK1- and JNK2-mediated signaling plays a positive and a negative role, respectively, in LPS-induced IL-12 production and PI3K p110beta controls LPS-induced JNK1 activation, not JNK2 activation, resulting in the positive regulation of IL-12 production in THP-1 macrophage cells.

Phase I study of oral S-1 plus Cisplatin with concurrent radiotherapy for locally advanced non-small-cell lung cancer.

PURPOSE To determine the maximum tolerated dose (MTD) and recommended dose (RD) of S-1 in combination with cisplatin and thoracic radiotherapy in patients with unresectable Stage III non-small-cell lung cancer (NSCLC). METHODS AND MATERIALS S-1 was administered orally twice daily for 14 days and cisplatin on Days 1 and 8 of each cycle; this was repeated every 3 weeks. Doses of each drug were planned as follows: level 0, 50/40; level 1, 60/40; level 2, 70/40; level 3, 80/40 (S-1 [mg/m(-2)/day(-1)]/cisplatin [mg/m(-2)/day(-1)]). Thoracic radiation therapy was administered in 2 Gy fractions five times weekly to a total dose of 60 Gy. RESULTS Ten patients were enrolled in this study. All patients received 60 Gy of thoracic radiotherapy and 7 (70%) patients received four cycles of chemotherapy. At level 1, 2 of 3 patients experienced a delay exceeding 10 days in the cisplatin administration of Day 29. Grade 4 neutropenia and Grade 3 fever occurred in 1 and 1 patients, respectively. Nonhematologic toxicities were mild. None developed >or=Grade 3 esophagitis or lung toxicity. At level 0, 2 of 7 patients developed dose-limiting toxicity. Thus, level 1 was considered the MTD and Level 0 was selected as the RD. Objective responses were seen in all patients. CONCLUSIONS The RD is the level 0 dose, and this regimen is a feasible and well-tolerated regimen for the treatment of patients with Stage III NSCLC.