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Maiko Kadowaki,1,* Koichi Sato,2,* Hisashi Kamio,3 Makoto Kumagai,3 Rikishi Sato,3 Takafumi Nyui,3 Yukihiro Umeda,1 Yuko Waseda,1 Masaki Anzai,1 Haruka Aoki-Saito,4 Yasuhiko Koga,4 Takeshi Hisada,5 Hideaki Tomura,6 Fumikazu Okajima,3 Tamotsu Ishizuka1 1Third Department of Internal Medicine, Faculty of Medical Sciences, University of Fukui, Fukui, 910-1193, Japan; 2Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebeshi, 371-8512, Japan; 3Laboratory of Signal Transduction, Faculty of Pharmaceutical Sciences, Aomori University, Aomori, 030-0943, Japan; 4Department of Respiratory Medicine, Gunma University Graduate School of Medicine, Maebeshi, 371-8511, Japan; 5Gunma University Graduate School of Health Sciences, Maebeshi, 371-8514, Japan; 6Laboratory of Cell Signaling Regulation, Division of Life Science, School of Agriculture, Meiji University, Kawasaki, 214-8571, Japan*These authors contributed equally to this workCorrespondence: Tamotsu IshizukaThird Department of Internal Medicine, Faculty of Medical Sciences, University of Fukui, 23-3 Matsuoka-Shimoaizuki, Eiheiji, Fukui, 910-1193, JapanTel +81-776-61-8355Fax +81-776-61-8111Email tamotsui@u-fukui.ac.jpPurpose: Human bronchial smooth muscle cells (BSMCs) contribute to airway obstruction and hyperresponsiveness in patients with bronchial asthma. BSMCs also generate cytokines and matricellular proteins in response to extracellular acidification through the ovarian cancer G protein-coupled receptor 1 (OGR1). Cobalt (Co) and nickel (Ni) are occupational agents, which cause occupational asthma. We examined the effects of Co and Ni on interleukin-6 (IL-6) secretion by human BSMCs because these metals may act as ligands of OGR1.Methods: Human BSMCs were incubated in Dulbecco’s Modified Eagle Medium (DMEM) containing 0.1% bovine serum albumin (BSA) (0.1% BSA-DMEM) for 16 hours and stimulated for the indicated time by exchanging the medium with 0.1% BSA-DMEM containing any of the metals or pH-adjusted 0.1% BSA-DMEM. IL-6 mRNA expression was quantified via reverse transcription polymerase chain reaction (RT-PCR) using the real-time TaqMan technology. IL-6 was measured using an enzyme-linked immunosorbent assay. Dexamethasone (DEX) was added 30 minutes before each stimulation. To knock down the expression of OGR1 in BSMCs, small interfering RNA (siRNA) targeting OGR1 (OGR1-siRNA) was transfected to the cells and non-targeting siRNA (NT-siRNA) was used as a control.Results: Co and Ni both significantly increased IL-6 secretion in human BSMCs at 300 μM. This significant increase in IL-6 mRNA expression was observed 5 hours after stimulation. BSMCs transfected with OGR1-siRNA produced less IL-6 than BSMCs transfected with NT-siRNA in response to either Co or Ni stimulation. DEX inhibited Co- and Ni-stimulated IL-6 secretion by human BSMCs as well as pH 6.3-stimulated IL-6 secretion in a dose-dependent manner. DEX did not decrease phosphorylation of ERK1/2, p38 MAP kinase, and NF-κB p65 induced by either Co or Ni stimulation.Conclusion: Co and Ni induce secretion of IL-6 in human BSMCs through activation of OGR1. Co- and Ni-stimulated IL-6 secretion is inhibited by DEX.Keywords: cobalt, nickel, proton, cytokine, glucocorticoid, asthma

Tamotsu Ishizuka,1,2 Takeshi Hisada,2 Yosuke Kamide,2 Haruka Aoki,2 Kaori Seki,2 Chisato Honjo,1 Hiroyuki Sakai,1 Maiko Kadowaki,1 Yukihiro Umeda,1 Miwa Morikawa,1 Masaki Anzai,1 Shingo Ameshima,1 Takeshi Ishizaki,1 Kunio Dobashi,2 Masanobu Yamada,2 Motoyasu Kusano3 1Third Department of Internal Medicine, Faculty of Medical Sciences, University of Fukui, Fukui, 2Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, 3Department of Endoscopy and Endoscopic Surgery, Gunma University Hospital, Maebashi, Japan Background: Losing the sense of smell, which suggests eosinophilic rhinosinusitis, is a subjective symptom, sometimes reported in asthmatic patients taking controller medication. Upper abdominal symptoms, suggesting gastroesophageal reflux disease (GERD) or functional dyspepsia, occur also in these patients. However, the relationship between these symptoms, concomitant with asthma, and the intensity of eosinophilic airway inflammation remains obscure. Objective: To assess the symptoms of asthma and rhinosinusitis, and to examine the relationship between the symptoms and bronchial inflammation, a new questionnaire, the G scale, was developed. To investigate the effects of GERD, dyspepsia, and rhinosinusitis on asthma symptoms and bronchial inflammation, the symptoms of asthma and rhinosinusitis obtained by the G scale, upper abdominal symptoms obtained by the modified F scale, a questionnaire for GERD and dyspepsia, and fractional exhaled nitric oxide (FeNO) were analyzed. Methods: A prospective, observational study was performed in four hospitals in Gunma prefecture, and a retrospective analysis was done using data obtained from five hospitals in Gunma prefecture and Fukui prefecture, Japan. A total of 252 patients diagnosed as having asthma participated in the prospective study. Results: The frequency of daytime phlegm or losing the sense of smell had a positive correlation with FeNO levels in asthmatic patients taking controller medication. Upper abdominal symptoms, as well as symptoms suggesting rhinitis, were well correlated with asthma symptoms. However, neither upper abdominal symptoms nor rhinitis symptoms increased FeNO levels, which reflect eosinophilic airway inflammation during treatment for asthma. On the other hand, the degree of upper abdominal symptoms or dyspepsia symptoms had a weak but significant negative correlation with FeNO levels. Conclusion: Daytime phlegm and losing the sense of smell suggest that eosinophilic airway inflammation persists, despite anti-inflammatory therapy, in patients with asthma. Although rhinitis and GERD made the subjective symptoms of asthma worse, they did not seem to enhance eosinophilic airway inflammation. Keywords: asthma symptoms, FeNO, rhinosinusitis, GERD, dyspepsia