Satoshi Matsukura

Expression of IL-6, IL-8, and RANTES on human bronchial epithelial cells, NCI-H292, induced by influenza virus A

Bronchial epithelial cells are primary sites of airway viral infection, and these cells may play an important role in the pathogenesis of respiratory diseases. It has recently been reported that bronchial epithelial cells express RANTES. RANTES attracts monocytes, T cells, eosinophils, and basophils; it can also activate eosinophils. To determine whether viral infection induces RANTES expression on bronchial epithelial cells, we infected a bronchial epithelial cell line, NCI-H292, with influenza virus A (H3N2). We then examined the concentration of RANTES in the culture medium of infected cells by ELISA and assessed expression of the gene for RANTES by the reverse-transcriptase polymerase chain reaction. We also investigated the concentrations of IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor in the medium of infected cells, because some virus infections have been reported to induce expression of these cytokines on bronchial epithelial cells, but there are few data concerning influenza virus infection. Small amounts of IL-6 and IL-8 were detected in the medium of uninfected cells. RANTES was not detected in the medium of uninfected cells. After influenza virus infection, significant amounts of IL-6, IL-8, and RANTES were released into the culture medium of infected cells, and RANTES messenger RNA was detected from infected cells. Granulocyte-macrophage colony-stimulating factor was not detected in the medium of uninfected and infected cells. These results suggest that influenza virus infection may stimulate production of IL-6, IL-8, and RANTES from human bronchial epithelial cells and that these cytokines may contribute to the pathogenesis of airway inflammatory diseases caused by influenza virus infection.

Synthetic double-stranded RNA induces multiple genes related to inflammation through Toll-like receptor 3 depending on NF-κB and/or IRF-3 in airway epithelial cells

Background We hypothesized that synthetic double‐stranded (ds)RNA may mimic viral infection and induce expression of genes related to inflammation in airway epithelial cells.

Expression of Cytokines on Human Bronchial Epithelial Cells Induced by Influenza Virus A

Bronchial epithelial cells play an important role in the pathogenesis of some inflammatory diseases of bronchial mucosa. Epithelial-cell-derived cytokines are important in the elucidation of the mechanism by which airway inflammation occurs, especially in respiratory virus infection, because these cells are the primary sites of viral infection. We infected bronchial epithelial cells, NCI-H292, with influenza virus A (H3N2) and examined the concentrations of cytokines, interleukin-6 (IL-6), IL-8 and regulated on activation, normal T cells, expressed and secreted (RANTES), in the culture media of infected cells using the enzyme-linked immunosorbent assay system and gene expression of RANTES on epithelial cells by the reverse-transcriptase-polymerase chain reaction method. We found that significant amounts of IL-6, IL-8 and RANTES were released. RANTES mRNA was also detected in infected bronchial epithelial cells. It is suggested that cytokine production in human bronchial epithelial cells may contribute to the pathogenesis of airway inflammatory disorders.

Functional Characterization of IL-17F as a Selective Neutrophil Attractant in Psoriasis

IL-17F is known to be involved in many inflammatory diseases, but its role in skin diseases has not been fully examined. Because IL-8 is involved in many skin diseases such as psoriasis, we investigated the production of IL-8 in normal human epidermal keratinocytes (NHEKs) stimulated by IL-17F, tumor necrosis factor-alpha (TNF-alpha), IL-17A, and control using real-time PCR and ELISA. The results showed that IL-17F induced production of IL-8 in NHEKs in a time-dependent manner. Interestingly, the amounts of IL-8 stimulated by IL-17F were much higher than those stimulated by TNF-alpha or IL-17A. Next, we confirmed that selective mitogen-activated protein kinase kinase inhibitors significantly inhibited IL-17F-induced IL-8 production. Moreover, mouse skin intradermally injected with IL-17F expressed high level of IL-8 mRNA and induced ERK1/2 phosphorylation. Histological examination of mouse skin that was injected with IL-17F revealed marked neutrophilia in dermis and the infiltration was significantly inhibited by anti-IL-8 antibody. Finally, IL-17F expression in skin biopsy samples from psoriasis patients were examined by western blotting and ELISA. IL-17F was upregulated in lesional psoriatic skin compared with nonlesional skin. These results indicate that IL-17F may be involved in psoriasis via, in part, the activation of ERK1/2 and the induction of IL-8 in keratinocytes.

Role of RIG-I, MDA-5, and PKR on the Expression of Inflammatory Chemokines Induced by Synthetic dsRNA in Airway Epithelial Cells

Background: We hypothesized that synthetic double-stranded (ds)RNA may mimic viral infection and reported that dsRNA stimulates expression of inflammatory chemokines through a receptor of dsRNA Toll-like receptor (TLR) 3 in airway epithelial cells. In this study, we focused our study on the role of other receptors for dsRNA, such as retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA-5), and double-stranded RNA-dependent protein kinase (PKR). Methods: Airway epithelial cell BEAS-2B was cultured in vitro. Expression of target RNA and protein were analyzed by PCR and ELISA. To analyze the role of receptors for dsRNA, knockdown of theses genes was performed with short interfering RNA (siRNA). Results: We first investigated the effects of chloroquine, an inhibitor of lysosomal acidification, on the expression of chemokines. Preincubation with 100 µM chloroquine significantly inhibited the expression of mRNA for RANTES, IP-10, and IL-8, stimulated by poly I:C, indicating that poly I:C may react with a receptor expressed inside the cells. RIG-I, MDA-5, and PKR are supposed to be expressed inside the airway epithelial cells. However, the expression of chemokines stimulated with poly I:C was not significantly inhibited for these putative receptors in the cells which were transfected with siRNA. Conclusions: Synthetic dsRNA poly I:C stimulates the expression of inflammatory chemokines in airway epithelial cells, but the putative receptors for dsRNA such as RIG-I, MDA-5, or PKR may not play pivotal roles in this process. TLR3 may play a major role as reported previously.

Double‐stranded RNA activates RANTES gene transcription through co‐operation of nuclear factor‐κB and interferon regulatory factors in human airway epithelial cells

Background Regulated on activation, normal T cells expressed and secreted (RANTES) is a member of the CC chemokine family and contributes to viral‐induced airway inflammation including exacerbations of asthma. Double‐stranded RNA (dsRNA) is known to be synthesized during replication of many viruses and a ligand of Toll‐like receptor 3. We hypothesized that dsRNA may mimic viral infection and induce RANTES expression in airway epithelial cells.

Expression of Interleukin-17F in a Mouse Model of Allergic Asthma

Background: Interleukin (IL)-17F is a recently discovered cytokine and is derived from a panel of limited cell types, such as activated CD4+ T cells, basophils, and mast cells. IL-17F is known to induce several cytokines and chemokines. However, its involvement in airway inflammation has not been well understood. To this end, the expression of IL-17F and the inhibitory effects of glucocorticoids on its expression in a mouse model of asthma were examined. Methods: Five-week-old BALB/c male mice were sensitized by intraperitoneal injection (i.p.) of ovalbumin (OVA) with alum, and challenged by daily inhalation of aerosolized 1% OVA. 24 h after last challenge (OVA/OVA), the expression of IL-17F was examined in lung tissues by immunohistochemistry and reverse-transcription polymerase chain reaction. Control mice were sensitized and challenged with saline (Sham/Sham). In addition, a group of OVA-sensitized mice received i.p. injection of water-soluble dexamethasone (DEX) in saline 1 h before OVA challenge (OVA/DEX). Results: In sham-challenged mice, IL-17F was not expressed in the lungs, while, in contrast, IL-17F was predominantly expressed in bronchial epithelial cells in addition to the infiltrating inflammatory cells in OVA/OVA mice. Further, the expression of IL-17 F was significantly attenuated by the treatment of mice with DEX. Conclusion: These results suggest that bronchial epithelium-derived IL-17F may represent a new pharmacological target for glucocorticoids and may play a role in allergic asthma.

Increase in Reactive Oxygen Metabolite Level in Acute Exacerbations of Asthma

Background: Oxidants including reactive oxygen species have been indicated to play an important role in the pathogenesis of asthma. Objective: We investigated oxidative status in patients with acute exacerbations of asthma and evaluated the therapeutic response using the D-ROM test which is simple to use and quick. Methods: We measured reactive oxygen metabolite (ROM) levels in the serum of 42 outpatients with acute exacerbations of asthma, 11 outpatients with stable asthma and 40 healthy subjects using the D-ROM test. Seven inpatients admitted due to acute exacerbations of asthma were also enrolled to evaluate the effects of treatment. Serum eosinophil cationic protein and plasma polymorphonuclear elastase were also measured by EIA or ELISA to evaluate the correlation between inflammation and oxidative status. Results: Serum ROM levels were significantly higher in patients with acute exacerbation of asthma than in patients with stable asthma or healthy subjects. Levels of serum eosinophil cationic protein and plasma polymorphonuclear elastase were increased in acute exacerbation and moderately correlated to ROM levels. Levels of ROM were significantly decreased after treatment with systemic steroids and bronchodilators. Conclusion: These findings suggest that acute exacerbation of asthma is associated with increased oxidative stress. Serum ROM levels would partly reflect the inflammation with eosinophils and neutrophils and may be useful as biomarkers of asthma.

Expression and effects of IL-33 and ST2 in allergic bronchial asthma: IL-33 induces eotaxin production in lung fibroblasts.

Background: Interleukin (IL)-33, a new member of the IL-1 cytokine family, has been recognized as a key cytokine that enhances T helper 2-balanced immune regulation through its receptor ST2; however, the function and relationship of the IL-33 and ST2 pathways in bronchial asthma are still unclear. We investigated the cellular origin and regulation of IL-33 and ST2 in allergic bronchial asthma in vivo and in vitro. Methods: BALB/c mice were sensitized by intraperitoneal injections of ovalbumin (OVA) with alum. Mice were exposed to aerosolized 1% OVA for 30 min a day for 7 days. These mice were then challenged with aerosolized 1% OVA 2 days after the last day of exposure. After the OVA challenge, the mice were sacrificed and their lung tissues were obtained. Mouse lung fibroblasts were cultured and treated with IL-33 or IL-13. Results: The levels of IL-33 mRNA and IL-33 protein in lung tissue increased after the OVA challenge. Most IL-33-expressing cells were CD11c+ cells and epithelial cells, and many ST2-expressing cells were stained lung fibroblasts and inflammatory cells. IL-33 induced eotaxin/CCL11 production in lung fibroblasts. IL-33 and IL-13 synergistically induced eotaxin expression. Conclusions: IL-33 may contribute to the induction and maintenance of eosinophilic inflammation in the airways by acting on lung fibroblasts. IL-33 and ST2 may play important roles in allergic bronchial asthma.

Expression of Eotaxin by Normal Airway Epithelial Cells after Influenza Virus A Infection

Background: Viral infection is known to cause lung inflammatory disease, including bronchial asthma. The mechanisms of inflammatory cell accumulation into the airways after viral infection are not well understood. Eotaxin is a CC chemokine which is a potent and specific agonist for CC chemokine receptor 3 (CCR3). CCR3 is expressed on eosinophils, basophils and T lymphocytes. These cells are known to be key cells in the pathogenesis of asthma. Although it has recently been demonstrated that airway epithelial cells express eotaxin in vivo and in vitro, there are few data about its epxression in viral infection. We hypothesized that eotaxin may play an important role in attracting inflammatory cells to the airways after viral infection, and analyzed whether viral infection attracts eotaxin in bronchial epithelial cells in vitro. Methods: Human airway epithelial cells obtained from bronchial tissue at lobectomy for lung cancer were infected with influenza virus A (subtype H3N2). The cells and cultured media were collected 8, 24, and 48 h after infection. Eotaxin mRNA was analyzed with reverse transcriptase-polymerase chain reaction. Eotaxin protein levels in the culture media were analyzed by enzyme-linked immunosorbent assay. We also studied a blocking assay to analyze the intervention of proinflammatory cytokines in its production induced by influenza virus. Results: Eotaxin mRNA appeared to be expressed constitutively in uninfected cells but was expressed more clearly in infected cells. Eotaxin protein release into culture media significantly increased after infection. Anti-TNF-α and anti-IL-1β antibodies did not alter the eotaxin protein levels after viral infection. Conclusions: These results suggest that influenza virus A infection in airway epithelial cells activates the expression of eotaxin and that eotaxin may participate in the pathogenesis of airway inflammatory disease caused by viral infection, such as infectious type asthma.