Hiroko Takeuchi

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.

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.

Vascular endothelial growth factor expression by activated synovial leukocytes in rheumatoid arthritis: critical involvement of the interaction with synovial fibroblasts.

OBJECTIVE To examine the expression and regulation of the angiogenic factor, vascular endothelial growth factor (VEGF), by fibroblast-like synoviocytes (FLS), monocytes, and polymorphonuclear neutrophils (PMNs) isolated from the synovial fluid (SF) of rheumatoid arthritis (RA) patients. METHODS Monocytes or PMNs obtained from RA SF were cocultured with unstimulated, semiconfluent RA FLS. Culture supernatants were assayed for the proliferation and in vitro tube formation of endothelial cells, and for the production of VEGF, by enzyme-linked immunosorbent assay. The expression of VEGF messenger RNA and protein was also determined by reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. RESULTS We found that the interaction of inflammatory, activated leukocytes with FLS resulted in synergistic increases in VEGF expression and secretion, which contributed to the proliferation of endothelial cells and to in vitro endothelial tube formation. The induction of VEGF was mediated via specific adhesion molecules, as indicated by the finding that anti-integrin antibodies significantly inhibited VEGF. Furthermore, the levels of VEGF secretion correlated with the expression of cell surface integrin (CD11b and CD18) on both monocytes and PMNs in the SF. CONCLUSION VEGF expression within inflamed joints thus appears to be regulated not only by inflammatory cytokines, but also by the physical interaction of activated leukocytes and FLS. Once expressed, VEGF likely plays a crucial role in the neovascularization of the pannus and the progressive joint destruction associated with the synovial inflammation of RA.

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.

Reactions of direct LDL-cholesterol assays with pure LDL fraction and IDL: comparison of three homogeneous methods

According to the definition of the Lipid Research Clinics protocol, low-density lipoprotein (LDL) refers to the lipoprotein of density (d)=1.006-1.063 g/ml which contains another atherogenic lipoprotein, IDL (d=1.006-1.019 g/ml). Because metabolic properties are largely different between LDL and IDL, LDL is now defined as the lipoprotein of d=1.019-1.063 g/ml. Recently direct LDL-cholesterol assay kits using novel surfactants (the homogeneous methods) have become commercially available and widely used in Japan. The aim of this study is to examine how three direct LDL-cholesterol assay kits, LDL-EX, Choletest-LDL and Determinor-L LDL, react with pure LDL (d=1. 019-1.063 g/ml) and IDL (1.006-1.019 g/ml) fractions isolated by ultracentrifugation. Thirty-one healthy subjects and one type III dysbetalipoproteinemic patient were enrolled in this study. All homogeneous methods highly correlated with LDL-cholesterol (r=0.95-0. 98), although the values for LDL-EX were closer to the values for ultracentrifugation than were those of the other two methods (95 vs. 86-87%, P<0.0001). Cross-reactivity with IDL was 31, 47 and 64% for LDL-EX, Choletest-LDL, and Determinor-L LDL, respectively. Similar results were obtained in the IDL from a type III dysbetalipoproteinemic patient. These results suggest that LDL-cholesterol measured by LDL-EX better reflects pure LDL fraction with weaker cross-reaction with IDL than other homogeneous methods.

Macrophage inflammatory protein 1 alpha expression by synovial fluid neutrophils in rheumatoid arthritis

OBJECTIVE To determine the contribution made by synovial fluid (SF) neutrophils to the augmented expression of macrophage inflammatory protein 1 α (MIP-1α) in rheumatoid arthritis (RA). METHODS Neutrophils were isolated from samples of SF from RA patients and peripheral blood (PB) samples from RA patients and healthy controls. Cell associated MIP-1α was visualised immunohistochemically, and cell associated MIP-1α as well as MIP-1α secreted into the SF was assayed by ELISA. Steady state expression of MIP-1α mRNA was assessed by reverse transcription polymerase chain reaction (RT-PCR). RESULTS Freshly isolated SF neutrophils contained significantly higher concentrations of both MIP-1α protein and its transcript than PB neutrophils from either RA patients or healthy controls; incubation in the absence or presence of tumour necrosis factor α for 24 hours resulted in a significant increase in MIP-1α secretion by RA SF neutrophils compared with neutrophils obtained from either normal PB or RA PB; and expression of MIP-1α by SF neutrophils was well correlated with both RA disease activity and SF mononuclear cell (MNC) counts. CONCLUSION Expression and secretion of MIP-1α by SF neutrophils may be indicative of local and systemic inflammation in RA. Moreover, this C-C chemokine may contribute to the recruitment of MNCs from the bloodstream into synovial joints and tissues.

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.