Akira Ishikawa

Interferon-γ Induces Retinoic Acid–Inducible Gene-I in Endothelial Cells

Interferon-γ (IFN-γ) induces expression of multiple genes in endothelial cells. Retinoic acid–inducible gene-I (RIG-I) encodes a protein belonging to the DExH-box family, but details of its physiological function are not clear. RIG-I is induced in leukemia cells by retinoic acid and in endothelial cells by lipopolysaccharide. In the present study, the authors found that IFN-γ also induces the expression of RIG-I in human umbilical vein endothelial cells. Induction of RIG-I mRNA by IFN-γ was not altered by the treatment with cycloheximide or interleukin-4. Fluorescent immunostaining and Western blot analysis revealed cytoplasmic distribution of RIG-I. The in situ endothelium in a normal lung tissue was also found to express RIG-I protein. Although the physiological function of RIG-I is still unknown, induction of RIG-I by IFN-γ may play an important role in inflammatory or immunological reactions in endothelial cells.

Double-stranded RNA enhances the expression of galectin-9 in vascular endothelial cells

Treatment of cells with double‐stranded RNA (dsRNA) in vitro mimics viral infection and regulates expression of various genes. We addressed the mechanisms of leucocyte traffic across the vascular endothelium induced by dsRNA. The present study focused on the expression of galectin‐9, which is one of key molecules in the regulation of the interaction between vascular wall and white blood cells. Human umbilical vein endothelial cells (HUVEC) in culture were treated with polyinosinic‐polycytidylic acid (poly IC), and expression of mRNA and protein of galectin‐9 was analysed by reverse transcription polymerase‐chain reaction (RT‐PCR) and Western blotting. Poly IC enhanced the expression of galectin‐9 mRNA and protein in concentration‐ and time‐dependent manners. This effect of poly IC was almost completely suppressed by the pretreatment with 2‐aminopurine, an inhibitor of dsRNA‐dependent kinase. Poly IC treatment of HUVEC also enhanced the adherence of EoL‐1 cells to the cells, which was inhibited by co‐treatment with lactose. We conclude that poly IC upregulates galectin‐9 expression in the vascular endothelium and this may explain part of the mechanism for leucocyte traffic through the vascular wall.

Expression of IP-10/CXCL10 Is Upregulated by Double-Stranded RNA in BEAS-2B Bronchial Epithelial Cells

Background: Interferon (IFN)-γ-inducible protein of 10 kDa (IP-10/CXCL10) is a potent chemoattractant for activated T and NK cells, and elevated levels of IP-10 are identified in bronchoalveolar lavage fluids from patients with pulmonary disorders related to Th-1-type immunity, which is a prerequisite for elimination of viral pathogens. Bronchial epithelial cells play an important role in respiratory infections as the initiator of airway inflammation by releasing chemokines and expressing cell surface membrane molecules involved in leukocyte adhesion. Polyinosinic-polycytidylic acid (poly IC) is a synthetic double-stranded RNA (dsRNA) and induces antiviral reactions in cells. Objectives: We investigated the regulation of IP-10 in BEAS-2B bronchial epithelial cells in response to poly IC, and also addressed the possible role of retinoic-acid-inducible gene-I (RIG-I) and IFN-regulatory factor 3 (IRF-3), two genes involved in the signaling induced by viral infection. Methods: The expressions of IP-10 mRNA and protein were analyzed by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. The overexpression of RIG-I or IRF-3 was performed by transfection of BEAS-2B cells with each cDNA. Results: Poly IC enhanced the expression of IP-10 mRNA and protein in concentration- and time-dependent manners. Overexpression of RIG-I or IRF-3 potentiated the poly-IC-induced upregulation of IP-10. Conclusions: IP-10 may contribute to antiviral activity through the activation of Th-1-type immunity, and RIG-I and IRF-3 may be involved in this reaction.

Double-Stranded RNA Induces the Synthesis of Retinoic Acid–Inducible Gene-I in Vascular Endothelial Cells

Viral infection induces various responses in vascular endothelial cells. Polyinosinic-polycytidylic acid (poly IC) is a synthetic double-stranded RNA (dsRNA), and treatment of cells with poly IC mimics the viral infection to the cells. Retinoic acid-inducible gene-I (RIG-I) is a protein belonging to the DExH-box family and designated as a putative RNA helicase. RIG-I is considered to play a role in antiviral responses through the regulation of gene expressions. In the present study, the authors treated human umbilical vein endothelial cells (HUVECs) with poly IC and found that poly IC induced the expression of RIG-I. The poly IC-induced RIG-I expression was inhibited by the preincubation of the cells with 2-aminopurine, an inhibitor of dsRNA-dependent protein kinase (PKR). Immunohistochemical examination revealed high levels of RIG-I immunoreactivity in vascular endothelial cells in the thalamus from rats inoculated with hantavirus. Induction of RIG-I by poly IC may be involved in the antiviral responses in endothelial cells.

Polyinosinic-polycytidylic acid induces the expression of GRO-α in BEAS-2B cells

Growth-related oncogene protein-α (GRO-α)/CXCLl is a chemokine that activates neutrophils and plays an important role in inflammatory reactions. Polyinosinic-polycytidylic acid (poly IC) is a synthetic double-stranded RNA (dsRNA), which is a ligand for Toll-like receptor-3. Poly IC mimics viral infection when applied to cells and induces inflammatory and immune responses. In the present study, we found the induction of GRO-α in BEAS-2B bronchial epithelial cells treated with poly IC. Pretreatment of cells with 2-aminopurine, an inhibitor for dsRNA-dependent protein kinase (PKR), inhibited the expression of GRO-α-induced by poly IC. Overexpression of interferon-regulatory factor-3 (IRF-3) or retinoic-acid inducible gene-I (RIG-I) enhanced the induction of GRO-α by poly IC. PKR, IRF-3, and RIG-I may be involved in the poly IC-induced expression of GRO-α in BEAS-2B cells. Airway viral infection may elicit GRO-α expression in the bronchial epithelium, which may be implicated in inflammatory and immune reactions.

Effect of Double-Stranded RNA on the Expression of Epithelial Neutrophil Activating Peptide-78/CXCL-5 in Human Endothelial Cells

Epithelial neutophil activating peptide-78 (ENA-78)/CXCL-5 is a member of CXC chemokines. ENA-78 was originally described as a factor produced by epithelial cells only. But other types of cells including vascular endothelial cells also produce it. ENA-78 production by endothelial cells may be important for the regulation of neutrophil activation in inflammatory reactions. Polyinosinic-polycytidylic acid (poly IC) is a synthetic double-stranded RNA, which mimics the viral infection when applied to cells and affects the expression of various genes related to inflammatory reactions. In the present study, we examined the effect of poly IC on the expression of ENA-78 in human umbilical vein endothelial cells (HUVEC). HUVEC in culture were treated with poly IC and the expression of ENA-78 mRNA and protein were analyzed by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Poly IC induced ENA-78 expression in time- and concentration-dependent manners. Th2-type cytokine IL-4 partially inhibited the induction of ENA-78 by poly IC. 2-Aminopurine, an inhibitor of dsRNA-dependent kinase, suppressed the induction of ENA-78 by poly IC. ENA-78 may be involved in the inflammatory reactions elicited by viral infection in endothelial cells.

DOUBLE-STRANDED RNA STIMULATES THE EXPRESSION OF MONOCYTE CHEMOATTRACTANT PROTEIN-1 IN BEAS-2B BRONCHIAL EPITHELIAL CELLS

BEAS-2B bronchial epithelial cells were treated with polyinosinic-polycytidylic acid (poly IC), a synthetic double-stranded RNA (dsRNA) analog, and the expressions of monocyte chemoattractant protein-1 (MCP-1) mRNA and protein were analyzed by reverse transcriptase–polymerase chain reaction and enzyme-linked immunosorbent assay. Poly IC enhanced the expression of MCP-1 and release of mononuclear cell chemotactic activity, which were inhibited by dexamethasone pretreatment. The poly IC–induced up-regulation of MCP-1 was blocked by 2-aminopurine, a specific inhibitor of dsRNA-dependent protein kinase, but not by nuclear factor (NF)-κB inhibitor SN50.