Mika Kumagai

Interferon-γ stimulates the expression of galectin-9 in cultured human endothelial cells

Galectin‐9 is a member of the galectin family and has been identified as an eosinophil chemoattractant produced by activated T lymphocytes. Vascular endothelial cells play an important role in the initial step of eosinophil recruitment and activation in immune and inflammatory responses. We have addressed the stimulation of galectin‐9 expression in endothelial cells. Galectin‐9 was detected in membrane and cytosolic fractions of human umbilical vein endothelial cells stimulated with interferon‐γ (IFN‐γ). IFN‐γ also enhanced the adhesion of human eosinophilic leukemia‐1 cells to endothelial monolayers, and it was inhibited by the presence of lactose. Interleukin‐4, which induces eotaxin expression, did not affect the expression of galectin‐9. The in situ endothelium from patients with inflammatory diseases was found to express galectin‐9. IFN‐γ‐induced production of galectin‐9 by endothelial cells may play an important role in immune responses by regulating interactions between the vascular wall and eosinophils.

Synergistic stimulation, by tumor necrosis factor-α and interferon-γ, of fractalkine expression in human astrocytes

Fractalkine is a CX3C chemonkine that appears to be a neuron-to-microglia signal molecule in the central nervous system. We studied the expression of fractalkine in normal human astrocytes in culture, by using semi-quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. We found that tumor-necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma) synergistically enhance the expression of fractalkine. The expression of both fractalkine mRNA and protein was increased in time- and concentration-dependent manners in the cells co-stimulated with TNF-alpha and IFN-gamma. Cycloheximide, an inhibitor of protein synthesis, and dexamethasone had no effect on the synergy of the stimulation of fractalkine expression. We conclude that normal human astrocytes produce fractalkine by co-stimulation with pro-inflammatory cytokines and it may serve as a potential signal for immune and inflammatory responses in the central nervous system.

Interleukin-1beta stimulates galectin-9 expression in human astrocytes.

Galectin-9 is an eosinophil chemoattractant produced by activated T lymphocytes. We have addressed expression of galectin-9 in normal human astrocytes in culture. Expression of galectin-9 mRNA and protein were examined by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescent staining. Interleukin-1β (IL-1β) was found to enhance the galectin-9 expression in time- and concentration-dependent manners. Galectin-9 protein was detected in the membrane fraction, 105 000 ×g precipitate, and immunofluorescent staining revealed diffuse cellular and perinuclear distributions. Dexamethasone pretreatment almost completely suppressed the production. We conclude that astrocytes produce galectin-9 in response to the stimulation with IL-1β, and this may contribute to inflammatory reactions in the CNS.

Proteasome inhibitor MG-132 enhances the expression of interleukin-6 in human umbilical vein endothelial cells: Involvement of MAP/ERK kinase

Interleukin‐6 (IL‐6) is a multifunctional cytokine that plays an important role in inflammatory reactions. We have addressed the possible regulation of IL‐6 expression by the ubiquitin‐protease system in human umbilical vein endothelial cells. Cultured endothelial cells were treated with MG‐132, a protease inhibitor, and the levels of IL‐6 mRNA and protein were measured by reverse transcription‐PCR and ELISA. MG‐132 increased the expression of IL‐6 mRNA and protein; and this effect was abolished by the pretreatment of the cells with U0126, an inhibitor of MAP or ERK kinases (MEK1/2). MG‐132 treatment was also found to enhance the level of phosphorylated MEK1/2. Treatment of the cells with actinomycin D inhibited IL‐6 expression in response to MG‐132, suggesting the transcriptional upregulation of IL‐6 under proteasomal inhibition. We conclude that a protease inhibitor MG‐132 upregulates IL‐6 expression in vascular endothelial cells, at least in part, through the activation of MEK1/2.

Platelet-activating factor enhances the expression of vascular endothelial growth factor in normal human astrocytes

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for vascular endothelial cells. To examine whether platelet-activating factor (PAF) induces the expression of VEGF in human astrocytes, we stimulated cultured normal astrocytes with PAF and performed semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) or real-time quantitative PCR for VEGF mRNA and enzyme-linked immunosorbent assay for VEGF protein. PAF increased the expression of VEGF in astrocytes in time- and dose-dependent manners. After 24-h stimulation, 10 nM PAF increased the levels of VEGF protein in astrocyte-conditioned medium by 1.3-fold. When the cells were subjected to hypoxia, the PAF-induced production of VEGF was enhanced by 6.7-fold as compared to the unstimulated cells incubated under normoxia. Dexamethasone was found to inhibit the enhanced VEGF production in response to the stimulation with PAF under hypoxia. We conclude that PAF induces VEGF gene expression in human astrocytes, and the PAF-induced increase in the expression of VEGF may modulate nervous tissue injury due to hypoxia.

15-Deoxy-D12,14-prostaglandin J2 inhibits CX3CL1/fractalkine expression in human endothelial cells.

Peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) is a member of nuclear hormone receptor superfamily, and is known to play a role in various biological processes including inflammatory responses and adipocyte differentiation. CX3CL1/fractalkine is a potent agonist for chemotaxis and adhesion of monocytes and lymphocytes. Endothelial cells produce fractalkine when stimulated with cytokines such as interleukin‐1 (IL‐1), tumour necrosis factor‐α and interferon‐γ (IFN‐γ). We herein report that 15‐deoxy‐n12,14 ‐prostaglandinJ2 (15d‐PGJ2), a PPAR‐γ agonist, inhibits the expression of fractalkine induced by IFN‐γ or IL‐1β in human endothelial cells. Agonist for PPAR‐α (WY14643) or PPAR‐γ (ciglitazone) did not inhibit the cytokine‐inducedfractalkine expression, and the effect of 15d‐PGJ2 maybe independent of PPAR. 15‐Deoxy‐D12,14 prostaglandin J2 also inhibited the adhesion of blood mononuclear cells to endothelial monolayers treated with IFN‐γ or IL‐1β. The data suggest that 15d‐PGJ2 regulates inflammatory reactions, at least in part, through the inhibition of fractalkine expression and leucocyte traffic through the endothelium.

15-Deoxy-Δ12,14-Prostaglandin J2 Inhibits IFN-γ-Induced Galectin-9 Expression in Cultured Human Umbilical Vein Endothelial Cells

Background: Galectin-9 is involved in chemotaxis and adhesion of eosinophils, and is induced in vascular endothelial cells by interferon-γ (IFN-γ). 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is a ligand for peroxisome proliferator-activated receptor-γ (PPAR-γ), and known to modulate the expression of various genes. Methods: We have studied the effect of 15d-PGJ2 on the IFN-γ-induced galectin-9 expression in human umbilical vein endothelial cells (HUVEC) in culture. Results: 15d-PGJ2 inhibited the IFN-γ-induced galectin-9 expression in a PPAR-γ-independent manner, and also inhibited the adhesion of EoL-1 cells to an HUVEC monolayer treated with IFN-γ. 15d-PGJ2 partially inhibited IFN-γ-induced phosphorylation of STAT-1 in HUVEC. Conclusions: 15d-PGJ2 may regulate inflammatory reactions through the inhibition of galectin-9 expression.

15-Deoxy-Δ12,14-prostaglandin J2 inhibits the expression of granulocyte-macrophage colony-stimulating factor in endothelial cells stimulated with lipopolysaccharide

Granulocyte-macrophage colony-stimulating factor (GM-CSF), one of major hematopoietic growth factors, activates mature leukocytes. GM-CSF is produced by endothelial cells stimulated with lipopolysaccharide (LPS), and the LPS-induced GM-CSF production may play an important role in the activation of neutrophils on the endothelial surface. 15-Deoxy-delta 12,14-prostaglandin J2 (15d-PGJ2) is a ligand for peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and modulates inflammatory reactions by regulating the expression of various genes. We studied the effect of 15d-PGJ2 on the LPS-induced GM-CSF expression in endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultured and the expressions of GM-CSF mRNA and protein were analyzed by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. 15d-PGJ2 inhibited the LPS-induced GM-CSF expression in a concentration-dependent manner; but ciglitazone, another agonist for PPAR-gamma, had no effect. This suggests that 15d-PGJ2 inhibits GM-CSF expression through a mechanism unrelated to PPAR-gamma. 15d-PGJ2 induced, by itself, the expression of interleukin-8, a potent proinflammatory chemokine, in HUVEC. 15d-PGJ2 may regulate inflammatory reactions by controlling the balance of various cytokines.

Effect of 15-deoxy-Δ12,14-prostaglandin J2 on IL-1β-induced expression of epithelial neutrophil-activating protein-78 in human endothelial cells ☆

Epithelial neutrophil-activating peptide-78 (ENA-78) is a member of CXC chemokines. It is produced by endothelial cells stimulated with interleukin-1 (IL-1), along with other CXC chemokines such as IL-8 and growth-related oncogene protein-alpha (GRO-alpha). IL-1-induced ENA-78 production by endothelial cells may be important for the regulation of neutrophil activation. 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a natural ligand for peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and affects the expression of various genes. We examined the effect of 15d-PGJ(2) on the expression of ENA-78 in cultured endothelial cells stimulated with IL-1beta. 15d-PGJ(2) inhibited the IL-1beta-induced expression of ENA-78, but not the expression of IL-8 or GRO-alpha in response to IL-1. Ciglitazone, another agonist for PPAR-gamma, had no effect on the expression of ENA-78, suggesting that 15d-PGJ(2) may inhibit the expression of ENA-78 in a PPAR-gamma-independent manner. 15d-PGJ(2) may modulate inflammatory reactions by regulating the balance of CXC chemokines 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.