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Lymphotoxin β receptor signaling induces IL-8 production in human bronchial epithelial cells.

Mikami Y, Matsuzaki H, Horie M, Noguchi S, Jo T, Narumoto O, Kohyama T, Takizawa H, Nagase T, Yamauchi Y - PLoS ONE (2014)

Bottom Line: LIGHT also induced luciferase activity of NF-κB response element, but not of activator protein-1 or serum response element.Specific inhibitors of phosphorylation of extracellular signal-regulated kinase (Erk) and that of inhibitor κB attenuated IL-8 production, suggesting that LIGHT-LTβR signaling induces IL-8 production via the Erk and NF-κB pathways.LIGHT, via LTβR signaling, may contribute to exacerbation of airway neutrophilic inflammation through cytokine and chemokine production by bronchial epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Asthma-related mortality has been decreasing due to inhaled corticosteroid use, but severe asthma remains a major clinical problem. One characteristic of severe asthma is resistance to steroid therapy, which is related to neutrophilic inflammation. Recently, the tumor necrosis factor superfamily member (TNFSF) 14/LIGHT has been recognized as a key mediator in severe asthmatic airway inflammation. However, the profiles and intracellular mechanisms of cytokine/chemokine production induced in cells by LIGHT are poorly understood. We aimed to elucidate the molecular mechanism of LIGHT-induced cytokine/chemokine production by bronchial epithelial cells. Human bronchial epithelial cells express lymphotoxin β receptor (LTβR), but not herpesvirus entry mediator, which are receptors for LIGHT. LIGHT induced various cytokines/chemokines, such as interleukin (IL)-6, oncostatin M, monocyte chemotactic protein-1, growth-regulated protein α and IL-8. Specific siRNA for LTβR attenuated IL-6 and IL-8 production by BEAS-2B and normal human bronchial epithelial cells. LIGHT activated intracellular signaling, such as mitogen-activated protein kinase and nuclear factor-κB (NF-κB) signaling. LIGHT also induced luciferase activity of NF-κB response element, but not of activator protein-1 or serum response element. Specific inhibitors of phosphorylation of extracellular signal-regulated kinase (Erk) and that of inhibitor κB attenuated IL-8 production, suggesting that LIGHT-LTβR signaling induces IL-8 production via the Erk and NF-κB pathways. LIGHT, via LTβR signaling, may contribute to exacerbation of airway neutrophilic inflammation through cytokine and chemokine production by bronchial epithelial cells.

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Effect of LTβR siRNA on IL-8 production by BEAS-2B cells.BEAS-2B cells were transfected with LTβR siRNA to knock down the receptor. (A) We purchased two types of siRNA (#1 and #2) and transfected them into BEAS-2B cells. We evaluated the knockdown efficacy of each siRNA 72 h later by qRT-PCR and western blotting. Lipofectamine reagent and the negative control siRNA (NC siRNA) did not affect LTβR mRNA expression, but siRNA#1 and #2 both significantly inhibited LTβR mRNA. (B) BEAS-2B cells were transfected with siRNA, and 72 h later they were stimulated with 50 ng/ml LIGHT. The IL-8 and IL-6 concentrations in the cell supernatants were determined by ELISA 24 h after stimulation. Both siRNA#1 and siRNA#2 significantly inhibited IL-8 and IL-6 production by BEAS-2B cells. n = 4 separate experiments. **: p<0.01. (C) BEAS-2B cells were pre-incubated with LTβR blocking antibody before stimulation with 50 ng/ml LIGHT. The blocking antibody for LTβR significantly attenuated both IL-8 mRNA expression and IL-8 production. n = 3 separate experiments. **: p<0.01.
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pone-0114791-g005: Effect of LTβR siRNA on IL-8 production by BEAS-2B cells.BEAS-2B cells were transfected with LTβR siRNA to knock down the receptor. (A) We purchased two types of siRNA (#1 and #2) and transfected them into BEAS-2B cells. We evaluated the knockdown efficacy of each siRNA 72 h later by qRT-PCR and western blotting. Lipofectamine reagent and the negative control siRNA (NC siRNA) did not affect LTβR mRNA expression, but siRNA#1 and #2 both significantly inhibited LTβR mRNA. (B) BEAS-2B cells were transfected with siRNA, and 72 h later they were stimulated with 50 ng/ml LIGHT. The IL-8 and IL-6 concentrations in the cell supernatants were determined by ELISA 24 h after stimulation. Both siRNA#1 and siRNA#2 significantly inhibited IL-8 and IL-6 production by BEAS-2B cells. n = 4 separate experiments. **: p<0.01. (C) BEAS-2B cells were pre-incubated with LTβR blocking antibody before stimulation with 50 ng/ml LIGHT. The blocking antibody for LTβR significantly attenuated both IL-8 mRNA expression and IL-8 production. n = 3 separate experiments. **: p<0.01.

Mentions: We used siRNA for LTβR to evaluate whether IL-8 production by BEAS-2B cells was induced by LTβR signaling. As shown in Fig. 5A, we found that siRNA #1 and #2 strongly inhibited LTβR expression. siRNA #1 and #2 also strongly inhibited IL-8 production by cells that were stimulated with LIGHT (Fig. 5B). In consideration of that result, we used siRNA #2 for subsequent experiments. These results indicated that LTβR signaling is important for LIGHT-induced cytokine production by BEAS-2B cells. We also evaluated the effect of blocking antibody for LTβR. As shown in Fig. 5C, the blocking antibody for LTβR significantly suppressed LIGHT-induced IL-8 mRNA and inhibited LIGHT-induced IL-8 production.


Lymphotoxin β receptor signaling induces IL-8 production in human bronchial epithelial cells.

Mikami Y, Matsuzaki H, Horie M, Noguchi S, Jo T, Narumoto O, Kohyama T, Takizawa H, Nagase T, Yamauchi Y - PLoS ONE (2014)

Effect of LTβR siRNA on IL-8 production by BEAS-2B cells.BEAS-2B cells were transfected with LTβR siRNA to knock down the receptor. (A) We purchased two types of siRNA (#1 and #2) and transfected them into BEAS-2B cells. We evaluated the knockdown efficacy of each siRNA 72 h later by qRT-PCR and western blotting. Lipofectamine reagent and the negative control siRNA (NC siRNA) did not affect LTβR mRNA expression, but siRNA#1 and #2 both significantly inhibited LTβR mRNA. (B) BEAS-2B cells were transfected with siRNA, and 72 h later they were stimulated with 50 ng/ml LIGHT. The IL-8 and IL-6 concentrations in the cell supernatants were determined by ELISA 24 h after stimulation. Both siRNA#1 and siRNA#2 significantly inhibited IL-8 and IL-6 production by BEAS-2B cells. n = 4 separate experiments. **: p<0.01. (C) BEAS-2B cells were pre-incubated with LTβR blocking antibody before stimulation with 50 ng/ml LIGHT. The blocking antibody for LTβR significantly attenuated both IL-8 mRNA expression and IL-8 production. n = 3 separate experiments. **: p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4263477&req=5

pone-0114791-g005: Effect of LTβR siRNA on IL-8 production by BEAS-2B cells.BEAS-2B cells were transfected with LTβR siRNA to knock down the receptor. (A) We purchased two types of siRNA (#1 and #2) and transfected them into BEAS-2B cells. We evaluated the knockdown efficacy of each siRNA 72 h later by qRT-PCR and western blotting. Lipofectamine reagent and the negative control siRNA (NC siRNA) did not affect LTβR mRNA expression, but siRNA#1 and #2 both significantly inhibited LTβR mRNA. (B) BEAS-2B cells were transfected with siRNA, and 72 h later they were stimulated with 50 ng/ml LIGHT. The IL-8 and IL-6 concentrations in the cell supernatants were determined by ELISA 24 h after stimulation. Both siRNA#1 and siRNA#2 significantly inhibited IL-8 and IL-6 production by BEAS-2B cells. n = 4 separate experiments. **: p<0.01. (C) BEAS-2B cells were pre-incubated with LTβR blocking antibody before stimulation with 50 ng/ml LIGHT. The blocking antibody for LTβR significantly attenuated both IL-8 mRNA expression and IL-8 production. n = 3 separate experiments. **: p<0.01.
Mentions: We used siRNA for LTβR to evaluate whether IL-8 production by BEAS-2B cells was induced by LTβR signaling. As shown in Fig. 5A, we found that siRNA #1 and #2 strongly inhibited LTβR expression. siRNA #1 and #2 also strongly inhibited IL-8 production by cells that were stimulated with LIGHT (Fig. 5B). In consideration of that result, we used siRNA #2 for subsequent experiments. These results indicated that LTβR signaling is important for LIGHT-induced cytokine production by BEAS-2B cells. We also evaluated the effect of blocking antibody for LTβR. As shown in Fig. 5C, the blocking antibody for LTβR significantly suppressed LIGHT-induced IL-8 mRNA and inhibited LIGHT-induced IL-8 production.

Bottom Line: LIGHT also induced luciferase activity of NF-κB response element, but not of activator protein-1 or serum response element.Specific inhibitors of phosphorylation of extracellular signal-regulated kinase (Erk) and that of inhibitor κB attenuated IL-8 production, suggesting that LIGHT-LTβR signaling induces IL-8 production via the Erk and NF-κB pathways.LIGHT, via LTβR signaling, may contribute to exacerbation of airway neutrophilic inflammation through cytokine and chemokine production by bronchial epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Asthma-related mortality has been decreasing due to inhaled corticosteroid use, but severe asthma remains a major clinical problem. One characteristic of severe asthma is resistance to steroid therapy, which is related to neutrophilic inflammation. Recently, the tumor necrosis factor superfamily member (TNFSF) 14/LIGHT has been recognized as a key mediator in severe asthmatic airway inflammation. However, the profiles and intracellular mechanisms of cytokine/chemokine production induced in cells by LIGHT are poorly understood. We aimed to elucidate the molecular mechanism of LIGHT-induced cytokine/chemokine production by bronchial epithelial cells. Human bronchial epithelial cells express lymphotoxin β receptor (LTβR), but not herpesvirus entry mediator, which are receptors for LIGHT. LIGHT induced various cytokines/chemokines, such as interleukin (IL)-6, oncostatin M, monocyte chemotactic protein-1, growth-regulated protein α and IL-8. Specific siRNA for LTβR attenuated IL-6 and IL-8 production by BEAS-2B and normal human bronchial epithelial cells. LIGHT activated intracellular signaling, such as mitogen-activated protein kinase and nuclear factor-κB (NF-κB) signaling. LIGHT also induced luciferase activity of NF-κB response element, but not of activator protein-1 or serum response element. Specific inhibitors of phosphorylation of extracellular signal-regulated kinase (Erk) and that of inhibitor κB attenuated IL-8 production, suggesting that LIGHT-LTβR signaling induces IL-8 production via the Erk and NF-κB pathways. LIGHT, via LTβR signaling, may contribute to exacerbation of airway neutrophilic inflammation through cytokine and chemokine production by bronchial epithelial cells.

Show MeSH
Related in: MedlinePlus