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

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.

Figure 1. Gene expression analysis of primary lung epithelial cells.

We analyzed gene expression of LTβR and HVEM in several types of primary cells using the ZENBU database, which analyzed CAGE (cap analysis gene expression) of 432 normal primary cells. The lung epithelial cells strongly expressed the LTβR gene (A), but not the HVEM gene (B).ZENBU database URL: http://fantom.gsc.riken.jp/zenbu/

Figure 2. Expression of LTβR and HVEM receptors on BEAS-2B cells.

BEAS-2B cells and THP-1 cells (positive control) were treated with anti-PE labeled LTβR antibody and anti-FITC labeled HVEM antibody. The fluorescence intensity was measured with a flow cytometer. THP-1 cells, a human monocytic cell line, were used as the positive control because they express both LTβR and HVEM on their cell surface. The fluorescence intensities of LTβR (A) and HVEM (B) suggest that BEAS-2B cells express LTβR but not HVEM.

Figure 3. Comprehensive analysis of LIGHT-induced cytokine and chemokine production.

BEAS-2B cells were stimulated with LIGHT (100 ng/ml) for 24 h, followed by determination of the protein levels of cytokines and chemokines by densitometry using a cytokine array. (A) The left image shows the unstimulated samples, while the right image shows the samples at 24 h after stimulation with LIGHT. (B) This table shows array mapping. The red color indicates cytokines that were upregulated more than twofold compared to the unstimulated sample. LIGHT induced inflammatory cytokines, such as GRO, GRO-α, oncostatin M, MCP-1, IL-6 and IL-8. (C) We investigated whether BEAS-2B cells produced LIGHT. THP-1 cells, which were used as a positive control, produced LIGHT when stimulated with PMA 50 ng/ml, but BEAS-2B cells did not.

Figure 4. Time-course and dose-dependent effects of LIGHT on bronchial epithelial (BEAS-2B) cells.

(A) BEAS-2B cells were stimulated with 50 ng/ml LIGHT and examined for the time-course effect on expression of mRNA for each of IL-8, IL-6, MCP-1 and RANTES. LIGHT significantly induced mRNA for each of IL-6, IL-8 and MCP-1. n = 4 separate experiments. *: p<0.05, **: p<0.01 vs 0 h. (B) BEAS-2B cells were stimulated with various concentrations of LIGHT (0, 1, 10, 100 ng/ml) for 1 h and evaluated for expression of mRNA for each of IL-8 and IL-6. LIGHT induced IL-8 and IL-6 mRNA dose-dependently. n = 4 separate experiments. ** and ††: p<0.01 vs 0 ng/ml. (C) BEAS-2B cells were stimulated with 100 ng/ml LIGHT for 24 h, and the protein concentrations in the cell supernatants were determined by ELISA. LIGHT significantly induced IL-8 and IL-6 proteins. n = 6 separate experiments. **: p<0.01.

Figure 5. 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.

Figure 6. LTβR signaling in bronchial epithelial cells induced by LIGHT.

BEAS-2B cells were stimulated with 50 ng/ml LIGHT, and cell lysates were prepared 0, 5, 15, 20, 30 and 90 minutes later to evaluate phosphorylation of Erk, JNK, p38 and IκB. The MAPKs were phosphorylated for up to 15 minutes, and their signaling was activated. IκB was also phosphorylated at the same time and induced NF-κB release and translocation to nucleus. (A) BEAS-2B cells were pre-treated for 1 h with various inhibitors of phosphorylation of MAPKs. U0126 significantly inhibited IL-8 production by the cells. (B) BEAS-2B cells were pre-treated for 1 h with BAY11-7082. BAY11-7082 significantly inhibited IL-8 production. (C) We performed the same experiments using NHBE cells, which are primary normal human bronchial epithelial cells. LTβR siRNA, U0126 and BAY11-7082 significantly inhibited IL-8 production by NHBE cells in the same manner as seen for BEAS-2B cells.

Figure 7. Erk1/2 signaling and NF-κB release.

(A) To evaluate the relationship between Erk1/2 signaling and NF-κB release, BEAS-2B cells were pretreated with U0126 (10 µM) 1 h before stimulation with LIGHT. U0126 did not inhibit IκBα phosphorylation or NF-κB translocation from the cytoplasm to the nucleus. (B) We evaluated the effect of LTβR knockdown on Erk1/2 signaling and NF-κB release. BEAS-2B cells were transfected with negative control siRNA (NC siRNA) or LTβR siRNA#2, stimulated with LIGHT (50 ng/ml) and analyzed by western blotting. The cells that were transfected with LTβR siRNA showed attenuation of Erk1/2 phosphorylation and NF-κB translocation induced by LIGHT.

Figure 8. Effect of LTβR signaling on transcription factor-driven luciferase activity by transient transfection of BEAS-2B cells.

BEAS-2B cells were transfected with a plasmid having luciferase as a reporter gene, controlled by a synthetic promoter containing NF-κB, AP-1 or serum response element (SRE). After 24 h, the cells were stimulated with LIGHT (50 ng/ml) or TNF-α (10 ng/ml) as a positive control, with/without U0126. The data are the mean ± SEM of four independent experiments performed in triplicate. Significant differences (p<0.01) are indicated with **.

Figure 9. Schematic summary of how LTβR signal transduction induces IL-8 expression in human bronchial epithelial cells.

LIGHT binding to LTβR may induce Erk1/2 or IκBα phosphorylation, which in turn induces NF-κB activation, and ultimately causes IL-8 release from the cells.