Lymphotoxin β Receptor Signaling Induces IL-8 Production in Human Bronchial Epithelial Cells

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.


Introduction
Bronchial asthma (BA) is a chronic airway inflammatory disease characterized by varying degrees of bronchial obstruction, airway hyperresponsiveness and airway remodeling. The standard therapy for asthma is administration of inhaled corticosteroids (ICS). A response to ICS predicts a good outcome [1].
The airway inflammation of BA is generally thought to be eosinophilic inflammation induced by T-helper lymphocytes following production of various levels of cytokines and chemokines by inflammatory cells [2]. Corticosteroids are generally effective for this airway inflammation [3]. However, the airway inflammation in some asthmatics resists corticosteroids and is considered to be severe/refractory asthma. Severe asthma is defined as asthma that requires treatment with a high-dose ICS plus a second controller and/or systemic corticosteroid to prevent it from becoming ''uncontrolled'', or asthma that remains ''uncontrolled'' despite this therapy [4].
Because the number of neutrophils in sputum is increased in severe asthma compared to mild and moderate asthma [5] and is negatively associated with lung function [6], it is thought that neutrophils play a key role in the pathogenesis of severe asthma. Moreover, interleukin (IL)-8, a major chemoattractant of neutrophils [7], is also increased in severe asthmatic airways [8]. Corticosteroids generally decrease airway eosinophils and are, as noted above, effective in treating eosinophilic inflammation (3), but neutrophils induce steroid-resistant inflammation, which leads to airway remodeling that consequently exacerbates the morbidity of asthma [9,10].
The tumor necrosis factor superfamily member (TNFSF) 14/LIGHT (homologous to lymphotoxins, exhibits inducible expression and competes with HSV glycoprotein D for herpesvirus entry mediator (HVEM), a receptor expressed by T lymphocytes [11]) is a type II membrane protein. LIGHT is produced by activated T cells and can bind to lymphotoxin b receptor (LTbR) and HVEM, both of which belong to the TNF receptor superfamily [12]. It is known that LIGHT-HVEM signaling regulates T-cell proliferation [13], while LIGHT-LTbR signaling induces apoptosis of cancer cells [14] and organization and maintenance of lymphoid structures [15]. Recently, LIGHT has been implicated in the pathogenesis of such inflammatory diseases as rheumatoid arthritis and inflammatory bowel disease [16,17]. We have already reported that LIGHT contributes to the pathogenesis of airway fibrosis through enhancement of epithelial mesenchymal transition [18]. Doherty et al. [19] showed that LIGHT is expressed on lung inflammatory cells after allergen exposure and that blockade of LIGHT suppresses expression of transforming growth factor (TGF)-b and IL-13 in the lung. TGF-b and IL-13 are cytokines that are implicated in airway remodeling. Moreover, a pharmacological inhibitor of LTbR reduced smooth muscle hyperplasia and airway hyperresponsiveness in house dust mite-induced mouse models of chronic asthma [19].
In clinical practice, LIGHT levels in the sputum of asthma patients were negatively associated with lung function, suggesting that LIGHT is associated with asthma severity [20]. Another study showed that LIGHT contributed to synovial inflammation and neutrophil accumulation via IL-8 production by fibroblasts [16]. Taken together, LIGHT might induce neutrophilic inflammation via IL-8 production. However, possible effects of LIGHT on IL-8 production by bronchial epithelial cells have not been studied, and the mechanism of IL-8 production by cells has not been fully elucidated.
Therefore, we first evaluated the cytokine profile, especially inflammatory cytokines such as IL-8 and IL-6, of bronchial epithelial cells exposed to LIGHT. We then investigated the mechanisms of LIGHT-induced IL-8 production by elucidating the intracellular signaling.

Cells and cell culture
BEAS-2B cells, an immortalized cell line established from normal human bronchial epithelium obtained by autopsy of non-cancerous individuals and widely used to study the functions of lung bronchial epithelial cells [21], were purchased from the American Type Culture Collection (Manassas, VA). Primary normal human bronchial epithelial cells (NHBE) were purchased from TAKARA BIO INC. (Tokyo, Japan). The cells were cultured in serum-free epithelial growth medium (BEGM; Cambrex, Walkersville, MD) supplemented with Bullet Kit (Cambrex) to contain 0.5 ng/ml human recombinant epidermal growth factor, 0.5 mg/ml hydrocortisone, 10 mg/ml transferrin, 0.5 mg/ml epinephrine, 5 mg/ml insulin, 50 mg/ml bovine pituitary extract, 0.1 ng/ml retinoic acid, 6.5 ng/ml triiodothyronine, 50 mg/ml gentamicin and 0.1 ng/ml amphotericin B at 37˚C in a humidified 5% CO 2 atmosphere. THP-1 cells, a human monocytic cell line, were purchased from the American Type Culture Collection. Cells were cultured in 10cm culture dishes in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37˚C in a humidified 5% CO 2 atmosphere.

Cell surface receptor analysis
Cell surface receptors were analyzed in accordance with the manufacturers' instructions. Briefly, phycoerythrin (PE)-conjugated mouse monoclonal antihuman LTbR antibody (R&D Systems Inc.) and fluorescein isothiocyanate (FITC)-labeled mouse monoclonal anti-human HVEM antibody (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) were used for detection of LTbR and HVEM, respectively, according to the manufacturers' instructions. Cells were run on an EPICS XL flow cytometer (Beckman Coulter Inc., Brea, CA) and analyzed using System II software.

Cytokine array
The experiments were performed using the RayBio Human Cytokine Antibody Briefly, cell supernatants were put on the membranes for 2 h. After washing, array antibody and HRP-conjugated streptavidin were added to the membrane for 2 h. Membranes were detected with 1X detection buffer C and D, and pictures were taken with a cold CCD camera. Densitometric quantification of blots was performed using CS Analyzer 3.0 (ATTO). Different membranes were normalized using backgrounds.

Quantitative reverse transcription-PCR (RT-PCR)
Total RNA was extracted from cells using an RNeasy Mini Kit (Qiagen, Tokyo, Japan). cDNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Quantification of mRNA levels was performed using Mx-3000P (Stratagene, La Jolla, CA) and QuantiTect SYBR Green PCR (Qiagen) according to the manufacturers' instructions. Relative mRNA expression was calculated using the DDCt method. Individual data were normalized against a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The specific primers for GAPDH, IL-8, LTbR, MCP-1, RANTES and IL-6 are shown in Table 1.

Western blot analysis
Cells were lysed in a lysis buffer solution (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS), followed by SDS gel-electrophoresis and semi-dry transfer of the proteins to polyvinylidene difluoride (PVDF) membranes. All sample protein concentrations were measured using the BCA Protein Assay Kit (Thermo Scientific, Waltham, MA), and the same amounts of protein were applied. Non-specific binding of proteins to the membranes was blocked by incubation in TBS-T buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl and 0.1% Tween-20) with 2% ECL Prime Blocking Reagent (GE Healthcare, Buckinghamshire, UK), and the membranes were then incubated with primary antibodies. The antibodies and dilutions used in these studies are described below. Immunodetection was performed with the ECL Prime Western Blotting Detection Kit (GE Healthcare). Pictures were taken with a cold CCD camera (EZ-Capture MG; ATTO, Tokyo, Japan).
Equal protein loading was confirmed by probing the blot with mouse anti-atubulin (Sigma-Aldrich) antibody.

Enzyme-linked immunosorbent assay (ELISA)
BEAS-2B and NHBE cells were seeded into each well of 24-well plates and stimulated with 50 ng/ml LIGHT for 24 h. IL-8 and IL-6 concentrations in the supernatant were measured using the PeliKine Compact human IL-8 ELISA kit and PeliKine Compact human IL-6 ELISA kit (Sanquin Blood Supply, Amsterdam, Netherlands) respectively, according to the manufacturer's instructions. The optical density was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA). The concentrations were calculated using a standard curve obtained with the recombinant kit standards. The data were analyzed using the Microplate Manager 6 software (Bio-Rad).
Transfection of small interfering RNA (siRNA) All siRNAs were purchased from Invitrogen (Tokyo, Japan). Knockdown of LTbR was performed using two specific siRNA duplex (LTbR #1-#2) sets (Stealth RNAi Pre-Designed siRNAs). The sequences of the RNA duplexes are shown in

Luciferase reporter assay
The transcription factor signal pathway from LTbR was determined using a pathway profiling system kit (#631911; BD Biosiences Clontech, UK). This kit was composed of several luciferase reporter vectors that contain a specific cisacting DNA sequence (enhancer element) upstream of the luciferase gene and one construct (pTAL) without any enhancer element upstream of the luciferase reporter gene, used as a negative control. The key cis-acting elements tested in the study were: activator protein-1 (AP-1), nuclear factor of kB (NF-kB) and serum response element (SRE). All these specific cis-acting DNA binding sequences were located upstream of the TATA-like promoter (TAL) region from the herpes simplex virus thymidine kinase (HSV-TK) promoter. The vector pTAL was used as a null vector, which did not have any cis-acting elements in its promoter region and was a negative control in the assay. To normalize the transfection efficiency, cells were co-transfected with pRL-TK Renilla luciferase (pRL-TK-Rluc).

Statistical analysis
Results were confirmed by repeating experiments on at least three separate occasions. Data shown in the figures are pooled data for each experiment and expressed as the mean ¡ SEM. Analyses were performed using JMP Pro (Version 11.1.1; SAS Institute Japan Ltd., Tokyo, Japan). Samples with multiple comparisons were analyzed for significance by analysis of variance (ANOVA). When ANOVA indicated a significant difference between groups, Tukey-Kramer's HSD was applied. P values of ,0.05 were considered to be significant.

LTbR and HVEM expression by bronchial epithelial cells
We first evaluated gene expression of LTbR and HVEM in various types of primary cells using the ''ZENBU'' database of CAGE (cap analysis gene expression) results for 432 normal primary cells [22]. The bronchial epithelial cells expressed the LTbR gene strongly, but the HVEM gene less so (Fig. 1). We also examined the receptors' expression on the bronchial epithelial cell surface by flow cytometry, and the results were the same as those for the ''ZENBU'' database (strong LTbR expression, weaker HEVM expression) (Fig. 2).

Comprehensive analysis of chemokine production by BEAS-2B cells
We used a cytokine array that detects 79 cytokines in order to analyze cytokines and chemokines secreted by bronchial epithelial cells (BEAS-2B) stimulated with LIGHT. As shown in Fig. 3B, LIGHT induced various cytokines, such as GRO, GRO-a, oncostatin M and MCP-1, and especially IL-6 and IL-8. These results indicate that LIGHT is a potent inducer of inflammatory cytokine and chemokine production by bronchial epithelial cells. We also demonstrated that BEAS-2B cells themselves did not produce LIGHT (Fig. 3C).

IL-8 and IL-6 expression by bronchial epithelial cells induced by LIGHT
To determine the effect of LIGHT on BEAS-2B cells, we analyzed the time-course effect on expression of mRNA for several inflammatory chemokines. As shown in Fig. 4A, LIGHT significantly induced IL-8, IL-6 and MCP-1 mRNA at 1 h after stimulation. Then the cells were stimulated with several concentrations of LIGHT, and IL-8 and IL-6 mRNA expression was measured after 1 h. LIGHT induced IL-8 and IL-6 mRNA dose-dependently (Fig. 4B). In addition, as shown in Fig. 4C, LIGHT had significantly induced IL-8 and IL-6 proteins in BEAS-2B cells at 24 h after stimulation. We also confirmed by MTT assay that LIGHT, at less than 100 ng/ml, did not show any cytotoxic effect on BEAS-2B cells (data not shown). As shown in Fig. 4A, LIGHT induced clear IL-8 production at a concentration of 50 ng/ml. In consideration of that result, we used LIGHT at that concentration for subsequent experiments.

Involvement of LTbR in LIGHT-induced IL-8 production
We used siRNA for LTbR to evaluate whether IL-8 production by BEAS-2B cells was induced by LTbR signaling. As shown in Fig. 5A, we found that siRNA #1 and #2 strongly inhibited LTbR 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 LTbR signaling is important for LIGHT-induced cytokine production by BEAS-2B cells. We also evaluated the effect of blocking antibody for LTbR. As shown in Fig. 5C, the blocking antibody for LTbR significantly suppressed LIGHT-induced IL-8 mRNA and inhibited LIGHT-induced IL-8 production.

LTbR signaling in bronchial epithelial cells induced by LIGHT
In order to determine the mechanism of LIGHT-induced IL-8 production by bronchial epithelial cells, we evaluated MAPKs signaling and NF-kB signaling. As shown in Fig. 6A, LIGHT potently induced MAPKs signals, and also IkBa phosphorylation. Moreover, to examine whether induction of MAPKs and NF-kB signaling contributed to production of IL-8, we used specific inhibitors of signaling. U0126, but not SP600125 or SB203580, significantly inhibited IL-8 production (Fig. 6B). These results suggest that LIGHT can induce Erk1/2, p38 and JNK signaling, but the major contributor to IL-8 production is Erk1/2 signaling. BAY11-7082 also inhibited IL-8 production by the cells (Fig. 6C). We also examined the mechanism of IL-8 production by NHBE cells. As shown in Fig. 6D, U0126, BAY11-7082 and siRNA for LTbR each inhibited IL-8 production by NHBE cells, similar to BEAS-2B cells.

Erk1/2 signaling and NF-kB release
Based on the above results, we examined whether Erk1/2 signaling could induce NF-kB for IL-8 production. U0126 did not inhibit IkBa phosphorylation or NF-kB translocation to the nucleus (Fig. 7A). In contrast, LTbR siRNA attenuated Erk1/2 phosphorylation and NF-kB translocation (Fig. 7B). These results indicate that Erk1/2 signaling and NF-kB signaling are down-stream of LTbR signaling, but NF-kB signaling is not directly down-stream of Erk1/2 signaling. We also  performed luciferase reporter assay to evaluate whether LTbR signaling could activate transcription factor response elements. As shown in Fig. 8, LIGHT induced luciferase activity of NF-kB response element, but not AP-1 or SRE. U0126 partially attenuated the luciferase activity of NF-kB response element induced by LIGHT, indicating that Erk1/2 signaling is partially involved in induction of NF-kB expression.

Discussion
We demonstrated that LIGHT induced several inflammatory cytokines and chemokines, including IL-8, in BEAS-2B and NHBE cells. Human bronchial epithelial cells expressed LTbR, a signal that induces the Erk and NF-kB pathways for IL-8 production. IL-8/C-X-C motif ligand (CXCL) 8 is produced by epithelial cells, airway smooth muscle cells and endothelial cells. It is a member of the a-CXC chemokine family. IL-8 is an inflammatory mediator that induces chemotaxis of neutrophils and other granulocytes, cells that release reactive oxygen species and elastase. Indeed, the number of neutrophils in sputum is higher in severe asthma compared to mild and moderate asthma [5]. Moreover, IL-8 is upregulated in the airway of severe asthmatics [23] and contributes to prolongation of steroid-resistant inflammation via accumulation of neutrophils [24,25]. IL-8 binds to G proteincoupled chemokine receptors (CXCR) 1 and CXCR2, which promote neutrophil influx into tissue sites of inflammation and induce acute lung injury [26]. In consideration of this background, IL-8 may play a key role in chronic airway inflammation in severe asthma through induction of neutrophilic inflammation [27].
We hypothesized that LIGHT and LTbR signaling contribute to severe asthma through induction of IL-8, which promotes accumulation of neutrophils and inflammation in the airway. First, we demonstrated cytokine production, especially IL-8, by BEAS-2B cells and NHBE cells when they were stimulated with LIGHT.
We examined expression of receptors for LIGHT using flow cytometry. It is known that the functional receptors of LIGHT are LTbR and HVEM [28]. LTbR is highly expressed in the lung, liver and kidney, and moderately expressed in the heart and testes. It is weakly expressed in the brain, thymus, spleen and lymph nodes [29]. On the other hand, HVEM expression is high in naïve and memory B cells, but it is not present on activated B cells in the germinal center [30,31]. Here, we found that BEAS-2B cells and NHBE cells expressed only LTbR. Based on that result, LTbR and its downstream signaling were considered as key players in cytokine production by bronchial epithelial cells. So we evaluated the profile of LTbR signaling-induced cytokines using not only a cytokine array but also qRT-PCR or ELISA. We showed that LIGHT induced various inflammatory cytokines, such as GRO, GRO-a, oncostatin M, MCP-1, IL-6 and IL-8, but not RANTES. IL-6 and IL-8 were induced dose-dependently.
Next, we analyzed the mechanism of cytokine production by the cells. Because previous studies showed MAPKs to be involved not only in TNF-a-induced IL-8 production [32], but also in steroid-resistant inflammation in severe asthma [33], we focused on this signaling. LIGHT induced not only MAPKs signaling, such as Erk1/2, p38 and JNK, but also NF-kB signaling. We used specific phosphorylation inhibitors to determine which pathways were most important for IL-8 production. U0126 and BAY11-7082-a MEK1/2 inhibitor and an IkBa phosphorylation inhibitor, respectively-both inhibited IL-8 production. On the other hand, SP600125 and SB20358-JNK and p38 phosphorylation inhibitorsdid not attenuate IL-8 production. In addition, we used a specific siRNA for LTbR, and it significantly attenuated IL-8 production and simultaneously attenuated Erk1/2 and IkBa phosphorylation. These results indicate that LIGHTinduced IL-8 production is due to activation of Erk1/2 and NF-kB via LTbR.
Some transcriptional factors, such as NF-kB and AP-1, were reported to regulate IL-8 expression [34]. It is thought that LIGHT induces IL-8 production through NF-kB release [35,36]. Our results showed that U0126 completely inhibited IL-8 production, so we investigated the possibility that the Erk1/2 pathway activated the NF-kB pathway. Indeed, U0126 partially attenuated LIGHT-induced luciferase activity of NF-kB response element, but it did not inhibit NF-kB release and translocation to the nucleus. As shown in Fig. 9, these results suggested that there were two pathways by which LIGHT induced IL-8: (1) via Erk1/2 and (2) directly via NF-kB. Although the mechanism by which Erk1/2 induces IL-8 gene expression is not clear, Erk2 (p44/42 MAPK) was reported to phosphorylate Elk-1 and activates SRE, leading to induction of c-fos, a component of AP-1 [37]. The AP-1 binding element is present in the promoter region of the IL-8 gene, and AP-1, a complex of c-fos and c-jun or jun B, plays an important role in transcriptional regulation of IL-8 mRNA expression [38][39][40]. On the other hand, it has been considered that LIGHT induces two types of NF-kB pathway. One is degradation of IkBa to release p50/RelA and p50/c-Rel heterodimers via TNF receptor-associated factors [41]. The other is involvement of LTbR in NF-kB-inducing kinase activation to promote p100 processing [42].
As described above, LTbR is a key receptor involved in airway inflammation through LIGHT-induced cytokine production. Blockade of LTbR by administering specific antibodies or siRNA might reduce excessive cytokine production. This might be a new therapeutic approach for, for example, severe asthma patients with a high sputum level of LIGHT. A limitation of this study is that we did not test our in vitro findings by performing in vivo experiments, such as in a murine asthma model. Such studies will be needed.

Conclusion
LIGHT, via LTbR signaling, may contribute to exacerbation of airway inflammation through cytokine and chemokine production by bronchial epithelial cells. The mechanism of LIGHT-induced cytokine production, especially for IL-8, is activation of LTbR signaling that activates the Erk and NF-kB pathways. Since LTbR is expressed on various airway epithelial cells, the LTbR signaling pathway might represent a new therapeutic target for severe asthma.
Ltd.; Tokyo) for English proofreading. Some of the results have previously been presented in abstract form at international conferences.