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The Role of IB Kinase 2, but Not Activation of NF-B, in the Release of CXCR3 Ligands from IFN--Stimulated Human Bronchial Epithelial Cells

Susan J. Tudhope^*, Matthew C. Catley^*, Peter S. Fenwick^*, Richard E. K. Russell, William L. Rumsey, Robert Newton, Peter J. Barnes^* and Louise E. Donnelly^1,*

^* Airway Disease, National Heart and Lung Institute, Imperial College, London, United Kingdom; Heatherwood and Wexham Park National Health Service Trust, Berkshire, United Kingdom; GlaxoSmithKline, Philadelphia, PA 19406 and Cell Biology and Anatomy, Respiratory Research Group, University of Calgary, Calgary, Alberta, Canada

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The severity of chronic obstructive pulmonary disease correlates with increased numbers of cytotoxic CD8⁺ T lymphocytes in the lung parenchyma. CD8⁺ T lymphocytes release IFN- which stimulates airway epithelial cells to produce CXCR3 chemokines leading to further recruitment of CD8⁺ T lymphocytes. To evaluate the signaling pathways involved in regulation of CXCR3 ligands, the human bronchial epithelial cell line BEAS-2B was stimulated with IFN- and the release of the CXCR3 ligands was measured by ELISA. The release of CXCL9, CXCL10, and CXCL11 was inhibited by an IB kinase 2 (IKK2) selective inhibitor 2-[(Aminocarbonyl)amino]-5-[4-fluorophenyl]-3-thiophenecarboxamide (TPCA-1) (EC₅₀ values were 0.50 ± 0.03, 0.17 ± 0.06, and 0.45 ± 0.10 µM, respectively (n = 6)) and an IKK1/2 selective inhibitor 2-amino-6-(2'cyclopropylemethoxy-6'-hydroxy-phenyl)-4-piperidin-3-yl-pyridine-3-carbonitrile (EC₅₀ values 0.74 ± 0.40, 0.27 ± 0.06, and 0.88 ± 0.29 µM, respectively (n = 6)). The glucocorticosteroid dexamethasone had no effect on CXCR3 ligand release. The release of CXCL10 was most sensitive to inhibition by IKK2 and a role for IKK2 in CXCL10 release was confirmed by overexpression of dominant-negative adenoviral constructs to IKK2 (68.2 ± 8.3% n = 5), but not of IKK1. Neither phosphorylation of IB, translocation of p65 to the nucleus, or activation of a NF-B-dependent reporter (Ad-NF-B-luc) were detected following stimulation of BEAS-2B cells with IFN-. These data suggest that IKK2 is also involved in the IFN--stimulated release of the CXCR3 ligands through a novel mechanism that is independent NF-B.

	Introduction

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Chronic obstructive pulmonary disease (COPD)² is the fourth commonest cause of death in developed countries. It is increasing in prevalence and will become the fifth commonest cause of disability by 2020 (1). Chronic lung inflammation is an important feature of COPD and comprises increased numbers of infiltrating macrophages, CD8⁺ T lymphocytes, and neutrophils (2, 3). The number of CD8⁺ T cells in the lung parenchyma correlates with airflow limitation, suggesting that these cells are important in the pathogenesis of COPD (2, 4). CD8⁺ T lymphocytes produce perforins and granzyme B (5) and these may contribute to the alveolar destruction observed in patients with COPD (6). Moreover, progression of COPD is associated with increased numbers of CD8⁺ T lymphocytes and B lymphocytes in the airways (7), further supporting a role for these cells in the pathophysiology of COPD.

In bronchial biopsies of patients with COPD, CD8⁺ T lymphocytes express the chemokine receptor CXCR3 together with IFN- (8). Furthermore, in the peripheral airways of COPD patients, the expression of T cell-associated CXCR3 is enhanced compared with healthy control subjects (8, 9). In chronic bronchitis, the numbers of IFN--positive T lymphocytes are increased compared with control subjects (10), and CD8⁺ T lymphocytes from COPD patients produce more IFN- compared with cells from control subjects (9). This may then cause an amplification of the observed lung inflammation. The importance of IFN-, in the pathophysiology of COPD, has been further demonstrated in transgenic mice, where overexpression of IFN- leads to alveolar enlargement and pulmonary emphysema that is associated with increased numbers of T lymphocytes and macrophages (11).

There are three chemokines that act as ligands at the CXCR3 receptor, namely monokine induced by IFN- (CXCL9), IFN-inducible protein of 10 kDa (CXCL10), and IFN-inducible T cell- chemoattractant (CXCL11) (12). Immunohistochemical studies have shown that the bronchial epithelium expresses the CXCR3 ligand CXCL10 (8), and that human primary airway epithelial cells (13, 14) and human airway smooth muscle cells (15) express these ligands following stimulation with IFN-. Similarly, macrophages from COPD patients also produce CXCR3 chemokines following stimulation with IFN- (16). It has also been reported that CXCL9 and CXCL10 can act directly on lung macrophages to secrete matrix metalloproteinase-12 that could further contribute to lung inflammation and tissue destruction in COPD (9).

Several new approaches to target the inflammation associated with COPD are being investigated, including the IB kinase (IKK) 2 (17). The IKK2-selective inhibitor 2-[(Aminocarbonyl)amino]-5-[4-fluorophenyl]-3-thiophenecarboxamide (TPCA-1) has been shown to reduce inflammatory cell accumulation in both rat models of asthma (18) and LPS-induced lung inflammation (19). IKK2 inhibitors also suppress cytokine release from human airway smooth muscle cells (20) and airway epithelial cells (21) following stimulation with IL-1 and may, therefore, be beneficial in reducing the inflammation in COPD.

Classically, the IKK complex is activated when cells are stimulated with proinflammatory cytokines such as IL-1 or TNF- (22). The use of adenoviral-mediated delivery of dominant-negative IKK2 to human pulmonary epithelial cells showed that IKK2 was critical for cytokine release mediated by both IL-1 and TNF- (23). IKK is thought to mediate its effects through the transcription factor, NF-B. In resting cells, NF-B is sequestered in the cytoplasm by the IB proteins, of which IB, IB, and IB are involved in NF-B activation (24). To date, NF-B has five family members, however, only two of these, p65 (also called RelA) and RelB, have transcriptional activity. NF-B can exist as a variety of homo- and heterodimers of which the most abundant form is the heterodimer of p65 and p50 (17). The IKK complex consists of IKK1 (also termed IKK) and IKK2 (also termed IKK) in conjunction with IKK. Both IKK1 and IKK2 have kinase activity, whereas IKK is a structural component lacking kinase activity. Activation of the IKK complex leads to the phosphorylation of IB proteins, the most studied of which is IB (22). Phosphorylated IB is then susceptible to polyubiquitination and subsequent degradation by the 26S proteosome. NF-B then translocates to the nucleus and activates proinflammatory genes (25). More recently, additional members of the IKK pathway have been cloned. Both TANK-binding kinase (TBK) and IKK- (also termed IKK-i) have kinase activity and can also phosphorylate IB- (26).

By contrast, IFN- signals through the JAKs that dock with the intracellular portion of the IFN- receptor. Phosphorylation of the JAKs leads to phosphorylation and homodimerization of the STAT-1 transcription factor (27). The stable homodimers of STAT-1 translocate to the nucleus where they bind to the IFN--responsive elements and -activated sequences thereby switching on IFN--dependent genes (27). STAT-1 can be phosphorylated at two different residues, Tyr⁷⁰¹ and Ser⁷²⁷ (28). Phosphorylation of both Tyr⁷⁰¹ and Ser⁷²⁷ is required for maximal transcriptional activation (29).

The aim of this study was to determine whether there was cross-talk between the IKK pathway and the IFN--stimulated release of the three CXCR3 chemokines. Consequently, we examined both selective small molecular inhibitors of IKK2 and dominant-negative constructs of key components in the IKK pathway. We describe the critical involvement of IKK2, but not activation of NF-B, in the IFN--dependent release of CXCL9, CXCL10, and CXCL11 from human bronchial epithelial cells.

	Materials and Methods

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Culture of BEAS-2B cells

The BEAS-2B cell line was purchased from American Type Culture Collection. Keratinocyte serum-free medium (K-SFM), bovine pituitary extract, and recombinant human epidermal growth factor were purchased from Invitrogen Life Technologies. The transformed human bronchial epithelial cell line BEAS-2B (passages 41–46) was cultured in K-SFM containing 50 µg/ml bovine pituitary extract and 5 ng/ml epidermal growth factor at 37°C in a humidified atmosphere containing 5% (v/v) CO₂ in air. Cells were growth factor starved for 24 h before stimulation.

Measurement of mRNA expression for the CXCR3 ligands

Total RNA was extracted from cells and reverse transcribed, as described previously (30). Gene expression was determined by TaqMan real-time PCR on a 7500 Real-Time PCR system (Applied Biosystems) using PCR Master Mix Reagent and "assays on demand" (Applied Biosystems). HPRT1 gene expression was used as the housekeeping gene. The data were analyzed using the cycle threshold method and expressed relative to the IFN- stimulation at 6 h (31).

Measurement of CXCR3 ligands

Cell-free supernatants were removed 20 h poststimulation with IFN- and assayed for CXCL9, CXCL10, and CXCL11 using Duoset ELISA kits (R&D Systems) according to the manufacturer’s instructions. The detection limits of these assays are 62, 31, and 7.8 pg/ml, respectively.

Cell viability

Cell viability was determined colorimetrically by measuring the reduction of MTT to formazan by mitochondrial dehydrogenases, as described previously (20).

IKK inhibitors and glucocorticosteroid

2-[(Aminocarbonyl)amino]-5-[4-fluorophenyl]-3-thiophenecarboxamide (TPCA-1) and APPC, compound 3o, in Murata et al. (Ref. 32) were provided by GlaxoSmithKline. Dexamethasone was obtained from Sigma-Aldrich. 2-amino-6-(2'cyclopropylmethoxy-6'-hydroxy-phenyl)-4- piperidin-3-yl-pyridine-3-carbonitrile (APPC) and TPCA-1 were both prepared in DMSO with a final concentration of 0.1% (v/v). Dexamethasone was prepared in HBSS and diluted in medium containing 0.1% (v/v) DMSO. These compounds did not alter cell viability.

Infection of BEAS-2B cells with adenoviral IKK dominant-negative and mutated constructs

BEAS-2B cells were cultured until 70% confluent and then infected with either the null virus (Ad5.CMV.Null), GFP (Ad5-GFP), dominant IBN, and dominant-negative IKK1(KM), IKK2(KA), TBK, and IKK-expressing viruses that have all been described previously (23, 33, 34, 35). All viruses were titered using a Quick Titer Adenovirus ELISA (Cell Biolabs) that uses an anti-hexon Ab to quantitate the infection of HEK 293 cells. Viruses were diluted in K-SFM to a multiplicity of infection (MOI) of 100 before infection of BEAS-2B cells for 20 h. The BEAS-2B cells were subsequently stimulated with IFN- (10 ng/ml) for a further 20 h in K-SFM containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Adenoviral infection at MOI 100 had no effect on cell viability.

Western blotting

ECL reagent and Hybond-ECL nitrocellulose were obtained from GE Healthcare. Bis-Tris SDS-PAGE (4–12%) gels and buffers were purchased from Invitrogen Life Technologies. Abs against phosphorylated and total IB and STAT-1 were purchased from New England Biolabs. BEAS-2B cells were treated with IFN- (5–60 min) or IL-1 (2–30) min in 6-well plates. Cells were scraped and lysed with lysis buffer (10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl, 2 mM EDTA, 1 mM sodium orthovanadate, 1% (v/v) Triton X-100, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) for 30 min on ice. The lysates were then centrifuged at 12,000 x g for 15 min. The protein concentration of the lysates was measured using the BCA protein assay (Bio-Rad). Equal amounts of protein (20 µg) were resolved with 4–12% Bis-Tris gels. After transferring the proteins onto a nitrocellulose membrane, the membranes were blocked with 5% (w/v) nonfat milk in TBS containing 0.1% (v/v) Tween 20 for 1 h at room temperature. The nitrocellulose was then incubated with Abs specific for either pIB or pSTAT-1 (Tyr⁷⁰¹ and Ser⁷²⁷) overnight at 4°C. The primary Ab was detected with peroxidase-conjugated secondary Abs and labeled proteins were detected by ECL.

Double-label immunoconfocal laser microscopy

BEAS-2B cells were cultured on 8-well glass Labtek slides (Nalge Nunc International). NF-B p65 expression was detected using a rabbit polyclonal Ab (sc-109; Santa Cruz Biotechnology). The cells were fixed in ice-cold methanol for 5 min and following three 5-min washes in PBS, cells were labeled with the rabbit anti-p65 Ab overnight in PBS/10% (v/v) normal human serum containing 0.1% (v/v) Triton-100 at 4°C. Following three 5-min washes in PBS, the cells were incubated with an Alexa 488-conjugated goat anti-rabbit IgG F(ab')₂ Ab (Molecular Probes) for 2 h in PBS/10% (v/v) normal human serum at room temperature. The slides were washed in PBS and subsequently incubated with 4',6-diamidino-2-phenyl-indole hydrochloride (DAPI) at 10 µM (Sigma-Aldrich) in HBSS for 3 min, washed again, and mounted using Citifluor. Cells (0.7-µm sections) were analyzed using a Leica TCS 4D confocal microscope (Leica Microsystems) equipped with argon and UV lasers. Confocal images were acquired at x20 magnification.

NF-B reporter assay

BEAS-2B cells were infected with MOI 100 of an adenovirus carrying a NF-B-dependent reporter (Ad-NF-B-luc). This construct was obtained by inserting the NF-B enhancer (five copies of the classical NF-B motif (underlined) 5'-TGG GGA CTT TCC GC-3'), TATA box, and luciferase gene from pNF-B-luc (Stratagene) into the Ad5 parent vector. After incubation for 24 h in K-SFM, the cells were then changed to fresh K-SFM and stimulated with IFN-, IL-1, or TNF- (0.1–100 ng/ml) for 6 h. Cells were then harvested and luciferase activity was measured as described previously (20).

STAT activation (TransAM)

STAT phosphorylation, nuclear translocation, and DNA binding was determined using TransAM assays (Actif Motif). BEAS-2B cells were treated with IKK inhibitors for 30 min before stimulation with IFN- (10 ng/ml) for 1 h. Nuclear extracts were prepared using the Actif Motif assay reagents. Briefly, the nuclear extracts (20 µg) were incubated in DNA-coated plates for 1 h. After washing, Abs specific for the active forms of STAT1, STAT3, STAT5A, and STAT5B were incubated for 1 h. The plates were washed and a secondary HRP-labeled anti-goat Ig Ab was incubated for a further 1 h. After washing, the assay was developed using the tetramethylbenzidine substrate provided and the reaction was stopped using 2 N sulfuric acid. The plates were read at 450 nm with a reference filter of 610 nm.

NF-B family nuclear localization and DNA binding (TransAM)

NF-B phosphorylation, nuclear translocation, and DNA binding were determined using TransAM assays (Actif Motif). BEAS-2B cells were treated with IFN- (10 ng/ml) or TNF- (1 ng/ml) for 15, 30, or 45 min. Nuclear extracts were prepared using the Actif Motif assay reagents. Briefly, the nuclear extracts (5 µg) were incubated in DNA-coated plates for 1 h. After washing, Abs specific for p65, p50, c-Rel, Rel B, and p52 were incubated for 1 h. The plates were washed and a secondary HRP-labeled anti-goat Ig Ab was incubated for a further 1 h. After washing, the assay was developed using the tetramethylbenzidine substrate provided and the reaction was stopped using 2 N sulfuric acid. The plates were read at 450 nm with a reference filter of 610 nm.

EMSA

EMSA was performed as described previously (36, 37). Briefly, nuclear proteins (5 µg) were used in binding reactions. The consensus NF-B probe (Promega) containing the decameric NF-B site (underlined) was 5'-AGT TGA GGG GAC TTT CCC AGG-3' (sense stand) was used in this assay. Specificity was determined by the prior addition of a 100-fold excess of unlabeled competitor consensus oligonucleotide. Reactions were separated on 6% nondenaturing acrylamide gels in Tris-buffered EDTA. Gels were dried, and protein-DNA complexes were visualized by autoradiography.

Statistical analyses

Data points, and values in the text and figure legends, represent the mean ± SEM of "n" independent determinations of BEAS-2B cells or using primary epithelial cells from different donors. Concentration-response curves were analyzed by least squares, nonlinear iterative regression with the "Graphpad Prism" curve fitting program (GraphPad software) and EC₅₀ values were subsequently interpolated from curves of best fit. Statistical differences were determined using the Kruskal-Wallis test followed by Dunn’s multiple comparison test or a Wilcoxon signed rank test as appropriate. Values of p < 0.05 were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Effect of IFN- on the release of the CXCR3 ligands from human BEAS-2B cells

BEAS-2B cells released CXCL9, CXCL10, and CXCL11 spontaneously with basal levels of 121.8 ± 39.2 (n = 6), 56.3 ± 24.4 (n = 6), and 65.7 ± 13.0 pg/ml (n = 6), respectively (Fig. 1A). Exposure to IFN- increased CXCL9, CXCL10, and CXCL11 release by BEAS-2B cells in a concentration-dependent manner with EC₅₀ values of 7.1 ± 4.4, 5.2 ± 2.3, and 6.9 ± 1.2 ng/ml, respectively (n = 6) (Fig. 1A). From these data, a submaximal concentration of IFN- (10 ng/ml), that caused the significant release of CXCL9, CXCL10, and CXCL11 (2.26 ± 0.77 (p < 0.05), 2.31 ± 0.60 (p < 0.05), and 1.41 ± 0.42 ng/ml (p < 0.05), respectively, n = 6), was selected for subsequent experiments. Similar experiments examined the time course of mRNA expression of CXCL9, CXCL10, and CXCL11 following stimulation of BEAS-2B cells with IFN-. Expression of mRNA for all three chemokines increased in a time-dependent manner with maximal expression at 8–12 h (Fig. 1B). A similar time-course experiment was also performed examining protein release indicating that IFN--induced protein release of all three CXCR3 ligands was not detectable until 8 h post exposure and increased up to 24 h (Fig. 1C).

View larger version (9K): [in this window] [in a new window]   FIGURE 1. Effect of IFN- on CXCR3 ligand release by BEAS-2B cells. A, BEAS-2B cells were stimulated with increasing concentrations of IFN- and supernatants harvested after 20 h, and the concentrations of CXCL9 (), CXCL10 (), and CXCL11 (•) were measured by ELISA. Data are presented as mean ± SEM; n = 6. B, BEAS-2B cells were stimulated with 10 ng/ml IFN- and cells lysed for mRNA analysis at time points up to 24 h. mRNA expression of CXCL9 (), CXCL10 (), and CXCL11 (•) were measured by TaqMan RT-PCR. Data are presented as mean ± SEM; n = 3. C, BEAS-2B cells were stimulated with 10 ng/ml IFN- and supernatants harvested at time points up to 24 h and the concentrations of CXCL9 (), CXCL10 (), and CXCL11 (•) were measured by ELISA. Data are presented as mean ± SEM; n = 3.

Effect of IKK2 selective inhibitor TPCA-1 and the glucocorticosteroid dexamethasone on IFN--stimulated chemokine mRNA expression by BEAS-2B cells

From these experiments, a submaximal time point of 6 h was selected to examine the effects of IKK2 inhibition on gene expression. The effect of an anti-inflammatory glucocorticosteroid was compared with the IKK2-selective inhibitor TPCA-1 on IFN--stimulated CXCL9, CXCL10, and CXCL11 mRNA expression by BEAS-2B cells. Dexamethasone did not inhibit IFN--stimulated mRNA expression of the CXCR3 chemokines in BEAS-2B cells (Fig. 2, A–C). However, 1 µM dexamethasone significantly increased CXCL9 mRNA expression (p < 0.05). By contrast the IKK2 inhibitor, TPCA-1, suppressed IFN--induced CXCL9, CXCL10, and CXCL11 mRNA expression by BEAS-2B cells in a concentration-dependent manner (Fig. 2, A–C). The EC₅₀ values of the inhibitory effect of TPCA-1 on expression of CXCL9, CXCL10, and CXCL11 mRNA were 1.20 ± 0.36, 0.12 ± 0.03, and 0.34 ± 0.08 µM, respectively (n = 5). These data suggested that CXCL10 mRNA expression was more susceptible to inhibition by TPCA-1 compared with CXCL9 (p < 0.05), although there was no difference with CXCL11.

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Effect of dexamethasone, APCC, and TPCA-1 on mRNA expression and release of CXCR3 ligands from BEAS-2B cells stimulated with IFN-. BEAS-2B cells were incubated with the steroid dexamethasone (•) and an IKK2-selective inhibitor TPCA-1 () for 30 min before stimulation with IFN- (10 ng/ml) and lysed after 6 h and gene expression of CXCL9 (A), CXCL10 (B), and CXCL11 (C) quantified by TaqMan RT-PCR. Data are presented as percentage inhibition ± SEM; n = 5 where 100% indicates the level of mRNA induced by IFN- in the absence of inhibitors. BEAS-2B cells were incubated with the steroid dexamethasone (•), an IKK-selective inhibitor APPC (), and an IKK2-selective inhibitor TPCA-1 () for 30 min before stimulation with IFN- (10 ng/ml). For CXCR3 ligand release supernatants were harvested after 20 h and CXCL9 (D), CXCL10 (E), and CXCL11 (F) release were measured by ELISA. Data are presented as percentage inhibition ± SEM; n = 6 where 100% indicates the level of protein released by IFN- in the absence of inhibitors.

Effect of IKK-selective small molecule inhibitors and the glucocorticosteroid dexamethasone on IFN--stimulated chemokine release from BEAS-2B cells

Similarly to the lack of inhibition on mRNA expression, dexamethasone had little effect on the release of the CXCR3 chemokines from these cells, whereas the IKK2 inhibitors TPCA-1 and APPC suppressed IFN--induced CXCL9, CXCL10, and CXCL11 release from BEAS-2B cells in a concentration-dependent manner (Fig. 2, D–F) (Table I). The EC₅₀ values of the inhibitory effect of TPCA-1 on release of CXCR3 ligands suggested that CXCL10 release was more susceptible to inhibition by TPCA-1 compared with CXCL9 (p < 0.05), although there was no difference with CXCL11 (Table I). By contrast, the second IKK1/2 inhibitor, APPC, had similar potency for all three ligands (Table I).

View this table: [in this window] [in a new window]   Table I. IC50 values and maximal inhibition values for the IKK inhibitors TPCA-1 and APPC and dexamethasone on IFN- (10 ng/ml) induced CXCR3 ligand release from BEAS-2B cells

Effect of IKK2-selective inhibitor TPCA-1 and the steroid dexamethasone on IFN--stimulated chemokine release from human primary bronchial epithelial cells

To support the use of the BEAS-2B cells, similar experiments were performed using primary human bronchial epithelial cells. Stimulation of primary bronchial epithelial cells with 10 ng/ml IFN- led to the release of 32.9 ± 2.0 (n = 6), 25.9 ± 1.9, and 25.7 ± 2.2 ng/ml CXCL9, CXCL10, and CXCL11, respectively. TPCA-1 (1 µM) significantly inhibited the IFN--induced release of CXCL9 (Fig. 3A), CXCL10 (Fig. 3B), and CXCL11 (Fig. 3C) by 52.0 ± 6.7%, 54.8 ± 3.3%, and 28.0 ± 8.1%, respectively; (n = 6). By contrast, 1 µM dexamethasone had no effect on the release of these chemokines (Fig. 3). However, previous studies have demonstrated that this concentration of dexamethasone has profound inhibitory activity on other stimuli in primary human bronchial epithelial cells (38).

View larger version (9K): [in this window] [in a new window]   FIGURE 3. Effect of TPCA-1 and dexamethasone on release of CXCR3 ligands from human primary bronchial epithelial cells stimulated with IFN-. Human primary bronchial epithelial cells were treated with the IKK-selective inhibitor TPCA-1 (1 µM) or dexamethasone (1 µM) for 30 min before stimulation with IFN- (10 ng/ml). Supernatants were harvested after 20 h and CXCL9 (A), CXCL10 (B), and CXCL11 (C) release were measured using Duoset ELISA. Data are presented as chemokine release ± SEM; n = 6, where *** represents p < 0.001; **, p < 0.01; *, p < 0.05 for differences between IFN- stimulation and TPCA-1 and also, where applicable between TPCA-1 and dexamethasone.

Validation of the IKK2-dependent release of CXCR3 ligands from IFN--stimulated BEAS-2B cells using dominant-negative adenoviral constructs of proteins from the IKK pathway

To confirm a role for IKK-2 in the release of the CXCR3 ligands from BEAS-2B cells, dominant-negative adenoviral constructs of proteins from the IKK pathway that lack kinase activity were overexpressed in these cells. Initial experiments were designed to validate the expression and activity of the dominant negative adenoviral constructs used in the subsequent experiments. Using EMSA, both IL-1- and TNF--dependent NF-B DNA binding was attenuated by overexpression of an IKK2 (KA) dominant-negative construct and a truncated version of IB (IBN), whereas overexpression of an IKK1 (KM) dominant construct and the null virus had no effect (Fig. 4A). In addition, we examined the effects of these construct in an adenoviral delivered NF-B reporter assay (Fig. 4B). Again, overexpression of the IKK2 dominant-negative construct and IBN inhibited both IL-1- and TNF--induced NF-B activation (Fig. 4B). It remained possible that the lack of effect of the IKK1 dominant-negative construct may be due to reduced protein expression within the BEAS-2B cells. To address this, Western blots were performed on the cells following infection with the adenoviral constructs and confirmed similar levels of protein expression of these constructs under the experimental conditions (Fig. 4C).

View larger version (60K): [in this window] [in a new window]   FIGURE 4. Effect of dominant-negative adenoviral constructs on NF-B DNA binding and NF-B activation. A, BEAS-2B cells were infected with adenoviral constructs (MOI 100) for 24 h before stimulation with IL-1 (1 ng/ml) or TNF- (10 ng/ml) for 5 min and nuclear extracts prepared for EMSA; 100x indicates reactions performed in the presence of 100-fold excess of unlabeled probe. The autoradiograph showing NF-B-DNA binding is representative of n = 4. B, BEAS-2B cells were infected with adenoviral constructs (MOI 100) for 24 h before stimulation with IL-1 (1 ng/ml) or TNF- (10 ng/ml) for a further 8 h and then harvested for luciferase assay. Data are presented as percentage stimulation ± SEM; with TNF- (10 ng/ml); n = 4. C, The cytoplasmic fraction of the BEAS-2B cell lysates used in A were analyzed by Western blotting to detect the hemagglutinin (HA)-tagged IKK1(KM), the FLAG-tagged IKK2 (KA) and IBN.

View larger version (9K): [in this window] [in a new window]   FIGURE 5. Effect of dominant-negative adenoviral constructs on release of CXCL10 from BEAS-2B cells stimulated with IFN-. BEAS-2B cells were treated with adenoviral constructs (IKK1, dominant-negative IKK1 construct; IKK2, dominant-negative IKK2 construct; IKK, dominant-negative IKK construct; IBN, truncated IB; TBK, TBK dominant-negative construct; GFP, GFP construct; Null, empty vector control construct) (MOI 100) for 24 h before stimulation with 10 ng/ml IFN-. Supernatants were harvested 20 h after stimulation and CXCL10 release were measured using Duoset ELISA. Data are presented as percentage inhibition ± SEM; n = 5, where *** represents p < 0.001; **, p < 0.01; *, p < 0.05 for differences from IFN- stimulation.

Effect of IFN- on IB phosphorylation in BEAS-2B cells

The data from the dominant-negative adenoviral construct for IKK2 provided support that IKK2 is important in IFN--dependent release of CXCL10. However, the lack of effect of IBN suggests that IB is not involved in CXCL10 release. To investigate this further, Western blotting was performed to determine whether IB was phosphorylated in response to IFN- stimulation. Incubation of BEAS-2B cells with 1 ng/ml IL-1 induced a time-dependent increase in expression of phosphorylated IB that was maximal at 20 min (Fig. 6A). However, there was no phosphorylation of IB with 10 ng/ml IFN-, over a time course of up to 60 min, although there is evidence of phosphorylated IB with 10 min treatment with IL-1 (Fig. 6B). Furthermore, there was no degradation of IB up to 60 min of treatment with IFN-, although IB had degraded with 10 min of treatment with IL-1 (Fig. 6C).

View larger version (13K): [in this window] [in a new window]   FIGURE 6. Western blot of phosphorylated and total IB. A, BEAS-2B cells were incubated with 1 ng/ml IL-1 for increasing periods of time up to 30 min. B, BEAS-2B cells were incubated with 10 ng/ml IFN- for increasing periods of time up to 60 min. A sample of BEAS-2B cells incubated for 10 min with IL-1 was used as a positive control on this gel. C, BEAS-2B cells were incubated with 10 ng/ml IFN- for increasing periods of time up to 60 min. A sample of BEAS-2B cells incubated for 10 min with IL-1 was used as a positive control on this gel. Cell lysates were prepared, insoluble proteins were removed, and 20 µg of soluble extract were denatured and subjected to electrophoresis on 4–12% (w/v) SDS polyacrylamide gels. Proteins were transferred to nitrocellulose and probed with an anti-phosphorylated IB Ab (A and B) or anti-IB Ab (C). The primary Abs were detected with peroxidase-conjugated secondary Abs and labeled proteins were detected by ECL. The blot is representative of n = 4 experiments and pIB indicates phosphorylated IB.

Effect of IFN- on translocation of the p65 subunit of NF-B in BEAS-2B cells

Although IB was not phosphorylated in response to IFN-, it remained possible that other members of this signal transduction pathway could play a role in IFN--dependent release of the CXCR3 ligands. Therefore, to determine whether p65 was involved, BEAS-2B cells were stimulated with IFN-, IL-1, or TNF- and the cellular localization of the p65 subunit of NF-B was monitored using confocal microscopy. When BEAS-2B cells were stimulated with IL-1 (1 ng/ml) or TNF- (1 ng/ml) for 30 min, p65 translocated to the nucleus (Fig. 7A, b and c). However, in resting cells (Fig. 7Aa) and those treated with IFN- (10 ng/ml; Fig. 7Ad) p65 remained predominantly cytoplasmic (Fig. 7A).

View larger version (41K): [in this window] [in a new window]   FIGURE 7. Cellular localization of p65 subunit of NF-B in BEAS-2B cells stimulated with IFN-, IL-1, or TNF-. A, BEAS-2B cells were incubated with either 1 ng/ml IL-1, 10 ng/ml TNF-, or 10 ng/ml IFN- for 30 min. The cells were fixed and stained with an anti-p65 Ab and detected with an Alexa 488-labeled secondary anti-Ig Ab, depicted in green (a–d), or DAPI depicted in blue (e–h). i–l, overlay of these two images. The images are representative of n = 5 experiments. B, BEAS-2B cells were incubated with either 10 ng/ml TNF- or 10 ng/ml IFN- for 15, 30, or 45 min. Levels of the nuclear NF-B subunit p65 were measured using a TransAM assay. Data are presented as OD at 450 nm ± SEM; n = 3.

Effect of IFN- on an adenoviral delivered NF-B reporter in BEAS-2B cells

Although the most abundant form of NF-B is p65, it remained feasible that other subunits, such as Rel B, could be involved in IFN--dependent release of the CXCR3 ligands. To determine whether stimulation with IFN- caused NF-B gene activation, or not, an adenoviral luciferase reporter for NF-B was transfected into BEAS-2B cells and the effect of cytokine stimulation was monitored after 6 h. IL-1 and TNF- both induced concentration- (Fig. 8A) and time-dependent (Fig. 8B) increases in NF-B-dependent luciferase activation, however, IFN- had no effect. Using this system, the effect of TPCA-1 and APPC were validated as IKK inhibitors against NF-B-dependent activation. TPCA-1 and APPC inhibited TNF--induced NF-B activation with EC₅₀ values of 0.096 ± 0.019 µM and 0.102 ± 0.014 µM, n = 5, respectively.

View larger version (11K): [in this window] [in a new window]   FIGURE 8. The effect of IFN- on activation of an NF-B-dependent luciferase reporter. BEAS-2B cells were infected with MOI 100 of an adenovirus carrying a NF-B-dependent reporter (Ad-NF-B-luc). After incubation for 24 h in K-SFM, the cells were then changed to fresh K-SFM. (A) BEAS-2B cells were stimulated with either IFN- (), IL-1 (•), or TNF- () for 6 h or (B) for up to 10 h with either 10 ng/ml IFN- (), 1 ng/ml IL-1 (•), or 1 ng/ml TNF- (), before harvesting and assaying for luciferase activity. Data are presented as luciferase activation (arbitrary units) ± SEM; n = 4.

Because the NF-B pathway did not appear to be involved in IFN- release of CXCR3 ligands, we examined whether the classical IFN--signaling pathway involving STAT-1 could be directly affected by IKK inhibitors. Incubation of BEAS-2B cells with IFN- (10 ng/ml) caused an increase in expression of phosphorylated STAT-1 at both Tyr⁷⁰¹ and Ser⁷²⁷ after 5 min that increased until 30 min (data not shown). The 91-kDa band detected for Tyr⁷⁰¹, comigrated with a band of comparable size from a commercial extract of phosphorylated STAT-1 (data not shown). Incubation of BEAS-2B cells with dexamethasone, or the IKK2-selective inhibitor TPCA-1 before stimulation with IFN- did not effect the expression of phosphorylated STAT-1 at Tyr⁷⁰¹ and Ser⁷²⁷ (Fig. 9A).

View larger version (11K): [in this window] [in a new window]   FIGURE 9. The effect of IFN- on phosphorylation of STAT-1 and STAT-1 DNA binding. A, BEAS-2B cells were treated with 10 ng/ml IFN- for increasing periods of time up to 30 min. Cell lysates were prepared, insoluble proteins were removed, and 20 µg of soluble extract were denatured and subjected to electrophoresis on 4–12% (w/v) SDS polyacrylamide gels. Proteins were transferred to nitrocellulose and probed with anti-phosphorylated STAT-1 Tyr701 and Ser727 Abs. The primary Abs were detected with peroxidase-conjugated secondary Abs and labeled proteins were detected by ECL. The blot is representative of n = 5 experiments and pSTAT-1 indicates phosphorylated STAT-1. B, BEAS-2B cells were treated with IKK-selective inhibitors or dexamethasone for 30 min before stimulation with 10 ng/ml IFN-. After 1 h, nuclear extracts of the cells were prepared and a TransAM assay for STAT-1 was performed. Data are presented as percentage of IFN- stimulation ± SEM; n = 4.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
CXCL9, CXCL10, and CXCL11 are chemotactic for human CD8⁺ T lymphocytes that express the specific receptor CXCR3 (12). In the COPD lung, there are increased numbers of these cells and it is thought that they contribute to the lung inflammation through the release of IFN- and to the emphysematous changes by the release of proteolytic enzymes, including perforin and granzyme B (5). Therefore, understanding the regulation and recruitment of these CTLs might offer novel therapeutic targets for treating this debilitating disease. In the present study we addressed the recruitment of these cells by examining the signaling transduction pathways that are activated to produce the CXCR3 chemokines. This study confirmed the work of Sauty et al. (13) and demonstrated that the bronchial epithelial cell was a likely source of IFN--stimulated CXCR3 chemokines. We have shown that there is a peak of mRNA expression for the CXCR3 ligands between 8 and 12 h poststimulation with IFN- and that there is a concomitant increase in protein release that is detectable from 8 h and increases up to 24 h. Classically, this pathway is thought to involve activation of STAT-1 and is resistant to glucocorticosteroids. This is pertinent to the inflammation observed in COPD, as administration of glucocorticosteroids does not alter the underlying inflammation profile of this disease (39, 40). This lack of steroid responsiveness was confirmed in the present study where dexamethasone failed to inhibit IFN--stimulated CXCR3 chemokine mRNA expression and release from primary bronchial epithelial cells or the BEAS-2B cell line. These data are also confirmed by Sauty et al. (13) who observed that dexamethasone did not inhibit CXCR3 ligand mRNA expression in human epithelial cells. By contrast, inhibition of IKK2, using two structurally different, highly selective, IKK inhibitors and overexpression of a dominant-negative construct for IKK2, abolished the release of CXCR3 ligands from IFN--stimulated BEAS-2B cells. TPCA-1 is highly selective for IKK2 with a –log IC₅₀ of 7.74 ± 0.18 for purified enzyme with a 22-fold selectivity over IKK-1 and >550-fold selectivity over other kinases and enzymes (18). APPC, is a different chemical class of compound compared with TPCA-1, has >100-fold selectivity over 50 other common kinases and enzymes, but has approximate equal selectivity for IKK1 and IKK2. Both compounds inhibit IFN--induced CXCR3 ligand production with comparable potency and efficacy suggesting that the observed effect is mediated by IKK2. The EC₅₀ values obtained for the inhibition of IFN--stimulated CXCR3 ligand production by TPCA-1 are similar to those for inhibition of TNF--induced activation of an NF-B-dependent luciferase reporter assay in BEAS-2B cells. These data also concur with previously reported EC₅₀ values for inhibition of LPS-induced human monocyte production of TNF-, IL-6, and IL-8 (41), and LPS-stimulated IL-6 production by myocytes (42).

The involvement of IKK2 was further supported by inhibition of CXCL10 release when BEAS-2B cells were transfected with the IKK2 dominant-negative adenoviral construct, but not that for IKK1. These data concur with those of Sizemore et al. (43) who showed that IFN--induced CXCL10 production by mouse embryo fibroblasts (MEFs) was IKK2, but not IKK1, dependent. The present study demonstrated that over expression of the dominant-negative construct for TBK also inhibited IFN--stimulated CXCL10 release from BEAS-2B cells by 30%. TBK has sequence similarity with IKK1 and IKK2, although it may have a role in the activation of the transcription factors, IFN regulatory factor (IRF)-3 and IRF7 (44, 45, 46), but is not thought to impact on the STAT-1 and IRF1 pathway. As the IKK inhibitors TPCA-1 and APPC do not affect TBK, the inhibition of CXCL10 ligand production by the small molecule inhibitors and adenoviral constructs, is therefore, attributed to IKK2.

Classically, activation of the IKK complex leads to phosphorylation of IB, leading to its degradation and the subsequent activation and nuclear translocation of NF-B (22). We have shown that IFN- does not induce IB phosphorylation, translocation of the p65 subunit of NF-B to the nucleus, or activation of an adenoviral-delivered luciferase construct for the classical NF-B reporter in BEAS-2B cells, when stimulated with IFN-. We suggest that IKK2 is involved in the IFN--stimulated release of CXCL9, CXCL10, and CXCL11 through a novel mechanism that is not mediated by NF-B. These data are supported by evidence in MEFs where IFN- failed to induce the NF-B promoter and did not induce NF-B activation in IKK1/2-deficient MEFs (43). Moreover, in p65/NF-B-deficient MEFs and cells containing a mutated form of IB lacking the phosphorylation sites, the IFN--stimulated production of CXCL10 was unaffected (43). The precise pathways involving IKK2 with IFN- signaling are unknown, although IKK2 has been implicated in a number of NF-B-independent pathways including proinflammatory, anti-apoptotic, proliferative, and tumor-promoting actions of this protein (47).

The classical signaling pathway of IKK and NF-B shows no overlap with signaling pathway involving JAK-STAT-1 and IFN--responsive element/-activated sequence. We found that IFN--induced Tyr⁷⁰¹ and Ser⁷²⁷ STAT-1 phosphorylation in BEAS-2B cells was unaffected by the kinase activity of IKK2 and that the IKK2-selective inhibitors had no effect on STAT-1 DNA binding. In support of our study, CXCL10 mRNA levels were suppressed in IKK1/2-deficient MEFS stimulated with IFN-, however, STAT-1 phosphorylation was found to be comparable to wild-type MEFs (43). We, therefore, postulate that IKK2 mediates IFN--induced release of the CXCR3 ligands from human epithelial cells via a STAT-1-independent mechanism.

In this study, we have identified a robust system to determine the effects of IFN- stimulation on the release of CXCR3 ligands. The involvement of IKK2 in IFN--stimulated release of the CXCR3 ligands could potentially have important implications for the treatment of COPD, as release of CXCR3 chemokines by human epithelial cells indicates that the mechanism is dependent upon IKK2, without involvement of NF-B and presents a novel mechanism for anti-inflammatory activity.

	Acknowledgments

 
IKK1 (KM), IKK2 (KA), and the null virus were a gift from Prof. Michael Karin. The IBN virus was a gift from Dr. Peter Loser. The TBK and IKK viruses were a gift from Dr. Anthony J. Coyle.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Address correspondence and reprint requests to Dr. Louise E. Donnelly, Airway Disease Section, Guy Scadding Building, National Heart and Lung Institute, Imperial College London, Dovehouse Street, London, SW3 6LY, U.K. E-mail address: l.donnelly{at}imperial.ac.uk

² Abbreviations used in this paper: COPD, chronic obstructive pulmonary disease; IKK, IB kinase; TPCA, 2-[(aminocarbonyl)amino]-5-[4-fluorophenyl]-3-thiophenecarboxamide; TBK, TANK-binding kinase; K-SFM, keratinocyte serum-free medium; MOI, multiplicity of infection; DAPI, 4',6-diamidino-2-phenyl-indole hydrochloride; MEF, mouse embryo fibroblast; IRF, IFN regulatory factor; APPC, 2-amino-6-(2'cyclopropylmethoxy-6'-hydroxy-phenyl)-4-piperidin-3-yl- pyridine-3-carbonitrile.

Received for publication April 13, 2007. Accepted for publication August 22, 2007.

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