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The Journal of Immunology, 2007, 179, 7030-7041
Copyright © 2007 by The American Association of Immunologists, Inc.
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Inhibition of NF-{kappa}B Activation Reduces the Tissue Effects of Transgenic IL-131

Svetlana P. Chapoval2,*, Amal Al-Garawi3,§, Jose M. Lora4,§, Ian Strickland{dagger}, Bing Ma*, Patty J. Lee*, Robert J. Homer{ddagger}, Sankar Ghosh{dagger}, Anthony J. Coyle5,§ and Jack A. Elias*

* Section of Pulmonary and Critical Care Medicine, {dagger} Section of Immunobiology, and {ddagger} Department of Pathology, Yale University School of Medicine, New Haven, CT 06520; and § Millennium Pharmaceuticals, Cambridge, MA 02142


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-13 is a major Th2 cytokine that is capable of inducing inflammation, excessive mucus production, airway hyperresponsiveness, alveolar remodeling, and fibrosis in the murine lung. Although IL-13 through its binding to IL-4R{alpha}/IL-13R{alpha}1 uses the canonical STAT6-signaling pathway to mediate these tissue responses, recent studies have demonstrated that other signaling pathways may also be involved. Previous studies from our laboratory demonstrated that IL-13 mediates its tissue effects by inducing a wide variety of downstream genes many of which are known to be regulated by NF-{kappa}B. As a result, we hypothesized that NF-{kappa}B activation plays a critical role in the pathogenesis of IL-13-induced tissue alterations. To test this hypothesis, we compared the effects of transgenic IL-13 in mice with normal and diminished levels of NF-{kappa}B activity. Three pharmacologic approaches were used to inhibit NF-{kappa}B including 1) PS1145, a small molecule inhibitor of I{kappa}B{alpha} kinase (IKK2), 2) antennapedia-linked NF-{kappa}B essential modulator-binding domain (NBD) peptide (wild-type NBD), and 3) an adenoviral construct expressing a dominant-negative version of IKK2. We also crossed IL-13-transgenic mice with mice with null mutations of p50 to generate mice that overproduced IL-13 in the presence and absence of this NF-{kappa}B component. These studies demonstrate that all these interventions reduced IL-13-induced tissue inflammation, fibrosis and alveolar remodeling. In addition, we show that both PS1145 and wild-type NBD inhibit lung inflammatory and structural cell apoptosis. PS1145 inhibits caspase activation and up-regulates inhibitor of apoptosis protein cellular-inhibitor of apoptosis protein 1 (c-IAP-1). Therefore, NF-{kappa}B is an attractive target for immunotherapy of IL-13-mediated diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Interleukin-13 is a key mediator of asthma, a complex chronic disease of the airways which is characterized by eosinophilic and lymphocytic airway inflammation, airway remodeling with subepithelial fibrosis, and mucus metaplasia (1, 2, 3, 4). In murine models of the asthmatic response, IL-13 has been shown to be both necessary and sufficient for the generation of similar asthma-like deleterious tissue alterations (1, 2, 3, 4). The mechanisms of these responses, however, have not been fully defined.

In addition to its asthma-like effects, IL-13 is also a powerful stimulator of alveolar remodeling and pulmonary parenchymal fibrosis (5, 6). This led to the belief that IL-13 plays important roles in the pathogenesis of pulmonary emphysema and/or asthmatic pseudoemphysema (5, 7) and diseases like idiopathic pulmonary fibrosis (8). Support for these concepts has been derived from studies that demonstrate that IL-13 is induced by cigarette smoke exposure (9), that polymorphisms of IL-13 correlate with the development of chronic obstructive pulmonary disease (COPD)6 (10, 11) and that prominent alterations in IL-13R expression can be seen in biopsies from patients with idiopathic pulmonary fibrosis (12). In accord with the importance of these responses, extensive studies of the mechanisms of these and other IL-13-induced responses have been undertaken. These studies demonstrated that IL-13 mediates its tissue effects by inducing a prominent downstream target gene cascade with alveolar remodeling being mediated, at least in part, by matrix metalloproteases (MMPs) and cathepsins (5) while the fibrosis is mediated, at least in part, by the induction and activation of TGF-β1 (6).

IL-13 binds to a polymeric receptor that contains IL-4R{alpha} and IL-13R{alpha}1 subunits. The majority of the studies of this receptor have focused on its ability to induce STAT-6 tyrosine phosphorylation and subsequent signaling. In murine models, IL-13 has been shown to mediate many of its asthma-relevant responses through the activation of the STAT-6 pathway in bronchial epithelial cells (13). However, there is reason to believe that other signal transduction pathways also contribute to the pathogenesis of asthma-like Th2 responses (14, 15). Importantly, studies from our laboratory recently demonstrated that ERK1/2 MAPK activation is required for optimal IL-13 stimulation of specific chemokines, MMPs, and protease inhibitors in the murine lung (15). The importance of other signaling pathways, however, has not been adequately addressed.

The transcription factor NF-{kappa}B is a critical mediator of a variety of inflammatory and remodeling responses that are central to innate and adaptive immunity The prototype of this family is a dimer made up of p50 (NF-{kappa}B1) and p65 (RelA) subunits. Under basal conditions, it is sequestered in the cytoplasm of the cell bound to I{kappa}B (16, 17). After activation I{kappa}B is targeted for degradation and the p50/p65 dimer enters the nucleus where it binds to consensus sequences in promoters of NF-{kappa}B-responsive genes (16, 17). NF-{kappa}B signaling has been implicated in the pathogenesis of asthma. This is based on studies highlighting exaggerated levels of NF-{kappa}B activation in tissues from asthmatics (18, 19) and airway epithelium from animal models of the disorder (20, 21). It is also based on studies that demonstrated that the NF-{kappa}B pathway plays an essential role in the induction of airway eosinophilic inflammation (22) and regulation of Th2 cytokine production by interfering with GATA-3 expression (23). Indeed, a variety of interventions that inhibit NF-{kappa}B activation including the administration of p65 antisense oligonucleotides (24), NF-{kappa}B decoy nucleotides (25), small molecule inhibitors of both NF-{kappa}B and AP-1 (26, 27), and I{kappa}B superrepressor constructs (CC10-I{kappa}B{alpha}SR-transgenic (tg) mice) (28) have all been shown to inhibit aspects of the experimental asthmatic response. Exaggerated NF-{kappa}B signaling has also been implicated in the pathogenesis of COPD (29, 30). However, despite the important roles that IL-13 is believed to play in asthma and other Th2 inflammatory and remodeling responses, the importance of NF-{kappa}B in the pathogenesis of IL-13-induced tissue responses has not been adequately addressed.

We hypothesized that NF-{kappa}B plays a critical role in the pathogenesis of IL-13-induced tissue alterations. To test this hypothesis, we compared the effects of lung-targeted tg IL-13 in mice in which NF-{kappa}B was regulated normally and mice in which a variety of interventions inhibited NF-{kappa}B activation. Four different NF-{kappa}B-based approaches were used in these studies including 1) the small molecule I{kappa}B kinase (IKK) 2 inhibitor PS1145, 2) the NF-{kappa}B essential modulator (NEMO)-binding domain (NBD) peptide NF-{kappa}B inhibitor, 3) virally administered IKK2-dominant-negative (DN) constructs, and 4) mice with null mutations of p50. These studies demonstrate that NF-{kappa}B plays an important role in IL-13-induced tissue responses because each of these interventions leads to a significant reduction of IL-13-induced tissue inflammation and remodeling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

The generation, characterization, and genotyping of CC10-IL-13 constitutive IL-13 (cIL-13) tg and CC10-rtTA-IL-13 inducible IL-13 (iIL-13) tg mice, and doxycyclin (DOX)-containing water administration for the inducible tg expression were described in details previously (1, 2, 5). Briefly, the Clara cell 10-kDa promoter was used in both tg systems that drive IL-13 expression in airway Clara cells. In the cIL-13 tg mice, gene expression is initiated in utero and continues throughout the animal life. In the iIL-13 tg mice, two constructs (2, 5) are used that allow the expression of IL-13 only when the tg-expressing animal is receiving DOX. This inducible system allows us to target IL-13 expression to animal adulthood and differentiate an adult-onset response from the development-dependent one. p50 knockout (KO) mice were purchased from The Jackson Laboratory; cIL-13 tg/p50 KO and iIL-13 tg/STAT6 KO (15) mice were generated by breeding. tg-negative littermates were used as wild-type (WT) controls in this study. Mice were bred and maintained under specific pathogen-free conditions within the animal facility at either the Yale University Medical School or Millennium Pharmaceutical until the day of sacrifice.

Materials

PS1145. IKK2 inhibitor PS1145 was synthesized at Millennium Pharmaceuticals (31). Specificity of PS1145 inhibition against IKK2 was demonstrated using 14 kinases, including protein kinase A, protein kinase C, casein kinase II, Src, Lck, calmodium-dependent kinase 2, IL-1R-associated kinase, stress-activated protein kinase 2{alpha} (p38{alpha}), MEK 1/2 (MAPK kinases), microtubule affinity regulating kinase-activated protein kinase 2, ERK2, JNK2, and epidermal growth factor receptor tyrosine kinase (31). The effectiveness and specificity of this compound was also determined in several B cell-like (activated B cell) lines (32). Treatment of activated B cell lines with PS1145 rapidly induced a series of gene expression changes similar to those induced by expression of genetic inhibitors of NF-{kappa}B.

Adenovirus vectors expressing GFP and either WT or mutant DN IKK2 (Ad-IKK2-WT or Ad-IKK2-DN, respectively) were constructed at the Millennium Pharmaceuticals as described previously (33). IKK2-DN has a lysine to methionine mutation at the amino acid 44 of the IKK2 ATP-binding site (34).

Antennapedia-linked NBD. The peptide sequences corresponding to the WT-NEMO-NBD and mutant µ-NBD of IKK2, their synthesis and storage have been described previously (35, 36). Briefly, the WT-NBD peptide contains the region of IKK2 from T735 to E745 synthesized in tandem with a membrane permeabilization sequence from the Drosophila antennapedia homeodomain protein. The µ-NBD peptide is identical except that W739 and W741 are replaced by alanines to render it biologically inactive. Each sequence was commercially synthesized and subsequently HPLC purified using a C18 reverse-phase column.

Experimental protocols

Four different ways of interference with NF-{kappa}B pathway activation in described above tg systems were used in this study.

(1, 2, 3) iIL-13 tg mice. 1) tg-positive and -negative littermates (5 wk old (w.o.), n = 4/group) were pretreated with oral gavage administration of PS1145 (50 mg/kg in 100 µl of 0.5% carboxymethylcellulose (CMC; Sigma-Aldrich) in sterile PBS (Invitrogen Life Technologies) per mouse using a 1-ml sterile latex-free syringe (BD Biosciences) with a sterile nonpyrogenic needle (Popper and Sons) 3 h before turning tg on with DOX water administration. Then mice were treated with PS1145 every 24 h thereafter by the same fashion for 7 consecutive days. On day 8, mice were euthanized and bronchoalveolar lavage (BAL) was performed as described previously (1, 2, 5). The dose and route of PS1145 for the treatment of mice were experimentally established at Millennium Pharmaceuticals in the delayed-type hypersensitivity study (Millennium Pharmaceuticals PS1145 efficacy summary statement, data not shown).

2) tg-positive and -negative littermates (5 w.o., n = 4/group) were pretreated by i.p. injection with 250 µg of WT-NBD peptide in 100 µl of 75% DMSO 6 h before DOX administration. A mutated version, µ-NBD peptide was used as a control. Mice were then treated with a relevant peptide every 24 h thereafter by the same fashion for 7 consecutive days. On day 8, mice were euthanized and BAL was performed as described. The optimal dose and route for WT-NBD administration were determined in the carrageenan-induced paw edema study (37).

3) tg-positive and -negative littermates (8 w.o., n = 3–5/group) were inoculated intranasally with adenovirus vectors expressing either GFP, IKK2-WT, or IKK2-DN in concentration of 109 viral particles/100 µl of PBS/mouse under anesthesia. The dose of adenovirus for intranasal administration to mice was experimentally determined previously (38). Twenty four hours later, mice were placed on DOX water for the tg induction. Control mice were kept on regular water. Mice were sacrificed on day 8 for the assessments.

cIL-13 tg mice. tg-positive and -negative littermates (6–8 w.o., n = 3–7/group) were dosed with oral gavage administration of PS1145 as described above for 17 consecutive days. Mice were euthanized 24 h after last administration of the compound for the assessments.

p50 KO mice. Six w.o. WT and p50 KO animals (n = 4/group) were treated intranasally with 5 µg/50 µl of PBS/mouse of recombinant mouse (rm) IL-13 (R&D Systems). Control animals (n = 2/group) received PBS instillations.

cIL-13 tg/p50 KO mice. To study the effect of p50 genetic ablation on IL-13-induced lung tissue phenotype, cIL-13 tg mice were bred to p50 KO animals.

The BAL levels of IL-13 were determined by ELISA (R&D Systems). Of note, neither PS1145 treatment nor adenovirus vectors administration had effect on BAL IL-13 levels in tg mice ranging from 400 to 725 pg/ml. The maximal mean differences of BAL IL-13 concentrations within the experimental group ranged from 19 to 209 pg/ml with maximal SEM differences from 75 to 158.

BAL immunoblot

Equal volumes of BAL were adsorbed onto trans-blot transfer medium nitrocellulose 0.2-µm membrane (Bio-Rad) using Minifold II dot-blot manifold (Schleicher & Schuell) (15). The membrane was dried, rehydrated, blocked with 5% nonfat milk (Nestle) in TBST, and incubated with anti-Muc-5AC Ab (Lab Vision) diluted 1/500 in blocking solution. After washing, membrane was incubated with HRP-conjugated goat anti-mouse IgG Ab (Sigma-Aldrich) diluted 1/1000 in blocking solution. Color development was done using ECL detection reagents kit (Amersham Biosciences) according to the manufacturer’s instruction. Image analysis was performed using the AlphaImager system (Alpha Innotech) with AlphaEaseFC software.

Histology and immunohistochemistry

The lungs were removed from euthanized mice on day 8 of experimental protocol. Sections were prepared as previously described (39) using 10% formalin for fixation and paraffin for embedding, and then stained either with H&E, periodic-acid Shiff (PAS) with diastase, or Masson’s trichrome. Stains were performed in the Research Pathology Laboratory at the Yale University.

TUNEL assay

TUNEL assay was performed on formalin-fixed lung tissue using a commercial kit as per the manufacturer’s instructions (In Situ Cell Death Detection kit; Roche).

EMSA

Lung tissue was obtained from iIL-13 tg mice being on DOX for 18 h, 48 h, and 8 days. Lung tissue from iIL-13 tg/STAT6 KO animals was obtained after 10 days of DOX administration. One-month-old cIL-13 tg mice were used in experiments. Tissues were snap-frozen, nuclear proteins were isolated and analyzed with radioactively labeled DNA probes containing the p65-binding site as described previously (40). Nuclear extracts from OVA-treated BALB/c mouse lungs were used as positive controls. Five micrograms of nuclear extract in 20 µl of the binding reaction was used for the time course of NF-{kappa}B induction study and 3 µg of extract/60 µl reaction was used in the experiments aiming to define the effect of STAT6 on IL-13-induced NF-{kappa}B activation.

OVA treatment

BALB/c mice were treated with OVA according to a previously described protocol with some modifications (41). Briefly, mice were sensitized and boosted by i.p. injection with 70 µg of chicken OVA (Sigma-Aldrich) in 200 µl of alum (Resorptar; Indergen). Then, on day 12, mice were anesthetized by i.p. injection of 100 mg/kg ketamine plus 10 mg/kg xylazine and challenged intranasally with 50 µg/50 µl of OVA/PBS. Tissue was isolated and processed for the assay 48 h after challenge.

Lung volume measurement and morphometric analysis

Lung volume was assessed via volume displacement and alveolar size was calculated from the mean of cord length of the airspace using the modified NIH Image program as described previously (5).

Airway obstruction measurements

Baseline enhanced pause (PenH) was measured using saline nebulization to the unrestrained mice in a whole body plethysmograph (Buxco) as previously described (1). Measurements were taken for 10 min and the average PenH response was plotted.

mRNA analysis

Total RNA was prepared from lung tissue; each sample was then subjected to RT-PCR with sense and antisense primers for specific genes as described previously (5, 6, 42).

Western blot

Whole lung lysate preparation, protein extraction, basic Western blot procedure, and protein detection were previously described (43). Primary Ab used in this study were as follows: anti-caspase-3 and -9 (clones 3G2 and 9504, respectively; Cell Signaling), anti-caspase-7 p20 and -8 p20 (clones N-17 and H-134, correspondingly; Santa Cruz Biotechnology), anti-c-IAP-1 (clone HIAP2; Travigen) and X-chromosome-linked inhibitor of apoptosis protein (R&D Systems). Appropriate secondary Ab were obtained from Santa Cruz Biotechnology. The ECL detection reagents kit (Amersham Biosciences) was used according to the manufacturer’s instruction.

Caspase activity measurements

Colorimetric kits were used for caspase-8 (APT171; Chemicon International) and caspase-3 (CaspACE; Promega) bioactivity detection according to manufacturer’s instructions.

Statistics

Data are summarized as mean ± SEM. To calculate significance levels between experimental groups, the Student t test (Microsoft Excel) was performed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
iIL-13 tg mice were generated as described in Materials and Methods. When DOX is administered to tg mice for 1 mo, the mice lungs were characterized by an asthma-like response with inflammatory cell infiltration, airway wall thickening, subepithelial fibrosis, and mucus metaplasia. In addition, IL-13 expression causes emphysema in these mice with enhanced lung volumes and tissue alveolar enlargement. Emphysema can be observed in tg mice after 7 days of DOX administration and continuously progress further. A significant inflammatory response in tg lungs was readily observed at 18 h of DOX administration as compared with WT mice. The inflammation was consisting primarily of macrophages and eosinophils (data not shown). The tissue effects observed in IL-13 tg mice were IL-13 dependent and IL-4 independent as no substantial IL-4 transcript and protein levels in IL-13 tg lungs were detected previously (1).

The time-course study of tg induction (24 h, 48 h, 1 wk, 1 mo, and 3 mo of DOX administration time points) demonstrated increased levels of BAL IL-13 within 24 h as compared with WT mice (129.4 ± 10.4 pg/ml vs 8.2 ± 1.5 pg/ml, correspondingly) and the levels stabilized within 48 h ranging from 534.9 to 598.8 pg/ml (data not shown).

Targeted expression of IL-13 to murine lung induces NF-{kappa}B activation

Using EMSA, we demonstrated an early transient increased level of NF-{kappa}B activity in lungs obtained from iIL-13 tg mice being on DOX for 18 h (Fig. 1A) followed by a decrease in its activity after 48 h of DOX administration. NF-{kappa}B activity in iIL-13 tg mouse lungs being on DOX water for 8 days was not different from that observed in control littermates without DOX exposure (Fig. 1B). We found much stronger NF-{kappa}B activation in the lungs of 1-mo-old cIL-13 tg mice as compared with DOX-driven iIL-13 tg at 18 h of DOX (Fig. 1A).


Figure 1
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  		FIGURE 1. Kinetic of IL-13 lung tissue expression-induced activation of NF-{kappa}B (A and B) and its negative regulation by STAT6 (B). A, Targeted expression of IL-13 to murine lung induces NF-{kappa}B activation in the inducible and constitutive IL-13 tg mice. Lung tissue nuclear extracts were isolated from mice being on DOX water for 18 h, 48 h, and 8 days. Nuclear extracts were analyzed using EMSA with radioactively labeled DNA probes containing {kappa}B-binding site as described in Materials and Methods. Nuclear extracts obtained from untreated and OVA-treated WT mice served as controls. Note an early transient increase in NF-{kappa}B activation in iIL-13 tg lungs (18 h of DOX). B, A course of transgene response and STAT6 down-regulate NF-{kappa}B activity in tg lungs. Lung tissue nuclear extracts were obtained from mice being on DOX water for 10 days and processed in a DNA-binding reaction as described in Materials and Methods. NF-{kappa}B activity was quantified by densitometry using GeneSnap software (Syngene). Data are expressed as mean ± SEM for iIL-13 tg mice with and without DOX exposure (n = 4–6 mice/group/experiment) and as a trend for mice with or without STAT6 deficiency which include WT (n = 2), STAT6 KO (n = 2), iIL-13 tg (n = 3), and iIL-13 tg/STAT6 KO (n = 4) mice.

 
Therefore, both the level of IL-13 stimulation and the phase of IL-13 responses affect tissue NF-{kappa}B activation. As STAT6 is the canonical signaling molecule for IL-13 in tg lungs, we examined the effect of STAT6 on the IL-13-induced NF-{kappa}B activation by using iIL-13 tg mice on either STAT6-sufficient or -deficient background for the EMSA. We observed an increase in NF-{kappa}B activity in iIL-13 tg/STAT6 KO lungs (Fig. 1B) as compared with iIL-13 tg littermates. The lungs of STAT6 KO mice also showed an increased trend of NF-{kappa}B activity when compared with WT mice. This demonstrates a possible down-regulatory role of STAT6 in IL-13-mediated NF-{kappa}B induction.

To understand the role of induced activation of NF-{kappa}B in the IL-13-induced lung phenotype, we have used four different ways of interference with NF-{kappa}B activation outlined in Materials and Methods and studied their effects on individual parameters of this phenotype.

Interference with NF-{kappa}B activation reduces IL-13-induced BAL and lung parenchyma inflammation

As we reported previously, either constitutive or inducible targeted expression of IL-13 in the lung of mice leads to the local inflammation comprising macrophages, eosinophils, and lymphocytes (1, 2, 5). This inflammatory response was noted around small and large airways and extended to lung parenchyma.

To test whether NF-{kappa}B inhibition will have an effect on IL-13-induced lung inflammation, iIL-13 tg mice and WT counterparts were treated with PS1145 as described in Materials and Methods. Vehicle-treated iIL-13 tg mice demonstrated a dramatic (eight times) increase in BAL total cell recovery over appropriate WT controls (Fig. 2A). Although in WT mice, macrophages were dominant cells in BAL (94.2 ± 2.1%), in the iIL-13 tg mice macrophages consisted of only 37.5 ± 5.5% of total cell recovery, with significant admixture of eosinophils (21.0 ± 3.0%) and lymphocytes (31.7 ± 4.8%). The IKK2 inhibitor had no effect on BAL cellularity in WT mice (data not shown), however, it significantly reduced inflammatory cell infiltration into BAL in the iIL-13 tg mice. In tg mice, neutrophilic inflammation was less prominent and thus no discernable affect of PS1145 treatment could be observed. The BAL IL-13 protein levels amounted to 600 pg/ml and were not affected by treatments. The BAL TNF-{alpha} levels were below the limit of detection of the assay, whereas IL-1β levels were low and ranged from 18 to 67 pg/ml in iIL-13 tg mice from different treatment groups. There was no significant difference in IL-1β content between treated and untreated tg mice within each experiment.


Figure 2
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  		FIGURE 2. Pharmacologic (PS1145, A and B), adenovirus-mediated gene transfer (C) or genetic (D) intervention with NF-{kappa}B activation reduces BAL cellular infiltration in either iIL-13 tg (A and C) or cIL-13 tg (B) mice, and p50 KO mice administered rmIL-13 (D). A–D, Mice were treated with compounds as described in Materials and Methods. Mean cell counts ± SEM are shown. The numbers of mice used are given in Materials and Methods unless specified here. BALs were collected 24 h after the last treatment. A, Data shown are from one of two representative experiments. *, p < 0.05, CMC-treated iIL-13 tg mice vs WT counterparts. **, p < 0.05, PS1145-treated vs CMC-treated tg mice. B, *, p < 0.05, CMC-treated tg vs WT mice. **, p < 0.05, PS1145- vs CMC-treated cIL-13 tg mice. C, Data shown are from two combined representative experiments, n = 6–9 mice/group. *, p < 0.05, WT mice vs PBS, GFP, IKK2-WT, or IKK2-DN treated iIL-13 tg. **, p < 0.05, IKK2-DN-treated iIL-13 tg mice vs tg mice with other treatments specified with exclusion for: macrophages, IKK2-DN vs GFP; eosinophils, IKK2-DN vs IKK2-WT; and lymphocytes, IKK2-DN vs PBS. D, *, p < 0.05, eosinophil numbers, rmIL-13-treated WT vs p50 KO animals.

 
To test whether an upstream inhibition of NF-{kappa}B activation with WT-NBD peptide will have an effect on IL-13-induced lung inflammation, iIL-13 tg mice and WT counterparts were treated with either WT-NBD peptide or control µ-NBD peptide as described in Materials and Methods. Marked influx of eosinophils in the BAL of tg mice was significantly (p < 0.00004) reduced with WT-NBD peptide treatment when compared with either DMSO or µ-NBD peptide-treated mice (data not shown). The protein expression levels of IL-13 in the BAL of tg lungs were not significantly affected by treatments and ranged from 515 to 725 pg/ml.

To determine whether NF-{kappa}B inhibition will have an effect on already established IL-13-induced lung inflammatory phenotype, cIL-13 tg mice and WT counterparts were treated with PS1145 as described above for more prolonged time. The IL-13-induced BAL inflammatory cell accumulation observed in tg mice was significantly reduced with this treatment (Fig. 2B). PS1145 showed a down-regulatory effect on the eosinophilic and lymphocytic components of assessed BAL cellular response.

To confirm that not only systemic but also local lung NF-{kappa}B activation inhibition effectively interferes with IL-13-induced lung inflammation, we used the adenovirus- mediated overexpression of either IKK2-WT or IKK2-DN in the lung epithelial cells. A recombinant adenovirus expressing GFP was used as a control virus. The expression of GFP was used as an indicator of transduction efficiency and was then assessed in the lung tissue by fluorescent microscopy 4 days after gene delivery. We have found that adenovirus efficiently transduces GFP into respiratory epithelial cells (data not shown). As expected, Ad-IKK2-WT transduction enhances mononuclear cell and specifically lymphocyte infiltration in BAL whereas the use of IKK2-DN leads to the inhibition of this inflammatory response (Fig. 2C). The BAL IL-13 protein expression levels of PBS or adenoviral vector-treated tg mice ranged from 404 to 452 pg/ml.

Short-term intranasal administration of rmIL-13 to WT mice by protocol described in Materials and Methods induced a prominent eosinophil infiltration into BAL (Fig. 2D). In contrast, rmIL-13 did not have the same effect in p50 KO mice. Although in cytokine-treated WT mice eosinophils were predominant type among BAL cells, in p50 KO mice eosinophil influx into BAL was significantly reduced.

The results of histological examination of the lung tissues paralleled the cell numbers in the BAL fluids. Furthermore, lung tissues of vehicle-treated iIL-13 tg mice demonstrated an inflammatory response around small and large airways (data not shown, for references, see 1 and 5) and in parenchyma (Fig. 3A). This inflammatory response was significantly reduced with either PS1145 (Fig. 3A) or WT-NBD (Fig. 3C) treatments. Higher magnification revealed that both treatments reduced the appearance of eosinophils and enlarged macrophages in the airways (Fig. 3, A and C). A marked inflammatory cell infiltration in the airways of vehicle-treated cIL-13 tg mice was observed at the time of tissue assessments (Fig. 3B). The administration of PS1145 significantly inhibited the cellular infiltrations. Similar inhibitory effects on IL-13-induced lung inflammatory response were observed in IKK2-DN-treated mice compared with PBS-, GFP, and AD-IKK2-WT-treated mice (data not shown). Therefore, both specific systemic or local inhibition of NF-{kappa}B activation or p50 genetic ablation reduce IL-13-induced BAL and lung parenchyma inflammation.


Figure 3
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  		FIGURE 3. Inhibition of lung tissue inflammations in the iIL-13 tg (A) and cIL-13 tg (B) mice treated with PS1145 and (C) iIL-13 tg mice treated with antennapedia-WT-NBD peptide. A and B, Mice were treated with either PS1145 or vehicle alone as described in Materials and Methods. A, Original magnification x10 and x100; B, original magnification x4. C, Mice were treated with either antennapedia-WT- or µ-NBD peptide, or vehicle alone as described in Materials and Methods, original magnification x4 and x100. Note specific inhibition of lung tissue eosinophilia in the iIL-13 tg mice with both types of treatments (A and C, original magnification x100). Arrowheads, Eosinophils.

 
Interference with NF-{kappa}B activation reduces IL-13-induced lung alveolar remodeling

Alveolar destruction with alveolar wall enlargement is characteristic feature of emphysema (43). Our previous studies have demonstrated that IL-13 expression in the lung of mice causes emphysema with enhanced lung volumes and cord length (5). To assess whether NF-{kappa}B inhibition will have an effect on IL-13-induced lung emphysema, iIL-13 tg mice were treated with IKK2 inhibitor PS1145 as described above and then the lungs were inflated with PBS at gradually increasing pressure for lung volume measurements. We observed a significant increase in lung volume in iIL-13 tg mice over WT counterparts (Fig. 4A). Vehicle treatment did not have an effect on lung volume whereas PS1145 treatment caused a substantial reduction in this feature of emphysema. The emphysema observed in the iIL-13 tg mice was also characterized by histological (Fig. 4B) and morphometric (Fig. 4C) evaluation of alveolar enlargement, where the down-regulatory effect of PS1145 on both features of lung tissue destruction could be appreciated.


Figure 4
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  		FIGURE 4. Pharmacologic (PS1145, A–D), genetic (E) or adenovirus-mediated gene transfer (F) intervention with NF-{kappa}B activation reduces alveolar remodeling in the iIL-13 tg (A–C and F) and cIL-13 tg (D and E) mice. Lung volume (A), alveolar histology (B, original magnification x4), and chord length (C, original magnification x10) were evaluated in the iIL-13 tg mice treated with either PS1145 or vehicle alone. Alveolar tissue destruction was examined histologically (original magnification x4) in the PS1145-treated (D) or bred to p50 KO (E) cIL-13 tg mice, and the iIL-13 tg mice with adenovirus-mediated IKK2-DN gene transfer (F). A, Mean lung volume ± SEM are shown, n = 3–7 mice/group. *, p < 0.005, WT mice vs nontreated or CMC-treated iIL-13 tg; **, p < 0.045, PS1145-treated vs nontreated or CMC-treated iIL-13 tg mice. C, Mean chord length ± SEM are shown. *, p < 0.003, WT vs treated and untreated iIL-13 tg mice; **, p < 0.02, PS1145-treated vs other groups of tg mice.

 
Comparison of histologic appearances of cIL-13 tg mice treated with either PS1145 or vehicle alone (Fig. 4D) or bred to p50 KO animals (Fig. 4E) demonstrated a significant down-regulatory effect on IL-13-induced alveolar wall enlargement. Similarly, interference with NF-{kappa}B activation by means of WT-NBD peptide also lead to similar effects on IL-13-induced alveolar remodeling (data not shown). Finally, the local inhibition of NF-{kappa}B activation with IKK2-DN had a profound effect on this tissue phenotype (Fig. 4F and data not shown). Therefore, both specific systemic or local delivery of NF-{kappa}B inhibitors, or indeed p50 genetic ablation, reduce IL-13-induced alveolar remodeling.

Interference with NF-{kappa}B activation does not affect IL-13-induced mucus production

Mucus hypersecretion is another prominent feature of IL-13 function in the lung (1, 5, 42). The effect of IKK2 inhibitors on IL-13-induced mucus production was examined using PAS staining of the lung tissue sections and the assessment of local Muc-5AC production by BAL immunoblot. tg IL-13 caused increases in goblet cell number in the lung epithelium and Muc-5AC concentration in BAL (Fig. 5, A and B). Neither PS1145 nor WT-NBD treatments altered the IL-13-induced MUC-5AC response (Fig. 5B). Therefore, both specific systemic or local inhibition of NF-{kappa}B activation, or p50 genetic ablation (data not shown), had no effect on IL-13-induced mucus production.


Figure 5
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  		FIGURE 5. The absence of PS1145 and WT-NBD effect on mucus production in the iIL-13 tg mice. Mucus was evaluated by histological examination (A, original magnification x20) and immunoblot for MUC 5AC in BAL (B).

 
p50 genetic ablation down-regulates IL-13-induced lung tissue fibrosis

Subepithelial fibrosis is another prominent feature of a selective IL-13 overexpression in the lung (1, 6). Masson’s trichrome stains of cIL-13 tg mouse lung tissues revealed enhanced collagen deposition in the subepithelial regions of large and small airways, as well as loosely packed collagen around blood vessels (Fig. 6A). The increase in lung collagen was significantly reduced in cIL-13 tg/p50 KO mice.


Figure 6
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  		FIGURE 6. Reduction of IL-13-induced lung tissue fibrosis (A) and airway obstruction (B) by genetic p50 ablation. The following number of mice was used in this study: 8 w.o., n = 4/group; 10/11 w.o., n = 4–7/group. A, Pictures represent the slides of trichrome-stained lungs. B, *, p < 0.05, baseline PenH of 11 w.o. cIL-13 tg vs cIL-13 tg/p50 KO mice and 8 w.o. counterparts.

 
p50 genetic ablation down-regulates IL-13-induced airway obstruction

Both diseases, asthma and COPD are characterized by increased airway resistance due to increased airway narrowing (44). As we reported previously, the baseline airway resistance of 4 w.o. cIL-13 tg and WT mice were similar (1). However, there was an increase in this parameter of lung obstruction in the 2- to 3-mo-old tg mice (1). The magnitude of airway obstruction in the aged cIL-13 tg mice was evaluated using a noninvasive assessment technique of pulmonary mechanics. There were clear PenH alterations in 6 w.o., 10 w.o., and 6-mo-old tg mice as compared with WT age controls (data not shown). Interestingly, this alteration was not observed in 8 w.o. tg (data not shown). Similarly, 8 w.o. WT, cIL-13 tg, and cIL-13 tg/p50 KO mice demonstrated an equal baseline PenH to PBS nebulization (Fig. 6B). However, 11 w.o. tg mice showed a significant increase in PenH which was eliminated with p50 genetic ablation. Thus, NF-{kappa}B activation plays a critical role in airway obstruction in part as a secondary event to inflammation.

The IKK2 inhibitor PS1145 has no effect on the expression of selected IL-13-regulated CC and CXC chemokines, proteases, and antiproteases

We previously demonstrated that IL-13 regulates chemokine production in the airways and defined the role of CCR1 and CCR2 signaling in the IL-13-induced lung phenotype (45, 46). As expected, IL-13 expression leads to potent stimulation of MCP-1, -2, -3, -5, MIP-1{alpha}, -3{alpha}, -3β, eotaxin (data not shown), and secondary lymphoid-tissue chemokine (6Ckine) (Fig. 7A). PS1145 treatment did not affect IL-13-stimulated expression of these chemokines. We also examined whether IL-13 induces CXC chemokine dysregulation in the lung (Fig. 7B). Compare with tg-negative littermates, we observed stimulation of the mRNA accumulation representing lipopolysaccharide-induced CXC-chemokine, IFN-{gamma}-induced protein, stromal cell-derived factor-1, and MIP-2g in the iIL-13 tg mice. Chemokine induced by IFN-{gamma}, monokine induced by IFN-{gamma} was down-regulated by transgene expression and was not affected by PS1145 treatment. Interference with NF-{kappa}B pathway with PS-1145 treatment did not alter the ability of IL-13 to stimulate the mRNA expression encoding these chemokines. In addition, this IKK2 inhibitor did not affect the expression of IL-13-regulated selected proteases/antiproteases and cathepsins in the murine lung (Fig. 7C).


Figure 7
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  		FIGURE 7. PS1145 has no effect on the selected IL-13-regulated CC (A) and CXC (B) chemokines, proteases, and antiproteases (C). Lung tissue RNA was obtained as described in Materials and Methods and examined for the levels of mRNA encoding selected markers by RT-PCR. Each evaluation is representative of two experiments.

 
The IKK2 inhibitor PS1145 reduces IL-13-induced lung inflammatory and structural cell apoptosis

To study the role of apoptosis in the IL-13-induced lung tissue phenotype and the effect of NF-{kappa}B inhibition on apoptosis, we performed TUNEL staining. IL-13 expression induces a significant increase in the number of TUNEL-positive nuclei compared with WT mouse lung tissue (10.5 ± 1.8% and 2.6 ± 0.3%, correspondingly, Fig. 8, A and B). Light microscopic evaluation revealed that the cells undergone apoptosis were the lung structural (bronchial epithelial and type I and II alveolar epithelial cells) and inflammatory cells (macrophages, eosinophils, and lymphocytes). Interference with NF-{kappa}B activation by PS1145 administration reduces lung inflammatory and structural cell apoptosis to 5.9 ± 0.8%. Similar results were obtained using WT-NBD treatment (data not shown).


Figure 8
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  		FIGURE 8. PS1145 reduces IL-13-induced lung inflammatory and structural cell apoptosis (A and B) by interfering with IL-13-induced activation of caspases -3, -7, and -8 (C and D) and up-regulating of cIAP-1 but not XIAP in the lungs (E) (upper panels, x10; lower panels, x40). A, Cell apoptosis was revealed using TUNEL stain of lung tissue histology slides, magnification x40. B, Apoptotic cells were counted in four representative high power fields for each slide and expressed as mean ± SEM. *, p < 0.007, WT vs CMC- or PS1145-treated iIL-13 tg mice. **, p < 0.025, PS1145- vs CMC-treated tg mice. C, Western blots were performed on lung lysates as described in Materials and Methods. Total form of proteins and they cleaved forms are marked with arrows showing an appropriate molecular mass. Note the absence of cleaved form of caspase 3 and diminished levels of cleaved caspases 7 and 8 in PS1145- vs CMC-treated mice. Data shown are representative of two to three experiments. D, Caspase activities in lung lysates were measured as described in Materials and Methods. Data shown are for two individual mice per experimental group. E, Expression of caspase inhibitors cIAP-1 and XIAP was evaluated using appropriate Ab in Western blot of lung lysates. Data shown are representative of two to three experiments.

 
The IKK2 inhibitor PS1145 down-regulates IL-13-induced activation of regulatory and effector caspases and up-regulates the lung expression of cIAP-1

To understand the mechanisms of IL-13-regulated apoptosis induction and PS1145-mediated apoptosis down-regulation in the lung, we performed the evaluation of caspases involved in death receptor or mitochondrial stress pathways. As can be seen from the Western blot analyses, IL-13 expression results in the induction of activating caspase-8 and effector caspases-3 and -7 (Fig. 8C). Activation of caspase-9 was not observed (data not shown). Evaluation of caspase-3 and -8 bioactivity paralleled the Western blot results (Fig. 8D). This suggests that IL-13 expression induces lung tissue apoptosis at least in part by activation of the extrinsic cell death pathway. The expression of "inhibitor of apoptosis protein" cIAP-1 but not XIAP was down-regulated in lung tissue homogenates obtained from iIL-13 tg mice and up-regulated with PS1145 treatment (Fig. 8E).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
IL-13 acting through its receptor IL-4R{alpha}/IL-13R{alpha}1 triggers a variety of the tissue effects in the lung (1, 2, 3, 4, 5, 6, 42, 45, 46). The most prominent of these effects is an airway inflammation composed of macrophages, eosinophils, and lymphocytes (1, 42). As NF-{kappa}B plays a central role in regulation of genes and effectors in the inflammatory response (16, 17), we hypothesized that interference with NF-{kappa}B activation would be beneficial in reducing the inflammatory effects of tg IL-13. Despite previous beliefs that IL-13 has an inhibitory effect on NF-{kappa}B (47, 48), we have found that a targeted IL-13 expression in the lung leads to the induction of local NF-{kappa}B activity (Fig. 1).

NF-{kappa}B belongs to a family of highly conserved transcription factors (16, 17) with the major form in cells being a p50/p65 heterodimeric complex. Phosphorylation and degradation of I{kappa}B{alpha} is a key step in the activation of NF-{kappa}B. A complex composed of IKKs (IKK{alpha}, IKKβ, and IKK{gamma}) is an essential component in NF-{kappa}B-signaling mechanisms participating in I{kappa}B{alpha} phosphorylation. Although IKK{alpha} (IKK1) and IKKβ (IKK2) are catalytic subunits, IKK{gamma} is a regulatory subunit. IKK2 is essential for activation of the classical NF-{kappa}B pathway through phosphorylation of serine residues 32 and 36 of I{kappa}B{alpha}, resulting in the ubiquitination and subsequent degradation of I{kappa}B{alpha} by the 26S proteasome. The degradation of I{kappa}B{alpha} exposes a nuclear translocation sequence facilitating translocation of NF-{kappa}B to the nucleus and activation of {kappa}B-responsive genes. In this work, we used four different ways of interference with NF-{kappa}B activation in our tg IL-13 model. First, three of them were specifically targeting IKK2 while the fourth one affected NF-{kappa}B.

First, we used a systemic administration of small molecule IKK2 inhibitor PS1145 (31, 32). Specificity of PS1145 against the IKK complex was determined previously (31) by testing the compound against 14 kinases and in B cell lines (32). Interestingly, a recent study by Fujioka et al. (49) proposed a mechanism of NF-{kappa}B-dependent regulation of AP-1 activity, thus PS1145 could potentially affect the AP-1-signaling pathway in the lung.

Second, we used a systemic administration of an upstream NF-{kappa}B inhibitor peptide, WT-NBD linked to a 16-aa peptide from antennapedia that facilitates the cytoplasmic uptake of the peptide (35, 36). This peptide competitively inhibits interaction between NEMO and IKK2. A mutated version of the peptide, µ-NBD that is unable to block this interaction was used as a control. As it has been shown previously, cell-permeable NBD peptides capable of blocking NEMO/IKK interactions inhibit the induction of NF-{kappa}B activation without altering its basal activity (35). Third, we used a local delivery of a DN mutant form of IKK2 (33). Lastly, we crossed cIL-13 tg mice with p50 KO mice and studied the effect of the complete absence of p65/p50 biological activity on IL-13-induced phenotype.

All four ways of interference with NF-{kappa}B activation lead to significant reduction of IL-13-induced inflammation in tg mouse lung tissues (Figs. 2 and 3). This stresses the importance of NF-{kappa}B signaling in IL-13-induced inflammatory diseases. Our results go in accord with other studies demonstrating down-regulatory effects of NF-{kappa}B inhibitions on asthmatic inflammation (24, 25, 26, 27, 28) and suppression of the inflammatory processes associated with other pathologies (31, 37, 50). In addition, individual NF-{kappa}B-based interventions lead to the reduction of other aspects of IL-13-induced airway remodeling characteristic of asthma (51), such as airway wall thickening (Figs. 3 and 4) and subepithelial fibrosis (Fig. 6). Airway wall thickening by itself is not only a simple result of airway inflammation. It has a more complex pathology that, in addition to inflammatory cell infiltration, includes an increase in airway smooth muscle, edema, and connective tissue deposition (51). The exact role of NF-{kappa}B signaling in mediating these aspects of disease is not understood. The effect of the absence of p50 on IL-13-induced tissue fibrosis was examined in cIL-13 tg mice where the transgene is expressed in utero and continues to be expressed with mouse age (1, 3). Subepithelial and adventitial airway fibrosis were readily appreciated in these tg mice at one month after birth (1, 3), whereas parenchymal fibrosis is appreciated later when the mice are at least 2–3 mo old (6). The p50 genetic ablation leads to significant down-regulation of lung tissue collagen deposition which was visualized using trichrome tissue staining (Fig. 6A). Lung tissue fibrosis changes were not evaluated in iIL-13 tg mice using NF-{kappa}B interventions. The endpoint of the experiments in iIL-13 tg mice was day 8 of DOX administration which is too early to observe a significant difference in this phenotype parameter between WT and tg mice. When DOX water is administered to iIL-13 tg for a more prolonged time (>1 mo), subepithelial fibrosis is observed in these tg mice (3).

Interestingly, systemic NF-{kappa}B inhibition with either PS1145 or WT-NBD did not have a significant effect on airway mucus production and BAL content of MUC5AC (Fig. 5) although more assessments are necessary for a definitive conclusion. A recent study by Broide et al. (52) had demonstrated that OVA-treated CC10-Cre tg/IKKβ{Delta}/{Delta} mice, in which NF-{kappa}B signaling is specifically ablated in airway epithelium, exhibited a significant reduction in both peribronchial fibrosis and mucus production when compared with their WT counterparts. The observed effect on fibrogenesis in these mice was related to indirect down-regulation of TGF-β1 in epithelial cells and especially eosinophils. Nevertheless, the effect on mucus was not mechanistically explained in this study. However, an assessment of the relative number of PAS-positive cells/bronchus showed an obvious difference. In the literature, there is no definitive conclusion about the regulation of mucus production. It is believed that IL-13 regulates mucus in part through the transcriptional up-regulation of the MUC-5a/c gene (53). In contrast, MUC-5a/c activation could be regulated by both Src- and epidermal growth factor receptor-kinase dependent pathways involving NF-{kappa}B signaling (54). As others treatments related to the NF-{kappa}B inhibition in the airways demonstrated also a down-regulatory effect on OVA-induced mucus production (27, 28) through down-regulation of IL-13, our NF-{kappa}B inhibition strategies failed to do so most probably because they did not interfere with tg expression. Levels of BAL IL-13 were not affected by these treatments.

To gain additional insight into the mechanism(s) of NF-{kappa}B regulation by IL-13, we measured BAL protein levels of two potent NF-{kappa}B inducers, TNF-{alpha} and IL-1β. We were unable to find any detectable TNF-{alpha} in BAL fluids in tg mice, and the levels of IL-1β were low and not affected by the treatments targeting NF-{kappa}B. Therefore, the NF-{kappa}B-mediated tissue effects of IL-13 are IL-1β independent.

We have shown previously that STAT-6 signaling is a critical event in mucus production through a direct effect of IL-13 on airway epithelial cells (15). Therefore, STAT-6 signaling is a key pathway in mucus production in this setting. However, STAT-6-deficient mice were able to mount an airway hyperreactivity and fibrosis in response to fungal sensitization (55) suggesting that these parameters in a fungal asthma are IL-13 dependent but STAT-6 independent. They as well could be mediated by NF-{kappa}B or other signaling molecules. The role of other signal transduction pathways in IL-13-mediated diseases is especially important to investigate considering a current observation that IL-13 induces totally different patterns of gene expression in three major resident cell types in the airway wall such as epithelial cells, smooth muscle cells, and fibroblasts (14).

Studies presented here also demonstrate that all the ways of interference with NF-{kappa}B activation lead to the inhibition of another prominent feature of targeted IL-13 expression in the lung, namely alveolar remodeling which is a part of COPD phenotype (Fig. 4). This is a novel and very important observation as the role of NF-{kappa}B in COPD pathogenesis and the beneficial effects of NF-{kappa}B inhibition in this disease were not addressed previously. In COPD patients and smokers, there is an increase in local number of inflammatory cells selectively expressing active NF-{kappa}B (29). In addition, the epithelial and endothelial nuclear expression of p65 was up-regulated suggesting that NF-{kappa}B activation is one of the key molecular mechanisms in the airways in this disease. However, the mechanisms leading to this pathology are still undefined. Our previous study has shown that IL-13-induced emphysema with enhanced lung volumes and compliance is cathepsin and MMP mediated (5). Comparison of lung tissue expression of selected cathepsins and MMPs in PS1145-treated and untreated mice did not show any effect of the inhibitor on some protease/antiprotease expressions (Fig. 7C). Therefore, either other molecules regulated by NF-{kappa}B pathway activation may play role in emphysema pathogenesis or other protease/antiprotease expressions may be affected by the NF-{kappa}B inhibitor.

Extensive studies of the mechanisms of IL-13 effects from our and other laboratories have demonstrated the role of selective cytokines (6), chemokines, and their receptors (45, 46) in lung tissue pathogenesis. As it is shown in Fig. 7, A and B, the IL-13-regulated expression of selected markers was not affected by PS1145.

It is well known that some patients with asthma have an enhanced decline in lung function over time. Our previous study of the effect of IL-13 on airway physiology also demonstrated this trend when the baseline resistance was compared between 1-mo-old and 3-mo-old WT and tg mice (1). The magnitude of airway obstruction paralleled the age of mice and, therefore, the time of tg expression and the resulted tissue effects (data not shown). In the aged cIL-13 tg mice with the genetic ablation of p50, both the index of airway obstruction and airway hyperresponsiveness to methacholine exposure are abrogated, suggesting a critical role of NF-{kappa}B in lung physiology.

Finally, we studied the mechanism of PS1145 effect on the IL-13-induced phenotype by assessing its role in apoptosis. It is accepted now that NF-{kappa}B activation can induce both pro- and antiapoptotic effects (56). NF-{kappa}B activation inhibits programmed cell death and promotes cell survival by the induction of several apoptosis inhibitor genes including the caspase inhibitor XIAP (54). Moreover, treatments targeting a constitutive and inducible NF-{kappa}B activity lead to the induction of apoptosis (57). However, little is known about the mechanism by which NF-{kappa}B induces apoptosis. One such possibility is a regulation of FasL expression. Two NF-{kappa}B-binding sites in the FasL promoter region were identified in T cells (58). One of these {kappa}B sites directly contributes to FasL gene expression. Therefore, blocking NF-{kappa}B nuclear translocation inhibits FasL gene expression and activation-induced cell death. Analysis of Fas and FasL protein expression in the lung tissue homogenates obtained from PS1145-treated and control tg mice did not demonstrate such inhibition (data not shown). Our observation suggests that this mechanism has no role in the proapoptotic effect of NF-{kappa}B in the whole lung tissue context.

As caspases are the key effectors in apoptosis, we analyzed specific caspase expression and activity. We found that IL-13 expression induced activation of caspase-3, -7, and-8, which were down-regulated by PS1145 treatment (Fig. 8, C and D). To delineate the mechanism of caspase regulation in our model, we studied the expression of the inhibitors of apoptosis proteins which prevent cell death by inhibiting active caspases. Despite the fact that both XIAP and cIAP-1 are widely expressed and similarly regulated by NF-{kappa}B (59), we found that PS1145 had no effect on IL-13-induced XIAP expression but up-regulates cIAP-1 expression. In addition, the absence of caspase-9 activation in the system could potentially explain the absence of XIAP up-regulation which is known to inhibit active processed caspase 9 (60). In contrast, c-IAP-1, a specific inhibitor of caspases 3 and 7 (61), was up-regulated by PS1145 treatment in parallel to down-regulation of caspase 3 and 7 activation.

We recently demonstrated the role of STAT6 in IL-13-mediated lung tissue effects (15) by examining the tissue response in IL-13 tg mice with null mutation of STAT6. The role of STAT6 in NF-{kappa}B-dependent and -independent IL-13 effects can be postulated based on this observation and our current study. First, there was a reduction in MIP-1a and MIP-2 expression at the transcript and protein levels in the iIL-13 tg/STAT6 KO mice (15) what was not observed in IKK2 inhibitor PS1145-treated iIL-13 tg mice (Fig. 7, A and B). Therefore, the expression of these IL-13-induced chemokines is STAT6 dependent and NF-{kappa}B independent. Second, interference with NF-{kappa}B activation does not affect IL-13-induced lung mucus production and goblet cell hyperplasia (Fig. 5) whereas these responses were down-regulated in the iIL-13 tg/STAT6 KO mice when compared with iIL-13 tg mice. Muc-5AC gene expression was also not affected by NF-{kappa}B inhibition in this tg model (Fig. 5), however, it was affected by STAT6 deficiency (15). In addition to NF-{kappa}B and STAT6, IL-13 is a potent activator of MAPKs in vitro and in vivo (15). Furthermore, analysis of the immune response to OVA in STAT6 KO mice has shown that MCP-5, SDF-1, and IP-10 were among 60 STAT6-independent genes (62). These genes were not regulated by NF-{kappa}B as the treatment with PS1145 did not change their lung tissue transcript expression compared with vehicle-treated tg mice (Fig. 7, A and B). Therefore, different cells participating in IL-13-induced lung tissue response may have: 1) a different STAT6 dependence because of the absence or presence of IL-4R{alpha} and/or STAT6; 2) a simultaneous induction of different signaling pathways such as STAT6 and ERK1/2 (15), etc., that either enhance or suppress the expression of target genes, especially genes containing composite sites; 3) a consecutive induction of different signaling pathways regulating each other. A possible negative effect of STAT6 on NF-{kappa}B activation has been reported in vitro (63) and in vivo (64). Moreover, phosphorylated STAT6 can bind both p50 and p65 subunits of NF-{kappa}B directly and this interaction substantially enhances STAT6 DNA-binding activity and synergistically activates IL-4-responsive gene transcription (65).

Previous study from our section demonstrated that IL-13 also stimulates ERK1/2 pathway in the lung and this stimulation is STAT6 independent (15). In part, ERK1/2 pathway was required for the optimal IL-13-induced MMP-2 and -12 activation. In addition, this pathway was important for IL-13 stimulation of certain chemokines such as MIP-1{alpha} and MIP-2. We did not find any regulation of the corresponding MMPs and chemokines by inhibitors of NF-{kappa}B in this tg model (Fig. 7, A and C). Therefore, the IL-13-induced expression of these molecules in the lungs of tg mice is ERK1/2 dependent and NF-{kappa}B independent.

Currently, there is no direct evidence for the induction of NF-{kappa}B activation in cells by IL-13. The molecular mechanisms of NF-{kappa}B activation induction by IL-13 in cells requires further detailed examination. Our data, however, provide substantial evidence for the direct effect of IL-13 expression in vivo on local tissue NF-{kappa}B activation. Our data show that NF-{kappa}B plays a critical role in IL-13-induced lung pathology. In addition, they show that IL-13 regulates the expression of certain molecules critical for the IL-13-induced lung pathology in an NF-{kappa}B-dependent manner.

Identifying mediators that control/promote the chronic nature of asthma and COPD is critical for the development of treatment strategies. NF-{kappa}B provides an attractive target for immunotherapy. Our studies presented here demonstrate that specific NF-{kappa}B inhibition may have a therapeutic implication in asthma, COPD, and other diseases of lungs. The use of small molecule inhibitor is especially attractive. For example, a prolonged administration of PS1145 caused no toxicity in our and other studies (66) and resulted in more complete protection in IL-13-induced lung phenotype and graft-vs-host disease as compared with other NF-{kappa}B-directed treatments.


   Acknowledgments
 
We thank Drs. C. G. Lee, M. J. Kang, and S. J. Cho (Section of Pulmonary Medicine, Yale University School of Medicine) for technical support with primers and reagents.


   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 This work was supported by National Institutes of Health Grants HL-64242, HL-78744, HL-66571, HL-56389 awarded to J.A.E. Back

2 Address correspondence and reprint requests to Dr. Svetlana P. Chapoval at the current address: Department of Microbiology and Immunology, Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, 800 West Baltimore Street, Baltimore, MD 21201. E-mail address: schapoval{at}som.umaryland.edu Back

3 Current address: Division of Respiratory Diseases and Allergy, McMaster University, Hamilton, Ontario, Canada. Back

4 Current address: Department of Inflammation, Autoimmunity and Transplantation, Roche Pharmaceuticals, Palo Alto, CA 94304. Back

5 Current address: Inflammation and Autoimmunity, MedImmune, Gaithersburg, MD 20878. Back

6 Abbreviations used in this paper: COPD, chronic obstructive pulmonary disease; MMP, matrix metalloprotease; IKK, I{kappa}B kinase; NEMO, NF-{kappa}B essential modulator; NBD, NEMO-binding domain; DN, dominant negative; tg, transgenic; DOX, doxycyclin; KO, knockout; WT, wild type; w.o., week old; CMC, carboxymethylcellulose; BAL, bronchoalveolar lavage; PAS, periodic-acid Schiff; PenH, enhanced pause; IAP, inhibitor of apoptosis protein; rm, recombinant mouse; iIL-13, inducible IL-13; cIL-13, constitutive IL-13. Back

Received for publication July 26, 2006. Accepted for publication August 30, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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