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Published ahead of print on November 14, 2003, doi:10.1165/rcmb.2003-0066OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 651-661, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0066OC

Immunostimulatory DNA Inhibits Transforming Growth Factor-ß Expression and Airway Remodeling

Jae Youn Cho, Marina Miller, Kwang Je Baek, Ji Won Han, Jyothi Nayar, Monica Rodriguez, Sook Young Lee, Kirsti McElwain, Shauna McElwain, Eyal Raz and David H. Broide

Department of Medicine, University of California San Diego School of Medicine, La Jolla, California

Address correspondence to: David H. Broide, M.B., Ch.B., University of California San Diego, Basic Science Building, Room 5090, 9500 Gilman Drive, La Jolla, CA 92093-0635. E-mail: dbroide{at}ucsd.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Immunostimulatory sequences of DNA (ISS) inhibit eosinophilic airway inflammation, Th2 responses, and airway hyperreactivity (AHR) in mouse models of acute ovalbumin (OVA)-induced airway inflammation. To determine whether ISS inhibits airway remodeling, we developed a mouse model of airway remodeling in which OVA-sensitized mice were repeatedly exposed to intranasal OVA administration for 1–6 mo. Mice chronically exposed to OVA developed sustained eosinophilic airway inflammation and sustained AHR to methacholine compared with control mice. In addition, the mice chronically exposed to OVA developed features of airway remodeling, including thickening of the peribronchial smooth muscle layer, peribronchial myofibroblast accumulation, expression of the profibrotic growth factor transforming growth factor-ß, and subepithelial collagen deposition (assessed by quantitation of the area of peribronchial trichrome staining using image analysis, and immunostaining with anti–collagen V antibodies). Administration of ISS systemically every other week significantly inhibited the development of AHR, eosinophilic inflammation, airway mucus production, and importantly, airway remodeling in mice chronically exposed to OVA for 3–6 mo. In addition, ISS significantly reduced bronchoalveolar lavage and lung levels of the profibrotic cytokine transforming growth factor-ß. These studies demonstrate that ISS prevents not only Th2-mediated airway inflammation in response to acute allergen challenge, but also airway remodeling associated with chronic allergen challenge.

Abbreviations: airway hyperresponsiveness, AHR • bronchoalveolar lavage, BAL • enzyme-linked immunosorbent assay, ELISA • interferon, IFN • interleukin, IL • immunostimulatory sequences, ISS • mutated oligodeoxynucleotide, M-ODN • oligodeoxynucleotide, ODN • ovalbumin, OVA • Periodic Acid Schiff, PAS • phosphate-buffered saline, PBS • Respiratory Syncutial Virus, RSV • transforming growth factor, TGF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
As several Th2 cytokines (interleukin [IL]-4, IL-5, IL-9, IL-13) are considered to play an important role in the pathogenesis of asthma, therapeutic strategies based on globally inhibiting Th2 cells, or alternatively inhibiting individual Th2 cytokines, are currently being investigated as novel therapies in mouse models of asthma as well as in human clinical trials (1). The importance of individual Th2 cytokines to asthma is suggested from their individual ability to induce many of the inflammatory features noted in the airways of individuals with allergic asthma. For example, IL-4 is a switch factor for IgE synthesis, IL-5 an eosinophil growth factor, IL-9 induces mucus secretion, and IL-13 induces airway hyperreactivity (1). One novel therapeutic strategy to inhibit Th2 responses in asthma is to use immunostimulatory sequences (ISS) of DNA. Several studies have demonstrated that ISS are effective in inhibiting Th2 cytokine responses, eosinophilic airway inflammation, mucus production, and airway reactivity to methacholine in mouse models of acute asthma (2–10). ISS inhibits Th2 responses in mice when administered as preventive therapy before airway antigen challenge (2–6), or as therapy to reverse already established airway eosinophilic inflammation (7, 9, 10). ISS is effective in inhibiting Th2-mediated eosinophilic inflammation in the lung when administered either systemically or delivered directly to the mucosa of the airway (2). A single systemic dose of ISS inhibits Th2 responses to ovalbumin (OVA) inhalation challenge for at least 4 wk (6).

The mechanism by which ISS inhibits Th2 responses and eosinophilic inflammation in mouse models of asthma is not completely understood. The effect of ISS on T lymphocytes is indirect, as these cells do not express toll receptor-9 (TLR-9), which bind and mediate ISS signaling (11). ISS activates cells of the innate immune system (mainly dendritic cells and macrophages) to generate cytokines such as interferon (IFN)-{gamma} and IL-12, which promote naive T lymphocytes to develop into Th1 cells (12). However, although it is evident that ISS both induces Th1 responses and inhibits Th2 responses in vivo, it is not clear that induction of the Th1 response is necessary for the inhibitory effect of ISS on Th2 responses. This is evident from studies demonstrating that adoptive transfer of antigen-specific Th1 cells aggravates antigen-induced Th2 responses in the airway (13), suggesting that induction of Th1 responses may not mediate the beneficial effect of ISS in vivo in mouse models of asthma. ISS may therefore have a broader range of anti-inflammatory effects than initially suggested from its initial description as a Th1 adjuvant.

In addition to the inhibitory effect of ISS on allergen-induced airway inflammation and airway hyperreactivity, ISS also demonstrates antiviral and anti-inflammatory effects in a mouse model of Respiratory Syncytial Virus (RSV) induced airway inflammation, suggesting that it might also protect against viral triggers of asthma (14). ISS inhibits both RSV viral load, as well as airway inflammation and mucus expression in mice challenged intranasally with RSV (14). The mechanism of the inhibitory effect of ISS is likely to be mediated by induction of antiviral cytokines, including IFN-{alpha}, IFN-ß, and IFN-{gamma}, which inhibit RSV replication in vitro (14).

Although these studies have demonstrated that ISS may have benefit as therapy in acute allergen- and acute viral-induced asthma, they do not address whether ISS can inhibit features of airway remodeling associated with more chronic asthma. Asthma represents chronic inflammation of the airways followed by repair. The end result of repeated cycles of inflammation and repair may be imperfect repair resulting in a structurally and functionally abnormal remodeling of the airways. The structural remodeling changes noted in asthmatic airway include subepithelial fibrosis, an increased smooth muscle mass, and an increase in mucous glands (15). Attempts to study the mechanism and significance of airway remodeling in asthma have been hindered in humans with asthma by the difficulties inherent in prospectively following sufficient numbers of individuals with asthma with chronic airway inflammation for sufficient time periods (? years, ? decades) to observe whether remodeling of the airways occurs. In addition, most individuals with asthma are on anti-inflammatory therapy, which may prevent remodeling of the airways. Mouse models of asthma have provided important insight into the mechanism of acute allergen-induced airway inflammation and airway hyperreactivity (AHR), but have been considered unsuitable for the study of airway remodeling as recurrent nebulized antigen challenge induces tolerance instead of chronic airway inflammation (which is considered to precede airway remodeling). Our laboratory and others (16) have successfully developed a method of allergen administration that induces chronic eosinophilic inflammation and airway remodeling in mice for at least 6 mo. Associated with the development of chronic eosinophilic inflammation, these mice repetitively challenged with allergen develop sustained AHR, increased collagen synthesis, and increased expression of the lung epithelial mucus gene Muc-5ac. In this study we investigated the ability of ISS to inhibit features of airway remodeling associated with repetitive antigen challenge in OVA-sensitized mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Induction of Chronic Pulmonary Eosinophilic Inflammation
Female BALB/c mice (16 mice/group; Jackson Laboratory, Bar Harbor, ME) were used when they reached 8–10 wk of age. Mice were immunized subcutaneously on Days 0, 7, 14, and 21 with 25 µg of OVA (grade V; Sigma, St. Louis, MO) adsorbed to 1 mg of alum (Aldrich) in 200 µl normal saline. Intranasal OVA challenges (20 ng/50 µl in phosphate-buffered saline [PBS]) were administered on Days 27, 29, and 31 under isoflurane (Vedco, Inc., St Joseph, MO) anesthesia. Intranasal OVA challenges were then repeated twice a week for 1, 3, or 6 mo (see Figure 1 for protocol). Age- and sex-matched control mice were sensitized but not challenged with OVA during the 1-, 3-, or 6-mo study. Mice were killed 24 h after the final OVA challenge and bronchoalveolar lavage (BAL) fluid (BALF) and lungs were analyzed. In selected experiments mice were killed on Day 32 24 h after completion of the acute OVA challenge protocol. All animal experimental protocols were approved by the University of California, San Diego Animal Subjects Committees.



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  		Figure 1. Mouse OVA experimental protocol. Mice were immunized subcutaneously on Days 0, 7, 14, and 21 with OVA. Intranasal OVA challenges were administered on Days 27, 29, and 31, and then repeated twice a week for 1, 3, or 6 mo. Age- and sex-matched control mice were sensitized but not challenged with OVA during the 1-, 3-, or 6-mo study. Mice were killed 24 h after the final OVA challenge, and BALF and lungs were analyzed. ISS or diluent control was administered intraperitoneally starting 1 d before the first intranasal OVA challenge on Day 26, and then continued every other week 1 d before intranasal challenges for 1, 3, or 6 mo.

 
Therapeutic Intervention with ISS
Different groups of mice (16 mice/group) were administered intraperitoneal endotoxin-free (< 1 ng/mg DNA) phosphorothioate ISS-ODN (5'-TGACTGTGAACGTTCGAGATGA-3') (100 µg in 100 µl of sterile, endotoxin-free PBS; Trilink, San Diego, CA), mutated oligodeoxynucleotide (M-ODN) (5'-TGACTGTGAAGGTTGGAGATGA-3') which lacks the CpG motif present in ISS, or diluent control starting 1 d before the first intranasal OVA challenge on Day 27, and then continuing every other week 1 d before intranasal challenges for 1, 3, or 6 mo. In selected experiments, in mice challenged repetitively with OVA for 3 mo, we ended the experiment either in the week in which mice had received ISS, or in the week that they had not received ISS. Previous studies in our laboratory have demonstrated that ISS, but not M-ODN, inhibits OVA-induced eosinophilic inflammation and AHR when administered 1 d before OVA challenge (2), and that this inhibitory effect lasts at least 4 wk (6).

Determination of Airway Responsiveness to MCh In Vivo
We measured airway responsiveness to MCh using noninvasive as well as invasive methods.

Noninvasive measurement of airway responsiveness. Airway responsiveness was assessed 24 h after the final OVA challenge (after 1, 3, or 6 mo of repetitive OVA challenges), using a single-chamber whole body plethysmograph obtained from Buxco (Troy, NY), as previously described in this laboratory (2). The enhanced pause (Penh) correlates closely with pulmonary resistance measured by conventional two-chamber plethysmography in ventilated mice as measured in our (9) and other laboratories (17). In the plethysmograph, mice were exposed for 3 min to nebulized PBS and subsequently to increasing concentrations of nebulized MCh (Sigma, St. Louis, MO) in PBS using an Aerosonic ultrasonic nebulizer (DeVilbiss, Somerset, PA). After each nebulization, recordings were taken for 3 min. The Penh values measured during each 3-min sequence were averaged and are expressed for each MCh concentration as the percentage of baseline Penh values following PBS exposure (2).

Measurements of airway responsiveness in intubated and ventilated mice. In selected experiments airway resistance was measured with an in-line pressure transducer and software program (Scireq, Montreal, PQ, Canada) in intubated and ventilated mice connected to a rodent ventilator as previously described in this laboratory (9). Airway resistance measurements were taken at baseline, after nebulized PBS, and after each increasing concentration of nebulized MCh (9).

Lung Eosinophil Counts
The killed mice had their tracheas surgically exposed and cannulated with 27-gauge silicon tubing attached to a 23-gauge needle on a 1-ml tuberculin syringe. Following instillation of 800 µl of sterile saline through the trachea into the lung, BALF was withdrawn and cytospun (3 min at 500 rpm) onto microscope slides. Eosinophil counts were performed as previously described (2).

Quantification of Airway Remodeling
Lungs in the different groups of mice were equivalently inflated with an intratracheal injection of a similar volume of 4% paraformaldehyde solution (Sigma) to preserve the pulmonary architecture. The inflated lungs were embedded in paraffin, stained with either hematoxylin and eosin, Periodic Acid Schiff (PAS), Trichrome stain, or processed for immunohistochemistry. The analyses were performed by investigators blinded to the treatment groups.

Peribronchial trichrome staining. The area of peribronchial trichrome staining in paraffin-embedded lung was outlined and quantified using a light microscope (Leica DMLS; Leica Microsystems Inc., De Pew, NY) attached to an image analysis system (Image-Pro Plus; Media Cybernetics, Silver Spring, MD). Results are expressed as the area of trichrome staining per micron length of basement membrane of bronchioles 150–200 µm of internal diameter. At least 10 bronchioles were counted in each slide.

Lung immunohistochemical staining ({alpha}-smooth muscle actin, collagen). Six-micron-thick sections of lung from each paraffin block were deparaffinized with xylene and hydrated in ethanol and PBS pH 7.4. Endogenous peroxidase activity was quenched by incubating lung sections with 0.3% hydrogen peroxide in anhydrous methanol for 5 min. After washing with PBS, the lung sections were incubated with 1% goat serum for 10 min to block nonspecific antibody binding.

For immunohistochemical detection of {alpha}-smooth muscle actin, the lung sections were incubated overnight at 4°C with either a primary monoclonal Ab directed against {alpha}-smooth muscle actin (Sigma), or as a negative control mouse serum instead of the primary antibody. Immunoreactivity was detected by sequential incubations of lung sections with a biotinylated secondary antibody, followed by peroxidase reagent and AEC chromogen (3-amino-9-ethylcarbazole). The lung sections were briefly incubated with hematoxylin counterstain for 30 s, and then mounted with aqueous mounting media. Similar methods were used for incubation of anti-collagen primary antibodies (anti-collagen subtypes I, III, V; Polyscience, Warrington, PA) for immunohistochemical detection of collagen. Mouse collagen subtypes were detected using a biotinylated secondary antibody, followed by peroxidase reagent and DAB (3,3'-diaminobenzidine) chromogen (Vector, Burlingame, CA).

The area of immunostaining ({alpha}-smooth muscle actin or collagen) in each paraffin embedded lung was outlined and quantified using a light microscope attached to an image analysis system. Results are expressed as the area of immunostaining per micron length of basement membrane of bronchioles 150–200 µm of internal diameter. At least 10 bronchioles were counted in each slide.

Peribronchial airway smooth muscle thickness. The thickness of the airway smooth muscle layer was measured using an image analysis system. Lungs that had been fixed in 3% gluteraldehyde and 1% osmium tetroxide were stained with Basic Fuchsin-Toluidine Blue, which allowed the best visualization of the peribronchial smooth muscle layer. The thickness of the peribronchial smooth muscle layer (the transverse diameter) was measured from the innermost aspect to the outermost aspect of the circumferential smooth muscle layer. The smooth muscle layer thickness in at least 10 bronchioles of similar size (150–200 µm) were counted on each slide.

Quantitation of airway mucus expression. To quantitate the level of mucus expression in the airway, the number of PAS-positive and PAS-negative epithelial cells in individual bronchioles were counted as previously described in this laboratory (14). At least 10 bronchioles were counted in each slide. Results are expressed as the % of PAS-positive cells/bronchiole, which is calculated from the number of PAS-positive epithelial cells per bronchus divided by the total number of epithelial cells of each bronchiole.

Measurement of BAL and Lung Cytokines Associated with Airway Remodeling (TGF-ß1, IL-13) and Lung Cytokines Associated with Th1 versus Th2 Responses (IFN-{gamma}, IL-5)
The concentrations of TGF-ß1 and IL-13 in BAL fluid were assayed by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). Before the TGF-ß1 assay, the BAL samples were treated with 2.5 N acetic acid to activate any latent TGF-ß1 to immunoreactive TGF-ß1 (18). Acidified samples were neutralized by 2.7 N NaOH. The TGF-ß1 and IL-13 ELISA assays each have a sensitivity of 61 pg/ml.

The concentrations of TGF-ß1 and IL-13, as well as IFN- {gamma} and IL-5, were also assayed in lung tissue by ELISA. Lungs homogenized in lysis buffer (0.5% Triton X-100, 150 mM NaCl, 15 mM Tris, 1 mM CaCl2, 1 mM MgCl2) were centrifuged at 10,000 x g for 20 min. After the lung supernatant was passaged through a 0.8 µm-pore-size filter, the lung supernatant was assayed for cytokines and protein content. The lung supernatant protein content was assayed using a Micro BCATM protein assay reagent kit (Pierce, Rockford, IL) which has a sensitivity of 0.5 µg/ml. Levels of cytokines in lung supernatants were measured by ELISA and results are expressed as pg cytokine/mg protein.

Statistical Analysis
Results in the different groups of mice were compared by ANOVA using the nonparametric Kruskal-Wallis test followed by post-testing using Dunn's multiple comparison of means. All results are presented as mean ± SEM. A statistical software package (Graph Pad Prism, San Diego, CA) was used for the analysis. P values of < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of ISS on Airway Responsiveness
Mice sensitized to OVA and challenged with repetitive intranasal administration of OVA developed sustained increases in airway responsiveness to MCh compared with control OVA-sensitized mice not repetitively challenged with OVA (Figure 2A). The increase in airway responsiveness was evident at all the time points studied, e.g., 1 mo (P = 0.05 versus control), 3 mo (P = 0.05 versus control), or 6 mo (P = 0.05 versus control) (Figure 2A). Systemic administration of ISS significantly reduced airway responsiveness to MCh in mice repetitively challenged with OVA compared with untreated mice repetitively challenged with OVA at 1 mo (P = 0.05), 3 mo (P = 0.05), and 6 mo (P = 0.05) (Figure 2A).



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  		Figure 2. ISS inhibits airway reactivity to MCh in mice repetitively challenged with OVA. (A) Noninvasive pulmonary function tests. Mice repetitively challenged with OVA for 1 mo (P = 0.05, OVA versus control), 3 mo (P = 0.05, OVA versus control), or 6 mo (P = 0.05, OVA versus control) developed increased airway responsiveness to MCh 48 mg/ml compared with control non–OVA-challenged mice as assessed by measurement of Penh. Systemic administration of ISS to mice repetitively challenged with OVA significantly reduced airway responsiveness to MCh 48 mg/ml compared with untreated mice repetitively challenged with OVA for 1 mo (P = 0.05 versus ISS + OVA versus OVA), 3 mo (P = 0.05 versus ISS + OVA versus OVA), or 6 mo (P = 0.05 versus ISS + OVA versus OVA). Triangles, OVA; inverted triangles, OVA + ISS; circles, no OVA. (B) Measurements of airway resistance in intubated and ventilated mice. Mice challenged repetitively with OVA for 3 mo developed significant increases in airway resistance in response to MCh challenge as compared with non–OVA-challenged control mice (P = 0.01, MCh 48 mg/ml). ISS significantly reduced airway resistance to MCh in OVA-challenged mice (P = 0.05, MCh 48 mg/ml), whereas M-ODN did not have any effect on inhibiting OVA-induced increases in airway resistance to MCh. Circles, M-ODN + OVA; triangles, OVA; inverted triangles, ISS + OVA; squares, no OVA.

 
We also measured airway responsiveness to MCh in intubated and ventilated mice. Mice challenged repetitively with OVA for 3 mo developed significant increases in airway resistance in response to MCh challenge as compared with non–OVA-challenged control mice (P = 0.01) (Figure 2B). ISS significantly reduced airway resistance to MCh in OVA-challenged mice (P = 0.05), whereas M-ODN did not have any effect on inhibiting OVA-induced increases in airway resistance to MCh (P = ns) (Figure 2B).

Effect of ISS on BAL Eosinophils
The absolute number of BAL eosinophils in mice sensitized to OVA and repetitively challenged with OVA was significantly greater than in control non–OVA-challenged mice at 1 mo (45.9 ± 5.55 x 103 versus 0.3 ± 0.1 x 103 BAL eosinophils) (P = 0.0001), 3 mo (34.9 ± 5.8 x 103 versus 0.10 ± 0.03 x 103 BAL eosinophils) (P = 0.0001), and 6 mo (8.9 ± 2.3 x 103 versus 0.1 ± 0.1 x 103 BAL eosinophils) (P = 0.0001) (Figure 3A). Although the number of BAL eosinophils in mice repetitively challenged with OVA were still significantly increased at 6 mo compared with control non–OVA-challenged mice (8.9 x 103 versus 0.1 x 103 BAL eosinophils), the number of BAL eosinophils in mice repetitively challenged with OVA was lower at 6 mo (8.9 x 103 BAL eosinophils) compared with mice repetitively challenged with OVA at 1 mo (45.9 x 103 BAL eosinophils) and 3 mo (34.9 x 103 BAL eosinophils).



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  		Figure 3. Effect of ISS on BAL cells. (A) Inhibition of BAL eosinophilia by ISS. Mice repetitively challenged with OVA for 1 mo (P = 0.0001, OVA versus no OVA), 3 mo (P = 0.0001, OVA versus no OVA), or 6 mo (P = 0.0001, OVA versus no OVA) had increased numbers of BAL eosinophils compared with control non–OVA-challenged mice. Systemic administration of ISS in mice repetitively challenged with OVA significantly reduced levels of BAL eosinophils, compared with untreated mice repetitively challenged with OVA for 1 mo (P = 0.001, ISS + OVA versus OVA) and 3 mo (P = 0.001, ISS + OVA versus OVA), whereas the reduction at 6 mo did not reach statistical significance (P = 0.15, ISS + OVA versus OVA). Open bars, no OVA; filled bars, OVA; diagonally striped bars, OVA + ISS. (B) Effect of M-ODN on BAL eosinophilia. Mice repetitively challenged with OVA for 3 mo had increased numbers of BAL eosinophils compared with control non–OVA-challenged mice (P = 0.001, OVA versus no OVA). Systemic administration of ISS in mice repetitively challenged with OVA significantly reduced levels of BAL eosinophils, compared with untreated mice repetitively challenged with OVA for 3 mo (P = 0.01, OVA + ISS versus OVA). Systemic administration of an M-ODN to mice repetitively challenged with OVA for 3 mo did not inhibit BAL eosinophila compared with mice challenged repetitively with OVA (P = ns, OVA + M-ODN versus OVA). (C) Effect of ISS on BAL mononuclear cells. Mice repetitively challenged with OVA for 1 mo (P = 0.01, OVA versus no OVA), 3 mo (P = 0.01, OVA versus no OVA), or 6 mo (P = 0.01, OVA versus no OVA) had increased numbers of BAL mononuclear cells compared with control non–OVA-challenged mice. Systemic administration of ISS before initiation of repetitive OVA challenges did not significantly change the absolute number of BAL mononuclear cells compared with untreated mice challenged repetitively with OVA at 1 mo, 3 mo, or 6 mo. Open bars, no OVA; filled bars, OVA; diagonally striped bars, OVA + ISS.

 
Systemic administration of ISS before initiation of repetitive OVA challenges significantly reduced the absolute number of BAL eosinophils compared with untreated mice challenged repetitively with OVA at 1 mo (16.87 ± 4.58 x 103 versus 45.9 ± 5.55 x 103 BAL eosinophils) (P = 0.001) and 3 mo (13.3 ± 3.9 x 103 versus 34.9 ± 5.8 x 103 BAL eosinophils) (P = 0.001), whereas the reduction at 6 mo did not reach statistical significance (4.1 ± 0.7 x 103 versus 8.9 ± 2.3 x 103 BAL eosinophils) (P = 0.15) (Figure 3A).

Systemic administration of an M-ODN to mice repetitively challenged with OVA for 3 mo did not inhibit BAL eosinophilia compared with untreated mice challenged repetitively with OVA (P = ns) (Figure 3B).

Effect of ISS on BAL Mononuclear Cells
The absolute number of BAL mononuclear cells in mice sensitized to OVA and repetitively challenged with OVA was significantly greater than in control non–OVA-challenged mice at 1 mo (25.4 ± 4.3 x 103 versus 4.5 ± 1.3 x 103 BAL mononuclear cells) (P = 0.01), 3 mo (29.1 ± 7.0 x 103 versus 2.9 ± 0.6 x 103 BAL mononuclear cells) (P = 0.01), and 6 mo (51.2 ± 8.6 x 103 versus 4.2 ± 1.0 x 103 BAL mononuclear cells) (P = 0.01) (Figure 3C).

Systemic administration of ISS before initiation of repetitive OVA challenges did not significantly change the absolute number of BAL mononuclear cells compared with untreated mice challenged repetitively with OVA at 1 mo (P = ns), 3 mo (P = ns), or 6 mo (P = ns) (Figure 3C).

ISS Reduces Peribronchial Fibrosis
We performed initial experiments to determine whether there are differences in peribronchial trichrome staining in mice following acute versus chronic OVA exposure. Acute OVA challenge in OVA-sensitized mice did not induce increased peribronchial trichrome staining (acute OVA versus no-OVA) (P = ns), whereas 3 mo of repetitive OVA challenges did induce significant increases in peribronchial trichrome staining (chronic OVA versus no-OVA) (P = 0.004) (Figure 4A).




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  		Figure 4. ISS reduces peribronchial fibrosis. (A) Peribronchial fibrosis: effect of acute versus chronic OVA challenges. Acute OVA challenge in OVA-sensitized mice did not induce increased peribronchial trichrome staining (acute OVA versus no-OVA) (P = ns), whereas 3 mo of repetitive OVA challenges did induce significant increases in peribronchial trichrome staining (chronic OVA versus no-OVA) (P = 0.004). (B) Peribronchial trichrome staining. Control bronchi derived from mice not challenged with OVA (panel A) exhibited minimal peribronchial trichrome staining (blue color). In contrast repetitive OVA challenge for 3 months induced circumferential peribronchial trichrome staining (panel B), which was significantly inhibited by ISS therapy (panel C). (C) Effect of ISS on area of peribronchial trichrome staining. Mice repetitively challenged with OVA for 3 months (P = 0.004, OVA versus no OVA), or 6 mo (P = 0.0001, OVA versus no OVA), but not 1 mo (P = ns, OVA versus no OVA), developed increased peribronchial trichrome staining compared with control non–OVA-challenged mice. Systemic administration of ISS significantly reduced levels of peribronchial trichrome staining in mice challenged repetitively with OVA, compared with untreated mice challenged repetitively with OVA for 3 mo (P = 0.0003, versus ISS + OVA versus OVA) or 6 mo (P = 0.0001, ISS + OVA versus OVA). Open bars, no OVA; filled bars, OVA; diagonally striped bars, OVA + ISS. (D) Effect of timing of administration of ISS and final OVA challenge on peribronchial trichrome staining. Mice repetitively challenged with OVA for 3 mo had increased peribronchial trichrome staining compared with control non–OVA-challenged mice (P = 0.004, OVA versus no OVA). Systemic administration of ISS every other week significantly reduced the levels of peribronchial trichrome staining in mice challenged repetitively with OVA for 3 mo, when ISS was administered either in the same week as the final OVA challenge (P = 0.001, OVA+ ISS versus OVA), or when ISS was administered 2 wk before the final OVA challenge (P = 0.003, OVA+ISS/off versus OVA). (E) Effect of the administration of ISS in the absence of OVA challenge on peribronchial trichrome staining. To investigate whether ISS directly affected peribronchial trichrome staining in the absence of allergen challenge, we administered ISS every other week without OVA challenge to mice for 3 mo. ISS did not significantly influence peribronchial trichrome staining compared with control non–OVA-challenged mice that were not treated with ISS. In contrast, systemic administration of ISS significantly reduced the levels of peribronchial trichrome staining in mice challenged repetitively with OVA for 3 mo (P = 0.01, OVA+ISS versus OVA). (F) Effect of M-ODN on peribronchial trichrome staining. Mice repetitively challenged with OVA for 3 mo had increased levels of peribronchial trichrome staining compared with control non–OVA-challenged mice (P = 0.001, OVA versus no OVA). Systemic administration of ISS to mice repetitively challenged with OVA significantly reduced levels of peribronchial trichrome staining compared with untreated mice repetitively challenged with OVA for 3 mo (P = 0.01, OVA + ISS versus OVA). Systemic administration of an M-ODN to mice repetitively challenged with OVA for 3 mo did not inhibit peribronchial trichrome staining compared with untreated mice challenged repetitively with OVA (P = ns, OVA + M-ODN versus OVA). (G) Collagen V immunostaining. Mice repetitively challenged with OVA for 3 mo developed increased peribronchial collagen V immunostaining compared with control non–OVA-challenged mice (P = 0.0001, OVA versus no OVA). Systemic administration of ISS significantly reduced levels of peribronchial collagen V immunostaining in mice challenged repetitively with OVA, compared with untreated mice challenged repetitively with OVA for 3 mo (P = 0.0001, ISS + OVA versus OVA).

 
We used two image analysis methods to quantitate peribronchial fibrosis, namely the area of peribronchial trichrome staining, and the area of peribronchial immunostaining with anti–collagen V Ab (expressed as the stained area in µm2/µm circumference of bronchiole). The area of peribronchial trichrome stain in mice which were repetitively challenged with OVA was significantly greater than in control non–OVA-challenged mice at 3 mo (0.60 ± 0.08 versus 0.26 ± 0.03 µm2/µm circumference of bronchiole) (P = 0.004), and 6 mo (0.79 ± 0.09 versus 0.36 ± 0.04 µm2/µm circumference of bronchiole) (P = 0.0001), whereas the increase at 1 mo did not reach statistical significance (0.58 ± 0.04 versus 0.32 ± 0.04 µm2/µm circumference of bronchiole) (P = 0.12) (Figures 4B and 4C).

Systemic administration of ISS to mice repetitively challenged with OVA significantly reduced the area of trichrome staining compared with untreated mice repetitively challenged with OVA at 3 mo (0.27 ± 0.03 versus 0.60 ± 0.08 µm2/µm circumference of bronchiole) (P = 0.0003), and 6 mo (0.40 ± 0.02 versus 0.79 ± 0.09 µm2/µm circumference of bronchiole) (P = 0.0001) (Figures 4B and 4C). Pretreatment with ISS did not significantly inhibit levels of trichrome staining at 1 mo in mice repetitively challenged with OVA compared with untreated mice repetitively challenged with OVA (0.58 ± 0.04 versus 0.55 ± 0.06 µm2/µm circumference of bronchiole) (P = ns) (Figure 4C).

The area of peribronchial trichrome staining noted in mice repetitively challenged with OVA and pretreated with ISS for 3 mo was reduced to levels of background peribronchial trichrome staining noted in non–OVA-challenged control mice (0.27 ± 0.04 versus 0.27 ± 0.03 µm2/µm circumference of bronchiole) (Figure 4C). Similar beneficial effects of ISS on reducing peribronchial trichrome staining to levels of non–OVA-challenged mice were also noted in mice treated with ISS for 6 mo (0.40 ± 0.02 versus 0.36 ± 0.04 µm2/µm circumference of bronchiole) (Figure 4C).

To determine whether the timing of the administration of ISS (ISS was administered every 2 wk) in relation to the final OVA challenge influences its effect on the extent of peribronchial trichrome staining, we evaluated the extent of peribronchial trichrome staining in mice challenged repetitively with OVA for 3 mo, ending the experiment either in the week mice had received ISS, or in the week that they had not received ISS. Systemic administration of ISS every other week significantly reduced the levels of peribronchial trichrome staining in mice challenged repetitively with OVA for 3 mo, when ISS was administered either in the same week as the final OVA challenge (P = 0.001, OVA+ ISS versus OVA), or when ISS was administered 2 wk before the final OVA challenge (P = 0.003, OVA+ ISS/off versus OVA) (Figure 4D).

To investigate whether ISS directly affected peribronchial trichrome staining in the absence of allergen challenge, we administered ISS every other week without OVA challenge to mice for 3 mo. ISS did not significantly influence peribronchial trichrome staining compared with non–OVA-challenged, non–ISS-treated control mice (Figure 4E).

Systemic administration of an M-ODN to mice repetitively challenged with OVA for 3 mo did not reduce peribronchial trichrome staining compared with mice challenged repetitively with OVA (Figure 4F).

Effect of ISS on Peribronchial Collagen Immunostaining
Pilot immunostaining studies of remodeled airways with anti–collagen I, III, and V Abs demonstrated that anti-collagen staining with the anti–collagen V Ab was reproducibly detected, whereas staining with anti–collagen I and III Abs was more variable in the lungs of mice repetitively challenged with OVA. Therefore we quantitated only anti–collagen V immunostaining. The area of peribronchial collagen V immunostaining in mice which were repetitively challenged with OVA was significantly greater than in control non–OVA-challenged mice at 3 mo (0.32 ± 0.02 versus 0.15 ± 0.02 µm2/µm circumference of bronchiole) (P = 0.0001) (Figure 4G). Systemic administration of ISS to mice repetitively challenged with OVA significantly reduced the area of collagen V immunostaining compared with untreated mice repetitively challenged with OVA at 3 mo (0.17 ± 0.02 versus 0.32 ± 0.02 µm2/µm circumference of bronchiole) (P = 0.0001) (Figure 4G).

Effect of ISS on Peribronchial Smooth Muscle Layer Thickness
The thickness of the peribronchial smooth muscle layer (measured in µm) in mice repetitively challenged with OVA was significantly greater than in control non–OVA-challenged mice at 1 mo (13.6 ± 0.4 versus 5.3 ± 0.5 µm) (P = 0.0001), 3 mo (14.3 ± 0.7 versus 8.9 ± 0.6 µm) (P = 0.0001), and 6 mo (15.0 ± 0.4 versus 8.3 ± 0.4 µm) (P = 0.0001) (Figure 5A).



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  		Figure 5. ISS inhibits thickness of peribronchial smooth muscle layer and myofibroblasts. (A) Peribronchial smooth muscle layer thickness. Mice repetitively challenged with OVA for 1 mo (P = 0.0001, OVA versus no OVA), 3 mo (P = 0.0001, OVA versus no OVA), or 6 mo (P = 0.0001, OVA versus no OVA), developed increased thickness of the peribronchial smooth muscle layer compared with control non–OVA-challenged mice. Systemic administration of ISS significantly reduced the peribronchial smooth muscle layer thickness in mice challenged repetitively with OVA, compared with untreated mice challenged repetitively with OVA for 1 mo (P = 0.0001, versus ISS + OVA versus OVA), 3 mo (P = 0.0001, versus ISS + OVA versus OVA), or 6 mo (P = 0.0001, ISS + OVA versus OVA). Open bars, no OVA; filled bars, OVA; diagonally striped bars, OVA + ISS. (B) Peribronchial {alpha}-smooth muscle actin immunostaining: effect of acute versus chronic OVA challenges. Acute OVA challenge in OVA-sensitized mice did not induce increased peribronchial {alpha}-smooth muscle actin immuonostaining (acute OVA versus no-OVA) (P = ns), whereas 3 mo of repetitive OVA challenges did induce significant increases in peribronchial {alpha}-smooth muscle actin immunostaining (chronic OVA versus no-OVA) (P = 0.0001). (C) Peribronchial myofibroblast immunostaining. Control bronchi derived from mice not challenged with OVA (panel a) exhibited peribronchial myofibroblast immunostaining (red color). Repetitive OVA challenge for 3 mo induced an increase in the area of myofibroblast immunostaining (panel b), which was significantly inhibited by ISS therapy (panel c). (D) ISS reduces peribronchial myofibroblast immunostaining. Mice repetitively challenged with OVA for 1 mo (P = 0.004, OVA versus no OVA), 3 mo (P = 0.0001, OVA versus no OVA), or 6 mo (P = 0.0001, OVA versus no OVA), developed an increase in the area of peribronchial myofibroblast immunostaining compared with control non–OVA-challenged mice. Systemic administration of ISS significantly reduced the area of peribronchial myofibroblast immunostaining in mice challenged repetitively with OVA, compared with untreated mice challenged repetitively with OVA for 3 mo (P = 0.001, ISS + OVA versus OVA) or 6 mo (P = 0.0001, ISS + OVA versus OVA), but not for 1 mo (P = ns, ISS + OVA versus OVA). Open bars, no OVA; filled bars, OVA; diagonally striped bars, OVA + ISS.

 
Systemic administration of ISS significantly reduced the peribronchial smooth muscle layer thickness in mice repetitively challenged with OVA compared with untreated mice repetitively challenged with OVA at 1 mo (7.5 ± 0.8 versus 13.6 ± 0.4 µm) (P = 0.0001), 3 mo (9.8 ± 0.5 versus 14.3 ± 0.7 µm) (P = 0.0001), and 6 mo (11.5 ± 0.3 versus 15.0 ± 0.4 µm) (P = 0.0001) (Figure 5A).

Effect of ISS on the Area of Peribronchial Myofibroblast {alpha}-Smooth Muscle Actin Immunostaining
The area of peribronchial myofibroblast {alpha}-smooth muscle actin immunostaining was quantified by image analysis and expressed as the stained area in µm2/µm circumference of a bronchiole. Acute OVA challenge in OVA-sensitized mice did not induce increased peribronchial {alpha}-smooth muscle actin immuonostaining (acute OVA versus no-OVA) (P = ns), whereas 3 mo of repetitive OVA challenges did induce significant increases in peribronchial {alpha}-smooth muscle actin immunostaining (chronic OVA versus no-OVA) (P = 0.0001) (Figure 5B). The area of peribronchial {alpha}-smooth muscle actin immunostaining in mice repetitively challenged with OVA was significantly greater than in control non–OVA-challenged mice at 1 mo (0.88 ± 0.06 versus 0.60 ± 0.09 µm2/µm circumference of bronchiole) (P = 0.004), 3 mo (0.97 ± 0.06 versus 0.49 ± 0.02 µm2/µm circumference of bronchiole) (P = 0.0001), and 6 mo (0.94 ± 0.07 versus 0.48 ± 0.03 µm2/µm circumference of bronchiole) (P = 0.0001) (Figures 5C and 5D).

Systemic administration of ISS before repetitive OVA challenges significantly reduced the area of {alpha}-smooth muscle actin immunostaining compared with untreated mice challenged repetitively with OVA at 3 mo (0.80 ± 0.11 versus 0.97 ± 0.06 µm2/µm circumference of bronchiole) (P = 0.0001), and 6 mo (0.72 ± 0.05 versus 0.94 ± 0.06 µm2/µm circumference of bronchiole) (P = 0.001) (Figures 5C and 5D). Pretreatment with ISS for 1 mo did not significantly inhibit the area of {alpha}-smooth muscle actin immunostaining in mice repetitively challenged with OVA, compared with untreated mice repetitively challenged with OVA at 1 mo (P = ns).

Effect of ISS on BALF TGF-ß1 and IL-13 Levels
As both TGF-ß1 and IL-13 are able to induce peribronchial fibrosis (19), we measured levels of these cytokines in BALF and lung tissue. Levels of BAL TGF-ß1 were significantly increased in mice exposed to repetitive OVA challenge compared with control non–OVA-challenged mice at 1 mo (300 ± 17 versus 135 ± 5 pg/ml TGF-ß1) (P = 0.03), 3 mo (356 ± 28 versus 156 ± 37 pg/ml TGF-ß1) (P = 0.03), and 6 mo (369 ± 74 versus 146 ± 8 pg/ml TGF-ß1) (P = 0.03) (Figure 6A). ISS significantly reduced levels of BAL TGF-ß1 in mice exposed to repetitive OVA challenge compared with untreated mice challenged repetitively with OVA at 3 mo (356 ± 28 versus 241 ± 19 pg/ml TGF-ß1) (P = 0.05), and at 6 mo (369 ± 74 versus 269 ± 37 pg/ml TGF-ß1) (P = 0.05), but not at 1 mo (Figure 6A).



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  		Figure 6. ISS reduces levels of TGF-ß1 in BAL fluid and lung. (A) BAL TGF-ß1. Mice repetitively challenged with OVA for 1 mo (P = 0.03, OVA versus no OVA), 3 mo (P = 0.03, OVA versus no OVA), or 6 mo (P = 0.03, OVA versus no OVA) had increased levels of BAL TGF-ß1 compared with control non–OVA-challenged mice. Systemic administration of ISS significantly reduced levels of BAL TGF-ß1 in mice challenged repetitively with OVA, compared with untreated mice challenged repetitively with OVA for 3 mo (P = 0.05, ISS + OVA versus OVA) or 6 mo (P = 0.05, ISS + OVA versus OVA), but not for 1 mo. Open bars, no OVA; filled bars, OVA; diagonally striped bars, OVA + ISS. (B) Lung TGF-ß1. Mice repetitively challenged with OVA for 3 mo had increased levels of lung TGF-ß1 compared with control non–OVA-challenged mice (P = 0.03, OVA versus no OVA). Systemic administration of ISS significantly reduced levels of lung TGF-ß1 in mice challenged repetitively with OVA, compared with untreated mice challenged repetitively with OVA for 3 mo (P = 0.05, versus ISS + OVA versus OVA). (C) BAL IL-13. Mice repetitively challenged with OVA for 1 mo (P = 0.05, OVA versus no OVA), 3 mo (P = 0.05, OVA versus no OVA), or 6 mo (P = 0.05, OVA versus no OVA), had increased levels of BAL IL-13 compared with control non–OVA-challenged mice. Systemic administration of ISS significantly reduced levels of BAL IL-13 in mice challenged repetitively with OVA for 1 mo (P = 0.05, ISS + OVA versus OVA). Although ISS reduced levels of BAL IL-13 in mice challenged repetitively with OVA for 3 or 6 mo, this was not statistically significant (P = ns, ISS + OVA versus OVA). Open bars, no OVA; filled bars, OVA; diagonally striped bars, OVA + ISS.

 
Similarly, levels of lung TGF-ß1 were significantly increased in mice exposed to repetitive OVA challenge for 3 mo compared with control non–OVA-challenged mice (1,946 ± 261 versus 664 ± 75 pg TGF-ß1/mg lung protein) (P = 0.02) (Figure 6B). ISS significantly reduced levels of lung TGF-ß1 in mice exposed to repetitive OVA challenge compared with untreated mice challenged repetitively with OVA (939 ± 171 versus 1,946 ± 261 pg TGF-ß1/mg lung protein) (P = 0.05) (Figure 6B).

Levels of BAL IL-13 were significantly increased in mice exposed to repetitive OVA challenge compared with control non–OVA-challenged mice at 1 mo (3,768 ± 245 versus 1,850 ± 112 pg/ml IL-13) (P = 0.05), 3 mo (4,189 ± 731 versus 1,597 ± 590 pg/ml IL-13) (P = 0.05), and 6 mo (3,341 ± 212 versus 1,910 ± 125 pg/ml IL-13) (P = 0.05) (Figure 6C). ISS significantly reduced levels of BAL IL-13 in mice exposed to repetitive OVA challenge at 1 mo (3,768 ± 245 versus 2,798 ± 100 pg/ml IL-13) (OVA versus OVA+ISS) (P = 0.05) (Figure 6C). Although ISS also reduced levels of BAL IL-13 in mice exposed to repetitive OVA challenge at 3 mo and 6 mo compared with untreated mice challenged repetitively with OVA, this reduction was not statistically significant (Figure 6C). ISS did not significantly reduce levels of lung IL-13 in mice exposed to repetitive OVA challenge compared with untreated mice challenged repetitively with OVA (data not shown).

Effect of ISS on Lung IL-5 and IFN-{gamma} Levels
Systemic administration of ISS to mice repetitively challenged with OVA for 3 mo significantly inhibited levels of lung IL-5 (511 ± 115, versus 310 ± 57 pg IL-5/mg lung protein) (OVA versus OVA + ISS) (P = 0.05). As anticipated repetitive OVA challenge for 3 mo induced a Th2 response (e.g., IL-5), but did not induce the Th1 cytokine IFN-{gamma} (< 9 pg IFN-{gamma}/mg lung protein). Interestingly, repetitive administration of ISS for 3 mo in OVA-challenged mice was associated with inhibition of Th2 responses, but not induction of Th1 responses in the lung (< 9 pg IFN-{gamma}/mg lung protein).

Effect of ISS on Airway Mucus Expression
The percentage of airway epithelium which stained positive with PAS in mice repetitively challenged with OVA was significantly greater than in control non–OVA-challenged mice at 1 mo (13.1 ± 1.6 versus 0.1 ± 0.01%) (P = 0.0001), 3 mo (22.1 ± 2.8 versus 0.1 ± 0.01%) (P = 0.0001), and 6 mo (22.9 ± 3.4 versus 0.2 ± 0.2%) (P = 0.0005) (Figures 7A and 7B).



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  		Figure 7. ISS reduces airway mucus. (A) PAS staining. Control bronchi derived from mice not challenged with OVA (panel a) exhibited minimal epithelial PAS staining (red color). In contrast repetitive OVA challenge for 3 mo induced circumferential epithelial PAS staining (panel b), which was significantly inhibited by ISS (panel c). (B) ISS inhibits PAS staining of airway epithelium. Mice repetitively challenged with OVA for 1 mo (P = 0.0001, OVA versus no OVA), 3 mo (P = 0.0001, OVA versus no OVA), or 6 mo (P = 0.0005, OVA versus no OVA), had increased PAS staining of airway epithelium compared with control non–OVA-challenged mice. Systemic administration of ISS significantly reduced the PAS staining of airway epithelium in mice challenged repetitively with OVA, compared with untreated mice challenged repetitively with OVA for 1 mo (P = 0.0005, ISS + OVA versus OVA), 3 mo (P = 0.0003, ISS + OVA versus OVA), or 6 mo (P = 0.02, ISS + OVA versus OVA). Open bars, no OVA; filled bars, OVA; diagonally striped bars, OVA + ISS. (C) Effect of M-ODN on peribronchial trichrome staining. Mice repetitively challenged with OVA for 3 mo developed increased levels of PAS staining of airway epithelium compared with control non–OVA-challenged mice (P = 0.0001, OVA versus no OVA). Systemic administration of ISS in mice repetitively challenged with OVA significantly reduced levels of PAS staining of airway epithelium compared with untreated mice repetitively challenged with OVA for 3 mo (P = 0.01, OVA + ISS versus OVA). Systemic administration of an M-ODN to mice repetitively challenged with OVA for 3 mo did not inhibit levels of PAS staining of airway epithelium compared with mice challenged repetitively with OVA (P = ns, OVA + M-ODN versus OVA).

 
Systemic administration of ISS in mice repetitively challenged with OVA significantly reduced the percentage of airway epithelium staining positively with PAS compared with untreated mice repetitively challenged with OVA at 1 mo (5.1 ± 0.9, versus 13.1 ± 1.6%) (P = 0.0005), 3 mo (7.4 ± 1.3 versus 22.1 ± 2.8%) (P = 0.0003), and 6 mo (8.4 ± 1.2 versus 22.9 ± 3.4%) (P = 0.02) (Figure 7B).

Systemic administration of an M-ODN to mice repetitively challenged with OVA for 3 mo did not reduce the percentage of airway epithelium staining positively with PAS compared with untreated mice challenged repetitively with OVA (Figure 7C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we demonstrate that repetitive airway allergen challenge in mice induces many of the features of airway remodeling noted in human asthma and that ISS significantly inhibits airway remodeling. Mice exposed to repetitive OVA challenge develop sustained airway hyperreactivity to MCh for up to 6 mo. This airway hyperreactivity is not only associated with Th2 cytokine expression, eosinophilic inflammation, and mucus expression, but also with features of airway remodeling including increased thickness of the peribronchial smooth muscle layer, peribronchial myofibroblast accumulation, increased levels of lung TGF-ß1, and peribronchial fibrosis. In contrast, acute OVA challenge did not induce the airway remodeling changes detected with repetitive OVA challenge. The time course studies demonstrate that the majority of airway remodeling in this model occurs during the first month of chronic allergen challenge. While many of the effects of ISS on airway remodeling are apparent at the 1-mo time point, some effects of ISS are only apparent at the 3-mo time point (Figure 4C, trichrome stain, and Figure 5D, {alpha}-smooth muscle actin immunostain, which are reduced by ISS at 3 mo, but not at 1 mo). Repeated systemic dosing with ISS every 2 wk results in sustained inhibition of airway hyperreactivity to MCh for at least 6 mo. In addition ISS administration significantly inhibits features of airway remodeling including mucus production, peribronchial myofibroblast accumulation, levels of the profibrotic cytokine TGF-ß1, peribronchial fibrosis, and reducing the thickness of the peribronchial smooth muscle layer.

This study shares similarities and differences with a recent study which demonstrated that CpG inhibited airway remodeling in a mouse model of asthma (20). Previous studies demonstrated that pretreatment with CpG inhibited lung eosinophilic inflammation, airway hyperreactivity to methacholine, peribronchial trichrome staining, and mucus expression in mice challenged with antigen for 6 wk (20). This study extended these observations by demonstrating that ISS inhibited an additional key component of the remodeled airway wall, namely peribronchial smooth muscle layer thickness. As smooth muscle plays a key role in airway hyperreactivity in asthma, the ability of ISS to reduce the thickness of the airway smooth muscle layer may be an important mechanism by which ISS inhibits airway hyperreactivity. In addition, we have made the novel observations that ISS inhibits accumulation of myofibroblasts, peribronchial collagen V deposition, ongoing Th2 cytokine responses, and demonstrated that repeated dosing with ISS could inhibit airway remodeling and airway hyperresponsiveness for up to 6 mo. Finally, our study is the first to demonstrate that ISS inhibits TGF-ß1 expression in both BALF and lung of the remodeled airway, and suggests that ISS mediated inhibition of lung TGF-ß1 expression is an important mechanism by which ISS inhibits airway remodeling.

In humans, the basement membrane of airway epithelium is comprised of two layers, the basal lamina (referred to as the true basement membrane and of normal thickness in asthma) and the lamina reticularis, which is thickened in asthma (15). The thickened lamina reticularis is comprised of collagen type I, III, V, and fibronectin which are likely produced by myofibroblasts (15). In addition to trichrome staining, we have used an anti–collagen V Ab in this study to demonstrate increased peribronchial collagen deposition in mice which develop airway remodeling in response to repetitive allergen challenge. The degree of thickening of the basement membrane in asthma has been correlated with the severity of asthma in some but not all studies (15). Differences in the results of these studies is probably due to study design, and the fact that for ethical reasons, it will never be possible to design the ideal experimental study in human asthma in which anti-inflammatory therapy is withheld in subjects with mild, moderate, and severe asthma for sufficient periods of time (? years) to determine whether severity of asthma correlates with features of airway remodeling.

The number of myofibroblasts in the subepithelium are increased in asthma and their numbers correlate with the thickness of the basement membrane (15). Myofibroblasts are a major source of collagenous and noncollagenous extracellular matrix molecules. In humans with asthma, allergen inhalation increases the number of myofibroblasts (21). The myofibroblast may contribute to airway remodeling by release of extracellular matrix components (fibronectin, laminin, elastin) and cytokines. Myofibroblast supernatants enhance eosinophil survival in vitro (22). Myofibroblasts can be detected by immunostaining lung sections with an anti–{alpha}-smooth muscle antibody (15). The number of peribronchial myofibroblasts increased in mice following chronic allergen administration suggesting that the increased numbers of myofibroblasts could be contributing to the deposition of collagen in the peribronchial region.

This study also demonstrated a significant increase in BALF and lung levels of the profibrotic cytokine TGF-ß1. TGF-ß1 stimulates fibroblasts to produce extracellular matrix proteins (collagen, fibronectin), decreases the production of enzymes that degrade the extracellular matrix (collagenase), and increases the production of proteins that inhibit enzymes that degrade the extracellular matrix (tissue inhibitor of metalloprotease, or TIMP) (23). The net effect is to increase the production of extracellular matrix proteins. In subjects with asthma, increased levels of TGF-ß1 have been reported in BAL and biopsy specimens (24, 25). TGF-ß1 expression correlates with the degree of subepithelial fibrosis, and levels of TGF-ß1 are significantly increased in patients with severe asthma who have prominent airway eosinophilic inflammation (24, 25). However, not all studies show an increase in TGF-ß1 in asthma (26). In the lung TGF-ß can be generated by multiple cell types (TGF-ß1, macrophages, eosinophils, epithelium, endothelium, connective tissue cells, hematpoetic cells; TGF-ß2, epithelial, neuronal cells; TGF-ß3, mesenchymal cells) (23).

IL-13 is postulated to be a key mediator of tissue fibrosis caused by Th2 lymphocytes. IL-13 is a potent stimulator of fibroblast proliferation and collagen production in vitro (19, 27). In vivo studies in a parasite model of liver fibrosis demonstrate that an IL-13 inhibitor blocks the development of hepatic fibrosis during this Th2-dominated inflammatory response (27). Similarly, recent in vivo studies with transgenic mice which overexpress IL-13 in airway epithelium demonstrate that the fibrogenic effects of IL-13 are mediated by TGF-ß1 (19). In vivo in these epithelial targeted IL-13 transgenic mice, IL-13 selectively stimulated TGF-ß1 (but not TGF-ß2 or TGF-ß3) production by macrophages in particular with lesser amounts of TGF-ß1 being produced by epithelium and eosinophils (19). As IL-13–induced lung fibrosis is significantly reduced by treatment with a TGF-ß1 antagonist (19), these studies suggest that the peribronchial fibrogenic effects of IL-13 are mediated to a great extent by this TGF-ß1 pathway. In this study we have demonstrated that peribronchial fibrosis is associated with increased levels of both IL-13 and TGF-ß1 suggesting that the IL-13/TGF-ß1/peribronchial fibrosis pathway may be operative in this chronic allergen-induced model of airway remodeling. We have also demonstrated that ISS significantly reduces peribronchial fibrosis as well as levels of TGF-ß1 with lesser inhibitory effects on levels of IL-13 suggesting that ISS may be reducing TGF-ß levels and peribronchial fibrosis by mechanisms other than reducing levels of the Th2 cytokine IL-13. Although this study has demonstrated that ISS both inhibits the expression of TGF-ß as well as inhibits airway remodeling, further studies are needed to determine whether this is an association or a cause-and-effect relationship.

Mucus hypersecretion and mucus plugging of the airways are characteristic features of human asthma (28). Acute allergen challenge in mouse models of asthma induces Muc-5ac mRNA and protein expression, as well as goblet cell metaplasia in murine airways (29). Adoptive transfer of Th2 cells induces airway mucus expression (30). When overexpressed in vivo, each Th2 cytokine (IL-4, IL-9, IL-13) induces goblet cell metaplasia in the murine lung (31). However, only IL-9 directly induces epithelial goblet cell metaplasia and human MUC5 gene expression (32). Neither IL-4 nor IL-13 directly induce Muc 5ac gene expression when administered to epithelial cells in vitro (31). Thus, it is assumed that the actions of IL-4 and IL-13 are upstream of IL-9 and act via indirect effects on as yet unknown cells or pathways (31). The mechanism by which ISS inhibits Muc 5ac mRNA and mucus expression in our chronic model of airway remodeling is likely due to the ability of ISS to inhibit Th2-derived cytokines known to be important in stimulating mouse airway mucus production, including IL-4 (31) and IL-9 (33).

Airway smooth muscle hyperplasia is an important component of the structural changes that constitute airway remodeling (15). Indeed, airway smooth muscle cells obtained by airway biopsy from subjects with asthma grow in vitro at twice the rate of airway smooth muscle cells derived from control subjects without asthma (34). Because exaggerated airway narrowing in response to a variety of stimuli is a characteristic feature of asthma, airway smooth muscle hyperplasia may play an important role in this exaggerated airway response. Extracellular matrix proteins such as collagen, fibronectin, and laminin can affect the growth and expression of contractile proteins in airway smooth muscle cells. For example, airway smooth muscle cells cultured on collagen produce a proliferative rather than a contractile phenotype (35). This study demonstrates that repetitive allergen challenge in mice induces an increased thickness of the peribronchial smooth muscle layer. Further studies are in progress to determine the levels of expression of extracellular matrix components and smooth muscle mitogens, which could contribute to the increased airway smooth muscle thickness and the mechanism by which ISS might inhibit this process.

In summary, this study demonstrates that repetitive allergen challenge induces sustained AHR for up to 6 mo in a mouse model of asthma. The sustained AHR is associated with increased levels of the profibrotic cytokine TGF-ß1 and airway remodeling. As ISS inhibits expression in the lung of several key Th2 cytokines including IL-4 (3), IL-5 (2), and IL-9 (33), ISS has the potential to inhibit many of the cytokine pathways important to the pathogenesis of asthma. Interestingly, repetitive administration of ISS for 3 mo in OVA-challenged mice was associated with inhibition of Th2 responses, but not induction of Th1 responses in the lung. This suggests that inhibition of Th2 responses rather than induction of Th1 responses may be a more important therapeutic mechanism of action of ISS in mouse models of airway inflammation and AHR. In mouse models, ISS is as effective as corticosteroids in preventing acute allergen-induced airway eosinophilic inflammation and AHR (2), and reversing established acute allergen induced airway eosinophilic inflammation and AHR (9). This study demonstrates that ISS can also prevent sustained AHR and airway remodeling in response to chronic allergen challenge. Further studies in humans will help to determine whether using ISS in the prevention and/or treatment of asthma will be safe and effective.


   Acknowledgments
 
This study was supported by NIH grants AI 33977 and AI 38425 (to D.B.) and NIH grant AI 40682 (to E.R.).

Received in original form February 28, 2003

Received in final form October 29, 2003


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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