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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 655-663, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.4853

Resolution of Airway Inflammation following Ovalbumin Inhalation

Comparison of ISS DNA and Corticosteroids

Reid K. Ikeda, Jyothi Nayar, Jae Youn Cho, Marina Miller, Monica Rodriguez, Eyal Raz and David H. Broide

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

Address correspondence to: Dr. 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
 
In this study we have compared the therapeutic effect of the administration of immunostimulatory DNA sequences (ISS) with that of corticosteroids on the resolution of airway inflammation and airway hyperreactivity (AHR) in a mouse model. Mice which had already developed significant levels of eosinophilic airway inflammation 24 h after allergen challenge were then treated with either ISS or corticosteroids, and the effect on AHR and airway inflammation assessed 6 d later. ISS inhibited AHR as effectively as corticosteroids. Combination therapy with ISS and corticosteroids was more effective than monotherapy with either ISS or corticosteroids in inhibiting AHR. In ovalbumin-challenged mice, levels of bronchoalveolar lavage (BAL) eosinophils were significantly reduced with either ISS or corticosteroids. ISS induced significant levels of BAL interferon-{gamma}, whereas corticosteroids did not induce expression of BAL interferon-{gamma}. Both ISS and corticosteroids significantly reduced levels of interleukin-5 in BAL, as well as the number of Periodic Acid Schiff–positive airway epithelial cells. Corticosteroids, but not ISS, increased the number of eosinophils in regional mediastinal lymph nodes. Very few apoptotic peribronchial cells were noted following ovalbumin challenge as assessed by TUNEL assay. Corticosteroids, but not ISS, induced an increase in the small number of apoptotic peribronchial cells. The mechanism by which either ISS or corticosteroids inhibit AHR is likely to be mediated by distinct and shared cellular pathways. The combination of the shared and distinct anti-inflammatory pathways may account for the additive effect of ISS and corticosteroids on inhibiting AHR.

Abbreviations: airway hyperresponsiveness, AHR • bronchoalveolar lavage, BAL • BAL fluid, BALF • dynamic compliance, Cdyn • diaminobenzidine, DAB • interferon, IFN • interleukin, IL • immunostimulatory DNA sequence, ISS • methacholine, MCh • ovalbumin, OVA • Periodic Acid Schiff, PAS • Toll-like receptor 9, TLR-9


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Allergic asthma is characterized by Th2 lymphocyte activation and infiltration of the airways with eosinophils following the inhalation of antigen. Th2 cytokines such as interleukin (IL)-5 promote airway eosinophilia through several different mechanisms, including increased bone marrow production of eosinophils and enhanced eosinophil tissue survival (1, 2). In addition to IL-5, several other Th2 cytokines, including IL-4, IL-9, and IL-13, play an important role in allergic inflammation through their effects on IgE synthesis, mast cell proliferation, endothelial adhesion molecule expression, mucus secretion, and airway responsiveness (3). Thus, it has become increasingly clear that the inflammatory cascade orchestrated by Th2 lymphocytes plays a pivotal role in the pathogenesis and propagation of allergic inflammation in asthma.

As targeting a single Th2 cytokine may not be as effective a therapeutic strategy as targeting multiple Th2 cytokines, we have investigated the ability of immunostimulatory DNA sequences (ISS) to globally inhibit Th2 responses in asthma. Our (4–6) and other (7, 8) laboratories have demonstrated that ISS containing CpG-rich motifs administered after allergen sensitization but before inhalation allergen challenge, inhibit Th2 responses and airway hyperreactivity (AHR) in mouse models of asthma. ISS inhibits IL-3 (4), IL-4 (7), IL-5 (4), and granulocyte macrophage–colony-stimulating factor (4) production by Th2 cells. In addition to inhibiting Th2 responses, ISS activates the innate immune response to release cytokines including interferon (IFN)-{alpha} (9), IFN-ß (9), IFN-{gamma} (10), IL-6 (11, 12), IL-12 (12, 13), and IL-18 (14). Although the intracellular pathways mediating ISS signaling are not completely understood, recent studies have demonstrated an important role for TLR 9 cell surface receptors (15) and activation of intracellular DNA-protein kinase (16), with the final common pathway of I-{kappa}-kinase induction, nuclear factor-{kappa}B activation, and modulation of immune response genes.

Whereas previous studies have demonstrated an important role for ISS as preventive therapy for asthma, in this study we were interested to compare how effective ISS therapy might be compared with systemic corticosteroids in the treatment of an acute exacerbation of asthma in a mouse model. We designed our current study to compare the therapeutic efficacy of ISS with that of systemic corticosteroids, the current standard therapy for acute exacerbations of asthma. As the mechanism by which inflammation resolves following an acute episode of asthma is incompletely understood, we also investigated the effects of ISS and corticosteroids on eosinophil trafficking into the lung, eosinophil clearance from the lung to regional lymph nodes, and apoptosis of peribronchial airway cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Oligonucleotides
Endotoxin-free (< 1 ng/mg DNA) phosphorothioate ISS-ODN (5'-TGACTGTGAACGTTCGAGATGA-3') (Trilink, San Diego, CA) synthesized as previously described (4), were used in the in vivo experiments described below.

Animals
Female BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) were used when they reached 8–10 wk of age. All animal experimental protocols were approved by the University of California, San Diego Animal Subjects Committees.

Ovalbumin Sensitization and Allergen Challenge
Mice were immunized subcutaneously on Days 0, 7, 14, and 21 with 25 µg of ovalbumin (OVA, grade V; Sigma, St. Louis, MO) adsorbed to 1 mg of alum (Aldrich, Milwaukee, WI) in 200 µl phosphate-buffered saline (PBS) (see Figure 1 outlining the experimental protocol) as previously described in this laboratory (4). On Days 26 and 31 OVA inhalation challenges were performed via the intranasal route, using 20 µg of OVA in 50 µl of PBS. To facilitate intranasal administration of allergen, mice were briefly anesthetized with isoflurane (Isosol; Abbott Laboratories, North Chicago, IL).



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  		Figure 1. Mouse experimental protocol. Mice were immunized with weekly subcutaneous OVA/Alum injections on Days 0, 7, 14, and 21. On Days 26 and 31, OVA inhalations were administered via the intranasal route. On Day 32, mice received either no treatment, ISS treatment intraperitoneally, or dexamethasone intraperitoneally. The mice were sacrificed on Day 38 after bronchial hyperreactivity was measured.

 
Therapeutic Intervention with ISS or Corticosteroids
In comparing the effect of ISS with that of corticosteroids on allergic airway inflammation, we administered a single systemic dose of the long-acting corticosteroid dexamethasone (0.5 mg/kg, the equivalent of 35 mg dexamethasone for a 70-kg patient) or a single 100-µg systemic dose of ISS 24 h after intranasal allergen exposures in sensitized mice (protocol summarized in Figure 1). One day after the final allergen challenge, different groups of mice received systemic therapy with either ISS (100 µg intraperitoneally), corticosteroids (dexamethasone, 0.5 mg/kg intrapeitoneally), the combination (ISS and corticosteroids), or placebo therapy (diluent).

We chose the dose of ISS and dexamethasone used in this study based on pilot dose response studies with ISS and dexamethasone. These studies demonstrated that administration of ISS (100 µg) or dexamethasone (0.5 mg/kg) induced maximal inhibition of airway eosinophil responses, with no additional effect noted with higher doses (data not shown). We therefore chose these doses of ISS and dexamethasone for our studies, as we wished to compare the effect of maximal inhibitory responses induced by each therapeutic agent.

We chose to analyze the effects of the different treatment modalities 6 d after therapy was instituted, a time point when untreated mice that are sensitized and challenged still have significant levels of bronchoalveolar lavage fluid (BALF) eosinophilia ({bsim} 40–50%) and AHR, allowing us to demonstrate a treatment effect if present. Previous studies from our laboratory (4) have demonstrated that the single dose of ISS or dexamethasone we have chosen for this study is biologically active for at least 6 d, and effectively inhibits the development of eosinophilic inflammation and AHR to methacholine (MCh) assessed 6 d after therapy was instituted. We therefore chose this 6 d post-therapy time point to study the effect of our therapeutic intervention on established eosinophilic inflammation and AHR to MCh.

Determination of Airway Responsiveness to MCh In Vivo
Airway responsiveness was assessed on Day 38, 6 d after therapeutic intervention, using a single-chamber whole body plethysmograph obtained from Buxco (Troy, NY), as previously described (4). In selected experiments measurements of peak inspiratory pressure and dynamic compliance in intubated and ventilated mice were also performed.

Noninvasive plethysmography. The enhanced pause (Penh), a dimensionless value which correlates well with pulmonary resistance measured by conventional two-chamber plethysmography in ventilated mice (17), was used to monitor airway responsiveness. In the plethysmograph, mice were exposed for 3 min to nebulized PBS to establish baseline Penh values, and were subsequently exposed to increasing concentrations of nebulized MCh (Sigma) in PBS using an Aerosonic ultrasonic nebulizer (DeVilbiss, Somerset, PA). Following 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 (17).

Measurement of dynamic compliance. Mouse intubation and ventilation. The mice were anesthetized with pentobarbital sodium (60–70 mg/kg intraperitoneally). An anterior, midcervical skin incision was made to expose the trachea, which was cannulated with an 18-gauge blunt needle. Mice were ventilated (Mouse Ventilator, model 683; Harvard Apparatus, Holliston, MA) initially with a tidal volume (VT) of 5–6 ml/kg at 120 breaths/min. The tidal volume was then adjusted and set to just prevent spontaneous respiration. Peak inspiratory pressure (PI) and peak expiratory pressure (PE) were recorded using Validyne pressure transducers.

MCh challenge.
MCh in PBS at a concentration of 48 mg/ml was aerosolized by an ultrasonic nebulizer (AeroSonic 5000D; DeVilbiss) and delivered to the airway opening through the inspiratory ventilator flow for 2 min at a tidal volume of {bsim} 0.4 ml.

Dynamic compliance measurement.
Dynamic compliance was measured before and after MCh challenge, at ambient tidal volume ({bsim} 0.4 ml) and frequency. Dynamic compliance was calculated using the following formula: VT/(PI - PE).

BAL, Lung, Bone Marrow, and Blood Eosinophil Counts
Immediately following the plethysmography on Day 38, the mice were killed by CO2 asphyxiation, and eosinophil counts of various tissues were performed.

BALF. The tracheas of the sacrificed mice were surgically exposed and cannulated with 27-gauge silicon tubing attached to a 23-gauge needle on a 1-ml tuberculin syringe. A quantity of 600 µl of sterile PBS was instilled through the trachea into the lung and withdrawn. The BALF was cytospun (3 min at 500 rpm) onto microscope slides and stained with Wright-Giemsa. The percentage of BALF eosinophils was obtained by counting 400 leukocytes on randomly selected portions of the slide by light microscopy (x40 magnification). BALF total white blood cell counts were performed using a hemacytometer.

Lung. Lung tissues embedded in TissueTek (O.C.T. Compound; Sakura, Torrance, CA) in 10 x 50 x 50 mm tissue wells were cryosectioned at 5 µ and acetone-fixed onto poly(L-lysine)-coated slides. Diaminobenzidine (DAB) staining was used to detect eosinophil peroxidase, and total eosinophil numbers were enumerated by light microscopy, as described in this laboratory (4). Slides were rinsed in PBS and incubated for 8 min with the peroxidase substrate DAB (Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin, air dried, and examined by light microscopy (x40 magnification). Five random fields were selected and eosinophils were counted to quantitate the number of eosinophils per mm2.

Peripheral blood. Blood was collected from the carotid and subclavian arteries. Red blood cells were lysed using a 1:10 solution of 100 mM potassium carbonate, 1.5 M ammonium chloride. The remaining cells were cytospun (3 min at 500 rpm) onto microscope slides and air dried before staining with Wright-Giemsa. Eosinophil counts were performed as described above. Total white blood cell counts were performed using an automated hematology analyzer (Cell-Dyn 4000; Abbot Diagnostics, Abbot Park, IL).

Bone marrow. Bone marrow cells were flushed from femurs with 1 ml PBS and cytospun onto microscope slides before staining with Wright-Giemsa. Eosinophil counts were performed as described above. Total bone marrow leukocyte counts were obtained by flushing all of the marrow from a standardized 1-cm length of femur in each mouse with 1 ml of PBS. Manual total white blood cell counts were subsequently performed using a hemacytometer.

Lymph nodes. Lymph nodes were dissected from the mediastinum in each mouse. The lymph nodes were embedded in OCT in 10 x 50 x 50 mm tissue wells and were cryosectioned and fixed onto poly(L-lysine)-coated slides. DAB staining was used to detect eosinophils in the lymph node cryosections. Light microscopy examination was performed at x40 magnification. Five random fields were selected and eosinophils were counted to determine the total eosinophil number per mm2.

Periodic Acid Schiff Staining of Airway Mucus Cells
Lungs were tied off at the trachea with surgical suture and were preserved in 10% buffered formalin (Sigma Diagnostics) before being embedded in paraffin. The paraffin-embedded lungs were sectioned at 5 µ onto microscope slides. The paraffin was removed from the lung sections using alcohol gradients and Citrosolv (Fisher Scientific, Tustin, CA). Periodic Acid Schiff (PAS) stained airway goblet cells were enumerated by light microscopy examination (x40 magnification).The number of PAS-positive or PAS-negative airway epithelial cells was assessed in ten randomly selected medium-sized bronchi (defined by having approximately 100–150 luminal airway epithelial cells). The percentage of PAS-positive airway epithelial cells was then calculated for each airway and the mean determined for each mouse as previously described (18).

Apoptosis Assays
An ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Intergen, Purchase, NY), based on a TUNEL assay method, was used to detect apoptotic cells in cryosectioned lungs. Digoxigenin-labeled nucleotides and terminal deoxynucleotidyl transferase (TdT) were added to the cryosectioned lungs to label the free 3' DNA ends of apoptotic cells (19, 20). An antidigoxigenin antibody conjugated to peroxidase was used to label the incorporated digoxigenin-labeled nucleotides, and developed with the substrate supplied by the manufacturer of the kit. The lung section was counterstained with methyl green (Vector Laboratories, Burlingame, CA) for 10 min to visualize lung cells. Apoptotic cells in the bronchus and peribronchial space were quantitated using light microscopy at x40 magnification in ten randomly selected medium-sized airways (defined by having approximately 100–150 luminal airway epithelial cells) in each mouse. The bronchus was centered in the high-power field, and all of the apoptotic cells in the field counted. The number of apoptotic cells was also counted in ten randomly selected interstitial compartments (defined by the absence of bronchi) at x40 magnification in each mouse.

Cytokine Assays
The levels of cytokines (IL-5 and IFN-{gamma}) in BALF supernatants obtained on the day of sacrifice were measured by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN), as was previously described (4). The sensitivity of the IL-5 assay was 9.6 pg/ml and the IFN-{gamma} assay was 9.4 pg/ml.

Statistical Analysis
Statistical analysis was performed with ANOVA, and individual groups were compared using an unpaired Mann-Whitney Test (4). A P value of < 0.05 was considered statistically significant. Results are expressed as the mean ± SEM unless otherwise indicated. All statistical analyses were performed with In-Stat software (GraphPad Software, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Resolution of Airway Eosinophilic Inflammation following Allergen Challenge
To determine the natural time course of the resolution of airway eosinophilic inflammation following OVA sensitization and allergen challenge, mice were sacrificed at different time points after the final allergen challenge, and the level of BAL eosinophilia was determined. The level of BAL eosinophilia peaked 2 d after the final allergen challenge (83.6 ± 0.7%) and was significantly greater than levels noted in unchallenged mice (< 1%). Six days after the final allergen challenge, the level of BAL eosinophilia was 49.6 ± 3.1%, 10 d after the final allergen challenge the level of BAL eosinophilia was 22.0 ± 3.5%, and 15 d after the final allergen challenge the level of BAL eosinophilia was 4.9 ± 1.2%. To determine the effect of therapy on the resolution of BAL eosinophila we thus chose a time point (6 d after institution of treatment with ISS or corticosteroids) during which there were still significant levels of BAL eosinophilia ({bsim} 40–50%) to assess the influence of therapy.

ISS Administered as Therapy following Allergen-Induced Airway Inflammation Inhibited Subsequent Development of Airway Hyperresponsiveness
In the absence of therapy, mice developed significant AHR to Mch as assessed by Penh measurements (Figure 2). ISS administered 1 d after allergen challenge was still able to significantly inhibit AHR to Mch measured 6 d after institution of therapy (ISS versus untreated: Mch 24 mg/ml, P = 0.001; Mch 48 mg/ml, P = 0.008) (n = 24–26 mice/group).



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  		Figure 2. Inhibition of allergen induced airway reactivity to methacholine by ISS, dexamethasone, and combined treatment. Treatment with ISS one day after the inhaled allergen challenge inhibited the methacholine (Mch) induced increase in Penh (n = 24; *P = 0.001 at 24 mg/ml Mch and P = 0.008 at 48 mg/ml Mch compared with untreated mice). Treatment with dexamethasone significantly inhibited allergen-induced airway reactivity to Mch compared with untreated mice at 24 mg/ml Mch only (n = 23; *P = 0.004 at 24 mg/ml Mch and P = 0.11 at 48 mg/ml Mch). Combined treatment with ISS and dexamethasone significantly inhibited airway responsiveness to Mch compared with ISS alone (Mch 24 mg/ml P = 0.002, Mch 48 mg/ml P = 0.0001) or dexamethasone alone (Mch 24 mg/ml P = 0.0001, Mch 48 mg/ml P < 0.0001) treatment alone. Results are expressed as the mean ± SEM.

 
Dexamethasone also significantly inhibited AHR to Mch to a similar degree, as noted with ISS therapy at Mch 24 mg/ml (dexamethasone versus untreated: Mch 24 mg/ml, P = 0.004) (n = 23–26 mice/group) (Figure 2). However, at a concentration of 48 mg/ml of Mch, dexamethasone reduced AHR, but this was not statistically significant (dexamethasone versus untreated: Mch 48 mg/ml, P = 0.11). Combination therapy with dexamethasone and ISS had an additive effect on the inhibition of AHR compared with monotherapy with dexamethasone alone (ISS and dexamethasone versus dexamethasone: Mch 48 mg/ml, P = <0.0001) (n = 21–26 mice/group) (Figure 2).

In selected experiments, we performed invasive measures of changes in dynamic compliance (Cdyn) in response to nebulized MCh in intubated ventilated mice. Mean baseline levels of Cdyn before MCh challenge were not significantly different in the OVA-challenged mice (0.0298 ± 0.0059 ml/cm H2O) compared with the OVA + ISS– (0.0275 ± 0.0056 ml/cm H2O), or the OVA + dexamethasone–treated mice (0.0276 ± 0.0017 ml/cm H2O). Measurement of Cdyn in OVA-challenged mice after MCh challenge (0.0109 ± 0.0023 ml/cm H2O) (66% reduction in Cdyn) demonstrated that both ISS (0.0160 ± 0.0026 ml/cm H2O, OVA versus OVA + ISS; P = 0.05) and corticosteroid therapy (0.0165 ± 0.0017 ml/cm H2O, OVA versus OVA + dexamethasone; P = 0.05) improved Cdyn measurements.

ISS Administered as Treatment for Airway Inflammation Inhibited BAL Eosinophilia as Effectively as Dexamethasone
In the absence of therapy, OVA-challenged mice developed significant BAL airway eosinophilia (402.4 ± 158.0 x 103 BAL eosinophils, n = 22 mice) compared with mice that had not been challenged with OVA (0.7 ± 0.2 x 103 BAL eosinophils, n = 11 mice; P < 0.0001). ISS administered 1 d after airway allergen challenge significantly reduced the absolute number of airway eosinophils (71.7 ± 24.2 x 103 BAL eosinophils, n = 21 mice; P = 0.001, Figure 3A) when compared with the group of OVA-challenged mice that received no treatment.




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  		Figure 3. Inhibition of lung eosinophilia by ISS and dexamethasone. (A) BAL Eosinophils. OVA challenge induced significant BAL eosinophilia in untreated mice compared with nonchallenged naive mice (***P < 0.0001). Treatment with ISS one day after the inhaled allergen challenge significantly reduced BAL fluid eosinophilia compared with untreated mice (**P = 0.001), as did treatment with dexamethasone (**P = 0.0002). Combined treatment with ISS and dexamethasone significantly decreased BAL fluid eosinophilia compared with untreated mice (***P < 0.0001). Results are expressed as the mean ± SEM. (B) Lung Eosinophils. OVA challenge induced significant lung eosinophilia in untreated mice compared with nonchallenged naive mice (***P < 0.0001). Treatment with ISS 1 d after the inhaled allergen challenge significantly decreased lung eosinophilia compared with untreated mice (***P < 0.0001), as did treatment with dexamethasone (**P = 0.0006). Combined treatment reduced lung eosinophilia significantly compared with dexamethasone monotherapy (*P < 0.05), but not compared with ISS monotherapy. Results are expressed as the mean ± SEM.

 
Dexamethasone, like ISS, significantly reduced the absolute number of airway eosinophils (39.0 ± 3.1 x 103 BAL eosinophils, n = 18 mice; P = 0.0002 versus no therapy 402.4 ± 158.0 x 103 BAL eosinophils). Combination therapy with ISS and dexamethasone led to a further reduction in the absolute number of airway eosinophils (24.1 ± 3.1 x 103 BAL eosinophils, n = 17 mice; P < 0.0001), which represented a statistically significant decrease compared with monotherapy with dexamethasone (P = 0.005) but not compared with ISS monotherapy (P = ns). However, as monotherapy with either ISS or dexamethasone inhibited airway eosinophilia by 80–90%, the effect of combination therapy on airway eosinophilia was not likely to be significantly better than monotherapy.

Therefore, to determine whether the combination of ISS and corticosteroids had an additive effect on inhibiting eosinophilic airway inflammation, we administered a suboptimal dose of ISS (10 µg) and a suboptimal dose of dexamethasone (0.005 mg/kg) alone or in combination to different groups of mice. These studies demonstrated that a suboptimal dose of ISS (10 µg) inhibited BAL eosinophilia by 42% (P = 0.05 versus no therapy, n = 8), and the suboptimal dose of dexamethasone (0.005 mg/kg) inhibited BAL eosinophila by 69% (P = 0.05 versus no therapy, n = 8), whereas the combination inhibited BAL eosinophilia by 73% (P = 0.05 versus no therapy, n = 8). The combination of the suboptimal doses of ISS and dexamethasone was therefore not more effective in inhibiting eosinophilic inflammation compared with a suboptimal dose of dexamethasone alone. The combination of the suboptimal doses of ISS and dexamethasone also inhibited eosinophilic inflammation less (73%) compared with an optimal dose of either dexamethasone or ISS alone (> 90% inhibition).

ISS Administered as Therapy for Airway Inflammation Inhibited Lung Eosinophilia as Effectively as Dexamethasone
In the absence of therapy, OVA-challenged mice developed significant lung eosinophilia (605.1 ± 41.9 eosinophils/mm2, n = 19 mice) compared with mice that had not been challenged with OVA (15.6 ± 2.5 eosinophils/mm2, n = 11 mice, P < 0.0001) (Figure 3B). ISS administered 1 d after airway allergen challenge significantly reduced the absolute number of lung eosinophils (258.4 ± 30.6 eosinophils/mm2, n = 23 mice, P < 0.0001) when compared with OVA-challenged mice that received no treatment.

Dexamethasone, like ISS, significantly reduced the absolute number of lung eosinophils (330.6 ± 53.5 eosinophils/mm2, n = 16 mice; P = 0.0006 versus no therapy, 605.1 ± 41.9 eosinophils/mm2) (Figure 3B). Combination therapy with ISS and dexamethasone led to a further reduction in the absolute number of lung eosinophils (213.8 ± 26.2 eosinophils/mm2, n = 18 mice), which represented a statistically significant decrease compared with monotherapy with dexamethasone (P < 0.05).

ISS Administered as Treatment for Allergic Inflammation Inhibited Bone Marrow and Peripheral Blood Eosinophilia More Effectively than Dexamethasone
To determine the mechanism of inhibition of airway eosinophilia, we analyzed the bone marrow production and lung clearance of eosinophils in mice treated with ISS or dexamethasone. Compared with the no-treatment group (614 ± 71 eosinophils/µl, n = 11), ISS significantly decreased bone marrow eosinophilia by 57% (262 ± 49 eosinophils/µl, n = 11; P < 0.0001). Dexamethasone also significantly decreased bone marrow eosinophils by 35% (400 ± 64 eosinophils/µl, n = 11; P = 0.01). Combination therapy with ISS and dexamethasone decreased bone marrow eosinophils by 66% (207 ± 32 eosinophils/µl, n = 14; P < 0.0001).

The reduction in bone marrow eosinophil counts induced by therapy with either ISS or dexamethasone was reflected in reduced peripheral blood eosinophil counts. Compared with the no-treatment group (518 ± 63 eosinophils/µl, n = 7), ISS reduced peripheral blood eosinophilia by 61% (201 ± 16 eosinophils/µl, n = 10; P = 0.001), whereas dexamethasone reduced peripheral blood eosinophilia by a statistically insignificant 20% (412 ± 43 eosinophils/µl, n = 11; P = ns), and combination therapy with ISS and dexamethasone reduced peripheral blood eosinophilia by 60% (206 ± 26 eosinophils/µl, n = 11; P = 0.0006), showing the same effect as ISS monotherapy.

ISS Administered as Therapy for Airway Inflammation Inhibits Generation of IL-5 and Induces Production of IFN-{gamma}
To investigate potential immunomodulatory mechanisms by which ISS and dexamethasone reduced airway eosinophilia, levels of BAL IL-5 were assessed by ELISA. Compared with the no-treatment group (BAL IL-5, 61.4 ± 6.3 pg/ml, n = 8), ISS significantly decreased BAL IL-5 by 50% (BAL IL-5, 30.7 ± 10.6 pg/ml, n = 8; (P = 0.01) (Figure 4A). Dexamethasone also significantly decreased BAL IL-5 by 35% (BAL IL-5, 40.1 ± 5.0 pg/ml, n = 8; P = 0.01). Combination therapy with ISS and dexamethasone (BAL IL-5, 29.8 ± 4.4 pg/ml, n = 8) did not reduce IL-5 production more than ISS monotherapy.




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  		Figure 4. Inhibition of BAL IL-5 and induction of BAL IFN-{gamma} by ISS. (A) BAL IL-5. OVA-challenged mice pretreated with ISS had significantly lower levels of BAL IL-5 than untreated OVA-challenged mice (*P = 0.01; untreated versus ISS). Similarly, OVA-challenged mice pretreated with dexamethasone had significantly lower levels of BAL IL-5 than untreated OVA-challenged mice (*P = 0.01; untreated versus dexamethasone). Combination therapy with ISS and dexamethasone did not reduce BAL IL-5 further (**P = 0.002; ISS and dexamethasone versus untreated mice). (B) BAL IFN-{gamma}. BAL IFN-{gamma} levels were below the sensitivity of the IFN-{gamma} assay in OVA-challenged untreated as well as OVA-challenged dexamethasone-treated mice. In OVA-challenged mice, ISS significantly induced BAL IFN-{gamma} (*P = 0.03; untreated versus ISS), as did the combination of ISS and dexamethasone (*P = 0.03; untreated versus ISS + dexamethasone). Dexamethasone in combination with ISS slightly reduced levels of BAL IFN-{gamma} compared with ISS therapy alone, but this was not statistically significant (P = ns; dexamethasone + ISS, versus ISS monotherapy).

 
IFN-{gamma} was not detected in the BAL of OVA-challenged mice (BAL IFN-{gamma}, < 9 pg/ml, n = 11). ISS induced significant levels of IFN-{gamma} (BAL IFN-{gamma}, 63.1 ± 23.5 pg/ml, n = 12; P = 0.03 versus no therapy) (Figure 4B). In contrast, dexamethasone did not induce IFN-{gamma} production. Combination therapy with ISS and dexamethasone induced less IFN-{gamma} (BAL IFN-{gamma}, 40.2 ± 17.3 pg/ml, n = 12) than ISS therapy alone, but this was not statistically significant.

Dexamethasone Therapy Increases Regional Lymph Node Eosinophilia
To investigate whether ISS or dexamethasone reduced lung and airway eosinophilia by increasing eosinophil clearance to regional lymph nodes, mediastinal lymph node eosinophil counts were performed (Figure 5). OVA challenge of untreated mice resulted in an increase in eosinophils in lymph nodes (OVA-challenged 45.5 ± 11.7 eosinophils/mm2, n = 13 versus nonchallenged 7.1 ± 1.9 eosinophils/mm2, n = 8). Compared with untreated mice, there was a marked increase in lymph node eosinophils in mice treated with dexamethasone (132.2 ± 27.6 eosinophils/mm2, n = 12; P = 0.003), as well as in mice treated with dexamethasone and ISS (160.9 ± 28.3 eosinophils/mm2, n = 15; P = 0.0001). There was no significant increase in the number of lymph node eosinophils in the ISS-treated group (64.7 ± 14.0 eosinophils/mm2, n = 13; P = ns versus untreated).



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  		Figure 5. Increase in regional lymph node eosinophilia with dexamethasone treatment. Mice exposed to inhaled OVA challenges had significantly higher numbers of mediastinal lymph node eosinophils compared with mice that were not challenged (*P = 0.03). Treatment with either dexamethasone (**P = 0.003 compared with untreated mice), or the combination of dexamethasone and ISS (***P < 0.0001 compared with untreated mice) further increased mediastinal lymph node eosinophilia, whereas ISS had no significant effect (P = ns) on regional lymph node eosinophilia. Combined treatment did not significantly increase lymph node eosinophilia compared with dexamethasone monotherapy (P = ns). Results are expressed as the mean ± SEM.

 
Dexamethasone Therapy Increases Apoptosis in the Peribronchial Region and Interstitial Space
To investigate whether ISS or dexamethasone may have reduced lung and airway inflammation through increased apoptosis, the level of peribronchial cellular apoptosis was assessed using a TUNEL assay (Figure 6A). In the absence of therapy, the number of apoptotic cells was increased in OVA-challenged compared with unchallenged mice in the peribronchial region (4.0 ± 0.8, versus 1.5 ± 0.3 apoptotic cells/mm2, n = 11; P = 0.001) (Figure 6B) and interstitial compartments (3.4 ± 0.7 versus 0.9 ± 0.2 apoptotic cells/mm2, n = 11; P = 0.004) (Figure 6C).





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  		Figure 6. Dexamethasone increased the number of apoptotic cells in the lung. (A) Peribronchial apoptosis. A demonstrates several peribronchial apoptotic cells in a dexamethasone-treated mouse at x40 magnification. (B) Peribronchial compartment. An increased number of apoptotic cells were detected in the peribronchial compartment of untreated OVA-challenged mice compared with naive mice that were not allergen-challenged (**P = 0.001). Dexamethasone increased the number of apoptotic cells in the peribronchial compartment compared with untreated mice (**P = 0.006). ISS (versus no treatment) had no effect on the number of apoptotic cells detected in the peribronchial compartment. (C) Interstitial compartment. An increased number of apoptotic cells were detected in the interstitial compartment of untreated OVA-challenged mice compared with naive mice that were not allergen-challenged (**P = 0.004). Dexamethasone increased the number of apoptotic cells in the interstitial compartment compared with untreated mice (*P = 0.01). ISS (versus no treatment) had no effect on the number of apoptotic cells detected in the interstitial compartment.

 
In the bronchus and the peribronchial space of OVA-challenged mice, the number of apoptotic cells was significantly higher in the dexamethasone-treated mice (8.8 ± 1.5 apoptotic cells/mm2, n = 11) compared with untreated mice (4.0 ± 0.8 apoptotic cells/mm2, n = 11; P = 0.006). The number of apoptotic cells was also noted to be higher in the interstitial compartment of OVA-challenged mice in the dexamethasone-treated mice (6.7 ± 1.2 apoptotic cells/mm2, n = 11) compared with untreated mice (3.4 ± 0.7 apoptotic cells/mm2, n = 11; P = 0.01). Although dexamethasone induced a statistically significant increase in the number of peribronchial and interstitial apoptotic cells, the absolute number of apoptotic cells detected was only a very small percentage of the total number of lung cells ({bsim} 1% of the cells examined). As a positive control for our TUNEL assay we used the positive control slides provided by the manufacturer of the apoptosis kit, which contained tissue (female rodent mammary gland obtained 3–5 d after the weaning of rat pups) known to have 1–2% apoptotic cells. We detected 1–2% apoptotic cells in the positive control tissue sections provided by the manufacturer of the apoptosis kit.

ISS did not change the number of apoptotic cells in the peribronchial (3.9 ± 1.0 apoptotic cells/mm2, n = 14; P = ns versus control) or interstitial compartments (2.4 ± 0.5 apoptotic cells/mm2, n = 14; P = ns versus control) (Figures 6B and 6C).

ISS and Dexamethasone Reduce the Percentage of PAS-Positive Airway Epithelial Cells
To examine whether ISS therapy following the induction of airway inflammation reduced mucus production, PAS staining was performed to quantitate the percentage of mucus-expressing airway epithelial cells (Figures 7A and 7B). Antigen challenge induced a significant increase in the percentage of PAS-positive cells in the airway compared with unchallenged mice (50.3 ± 2.6% PAS-positive cells/bronchus, n = 9; versus 0.4 ± 0.1% PAS-positive cells/bronchus, n = 11; P < 0.0001) (Figure 7C). Both ISS (23.6 ± 1.6% PAS-positive cells/bronchus, n = 10; P < 0.0001 versus untreated) and dexamethasone (21.6 ± 5.0% PAS-positive cells/bronchus, n = 8; P = 0.0008 versus untreated) treated mice had a significantly lower percentage of PAS-positive cells per bronchus than the mice receiving no treatment. Combination therapy with ISS and dexamethasone (15.1 ± 2.2% PAS-positive cells/bronchus, n = 7) significantly reduced the percentage of PAS-positive cells per bronchus compared with ISS monotherapy (P = 0.005), whereas it did not significantly reduce the percentage of PAS-positive cells per bronchus compared with corticosteroid monotherapy (P = ns).





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  		Figure 7. Reduction in the percentage of PAS-positive airway cells induced by ISS and dexamethasone. (A and B) PAS stain of OVA-challenged untreated and ISS-treated airways. Figure is an example of PAS staining of OVA-challenged airway in untreated (A) and ISS-treated (B) mice at x40 magnification. (C) PAS-positive bronchioles. OVA-challenged untreated mice had significantly more PAS-positive airway cells compared with naive unchallenged mice (***P < 0.0001). ISS significantly reduced the percentage of PAS-positive airway cells (***P < 0.0001 compared with untreated OVA-challenged mice), whereas dexamethasone similarly decreased the percentage of PAS-positive airway cells (**P = 0.0008). Combined treatment decreased the percentage of PAS-positive airway cells further (***P < 0.0001 compared with no treatment).

 
Effect of ISS and Corticosteroids on the Total Number of BAL Lymphocytes
The total number of BAL T lymphocytes following OVA challenge (3.1 ± 2.6 x 104 BAL lymphocytes) was significantly reduced in ISS-treated mice (1.3 ± 0.7 x 104 BAL lymphocytes, n = 8; P < 0.001, ISS + OVA, versus untreated OVA challenge), and in dexamethasone-treated mice (2.1 ± 1.4 x 104 BAL lymphocytes, n = 8; P = 0.008, dexamethasone + OVA, versus untreated OVA challenge). The combination of ISS and dexamethasone treatment (1.2 ± 0.7 x 104 BAL lymphocytes) did not reduce the total number of BAL lymphocytes more than ISS alone (1.3 ± 0.7 x 104 BAL lymphocytes, n = 8; P = ns).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have demonstrated that ISS administered as therapy for allergic airway inflammation and AHR, such as might occur during an acute episode of asthma, decreases AHR as effectively as dexamethasone, and that the combination of ISS and dexamethasone is more effective than monotherapy with either agent alone in reducing AHR. The mechanism by which either ISS or corticosteroids inhibit AHR is likely to be mediated by distinct and shared cellular pathways. Distinct anti-inflammatory pathways are suggested by the fact that ISS and corticosteroids differ in their mechanism of eosinophil clearance to regional lymph nodes, induction of cellular apoptosis in peribronchial regions, and induction of the Th1 cytokine IFN-{gamma}, which has antieosinophilic effects (21). Shared anti-inflammatory pathways are suggested by the fact that ISS and corticosteroids both inhibit Th2 cytokine responses (IL-5), the total number of BAL T lymphocytes, and mucus production to a similar degree. The combination of the shared and distinct anti-inflammatory pathways (some of which we have identified), may account for the additive effect of ISS and corticosteroids on inhibiting AHR.

As eosinophilic inflammation is one cellular pathway leading to AHR, we have investigated the effect of ISS and corticosteroids on eosinophil production, trafficking into the lung, and clearance from the lung. Both ISS and corticosteroids significantly inhibited airway eosinophilia. ISS was more effective than corticosteroids in inhibiting bone marrow and blood eosinophilia, suggesting that ISS exerted a greater inhibitory effect on the bone marrow production and release of eosinophils into the circulation. However, corticosteroids differed from ISS in that corticosteroids, but not ISS, increased clearance of eosinophils to regional lymph nodes. Previous studies have demonstrated that fluorescently-labeled eosinophils, recovered from both antigen-challenged airways and from the peritoneal cavity of IL-5 transgenic mice, when instilled into the tracheal lumen of normal mice, homed to the peritracheal lymph nodes (10% within 24 h) and were able to stimulate antigen-specific CD4+, but not CD8+, T cell proliferation (22). Our study suggests that corticosteroids might augment the migration of eosinophils from the airway mucosa to regional lymph nodes and thus reduce eosinophilic inflammation in the lung.

Apoptosis of recruited airway inflammatory cells such as eosinophils is one mechanism by which eosinophilic inflammation may resolve in asthma. Corticosteroids are known to induce eosinophil apoptosis in vitro (23). Corticosteroid-treated patients with asthma have decreased airway eosinophilia, increased eosinophil apoptosis, and increased expression of Bcl-2, Fas, and Fas ligand (24). Sputum samples obtained during an asthma exacerbation in corticosteroid-treated subjects demonstrate decreased airway eosinophilia and increased eosinophil apoptosis (25). Furthermore, the number of apoptotic eosinophils and macrophages in bronchial biopsy specimens from subjects with asthma is inversely correlated with the severity of asthma symptoms (26). Delayed eosinophil apoptosis (27) may contribute to tissue eosinophilia at sites of allergic inflammation (28). In mouse models of asthma lung eosinophils lavaged from aerosol allergen-challenged mice express the Fas receptor, and when activated in vitro with an anti-Fas mAb induces eosinophil death by apoptosis (29). In vivo administration of an anti-Fas antibody to mice that were sensitized and allergen-challenged with OVA-induced eosinophil apoptosis, and also suppressed AHR (30).

We observed an increased number of apoptotic cells in the peribronchial compartment of allergen-challenged compared with naive mice. As the number of lung eosinophils following allergen challenge ({bsim} 600 eosinophils/mm2) far exceeded the number of peribronchial apoptotic cells ({bsim} 4 cells/mm2), the vast majority of eosinophils were not apoptotic 7 d after inhalation of OVA allergen. As levels of IL-5 were still significantly elevated at this time point, the antiapoptotic effect of IL-5 could account for the small number of apoptotic eosinophils. We did, however, note that dexamethasone induced a significant increase in the number of peribronchial apoptotic cells in OVA-challenged mice. However, the number of apoptotic cells induced by corticosteroid therapy ({bsim} 9 cells/mm2) was also small in comparison to the number of peribronchial eosinophils ({bsim} 600 eosinophils/mm2). In contrast to corticosteroids, ISS did not induce peribronchial cellular apoptosis. We have previously demonstrated that ISS has no direct effect on inducing or inhibiting eosinophil apoptosis in vitro (4).

Several groups (7, 8), including ours (4), have demonstrated that ISS is an effective preventive therapy in mouse models of asthma. The ability of ISS to inhibit airway inflammation and AHR was suggested in a previous study (31), but that study design in which ISS was also administered between two inhalation allergen challenges (preventive therapy for the second inhalation) incorporated components of both preventive therapy (ISS administered after first inhalation of OVA is preventive therapy for the second OVA inhalation 6 d later) and acute therapy following allergen exposure, and precluded definitively assessing whether ISS administered only as therapy after allergen challenge is effective. In this study, we have only administered ISS after both inhalation challenges had been completed to assure that no preventive therapy was incorporated into the study design. In addition, we have compared for the first time the efficacy of ISS compared with systemic corticosteroids, the standard therapy for acute exacerbations of asthma.

In summary, we have demonstrated that ISS administered as therapy can significantly reverse allergic airway inflammation and AHR. Combination therapy with ISS and dexamethasone had an additive effect on the inhibition of AHR. The mechanisms by which ISS and dexamethasone inhibit AHR likely use shared and distinct pathways, suggesting that combination therapy might reduce AHR more effectively than monotherapy with either therapy alone.


   Acknowledgments
 
The authors thank Peter Wagner, M.D. (UCSD) for performing measurements of dynamic compliance. This study was supported by an NIH grant T32 HL07022 (UCSD Pulmonary Division Training Grant) and an American Lung Association of California Research Training Fellowship Award (to R.I.), NIH grants AI 33977 and AI 38425 (to D.H.B.), and NIH grant AI 40682 and Dynavax (to E.R.).

Received in original form February 28, 2002


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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