This Article Abstract Full Text (PDF) Online Supplement All Versions of this Article: 2005-0456OCv1 36/3/276    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Karras, J. G. Articles by Gregory, S. A. Search for Related Content PubMed PubMed Citation Articles by Karras, J. G. Articles by Gregory, S. A.

Anti-Inflammatory Activity of Inhaled IL-4 Receptor- Antisense Oligonucleotide in Mice

Departments of Clinical Development, Pharmacokinetics, and Antisense Drug Discovery, Isis Pharmaceuticals, Carlsbad, California

Correspondence and requests for reprints should be addressed to James G. Karras, Ph.D., Department of Clinical Development, Isis Pharmaceuticals, 1896 Rutherford Road, Carlsbad, CA 92008. E-mail: jkarras{at}isisph.com

	Abstract

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
The Th2 cytokines IL-4 and IL-13 mediate allergic pulmonary inflammation and airways hyperreactivity (AHR) in asthma models through signaling dependent upon the IL-4 receptor- chain (IL-4R). IL-13 has been further implicated in the overproduction of mucus by the airway epithelium and in lung remodeling that commonly accompanies chronic inflammation. IL-4R–deficient mice are resistant to allergen-induced asthma, highlighting the therapeutic promise of selective molecular inhibitors of IL-4R. We designed a chemically modified IL-4R antisense oligonucleotide (IL-4R ASO) that specifically inhibits IL-4R protein expression in lung eosinophils, macrophages, dendritic cells, and airway epithelium after inhalation in allergen-challenged mice. Inhalation of IL-4R ASO attenuated allergen-induced AHR, suppressed airway eosinophilia and neutrophilia, and inhibited production of airway Th2 cytokines and chemokines in previously allergen-primed and -challenged mice. Histologic analysis of lungs from these animals demonstrated reduced goblet cell metaplasia and mucus staining that correlated with inhibition of Muc5AC gene expression in lung tissue. Therapeutic administration of inhaled IL-4R ASO in chronically allergen-challenged mice produced a spectrum of anti-inflammatory activity similar to that of systemically administered Dexamethasone with the added benefit of reduced airway neutrophilia. These data support the potential utility of a dual IL-4 and IL-13 oligonucleotide inhibitor in allergy/asthma, and suggest that local inhibition of IL-4R in the lung is sufficient to suppress allergen-induced pulmonary inflammation and AHR.

Key Words: allergy • chronic asthma • antisense oligonucleotide

CLINICAL RELEVANCE Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References   IL-4 receptor reduction restricted to the pulmonary compartment is sufficient to suppress lung inflammation and airways hyperresponsiveness in an asthma model. These findings support an inhaled IL-4 receptor antisense oligonucleotide therapeutic strategy for asthma.

 
The etiology of allergy and asthma is complex and multi-factorial, with evidence supporting roles for both genetic predisposition and environmental influence (1). Both disorders are characterized by nonproductive immune responses to normally innocuous environmental antigens. Despite the heterogeneity of these diseases, a unifying mechanism of dysregulated Th2 cytokine–mediated allergic inflammation is generally acknowledged (2). The presence of CD4+ T cells producing IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid and in airway epithelial biopsies of individuals with asthma has been clearly documented (3, 4). IL-5 has been linked to allergic eosinophilia but not AHR in humans (5), while IL-4 and IL-13 have been implicated in multiple pathologies of allergy and asthma, including elevated serum IgE via regulation of the Ig isotype switch to the epsilon heavy chain, increased cellular VCAM-1 expression, airway eosinophilia, the development of AHR, lung remodeling, and promotion of the secretory phenotype of the inflamed airway epithelium (reviewed in Ref. 6). These observations underscore the potential therapeutic value of inhibitors that are capable of blocking both IL-4 and IL-13 signaling.

As a common signaling chain of both the IL-4 and IL-13 receptors, IL-4R functions as a key intermediary protein in Th2 cytokine responses (7). IL-4R pairs with the common chain first identified as a component of the IL-2 receptor on cells of hematopoietic and nonhematopoietic origin to form the type I IL-4R that is exclusively used by IL-4. IL-13 signals through the type II IL-4R also expressed on both hematopoietic and nonhematopoietic cells. The type II IL-4R contains the IL-13 R1 chain and IL-4R in a heterodimeric complex that converts the normally low-affinity IL-13 R1 receptor to a high-affinity form. Recent studies suggest that the type II IL-4R may also allow signaling by IL-4 in nonhematopoietic cells (8). A second IL-13 receptor, IL-13 R2, exists as a single chain, binds IL-13 with high affinity, and is thought to act as a decoy receptor to negatively regulate IL-13 signaling (9, 10). Ligated type I and type II IL-4 receptors activate the Jak-Stat pathway, insulin–IL-4 receptor (I4R) motif–associated factors such as the insulin receptor substrate family of proteins, and SH2-containing tyrosine phosphatases (6). Stat6 mediates many of the functional activities of these receptors, including Th2 cell differentiation and IgE production, although I4R proteins appear to regulate IL-4–induced mitogenesis and anti-apoptosis (reviewed in Ref. 7). Other Stats, such as Stat1 and Stat3, have been implicated in IL-13R1–mediated activation of the 15-lipoxygenase pathway via p38 (11).

Genetic and pharmacologic studies have demonstrated that the IL-4R/Stat6 signaling pathway is required for allergen-induced pulmonary inflammation and AHR in mice (12, 13, and references therein). Both IL-4R– and Stat6-deficient mice do not develop AHR, lung inflammation, or mucus production in response to allergen challenge. While protein inhibitors of either IL-4 or IL-13 have produced anti-inflammatory effects in mouse pulmonary inflammation models or in clinical trials (13–19) and are currently being pursued as novel therapeutics for allergy and asthma, it is not clear whether protein-based biologicals that require parenteral administration display the necessary distribution properties within the lung and stability profile in vivo to succeed clinically. Since the pharmacokinetics, clinical safety, and preclinical and clinical efficacy of inhaled antisense molecules has been previously demonstrated (20, and references therein; 21), we hypothesized that inhibition of IL-4R substantially confined to the pulmonary tissues using a locally administered nucleic acid–based inhibitor would down-regulate the inflammatory Th2 immune response triggered by allergen challenge and improve airway performance in mice. In this study, we used an inhaled antisense oligonucleotide inhibitor to address the sufficiency of pulmonary IL-4R to mediate allergic inflammation in mouse models of asthma. Our results indicate that suppression of pulmonary IL-4R decreases the allergen-induced inflammatory response and suggests that inhaled IL-4R antisense is an effective anti-inflammatory approach for respiratory diseases.

	MATERIALS AND METHODS

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
Cell Culture, Oligonucleotides, and mRNA Analysis
Mouse brain endothelial cells (bEND.3; American Type Culture Collection, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS (Invitrogen, Carlsbad, CA). All oligonucleotides were synthesized with uniform phosphorothioate backbones and chimeric design, with 2'-O-methoxyethylribose (MOE)-modification on bases 1–5 and 16–20, termed "second generation" designs, differentiating them from "first generation" oligodeoxyribonucleotide phosphorothioates. The oligonucleotides were synthesized using an Applied Biosystems 380B automated DNA synthesizer (Perkin Elmer-Applied Biosystems, Foster City, CA) and purified as described (22). The sequences of the murine IL-4R antisense oligonucleotide (IL-4R ASO) and control oligonucleotides with 1-, 3-, 5-, or 7-base mismatches are shown in Table 1. All oligonucleotide sequences used were devoid of murine immune-stimulatory motifs and all cytosine residues were methylated. Oligonucleotide transfections were performed as previously described (22). Total RNA was purified 24 h after ASO transfection by elution into 100 µl of water using the RNeasy 96 Plate kit (Qiagen, Valencia, CA). RNA levels were quantified using the Perkin-Elmer (PE) ABI PRISM 7700 Sequence Detection System by real-time fluorescence PCR detection, as described previously (23). The IL-4R primer/probe sequences used were: forward primer, 5'-TCCCATTTTGTCCACCGAATA-3'; reverse primer, 5'-GTTTCTAGGCCCAGCTTCCA-3'; and probe, 5'-56-FAM-TGTCACTCAAGGCTCTCAGCGGTCC-36-TAMTph/-3'. For in vivo Muc5AC mRNA analysis, lungs were harvested from mice on Day 68, 6 h after secondary allergen challenge. RNA was extracted from 100 mg of lung tissue using Qiagen columns (Qiagen) and quantitative RT-PCR performed as described above. The primer/probe sequences used for detection of Muc5AC were: forward primer, 5'-CCTACCTGCCGCTTCCTCT-3'; reverse primer, 5'-GGGCCTCTCCTACCTCCAAG-3'; and probe, 5'-/56-FAM-CCAGAGGGACATCGACCCCAGATGG-36-TAMTph/-3'. For -actin: forward primer, 5'-ATTGCTGACAGGATGCAGAA-3'; reverse primer, 5'-GCTGATCCACATCTGCTGGAA-3'; and probe, 5'-/56-FAM-CAAGATCATTGCTCCTCCTGAGCGCA-36-TAMTph/-3'. Expression of Muc5AC was normalized to the level of -actin mRNA.

ASO suspended in 0.9% sodium chloride (Baxter Healthcare Corporation, Deerfield, IL) was delivered via inhalation using a nose-only delivery system as described previously (26). A Lovelace nebulizer (model 01–100), was used to deliver the ASO at a flow rate of 1 liter/min. The total flow rate of the delivery system was 10 liters/min. The mass median aerodynamic size range of the generated particles was determined to be 1.63–1.91 µm, within the respirable range for the mouse. The exposure chamber was equilibrated with the oligonucleotide aerosol suspension for 5 min before mice were placed in restraint tubes and attached to the chamber. Restrained mice were treated for a total of 10 min. In the secondary allergen challenge model, mice were given five aerosolized doses of ASO on Days 59, 61, 63, 66, and 68. Inhalation of aerosolized oligonucleotides in mice has previously been shown to result in broad distribution to multiple cell types within the lung (20, and references therein). We observed lung concentrations of 89.3, 200, and 279 ng/g of total lung at estimated inhaled doses (EID) of 10, 100, and 500 µg/kg, respectively, after ASO inhalation. In the chronic model, mice received therapeutic aerosolized ASO treatment on Days 31, 38, 45, 52, 59, 66, and 73. Control groups received Dexamethasone (Dex, 2.5 mg/kg; Sigma) in PBS injected intraperitoneally (0.2 ml) 1 h before each nebulized OVA challenge in the secondary allergen challenge model (Days 66 and 67) or 1 h before each intranasal challenge in the chronic model (Days 47, 62, 73, 74, and 75).

Determination of Inflammatory Cells in Bronchial Alveolar Lavage Fluid and ELISA Analysis
Mouse lungs were lavaged two times with 0.5 ml of PBS containing 2% fetal calf serum (FCS). Bronchoalveolar lavage (BAL) fluid samples were centrifuged to generate a cell pellet and a cell-free supernatant. The recovered airway cells were resuspended in PBS-2% FCS, and a cytospin was performed. Cells were stained with Diff-Quik stain (Dade Behring, Newark, DE). Data are presented as the percent of each inflammatory cell type present in the total recovered BAL cell population. Cell-free lavage fluid was frozen and stored at –80°C until analyzed. ELISA assays for IL-13, IL-5, CXCL8 (CXCL1 or "KC" in the mouse) and CCL2 (monocyte chemotactic protein [MCP]-1) were performed on the BAL supernatants using cytokine and chemokine ELISA kits from R&D Systems (Minneapolis, MN).

Measurement of AHR
AHR was determined by inducing bronchoconstriction with methacholine aerosol at escalating doses (27). Total pulmonary airflow in unrestrained mice was estimated using a whole-body plethysmograph (Buxco Electronics, Sharon, CT). Pressure differences between a chamber containing an individual mouse and a reference chamber were used to extrapolate the enhanced pause (Penh). Penh is a dimensionless parameter that is a function of total pulmonary airflow in mice during each respiratory cycle. This parameter closely correlates with airway resistance as measured by traditional invasive techniques using ventilated mice (27). Values for all measurements were obtained using Excel software and are expressed as the mean ± SEM. For invasive lung measurements, mice were first weighed and anesthetized with ketamine (150 mg/kg) mixed with xylazine (10 mg/kg). A tracheostomy was performed and the mice were ventilated using the Flexivent system (SCIREQ, Montreal, PQ, Canada) using traditional mouse parameters (28). Increasing concentrations of methacholine were aerosolized using the Flexivent system with an Aeroneb lab nebulizer system, and resistance (RL) and compliance (CL) were measured.

Assessment of Mucus Production
Lungs were inflated with 10% formalin overnight before embedding in paraffin. Mucus cell development along the airway epithelium was assessed in paraffin-embedded tissue sections (4 µm) stained with periodic acid-Schiff's reagent (PAS). The number of PAS-positive airways present in each lung section was determined. Parasagittal tissue sections were analyzed by bright field microscopy, and images were collected. Images were analyzed using ImagePro Plus (Media Cybernetics, Silver Spring, MD) to derive an airway mucus index (MI) reflective of both the amount of mucus per airway and the number of airways affected (29). The mucus content of all the airways per section (proximal to distal) was measured from groups of five animals. Image ProPlus was used to quantify the area and intensity of PAS staining per airway. The data were quantified as follows: MI = (average PAS staining intensity of airway epithelium) x (area of airway epithelium staining with PAS)/(total area of conducting airway epithelium) x (total number of airways assessed).

Determination of IL-4R Expression on Inflammatory and Epithelial Cells from Lungs of Allergen-Rechallenged Mice
Lungs were digested with type 1A collagenase as previously described (30). Briefly, whole lungs were collected in warm (37°C) RPMI-1640 basal media on Day 68, 24 h after the last secondary OVA challenge. Lungs were minced in 4 ml RPMI media containing 175 U/ml of type 1A collagenase (Sigma), penicillin (100 U/ml), streptomycin (100 µg/ml), and DNase (413 U/ml) (each from Invitrogen). Digestion was performed at 37°C for 20 min in an orbital incubator shaker (New Brunswick Scientific, New Brunswick, NJ). The tissue was sheared through a 20-guage needle and filtered through a sterile 70 µm cell strainer (BD Biosciences, Bedford, MA). The filtered cell suspensions were centrifuged at 1,200 rpm for 10 min at 4°C, supernatant decanted and red blood cells in the pellets lysed using cold erythrocyte lysis buffer (Qiagen). Samples were centrifuged at 1,200 rpm for 10 min at 4°C and washed once with PBS/0.5%BSA. 1–2E5 cells were used for each staining combination. Cells were incubated with the appropriate antibody combinations used to identify surface markers specific for granulocytes (CD11b+GR-1 hiSSChi), eosinophils (CD11b+Gr-1loCD49 d+), macrophages (CD11bloSSCloF4/80+), DCs (I-A^d+CD11chiCD83+) and epithelial cells (E-Cadh+CD11b-CD45-) using standard vendor protocols. To identify expression of IL-4R on the different cell types from collagenase digested lungs, cells were stained with anti-IL-4RPE antibody along with the cell type specific markers. Cells were analyzed on the FACSCalibur using Cell Quest software (BD Biosciences). The following antibodies were purchased from BD Pharmingen (San Diego, CA): anti-CD11bFITC (_M chain, Mac-1 -chain, clone M1/70), anti-GR-1PerCP (Ly-6G and Ly-6C, clone RB6–8C5), anti-CD11cAPC (_X chain, clone HL3), anti-I-A^d FITC (clone AMS 32.1), anti-E CadherinFITC, anti-CD49dPE (clone 9C10), and anti-IL-4RaPE (clone mIL-4R-M1). Anti-F4/80FITC (RM2901) was purchased from Caltag Laboratories (Burlingame, CA) and anti-CD83FITC antibody was purchased from Impact Biotechnology (Carlsbad, CA). Appropriate isotype control antibodies were used for all staining combinations.

Statistics
Analysis of group differences in Penh response was performed using two-way repeated measures ANOVA using JMP statistical discovery software (SAS, Cary, NC). Analysis of group differences in all other endpoints was performed using a Student's t test.

	RESULTS

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
Cell Surface IL-4R Protein Expression on Pulmonary Eosinophils, Macrophages, Dendritic Cells, and Epithelial Cells Is Reduced by Inhaled IL-4R ASO in a Secondary Allergen Challenge Mouse Model
Murine IL-4R ASOs were designed in silico and screened for inhibition of target mRNA in b.END3 cells using a high-throughput qRT-PCR approach. Lead compounds were initially characterized by dose response analysis in b.END3 cells, and the most potent ASO was evaluated for sequence-dependent down-regulation of IL-4R mRNA (Figure 1A). b.END3 cells were transfected with a series of oligonucleotides containing either 1-, 3-, 5-, or 7-base mismatches to the IL-4R mRNA sequence, and IL-4R mRNA levels were determined (see Table 1 for sequences). Only the one base mismatched oligonucleotide reduced IL-4R mRNA expression, with no effect of the 3-, 5-, or 7-base mismatch oligonucleotides observed. These results provide evidence that Watson-Crick hybridization is necessary for the activity of the parent IL-4R ASO, consistent with an antisense mechanism of action.

We evaluated IL-4R protein expression on pulmonary immune and structural cells in naïve and allergic mice and assessed the effects of inhaled IL-4R ASO treatment in the secondary allergen challenge model using multicolor flow cytometry. IL-4R protein expression on the CD11b+GR-1lo mixed eosinophil and macrophage population was quantitated as the mean fluorescence intensity (MFI) of anti–IL-4R staining relative to isotype control antibody for each treatment group and was significantly reduced at each dose of inhaled ASO tested (10–500 µg/kg estimated inhaled dose, EID; Figure 1B, upper panel). The MFI for the mixed population of eosinophils and macrophages was reduced by 34, 30, and 38% by IL-4R ASO treatment at 10, 100, and 500 µg/kg, respectively (P 0.05 at each dose level). The MFI of MHC II^loCD11c^hi DCs staining for IL-4R was also significantly decreased after inhaled IL-4R ASO treatment but only at the 500 µg/kg EID (Figure 1B, middle panel; 31% reduction, resulting in a level of expression below that on dendritic cells from naïve animals). However, DCs isolated from mediastinal lymph nodes after inhaled IL-4R ASO treatment did not show reduced levels of IL-4R expression. The lack of observed IL-4R protein reduction in draining lymph nodes of the lung suggests that systemic exposure is limited at efficacious doses. In other studies, we have demonstrated that absorption of inhaled oligonucleotide into the systemic circulation is less than 1% of the estimated lung deposited dose (R. Yu and R. Geary, unpublished observations).

View larger version (28K): [in this window] [in a new window]   Figure 1. Characterization of IL-4R ASO target-directed activity in vitro and after inhalation in mice. (A) Dose- and hybridization-dependent reduction of IL-4R mRNA expression in vitro after IL-4R ASO transfection. Demonstration of sequence-specific IL-4R mRNA reduction in b.END3 cells by quantitative RT-PCR 24 h after transfection with IL-4R ASO or 1-, 3-, 5-, or 7-base pair mismatch control oligonucleotides. (B) Reduction of cell surface IL-4R protein on pulmonary epithelial and antigen presenting cells from allergic mice that inhaled IL-4R ASO. OVA-sensitized and -challenged mice were rested for 1 mo before treatment with aerosolized oligonucleotides and subsequent rechallenge with nebulized OVA. Lungs were harvested 6 h after the second nebulized OVA rechallenge on Day 67 from mice treated with IL-4R ASO or its 7-base MM control oligonucleotide (MM; 100 and 500 µg/kg EID, administered on Days 59, 61, 63, 66, and 68). Lung cells were recovered after collagenase treatment of the tissue and analyzed by multi-parametric flow cytometry. IL-4R protein expression was measured on a mixed population of lung eosinophils and macrophages (CD11b-positive, GR-1 negative or low; upper panel), CD11c-positive and MHC class II–positive dendritic cells (middle panel), and E-cadherin–positive epithelial cells (lower panel). Data are expressed as the group mean percent of vehicle control treated cells (A) or the group mean fluorescence intensity (MFI; B) ± SE (n = 4/group). *P < 0.05, Student's t test. NA, naïve; MM, mismatched base control oligonucleotide; VH, vehicle.

Normalization of AHR and Inhibition of Airway Inflammation after Secondary Allergen Challenge in Mice Treated with Inhaled IL-4R ASO
The role of IL-4R in mediating the response of T cells to allergen challenge in previously primed mice was assessed. OVA-sensitized and -challenged mice were rested for 1 mo before treatment with inhaled IL-4R ASO and subsequent rechallenge to induce pulmonary inflammation and AHR. The estimated inhaled dose of IL-4R ASO sufficient to inhibit IL-4R protein expression on lung immune and structural cells (100 µg/kg) resulted in suppression of the OVA-induced Penh response in mice (Figure 2A). Systemically administered Dex (2.5 mg/kg intraperitoneally 1 h before each allergen rechallenge), in contrast, did not significantly inhibit Penh (Figure 2A). Similar exposure to a control oligonucleotide of the same chemistry but containing 7-base mismatches to the IL-4R mRNA target sequence was without effect. The minimal effective dose of IL-4R ASO was demonstrated to be > 0.1 µg/kg (Figure 2B; acutely challenged mice). In separate experiments in OVA-rechallenged mice, inhaled IL-4R ASO reduced RL and improved CL in anaestitized, tracheotomized, intubated, and mechanically ventilated mice, in the absence of similar effects by the mismatched control oligonucleotide (Figures 2C and 2D).

View larger version (15K): [in this window] [in a new window]   Figure 2. Inhaled IL-4R ASO suppresses AHR in previously allergen-primed and -challenged mice. (A) Methacholine-induced Penh dose–response curves from Balb/c mice that inhaled IL-4R ASO or a 7-base mismatched control oligonucleotide (as described in Figure 1) or saline before secondary challenge with nebulized OVA. A separate group of mice received Dexamethasone (Dex; 2.5 mg/kg intraperitoneally 24 h before each of two secondary allergen challenges). Responses of naïve mice (open triangles), saline vehicle–treated mice (open circles), vehicle plus Dex–treated mice (solid triangles), IL-4R ASO (100 µg/kg)–treated mice (open squares), and mismatch control oligonucleotide (100 µg/kg)–treated mice (open diamonds) are shown. The areas under the curves for the naïve and IL-4R ASO treatment groups are significantly different from that of the vehicle-treated group. (B) Dose–response of inhaled IL-4R ASO treatment in acutely OVA-challenged mice exposed to 100 mg/ml methacholine. (C and D) Measures of RL and CL in naïve or allergen-rechallenged mice treated with IL-4R ASO, MM, or saline and evaluated after anesthesia, tracheostomy, intubation, and mechanical ventilation, using a Flexivent apparatus. Data are presented as group means ± SE, n = 10/group except RL and CL, where n = 4–6/group. *P < 0.05, Student's t test.

View larger version (19K): [in this window] [in a new window]   Figure 3. Suppression of pulmonary inflammation in OVA-rechallenged mice treated with inhaled IL-4R ASO. Airway cells were recovered by BAL either 6 h after the second nebulized OVA rechallenge on Day 67 or immediately after measurement of AHR on Day 69 (rechallenge model) or Day 28 (acute model) and evaluated by cytospin and differential cell count analysis. Kinetic studies demonstrated peak neutrophil and eosinophil recruitment to the airways at these respective time points in the models (data not shown). (A) Dose response of inhaled IL-4R ASO treatment on airway eosinophilia in acutely OVA-challenged mice. (B) Effect of inhaled IL-4R ASO treatment on airway neutrophilia in OVA-rechallenged mice at Day 67. (C) Effect of inhaled IL-4R ASO treatment on airway eosinophilia in OVA-rechallenged mice at Day 69. For each treatment group, percent macrophages (dark gray bars), lymphocytes (open bars), eosinophils (light gray bars), and neutrophils (solid bars) are shown. Data are presented as group means ± SE, n = 10/group. *P < 0.05, Student's t test.

Aerosolized IL-4R ASO Suppresses Allergen-Induced Production of Airway Th2 Cytokines and Chemokines and Inhibits Mucin Gene Expression and Goblet Cell Metaplasia

View larger version (18K): [in this window] [in a new window]   Figure 4. Inhibition of BALF Th2 cytokines and chemokines in aerosolized IL-4R ASO but not MM control oligonucleotide treated OVA-rechallenged mice. BALF cytokine levels of IL-13 (A), IL-5 (B), CXCL8 (C), and CCL2 (D) were quantitated by ELISA on Day 67, 6 h after the second nebulized OVA challenge. Cytokine concentrations were determined from linear regression analysis of multi-point standard curves. Data are expressed as the group mean ± SE, n = 4/group. *P < 0.05, Student's t test.

Mucus overproduction in experimental models of asthma is regulated in part by Th2 cytokines and in particular by IL-13 (13, and references therein). Expression of Muc5AC mRNA was markedly induced after OVA rechallenge (Figure E2 and Figure 5A) and significantly suppressed in lung tissue from mice that inhaled IL-4R ASO compared with saline, while inhalation of a mismatched control oligonucleotide had no significant effect (Figure 5A). Image analysis of mucus-containing goblet cells in PAS-stained lung tissue demonstrated an increase in mucus in mice treated with inhaled saline or control oligonucleotide before OVA rechallenge compared with mice that were treated with inhaled saline but were not rechallenged with OVA (Figure 5B). Treatment with inhaled IL-4R ASO produced a significant decrease in PAS staining in OVA-rechallenged mice compared with inhalation of saline (Figure 5B). Goblet cell metaplasia, thickening of the airway epithelium with evident subepithelial fibrosis, and cellular infiltrates in the alveolar space were notable in PAS-stained lung tissue from saline and control oligonucleotide–treated mice, and appeared to be less prominent in mice that inhaled IL-4R ASO (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 5. Aerosolized IL-4R ASO reduces Muc5AC gene expression and goblet cell metaplasia in the airways of OVA-rechallenged mice. (A) Evaluation of Muc5AC mRNA by quantitative RT-PCR in lung extracts from allergen-rechallenged mice. Lung tissue was harvested on Day 69. Data are expressed as the ratio of Muc5AC to -actin mRNA expression. (B) Quantitation of mucus by digital imaging of PAS-stained lungs from mice treated with either inhaled saline, IL-4R ASO, or MM control oligonucleotide and subsequently rechallenged with OVA (see MATERIALS AND METHODS for details). Mucus analysis was performed in mice not subjected to AHR or BAL procedures. Data are expressed as the group mean ± SE, n = 4/group. *P < 0.05, Student's t test.

Therapeutic Efficacy of Topically Delivered IL-4R ASO in Mice Chronically Challenged with Allergen

View larger version (211K): [in this window] [in a new window]   Figure 6. Effects of IL-4R ASO on lung inflammation and goblet cell metaplasia in chronically OVA-challenged mice. (A) Schedule for intranasal OVA challenge, aerosolized oligonucleotide exposure, and systemic Dex treatment in a chronic model of allergic lung inflammation in mice. (B) Analysis of OVA-induced chronic lung inflammation in formalin-fixed, paraffin-embedded lung stained with PAS reagent. A representative photograph of PAS-stained airways is presented. Enhanced goblet cell metaplasia and inflammatory cell infiltration are evident. (C) Representative images of lung histology and PAS staining analysis in vehicle-, Dex-, and IL-4R ASO–treated animals at Day 76.

View larger version (21K): [in this window] [in a new window]   Figure 7. Suppression of lung inflammation by inhaled IL-4R ASO using a therapeutic treatment regimen in chronically OVA-challenged mice. (A) Methacholine-induced Penh dose–response curves from Balb/c mice exposed to inhaled IL-4R ASO (5 and 500 µg/kg EID administered as described in Figure 6A) following the development of established lung inflammation (see Figure 6B). Additional groups of mice were exposed to nebulized saline or injected with Dex (2.5 mg/kg intraperitoneally on Days 47, 62, 73–75). Responses of naïve mice (open triangles), saline vehicle–treated mice (open circles), vehicle plus Dex–treated mice (solid triangles), IL-4R ASO (5 µg/kg)–treated mice (open squares), and IL-4R ASO (500 µg/kg)–treated mice (filled squares) are shown. The areas under the curves for the naïve, Dex, and IL-4R ASO treatment groups are significantly different from that of the vehicle-treated group. Airway cells were recovered by BAL and analyzed as described in Figure 1 on Day 62 for neutrophils (B) or immediately after measurement of AHR on Day 76 for eosinophils (C). For each treatment group, percent macrophages (dark gray bars), lymphocytes (open bars), eosinophils (light gray bars), and neutrophils (solid bars) are shown. Data are presented as group means ± SE, n = 10/group for Penh and n = 7/group for cell differentials. *P < 0.05, Student's t test.

	DISCUSSION

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

Our findings are consistent with suppression of IL-13 generation and bioactivity within the lung. IL-13 protein was highly expressed in the secondary OVA challenge model and inhaled IL-4R ASO treatment significantly suppressed IL-13 levels in BAL fluid. Locally administered IL-4R ASO also inhibited the IL-13–mediated promotion of the epithelial secretory phenotype, including the induction of Muc5AC gene expression, CCL2 (MCP-1) elaboration, and goblet cell metaplasia. While we did not detect significant levels of IL-4 in BALF, consistent with other studies, we cannot exclude the potential contribution of inhibition of IL-4 signaling to the IL-4R ASO-induced phenotype. We conclude that interference with Th2 cytokine and chemokine pathways by suppressing expression of IL-4R on airway epithelium and pulmonary nonlymphoid innate immune cells reduces the orchestrated inflammatory and mucus overproduction response characteristic of allergen challenge models of asthma.

We propose that inhibition of IL-4R surface protein levels on nonlymphoid immune cells and perhaps airway epithelium after IL-4R ASO inhalation contributes to the reduced response of allergen-primed mice to nebulized OVA challenge. Recent studies have implicated the nonlymphoid resident airway tissues in the development of goblet cell metaplasia and mucus overproduction. Instillation of IL-13 into the airways of mice produces AHR that precedes the appearance of an inflammatory cell infiltrate (6). Stat6-deficient mice fail to develop goblet cell metaplasia in response to IL-13 instillation, and this response can be rescued by epithelial-directed expression of a Stat6 transgene (13, and references therein). Other studies also evaluated the role of pulmonary structural cells in the inflammatory response to allergen, using a murine radiation bone marrow chimera model. Irradiated, bone marrow–depleted genetically intact mice reconstituted with bone marrow from IL-4R–deficient mice developed goblet cell metaplasia after allergen challenge. Conversely, IL-4R–deficient mice that were reconstituted with nonirradiated bone marrow from IL-4R–expressing mice failed to manifest allergen-induced goblet cell metaplasia, even though Th2 cells and eosinophils were present in the airways and alveolar space (32). Kelly-Welch and coworkers also reported evidence for a non–lymphoid-derived bone marrow cell type that enhances the pulmonary mucosal allergic response (32). The authors suggested that this cell may be involved in amplifying the Th2 response or in providing distinct effector functions beyond that of Th2 cells. Among the cell types noted in this category—for example, dendritic cells, mast cells, eosinophils, and monocyte/macrophages—we have demonstrated IL-4R ASO-mediated reduction in IL-4R levels (DCs, mixed eosinophil/macrophage/monocyte population) in all except mast cells, which are rare in our models.

IL-13 plays a contributory role in the development of subepithelial pulmonary fibrosis in mice and transforms bronchial fibroblasts from individuals with asthma into a fibrotic phenotype via its promotion of TGF- synthesis and activation (33, 34). The release of IL-4– or IL-13–induced TGF-2 from asthmatic bronchial fibroblasts was found to be insensitive to corticosteroid inhibition (34). In our studies, widening of the airway epithelial basement membrane was observed after secondary allergen challenge as well as in chronically OVA-challenged mice and inhalation of the IL-4R ASO appeared to lessen this response. The effects of ectopic expression of IL-13 in the lung include significant increases in baseline airway resistance and methacholine-induced AHR (6, and references therein). It is likely that IL-13–induced lung remodeling responses are important in establishing the epithelial–mesenchymal trophic unit (34) believed to contribute to altered lung function in chronic lung inflammatory disease states. The contribution of subepithelial fibrosis and collagen deposition to enhanced allergen-induced AHR in our mouse models is not clear; however, moderation of these changes would be expected to increase airway compliance.

IL-4R is a logical target for multiple allergic respiratory disorders in addition to asthma, including allergic rhinitis and nasal polyposis, and may be involved in chronic inflammatory lung diseases, such as COPD and fibrosis. ASOs are amenable to formulation for either inhalation or intranasal spray applications. The biology of the target supports the notion that early intervention or prophylaxis in allergic disorders could potentially stall these diseases in the "initiation phase," before the onset of lung remodeling that is linked with their enhanced severity and chronicity (35). Our data indicate multiple anti-inflammatory modes of action, including the unexpected inhibition of neutrophil recruitment. CXCL8 (KC) is known to attract neutrophils, and the IL-4R ASO-mediated decrease in CXCL8 (KC) may explain this effect. In contrast, Dex treatment did not affect airway neutrophilia but did inhibit BALF CXCL8, suggesting that other activities of Dex may mask its effect on CXCL8 in this model. Further studies will be required to uncover the role of IL-4R in neutrophil attraction, but these results suggest the potential for additive or synergistic anti-inflammatory effects in combination with inhaled corticosteroids.

IL-13 targeting or dual blockade of IL-4 and IL-13 signaling is currently being pursued using protein biological-based approaches (16–18). These compounds show clinical promise as systemically administered agents. Our data in chronically allergen-challenged mice demonstrate pharmacologic activity at inhaled EIDs below 1 mg/kg IL-4R ASO administered weekly that is similar to that demonstrated in allergic mice with a systemically delivered IL-13 mAb (36). Single-dose inhalation kinetic studies using ASOs of identical chemistry estimate the half-life of these compounds to be 4 d in mouse lung. These results suggest clinical feasibility, and we speculate that an inhaled ASO approach may avoid distribution and stability issues that are potential limiting factors for systemically delivered agents that need to traffic to and/or penetrate the mucosal surface of the pulmonary epithelium. In addition, ASOs do not elicit humoral immune responses that can sometimes hinder protein-based therapeutic approaches. Exposure of mice to an EID of IL-4R ASO up to 1 mg/kg three times per week for 3 wk did not produce overt signs of toxicity with no evidence of macrophage/monocyte infiltrates (lung ASO concentration of 32.4 ± 4.3 µg/g lung tissue; R. Yu and R. Geary, unpublished observations). Inhaled ASOs of this chemical class were also well tolerated in a nonhuman primate study, with no evidence of increased inflammatory cells in BAL evident in allergic animals at doses up to 0.5 mg/kg EID administered once every 3 d for 36 d (37). Further primate and clinical studies are clearly needed to define potency and tolerability and to assess optimal treatment frequency for inhaled ASOs of this chemical class.

In summary, we show that inhalation of a chemically optimized IL-4R ASO reduces IL-4R surface protein expression on lung dendritic cells, eosinophils, and alveolar macrophages, as well as on pulmonary epithelial cells, and decreases allergen-induced lung inflammation and AHR. Importantly, Th2 cytokines, including IL-5 and IL-13 as well as proinflammatory chemokines such as CXCL8 (KC) and CCL2 (MCP-1), are inhibited in the airway tissues subsequent to IL-4R ASO treatment and the systemic corticosteroid–insensitive airway neutrophil recruitment response is suppressed. Weekly IL-4R ASO inhalation results in therapeutic efficacy in mice exhibiting chronic pulmonary inflammation. Collectively, these findings suggest that local administration of an IL-4R ASO is a novel approach for the therapy of inflammatory respiratory disease.

	Acknowledgments

 
The authors thank Drs. Rosie Yu, Luis Dellamary, and Nora Chew, as well as Dominic Kowalski, Scott Cooper, Donna Witchell, Jinsoo Kim, John Matson, Chad Arberg, and Brianna Bender for their expertise and advice. They also thank Drs. Steven Zuckerman and David Snyder of Eli Lilly for their contributions and Dr. C. Frank Bennett for critical reading of the manuscript.

	Footnotes

 
This article has an online supplement, which is available from this issue's table of contents at www.atsjournals.org.

Originally Published in Press as DOI: 10.1165/rcmb.2005-0456OC on September 21, 2006

Conflict of Interest Statement: J.G.K. is an employee of Isis Pharmaceuticals, is named as an inventor on a pending U.S. patent application citing the use of an interleukin-4 antisense oligonucleotide for the treatment of respiratory disease, and owns company stock and stock options. J.R.C. is an employee of Isis Pharmaceuticals, is named as an inventor on a pending U.S. patent application citing the use of an interleukin-4 antisense oligonucleotide for the treatment of respiratory disease, and owns company stock and stock options. M.G. has left Isis, but owns company stock and is named as an inventor on a pending U.S. patent application. D.T. was an employee of Isis from 2002–2005 but does not currently have any financial contact with Isis. D.A.M. was employed by Isis Pharmaceuticals from July 2000 through June 2005. W.A.G. is an employee of Isis Pharmaceuticals, and owns company stock and stock options. R.S.G. is an employee of Isis Pharmaceuticals and owns company stock and stock options. B.P.M. is an employee of Isis Pharmaceuticals, is named as an inventor on a pending U.S. patent application citing the use of an interleukin-4 antisense oligonucleotide for the treatment of respiratory disease, and owns company stock and stock options. S.A.G. is an employee of Isis Pharmaceuticals, is named as an inventor on a pending U.S. patent application citing the use of an interleukin-4 antisense oligonucleotide for the treatment of respiratory disease, has been reimbursed by Isis for attending several scientific and medical conferences and training courses, and owns company stock and stock options.

Received in original form December 12, 2005

Accepted in final form August 31, 2006

	References

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 

1. Barnes KC, Marsh DG. The genetics and complexity of allergy and asthma. Immunol Today 1998;19:325–332.[CrossRef][Medline]
2. Kay AB. T lymphocytes and their products in atopic allergy and asthma. Int Arch Allergy Appl Immunol 1991;94:189–193.[Medline]
3. Walker C, Bode E, Boer L, Hansel TT, Blaser K, Virchow J Jr. Allergic and non-allergic asthmatics have distinct patterns of T-cell activation and cytokine production in peripheral blood and bronchoalveolar lavage. Am Rev Respir Dis 1992;146:109–115.[Medline]
4. Robinson DS, Hamid Q, Ying S, Tsicopoulos A, Barkans J, Bentley AM, Corrigan C, Durham SR, Kay AB. Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma. N Engl J Med 1992;326:298–304.[Abstract]
5. Leckie MJ, ten Brinke A, Khan J, Diamant Z, O'Connor BJ, Walls CM, Mathur AK, Cowley HC, Chung KF, Djukanovic R, et al. Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyper-responsiveness, and the late asthmatic response. Lancet 2000;356:2144–2148.[CrossRef][Medline]
6. Chatila TA. Interleukin-4 receptor signaling pathways in asthma pathogenesis. Trends Mol Med 2004;10:493–499.[CrossRef][Medline]
7. Nelms K, Keegan AD, Zamorano J, Ryan JJ, Paul WE. 1999. The IL-4 receptor: signaling mechanisms and biologic functions. Annu Rev Immunol 1999;17:701–738.[CrossRef][Medline]
8. Obiri NI, Debinski W, Leonard WJ, Puri RK. Receptor for interleukin 13. Interaction with interleukin 4 by a mechanism that does not involve the common gamma chain shared by receptors for interleukins -2, -4, -7, -9, and -15. J Biol Chem 1995;270:8797–8804.[Abstract/Free Full Text]
9. Chiaramonte MG, Mentink-Kane M, Jacobson BA, Cheever AW, Whitters MJ, Goad ME, Wong A, Collins M, Donaldson DD, Grusby MJ, et al. Regulation and function of the interleukin-13 receptor alpha 2 during a T helper cell type 2-dominant immune response. J Exp Med 2003;197:687–701.[Abstract/Free Full Text]
10. Wood N, Whitters MJ, Jacobson BA, Witek J, Sypek JP, Kasaian M, Eppihimer MJ, Unger M, Tanaka T, Goldman SJ, et al. Enhanced interleukin (IL)-13 responses in mice lacking IL-13 receptor alpha 2. J Exp Med 2003;197:703–709.[Abstract/Free Full Text]
11. Xu B, Bhattacharjee A, Roy B, Xu HM, Anthony D, Frank DA, Feldman GM, Cathcart MK. Interleukin-13 induction of 15-lipoxygenase gene expression requires p38 mitogen-activated protein kinase-mediated serine 727 phosphorylation of Stat1 and Stat3. Mol Cell Biol 2003;23:3918–3928.[Abstract/Free Full Text]
12. Hytonen AM, Lowhagen O, Arvidsson M, Balder B, Bjork AL, Lindgren S, Hahn-Zoric M, Hanson LA, Padyukov L. Haplotypes of the interleukin-4 receptor alpha chain gene associate with susceptibility to and severity of atopic asthma. Clin Exp Allergy 2004;34:1570–1575.[CrossRef][Medline]
13. Wills-Karp M. Interleukin-13 in asthma pathogenesis. Immunol Rev 2004;202:175–190.[CrossRef][Medline]
14. Grunewald SM, Werthmann A, Schnarr B, Klein CE, Brocker EB, Mohrs M, Brombacher F, Sebald W, Duschl A. An antagonistic IL-4 mutant prevents type I allergy in the mouse: inhibition of the IL-4/IL-13 receptor system completely abrogates humoral immune response to allergen and development of allergic symptoms in vivo. J Immunol 1998;160:4004–4009.[Abstract/Free Full Text]
15. Lindell D, Gundel R, Fitch N, Harris P. The IL-4 Receptor Antagonist (Bay 16–9996) Reverses Airway Hyperresponsiveness in a Primate Model of Asthma. Am J Respir Crit Care Med 1999;159:A230. (Abstr.).
16. Economides AN, Carpenter LR, Rudge JS, Wong V, Koehler-Stec EM, Hartnett C, Pyles EA, Xu X, Daly TJ, Young MR, et al. Cytokine traps: multi-component, high-affinity blockers of cytokine action. Nat Med 2003;9:47–52.[CrossRef][Medline]
17. Kumar RK, Herbert C, Webb DC, Li L, Foster PS. Effects of anti-cytokine therapy in a mouse model of chronic asthma. Am J Respir Crit Care Med 2004;170:1043–1048.[Abstract/Free Full Text]
18. Yang G, Volk A, Petley T, Emmell E, Giles-Komar J, Shang X, Li J, Das AM, Shealy D, Griswold DE, et al. Anti-IL-13 monoclonal antibody inhibits airway hyperresponsiveness, inflammation and airway remodeling. Cytokine 2004;28:224–232.[CrossRef][Medline]
19. Borish LC, Nelson HS, Lanz MJ, Claussen L, Whitmore JB, Agosti JM, Garrison L. Interleukin-4 receptor in moderate atopic asthma: a phase I/II randomized, placebo-controlled trial. Am J Respir Crit Care Med 1999;160:1816–1823.[Abstract/Free Full Text]
20. Duan W, Chan JH, McKay K, Crosby JR, Choo HH, Leung BP, Karras JG, Wong WS. Inhaled p38alpha mitogen-activated protein kinase antisense oligonucleotide attenuates asthma in mice. Am J Respir Crit Care Med 2005;171:571–578.[Abstract/Free Full Text]
21. Ball HA, Sandrasagra A, Tang L, Van Scott M, Wild J, Nyce JW. Clinical potential of respirable antisense oligonucleotides (RASONs) in asthma. Am J Pharmacogenomics 2003;3:97–106.[CrossRef][Medline]
22. Baker BF, Lot SS, Condon TP, Cheng-Flournoy S, Lesnik EA, Sasmor HM, Bennett CF. 2'-O-(2-Methoxy)ethyl-modified anti-intercellular adhesion molecule 1 (ICAM-1) oligonucleotides selectively increase the ICAM-1 mRNA level and inhibit formation of the ICAM-1 translation initiation complex in human umbilical vein endothelial cells. J Biol Chem 1997;272:11994–12000.[Abstract/Free Full Text]
23. Condon T, Flournoy S, Sawyer GJ, Baker BF, Kishimoto TK, Bennett CF. Adam 17 but not adam 10 mediates tumor necrosis factor-alpha and 1-selectin shedding from leukocyte membranes. Antisense Nucleic Acid Drug Dev 2001;11:107–116.[CrossRef][Medline]
24. Taube C, Duez C, Cui ZH, Takeda K, Rha YH, Park JW, Balhorn A, Donaldson DD, Dakhama A, Gelfand EW. The role of IL-13 in established allergic airway disease. J Immunol 2002;169:6482–6489.[Abstract/Free Full Text]
25. Henderson WR Jr, Tang LO, Chu SJ, Tsao SM, Chiang GK, Jones F, Jonas M, Pae C, Wang H, Chi EY. A role for cysteinyl leukotrienes in airway remodeling in a mouse asthma model. Am J Respir Crit Care Med 2002;165:108–116.[Abstract/Free Full Text]
26. Silbaugh SA, Stengel PW, Dillard RD, Bemis KG. Pulmonary gas trapping in the guinea pig and its application in pharmacological testing. J Pharmacol Methods 1987;18:295–303.[CrossRef][Medline]
27. Hamelmann E, Schwarze J, Takeda K, Oshiba A, Larsen GL, Irvin CG, Gelfand EW. Non-invasive measurement of airway responsiveness in allergic mice using barometric plethysmography. Am J Respir Crit Care Med 1997;156:766–775.[Abstract/Free Full Text]
28. Adler A, Cieslewicz G, Irvin CG. Unrestrained plethysmography is an unreliable measure of airway responsiveness in BALB/c and C57BL/6 mice. J Appl Physiol 2004;97:286–292.[Abstract/Free Full Text]
29. Justice JP, Crosby JR, Borchers MT, Hines EM, Tomkinson A, Lee JJ, Lee NA. CD4⁺ T cell-dependent airway mucus production occurs in response to IL-5 expression in the lung. American Journal of Physiology LCMP 2002;282:L1066–L1074.
30. Hamelmann E, Oshiba A, Paluh J, Bradley K, Loader J, Potter TA, Larsen GL, Gelfand EW. Requirement for CD8(+) T cells in the development of airway hyperresponsiveness in a murine model of airway sensitization. J Exp Med 1996;183:1719–1729.[Abstract/Free Full Text]
31. Butler M, McKay RA, Popoff IJ, Gaarde WA, Witchell D, Murray SF, Dean NM, Bhanot S, Monia BP. Specific inhibition of PTEN expression reverses hyperglycemia in diabetic mice. Diabetes 2002;51:1028–1034.[Abstract/Free Full Text]
32. Kelly-Welch AE, Melo ME, Smith E, Ford AQ, Haudenschild C, Noben-Trauth N, Keegan AD. Complex role of the IL-4 receptor alpha in a murine model of airway inflammation: expression of the IL-4 receptor alpha on non-lymphoid cells of bone marrow origin contributes to severity of inflammation. J Immunol 2004;172:4545–4555.[Abstract/Free Full Text]
33. Lee CG, Homer RJ, Zhu Z, Lanone S, Wang X, Koteliansky V, Shipley JM, Gotwals P, Noble P, Chen Q, et al. Interleukin-13 induces tissue fibrosis by selectively stimulating and activating transforming growth factor beta(1). J Exp Med 2001;194:809–821.[Abstract/Free Full Text]
34. Richter A, Puddicombe SM, Lordan JL, Bucchieri F, Wilson SJ, Djukanovic R, Dent G, Holgate ST, Davies DE. The contribution of interleukin (IL)-4 and IL-13 to the epithelial-mesenchymal trophic unit in asthma. Am J Respir Cell Mol Biol 2001;25:385–391.[Abstract/Free Full Text]
35. Holt PG, Sly PD, Martinez FD, Weiss ST, Bjorksten B, von Mutius E, Wahn U. Drug development strategies for asthma: in search of a new paradigm. Nat Immunol 2004;5:695–698.[CrossRef][Medline]
36. Yang G, Li L, Volk A, Emmell E, Petley T, Giles-Komar J, Rafferty P, Lakshminarayanan M, Griswold DE, Bugelski PJ, et al. Therapeutic dosing with anti-interleukin-13 monoclonal antibody inhibits asthma progression in mice. J Pharmacol Exp Ther 2005;313:8–15.[Abstract/Free Full Text]
37. Karras JG, Geary RS, Gregory SA. Inhaled antisense oligonucleotide therapies: inspiration and progress. Drug Discovery Today: Therapeutic Strategies, Inflammation 2006;3:335–341.[CrossRef]

This article has been cited by other articles:

				B. Zhou, M. B. Headley, T. Aye, J. Tocker, M. R. Comeau, and S. F. Ziegler Reversal of Thymic Stromal Lymphopoietin-Induced Airway Inflammation through Inhibition of Th2 Responses J. Immunol., November 1, 2008; 181(9): 6557 - 6562. [Abstract] [Full Text] [PDF]

				E. M. Wagner, J. Sanchez, J. Y. McClintock, J. Jenkins, and A. Moldobaeva Inflammation and ischemia-induced lung angiogenesis Am J Physiol Lung Cell Mol Physiol, February 1, 2008; 294(2): L351 - L357. [Abstract] [Full Text] [PDF]

				J. R. Crosby, M. Guha, D. Tung, D. A. Miller, B. Bender, T. P. Condon, C. York-DeFalco, R. S. Geary, B. P. Monia, J. G. Karras, et al. Inhaled CD86 Antisense Oligonucleotide Suppresses Pulmonary Inflammation and Airway Hyper-Responsiveness in Allergic Mice J. Pharmacol. Exp. Ther., June 1, 2007; 321(3): 938 - 946. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Online Supplement All Versions of this Article: 2005-0456OCv1 36/3/276    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Karras, J. G. Articles by Gregory, S. A. Search for Related Content PubMed PubMed Citation Articles by Karras, J. G. Articles by Gregory, S. A.