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Published ahead of print on August 29, 2007, doi:10.1164/rccm.200702-334OC
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American Journal of Respiratory and Critical Care Medicine Vol 176. pp. 974-982, (2007)
© 2007 American Thoracic Society
doi: 10.1164/rccm.200702-334OC

Original Article

Transforming Growth Factor-beta1 Suppresses Airway Hyperresponsiveness in Allergic Airway Disease

John F. Alcorn1, Lisa M. Rinaldi2, Elizabeth F. Jaffe2, Mirjam van Loon2, Jason H. T. Bates2, Yvonne M. W. Janssen-Heininger1 and Charles G. Irvin2

1 Department of Pathology and 2 Department of Medicine, University of Vermont, Burlington, Vermont

Correspondence and requests for reprints should be addressed to John F. Alcorn, Ph.D., 223 HSRF, Department of Pathology, University of Vermont, Burlington, VT 05405. E-mail: john.alcorn{at}med.uvm.edu


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Asthma is characterized by increases in airway resistance, pulmonary remodeling, and lung inflammation. The cytokine transforming growth factor (TGF)-beta has been shown to have a central role in asthma pathogenesis and in mouse models of allergic airway disease.

Objectives: To determine the contribution of TGF-beta to airway hyperresponsiveness (AHR), we examined the time course, source, and isoform specificity of TGF-beta production in an in vivo mouse asthma model. To then elucidate the function of TGF-beta in AHR, inflammation, and pulmonary fibrosis, we examined the effects of blocking TGF-beta signaling with neutralizing antibody.

Methods: Mice were sensitized and challenged with ovalbumin (OVA) to establish allergic airway disease. TGF-beta activity was neutralized by intranasal administration of monoclonal antibody.

Measurements and Main Results: TGF-beta1 protein levels were increased in OVA-challenged lungs versus naive controls, and airway epithelial cells were shown to be a likely source of TGF-beta1. In addition, TGF-beta1 levels were elevated in OVA-exposed IL-5–null mice, which fail to recruit eosinophils into the airways. Neutralization of TGF-beta1 with specific antibody had no significant effect on airway inflammation and eosinophilia, although anti–TGF-beta1 antibody enhanced OVA-induced AHR and suppressed pulmonary fibrosis.

Conclusions: These data show that TGF-beta1 is the main TGF-beta isoform produced after OVA challenge, with a likely cellular source being the airway epithelium. The effects of blocking TGF-beta1 signaling had differential effects on AHR, fibrosis, and inflammation. While TGF-beta neutralization may be beneficial to abrogating airway remodeling, it may be detrimental to lung function by increasing AHR.

Key Words: lung • mice • hypersensitivity • cytokines



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Transforming growth factor (TGF)-beta1 is known to play a critical role in promoting pulmonary remodeling; however, its contribution to airway hyperresponsiveness (AHR) is less well defined.

What This Study Adds to the Field
TGF-beta1 is the predominant TGF-beta isoform produced in a mouse asthma model and neutralization of TGF-beta1 results in less fibrosis and increased AHR.

 
Transforming growth factor (TGF)-beta has emerged as a key mediator of pulmonary fibrosis, and accordingly a good candidate responsible for the subepithelial fibrosis observed in asthma (1, 2). The cellular source of TGF-beta in asthma is unclear; TGF-beta1 and TGF-beta3 have been shown to be expressed in the bronchial epithelium (3), whereas TGF-beta1 production has been reported in eosinophils and fibroblasts (4, 5). Moreover, patients with asthma express increased TGF-beta1 in the bronchoalveolar lavage (BAL) fluid in response to segmental allergen challenge and TGF-beta1 levels in the airway epithelium and submucosa correlate to airway basement membrane thickness, suggesting a direct role for TGF-beta1 in airway remodeling (6, 7). In addition, TGF-beta1 promoter polymorphisms have been linked to asthma susceptibility (8). TGF-beta1 instillation into mouse lungs and TGF-beta1 adenoviral expression or transgenic overexpression in the airway epithelium induce airway collagen mRNA and protein deposition, indicating that TGF-beta1 is sufficient to induce fibrosis (9–11). In addition, a causal role for TGF-beta1 has been elucidated in IL-13–mediated fibrosis (12).

Recent studies have clarified some of the mechanisms involved in TGF-beta1 stimulation of airway remodeling. TGF-beta1 activates gene transcription via binding to a heterodimeric receptor, part of the activin receptor-like kinase (ALK) family. TGF-beta receptor I (ALK5) phosphorylates members of the Smad (Similar to mothers against decapentaplegic) protein family to initiate nuclear translocation and transcription (13). Blockade of ALK5 kinase activity by an oral drug inhibits adenoviral TGF-beta1–induced lung fibrosis (14). Furthermore, anti–TGF-beta antibody has been shown to inhibit airway remodeling in an ovalbumin (OVA) model by blocking activation of the Smad signaling cascade (15). In the latter study, an antibody specific for all three TGF-beta isoforms did not affect pulmonary inflammation but significantly reduced collagen deposition, smooth muscle cell proliferation, and goblet cell mucus production. This raises the interesting question as to whether blocking the actions of TGF-beta might ameliorate the airway hyperresponsiveness (AHR) associated with allergic inflammation. Reduction of smooth muscle or airway mucus would be expected to potentially reduce AHR (16); however, fibrotic remodeling of the airway wall may actually reduce AHR by stiffening the airway and making it more resistant to constriction (17).

The goal of the present study was to examine the effects of TGF-beta1 neutralizing antibody on lung pathophysiology in terms of fibrosis, inflammation, and lung function. We also sought to elucidate the specific isoform and cellular source of TGF-beta in a common allergic inflammation mouse model. We report that anti-TGF-beta1 antibody suppressed airway fibrosis induced by OVA challenge, while on the other hand, anti–TGF-beta1 antibody increased AHR to inhaled methacholine (Mch). These results suggest that TGF-beta1, although implicated in promoting structural remodeling of the airway wall, plays a suppressive role in the pathogenesis of airway responsiveness induced by antigen.


   METHODS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Reagents
Female BALB/c mice, C57BL/6 mice, or IL-5–/– mice ages 2–4 months were purchased from Jackson Laboratories (Bar Harbor, ME). All chemicals used were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. All animal studies were approved by the Institutional Animal Care and Use Committee at the University of Vermont. Antibody to human TGF-beta1, TGF-beta2, and TGF-beta3, and nonspecific IgG1 were purchased from R&D Systems (Minneapolis, MN) and total TGF-beta protein levels were measured by ELISA (R&D Systems). Mouse monoclonal anti–TGF-beta1 antibody was obtained from R&D Systems (catalog no. MAB240, clone 9016). Phospho-Smad 2 antibody was purchased from Cell Signaling Technology (Danvers, MA).

Model of Allergic Airway Disease
Allergic airway inflammation was induced as previously reported (18). Briefly, mice were injected intraperitoneally with 40 µg of OVA in the adjuvant aluminum hydroxide (Alum) (Pierce Chemical, Rockford, IL) on Days 1 and 14 to induce sensitization. Sham-sensitized mice received Alum alone. Mice were then exposed to an aerosolized 1% OVA solution in sterile phosphate-buffered saline (PBS) for 30 minutes on Day 21 (1x), Days 21 through 23 (3x), or Days 21 through 26 (6x). Lung tissues were harvested 2 days after the final OVA challenge, except in TGF-beta1 time-course studies where tissues were collected as indicated (Figure E1 of the online supplement).

TGF-beta Blocking Antibody Studies
TGF-beta1 neutralizing antibody (75 µg) in sterile PBS was delivered 1 hour before OVA aerosol challenges via intranasal administration. Mice received four total doses of TGF-beta1 antibody, three doses preceding the three OVA challenges, and one dose on the day before lung function was assessed.

Respiratory Mechanics
Airway resistance and tissue elastance were measured as previously described (19). Multiple linear regression was used to fit measured pressure and volume in each individual mouse to the model of linear motion of the lung (20, 21). See online supplement for details of AHR measurements.

Lung Histology and Immunohistochemistry
Lungs were inflated and fixed with 4% paraformaldehyde, followed by paraffin embedding. Blocks were cut into 5-µm sections. Airway inflammation was assessed by hematoxylin-and-eosin staining or TGF-beta1 immunohistochemistry was performed (see online supplement). Phospho-Smad 2 staining was performed according to the antibody manufacturer's instructions.

Assessment of Pulmonary Fibrosis
Lung sections of 5 µm were then stained with picosirius red reagent that selectively stains collagen when visualized by polarized light microscopy (22). Slides were then scored using a scale of 0 to 3 (0 being the least stain intensity, 3 the highest intensity) for airway-associated collagen deposition by two independent, blinded observers. The cumulative score from each mouse was then averaged according to treatment group.

Digital Image Analysis
Digital images of immunohistochemistry and picosirius red–stained slides were captured using a Zeiss Axioskop2 plus microscope and the Zeiss Axiocam digital camera (Carl Zeiss Microimaging, Thornwood, NY). Color photos were then converted to 8-bit gray-scale images and mean pixel density was measured using NIH Image J software (National Institutes of Health, Bethesda, MD). Three sections of the airway wall were sampled per image. Images were obtained from a minimum of three different mice per group.

Cytokine Analyses in BAL
BAL samples from OVA-exposed mice were analyzed using the Bioplex System and a 23-plex cytokine array (BioRad Laboratories, Hercules, CA).

Statistical Analyses
Data presented in the figures were subjected to one-way analysis of variance followed by Tukey test for multiple comparisons. Comparisons of two means were conducted by unpaired Student's t test assuming unequal variance. Analyses with resultant P values less than 0.05 were determined significant, except where noted. Statistics were performed (Microsoft Excel software package; Microsoft Corp., Redmond, WA), and data are presented as mean values ± SEM.


   RESULTS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TGF-beta1 Is the Primary Isoform of TGF-beta Produced after OVA Challenge
To determine the effects of OVA challenge on TGF-beta production, mice were challenged with OVA for 6 consecutive days followed by 2 to 180 days of recovery. Specific TGF-beta isoform levels were measured in the BAL fluid by ELISA. TGF-beta1 was the only isoform of TGF-beta significantly increased by OVA challenge in the cell-free BAL fluid; total TGF-beta1 protein reached a peak level of 1,745.3 ± 188.2 pg/ml 2 days after the final OVA challenge (7 d after the first OVA challenge) and remained elevated for 1 week after OVA exposure (Figure 1A). TGF-beta1 levels were not significantly elevated above controls at later time points. Levels of active TGF-beta1 protein in BAL fluid were undetectable at the time points analyzed. No significant changes in the levels of TGF-beta2 or TGF-beta3 were found, nor were changes identified in the levels of TGF-beta in BAL cell lysates (Figure 1B).


Figure 1
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  		Figure 1. Detection of transforming growth factor (TGF)-beta isoforms in the bronchoalveolar lavage (BAL) fluid after ovalbumin (OVA) sensitization and challenge. BALB/c mice were sensitized to OVA and challenged with 1% aerosolized OVA on 6 consecutive days, and BAL samples were analyzed at the indicated time points after the last challenge (n = 4 mice each). Control mice were not sensitized or challenged with OVA. TGF-beta levels were measured by isoform-specific ELISA on the BAL supernatant (A) or cell lysate (B). TGF-beta1 levels in the BAL fluid were measured at the indicated times after one, three, or six OVA challenges (C). *P < 0.05.

 
We next examined the kinetics of TGF-beta1 release into the BAL fluid. Peak TGF-beta1 production was observed 7 days from the first OVA exposure regardless of the number (one, three, or six) of OVA challenges (Figure 1C). Additional OVA challenges increased the maximal release of TGF-beta1 from 803.9 pg/ml after one challenge, to 1,000.0 and 1,359.8 pg/ml for three and six challenges, respectively. Collectively, these data indicate that TGF-beta1 is perhaps the most prominent isoform of TGF-beta in the OVA mouse model of allergic airway disease and that the timing of peak TGF-beta1 release appears to be independent of the OVA challenge protocol.

The Airway Epithelium Is an Important Source of TGF-beta1 Production in Allergic Airway Inflammation
To identify the source of TGF-beta1 in mouse lungs, in situ, immunohistochemical analyses for TGF-beta1 protein were performed on lung sections. In naive mice, marked TGF-beta1 immunoreactivity occurred in the bronchial airway epithelium of control animals (Figure 2A). Significantly, after antigen challenge in sensitized mice, the TGF-beta1 immunoreactivity of the epithelium was lost, potentially indicating release of TGF-beta1 into the airspaces, which is supported by the timing of the loss of staining in the epithelium coinciding with the increase in BAL fluid TGF-beta1 protein presented in Figure 1. TGF-beta1 immunoreactivity was quantified by digital analysis and revealed a significant reduction in TGF-beta1 protein levels after OVA sensitization and challenge (Figure 2B).


Figure 2
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  		Figure 2. Transforming growth factor (TGF)-beta1 protein colocalizes with the bronchial epithelium and is decreased upon ovalbumin (OVA) sensitization and challenge. BALB/c mice were challenged six times with 1% aerosolized OVA, and paraffin-embedded sections were prepared. Immunohistochemistry specific for TGF-beta1 was then performed as described in METHODS (A) (n = 5 mice each). Alum/OVA indicates control mice that were sham sensitized then challenged with OVA, OVA/OVA indicates OVA-sensitized and -challenged mice, No Ab indicates a no primary antibody control. TGF-beta1 is indicated by red staining at x200 original magnification; scale bar equals 25 µm. TGF-beta1 staining was then quantified by mean pixel density (B) (n = 12), OVA/OVA-treated mice had decreased TGF-beta1 immunoreactivity compared with Alum/OVA. *P < 0.05.

 
The time course of TGF-beta1 protein increases also mirrored the OVA-induced eosinophilic inflammation. To examine the potential role of eosinophils in OVA-induced TGF-beta1 production, we measured the BAL fluid levels of TGF-beta1 in wild-type C57Bl/6 or IL-5–deficient mice after OVA challenge. It has been previously shown that IL-5–deficient mice fail to recruit eosinophils after OVA challenge (23). We therefore exposed IL-5–null mice and their background controls, C57Bl/6 mice, to six OVA challenges and assessed airway inflammation and TGF-beta1 BAL levels. IL-5–null mice indeed failed to exhibit airway eosinophilia (Figure 3A). Despite the absence of airway eosinophils, IL-5–null mice produced similar or higher levels of BAL TGF-beta1 (787.7 ± 129.4 pg/ml) compared with C57Bl/6 control mice (596.0 ± 148.2 pg/ml) (Figure 3B). These data suggest that TGF-beta1 levels in BAL fluid increase independently of IL-5 or eosinophilic inflammation in response to OVA antigen.


Figure 3
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  		Figure 3. IL-5–null mice produce similar transforming growth factor (TGF)-beta1 levels to control animals after ovalbumin (OVA) challenge despite decreased eosinophilia. IL-5–null mice or C57BL/6 controls were sensitized to OVA and challenged six times with 1% aerosolized OVA. Bronchoalveolar lavage (BAL) samples were then taken and analyzed for cell number, identity, and TGF-beta1 levels (n = 3 mice each). IL-5–null mice failed to recruit eosinophils into the airspaces compared with controls (A). Both control and IL-5–null mice produce similar levels of TGF-beta1 protein in the BAL fluid (B). *P = 0.058.

 
Anti–TGF-beta Antibody Does Not Inhibit OVA–induced Airway Inflammation
To understand the functional significance of increased airway TGF-beta1 in mice with allergic airway disease, we next interfered with the effects of TGF-beta1 by neutralizing TGF-beta1 activity using intratracheal administration of a TGF-beta1–specific antibody. To confirm that our instilled anti–TGF-beta1 antibody blocked TGF-beta1 activity and signaling in the airways, we challenged mice three times with OVA 30 minutes after instillation of anti–TGF-beta1 neutralizing antibody. The transcriptional regulator Smad 2 is activated by phosphorylation after TGF-beta1 receptor binding, leading to nuclear accumulation (13). We stained OVA-treated lung sections for phospho-Smad 2 levels (Figure 4A). TGF-beta1 neutralizing antibody blocked phospho-Smad 2 nuclear accumulation in the airways of OVA-treated mice; control nonspecific IgG1 had no effect. Phospho-Smad 2 levels were quantified by digital pixel density, which confirmed that anti–TGF-beta1 treatment did indeed significantly decrease phospho-Smad 2 levels (Figure 4B). These data confirm that anti–TGF-beta1 antibody effectively abrogates TGF-beta signaling in vivo.


Figure 4
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  		Figure 4. Ovalbumin (OVA)-induced phosphorylation of Smad 2 is attenuated by anti–transforming growth factor (TGF)-beta1 antibody treatment. BALB/c mice were sensitized to OVA and challenged on 3 consecutive days with 1% aerosolized OVA. TGF-beta1 neutralizing antibodies (75 µg) or control nonspecific IgG1 were administered once daily for 4 days starting the day of the first OVA challenge. Paraffin-embedded lung sections were then stained for phospho-Smad 2 (red) and counterstained with the nuclear dye Sytox Green (green) (A). Nuclear phospho-Smad 2 appears yellow due to the overlapping stains. Images were taken at x200 original magnification by confocal microscopy; scale bar equals 25 µm. Phospho-Smad 2 staining was then quantified by mean pixel density (B) (n = 21, 24, 24, respectively). Anti–TGF-beta1 significantly decreased phospho-Smad 2 immunoreactivity. *P < 0.05.

 
To next address the functional consequence of antibody instillation on airway inflammation induced by OVA, we analyzed BAL cell numbers, differential cell counts, and BAL cytokine levels. Administration of anti–TGF-beta1 antibody did not significantly alter OVA-induced airway eosinophilia (Figure 5A). In addition, lung histology revealed peribronchial and perivascular tissue inflammation in both antibody-treated and control mice (Figure 5B). Finally, anti–TGF-beta1 antibody treatment significantly enhanced Th2 cytokine levels of IL-4 and IL-13, as well as nonsignificantly increased IL-5 and IL-6 (Figure 5C). However, these data indicate that TGF-beta1 activity is not required for inflammatory cell recruitment to the airways in OVA allergic airway disease, because TGF-beta1 neutralization did not alter inflammatory cell numbers and it appears that TGF-beta1 may have a suppressive effect on Th2 cytokine production.


Figure 5
Figure 5
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  		Figure 5. Neutralization of transforming growth factor (TGF)-beta1 does not alter pulmonary inflammation in mice subjected to ovalbumin (OVA) sensitization and challenge. BALB/c mice were sensitized to OVA and challenged on 3 consecutive days with 1% aerosolized OVA. TGF-beta1 neutralizing antibodies (75 µg) were administered once daily for 4 days starting the day of the first OVA challenge. Cell number and identity were determined in the bronchoalveolar lavage (BAL) (n = 4, 8, 9 mice, respectively) (A). Whole lung sections were prepared by paraffin embedding and stained with hematoxylin and eosin to examine the tissue inflammation in the treatment groups (B), x200 original magnification. BAL cytokine profiles were assessed by Bioplex analysis (n = 7, 8, 8, respectively) (C). *P < 0.06 versus OVA mice; **P < 0.05 versus OVA mice.

 
TGF-beta Antibody Neutralization Inhibits Airway Collagen Deposition
To confirm that TGF-beta neutralization in our study reduced antigen-induced airway fibrosis, as previously reported (15), we quantified the amount of subepithelial collagen deposition after OVA challenge. Histologic scoring of bronchial collagen deposition was performed after OVA challenge with or without anti–TGF-beta1 antibody administration. OVA-induced collagen deposition in control animals significantly increased by 132.8 ± 15.0%, and this increase in airway collagen deposition was partially inhibited by anti–TGF-beta1 antibody, 118.5 ± 13.4% (Figures 6A, top, and 6B). Digital pixel density analysis of subepithelial collagen deposition showed that OVA treatment significantly increased peribronchial collagen levels compared with controls (optical density [o.d.], 35.3 ± 2.4 vs. 22.0 ± 1.7, respectively), and anti–TGF-beta1 significantly decreased this response (o.d., 24.0 ± 1.5) (Figure 6A, bottom). Subepithelial collagen deposition was not statistically different between control mice and mice treated with anti–TGF-beta1. These data confirm that TGF-beta1 neutralization blocks OVA-induced peribronchial collagen deposition.


Figure 6
Figure 6
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  		Figure 6. Neutralization of transforming growth factor (TGF)-beta1 decreases subepithelial collagen deposition after ovalbumin (OVA) challenge. BALB/c mice were sensitized to OVA and challenged on 3 consecutive days with 1% aerosolized OVA. TGF-beta1 neutralizing antibodies (75 µg) were administered once daily for 4 days starting the day of the first OVA challenge. Picosirius red–stained paraffin sections were assessed by blinded scorers for collagen deposition (A, top) (n = 6, 12, 15 mice, respectively). Picosirius red images were then analyzed by mean pixel density for collagen deposition (A, bottom) (n = 27, 27, 30 airways analyzed, respectively). Differential interference contrast images of Picosirius red–stained lung sections (B, x200 original magnification, scale bar = 25 µm). Insets depict a portion of the airway wall at increased magnification. (A, top) *P = 0.059 versus control mice (histologic scoring); (A, bottom) *P < 0.05 versus control; **P < 0.05 versus OVA (digital imaging); OVA + anti–TGF-beta1 were not statistically different from control.

 
Anti–TGF-beta1 Antibody Enhances AHR in Response to Inhaled Mch in the OVA Model
Because AHR is a significant functional outcome of allergic airway disease, we assessed the effects of TGF-beta neutralization on OVA-induced AHR. Anti–TGF-beta1 antibody was administered to mice during three OVA challenges. OVA plus control IgG or OVA plus anti–TGF-beta1 antibody instillation did not alter baseline lung mechanical values from those of OVA controls (Table 1). TGF-beta1 blockade significantly enhanced AHR induced by OVA in terms of tissue resistance (G) and tissue elastance (H) throughout the Mch dose–response curve. Peak G responses after Mch challenge were significantly elevated by anti–TGF-beta1 treatment compared with OVA controls (11.57 ± 2.18 vs. 6.46 ± 1.06 at 12.5 mg/ml Mch and 26.57 ± 6.00 vs. 16.64 ± 3.71 at 50 mg/ml Mch). Peak H responses were also significantly increased by TGF-beta1 neutralization (37.2 ± 4.4 vs. 26.4 ± 2.3 at 12.5 mg/ml Mch and 99.6 ± 15.7 vs. 46.8 ± 9.7 at 50 mg/ml Mch) (Figure 7A). AHR as assessed by G and H parameters remained significantly elevated between Mch challenges in mice treated with anti–TGF-beta1 compared with OVA controls (Figure 7B). TGF-beta1 neutralization had no significant effect on airway resistance (RN). Control IgG antibody treatment had no significant effects on OVA-induced AHR. Anti–TGF-beta1 antibody in the absence of OVA challenge did not alter AHR from control levels (data not shown). These data show that neutralization of TGF-beta1 results in significantly increased AHR in the mouse OVA model of asthma.


Figure 7
Figure 7
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  		Figure 7. Neutralization of transforming growth factor (TGF)-beta1 enhances ovalbumin (OVA)-induced airway hyperresponsiveness (AHR). BALB/c mice were sensitized to OVA and challenged on 3 consecutive days with 1% aerosolized OVA. TGF-beta1 neutralizing antibodies (75 µg) were administered once daily for 4 days starting the day of the first OVA challenge. After challenge, airway function was assessed by Flexivent (SCIREQ, Montreal, PQ, Canada). TGF-beta1 neutralization enhanced OVA sensitization and challenge induced AHR (A) (peak response; n = 7, 8, 8 mice, respectively). In addition to increasing peak methacholine (Mch) responses, TGF-beta1 antibody enhanced prolonged AHR between Mch doses (B). There were no effects of control IgG treatment on OVA-induced AHR. AHR as measured by the G (tissue resistance) and H (tissue elastance) parameters were significantly increased. *P < 0.05 versus OVA mice.

 

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  		TABLE 1. BASELINE LUNG FUNCTION VALUES FOR MICE BEFORE METHACHOLINE TREATMENT

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
TGF-beta has been suggested to be an important cytokine in the genesis of airway remodeling that underlies diverse pulmonary diseases, and which has been shown to be sufficient in mouse models to drive airway fibrosis. Numerous studies in vitro and in vivo have shown that TGF-beta1 stimulates production of fibrotic genes, such as collagen, connective tissue growth factor (CTGF), or plasminogen activator inhibitor (PAI)-1, in diverse cell types (1). In addition, the mouse has been reported to have TGF-beta1, TGF-beta2, and TGF-beta3 protein expression in the lung (24). Therefore, we investigated the production of specific TGF-beta isoforms in an OVA model of allergic airway disease and showed that TGF-beta1 is the predominant isoform released into the airspaces. Time-course experiments indicated that TGF-beta1 release is initiated early in the disease process after OVA challenge and peaks 7 days after the first OVA exposure. We also demonstrated that the bronchial epithelium of the conducting airways is a key localization of TGF-beta1 protein in control mice, its expression is decreased in the OVA model, and eosinophil recruitment is not required for TGF-beta1 production.

The functional role of TGF-beta signaling in the lung was then assessed using anti–TGF-beta1 antibody. TGF-beta1 antibody neutralization indeed blocked downstream phospho-Smad 2 accumulation in the airways, confirming that the approach used attenuates canonical TGF-beta1 signaling. Anti–TGF-beta1 antibody treatment failed to effect the establishment of eosinophilic or monocytic inflammation in the lung; however, it did increase Th2 cytokine profiles and it enhanced OVA-induced AHR, whereas it inhibited subepithelial collagen deposition. These data show that, although inhibition of pulmonary fibrosis with TGF-beta1 neutralizing antibody may be a possible outcome, anti–TGF-beta1 antibody may, in fact, exacerbate asthma pathology in terms of enhancing AHR. Furthermore, these data suggest a complex and apparently paradoxical role for TGF-beta1 in disease pathophysiology: on the one hand, it may contribute to the fibrosis, whereas on the other hand, it has an ameliorating role in regard to the genesis of AHR.

The role of TGF-beta signaling in lung fibrosis has been elucidated in recent years. Indeed, inhibition of TGF-beta1 signaling via downstream ALK5 receptor inhibition blocks lung expression of several fibrotic genes, including type I collagen and PAI-1 (14). Conversely, gain of function mutations in TGF-beta receptors correlate to increased CTGF gene expression (25). Treatment of OVA-induced mice with pan–TGF-beta antibody reduced airway collagen deposition, epithelial mucus metaplasia, and smooth muscle cell proliferation (15). It is therefore not surprising that, in the current study, anti–TGF-beta1 antibody was effective in inhibiting pulmonary fibrosis; however, the above studies did not assess the impact of the inhibition of fibrosis on either airway function or AHR.

The observation that immunoreactive TGF-beta1 protein is primarily present in airway epithelial cells is supported by recent studies using laser capture microdissection to identify the source of OVA-induced fibrotic cytokines (26). In those studies, TGF-beta1, CTGF, and PAI-1 mRNA levels were all specifically enriched in the epithelial layer and not in the underlying smooth muscle. In addition, recent studies have shown activation of signaling pathways downstream of TGF-beta receptor engagement in the bronchial epithelium in both animal models and biopsies from humans with asthma (15, 26–29). Specifically, phospho-Smad 2, the activated receptor Smad, colocalizes with the epithelium in both antigen-challenged mice (including in this study) and humans with asthma. Taken together, these data implicate TGF-beta1 as potentially playing an autocrine role in the airway epithelium to induce pulmonary remodeling and other pulmonary pathologies. The relative contribution of inflammatory cells to TGF-beta1 production may be an important difference between human and mouse allergic airway disease, and we cannot exclude the possibility that eosinophils produced and released TGF-beta1 at time points different to those studied in our model.

In the current study, anti–TGF-beta1 antibody had no effect on eosinophilic inflammation but did enhance Th2 cytokine profiles induced by OVA challenge. The finding that inflammatory cell recruitment is unaltered by TGF-beta1 neutralization is consistent with two recent studies using anti–pan-TGF-beta antibody or ALK5 inhibition (14, 15). Further support for these results comes from an additional study using anti–TGF-beta antibody followed by a detailed assessment of systemic immune cell function. These authors found that anti–pan-TGF-beta antibody had no effect on lymphocyte proliferation, phagocytic activity, cytokine production, or immunoglobulin production (30). However, studies conducted using the OVA model in TGF-beta1 heterozygous mice, systemically lacking an allele of the TGF-beta1 gene, showed increased eosinophil accumulation and increased Th2 cytokine production (31). In these mice, TGF-beta1 protein levels in the lung were reduced to 30% of normal wild-type levels after OVA challenge and it is unclear why this study produced significantly different results from our current work, although we did observe increased Th2 cytokine production similar to the results found in TGF-beta1 heterozygous mice. It is important to note that disruption of the TGF-beta1 gene has been shown to produce partial lethality and severe inflammatory abnormalities in mice (32–34). Elevated Th2 cytokine levels have been linked with AHR and allergic airway disease severity in numerous studies, suggesting that TGF-beta1 neutralization may impact AHR through modulation of Th2 cell responses (35, 36).

Interference with TGF-beta1 function using neutralizing antibody had a significant effect on the pathophysiology induced by antigen challenge. Although anti–TGF-beta1 antibody increased all measures of baseline (pre-Mch) lung mechanics, this effect was not significant (Table 1). However, anti–TGF-beta1 antibody did significantly enhance AHR as assessed with G (tissue resistance) and H (elastance) but not RN (airway resistance), which is consistent with a peripheral (small) airway effect. These findings are both surprising and at first glance inconsistent with the commonly held view that airway remodeling is a major cause of AHR.

Some potential explanations for this outcome can be considered. One possible explanation for the results is the well-known effect that TGF-beta1 has on T-cell regulation. TGF-beta1 overexpression in helper T cells has been shown to inhibit OVA-induced AHR in mice (37). Moreover, in two recent studies, one in which TGF-beta1 was overexpressed in airway epithelial cells (38) and another in which TGF-beta1 was administered intratracheally (39), both treatments were associated with increased TGF-beta1 and a decrease in AHR. On the other hand, there are other reports where reduced levels of TGF-beta1 (31) or ectopically administered TGF-beta1 (9) did not alter airway resistance or AHR. Unfortunately, the determination of the mechanical response and AHR in all of the above studies was obtained noninvasively with a whole body plethysmographic technique, a measurement that has been shown to have severe shortcomings and uncertain interpretation (40).

Another potential immunologic mechanism by which TGF-beta1 may decrease antigen-induced AHR is through effects on regulatory T lymphocyte (Treg) activity. Treg cells were initially identified as a specific class of TGF-beta1–producing T cells that make up a small percentage of the T-cell pool (41). The Treg cells are important in the regulation of Th2 immunity and are believed to play an important role in the control of allergic responses because Treg cells have been shown to suppress OVA-induced AHR in mice, via a TGF-beta1–dependent mechanism (42). In that study, anti–TGF-beta antibody blocked the inhibitory effects of Tregs on AHR, analogous to our findings. In addition, Treg cells have been shown to induce Th2 cell apoptosis (43) and increased tolerance to inhaled antigen (44), promoting resolution of AHR. Recent work has shown that TGF-beta1 induces maturation of naive T cells to a Treg phenotype via expression of the Treg-specific transcription factor Foxp3 (45). These data suggest that TGF-beta1 activity is required for normal Treg function in the lung. By inhibiting TGF-beta1 activity, it is possible that lung T-cell function is skewed toward an exacerbated allergic phenotype, which would include increased AHR. The role of Treg activity in the structure/function relationships in the lung and the link to TGF-beta1 remain to be determined.

Last, it is likely that, during the inflammatory events caused by antigen challenge, elaboration of TGF-beta1 inhibits AHR by a structural mechanism. Thickening of the airway walls of patients with asthma is related to asthma severity (46, 47) and has shown to be greatest in those who die of asthma (48, 49). Mathematical modeling (50, 51) suggests that thickening of the airway wall contributes to AHR in vivo by a geometric mechanism, in which a thickened airway wall internal to the airway smooth muscle (ASM) will narrow the airway lumen more for any given stimuli to the ASM. MacParland and colleagues (17) have recently reviewed the evidence that structural remodeling of the airway could hypothetically either contribute to AHR or ameliorate AHR; however, the evidence directly linking AHR to airway wall alterations is both limited and inconsistent (52). To explain the present findings, we suggest that antigen-induced inflammation leads to the well-documented elaboration of mediators (e.g., leukotrienes and interleukins) and, in this case, TGF-beta1 (Figure 1), which, in turn, causes subepithelial fibrosis (Figure 6). This airway wall fibrosis would then result in any one or combination of events, including the following: (1) stiffening of the wall preventing the ASM from narrowing the lumen in response to Mch; (2) as the fibrosis occurs within and between the cells of the ASM, this would increase the parallel elastic load to ASM which prevents shortening; or (3) increasing the series load against which the ASM must contract (17, 53). It has been noted that chronic antigen challenge leads to more substantial airway wall remodeling but a decline in AHR (54), consistent with this proposed mechanism of an ameliorating effect of airway fibrosis. Consistent with this explanation is the spatial location of the antigen-induced deposition of collagen with and outside of the ASM (Figure 6B) and its reduction by an anti–TGF-beta1 antibody. Moreover, the pattern of the temporal response to a single dose of inhaled Mch of both elastance (H) and tissue resistance (G) (Figure 7B) shows a markedly enhanced peak response consistent with enhanced ASM narrowing. Anti–TGF-beta1 antibody, by decreasing the amount of submucosal collagen, would allow ASM to contract more, leading to a marked increase in the peak response of H, a temporal pattern that we have not observed previously even with antigen challenge (55, 56). In this way, airway fibrosis would serve to lessen airway narrowing, but prevention of its formation by blocking the effects of TGF-beta1 results in enhanced airway narrowing.

In conclusion, the data presented in this study show that, although anti–TGF-beta antibody strategies may indeed provide desired antifibrotic potential, the effects of anti–TGF-beta1 antibody in fact exacerbates the AHR associated with the allergic airway response. The mechanism by which TGF-beta1 regulates AHR remains unclear and further study is warranted to elucidate the pathways involved. In addition, we identify TGF-beta1 as the primary TGF-beta isoform produced in the antigen challenge model and the airway epithelium as a potential source of TGF-beta. We also demonstrate that the mechanisms of antigen-induced inflammation, remodeling, and AHR appear to be independent with regard to the TGF-beta signaling pathway. We conclude that the function of TGF-beta1 in allergic airway disease is to shift the lung toward a fibrotic phenotype while dampening the AHR induced by antigen challenge, perhaps by either an immunologic or mechanical mechanism. Collectively, these findings suggest that the commonly held notion that airway remodeling is directly involved in the genesis of AHR may need to be reconsidered.


   Acknowledgments
 
The authors thank Amy L. Brown and Jennifer L. Ather for technical assistance in these studies.


   FOOTNOTES
 
Supported by grants from the National Institute of Health HL P01-67004 and P20 RL 15557 National Center for Research Resources, Centers of Biomedical Research Excellence.

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: 10.1164/rccm.200702-334OC on August 29, 2007

Conflict of Interest Statement: J.F.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. L.M.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.F.J. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.v.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.H.T.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Y.M.W.J.-H. received $1,500 from Sepracor for a lecture given in January 2005. C.G.I. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form February 27, 2007; accepted in final form August 24, 2007


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
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 DISCUSSION
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