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Rationale: Asthma is characterized by increases in airway resistance, pulmonary remodeling, and lung inflammation. The cytokine transforming growth factor (TGF)-β 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-β to airway hyperresponsiveness (AHR), we examined the time course, source, and isoform specificity of TGF-β production in an in vivo mouse asthma model. To then elucidate the function of TGF-β in AHR, inflammation, and pulmonary fibrosis, we examined the effects of blocking TGF-β signaling with neutralizing antibody.
Methods: Mice were sensitized and challenged with ovalbumin (OVA) to establish allergic airway disease. TGF-β activity was neutralized by intranasal administration of monoclonal antibody.
Measurements and Main Results: TGF-β1 protein levels were increased in OVA-challenged lungs versus naive controls, and airway epithelial cells were shown to be a likely source of TGF-β1. In addition, TGF-β1 levels were elevated in OVA-exposed IL-5–null mice, which fail to recruit eosinophils into the airways. Neutralization of TGF-β1 with specific antibody had no significant effect on airway inflammation and eosinophilia, although anti–TGF-β1 antibody enhanced OVA-induced AHR and suppressed pulmonary fibrosis.
Conclusions: These data show that TGF-β1 is the main TGF-β isoform produced after OVA challenge, with a likely cellular source being the airway epithelium. The effects of blocking TGF-β1 signaling had differential effects on AHR, fibrosis, and inflammation. While TGF-β neutralization may be beneficial to abrogating airway remodeling, it may be detrimental to lung function by increasing AHR.
Transforming growth factor (TGF)-β1 is known to play a critical role in promoting pulmonary remodeling; however, its contribution to airway hyperresponsiveness (AHR) is less well defined.
TGF-β1 is the predominant TGF-β isoform produced in a mouse asthma model and neutralization of TGF-β1 results in less fibrosis and increased AHR.
Transforming growth factor (TGF)-β 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-β in asthma is unclear; TGF-β1 and TGF-β3 have been shown to be expressed in the bronchial epithelium (3), whereas TGF-β1 production has been reported in eosinophils and fibroblasts (4, 5). Moreover, patients with asthma express increased TGF-β1 in the bronchoalveolar lavage (BAL) fluid in response to segmental allergen challenge and TGF-β1 levels in the airway epithelium and submucosa correlate to airway basement membrane thickness, suggesting a direct role for TGF-β1 in airway remodeling (6, 7). In addition, TGF-β1 promoter polymorphisms have been linked to asthma susceptibility (8). TGF-β1 instillation into mouse lungs and TGF-β1 adenoviral expression or transgenic overexpression in the airway epithelium induce airway collagen mRNA and protein deposition, indicating that TGF-β1 is sufficient to induce fibrosis (9–11). In addition, a causal role for TGF-β1 has been elucidated in IL-13–mediated fibrosis (12).
Recent studies have clarified some of the mechanisms involved in TGF-β1 stimulation of airway remodeling. TGF-β1 activates gene transcription via binding to a heterodimeric receptor, part of the activin receptor-like kinase (ALK) family. TGF-β 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-β1–induced lung fibrosis (14). Furthermore, anti–TGF-β 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-β 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-β 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-β1 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-β in a common allergic inflammation mouse model. We report that anti-TGF-β1 antibody suppressed airway fibrosis induced by OVA challenge, while on the other hand, anti–TGF-β1 antibody increased AHR to inhaled methacholine (Mch). These results suggest that TGF-β1, although implicated in promoting structural remodeling of the airway wall, plays a suppressive role in the pathogenesis of airway responsiveness induced by antigen.
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-β1, TGF-β2, and TGF-β3, and nonspecific IgG1 were purchased from R&D Systems (Minneapolis, MN) and total TGF-β protein levels were measured by ELISA (R&D Systems). Mouse monoclonal anti–TGF-β1 antibody was obtained from R&D Systems (catalog no. MAB240, clone 9016). Phospho-Smad 2 antibody was purchased from Cell Signaling Technology (Danvers, MA).
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 (1×), Days 21 through 23 (3×), or Days 21 through 26 (6×). Lung tissues were harvested 2 days after the final OVA challenge, except in TGF-β1 time-course studies where tissues were collected as indicated (Figure E1 of the online supplement).
TGF-β1 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-β1 antibody, three doses preceding the three OVA challenges, and one dose on the day before lung function was assessed.
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.
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-β1 immunohistochemistry was performed (see online supplement). Phospho-Smad 2 staining was performed according to the antibody manufacturer's instructions.
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 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.
BAL samples from OVA-exposed mice were analyzed using the Bioplex System and a 23-plex cytokine array (BioRad Laboratories, Hercules, CA).
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.
To determine the effects of OVA challenge on TGF-β production, mice were challenged with OVA for 6 consecutive days followed by 2 to 180 days of recovery. Specific TGF-β isoform levels were measured in the BAL fluid by ELISA. TGF-β1 was the only isoform of TGF-β significantly increased by OVA challenge in the cell-free BAL fluid; total TGF-β1 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-β1 levels were not significantly elevated above controls at later time points. Levels of active TGF-β1 protein in BAL fluid were undetectable at the time points analyzed. No significant changes in the levels of TGF-β2 or TGF-β3 were found, nor were changes identified in the levels of TGF-β in BAL cell lysates (Figure 1B).
We next examined the kinetics of TGF-β1 release into the BAL fluid. Peak TGF-β1 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-β1 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-β1 is perhaps the most prominent isoform of TGF-β in the OVA mouse model of allergic airway disease and that the timing of peak TGF-β1 release appears to be independent of the OVA challenge protocol.
To identify the source of TGF-β1 in mouse lungs, in situ, immunohistochemical analyses for TGF-β1 protein were performed on lung sections. In naive mice, marked TGF-β1 immunoreactivity occurred in the bronchial airway epithelium of control animals (Figure 2A). Significantly, after antigen challenge in sensitized mice, the TGF-β1 immunoreactivity of the epithelium was lost, potentially indicating release of TGF-β1 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-β1 protein presented in Figure 1. TGF-β1 immunoreactivity was quantified by digital analysis and revealed a significant reduction in TGF-β1 protein levels after OVA sensitization and challenge (Figure 2B).
The time course of TGF-β1 protein increases also mirrored the OVA-induced eosinophilic inflammation. To examine the potential role of eosinophils in OVA-induced TGF-β1 production, we measured the BAL fluid levels of TGF-β1 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-β1 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-β1 (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-β1 levels in BAL fluid increase independently of IL-5 or eosinophilic inflammation in response to OVA antigen.
To understand the functional significance of increased airway TGF-β1 in mice with allergic airway disease, we next interfered with the effects of TGF-β1 by neutralizing TGF-β1 activity using intratracheal administration of a TGF-β1–specific antibody. To confirm that our instilled anti–TGF-β1 antibody blocked TGF-β1 activity and signaling in the airways, we challenged mice three times with OVA 30 minutes after instillation of anti–TGF-β1 neutralizing antibody. The transcriptional regulator Smad 2 is activated by phosphorylation after TGF-β1 receptor binding, leading to nuclear accumulation (13). We stained OVA-treated lung sections for phospho-Smad 2 levels (Figure 4A). TGF-β1 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-β1 treatment did indeed significantly decrease phospho-Smad 2 levels (Figure 4B). These data confirm that anti–TGF-β1 antibody effectively abrogates TGF-β signaling in vivo.
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-β1 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-β1 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-β1 activity is not required for inflammatory cell recruitment to the airways in OVA allergic airway disease, because TGF-β1 neutralization did not alter inflammatory cell numbers and it appears that TGF-β1 may have a suppressive effect on Th2 cytokine production.
To confirm that TGF-β 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-β1 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-β1 antibody, 118.5 ± 13.4% (Figures 6A, top, and and6B).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-β1 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-β1. These data confirm that TGF-β1 neutralization blocks OVA-induced peribronchial collagen deposition.
Because AHR is a significant functional outcome of allergic airway disease, we assessed the effects of TGF-β neutralization on OVA-induced AHR. Anti–TGF-β1 antibody was administered to mice during three OVA challenges. OVA plus control IgG or OVA plus anti–TGF-β1 antibody instillation did not alter baseline lung mechanical values from those of OVA controls (Table 1). TGF-β1 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-β1 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-β1 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-β1 compared with OVA controls (Figure 7B). TGF-β1 neutralization had no significant effect on airway resistance (RN). Control IgG antibody treatment had no significant effects on OVA-induced AHR. Anti–TGF-β1 antibody in the absence of OVA challenge did not alter AHR from control levels (data not shown). These data show that neutralization of TGF-β1 results in significantly increased AHR in the mouse OVA model of asthma.
TGF-β 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-β1 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-β1, TGF-β2, and TGF-β3 protein expression in the lung (24). Therefore, we investigated the production of specific TGF-β isoforms in an OVA model of allergic airway disease and showed that TGF-β1 is the predominant isoform released into the airspaces. Time-course experiments indicated that TGF-β1 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-β1 protein in control mice, its expression is decreased in the OVA model, and eosinophil recruitment is not required for TGF-β1 production.
The functional role of TGF-β signaling in the lung was then assessed using anti–TGF-β1 antibody. TGF-β1 antibody neutralization indeed blocked downstream phospho-Smad 2 accumulation in the airways, confirming that the approach used attenuates canonical TGF-β1 signaling. Anti–TGF-β1 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-β1 neutralizing antibody may be a possible outcome, anti–TGF-β1 antibody may, in fact, exacerbate asthma pathology in terms of enhancing AHR. Furthermore, these data suggest a complex and apparently paradoxical role for TGF-β1 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-β signaling in lung fibrosis has been elucidated in recent years. Indeed, inhibition of TGF-β1 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-β receptors correlate to increased CTGF gene expression (25). Treatment of OVA-induced mice with pan–TGF-β 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-β1 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-β1 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-β1, 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-β 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-β1 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-β1 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-β1 at time points different to those studied in our model.
In the current study, anti–TGF-β1 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-β1 neutralization is consistent with two recent studies using anti–pan-TGF-β antibody or ALK5 inhibition (14, 15). Further support for these results comes from an additional study using anti–TGF-β antibody followed by a detailed assessment of systemic immune cell function. These authors found that anti–pan-TGF-β antibody had no effect on lymphocyte proliferation, phagocytic activity, cytokine production, or immunoglobulin production (30). However, studies conducted using the OVA model in TGF-β1 heterozygous mice, systemically lacking an allele of the TGF-β1 gene, showed increased eosinophil accumulation and increased Th2 cytokine production (31). In these mice, TGF-β1 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-β1 heterozygous mice. It is important to note that disruption of the TGF-β1 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-β1 neutralization may impact AHR through modulation of Th2 cell responses (35, 36).
Interference with TGF-β1 function using neutralizing antibody had a significant effect on the pathophysiology induced by antigen challenge. Although anti–TGF-β1 antibody increased all measures of baseline (pre-Mch) lung mechanics, this effect was not significant (Table 1). However, anti–TGF-β1 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-β1 has on T-cell regulation. TGF-β1 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-β1 was overexpressed in airway epithelial cells (38) and another in which TGF-β1 was administered intratracheally (39), both treatments were associated with increased TGF-β1 and a decrease in AHR. On the other hand, there are other reports where reduced levels of TGF-β1 (31) or ectopically administered TGF-β1 (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-β1 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-β1–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-β1–dependent mechanism (42). In that study, anti–TGF-β 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-β1 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-β1 activity is required for normal Treg function in the lung. By inhibiting TGF-β1 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-β1 remain to be determined.
Last, it is likely that, during the inflammatory events caused by antigen challenge, elaboration of TGF-β1 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-β1 (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-β1 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-β1 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-β1 results in enhanced airway narrowing.
In conclusion, the data presented in this study show that, although anti–TGF-β antibody strategies may indeed provide desired antifibrotic potential, the effects of anti–TGF-β1 antibody in fact exacerbates the AHR associated with the allergic airway response. The mechanism by which TGF-β1 regulates AHR remains unclear and further study is warranted to elucidate the pathways involved. In addition, we identify TGF-β1 as the primary TGF-β isoform produced in the antigen challenge model and the airway epithelium as a potential source of TGF-β. We also demonstrate that the mechanisms of antigen-induced inflammation, remodeling, and AHR appear to be independent with regard to the TGF-β signaling pathway. We conclude that the function of TGF-β1 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.
The authors thank Amy L. Brown and Jennifer L. Ather for technical assistance in these studies.
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.