Published ahead of print on January 3, 2008, doi:10.1164/rccm.200706-958OC
© 2008 American Thoracic Society doi: 10.1164/rccm.200706-958OC
Transforming Growth Factor-β Regulates House Dust Mite–induced Allergic Airway Inflammation but Not Airway Remodeling1 Division of Respiratory Diseases and Allergy, Centre for Gene Therapeutics, and Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada; and 2 Department of Cytokine Biology, Cell and Protein Therapeutics, Genzyme Corporation, Framingham, Massachusetts Correspondence and requests for reprints should be addressed to Manel Jordana, M.D., Ph.D., Professor, Department of Pathology and Molecular Medicine, Head, Division of Respiratory Diseases and Allergy, MDCL 4013, McMaster University, 1200 Main Street West, Hamilton, ON, L8N 3Z5, Canada. E-mail: jordanam{at}mcmaster.ca
Rationale: It is now believed that both chronic airway inflammation and remodeling contribute significantly to airway dysfunction and clinical symptoms in allergic asthma. Transforming growth factor (TGF)-β is a powerful regulator of both the tissue repair and inflammatory responses, and numerous experimental and clinical studies suggest that it may play an integral role in the pathogenesis of asthma. Objectives: We investigated the role of TGF-β in the regulation of allergic airway inflammation and remodeling using a mouse model of house dust mite (HDM)–induced chronic allergic airway disease. Methods: We have previously shown that intranasal administration of an HDM extract (5 d/wk for 5 wk) elicits robust Th2-polarized airway inflammation and remodeling that is associated with increased airway hyperreactivity. Here, Balb/c mice were similarly exposed to HDM and concurrently treated with a pan-specific TGF-β neutralizing antibody. Measurements and Main Results: We observed that anti–TGF-β treatment in the context of either continuous or intermittent HDM exposure had no effect on the development of HDM-induced airway remodeling. To further confirm these findings, we also subjected SMAD3 knockout mice to 5 weeks of HDM and observed that knockout mice developed airway remodeling to the same extent as HDM-exposed littermate controls. Notably, TGF-β neutralization exacerbated the eosinophilic infiltrate and led to increased airway hyperreactivity. Conclusions: Collectively, these data suggest that TGF-β regulates HDM-induced chronic airway inflammation but not remodeling, and furthermore, caution against the use of therapeutic strategies aimed at interfering with TGF-β activity in the treatment of this disease.
Key Words: immunology • allergic asthma • mouse model • lung
Asthma is a chronic disease that is characterized clinically by variable airflow obstruction and a decline in airway function (1). It is now believed that these outcomes are the consequences of two distinct, although likely connected, processes—namely, allergic airway inflammation and airway remodeling (2–5). Experimental studies in animal models of allergic airway disease have afforded considerable understanding with respect to the nature and mechanisms that drive allergic inflammation; however, relatively speaking, our knowledge of the mechanistic processes that underlie the development of airway remodeling remains limited and in some cases controversial. Moreover, although it is generally accepted that inflammation-associated damage consequently triggers tissue reparative responses as a means of restoring tissue integrity, whether and how this is coordinately regulated in allergic airway disease are also poorly understood. In the context of allergic asthma, the pathologic hallmarks of airway remodeling include increased subepithelial extracellular matrix deposition, alterations in the airway epithelium (goblet cell hyperplasia) leading to excessive mucus production, and airway smooth muscle thickening resulting from smooth muscle cell hypertrophy and hyperplasia (6, 7). Among the various factors that are believed to be involved, transforming growth factor (TGF)-β, a powerful regulator of the tissue repair response, has been strongly implicated in the development of airway remodeling. Clinical studies have documented increased TGF-β expression on both the level of mRNA and protein in bronchial biopsies and in the bronchoalveolar lavage (BAL) of humans with asthma compared with control subjects (8–12). Similarly, TGF-β expression has also been shown to be increased in animal models of allergic airway disease (13–17). In addition, the number of TGF-β–expressing epithelial or submucosal cells has been correlated with the basement membrane thickness in patients with asthma (12). Direct evidence in support of a role for TGF-β in the development of allergic airway remodeling comes from a study by McMillan and colleagues, who showed that TGF-β blockade using a neutralizing antibody (Ab) reduced the extent of ovalbumin (OVA)-induced remodeling in a chronic mouse model (18). In addition, two very recent studies, one using SMAD3 knockout (KO) animals (19) and the other using blocking Abs (20), have also demonstrated that TGF-β contributes to the development of airway remodeling in similar OVA-based systems. Animal studies demonstrating a critical role for TGF-β in the pathogenesis of related diseases involving aberrant tissue repair, such as liver, kidney, and lung fibrosis, further support the notion of TGF-β involvement in allergic airway remodeling (21–24). Importantly, it is clear that TGF-β is also one of the most potent negative regulators of inflammation (25). Indeed, TGF-β–deficient mice die soon after birth due to rampant, multifocal inflammation (26). This activity may also be of particular relevance to allergic airway disease as TGF-β is believed to contribute, at least in part, to the regulation of the Th2-polarized airway inflammatory response that is characteristic of this disease. Numerous in vitro studies have collectively shown that TGF-β suppresses the activity and/or proliferation of various inflammatory cells, including Th2 cells, B cells, macrophages, and eosinophils (25, 27–29), and moreover, TGF-β can promote their apoptosis (30–32). Three separate studies have investigated the impact of genetically interfering with TGF-β activity on allergic sensitization and airway inflammation in acute models of allergic airway disease using OVA. Their findings consistently demonstrate that abrogation of TGF-β or TGF-β signaling pathways substantially potentiates the immune response and exacerbates the allergic inflammatory response (33–35).
In light of these reports, we and others have proposed that TGF-β may play a dual role in asthma by coordinately regulating both the inflammatory and reparative responses; specifically, that TGF-β may dampen the inflammatory response elicited upon aeroallergen exposure in a sensitized individual while subsequently initiating tissue repair processes. In this study, we investigated the role of TGF-β in the regulation of chronic allergic airway inflammation and remodeling. To address this issue experimentally, we conducted a series of loss-of-function experiments in a model of chronic allergic airway disease induced by respiratory mucosal exposure to a clinically relevant aeroallergen, house dust mite (HDM). We have previously shown that repeated exposure to an HDM extract elicits robust Th2-polarized airway inflammation and remodeling that are associated with increased bronchial hyperreactivity (36). Here we report that mice exposed to HDM and concurrently treated with a pan-neutralizing anti–TGF-β ( Some of the results of these studies have been previously reported in the form of an abstract (37).
Animals Female Balb/c mice (6–8 wk old) were purchased from Charles River Laboratories (Ottawa, ON, Canada). SMAD3 heterozygous mice (129SV/EV x C57B/6 background; courtesy of Dr. A. Roberts, National Institutes of Health, Bethesda, MD) (38) were bred in-house. The genotypes of both WT littermate and SMAD3 KO mice were determined by polymerase chain reaction analysis on tail DNA obtained from 3-week-old animals. All mice were housed under specific pathogen–free conditions and maintained on a 12-hour light:dark cycle, with food and water ad libitum. All experiments described in this study were approved by the Animal Research Ethics Board of McMaster University (Hamilton, ON, Canada).
Sensitization Protocols
Ab administration.
Continuous HDM exposure protocol.
Intermittent HDM exposure protocol. Separate groups of mice were exposed daily to HDM or saline for 10 consecutive days followed by approximately 30 days of rest. We have previously shown that 10 days of HDM exposure elicits robust Th2-polarized allergic sensitization and airway inflammation (39). Saline- and HDM-sensitized mice were subsequently subjected to six cycles of saline or HDM reexposure, respectively; each cycle consisted of daily intranasal exposure for 3 consecutive days followed by 12 days of rest. In addition, mice were concurrently treated every other day with  TGF-β or control IgG over each of the six reexposure cycles beginning 2 days before the first saline/HDM exposure and ending on the second day after the last (i.e., third) saline/HDM exposure, for a total of four Ab administrations per cycle. Mice were killed 72 hours after the last challenge (Figure 1C).
Collection and Measurement of Specimens
Preparation of Lung Tissue Homogenate and Hydroxyproline Measurement
Histology and Immunohistochemistry
Morphometric Analysis
Splenocyte Culture
Bioassay for TGF-β
Cytokine and Immunoglobulin Measurements by ELISA
Airway Responsiveness Measurements
Data Analysis
Kinetic Analysis of HDM-induced Responses in the Lung Consistent with our previous report, we observed that Balb/c mice exposed via the respiratory mucosa to HDM for 5 weeks develop robust Th2-polarized airway inflammation (36). Kinetic analysis of the BAL revealed that an increase in the total cell number is evident after just 1 week of HDM, peaks at 3 weeks, and is maintained at this level throughout the course of allergen exposure (Figure 2A). Furthermore, we observed that this process is associated with a progressive airway reparative response that results in altered airway structure (Figure 2A and Reference 36). These structural changes are incipient after 3 weeks of HDM exposure and overt after 5 weeks, and include increased subepithelial collagen deposition and smooth muscle thickening. The concept that inflammation and repair are related strongly implicates TGF-β. Thus, we examined the expression of TGF-β in the BAL of mice exposed to 1, 3, or 5 weeks of HDM. We observed that the concentration of active TGF-β1 was substantially elevated after 3 and 5 weeks of HDM compared with untreated (0 wk) and saline exposed control animals (data not shown), and appeared to be increased after just 1 week of HDM (Figure 2B). Interestingly, we noted that the increase in active TGF-β1 at 3 and 5 weeks corresponded with both the plateau in inflammation and the airway remodeling response. These findings supported our hypothesis that TGF-β may be dampening the chronic allergic inflammatory response while at the same initiating reparative processes.
To directly investigate this in vivo we used a pan-neutralizing TGF-β Ab that is specific for the active form of all three mammalian TGF-β isoforms. This Ab has been previously shown to reduce the extent of OVA-induced allergic airway remodeling (18). Although the characteristics and functional properties of this Ab have been extensively assessed, we reconfirmed the functionality of each batch of Ab by testing its ability to block TGF-β1 detection by ELISA (Figure 2C) and TGF-β–induced luciferase expression in the plasminogen activator inhibitor-1 luciferase-based (PAI-L) bioassay (data not shown). To determine whether the Ab is able to reach the airway walls and subepithelial space, we also tracked its kinetics and distribution in vivo, in three separate compartments: the serum, lung tissue, and airway lumen. Separate groups of mice were given a single intraperitoneal administration of TGF-β and then killed 2, 6, and 12 hours postinjection. We observed that the TGF-β Ab was already present at considerable levels in the serum and lung tissue 2 hours postinjection, could be detected in the airway lumen in as little as 6 hours, and that concentrations steadily increased or were maintained thereafter (Figure 2D). This demonstrated that TGF-β was indeed able to reach critical target regions in the lung.
Impact of TGF-β Blockade on the Development of HDM-induced Allergic Airway Remodeling
We proceeded to examine whether TGF-β blockade was ineffective in the above experimental setting due to the timing of treatment initiation. Given that the levels of active TGF-β1 are increased early on during HDM exposure (1–2 wk; Figure 2B), albeit to a lesser extent than at later time points, we questioned whether this early expression may have been sufficient to trigger a cascade of events that subsequently drove the remodeling response in a manner that was then independent of TGF-β. To address this, we subjected mice to the continuous HDM exposure protocol and treated them with TGF-β or the control IgG over nearly the entire course of exposure. Even under this extended treatment regimen and using the 100-µg dose, we observed that HDM-exposed TGF-β–treated mice developed equivalent increases in subepithelial collagen, mucus production, and smooth muscle thickening as HDM-exposed IgG-treated mice (Figure 4). The similarity in the extent of collagen accumulation between HDM-exposed TGF-β– and IgG-treated groups was further confirmed by a hydroxyproline assay (Figure E2).
Next, we ascertained whether the inability of TGF-β neutralization to inhibit airway remodeling was due to the severity of the inflammatory response that is elicited by this protocol. To this end, we developed a model of intermittent HDM exposure that was based on short, recurrent HDM rechallenges separated by 12-day rest periods (Figure 1C), rather than continuous HDM exposure. In this model, we observed that each 3-day HDM rechallenge elicited an eosinophilic inflammatory response, which then partially resolved over the subsequent rest period; total cells and eosinophils were decreased by approximately 60 and 65%, respectively, before subsequent rechallenge. Thus, the structural alterations that develop in this experimental protocol are associated with a less severe inflammatory response. We subjected mice to this intermittent exposure protocol and treated them with four 100-µg administrations of TGF-β or the IgG control Ab during each HDM rechallenge (Figure 1C). We documented that TGF-β–treated HDM-exposed mice developed identical increases in subepithelial collagen deposition as HDM-exposed IgG-treated control animals (Figures 5A–5D), demonstrating that, even under these less severe conditions, TGF-β blockade had no effect on the development of HDM-induced airway remodeling.
Latent TGF-β protein is embedded throughout the extracellular matrix of most organs, especially the lungs, and it is believed that, once activated, TGF-β acts rapidly on only those cells that are in direct contact. Thus, we questioned whether such a mechanism would allow at least some TGF-β to escape neutralization by the blocking Ab. Importantly, we note that, in other experimental models of fibrosis, in which such a mechanism is believed to mediate fibrogenesis (44), treatment with the TGF-β Ab used here significantly abrogated the development of fibrosis (Reference 45 and S. Lonning, personal communication). To confirm our results given this possibility, we used a genetically based approach to interfere with TGF-β activity. The use of TGF-β–deficient mice, although ideal, is effectively impossible given their extremely short life spans. As an alternative, we used SMAD3 KO mice because SMAD3 is a signaling molecule that has been shown to be a critical mediator of TGF-β–induced remodeling-associated gene expression (46). We subjected SMAD3 KO mice and their WT littermate controls to the continuous exposure protocol. After 5 weeks of HDM administration, we evaluated the development of airway remodeling in these mice. Morphometric analysis showed significant increases in collagen deposition (Figure 6A) and smooth muscle thickening (Figure 6B) in both WT and KO animals exposed to HDM compared with their respective saline-treated controls. Moreover, we found that the structural alterations observed in HDM-exposed SMAD3 KO mice were equivalent in magnitude to HDM-exposed WT control animals (Figure 6).
Impact of TGF-β Blockade on the Regulation of Chronic Allergic Airway Inflammation Induced by HDM Given that TGF-β is a potent negative regulator of inflammation and has been shown to inhibit the activity, differentiation, and proliferation of various Th2-associated inflammatory cell types in vitro, we explored whether TGF-β blockade had any impact on the regulation of established chronic allergic airway inflammation. We exposed mice to the continuous HDM protocol and blocked TGF-β during the last 3 weeks of exposure using the neutralizing TGF-β Ab. Treatment was initiated at this time to allow sensitization and the inflammatory response to become fully established. Analysis of the cellular infiltrate in the BAL revealed that mice exposed to HDM and treated with 10 µg of TGF-β had a slight, but nonsignificant, increase in the number of total cells and eosinophils compared with IgG-treated control animals (Figure 7A). Treatment with a higher dose of TGF-β Ab (100 µg/dose) significantly increased the number of total cells present in the BAL compared with HDM-exposed IgG-treated mice and, interestingly, this increase was due exclusively to a greater number of eosinophils (Figure 7A). Indeed, the proportion of eosinophils present in the BAL of mice exposed to HDM and treated with 100 µg of TGF-β was nearly double that of IgG control animals. We further examined the effect of TGF-β neutralization on the inflammatory response in the context of extended Ab treatment and intermittent HDM exposure. We observed that treatment with TGF-β over nearly the entire 5-week course of HDM administration also exacerbated the proportion of eosinophils in the BAL (Figure 7B). Notably, this extended treatment also resulted in a considerable decrease in the number of mononuclear cells (data not shown); this was not unexpected because TGF-β is known to be a potent monocyte chemoattractant factor (47). Finally, consistent with the findings above, TGF-β blockade also noticeably increased the number of total cells (data not shown) and the extent of eosinophilia in the BAL of mice subjected to the intermittent HDM exposure protocol (Figure 7C), although these differences did not reach statistical significance.
Impact of TGF-β Blockade on HDM-specific Adaptive Immunity Because TGF-β blockade led to increased airway eosinophilic inflammation, we explored whether this may have been a consequence of altered T-cell responsiveness. To evaluate T-cell function, we harvested splenocytes and measured the production of Th2-associated cytokines after stimulation with HDM in vitro. We observed a significant increase in the production of IL-5 by splenocytes taken from mice exposed to HDM for 5 weeks and concurrently treated with TGF-β (100-µg dose) during the final 3 weeks compared with HDM-exposed IgG-treated control animals (Figure 8A). We also documented significantly greater IL-5 production when the duration of TGF-β administration was increased (Figure 8B). In addition, we further noted a trend toward augmented IL-13 production in both of these experimental protocols; however, this did not reach significance (Figures 8A and 8B). No differences were observed in HDM-specific IL-4 production.
We also examined the impact of TGF-β blockade on humoral immunity. Similar to the findings above, mice treated with TGF-β Ab during the last 3 weeks or over almost the entire course of the continuous HDM exposure protocol possessed significantly elevated serum levels of HDM-specific IgE and IgG1 compared with the HDM-exposed IgG-treated group (Figures 8C and 8D).
Impact of TGF-β Neutralization on Lung Function
In light of the mounting evidence implicating TGF-β in the pathogenesis of asthma, we investigated the impact of interfering with TGF-β activity on the regulation of chronic allergic airway inflammation and remodeling induced by exposure to HDM. We observed that treatment with a neutralizing TGF-β Ab had no effect on the development of airway remodeling, even if the dose of Ab was increased 10-fold and treatment was extended over nearly the entire course of HDM exposure. These observations suggested to us that the remodeling response, at least when triggered by continuous HDM exposure, could occur independently of TGF-β activity. Similarly, treatment with TGF-β in the context of an intermittent HDM exposure protocol, in which the ensuing inflammatory response is recurrent and therefore considerably lower, also had no effect. This argued against the possibility that TGF-β was dispensable in the continuous HDM exposure protocol simply because that protocol elicits a robust inflammatory response that is sustained over several weeks. Moreover, these findings suggest that other factors, such as the nature of the eliciting agent and/or the type of the inflammatory response, may ultimately influence whether TGF-β is required in a given reparative/fibrotic response. Indeed, that the type of inflammation impacts whether a reparative/fibrotic response will develop has been previously demonstrated (14, 48, 49). To further confirm the observations made with the blocking Ab, we used a second approach, the use of SMAD3 KO mice, to evaluate the role of TGF-β in HDM-induced remodeling. The SMAD signaling pathway is not the only pathway by which the fibrogenic effects of TGF-β may be mediated (50); however, SMAD3 has been clearly shown to play a fundamental role in the signal transduction pathways associated with TGF-β–mediated wound healing and fibrosis (46). In agreement with the findings above, we observed that HDM-exposed SMAD3 KO mice developed airway remodeling to the same extent as WT littermate controls. Thus, when taken together, the findings we report here demonstrate that HDM-induced allergic airway remodeling can develop independently of TGF-β. Although our data may initially appear to be at variance with the prevailing notion that TGF-β mediates, in all circumstances, tissue repair, careful consideration of the differences between the various systems used may in fact account for these seemingly divergent findings. In the case of fibrogenesis, a common feature of models of inflammation and fibrosis is that there is a single eliciting event (e.g., bleomycin, radiation, administration of TGF-β). This generates an acute and self-limited inflammatory process that is associated with the development of fibrosis. These approaches remarkably contrast with our model in that the eliciting agent (HDM) is delivered continuously for a considerable period of time. In addition, the type of inflammation (Th2 polarized) is yet another central difference between our model and most models of fibrosis. The findings by McMillan and colleagues warrant particular consideration as they investigated the impact of TGF-β neutralization, using the same Ab as the one used here, in a model of chronic allergic airway disease (18). They showed that TGF-β neutralization prevented the progression of airway remodeling after repeated OVA challenge. However, there are several important considerations that must be taken into account. First, one should note that, whereas this treatment indeed prevented airway remodeling at an early time point (Day 35) of their protocol, the effects of TGF-β neutralization on the extent of remodeling at a later time point (Day 55) were substantially reduced, suggesting, as the authors noted, that additional factors are likely contributing to the development of airway remodeling in their model. Second, as is well known in models using OVA, repeated OVA challenge leads to a diminution of the inflammatory response, as certainly occurred in McMillan and colleagues' protocol. This is in sharp contrast with the model that we used in which we maintain robust inflammation (30–35% eosinophils in BAL) for several weeks. Third, it may be particularly important that the antigen used in this model was OVA, a prototypic innocuous antigen with fundamental biochemical differences to HDM. Indeed, whereas OVA preparations are essentially pure, HDM is, in stark contrast, a complex material consisting of numerous protein and nonprotein components, with considerable proteolytic activity. This added complexity may be of considerable relevance because the biochemical and immunogenic profile of HDM will likely draw on a distinct network of molecular responses. Therefore, we argue that, whereas OVA can, under certain conditions (e.g., intraperitoneal sensitization with intermittent exposure) lead to remodeling, achieving the same outcome using HDM and a route of mucosal sensitization does not imply the same underlying biochemical or immunologic process; nor can this knowledge be universally applied to the responses elicited by other aeroallergen families. Thus, from this perspective, we do not view our findings to be in contrast with those described above but instead to be a reflection of the differences in the contexts in which the role of TGF-β is being explored. Particular attention should also be drawn to the study by Kaviratne and coworkers (51). There they comprehensively investigated the requirement for TGF-β in the generation of liver fibrosis induced by Schistosoma mansoni infection. Most interesting, this fibrotic response develops as a consequence of a Th2-polarized inflammatory response directed against schistosome eggs, which get trapped in the liver. Using an exhaustive array of methodologies, the authors conclusively demonstrated that the fibrotic response induced by schistosome infection was TGF-β independent. Notably, IL-13, a prototypical Th2-associated cytokine, was shown to be an indispensable mediator of hepatic fibrosis in this model (52). Interestingly, constitutive IL-13 overexpression in the lung has been shown to trigger a Th2-like response involving airway eosinophilia and hyperreactivity that was furthermore associated with the development of subepithelial airway fibrosis (53). However, in contrast to the observations made in the schistosome model, the fibrotic response that develops as a consequence of IL-13 overexpression was found to be mediated by TGF-β, because administration of a soluble TGF-β receptor–Fc molecule ameliorated IL-13–induced fibrosis (54). The difference in the requirement for TGF-β between these two models may reflect the fact that IL-13 overexpression does not fully recapitulate a Th2-polarized immune response. Indeed, many of the other hallmark Th2-associated cell types and molecules (B cells, Th2 cells, IL-4, IL-5, IL-9, and IgE) were absent in the transgenic IL-13 model. These additional components are likely to critically influence the lung microenvironment and therefore may alter the requirement for TGF-β in the generation of a fibrotic response that develops in the context of an antigen-dependent Th2 immune response. Taken together, these studies support the concept that some Th2-associated airway reparative responses occur independently of TGF-β, and moreover, that the molecular signatures underlying the development of a "fibrotic event" may be critically influenced by the type of inflammatory response, and therefore by the nature of the eliciting agent (e.g., bleomycin, radiation, schistosoma, OVA, HDM). Thus, it follows that there may be several distinct pathways leading to fibrosis—that is, a TGF-β–dependent pathway and an IL-13–dependent TGF-β–independent pathway, possibly among others. In addition, there exists a panoply of other molecules that display powerful fibrogenic activity. These include platelet-derived growth factor, vascular endothelial growth factor, connective tissue growth factor, and oncostatin M, among several others (55, 56). The precise contribution of these molecules, either alone or in concert, in mediating tissue reparative responses associated with Th2-polarized airway inflammation remains to be fully dissected.
We also investigated the impact of interfering with TGF-β activity on the allergic inflammatory response. We observed that TGF-β neutralization, when initiated at a time when the inflammatory response was already ongoing (2 wk of HDM exposure), led to a significant increase in inflammation that was characterized exclusively by an increase in eosinophils; similar effects were also evident when TGF-β neutralization was initiated early on during continuous HDM exposure (Day 4) and in the intermittent exposure protocol, although the increase did not reach statistical significance in the latter. These increases could be the result of augmented IL-5 production, although it is also plausible that Several related studies using acute conventional OVA models (33–35) have also documented increases in airway inflammation attributable in large part to the following: enhanced eosinophilia; elevated levels of Th2-associated cytokines, including IL-4, IL-5, and IL-13; and increased serum IgE and IgG1 in mice in which TGF-β levels or activity had been impaired. Although it was not possible to clearly identify the effects on established inflammation alone given the genetically based methods these authors used, their data implicate TGF-β as an important regulator of allergic airway inflammation. Interestingly, in both the McMillan and Alcorn studies, TGF-β neutralization in their models had no effect on established inflammation. We suspect that this observation may be related to the dose of Ab used and/or the system used and not necessarily a reflection of the role of TGF-β in the regulation of chronic allergic inflammation. Using a similar dose of Ab used in McMillan and colleagues' protocol, we observed a clear trend toward enhanced eosinophilia. Administration of a higher dose of Ab conclusively demonstrated, at least in our system, that TGF-β is a critical regulator of HDM-induced chronic allergic airway inflammation. In our view, the findings we present here do not question the importance of TGF-β to the tissue repair response. Rather, they compel us to consider the notion that the role of a given molecule in a process is contextual. In the context of robust airway inflammation elicited by persistent exposure to a common aeroallergen (i.e., HDM), the research we present here demonstrates that TGF-β does not play a critical role in airway remodeling, which, we understand, may be divergent from the prevailing dogma. In addition, our data show that interference with TGF-β leads to worsened airway inflammation and function. We surmise that these findings are, at least teleologically, in accord with the principal goal of the immune response: survival of the host. That is, that the key role of TGF-β under these conditions may be to protect the host from devastating inflammation and tissue damage. From this perspective, an important concern arises with respect to the proposition of therapeutic interference with TGF-β as a means of limiting the progression of airway remodeling, not only that it may not work but also that the treatment may lead to a loss of control of the inflammatory response and, ultimately, the disease.
The authors gratefully acknowledge the technical help of Mary-Jo Smith, Mary Bruni, and Marcia Fattouh. They also thank Dr. Mark Inman and Jennifer Wattie for their excellent help with measuring airway reactivity and Dr. Gail Martin for her help with the PAI-1/L bioassay and valuable discussion. They are also indebted to Mary Kiriakopoulos for secretarial assistance.
Supported by the Ontario Thoracic Society. R.F. was supported by a Canadian Institutes for Health Research (CIHR) Doctoral Canadian Graduate Scholarship and is currently supported by an Ontario Graduate Scholarship in Science and Technology; J.R.J holds a CIHR Doctoral award; M.J. is a Senior Canada Research Chair. This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200706-958OC on January 3, 2008 Conflict of Interest Statement: R.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. N.G.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.R.J. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. T.D.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.P.S. is an employee of Genzyme Corporation and owns shares of Genzyme Corporation common stock. R.C.G. is an employee of Genzyme Corporation and owns shares of Genzyme Corporation common stock. S.L. is a salaried employee of Genzyme Corporation and has shares of Genzyme Corporation common stock. J.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.J. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form June 28, 2007; accepted in final form January 2, 2008
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ABSTRACT
AT A GLANCE COMMENTARY
0.5 and 5 mg/kg, respectively) in 0.5 ml of sterile phosphate-buffered saline immediately before HDM administration. 
1- week HDM-exposed groups, respectively.
HDM + 
