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Published ahead of print on February 21, 2008, doi:10.1164/rccm.200709-1376OC
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American Journal of Respiratory and Critical Care Medicine Vol 177. pp. 1074-1081, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200709-1376OC

Original Article

Basal Expression of Bone Morphogenetic Protein Receptor Is Reduced in Mild Asthma

Harsha H. Kariyawasam1,2,3, Georgina Xanthou4, Julia Barkans1,3, Maxine Aizen1,3, A. Barry Kay2,3 and Douglas S. Robinson1,2,3

1 Allergy and Clinical Immunology Section, 2 Leukocyte Biology Section, and 3 MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London, United Kingdom; and 4 Biomedical Research Foundation of the Academy of Athens, Athens, Greece

Correspondence and requests for reprints should be addressed to Dr. A. B. Kay, M.D., Ph.D., Emeritus Professor of Allergy and Clinical Immunology, Sir Alexander Fleming Building, Leukocyte Biology Section, Imperial College, South Kensington Campus, London, SW7 2AZ UK. E-mail: a.b.kay{at}imperial.ac.uk


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Despite increasing recognition of bone morphogenetic protein (BMP) signaling in tissue remodeling, the expression pattern of ligands and signaling pathways remain undefined in the asthmatic airway.

Objectives: To determine expression of BMP ligands (BMP-2, BMP-4, and BMP-7) and type I and type II receptors (ALK-2, ALK-3, ALK-6, and BMPRII) as well as evidence for activation of BMP signaling via detection of phosphorylated Smad1/5 (pSmad1/5) expression in asthmatic airways at baseline (compared with nonasthmatic controls), and after allergen challenge.

Methods: Bronchial biopsies were obtained from 6 nonasthmatic control volunteers, and 15 atopic patients with asthma (median age, 25 yr; median FEV1% predicted, 97%) at baseline, then at 24 hours and 7 days after allergen challenge. Expression of BMP ligands, receptors, and signaling was analyzed using immunohistochemistry.

Measurements and Main Results: BMP ligand expression did not differ between asthmatic and control airways at baseline. Compared with the normal airway, there was significant down-regulation of ALK-2 (P = 0.001), ALK-6 (P = 0.0009), and BMPRII (P = 0.009) expression in asthma. Allergen challenge was associated with marked and sustained up-regulation of BMP-7 in airway epithelium (P = 0.017) and infiltrating inflammatory cells (P = 0.071) (predominantly in eosinophils, but also CD4+ T cells, mast cells, and macrophages). Up-regulation of pSmad1/5 expression (P = 0.031), ALK-2 (P = 0.002), and ALK-6 (P < 0.001) was observed indicating active signaling.

Conclusions: BMP receptor expression is down-regulated in the asthmatic airway, which may impede repair responses. Allergen provocation increases expression of the regulatory ligand BMP-7, activates BMP signaling, and increases receptor expression, all of which may contribute to repair and control of inflammation.

Key Words: bone morphogenetic protein • BMP ligands • BMP receptors • asthma • signaling



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Bone morphogenetic proteins (BMPs) have an essential role in organogenesis and tissue repair, suggesting an important role in airway remodeling. The expression of BMP ligands and signaling pathways are undefined in the normal airway and asthma.

What This Study Adds to the Field
BMP receptor expression is markedly different in the asthmatic airway compared with that of normal airways. Furthermore, BMP signaling is rapidly modulated in response to allergen challenge, suggesting a role in the disease process.

 
The transforming growth factor (TGF)-β superfamily of ligands comprises more than 35 members in mammals of which the bone morphogenetic proteins (BMPs) are the largest subgroup of structurally and functionally related proteins (1). Consistent with their important role in embryonic development and tissue homeostasis, BMPs are highly conserved between species (2).

BMP ligands signal via a constitutively active serine-threonine kinase–specific type II receptor that complexes with a type I receptor, which then propagates the signal downstream by phosphorylating receptor-regulated Smads (R-Smads). R-Smads then translocate to the nucleus in association with the common Smad4 to initiate gene transcription (3). BMP signaling is predominantly through the type II receptor BMPRII, and three type I receptors, ALK-2, ALK-3, and ALK-6. TGF-β proteins are regulated by synthesis but are also bound to the extracellular matrix (ECM) as inactive forms. Thus, signaling analysis is required to detect activity of these factors (e.g., detection of phosphorylated R-Smads).

A vital function of BMPs in the process of epithelial–mesenchymal interactions during organ development and in regeneration is suggested by the failure of organ systems with layered tissue structure to develop in the absence of BMP signaling. BMP-2 knockout mice exhibit severe heart abnormalities that are embryonically lethal (4), and BMP-4 knockout mice display failure of mesodermal induction (5). In lung branching morphogenesis, BMP-4 expression is expressed predominantly in the distal epithelium of the bud outgrowth (branching tips) that grows into the mesenchymal tissue. Mesenchyme-derived fibroblast growth factor-10 can induce this expression of BMP-4 (6). BMP-4 directly increases the number of {alpha}-smooth muscle actin ({alpha}-SMA)–positive parabronchial cells in an in vivo lung explant system (7).

BMP-4 has been demonstrated to act to inhibit fibroblast proliferation but promote {alpha}-SMA expression and differentiation of fibroblasts (8).

BMP-7 (also known as osteogenic protein [OP]-1), identified originally as a potent osteogenic factor from bone, is an antifibrotic factor and can antagonize the effects of TGF-β1. BMP-7 expression is seen at sites of epithelial–mesenchymal tissue interactions with regulatory effects on branching morphogenesis. During lung formation, expression is most prominent along the basement membrane (9). In kidney development, BMP-7 regulates branching morphogenesis and serves as a survival factor for epithelium (10, 11). End-stage kidney disease is characterized by massive fibrosis driven by TGF-β1 signaling associated with decreased BMP-7 expression (12, 13). Exogenous administration of BMP-7 into experimental systems is able to reverse the fibrotic effects of TGF- β1, through counteracting TGF-β1–induced epithelial–mesenchymal transition, and is associated with decreased expression of type I collagen by fibroblasts (14, 15). In lung myofibroblasts, BMP-7 inhibits TGF-β1–mediated collagen, {alpha}-SMA, and tissue inhibitor of metalloproteinases (TIMP)-2 expression (16). It has been demonstrated in both the kidney (17) and the gut (18) that BMP-7 potently reduces inflammation and fibrosis. Such findings suggest that BMPs may play a significant role in epithelial–mesenchymal interactions during tissue remodeling.

We have demonstrated previously that allergen inhalation challenge in asthma is associated with activation of TGF-β signaling, with acute remodeling changes within 24 hours (19). Currently, the expression patterns of BMP ligands and their pathway signaling components remain undefined in human asthma. In an ovalbumin-induced murine model of airway inflammation, there was rapid induction of BMP signaling as evidenced by increased phosphorylated Smad1/5 (pSmad1/5) expression together with induction of activin-like kinase (ALK)-2 and ALK-6 mRNA and protein after inhaled allergen (20). In addition, we have recently shown that there is rapid and sustained up-regulation of markers of airway remodeling at 24 hours and 7 days post–allergen challenge (21). Given the essential role of BMP signaling in tissue regeneration and repair in the kidneys and gut, we hypothesized that there would be comparable sustained increases in BMP signaling in the asthmatic airway in response to allergen challenge. Some of the results from this study have previously been published in abstract form (22).


   METHODS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Volunteer Details and Study Design
The study was approved by the Royal Brompton and Harefield Hospital Ethics Committee and volunteers gave written, informed consent. Fifteen volunteers with a history of atopic asthma with either a 15% increase in FEV1 to β2-agonist or methacholine provocative concentration causing a 20% fall in FEV1 (PC20) of 8 mg/ml or less were recruited. The median age was 25 (range, 19–46) years with an FEV1% predicted of 97% (range, 75.4–125.7%) at study entry with a methacholine PC20 of 2.1 (1.2–3.6) mg/ml (geometric mean ± 95% confidence interval). All subjects demonstrated positive skin prick tests (wheal size, >=3 mm) to one or more of the aeroallergens house dust mite, cat dander, or grass (ALK-Abelló, Hørsholm, Denmark). Volunteers sensitive to pollens were studied outside of the season. Volunteers were controlled with only rescue β2-agonists at the time of study and had no clinical features of infection for at least 4 weeks before starting the study and none throughout the study period. The study design has been previously described (21). Briefly, fiberoptic bronchoscopy with bronchial biopsies was performed as previously described (19) at baseline, then 24 hours and 7 days after inhalational allergen challenge. Six normal volunteers (4 males and 2 females) of a median age of 30.5 (range, 27–42) years, an FEV1% predicted of 100.4% (range, 80–104.3%), with a methacholine PC20 of more than 16 mg/ml underwent a single baseline bronchoscopy. There was no history of asthma or atopy, with negative skin prick tests and IgE RAST to common aeroallergens, no significant past medical history, and no medication use in the normal volunteers. All volunteers were nonsmokers. Apart from allergen challenge, normal volunteers underwent the same study protocol, including methacholine challenge testing followed by 5 mg nebulized albuterol before bronchoscopy. All bronchoscopies were performed between 8.30 and 9:00 A.M.

Immunohistochemistry
Tissue processing and immunostaining were performed as previously described (23, 24) as was the alkaline phosphatase–antialkaline phosphatase (APAAP) method (23) to enumerate cells binding antibodies, with the reaction visualized using Fast Red chromogen. BMP-2 expression was detected using a mouse monoclonal antibody (Clone 100221; R&D Systems, Minneapolis, MN), whereas BMP-4 and BMP-7 were detected using goat polyclonal antibodies (R&D Systems). Cellular localization of BMP-7 was through a double staining technique as previously described, but the reaction developed using 3,3'-diaminobenzidine chromogen, which produces a brown end product. Incubation of tissue sections with an irrelevant species–specific IgG antibody served as a negative control. The antibodies directed against the type I and type II receptors and Smads were a kind gift from Prof. P. Sideras, Athens Biomedical Institute, Greece. Briefly, polyclonal antibodies were raised in rabbits against synthetic polypeptides and then tested for specificity by immunoprecipitation and Western blotting as previously described (20, 25). These antibodies have been previously been validated in human tissue (26). Cells counts were done as previously described (19). All counts were done in a blinded fashion using an Olympus BH-2 microscope (Olympus Corp., Lake Success, NY).

Statistical Methods
Cell counts are presented as median ± interquartile range. The Mann-Whitney U test was used to compare baseline expression between normal volunteers and volunteers with asthma. Data were analyzed using GraphPad Prism version 4 (GraphPad Software, Inc., San Diego, CA). The time course data were analyzed using a linear mixed model to assess the change over time (allowing us to incorporate the data of one volunteer who did not complete the final visit) using the statistical program Stata version 9.2 (StataCorp LP, College Station, TX). In this model, patients were entered as a random effect, with time as a fixed effect (27). Significance was accepted as P < 0.05. Results are summarized in Table 3.


   RESULTS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Baseline BMP Receptor Expression Is Down-regulated in Asthma
Numbers of epithelial cells expressing ALK-2 and ALK-6 expression were significantly less in the asthmatic airway compared with normal airways (P = 0.001 and P = 0.0009, respectively) (Figures 1A and 1C). ALK-2 and ALK-6 expression was predominantly localized to the airway epithelium. ALK-3 expression was evident on submucosal inflammatory-like cells and airway epithelium and did not differ between the asthmatic and normal airway (Figure 1B). Representative photomicrographs of type I receptor expression are shown in Figures 2A–2D.


Figure 1
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  		Figure 1. Summary of bone morphogenetic protein (BMP) receptor (A–D) and pSmad1/5 (E) expression in the normal airway compared with asthma. The number of cells expressing each receptor is expressed as a percentage of the total number of epithelial cells present when comparing expression with the normal airway. Other than ALK-3, all other BMP receptors demonstrate down-regulation of expression in the asthmatic airway. ALK = activin-like kinase.

 

Figure 2
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  		Figure 2. Representative photomicrographs of bone morphogenetic protein (BMP) receptor and pSmad1/5 expression Normal volunteers demonstrate increased intensity of ALK-2 and ALK-6 expression in epithelium, as well in some inflammatory-like cells below the basement membrane (A–B and C–D, respectively). Similarly, normal volunteers (E) demonstrate marked immunostaining for the BMP type receptor BMPRII compared with the asthmatic airway (F). Normal volunteers demonstrate expression of pSmad1/5 in epithelium and inflammatory cells (G). Subjects with asthma demonstrate less epithelial expression of pSmad1/5 (H), suggesting active BMP signaling may be down-regulated in the asthmatic airway.

 
BMPRII expression was also detected on significantly fewer cells in the asthmatic airway epithelium compared with the normal airway (P = 0.009) (Figure 1D). A representative photomicrograph of BMPRII expression in the normal and asthmatic airway is presented (Figures 2E and 2F).

BMP Ligand Expression Is Similar in Normal and Asthmatic Airways
The expression of BMP-2, BMP-4, and BMP-7 ligands in the normal airway was similar to that of the asthmatic airway at baseline (Table 1). BMP-2 expression was confined to only the airway epithelium. Both BMP-4 and BMP-7 were expressed throughout the airway epithelium, although submucosal cells with inflammatory cell-like morphology also expressed BMP-4 and BMP-7.


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  		TABLE 1. SUMMARY OF THE PERCENTAGE OF EPITHELIAL CELLS EXPRESSING EACH BONE MORPHOGENETIC PROTEIN LIGAND IN THE NORMAL AIRWAY AND IN BASELINE ASTHMA

 
Evidence for activation of BMP ligand signaling was obtained by counting the number of pSmad1/5-positive epithelial cells. Immunostaining was confined predominantly to the airway epithelium. The median percentage of cells staining for pSmad1/5 in the normal airway was 40% (16.40–57% interquartile range) versus 3.8% (0–72.22%) in the asthmatic airway, although this was not statistically different (Figure 1E). Representative photomicrographs of pSmad1/5 expression in the normal airway versus the asthmatic airway are presented in Figures 2G and 2H.

Effect of Allergen Challenge
Type I receptor expression is modulated in response to allergen challenge.
Allergen challenge was associated with marked up-regulation of ALK-2 and ALK-6 postallergen and with further increases at the 7-day time point (P = 0.002 and P < 0.001, respectively) (Figures 3A and 3C, respectively). No significant modulation of BMPRII expression was seen in response to allergen challenge (Figure 3D).


Figure 3
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  		Figure 3. Summary of expression of bone morphogenetic protein (BMP) activated receptors and pSmad1/5 post–allergen challenge in asthma. The number of epithelial cells expressing each protein is expressed as the number per unit length of basement membrane (BM) (cells/mm BM) when comparing modulation of expression in the asthmatic airway. Allergen challenge is associated with increased expression of ALK-2 and ALK-6 at 24 hours and this was sustained at the 7-d time (A and C) Activation of BMP-activated pSmad1/5 signaling was evident at both 24 hours and 7 days after allergen challenge (E).

 
BMP signaling is activated in response to allergen challenge.
Allergen challenge was associated with significantly increased and sustained expression of pSmad1/5 at the 7-day time point (P = 0.031) (Figure 3E).

BMP-7 expression is modulated in response to allergen challenge.
There were no allergen-induced changes in BMP-2 or BMP-4 expression (Table 2). Allergen challenge was associated with significantly increased numbers of epithelial cells expressing BMP-7 at the 7-day postallergen time point (P = 0.017) (Figure 4A), whereas there was a noticeable trend in increased number of inflammatory cells postallergen expressing BMP-7 (P = 0.07) (Figure 4B).


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  		TABLE 2. SUMMARY OF EPITHELIAL BONE MORPHOGENETIC PROTEIN LIGAND EXPRESSION IN RESPONSE TO ALLERGEN CHALLENGE IN ASTHMA

 

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  		TABLE 3. ANALYSIS OF DATA USING A MIXED MODELING APPROACH (REPEATED MEASURES ANALYSIS)

 

Figure 4
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  		Figure 4. Summary of bone morphogenetic protein (BMP)-7 expression in the asthmatic airway in response to allergen challenge. Localization of BMP-7 expression to inflammatory cell types (C) and a representative photomicrograph of colocalization of BMP-7 to major basic protein (MBP+) eosinophils (original magnification, x40 [D] and x100 [E]). The number of epithelial cells expressing each protein is expressed as the number per unit length of basement membrane (BM) (cells/mm BM). Positive inflammatory cells present are expressed as cells/mm2. BMP-7 expression was up-regulated in epithelium at 7 days postallergen (A), whereas increased numbers of inflammatory cells were seen at both 24 hours and 7 days (B). Using double-staining technique immunohistochemistry, BMP-7 expression was localized to inflammatory cell phenotypes in tissue sections obtained at the 24-hour postallergen time point (C). Cells counts are expressed as the percentage of double positive cells. BMP-7 is stained brown via 3,3'-diaminobenzidine, and the cell phenotyped using the chromogen Fast Red, staining red. Colocalization is seen as a darker red-brown color in cells (arrows) (D and E).

 
Eosinophils are the predominant inflammatory cell source of BMP-7 in asthma.
Immunohistochemical double staining of BMP-7 expression to inflammatory cell phenotypes in n = 6 volunteers 24 hours postallergen confirmed that eosinophils were the predominant source of inflammatory cells producing BMP-7, with a median percentage of 58% (38.58–72.20% interquartile range) of the population of eosinophils staining positive for BMP-7 (Figure 4C). A representative photomicrograph of BMP-7 colocalizing to major basic protein (MBP+) eosinophils is shown (Figures 4D and 4E). CD4+ T cells, CD68+ macrophages, and tryptase-positive mast cells also double-stained for BMP-7, albeit at a markedly lower frequency compared with the eosinophil population.


   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here we have defined the expression of BMP ligands and receptors as well as activation of signaling in asthmatic airways at baseline in comparison to nonasthmatics, and after allergen challenge of patients with mild atopic asthma. For the first time, we show a down-regulation of BMP receptor expression in the airways of patients with mild asymptomatic asthma. In contrast, allergen challenge was associated with increased expression of BMP-7, swift functional activation of BMP ligand signaling, together with rapid restitution of type I receptor expression that was sustained for at least 7 days after challenge.

Until this study, the expression of BMP ligands has remained mostly undefined in the normal and diseased lung. This is despite increasing awareness that the BMP ligand system represents a major developmental signaling pathway critical for organ and tissue generation in early development. The importance of this work is that it allows further hypotheses regarding the role of BMP signaling in asthma to be constructed.

In adult tissue and organ systems, BMP signaling may be reactivated to promote and regulate tissue regeneration, repair, and maintenance. In diseases in which dysregulated BMP signaling is present, such as in primary pulmonary hypertension (due to vascular smooth muscle proliferation) and non–small cell lung cancer (28), the loss of BMP-regulated signaling allows expression of other pro–growth factor signaling pathways. Our previous work has confirmed that markers of airway remodeling are increased in baseline symptomatic asthma (29). If BMP-mediated signaling has functional consequences for regulation of airway inflammatory and remodeling processes, our present findings suggest that down-regulated BMP signaling pathways in the airway of patients with mild asymptomatic asthma may contribute to the increased activity of remodeling processes in symptomatic asthma.

In this study, the levels of BMP-2, BMP-4, and BMP-7 expression were similar between the normal and mild asthmatic airway, suggesting that the BMPs have an important role in basal airway homeostasis and that there is a readily accessible reservoir of ligand that can be activated on demand. In our group of 15 volunteers with asthma, only 6 individuals demonstrated increases in epithelial pSmad1/5 expression at baseline. Despite not demonstrating a statistically significant difference in the basal level of active BMP signaling, it can be seen from the raw data illustrated in Figure 1E that the overall trend for the degree of pSmad1/5 signaling appears to be less than that in the baseline normal airway epithelium. In response to allergen challenge, there was up-regulation of pSmad1/5 expression in the airways of patients with mild asthma, which was sustained at 7 days. Predominant localization to the airway epithelium was consistent with the increasingly recognized role of epithelium in the airway injury–repair response. Our finding of six volunteers with asthma and with evidence of increased activation of BMP signaling at baseline reflects induction of signaling and highlights the concept that there are probably multiple factors that can activate signaling, particularly in a complex heterogeneous disease such as asthma. It is interesting that there are further increases in BMP signaling in response to allergen challenge in these six volunteers.

Of the BMP ligands, BMP-7 alone was significantly up-regulated in response to allergen challenge. The activation of BMP signaling may reflect this increased expression of BMP-7 but could also be due to signaling by other BMP ligands because these may be activated without de novo expression. The significant and sustained increase in BMP-7 expression in both epithelium and inflammatory cells may be related to the role of BMP-7 in epithelial–mesenchymal tissue interaction (9) required for branching morphogenesis, a process that is reactivated in tissue repair. Such induction may also be related to the role of BMP-7 in the down-regulation of inflammation (18). Therefore, BMP-7 expression may be an attempt to regulate both the inflammatory and repair response in asthma. The finding of BMP-7 expression in 58% of infiltrating eosinophils is consistent with the evolving view that these cells are important tissue-repair cells (30, 31). The functional significance of eosinophil-derived BMP-7 remains to be determined, but it may be predicted that it is related to regulating repair through interaction with other TGF-β signaling pathways. For example, BMP-7 may act to control profibrotic effects of TGF-β1, which was also localized to eosinophils after allergen challenge (32). Infiltrating inflammatory cells expressing BMP-7 were localized to the submucosa, with minimal infiltration of the epithelium (data not shown).

TGF-β receptors are complex structures that are regulated at a number of different levels. Type II receptors are constitutively expressed. It is the type I receptor that directly activates the canonical Smad signaling pathway and it is therefore expected that type I receptor modulation will serve as a point of pathway regulation. We found a marked difference in the levels of BMPRII, ALK-2, and ALK-6 expression between the normal and asthmatic airways. We quantified the numbers and percentage of cells with positive staining for the various receptors, ligands, and signaling elements. We assume this is related to receptor density per cell but cannot equate staining with absolute receptor numbers or strictly compare between receptors using different antibodies. Although such differences may relate to differences in epithelial turnover and maturity, it is tempting to speculate that such receptor down-regulation leads to functional consequences for BMP-mediated signaling responses. Whether such differences in receptor expression are a fundamental property of the asthmatic airway or whether they may reflect an intrinsic defect in the way an asthmatic airway epithelium can respond to injury remains an important point of discussion. It is possible that such receptor down-regulation could also be a consequence of airway inflammatory pathways interacting with BMP signaling components. It is also possible that the mechanisms of receptor processing are different in the asthmatic airway. Mechanisms of ligand-induced receptor down-regulation that occur as a receptor regulatory response, as has been observed in other signaling systems such as the IL-5 receptor on eosinophils (33), are beginning to be understood for the TGF-β family of receptors. For example, with the TGF-β1–3 isoform type I receptor ALK-5, ligand–receptor interaction leads to rapid receptor internalization, leading to either receptor degradation or recycling back onto the cell surface (34, 35).

It is important to note that TGF-β1 itself can activate pSmad1/5 signaling, albeit through the type I receptor ALK-1 (36). The complexity and versatility of the signaling system are further enhanced by the ability of several non–TGF-β signaling pathways that can converge on the Smad pathway (37). For example, in asthmatic epithelium, it is shown that there is extensive up-regulation of epidermal growth factor receptor (EGFR) expression leading to continuous activation of extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) signaling (38, 39). ERK signaling activation in in vitro experiments leads to phosphorylation of Smad1 at the linker region, leading to inhibition of the BMP signaling pathway (40). It is possible that the observed down-regulation of type I receptors ALK-2 and ALK-6 as well as the type II receptor BMPRII is a reflection of other cellular pathways down-regulating BMP signaling in the asthmatic airway. Whether Th2 cytokines or other mediators of inflammation in asthma affect TGF-β receptor regulation remains to be determined.

We have previously documented the time course of inflammatory cell infiltration to the bronchial mucosa in these same subjects after allergen challenge: essentially, inflammation peaked at 24 hours and largely returned to baseline at 7 days after challenge (21). In contrast, markers of activation of collagen synthesis, such as heat shock protein 47, were increased at 24 hours and remained elevated at 7 days after challenge, whereas others markers, such as tenascin (ECM), increased at 24 hours and returned toward baseline at 7 days. We speculate that some elements of remodeling, such as ECM deposition, are closely related to inflammation, whereas others, such as collagen synthesis and activation of TGF-β superfamily ligand signaling pathways, have a more delayed time course. In the current study, we show reduced expression of BMP receptors at baseline in subjects with mild asthma compared with control subjects, which may relate to differences in epithelial turnover and maturity, followed by changes in BMP-7 ligand expression directly linked to infiltration of inflammatory cells after allergen challenge; in addition, there was a tendency to restore receptor numbers for ALK-2 and ALK-6, which may relate to inflammation or from activation of the repair process including BMP and other TGF-β superfamily ligand signaling. We would argue that BMP signaling, eosinophilic inflammation, and activation of collagen synthesis and ECM deposition via TGF-β signaling are distinct, although related, aspects of the airway remodeling process in asthma. From our data here we can formulate the hypothesis that, in patients with mild asymptomatic asthma, there is a relative deficiency of BMP receptors, which may render the airway susceptible to activation of remodeling (through eosinophils and TGF-β) upon allergen challenge through lack of protective, antifibrotic BMP responses. After allergen challenge, there was a BMP-7 response in infiltrating cells with evidence of active BMP signaling and restoration of receptor numbers. We would thus also hypothesize that there is a protective BMP response to allergen challenge in asthma, but this may be delayed because of the relative lack of BMP receptors at baseline. Clearly, intervention studies in animal models will be of importance to address this further.

Scattered inflammatory-like cells also stained positive for pSmad1/5, confirming that BMP ligands have functional consequences on airway inflammatory cells. To our knowledge, there have been no studies published looking at the role of BMP ligands and their signaling pathways in airway inflammatory processes. BMP-2 expression is activated by the proinflammatory cytokines IL-1 and tumor necrosis factor (TNF)-{alpha} (41), and BMP-7 at least can modulate inflammatory processes, such as inhibiting macrophage trafficking and IL-6 expression, and modulating TNF-{alpha}–induced proinflammatory gene expression (17, 18). Further focus is now required to help define the functional consequences of the BMP ligand–mediated signaling on airway inflammatory and remodeling processes in the context of asthma.

Thus far, much of the understanding of BMP signaling and its functional consequences in vivo has come from work in developmental biology and animal models of disease. For example, in a transgenic mouse model, it has been shown that lung BMP-4 overexpression leads to inhibition of epithelial cell proliferation and a reduction of type II epithelial cell progenitor cells required to repopulate injured airway epithelium. Similarly, the overexpression of either dominant negative ALK-6 or Noggin (a physiologic inhibitor of BMP signaling) in the lung was associated with inhibition of epithelial cell proliferation (42). By inference, BMP signaling is required for epithelial restoration. Impaired epithelial proliferation is an important feature of the remodeled asthmatic airway. Such "injured" epithelium is implicated as one of the factors that contribute to the "impaired wound" scenario of the asthma phenotype, which is believed to sustain aberrant epithelial–mesenchymal signaling in the airway that may drive the airway remodeling process (43). In this study, there was minimal expression of ALK-6 in the asthmatic epithelium at baseline, and therefore absent ALK-6–mediated signaling may contribute to the abnormally remodeled phenotype in asthma. Rapid and sustained ALK-6 expression after allergen-induced airway damage may then be in response to the process of airway repair. This could also explain the modulation of ALK-2 expression postallergen. Thus, after allergen challenge, BMP signaling may occur via ALK-2 and ALK-6 in addition to ALK-3, although it is of note that the expression of BMPRII levels in patients with asthma did not reach the level of expression seen in normal volunteers at any stage. The functional consequences of use of different type I receptors remain to be defined.

The only ovalbumin-induced murine model of airway inflammation investigating BMP ligand and signaling pathway expression supports a role for BMP signaling in allergen-induced airway injury (20). Here there was also rapid induction of BMP signaling as evidenced by increased pSmad1/5 expression together with induction of ALK-2 and ALK-6 mRNA and protein. Neither ALK-3 mRNA nor BMPRII mRNA and protein expression were modulated in response to allergen challenge. In contrast to our findings, there was markedly decreased BMP-7 mRNA and protein expression in this animal model of acute allergen-induced airway injury. It will be of interest to study BMP regulation in chronic airway challenge models. What this murine model also suggests is that it is the type I receptor that is modulated in response to airway injury and which is therefore the important site of BMP signaling regulation. Thus, manipulation of these receptors may identify novel disease-modifying strategies for the future.

In summary, this study confirms that TGF-β signaling pathways other than that of TGF-β1 are operative in the human airway and that the TGF-β signaling response to airway injury is a complex and coordinated response. The asthmatic airway demonstrated marked differences in the expression of BMP ligand signaling pathway components, suggesting that dysregulated BMP signaling may contribute to the asthma phenotype. Allergen challenge was associated with rapid induction and functional activation of potential antiinflammatory and antifibrotic BMP signaling pathways. Further understanding of the functional significance of these findings will be important.


   Acknowledgments
 
The authors thank Prof. Paschalis Sideras, Athens Biomedical Institute, for the gift of TGF-β family signalling antibodies used in this study, and Michael Roughton, NHLI Division, Imperial College London, for statistical advice.


   FOOTNOTES
 
Supported by the Imperial College Trust Fund.

Originally Published in Press as DOI: 10.1164/rccm.200709-1376OC on February 21, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form September 17, 2007; accepted in final form February 14, 2008


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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