Rationale: Bronchial remodeling, including increased bronchial smooth muscle (BSM) mass, contributes to bronchial obstruction in asthma. However, its mechanisms are complex and remain controversial. Recently, a role of the chitinase 3-like 1 protein (YKL-40) has been evoked in asthma. Indeed, YKL-40 concentration was increased in asthmatic serum, and correlated with asthma severity and subepithelial membrane thickness. Nevertheless, the role of YKL-40 on BSM cells remains to be investigated.
Objectives: To evaluate whether YKL-40 altered the physiologic properties of BSM cells in asthma in vitro and ex vivo.
Methods: We enrolled 40 subjects with asthma, 13 nonsmokers, and 16 smokers. BSM cells were derived from bronchial specimens obtained by either fiberoptic bronchoscopy or lobectomy. We assessed cell proliferation using BrdU, flow cytometry, and cell count; signaling intermediates using Western blot; cell migration using inserts, wound healing, and phalloidin staining; and cell synthesis using ELISA and Western blot. The involvement of protease activated receptor (PAR)-2 was evaluated using blocking antibody and dedicated lentiviral small hairpin RNA. We also determined the BSM area and the YKL-40 staining ex vivo using immunohistochemistry on biopsies from subjects with asthma and control subjects.
Measurements and Main Results: We demonstrated that YKL-40 increased BSM cell proliferation and migration through PAR-2–, AKT-, ERK-, and p38-dependent mechanisms. The increased cell migration was higher in BSM cells of subjects with asthma than that of control subjects. Furthermore, YKL-40 epithelial expression was positively correlated with BSM mass in asthma.
Conclusions: This study indicates that YKL-40 promotes BSM cell proliferation and migration through a PAR-2–dependent mechanism.
YKL-40 is expressed by the asthmatic epithelium and its serum concentration is increased and correlated with subepithelial membrane thickness in patients with asthma. However, the role of YKL-40 in bronchial smooth muscle remodeling has not been explored.
In bronchial smooth muscle cells derived from patients with asthma, we found that YKL-40 increases bronchial smooth muscle cell proliferation and migration through protease activated receptor-2–, AKT-, ERK-, and p38-dependent pathways. Moreover, its epithelial expression is positively correlated with bronchial smooth muscle mass in patients with asthma.
Asthma is a disease seen frequently that is characterized by bronchial inflammation and remodeling (1). Bronchial remodeling corresponds to an increased thickening of the bronchial wall caused by various structural alterations including abnormal epithelium, subepithelial membrane thickening, alteration in extracellular matrix deposition, mucus gland hypertrophy, and increased mass of bronchial smooth muscle (BSM) (2). This latter feature has a crucial prognostic role in asthma because it correlates with the decrease in lung function (3). However, the mechanisms of increased BSM mass are complex and remain controversial (2). For instance, increased proliferation of BSM cells (4, 5), increased BSM cell size (6, 7), and migration of BSM cells (8) have been proposed as potential mechanisms of BSM remodeling in asthma.
The role of the chitinase-like protein YKL-40 in bronchial remodeling has been recently evoked because its concentration in the serum of patients with asthma is increased and correlated with subepithelial membrane thickness (9). YKL-40 is secreted by epithelial cells and macrophages particularly in people with severe asthma (9). In addition, a single nucleotide polymorphism of CHI3L1, the gene encoding YKL-40 protein, also called human cartilage glycoprotein (HCgp-39) or chitinase 3-like 1, was associated with elevated serum YKL-40 levels and asthma (10). These findings could argue for either a causative or a sentinel role of YKL-40 in asthma. However, there are some lines of evidence suggesting a causative role of YKL-40. CHI3L1 gene was also associated with bronchial hyperresponsiveness and reduced lung function in asthma (10), two characteristics that have been related to BSM remodeling (3, 11). However, YKL-40 has been shown to induce the proliferation of mesenchymal cells, such as human chondrocytes, synovial cells, skin or fetal lung fibroblasts (12), and the migration and adhesion of vascular smooth muscle cells (13). More recently, YKL-40 has been implicated in the initiation and effector phases of Th2 inflammation using BRP-39 (i.e., the murine homologue of YKL-40) knockout mice and allergen sensitization (14). Nevertheless, with respect to asthma pathophysiology, the effect of YKL-40 on BSM remains largely unknown. The aim of the present study was to determine whether YKL-40 alters the physiologic properties of BSM cells in asthma. For this purpose, we have investigated the effects of YKL-40 on cell proliferation, apoptosis, migration, and synthesis using primary cultured BSM cells from patients with asthma and control subjects. We have also correlated YKL-40 epithelial expression with BSM mass as a surrogate of bronchial remodeling in asthma. Some of the results of this study have been previously reported in the form of abstracts (15, 16).
A detailed description of these methods can be found in the online supplement.
A total of 40 patients with mild to severe persistent asthma, 13 nonsmokers, and 16 smokers were prospectively recruited from the Centre Hospitalier Universitaire of Bordeaux, France, according to the Global Initiative for Asthma (17). All subjects gave their written informed consent to participate to the study, after the nature of the procedure had been fully explained. The study received approval from the local ethics committee. Bronchial specimens from all subjects were obtained by either fiberoptic bronchoscopy or lobectomy, as previously described (5).
Human BSM cells were derived from bronchial specimens as described previously (18, 19). All experiments were performed on phenotypically confirmed BSM cells between passages 2 and 5 (5). BSM cells were serum deprived for 24 hours and stimulated with YKL-40 (MedImmune, Gaithersburg, MD) in a time- or concentration-dependent manner, with protease activated receptor type 2 (PAR-2) specific agonist (i.e., SLIGKV-NH2) or with a PAR-2–independent agonist (i.e., platelet-derived growth factor [PDGF]). The involvement of PAR-2 was evaluated using blocking anti–PAR-2 antibody or dedicated lentivirus producing shRNA directed against PAR-2. Downstream transduction mechanisms were analyzed using Western blot designed to measure phosphorylated AKT, ERK, or p38.
Cell proliferation was evaluated using BrdU incorporation and cell counting as described previously (5). Cell cycle was assessed by flow cytometry (20). Briefly, cells were stained with Ki67 and propidium iodide. G0 or G1 phase cells were then gated and cell size was determined using forward scatter measurement on FACSCanto (BD Biosciences, Pont de Claix, France). Cell apoptosis was assessed by flow cytometry using FITC-Annexin V kit (BD Biosciences).
We assessed cell migration of BSM cells against YKL-40 using modified Boyden chamber method and wound healing assay, as described previously (21–23). Actin reorganization was assessed using phalloidin (24).
Immunoblotting was performed on cell protein extracts (25) using primary antibodies (Abs) directed against α-smooth muscle actin (SMA) (Sigma-Aldrich, Saint Quentin-Fallavier, France), collagen IA (Santa Cruz Biotechnology, Santa Cruz, CA), or β-actin (Sigma-Aldrich). Levels of immunoreactive transforming growth factor-β1, inducible protein-10, IL-8, or stem cell factor were assayed in the cell supernatant by ELISA according to the manufacturer's instructions (R&D Systems, Abingdon Oxon, UK).
Specimens were embedded in paraffin and processed for immunohistochemistry as previously described (5). Primary Abs included mouse anti–α-SMA (Sigma-Aldrich), rat anti–YKL-40 (MedImmune), or appropriate unrelated Ab. The normalized epithelial staining intensity of anti–YKL-40 and the area of BSM (positive for anti–α-SMA Ab) were automatically assessed by Quancoul software at a magnification of ×400 and ×100, respectively (24, 26).
YKL-40 serum concentration was measured in patients with asthma using ELISA on the Meso Scale Discovery Platform (Gaithersburg, MD).
The statistical analysis was performed with NCSS software (NCSS 2001; NCSS Statistical Software, Kaysville, UT). Values are presented as the mean ± the SEM. Statistical significance was analyzed by paired t tests, Wilcoxon tests, Kruskall-Wallis and z tests, chi-square, analysis of variance, and Bonferroni tests. Spearman coefficient was used to evaluate the correlation between YKL-40 normalized epithelial staining intensity and BSM area. A P value less than 0.05 was considered statistically significant.
The clinical characteristics of all subjects are shown in Table 1. The entire asthmatic population (n = 40) includes 19 subjects with nonsevere asthma and 21 subjects with severe persistent asthma. They received stable treatments, including oral or inhaled corticosteroids and β2 agonists. None of the patients with asthma experienced a recent (<3 mo) exacerbation of the disease. The mean duration of the disease in patients with asthma was 32 ± 2.9 years. The control population includes 13 nonsmokers and 16 smokers who received no treatment. Compared with subjects with asthma, nonsmokers were not different in terms of age, sex, body mass index, and smoking pack-years, whereas smokers were not different in terms of age, body mass index, FEV1, FEV1/FVC ratio, and forced expiratory flow midexpiratory phase.
|Characteristics||Nonsmokers||Smokers||Subjects with Asthma||P Value|
|No. of patients||13||16||40|
|Age, yr||50 ± 6||55 ± 2.8||47 ± 2.6||0.2|
|Body mass index, kg/m2||27 ± 1.3||24.5 ± 1||26.9 ± 1||0.4|
|Pack-years||0 ± 0*||35 ± 4.8†||0.98 ± 0.5*||<0.001|
|Nonsmoker, no. of patients||13||0||35|
|Current, no. of patients||0||11||0|
|Former, no. of patients||0||5||5|
|Years since quitting||0 ± 0||8.2 ± 2.6||9.8 ± 6.9|
|LABA, no. of patients||0||0||29|
|ICS, no. of patients||0||0||34|
|OCS, no. of patients||0||0||7|
|Liters||3 ± 0.2||2.6 ± 0.2||2.3 ± 0.2||0.05|
|Percentage of predicted value||100 ± 5.3||85.1 ± 3.7||80.7 ± 3.2†||0.01|
|FEV1/FVC ratio, % of FVC||80.8 ± 1.5||73.2 ± 2||70.5 ± 2†||0.02|
|Liters per second||3.3 ± 0.2||2.5 ± 0.3||2 ± 0.2†||0.002|
|Percentage of predicted value||94.5 ± 7.4||58.6 ± 6†||50.8 ± 4.2†||<0.001|
YKL-40 increased the number of BSM cells obtained from nonsmoking individuals in a time-dependent manner (Figure 1A). This effect was not related to a decrease in cell apoptosis (see Figure E1 in the online supplement), but to an increased DNA synthesis (Figure 1B). DNA synthesis started at 24 hours and preceded the increased cell number, which occurred later at 48 hours. YKL-40–induced BSM cell proliferation also occurred in a concentration-dependent manner with a maximal effect for the concentration of 300 ng/ml (Figure 1C). Such an effect was similar to that induced by an optimal concentration of PDGF (Figure 1C). YKL-40 also increased DNA synthesis in asthmatic BSM cells (Figure 1D) and this effect was quantitatively similar to that in the BSM cells of nonsmokers and smokers.
Regarding the transduction mechanism, YKL-40–induced BSM cell proliferation first involved the PAR-2. Indeed, a blocking anti–PAR-2 antibody significantly decreased BSM cell proliferation induced by YKL-40 and SLIGKV-NH2 but not that induced by PDGF (Figure 2A). In another set of experiments, we observed that this anti–PAR-2 antibody-mediated inhibition of YKL-40–dependent BSM cell proliferation was similar within the three populations of subjects (Table 2). Moreover, to further confirm the PAR-2 involvement, we designed dedicated lentivirus able to produce shRNA directed against PAR-2. BSM cells were efficiently transduced by such a lentivirus, as shown by the dramatic increased expression of GFP reporter (Figure 2B) and by a significant decrease in PAR-2 mRNA (Figure 2C). As for the antibody, knocking down PAR-2 using this shRNA lentivirus significantly decreased YKL-40 and SLIGKV-NH2–induced BSM cell proliferation but not that induced by PDGF (Figure 2D). Downstream transduction mechanism of YKL-40–induced BSM cell proliferation involved activation of pertussis toxin–, PI3 kinase-, PKC-, ERK-, and p38 MAP kinase-dependent pathways (Table 2). The effect of a series of relevant inhibitors on YKL-40–induced BSM cell proliferation was concentration dependent (see Figure E2). This transduction mechanism was similar within the three populations of subjects (Table 2). The same inhibitors also abolished SLIGKV-NH2–induced BSM cell proliferation (see Figure E3), confirming that PAR-2 and YKL-40 activate the same transduction pathways. Furthermore, we also analyzed the signaling intermediates in BSM cells. YKL-40 and SLIGKV-NH2 phosphorylated AKT, ERK, and p38 to the same extent as PDGF (Figure 3). As for proliferation, blocking anti–PAR-2 abolished the phosphorylation of AKT, ERK, and p38 induced by YKL-40 and SLIGKV-NH2 (data not shown).
|Nonsmokers||Smokers||Subjects with Asthma|
|Irrelevant Ab||1.87 ± 0.1||1.82 ± 0.1||1.75 ± 0.1|
|Anti–PAR-2 Ab||1.21 ± 0.0*||1.23 ± 0.1*||1.36 ± 0.0*|
|DMEM||1.82 ± 0.1||1.81 ± 0.1||1.81 ± 0.1|
|Pertussis toxin||1.20 ± 0.0†||1.15 ± 0.1†||1.34 ± 0.1†|
|0.1% DMSO||1.89 ± 0.1||1.80 ± 0.1||1.71 ± 0.1|
|LY 294002||0.85 ± 0.1‡||0.90 ± 0.1‡||0.91 ± 0.0‡|
|Calphostin C||0.53 ± 0.1‡||0.44 ± 0.1‡||0.58 ± 0.1‡|
|PD 98059||1.07 ± 0.1‡||1.04 ± 0.0‡||1.05 ± 0.0‡|
|SB 203580||0.92 ± 0.1‡||0.85 ± 0.1‡||0.987 ± 0.1‡|
Because increasing cell size during G1 phase is a key event to continue cell proliferation through the checkpoint at the end of G1 of the cell cycle, we also analyzed the size of BSM cells using flow cytometry (see Figures E4A–E4C). YKL-40 significantly increased the BSM cell size during G1 phase within the three populations of subjects (see Figure E4D). The cell size effect of YKL-40 was not statistically different between the BSM cells of subjects with asthma and nonsmokers and smokers. Moreover, such an effect was not related to cell hypertrophy per se because cell size was not altered during G0 phase (see Figure E4D) and YKL-40 did not alter α-SMA and collagen I contents in BSM cells from the three populations (see Figure E5).
We then assessed cell migration up to 24 hours of YKL-40 incubation. YKL-40 significantly increased the migration of nonsmokers’ BSM cells with a maximal effect occurring at 24 hours (Figure 4A). Such an effect cannot be related with the proliferative effect of YKL-40 because at this time point the cell number was not increased (Figure 1A). Furthermore, using the wound healing assay, YKL-40 induced a significant cell migration after only 6 hours (see Figure E6). The migration of BSM cells induced by YKL-40 was not significantly different between nonsmokers and smokers (Figure 4B; see Figure E6) and was within the same range as that induced by an optimal time and concentration of PDGF (Figure 4B). Interestingly, the effect induced by YKL-40 was significantly larger in BSM cells of subjects with asthma than in nonsmokers and smokers (Figure 4B). In addition, the migration induced by YKL-40 was related to chemotaxis and not chemokinesis in asthmatic BSM cells, whereas an association of chemotaxis and chemokinesis was observed in BSM cells from nonsmokers and smokers (Figure 4C). We then analyzed the effect of YKL-40 on BSM cytoskeleton reorganization and cell shape changes using flow cytometry and confocal microscopy. YKL-40 significantly increased F-actin content in BSM cells from both nonsmokers and smokers, and in asthmatic BSM cells (Figure 4D). YKL-40 also promoted the development of small pseudopods associated with an intense fluorescence (Figures 4E and 4F).
The transduction mechanism of YKL-40–induced BSM cell migration also involved PAR-2. Blocking anti–PAR-2 antibody and the dedicated shRNA lentivirus directed against PAR-2 significantly decreased BSM cell migration induced by either YKL-40 or SLIGKV-NH2 but not that induced by PDGF (Figures 5A and 5B). Regarding the downstream transduction mechanisms, a pertussis toxin–dependent pathway was involved only in asthmatic BSM cells (Table 3). However, a common pathway was also implicated within the three populations of subjects, and included a subsequent activation of PI3 kinase, ERK, and p38 MAP kinase (Table 3). As for proliferation, the inhibitory effect of LY294002, PD98059, and SB203580 on YKL-40–induced BSM cell migration was concentration dependent (see Figure E7). These inhibitors similarly abolished SLIGKV-NH2–induced BSM cell migration (see Figure E8).
|Nonsmokers||Smokers||Subjects with Asthma|
|Irrelevant Ab||1.71 ± 0.1||1.60 ± 0.1||2.30 ± 0.1|
|Anti–PAR-2 Ab||1.11 ± 0.0*||0.95 ± 0.0*||0.95 ± 0.1*|
|DMEM||1.72 ± 0.1||1.80 ± 0.1||2.50 ± 0.0|
|Pertussis toxin||1.40 ± 0.1||1.60 ± 0.4||1.35 ± 0.1†|
|0.1% DMSO||1.72 ± 0.0||1.84 ± 0.2||2.40 ± 0.1|
|LY 294002||1.35 ± 0.1‡||1.00 ± 0.0‡||1.05 ± 0.1‡|
|Calphostin C||1.30 ± 0.3||1.65 ± 0.2||1.50 ± 0.3|
|PD 98059||1.12 ± 0.2‡||0.93 ± 0.1‡||0.90 ± 0.1‡|
|SB 203580||1.03 ± 0.2‡||0.95 ± 0.2‡||1.10 ± 0.1‡|
Furthermore, YKL-40 did not alter the production of various cytokines known to induce cell chemotaxis, such as transforming growth factor-β1, IL-8, inducible protein-10, and stem cell factor in BSM cells from nonsmokers and smokers, and in asthmatic BSM cells (see Figure E9).
We assessed the expression of YKL-40 in bronchial biopsies from subjects with mild to severe asthma using immunohistochemistry. Whereas YKL-40 was not expressed by BSM, it was expressed by asthmatic epithelial cells, although at various intensities (Figures 6A and 6B). In this connection, YKL-40–normalized epithelial expression was higher in subjects with severe asthma (3.84 ± 0.58; n = 18) than in subjects with nonsevere asthma (1.79 ± 0.46; n = 11) and nonsmoking subjects (0.99 ± 0.30; n = 9; P = 0.0003; Kruskall-Wallis analysis of variance and z tests). More interestingly, with respect to bronchial remodeling, YKL-40–normalized epithelial expression was significantly correlated with the BSM mass in subjects with asthma (Spearman r = 0.37; P = 0.04) (Figure 6C). Finally, we measured the concentration of circulating YKL-40 using ELISA. Mean serum YKL-40 concentration was higher in subjects with severe asthma (909 ± 315 ng/ml; n = 9) than with nonsevere asthma (340 ± 45 ng/ml; n = 11; P =0.04; two samples Wilcoxon test). However, serum YKL-40 concentration was correlated with neither YKL-40 normalized epithelial expression nor BSM mass in subjects with asthma.
In this study, we have demonstrated that YKL-40 increases BSM cell proliferation and migration through PAR-2–, AKT-, ERK-, and p38-dependent mechanisms, and that its epithelial expression is positively correlated with BSM mass in asthma.
These results indicate that YKL-40 is more than a simple biomarker in asthma but can be considered as an active player, at least for BSM remodeling in asthma. In the present experiments, the optimal concentration of YKL-40 was 300 ng/ml for whatever effect, such as increasing cell proliferation and migration. Such a concentration is in agreement with what has been measured in the serum of people with asthma (9) including the present study, particularly during exacerbation (27). YKL-40 has been shown to be secreted by bronchial macrophages and epithelium particularly in people with severe asthma (9) and allergen-sensitized mice (14). Whereas we confirm these findings, at least for epithelial cells, we also demonstrate that YKL-40 is not secreted by the BSM ex vivo. Moreover, because the epithelial YKL-40 expression was correlated with the BSM mass, it may be suggested that the BSM layer is a target for YKL-40. Incidentally, the present YKL-40 epithelial staining intensity seems lower than that of the study by Chupp and coworkers (9) even in severe asthma. However, it is noteworthy that we used a rat primary Ab suitable for paraffin-embedded sections, whereas Chupp and coworkers performed their study using a rabbit Ab on frozen sections. Conversely, the present serum YKL-40 concentrations seem higher than those reported in the study by Chupp and coworkers (9). However, we used a different set of antibodies for ELISA.
Regarding BSM cell proliferation, the effect induced by YKL-40 was quantitatively similar to that induced by PDGF, which is one of the most potent proliferating growth factors (28). It is now well accepted that BSM cell proliferation in response to fetal calf serum is increased in asthma compared with controls and patients with chronic obstructive pulmonary disease (4, 5, 29, 30). In our study, the increased proliferation of BSM cells induced by YKL-40 was similar in control subjects and subjects with asthma. Similar findings have been obtained using PDGF (data not shown). We also analyzed the effect of YKL-40 on cell size in G1 phase as another marker of the proliferative effect of YKL-40. We paid special attention to also analyze cell size in nonproliferating G0 cells as described previously (20). YKL-40 increased BSM cell size in G1 phase only within the three populations of subjects.
To date, few studies have evaluated the migration of nonasthmatic BSM cells (2). Here we demonstrate that YKL-40 increases the migration of BSM cells to the same extent as that induced by PDGF, although BSM migration was slower than that of more specialized inflammatory cells. BSM migration was also compared between asthmatic and nonasthmatic cells and revealed that the effect induced by YKL-40 was amplified in asthmatic BSM cells. These findings suggest that YKL-40 is more potent in asthma. In addition, the type of migration seems different in asthmatic and nonasthmatic BSM cells. Indeed, in asthmatic BSM cells, the effect of YKL-40 is likely to be restricted to chemotaxis. Thus, because of such difference, it seems important to specifically use asthmatic BSM cells when evaluating new agonists or testing the efficacy of new drugs (2).
The mechanism by which YKL-40 mediates its biologic effects remains largely unknown. YKL-40 binds chitin but lacks chitinase activity (31). However, mammals including humans do not produce chitin (31). Chitin, a polymer of N-acetylglucosamine, is the second most abundant polysaccharide in nature. It is found in the exoskeleton of crustaceans and insects, the walls of fungi, and the microfilarial sheath of parasitic nematodes (31). A role for YKL-40 has been suggested in inflammation and tissue remodeling particularly in asthma (9), but also in rheumatoid arthritis, osteoarthritis, breast or lung cancer, and hepatic fibrosis (31). In this study, we determined, for the first time, the transduction mechanism induced by YKL-40 in human BSM cell proliferation and migration. We provide evidence that, for both effects, YKL-40 activates PAR-2 by means of two complementary approaches. Both a blocking anti–PAR-2 Ab and a lentiviral shRNA knocking down PAR-2 were able to decrease YKL-40–induced BSM cell proliferation and migration. These two approaches seem PAR-2–specific because they also decreased the effects induced by the synthetic peptide SLIGKV-NH2, which specifically activates PAR-2 in human, whereas they did not alter those induced by PDGF, which activates BSM cell proliferation and migration through a PAR-2–independent mechanism. This lentiviral strategy was developed because the time course of the siRNA directed against PAR-2 strategy we previously set up does not allow evaluating BSM cell proliferation or migration (18, 19). Such a PAR-2 activation has never been demonstrated earlier for YKL-40, which was not supposed to be a protease. However, these results are in agreement with data showing that exogenous chitinases, such as Streptomyces griseus, activate PAR-2 within bronchial epithelial cells (32). The downstream transduction pathways are similar to those previously determined in response to a direct PAR-2 activation of BSM cell proliferation using SLIGKV-NH2 (33). However, pharmacologic inhibitors of PI3 kinase, ERK, and p38 MAP kinase concentration-dependently reduce YKL-40–induced BSM cell proliferation and migration, and YKL-40 induces AKT, ERK, and p38 phosphorylation in these cells. We conclude that YKL-40 may be implicated in bronchial remodeling in asthma by way of an activation of PAR-2.
The authors thank J. M. Vernejoux for performing the fiberoptic bronchoscopy, the staff of Service de Chirurgie Thoracique for the supply of human lung tissue, and Mélanie Willème and David Legros for technical assistance.
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Supported by a grant from the Agence Nationale de la Recherche (No. 2010 CESA 001 01 [2010-0145]) and the Fondation pour la Recherche Médicale. I.B. was funded by the Société de Pneumologie de Langue Française. J.C. was funded by the Fondation pour la Recherche Médicale. R.K., A.J.C., and J.W. were employees of MedImmune.
Author Contributions: Conception and design, I.B., R.K., A.J.C., J.-M.T.d.L., R.M., and P.B.; analysis and interpretation, I.B., A.O., P.-O.G., G.C., J.C., H.B., M.T., O.O., J.W., and P.B.; drafting the manuscript for important intellectual content, I.B., R.K., A.J.C., J.-M.T.d.L., R.M., and P.B.; revising the manuscript for important intellectual content, A.O., P.-O.G., J.C., H.B., and M.T.; and final approval of the manuscript, I.B., A.O., P.-O.G., G.C., J.C., H.B., M.T., O.O., R.K., A.J.C., J.W., J.-M.T.d.L., R.M., and P.B.
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Originally Published in Press as DOI: 10.1164/rccm.201105-0915OC on January 26, 2012