Published ahead of print on September 28, 2006, doi:10.1165/rcmb.2006-0270OC
© 2007 American Thoracic Society DOI: 10.1165/rcmb.2006-0270OC Trefoil Factor Family 3 Peptide Promotes Human Airway Epithelial Ciliated Cell DifferentiationINSERM U514, Université Reims Champagne Ardenne, and CHU Reims, Hôpital Maison Blanche, Reims; INSERM U560, Lille, France; Otto-von-Guericke-Universität, Institut für Molekularbiologie und Medizinische Chemie, Magdeburg, Germany; and Washington University School of Medicine, St. Louis, Missouri Correspondence and requests for reprints should be addressed to Edith Puchelle, INSERM U514, 45 rue Cognacq Jay, F-51092 Reims Cedex, France. E-mail: edith.puchelle{at}univ-reims.fr
Human airway surface epithelium is frequently damaged by inhaled factors (viruses, bacteria, xenobiotic substances) as well as by inflammatory mediators that contribute to the shedding of surface epithelial cells. To regain its protective function, the epithelium must rapidly repair and redifferentiate. The Trefoil Factor Family (TFF) peptides are secretory products of many mucous cells. TFF3, the major TFF in the airways, is able to enhance airway epithelial cell migration, but the role of this protein in differentiation has not been defined. To identify the specific role of TFF3 in the differentiation of the human airway surface epithelium, we analyzed the temporal expression pattern of TFF3, MUC5AC, and MUC5B mucins (goblet cells) and ciliated cell markers  -tubulin (cilia) and FOXJ1 (ciliogenesis) during human airway epithelial regeneration using in vivo humanized airway xenograft and in vitro air–liquid interface (ALI) culture models. We observed that TFF3, MUC5AC, MUC5B, and ciliated cell markers were expressed in well-differentiated airway epithelium. The addition of exogenous recombinant human TFF3 to epithelial cell cultures before the initiation of differentiation resulted in no change in MUC5AC or cytokeratin 13 (CK13, basal cell marker)–positive cells, but induced an increase in the number of FOXJ1-positive cells and in the number of -tubulin-positive ciliated cells (P < 0.05). Furthermore, this effect on ciliated cell differentiation could be reversed by specific epidermal growth factor (EGF) receptor (EGF-R) inhibition. These results indicate that TFF3 is able to induce ciliogenesis and to promote airway epithelial ciliated cell differentiation, in part through an EGF-R–dependent pathway.
Key Words: airway epithelium • ciliated cell differentiation • TFF3 • mucins • FOXJ1
Surface airway epithelium is repeatedly damaged by inhaled noxious agents, such as viruses, bacteria, or xenobiotic substances, as well as by the associated inflammatory response. This results in epithelial remodeling and desquamation, leading to denuding of the basal lamina and loss of the epithelial barrier function, which has to be restored rapidly.
In mammals, the Trefoil Factor Family (TFF) consists of three low–molecular weight peptides (TFF1: Although a role for mucins (8) or epidermal growth factor (EGF) receptors (EGF-R) (9, 10) have been proposed in TFF signal transduction, there are no molecular data published that unambiguously describe TFF receptors. Mucins (MUCs) are very high–molecular weight glycoproteins that can be classified in two main categories: membrane-bound mucins (mainly MUC1 and MUC4) and secreted, mucus-forming mucins (mainly MUC2, MUC5AC, and MUC5B in the respiratory tract) (review in Ref. 11). Secretory MUCs are responsible for the mucus rheological properties that facilitate the elimination of pathogens through mucociliary clearance (12), but they may also act as reservoir or as co-receptors as their heavy glycosylation, von Willebrand–type domains and receptor-like domains provide docking sites for lectins (13) or growth factors (14, 15). The aim of this study was to analyze the temporal expression and secretion patterns of TTF3 relative to MUC5AC and MUC5B during airway epithelial regeneration and to determine the specific role of TFF3 in epithelial differentiation. To reproduce human airway epithelial regeneration and differentiation, we used two different models: an in vivo open humanized airway xenograft model and an in vitro air–liquid interface (ALI) primary human epithelial cell culture model, both mimicking the in vivo dynamics of epithelial regeneration and allowing complete epithelial differentiation. In these models, as previously demonstrated in animal models in vivo (16), epithelial cells migrate to cover the denuded area, then they proliferate and form a pluristratified (multilayered undifferentiated) epithelium, and finally re-differentiate into a columnar, pseudostratified mucociliary epithelium. We show in the present study that, in these models, TFF3 and secretory MUCs are increasingly expressed and secreted by airway surface epithelial mucous cells, in which they are partially co-localized. We also report that the addition of recombinant human TFF3 in the basal culture medium promotes ciliated cell differentiation and ciliogenesis in the ALI cultures, as assessed by the appearance of ultrastructural markers of ciliogenesis associated to an increase in the number of cells expressing the ciliogenesis-related transcription factor FOXJ1 (17) in their nuclei. Furthermore, we demonstrate that the specific EGF-R inhibitor 4-(3-Chloroanilino)-6,7-dimethoxyquinazoline (tyrphostin AG-1478) could reverse TFF3-dependent induction of ciliated cell differentiation. Taken together, these observations support the hypothesis that TFF3 is capable of promoting ciliated cell differentiation in human airway epithelial cells through EGF-R and FOXJ1 signaling pathways.
Human Surface Airway Epithelial Cell Isolation Human surface airway epithelial cells were dissociated from bronchi or nasal polyps as previously described (18). Briefly, surgical pieces (obtained according to the L1245–2 article of Bioethical Law 94–654, new Public Health Code) were carefully rinsed with HEPES-buffered RPMI 1640 culture medium (Gibco BRL, Paisley, UK) supplemented with penicillin (200 UI/ml) and streptomycin (200 µg/ml). Airway tissues were incubated with 0.1% type XIV collagenase (Pronase E; Sigma-Aldrich, St. Louis, MO) in RPMI 1640–HEPES overnight at 4°C. Epithelial cells were dissociated by agitation, numbered, and either used in the in vitro ALI culture model or amplified on plastic culture dishes in proliferation medium consisting of Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12, 3/1 (Gibco BRL) supplemented with 0.87 µM bovine insulin, 65 nM human transferrin, 1.6 nM rhEGF, 1.38 µM hydrocortisone, 30 nM retinyl acetate, 9.7 µM 3,3',5-triiodo-L-thyronine, 2.7 µM epinephrine, 35 µg/ml bovine pituitary extract (BPE), 5 µM ethanolamine, 5 µM O-phosphorylethanolamine, 30 nM sodium selenite, 1 nM manganese chloride tetrahydrate, 5 nM ammonium vanadate, 1 nM nickel sulfate hexahydrate, and 0.5 nM stannous chloride dihydrate (Sigma Aldrich), then seeded in the in vivo xenograft model described below.
Humanized Airway Xenografts
ALI Culture To investigate the effects of exogenous TFF3 addition in the ALI culture model, recombinant TFF3 (2 µM) (kindly provided by D. K. Podolsky, Massachusetts General Hospital, Boston, MA) was added to the medium of ALI cultures from three different patients and to EGF-free medium of ALI cultures from four different patients, at ALI Day 0. Membranes were processed for either immunohistologic analysis and RNA extraction or for transmission electron microscopy (TEM) examination. To study the role of EGF-R in the induction of ciliated cell differentiation by TFF3, we used a specific EGF-R inhibitor, 4-(3-Chloroanilino)-6,7-dimethoxyquinazoline (tyrphostin AG1478, 100 nM; Calbiochem, Merck, Darmstadt, Germany): in ALI cultures from three different patients, cells were cultured in EGF-free medium alone (control), in EGF-free medium with AG1478, in EGF-free medium with TFF3 (2 µM), or in EGF-free medium with both TFF3 and AG1478. Epithelial differentiation was evaluated at Days 12 and 15, corresponding to intermediate steps of differentiation between step 2 (Day 10, undifferentiated pluristratified epithelium) and step 3 (Day 30, mature pseudostratified differentiated epithelium).
RNA Extraction and RT-PCR Analysis The following TFF3 primers were used: forward, 5'-GCTGCTGCTTTGACTCCAGGAT-3'; reverse, 5'-CGTTAAGACATCAGGCTCCAGAT-3' (expected amplicon size: 230 bp). RT was performed at 70°C (15 min), followed by cDNA denaturation for 2 min at 95°C and amplified by 35 PCR cycles using the protocol as follows: 90°C, 20 s; 65°C, 30 s; 72°C, 30 s. A final elongation step (72°C, 2 min) was performed before PAGE analysis. Resulting bands were quantified by fluorescence scanning (LAS-1000; Fujifilm, Tokyo, Japan) and normalized to GAPDH (forward and reverse primers: 5'-CCAGGGCTGCTTTTAACTCTGGTA-3' and 5'-GAGGGATCTCGCTCCTGGAAGAT-3') for xenografts and 28S RNA (forward and reverse primers: 5'-GTTCACCCACTAATAGGGAACGTGA-3' and 5'-GGATTCTGACTTAGAGGCGTTCAGT-3') for ALI cultures. For MUCs transcription analysis in the ALI culture model, 0.5 µg of total RNA was used to prepare cDNA using oligod(T) (1 µl) and recombinant reverse transcriptase M-MLV (1 µl) from Promega (Charbonnières, France). PCR was performed using Taq Polymerase (Roche) on 5 µl of cDNA using specific pairs of primers for MUC5AC and MUC5B genes (described in Ref. 19). PCR reactions were performed in 50-µl final solutions as previously described (20). The ribosomal RNA 28S subunit was used as the internal control. PCR products (10 µl) were separated on a 1.5% agarose gel containing ethidium bromide run in 1x Tris-borate-ethylenediaminetetraacetic acid buffer. The mucin gene/28S ratio was calculated after scanning DNA bands with GelAnalyst-GelSmart software (Claravision, Orsay, France). Stomach mRNA was used as a positive control. For MUCs transcription analysis in the xenograft model, the same protocol was used except that the RT was performed using the Clontech advantage RT-for-PCR kit with 50 ng of total RNA and oligod(T). The following program was used: 94°C for 4 min, then 35 cycles (MUC5B) or 40 cycles (MUC5AC): 94°C 30 s, 58°C 30 s, 72°C 1 min, followed by a a final elongation step (72°C for 7 min), and the results were normalized to GAPDH.
Western Blotting Analysis Methods used for TFF3 Western blot analysis under reducing conditions were essentially as described previously (22). In brief, centrifuged culture supernatants were analyzed on 15% SDS-polyacrylamide gels (45 µl supernatant per lane). After electrophoretic transfer to nitrocellulose membranes and fixation with 0.2% glutaraldehyde, the membrane was treated with the affinity-purified polyclonal rabbit antiserum (anti–hTFF3-2) directed against the peptide FKPLQEAECTF representing the C-terminus of human TFF3 (6) or with the monoclonal antiserum anti–TFF3-15C6 raised against the N-terminus of mature human TFF3 (nanoTools Antikörpertechnik, Teningen, Germany). As secondary antibody, a peroxidase-conjugated anti-rabbit antibody (1:2,000 dilution; Vector Laboratories Inc., Burlingame, CA) or a peroxidase-conjugated anti-mouse antibody (Vector Laboratories) were used, respectively. Detection was with the help of the enhanced chemiluminescence (ECL) system and Kodak BioMax Light Film (Kodak Industrie, Chalon-sur-Saône, France). For all Western blotting analyses BEBM/DMEM medium alone and nasal polyp protein extracts were used as negative and positive controls, respectively.
Immunohistochemistry Immunohistology for TFF3, MUC5AC, and MUC5B was performed as follows: paraffin-embedded and frozen sections were incubated with 3% BSA in PBS (0.1 M, pH 7.2; Sigma-Aldrich) to prevent unspecific binding, then primary antibody was added for 1 h at RT, slides were rinsed twice in PBS and once in PBS + 1% BSA, and detection was performed using Alexa-conjugated secondary antibodies (Interchim, Eugene, OR). Slides were counterstained using Harris hematoxylin and coverslip mounted using Biomeda GEL/MOUNT medium (Biomeda, Foster City, CA). MUC/TFF co-immunostaining was performed by adding both primary antibodies (mouse and rabbit antibodies) then both secondary antibodies (Alexa 488–conjugated anti-mouse and Alexa 594–conjugated anti-rabbit antibodies), and were then analyzed by confocal microscopy.
FOXJ1/
The following antibodies (at the indicated dilution) were used: anti–
TEM
Ciliary Beat Frequency
Statistical Analysis
TFF3 and MUCs Localization during Regeneration Regeneration of a functional, fully differentiated human airway epithelium in the xenograft model has already been described (18). A similar regeneration profile was observed in the ALI culture model. At step 1, the cells flattened and migrated to cover the whole denuded rat trachea or microporous membrane in xenograft or ALI culture, respectively (Figures 1A and 1G). At step 2 the cells proliferated, as identified by nuclear KI-67 positive immunostaining (data not shown), and formed a pluristratified epithelium (Figures 1B and 1H). Finally, at step 3, they differentiated into a normal pseudostratified epithelium characterized by basal, ciliated, and goblet cells (Figures 1C and 1I). MUC and TFF3 expression was characterized at these three regeneration steps using immunohistochemistry. At steps 1 and 2, no TFF3, MUC5AC, or MUC5B staining was observed (data not shown). At step 3, all goblet cells (as identified by Alcian blue-periodic acid-Schiff staining, not shown) expressed MUC5AC antibody in both models (Figures 1D and 1J). MUC5B was detectable in a subset of goblet cells (Figures 1E and 1K). TFF3 expression was identified in a subset of goblet cells (Figures 1F and 1L).
MUCs and TFF3 Are Partially Colocalized To define the cell types that expressed TFF3, we examined ALI cultures and xenografts for TFF3 and MUCs, and we colocalized MUC5AC/TFF3 and MUC5B/TFF3 by immunostaining analyzed using confocal microscopy. MUC5AC expression was present in all observed secretory granules of all goblet cells, whereas TFF3 antibody stained a subcategory of MUC5AC-positive cells (data not shown). There was partial overlap between cells that expressed TFF3 and MUC5B, suggesting that some cells expressed both markers, whereas some other expressed only one. Moreover, in cells expressing both markers, some granules expressed only a single marker, whereas some others expressed both TFF3 and MUC5B (Figure 2).
MUCs and TFF3 Expression and Secretion Increase with Differentiation Secretory MUCs and TFF3 gene expression during regeneration was assessed by RT-PCR analysis. In the xenograft model, TFF3 expression was low at steps 1 and 2, and a significant (P < 0.005) increase was observed at step 3 (67.9 [45.0; 92.7]) as compared with step 1 (0.5 [0.1; 1.8]) (Figure 3A). Similarly, MUC5AC and MUC5B expression significantly increased at the differentiated step (step 3, as compared with step 1: 40.7 [38.1; 45.9] versus 4.9 [3.8; 12.4], P < 0.05 and 91.0 [89.9; 95.5] versus 13.6 [13.1; 21.4], P < 0.005, respectively) (Figure 3B). In the ALI culture model, TFF3 expression was low at step 1 and markedly increased by step 3. This increase was statistically significant at step 3 as compared with step 1 (42.1 [16.7; 56.9] versus 6.1 [4.8; 16.7], P < 0.05) (Figure 3C). MUC5B transcription followed the same profile as TFF3, whereas the MUC5AC mRNAs were barely detectable (Figure 3D).
At the final step 3 of differentiation, epithelia synthesized MUCs and TFF3, as shown in Figure 1. Western blot analysis of the ALI culture supernatants allowed us to investigate products that were secreted in the upper compartment. The undifferentiated epithelium (step 1) did not secrete TFF3, MUC5AC, or MUC5B. In contrast, these proteins could be detected in the secretions obtained from well-differentiated epithelia (step 3) (Figure 4) although in several samples of apical ALI surface liquid the secretion was very low or even absent.
TFF3 Promotes Ciliated Cell Differentiation in ALI Cultures We next sought to determine if TFF3 might have a role in the differentiation of airway epithelial cells. In ALI cultures, ciliated cell differentiation (FOXJ1 and apical -tubulin expression) was detectable as soon as Day 12, and nuclear FOXJ1 was detectable in some cells devoid of apical -tubulin staining (data not shown). However, the number of ciliated cells was relatively low at Day 12, precluding quantification. Accordingly, ALI Day 15, a time at which ciliogenesis was initiated, but not complete, was selected to analyze TFF3 effects on human airway epithelial differentiation. When the cells were cultured in the fully complemented medium, the addition of TFF3 peptide did not significantly modify the number of ciliated cells (+TFF3: 26.4 [15.21; 29.42] versus control: 25.44 [14.81; 31.11] ciliated cells/mm, P = 0.41). We therefore removed the EGF from the medium to avoid interferences.
The addition of TFF3 to EGF-deprived ALI cultures at ALI Day 0 did not change the number of goblet cells or basal cells at Day 15 (1.4 [0.6; 2] versus 1.8 [1.4; 2.9] goblet cells/mm, P = 0.26 and 52.4 [40.0; 68.5] versus 38.9 [31.6; 50.7] basal cells/mm, P = 0.28, respectively). However, the continuous addition of TFF3 resulted in a significant increase (2.8 [2.4; 2.9]-fold, P < 0.05) in the number of
Light microscopy observation before culture fixation showed that the cilia were functional; moreover, the ciliary beating frequency has been used as an index of ciliated cell maturation in culture models. In this regard, the ciliary beating frequency was significantly higher in TFF3-treated than in control cultures (8.9 [7.9; 9.9] Hz versus 7.8 [7.4; 8.6] Hz in control, P < 0.05).
TFF3 Signal Transduction Requires EGF-R Activation
Several studies have reported that TFF peptides are up-regulated at the sites of mucosal injury and participate in mucosal repair in the gastrointestinal tract, mainly related to an increased migration of cells at the wounding edge (2–4). We demonstrate herein, for the first time, that apart from its motogenic activity, TFF3 plays a major role in human airway epithelial regeneration by promoting ciliated cell differentiation. We analyzed the temporal expression pattern of secretory mucins MUC5AC and MUC5B and of TFF3, which are secreted in the apical airway liquid collected from the ALI cultures after complete epithelial differentiation. At this step, the epithelium is a pseudostratified mucociliary epithelium similar to that observed in well-polarized human airway surface epithelial tissues. This result is consistent with a previous study showing that MUC transcription was activated concomitantly to mucous cell differentiation in an airway epithelial cell ALI culture model (24). MUCs and TFF3 localization in mucous cells is in agreement with the results previously reported by Groneberg and coworkers (25), Wiede and colleagues (5), and dos Santos Silva and associates (6). Nevertheless, though mucins and TFF3 may co-localize inside the secretory granules, their association is not an exclusive feature. In ALI culture, MUC5AC transcripts were barely detectable in the first two steps of the regenerative process, whereas the corresponding protein was expressed and secreted at a later stage (step 3). Moreover, in the xenograft model, mucin mRNAs were detectable at a low level at step 2, but the corresponding proteins were not detectable. This suggests that mucins, in particular MUC5AC, are transcribed early during the regeneration process, allowing a pool of mRNAs to be present in the cell and translated at a later stage of mucous differentiation. The fact that MUC5AC mRNA was hardly detectable in the ALI culture model, though MUC5AC protein was expressed and secreted, suggests a very strong positive post-transcriptional regulation. This is supported by the fact that we had to apply 40 PCR cycles to detect this mRNA in the xenograft model, to be compared with the strong MUC5B signal obtained at 35 PCR cycles. Although MUCs and TFF3 co-localized in secretory granules, the pattern of MUC expression was distinct from TFF3, suggesting that these factors were independently regulated. In this ALI model, devoid of inflammatory factors, mucous cells are small and sparse. This may explain the weak MUC5AC expression in ALI cultures as compared with the high MUC5AC mRNA level and the numerous goblet cells in the xenograft model, which could be specifically induced by immune cells (26) or other signals absent in the ALI culture model. MUC5B gene was more highly transcribed, but the corresponding protein was present in fewer cells. This is consistent with differential regulations of MUCs and TFF3 expression, suggesting that these molecules could play important and independent roles in the physiology of human airway surface epithelium.
We also demonstrate here that addition of exogenous TFF3 to ALI cultures at a very early step, when ciliated cells are not present, can favor ciliated cell differentiation, as shown by the increase in the number of Interestingly, TFF3 also increased ciliary beating frequency at ALI Day 15. It is difficult to directly link dynamic functional parameters, such as ciliary beating frequency, with histologic or cytological observations. However, it has been reported that ciliary beating frequency (CBF) increases with differentiation in the ALI culture model (30). The CBF increase that we observed can be interpreted as the marker of a higher differentiation stage in the TFF3-treated cultures, further supporting the role of TFF3 peptide in airway epithelial ciliated cell differentiation. Taken together, these results suggest that TFF3 signaling plays an early role in the ciliogenesis process, upstream to axoneme assembly. TFF3 appears to specifically enhance ciliated cell differentiation, as the number of goblet or basal cells did not significantly change. Nevertheless, we observed a tendency toward a higher number of goblet cells. The lack of statistical significance may be explained by the overall low number of goblet cells in the ALI culture model. This model system is devoid of immune cells, normally present in the mesenchyme, and may therefore lack additional signals necessary for mucous differentiation of the airway surface epithelium. This view is supported by the higher number of goblet cells observed in the xenograft model as well as by reports showing that inflammatory factors such as chemokines and cytokines are responsible for mucous metaplasia (review in Ref. 11). TFF3 peptide did not increase the number of ciliated cells when added in the fully complemented ALI medium. This suggests that TFF3 and EGF may share common signaling pathways, as TFF3 effects are dependent on EGF-R activation. Indeed, we also demonstrated that TFF3 effects could be reversed by tyrphostin AG1478 addition. This molecule is a potent HER1 inhibitor (IC50 = 3 nM) but does not affect other receptors at low concentration (PDGF-R and HER2 IC50 > 100 µM). At the concentration used (100 nM), the addition of the EGF-R inhibitor did not induce any change in EGF-free culture differentiation. Liu and coworkers demonstrated that rat TFF3 can enhance EGF-R phosphorylation in the colonic cell line HT29 (9); Rivat and colleagues showed that STAT3 activation is necessary to TFF3 pro-invasive effect on colonic cells, but the upstream pathway was not elucidated (31). Our results strongly suggest that TFF3 signal transduction and pro-differentiation effect in ALI-cultured primary airway epithelial cells require EGF-R (HER1) activation. This is also supported by the fact that we had to remove EGF from the medium to unravel the effects of TFF3 addition. Furthermore, a recent case report from Vermeer and coworkers (32) showed that EGF-R family receptor ErbB2 blockade could impair airway epithelial differentiation, further emphasizing the importance of this family in the differentiation of the respiratory epithelium. It is not clear yet whether TFF3 can bind to EGF-R. TFF3 effects could depend on endogenous synthesis of an EGF-R ligand, or a co-receptor may be needed. As TFF3 is present in a wide variety of tissues, specific co-receptors could modulate its activity and allow tissue-specific effects. TFFs can bind to MUCs (33), with which they are specifically co-expressed in various organs (review in Ref. 34), thus mucins could play this regulatory role. Modulation of TFF signals by specific association with mucins would allow a fine-tuned regulation of local cell signaling by allowing local variations in TFFs/MUCs association, and thus local variations in signal transduction. We show here that TFF3 is not endogenously produced by undifferentiated regenerating cells, but is secreted by well-differentiated mucous cells. Thus, by adding TFF3 at the beginning of the regeneration, we presumably mimic the role of the secretory cells surrounding a lesion in vivo. We propose that, in addition to its role in mucus rheology, it could act both as a lesion sensor and a regeneration enhancer. When the airway epithelium is damaged, TFF3 present in the mucus layer could conceivably bind to EGF-R present at the membrane of injured cells and promote cell migration, as well as differentiation, leading to the faster restoration of a fully functional airway epithelium. TFF3 would thus be a key peptide in the in vivo airway epithelial regeneration. A role for TFF3 as a differentiation factor would be in line with a previous report on the altered differentiation of gastric cells in TFF1-deficient mice (35). Cross-talk with the ciliogenesis signaling pathway is likely to occur early, at or before the axoneme assembly step. Elucidating the mechanisms involved in TFF3 expression may provide insights into the epithelial diseases related to abnormal repair and regeneration after injury. Whether defects in TFF3 expression and release at the airway epithelial level could lead to impaired restoration of functional epithelium and may occur in chronic airway diseases is unknown, but the frequent extensive remodeling observed in pathologies such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, is likely to impair TFF3 secretion, and this mechanism could participate in the pathologic process. Pro-regenerative molecules such as TFF3 may represent novel therapeutic approaches in chronic and acute airway diseases involving epithelial injury and repair.
The authors thank Dr. C. Martinella-Catusse (INSERM UMRS 514, Reims, France), who started this project during her post-doctoral stage; Dr. J.-M. Zahm (INSERM UMRS 514, Reims, France) for CBF measurements and assistance with statistical analysis; Dr. H. Kaplan (IFR 53, Reims, France) for the confocal microscopy analysis; Prof. P. Birembaut (INSERM UMRS 514/Laboratoire Pol Bouin, Department of Histology and Cytology, Maison Blanche Hospital, Reims) for his support to the histologic techniques; and M.-P. Ducourouble (INSERM U 560, Lille, France) for her excellent technical help.
This work was supported by a PhD grant from INSERM/Région Champagne-Ardenne, by Adult Stem Cell Concerted Action INSERM/Ministry of Research, by the French association Vaincre la Mucoviscidose and by Cancéropôle Grand Est. Originally Published in Press as DOI: 10.1165/rcmb.2006-0270OC on September 28, 2006 Conflict of Interest Statement: P.L does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. I.v.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.-P.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.-C.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. W.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.L.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.P. has received research grants to support work carried out in her laboratory from GSK (UK) and by ARDS/Soliance (France). Received in original form July 27, 2006 Accepted in final form September 19, 2006
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Abstract
CLINICAL RELEVANCE
6.6 kD; TFF2: 
P < 0.05 as compared with TFF3 treatment.