Down-regulation of the Anti-inflammatory Protein Annexin A1 in Cystic Fibrosis Knock-out Mice and Patients*,S
Noura Bensalema,b,
Ana Paula Venturab,c,
Benoît Valléea,
Joanna Lipeckaa,d,
Danielle Tondeliera,
Noélie Davezaca,
Alexandre Dos Santosd,e,
Mauro Perrettif,g,
Anne Fajach,
Isabelle Sermet-Gaudelusa,i,
Michel Renouilj,
Jean-François Lesurek,
Frédéric Halgandc,l,
Olivier Laprévotec and
Aleksander Edelmana,d,m
From the a INSERM U467, d Proteomic Core Facilities, and e INSERM U370, Faculté de médecine Necker, Université Paris-Descartes, 156 rue de Vaugirard, Paris F-75015, France, c Laboratoire de spectrométrie de masse, Institut de Chimie des Substances Naturelles CNRS, Gif-sur-Yvette F-91198, France, f The William Harvey Research Institute, Centre of Biochemical Pharmacology, Barts and The London Queen Marys School of Medicine and Dentistry, London EC1M 6BQ, United Kingdom, h Service dHistologie-Biologie Tumorale, Unité Propre Enseignement Supérieur-Equip dAcceuil 3499, Hôpital Tenon, 4 rue de Chine, Paris F-75020, France, i Service de Pédiatrie Générale, Hôpital Necker-Enfants Malades, 149 rue de Sèvres, Paris F-75015, France, j Centre de Ressources et de Compétence en Mucoviscidose, Groupe Hospitalier Sud Réunion, BP 350, Saint Pierre F-97448, France, and k Centre de Ressources et de Compétence en Mucoviscidose, Hôpital dEnfants, Saint Denis F-97476, France
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ABSTRACT
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Cystic fibrosis is a fatal human genetic disease caused by mutations in the CFTR gene encoding a cAMP-activated chloride channel. It is characterized by abnormal fluid transport across secretory epithelia and chronic inflammation in lung, pancreas, and intestine. Because cystic fibrosis (CF) pathophysiology cannot be explained solely by dysfunction of cystic fibrosis transmembrane conductance regulator (CFTR), we applied a proteomic approach (bidimensional electrophoresis and mass spectrometry) to search for differentially expressed proteins between mice lacking cftr (cftrtm1Unc, cftr/) and controls using colonic crypts from young animals, i.e. prior to the development of intestinal inflammation. By analyzing total proteins separated in the range of pH 611, we detected 24 differentially expressed proteins (>2-fold). In this work, we focused on one of these proteins that was absent in two-dimensional gels from cftr/ mice. This protein spot (molecular mass, 37 kDa; pI 7) was identified by mass spectrometry as annexin A1, an anti-inflammatory protein. Interestingly, annexin A1 was also undetectable in lungs and pancreas of cftr/ mice, tissues known to express CFTR. Absence of this inhibitory mediator of the host inflammatory response was associated with colonic up-regulation of the proinflammatory cytosolic phospholipase A2. More importantly, annexin A1 was down-regulated in nasal epithelial cells from CF patients bearing homozygous nonsense mutations in the CFTR gene (Y122X, 489delC) and differentially expressed in F508del patients. These results suggest that annexin A1 may be a key protein involved in CF pathogenesis especially in relation to the not well defined field of inflammation in CF. We suggest that decreased expression of annexin A1 contributes to the worsening of the CF phenotype.
Cystic fibrosis (CF)1 is a lethal genetic disease characterized by pulmonary, pancreatic, and intestinal disorders and chronic inflammation (1). CF is caused by mutations in the CFTR gene encoding the CF transmembrane conductance regulator protein (CFTR). CFTR is a cAMP-dependent chloride channel but also displays other functions ranging between the regulation of activity of other transporters and the modulation of gene expression (2). For example, CFTR was shown to modulate the activity of Na+ channels (3), to regulate the expression of nitric-oxide synthase (4), and through interaction with bacteria to be involved in the mechanisms of susceptibility to Pseudomonas aeruginosa infections in CF (5). Numerous studies have demonstrated that proteins interacting with CFTR regulate its traffic and/or Cl channel activity (2, 68) and that some of these proteins may be involved in the traffic of mutated CFTR (9).
Clinical observations strongly suggest that CF pathogenesis cannot be explained solely by dysfunction of CFTR. The main observation leading to this statement is the existence of a large spectrum of CF phenotypes in CF patients even among individuals bearing the same mutation (10). It has been postulated that proteins such as multidrug resistance-associated protein-1, a member of the ATP-binding cassette superfamily, may be expressed in CF cells to take over one of the functions of abnormal CFTR (11). It is clear that other factors than the mutated CFTR (modifier genes, environmental factors, and protein complexes) might modulate CF pathogenesis (12). To get some insights in CF pathophysiology we searched for differentially expressed proteins in the well established cftr knock-out mouse model cftrtm1Unc (cftr/ (13)). This CF murine model represents a useful tool for such investigations because the mice spontaneously develop an intestinal obstructive defect due to the absence of CFTR in intestinal crypts leading to abnormal chloride secretion and death of most animals within 5 weeks of age. This behavior resembles the meconium ileus present in 1015% of CF patients (14). When bred under a special diet, these mice survive up to 24 months, developing lung inflammation (15) and increased susceptibility to lung bacterial colonization (16). To identify the initial protein changes before development of any inflammation, we focused the analysis on young animals (34 weeks old). It is also important to note that investigating the properties of cftr/ mice raises the possibility to discover the primary consequences of the absence of CFTR protein.
We chose intestinal colonic crypts, which are the major site of functional defect of CFTR in cftr/ mice (17), to initiate the study aimed to compare the protein expression pattern in tissues that express or do not express CFTR. We applied a proteomic strategy (bidimensional electrophoresis and the identification of proteins of interest by MS) to decipher the differential protein expression in colonic crypts of normal and cftr/ mice. Image software analysis performed on total proteins separated by bidimensional (2D) gels between pH 611 unmasked 24 differentially expressed proteins (>2-fold). We analyzed in more detail one of these proteins that was missing in 2D gels from cftr/ mice. This protein was identified as annexin A1, also called lipocortin-1, and was absent in colon, lungs, and pancreas (i.e. in tissues affected in CF) from cftr/ mice. We also observed that annexin A1 was not detected in nasal ciliated cells from CF patients bearing homozygous nonsense mutations in the CFTR gene and that its expression could also vary in patients with F508del mutation, the most frequent mutation in CF patients.
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EXPERIMENTAL PROCEDURES
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Materials
All reagents necessary for sample preparation, 2D gels, and mass spectrometry were of the highest grade available (urea, thiourea, DTT, and CHAPS were from Sigma; SDS was from Invitrogen). Silver nitrate was from Prolabo (Fontenay-sous-Bois, France). MALDI matrices were obtained from Sigma. The standard peptide mixture used for sample calibration was purchased from LaserBiolabs (Sophia-Antipolis, Nice, France). Anti-annexin A1 antibodies (i) mouse monoclonal antibody (EH17A), (ii) rabbit polyclonal antibody (H-65), and (iii) goat polyclonal antibody (N-19) and rabbit polyclonal anti-actin were all purchased from Santa Cruz Biotechnology, Inc./Tebu-Bio SA (Paris, France). The rabbit polyclonal anti-CFTR antibody raised against the carboxyl terminus of CFTR was a gift from Dr. C. Figarella (Marseille, France). The rabbit polyclonal anti-cytosolic phospholipase A2 antibody was a gift from Dr. L. Touqui (Paris, France).
Mice
Mice lacking cftr expression (cftrtm1Unc, cftr/ (13)), backcrossed onto a C57BL/6 background in the Animal Core Facility at Centre de Distribution, Typage and Archivage animal (Service CNRS, Orléans, France), and C57BL/6 (cftr+/+) mice were obtained from the same Core Facility. cftr+/+ and cftr+/ (heterozygous) mice were siblings of cftr/ mice. Each mouse was genotyped before use. All mice used were between 3 and 4 weeks of age. Only males were used for this study. All experiments were approved by INSERM.
Nasal Epithelial Cells from CF Patients
Six CF patients with a Y122X/Y122X nonsense mutation were followed up in the CF centers at the Saint Pierre Children Hospital and at Saint Denis Children Hospital (La Réunion, France). One patient homozygous for 489delC nonsense mutation and three patients homozygous for the F508del mutation were followed in the Centre de Ressources et de Competences de la Mucoviscidose at Necker Hospital (Paris, France). The diagnosis of CF was based on elevated sweat chloride level: all patients had Cl concentration values >80 meq/liter. Sodium and chloride conductances of nasal epithelium were evaluated by nasal potential difference according to the set-up and protocol established by Knowles et al. (18). The clinical characteristics of CF patients are summarized in Table I.
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TABLE I Description of CF patients and summary of annexin A1 staining in nasal epithelial cells
For annexin A1, "++" indicates staining much lower than in healthy subject, "+" indicates slight staining, "+/" indicates slight punctuate staining, and "" indicates non-detectable staining. ND, not determined.
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Nasal epithelial cells were obtained by gentle brushing of the middle turbinate. Cells were immediately spread on slides, fixed in acetone for 10 min at room temperature, and stored at 20 °C until further immunocytochemical analysis.
Crypt Isolation and Total Protein Preparation for 2D Gels
Animals were sacrificed by cervical dislocation, and their distal colon was removed and immediately rinsed with a cold HEPES-buffered solution (140 mM NaCl, 4.7 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, 10 mM HEPES, pH 7.2). For the preparation of crypts slight modifications of the Morris method were used (19). Briefly, the colons were opened longitudinally with scissors and immersed in a Ca2+-free solution containing 107 mM NaCl, 4.5 mM KCl, 0.2 mM NaH2PO4, 1.8 mM Na2HPO4, 10 mM glucose, 10 mM EDTA, pH 7.2. Crypts were then separated from connective tissue and muscle layers at 4 °C for 2030 min by mechanical vibration using a vortex. The isolation of crypts was verified by microscopy. Isolated crypts were centrifuged for 1 min at 1000 x g and immediately solubilized. Total proteins from colonic crypts were solubilized using the method described by Harder et al. (20). 200 µl of buffer containing 1% SDS, 100 mM Tris-HCl, pH 7.0 warmed at 95 °C were added to the crypts present in the pellet, and the preparation was sonicated for 5 min. The use of SDS buffer at 95 °C lyses cells instantaneously, inhibits the activity of proteases, diminishes the viscosity due to DNA, and solubilizes proteins. Next, protein samples were heated again at 100 °C for 5 min, cooled on ice, diluted with 500 µl of solubilization buffer (2.5 M thiourea, 8 M urea, 4% (w/v) CHAPS, 50 mM DTT), and vigorously shaken for 1 h. Then the extracts were centrifuged at 16,000 x g and 4 °C for 20 min, and the pellet was discarded. The concentration of solubilized proteins was determined using the reducing agent compatible detergent compatible kit (Bio-Rad). Samples were aliquoted and frozen at 80 °C. The 2D gel experiments were done within a week.
2D Gel Electrophoresis and Mass Spectrometry
For isoelectric focusing, 80 µg (or 200 µg for MS analysis) of each sample were diluted in 8 M urea, 2.5 M thiourea, 4% (w/v) CHAPS, and 5 mM DTT to get a 100-µl final volume. The samples were filtered through 0.45-µm Vectaspin microfilters (Whatman), and 0.5% ampholytes were added. Aliquots of total extracted proteins were loaded on 18-cm linear pH 611 strips in an IPGphor system (Amersham Biosciences). SDS-PAGE (10% polyacrylamide gels) was performed using Ettan DALT II (Amersham Biosciences) following the manufacturers instructions. A standard silver staining was used to reveal protein spots after 2D gel electrophoresis. MS-compatible silver staining was used for MS experiments (21). In the first series of experiments, 2D PAGE of total cryptic proteins prepared from individual animals (two cftr+/+ and two cftr/) was performed (three gels for each mouse). In the second series of experiments, cryptic proteins from three animals of the same group were pooled, and the 2D gel analysis was performed (cftr+/+ versus cftr/, three gels per pooled sample). The analysis of differentially expressed proteins was done by eye and using the ImageMaster 2D Platinum software package (version 5.0, Amersham Biosciences) on nine gels per genotype. For the latter analysis, gels were scanned using the Image Master Labscan version V3.00. Silver-stained spots were quantified on the basis of their relative volume (the spot volume divided by the total volume over the whole set of gel spots, according to the manufacturers instructions). Only spots with a relative volume >0.1 were considered for differential analysis. Spots presenting saturated relative volume were excluded from the analysis. Statistical analysis was performed using unpaired Students t test. A factor greater than 2-fold difference in the average spot volume between cftr+/+ and cftr/ was reported as up- (or down)-regulation at protein level.
Identification of the protein of interest was conducted according to a standard procedure consisting of an "in-gel" digestion after a 2D gel electrophoresis separation (for a review, see Ref. 22). In-gel tryptic digestion was performed by adding 10 µl of a 6 ng/µl trypsin solution (Promega, Lyon, France) to cover gel pieces. After 30 min the remaining trypsin solution was discarded to avoid autolysis. Digestion was then performed overnight. To improve peptide recovery argentic salts were removed using the Silver Quest decoloration kit (Invitrogen).
Mass spectra of the tryptic peptide map of the proteins of interest were recorded on a MALDI-TOF instrument (Voyager DE STR, Applied Biosystems, Les Ulis, France). The acquisition parameters were the following: accelerating voltage was 20 kV, the grid was set at 65%, and the delay extraction was optimized at a value of 400 ns. Mass spectra were calibrated in the close extern mode using the Pepmix 4 standard peptide mixture (LaserBiolabs, Sophia Antipolis, France). Identification of the protein of interest was performed in several databases (National Center for Biotechnology Information (NCBI), Owl, and Swiss-Prot) with three different search engines (MASCOT, Profound, and MSFit).
Immunoblotting
Colon crypts of control and cftr/ mice were lysed for 30 min in a buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS plus protease inhibitors ("Complete" protease inhibitor mixture, Roche Applied Science). Cell lysates were centrifuged at 20,000 x g at 4 °C for 15 min. The supernatant corresponding to total proteins was obtained, the protein concentration was determined using the detergent compatible kit (Bio-Rad), and protein samples were then aliquoted and stored at 80 °C.
Total protein extracts (50 µg) from distal colon crypts of controls and cftr/ mice were separated by 10% SDS-PAGE and transferred to nitrocellulose filters (Bio-Rad). Free binding sites were blocked with 1% nonfat dry milk, 1% BSA, 0.05% Tween 20 in PBS (pH 7.4), and the membranes were probed with the rabbit anti-annexin A1 antibody (diluted 1:1000), the anti-cytosolic phospholipase A2 (cPLA2) antibody (diluted 1:1000), or the anti-actin antibody (diluted 1:1000) and incubated overnight at 4 °C. Nitrocellulose membranes were washed three times with PBS-T (0.05% Tween 20 in PBS) and incubated with goat anti-rabbit IgG (heavy + light) horseradish peroxidase-conjugated secondary antibody (diluted 1:1000) from AbCys (Paris, France). Proteins were detected by incubating the nitrocellulose filters (Bio-Rad) in ECL PlusTM reagent (Amersham Biosciences) according to the manufacturers instructions and exposing them to Kodak x-ray film.
Immunohistochemistry
Mouse intestinal distal colon, pancreas, and muscle samples were rapidly rinsed in PBS, recovered with Bright Cryo-M-Bed (myNeurolab, Saint Louis, MO), and immediately frozen in liquid nitrogen. Lungs were first flushed with PBS containing 50% Bright Cryo-M-Bed, recovered with 100% Bright Cryo-M-Bed, and frozen in liquid nitrogen. Six-micrometer cryosections of mouse tissues or human nasal cells were fixed with cold acetone for 10 min. The immunohistochemical procedures were performed as described elsewhere (23). Briefly, all the steps were done in a humid chamber. Mouse sections or human cells were rehydrated in PBS (pH 7.4) and permeabilized with 0.25% Triton X-100 in PBS. Nonspecific binding sites were blocked with 10% FCS, 3% BSA, 0.01% Triton X-100 in PBS for 1 h at room temperature. Sections were incubated with a primary anti-annexin A1 antibody alone or combined with an anti-CFTR antibody in PBS containing 10% FCS, 1% BSA, and 0.01% Triton X-100 overnight at 4 °C (working dilutions were as follows: 1:100 for anti-CFTR, 1:50 for rabbit anti-annexin A1, 1:25 for goat anti-annexin A1, and 1:100 for mouse anti-annexin A1). CFTR was visualized with Alexa 594-conjugated goat anti-rabbit IgG (heavy + light) secondary antibody (Molecular Probes, Leiden, Netherlands) diluted 1:1000 in 0.01% Triton X-100 in PBS after a 1-h incubation at room temperature. Annexin A1 was visualized with Alexa 488-conjugated donkey anti-goat IgG, goat anti-rabbit IgG, or goat anti-mouse IgG secondary antibody (Molecular Probes) diluted 1:1000 in 0.01% Triton X-100 in PBS after a 1-h incubation at room temperature. The nuclei were visualized with propidium iodide contained in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Tissue sections were examined under a Zeiss confocal laser screening microscope with argon ion lasers appropriate for Alexa 488 or 594 fluorochromes and propidium iodide. Images were collected with Zeiss x40 or x100 oil objectives.
Morphology
The morphological analysis of colon, lungs, and pancreas was performed in two cftr/ and two cftr+/+ mice. Colon and pancreas were excised, rinsed in PBS, and immersed in paraformaldehyde for 5 days before embedding in paraffin. For lung preparations, after exsanguination, the chest was opened, the trachea was exposed, and lungs were perfused in situ with 10% (v/v) neutral buffered formalin under the same pressure for all analyzed lungs. Thereafter the trachea was ligated, and the lungs were excised and immersed in paraformaldehyde for 5 days before embedding in paraffin. Four-micrometer serial sections of the analyzed organs were cut and stained with Massons trichrome.
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RESULTS
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2D Gel Analysis and MS Identification
We performed 2D gel analysis on total protein extracts obtained from colonic crypts isolated from cftr/ mice and controls (Fig. 1). In preliminary experiments using the pH range 310 for isoelectric focusing we noticed that one protein spot in the pH range around 7 and with molecular mass close to 37 kDa was lacking in cftr/ (not shown). The subsequent experiments were performed in the pH range 611, and 2D gel analysis was first done by eye (two mice for each genotype, three gels per mouse, and one experiment in which proteins from three cftr/ and three cftr+/+ mice were pooled). The analysis of all gels confirmed the absence of the 37-kDa, pI 7 protein spot in cftr/ mice spot A1,Fig. 1. Fig. 1 shows two representative gels, one for each mouse genotype. The subsequent analysis of gels from cftr+/+ and cftr/ animals by image software indicated that, in addition to the protein mentioned above, another one was not detectable in cftr/, whereas five proteins were absent in cftr+/+ mice. Moreover, 17 proteins were differentially expressed in cftr/, showing at least a 2-fold change (increase or decrease) with respect to controls: 14 proteins were down-regulated, and three were up-regulated (Supplemental Fig. 1). All these proteins were not further characterized in the present study; here we focused on the spot (A1) most visibly lacking in cftr/ mice.

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FIG. 1. Example of two-dimensional gel electrophoresis. A, 80 µg of total protein extracted from colonic crypts of cftr/ and cftr+/+ mice were separated on linear strips with a pH range of 611 followed by protein separation by 10% SDS-PAGE. In an enlarged area of 2D PAGE the lacking protein spot (spot A1) is indicated on the left, and on the right of each enlarged area are the relative volumes in a three-dimensional representation. B, the semiquantification of protein spot A1 was performed using ImageMaster Platinum software quantified on the basis of its relative volume. Data are the means of nine independent experiments per genotype (see "Experimental Procedures" for details). The protein spots 291 and 292 are down-regulated in cftr/ by a factor lower than 2-fold. ***, p < 0.001; **, p < 0.01 (unpaired Students t test).
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The most noticeably absent protein spot from cftr/ mice ran on 2D gels in the pH range of 6.57 and had a molecular mass of
37 kDa. This protein was identified by the MALDI-TOF MS technique. According to the results obtained with different databases by applying several computer search engines, only one protein candidate matched (Fig. 2A; the MS spectrum is shown in Supplemental Fig. 2). This molecule was annexin A1 (also called phospholipase A2-inhibitory protein, Swiss-Prot accession number P10107), a known member of the endogenous anti-inflammatory portfolio of homeostatic mediators (24). The percentage of sequence coverage was between 80 and 86% depending on the computer search engine used with an average error of 20 ppm. The theoretical molecular mass was 38.6 kDa with a theoretical pI of 7.15; both values are close to those experimentally determined in the 2D gel analysis.

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FIG. 2. Identification of annexin A1 by mass spectrometry and immunoblot. A, annexin A1 sequence (Fig. 1, spot A1) identified by MALDI-TOF mass spectrometry. Peptides that allowed the identification of annexin A1 are labeled in bold along the amino acid sequence. B, immunoblot analysis of total proteins (50 µg/lane) from colonic crypts of cftr/ and cftr+/+ mice. Top, using anti-annexin A1 antibody, annexin A1 is detected in cftr+/+ but not in cftr/ mice. Middle, the same blot was reprobed with anti cPLA2 antibody. cPLA2 was detected in both samples, but an increased quantity was observed in cftr/ mice (semiquantification was done using ImageMaster 1 software, Amersham Biosciences) in arbitrary units: cftr+/+, 50,442; cftr/, 138,719 (i.e. a 2.75-fold increase of cPLA2 in the cftr/ sample was observed). Bottom, the same blot was reprobed with anti-actin antibody.
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This result was confirmed by immunoblotting analysis of total proteins prepared from colonic crypts from cftr+/+ and cftr/ mice using specific anti-annexin A1 antibodies. Lack of the annexin A1 band was observed in cftr/ samples (Fig. 2B).
Expression of Annexin A1 in Murine Tissues
Immunohistochemical analysis confirmed these marked differences in annexin A1 expression in the colon and also in other tissues relevant to CF, i.e. known to physiologically express CFTR and to be affected in CF, namely lungs and pancreas.
Colon
No major morphological differences were found between colons from 34-week-old cftr+/+ and cftr/ mice (Fig. 3, top, left panels) with tissues not showing any signs of intestinal inflammation because young mice were used. Colonic annexin A1 was not detectable in the three cftr/ mice analyzed, whereas it was unambiguously detected in cftr+/+ mice (Fig. 3). The protein was expressed all along the crypts and by the surface cells, whereas CFTR was present in the apical side of cryptic cells of cftr+/+ mice (as reported, see Ref. 17). Co-distribution of the two proteins was observed in some crypts (Fig. 3e).

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FIG. 3. Localization of annexin A1 in colon and lungs of cftr/ and cftr+/+ mice. Top, colon. Left panels, optical microscopy of cftr+/+ and cftr/ colon sections. Tissues were immersed in paraformaldehyde before being embedded in paraffin and stained with Massons trichrome (for more details see "Experimental Procedures"). af, cftr+/+ mice. a, phase-contrast image of b. b, annexin A1 (green) is localized in the apical side of crypts and surface cells (nuclei were stained with propidium iodide, red). c, control image without anti-annexin A1 antibody (nuclei in red). d, phase-contrast image of e and f. e, co-localization of CFTR (red) and annexin A1 (green); the two proteins may co-localize in crypts at the apical side (yellow). f, control image without anti-CFTR and anti-annexin A1 antibodies. gi, cftr/ mice. g, phase-contrast image of h. h, annexin A1 is not detected; nuclei are stained with propidium iodide (red). i, control image without anti-annexin A1 antibody. Bottom, lungs. Left panels, optical microscopy of cftr+/+ and cftr/ lung sections. Lungs were immersed in paraformaldehyde, embedded in paraffin, and stained with Massons trichrome. jo, cftr+/+ mice. j, phase-contrast image of k. k, annexin A1 (green) is localized to the apical side of airways; nuclei are stained with propidium iodide (red). l, control without annexin A1 antibody. m, phase-contrast image of n. n, localization of CFTR (red) and annexin A1 (green); the two proteins co-localize at the apical side of bronchi. o, control without anti-CFTR and anti-annexin A1 antibodies. pr, cftr/ mice. p, phase-contrast image of q. q, annexin A1 is not detected (nuclei in red). r, control image without anti-annexin A1 antibody.
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Lungs
No infection, inflammation, or fibrosis of intrapulmonary airways, alveoli, and lung parenchyma was observed in both cftr+/+ and cftr/ mice (Fig. 3, bottom, left panel). However, enlargement of alveolar spaces was observed in cftr/ mice as compared with cftr+/+ as previously reported in this model (25). In cftr/ mice, no staining for annexin A1 was detected in the lungs (Fig. 2l, n = 3). In cftr+/+ mice, annexin A1 was detected in the apical region of airway cells and in alveoli (Fig. 3k), whereas CFTR was present in airways (Fig. 3n) but not in alveoli (data not shown). Annexin A1 and CFTR were co-expressed at the apical surface of the same cells (Fig. 3n).
Pancreas
Major morphological differences were seen in the pancreas of cftr+/+ and cftr/ mice. In cftr/ mice, less exocrine pancreas tissue was observed, whereas the islets of Langerhans were still present (Fig. 4, top, left panels). Acini and ducts were often dilated and surrounded by significant fibrosis, clear signs of pancreatic dysfunction in cftr/ mice. Annexin A1 was absent in the pancreas of cftr/ mice (Fig. 4c), whereas it was present in small ducts, especially intercalated ducts, of cftr+/+ mice (Fig. 4a).

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FIG. 4. Localization of annexin A1 in pancreas and skeletal muscle from cftr/ and cftr+/+ mice. Top, pancreas. Left panels, optical microscopy of cftr+/+ and cftr/ pancreas. Tissues were immersed in paraformaldehyde before being embedded in paraffin and stained with Massons trichrome. Acini and ducts were often dilated, and important fibrosis surrounded them. a and b, cftr+/+ mice. a, annexin A1 (green) is present in the apical side of ductal cells of cftr+/+ mouse; nuclei are shown in red. b, control without anti-annexin A1 antibody. c and d, cftr/ mice. c, annexin A1 is not detected in pancreas of cftr/ mice. d, control without anti-annexin A1 antibody. Bottom, skeletal muscle. e and f, cftr+/+. g and h, cftr/ mice. e and g, dual labeling with a polyclonal antibody against annexin A1 (green) that delineates the skeletal muscle cells and a polyclonal antibody against CFTR (which should be seen in red). The lack of staining confirms that CFTR is not expressed in skeletal muscle. f and h, control without anti-annexin A1 and CFTR antibodies.
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Skeletal Muscle
To substantiate the patterns of co-expression for CFTR and annexin A1, we analyzed skeletal muscle, known to not express CFTR. This time, annexin A1 was found both in cftr+/+ and cftr/ mice (Fig. 4, bottom, e and g), whereas, as expected, no CFTR protein was detected in both cftr+/+ and cftr/ mice (Fig. 4, f and h). Altogether the present results show that the anti-inflammatory protein annexin A1 is lacking in tissues affected in CF (colon, lungs, and pancreas) from cftr/ mice.
Expression of Annexin A1 in Nasal Epithelial Cells from CF Patients
To give pathological relevance to these findings, next we set out to investigate annexin A1 expression in CF patients. Six CF patients, bearing the Y122X/Y122X nonsense mutation and one homozygous for 489delC, both predicted to encode a truncated protein ending at part of the second transmembrane domain of the CFTR protein, were analyzed. We reasoned that expression of this very short CFTR protein might induce protein expression changes similar to those associated with lack of CFTR in cftr/ mice. We analyzed nasal epithelial cells as they are commonly used in human CF studies. Indeed, these cells are obtainable by a non-invasive procedure and are considered to mimic the alterations in lung epithelial cells. First, we conducted an immunocytochemistry investigation to confirm the absence of CFTR in nasal epithelial cells from Y122X/Y122X and 489delC patients (data not shown). Second, we could demonstrate that annexin A1 expression was absent or markedly decreased in nasal epithelial cells from these patients as compared with two healthy controls where, in line with previous reports (e.g. see Ref. 26), it was found predominantly in the cytosol (Fig. 5 and Table I). Last, we performed immunocytochemistry experiments on nasal cells from three patients homozygous for the F508del mutation (the most frequent mutation, present in 70% of patients). Two patients developed a mild phenotype of CF, and the third one displayed a severe phenotype. Annexin A1 was detected in all three patients; however, the immunostaining was markedly reduced when compared with healthy subjects (Fig. 6). When differences among F508del patients were analyzed, we found that Annexin A1 expression was less attenuated in those with mild phenotype than in the one with severe phenotype (Fig. 6, b and c, and Table I). For patients with mild disease, the F508delCFTR protein was detected in the intracellular compartment and in the vicinity of apical membrane of nasal cells almost similar to healthy subjects (Fig. 6, d and e). For the patient with severe disease F508delCFTR was present only in the intracellular compartment (Fig. 6f).

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FIG. 5. Annexin A1 expression in human nasal cells in patients homozygous for nonsense mutation. a, control without primary antibody; nuclei are stained by propidium iodide (red). b and c, annexin A1 staining (green) in nasal ciliated cells from healthy subjects. df, annexin A1 staining could not be detected in CF patients bearing the Y122X/Y122X mutation (d and f); slight staining was observed in another Y122X/Y122X CF patient (e).
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FIG. 6. Annexin A1 expression in human nasal cells in patients homozygous for F508del mutation. Staining of annexin A1 (green) in nasal epithelial cells of healthy subjects (a) and F508del homozygous patients (b and c) is shown. The annexin A1 signal is weaker in CF patients. Furthermore it decreases with CF phenotype severity. d, e, and f, localization and expression of CFTR protein (red) in nasal cells from healthy individuals (d) showing a strong label in the region of the apical membrane that is also detected in cells from F508del/F508del patients with mild phenotype (e) but disappears for the F508del/F508del patient with severe respiratory disease (f). White arrows indicate the vicinity of apical membrane.
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Expression of cPLA2 in Murine Colon
In the last set of experiments we questioned whether down-regulation of an important anti-inflammatory mediator would be associated with up-regulation of proinflammatory molecules. It is already known that cftr/ mice may develop intestine inflammation characterized by mast cell and neutrophil infiltration (27), an increase in arachidonic acid (28) and, in the lung, a deficiency of interleukin-10 secretion (29). Here we examined cPLA2 because it has been found to be up-regulated in annexin A1 null cells (30) and mice (31). cPLA2 catalyzes the release of fatty acids from membrane phospholipids leading to the synthesis of bioactive lipids involved in the inflammation process (32). Immunoblotting analysis of proteins obtained from murine colonic crypts of cftr/ mice showed a significant increase in cPLA2 (85 kDa) as compared with control cftr+/+ animals (Fig. 2B). These results add cPLA2 to the list of proinflammatory molecules overexpressed in this model of CF and demonstrate, as a proof of concept, a yin/yang regulation of anti-inflammatory and proinflammatory mediators in mice that will ultimately develop fibrosis.
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DISCUSSION
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The new data we present here demonstrate a down-regulation of annexin A1 protein in CFTR-expressing tissues from cftr/ mice as well as in epithelial cells of CF patients bearing a homozygous nonsense mutation in the CFTR gene. The proteomic approach we applied led to the identification of annexin A1, although the total number of differentially expressed proteins detected on 2D gels by software analysis was 24. This number is lower that of differentially expressed transcripts reported for the intestine of the same mouse model. Several reasons may explain this difference. We note the following. (i) Our analysis is limited to proteins with pI ranging from 6 to 11, whereas transcript analysis by microarray covers several thousands of genes (27). (ii) Silver staining allows the detection of amounts of proteins >1 ng (i.e. high abundance proteins, which represent 515% of total proteins present in the cell; we believe that many other differentially expressed proteins might correspond to low abundance proteins, which are expressed at (or below) attomole concentrations, which can be detected by radioisotopic methods (22)). On the other hand, the silver staining detection is linear within 1100 ng, which precludes the analysis of proteins very highly expressed (33). (iii) The protein analysis presented here corresponds to colonic crypts, whereas transcript analyses were done in whole intestine. (iv) The mice in our study were fed normal food, whereas the mice used for transcript analysis were under special diet for several months (27).
We have recently reported that the expression and the intracellular distribution of cytokeratins 8 and 18 are modified in cells expressing mutated CFTR (9). In the present study we did not find any marked difference in protein expression pattern in the region of 2D gels corresponding to the molecular masses and pI values of these cytokeratins, i.e. molecular mass of 4852 kDa, pH 56.5. The absence of differential expression of cytokeratins may result from the differences in the experimental models used (HeLa cells in the previous study (9) versus mice in this study). Alternatively, protein expression may be different when mutated CFTR is expressed (previous work (9)) or if CFTR is lacking (present work). To verify the expression levels and the network of cytokeratins 8/18, the investigation of F508delCFTR mice and/or cells from CF patients bearing the F508del mutation should be performed.
The first differentially expressed protein that we visually detected by eye was unambiguously identified by mass spectrometry as annexin A1. It is a key molecule in the regulation of inflammation (34). Annexin A1 belongs to a family of proteins characterized by their ability to bind phospholipids and is known to negatively modulate leukocyte activation in response to inflammation. Together with the observations showing the lack of annexin A1 in nasal ciliated cells of CF patients bearing a nonsense mutation and the differential expression in CF patients with F508del mutation displaying different phenotypes, this leads us to propose that the CFTR-related down-regulation of annexin A1 might be functionally relevant to the inflammatory status of CF patients. In further support of this view is the increased expression of proinflammatory cPLA2 concomitant with the down-regulation of annexin A1 as detected in the colon from cftr/ mice (Fig. 2B). However, inflammation was not observed in colon or lungs from cftr/ mice. It must be pointed out that the animals analyzed here were young (34 weeks old). Others have reported the lack of neutrophil infiltration in the lungs of young cftr/ mice (25) and that intestine inflammation appears only in older animals maintained under special diet (27). In CF, inflammatory events are correlated with changes in inflammatory molecules such as interleukin-10, arachidonic acid, leukotriene B4, and prostaglandin E2 (3537). Leukotriene B4 and prostaglandin E2 generation could be modulated by cellular annexin A1 (38). Although all these molecules are part of the delayed response of inflammation, the down-regulation of annexin A1 expression we observed appears to be among the first events that trigger the inflammatory response in CF. Of note, annexin A1 knock-out mice are viable and normal under physiological conditions, but they display higher acute and chronic inflammatory responses characterized by a higher degree of neutrophil trafficking (31). On the other hand, CF lung disease is accompanied by an increased number of neutrophils, which dominate the inflammatory response (39). Thus, systematic investigations of inflammation-related phenomena in cftr/ and annexin A1 knock-out mice will allow a better understanding of the inflammation in CF. At present, however, it is difficult to compare the cftr/ with the annexin A1 null mouse because the two strains are different.
The down-regulation of annexin A1 in cftr/ mice in tissues known to express CFTR (lungs, colon, and pancreas) and in nasal epithelial cells from CF patients with nonsense mutations suggests that the expression and functions of annexin A1 and CFTR might be linked, both being related to transepithelial ion transport and inflammatory processes in CF. Annexin A1 belongs to a family of proteins characterized by their ability to bind phospholipids, modulate Ca2+ fluxes (40), and intervene in the process of neutrophil activation in inflammation (34).
On the one hand, CFTR is a rate-limiting step for fluid absorption and is related to an inflammatory state with up-regulation of components of the innate immune system in intestine (27). On the other hand, annexin A1 may influence ion transport (41) through regulation of the cytoskeletal dynamic network by its association, among others, with cytokeratin 8. Because expression of the latter protein is correlated with CFTR (9), it is tempting to hypothesize that annexin A1 and CFTR may influence reciprocally their functions either to assure the correct localization of CFTR in the plasma membrane or to control the level of expression of annexin A1 providing an appropriate cell answer to inflammation. Moreover the differential expression of annexin A1 in F508del patients displaying different CF phenotypes suggests that annexin A1 may be a marker of inflammation in CF.
A key question is how CFTR regulates annexin A1 expression. In preliminary experiments, we found no major difference in annexin A1 transcript levels in different tissues between cftr/ and control mice, suggesting that annexin A1 regulation by CFTR is post-transcriptional. Knowing that annexin A1 phosphorylation depends on chloride (42) and that the phosphorylation status favors its subsequent proteolysis (43), we hypothesize that modifications in intracellular chloride concentrations occur in cftr/ mice, thus modifying annexin A1 into a conformation more susceptible to proteolysis.
In conclusion, we identified at least one fundamental alteration in the absence of CFTR, which is the down-regulation of annexin A1, a critical anti-inflammatory/protective protein crucial for maintaining homeostasis and tissue repair after an inflammatory episode. These findings might give rise to new therapeutic approaches for CF patients aiming at increasing cell-associated expression of annexin A1. In support of our results, recent clinical analyses indicate that dexamethasone, known to act as a positive regulator of annexin A1 protein expression, is effective in slowing down the progression of CF lung disease (44).
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ACKNOWLEDGMENTS
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We thank Baya Cherif-Zahar and Maryvonne Baudouin-Legros for help in the different experiments, Nicole Maurin for morphology experiments, Meriem Garfa for confocal microcopy analysis, Philippe Hulin for help in statistical analysis, Jean-Yves Lallemand for initiating the mass spectrometry identification, and Drs. Marc Renouil and Jean-François Lesure for help in characterizing CF patients at La Réunion. We thank Mario Ollero for English editing.
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FOOTNOTES |
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Received, January 17, 2005, and in revised form, June 7, 2005.
Published, MCP Papers in Press, July 12, 2005, DOI 10.1074/mcp.M500019-MCP200
1 The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; cftr/ and cftrtm1Unc, cystic fibrosis knock-out mice; cftr+/+, normal C57BL/6 mice; 2D, bidimensional; cPLA2, cytosolic phospholipase A2. 
* This work was supported by grants from the European Community (Grant QLG2-CT-2001-01335), INSERM, and the French cystic fibrosis associations Vaincre la Mucoviscidose and ABC Proteines. 
S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. 
b Both authors contributed equally to this study. 
g Supported by the Arthritis Research Campaign UK. 
l To whom correspondence may be addressed. Tel.: 33-1-69-82-30-32; Fax: 33-1-69-07-72-47; E-mail: halgand{at}mailhost.icsn.cnrs-gif.fr
m To whom correspondence may be addressed: INSERM U467, Faculté de médecine, Université Paris-Descartes, 156 rue de Vaugirard, Paris F-75015, France. Tel.: 33-1-40-61-56-21; Fax: 33-1-40-61-55-91; E-mail: edelman@necker.fr
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