Cystic fibrosis (CF) is due to mutations in the CF transmembrane conductance regulator gene CFTR. CF is characterised by mucus dehydration, chronic bacterial infection and inflammation, and increased levels of cytosolic phospholipase A2α (cPLA2α) products in airways.
We aimed to examine the role of cPLA2α in the modulation of mucus production and inflammation in CFTR-deficient mice and epithelial cells. Mucus production was assessed using histological analyses, immuno-histochemistry and MUC5AC ELISA. cPLA2α activation was measured using an enzymatic assay and lung inflammation determined by histological analyses and polymorphonuclear neutrophil counts in bronchoalveolar lavages.
In lungs from Cftr-/- mice, lipopolysaccharide induced mucus overproduction and MUC5AC expression associated with an increased cPLA2α activity. Mucus overproduction was mimicked by instillation of the cPLA2α product arachidonic acid, and abolished by either a cPLA2α null mutation or pharmacological inhibition. An increased cPLA2α activity was observed in bronchial explants from CF patients. CFTR silencing induced cPLA2α activation and MUC5AC expression in bronchial human epithelial cells. This expression was enhanced by arachidonic acid and reduced by cPLA2α inhibition. However, inhibition of CFTR chloride transport function had no effect on MUC5AC expression.
Reduction of CFTR expression increased cPLA2α activity. This led to an enhanced mucus production in airway epithelia independent of CFTR chloride transport function. cPLA2α represents a suitable new target for therapeutic intervention in CF.
Cystic fibrosis (CF) is a common recessively inherited disorder in the Caucasian population due to mutations in the CF transmembrane conductance regulator gene CFTR. The most common mutation results in the absence of a phenylalanine residue at amino-acid position 508 (F508del) 1. Mutations of CFTR cause dysfunction of chloride and sodium channels leading to airway mucus abnormality, a critical pathophysiological feature of CF 2. This leads to airway obstruction, chronic bacterial infection, in particular by Pseudomonas aeruginosa, and inflammation that results in a dramatic respiratory insufficiency, the major cause of mortality in CF patients 3–5.
Normally, the epithelium of conducting airways is covered with a thin layer of mucus that plays important roles in airway defence against inhaled pathogens by facilitating their clearance via mucociliary clearance to the upper airways 6–8. Mucins are large glycoproteins secreted in the airway lumen by epithelial and submucosal gland cells. Mucins MUC5AC and MUC5B have been identified as important components of airway mucus in normal subjects 9. In patients with CF, airway disease is characterised by progressive airway obstruction by mucous secretions 10. Studies of mucins in CF airways excised at the time of transplantation have shown that: 1) MUC5AC mRNA expression is increased in CF epithelium compared to the epithelium of control subjects 11, 2) immunostaining for MUC5AC protein is increased in CF epithelium compared with control subjects 12, 13, 3) MUC5AC protein is present in the airway lumen of CF subjects, contributing to airway plugging 12, 13 and 4) MUC5B is also increased in CF airway epithelium and lumen 12, 13. Altogether, these data suggest that overproduction and secretion of MUC5AC and MUC5B mucins may occur in CF airways and contribute to their progressive plugging. A recent paper by Garcia et al. 14 showed that CFTR activity is required for normal hydration and secretion of intestinal mucin in a mouse model of CF. It is inferred that a similar mechanism may play a role in CF airways 14. This does not exclude mucus hypersecretion and goblet cell hyperplasia as contributing factors.
Many studies have examined the mechanisms involved in the induction of pulmonary mucus hypersecretion and mucin expression in various animal models of asthma 15, 16 and chronic obstructive pulmonary disease 17. Although CFTR mutations have been shown to induce mucus dehydration and accumulation, the impact of these mutations on mucin expression is still unclear. This expression can be a direct consequence of CFTR alteration or secondary to the exacerbated inflammation that CFTR alteration induces in the lung. Although increased production of various arachidonic acid metabolites, including leukotriene B4, has been reported in airways of CF patients 18 the levels of cysteinyl leukotrienes, known to induce mucus production 16, 19, remained unknown. We have recently shown that a key enzyme involved in the release of arachidonic acid, cytosolic phospholipase (cPLA) A2α , forms complexes with CFTR, and therefore may play a critical role in the pathogenesis of CF 20. cPLA2α hydrolyses membrane phospholipids at the sn-2 position leading to a selective release of arachidonic acid 21, 22. The latter is further converted by cyclooxygenase and lipoxygenase into prostaglandins (PG) and leukotrienes (LT), respectively 23, 24, among other mediators. The implication of cPLA2α in the development of lung inflammation has been extensively examined in various animal models of lung inflammatory diseases 25–27 but its involvement in CF, and in particular, in airway mucus secretion has not been addressed. The present study aimed to investigate the role of cPLA2α in lipopolysaccharide (LPS)-induced lung mucus production in a mouse model of CF (Cftr-/- mice) and CFTR-deficient human bronchial epithelial cells.
MATERIALS AND METHODS
Cftr-/- (C57BL/6J Cftrm1UNC), established by gene targeting 28, were obtained from the “Centre de Distribution, de Typage et d'Archivage Animal” (Orleans, France). After weaning, a commercial osmotic laxative (Movicol®; Norgine, Middlesex, UK) was provided in the drinking water to increase the survival of Cftr-/- mice. C57BL/6J/cPLA2α-null mice were established as previously reported 27 and fed a standard laboratory diet and water. Experiments were performed on 8–12-week-old mice, using at least six mice in each group. Mice were cared for in accordance with Pasteur Institute, Paris, France guidelines in compliance with European animal welfare regulations.
LPS and arachidonyl trifluoromethyl ketone instillations
Mice were anesthetised with xylazine 2% (8 mg·kg−1) (Rompum, Bayer Healthcare, Puteaux France) and Ketamine 1000 (40 mg·kg−1) (Imalgène1000 Merial, Lyon, France) and treated with the cPLA2α inhibitor, arachidonyl trifluoromethyl ketone (ATK) (Sigma, St. Louis, MO, USA) as previously described 29. Briefly, mice received intraperitoneal instillation of ATK (20 mg·kg−1) in 20% ethanol solution, or the same volume of this solution. After 1 h, mice received intratracheal instillation of P. aeruginosa LPS (330 μg·kg−1 in 20 μL PBS) (serotype 10; Sigma). Mice received a second intraperitoneal instillation of ATK (20 mg·kg−1) 24 h later. Bronchoalveolar lavages (BALs) were performed with PBS, 24 h or 4 days after LPS instillation. PBS was introduced slowly over 1 min to minimise trauma and hence red blood cell contamination. The extent of inflammation was assessed by measuring polymorphonuclear neutrophil (PMN) counts performed as previously described 30.
Tissue fixation and histological staining of tissue sections
Lungs were flushed to remove blood, immersed in 4% formaldehyde for 48 h at 4°C and processed for paraffin inclusion. Analyses of cells present in the epithelia lining the small and the large intrapulmonary bronchi were performed as previously described 31. Longitudinal and transversal sections of the major intrapulmonary bronchi of 5-μm thickness were prepared and stained with Haematoxylin/eosin (H&E), periodic acid-Schiff (PAS) or Alcian blue (AB; pH 2.4). Sections (five sections per mouse) were made to visualise large and small airways in all experimental groups. Mucus scores were established in a double-blinded fashion by two independent observers under the supervision of a pathologist counting AB-positive cells, in 30 fields per mouse on 400× magnification, as previously reported 31, 32. The score analyses are shown (table 1 in the online supplementary material) and their statistical analyses performed as indicated in the section “statistical analyses”.
Paraffin sections (5 μm) of mouse lungs were stained with a specific monoclonal antibody directed against MUC5AC (clone 45 M1; Neomarkers, Fermont, CA, USA). After 2 h at room temperature, sections were washed with PBS containing 2% bovine serum albumin (BSA) for 30 min and incubated with an immunoglobulin (Ig) G conjugated horseradish peroxidase anti-mouse (Dako Cytomation Envision System, Carpinteria, CA, USA). Sections were then washed and stained with amino ethyl carbazol (Sigma).
5-μm sections of human bronchial explants were incubated with a specific rabbit polyclonal antibody raised against phospho-cPLA2α (1:50 dilution; Cell signalling, Boston, MA, USA) at room temperature for 1 h. After washing with PBS, sections were incubated with a biotinylated horse anti-rabbit antibody (1:200 dilution; Vector laboratories, Burlingame, CA, USA). The results were revealed with avidin–biotin–peroxidase complex method (Elite ABC kit; Vector laboratories).
Epithelial cell incubations
NCI-H292 cells were grown in RPMI-1640 medium as previously described 33, pre-incubated with cPLA2α inhibitors methyl arachidonyl fluorophosphonate (MAFP) or Pyrrolidine-1 for 1 h before stimulation with LPS, transformin growth factor (TGF)-α or phorbol myristate acetate (PMA). The concentrations of these compounds were adopted based on previous publications. In other studies, cells were transfected with CFTR siRNA (Sigma) and corresponding siRNA control using TransIT-siQUEST® transfection reagent according to the manufacturer's instructions (Mirus, Madison, WI, USA). The cells were then incubated for 1 h with MAFP followed by 24 h stimulation with PMA.
Measurements of cPLA2α activity and free arachidonic acid
Lung tissues and NCI-H292 cells were lysed according to Filgueiras and Possmayer 34 and centrifuged for 5 min at 1,000 xg to remove debris. Protein concentrations in pellets were measured by using a kit from Pierce (Thermo Scientific, Rockford, IL, USA). Extracts with equivalent protein contents were incubated for 30 min with 1 mL vesicles containing 6 nmoles of 1-palmitoyl-2[14C]arachidonoyl-sn-glycero-3-phosphorylcholine (>53 mCi·mmol−1) (Perkin-Elmer, Boston, MA, USA) and 4 nmoles of diacylglycerol (Sigma), in the presence of 5 mM CaCl2 and 1 mM 2-mercaptoethanol 35. This assay detects selectively cPLA2α activity as iPLA2 (calcium independent phospholipase A2) activity does not require calcium, and 2-mercaptoethanol inhibits sPLA2 (secretory phospholipase A2) but not cPLA2α activity 35. Then, measurements and calculations of cPLA2α activity were measured 35. The levels of free arachidonic acid were measured in BAL fluid (BALF) by gas chromatography/mass spectrometry as previously reported 36.
Aliquots from cell lysates or cell cultures were incubated with bicarbonate-carbonate buffer (50 μL) at 40°C in a 96-well plate (Nalge Nunc International, Rochester, NY, USA) until dry. MUC5AC levels were measured by ELISA using a mouse monoclonal antibody directed against MUC5AC (clone 45 M1, Neomarkers) as previously reported 37. This antibody is specific for MUC5AC.
Interleukin-8 and PGE2 measurements
Interleukin (IL)-8 and PGE2 levels in cell cultures were determined using commercial ELISA and EIA kits from R&D Systems (Minneapolis, MN, USA) and Cayman Chemicals (Ann Arbor, MI, USA).
Western blotting analysis
Lung tissues and NCI-H292 cells were lysed in lysis buffer (Buffer RLT from Qiagen, Courtaboeuf, France) and RIPA buffer, respectively. Equivalent amounts of proteins per sample were loaded onto 7.5% Tris/glycine/SDS-polyacrylamide gels. Blots were then incubated for 1 h at room temperature with 1:200 dilution of a goat antibody specific for human cPLA2α (sc-454, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or a 1:1000 dilution of a mouse monoclonal antibody specific for human CFTR (ab2784; Abcam, Cambridge, MA, USA).
Bronchial explants of CF and non-CF patients
Bronchial explants were obtained at transplantation from 10 adults with CF with F508del mutation and seven non-CF patients with lung cancer (table 2 in the online supplementary material), as previously reported 12. None of these patients required invasive mechanical ventilation before the lung transplantation procedure. The study conformed to the Declaration of Helsinki and to the rules of the Committee on Human Research of Hôpital Cochin, Paris, France.
We performed comparisons among all groups using ANOVA, and between two subject groups by the two-tailed t-test using SPSS software. Levene's test was used to test the homogeneity of variance. Data are expressed as means±sem and a p-value <0.05 was considered significant.
Cftr-/- mice exhibit increased pulmonary mucus production and MUC5AC expression
Histological analyses of lung sections showed a normal structure, no inflammatory cells and no mucus-positive cells in Cftr+/+ mice. However, in Cftr-/- mice, increased numbers of mucus-positive cells (fig. 1a-l) were detected compared with Cftr+/+ mice (see also mucus scores in fig. 1s, p<0.05). Intratracheal instillation of P. aeruginosa LPS (330 μg·kg−1) induced an intense bronchopneumonia (characterised by an accumulation of inflammatory cells inside the bronchial lumen and parenchyma) to a similar extent in the two mouse strains (fig. 1a–l). This dose of LPS (330 μg·kg−1) has been shown to induce an optimal lung inflammation 30. No significant differences in PMN counts (fig. 1a in the online supplementary material) and macrophage inflammatory protein-2 concentration (data not shown) were observed in BAL fluids from the two strains. However, LPS increased the numbers of mucus-positive cells (fig. 1a–l) in Cftr-/- compared with Cftr+/+ mice (see mucus scores in fig. 1s, p<0.01). Enhanced mucus production in Cftr-/- mice was more evident with AB than with PAS staining. Immuno-histochemical analyses revealed a higher number of MUC5AC-positive cells in the lungs of Cftr-/- compared to Cftr+/+ mice (fig. 1m–r).
Cftr-/- mice display an increased pulmonary cPLA2α activity and arachidonic acid release
An increased cPLA2α activity was observed in lung homogenates of Cftr-/- compared with Cftr+/+ mice, both in basal conditions (p<0.05) or upon LPS challenge (p<0.01) (fig. 2a in the online supplementary material). This was accompanied by an increase in the levels of free arachidonic acid in Cftr-/- compared with Cftr+/+ mice (p<0.05) (fig. 2b in the online supplementary material). Similar levels of cPLA2α protein (fig. 2c in the online supplementary material) were observed in lungs of Cftr-/- and Cftr+/+ mice, before and after LPS challenge. The specific cPLA2α inhibitor ATK 29 reduced cPLA2α activity in lung homogenates by >80% (p<0.01) (fig. 2d in the online supplementary material). Thus, the observed increase in PLA2 activity is likely due (in large part) to an enhanced stimulation of cPLA2α activity.
cPLA2α regulates bronchial mucus production in Cftr-/- mice
Intraperitoneal injection of ATK before intratracheal LPS instillation reduced AB staining (fig. 2a–l versus fig. 1a–l) in both Cftr-/- and normal littermate mice (see mucus scores in fig. 1s; p<0.01). ATK also abrogated the number of MUC5AC positive cells (fig. 2m–p). However, ATK had no effect on the inflammatory status as shown by histological analyses (fig. 2a–l) and PMN counts in BAL (fig. 1b in the online supplementary material). In contrast to cPLA2α+/+ mice, no detectable mucus-positive cells were observed in cPLA2α-/- mice, both under basal conditions and after LPS instillation (fig. 3a–l and fig. 3s; p<0.01). Intratracheal instillation of LPS induced an intense bronchopneumonia (fig. 3a–l). This was accompanied by increased PMN counts in BAL (fig. 3 in the online supplementary material). These processes were observed at similar intensities in wild-type and cPLA2α-/- mice. Intratracheal instillation of arachidonic acid to C57/Bl6 wild-type mice increased the number of mucus-positive cells (fig. 3m–r and t; p<0.01). However, arachidonic acid had no effect on PMN infiltration in lung tissues as shown by H&E staining (fig. 3m–r).
CF patients exhibit an increased bronchial cPLA2α activity
A marked immuno-staining of the phosphorylated (active) form of cPLA2α was observed in all sections from CF compared with non-CF patients. Positive staining was observed in the nuclei and plasma membranes of airway epithelial cells. Infiltrating inflammatory cells (e.g. neutrophils) were also positively stained (fig. 4a–c). Higher levels of cPLA2α activity were found in homogenates of bronchial explants from CF compared with non-CF patients (fig. 4d; p<0.01). This activity was significantly reduced by treating homogenates from CF patients with ATK (8,422±300 versus 2,415±187 dpm·mg−1, mean±sem, n = 6, in control and ATK-treated homogenates, respectively; p<0.01).
CFTR modulates MUC5AC expression in NCI-H292 cells via a cPLA2α−dependent mechanism
We examined the role of cPLA2α and CFTR in MUC5AC expression in the human lung epithelial cell line NCI-H292, which expresses both CFTR and cPLA2α (fig. 4a and b in the online supplementary material). LPS, TGF-α and PMA induced an increase of MU5AC levels in cell lysates (fig. 5a and b) (p<0.01). PMA is known to induce MUC5AC expression in NCI-H292 cells via matrix metalloprotease-mediated release of TGF-α 38. MUC5AC levels were reduced by cPLA2α inhibitors MAFP and Pyrrolydine-1 (fig. 5b) (p<0.01), and enhanced by arachidonic acid (fig. 5c) (p<0.01). Neither cPLA2α inhibitors nor arachidonic acid interfered with IL-8 secretion (data not shown).
Silencing of CFTR expression by siRNA reduced the levels of CFTR protein by 72±7.5% (p<0.01) and 62±5.5% (p<0.05) compared with negative siRNA-treated and with untreated cells, respectively (fig. 4 b and c in the online supplementary material). This led to an increased cPLA2 activity (fig. 6a) (p<0.01) and PGE2 release (fig. 5a in the online supplementary material). This was accompanied by increased MUC5AC levels in cell extracts (fig. 6b) (p<0.01) and culture media (fig. 5b in the online supplementary material), both of which were reduced by MAFP. In contrast, pre-treating cells with the specific CFTR functional inhibitor CFTRinh-172 39 failed to increase MUC5AC expression and even decreased it (fig. 6c). As a positive control, we showed that CFTRinh-172 induced an increased IL-8 secretion under LPS (p<0.05) and PMA (p<0.01) stimulation (fig. 6d), which is considered as evidence of effective inhibition of CFTR function by CFTRinh-172. At this concentration CFTRinh-172 has been shown to inhibit specifically the CFTR Cl-function without interfering with the activity of other Cl- channels 39. We conclude that the level of CFTR protein expression, but not CFTR transport activity, regulates cPLA2α activity, and subsequently MUC5AC expression.
The present study shows that Cftr-/- mice exhibit enhanced bronchial mucus production which is exacerbated by P. aeruginosa LPS. This occurs through a process involving, at least in part, an up-regulation of cPLA2α activity. In support of this, we showed that mucus production was abolished by a cPLA2α null mutation or by a specific cPLA2α inhibitor. Airway explants from CF patients also showed enhanced cPLA2α activity mainly located in epithelial cells that have been shown to exhibit increased MUC5AC expression 12. In a cell model, we showed that cPLA2α activity and MUC5AC expression increase after reduction of CFTR expression. This MUC5AC expression was abrogated by cPLA2α inhibition. However, cPLA2α appears to play a minor role in LPS-induced inflammation in our experimental models. Indeed, neither cPLA2α knock-out nor its pharmacological inhibition had any effect on PMN influx in the airways, or on IL-8 expression in epithelial cells. Although our studies clearly indicate that cPLA2α plays a key role in mucus production, the implication of other PLA2 in this process cannot be excluded. Indeed, it has been shown that secreted PLA2 stimulate mucus secretion in ferret trachea 40.
Our studies demonstrated an impact of CFTR dysfunction on mucus production in airway epithelial cells, both in vitro and in vivo, using three different methods (Immuno-histochemistry, histological staining and ELISA). Our findings showing that Cftr-/- mice exhibit exacerbated bronchial mucus production is in agreement with a previous report 41 although this was contested in another one 42. Differences in animal husbandry, experimental protocols or genetic background may explain this apparent discrepancy.
The enhanced cPLA2α activity in Cftr-/- mouse lung and human CF bronchial explants observed in the present work is in agreement with previous studies in human cell lines bearing the F508del mutation 43, 44. In these cells, an abnormal cPLA2α activity has been suggested to be a direct consequence of CFTR alteration. One plausible scenario is that CFTR acts as an inhibitor of cPLA2α activity through a domain with high homology to annexin-1 45, a cPLA2α inhibitory protein 46, 47. Consequently, the conditions that reduce CFTR levels would be expected to enhance cPLA2α activity. It is established that F508del mutation leads to endoplasmic reticulum retention and rapid degradation of CFTR 48, 49. The present study shows that CFTR silencing increases cPLA2α activity in epithelial cells. In a recent work 20, we presented evidence that cPLA2α and CFTR form a complex, apparently through interaction with S100A10/annexin-1 and that the integrity of this complex may affect cPLA2α activity. This is in agreement with previous reports demonstrating the ability of CFTR to form a complex with annexins II 50 and V 51. Decreased annexin-1 levels have been reported in a mouse model of asthma 52 with a parallel stimulation of cPLA2α activity. The expression of annexin-1 is strongly diminished in nasal cells from CF patients, as well as in lung of Cftr-/- mice 53. Based on these reports and our findings that cPLA2α activity is increased in lungs of Cftr-/- mice and CF patients, we hypothesise that alteration of levels of one of the components forming this putative complex might impact cPLA2α activity. Whether this activity is increased in CF epithelial cells as a consequence of alteration of the levels of either CFTR or annexin-1, or both, remains to be elucidated.
We also showed that the CFTR functional inhibitor (CFTRinh-172) failed to induce MUC5AC expression in NCI-H292 cells, suggesting that CFTR transport function may not play a major role in MUC5AC expression in this cell model. We suggest that in addition to the effects of CFTR chloride transport function on mucus properties 14, reduction of CFTR levels may modulate MUC5AC expression via a cPLA2α-dependent mechanism. The fact that cPLA2α inhibition abrogates PMA- and TGF-α-induced MUC5AC expression suggests that epidermal growth factor receptor (EGFR)-mediated mucus production occurs, at least in part through cPLA2α activation. Indeed, PMA is known to induce MUC5AC expression in NCI-H292 cells via matrix metalloprotease-mediated release of TGF-α and EGFR activation 38.
The stimulatory effect of CFTR transcriptional silencing on cPLA2α activity and MUC5AC expression in epithelial cells suggests that MUC5AC expression in Cftr-/- mice may occur in epithelial cells through an autocrine modulation of cPLA2α. Alternatively, MUC5AC induction can occur in goblet cells by a paracrine stimulation by aracgidonic acid released from other epithelial cells. We hypothesise that cPLA2α may play a role in mucus overproduction during the episodes of P. aeruginosa infection in CF patients. Indeed, we showed that: 1) cPLA2α mediates P. aeruginosa LPS-induced mucus overproduction in Cftr-/- mouse lungs, 2) this enzyme plays a role in MUC5AC expression in CFTR-deficient human bronchial epithelial cells and 3) cPLA2α activity increases in bronchial explants from CF patients. It should be stressed that study of non-infected lower airway tissues from living CF subjects is still a major challenge, since most patients will have chronic airway infection/colonisation during the course of the disease. Thus, although bronchial explants do not reflect basal state of these airways, it accurately reflects the in vivo state of airways in chronically infected/inflamed CF airways. Therefore, although our findings suggest a role for cPLA2α in increased mucus production in CF patients, this increase can also be a secondary response to inflammation.
We conclude that reduction of CFTR expression in CF lungs leads to an enhanced activation of cPLA2α, which in turn induces mucus overproduction. Induction of mucus production by cPLA2α may contribute to the mucus-related pathology seen in CF. A pharmacological approach based on the use of a cPLA2α inhibitor attenuated mucus overproduction in both human epithelial cells and in Cftr-/- mice. We propose a potential therapeutic role for cPLA2α inhibitors in reducing mucus accumulation in CF.
The authors would like to thank V. Balloy (Unite de Défense Innée et Inflammation/Unité INSERM U. 874, Institut Pasteur, Paris, France) for her helpful assistance for animal instillations. We are grateful to M. Huerre (Unité de Recherche et d'Expertise Histotechnologie et Pathologie, Institut Pasteur) for expert advice and criticisms and P. Ave and S. Bergère (Unité de Recherche et d'Expertise Histotechnologie et Pathologie, Institut Pasteur) for help on histological analyses. M. Wilke (Erasmus MC, Biochemistry Dept, Rotterdam, The Netherlands) and R. Buijs-Offerman (Erasmus MC, Cell Biology Dept) are acknowledged for assistance in preparing the lung tissues and BALF samples.
For editorial comments see page 991.
This article has supplementary material available from www.erj.ersjournals.com
This work was supported in part by a Contrat de Recherche with the French Associations “Vaincre la Mucoviscidose” and DARRI, Institut Pasteur. F. Dif was supported by “Vaincre la Mucoviscidose”. J. Aarbiou and R. Buijs-Offerman (Erasmus MC, Cell Biology Dept, Rotterdam, The Netherlands) were supported by grants STW 6565 and EU FP6IMPROVED PRECISION LSHB-CT-2004-005213.
Statement of Interest
- Received November 18, 2009.
- Accepted March 31, 2010.
- ©ERS 2010