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Published ahead of print on July 15, 2004, doi:10.1164/rccm.200404-507OC
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American Journal of Respiratory and Critical Care Medicine Vol 170. pp. 1049-1056, (2004)
© 2004 American Thoracic Society
doi: 10.1164/rccm.200404-507OC

Original Article

Presence of Activated Mobile Fibroblasts in Bronchoalveolar Lavage from Patients with Mild Asthma

Kristoffer Larsen, Ellen Tufvesson, Johan Malmström, Matthias Mörgelin, Marie Wildt, Annika Andersson, Anna Lindström, Anders Malmström, Claes-Göran Löfdahl, György Marko-Varga, Leif Bjermer and Gunilla Westergren-Thorsson

Department of Cell and Molecular Biology, and Department of Analytical Chemistry, Lund University; and Department of Respiratory Medicine and Allergology, Lund University Hospital, Lund, Sweden

Correspondence and requests for reprints should be addressed to Kristoffer Larsen, BMC C13, S-221 84 Lund, Sweden. E-mail: kristoffer.larsen{at}medkem.lu.se


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activated fibroblasts are suggested to be involved in the deposition of extracellular matrix in the formation of peribronchial fibrosis in asthma. We report the novel finding of activated elongated fibroblasts accompanied by elevated numbers of eosinophils in bronchoalveolar lavage fluid from 5 out of 12 patients with mild asthma (= 42%), whereas no fibroblasts were observed in the control subjects without asthma (n = 17). The elongated fibroblasts migrated twice as far when compared with fibroblasts from corresponding bronchial biopsies from the same patients, accompanied by an induced expression of RhoA and Rac1, indicating that the increased expression of these proteins are linked to increased migratory capabilities. Moreover, the elongated fibroblasts had an elevated production of the proteoglycans biglycan, versican, perlecan, and decorin, which correlated to an active cytoplasm in these cells. Differential expression patterns between the two fibroblast groups in motility-regulating proteins, such as cofilin, nuclear chloride ion channel protein, and heat-shock protein 20, were identified by two-dimensional electrophoresis and mass spectrometry. These findings indicate the presence of activated and mobile fibroblasts accompanied by an induced inflammatory response outside the airway epithelium in patients with mild asthma, results that may play a role in formation of airway fibrosis.

Key Words: asthma • bronchoalveolar lavage fluid • fibroblast • fibrosis • migration

Remodeling of the extracellular matrix (ECM) is considered an important factor in respiratory disorders such as asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and systemic sclerosis (1–3). Shedding of the epithelial layer in the large airways is a common feature associated with asthma, which induces a wound healing process (4). This process has been proposed to be abnormal, resulting in chronic inflammation and subepithelial remodeling of the airways leading to a deposition of ECM components such as collagens and proteoglycans (5–7). Activated fibroblast-like cells, such as myofibroblasts and protomyofibroblasts, are present in abundance. These fibroblastic cells migrate to the tissues undergoing remodeling and are proposed to play a role in the control synthesis and breakdown of ECM components (8). This migration process is facilitated by actin cytoskeletal–associated proteins and intracellular signaling pathways involving small GTPases such as RhoA and Rac1, which induce formation of stress fibers and focal adhesions (9). The activated fibroblast acquires a myofibroblast phenotype, which is characterized by an induced expression of {alpha}-smooth muscle actin ({alpha}-SMA) and increased secretion of ECM components such as proteoglycans (10). Importantly, the proteoglycans biglycan, decorin, perlecan, and versican have been shown to be associated with different stages in the remodeling process in asthmatic patients, where biglycan, perlecan, and versican are highly expressed during the early phases and decorin is expressed during the later stages (11). Moreover, alternatively spliced fibronectin, platelet-derived growth factor, and transforming growth factor-ß have also been suggested as important factors in the recruitment and differentiation of myofibroblasts (12–14).

By culturing fibroblasts from bronchial biopsies and using bronchoalveolar lavage fluid (BALF) from patients with asthma, new insights into the remodeling process of the asthmatic lung can be achieved (15–17). Cultured fibroblasts from bronchial biopsies have been shown to display different characteristics between distinct central areas within the lung of patients with asthma, suggesting a heterogenic environment within the lung (16).

In this study, we hypothesized that BALF from patients with mild, untreated asthma and clear bronchial hyperresponsiveness might contain activated fibroblasts with differing characteristics than those from bronchial biopsy fibroblasts from the same patients. Because mesenchymal cells such as fibroblasts are associated with areas beneath the basement membrane, we aimed to characterize the migratory and cellular characteristics of BALF fibroblasts when compared with fibroblasts from corresponding bronchial biopsies.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects, Bronchoalveolar Lavage, and Sampling of Lung Tissue
Patients (n = 12) suffering from mild asthma and definite bronchial hyperresponsiveness with the following inclusion criteria were included in the study: 18 to 70 years of age, positive phadiotope staining, PD20 < 2 mg/ml of methacholine stimulation, stable asthmatic conditions, free of infections 6 weeks before bronchoscopy, and no corticosteroid treatment 6 months before the study (Table 1). The exclusion criteria were: planned or current pregnancy, diabetes, smoker, and/or allergy sufferer.


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  		TABLE 1. Subjects with asthma and control subjects included in the study

 
As control subjects, 17 normal healthy volunteers (10 male, 7 female; 24 to 58 years of age) were used, with a reversibility in FEV1 < 12% and no asthmatic or allergic symptoms. Control subjects did not responded to doses of methacholine lower than 2 mg/ml. BAL was performed by flushing the airways with up to 140 ml of 0.9 M NaCl, and the resulting fluid was used for analysis. Bronchial biopsies were taken from the right lung and collected as previously described (16). This study was fully approved by the Swedish Research Ethical Committee (LU339–00).

Cell Cultures, Morphologic Characterization, and 4-Hydroxylase Assay
Fibroblast cells from BALF were isolated by separating the cells from the lavage fluid and culturing them in a tissue culture flask until the outgrowth of fibroblast-like cells reached confluence (5–6 days). To ensure a pure fibroblast population, the cultures were trypsinized and used in passages 5–7. Crystal violet staining was performed as previously described (18). For further details on cell cultures, prolyl 4-hydroxylase, and {alpha}-SMA assays, see the online supplement.

Cytospin Analysis
Cells in the BALF from the subjects described above were collected using a cytocentrifuge (Shandon Southern Products Ltd, Runcorn, Cheshire, UK). For details, see the online supplement.

Electron Microscopic Analysis
Fibroblasts from BALF and biopsies were studied in a JEOL JEM 1230 electron microscope (JEOL, Peabody, MA). See the online supplement for further details.

Western Blot
RhoA and Rac1 antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA) and Transduction Labs (Lexington, KY), respectively. Western blots were performed as previously described (18).

Proteoglycan Assays
The assays used to quantify the total proteoglycan production and the specific production of versican, perlecan, biglycan, and decorin were performed as previously described (19).

Migration and Stress Fiber Assay
Lung fibroblasts from BALF and bronchial biopsies (30,000) were cultured for 6 hours within a cloning cylinder and the fibroblasts were allowed to migrate after removing the cylinder. For additional details, see the online supplement. Stress fibers in fibroblasts from BALF and biopsies were analyzed as previously described (18).

Protein Expression Analysis
Lysed cells were separated using 180 mm pH 4–7 Immobiline DryStrips (Amersham Biosciences, Uppsala, Sweden). A Hoefer DALT gel apparatus (Amersham Biosciences, San Fransisco, CA) was used for gel electrophoresis. Image analysis was performed using PDQuest 7.01 (Bio-Rad, Sundbyberg, Sweden). For further details, see the online supplement.

Statistical Methods
Mean values ± SEM were calculated and the Student's t test was used for analyses of statistical significance. All values of p < 0.05 (*) (Figures 1, 2, 4, 5) and p < 0.01 (**) (Figure 6) were considered significant.



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  		Figure 1. Cellular profile of the bronchoalveolar lavage fluid (BALF) from patients with asthma and control subjects in the study. Cells from BALF were precipitated by cytospin and stained using the May-Grünwald/Giemsa method described in METHODS before being counted. Data included are from control subjects and patients with asthma with (+) or without (–) fibroblasts in the BALF (n = 12–17 patients) and are presented as a percentage of total cell numbers. Values are presented as means ± SEM. *Significant difference when compared with patients without BALF fibroblasts (p < 0.05).

 



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  		Figure 2. Morphologic characterization of fibroblast phenotypes from subjects with asthma. Fibroblasts were cultured from BALF and bronchial biopsies. Both phenotypes were stained with crystal violet (A and B) and displayed an expression of the fibroblast marker prolyl 4-hydroxylase (C and D), an enzyme involved in the production of collagen. Moreover, the cells were stained for the myofibroblast marker {alpha}-smooth muscle actin ({alpha}-SMA) (F–H). Comparable magnification was used for A and B, C and D, and F and G, respectively. Two hundred cells/sample for each patient were counted. The ratio of the length:width of the cells was measured and the results presented as mean ± SEM for n = 5–7 patients (E). *Significant difference when compared with subjects with no fibroblasts in BALF (p < 0.05).

 

   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cellular Profile in BALF of Patients with Asthma and Control Subjects in the Study
In 42% of the patients with asthma, fibroblasts could be isolated from the BALF and cultured, whereas in control subjects no fibroblasts were present (Table 1). To study the cellular profile in the BALF from the patients with asthma and control subjects, cytospin analysis was performed followed by May-Grünwald/Giemsa staining. Macrophages, lymphocytes, neutrophils, and eosinophils were counted. The total number of cells (106 cells) present in the BALF for the control subjects was 3.59 ± 0.97, for patients with asthma with no BALF fibroblasts 3.58 ± 3.15, and for the patients with asthma with BALF fibroblasts 5.68 ± 4.17. Patients with asthma with fibroblasts present in the BALF had a fivefold increase of eosinophils in their cellular profile when compared with the control subjects (p < 0.05) and a threefold higher percentage (p < 0.05) when compared with patients with asthma with no BALF fibroblasts (Figure 1). Furthermore, patients with asthma with no BALF fibroblasts displayed a twofold (p < 0.05) increase in eosinophils compared with control levels. The cellular profile of macrophages in BALF (106 cells) was for control subjects, patients with asthma with no BALF fibroblasts, and patients with asthma with BALF fibroblasts 71 ± 4, 72 ± 4, and 69 ± 3, respectively. Lymphocytes and neutrophils were similar between each patient group. Furthermore, the two groups of patients with asthma showed similar responses when stimulated with methacholine (PD20) (Table 1). To characterize the BALF fibroblasts, the following set of experiments were performed aimed at studying several important factors involved in the remodeling process such as cell morphology, migration, ECM production, and protein expression pattern.

Fibroblasts Cultured from BALF Display a Different Cellular Phenotype
Fibroblasts from the BALF displayed a thin, elongated phenotype, whereas the corresponding fibroblast phenotype cultured from the biopsy displayed a more compact phenotype (Figures 2A and 2B). Importantly, this phenotype was stable throughout the different passages (passages 3–7, data not shown). Furthermore, BALF fibroblasts and biopsy fibroblasts stained for the fibroblast marker prolyl 4-hydroxylase (Figures 2C and 2D), indicating that they are collagen-producing cells. The mean ratio of length to width was 7.81 ± 1.79 for BALF fibroblasts and 4.62 ± 1.23 for fibroblasts from biopsies (p < 0.05), which demonstrates that fibroblasts from BALF have a more elongated cell shape (Figure 2E). No difference in length to width was observed between biopsy fibroblasts from patients with asthma with or without BALF fibroblasts (data not shown). Importantly, BALF fibroblast and biopsy fibroblasts were both stained for the myofibroblasts marker {alpha}-SMA (Figures 2F and 2G), suggesting that these cells had acquired an activated myofibroblast phenotype with indications that BALF fibroblasts express more {alpha}-SMA than biopsy fibroblasts (Figure 2H).

The BALF fibroblasts expressed microvilli and filopodia, and displayed intracellular and secreted vesicles when studied by electron microscopy (Figure 3A). These vesicles may indicate a high rate of cellular transportation, possibly of different ECM molecules. Microvilli are characterized as being smaller and thinner than filopodia and are expressed at the cell surface of BALF fibroblasts. In the biopsy fibroblasts, the endoplasmic reticulum (ER) is clearly visible together with an irregularly shaped nuclear envelope (NE) (Figure 3B). Both of these observations are typical of myofibroblasts (20).



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  		Figure 3. Intracellular profile of fibroblasts from BALF and lung biopsies. This figure shows representative electron microscopy pictures of fibroblasts from BALF (A) and corresponding lung biopsies (B). The magnifications used in this experiment were x10,000 and x50,000, respectively. ER = endoplasmic reticulum; F = filopodium; NE = nuclear envelope; N = nucleus; V = vesicle.

 
BALF Fibroblasts Are More Mobile than Fibroblasts from Bronchial Biopsies
The fibroblasts from BALF had migrated double the distance compared with the fibroblasts from the biopsies (p < 0.05) (Figures 4A and 4B), indicating a more mobile phenotype. This finding was further supported by the induced expression in BALF fibroblasts of the small GTPases RhoA (Figure 4C) and Rac1 (Figure 4D) (p < 0.05), which are known to be of importance in migration. To examine how the migratory changes were supported by the cytoskeleton, cells were stained to visualize the stress fibers (Figures 4E and 4F). Fibroblasts from BALF contained long, extended actin bundles compared with cells from bronchial biopsies (Figures 4G and 4H).




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  		Figure 4. Migration of fibroblast cells from BALF and lung biopsies. Fibroblasts from BALF and lung biopsies were cultured in a "clone-cylinder". The cylinder was removed after 24 hours and the distance from the border of the removed cylinder covered by the cells was measured after an additional 48 hours (A). The quantified difference between the two phenotypes was significantly larger for the BALF fibroblast (B). The level of expression of RhoA and Rac1 was determined by Western Blotting and further quantified by measuring the optical density of the bands for RhoA (C) and Rac1 (D). Values are presented in B, C, and D as means ± SEM for n = 5–7 patients. *Significant difference when compared with fibroblasts from biopsies (p < 0.05). For stress fiber expression, cells were seeded on four-well chamber slides (5,000 cells/well) and stained with Alexa Fluor 488 phalloidin to show the F-actin, and analyzed using a light scanning microscope (E and F). Areas of higher magnification detail the organization of the stress fibers (G and H).

 
BALF Fibroblasts Display an Increased Production of ECM Molecules
According to the observations from the electron microscope study described above, BALF fibroblasts and biopsy fibroblasts displayed a distinct intracellular vesicular pattern. This set of experiments aimed at comparing the production and secretion of ECM components between these cell phenotypes. The total production of proteoglycans increased fivefold (p < 0.05) for the BALF fibroblast cultures (Figure 5A) when compared with biopsy fibroblasts. The proteoglycan fractions obtained were further digested by chondroitin avidin-biotin-peroxidase complex (ABC) lyase, which cleaves condroitin and dermatan sulfate chains. After separation on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), specific proteoglycans could be determined: versican (Mr > 400 kD), the ABC lyase insensitive perlecan ({approx} 300 kD), biglycan ({approx} 200 kD) and decorin ({approx} 97 kD) (Figure 5B). Our observations showed that the fibroblasts from BALF produced significantly more biglycan (3.5-fold increase, p < 0.05), decorin (1.5-fold increase, p < 0.05), versican (twofold increase, p < 0.05) and perlecan (threefold increase, p < 0.05) than the corresponding biopsy phenotype (Figure 5C). Moreover, the increase observed in the different proteoglycan populations was similar in the different passages used (data not shown).



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  		Figure 5. Proteoglycan production in fibroblasts from BALF and lung biopsies. Cells were grown in [35S]-supplemented medium and diethylaminoethyl (DEAE)-52 columns were used to separate the proteoglycans with incorporated 35S from the cell medium. The quantification of total proteoglycan production was determined using a scintillation counter and is presented as dpm/µg protein (A). Versican, perlecan, biglycan, and decorin production was determined by further separation on a SDS-PAGE (B), before (–) and after (+) treatment with chondroitinase ABC lyase (c'ase ABC), and the intensity of the bands were measured and related to protein expression (C). Values are presented as means ± SEM for n = 5–7 patients. *Significant difference when compared with fibroblasts from biopsies (p < 0.05).

 
The Differential Protein Expression Pattern in BALF Fibroblasts Involves Migration-Regulating Proteins
To analyze the mechanisms behind the characteristics of the BALF fibroblasts on a molecular level, we analyzed the protein expression pattern by applying gel-based proteomics in the pH range of 4–7. A series of triplicate gels were studied where the master gel for each fibroblast group comprised of 500 unique spots; nine of these proteins showed a statistically significant change in expression when BALF fibroblasts were compared with biopsy fibroblasts (Figure 6A). These proteins were identified by mass spectrometry as: Cofilin, endozepine, 40S ribosomal protein SA (P40) laminin-binding protein, myosin V, nucleophosmin, heat-shock protein 20, stathmin, nucleoside diphosphate kinase, and nuclear chloride ion channel protein. The largest differences in expression when comparing the BALF fibroblasts with fibroblasts from bronchial biopsies were observed in proteins involved in the migration process of the fibroblast, e.g., heat-shock protein 20 (20-fold increase in BALF fibroblasts, p < 0.01) (Figure 6B), cofilin (2-fold increase in biopsy fibroblasts, p < 0.01) (Figure 6C), nuclear chloride intracellular ion channel protein (2.5-fold increase in biopsy fibroblasts, p < 0.01) (Figure 6D). Moreover, the proteomic profiles of the different phenotypes were similar when comparing cell cultures from different passages (data not shown).




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  		Figure 6. Protein expression pattern in fibroblasts from BALF and lung biopsies. Cells were cultured in six-well plates and harvested in solubilization solution: 7 M urea, 2 M thiourea, 2% ((chloamidopropyl) dimethylammonio)propanesulfonate (CHAPS). The lysed cells were separated by two-dimensional electrophoresis in a pH-range of 4–7. The protein expression pattern for the BALF and biopsy fibroblast master gels is presented, and the zoom-boxes show the marked area for the proteome in the low-molecular region (A). Arrows in the figure indicate identified proteins that displayed a differential expression pattern between the two different fibroblast phenotypes. The quantitative expression pattern of heat-shock protein 20 (B), nuclear chloride ion channel protein (C), and cofilin (D) is displayed for the two different fibroblast phenotypes. Values are presented as means ± SEM for n = 5–6 cell cultures. **Significant difference when comparing the expression between BALF fibroblasts and fibroblasts from bronchial biopsies (p < 0.01).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we report the novel finding of the presence of migratory BALF fibroblasts in 42% of subjects with mild asthma and definite bronchial hyperresponsiveness. No fibroblasts from BALF were reported in any of the control subjects without asthma. The patients with asthma with BALF fibroblasts had elevated numbers of eosinophils when compared with patients with asthma without BALF fibroblasts and control subjects, indicating an active inflammatory process in the airways of these patients (1). The experiments indicate that BALF fibroblasts are activated myofibroblasts and migratory, because the cells seen are elongated and express {alpha}-SMA and migration-facilitating filopodia. Fibroblasts have never been reported in BALF from patients with asthma before. They have, however, been reported earlier in BALF from patients with systemic sclerosis (21), in whom elevated levels of fibroblast activators such as transforming growth factor-ß, platelet-derived growth factor, and thrombin have been observed in BALF (22).

In this study, BALF fibroblasts displayed a higher production of ECM molecules such as proteoglycans, and a unique protein expression pattern that can be linked to a characteristic cell phenotype. There can be several potential possibilities with respect to where the BALF fibroblasts originate. The elevated numbers of eosinophils observed in the patients with BALF fibroblasts might be accompanied by epithelial shedding (23). Furthermore, differentiation of fibroblast progenitor cells, e.g., fibrocytes from the airway mucosa and epithelial mesenchymal transition, may be other possible sources of the BALF fibroblasts (24, 25).

An excellent tool when studying different cell phenotypes on a molecular level is protein expression profiling by two-dimensional electrophoresis and protein identification by mass spectrometry. When comparing BALF fibroblasts to biopsy fibroblasts, nine unique proteins showed a significant difference in the levels of their expression. Nuclear chloride ion channel protein is a member of the CLIC family of chloride channels and has earlier been shown to be induced in myofibroblasts as opposed to normal fibroblasts (26). Furthermore, the same study showed in a migration assay that certain CLIC members, such as CLIC4, reduced fibroblast motility. This might explain on a molecular level why fibroblasts from bronchial biopsies showed less cell mobility when compared with the BALF fibroblasts. Furthermore, heat-shock protein 20, which was induced in BALF fibroblasts, has recently been suggested to induce relaxation of smooth muscle cells and be dynamically associated with the actin cross-linking protein actinin (27, 28). Dynamic reorganization of the actin cytoskeleton is considered as one of the cornerstones in cell migration, which therefore suggests that heat-shock protein 20 is involved in the mobility process. Other migration-affecting protein are the small GTPases RhoA and Rac1, which are highly expressed in BALF fibroblasts and induce the formation of stress fibers as well as the formation and maintenance of focal adhesions (29). RhoA and Rac1 affect each other, which results in the formation of new focal adhesion sites and filament assembly in the advancing lamellipodia. These processes, together with formation of stress fibers and contraction, cause the cell to move forward (30). The induced expression of RhoA and Rac1 in BALF fibroblasts may also be linked to the observed increase in the production of proteoglycans in these cells. These ECM molecules are secreted in large amounts by moving cells, where they function to provide a site of attachment for cells in their adjacent environment. The elevated levels of biglycan produced by the BALF fibroblasts are interesting since this proteoglycan can be linked to the migratory and morphologic properties involving RhoA and Rac1 signaling in these cells (18).

Cofilin, induced in fibroblasts from biopsies, is also involved in cell movement, where it inhibits actin depolymerization and stabilizes actin filaments (31). Thus, its induction could also reflect the migratory characteristics of the BALF fibroblasts in this set of experiments.

The increased production of proteoglycans in BALF fibroblasts further implicates their involvement in the inflammatory repair process. Many cytokines such as transforming growth factor-ß can induce the expression of versican suggesting that these findings, in combination with the induction of biglycan, are related to a profibrotic environment within the areas accessible to the BALF fibroblasts (32). Hence, the observations of BALF fibroblasts in the patients with asthma may be an indicator of an early repair phase and the cells may be an important target cell for future therapy. The ratio of biglycan to decorin could thus provide a readout for the status of the cells (33). Moreover, the data displays the importance of performing both BAL and biopsies when studying respiratory disorders in the human lung. BAL is considered the more adequate sampling technique due to its coverage including the small airways, whereas biopsies are used for specific sampling preferably in the central parts of the lung. Asthma is mostly manifested in the smaller airways, though the inflammation in the central airways is also considered to be associated with peripheral observations of the asthmatic lung (34, 35). However, it is difficult to show the degree of severity in the remodeling process, and these findings emphasize asthma as a heterogenic disease where some patients are more susceptible to remodeling than others.

In conclusion, the observation of activated and mobile fibroblasts outside the airway epithelium in patients with mild asthma and active airway inflammation leads to new perspectives regarding the fibrotic process.


   Acknowledgments
 
The authors thank Kristian Nilsson for laboratory and technical skills. They also thank Maria Baumgarten for technical skills regarding the electron microscopy analysis, and David Bohgard, Johan Flank, and Massih Naisan for technical assistance.


   FOOTNOTES
 
Supported by grants from the Swedish Medical Research Council (11,550), Heart-Lung Foundation, CFN Centrala Försökdjursnämden, Greta and John Kock, Alfred Österlund, Anna-Greta Crafoord Foundations, Riksföreningen mot Reumatism, Gustaf V:s 80 Årsfond, and the Medical Faculty, Lund University.

This article has an online data supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Conflict of Interest Statement: K.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; E.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; C.-G.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; G.M.-V. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; L.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; G.W.-T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form April 16, 2004; accepted in final form July 14, 2004


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 DISCUSSION
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