1 Medical Research Service, Seattle Veterans Affairs Medical Center, Seattle 98108-1597; 2 Division of Pulmonary and Critical Care Medicine and 3 Division of Infectious Diseases, Department of Medicine, University of Washington School of Medicine, Seattle, Washington 98195; and 4 Department of Immunology and Inflammation, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08540
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ABSTRACT |
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This study investigated whether recombinant human soluble Fas ligand (rh-sFasL) induces apoptosis of primary type II pneumocytes in vitro and lung injury in vivo. Type II cells isolated from normal rabbit lung expressed Fas on their surface and became apoptotic after an 18-h incubation with rh-sFasL. Fas expression in normal rabbit lungs was localized by immunohistochemistry to alveolar and airway epithelia and alveolar macrophages. The administration of 10 µg of rh-sFasL into the right lungs of rabbits resulted 24 h later in both significantly more bronchoalveolar lavage fluid total protein and significantly more tissue changes compared with those in the left lungs, which received rh-sFasL plus Fas:Ig (a fusion protein that binds and blocks sFasL). Tissue changes included thickening of the alveolar walls, neutrophilic infiltrates, apoptotic (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive) cells in the alveolar walls, and increased expression of interleukin-8 by alveolar macrophages (as determined by immunohistochemistry). We conclude that the alveolar epithelium of normal rabbits expresses Fas and that sFasL induces lung injury and inflammation in rabbits.
pneumocytes; neutrophils; interleukin-8; macrophages
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INTRODUCTION |
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ACUTE RESPIRATORY DISTRESS SYNDROME (ARDS) is characterized histopathologically by neutrophilic inflammation and severe damage to the alveolar epithelium and capillary endothelium (2, 3). The mechanisms responsible for alveolar epithelial damage and its relationship to the inflammatory response remain unclear.
Recent evidence suggests that apoptosis may play a significant role in the pathogenesis of epithelial injury. Features consistent with apoptosis can be identified in the alveoli of humans dying with lung injury (5). The alveolar epithelium from humans with diffuse alveolar damage shows increased expression of the apoptotic promoter Bax, a homolog of Bcl-2 (14). Apoptosis of the alveolar epithelium has been associated with the development of pulmonary fibrosis in humans and mice (15, 24, 26, 44). Moreover, mediators such as Fas ligand (FasL) and angiotensin II can induce apoptosis in cell lines derived from type II cells (10, 47).
The Fas-FasL system is composed of the membrane receptor Fas (CD95), a
45-kDa membrane receptor that belongs to the tumor necrosis factor-
family of proteins, and its natural ligand, FasL (19, 41).
FasL exists as a membrane-bound form and a soluble form (sFasL), both
of which can activate Fas (20, 30, 43). Binding of Fas to
FasL can also lead to activation of the nuclear factor (NF)-
B and
release of inflammatory cytokines (34, 38, 39).
Fas antigen is expressed in the lungs, and its expression has been localized to alveolar and bronchial epithelial cells, Clara cells, alveolar macrophages, and parenchymal cells such as myofibroblasts (6, 8, 9, 11, 12, 16, 17, 23, 26, 36, 48). Both Fas and sFasL have been associated with human lung disease. Fas expression is upregulated in bronchiolar and alveolar epithelial cells of patients with interstitial pneumonia associated with collagen vascular diseases or hypersensitivity pneumonitis (22). Recently, Matute-Bello et al. (32) found that sFasL is detectable in bronchoalveolar lavage (BAL) fluids from patients with ARDS and that patients with ARDS who die have higher concentrations of sFasL. Furthermore, BAL in patients with ARDS induces Fas-mediated apoptosis of distal lung epithelial cells (DLECs), which are primary cells closely resembling alveolar pneumocytes (32). sFasL is present in other human lung diseases characterized by epithelial damage, such as bronchiolitis obliterans organizing pneumonia (BOOP), idiopathic pulmonary fibrosis, and interstitial pneumonitis associated with collagen vascular diseases (25). In animals, a one-time administration of the Fas-activating antibody Jo2 into the lungs of mice results in acute pulmonary inflammation and type II cell apoptosis, whereas repeated administration of Jo2 results in pulmonary fibrosis (10, 13, 15, 33). Thus it has been proposed that the Fas/FasL system could play a role in lung injury by inducing apoptosis of alveolar epithelial cells.
However, the biological significance of sFasL as a relevant proapoptotic molecule has been questioned. Both Tanaka et al. (42) and Schneider et al. (40) have suggested that shedding of sFasL may actually downregulate membrane-bound FasL. Schneider et al. note that the proapoptotic activity of sFasL appears to be highly dependent on the aggregation of sFasL trimers. Moreover, Liu et al. (29) found that type II cells, despite bearing functional Fas on their membrane surface, are not sensitive to T cell-bound membrane FasL. Because all animal studies to date studying activation of the Fas system in the lungs have used activating antibodies instead of FasL, the controversy remains unresolved.
The main goal of this study was to determine whether rabbit type II cells become apoptotic when exposed to recombinant human sFasL (rh-sFasL), which binds to and activates rabbit Fas, and to determine whether the administration of rh-sFasL to the lungs of rabbits results in pulmonary inflammation. A secondary goal was to determine whether Fas activation induces an inflammatory phenotype in resident alveolar macrophages.
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METHODS |
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Reagents
rh-sFasL and the fusion protein Fas:Ig were prepared as previously described (28). Both rh-sFasL and Fas:Ig were found to be lipopolysaccharide (LPS) free by the Limulus amebocyte assay. Fas expression was detected by flow cytometry with an anti-Fas phycoerythrin (PE)-conjugated mouse IgG1 monoclonal antibody (clone DX2) and a PE-conjugated mouse IgG1 monoclonal antibody (clone MOPC-21) as an isotype control antibody (both from PharMingen, San Diego, CA). Fas expression in tissues was detected with a polyclonal goat anti-human Fas antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Interleukin (IL)-8 expression in lung tissues was detected with goat anti-rabbit IL-8 as previously described (21).Animal Preparation
The animal protocols were approved by the Animal Research Committee of the Veterans Affairs Puget Sound Health Care System (Seattle, WA). We used specific pathogen-free female New Zealand White rabbits weighing 3.0-3.5 kg (Western Oregon Rabbit, Philomath, OR). The animals were housed in the animal facility at the Seattle Veterans Affairs Medical Center until the day of the experiment. Briefly, each rabbit was lightly anesthetized with xylazine (0.33 mg/kg) and ketamine (15 mg/kg). Oxygen was delivered via face mask at 6 l/min for 5 min, and then a fiber-optic bronchoscope (Pentax FI-10P2, 3.4-mm outer diameter) was introduced orally. The bronchoscope was wedged in the right main stem bronchus, and 10 µg of rh-sFasL (10 µg/ml) were instilled into the lung via the suction channel. The bronchoscope was then removed, the suction channel was rinsed with 0.9% NaCl, and the bronchoscope was reintroduced and wedged in the left main stem bronchus. Then 1.0 ml of a solution containing sFasL (10 µg) and Fas:Ig (1 mg) was instilled into the left lung. The bronchoscope was removed, and the animal was returned to its cage and allowed free access to water and food.At 24 h, the animals were euthanized with intravenous pentobarbital sodium and exsanguinated by cardiac puncture. The thorax was opened rapidly, the lungs and heart were removed en bloc, and the lungs were dissected free. The trachea was cannulated, and each lung was lavaged with 5 separate 15-ml aliquots of 0.9% NaCl containing 0.6 mM EDTA. The lungs were fixed with 10% neutral buffered formalin at a transpulmonary pressure of 15 cmH2O, embedded in formalin, and processed for histological analyses.
Aliquots of the BAL fluid were removed for total and differential cell
counts with a hemacytometer. The remainder of the fluid was spun at 200 g to pellet cells. Cell-free aliquots of the BAL fluid were
stored at 70°C. The total protein concentration of the BAL fluid
was determined in thawed samples with the bicinchoninic acid method
(BCA assay, Pierce, Rockford, IL).
Rabbit Type II Cell Isolation and Culture
Normal New Zealand White rabbits weighing 3.2 kg were anesthetized with ketamine-xylazine as described in Animal Preparation, intubated endotracheally with a 3.0-mm tube, and ventilated with a Harvard apparatus (Holliston, MA) at the following settings: inspired O2 fraction of 0.21, ventilatory rate of 32 beats/min, and tidal volume of 20 ml. Heparin (800 U) was administered intravenously. The rabbit was euthanized with intravenous pentobarbital sodium, the chest was rapidly opened, and the animal was exsanguinated by direct cardiac puncture. The pulmonary circulation was perfused with ice-cold Finkelstein's balanced salt solution (FBSS; 137.0 mM NaCl, 6.0 mM KCl, 0.7 mM Na2HPO4, 10 mM HEPES, 1.2 mM MgSO4 · 7H2O, 5.5 mM glucose, 2.0 mM CaCl2, 10,000 U/ml of penicillin, and 10 mg/ml of streptomycin, pH 7.4). The perfusate circulated by gravity to a maximum pressure of 60 cmH2O until all the blood was removed. The lungs were dissected free and lavaged with five 150-ml aliquots of 0.9% NaCl-0.6% EDTA at 37°C to remove alveolar macrophages. Next, the lungs were lavaged three times with ice-cold FBSS alternating with cold saline solution (0.85% NaCl, 3.0 mM K2HPO4, 5.0 mM Tris, 3.0 mM EDTA, 6.0 mM glucose, and 10 mM HEPES, pH 7.4; 50 ml/lavage). After the lavages, 60 ml of protease solution at 37°C (elastase and DNase I) were instilled into the trachea, and the lungs were incubated for 35 min in a 37°C water bath. Protease activity was stopped by five to six instillations of FBSS supplemented with 10% FCS and 10 µg/ml of DNase I (FBSS II). Next, the trachea and major bronchi were removed, and the remaining lung tissue was homogenized in FBSS II for 10 min at 4°C. The homogenate was filtered through a series of nylon meshes ranging from 40 to 160 µm, reconstituted to 400 ml with FBSS II, and spun at 200 g for 10 min. The pellet was washed twice in Dulbecco's modified Eagle's medium nutrient mixture Ham's F-12 (DME-F-12; Sigma) supplemented with L-glutamine (292 µg/ml), penicillin (10,000 U/ml), streptomycin (10 mg/ml), and gentamicin (48 µg/ml). Residual erythrocytes were removed by hypotonic lysis, and then the cells were washed twice with DME-F-12, filtered through 70-µm nylon mesh, and washed again in DME-F-12. Residual alveolar macrophages were removed by incubating the cells for 1 h at 37°C in 5% CO2 on tissue culture dishes coated with rabbit IgG. After incubation, the supernatant was spun at 200 g for 15 min. The resulting type II cell pellet was resuspended in DME-F-12 supplemented with 12% FCS, plated onto tissue culture dishes coated with collagen IV (Sigma), and incubated at 37°C in 5% CO2. The cells were split on reaching 80-90% confluence. The identity of the cells was verified by modified Papanicolaou stain, cytokeratin, and vimentin immunocytochemistry and electron microscopy. Only cultures containing >99% type II cells as determined by cytokeratin and vimentin stains were used for the experiments. The cells were used before passage 3.Determination of Fas Expression by Flow Cytometry
Briefly, rabbit type II cells were cultured in complete medium until they reached 70-90% confluence, detached with 0.025% trypsin containing 0.26 mM EDTA (GIBCO BRL, Life Technologies, Gaithersburg, MD), and washed with PBS. The cell pellet was resuspended at 4 × 106 cells/ml in PBS with 10% FCS (HyClone, Logan, UT) and incubated for 45 min at 4°C in the dark with 10 µl of either anti-Fas R-PE or control antibody (0.5 mg/ml) per 106 cells. After incubation, the cells were washed twice, resuspended in 250 µl of PBS, and then analyzed by flow cytometry with a FACScan instrument (Becton Dickinson, San Jose, CA).Determination of Apoptosis
Rabbit type II cell viability in vitro was determined by Alamar blue reduction, and apoptosis was confirmed by acridine orange staining (4, 32). Apoptotic cells in tissue sections were identified by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) method as previously described (32).Histopathology Protocols
Lung injury score. Each slide was evaluated without knowledge of the treatment group as previously described (31). Briefly, a total of 300 alveoli were counted on each slide at ×400 magnification. Within each field, points were assigned according to predetermined criteria as previously published (31). All the points for each category were added and weighed. The injury score was calculated according to the following formula: injury score = [(alveolar hemorrhage points/no. of fields) + 2(alveolar infiltrate points/no. of fields) + 3(fibrin points/no. of fields) + (alveolar septal congestion/no. of fields)]/total number of alveoli counted.
Fas immunohistochemistry. Immunohistochemistry was performed with Vector Elite ABC-HP kit (Vector Laboratories, Burlingame, CA). Briefly, the slides were deparaffinized by washing twice in xylene for 5 min and rehydrated by washing twice in 100% ethanol for 3 min, twice in 95% ethanol for 3 min, and once in distilled H2O for 5 min. The slides were rinsed twice with PBS for 5 min, and the samples were digested with pepsin for 10 min (Digest-All kit, Zymed, San Francisco, CA). After digestion, the slides were rinsed twice with PBS for 5 min and blocked with 10% normal rabbit serum for 60 min at room temperature. Then the samples were labeled with goat anti-human Fas overnight in a moist chamber at 4°C. Next, the slides were rinsed twice with PBS and labeled with anti-goat biotinylated antibody for 2 h at 4°C. The slides were rinsed twice with PBS and incubated with 0.3% H2O2 in methanol for 30 min to block endogenous peroxidases and then rinsed twice with PBS. The samples were labeled with ABC-HP and incubated in a moist chamber for 60 min at room temperature, rinsed twice with PBS, and developed in a moist chamber with VIP substrate (Vector) for 1-15 min in the dark at room temperature. The slides were rinsed with running deionized H2O for 5 min and counterstained with 1% methyl green for 5 min. The slides were dehydrated with ethanol, incubated in xylene for 5 min, and mounted with Cytoseal XYL.
Immunohistochemistry for IL-8. IL-8 was detected with goat-anti rabbit IL-8 with the same protocol described in Fas immunochemistry with the following differences: 1) the blocking buffer was 5% nonfat milk in 3% rabbit serum and 2) the signal was developed with diaminobenzidine (SigmaFast DAB tablets).
Statistical Analysis
Comparisons between two unpaired groups were done with the Mann-Whitney U-test. Comparisons between paired data were done with the Wilcoxon rank sum test. A P value of <0.05 was considered significant. Data are means ± SE. ![]() |
RESULTS |
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Cell Features
To determine whether primary rabbit alveolar type II cells express Fas and become apoptotic in response to rh-sFasL in vitro, type II cells were isolated from normal rabbit lungs. The cells exhibited multiple features characteristic of type II pneumocytes. Specifically, the cells were nonciliated cells grown as flat monolayers (Fig. 1A) and contained dense blue granules by the Papanicolaou stain (Fig. 1B). Moreover, the cells stained positive for cytokeratin (Fig. 1C), and electron microscopy demonstrated the presence of lamellar bodies (Fig. 1D). Neurosecretory granules and myofibrils were absent.
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Fas Expression in Cultured Type II Cells and in Lung Sections
Cultured rabbit type II cells expressed Fas on the membrane surface as determined by flow cytometry (Fig. 2). To confirm that type II cells express Fas in vivo, rabbit lung sections were stained for Fas with immunohistochemistry. Fas expression was localized to the alveolar epithelium, the membrane of alveolar macrophages, and the airway epithelium (Fig. 3).
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Proapoptotic Effects of FasL In Vitro
To determine whether alveolar cells become apoptotic in response to Fas ligation, primary rabbit type II cells and alveolar macrophages recovered from the lungs of normal rabbits were incubated for 18 h in medium supplemented with serial dilutions of rh-sFasL ranging from 31.25 to 500 ng/ml. Cell viability was determined with the fluorescent Alamar blue assay, and the results are shown as percent fluorescence of live control cells. Type II cells showed a dose-related decrease in viability in response to sFasL, from 70.3 ± 3.4% at a rh-sFasL dose of 32.5 ng/ml (P < 0.05 compared with live control cells) to 49.5 ± 5.2% at a rh-sFasL dose of 500 ng/ml (Fig. 4). In contrast, rabbit alveolar macrophages did not show decreases in viability in response to rh-sFasL (Fig. 4). Apoptosis was confirmed morphologically in type II cells by acridine orange staining (Fig. 5).
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Biological Relevance of the In Vitro Findings
To determine biological relevance of the in vitro findings, rh-sFasL was instilled in the right lung of three rabbits with a pediatric bronchoscope. As a control, rh-sFasL plus Fas:Ig was instilled into the left lung of each rabbit. The Fas:Ig is a fusion protein that binds and neutralizes sFasL. Twenty-four hours after instillation, the BAL fluid from the right lungs contained a significantly higher total protein concentration than the fluid from the left (control) lungs (80.1 ± 7.9 and 60.8 ± 4.6 µg/ml, respectively; P < 0.01; Fig. 6A). There was a trend toward higher numbers of total BAL fluid polymorphonuclear neutrophils in the right lungs compared with the left (control) lungs (9.2 ± 4.5 × 105 and 1.5 ± 0.6 × 105 cells, respectively; P = not significant; Fig. 6B).
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The right lungs showed neutrophilic infiltrates, whereas the left lungs
appeared normal (Fig. 7, A and
B). A semiquantitative histopathological analysis
showed a significantly higher lung injury score in the right lungs
(137 ± 107 for the right lungs and 17.2 ± 5.4 for the left;
P < 0.05). TUNEL assays revealed the presence of
apoptotic cells in the alveolar walls (Fig. 7, C and
D), and immunohistochemistry for IL-8 showed staining in alveolar macrophages in the rh-sFasL-treated lungs (Fig. 7,
E and F).
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DISCUSSION |
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The goal of this study was to determine whether rh-sFasL can induce apoptosis of alveolar pneumocytes and lung injury in rabbits. The main findings are that rabbit alveolar epithelial type II cells express Fas in vivo and in vitro and that rabbit type II cells undergo apoptosis when exposed to rh-sFasL in vitro. In addition, rabbits treated with intrabronchial rh-sFasL developed lung injury characterized by neutrophilic infiltrates, increased total protein in the lung lavage, and local expression of the proinflammatory cytokine IL-8.
The finding that primary rabbit type II pneumocytes are susceptible to apoptosis mediated by rh-sFasL agrees with previous findings in primary rat type II cells (45, 46). The relevance of these in vitro studies was shown in vivo because the administration of rh-sFasL to the lungs of rabbits resulted in lung injury characterized by neutrophilic inflammation, swelling of the alveolar walls, and increased BAL fluid total protein. Previous studies (10, 13, 15, 33) in rodents had shown that the administration of antibodies that activate Fas to the lungs leads to apoptosis of type II cells and development of neutrophilic lung injury in the short term and pulmonary fibrosis in the long term. However, the significance of those studies has been questioned because some authors (40, 42) have suggested that sFasL is not a biologically relevant activator of Fas. Here we show that rh-sFasL induces apoptosis of alveolar type II cells in vitro and in vivo as well as neutrophilic lung injury with increased BAL fluid total protein concentration. The most likely explanation for the increase in BAL fluid total protein is an increase in the permeability of the alveolar epithelium. These findings cannot be ascribed to reagent contamination with LPS because first, rh-sFasL was determined to be LPS free, and second, Fas:Ig completely blocked the effects of rh-sFasL.
The observation that rh-sFasL can induce lung injury is an important observation because elevated concentrations of sFasL are present in the alveoli of patients with several lung diseases associated with alveolar epithelial injury, including ARDS, idiopathic pulmonary fibrosis, BOOP, and interstitial pneumonia associated with collagen vascular diseases (25, 32). In patients with early ARDS, the patients who died had significantly higher concentrations of sFasL in their lungs compared with patients who survived (32). More recently, Liu et al. (29) found that membrane-bound FasL does not induce apoptosis in the alveolar epithelial cell line MLE-15.
It should be emphasized, however, that further studies are needed to
determine whether differences in aggregation or the presence of
inhibitors modulate the activity of sFasL in human diseases. For
example, in humans with early ARDS, the BAL fluid contained significant
concentrations of sFasL (mean 215.4 pg/ml) (32). The sFasL
contained in these BAL fluid samples was capable of inducing
Fas-mediated apoptosis in human DLECs. However, the BAL fluid
from patients at risk for ARDS contained similar concentrations of
sFasL (mean 216.1 pg/ml) yet failed to induce apoptosis in DLECs. This was not due to changes in the expression of Fas in DLECs.
It is possible that the apoptotic effect of sFasL is dependent on
complex interactions between agents that promote sFasL aggregation, soluble inhibitors, and intracellular factors. Although the mechanisms that promote aggregation of sFasL remain unclear, soluble inhibitors of
sFasL, such as soluble Fas, are also present in patients with BOOP
(25). Soluble Fas results from cleavage of membrane Fas by
metalloproteinases and can act as a "sink" for sFasL (23, 35). Another potential inhibitor is the Fas decoy receptor DCR3 (TR6), which binds and inactivates sFasL (49). Additional
factors that could modify the effects of Fas include the presence of
reactive oxygen species and preexposure to inflammatory cytokines such as IL-1 and tumor necrosis factor-
(1, 7, 18).
Finally, intracellular factors could also play a role. Fas-associated
phosphatase-1, an intracellular phosphatase that interacts with the
cytosolic domain of Fas and inhibits Fas-mediated apoptosis, is
expressed in bronchial epithelial cells (27).
The expression of Fas in rabbit lungs was localized by
immunohistochemistry to the alveolar walls, the bronchial epithelium, and the membrane of alveolar macrophages. In addition to demonstrating that the alveolar epithelium of rabbits expresses Fas in vivo, the
finding that resident alveolar macrophages express Fas yet are
resistant to Fas-mediated death has important implications. The
potential role of the Fas/FasL system in the pathogenesis of acute lung
injury may go beyond the induction of apoptosis. Binding of
FasL to Fas can lead to activation of NF-B and release of
inflammatory cytokines (34, 38, 39). Human alveolar
macrophages release IL-8 when exposed to FasL (37), and we
identified local IL-8 production at the site of rh-sFasL instillation
in rabbit lungs. Furthermore, neutrophil recruitment to the lungs in
response to inhaled LPS is depressed in mice deficient in Fas
expression (lpr mice; Matute-Bello, unpublished data). It is
generally thought that within a given cell, Fas ligation can lead to
either apoptosis or NF-
B activation but not to both
simultaneously. Thus activation of the Fas/FasL system in the lungs
could result in both apoptosis of alveolar epithelial cells and
release of proinflammatory cytokines by alveolar macrophages,
ultimately triggering an inflammatory response. Supporting this
concept, the intrabronchial administration of sFasL resulted in both
increased BAL fluid total protein and polymorphonuclear neutrophil recruitment.
In summary, we have demonstrated that the alveolar epithelium of normal rabbits expresses Fas and that sFasL induces lung injury in vivo in rabbits. We conclude that the Fas/FasL system may play a role in the pathogenesis of lung injury by playing a dual role: first, by inducing apoptosis of alveolar epithelial cells, and second, by inducing an inflammatory phenotype in resident alveolar macrophages, thus contributing to a neutrophilic inflammatory response.
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ACKNOWLEDGEMENTS |
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We thank Drs. Emil Chi and Mechtild Jonas, who kindly generated the electron microscopy images, and Krystine Wynant for expert technical assistance.
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FOOTNOTES |
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This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-30542, HL-65892 (both to T. R. Martin), and HL-65649 (to W. C. Liles).
Address for reprint requests and other correspondence: T. R. Martin, Seattle VA Medical Center, 151L, 1660 South Columbian Way, Seattle WA 98108-1597 (E-mail: trmartin{at}u.washington.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 December 2000; accepted in final form 21 February 2001.
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