Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Surfactant protein (SP) D is a pulmonary surfactant-associated protein that may function in lung host defense. SP-D is produced by alveolar type II cells and nonciliated bronchiolar epithelial (Clara) cells of the airway and is secreted into the air space. Here we investigated whether alveolar macrophages degraded SP-D in vitro. We also examined the effects of SP-A and lipids on SP-D metabolism. The results showed that alveolar macrophages bound and degraded SP-D in a time- and temperature-dependent fashion. After 100 min of incubation, the formation of trichloroacetic acid-soluble degradation products increased 4-fold in the medium and 30-fold in the cells. The degradation of SP-D was via a cell-associated process because SP-D was not degraded when incubated in medium previously conditioned by alveolar macrophages. Gel autoradiography of cell lysate samples after incubation with 125I-labeled SP-D demonstrated an increase in degradation products, further confirming the degradation of SP-D by alveolar macrophages. In addition, the degradation of SP-D was not affected by coincubation with SP-A or surfactant-like liposomes containing either phosphatidylglycerol or phosphatidylinositol. In conclusion, alveolar macrophages rapidly degrade SP-D and may play an important role in SP-D turnover and clearance.
alveolar surfactant; collectin; C-type lectin; alveolar type II cells
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INTRODUCTION |
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SURFACTANT PROTEIN (SP) D is one of the four surfactant-associated proteins. Although a role for SP-D in reduction of alveolar surface tension or regulation of surfactant metabolism has not been defined, several studies suggest that SP-D may function in pulmonary host defense. For example, SP-D binds and aggregates various gram-negative bacteria (11), interacts with Pneumocystis carinii and mediates organism adherence to alveolar macrophages (19), inhibits the hemagglutination activity of influenza virus (6), and enhances the production of superoxide by alveolar macrophages (23). Many of these host defense-related functions appear to involve alveolar macrophages, at least in vitro.
Because alveolar macrophages are the predominant phagocytic cell in the air space in healthy animals, they may play a role in SP-D metabolism. Alveolar macrophages have been shown to participate in various aspects of surfactant metabolism. For example, alveolar macrophages internalize and degrade surfactant lipids (5, 16, 28) and SP-A (1, 28) and contribute significantly to the clearance of SP-A and surfactant lipids (28). Immunocytochemistry studies showed that Clara cells, type II cells, and alveolar macrophages contained immunoreactive SP-D (24), but only type II cells and Clara cells contained SP-D mRNA by in situ hybridization (25). In addition, SP-D was shown to bind to alveolar macrophages specifically (10), which is consistent with the idea that SP-D binds to receptors on alveolar macrophage membranes. Furthermore, immunocytochemistry and electron microscopy studies showed that in addition to localization on the cell surface, immunoreactive SP-D was detected intracellularly, predominantly in the vacuolar or vesicular compartments (10) of alveolar macrophages, indicating that SP-D may be internalized and degraded in alveolar macrophages in vivo.
Although SP-A has been implicated in regulation of the metabolism of surfactant lipids, the role of SP-D in the process has not been reported. It is reasonable to speculate that SP-D may be involved in lipid metabolism because it has been reported that lipids coisolate with SP-D from the lavage of silica-treated rats (13). Furthermore, SP-D binds to phosphatidylinositol (PI) and aggregates PI-containing liposomes (20). Although PI is a minor component of surfactant lipids isolated from healthy animals (<5% of total surfactant lipids; see Ref. 27), there can be an increase in PI and a decrease in phosphatidylglycerol (PG) in surfactant lipids under certain pathological conditions (7, 8). Moreover, SP-D coexists with other SPs in the air space, and it has been shown that SP-D binds to SP-A (18) in vitro. Therefore, it would be interesting and important to examine the effects of surfactant-like lipids and SP-A on SP-D metabolism.
In the current study, we investigated whether SP-D was degraded by alveolar macrophages in vitro. In addition, we examined the effects of SP-A and surfactant-like lipids on SP-D metabolism by alveolar macrophages. These studies provide direct evidence of degradation of SP-D by alveolar macrophages for the first time.
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MATERIALS AND METHODS |
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Materials.
Lipids (L--PG,
L-
-dipalmitoylphosphocholine,
L-
-PI, and cholesterol) were
purchased from Avanti Polar Lipids (Birmingham, AL).
L-[
-
14C]dipalmitoylphosphatidylcholine
(DPPC; 110 µCi/mmol) was purchased from NEN (Boston, MA). Dulbecco's
phosphate-buffered saline, F-12 medium, and lipofectamine were
purchased from GIBCO BRL (Gaithersburg, MD).
-Eagle's minimum
essential medium (MEM) medium and HB-CHO serum-free medium
were from Irvine Scientific (Santa Ana, CA). The pEE14 vector was
purchased from Celltech Therapeutics (Berkshire, UK). The bicinchoninic
acid (BCA) protein assay reagent was purchased from Pierce Chemical
(Rockford, IL). Ecolite liquid scintillation cocktail was from ICN
(Costa Mesa, CA). Bolton-Hunter reagent and Amplify solution were
purchased from Amersham (Little Chalfont, Buckinghamshire, UK). Other
chemicals were from Sigma Chemical (St. Louis, MO). Polyclonal rabbit
anti-rat SP-D antibody was kindly provided by Dr. Samuel Hawgood,
University of California, San Francisco.
Expression of recombinant rat SP-D in Chinese hamster ovary cells. A full-length rat SP-D cDNA construct was kindly provided by Dr. James H. Fisher, Wayne State University. The cDNA was religated into the pEE14 vector, and the orientation of the subclones was determined by restriction mapping.
The Chinese hamster ovary (CHO-K1) cells in F-12 medium were transfected with pEE14-rat SP-D or were mock transfected with pEE14 using lipofectamine. The cells were incubated for 4 h, then 10% (vol/vol) dialyzed fetal calf serum was added, and the incubation continued overnight. The next day, the medium was changed toPurification of recombinant SP-D. The serum-free HB-CHO medium was collected after ~8 days of culture and was dialyzed at room temperature against 25 mM tris(hydroxymethyl)aminomethane (Tris), 140 mM NaCl, and 2 mM CaCl2 with four changes. Subsequently, the medium was applied to a maltose column. After washing with dialysis buffer, SP-D was eluted from the column with 25 mM Tris, 140 mM NaCl, and 2 mM EDTA (pH 7.4). The purified SP-D was analyzed by Coomassie blue staining and Western blot, and the concentration of purified SP-D was measured by BCA assay.
3H labeling of recombinant rat SP-D.
The rat SP-D CHO-K1 cells were cultured in serum-free medium as
described previously except that 20 µCi/ml
[3H]proline were
included in the medium. The medium was collected after ~1 wk, and
SP-D was purified from the medium as described above. The
[3H]SP-D was evaluated
by Western blot, Coomassie blue staining, and gel autoradiography. The
specific activity was ~6 × 104
counts · min1
(cpm) · µg
protein
1.
Iodination of recombinant SP-D. Iodination was performed using the Bolton-Hunter reagent according to the method described by Kuan et al. (10). Approximately 20 µg of recombinant rat SP-D were incubated with dried Bolton-Hunter reagent in a final volume of 0.2 ml of 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-150 mM NaCl (HBS; pH 7.4) containing 2 mM CaCl2, 0.1% Triton X-100, and 30 mM maltose at 4°C for 15 min. The iodinated SP-D was separated from free iodine by a P-2 column (Bio-Rad) in HBS containing 0.1% Triton X-100 at room temperature. The specific activity of the protein was ~4 × 106 cpm/µg protein. The protein was subsequently dialyzed against HBS containing 2 mM EDTA at room temperature to decrease further the free iodine associated with the protein. Iodinated SP-D was used within 24 h after the labeling.
Preparation of liposomes. Small unilamellar liposomes were prepared by extrusion from a French pressure cell as described previously (28). The PG-containing liposomes consisted of (by weight) 54% DPPC, 27% egg PC, 11% egg PG, and 8% cholesterol, and the PI-containing liposomes were the same as PG-containing liposomes except that PI (13% by weight) was substituted for PG.
Isolation of cells. Alveolar macrophages were isolated by lung lavage of 250- to 300-g male Sprague-Dawley rats (Charles River Laboratory, Raleigh, NC). Briefly, rats were killed by injection of Nembutal and exsanguination. The lungs were removed from the chest and were lavaged eight times with phosphate-buffered saline (PBS) containing 0.2 mM EDTA. The alveolar macrophages were collected by centrifugation at 200 g for 10 min. The purity and viability of the cells were routinely >95%.
Degradation of [3H]SP-D by alveolar macrophages. The isolated alveolar macrophages (2.5 × 106) were resuspended in 0.5 ml of incubation buffer (PBS with 0.9 mM CaCl2 and 0.1% bovine serum albumin) and various concentrations of [3H]SP-D. In some experiments, primary cultured rat lung fibroblasts (15) were used in the degradation assay. To study the effects of lipids and SP-A on SP-D metabolism, human SP-A (purified from proteinosis patient lavage) or PI- or PG-containing liposomes were coincubated with [3H]SP-D and cells in the experiments. In gel autoradiography experiments, 125I-labeled SP-D was used instead of [3H]SP-D to increase the sensitivity. The cells were incubated at 37 or 4°C with gentle shaking for various amounts of time. After incubation, the cells were collected by centrifugation at 200 g for 10 min (or 500 g for 5 min), and the medium was saved for further analysis. The cells were washed one time and then were transferred to a new tube to minimize nonspecific binding of radioactivity to the tubes, followed by two washes. For binding experiments, the cells were lysed in 0.2 ml of cell lysis buffer (150 mM NaCl, 50 mM phosphate buffer, 0.5% Nonidet P-40, and 2 mM EDTA), of which 0.15 ml was analyzed for radioactivity in an LS 1800 scintillation counter (Beckman, Fullerton, CA), and the remaining 0.05 ml was assayed for protein using BCA assay. For protein degradation studies, the cells were resuspended into 0.5 ml of the incubation buffer containing 0.1% BSA, and trichloroacetic acid (TCA) was added to a final concentration of 10% to both the cells and medium. The samples were then incubated on ice for 25 min and were centrifuged at 9,000 g for 10 min at 4°C. The supernatants were transferred to scintillation vials, and the pellets were resuspended in PBS containing 0.1% BSA and were transferred to scintillation vials. Four milliliters of scintillation cocktail were added to each sample.
In some experiments, alveolar macrophages were first preincubated with 2 µg/ml [3H]SP-D at 4°C for 2 h. Then after being washed and transferred to new tubes, the cells were warmed to 37°C and were incubated at 37°C for another 100 min, followed by TCA precipitation of medium and cell samples. Control experiments were performed by preincubation of cells without SP-D at 4°C for 2 h, addition of 2 µg/ml [3H]SP-D to the cells after they were warmed to 37°C, and continuation of the incubation for another 100 min.Gel autoradiography.
Samples with 125I-SP-D were
analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis (PAGE) and were stained with Coomassie blue. After
being destained, the gels were dried and exposed to X-OMAT film (Sigma
Chemical) for various amounts of time at 80°C depending on
the radioactivity of the samples loaded on the gels.
Statistical analysis. Most of the results are shown as means ± SE. Student's t-test was performed for the comparison of different values with the corresponding controls for significance, which was accepted at P < 0.05.
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RESULTS |
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3H-labeled recombinant rat SP-D. Recombinant rat SP-D was expressed in CHO-K1 cells as described in MATERIALS AND METHODS. To radiolabel SP-D, CHO cells were incubated with [3H]proline-supplemented serum-free medium. The medium was collected after 7-8 days of culture and was applied to a maltose-affinity column to purify the [3H]SP-D. The results (Fig. 1, lanes 1 and 2) of immunoblotting demonstrated the comparison of recombinant [3H]SP-D and wild-type SP-D. The major form of SP-D is the high-molecular-mass form of ~43 kDa; the low-molecular-mass form is probably the deglycosylated form of SP-D because the 43-kDa form shifted to the low-molecular-mass form after treatment with N-glycosidase. On nonreducing gels, the major form of both recombinant and wild-type SP-D was ~130 kDa (data not shown). Coomassie blue staining (Fig. 1, lane 3) showed that SP-D was the only detectable protein that eluted from the column. This was confirmed by gel autoradiography (Fig. 1, lane 4), demonstrating that [3H]SP-D was the only tritiated protein purified from the column. The specific activity of the [3H]SP-D used in the experiments was ~6 × 104 cpm/µg SP-D. The [3H]SP-D was stable for at least 1-2 mo after the labeling when tested by TCA precipitation and gel autoradiography.
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Degradation of [3H]SP-D by alveolar macrophages. At 4°C (Fig. 2A), the recombinant [3H]SP-D bound to isolated alveolar macrophages in a Ca2+- and concentration-dependent manner. The binding was saturated at an ~1 µg/ml SP-D concentration. Binding of [3H]SP-D to isolated alveolar macrophages was also observed at 37°C (Fig. 2B), although it did not reach saturation. The reason for the lack of saturation at 37°C may be due to the simultaneous uptake and degradation of SP-D by alveolar macrophages at 37°C.
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Effects of SP-A and lipids on [3H]SP-D metabolism by alveolar macrophages. Because SP-D may interact with other surfactant components, including SP-A and surfactant lipids, the effects of SP-A and surfactant-like liposomes containing either PG or PI on SP-D metabolism were evaluated. As shown in Table 3, coincubation of 10 µg/ml SP-A with 1 µg/ml [3H]SP-D did not affect the rapid degradation of [3H]SP-D by alveolar macrophages. Furthermore, as shown in Tables 4 and 5, the formation of TCA-soluble radioactivity was similar in both the medium and the cells in the presence or absence of either type of liposome, indicating that the degradation of [3H]SP-D was not affected by either PG- or PI-containing liposomes.
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DISCUSSION |
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The major findings of this study are summarized as follows. First, SP-D is degraded by isolated alveolar macrophages by a relatively rapid process. Second, the degradation is via a cell-associated process. Third, neither SP-A nor surfactant lipids affect SP-D degradation by alveolar macrophages under current conditions.
Initially, both wild-type and recombinant rat SP-D were radiolabeled with 125I using either Iodo beads (26) or the Bolton-Hunter method (see Ref. 10). However, the 125I-SP-D was not stable for routine metabolism studies because it underwent rapid and spontaneous degradation as assessed by the appearance of TCA-soluble iodinated products. Therefore, we labeled the recombinant SP-D by incorporating tritiated proline into the newly synthesized SP-D in CHO cells. The [3H]SP-D was stable for at least 1-2 mo, and the specific activities were high enough for us to carry out most of the in vitro degradation experiments.
Because recombinant SP-D was used in the metabolic study, there is a concern that it may be metabolized differently than the wild-type SP-D. At present, we cannot rule out this possibility. A direct comparison of recombinant and wild-type SP-D is confounded by the relative instability of the iodinated SP-D. However, recombinant rat SP-D has been very well characterized and has been shown to be quite similar to wild-type SP-D both structurally and functionally (3). For example, recombinant SP-D aggregates gram-negative bacteria as does wild-type SP-D (3), and it binds to PI as does wild-type SP-D (9). Our recombinant SP-D has been characterized with similar experiments, and results showed that the major form of both recombinant and wild-type SP-D was ~130 kDa under nonreducing conditions in SDS-PAGE, and the major form was shifted to a low-molecular-mass form after N-glycosidase digestion. Furthermore, the recombinant SP-D aggregated Escherichia coli and bound to PI-containing liposomes in a similar way as wild-type SP-D (data not shown). Taken together, these results demonstrated that our recombinant SP-D behaves similarly to wild-type SP-D in these functional assays.
There was a significant increase in TCA-soluble SP-D degradation products both in the medium and associated with the alveolar macrophages with increasing incubation time as shown in Fig. 3. This was further confirmed by the gel autoradiography that showed the increase of small degradation products and the changes in the intensity of high- and low-molecular-mass forms of SP-D (Fig. 5). The low-molecular-mass form of SP-D is most likely deglycosylated SP-D because a significant increase in the low-molecular-mass form was detected when SP-D was treated with N-glycosidase (data not shown).
The results with alveolar macrophages in conditioned medium fail to show the degradation of SP-D. This suggests that the cells do not degrade the protein by releasing proteolytic enzymes to the medium but supports a degradative pathway in which SP-D is internalized and degraded inside the cells.
Because of the concern that the background TCA-soluble radioactivity in [3H]SP-D preparations may complicate the measurement of the degradation of SP-D, [3H]SP-D was prebound to alveolar macrophages at 4°C. After the cells were washed and incubated at 37°C for an additional 100 min, the degradation was analyzed by TCA precipitation. The results (Table 1) confirmed that alveolar macrophages degraded SP-D and released most of the TCA-soluble degradation products in the medium.
Based on the results shown in Figs. 2 and 3, A and B, we can estimate the distribution of total TCA-soluble radioactivity. At 75 min, ~2 ng of SP-D (or 120 cpm/0.1 mg cell protein) are associated with the cells at 1 µg/ml SP-D based on the results shown in Fig. 2B. Because ~20% of radioactivity in cells is TCA soluble at 75 min of incubation (Table 5), the amount of TCA-soluble radioactivity represents 0.4 ng of SP-D (2 ng × 0.2). If we assume that the total amount of protein per 106 cells is 0.25 mg (14) and because there are 2.5 × 106 cells in each sample, the total TCA-soluble radioactivity represents ~2.5 ng of SP-D [(0.4 ng SP-D/0.1 mg) × (0.25 mg/106 cells) × (2.5 × 106 cells)]. After 75 min of incubation, the amount of TCA-soluble radioactivity in medium is ~0.85% of total radioactivity (Table 4; 1.35% at 75 min, 0.50% at 5-15 s), representing ~4.25 ng of [3H]SP-D. Therefore, ~63% of the SP-D degradation products are found in the medium [4.25 ng in medium/(4.25 ng in medium + 2.5 ng in cells)]. This indicated that the cells degraded SP-D and released most of the degradation products into the medium.
The comparison of the degradation of SP-D by alveolar macrophages with that by primary cultured lung fibroblasts (Table 2) demonstrates that, in contrast to alveolar macrophages, there was little increase in TCA-soluble radioactivity in the medium or in the cells with fibroblasts after incubation. This result indicates that the rapid degradation of SP-D by alveolar macrophages is not a general process carried out by all types of cells.
We are able to estimate the percentage of SP-D that is degraded by alveolar macrophages and the overall turnover and clearance of SP-D in vivo by making a few assumptions. If we assume that the total number of alveolar macrophages is 27 × 106/rat lung (2), the total volume of hypophase fluid is ~0.5 ml/rat lung (22), and the concentration of SP-D in the air space of rats is 3 µg/ml (4), then using the results we obtained in this study, ~105 ng of SP-D would be degraded in 100 min, which is ~7% of the total SP-D pool in the lung. Based on this calculation, if we assume that SP-D is degraded predominantly by alveolar macrophages in the lung, the turnover time of SP-D would be ~24 h. Because free amino acids could be released from SP-D during the degradation, some of the [3H]proline might be released and reutilized within the incubation period. Therefore, the degradation of SP-D could be underestimated. In addition, these results are based on in vitro analyses and some perhaps oversimplified assumptions. Nevertheless, this is a general estimate of the capability of alveolar macrophages to degrade SP-D. Electron microscopy studies demonstrated that alveolar macrophages were one of the major sites for SP-D localization in the alveolar space, and no obvious staining was detectable in the alveolar lining (10). Taken together, our results, as well as the above observations, support the possibility that alveolar macrophages play a major role in SP-D degradation in the lung. Further studies with other pulmonary cells such as type II cells and in vivo turnover studies with SP-D should help to understand further the metabolic process of SP-D in the lung.
Based on the calculation from actual counts and the specific activities
of [3H]SP-D, ~10 ng
of SP-D were degraded by 2.5 × 106 alveolar macrophages after a
100-min incubation. This accounts for 2% of SP-D added to the medium
(Table 4). In contrast, ~15% of added SP-A was degraded after a
100-min incubation (28). The difference in the degradation of SP-A and
SP-D may be due to the distinct metabolic pathway of these two
proteins. It may also depend on the different binding properties of
SP-A and SP-D to alveolar macrophages. It has been shown that SP-D
binds to alveolar macrophages with a dissociation constant
(Kd) of 1.4 × 106 M (10), whereas
SP-A binds to alveolar macrophages with a
Kd of 4 × 10
9 M (21). Therefore, the
binding affinity of SP-D to alveolar macrophages is much lower than
that of SP-A. In addition, cell-associated partial degradation products
were detected (Fig. 5), and there was a rapid increase in TCA-soluble
radioactivity associated with the cells after incubation with SP-D
(Fig. 3A). In contrast, there were
no detectable partial degradation products (data not shown) and very
little TCA-soluble radioactivity in the cells (28) after incubation
with SP-A.
The effects of SP-A on SP-D metabolism were also studied. SP-D has been shown to interact with SP-A and to counteract the inhibition of type II cell lipid secretion by SP-A (12). However, our results indicated that under the current experimental conditions SP-A did not affect SP-D metabolism. It would be interesting to test if the coincubation of SP-D and SP-A would affect SP-A degradation.
Although the majority of SP-D does not seem to be associated with lipids in the lavage fluid, some of the SP-D may associate with surfactant lipids and other surfactant components (13). SP-D has been shown to bind to PI and to aggregate PI-containing liposomes in vitro (20). Therefore, SP-D may interact with surfactant lipids via PI, especially under circumstances in which PI levels are elevated, such as in alveolar proteinosis and silicosis. We speculated, therefore, that the metabolism of SP-D might be affected by PI-containing liposomes to a greater extent than by PG-containing liposomes. However, neither PG- nor PI- containing liposomes affected the degradation process of SP-D by alveolar macrophages (Tables 4 and 5). The lack of any effect of lipids on SP-D metabolism could be due to the lack of aggregation of lipids by SP-D. Our results with the liposomes used in these studies showed that although SP-D bound to PI-containing liposomes, it did not aggregate either PG- or PI- containing liposomes. The disparity in the lipid aggregation results may be due to the different experimental conditions. In our experiments, we used PBS containing a lower Ca2+ concentration (1 mM), which is more physiologically relevant, and used liposomes with a lipid composition similar to that of native surfactant. Under the same conditions, SP-A dramatically enhanced lipid aggregation (data not shown). Therefore, the finding that the degradation of SP-A by alveolar macrophages is reduced by the presence of lipids (28) may be a result of the aggregation of lipids by SP-A.
It is important to understand the metabolic pathways of surfactant components because the maintenance of appropriate, functional pools of surfactant is required for normal lung function and possibly maintenance of host defense in the lung. Under certain pathological conditions such as silicosis and proteinosis, there is an accumulation of surfactant components, including SP-D (4). Although in both cases there is significantly more surfactant in the lung, the function of the lung is impaired, indicating that maintaining homeostasis is critical for the normal function of the lung. Whether alveolar macrophages are malfunctional in the above pathological conditions, thereby causing the accumulation of surfactant, including SP-D, is not clear. The mechanism by which alveolar macrophages degrade SP-D under normal and pathological conditions requires further investigation.
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ACKNOWLEDGEMENTS |
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We thank Sabrena Mervin-Blake for preparation of [3H]surfactant protein D (SP-D), Dr. James H. Fisher (Wayne State University School of Medicine) for providing the SP-D cDNA construct, and Dr. James Clarke McIntosh (Duke Hospital, Duke University Medical Center) for providing the primary cultured lung fibroblasts and helpful suggestions. We also thank Dr. Stephen Young (Duke University) for valuable discussions.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-30923.
Address for reprint requests: J. R. Wright, Box 3709, Dept. of Cell Biology, Duke Univ. Medical Center, Durham, NC 27710.
Received 28 February 1997; accepted in final form 29 September 1997.
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