Hypertrophic Alveolar Type II Cells from Silica-treated Rats Are Committed to DNA Synthesis In Vitro Ralph J. Panos, Akira Suwabe, Christina C. Leslie, and Robert J. Mason Departments of Medicine and Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado
Alveolar type II cell hyperplasia and hypertrophy are common reparative responses of the alveolar epithelium after silica-induced lung injury. We studied in vitro DNA synthesis in type II cells isolated after silica instillation in the rat to determine the proliferative potential of silica type II cells in primary culture and to correlate alveolar type II cell size with the level of in vitro DNA synthesis. To determine if the alveolar lining fluid is a source of growth factors that stimulate alveolar type II cell proliferation, we also examined the mitogenic effect of bronchoalveolar lavage fluid (BALF) from silica-treated rats on type II cells in primary culture. Alveolar type II cells were isolated from rats 1, 2, 3, and 4 wk after intratracheal silica instillation, cultured in DME supplemented with 10% fetal bovine serum, and labeled with pH]thymidine from day 1 to day 3 in culture. DNA synthesis was determined by pH]thymidine incorporation and autoradiographic labeling index. The level of thymidine incorporation increased progressively from 22.3 ± 5.4 x 103 dpm/well 7 d after silica instillation to 34.4 ± 5.0 x 103 dpm/well at 28 d. Type II cells isolated 14 d after silica instillation were separated into groups of increasing cell size by centrifugal elutriation. The plating efficiency and alveolar type II cell purity (> 88 %) were the same in all groups of elutriated cells. The hypertrophic type II cells had a higher level of thymidine incorporation (22.0 ± 2.8 X 103 dpm/well) than the normotrophic type II cells (1Ll ± 0.7 X 103 dpm/well) [P < 0.01]). Each group of elutriated type II cells was capable of responding to the same level of stimulated DNA synthesis in the presence of insulin, epidermal growth factor, and cholera toxin. The autoradiographic labeling index increased from 6.2 ± 0.8% in the normotrophic type II cells to 15.8 ± 2.1% in the hypertrophic cells compared to 1.5 ± % in type II cells isolated from saline control animals. BALF from silica-treated rats stimulated the same level of DNA synthesis as that induced by BALF from untreated animals. We conclude that hypertrophic type II cells are a subgroup of alveolar type II cells that are committed to DNA synthesis in primary culture and that the mitogenic factor(s) for alveolar type II cells found in BALF from normal rats is not increased after silica instillation.
om
Acute lung injury frequently results in damage to type I cells and disruption of the alveolar epithelial lining. Repair of the injury occurs when alveolar type II cells proliferate and differentiate into type I cells, restoring the integrity of the alveolar epithelium (1-4). The type I cell is more readily damaged than the type II cell. The type II cell retains its potential for cellular division and subsequent differentiation into a flattened type I cell structured for gas exchange (3). The cell turnover time for alveolar epithelial cells in normal Key lJbrds: type II cell, proliferation, silica, rat (Received in original form October 19, 1989 and in revised form January 25, 1990) Address correspondence to: Ralph 1. Panos, Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson Street, Denver, CO 80206. Abbreviations: bronchoalveolar lavage fluid, BALF; specific pathogen free, SPF. Am. J. Respir. Cell Mol. BioI. Vol. 3. pp. 51-59, 1990
animals is 4 to 5 wk. However, after lung injury caused by oxygen or nitrogen dioxide exposure, the alveolar type II cell turnover time decreases to 3 d (5). During lung growth and after lung injury, both the number and the size of alveolar type II cells increase (6-14). This hyperplasia and hypertrophy of type II cells has been observed in many types of pulmonary disease and lung injury models and has been documented in detail after pneumonectomy and after asbestos or silica exposure in the rat (6, 7, 12-16). Seven days after pneumonectomy in rats, type II cells isolated from the remaining lung are hypertrophic and have an increased level of thymidine incorporation in vitro (7). After 15 d, alveolar type II cell size is normal and the level of thymidine incorporation returns to basal levels. In vivo autoradiographic studies have demonstrated an increased nuclear labeling index of alveolar type II cells in rats after silica instillation or asbestos inhalation (17, 18). After silica instillation in the rat, there is a time- and dosedependent increase in the percentage of hypertrophic type II
52
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 3 1990
cells (13). Although both hyperplasia and hypertrophy of alveolar type II cells have been reported in numerous models of lung injury and appear to be common responses in alveolar epithelial repair, it is not established that the hypertrophic cells are the actual proliferating cells. The regulation of alveolar type II cell proliferation is poorly understood. Leslie and coworkers (19, 20) have demonstrated that mitogenic factors, such as epidermal growth factor, insulin, and cholera toxin, and conditioned media from macrophages all induce type II cell DNA synthesis in vitro. In addition, Brandes and Finkelstein (21) recently demonstrated that stimulated macrophages secrete a factor that stimulates the proliferation of rabbit type II cells. Leslie and colleagues (22) also described a factor(s) in bronchoalveolar lavage fluid (BALF) from normal rats that stimulates DNA synthesis in alveolar type II cells in primary culture. Therefore, BALF recovered from injured lung may be a source of growth factors that can stimulate alveolar type II cell proliferation. Since alveolar type II cell hypertrophy and hyperplasia occur after silica instillation in the rat and the hypertrophic cells can be isolated, we studied the proliferative characteristics of these cells in primary culture. Specifically, we sought (1) to determine the time course of in vitro pH]thymidine incorporation of alveolar type II cells isolated after silica instillation in the rat, (2) to correlate alveolar type II cell size with the level of in vitro DNA synthesis, and (3) to determine if the alveolar lining fluid is a source of growth factors that stimulate alveolar type II cell proliferation after silicainduced lung injury.
Materials and Methods Animals and Silica Instillation Specific pathogen-free (SPF) male Sprague-Dawley rats weighing 175 to 250 g were obtained from Bantin and Kingman (Fremont, CA). SPF rats were housed in a horizontal laminar air flow hood and were transferred to conventional animal quarters after silica or saline instillation. Crystalline silica (Berkeley Min-U-Sil [less than 5-!-'m particle size]; Pennsylvania Glass Sand Corporation, Pittsburgh, PA) was prepared as previously described (23) to remove ferrous contaminants, suspended in sterile 0.15 M NaCI, and sonicated for 10 min prior to use. Rats were anesthetized by i.p. injection of 1.25 ml/kg of an anesthetic solution (ketamine hydrochloride [Bristol Lab, Syracuse, NY}, xylazine [Miles Lab, Shaunee, KS], and 0.15 M NaCI [2: 1:I, vol/vol/vol]). After stage III anesthesia was achieved, the animals were placed in a supine position and orally intubated with an 18-gauge Teflon catheter while the posterior pharynx was illuminated with a fiberoptic light source (Schott Fiber Optics, Southbridge, MA). Once the correct catheter position was ascertained by lung insufflation, the rat was held in a vertical position and silica (100 mg/kg) suspended in 0.5 ml of sterile 0.15 M NaCI was instilled intratracheally followed by 5 ml of air. Saline control animals received 0.5 ml of sterile 0.15 M NaCI in the same manner. After instillation, the animals were ventilated with a Harvard small rodent ventilator (Harvard Apparatus Company, Newport Beach, CA) until they resumed spontaneous respirations.
Bronchoalveolar Lavage Rats were killed 7, 14, 21, and 28 d after silica or saline instillation. After anesthesia with pentobarbital (Abbott Laboratories, North Chicago, IL) and exsanguination, the lungs were perfused and excised as previously described (24). A buffered salt solution (0.15 M NaCI, 0.1 M sodium phosphate buffer, 0.2 M Hepes buffer, 0.15 M KCl, 100:3:6:4, vol/vol! vol/vol, pH 7.4) containing 1 mg/ml glucose and 10 p,g/ml gentamicin was used to lavage the lungs through a tracheal catheter. Five 12-ml aliquots of the balanced salt solution were instilled and aspirated 4 times and then collected. Lavage recovery was approximately 75 % in all groups. Lavage fluid was centrifuged at 230 X g for 10 min at 4 0 C to remove cells and larger silica particles. The pellet was resuspended in sterile 0.15 M saline; cells were counted in a hemacytometer and stained with Diff-Quik" (American Scientific Products, McGraw Park, IL) for differential cell counts. Aliquots of the supernatant were frozen at -20 0 C for protein determination (25). The supernatant was centrifuged at 180,000 x g for 4 h at 4 0 C to remove pulmonary surface active material and instilled silica. The resulting supernatant was concentrated to one-tenth the original volume by Amicon ultrafiltration with a YMIO filter (w. R. Grace & Co., Danvers, MA) and then dialyzed against DME in dialysis tubing with a molecular weight cutoff of 3,500 (Spectra/Por; Spectrum Medical Industries, Los Angeles, CA). This dialyzed material is referred to as 10 X concentrated BALF. The mitogenic activity for type II cells in primary culture is found in the supernatant fraction of the lavage after the surfactant is sedimented (22). BALF from untreated animals was processed in a similar manner. Isolation and Elutriation of Alveolar Type II Cells After lavage, alveolar type II cells were isolated from the excised lungs by elastase dissociation and purification on a discontinuous metrizamide density gradient as described in detail previously (24). Type II cells were isolated from silica- and saline-treated animals 7, 14,21, and 28 d after instillation as well as from untreated SPF rats. Cell viability was determined by erythrosin B exclusion and the purity of alveolar type II cells was assessed by modified Papanicolaou staining (26). For the elutriation experiments, type II cells were isolated from rats 14 d after silica instillation and then suspended in HBSS (GIBCO, Grand Island, NY) containing 3.75 mM Hepes, 1 mg/ml BSA (Sigma, St. Louis, MO), 1 mg/ml deoxyribonuclease I (Sigma), 100 U/ml penicillin, and 100 !-,g/ml streptomycin. The cell suspension was loaded into the elutriator mixing chamber and pumped into the rotor separation chamber at a flow rate of 9 ml/min with a rotor speed of 2,000 rpm (Beckman model J2-21M centrifuge with a JE6B elutriator rotor; Beckman, Palo Alto, CA). Cells were eluted by stepwise increases in flow rate (27, 28). The cells in each fraction were centrifuged at 230 x g rpm for 8 min at 4 0 C, and counted in a hemacytometer. Cell Culture, pH]thymidine Incorporation, and Autoradiography Isolated alveolar type II cells suspended in DME containing 10% FBS, 2 mM glutamine, 10 !-,g/ml gentamicin, 2.5
Panos, Suwabe, Leslie et al.: Hypertrophic Type II Cells
1/-g/ml amphotericin B, 100 V/ml penicillin G, and 100 1/-g/rnl streptomycin were plated onto 48-well plates (Costar, Cambridge, MA) at 2 X 105 cells/well. The cells were maintained at 3r C in a humidified incubator containing 90% air/lO% CO2 , pH]thymidine incorporation was assayed as described previously (20). Briefly, 24 h after plating, the nonadherent cells and media were removed and 0.5 ml of varying concentrations of iox concentrated, dialyzed BALF (0%, 0.5%, 1%, 2%, 5%, 10%, 20%, 50%, and 100%), 0.5 1/-Ci/ml pH]thymidine (adjusted to a specific activity of 1.12 1/-Ci/ nmol) , and FBS to a final concentration of 10% were added and the cells were incubated for 48 h. To determine the effect of mitogenic stimulation, cells were cultured in DME containing 10% FBS and 10 1/-g/ml insulin (Collaborative Research, Bedford, MA), 10 ng/ml epidermal growth factor (Collaborative Research), and 1 1/-g/ml cholera toxin (Schwarz Mann, Orangeburg, NY). Each condition was tested in triplicate. To determine pH]thymidine incorporation, cells were washed with cold 10% TeA, solubilized with 1.0M sodium hydroxide, and aliquots counted by liquid scintillation spectrometry. To determine cell number, cells were harvested with 0.05% trypsin in 0.053 mM EDTA (GIBCO). Trypsin was inactivated by an equal volume ofDME containing 10% FBS, and the cells were counted in a hemacytometer. Additionally, cells were harvested by trypsin/EDTA dissociation, precipitated in 10% TeA, and DNA content was determined by fluorometric assay (29, 30). For the autoradiography experiments, cells were plated onto 35-mm tissue culture dishes (Falcon; Becton Dickson Labware, Lincoln Park, NJ) at 2 X 106 cells/dish. Twentyfour hours after plating, the media was changed to DME containing 10% FBS and 0.5 1/-Ci/ml pH]thymidine (adjusted to a specific activity of 1.12 1/-Ci/nmol). After 48 h, the cells were fixed in 1.5% glutaraldehyde in 0.1 M cacodylate buffer, stained with osmium tetroxide and then tannic acid, coated with a 1:1 dilution of Kodak Nuclear Track Emulsion (Eastman Kodak Co., Rochester, NY), and exposed for 10 to 14 d. Autoradiographs were developed and stained with a polychrome stain (20, 31). Cells containing two or more densely stained inclusions were counted as type II cells. Labeling index was defined as the number of cells that had incorporated (3H]thymidine divided by the total number of counted type II cells. Five hundred cells per dish were counted. Statistics Data are expressed as mean ± SEM. Statistical analysis was performed by Student's t test and significance was set at P < 0.05 (StatWorks; Cricket Software, Philadelphia, PA).
Results Lung Histopathology and Alveolar Type II Cell Recovery and Morphology The morphologic and histopathologic changes after silica instillation were similar to previously described findings of silica-induced lung injury (32-35). One week after silica instillation, subpleural nodules were observed grossly in the excised lungs. Pleural adhesions and mediastinal and hilar
53
TABLE 1
Characteristics of elutriated type II cells isolated 14 d after silica instillation * Elutriation Fraction (ml/min)
9 14 18 22 28 34
Total Cells Recovered after Elutriation
Type II Cell Purity
(%)
(%)
14.0 ± 2.4 20.6 ± 1.3 21.1 ± 1.1 21.5 ± 0.7 19.0 ± 2.5 3.0 ± 0.4
11.6 47.4 88.5 95.4 93.7 92.0
± ± ± ± ± ±
Total Type II Cells Recovered after Elutriation ,
2.4 4.3 2.2 0.6 1.2 1.6
(%)
2.6 ±0.8 13.7 ± 1.6 25.9 ± 1.6 28.5 ± 1.2 24.4 ± 3.0 4.9 ± 0.5
* Animals received silica (100 mg/kg) by intratracheal instillation. Fourteen days later, alveolar type II cells were isolated by elastase dissociation and centrifugation on a discontinuous metrizantide gradient. Silica type II cells were separated by centrifugal elutriation into groups of increasing cell size. Increasing elutriation flow rate corresponds to increasing cell size. Cells were counted in a hemacytometer and the purity of alveolar type II cells was assessed by modified Papanicolaou staining (26). Values are the mean ± SEM of seven experiments.
adenopathy were present 3 to 4 wk after silica instillation. Microscopically, numerous small granulomas and macrophage aggregates were localized around the airways. Large coalescing granulomas and silicotic nodules were distributed heterogeneously throughout the lung from 2 to 4 wk after silica instillation. The alveoli contained numerous neutrophils, large foamy macrophages, cellular debris, and acellular proteinaceous material and were lined by hyperplastic type II cells (data not shown). Alveolar type II cells were isolated 7, 14, 21, and 28 d after silica instillation. The total number of cells isolated per rat was 48.3 ± 3.3 x 106 at day 7, 49.8 ± 8.7 x 106 at day 14, 54.3 ± 9.7 x 106 at day 21, and 53.1 ± 7.9 x 106 at day 28. Most of the cells (78.2 ± 1.8%) excluded the vital dye erythrosin B. Alveolar type II cell purity was 64.4 ± 2.7% (range, 48 to 72%) on the day of isolation, 86.5 ± 2.3 % (range, 72 to 94 %) after 1 d in culture, and 92.0 ± 0.8% (range, 86 to 97 %) after 3 d in culture. Ranges are from alveolar type II cell isolations at all time points after silica instillation. There were no differences in alveolar type II cell purity with time after silica instillation. The total number of cells isolated per rat after saline instillation was 26.6 ± 4.7 x 106 and the viability was 86.4 ± 2.3 %. The alveolar type II cell purity was 73.6 ± 3.0% on the day of isolation and 90.4 ± 0.7% after 1 d in culture. Macrophages and lymphocytes were the major contaminating cell types in the type II cell preparations isolated after either silica or saline instillation. To determine the relationship between alveolar type II cell size and level of DNA synthesis, we separated type II cells isolated from rats 14 d after silica instillation into four groups of increasing cell size by centrifugal elutriation. Previous studies have shown that alveolar type II cells isolated from untreated SPF rats by centrifugal elutriation have a narrow size distribution, with greater than 90 % of type II cells elutriating at flow rates of 18 and 22 ml/min (27, 28). Therefore, cells elutriating at these flow rates were considered normotrophic, whereas type II cells elutriating at higher flow rates were considered hypertrophic (28). The alveolar
54
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 3 1990
40
....:l
30
•
Saline
EJ
Silica
instillation to 34.4 ± 5.0 x 103 dpm/well at 28 d. The level of thymidine incorporation in type II cells at each time point after saline instillation was not significantly different from the level in type II cells from untreated rats (P > 0.08), and the level of thymidine incorporation did not change with time after saline instillation.
o SPF
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Thymidine Incorporation and Autoradiography of Elutriated Groups of Alveolar Type II Cells
P-.8
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10
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7
14
21
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TIME AFTER INSTILLATION (DAYS) Figure 1. Thymidine incorporation in alveolar type II cells isolated from silica-treated rats increases with time after instillation. Rats received either silica (100 mg/kg) or an equivalent volume of saline by intratracheal instillation. SPF are specific pathogen-free rats that were not treated. Type II cells were isolated by elastase dissociation and discontinuous metrizamide gradient purification at weekly intervals for 4 wk, cultured in DME supplemented with 10% FRS, and incubated with ['H]thymidine from day 1 to day 3 of culture. Values are mean ± SEM of six experiments for each silica time point, four experiments for each saline time point, and six experiments for the type II cells isolated from SPF rats.
type II cells recovered at flow rates of28 and 34 ml/min were larger than the cells isolated at flow rates of 18 and 22 ml/min by light microscopy and by flow cytometric analysis of forward light scatter (data not shown). In a previous study, these hypertrophic cells had more protein and phospholipid per cell than the normotrophic cells (28). The cellular distribution, alveolar type II cell purity, and viability of each elutriated fraction are presented in Table 1. Cell recovery after elutriation was 77.8 ± 5.6% (n = 7). Cells elutriating at a flow rate of 9 ml/min consisted primarily of lymphocytes, and the cells separating at 14 mllmin consisted of polymorphonuclear leukocytes and mononuclear cells, including lymphocytes and macrophages, as well as type II cells. Because the fractions elutriating at flow rates less than 18 ml/min contained less than 50% alveolar type II cells, only those cells elutriating at flow rates of 18, 22, 28, and 34 were cultured. The purity of alveolar type II cells in these fractions was greater than 88 % and the viability was greater than 88 %. Time Course of Thymidine Incorporation after Silica Instillation Type II cells were isolated every 7 d for 4 wk after silica instillation and cultured on tissue culture plastic in DME containing 10 % FBS but no other added growth factors. Plating efficiency after 24 h was 31.6 ± 5.6 % at 7 d after silica instillation and 37.5 ± 6.0% at 28 d compared to 30.5 ± 4.2 % for type II cells isolated from untreated rats. There was a time-dependent increase in thymidine incorporation (Figure 1). The level of thymidine incorporation increased progressively from 22.3 ± 5.4 x 103 dpm/well at 7 d after silica
Miller and colleagues (12-14) have demonstrated a time- and dose-dependent hyperplasia and hypertrophy of rat alveolar type II cells after silica-induced lung injury. To determine if the hypertrophic silica alveolar type II cells might account for the increased level of thymidine incorporation, we separated type II cells isolated 14 d after silica instillation into groups of increasing cell size by centrifugal elutriation. As demonstrated in the upper panel of Figure 2, cell size, which is indicated by the flow rate required for collection (28), correlated with thymidine incorporation in the groups of elutriated type II cells. Larger alveolar type II cells incorporated a higher level of thymidine than smaller type II cells. The hypertrophic silica type II cells (fractions 28 and 34) had a higher level of thymidine incorporation (22.0 ± 2.8 x 103 dpm/well) compared to 11.1 ± 0.7 x 103 dpm/ well in the normotrophic silica cells (fractions 18 and 22) (P < 0.01). Thymidine incorporation normalized to DNA content per well for the elutriated silica type II cells in fractions 18, 22, 28, and 34 was 28.1 ± 5.2 x 103 , 32.5 ± 0.5 x 103 , 38.4 ± 3.9 x 103, and 68.7 ± 5.4 x 103 dpm/ug DNA, respectively (n = 3). The cellular DNA content was 5.66 ± 1.37, 5.35 ± 1.00, 5.57 ± 1.21, and 5.28 ± 0.66 Jlg DNA/1 x lQ6 cells for type II cells in fractions 18, 22, 28, and 34, respectively, on day 3 in culture. The level of thymidine incorporation in the normotrophic silica type II cells was not statistically different from the level in type II cells isolated from rats 14 d after saline instillation (P > 0.3). The response of each of the elutriated groups of silica cells to mitogenic stimulation by insulin, epidermal growth factor, and cholera toxin was the same (Figure 2, lower panel). Thus, each group of elutriated type II cells was capable of responding to the same level of §titnulated DNA synthesis in the presence of these mitogens. To determine if there was an increase in cell number during the labeling period, cell counts were performed on day 1 and day 3 of culture. Plating efficiency at 24 h for the elutriated groups of silica type II cells in fractions 18, 22, 28, and 34 was 36.7 ± 5.1, 31.8 ± 6.3, 28.3 ± 6.6, and 30.0 ± 5.6 %, respectively. The cell number per well did not change from day 1 to day 3 in any of the groups of elutriated silica type II cells. However, in the largest silica type II cells, fraction 34, there was a trend toward increasing numbers of cells during the 48-h labeling period (59.9 ± 11.1 x 103 cells/well on day 1 and 73.3 ± 10.2 x 103 cells/well on day 3), but this increase was not statistically significant. To determine the fraction of cells undergoing DNA synthesis and to verify that thymidine incorporation was occurring in type II cells, the nuclear labeling index was determined by autoradiography using the cultured groups of elutriated silica type II cells. The labeling index of elutriated type II cells isolated 14 d after silica treatment increased
Panos, Suwabe, Leslie et al.: Hypertrophic Type II Cells
55
TABLE 2
Autoradiographic labeling index of elutriated silica type II cells correlates with increasing cell size"
30 --J --J
Elutriation Fraction (ml/min)
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20
18
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10
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SAL
18
22
28
34
ELUTRIATION FRACTION (rn l/rn tn) 100.-----------------...,
80
Labeling Index (%)
6.2 9.7 11.8 15.8
± ± ± ±
Binucleated Type II Cells (%)
0.8 1.9 1.3 2.1
1.4 4.5 8.3 11.1
± ± ± ±
0.3 1.1 0.6 1.9
* Animals received silica (100 mg/kg) by intratracheal instillation. Fourteen days later, alveolar type II cells were isolated by elastase dissociation and centrifugation on a discontinuous metrizamide gradient. Silica type II cells were separated by centrifugal elutriation into groups of increasing cell size. Increasing elutriation flow rate corresponds to increasing cell size. Cells were cultured in DME supplemented with 10% FBS and incubated with [3Hlthymidine from day 1 to day 3 of culture. The cells were fixed in glutaraldehyde in cacodylate buffer, stained with osmium tetroxide and then tannic acid, coated with a photographic emulsion, and exposed for 10 to 14 d. Autoradiographs were developed and stained with a polychrome stain (20, 31). Cells containing two or more densely stained inclusions were counted as type II cells. Labeling index was defined as the number of cells that had incorporated [3H]thymidine divided by the total number of counted type II cells. Five hundred cells per dish were counted. Values are the mean ± SEM of five experiments.•
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d;:::;
60
isolated 14 d after saline instillation (1.5
3:' ...... w
LO
0-
X
o~
±
0.01% [n = 4;
P < 0.01]) or the less than 1% labeling index observed by Les40
lie and colleagues (20) for rat alveolar type II cells isolated from normal lungs, cultured and labeled under the same conditions. The percentage ofbinucleated type II cells increased from 1.4 ± 0.3% to 11.1 ± 1.9% from the smallest to the largest group of elutriated silica type II cells (P < 0.001).
20
o 18
22
28
34
ELUTRIATION FRACTION (rn l/min) Figure 2. Upper panel: Thymidine incorporation correlates with
cell size of elutriatedtype II cells isolated from silica-treated rats. Animalsreceived either silica (100 mg/kg) or an equivalent volume of saline by intratracheal instillation. Fourteen days later, alveolar type II cells were isolatedby elastase dissociation and centrifugation on a discontinuous metrizamide gradient. Silica type II cells were separatedby centrifugal elutriation into groups of increasing cell size. (Increasing elutriationflow rate corresponds to increasing cell size.) Cells Were cultured in DME supplemented with 10% FBS and incubated with pH]thymidine from day I to day 3 of culture. Values are mean ± SEM of four experiments for the cells isolated from saline-treated animals (SAL), five experiments for each silica type II cell elutriated fraction, and six experiments for the cells from untreated, specific pathogen-free (SPF) animals. Lower panel: Each group of elutriatedtype II cells from silica-treated rats is capable of responding to the same level of stimulated DNA synthesis in the presenceof mitogens. TypeII cells were isolatedfrom silica-treated animals as in the upper panel and cultured in DME supplemented with 10% FBS and incubated with 10 ~g/ml insulin, 10 ~g/ml epidermal growth factor, I ~g/ml cholera toxin, and pH]thymidine from day I to day 3 of culture. Values are mean ± SEM of five experiments for each fraction of elutriated silica type II cells. from 6.2 ± 0.8% in fraction 18 cells (normotrophic silica cells) to 15.8 ± 2.1% in fraction 34 (hypertrophic silica cells) (P < 0.001) (Table 2). All labeling indices were significantly greater than the labeling index of type II cells
Bronchoalveolar Lavage Fluid after Silica-induced Lung Injury Cellular and protein characteristics. The total number of cells recovered in bronchoalveolar lavage increased significantly 7, 14, 21, and 28 d after silica instillation compared to saline-treated and untreated animals (P < 0.001). Total cell count was 1.6 ± 0.3 X 106 cells/rat for untreated animals and 2.7 ± 0.5 x 106 cells/rat for saline-treated animals. The total cell count and differential did not change with time after saline instillation and, therefore, values from all time points are expressed together. In the silica-treated animals, the total cell count reached a maximum 21 dafter silica instillation, 38.7 ± 2.1 X 106 cells/rat, and then decreased slightly at 28 d to 36.7 ± 2.3 X 106 cells/rat (Figure 3). The differential cell count showed a significant increase in the number of neutrophils at 7, 14, 21, and 28 d after silica instillation compared to saline-treated and untreated animals (P< 0.001) (Figure 3). BALF cells consisted of approximately 64 % neutrophils, 30 % macrophages, and 5 % lymphocytes 7 d after silica instillation. The percentage of neutrophils increased to 78 % by day 14 and then decreased to 70% by day 28, whereas the percentage of macrophages reached a nadir of 18% on day 14 and then rose to 26 % by day 28. The differential cell count of the salinetreated animals was the same as that of the untreated animals: 95 % macrophages and 3 % neutrophils. BALF protein content was 0.28 ± 0.02 mg/ml in untreated SPF rats and 0.19 ± 0.01 mg/ml in saline-treated animals, and rapidly rose to greater than 1.0 mg/ml 14 dafter silica instillation. As shown in Figure 4, BALF protein con-
56
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 3 1990
30 ן-
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Alveolar type II cell hyperplasia and hypertrophy are common reparative responses of the alveolar epithelium after silica-induced lung injury. We studied in vitro DNA synthesis in type II cells isolated after silica instillation in the rat to determine the proliferative potential of silica type II cells in primary culture and to correlate alveolar type II cell size with the level of in vitro DNA synthesis. To determine if the alveolar lining fluid is a source of growth factors that stimulate alveolar type II cell proliferation, we also examined the mitogenic effect of bronchoalveolar lavage fluid (BALF) from silica-treated rats on type II cells in primary culture. Alveolar type II cells were isolated from rats 1, 2, 3, and 4 wk after intratracheal silica instillation, cultured in DME supplemented with 10% fetal bovine serum, and labeled with pH]thymidine from day 1 to day 3 in culture. DNA synthesis was determined by pH]thymidine incorporation and autoradiographic labeling index. The level of thymidine incorporation increased progressively from 22.3 ± 5.4 x 103 dpm/well 7 d after silica instillation to 34.4 ± 5.0 x 103 dpm/well at 28 d. Type II cells isolated 14 d after silica instillation were separated into groups of increasing cell size by centrifugal elutriation. The plating efficiency and alveolar type II cell purity (> 88 %) were the same in all groups of elutriated cells. The hypertrophic type II cells had a higher level of thymidine incorporation (22.0 ± 2.8 X 103 dpm/well) than the normotrophic type II cells (1Ll ± 0.7 X 103 dpm/well) [P < 0.01]). Each group of elutriated type II cells was capable of responding to the same level of stimulated DNA synthesis in the presence of insulin, epidermal growth factor, and cholera toxin. The autoradiographic labeling index increased from 6.2 ± 0.8% in the normotrophic type II cells to 15.8 ± 2.1% in the hypertrophic cells compared to 1.5 ± % in type II cells isolated from saline control animals. BALF from silica-treated rats stimulated the same level of DNA synthesis as that induced by BALF from untreated animals. We conclude that hypertrophic type II cells are a subgroup of alveolar type II cells that are committed to DNA synthesis in primary culture and that the mitogenic factor(s) for alveolar type II cells found in BALF from normal rats is not increased after silica instillation.
om
Acute lung injury frequently results in damage to type I cells and disruption of the alveolar epithelial lining. Repair of the injury occurs when alveolar type II cells proliferate and differentiate into type I cells, restoring the integrity of the alveolar epithelium (1-4). The type I cell is more readily damaged than the type II cell. The type II cell retains its potential for cellular division and subsequent differentiation into a flattened type I cell structured for gas exchange (3). The cell turnover time for alveolar epithelial cells in normal Key lJbrds: type II cell, proliferation, silica, rat (Received in original form October 19, 1989 and in revised form January 25, 1990) Address correspondence to: Ralph 1. Panos, Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson Street, Denver, CO 80206. Abbreviations: bronchoalveolar lavage fluid, BALF; specific pathogen free, SPF. Am. J. Respir. Cell Mol. BioI. Vol. 3. pp. 51-59, 1990
animals is 4 to 5 wk. However, after lung injury caused by oxygen or nitrogen dioxide exposure, the alveolar type II cell turnover time decreases to 3 d (5). During lung growth and after lung injury, both the number and the size of alveolar type II cells increase (6-14). This hyperplasia and hypertrophy of type II cells has been observed in many types of pulmonary disease and lung injury models and has been documented in detail after pneumonectomy and after asbestos or silica exposure in the rat (6, 7, 12-16). Seven days after pneumonectomy in rats, type II cells isolated from the remaining lung are hypertrophic and have an increased level of thymidine incorporation in vitro (7). After 15 d, alveolar type II cell size is normal and the level of thymidine incorporation returns to basal levels. In vivo autoradiographic studies have demonstrated an increased nuclear labeling index of alveolar type II cells in rats after silica instillation or asbestos inhalation (17, 18). After silica instillation in the rat, there is a time- and dosedependent increase in the percentage of hypertrophic type II
52
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 3 1990
cells (13). Although both hyperplasia and hypertrophy of alveolar type II cells have been reported in numerous models of lung injury and appear to be common responses in alveolar epithelial repair, it is not established that the hypertrophic cells are the actual proliferating cells. The regulation of alveolar type II cell proliferation is poorly understood. Leslie and coworkers (19, 20) have demonstrated that mitogenic factors, such as epidermal growth factor, insulin, and cholera toxin, and conditioned media from macrophages all induce type II cell DNA synthesis in vitro. In addition, Brandes and Finkelstein (21) recently demonstrated that stimulated macrophages secrete a factor that stimulates the proliferation of rabbit type II cells. Leslie and colleagues (22) also described a factor(s) in bronchoalveolar lavage fluid (BALF) from normal rats that stimulates DNA synthesis in alveolar type II cells in primary culture. Therefore, BALF recovered from injured lung may be a source of growth factors that can stimulate alveolar type II cell proliferation. Since alveolar type II cell hypertrophy and hyperplasia occur after silica instillation in the rat and the hypertrophic cells can be isolated, we studied the proliferative characteristics of these cells in primary culture. Specifically, we sought (1) to determine the time course of in vitro pH]thymidine incorporation of alveolar type II cells isolated after silica instillation in the rat, (2) to correlate alveolar type II cell size with the level of in vitro DNA synthesis, and (3) to determine if the alveolar lining fluid is a source of growth factors that stimulate alveolar type II cell proliferation after silicainduced lung injury.
Materials and Methods Animals and Silica Instillation Specific pathogen-free (SPF) male Sprague-Dawley rats weighing 175 to 250 g were obtained from Bantin and Kingman (Fremont, CA). SPF rats were housed in a horizontal laminar air flow hood and were transferred to conventional animal quarters after silica or saline instillation. Crystalline silica (Berkeley Min-U-Sil [less than 5-!-'m particle size]; Pennsylvania Glass Sand Corporation, Pittsburgh, PA) was prepared as previously described (23) to remove ferrous contaminants, suspended in sterile 0.15 M NaCI, and sonicated for 10 min prior to use. Rats were anesthetized by i.p. injection of 1.25 ml/kg of an anesthetic solution (ketamine hydrochloride [Bristol Lab, Syracuse, NY}, xylazine [Miles Lab, Shaunee, KS], and 0.15 M NaCI [2: 1:I, vol/vol/vol]). After stage III anesthesia was achieved, the animals were placed in a supine position and orally intubated with an 18-gauge Teflon catheter while the posterior pharynx was illuminated with a fiberoptic light source (Schott Fiber Optics, Southbridge, MA). Once the correct catheter position was ascertained by lung insufflation, the rat was held in a vertical position and silica (100 mg/kg) suspended in 0.5 ml of sterile 0.15 M NaCI was instilled intratracheally followed by 5 ml of air. Saline control animals received 0.5 ml of sterile 0.15 M NaCI in the same manner. After instillation, the animals were ventilated with a Harvard small rodent ventilator (Harvard Apparatus Company, Newport Beach, CA) until they resumed spontaneous respirations.
Bronchoalveolar Lavage Rats were killed 7, 14, 21, and 28 d after silica or saline instillation. After anesthesia with pentobarbital (Abbott Laboratories, North Chicago, IL) and exsanguination, the lungs were perfused and excised as previously described (24). A buffered salt solution (0.15 M NaCI, 0.1 M sodium phosphate buffer, 0.2 M Hepes buffer, 0.15 M KCl, 100:3:6:4, vol/vol! vol/vol, pH 7.4) containing 1 mg/ml glucose and 10 p,g/ml gentamicin was used to lavage the lungs through a tracheal catheter. Five 12-ml aliquots of the balanced salt solution were instilled and aspirated 4 times and then collected. Lavage recovery was approximately 75 % in all groups. Lavage fluid was centrifuged at 230 X g for 10 min at 4 0 C to remove cells and larger silica particles. The pellet was resuspended in sterile 0.15 M saline; cells were counted in a hemacytometer and stained with Diff-Quik" (American Scientific Products, McGraw Park, IL) for differential cell counts. Aliquots of the supernatant were frozen at -20 0 C for protein determination (25). The supernatant was centrifuged at 180,000 x g for 4 h at 4 0 C to remove pulmonary surface active material and instilled silica. The resulting supernatant was concentrated to one-tenth the original volume by Amicon ultrafiltration with a YMIO filter (w. R. Grace & Co., Danvers, MA) and then dialyzed against DME in dialysis tubing with a molecular weight cutoff of 3,500 (Spectra/Por; Spectrum Medical Industries, Los Angeles, CA). This dialyzed material is referred to as 10 X concentrated BALF. The mitogenic activity for type II cells in primary culture is found in the supernatant fraction of the lavage after the surfactant is sedimented (22). BALF from untreated animals was processed in a similar manner. Isolation and Elutriation of Alveolar Type II Cells After lavage, alveolar type II cells were isolated from the excised lungs by elastase dissociation and purification on a discontinuous metrizamide density gradient as described in detail previously (24). Type II cells were isolated from silica- and saline-treated animals 7, 14,21, and 28 d after instillation as well as from untreated SPF rats. Cell viability was determined by erythrosin B exclusion and the purity of alveolar type II cells was assessed by modified Papanicolaou staining (26). For the elutriation experiments, type II cells were isolated from rats 14 d after silica instillation and then suspended in HBSS (GIBCO, Grand Island, NY) containing 3.75 mM Hepes, 1 mg/ml BSA (Sigma, St. Louis, MO), 1 mg/ml deoxyribonuclease I (Sigma), 100 U/ml penicillin, and 100 !-,g/ml streptomycin. The cell suspension was loaded into the elutriator mixing chamber and pumped into the rotor separation chamber at a flow rate of 9 ml/min with a rotor speed of 2,000 rpm (Beckman model J2-21M centrifuge with a JE6B elutriator rotor; Beckman, Palo Alto, CA). Cells were eluted by stepwise increases in flow rate (27, 28). The cells in each fraction were centrifuged at 230 x g rpm for 8 min at 4 0 C, and counted in a hemacytometer. Cell Culture, pH]thymidine Incorporation, and Autoradiography Isolated alveolar type II cells suspended in DME containing 10% FBS, 2 mM glutamine, 10 !-,g/ml gentamicin, 2.5
Panos, Suwabe, Leslie et al.: Hypertrophic Type II Cells
1/-g/ml amphotericin B, 100 V/ml penicillin G, and 100 1/-g/rnl streptomycin were plated onto 48-well plates (Costar, Cambridge, MA) at 2 X 105 cells/well. The cells were maintained at 3r C in a humidified incubator containing 90% air/lO% CO2 , pH]thymidine incorporation was assayed as described previously (20). Briefly, 24 h after plating, the nonadherent cells and media were removed and 0.5 ml of varying concentrations of iox concentrated, dialyzed BALF (0%, 0.5%, 1%, 2%, 5%, 10%, 20%, 50%, and 100%), 0.5 1/-Ci/ml pH]thymidine (adjusted to a specific activity of 1.12 1/-Ci/ nmol) , and FBS to a final concentration of 10% were added and the cells were incubated for 48 h. To determine the effect of mitogenic stimulation, cells were cultured in DME containing 10% FBS and 10 1/-g/ml insulin (Collaborative Research, Bedford, MA), 10 ng/ml epidermal growth factor (Collaborative Research), and 1 1/-g/ml cholera toxin (Schwarz Mann, Orangeburg, NY). Each condition was tested in triplicate. To determine pH]thymidine incorporation, cells were washed with cold 10% TeA, solubilized with 1.0M sodium hydroxide, and aliquots counted by liquid scintillation spectrometry. To determine cell number, cells were harvested with 0.05% trypsin in 0.053 mM EDTA (GIBCO). Trypsin was inactivated by an equal volume ofDME containing 10% FBS, and the cells were counted in a hemacytometer. Additionally, cells were harvested by trypsin/EDTA dissociation, precipitated in 10% TeA, and DNA content was determined by fluorometric assay (29, 30). For the autoradiography experiments, cells were plated onto 35-mm tissue culture dishes (Falcon; Becton Dickson Labware, Lincoln Park, NJ) at 2 X 106 cells/dish. Twentyfour hours after plating, the media was changed to DME containing 10% FBS and 0.5 1/-Ci/ml pH]thymidine (adjusted to a specific activity of 1.12 1/-Ci/nmol). After 48 h, the cells were fixed in 1.5% glutaraldehyde in 0.1 M cacodylate buffer, stained with osmium tetroxide and then tannic acid, coated with a 1:1 dilution of Kodak Nuclear Track Emulsion (Eastman Kodak Co., Rochester, NY), and exposed for 10 to 14 d. Autoradiographs were developed and stained with a polychrome stain (20, 31). Cells containing two or more densely stained inclusions were counted as type II cells. Labeling index was defined as the number of cells that had incorporated (3H]thymidine divided by the total number of counted type II cells. Five hundred cells per dish were counted. Statistics Data are expressed as mean ± SEM. Statistical analysis was performed by Student's t test and significance was set at P < 0.05 (StatWorks; Cricket Software, Philadelphia, PA).
Results Lung Histopathology and Alveolar Type II Cell Recovery and Morphology The morphologic and histopathologic changes after silica instillation were similar to previously described findings of silica-induced lung injury (32-35). One week after silica instillation, subpleural nodules were observed grossly in the excised lungs. Pleural adhesions and mediastinal and hilar
53
TABLE 1
Characteristics of elutriated type II cells isolated 14 d after silica instillation * Elutriation Fraction (ml/min)
9 14 18 22 28 34
Total Cells Recovered after Elutriation
Type II Cell Purity
(%)
(%)
14.0 ± 2.4 20.6 ± 1.3 21.1 ± 1.1 21.5 ± 0.7 19.0 ± 2.5 3.0 ± 0.4
11.6 47.4 88.5 95.4 93.7 92.0
± ± ± ± ± ±
Total Type II Cells Recovered after Elutriation ,
2.4 4.3 2.2 0.6 1.2 1.6
(%)
2.6 ±0.8 13.7 ± 1.6 25.9 ± 1.6 28.5 ± 1.2 24.4 ± 3.0 4.9 ± 0.5
* Animals received silica (100 mg/kg) by intratracheal instillation. Fourteen days later, alveolar type II cells were isolated by elastase dissociation and centrifugation on a discontinuous metrizantide gradient. Silica type II cells were separated by centrifugal elutriation into groups of increasing cell size. Increasing elutriation flow rate corresponds to increasing cell size. Cells were counted in a hemacytometer and the purity of alveolar type II cells was assessed by modified Papanicolaou staining (26). Values are the mean ± SEM of seven experiments.
adenopathy were present 3 to 4 wk after silica instillation. Microscopically, numerous small granulomas and macrophage aggregates were localized around the airways. Large coalescing granulomas and silicotic nodules were distributed heterogeneously throughout the lung from 2 to 4 wk after silica instillation. The alveoli contained numerous neutrophils, large foamy macrophages, cellular debris, and acellular proteinaceous material and were lined by hyperplastic type II cells (data not shown). Alveolar type II cells were isolated 7, 14, 21, and 28 d after silica instillation. The total number of cells isolated per rat was 48.3 ± 3.3 x 106 at day 7, 49.8 ± 8.7 x 106 at day 14, 54.3 ± 9.7 x 106 at day 21, and 53.1 ± 7.9 x 106 at day 28. Most of the cells (78.2 ± 1.8%) excluded the vital dye erythrosin B. Alveolar type II cell purity was 64.4 ± 2.7% (range, 48 to 72%) on the day of isolation, 86.5 ± 2.3 % (range, 72 to 94 %) after 1 d in culture, and 92.0 ± 0.8% (range, 86 to 97 %) after 3 d in culture. Ranges are from alveolar type II cell isolations at all time points after silica instillation. There were no differences in alveolar type II cell purity with time after silica instillation. The total number of cells isolated per rat after saline instillation was 26.6 ± 4.7 x 106 and the viability was 86.4 ± 2.3 %. The alveolar type II cell purity was 73.6 ± 3.0% on the day of isolation and 90.4 ± 0.7% after 1 d in culture. Macrophages and lymphocytes were the major contaminating cell types in the type II cell preparations isolated after either silica or saline instillation. To determine the relationship between alveolar type II cell size and level of DNA synthesis, we separated type II cells isolated from rats 14 d after silica instillation into four groups of increasing cell size by centrifugal elutriation. Previous studies have shown that alveolar type II cells isolated from untreated SPF rats by centrifugal elutriation have a narrow size distribution, with greater than 90 % of type II cells elutriating at flow rates of 18 and 22 ml/min (27, 28). Therefore, cells elutriating at these flow rates were considered normotrophic, whereas type II cells elutriating at higher flow rates were considered hypertrophic (28). The alveolar
54
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 3 1990
40
....:l
30
•
Saline
EJ
Silica
instillation to 34.4 ± 5.0 x 103 dpm/well at 28 d. The level of thymidine incorporation in type II cells at each time point after saline instillation was not significantly different from the level in type II cells from untreated rats (P > 0.08), and the level of thymidine incorporation did not change with time after saline instillation.
o SPF
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,0
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20
Thymidine Incorporation and Autoradiography of Elutriated Groups of Alveolar Type II Cells
P-.8
0
10
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7
14
21
28
TIME AFTER INSTILLATION (DAYS) Figure 1. Thymidine incorporation in alveolar type II cells isolated from silica-treated rats increases with time after instillation. Rats received either silica (100 mg/kg) or an equivalent volume of saline by intratracheal instillation. SPF are specific pathogen-free rats that were not treated. Type II cells were isolated by elastase dissociation and discontinuous metrizamide gradient purification at weekly intervals for 4 wk, cultured in DME supplemented with 10% FRS, and incubated with ['H]thymidine from day 1 to day 3 of culture. Values are mean ± SEM of six experiments for each silica time point, four experiments for each saline time point, and six experiments for the type II cells isolated from SPF rats.
type II cells recovered at flow rates of28 and 34 ml/min were larger than the cells isolated at flow rates of 18 and 22 ml/min by light microscopy and by flow cytometric analysis of forward light scatter (data not shown). In a previous study, these hypertrophic cells had more protein and phospholipid per cell than the normotrophic cells (28). The cellular distribution, alveolar type II cell purity, and viability of each elutriated fraction are presented in Table 1. Cell recovery after elutriation was 77.8 ± 5.6% (n = 7). Cells elutriating at a flow rate of 9 ml/min consisted primarily of lymphocytes, and the cells separating at 14 mllmin consisted of polymorphonuclear leukocytes and mononuclear cells, including lymphocytes and macrophages, as well as type II cells. Because the fractions elutriating at flow rates less than 18 ml/min contained less than 50% alveolar type II cells, only those cells elutriating at flow rates of 18, 22, 28, and 34 were cultured. The purity of alveolar type II cells in these fractions was greater than 88 % and the viability was greater than 88 %. Time Course of Thymidine Incorporation after Silica Instillation Type II cells were isolated every 7 d for 4 wk after silica instillation and cultured on tissue culture plastic in DME containing 10 % FBS but no other added growth factors. Plating efficiency after 24 h was 31.6 ± 5.6 % at 7 d after silica instillation and 37.5 ± 6.0% at 28 d compared to 30.5 ± 4.2 % for type II cells isolated from untreated rats. There was a time-dependent increase in thymidine incorporation (Figure 1). The level of thymidine incorporation increased progressively from 22.3 ± 5.4 x 103 dpm/well at 7 d after silica
Miller and colleagues (12-14) have demonstrated a time- and dose-dependent hyperplasia and hypertrophy of rat alveolar type II cells after silica-induced lung injury. To determine if the hypertrophic silica alveolar type II cells might account for the increased level of thymidine incorporation, we separated type II cells isolated 14 d after silica instillation into groups of increasing cell size by centrifugal elutriation. As demonstrated in the upper panel of Figure 2, cell size, which is indicated by the flow rate required for collection (28), correlated with thymidine incorporation in the groups of elutriated type II cells. Larger alveolar type II cells incorporated a higher level of thymidine than smaller type II cells. The hypertrophic silica type II cells (fractions 28 and 34) had a higher level of thymidine incorporation (22.0 ± 2.8 x 103 dpm/well) compared to 11.1 ± 0.7 x 103 dpm/ well in the normotrophic silica cells (fractions 18 and 22) (P < 0.01). Thymidine incorporation normalized to DNA content per well for the elutriated silica type II cells in fractions 18, 22, 28, and 34 was 28.1 ± 5.2 x 103 , 32.5 ± 0.5 x 103 , 38.4 ± 3.9 x 103, and 68.7 ± 5.4 x 103 dpm/ug DNA, respectively (n = 3). The cellular DNA content was 5.66 ± 1.37, 5.35 ± 1.00, 5.57 ± 1.21, and 5.28 ± 0.66 Jlg DNA/1 x lQ6 cells for type II cells in fractions 18, 22, 28, and 34, respectively, on day 3 in culture. The level of thymidine incorporation in the normotrophic silica type II cells was not statistically different from the level in type II cells isolated from rats 14 d after saline instillation (P > 0.3). The response of each of the elutriated groups of silica cells to mitogenic stimulation by insulin, epidermal growth factor, and cholera toxin was the same (Figure 2, lower panel). Thus, each group of elutriated type II cells was capable of responding to the same level of §titnulated DNA synthesis in the presence of these mitogens. To determine if there was an increase in cell number during the labeling period, cell counts were performed on day 1 and day 3 of culture. Plating efficiency at 24 h for the elutriated groups of silica type II cells in fractions 18, 22, 28, and 34 was 36.7 ± 5.1, 31.8 ± 6.3, 28.3 ± 6.6, and 30.0 ± 5.6 %, respectively. The cell number per well did not change from day 1 to day 3 in any of the groups of elutriated silica type II cells. However, in the largest silica type II cells, fraction 34, there was a trend toward increasing numbers of cells during the 48-h labeling period (59.9 ± 11.1 x 103 cells/well on day 1 and 73.3 ± 10.2 x 103 cells/well on day 3), but this increase was not statistically significant. To determine the fraction of cells undergoing DNA synthesis and to verify that thymidine incorporation was occurring in type II cells, the nuclear labeling index was determined by autoradiography using the cultured groups of elutriated silica type II cells. The labeling index of elutriated type II cells isolated 14 d after silica treatment increased
Panos, Suwabe, Leslie et al.: Hypertrophic Type II Cells
55
TABLE 2
Autoradiographic labeling index of elutriated silica type II cells correlates with increasing cell size"
30 --J --J
Elutriation Fraction (ml/min)
~
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20
18
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22
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28 34
10
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SAL
18
22
28
34
ELUTRIATION FRACTION (rn l/rn tn) 100.-----------------...,
80
Labeling Index (%)
6.2 9.7 11.8 15.8
± ± ± ±
Binucleated Type II Cells (%)
0.8 1.9 1.3 2.1
1.4 4.5 8.3 11.1
± ± ± ±
0.3 1.1 0.6 1.9
* Animals received silica (100 mg/kg) by intratracheal instillation. Fourteen days later, alveolar type II cells were isolated by elastase dissociation and centrifugation on a discontinuous metrizamide gradient. Silica type II cells were separated by centrifugal elutriation into groups of increasing cell size. Increasing elutriation flow rate corresponds to increasing cell size. Cells were cultured in DME supplemented with 10% FBS and incubated with [3Hlthymidine from day 1 to day 3 of culture. The cells were fixed in glutaraldehyde in cacodylate buffer, stained with osmium tetroxide and then tannic acid, coated with a photographic emulsion, and exposed for 10 to 14 d. Autoradiographs were developed and stained with a polychrome stain (20, 31). Cells containing two or more densely stained inclusions were counted as type II cells. Labeling index was defined as the number of cells that had incorporated [3H]thymidine divided by the total number of counted type II cells. Five hundred cells per dish were counted. Values are the mean ± SEM of five experiments.•
--J
d;:::;
60
isolated 14 d after saline instillation (1.5
3:' ...... w
LO
0-
X
o~
±
0.01% [n = 4;
P < 0.01]) or the less than 1% labeling index observed by Les40
lie and colleagues (20) for rat alveolar type II cells isolated from normal lungs, cultured and labeled under the same conditions. The percentage ofbinucleated type II cells increased from 1.4 ± 0.3% to 11.1 ± 1.9% from the smallest to the largest group of elutriated silica type II cells (P < 0.001).
20
o 18
22
28
34
ELUTRIATION FRACTION (rn l/min) Figure 2. Upper panel: Thymidine incorporation correlates with
cell size of elutriatedtype II cells isolated from silica-treated rats. Animalsreceived either silica (100 mg/kg) or an equivalent volume of saline by intratracheal instillation. Fourteen days later, alveolar type II cells were isolatedby elastase dissociation and centrifugation on a discontinuous metrizamide gradient. Silica type II cells were separatedby centrifugal elutriation into groups of increasing cell size. (Increasing elutriationflow rate corresponds to increasing cell size.) Cells Were cultured in DME supplemented with 10% FBS and incubated with pH]thymidine from day I to day 3 of culture. Values are mean ± SEM of four experiments for the cells isolated from saline-treated animals (SAL), five experiments for each silica type II cell elutriated fraction, and six experiments for the cells from untreated, specific pathogen-free (SPF) animals. Lower panel: Each group of elutriatedtype II cells from silica-treated rats is capable of responding to the same level of stimulated DNA synthesis in the presenceof mitogens. TypeII cells were isolatedfrom silica-treated animals as in the upper panel and cultured in DME supplemented with 10% FBS and incubated with 10 ~g/ml insulin, 10 ~g/ml epidermal growth factor, I ~g/ml cholera toxin, and pH]thymidine from day I to day 3 of culture. Values are mean ± SEM of five experiments for each fraction of elutriated silica type II cells. from 6.2 ± 0.8% in fraction 18 cells (normotrophic silica cells) to 15.8 ± 2.1% in fraction 34 (hypertrophic silica cells) (P < 0.001) (Table 2). All labeling indices were significantly greater than the labeling index of type II cells
Bronchoalveolar Lavage Fluid after Silica-induced Lung Injury Cellular and protein characteristics. The total number of cells recovered in bronchoalveolar lavage increased significantly 7, 14, 21, and 28 d after silica instillation compared to saline-treated and untreated animals (P < 0.001). Total cell count was 1.6 ± 0.3 X 106 cells/rat for untreated animals and 2.7 ± 0.5 x 106 cells/rat for saline-treated animals. The total cell count and differential did not change with time after saline instillation and, therefore, values from all time points are expressed together. In the silica-treated animals, the total cell count reached a maximum 21 dafter silica instillation, 38.7 ± 2.1 X 106 cells/rat, and then decreased slightly at 28 d to 36.7 ± 2.3 X 106 cells/rat (Figure 3). The differential cell count showed a significant increase in the number of neutrophils at 7, 14, 21, and 28 d after silica instillation compared to saline-treated and untreated animals (P< 0.001) (Figure 3). BALF cells consisted of approximately 64 % neutrophils, 30 % macrophages, and 5 % lymphocytes 7 d after silica instillation. The percentage of neutrophils increased to 78 % by day 14 and then decreased to 70% by day 28, whereas the percentage of macrophages reached a nadir of 18% on day 14 and then rose to 26 % by day 28. The differential cell count of the salinetreated animals was the same as that of the untreated animals: 95 % macrophages and 3 % neutrophils. BALF protein content was 0.28 ± 0.02 mg/ml in untreated SPF rats and 0.19 ± 0.01 mg/ml in saline-treated animals, and rapidly rose to greater than 1.0 mg/ml 14 dafter silica instillation. As shown in Figure 4, BALF protein con-
56
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 3 1990
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