Isolation and Properties of Type II Alveolar Cells from Rat Lung 1 2 •
ROBERT J. MASON,3 MARY C. WILLIAMS, 4 ROBERT D. GREENLEAF,5 and JOHN A. CLEMENTS6
SUMMARY __________________________________________ ______________ Type II alveolar cells can be isolated and partially purified from adult rat lung by a series of steps that includes enzymatic digestion of the lung with trypsin and separation of cells on a discontinuous albumin density gradient. The yield of the isolated type II cells depends on the supplier and the housing of the rats used to prepare the cells. With specific pathogen-free rats housed in a laminar flow hood, the yield was 20.3 X 106 cells per rat, of which 60 per cent were type II cells. With rats from 2 other suppliers and no special housing, the yields were 8.8 and 8.3 X 106 cells per rat, of which 67 and 65 per cent were type II cells. The ultrastructural appearance of the isolated cells was similar to that of cells from intact lung, except for some dilatation of the endoplasmic reticulum and the perinuclear space. Most cells (92 ± 5 per cent) excluded the vital dye, trypan blue. The cells consumed 0 2 at the rate of 76 ± 12 nmole per 106 cells per hour and released only 5.7 ± 2.0 per cent of their lactate dehydrogenase, a cytoplasmic enzyme, into the medium after l hour of incubation. The isolated type II cells contained disaturated phosphatidylcholine, a major component of purified surface-active material. The cells, however, had a low glucose utilization compared to their 0 2 consumption, which may indicate an abnormality in the metabolism of glucose. This population of cells could be further purified to 89 per cent type II cells by unit gravity velocity sedimentation.
Introduction The cellular heterogeneity of the lung makes assignment of specific metabolic functions to individual cell types very difficult. One approach that circumvents the problem of cellular heterogeneity is to study preparations of a single cell (Received in original form November 24, 1976 and in revised form February 2, 1977) 1 From the Cardiovascular Research Institute, University of California Medical Center, San Francisco Ca. 94143. 2 This work was supported by Program Project grant HL-06285 and Pulmonary SCOR grant HL14201 from the U.S. Public Health Service. 3 R. J. Mason is an Established Investigator of the American Heart Association. 4 Mary C. Williams is the recipient of a Research Car.?..!r Development Award (HL-00221) from the National Heart, Lung and Blood Institute. 5 Robert D. Greenleaf is a tr'linee supported by
type. Extensive evidence indicates that type II alveolar cells synthesize and secrete pulmonary surface-active material (1-4). Because alterations in the metabolism of this complex substance have been implicated in numerous disease processes, especially in respiratory distress syndrome of the newborn, a detailed study of the synthesis and secretion of surface-active material by isolated type II cells may help elucidate the pathogenesis of certain diseases and suggest new means of therapy. Before detailed biochemical studies can be performed, methods for isolating type II cells need to be devised. This report describes our method for isolating a partially purified population of type II cells from adult rat lung and some of the metabolic grant HL-05251 from the National Heart, Lung and Blood Institute. 6 John A. Clements is a Career Investigator of the American Heart Association.
AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME 115, 1977
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MASON, WILLIAMS, GREENLEAF, AND
properties of these cells. Kikkawa and associates (2) and Kikkawa and Yoneda (5) have previously reported a method for isolating type II cells from the lungs of rats and rabbits, and their data provide a basis for comparisons of yield, purity, and function. A preliminary report of our method has been published (6). Materials and Methods Solutions for the isolation of type II cells. To make the isolation procedure easier to describe, we list the composition of the solutions used at the beginning. The main balanced salt solution designated, solution A, contained 128 mM sodium chloride (NaCl), 5 mM potassium chloride (KCl), 2.5 mM sodium phosphate buffer, 1.9 mM calcium chloride (CaC1 2 ), 1.2 mM magnesium sulfate (MgSO 4 ), 17 mM HEPES (N-2-hydroxyethyl piperazine N-2-ethane-sulfonic acid), 5.5 mM glucose, and lO p.g of gentamicin per mi. Solution B was the same as solution A, except that CaC1 2 and MgSO 4 were omitted. The pH of solution A was adjusted to 7.4 at room temperature (22° C), but it should be noted that the pH of HEPES buffers is temperature dependent (7). The osmolality of solution A was 290 mOsm, as measured by freezing point depression. An emulsion of fluorocarbon and albumin was prepared for instillation into the lung to facilitate the removal of macrophages by increasing their density. The amount was sufficient for the 4 rats that we routinely used in each experiment. Four milliliters of fluorocarbon (FC-75, Minnesota Mining Co., St. Paul, Minn.) and 12 ml of either defatted bovine serum albumin (10 mg per ml) (Sigma Chemical Co., St. Louis, Mo.) or fraction V bovine serum albumin (10 mg per ml) (Miles Laboratory, Kankakee, III.) dissolved in solution A were sonicated together. The emulsion was diluted with an additional 44 ml of the albumin solution before use. The defatted albumin appeared to make a slightly more stable emulsion, but both albumin solutions were satisfactory. The solution containing trypsin was made by dissolving recrystallized bovine trypsin (3 mg per ml) (Sigma Chemical Co., St. Louis, Mo.) and 30 p.g of deoxyribonuclease (DNase) per ml (Sigma Chemical Co. or P. L. Biochemicals Inc., Milwaukee, Wis.) in solution A. The solution with trypsin inhibitor was made by dissolving I mg of soybean trypsin inhibitor per ml (Sigma Chemical Co., St. Louis, Mo.) and 30 p.g of Dl':ase per ml in solution A. A discontinuous albumin density gradient was made with Pathocyte 4 (Miles Lab., Kankakee, Ill.) diluted to densities of 1.080 and 1.040 with 0.15 M l':aCI. The densities of the albumin solutions were measured in a column of kerosene and carbon tetrachloride, which was calibrated with solutions of sucrose (8). Isolation of cells. Male and female SpragueDawley rats weighing 180 to 250 g were purchased
CLE.~IENTS
from 3 suppliers. The rats from Simonsen (Gilroy, Ca.) and Charles River Breeding Laboratories, Inc. (Wilmington Mass.) were housed at the University of California in animal quarters shared by a number of investigators. No special precautions were taken to prevent pulmonary infections. Animals with grossly visible pulmonary infections at the time of cell preparation were discarded, but obvious pneumonia was an infrequent occurrence ( < 2 per cent). Because of the ubiquitous problem of clinically inapparent murine pneumonia, special precautions were taken with specific pathogen-free rats obtained from Hilltop Lab Animals, Scottdale, Pa. (9 10). Pathogen-free animals were shipped in cages with special filters, housed in a laminar flow hood, and given autoclaved food, water, and bedding. Cultures of the lungs from 2 rats housed in this manner for 2 weeks did not reveal any bacteria or Mycoplasma, and histologic examination failed to show any changes of murine pneumonia. The rats were fed ad libitum until killed. They were anesthetized and anticoagulated by a single intraperitoneal injection containing 250 units of sodium heparin per 100 g of body weight and 5 mg of sodium pentobarbital per 100 g of body weight. Fifteen minutes later, the aorta was transected, a tracheostomy was performed, and the lungs were ventilated 3 times with 7 ml of 0 2 or air. The chest was then opened, and the lungs were briefly perfused with solution B through a catheter placed in the pulmonary artery via the right ventricle. During the perfusion, the lungs were ventilated 3 times with 7 ml of 0 2 or air. We initially used 0 2 for the ventilation, but then arbitrarily switched to air, with no apparent change in the experimental results. The lungs were removed from the thorax and were instilled with solution B (5 times with 10 ml per lavage). The fluid from the lavage was saved to isolate macrophages for the metabolic studies. The lungs were then instilled with 12 ml of the fluorocarbon-albumin emulsion, and immersed in 0.15 M NaCl for 20 min at 37° C to allow remaining macrophages to ingest the heavy emulsion. The lungs were then removed from the saline bath and instilled 5 times with solution B and once with the trypsin solution. This solution was discarded. The lungs were then instilled with the trypsin solution and were incubated at 37o C in the saline bath. After lO min, the lungs collapsed; they were filled with additional trypsin solution and were incubated for an additional 10 min. The lungs were then removed and filled with the solution of soybean trypsin inhibitor. The trachea and visible hilar structures were cut away. The lobes were allowed to collapse for 5 min and then were minced for 45 sec with very sharp scissors. The pieces and mincing solution from each lung were brought to a volume of 20 ml in a plastic graduated centrifuge tube by the addition of the solution with soybean trypsin inhibitor and were transferred to siliconized
TYPE II ALVEOLAR CELLS
250-ml Erlenmeyer flasks_ The flasks were shaken for lO min in a reciprocating water bath at 100 cycles per min at 37• C. The minces and freed cells were decanted through a series of filters (cotton gauze, 100-,um nylon mesh, and 20,um nylon mesh) (Tobler, Ernst, and Trubler, Inc., Elmsford, N. Y.). The cells were layered over a discontinuous gradient composed of 10 ml of albumin with a density of 1.040 and 10 ml of albumin with density of 1.080 in a 50-ml centrifuge tube. The interfaces of the albumin gradient were slightly mixed either by rapid layering or by intentional mixing (II). The gradient was centrifuged at 315 g (1,200 rpm) for 20 min at 4• C. The band between the densities 1.040 and 1.080 was aspirated, and the cells were washed twice with solution A. The time from the onset of anesthesia to obtaining the final cell suspension was approximately 3 hours. Macrophages were collected from the fluid obtained from the initial lavage, washed in solution A, stored at 4• C, and assayed at the same time as the type II cells. Identification. Type II cells were identified by fluorescence microscopy with phosphine 3R (12) (Roboz Surgical Instrument Co., Washington, D.C.) by a modified Papanicolaou stain, and by transmission electron microscopy. For fluorescence microscopy, unfixed cells were suspended in solutiGn A with phosphine 3R (approximately I ,ug per ml) and viewed through a Zeiss fluorescence microscope ·.vith a BG-12 primary filter and a 53 barrier filter. Phosphine 3R has an excitation maximum of 466 nm and an emission maximum of 512 nm. Airdried smears made by hand or with a cytocentrifuge (Cytopsin, Shandon Southern Instruments Ltd., Camberley, England) were stained with a Papanicolaou stain that was modified by avoiding any fixation with organic solvents before the staining procedure and by omitting an acid alcohol step in the decolorization (5). Cells processed for electron microscopy were fixed with 2 per cent glutaraldehyde and I per cent paraformaldehyde in phosphate buffer, postfixed with 1.5 per cent osmium tetroxide in verona! acetate buffer, stained en bloc with 1.5 per cent uranyl acetate, and dehydrated in acetone. They were then embedded in Epon®, sectioned, stained with uranyl acetate and lead citrate, and examined in a Zeiss EM-10 transmission electron microscope (13). Alveolar macrophages were identified simply as large mononuclear cells stained with Giemsa's stain (Giemsa's Blood Stain, Matheson, Coleman, and Bell, Norwood, Ohio). Buoyant density centrifugation. To determine the buoyant density of type II cells and to select our discontinuous gradients, we performed several experiments with continuous albumin gradients under conditions similar to those used for the discontinuous gradients. Cells were prepared in the usual manner and then were filtered through 10-,um
1017
mesh to remove any cellular aggregates. Thirty-five milliliters of the cellular suspension combined from the lungs of 2 rats were placed on top of a 50-ml linear gradient (density range, 1.040 to 1.100) and a 10-ml cushion of albumin (density, l.IOO) in 100-ml tubes (II) and were centrifuged at 1,300 rpm (300 g at the interface of the cell suspension and the density gradient) for 20 min at 4• C. Fractions of 5 ml were collected with a special capping device (Halpro Inc., Rockville, Md.) (II). Aliquots of each fraction were used for cell counts, differential counts, and density determination. Unit gravity velocity sedimentation. Unit gravity velocity sedimentation was chosen as a means of purifying the population enriched in type II cells recovered from the discontinuous albumin density gradients. We use a 3-liter chamber similar to the one described by Brubaker and Evans (14). The gradient was generated from a mixing chamber (300 ml, 0.35 per cent bovine serum albumin in Hank's balanced salt solution without calcium and magnesium) and from two 1,500-ml chambers for making the linear portion of the gradient that contained 1 and 2 per cent bovine serum albumin in Hank's balanced salt solution without calcium and magnesium. The solutions used for the gradient were passed through 0.45-,um filters to remove particles that would interfere with the use of a Coulter counter (Coulter Electronics, Inc., Hialeah, Fla.). To remove any cell aggregates, the cell suspension was passed through a 10-,um nylon filter immediately before it was placed on the gradient. The separation was performed at room temperature. Fractions of 60-ml volume were collected, and the number of cells in each fraction was counted. Based on the cell counts, fractions were pooled and smears were made for the Papanicolaou stain. A typical run took 45 min to load, 3 hours to settle, and 45 min to unload. Cell measurements. Cell number was determined with a hemacytometer, except for experiments in which cells were separated by unit gravity velocity sedimentation or continuous density centrifugation. Because of the large number of fractions to be counted in the latter experiments, a Coulter counter was used. The percentage of type II cells was calculated by counting at least 200 cells on smears stained by the Papanicolaou method. To test the ability of the cells to exclude a vital dye, the cells were resuspended in solution A without protein and incubated for 4 min with trypan blue (final concentration, 360 ,ug per ml) (15). Biochemical measurements. The biochemical measurements were designed to provide a compositional analysis of the isolated cells and to evaluate their metabolic activity for comparison with other isolation procedures and future modifications of this procedure. Lipid extraction and phospholipid determination were performed as described previously (16). Disaturated phosphatidylcholine was
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MASO.N, WILLIAMS, GREENLEAF, AND CLEMENTS
isolated by alumina column chromatography after reacting the total lipid extract with osmium tetroxide (17). Dipalmitoylphosphatidylcholine labeled with carbon-14 was added as an internal standard for calculating recoveries (17). Cell protein was measured by the method of Lowry and associates (18) with bovine serum albumin as the standard. Because HEPES buffer produces some color with reagents for the Lowry procedure, buffer solutions were routinely analyzed for correcting the results with cells (19). To calculate the number of cells in whole lung, DNA was measured by the method of Burton (20), with calf thymus DNA (Sigma Chemical Co., St. Louis, Mo.) as a standard. The metabolic capabilities of the isolated cells were evaluated by measurement of 0 2 consumption, glucose utilization, lactate production, and content and leakage of lactate dehydrogenase, a cytoplasmic enzyme. Oxygen consumption was mea· sured polarographically in a sealed 4-ml chamber at 37• C; cells (11 to 20 X 106 cells per 4 ml) were suspended in solution A that contained 1 mg of bovine serum albumin per ml, but no glucose. Oxygen consumption was determined during 2 8-min periods and was linear throughout this time. Glucose utilization, lactate production, and release of lactate . dehydrogenase were determined with cells in the same medium, except that 2.7 mM glucose was added. For these latter measurements, the cells were allowed to recover from the separation procedure for 30 min at 37• C. They were then centrifuged; the medium was discarded; and the cells were resuspended in fresh medium. Measurements were made after resuspension and after 1 or 2 hours of incubation. Cells were separated from medium by centrifugation at 150 g for 6 min. Lactate was measured in the medium by an enzymatic fluorometric method (21). Glucose in the medium was measured enzymatically by coupled hexokinase and glucose-6-phosphate dehydrogenase reactions (Gluose Stat-Pack, Calbiochem, La Jolla, Ca.). In sep"rate experiments, oxidation of [l-14C]glucose was found to be linear with time for 2 hours and to be directly proportional to the cell concentrations used in these experiments. Lactate dehydrogenase was measured in the reverse reaction (LDH-L Stat-Pack, Calbiochem, La Jolla Ca.). Results
Identification. Light microscopy. Type II cells are readily identified by the presence of dark cytoplasmic inclusions stained with the modified Papanicolaou stain. These inclusions are absent from cells extracted with lipid solvents, i.e., chloroform-methanol, 2: I (voljvol), or acetone at room temperature. The fluorescent compound, phosphine 3R, is useful for monitoring cell separation procedures and for examining unfixed slices of lung for type II cells. Type II
cells have brightly fluorescent cytoplasmic inclusions (22). Most macrophages from adult animals and other lung cells fluoresce diffusely green with this compound and do not contain the large inclusions of type II cells. Some alveolar macrophages from newborn animals, however, also contain large inclusions both with phosphine 3R and the· modified Papanicolaou stain. By ultrastructural criteria, these cells are believed to be macrophages, rather than desquamated type II cells. These macrophages, however, contain more disaturated phosphatidylcholine than similar cells from adult animals and have multiple lamella ted inclusions by transmission electron microscopy (data not shown). Although phosphine 3R provides a method for rapid assessment of isolated cells, the fluorescence fades during illumination, and differential cell counts are difficult to perform. With the same cell preparations, the results with phosphine 3R and the Papanicolaou stain correlate with each other and with the results from transmission electron microscopy, but we have not been able to study the same cell by the 3 different techniques. Air-dried and fixed cells do not show the specific fluorescence with phosphine 3R. Electron microscopy. The ultrastructural appearance of an isolated type II cell and that of a group of isolated cells are shown in figures I and 2. The lamellar bodies are well preserved and appear to be similar to those of type II cells in intact lung. The cells show some evidence of damage, as indicated by areas of dilated endoplasmic reticulum and cytoplasmic vacuoles. Most of the subcellular organelles are well preserved. In other micrographs, it was clear that most of the cells in our preparation that were not type II cells were lymphocytes and macrophages. Ciliated cells and other cells that were tentatively identified as type I cells or endothelial cells were also present. Yield and purity. The data for yield and purity of the cell preparations for rats from 3 suppliers are shown in table I. There were no differences between the rats from the first 2 suppliers. The cell yield from the pathogen-free rats was significantly greater than yields from rats from the other suppliers (P < 0.01), although the purity was slightly less. In 55 experiments, the yield ranged from 3 to 30 X I oo cells per rat and from 42 to 92 per cent type II cells. Rats of approximately the same size contained 7.5 mg of DNA per lung (24), and based on 6.2 pg of DNA per rat lung cell (25), the yield with spe-
TYPE II ALVEOLAR CELLS
1019
Fig. 1. An electron micrograph of an isolated type II cell. The cellular organelles appear to be relatively intact, and the characteristic lamellar inclusion bodies are apparent. There is some dilatation of the endoplasmic reticulum and perinuclear space. The inset bar is 1 ~Jm (original magnification: X 12,000).
cific pathogen-free rats was 1.7 per cent of total lung cells. If type II cells comprised 14 per cent of total lung cells, we isolated 12 per cent of the type II cells in rat lung (26). The distributions of total cells and type II cells on the discontinuous albumin gradient were analyzed. In 7 experiments, 1 ± 1 per cent of the recovered cells were located above the 1.040-to· 1.080 interface; 31 ± 6 per cent at the interface; and 68 ± 8 per cent below the interface. Approximately 75 per cent of the cells applied to the gradient were recovered. Approximately 40 per cent of the type II cells passed through the 1.040· to-1.080 interface. Precise differential counts were difficult to perform on the portion of the gradient below the interface, because of the mass of the uningested fluorocarbo n, but 25 to 50 per cent of the cells were type II cells. When the density of the heavier albumin was decreased, the yield of cells at the interface decreased, but the purity was not greatly increased. When the cells collected from the 1.040-to-1.080 interface were centrifuged on a second identical gradient, the purity was not substantially increased. When the initial gradient was centrifuged at 2,700 rpm
(1500 g) for 20 min, the purity was also not improved, but the yield was slightly less. Continuous albumin gradients were used to separate type II cells and to help select densities for the discontinuous gradients. A representative experiment is shown in figure 3. Most type II cells have a density of 1.060 to 1.085. The per· centage of type II cells was also slightly greater in this density range, but the actual purification of type II cells on the gradient was modest. As figure 3 suggests, the yield of cells can be in· creased by increasing the bottom density of the discontinuous gradient from 1.080 to 1.085. Under our conditions of tissue dissociation and cell isolation, very few type II cells h ad a density less than 1.060. Composition and m etabolic measurements. Only studies of preparations that contained more than 60 per cent type II cells are reported here; all rats were from Simonsen (table 1). Macrophages were obtained from the same ani· mals that were used for the isolation of type II cells. The composi tion of type II cells and macrophages is shown in table 2. Type II cells were
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MASON, WILLIAMS, GREENLEAF, AND CLEMENTS
Fig. 2. A low-power electron micrograph of isolated type II cells. This preparation was 85 p er cent type II cells , as determined by differential counts ofPapanicolaou-stained smears. The inse t bar is 1 J,Lm (original m agnification: X 5,000).
1021
TYPE II ALVEOLAR CELLS
TABLE 1 YIELD AND PURITY OF TYPE II CELLS Supplier Simonsen
N 33 33
No. of cells X 106/Rat
Simonsen Charles River Hilltop Simonsen
33 11 11 33 33
8.8 ±4.3 8.3 ±3.0 20.3 ±4.0
% Type II Cells 67 ± 11 65 ± 10 60± 9
Mean ±SO values are reported; N is the number of separate experiments (a group of 4 rats). All rats were Sprague- Dawley strain, but they were housed differently, as explained in the text. Analysis of variance showed a significant difference in yields between the 0.001 by F test), but no difference in the per cent of type II cells (23). By groups (P the unpaired "Student's" t test, the yield of cells from the rats from Hilltop Lab Animals was significantly greater than the yields from rats from the other 2 suppliers (P < 0.01) (23).
ROBERT J. MASON,3 MARY C. WILLIAMS, 4 ROBERT D. GREENLEAF,5 and JOHN A. CLEMENTS6
SUMMARY __________________________________________ ______________ Type II alveolar cells can be isolated and partially purified from adult rat lung by a series of steps that includes enzymatic digestion of the lung with trypsin and separation of cells on a discontinuous albumin density gradient. The yield of the isolated type II cells depends on the supplier and the housing of the rats used to prepare the cells. With specific pathogen-free rats housed in a laminar flow hood, the yield was 20.3 X 106 cells per rat, of which 60 per cent were type II cells. With rats from 2 other suppliers and no special housing, the yields were 8.8 and 8.3 X 106 cells per rat, of which 67 and 65 per cent were type II cells. The ultrastructural appearance of the isolated cells was similar to that of cells from intact lung, except for some dilatation of the endoplasmic reticulum and the perinuclear space. Most cells (92 ± 5 per cent) excluded the vital dye, trypan blue. The cells consumed 0 2 at the rate of 76 ± 12 nmole per 106 cells per hour and released only 5.7 ± 2.0 per cent of their lactate dehydrogenase, a cytoplasmic enzyme, into the medium after l hour of incubation. The isolated type II cells contained disaturated phosphatidylcholine, a major component of purified surface-active material. The cells, however, had a low glucose utilization compared to their 0 2 consumption, which may indicate an abnormality in the metabolism of glucose. This population of cells could be further purified to 89 per cent type II cells by unit gravity velocity sedimentation.
Introduction The cellular heterogeneity of the lung makes assignment of specific metabolic functions to individual cell types very difficult. One approach that circumvents the problem of cellular heterogeneity is to study preparations of a single cell (Received in original form November 24, 1976 and in revised form February 2, 1977) 1 From the Cardiovascular Research Institute, University of California Medical Center, San Francisco Ca. 94143. 2 This work was supported by Program Project grant HL-06285 and Pulmonary SCOR grant HL14201 from the U.S. Public Health Service. 3 R. J. Mason is an Established Investigator of the American Heart Association. 4 Mary C. Williams is the recipient of a Research Car.?..!r Development Award (HL-00221) from the National Heart, Lung and Blood Institute. 5 Robert D. Greenleaf is a tr'linee supported by
type. Extensive evidence indicates that type II alveolar cells synthesize and secrete pulmonary surface-active material (1-4). Because alterations in the metabolism of this complex substance have been implicated in numerous disease processes, especially in respiratory distress syndrome of the newborn, a detailed study of the synthesis and secretion of surface-active material by isolated type II cells may help elucidate the pathogenesis of certain diseases and suggest new means of therapy. Before detailed biochemical studies can be performed, methods for isolating type II cells need to be devised. This report describes our method for isolating a partially purified population of type II cells from adult rat lung and some of the metabolic grant HL-05251 from the National Heart, Lung and Blood Institute. 6 John A. Clements is a Career Investigator of the American Heart Association.
AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME 115, 1977
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MASON, WILLIAMS, GREENLEAF, AND
properties of these cells. Kikkawa and associates (2) and Kikkawa and Yoneda (5) have previously reported a method for isolating type II cells from the lungs of rats and rabbits, and their data provide a basis for comparisons of yield, purity, and function. A preliminary report of our method has been published (6). Materials and Methods Solutions for the isolation of type II cells. To make the isolation procedure easier to describe, we list the composition of the solutions used at the beginning. The main balanced salt solution designated, solution A, contained 128 mM sodium chloride (NaCl), 5 mM potassium chloride (KCl), 2.5 mM sodium phosphate buffer, 1.9 mM calcium chloride (CaC1 2 ), 1.2 mM magnesium sulfate (MgSO 4 ), 17 mM HEPES (N-2-hydroxyethyl piperazine N-2-ethane-sulfonic acid), 5.5 mM glucose, and lO p.g of gentamicin per mi. Solution B was the same as solution A, except that CaC1 2 and MgSO 4 were omitted. The pH of solution A was adjusted to 7.4 at room temperature (22° C), but it should be noted that the pH of HEPES buffers is temperature dependent (7). The osmolality of solution A was 290 mOsm, as measured by freezing point depression. An emulsion of fluorocarbon and albumin was prepared for instillation into the lung to facilitate the removal of macrophages by increasing their density. The amount was sufficient for the 4 rats that we routinely used in each experiment. Four milliliters of fluorocarbon (FC-75, Minnesota Mining Co., St. Paul, Minn.) and 12 ml of either defatted bovine serum albumin (10 mg per ml) (Sigma Chemical Co., St. Louis, Mo.) or fraction V bovine serum albumin (10 mg per ml) (Miles Laboratory, Kankakee, III.) dissolved in solution A were sonicated together. The emulsion was diluted with an additional 44 ml of the albumin solution before use. The defatted albumin appeared to make a slightly more stable emulsion, but both albumin solutions were satisfactory. The solution containing trypsin was made by dissolving recrystallized bovine trypsin (3 mg per ml) (Sigma Chemical Co., St. Louis, Mo.) and 30 p.g of deoxyribonuclease (DNase) per ml (Sigma Chemical Co. or P. L. Biochemicals Inc., Milwaukee, Wis.) in solution A. The solution with trypsin inhibitor was made by dissolving I mg of soybean trypsin inhibitor per ml (Sigma Chemical Co., St. Louis, Mo.) and 30 p.g of Dl':ase per ml in solution A. A discontinuous albumin density gradient was made with Pathocyte 4 (Miles Lab., Kankakee, Ill.) diluted to densities of 1.080 and 1.040 with 0.15 M l':aCI. The densities of the albumin solutions were measured in a column of kerosene and carbon tetrachloride, which was calibrated with solutions of sucrose (8). Isolation of cells. Male and female SpragueDawley rats weighing 180 to 250 g were purchased
CLE.~IENTS
from 3 suppliers. The rats from Simonsen (Gilroy, Ca.) and Charles River Breeding Laboratories, Inc. (Wilmington Mass.) were housed at the University of California in animal quarters shared by a number of investigators. No special precautions were taken to prevent pulmonary infections. Animals with grossly visible pulmonary infections at the time of cell preparation were discarded, but obvious pneumonia was an infrequent occurrence ( < 2 per cent). Because of the ubiquitous problem of clinically inapparent murine pneumonia, special precautions were taken with specific pathogen-free rats obtained from Hilltop Lab Animals, Scottdale, Pa. (9 10). Pathogen-free animals were shipped in cages with special filters, housed in a laminar flow hood, and given autoclaved food, water, and bedding. Cultures of the lungs from 2 rats housed in this manner for 2 weeks did not reveal any bacteria or Mycoplasma, and histologic examination failed to show any changes of murine pneumonia. The rats were fed ad libitum until killed. They were anesthetized and anticoagulated by a single intraperitoneal injection containing 250 units of sodium heparin per 100 g of body weight and 5 mg of sodium pentobarbital per 100 g of body weight. Fifteen minutes later, the aorta was transected, a tracheostomy was performed, and the lungs were ventilated 3 times with 7 ml of 0 2 or air. The chest was then opened, and the lungs were briefly perfused with solution B through a catheter placed in the pulmonary artery via the right ventricle. During the perfusion, the lungs were ventilated 3 times with 7 ml of 0 2 or air. We initially used 0 2 for the ventilation, but then arbitrarily switched to air, with no apparent change in the experimental results. The lungs were removed from the thorax and were instilled with solution B (5 times with 10 ml per lavage). The fluid from the lavage was saved to isolate macrophages for the metabolic studies. The lungs were then instilled with 12 ml of the fluorocarbon-albumin emulsion, and immersed in 0.15 M NaCl for 20 min at 37° C to allow remaining macrophages to ingest the heavy emulsion. The lungs were then removed from the saline bath and instilled 5 times with solution B and once with the trypsin solution. This solution was discarded. The lungs were then instilled with the trypsin solution and were incubated at 37o C in the saline bath. After lO min, the lungs collapsed; they were filled with additional trypsin solution and were incubated for an additional 10 min. The lungs were then removed and filled with the solution of soybean trypsin inhibitor. The trachea and visible hilar structures were cut away. The lobes were allowed to collapse for 5 min and then were minced for 45 sec with very sharp scissors. The pieces and mincing solution from each lung were brought to a volume of 20 ml in a plastic graduated centrifuge tube by the addition of the solution with soybean trypsin inhibitor and were transferred to siliconized
TYPE II ALVEOLAR CELLS
250-ml Erlenmeyer flasks_ The flasks were shaken for lO min in a reciprocating water bath at 100 cycles per min at 37• C. The minces and freed cells were decanted through a series of filters (cotton gauze, 100-,um nylon mesh, and 20,um nylon mesh) (Tobler, Ernst, and Trubler, Inc., Elmsford, N. Y.). The cells were layered over a discontinuous gradient composed of 10 ml of albumin with a density of 1.040 and 10 ml of albumin with density of 1.080 in a 50-ml centrifuge tube. The interfaces of the albumin gradient were slightly mixed either by rapid layering or by intentional mixing (II). The gradient was centrifuged at 315 g (1,200 rpm) for 20 min at 4• C. The band between the densities 1.040 and 1.080 was aspirated, and the cells were washed twice with solution A. The time from the onset of anesthesia to obtaining the final cell suspension was approximately 3 hours. Macrophages were collected from the fluid obtained from the initial lavage, washed in solution A, stored at 4• C, and assayed at the same time as the type II cells. Identification. Type II cells were identified by fluorescence microscopy with phosphine 3R (12) (Roboz Surgical Instrument Co., Washington, D.C.) by a modified Papanicolaou stain, and by transmission electron microscopy. For fluorescence microscopy, unfixed cells were suspended in solutiGn A with phosphine 3R (approximately I ,ug per ml) and viewed through a Zeiss fluorescence microscope ·.vith a BG-12 primary filter and a 53 barrier filter. Phosphine 3R has an excitation maximum of 466 nm and an emission maximum of 512 nm. Airdried smears made by hand or with a cytocentrifuge (Cytopsin, Shandon Southern Instruments Ltd., Camberley, England) were stained with a Papanicolaou stain that was modified by avoiding any fixation with organic solvents before the staining procedure and by omitting an acid alcohol step in the decolorization (5). Cells processed for electron microscopy were fixed with 2 per cent glutaraldehyde and I per cent paraformaldehyde in phosphate buffer, postfixed with 1.5 per cent osmium tetroxide in verona! acetate buffer, stained en bloc with 1.5 per cent uranyl acetate, and dehydrated in acetone. They were then embedded in Epon®, sectioned, stained with uranyl acetate and lead citrate, and examined in a Zeiss EM-10 transmission electron microscope (13). Alveolar macrophages were identified simply as large mononuclear cells stained with Giemsa's stain (Giemsa's Blood Stain, Matheson, Coleman, and Bell, Norwood, Ohio). Buoyant density centrifugation. To determine the buoyant density of type II cells and to select our discontinuous gradients, we performed several experiments with continuous albumin gradients under conditions similar to those used for the discontinuous gradients. Cells were prepared in the usual manner and then were filtered through 10-,um
1017
mesh to remove any cellular aggregates. Thirty-five milliliters of the cellular suspension combined from the lungs of 2 rats were placed on top of a 50-ml linear gradient (density range, 1.040 to 1.100) and a 10-ml cushion of albumin (density, l.IOO) in 100-ml tubes (II) and were centrifuged at 1,300 rpm (300 g at the interface of the cell suspension and the density gradient) for 20 min at 4• C. Fractions of 5 ml were collected with a special capping device (Halpro Inc., Rockville, Md.) (II). Aliquots of each fraction were used for cell counts, differential counts, and density determination. Unit gravity velocity sedimentation. Unit gravity velocity sedimentation was chosen as a means of purifying the population enriched in type II cells recovered from the discontinuous albumin density gradients. We use a 3-liter chamber similar to the one described by Brubaker and Evans (14). The gradient was generated from a mixing chamber (300 ml, 0.35 per cent bovine serum albumin in Hank's balanced salt solution without calcium and magnesium) and from two 1,500-ml chambers for making the linear portion of the gradient that contained 1 and 2 per cent bovine serum albumin in Hank's balanced salt solution without calcium and magnesium. The solutions used for the gradient were passed through 0.45-,um filters to remove particles that would interfere with the use of a Coulter counter (Coulter Electronics, Inc., Hialeah, Fla.). To remove any cell aggregates, the cell suspension was passed through a 10-,um nylon filter immediately before it was placed on the gradient. The separation was performed at room temperature. Fractions of 60-ml volume were collected, and the number of cells in each fraction was counted. Based on the cell counts, fractions were pooled and smears were made for the Papanicolaou stain. A typical run took 45 min to load, 3 hours to settle, and 45 min to unload. Cell measurements. Cell number was determined with a hemacytometer, except for experiments in which cells were separated by unit gravity velocity sedimentation or continuous density centrifugation. Because of the large number of fractions to be counted in the latter experiments, a Coulter counter was used. The percentage of type II cells was calculated by counting at least 200 cells on smears stained by the Papanicolaou method. To test the ability of the cells to exclude a vital dye, the cells were resuspended in solution A without protein and incubated for 4 min with trypan blue (final concentration, 360 ,ug per ml) (15). Biochemical measurements. The biochemical measurements were designed to provide a compositional analysis of the isolated cells and to evaluate their metabolic activity for comparison with other isolation procedures and future modifications of this procedure. Lipid extraction and phospholipid determination were performed as described previously (16). Disaturated phosphatidylcholine was
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MASO.N, WILLIAMS, GREENLEAF, AND CLEMENTS
isolated by alumina column chromatography after reacting the total lipid extract with osmium tetroxide (17). Dipalmitoylphosphatidylcholine labeled with carbon-14 was added as an internal standard for calculating recoveries (17). Cell protein was measured by the method of Lowry and associates (18) with bovine serum albumin as the standard. Because HEPES buffer produces some color with reagents for the Lowry procedure, buffer solutions were routinely analyzed for correcting the results with cells (19). To calculate the number of cells in whole lung, DNA was measured by the method of Burton (20), with calf thymus DNA (Sigma Chemical Co., St. Louis, Mo.) as a standard. The metabolic capabilities of the isolated cells were evaluated by measurement of 0 2 consumption, glucose utilization, lactate production, and content and leakage of lactate dehydrogenase, a cytoplasmic enzyme. Oxygen consumption was mea· sured polarographically in a sealed 4-ml chamber at 37• C; cells (11 to 20 X 106 cells per 4 ml) were suspended in solution A that contained 1 mg of bovine serum albumin per ml, but no glucose. Oxygen consumption was determined during 2 8-min periods and was linear throughout this time. Glucose utilization, lactate production, and release of lactate . dehydrogenase were determined with cells in the same medium, except that 2.7 mM glucose was added. For these latter measurements, the cells were allowed to recover from the separation procedure for 30 min at 37• C. They were then centrifuged; the medium was discarded; and the cells were resuspended in fresh medium. Measurements were made after resuspension and after 1 or 2 hours of incubation. Cells were separated from medium by centrifugation at 150 g for 6 min. Lactate was measured in the medium by an enzymatic fluorometric method (21). Glucose in the medium was measured enzymatically by coupled hexokinase and glucose-6-phosphate dehydrogenase reactions (Gluose Stat-Pack, Calbiochem, La Jolla, Ca.). In sep"rate experiments, oxidation of [l-14C]glucose was found to be linear with time for 2 hours and to be directly proportional to the cell concentrations used in these experiments. Lactate dehydrogenase was measured in the reverse reaction (LDH-L Stat-Pack, Calbiochem, La Jolla Ca.). Results
Identification. Light microscopy. Type II cells are readily identified by the presence of dark cytoplasmic inclusions stained with the modified Papanicolaou stain. These inclusions are absent from cells extracted with lipid solvents, i.e., chloroform-methanol, 2: I (voljvol), or acetone at room temperature. The fluorescent compound, phosphine 3R, is useful for monitoring cell separation procedures and for examining unfixed slices of lung for type II cells. Type II
cells have brightly fluorescent cytoplasmic inclusions (22). Most macrophages from adult animals and other lung cells fluoresce diffusely green with this compound and do not contain the large inclusions of type II cells. Some alveolar macrophages from newborn animals, however, also contain large inclusions both with phosphine 3R and the· modified Papanicolaou stain. By ultrastructural criteria, these cells are believed to be macrophages, rather than desquamated type II cells. These macrophages, however, contain more disaturated phosphatidylcholine than similar cells from adult animals and have multiple lamella ted inclusions by transmission electron microscopy (data not shown). Although phosphine 3R provides a method for rapid assessment of isolated cells, the fluorescence fades during illumination, and differential cell counts are difficult to perform. With the same cell preparations, the results with phosphine 3R and the Papanicolaou stain correlate with each other and with the results from transmission electron microscopy, but we have not been able to study the same cell by the 3 different techniques. Air-dried and fixed cells do not show the specific fluorescence with phosphine 3R. Electron microscopy. The ultrastructural appearance of an isolated type II cell and that of a group of isolated cells are shown in figures I and 2. The lamellar bodies are well preserved and appear to be similar to those of type II cells in intact lung. The cells show some evidence of damage, as indicated by areas of dilated endoplasmic reticulum and cytoplasmic vacuoles. Most of the subcellular organelles are well preserved. In other micrographs, it was clear that most of the cells in our preparation that were not type II cells were lymphocytes and macrophages. Ciliated cells and other cells that were tentatively identified as type I cells or endothelial cells were also present. Yield and purity. The data for yield and purity of the cell preparations for rats from 3 suppliers are shown in table I. There were no differences between the rats from the first 2 suppliers. The cell yield from the pathogen-free rats was significantly greater than yields from rats from the other suppliers (P < 0.01), although the purity was slightly less. In 55 experiments, the yield ranged from 3 to 30 X I oo cells per rat and from 42 to 92 per cent type II cells. Rats of approximately the same size contained 7.5 mg of DNA per lung (24), and based on 6.2 pg of DNA per rat lung cell (25), the yield with spe-
TYPE II ALVEOLAR CELLS
1019
Fig. 1. An electron micrograph of an isolated type II cell. The cellular organelles appear to be relatively intact, and the characteristic lamellar inclusion bodies are apparent. There is some dilatation of the endoplasmic reticulum and perinuclear space. The inset bar is 1 ~Jm (original magnification: X 12,000).
cific pathogen-free rats was 1.7 per cent of total lung cells. If type II cells comprised 14 per cent of total lung cells, we isolated 12 per cent of the type II cells in rat lung (26). The distributions of total cells and type II cells on the discontinuous albumin gradient were analyzed. In 7 experiments, 1 ± 1 per cent of the recovered cells were located above the 1.040-to· 1.080 interface; 31 ± 6 per cent at the interface; and 68 ± 8 per cent below the interface. Approximately 75 per cent of the cells applied to the gradient were recovered. Approximately 40 per cent of the type II cells passed through the 1.040· to-1.080 interface. Precise differential counts were difficult to perform on the portion of the gradient below the interface, because of the mass of the uningested fluorocarbo n, but 25 to 50 per cent of the cells were type II cells. When the density of the heavier albumin was decreased, the yield of cells at the interface decreased, but the purity was not greatly increased. When the cells collected from the 1.040-to-1.080 interface were centrifuged on a second identical gradient, the purity was not substantially increased. When the initial gradient was centrifuged at 2,700 rpm
(1500 g) for 20 min, the purity was also not improved, but the yield was slightly less. Continuous albumin gradients were used to separate type II cells and to help select densities for the discontinuous gradients. A representative experiment is shown in figure 3. Most type II cells have a density of 1.060 to 1.085. The per· centage of type II cells was also slightly greater in this density range, but the actual purification of type II cells on the gradient was modest. As figure 3 suggests, the yield of cells can be in· creased by increasing the bottom density of the discontinuous gradient from 1.080 to 1.085. Under our conditions of tissue dissociation and cell isolation, very few type II cells h ad a density less than 1.060. Composition and m etabolic measurements. Only studies of preparations that contained more than 60 per cent type II cells are reported here; all rats were from Simonsen (table 1). Macrophages were obtained from the same ani· mals that were used for the isolation of type II cells. The composi tion of type II cells and macrophages is shown in table 2. Type II cells were
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MASON, WILLIAMS, GREENLEAF, AND CLEMENTS
Fig. 2. A low-power electron micrograph of isolated type II cells. This preparation was 85 p er cent type II cells , as determined by differential counts ofPapanicolaou-stained smears. The inse t bar is 1 J,Lm (original m agnification: X 5,000).
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TYPE II ALVEOLAR CELLS
TABLE 1 YIELD AND PURITY OF TYPE II CELLS Supplier Simonsen
N 33 33
No. of cells X 106/Rat
Simonsen Charles River Hilltop Simonsen
33 11 11 33 33
8.8 ±4.3 8.3 ±3.0 20.3 ±4.0
% Type II Cells 67 ± 11 65 ± 10 60± 9
Mean ±SO values are reported; N is the number of separate experiments (a group of 4 rats). All rats were Sprague- Dawley strain, but they were housed differently, as explained in the text. Analysis of variance showed a significant difference in yields between the 0.001 by F test), but no difference in the per cent of type II cells (23). By groups (P the unpaired "Student's" t test, the yield of cells from the rats from Hilltop Lab Animals was significantly greater than the yields from rats from the other 2 suppliers (P < 0.01) (23).
