a m e r e n t cell characteristic, such as size, surface charge, or immunologic activity, will be utilized. Discussion
Dr. Daniek: I noticed that your major peak came at
1.077, which is the optimal density used to separate lymphocytes. Did you h d a large number of lymphocytes in that peak? Dr. Btenkwski: Yes, there are a large number of lymphocytes in that peak. Dr. Ffsher: The rat is very susceptible to pulmonary infections causing lymphocytic infiltration of the lung. Have you done any work with noninfected rats? Dr. Bienkowski: No. Dr. Massaro: Have you studied what Ludox-PVP does to oxygen consumption of your cells? Dr. Bienkowski: No, but we have assessed viability by dye exclusion techniques and have found greater than 90 percent viability.
Isolation and Identification of Type 2 Alveolar Epithelial cells* Robert Mason, M.D., Mazy C. Williams, M.D. cmd John A. Ckments, M.D.
T
ype 2 alveolar epithelial cells are thought to be the source of pulmonary surface active material. This important substance is necessary for preventing alveolar collapse at low lung volumes in normal lungs, and a deficiency or inactivation of this material has been reported in most diseases characterized by atelectasis such as respiratory distress syndrome of the newborn, infectious and chemical pneumonitis, pulmonary infarction, and pulmonary edema. Although it would be valuable to understand the metabolism of surface active material in greater detail, metabolic studies of intact lung are difficult to interpret because of the cellular heterogeneity of the tissue. To overcome this difficulty, we have developed a technique to isolate type 2 cells for metabolic studies.
The method is designed to remove blood cells and macm phages, to digest the lung in a manner to obtain a population enriched in type 2 cells, and to separate type 2 cells from other lung cells by buoyant density centrifugation. In brief, 250 mg Sprague-Dawley rats of either sex are anesthetized and anticoagulated by an intraperitoneal injection of pentobarbital and heparin. In each rat, we transect the aorta, place a cannula in the trachea, ventilate the lungs with oxygen, perform a thoracotomy and perfuse the pulmonary circulation. The lungs are removed and washed out with warm buffered saline solution to remove some macrophages. The *Cardiovascular Research Institute and the Specialized Center for Research in Pulmonary Disease, University of Califomia, San Francisco.
38S 17TH ASPEN LUNG CONFERENCE
RCURE 1. Isolated type 2 alveolar epithelial cell. The bar in the lower left corner indicates one micron. lungs are then filled with an emulsion and incubated at 37% for U) minutes to allow reniaining macrophages to ingest this heavy material. The em&m is preparated by sonicathg 1 ml of a fluorocahn (FC-7!5, density 1.77, Minnesota Mining Co) with 3 ml of albumin (10 mg/ml) in Hank's balanced salt solution supplemented with 10 mM HEPES buffer (pH 7.4). The emulsion is diluted to 12 ml with the a l b d solution before being placed in the lungs. The lungs are then washed out, fiUed with a buff= containing trypsin( 3 mg/ml) and incubated again for 20 minutes at 37.C. The incubation is terminated by instilling buffer containing soy bean trypsin inhibitor ( 1 mg/ml). Thelungs are minced with a scia9ors, and the pieces agitated for ten minutes in flflaPks in a shaking water bath. The freed cells are filtered through gauze and nylon mesh. The cellular suspension (25 ml) is layered on a discontinuous albumin density gradient which is composed of 10 ml of density 1.040 and 10 ml of density 1.080. The gradient is centdiged for U) minutes at 800g at 4%. The type 2 cells are collected from the 1.0110-1.080 interface. In ten experiments, this band contained 11.4x10a cells (mean, range 56-24.2xlW) of which 76 percent (mean, range 56-86 percent) were type 2 cells as judged by the Papanicdam stain. Their viability was tested by the exclusion of vital dye (trypan blue and e& Y ) and 96 percent (mean, range 9892 percent) of the cells excluded the dyes. The band between the original cell suspension and the albumin of density 1.040 contaius mostly debris, and the pellet below 1.080 con* uningested Fuorocarbon, &hagladen with fluorocarbon, and other lung ceh including some type 2 cells.
Identification of type 2 d s in a population of dispersed cells is a major problem and is crucial for the development of cell separation techniques. Ultimate identification relies on electron microscopy. The cell
shown in Figure 1was isolated by the above method and is a representative type 2 cell with characteristic lamellar inclusions. The cell shows minimal morphologic evidence of damage attributable to the isolation procedure. Type 2 cells can also be identified by light microscopy in 0.5 micron sections of plastic embedded material which is stained by a polychrome stain of methylene blue, azure 2, and basic fuchsin.l The lamellar bodies appear as pink homogeneous inclusions. Because of layering of the Merent types of cells within the pellet processed for
CHEST 67: 2, FEBRUARY, 1975 SUPPLEMENT
electron micmscopy, blocks of the embedded cells must be sampled at different levels. The amount of time for processing and difficulty in quantitation restrict the routine use of these methods. To overcome these probl&s, Kikkawa developed a stain for air dried smears of type 2 ceh by modlfymg the Papanicolaou stain (eg avoiding fixation in organic solvents and omitting the acid-alcohol wash during the staining s e q ~ e n c e ) Lamellar .~ bodies appear as dark blue inclusions by light microscopy. This method, however, requires dried smears and takes several hows to perform. We discovered that a fluorescent compound phosphine 3R9 causes the lamellar inclusions of unfixed type 2 cells in suspension to fluoresce intensely and pennits differentiation from other lung cell types. Staining with phosphine 3R is simple and rapid and, therefore, can be used to monitor cell separation procedures dwing the course of an experiment. A cautionary note should be added about identifying type 2 cells by light microscopy. During the develop ment of this method, we found that alveolar macrcF phages from newborn lambs and dog contained inclusions which could not be readily differentiated from those of type 2 cells by staining with phosphine 3R or the Papanicolaou technique. These particular macrophages also differed from those from adult animals in that they contained a greater content of disaturated phosphatidylcholine, possibly derived from ingested surface active material. Identification of type 2 cells by light microscopy, thus, requires c o n h a t i o n by electron microscopy in each species and in each pathologic or physie logic state.
1 Madtay GR, Mead ML: A simple dichromatic stain for
plastic embedded tissues. Proceedings of the Electron Microscopy Society of America Claude J. Arceneaux (ed). Baton Rouge, 4 Claitor's Publishing Division, 1974, 296297 2 Kikkawa Y, Yoneda K: The type 2 epithelial cell of the lung 1. Method of isolation. Lab Invest 30:7&84.1974 3 Popper H: Histologic distribution of vitamin A in human organs under normal and under pathologic conditions. Arch Path 31:766-802.1941 Discussion
Dr. Fisher: Why don't the alveolar macrophages stain
with phospine 3R? Previous studies have shown that they contain the same lipids as type 2 cells. Dr. Mason: From 0 to 6 percent of alveolar macrophages in adult rats do stain. Dr. Massaro: Have you shown that cells not staining with phosphine 3R,that you are calling macrophages, do in fact ingest particles? Dr. Mason: No. Dr. Taewch: Perhaps your separation technique could get too good because it would not identify type 2 cells that had discharged their inclusions. Dr. Mason: True, there is no way to pick up false negatives
CHEST 67: 2, FEBRUARY, 1975 SUPPLEMENT
Phospholipid Metabolism in Lung Adenomas: A Model of Surface Active Material Synthesis* Danfel Klass, M.D., Esther Lesperance and Arnold NcJmMk, M.D.
L
ung cells produce a lipoprotein, pulmonary surface active material (PSAM) , which maintains alveolar stability. Morphologic evidence would suggest that the alveolar type 2 cell is the site of synthesis of this substance, but difficulties encountered in preparing large samples of this cell type have made biochemical confirmation of this suggestion impossible. Pulmonary adenomas can be induced in mouse lungs by the injection of urethane, and the cells comprising these tumors have striking morphologic similarity to type 2 cells.'J We have, therefore, asked the question, "Does the function of these tumor cells bear any relationship to the synthesis of PSAM, as their appearance would suggest?" If so, the ease of obtaining large numbers of cells from these tumors might provide a convenient model for the study of PSAM metabolism.
We first prepared mouse PSAM by the technique of King and Clementss using material obtained by lung mincing and saline lavage. We found that this material was similar in physical and chemical properties to that found in the dog (Table 1). Because the mouse PSAM contained an unusually high proportion of fully saturated phospholipid, we studied the metabolism of this lipid class as an index of PSAM synthesizing capacity. The tumors were induced by injecting 1 mg/kg of urethane intraperioneally into young Swiss-Webster mice. Tumors began to appear after four to five months. They were easily separated from the underlying lung tissue. Tumors and lung tissue from tirne-matched normal mice who were injected with saline were minced and incubated in a standard medium containing 14C labelled palmitate. After one hour's incubation, the tissue was removed and homogenized in chloroform-methanol. Lipid* and phospholipid6 in the tissues were separated, and the fully saturated phosphatidyl choline fraction was isolated by mercuric acetate adduction.6 Protein7, phosphorus8 and 14C incorporation were measured by standard techniques.
Electron microscopy revealed that the tumors were composed of cells with the appearance of the type 2 alveolar pnewnonocyte. Lamellar inclusions were seen intra- and extra-cellularly ( Fig 1) . In comparison to normal lung tissue, we found the tumors, on a weight basis, to have similar levels of phospholipid, and saturated phospholipid, and we found that they incorporated palmitate into these fractions *From the Respiratory Investigation Unit, Department of Medicine and Department of Physiology, University of Manitoba, Faculty of Medicine, Winnipeg, Canada. Supported by the Medical Research Council of Canada.
17TH ASPEN LUNG CONFERENCE 37s
Dr. Daniek: I noticed that your major peak came at
1.077, which is the optimal density used to separate lymphocytes. Did you h d a large number of lymphocytes in that peak? Dr. Btenkwski: Yes, there are a large number of lymphocytes in that peak. Dr. Ffsher: The rat is very susceptible to pulmonary infections causing lymphocytic infiltration of the lung. Have you done any work with noninfected rats? Dr. Bienkowski: No. Dr. Massaro: Have you studied what Ludox-PVP does to oxygen consumption of your cells? Dr. Bienkowski: No, but we have assessed viability by dye exclusion techniques and have found greater than 90 percent viability.
Isolation and Identification of Type 2 Alveolar Epithelial cells* Robert Mason, M.D., Mazy C. Williams, M.D. cmd John A. Ckments, M.D.
T
ype 2 alveolar epithelial cells are thought to be the source of pulmonary surface active material. This important substance is necessary for preventing alveolar collapse at low lung volumes in normal lungs, and a deficiency or inactivation of this material has been reported in most diseases characterized by atelectasis such as respiratory distress syndrome of the newborn, infectious and chemical pneumonitis, pulmonary infarction, and pulmonary edema. Although it would be valuable to understand the metabolism of surface active material in greater detail, metabolic studies of intact lung are difficult to interpret because of the cellular heterogeneity of the tissue. To overcome this difficulty, we have developed a technique to isolate type 2 cells for metabolic studies.
The method is designed to remove blood cells and macm phages, to digest the lung in a manner to obtain a population enriched in type 2 cells, and to separate type 2 cells from other lung cells by buoyant density centrifugation. In brief, 250 mg Sprague-Dawley rats of either sex are anesthetized and anticoagulated by an intraperitoneal injection of pentobarbital and heparin. In each rat, we transect the aorta, place a cannula in the trachea, ventilate the lungs with oxygen, perform a thoracotomy and perfuse the pulmonary circulation. The lungs are removed and washed out with warm buffered saline solution to remove some macrophages. The *Cardiovascular Research Institute and the Specialized Center for Research in Pulmonary Disease, University of Califomia, San Francisco.
38S 17TH ASPEN LUNG CONFERENCE
RCURE 1. Isolated type 2 alveolar epithelial cell. The bar in the lower left corner indicates one micron. lungs are then filled with an emulsion and incubated at 37% for U) minutes to allow reniaining macrophages to ingest this heavy material. The em&m is preparated by sonicathg 1 ml of a fluorocahn (FC-7!5, density 1.77, Minnesota Mining Co) with 3 ml of albumin (10 mg/ml) in Hank's balanced salt solution supplemented with 10 mM HEPES buffer (pH 7.4). The emulsion is diluted to 12 ml with the a l b d solution before being placed in the lungs. The lungs are then washed out, fiUed with a buff= containing trypsin( 3 mg/ml) and incubated again for 20 minutes at 37.C. The incubation is terminated by instilling buffer containing soy bean trypsin inhibitor ( 1 mg/ml). Thelungs are minced with a scia9ors, and the pieces agitated for ten minutes in flflaPks in a shaking water bath. The freed cells are filtered through gauze and nylon mesh. The cellular suspension (25 ml) is layered on a discontinuous albumin density gradient which is composed of 10 ml of density 1.040 and 10 ml of density 1.080. The gradient is centdiged for U) minutes at 800g at 4%. The type 2 cells are collected from the 1.0110-1.080 interface. In ten experiments, this band contained 11.4x10a cells (mean, range 56-24.2xlW) of which 76 percent (mean, range 56-86 percent) were type 2 cells as judged by the Papanicdam stain. Their viability was tested by the exclusion of vital dye (trypan blue and e& Y ) and 96 percent (mean, range 9892 percent) of the cells excluded the dyes. The band between the original cell suspension and the albumin of density 1.040 contaius mostly debris, and the pellet below 1.080 con* uningested Fuorocarbon, &hagladen with fluorocarbon, and other lung ceh including some type 2 cells.
Identification of type 2 d s in a population of dispersed cells is a major problem and is crucial for the development of cell separation techniques. Ultimate identification relies on electron microscopy. The cell
shown in Figure 1was isolated by the above method and is a representative type 2 cell with characteristic lamellar inclusions. The cell shows minimal morphologic evidence of damage attributable to the isolation procedure. Type 2 cells can also be identified by light microscopy in 0.5 micron sections of plastic embedded material which is stained by a polychrome stain of methylene blue, azure 2, and basic fuchsin.l The lamellar bodies appear as pink homogeneous inclusions. Because of layering of the Merent types of cells within the pellet processed for
CHEST 67: 2, FEBRUARY, 1975 SUPPLEMENT
electron micmscopy, blocks of the embedded cells must be sampled at different levels. The amount of time for processing and difficulty in quantitation restrict the routine use of these methods. To overcome these probl&s, Kikkawa developed a stain for air dried smears of type 2 ceh by modlfymg the Papanicolaou stain (eg avoiding fixation in organic solvents and omitting the acid-alcohol wash during the staining s e q ~ e n c e ) Lamellar .~ bodies appear as dark blue inclusions by light microscopy. This method, however, requires dried smears and takes several hows to perform. We discovered that a fluorescent compound phosphine 3R9 causes the lamellar inclusions of unfixed type 2 cells in suspension to fluoresce intensely and pennits differentiation from other lung cell types. Staining with phosphine 3R is simple and rapid and, therefore, can be used to monitor cell separation procedures dwing the course of an experiment. A cautionary note should be added about identifying type 2 cells by light microscopy. During the develop ment of this method, we found that alveolar macrcF phages from newborn lambs and dog contained inclusions which could not be readily differentiated from those of type 2 cells by staining with phosphine 3R or the Papanicolaou technique. These particular macrophages also differed from those from adult animals in that they contained a greater content of disaturated phosphatidylcholine, possibly derived from ingested surface active material. Identification of type 2 cells by light microscopy, thus, requires c o n h a t i o n by electron microscopy in each species and in each pathologic or physie logic state.
1 Madtay GR, Mead ML: A simple dichromatic stain for
plastic embedded tissues. Proceedings of the Electron Microscopy Society of America Claude J. Arceneaux (ed). Baton Rouge, 4 Claitor's Publishing Division, 1974, 296297 2 Kikkawa Y, Yoneda K: The type 2 epithelial cell of the lung 1. Method of isolation. Lab Invest 30:7&84.1974 3 Popper H: Histologic distribution of vitamin A in human organs under normal and under pathologic conditions. Arch Path 31:766-802.1941 Discussion
Dr. Fisher: Why don't the alveolar macrophages stain
with phospine 3R? Previous studies have shown that they contain the same lipids as type 2 cells. Dr. Mason: From 0 to 6 percent of alveolar macrophages in adult rats do stain. Dr. Massaro: Have you shown that cells not staining with phosphine 3R,that you are calling macrophages, do in fact ingest particles? Dr. Mason: No. Dr. Taewch: Perhaps your separation technique could get too good because it would not identify type 2 cells that had discharged their inclusions. Dr. Mason: True, there is no way to pick up false negatives
CHEST 67: 2, FEBRUARY, 1975 SUPPLEMENT
Phospholipid Metabolism in Lung Adenomas: A Model of Surface Active Material Synthesis* Danfel Klass, M.D., Esther Lesperance and Arnold NcJmMk, M.D.
L
ung cells produce a lipoprotein, pulmonary surface active material (PSAM) , which maintains alveolar stability. Morphologic evidence would suggest that the alveolar type 2 cell is the site of synthesis of this substance, but difficulties encountered in preparing large samples of this cell type have made biochemical confirmation of this suggestion impossible. Pulmonary adenomas can be induced in mouse lungs by the injection of urethane, and the cells comprising these tumors have striking morphologic similarity to type 2 cells.'J We have, therefore, asked the question, "Does the function of these tumor cells bear any relationship to the synthesis of PSAM, as their appearance would suggest?" If so, the ease of obtaining large numbers of cells from these tumors might provide a convenient model for the study of PSAM metabolism.
We first prepared mouse PSAM by the technique of King and Clementss using material obtained by lung mincing and saline lavage. We found that this material was similar in physical and chemical properties to that found in the dog (Table 1). Because the mouse PSAM contained an unusually high proportion of fully saturated phospholipid, we studied the metabolism of this lipid class as an index of PSAM synthesizing capacity. The tumors were induced by injecting 1 mg/kg of urethane intraperioneally into young Swiss-Webster mice. Tumors began to appear after four to five months. They were easily separated from the underlying lung tissue. Tumors and lung tissue from tirne-matched normal mice who were injected with saline were minced and incubated in a standard medium containing 14C labelled palmitate. After one hour's incubation, the tissue was removed and homogenized in chloroform-methanol. Lipid* and phospholipid6 in the tissues were separated, and the fully saturated phosphatidyl choline fraction was isolated by mercuric acetate adduction.6 Protein7, phosphorus8 and 14C incorporation were measured by standard techniques.
Electron microscopy revealed that the tumors were composed of cells with the appearance of the type 2 alveolar pnewnonocyte. Lamellar inclusions were seen intra- and extra-cellularly ( Fig 1) . In comparison to normal lung tissue, we found the tumors, on a weight basis, to have similar levels of phospholipid, and saturated phospholipid, and we found that they incorporated palmitate into these fractions *From the Respiratory Investigation Unit, Department of Medicine and Department of Physiology, University of Manitoba, Faculty of Medicine, Winnipeg, Canada. Supported by the Medical Research Council of Canada.
17TH ASPEN LUNG CONFERENCE 37s
