EXPERIMENTAL

Type

AND

MOLECULAR

II Epithelial

PATHOLOGY

27, 152-166

( 1977)

Cells from the Lung of Syrian Isolation and Metabolism1 RAYMOND

C.

Hamsters:

PFLECER

Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Research Institute, P.O. Box 5890, Albuquerque, New Mexico 87115 Received August 17, 1976, and in revised form January 4, 1977 A population of pulmonary cells enriched in epithelial Type II pneumonocytes was isolated from Syrian hamsters using modifications of existing technology. The animals were euthanized, the heart-lung-trachea system was removed, and the lung was perfused intravascularly and intratracheally with saline. A fluorochemical-albumin emulsion was instilled into the lung via the trachea, and after 20 min of incubation at 37°C the material was removed and the lungs were relavaged 10 additional times. The lungs were trypsinized, the trypsin was deactivated, the lungs were minced, the cell suspension was filtered, and the cells were concentrated by centrifugation before separation on a discontinuous Ficoll gradient. The cell types were identified by light bright-field microscopy with the Papanicolaou stain, by fluorescent microscopy with Phosphine 3R, and by transmission electron microscopy. The enriched population of epithelial Type II cells (67-72s) concentrated at the buffer-l.041 g/ml of interface; 4 X lo6 viable Type II cells were recovered per animal. When radiolabeled palmitic acid was injected 4 hr before sacrifice, the label was taken up by Type II cells and incorporated into surface-active phosphatidyl glycerol.

INTRODUCTION The lung represents a particular problem for cellular biochemical studies because it contains abundant comrective tissue and widely divergent cell types. Although the lung consists of more than 40 different cell types, no one type comprises a major portion of the mass of the lung ( Sorokin, 1970). Since the cell types making up the lung may change in both number and function in animals exposed to potentially injurious substances, traditional biochemical methods involving tissue slices or whole lung homogenates may be relatively insensitive to detecting subtle local cellular alterations. Methods by which lung cells can be isolated, identified, and studied metabolically are needed in order to define the nature of the cellular changes which are associated with lung injuries. Adamson and Bowden (1974) showed that oxygen-induced epithelial injury selectively involved Type I cells, yet the recovery phase was characterized by proliferation of Type II cells which are thought to 1 Research performed under U.S. Energy Research and Development Administration Contract No. EY-76-C-04-1013 and conducted in facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care. Portions of this paper were read at the 59th Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, April 13-18, 1975.

152 Copyright All rights

0 1977 by Academic I’ress, Inc. of reproduction in any form reserved.

ISSN

0014

lSO0

TYPE

II PULMONARY

EPITHELIAL

CELLS

153

synthesize the highly surface-active pulmonary surfactant (Mackhn, 1954). Since pulmonary injury may arise from alveolar deposition of a great variety of airborne pollutants (Evans et al., 1973) and Type II cells may be involved in the injury and repair processes, we have initially concentrated our efforts on developing methods for isolation and separation of alveolar Type II cells. Further, an understanding of normal Type II cell metabolism will provide an insight into the pathogenesis of pulmonary disease processes. This report describes the technique of isolation of an enriched population of pulmonary epithelial Type II cells from the lungs of Syrian hamsters and demonstrates the in viuo incorporation of palmitic acid into isolated surface-active phosphatidyl glycerol. Studies in progress are directed at alterations in Type II cell metabolism following exposure to environmental pollutants. These procedures, which yielded a relatively consistent number of Type II cells for metabolism studies, are the latest modifications of our preliminary studies (Pfleger, 1975). The techniques of Kikkawa and Yoneda (1974) failed to consistently yield a satisfactory population or a sufficient number of Type II cells from Syrian hamsters. In pulmonary research, a species relatively free of lung infection is desirable. For this reason, the Syrian hamster is preferable to conventionally raised rats which are prone to chronic murine pneumonia. In addition, Syrian hamsters have been used extensively in the past decade in biomedical research and have served as a model for chemical carcinogenesis of the lung (Saffiotti et al., 1968). Thus efforts were directed toward the development of an enriched population of Type II cells derived from Syrian hamsters. The Type II epithelial pneumonocytes were identified by light bright-field microscopy (Phosphine 3R) and transusing the Papanicolaou stain, and by fluorescence mission electron microscopy. In oioo uptake of radiolabeled palmitic acid and thymidine by pulmonary Type II cells was also examined. METHODS Type II Cell Separation Approximately equal numbers of male and female Syrian hamsters (Sch:SYR)? were caged individually or in pairs and given food 3 and water ad libitum. All animals were between 100 and 200 days of age and weighed 90 to I50 g when anesthetized by intraperitoneal injection of pentobarbitol sodium. The heart and lungs were removed intact within 1 min after inducing anesthesia. The lungs were perfused with saline intravascularly via the pulmonary artery until the organ became blanched. A Teflon catheter (Cathlon IV, Jelco Laboratories, Raritan, New Jersey; 16 gauge, 6.4 cm) was inserted into the trachea and tied, and the lungs were lavaged IO times; first with 6 ml of normal saline at room temperature and thereafter with 5-ml aliquots to remove free macrophages. The vasculature was perfused a second time with 20 ml of normal saline. Five milliliters of a fluorochemical liquid-albumin suspension was introduced into the lung via the tracheal catheter. Macrophages increased their density by phagocytizing the dense material which aided in their separation from the Type II epithelial cells upon 2 ARS Sprague-Dawley, 3 Wayne Lab-Blox,

Allied

Madison, Mills,

Wisconsin. Inc., Chicago,

Illinois.

154

RAYMOND

C. PFLECER

centrifugation. The fluorochemical emulsion was prepared by sonicating 1 ml of Mediflor fluorochemical liquid (FC-80, density = 1.76 g/ml; 3M Co., St. Paul, Minnesota) and 15 ml of a 0.0057, bovine albumin solution at 5-g pressure, The lungs were placed in 5 ml of Krebs Ringer-bicarbonate-0.4% citrate buffer, pH 7.2, and incubated for 20 min at 37°C. This buffer was used throughout the procedures and was sterilized through a 0.3-pm membrane. The fluorochemical liquid and albumin were then withdrawn, and the lung was lavaged 10 times with 5 ml of fresh normal saline. A 0.3% trypsin ( 1:250, Difco Laboratories, Detroit, Michigan) solution (5 ml) was introduced through the tracheal catheter into the lungs, which were immersed in 5 ml of buffer and incubated for 20 min at 37°C. After incubation, a 0.170 solution of Soybean Trypsin Inhibitor (Miles Laboratories, Elkhart, Indiana) was instilled into the lungs via the trachea. The lobes were then dissected free of the major airways and minced into 0.5-mm fragments. The lung mince was filtered through nylon bolting cloth (HC-3-160, 115 mesh; Tetco, Inc., Elmsford, New York) with agitation and washed with “250 ml of cold buffer into a flask containing deoxyribonuclease (Worthington, Freehold, New Jersey). The cell filtrate and DNase were mixed well to inhibit cell clumping. The cold lung cell suspensionwas filtered again, first through 350-mesh nylon bolting cloth and then through 500-mesh nylon into 50-ml plastic centrifuge tubes. Each cloth was rinsed with cold buffer. The cell suspension was centrifuged for 15 min at 275 gmx at 4°C. After centrifugation, the buffer was aspirated, the cells were resuspended in fresh buffer, the cells were counted in a standard hemocytometer, and cell viability was determined by trypan blue exclusion. Discontinuous gradients of Ficoll fortified with 100 units of penicillin G, 100 pg of streptomycin sulfate, and 100 pg of neomycin sulfate/ml were made in thick-wall (3 mm) Sorvall centrifuge tubes (23 mm i.d. x 10.2 cm long). First, 6 ml of a 16.67~ ( w / v ) so1u t ion (density = 1.061 g/ml) of Ficoll was pipetted into the conical area, and then sequentially 7 ml each of 15 (density = 1.055 g/cc) and II.370 (w/v) (d ensity = 1.041 g/ml) solutions of Ficoll were added. The gradients were refrigerated for at least 30 min before the cell suspension (density = 1.003 g/ml) was carefully layered on the 11.3’j/ Ficoll. The cell suspension was diluted so that no more than 10 ml or approximately lo7 total cells were applied to the discontinuous gradient. Discontinuous gradients were centrifuged for 30 min at 275 g,,,, at 4°C. From Stokes’ equation (Glasstone and Lewis, 1960)) cells of density < 1.041 but > 1.003 g/ml theoretically should have reached the buffer-11.3% Ficoll interface in 0.5 min. After centrifugation, the buffer was aspirated and the cells were removed from the three interfaces along with some of the Ficoll solution. The cell suspensions were placed in centrifuge tubes, diluted to 50 ml with buffer, thoroughly mixed, and centrifuged for 30 min at 275 gmaJat 4°C. The medium was aspirated, the cells were resuspended in buffer and examined cytologically, and counted, and their viability was determined as previously described. To determine density profiles for the production of discontinuous gradients, the trypsin-released cell suspensions were centrifuged on chilled continuous gradients. These were prepared by constructing a continuous gradient apparatus similar to that described by Pretlow and Boone (1969). The cells were allowed to achieve their own isopyknic bouyant density by centrifugation for I5 min at

TYPE II PULMONARY

EPITHELIAL

CELLS

155

325 g,,, at 4°C. For a 30-ml continuous gradient in the 60-ml tubes, 55 X 10F cells in 10 ml of buffer was the maximum number of cells which permitted distinct banding. The gradient was unloaded via the special Ultramax valve in consecutive X-drop samples. The percentage of solids (Ficoll) in each sample was determined using a refractometer. A total cell count and viability test were determined on each sample. IO Vivo Uptake of [14C]PaZnlitic Acid and [SM]l’hymidine The metabolic activity of the Type II cell population and its ability to synthesize surface-active phospholipids was studied by injecting four adult Syrian hamsters intraperitoneally with 50 &i of [ 1-‘*C]palmitic acid (57.9 mCi/mmole) in 0.1 ml of ethanol and 100 &i of [6-3H]thymidine (23 Ci/mmole) in 0.1 ml of water 4 hr before sacrifice. Carbon-14 and 3H were assayed by standard techniques in a liquid scintillation spectrometer. The Type II cell population was isolated as described above, and total lipids were extracted (Bligh and Dyer, 1959) and quantitated as previously described (Pfleger and Thomas, 1971). Phosphatidyl glycerol was isolated and separated into the disaturated molecular species by the cryochromatography procedure developed in this laboratory (Henderson and Clayton, 1976). Light Bright-Field

Microscopy

Smears of cells were prepared in a cytocentrifuge for examination by light microscopy. Air-dried unfixed specimens were stained by the Papanicolaou method without an alcohol fixation and with a 30-set rinse in 0.05% HCl and 0.5% sodium acetate solutions which specifically stained the inclusion bodies of Type II cells dark blue. Fluorescence Microscopy Type II cells in buffer were treated with a 0.1% solution (4:1, v/v) of Phosphine 3R (Roboz Surgical Instrument Co., Inc., Washington, D.C.) and examined under a Zeiss microscope using fluorescent techniques. The cells fluoresce a brilliant yellow. The preparation was wet-mounted on a glass slide and the Type II cells were counted. Excitation filter BG 12 with barrier filters 53 and 41 was used. Transmission Electron Microscopy Cells for electron microscopy studies were pelleted and fixed with 2% glutaraldehyde buffered ( pH 7.4 ) with 0.2 M sodium cacodylate. After postfixation in cold 1% buffered osmium tetroxide, they were incubated overnight in cold 0.5% uranyl acetate, dehydrated in alcohol, and embedded in Epon. Thin sections were doubly stained with an aqueous solution of uranyl acetate and lead citrate according to Reynolds ( 1963) and examined for cell identification, integrity, and size with a Hitachi HU 11C electron microscope. RESULTS Removal of Lung Cells by Bronchopulmonary

Lavage

The efficacy of the pulmonary vasculature perfusion was judged by the production of white unblemished lung tissue, the absence of red blood cells, and a

156

RAYMOND

C. PFLEGER

-8

41.

.

I

I

PERCENT

, 32

FICOLL

FIG. 1. Separation, on continuous linear gradients (dotted lines) of Ficoll, of cells that were obtained by pulmonary lavage of perfused lungs of Syrian hamsters. (A) A population of obtained by lavage immedi3.5 x 10’ viable cells, with 91% viability (957 0 macrophages), ately after the lungs were perfused was layered in 2 ml of buffer above a gradient (30 ml, density = 1.003 to 1.090 g/ml) and centrifuged for 15 min at 325 g,,,. (B) A population of 0 macrophages), obtained by lavage im3.2 X 10” viable cells, with 53.1% viability (“757 mediately after the lungs were treated with the fluorochemical-albumin mixture and incubated for 20 min was layered above a gradient (30 ml, density = 1.003 to 1.112 g/ml) and centrifuged for 15 min at 325 g,,,. A large number of cells appeared at the bottom of the gradient and had a density >1.115 g/ml. This is shown in the figure by the peak at 31% solids.

reduction in the white blood cell elements in the final Type II cell population. The number of cells removed with the saline lavage decreased after the second lavage in a manner similar to that reported by Felicetti et al. (1975) in dogs. The average yield of cells, predominantly macrophages (>95%), from 10 bronchopulmonary lavages was 3.45 + 1.01 x lo6 cells per animal (66 animals) with a viability of 91.0 * 7.9%. Wh en the cells removed by lavage were placed on a continuous gradient of Ficoll and centrifuged, essentially one peak was obtained (Fig. 1A). The density of these cells ranged from 1.058 to 1.077 g/ml (15.6 and 20.8% Ficoll) and had an average diameter of 19.5 pm (range 16.6 to 23.9 pm). Following the fluorochemical treatment, the 10 lavages yielded an average total of 4.8 x lo6 cells (>757 0 macrophages) per animal with a viability of 56.4 * 2.9% based on samples from eight animals. The decreased total cell viability after the fluorochemical treatment was possibly due to the presence of

TYPE

II PULMONARY

EPITHELIAL

I 0 3-

I

l ** .

2-

I

CELLS

157

30

-24 .

.

.

..

-16 .

.

.

.24

.6

FIG. 2. Separation, on a continuous linear gradient (dotted line) of Ficoll (density = 1.003 to 1.092 g/ml), of the initial enzyme-liberated cell population obtained from the perfused lungs of Syrian hamsters. The cells were centrifuged for 15 min at 325 g,,,. (C) The cells (66.0 X 10” total, 84% viability) appear to separate into six principle bands with the Type II cells banding between 12.2 and 16.8% Ficoll (d ensity = 1.044 and 1.059 g/ml) and the fluorocarbon-laden (dense) macrophages banding at 17.8 and 19.6% Ficoll (density = 1.065 and 1.072 g/ml). (D) The cells (138 X 10’ total, 76.4y 0 viability) appear to separate into six principle bands with the Type II cell peak (density = 1.044 g/ml) not clearly separated from the macrophage peaks.

the fluorochemical-albumin suspension and/or lack of oxygen. When this mixture of cells was placed on a continuous gradient of Ficoll and centrifuged, a variety of cell types were obtained ranging in density from 1.013 to >1.115 g/ml (Fig. 1B). Most of the cells, however, were observed in the fraction >I115 g/ml (>31% Ficoll), indicating probable phagocytosis of the fluorochemical liquid. In comparison to the macrophages obtained in the first series of lavages, these cells were heavier, had a smaller diameter, and ranged in size from 14.5 to 21.8 pm with an average of 17.0 pm. Isolation of Type II Alveolar Epithetid Lung Cells Centrifugation of the initial enzyme-liberated cell suspension (WOO ml/ animal) yielded an average of 50 * 17 x lo6 total viable cells per animal with a viability of 81.2 * 4.0% (23 animals). When 66 x 10e total cells were separated on a continuous gradient, the profile (Fig. 2C ) showed three principle

RAYMOND

158

C. PFLEGER

a Type II cell peak between 12.2 and 16.8% Ficoll (density =1,0441.059 g/ml) and two macrophage peaks, one at 17.8 (density = 1.065 g/ml) and another at 19.6% (density = 1.072 g/ml) Ficoll. The viability of cells recovered from the continuous gradient was 56%. When 31.0 x lo6 viable cells were recovered, 7.7 X lo6 cells were in the Type II cell peak and of these >70% were Type II pneumonocytes. The dotted line shows the linear nature of the gradient (Fig. 2C). When 138 x lo6 total cells were applied to a continuous gradient of Ficoll, a separation of the macrophages from the Type II cells was not achieved (Fig. 2D). This indicated an overloading of the gradient and interference with cell bouyant density. For separation of the initial enzyme-liberated cell suspension on discontinuous gradients of Ficoll, no more than 12 X IO6 total cells were applied, After centrifugation, the cells at the three interfaces were counted and characterized as described. At the buffer-11.3% Ficoll interface (density = 1.003-1.041 g/ml), peaks:

“56%

of the recovered

cells

banded

and

of these

>70%

appeared

by light

microscopy (Papanicolaou stain) as Type II pneumonocytes. At the 11.3-15% Ficoll interface, “32% of the initial cell population was found and of these 36% were Type II cells. At the 15-16.67, Ficoll interface, slightly >lO% of I 3.

I

I

.30

.

.24

.I6

.I2

.6

c0s=

% x

I

Y

. . . :

ii m222 20-

30 F5

l.

&‘*.

IS-

1 I

-24

.

/ .

-16

. .

-12

IO-

b

.

0

I0

I 8 PERCENT

2 e

1 16 FICOLL

-6 .

. .

2s

. 32O

FIG. 3. Separation, on a continuous linear gradient (dotted line) of Ficoll (density = 1.003 to 1.092 g/ml), of a population of Syrian hamster Type II cells that was obtained from the 10%17.6% Ficoll interface of a discontinuous gradient. The cells were centrifuged for 15 (E) The cells (20.1 X lo8 viable and 79.9% viability) peak at a density = min at 325 g,.,. 1.059 g/ml. However, a wide range in cell density ( 10% solids, density = 1.036 g/ml to 25% solids, density cl.092 g/ml) is observed. (F) The cells (2.22 X 10’ viable cells, 62.8% viability) peak at a density = 1.054 g/ml and show a relatively small range in density.

TYPE

II

PULMONARY

EPITHELIAL

CELLS

159

FIG. 4. A sample of a cytocentrifuged preparation of the enriched Type II cell population from Syrian hamsters. Most of the cells are Type II pneumonocytes (II) with the exception of a few macrophages ( M ), lymphocytes ( L), and degenerate cells. The cells were obtained from the buffer-11.3% Ficoll interface. The Type II cells show clearly an abundance of welldefined inclusion bodies. Not all cells are in the same focal plane. Modified Papanicolaou stain. FIG. 5. Electron micrograph of the pellet from the Type II cell fraction corresponding to that shown in Fig. 4. The micrograph demonstrates the well-preserved Type II cells. Uranyl acetate and lead citrate.

160

RAYMOND

C. PFLEGER

the initial cell population was found, but most of these cells were macrophages with very few Type II cells present. The density of these cells ( >1.065 g/ml) can probably be attributed to phagocytosis of the fluorochemical liquid. Macrophages, cellular debris, fluorochemical liquid, and albumin were found at the bottom of the centrifuge tube, The cells, banding at the 10.8-17.6c/r Ficoll interface (25.1 x lo6 cells) from two discontinuous gradients each loaded initially with 50.3 x lo6 cells, were reloaded on a continuous gradient, and the profile shown in Fig. 3E was obtained. However, by reducing the total number of cells initially applied to discontinuous gradients to 12.7 x lo”, the cells (3.5 x 106) from the 10%17.6% Ficoll interface (Fig. 3F) when applied to a continuous gradient produced a much narrower and more defined profile than the profile of Fig. 3E. This overloading phenomena emphasizes the interference with isopyknic bouyant density that will occur with too many cells on a gradient. The cells shown in Fig. 4 were a typical population of the pulmonary alveolar Type II pneumonocytes that can be isolated by our procedure. The lamellar bodies appeared as dark blue inclusions by light microscopy and were seen as black dots in the cells (Papanicolaou stain). Macrophages and lymphocytes were readily distinguished from the Type II cells. An example of a binucleated cell described previously ( Kikkawa and Yoneda, 1974) was also evident. The cells in the electron micrographs (Figs. 5-8) were typical of the Type II cells isolated by this procedure. All show the characteristic lamellar inclusion bodies. The different pattern of the intravesicle lipid-protein complex was probably due to the different stages of inclusion body maturation as suggested by Ryan et ~2. ( 1975). The cells assumed indefinite shapes, and extracellular surfactant was observed which suggests some membrane damage and possibly some cell death. A pulmonary alveolar macrophage (Fig. 9) and those cells accompanying it were recovered from the 15-16.6(;/, Ficoll interface of a discontinuous gradient and were part of the initially isolated cell population. The characteristic appearance of this macrophage readily distinguishes it from the pulmonary alveolar epithelial Type II cells. Macrophages dyed with Phosphine 3R (Mason et al., 1975) fluoresced bright yellow with the granular bodies and nucleus brighter than the cytoplasm. The nucleus and fine inclusions in viable Type II cells fluoresced a brillant yellow, and in some instances the great number of such inclusions resulted in total cytoplasmic fluorescence (Fig. 10). On the basis of photographic exposure times, Type II cells exhibited 15 times the fluorescent brilliance of macrophages. Table I provides a summary of the results from the lung cells isolated from Syrian hamsters. The number of viable cells recovered per animal varied somewhat but an average of 50 x 10F was recovered. Inevitably, a large percentage (“75% ) of the cells was lost on the discontinuous gradients. The recovery of 4 x lo6 cells in the enriched Type II cell population was tabulated from over 70 experiments that involved approximately 300 animals. In Vivo Metabolism of Pulmonary Alveolar Epithelial Type II Cells The in vivo uptake of the [1-**C]palmitic acid into the initial enzyme-liberated cell and enriched Type II cell populations shows that the Type II cells rapidly metabolize surfactant precursor material and are rich in lipids compared with the total population (Table I). The Type II cells actively incorporated the

TYPE

II

PULMONARY

EPITHELIAL

CELLS

161

162

RAYMOND

C. PFLEGER

FIGS. 6-8. Electron micrographs from three different preparations of Type difference in inclusion body appearance may be due to variations in the degree Uranyl acetate and lead citrate.

II cells. The of maturation.

surfactant precursor into phosphatidyl glycerol, a compound we have previously shown to be an important surface-active component of pulmonary surfactant (Pfleger et al., 1972). The largest class of surface-active lipid, phosphatidyl choline, was also labeled with [“C]palmitate but quantitative separation of the dipalmitoyl lecithin from the unsaturated lecithins was not achieved. The fourfold greater uptake of the [6-3H]thymidine into the Type II cell fraction as compared to the initial enzyme-liberated cell population suggests that indeed these active cells divide at four times the rate of the initial isolated population. DISCUSSION Identification of Type II cells is based upon their unusual fluorescence with Phosphine 3R, their characteristic display of lamellar bodies (Papanicolaou stain and electron microscopy), their size ( 12 pm), their buoyant density ( < 1.055 g/ml), and their ability to synthesize disaturated surface-active phospholipids. The lung perfusion was completed before coagulation set in so as to minimize blood elements in the initially isolated cell population. Attempts to shorten the procedure by eliminating the first lavage treatment and by using only five lavages following the incubation with the fluorochemical-albumin suspension resulted in an enzyme-liberated cell suspension that behaved similarly to the results

TYPE

FIG. 9. Electron lated cell population Ficoll interface.

II PULMONARY

micrograph of a pulmonary that was recovered from

EPITHELIAL

CELLS

alveolar macrophage from a discontinuous gradient

163

the initially isoat the 15-16.6s

shown in Fig. 2D. Upon centrifugation with a discontinuous gradient, such a cell suspension would only yield a population of cells 30% enriched in Type II pneumonocytes. The time of fluorochemical liquid-albumin residence in the lung was identical to that reported by Mason et al. ( 1975). However, when the concentration of fluorochemical liquid and albumin reported by Mason et al. (1975) was used, the number of cells obtained in the second lavage treatment isolated enzymeincreased to 9.3 x 106, but the viability of the subsequently liberated cell suspension was depressed to 31% and the viable cell yield decreased to 2 x 106. Whether hypoxia or another effect was responsible for this is unclear. In addition, the large amount of albumin that was suggested (50 mg/animal), interfered with the separation of the Type II cell band on both the continuous and the discontinuous gradients, similar to that shown in Fig. 2D. The macrophage yield following the fluorochemical liquid-albumin suspension treatment was greater than that obtained from the first lavage treatment and probably reflects the removal of those cells that were in the interstitium which migrated to the surface and became removable by lavage (Bowden and Adamson, 1976). Without the fluorochemical liquid treatment, the macrophages migrated with the Type II cells in both gradients which decreased the enrichment factor significantly. We selected the lowest possible percentage of trypsin and shortest time of

164

RAYMOND

C. PFLEGER

trypsin incubation consistent with high epithelial cell yield and viability while leaving the interstitium and endothelium intact. A 0.3% trypsin solution combined with a 20-min incubation yielded a sufficient number of cells with intact membranes without adverse effects (Phillips and Skank, 1964). The residual albumin in the lung from the fluorochemical liquid-albumin treatment may inhibit a certain percentage of the enzyme activity. Our technique of exposing the lung epithelial lining to the enzyme to achieve a stripping action seemed to assure a more complete exposure of the total epithelium to the trypsin than use of a lung mince ( Kikkawa and Yoneda, 1974). Also in the intact lung, by maintaining vascular integrity, we did not observe fibroblasts or endothelial components in our enzyme-isolated cell population. Data from Kikkawa and data from our laboratory suggest that Yoneda (1974) and recent preliminary the Type I cells were killed by the trypsinization procedure. Separation of cells in a Ficoll medium has been reported to cause few, if any, adverse cellular effects (Warters and Hofer, 1974). The yield of Type II cells per animal was less in this system than reported in the note by Mason et al. (1975) but was an order of magnitude greater than that of the first report of Type II cell isolaion by Kikkawa and Yoneda ( 1974).

FIG. 10. A photomicrograph of this cell suspension displays the brighter yellow fluorescence of the Type II cells in contrast to the less bright macrophages. The cell sample was recovered from a buffer-Ficoll interface of a discontinuous gradient. The cells in Phosphine 3R were examined using fluorescent techniques. All cells are not in the same focal plane.

TYPE

II

PULMONARY

EPITHELIAL TABLE

Recovery

of Viable

Lung

CELLS

165

I

Cells

from

Syrian

Hamsters

Initial enzymeliberated cell population Number recovered per animal ( X 10’) Percentage recovered from gradient* W per lo6 cellsd (dpm) ‘4C-labeled Lipid per 106 cells (dpm) Lipid per 106 cells bg) W-labeled Lipid per microgram of lipid (dpm) Percentage of [%]phosphatidyl disaturated fatty acid esters 3H per lo6 cellse (dpm)

50 f 17. 25f 6 686 f 92 518 f 201 28f 2 per

6f 3310 f 2089 f 347 f

lc 543 477 23

lo6 cells 18f

glycerol

Enriched population of type II cells

8

6f

1

with 16f 660 f

4 110

56f 2589 f

1 404

* Mean f SE. b Centrifugation of 9.5-lo..5 X lo6 viable cells on a discontinous gradient for 30 min at 275 g,,,==. c 70y0 (range = 67-727,) of the cell population at the buffer-11.3% Ficoll interface was viable Type II cells. Macrophages (16%), lymphocytes (7.5%), ciliated (0.2%), and other cells (6.5%) were also present. d [l-W]Palmitic acid (50 kCi) injected ip 4 hr before sacrifice. e [6-3H]Thymidine (100 &i) injected ip 4 hr before sacrifice.

Subsequent analysis showed that the enriched population of Type II cells had actively taken up the palmitic acid into the surface-active phosphatidyl glycerol at a greater rate than the initial enzyme-liberated cell population. These data are the first demonstration of a pulmonary surfactant precursor incorporation in viva into surface-active phosphatidyl glycerol. Our previously reported work showed the in vitro uptake of a-glycerol phosphate into triglycerides, phosphatidyl choline, and phosphatidyl glycerol (Pfleger, 1975). It is now generally accepted that the pulmonary alveolar Type I cells, lining most of the epithelial alveolar surface, are labile entities when exposed to various noxious agents and/or environmental pollutants. It is recognized further that, following Type I cell death, proliferation of Type II pneumonocytes, which in a few hours transform into Type I cells (Evans et al., 1975), quickly occurs. The proliferative response has been observed following lung damage by various pulmonary toxicants (Freeman et al., 1974; Kapanci et al., 1969; Bingham et al., 1976). Neoplastic proliferation of Type II cells has been observed in mice (Brooks, 1968), sheep (Nisbet et al., 1971), and man (Nash et al., 1972). Thus an understanding of the normal biological behavior of Type II pneumonocytes, as well as alterations in the hyperplastic and neoplastic analogs, may provide insight into the pathogenesis of pulmonary disease following inhalation of toxic materials. ACKNOWLEDGMENTS I am grateful to members of our professional staff for their assistance in cell characterization to M. M. Sturm for the electron microscopy, and to J. J. and in manuscript preparation, Waide, M. Binette, and W. D. Roddy for exceptional technical assistance.

REFERENCES ADAMSON,

epithelial

I. Y. R., and regeneration.

BOWDEN,

D.

H.

(1974).

Lab. Inuest. 30, 35-42.

The

type

2 cell

as progenitor

of

alveolar

166

RAYMOND

C. PFLEGER

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Type II epithelial cells from the lung of Syrian hamsters: isolation and metabolism.

EXPERIMENTAL Type AND MOLECULAR II Epithelial PATHOLOGY 27, 152-166 ( 1977) Cells from the Lung of Syrian Isolation and Metabolism1 RAYMOND C...
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