Metabolic properties and ultrastructure of alveolar type II cells isolated with elastase

Metabolic properties and ultrastructure of alveolar type II cells isolated with elastase

510 Biochimica et Biophysics Acta, 618 (1980) 510-523 0 Elsevier/North-Holland Biomedical Press BBA 57580 METABOLIC PROPERTIES AND ULTRASTRUCTURE T...

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510

Biochimica et Biophysics Acta, 618 (1980) 510-523 0 Elsevier/North-Holland Biomedical Press

BBA 57580

METABOLIC PROPERTIES AND ULTRASTRUCTURE TYPE II CELLS ISOLATED WITH ELASTASE

OF ALVEOLAR

LELAND G. DOBBS, EUGENE F. GEPPERT, MARY C. WILLIAMS, ROBERT D. GREENLEAF and ROBERT J. MASON * Cardiovascular Research Institute, University of California, San Francisco, (U.S.A.)

CA 94143

(Received September 17th, 1979)

Key words: Type I1 cell metabolism; metabolism; (Rat lung)

Ultrastructure; Elastase isolation; Fatty acid

summaly

We used porcine pancreatic elastase to isolate type II cells from the lungs of rats; the yield and purity of the type II cells was better than that obtained by methods using trypsin. In 102 experiments we obtained 82 + 23 - IO6 cells/rat, 68 + 11% (mean + S.D.) of which were type II cells. This preparation of cells, when centrifuged over a discontinuous density gradient, yielded 25 + 10 - lo6 cells/rat, 80 f 13% of which were type II cells (n = 102). The cells, after density gradient centrifugation, could be further purified by centrifugal elutriation (94 + 3% type II cells, n = 22) or adherence in primary culture (94 * 2% type II cells, n = 34). Type II cells isolated with elastase are similar morphologically and biochemically to type II cells isolated from rats with trypsin. The preparations of cells appeared healthy by several different criteria: ultrastructure, exclusion of vital dye, lack of stimulation of oxygen consumption by exogenous sodium succinate, and linear rates of oxidation of [l-14C]palmitic acid and of incorporation of [1-14C]acetate into fatty acids. Type II cells consumed 75 + 20 nmol 02/106 cell per h, oxidized [l-‘4C]palmitic acid at a rate of 0.4 nmol/106 cells per h, and incorporated [l-14C]acetate into fatty acids at a rate of 7.5 nmol/106 cells per h. Introduction Many methods [l-5] the ability to produce

of isolating type II alveolar cells (the cells that have surface-active material and reline damaged alveolar

* To whom correspondence should be sent at: CVRI, M1315, San Francisco. CA 94143, U.S.A. Abbreviation: Hepes. N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic

University acid.

of California

Medical

Center,

511

surfaces) have utilized the enzyme .trypsm to dissociate cells from the lung. Because treatment with trypsin can&r b&h till receptors [6-6] and cell rne~~m [9,10], we were interestedin developing a method of isolatingtype II cells that did not require trypsin. We have found that porcine pancreatic elastase can be used instead of trypsin to isolate type II cells and report here the morphologic and biochemical characteristicsof type II cells isolated from rat lungs with elastase.Some of these data have been reported in preliminary form [11,12]. Materials and Methods

Because there are numerous differences (as well as similarities)between this and methods for isolating type II cells which we have described previously, we have described this method in detail Animnls. We have used two strains of male rats (150-400 g) from three different sources: specific pathogen-thee Sprague-Dawleyrats from Hilltop Labs, Scottdale, PA (n = 72 experiments); and Long-Evansrats from a colony at the Universityof California which was maintaineduntil June, 1977 (n = 11) and from Simonsen, Gilroy, CA (n = 19). We have not observed consistent variationsin cell yield or purity that we could attributeto the different sources of the animals.From two to eight rats were used in each experiment., Solutions used to iaolute type II cells. Ail solutions were made up with double glassdistilled water. Solution I comprised 136 mM NaCl, 5.3 mM KCI, 5.6 mM glucose, 2.6 mM sodium phosphate buffer, IO mM Hepes buffer (pH 7.40 at 22°C). Solution II comprised solution I with 1.9 mM CaC&and 1.3 mM MgSG,. The ffuoroc~bon/~bum~ emulsion was made by sonicating (‘L converter’, Branson Sonic Power, Danbury, CT) 2 ml of fluorocarbon FC-75 (3M Co., St Paul, MN) with 6 ml of an albumin solution (fatty acid free albumin, Sigma Chemical Company, St. Louis, MO, 10 mg/ml in solution II) for 2 min at 75 W and adding this emulsion to 24 ml of the albuminsolution. This volume was sufficient for the lungs from two rats. Elastasesolution contained elastase 40 orcein-elastinunits (U)/ml in solution II (porcine pancreas, twice crystallized, aqueous suspension, Worthington Biochemical Corp., Freehold, NJ). We verified the stated activity of the elastaseby one of two assays 113,141. We measuredtryptic [15] and chymotryptic activity 116,171 in seven out of the 13 lots of elastaseused in these experiments.These was no measureable tryptic activity (less than O.Ol%, w/w; less than 100 ng/ml in elastasesolution) and a variable but small amount of chymotryptic activity (less than 0.04% w/w; less than 600 ng/ml in elastasesolution) in the lots we tested. We tested the effect of varying the concentration of elastasein five different lots of elastaseon the yield, purity, and viability of type II cells. We found that in three lots of elastasewe could obtain equivalentresultsto 40 U elastase/lwith con~n~tions as low as 20 U/ml. Becausewe obtained lower yields in two lots of elastasewith 20 U/ml than with 40 U/ml, we elected .to use 40 U/ml as our standard concentration of elastase. All experiments reported in this paper were performed with 40 U/ml. isolation of type II cells with elustase. We used plastic or freshly siliconixed glassware ~rou~out the cell isolation procedure. Each .rat was injected

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intmperitoneally with 400 U heparin/lOO g wt. and 10 mg pentobarbital/lOO g wt. After the rats were anesthetized, we perfused the lungs via the pulmonary artery and then excised the lungs as previously described [ 21. The excised lungs were lavaged through the tracheal cannula with solution I, 8-15 ml (to reach total lung capacity) of the fluorocarbon/albumin emulsion was instilled, and the lungs were incubated in 154 mM NaCl at 37°C for 20 min. The lungs were removed from the saline, lavaged eight times with solution I to remove most of the fluorocarbon emulsion, and then lavaged once with 8 ml of the elastase solution. We next added the elastase solution to total lung capacity (8-15 ml) and incubated the lungs in 154 mM NaCl at 37°C for 20 min, during which time we maintained the lungs at total lung capacity by continually filling them with the elastase solution. After this, the lungs were minced with scissors for 60 s (200 s&sons strokes) until the final size of the pieces of lung was 1-2 mm3. (Instilling the enzyme via the trachea and then mincing the lung as described yielded more type II cells than performing the procedures in reverse order; i.e. mincing the lung tissue fit and then incubating the minced pieces with enzyme.) We added 5 ml of fetal calf serum and solution II to a final volume of 20 ml, poured the minced lung tissue and cell suspension into a 250 ml Erlenmeyer flask, and shook the flask in a reciprocating water bath at 130 cycles/min for 2 min at 37°C. The suspension was filtered sequentially through three filters: cotton gauze, 100 pm nylon mesh, and 20 pm nylon mesh (Tobler, Ernst and Traber, Inc., Elmsford, NY). We refer to the cell suspension at this point in the isolation procedure as ‘before gradient’. A discontinuous density gradient was made with either albumin (Path-OCyte 4, Miles Laboratories, Kankakee, IL) or metrizamide (Accurate Chemical and Scientific Corp., Hicksville, NY), by layering 10 ml of density 1.0361.041 (range) on top of 10 ml of density 1.085-1.091 (range). We layered 20 ml of the cell suspension on top of each gradient and centrifuged the gradients for 20 min at 200 X g (1.040-1.090 interface) (1000 rev./min) in an International PR-2 centrifuge (International Equipment Co., Needham Heights, MA) at 4°C. The top 26 ml contained few cells and was discarded; the next 13 ml, which contained the interface between the solutions of approx. 1.040 and approx. 1.090 density was carefully removed and placed into a 50 ml centrifuge tube. We added solution II to a volume of 45 ml, centrifuged the cells at 200 X g for 10 min, removed the supematant liquid, added solution II to 45 ml, and centrifuged the cells at 160 X g for 10 min. We refer to the cell preparation at this point as ‘after gradient’. Alveolar type II cells could be purified further by centrifugal elutriation [18] or by adherence in primary culture [ 12,191. Cells were elutriated as previously described [ 181. For primary culture, cells were plated at a density of 106 cell/ml in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 50 pg/ml gentamicin, 2 mM glutamine, and 100 U/ml penicillin G. Seeding efficiency was determined by comparing the DNA content [20] of the original cell suspension to that of the cells adherent to the culture dish after 22 h of culture. Identification of type II cells. Type II cells were routinely identified by the modified Papanicolaou stain [ 11. Morphology. In three experiments we purified cells isolated with elastase to

613

the step designated ‘after gradient’. T&se & were prepared far electron microscopy by methods we have used previously [2]. To assess the effects of the ela&ase treatment on lung structure, we examined minces of lungs that had been treated with elastase to isohte type IX cells and minces of lungr that had been treated in an analogous fashion but without elastase. These minces of hmg tissue wene prepared for electron microscopy by methods similar to those used to prepare type II cells, To stain for elastic fibers. we also embedded minces of lung tissue in glycolmethacryiak, sectioned the tissue at 2 pm thickness, and stained the sections with Verhoff stain. Vital dye exclusion. Exclusion of the vital dye erytbrosin B was determined by a previously described method [ 211. Lipid composition. Five different preparations of type II cells were purified by centrifugal elutriation; the preparations used for analysis of lipid composition were 93. k 2% (mean f S.D.) type II cells. Each preparation of cells was treated separately. Lipids were extracted by the procedure of Folch et al. [22] and the mixed lipids were separated by two-dimensional thin-layer chromatography [23]. Spota were identified with 60% ammonium bisulfate, scraped, and phosphorus was determined by the method of Bartlett [ 24 1. Recoveries, determined in four out of five experiments, were 98 f 3% fn = 4). For determination of the percent of ph~phatidylcho~e that was saturated, spots were identified by brief exposure to iodine vapor and then scraped. Total phosphatidylcholines were eluted from the silica with CHCl&!H30H/HzO/C!H&XXX-I (SO : 60 : 2 : 1) followed by CHCIJ/CH~OH/H1O (30 : 60 : Ei), extracted [22], and then reacted with OsO, in CC& [2S]. Saturated phosphatidylcholine was separated from species of phospha~dyl~ho~e containing unsaturated fatty acids by thinlayer c~rna~~phy on silica gel G plates impregnated with boric acid in a solvent system of CHClJ/CH30H/14 N N&OH/Hz0 (75 : 25 : 1 : 2). Oxygen consumption. Eight different preparations of type II cells were purified by centrifugal elutriation; 92 f 2% of the cells were type II cells and 91 f 6% (mean + S.D.) excluded the vital dye erythrosin B. Oxygen consumption was measured as described previously [ 181. Because the value of oxygen consumption for type II cells isolated with elastase and purified by centrifugal eiutriation was somewhat lower than we had previousiy &ported for cells isolated with trypsin and purified by centrifugal elutriation [ 181, we felt that it was important to repeat the earlier experiments. We therefore isolated type II cells with 3 mg/ml trypsin and purified them by centrifugal elutition as we had previously [18] ; the cells were 88 f 5% type II cells and 93 -+4% excluded erythrosin B. Oxidation of [l-“Clpalmitic acid. Type II cells ware purified by centrifugal elutriation for the studies of oxidation of [l-14C]palmitic acid and incorporation of (l-“Clacetate; the cell preparations were 94 k 3% (mean i S.D.; n = 4) type II cells, 93 f 7% of which excluded erythrosin B. Cells were suspended (4 * 10’ cells/ml) in minimal essential medium (Eagle’s) containing Hank’s sat& 26 mM Hepes buffer, 100 U/ml panicill& 50 &ml gentamicin, 2mM glutamine, and [ l-14C]palmitic acid (New Engiand Nuclear Corp., Boston, MA) bound to Mty acid-poor bovine albumin (Sigma Chemical) to give final concentrations of 0.1 mM pa&tic acid (spec. act. 0.6 mCi/&ol) and 10 mg/ml albumin, with a palmitic acid/albumin molar ratio of 0.7. We placed 2.5 ml of

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the cell suspension(lo6 cells) into each of severalsiliconized glassscintillation counting vials and sealed the vials with rubber stoppers equipped with center wells for collecting CO, [ 261. Vials that contained radioactivemedium wi~out cells were incmbatedas blanks. The incubation vials were placed in a reciprocating water bath at 37°C at 35 cycles/min. At the end of the incubation period, 0.25 ml of hyamine hydroxide was injected through the rubber seal into the center well and 0.3 ml of 3% HzSQ was injected into the cell suspension 1261. The sealed vials were allowed to stand at room temperatureovernight; we found, in preliminaryexperiments with NaHS4C03,that this procedure resulted in recovery of 99% of 14C02in the center well. We put the center wells into counting vials containing 20 ml of toluene/CHJOH/Liquifluor(New EnglandNuclear)(2 : 1 : 0.125) for liquid scintillationcounting. Incorpomtion of [l-‘4Cf~cetute into fatty acids. We suspended cells (4-8 * 10’ cells/ml) in modified Eagle’s medium with Earle’s salts, 2 mM glutamine, 100 U/ml penicillin, 50 M/ml gentamicin, 3 mglml fatty acid-poor bovine serum albumin and 2.1 mM [1-‘4C]acetate (New EnglandNuclear;2.4 Ci/mol), 2 ml of this cell suspension (containing 0.8-1.6 . lo6 cells) was placed in each of severalsiliconized scintillation counting vialsand the vials were incubated at 37°C in an incubator containing 5% CO* in air; the incubatingvials were swirled by hand every 15 min. At the end of the fruition period, the cell suspension was transferredinto tubes at 4°C which contained 20 ml of a solution of 1 mM N-ethylmaleimidein 154 mM NaCl. We collected the cells as a pellet by centrifugation at 250 X g for 10 min and washed the pellet three times. We added 0.6 mg of mixed lipids from dog lung as carriersand extracted the lipids from the cell pellets by the method of Folch et al. 1221, usingan aqueous solution that contained 100 mM KCl and 100 mM sodium acetate. The lipids were saponified with etbanolic KOH. The non-saponifiable,lipids were removed by three extractions with hexane. We then acidified the saponified lipids, extracted the fatty acids by three extractions with hexane, and backwashedthe fatty acid samples with 5% acetic acid. The fatty acid sampleswere evaporated to dryness and redissolved in CHCIJ/CHSOH(2 : 1, v/v). An aliquot was removed for determinationof radioactivi~ 1271. In order to verify that we had isolated fatty acids, we chromatographedOUT samples on silica gel G thin-layerplates in a solvent system of hexane/diethyl ether/glacialacetic acid (50 : 50 : 1, v/v) [ 281 using oleic acid, cholesterol and cholesterol palmitate as standards.We visualizedthe lipids with iodine vapor, scraped the spots into coun~g vials, added dioxanelwater sc~~ation fluid, and determined the radioactivity in the samples f27 1. We tested four samples from two separateexperiments and in each case found that more than 98% of the recovered counts cochromatographedwith the oleic acid standard. ReSUltS

Yield, purity, and exclusion of vital dye The yield, purity, and vital dye exclusion of our preparation of cells before and after centrifugation over a discontinuous density gradient are described in Table I. The cell population after the gradient could be further purified either by centrifugal elutxiation or by adherence in primary culture. The results of

516 TABLE I ISOLATION OF TYPE II CELLS D&e exe mean f SD. ad..

not done. Before

CaBe holeted with 40 U/ml ekrba No. of cells (xxo6)/?8t % of type II c&s % of c&e sxaiudlng erHhrodn

Aftax SNdiMnt

VI - 102) 25*10 80 f 13 92% 6

32 + 28 68ill 90 * 10

B

tulle holatod with 3 m3/ml CrYwe No. of cella (X lO*)lrat % of type II cellJ Cells ieolated with 0.1 ml/ml No. of cells (Xlti)/ret % of type If c6lis

gxxdirnt

trYpdn

@I f

86) 11* 6 63 * 10

n.d. n.d.

crwMl.tne tnPh

(n= a.d. n.d.

26) ri* 2 60 * 10

centrifugalelutition are depicted in Table II. We loaded 6-20 - 10’ cells into the elutriator and recovered 48 -+5% (n = 7) of the loaded cells in the two best fractions; these two fractions were 93 f 3% type II cells (mean f: S.D.; n = 7). In an additional 15 experiments with centrifugalelutriation, we did not determine the yield and purity of the cells at each flow rate; for all experiments (n = 22) employing elutriation, the pooled cella obtained at flow rates of 18 ml/min and 22 ml/min were 94 f 3% type II cells (m&m f S.D.). Adherence in primary culture produced, after 22 h in culture, a cell population that was 94 f 2% (n = 34) type II cells. Seeding e?ficiency, as determined by DNA, was 68*8%@=9)in22h. For purposes of comparkon, we have included in Table I our cumulative experience in isolatingtype II cells with trypsin by methods we have previously described [!&19]. We did not routinely determineviability during the cell iaolation procedures nor did we examine the preparation of cells before gradient cen~~tion,

TABLE II FUBTWER

PURIFICATION

OF TYPE II CELLS BY CENTRIFUGAL

ELUTRIATION

The a& a? the gradient wem fwtber ~urificd by centrifugal elutrietion 1181. The cells were collected et ds fior MU, rangins from 7 to 34 ml/min. In ordar to preeent eii the expezimsnte. the yield et eeeb flow rnk k given eJ e Percent of tow celle loaded into the elutdation chunk. In aeven extMKlmenta. the number of cells loaded ranged f&m 5 to 20 - 10’ aells: the aelb were 02 16% type II calla(meant SD.). After elutrhtion, there were come aella rem&&g In both tbe mlxin# uxl alutdatlon cbamw The mixina chamber contained 0.6 f 0.2% of the eelle tht wem loaded into the elutchtor: 30 * 12% of them were type II ealle, The elutriator dumber coateined 2.8 f 0.8% of the celle that wereloaded; thaw! were US f 10% typa 11 aelk Flow ate (mi/min)

Celh

8 cdl8 loeded iota 96 type II ceJh

e1utri8t02

7

14

18

22

28

34

1*1 7a7

10 * 5 63 f 4

21 f 8 94 * 3

22 f 4 93 f 4

8*4 85 f 0

3* 8 71 f 10

516

F&L 1. This low-power electron micrographiJiustratesthat elastase-isolatedtype II cells are structurally intact, have cytophsmic and nucleardensitieswhich are idmikuto those in intact lungs. and contain 8 full complement of organelles. In a few cells (arrows). the endoplasmic reticulum is dilated. All cel%ain the field except one leucocyte (*) are type II cells. X3750.

517

As shown in Figs. 1 and 2, type II cells isolated with elastase have a wellt&awhu&re. In some cells (Fig. I), the rough endoplasmic retieulunr is somewhat dilated and membranes of lamellar bodies are less densely pack& than those in intact lung tissue. other oqanelles, especially mitochondria (Fig. 2), appear, relatively undamaged by the isolation procedure. Cells isolated with elastase cannot be distinguished from cells isolated with trypsin by morphologic methods. In order to evaluate possible sites at which elastase acts to dissociate the alveolar epithelium, we examined lungs treated with elastase by electron microscopy. As shown in Figs. 3 and 4, elastase facilitates the detachment of epithelial cells from the underlying basal lamina; the remaining cells are solely within the interstitial and vascular compartments. The denuded basal lamina remains tightly apposed to the interstitial tissue. Interstitial elastic fibers and elastic laminae of alveolar blood vessels are, however, also digested by the elastase treatment, as judged both by electron microscopy (Figs. 3 and 4) and by the lack of specific staining for elastin (Figs. 5 and 6).

P

Phospholipia!composition The phospholipid composition

of type II cells is shown in Table III. The

Fig. 2. Mitochondria ([email protected]), Golgl corn&x (G). lemeller bodier (LDe). end other orgenellee of thie ehtaeeieoleted cell eppeer unaltered by the isolation procedure. X11 600.

--

_-.

Fig. 3. An electron micrograph of mimed tissue lbrom lungs treatwl with elastaae demonatratsr that elastsse remows or ioooeas the shraolar s~i#eliu~ From the u+ieri~ Iwai kmfna fa a reasonably sek&i~e fashion, ‘P&e remaiaing c&s in this fi&d am entkely tpitfrin the limits of the baa& laxnina (arrow) on either side of the alveolar septae. Bundles of collagenous fibers CC) are eke&y seen in tba inter&t&m; eiastie fibers are absent. X5500.

Ffg. 4. The reiatimmhip of the fuzzy basei &a&a (BL) to msldual alveolar set?talCells can be better v-in& ized in this &gber magnification electron micrograph. X11 000.

Fig. 5. DarWy staking eIastfc fibers @row) are readily sesn in this light micrograph of minced lung tissue. The lung was prepared by the methods described in the text except eiastase was omitted. The interna! and external elastiu laminae of a small arteriole (A) are also positively stained. Verboff stain. X400. Fig. 6. In contrast, elastic fibers appear to be completely absent from the elastase-treated tissues seen here. The partial collapse of alveolar structure gives the impression that the cellukuity is increased, rather than decreased. Verhoff stain. X400.

percent of ~ho~hatidylcho~e (n = 3)*

that

was fully saturated

was 44.7 + 1.5%

Oxygen consumption

Type II ceils after centrifugal elutriation consumed 75 * 20 nmoE C&/lo6 ceuls per h (n = 7); the addition of 10 mM sodium succinate did not significantly alter oxygen consumption (‘9 + 15%; ft = 7). Concurrent experiments with type II cells isolated with 3 mg/ml trypsin instead of elastase and purified by centrifugal elutriation consumed 79 + 25% nmol O&O6 cells per h (n = 9); sodium sue&ate slightly increased (+17% + 13%; n = 7) basal oxygen consumption. Oxidation of [1-14C]palmitate and incorporation of ~l-14C~~cetate into fatty acid The oxidation of pahnitic acid was linear for the 4 h incubation period at a rate of 0.4 nmol/104 cells per h (n = 4). This rate assumes that palmitic acid is

521 TABLE III PHOSPPHOLiPIII CObfPOSITION OF TYPE II CELLS n.d.. not detected. Percent (melln f S9.J PhveM&drlchoUne PhosphrtidH#lycerol ~o~ha~dyle~v~~~e ~o~ffdy~o~tol PhosMatidrlserfne SpinsomYeUn Lyropho8phatidylcholine Other

68.0 9.6 13.6 1.6 2.0 2.3 n.d. 2.1

* 1.3 * 1.5 * 1.3 l 0.5 * 0.8 f 0.8 f 0.6

completely oxidized to CO, ; some of the acetyl-CoA units may have also been reutilized and not converted to COZ. hype II cells incorporated [l-14C]acetate into fatty acid linearly during the period ‘of 0.5-4 h of incubation at a rate of 7.5 nmol/[email protected] cells per h (n = 4). Less than 2% of the [1-14Cfacetate was inco~om~d into the non~pon~iable lipids. Discussion The choice of which proteolytic enzyme to use in a cell isolation procedure is largely empiric. Tkypsin, which has been used more frequently than other enzymes to disaggregate tissue [29], was the enzyme initially used to isolate type II cells. Because there is now ample evidence that trypsin can alter cellular functions, we became interested in having an alternative method to that of tryptic digestion of lung for isolating alveolar type II cells. We chose elastase, which has been used infrequently to isolate cells 129-32 J, as the proteolytic enzyme because, in screening other enzymes, we obtained more type II cells with elastase than we did with chymotrypsin or with collagenase (dam not shown). The mechanism by which proteolytic enzymes in general dissociate tissue is not well understood [29]. We believe that the efficacy of elastase in isolating type II cells from whole lung tissue is not dependent upon the presence of con~~a~g trypsin or chymotrypsiu in the elastase preparations, since we found no tryptic and only trace ~0~~ of ~yrno~ti~ activity in the lots of elastase that we used in these experiments. We did not screen for the presence of other proteolytic enzymes. We found that the alveolar epithelium was removed by elastase, leaving in many places an intact hasement membrane overlying an intact interstitium (Figs. 3 and 4). It would therefore appear that our preparations of elastase disrupted both intercelhiiar connections and connections between cells and the basement membrane. In addition, elastase disrupted or dissolved the elastic fibers in both the interstitial and perivascular spaces (Fig. 6); the finding is compatible with the report [ 331 that elastase administered intratracheally could be demonstrated to be associated with lung connective tissue. Although the entire epithelium appeared to be removed by treatment with

622

elastase, we recovered a cell population that was striking for its high proportion of type II cells. Weibel et al. 134) have calculated that the adult rat lung contains 14% type II cells; we obtained, after treatment of lungs for 20 min with elastase, a cell population #at was 68 ? 11% type II cells (Table I). According to recent morphometric data [35], our yield represents almost twothirds of the type II cells in the lungs of 250% rats. We could achieve higher purity of type II cells, albeit with cell loss, by density gradient centrifugation, centrifugal elutriation, and/or primary culture. In our laboratory, the yield and purity of type II cells isolated with elastase has been reliable and reproducible; the yield of cells is considerably greater and the purity is comparable to or better than other methods, all of which have employed trypsin as the proteolytic enzyme. Type II cells isolated with elastase are similar both morphologically and biochemically to type II cells isolated from the lungs of rats with trypsin. The ultrastructure of freshly isolated cells prepared with trypsin or elastase is well preserved, aside from the dilatation of the endoplasmic reticulum in some cells [2,18]. The phospholipid composition of type II cells isolated with elastase is similar to that of type II cells isolated with trypsin and to that of pulmonary surface-active material. Noteworthy is the high percentage of phosphatidylglycerol and saturated phosphatidylcho~e. In concurrent experiments, we found the rate of oxygen consumption to be the same for type II cells isolated with elastase or crystalline trypsin. The rate is similar to that reported for rat type II cells isolated with crude trypsin [ 31. Our preparations of type II cells appear healthy by analyses of single cells (ultrastructure and exclusion of vital dye) and by metabolic properties of the cell population (lack of suction of oxygen con~mp~on by exogenous sodium succinate [36] and linear rates of both oxidation of palmitic acid and incorporation of acetate into fatty acids). The length of time that cells synthesize macromolecules such as protein has been used by others [37] as a sensitive way of determining the viabiMy of a cell preparation. We chose to measure both a biosynthetic property of the cytoplasm, i.e. fatty acid synthesis, and an energy production pathway of mitochondria, i.e. fatty acid oxidation; both these processes require activation of the precursor and an intact multienzyme complex. The rates of both fatty acid synthesis and oxidation were linear for 4 h. The rate of incorporation of acetate was similar to that found by Batenburg et al. [38] for cells isolated with trypsin and maintained in primary culture for one day. Almost all of the exogenous [l-14C]acetate was converted into fatty acids rather than into non-saponifiable lipid such as cholesterol. Type II cells isolated with elastase also synthesize a high percentage of saturated phosphatidylcholine (data not shown) and secrete saturated phosphatidylcholine in response to chemical stimuli [ 121. Since treatment with elastase reproducibly provides a greater number of type II cells which are at least as pure preparations of type II cells as those obtained by treatment with trypsin, since the cells have the morphological and biochemical characteristics of type II cells, and since the cells are healthy by several different criteria, we believe that the method described in this paper is a useful way of obtaining isolated type II cells.

523

Acknowkdgements The authors thank Mr. Leonard Berry, Ms. Jean Nellenbogen and Ms. Yuki Kubo-Hendricks for technical aseiatance, and Dr. John A. Clements and Dr. Bradley Benson for advice during the execution of these experiments. This research was performed while L.G.D. was the recipient of a Young Investigator Award (HL-19518), E.F.G. of the Medical School Pulmonary Faculty Training Award (HL-O7159), and M.C.W. of a Research Career Development Award (HL-O0221), from the National Heart, Lung and Blood Institute, U.S.A. R.J.M. was an Established Investigator of the American Heart Association. This research was supported by Program Project Grant HL-6285 and Pulmonary SCOR Grant HL-19185, from the National Heart, Lung and Blood Institute. References 1 Kfkkawa, Y. and Yoneda, K. (1974) Lab. Invest. 30.76-84 R.D. and Clements. J.A. (1977) Am. Rev. Respfr. Dis. 116. 2 Mawm. R.J.. Williamr. M.C.. Greedeat. 1016-1026 King. R.J. (1977) Am. Rev. Reapir. Dir. 115,part 2, 73-79 Pfleger, R.C. (1977) Exp. Mol. Pathol. 27,152-166 Ffahar. A.B. and Furh, L. (1977) Lun6 154.155-166 Kono. T. (196s) J. Biol. Chem. 244, 5777-5784 Mango& J.A.. Mc8herry. N.R.. Butcher, F., Irwin. K. and Barber, T. (1975) Am. J. Physiol. 229, 563569 8 Lefkowitz, R.J.. Mukherjee. C., Ltmbird. L.E.. Caron. M.G.. WiUams. L.T.. Alexander. R.W.. Mickey, J.V. and Tote. R. (1976) Rec. Rog. Harm. Ru. 32. 597-630 a Bobo, B. and Drewfnko. B. (1978) Rot. Sot. Exp. Biol. Med. 168.666-670 10 Weksler. B.B.. Ley. C.W. and Jaffe. E.A. (1978) J. Clin. fnve& 62.923-930 11 Dobbr. L.G., Geppert. E.F.. Greenleaf. R.D.. Benson. B.J.. Williams. M.C. and Mason. R.J. (1979) Am. Rev. Respk. Dfs. 119. 304 (abstr.) 12 Dobbs, L.C. and Mason, R.J. (1979) J. Clin. Invest. 63.378-387 13 8achar. L.A., Wfnter. K.K., Sicher, N. and Frankel. S. (1955) Sot. Exp. Biol. Med. 90.323-326 14 Bleth. J.. Spleu. B. and Wermuth, C.G. (1974) Biochem. Med. 11.350-357 16 Erlanger, B.F.. Kokowsky. N. and Cohen, W. (1961) Arch. Biochem. Biophys. 96.271-278 16 Hummel. B.C.W. (1969) Can. J. Biochem. Phyriol. 37. 1393-1399 17 Rinderknecht. Hand Fleming. R.M. (1975) Chn. Chim. Acta 59.139-146 18 Greenleaf. RD.. Mason. R.J. and Wffliamr. MC. (1979) In Vitro 15. 673-684 19 Mason, R.J., WBBams, MC. and Dobbs. L.G. (1977) in Pulmonary Macrophage and Epithelfal Cells, Rot. 16th Ann. Hanford Biol. Symp. (Sandem, C.L.. Schneider. R.P.. Dagfe. G.E. and Ra~an. H.A.. eda.), pp. 280-297. Technical Information Center, Energy Reseerch and Development AdmtnistraUon. Sprfn6field. VA 20 Setaro. F. and Morley. C.G.D. (1976) Anal. Biochem. 71.313-317 21 Phillfpr, H.J. (1973) in Tinue Culture. Methoda and Applications (Kruse. P.F.. Jr. and Patterson. M.K.. Jr., eds.), PP. 406408. Academtc Preaa, Inc., New York 22 Folch. J.. Leer. M. and Stanley, G.H.S. (1957) J. Biol. Chem. 226.497-509 23 Poorthub. J.H.M.. Yauki, P.J. and Hostetler. K.Y. (1976) J. L&id Res. 17. 433437 24 Rutlett. G.R. (1959) J. Biol. Chem. 234.466 25 Mason, R.J.. Nellenbogen. J. and Clement% J.A. (1976) J. Lipid Res. 17.281-284 26 Ho. R.J. and Jeanrenaud. B. (1967) Biochfm. Biophys. Acta 144.61 27 Snyder, F. (1964) Anal. Biochem. 8.183-196 28 Renkonen. 0. (1966) Biochtm. Biophys. Acta 126.288-309 2s Waymouth. C. (1973) In Vitro 10.97-111 30 Day, A.J.. PhtL D.. Newman. H.A.I. and Eflvemmit. D.B. (1966) Ctrc. Rea. IS. 122-131 31 Peterr, T.J., MuUer. J.M. and DeDuve, C.J. (1972) J. EXP. Med. 136. 1117-1139 32 Phtlifps. H.J. (1972) In Vitro 8.101-106 33 8andhau8, R.A. and Janoff. A. (1976) Am. Rev. Rerpir. Dir. 113. 214 (Abstr.) 34 Welbel. E.R.. Gehr, P.. Hater. D.. oil. J. and Bachofen. hf. (1976) in Lung Cefh in Dfseaae (Bouhuys. A.. ad.). Pp. 3-16. North-HoBand. Amsterdam 35 Brumley, G.. Tussle. R.. Luner, L. and Crapo. J. (1978) Am. Rev. Respir. Dts. 119. 461470 36 Berry. MN. (1962) J.%eB Biol. 15. l-6 37 Wa6le. S.R. (1876) Life Sci. 17.260-277 38 Batenburs. J.J.. Loncmore. W.J. and van Golde. L.M.G. (1978) Biochtm. Biophys. Acta 529. 160170