Lung 154, 155-165 (1977)
®El; Spri~ger-V~
Isolation and Metabolism of Granular Pneumocytes from Rat Lungs A r o n B. Fisher and Linda Furia Department of Physiology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19174, U S A
A b s t r a c t. A population of rat lung cells enriched in granular pneumonocytes (type II cells) was prepared by modification of the m e t h o d of Kikkawa (Lab. Invest. 30, 76, 1974). Lungs were minced, mechanically agitated and incubated with a fluorochemical emulsion and 1% trypsin. The isolated cells w e r e centrifuged in a discontinuous Ficoll gradient. Granular pneurnonocytes were identified by the presence of cytoplasmic inclusions on Papanicolaou and acid phosphatase staining and a strongly positive alkaline phosphatase reaction. The yield at the interface between 1.058 and I.i00 density Ficoll was (4.0 _+ 1.5) x 106 (mean -+ SD) cells per g of lung tissue consisting of 72.7 + 9.0% granular pneumonocytes and 20.3 + 8.4% alveolar macrophages. 0 2 uptake by the cells at r o o m temperature w a s 46.9 +- 5.8 n m o l / h per 106 cells (mean + SE; n=5). Respiration was inhibited by oligom y c i n and subsequently stimulated by an uncoupler of oxidative phosphorylation. During incubation with [U-14C] glucose, the cells produced 14CO2, lactate and pyruvate at rates of 17.0 +- 2.9, 17.i + 1.6 and 8.7 + 0.8 nmol/h/ 106 cells, respectively. These results s h o w that a cell population enriched in granular pneumonocytes can be isolated by lung trypsinization. The cells in this preparation show respiration that is coupled to oxidative phosphorylation and intact glycolytic pathways. K e y ~ v o r d s : Trypsinization - Density gradient centrifugation - Histochemistry - Oxygen uptake
INTR OD UC TION Valuable information with respect to lung metabolism has been obtained through studies with the isolated perfused lung, lung slices and lung h o m o genates. However, interpretation of these results is h a m p e r e d by the cellular heterogeneity of the p u l m o n a r y parenchyrna. Investigation of the metabolic properties of the lung would be facilitated if pure populations of lung
156
cell types were available for study. The initial step in this process is dissociation of the lung into free and viable cells. Several groups have shown that dissociation of pulmonary parenchyma is possible with the use of collagenase, elastase or various other proteolytic enzymes [i,4,5,11,14]. However, this treatment has resulted in a complex mixture of cells that have not been further resolved into homogeneous populations. Recently, Kikkawa [7 ]and, subsequently, Mason[10] have demonstrated relatively selective release of granular pneumonocytes (type 2 alveolar cells) from minced lung tissue by treatment with trypsin. The cell mixture was then treated by gradient centrifugation for further separation of the cell types. Granular pneumonocytes isolated in this way retained the necessary enzymes to synthesize disaturated lecithins [8 ~ In the present study, the cell isolation procedures of Kikkawa were slightly modified and the preparation was evaluated to determine whether the pathways of glycolysis and oxidative phosphorylation are functional in the trypsinized cells.
METHODS Materials. Substrates, enzymes, inhibitors and histochemical stains were obtained from Sigma Chemical Co. (St. Louis, MO). Fluorochemical FC-80 was obtained from 3M Co. (Minneapolis, MN). Dr. P. Heytler (DuPorft Corp., Wilmington, DE) donated the 1,3-bis[hexafluoroacetonyl] acetone (compound "1799"). [U-14C]glucose was obtained from New England Nuclear Corp., Boston, MA. Nylon bolting cloth was obtained from Tetko, Elmsford, NY. Animals. Male Sprague-Dawley rats weighing 200-300 g were obtained from Hilltop Lab Animals (Scottdale, PA) and housed six to a cage in an environmental hood supplied with HEPA filtered air. Rats were given free access to food (Wayne Lab Blox, Allied Mills, Chicago, IL) and water.
Preparation of Lung Cells. Rats were decapitated and the lungs were removed from the thorax and rinsed in ice-cold Krebs-Ringer Tris (KRT) solution consisting of 118 mM NaCI, 4.7 mM KCI, 1.2 mM CaCI 2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 20 mM Tris CI, pH 7.4 (at 23o). This solution was 290 mOsmolar as measured by freezing point depression (Osmette, Precision Systems, Inc. ). Bronchi and major vessels were trimmed and the lungs were minced with scissors into pieces of 1-3 mm in diameter. The lung mince from six or more rats was pooled for preparation of lung cells. The mince was mechanically agitated for 2 rain using a vortex mixer set at a medium speed and was then filtered through HC-3-160 nylon bolting cloth. The mince was resuspended in isolation medium (1:6, v/v) that contained 0.1% fluoroehemical (FC-80) emulsion. The fluorochemical emulsion was freshly prepared from a i: i00 mixture (v/v) of FC-80 and 5% human serum albumin in saline by sonication at ii0 watts for 15-20 sec. The flask containing the lung mince was degassed by gentle suc-
157
tion and mechanically agitated with a magnetic stirrer during 20 min incubation at 37 ° C. The flask was then gassed with 02 for 2 rain, sufficient trypsin (2 x crystallized) was added to make a 1% solution, and incubation was continued for an additional 30 rain. The reaction was stopped by the addition of crude soybean trypsin inhibitor according to the direction of the supplier (Sigma Chemical Co. ). The suspension was filtered sequentially through HC-3-160, HC-3-37 and HC-3-15 bolting cloth. DNAse was added to the filtrate in a concentration of approximately I0 ug/ml to inhibit cell clumping. This suspension was designated the crude cell preparation. For further purification of cell populations, the crude cell preparation was centrifiged on a 3-1ayer discontinuous gradient consisting of 7 ml of 11.5% Ficollj 7 ml of 13.2% Ficoll and 21 ml of 23.0% Ficoll, as described by Kikkawa[7]. Ficoll was dissolved in KRT solution. The densities of these three layers measured at 25 ° by refractive index were 1.048, 1.058 and i. I00. The gradient was formed in 50 ml polycarbonate centrifuge tubes and 10-12 ml of the cell mix containing not more than 6 x 107 cells was carefully layered on top. The tubes were centrifuged at 800 x g for 30 rain using an HC-4 rotor withRC-2B centrifuge (Sorvall Inc., Norwalk, CT) maintained at 4 ° C. Cell fractions were aspirated from each of the three interfaces and the pellet. These fractions were centrifuged at 550 x g for 5 rain, washed and recentrifuged 3 times with KRT, and then suspended in KRT for subsequent use. Cell Identification and Histoch'emistry. Cells were counted using a hemocytometer and identified in air-dried thin smears stained by a modified Papanicolaou technique as described byKikkawa[7]. Phosphatase activity was evaluated in air-dried smears using naphthol AS-MX phosphate as substrate at either pH 5.2 or pH 8.6 [12]. ~-Oalactosidase activity was evaluated using 6 -bromo2 -naphthol- 8 -D -galactopyrano side as substrate[15]. Slides were photographed through a Zeiss microscope with oil immersion objective using Kodachrome-X (5500 OK) film with 80A and 82A filters. Metabolic Assays. All metabolic studies were carried out on freshly isolated cells. Oxygen consumption by the cell preparation was measured polarographically at 23 ° using a stirred incubation cuvette of 0.25 ml volume and a membrane-covered silver wire electrode [6]. Lactate, pyruvate and 14CO2 production from 5.5 mM [U-14C] glucose was measured during incubation at 370 with a Dubnoff metabolic shaking incubator, as previously described [I]. For each experiment, cells were incubated in individual 25 ml capped Ehrlenmeyer flasks for I0, 20 or 30 min. All incubations were carried out in duplicate. Lactate and pyruvate in the cell-free supernatant were assayed by enzyme methods [2], and 14CO 2 production was quantitated by scintillation counting (Packard Tri Carb. • Downer' s Grove, IL) using the channels ratio method based on internal standards to correct for quenching.
mixture
Crude
+ SD
for the number
-+ 1.5
-+ 9.0
20.3
± 6.8
41.6
AM
i.i00
density
± 4.2
4.8
-+ 3.2
14.8
LY
± 1.8
0.6
± 2.1
0.6
BR
Ficoll
after gradient
= polymorphonuclear
in parentheses
-+ 1.2
1.6
-+ 2.5
5.5
PMN
PMN
indicated
-+ 8.4
macrophages, cells
of preparations
= granular pneumonocytes, AM = alveolar = lymphocytes, BR = ciliated bronchiolar
are mean
b (9)
72.7
± 6.8
4.0
37.5
GP
% of nucleated
cells
of rat lung
Cell distribution
-+ 14.4
cells
45.1
lung
of nucleated
of cells after trypsinization
106cells/g
Yield
distribution
a (13)
and
a Cells obtained by trypsinization of the lung mince b Cells obtained at the interface between 1.058 and of the crude mixture
GP LY
Values
pneumonocytes
Granular
i. Yield
Table
cells
centrifugation
leukocytes,
± 0.7
1.2
nucleated
Erythrocytes
159
RESULTS Cell Yield and Distribution. Trypsinization of the hng yielded 45 x 106 nucleated cells per g of lung tissue (Table i), corresponding to approximately i00 x 108 nucleated cells from the lungs of one rat. Erythrocytes in this crude cell preparation slightly exceeded the number of nucleated cells (Table i). Identification of granular pneumonocytes was made by the characteristic size of approximately 12-15 u diameter cells with central nucleus and cytoplasm containing multiple deeply stained inclusions with a surrounding clear area on Papanicolaou stain. Alveolar macrophages, the m a j o r contaminant, w e r e differentiated by their larger size but smaller inclusions which lacked the surrounding clear area. Small n u m b e r s of lymphocytes and polymorphonuclear leukocyles w e r e present. Ciliated bronchiolar cells w e r e rarely seen. Approximately 10% of cells could not be reliably identified and w e r e excluded f r o m the differential count. Following gradient centrifugation, approximately 9% of the original nucleated cells were recovered at the interface between 1.058 and i.i00 densities Ficoll (Table i). The enrichment of granular pneumonocytes in this fraction was approximately twofold over the starting mixture. Alveolar macrophages remained the major contaminant; erythrocytes were not present. The interface between 1.048 and 1.058 densityFicoll consisted of debris and swollen cells. The pellet consisted of fluorochemical, erythrocytes and distorted and damaged cells. H{stochemical Staining Characteristics of Lung Cells. Four preparations that consisted of 81.2 -+ 3.2% cells with the characteristics of granular pneumonocytes were stained for histochemical activity. These cells were strongly positive to alkaline phosphatase and weakly positive or negative with ~-galactosidase (Fig. I). Acid phosphatase staining showed positively reacting cytoplasmic granules that probably correspond to the lamellar bodies. Alveolar macrophages obtained from the lung mince were strongly positive for acid phosphatase and ~-galactosidase but weakly positive or negative for alkaline phosphatase (Fig. i). Oxygen Uptake and Glucose Utilization of Lung Cells. Granular pneumonocytes suspended in KRT buffer at room temperatur showed linear rates of 02 utilization without added substrate (Fig. 2A). The mean rate of 02 uptake {_+ SE) in five experiments was 46.9 -+ 5.8 nmol/h per i08 cells. Addition of glucose did not significantly alter the rate of 02 utilization. Addition of succinate stimulated 0 2 consumption approximately 20-30%. The rate of oxidation of these substrates was linear until the PO2 of the suspending medium reached approximately 5 mm Hg. These results indicate functioning of mitochondrial pathways for 02 utilization. In order to determine whether oxygen utilization was coupled to oxidative phosphorylation, we evaluated the effects of several inhibitors on cell respiration.
I60
g ,
!
L
I
A
ll..
C
D Fig. i. Histochemical staining of the isolated granular p n e u m o n o c y t e preparation c o m p a r e d with alveolar rnacrophages obtained from the lung mince. A. Granular pneumonocytes showing positive reaction to alkaline phosphatase. B. Alveolar m a c r o p h a g e s showing negative reaction to alkaline phosphatase. Note the presence of alkaline phosphatase positiye debris. C. Granular pneumonocyles with acid phosphatase positive inclusions. D. Alveolar m a c r o p h a g e s strongly positive to acid phosphatase. ( , ' = I0 M m )
Addition of oligomycin to the respiring cells resulted in approximately 50% inhibition of respiration (Fig. 2B). Respiration was restored to control levels by addition of c o m p o u n d "1799" (l,3-bis [hexafluoroacetonyl] acetone), an uncoupler of oxidative phosphorylation. Addition of antimycin As* an inhibitor of the mitochondrial respiratory chains resulted in nearly total inhibition of respiration. These results indicate that the rnitochondrial pathways of 0 2 utilization were sensitive to n o r m a l regulatory m e c h a n i s m s . T h e y further indicate that the necessary co-factors for mitochondrial respiration (e.g. A D P , N A D +) were retained in the trypsinized cells. Granular pneumonocytes incubated at 37oc with I0 m M [U-14 C]glucose produced lactate~ pyruvate and 1 4 C O 2 at linear rates during a 30. m i n incubation (Fig. S). Approximately twice as m u c h lactate was produced as pyruvate (Table 2). The flux of glucose carbon atoms to lactate plus pyru-
161 Type rr cells .~' 1.5 x 107 cetls/mt
150
~
'~GLUCOSE
100 P02,
~ SUCCINATE
mm HG 50
q
" ~
mM
]20~M o2 15 min j
0
~
'~
Type 1"-[" Aveoar ce s
1.9 X 107 cellsiml
\ k'~
OIkjomycm l 0.4 ~g/rnl
"IT99" j2~M
2~M 02 rain
\
Antimycin A
0.4 .ug/rnl
Fig. 2. Polarographic measurement of oxygen consumption by isolated granular pneumonocytes from rat lung. Cells were suspended in Krebs-Ringer Tris buffer at 23 ° C in a stirred cuvette of 0.25 ml volume. Additions are indicated by the arrows. A. Effect of added substrates on 02 utilization. In this experiment, the rate of 02 utilization was 0.73 nmol/min/106 cells with endogenous substrate, 0.76 nmol/min/106 cells in the presence of glucose, and 0.90 nmol/min/106 cells after addition of succinate. B. Effect of metabolic inhibitors on oxygen utilization. The buffer contained 5 mM glucose as substrate. In this experiment, the control 02 utilization was 0.25 nmol/ min/106 cells. The output of the electrode was electrically expanded fivefold for recording of oxygen uptake
162 ~'
10-
/.
° 4 Lactate
~-oPyruvate •--.14co2
E ~--
6-
"6
4-
/
/
,"
~2 O~ 0
time of incubation, rains
Fig. 3. Results from a typical experiment for the study of metabolism of [U-14C] glucose by a granular pneumonocyle preparation from rat lung. Cells were incubated for i0, 20 or 30 rain, and production of lactate, pyrurate and 14CO2 was measured. A total of six incubation vessels (two at each time point) were used for this experiment
Table 2. Metabolite production
production
by isolated rat lung granular
14 CO2
lactate
pyruvat e
production
production
production
pneumonocyte
lactate production pyruvate
ru-nol/h/106 cells
nmol/h/lO 8 ceils
production
n - m o l / h / l O 8 cells
17.0
]7.1
8.7
2.2
± 2.9
± 1.6
f 0.8
e 0.3
V a l u e s are m e a n + S E for 5 preparations of cells. A p p r o x i m a t e l y 2 x 106 cells per m l w e r e incubated for 10-30 rain with 5 mlV[ U - 1 4 C glucose at 37 °. Rates of production w e r e determined f r o m the least m e a n squares relationship bet w e e n metabolic concentration and t i m e of incubation as indicated in Figure 3
163
vate was 4.5 times the flux to CO2, i.e. 18% of the carbon atoms in these three products of glucose catabolism were recovered in the CO2 fraction. These results indicate retention of transport systems for glucose and enzymes for glycolysis by the trypsinized cells.
DISCUSSION
These results show that an enriched population of cells with characteristic morphology by Papanicolaou staining can be obtained by trypsinization of the lung and gradient centrifugation. These cells were strongly alkaline phosphatase positive, had acid phosphatase positive inclusions, and were negative or weakly positive to 8-galactosidase. This pattern of histochemical staining is similar to that found for granular pneumonocyles in the intact lung [9, 13]. Kikkawa[7]and Mason [i0] have shown good correlation between the presence of granules on Papanicolaou staining and the presence of lamellar bodies and other ultrastructural characteristics of granular pneumonocytes by electron microscopy. Therefore, this enriched population of cells has morphologic and histochemical characteristics of granular pneumonocytes. Differentiation from alveolar macrophages, the major contaminant, can be made by cell size and characteristics of the inclusions. In addition, histochemical studies of alveolar macrophages show strongly positive acid phosphatase and 8-galactosidase reactions and a weakly positive or negative reaction to alkaline phosphatase. Other contaminating cells are easily recognized on routine microscopy. The method utilized for granular pneumonocytes preparation resulted in a population that varied from 58-90% purity. A major factor in this variability may be related to properties of the trypsin utilized for lung dissociation. This was evident in a subsequent series of experiments when trypsinization yielded cells that appeared swollen on light microscopy and banded during centrifugation at a density less than 1.058. These cells had no demonstrable 0 2 uptake or glycolytic activity. A decrease in trypsin concentration again yielded viable cells. These results indicate the need for attention to variations in potency among various batches of trypsin in order to achieve reproducible results for lung cell separation. The goal of the present study was to determine whether the isolated cells were able to carry out basic functions of oxidation and glycolysis. The results indicate that pathways of intermediary metabolism were intact and responsive to biochemical control mechanisms. Thus, the cells were capable of glucose transport with conversion to lactate and pyruvate and oxidation to 14CO2. The ratio of lactate to pyruvate was low, indicating normal redox state. The cells demonstrated oxygen utilization. Coupling of oxygen utilization to ATP synthesis was suggested by decreased respiration with oligomycin (an inhibitor of "coupled" respiration) and subsequent increase with an uncoupler of oxidative phosphorylation. The rate of 02 utilization by the lung cells (59 nmol/min/mg dry wt, assuming that ~he dry weight of 106 lung cells is 0.8 rag) [i] is the same order of magnitude as previouly reported
164
for rat lung slices (approximately 40 nrnol/rnin/mg dry wt) [3]. These results indicate that lung cells obtained by the trypsinization procedure retain the enzymes and transport processes necessary for the uptake of glucose and its rnetabolsm by both cytoplasmic and rnitochondrial pathways.
Acknowledgements. We appreciate the cooperation and suggestions of Dr. Bayard Storey for measurement of oxygen consumption. Gary A. Huber provided excellent technical assistance. Results have been presented in part to the Fed. Amer. Soc. for Exp. Biol. in Anaheim, CA, April 1976 (Fed. Proc. 35, 479, 1976). Supported by HL 15013 and HL 15061 (SCOR). Dr. Fisher was a Clinical Investigator of the Veterans Administration Research Service (1973-1976).
REFERENCES i. Ayuso, M.S., Fisher, A.B., Parilla, R., Williamson, J.R.:Glucose metabolism by isolated rat lung cells. Arner. J. Physiol. 225, 1153 1160 (1973) 2. Bergmeyer, H.V.: Methods of Enzymatic Analysis, p. 253-259, 266277. Weinheirn-New York: Verlag Chemie-Academic Press 1965 3. Fisher, A. ]B. : Oxygen utilization and energy production. In: Biomedical Basis of Lung Function, p. 75-104 (ed. R. Crystal). New York: Marcel Dekker 1976 4. Frazier, M.E., Hodley, J.G., Andrews, T.K., Drucker, H.:Use of thermolysin for the dissociation of lung tissue into cellular components. Lab. Invest. 33, 231-238 (1975) 5. Gould, K.G. Jr., Clernents, A.J., Jones, A.L., Felts, J.M.: Dispersal of rabbit lung into individual viable cells. A new model for the study of lung metabolism. Science 178, 1209-1210 (1972) 6. Keyhanij E., Storey, B.T.: Energy conservation capacity andmorphologic integrity of rnitochondria in hypotonically treated rabbit epididyrnal spermatozoa. Biochem. Biophys. Acta 305, 557-569 (1975) 7. Kikkawa, Y. , Yoneda, K. : The type II epithelial Cell of the lung. I. Method of isolation. Lab. Invest. 30, 76 (1974) 8. Kikkawa, Y., Yoneda, K., Smith,--F., Packard, B., Suzuki, K.: The type II epithelial cells of the lung. If. Chemical composition and phospholipid synthesis. Lab. Invest. 32, 295-302 (1975) 9. Kuhn, C. III: Cylochemistry of pu~m~onary alveolar epithelial cells. Amer. J. Pathol. 53, 809-833 (1968) i0. Mason, R., Williams, M.C., Clements, J.A.:Isolation and identification of type II alveolar epithelial cells. Chest 67, 36S-37S (1975) 1 I. Phillips, If. J. : Dissociation of single cells from l---ungor kidney tissue with elastase. In Vitro 8, 101-105 (1972) 12. Sigma Technical Bullet~n No. 85: The histochemical demonstration of acid and alkaline phosphatase. Sigma Chemical Co., St. Louis, MO, revised 1971
t 65
13. 14.
15.
Sorokin, S. P. : A rnorphologic and cytochernical study on the great alveolar cell. J. Histochem. Cytochem. 14, 884-897 (1967) Wolfe, B.M.J., Rubinstein, D., Beck, J.C.:The metabolism of isolated pneurnonoeytes from rabbit lung. Canad. J. Bioehem. 46, 151 154 (1968) Yarborough, D.J., Myer, O.T., Dannenberg, A.M., Jr., Pearson, B.: Histochemistry of macrophage hydrolases. 3. Studies on B-galactosidase, 8-glueuronidase and arninopeptidase with indolyl and naphthyl substrates. J. Reticuloend. Soc. 4, 390-408 (1967)
Accepted May 23,
for publication: 1977
Aron B. Fisher, IVf.D. Department of Physiology/G4 University of Pennsylvania School of Medicine
Philadelphia, Pennsylvania 19174, U S A
®El; Spri~ger-V~
Isolation and Metabolism of Granular Pneumocytes from Rat Lungs A r o n B. Fisher and Linda Furia Department of Physiology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19174, U S A
A b s t r a c t. A population of rat lung cells enriched in granular pneumonocytes (type II cells) was prepared by modification of the m e t h o d of Kikkawa (Lab. Invest. 30, 76, 1974). Lungs were minced, mechanically agitated and incubated with a fluorochemical emulsion and 1% trypsin. The isolated cells w e r e centrifuged in a discontinuous Ficoll gradient. Granular pneurnonocytes were identified by the presence of cytoplasmic inclusions on Papanicolaou and acid phosphatase staining and a strongly positive alkaline phosphatase reaction. The yield at the interface between 1.058 and I.i00 density Ficoll was (4.0 _+ 1.5) x 106 (mean -+ SD) cells per g of lung tissue consisting of 72.7 + 9.0% granular pneumonocytes and 20.3 + 8.4% alveolar macrophages. 0 2 uptake by the cells at r o o m temperature w a s 46.9 +- 5.8 n m o l / h per 106 cells (mean + SE; n=5). Respiration was inhibited by oligom y c i n and subsequently stimulated by an uncoupler of oxidative phosphorylation. During incubation with [U-14C] glucose, the cells produced 14CO2, lactate and pyruvate at rates of 17.0 +- 2.9, 17.i + 1.6 and 8.7 + 0.8 nmol/h/ 106 cells, respectively. These results s h o w that a cell population enriched in granular pneumonocytes can be isolated by lung trypsinization. The cells in this preparation show respiration that is coupled to oxidative phosphorylation and intact glycolytic pathways. K e y ~ v o r d s : Trypsinization - Density gradient centrifugation - Histochemistry - Oxygen uptake
INTR OD UC TION Valuable information with respect to lung metabolism has been obtained through studies with the isolated perfused lung, lung slices and lung h o m o genates. However, interpretation of these results is h a m p e r e d by the cellular heterogeneity of the p u l m o n a r y parenchyrna. Investigation of the metabolic properties of the lung would be facilitated if pure populations of lung
156
cell types were available for study. The initial step in this process is dissociation of the lung into free and viable cells. Several groups have shown that dissociation of pulmonary parenchyma is possible with the use of collagenase, elastase or various other proteolytic enzymes [i,4,5,11,14]. However, this treatment has resulted in a complex mixture of cells that have not been further resolved into homogeneous populations. Recently, Kikkawa [7 ]and, subsequently, Mason[10] have demonstrated relatively selective release of granular pneumonocytes (type 2 alveolar cells) from minced lung tissue by treatment with trypsin. The cell mixture was then treated by gradient centrifugation for further separation of the cell types. Granular pneumonocytes isolated in this way retained the necessary enzymes to synthesize disaturated lecithins [8 ~ In the present study, the cell isolation procedures of Kikkawa were slightly modified and the preparation was evaluated to determine whether the pathways of glycolysis and oxidative phosphorylation are functional in the trypsinized cells.
METHODS Materials. Substrates, enzymes, inhibitors and histochemical stains were obtained from Sigma Chemical Co. (St. Louis, MO). Fluorochemical FC-80 was obtained from 3M Co. (Minneapolis, MN). Dr. P. Heytler (DuPorft Corp., Wilmington, DE) donated the 1,3-bis[hexafluoroacetonyl] acetone (compound "1799"). [U-14C]glucose was obtained from New England Nuclear Corp., Boston, MA. Nylon bolting cloth was obtained from Tetko, Elmsford, NY. Animals. Male Sprague-Dawley rats weighing 200-300 g were obtained from Hilltop Lab Animals (Scottdale, PA) and housed six to a cage in an environmental hood supplied with HEPA filtered air. Rats were given free access to food (Wayne Lab Blox, Allied Mills, Chicago, IL) and water.
Preparation of Lung Cells. Rats were decapitated and the lungs were removed from the thorax and rinsed in ice-cold Krebs-Ringer Tris (KRT) solution consisting of 118 mM NaCI, 4.7 mM KCI, 1.2 mM CaCI 2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 20 mM Tris CI, pH 7.4 (at 23o). This solution was 290 mOsmolar as measured by freezing point depression (Osmette, Precision Systems, Inc. ). Bronchi and major vessels were trimmed and the lungs were minced with scissors into pieces of 1-3 mm in diameter. The lung mince from six or more rats was pooled for preparation of lung cells. The mince was mechanically agitated for 2 rain using a vortex mixer set at a medium speed and was then filtered through HC-3-160 nylon bolting cloth. The mince was resuspended in isolation medium (1:6, v/v) that contained 0.1% fluoroehemical (FC-80) emulsion. The fluorochemical emulsion was freshly prepared from a i: i00 mixture (v/v) of FC-80 and 5% human serum albumin in saline by sonication at ii0 watts for 15-20 sec. The flask containing the lung mince was degassed by gentle suc-
157
tion and mechanically agitated with a magnetic stirrer during 20 min incubation at 37 ° C. The flask was then gassed with 02 for 2 rain, sufficient trypsin (2 x crystallized) was added to make a 1% solution, and incubation was continued for an additional 30 rain. The reaction was stopped by the addition of crude soybean trypsin inhibitor according to the direction of the supplier (Sigma Chemical Co. ). The suspension was filtered sequentially through HC-3-160, HC-3-37 and HC-3-15 bolting cloth. DNAse was added to the filtrate in a concentration of approximately I0 ug/ml to inhibit cell clumping. This suspension was designated the crude cell preparation. For further purification of cell populations, the crude cell preparation was centrifiged on a 3-1ayer discontinuous gradient consisting of 7 ml of 11.5% Ficollj 7 ml of 13.2% Ficoll and 21 ml of 23.0% Ficoll, as described by Kikkawa[7]. Ficoll was dissolved in KRT solution. The densities of these three layers measured at 25 ° by refractive index were 1.048, 1.058 and i. I00. The gradient was formed in 50 ml polycarbonate centrifuge tubes and 10-12 ml of the cell mix containing not more than 6 x 107 cells was carefully layered on top. The tubes were centrifuged at 800 x g for 30 rain using an HC-4 rotor withRC-2B centrifuge (Sorvall Inc., Norwalk, CT) maintained at 4 ° C. Cell fractions were aspirated from each of the three interfaces and the pellet. These fractions were centrifuged at 550 x g for 5 rain, washed and recentrifuged 3 times with KRT, and then suspended in KRT for subsequent use. Cell Identification and Histoch'emistry. Cells were counted using a hemocytometer and identified in air-dried thin smears stained by a modified Papanicolaou technique as described byKikkawa[7]. Phosphatase activity was evaluated in air-dried smears using naphthol AS-MX phosphate as substrate at either pH 5.2 or pH 8.6 [12]. ~-Oalactosidase activity was evaluated using 6 -bromo2 -naphthol- 8 -D -galactopyrano side as substrate[15]. Slides were photographed through a Zeiss microscope with oil immersion objective using Kodachrome-X (5500 OK) film with 80A and 82A filters. Metabolic Assays. All metabolic studies were carried out on freshly isolated cells. Oxygen consumption by the cell preparation was measured polarographically at 23 ° using a stirred incubation cuvette of 0.25 ml volume and a membrane-covered silver wire electrode [6]. Lactate, pyruvate and 14CO2 production from 5.5 mM [U-14C] glucose was measured during incubation at 370 with a Dubnoff metabolic shaking incubator, as previously described [I]. For each experiment, cells were incubated in individual 25 ml capped Ehrlenmeyer flasks for I0, 20 or 30 min. All incubations were carried out in duplicate. Lactate and pyruvate in the cell-free supernatant were assayed by enzyme methods [2], and 14CO 2 production was quantitated by scintillation counting (Packard Tri Carb. • Downer' s Grove, IL) using the channels ratio method based on internal standards to correct for quenching.
mixture
Crude
+ SD
for the number
-+ 1.5
-+ 9.0
20.3
± 6.8
41.6
AM
i.i00
density
± 4.2
4.8
-+ 3.2
14.8
LY
± 1.8
0.6
± 2.1
0.6
BR
Ficoll
after gradient
= polymorphonuclear
in parentheses
-+ 1.2
1.6
-+ 2.5
5.5
PMN
PMN
indicated
-+ 8.4
macrophages, cells
of preparations
= granular pneumonocytes, AM = alveolar = lymphocytes, BR = ciliated bronchiolar
are mean
b (9)
72.7
± 6.8
4.0
37.5
GP
% of nucleated
cells
of rat lung
Cell distribution
-+ 14.4
cells
45.1
lung
of nucleated
of cells after trypsinization
106cells/g
Yield
distribution
a (13)
and
a Cells obtained by trypsinization of the lung mince b Cells obtained at the interface between 1.058 and of the crude mixture
GP LY
Values
pneumonocytes
Granular
i. Yield
Table
cells
centrifugation
leukocytes,
± 0.7
1.2
nucleated
Erythrocytes
159
RESULTS Cell Yield and Distribution. Trypsinization of the hng yielded 45 x 106 nucleated cells per g of lung tissue (Table i), corresponding to approximately i00 x 108 nucleated cells from the lungs of one rat. Erythrocytes in this crude cell preparation slightly exceeded the number of nucleated cells (Table i). Identification of granular pneumonocytes was made by the characteristic size of approximately 12-15 u diameter cells with central nucleus and cytoplasm containing multiple deeply stained inclusions with a surrounding clear area on Papanicolaou stain. Alveolar macrophages, the m a j o r contaminant, w e r e differentiated by their larger size but smaller inclusions which lacked the surrounding clear area. Small n u m b e r s of lymphocytes and polymorphonuclear leukocyles w e r e present. Ciliated bronchiolar cells w e r e rarely seen. Approximately 10% of cells could not be reliably identified and w e r e excluded f r o m the differential count. Following gradient centrifugation, approximately 9% of the original nucleated cells were recovered at the interface between 1.058 and i.i00 densities Ficoll (Table i). The enrichment of granular pneumonocytes in this fraction was approximately twofold over the starting mixture. Alveolar macrophages remained the major contaminant; erythrocytes were not present. The interface between 1.048 and 1.058 densityFicoll consisted of debris and swollen cells. The pellet consisted of fluorochemical, erythrocytes and distorted and damaged cells. H{stochemical Staining Characteristics of Lung Cells. Four preparations that consisted of 81.2 -+ 3.2% cells with the characteristics of granular pneumonocytes were stained for histochemical activity. These cells were strongly positive to alkaline phosphatase and weakly positive or negative with ~-galactosidase (Fig. I). Acid phosphatase staining showed positively reacting cytoplasmic granules that probably correspond to the lamellar bodies. Alveolar macrophages obtained from the lung mince were strongly positive for acid phosphatase and ~-galactosidase but weakly positive or negative for alkaline phosphatase (Fig. i). Oxygen Uptake and Glucose Utilization of Lung Cells. Granular pneumonocytes suspended in KRT buffer at room temperatur showed linear rates of 02 utilization without added substrate (Fig. 2A). The mean rate of 02 uptake {_+ SE) in five experiments was 46.9 -+ 5.8 nmol/h per i08 cells. Addition of glucose did not significantly alter the rate of 02 utilization. Addition of succinate stimulated 0 2 consumption approximately 20-30%. The rate of oxidation of these substrates was linear until the PO2 of the suspending medium reached approximately 5 mm Hg. These results indicate functioning of mitochondrial pathways for 02 utilization. In order to determine whether oxygen utilization was coupled to oxidative phosphorylation, we evaluated the effects of several inhibitors on cell respiration.
I60
g ,
!
L
I
A
ll..
C
D Fig. i. Histochemical staining of the isolated granular p n e u m o n o c y t e preparation c o m p a r e d with alveolar rnacrophages obtained from the lung mince. A. Granular pneumonocytes showing positive reaction to alkaline phosphatase. B. Alveolar m a c r o p h a g e s showing negative reaction to alkaline phosphatase. Note the presence of alkaline phosphatase positiye debris. C. Granular pneumonocyles with acid phosphatase positive inclusions. D. Alveolar m a c r o p h a g e s strongly positive to acid phosphatase. ( , ' = I0 M m )
Addition of oligomycin to the respiring cells resulted in approximately 50% inhibition of respiration (Fig. 2B). Respiration was restored to control levels by addition of c o m p o u n d "1799" (l,3-bis [hexafluoroacetonyl] acetone), an uncoupler of oxidative phosphorylation. Addition of antimycin As* an inhibitor of the mitochondrial respiratory chains resulted in nearly total inhibition of respiration. These results indicate that the rnitochondrial pathways of 0 2 utilization were sensitive to n o r m a l regulatory m e c h a n i s m s . T h e y further indicate that the necessary co-factors for mitochondrial respiration (e.g. A D P , N A D +) were retained in the trypsinized cells. Granular pneumonocytes incubated at 37oc with I0 m M [U-14 C]glucose produced lactate~ pyruvate and 1 4 C O 2 at linear rates during a 30. m i n incubation (Fig. S). Approximately twice as m u c h lactate was produced as pyruvate (Table 2). The flux of glucose carbon atoms to lactate plus pyru-
161 Type rr cells .~' 1.5 x 107 cetls/mt
150
~
'~GLUCOSE
100 P02,
~ SUCCINATE
mm HG 50
q
" ~
mM
]20~M o2 15 min j
0
~
'~
Type 1"-[" Aveoar ce s
1.9 X 107 cellsiml
\ k'~
OIkjomycm l 0.4 ~g/rnl
"IT99" j2~M
2~M 02 rain
\
Antimycin A
0.4 .ug/rnl
Fig. 2. Polarographic measurement of oxygen consumption by isolated granular pneumonocytes from rat lung. Cells were suspended in Krebs-Ringer Tris buffer at 23 ° C in a stirred cuvette of 0.25 ml volume. Additions are indicated by the arrows. A. Effect of added substrates on 02 utilization. In this experiment, the rate of 02 utilization was 0.73 nmol/min/106 cells with endogenous substrate, 0.76 nmol/min/106 cells in the presence of glucose, and 0.90 nmol/min/106 cells after addition of succinate. B. Effect of metabolic inhibitors on oxygen utilization. The buffer contained 5 mM glucose as substrate. In this experiment, the control 02 utilization was 0.25 nmol/ min/106 cells. The output of the electrode was electrically expanded fivefold for recording of oxygen uptake
162 ~'
10-
/.
° 4 Lactate
~-oPyruvate •--.14co2
E ~--
6-
"6
4-
/
/
,"
~2 O~ 0
time of incubation, rains
Fig. 3. Results from a typical experiment for the study of metabolism of [U-14C] glucose by a granular pneumonocyle preparation from rat lung. Cells were incubated for i0, 20 or 30 rain, and production of lactate, pyrurate and 14CO2 was measured. A total of six incubation vessels (two at each time point) were used for this experiment
Table 2. Metabolite production
production
by isolated rat lung granular
14 CO2
lactate
pyruvat e
production
production
production
pneumonocyte
lactate production pyruvate
ru-nol/h/106 cells
nmol/h/lO 8 ceils
production
n - m o l / h / l O 8 cells
17.0
]7.1
8.7
2.2
± 2.9
± 1.6
f 0.8
e 0.3
V a l u e s are m e a n + S E for 5 preparations of cells. A p p r o x i m a t e l y 2 x 106 cells per m l w e r e incubated for 10-30 rain with 5 mlV[ U - 1 4 C glucose at 37 °. Rates of production w e r e determined f r o m the least m e a n squares relationship bet w e e n metabolic concentration and t i m e of incubation as indicated in Figure 3
163
vate was 4.5 times the flux to CO2, i.e. 18% of the carbon atoms in these three products of glucose catabolism were recovered in the CO2 fraction. These results indicate retention of transport systems for glucose and enzymes for glycolysis by the trypsinized cells.
DISCUSSION
These results show that an enriched population of cells with characteristic morphology by Papanicolaou staining can be obtained by trypsinization of the lung and gradient centrifugation. These cells were strongly alkaline phosphatase positive, had acid phosphatase positive inclusions, and were negative or weakly positive to 8-galactosidase. This pattern of histochemical staining is similar to that found for granular pneumonocyles in the intact lung [9, 13]. Kikkawa[7]and Mason [i0] have shown good correlation between the presence of granules on Papanicolaou staining and the presence of lamellar bodies and other ultrastructural characteristics of granular pneumonocytes by electron microscopy. Therefore, this enriched population of cells has morphologic and histochemical characteristics of granular pneumonocytes. Differentiation from alveolar macrophages, the major contaminant, can be made by cell size and characteristics of the inclusions. In addition, histochemical studies of alveolar macrophages show strongly positive acid phosphatase and 8-galactosidase reactions and a weakly positive or negative reaction to alkaline phosphatase. Other contaminating cells are easily recognized on routine microscopy. The method utilized for granular pneumonocytes preparation resulted in a population that varied from 58-90% purity. A major factor in this variability may be related to properties of the trypsin utilized for lung dissociation. This was evident in a subsequent series of experiments when trypsinization yielded cells that appeared swollen on light microscopy and banded during centrifugation at a density less than 1.058. These cells had no demonstrable 0 2 uptake or glycolytic activity. A decrease in trypsin concentration again yielded viable cells. These results indicate the need for attention to variations in potency among various batches of trypsin in order to achieve reproducible results for lung cell separation. The goal of the present study was to determine whether the isolated cells were able to carry out basic functions of oxidation and glycolysis. The results indicate that pathways of intermediary metabolism were intact and responsive to biochemical control mechanisms. Thus, the cells were capable of glucose transport with conversion to lactate and pyruvate and oxidation to 14CO2. The ratio of lactate to pyruvate was low, indicating normal redox state. The cells demonstrated oxygen utilization. Coupling of oxygen utilization to ATP synthesis was suggested by decreased respiration with oligomycin (an inhibitor of "coupled" respiration) and subsequent increase with an uncoupler of oxidative phosphorylation. The rate of 02 utilization by the lung cells (59 nmol/min/mg dry wt, assuming that ~he dry weight of 106 lung cells is 0.8 rag) [i] is the same order of magnitude as previouly reported
164
for rat lung slices (approximately 40 nrnol/rnin/mg dry wt) [3]. These results indicate that lung cells obtained by the trypsinization procedure retain the enzymes and transport processes necessary for the uptake of glucose and its rnetabolsm by both cytoplasmic and rnitochondrial pathways.
Acknowledgements. We appreciate the cooperation and suggestions of Dr. Bayard Storey for measurement of oxygen consumption. Gary A. Huber provided excellent technical assistance. Results have been presented in part to the Fed. Amer. Soc. for Exp. Biol. in Anaheim, CA, April 1976 (Fed. Proc. 35, 479, 1976). Supported by HL 15013 and HL 15061 (SCOR). Dr. Fisher was a Clinical Investigator of the Veterans Administration Research Service (1973-1976).
REFERENCES i. Ayuso, M.S., Fisher, A.B., Parilla, R., Williamson, J.R.:Glucose metabolism by isolated rat lung cells. Arner. J. Physiol. 225, 1153 1160 (1973) 2. Bergmeyer, H.V.: Methods of Enzymatic Analysis, p. 253-259, 266277. Weinheirn-New York: Verlag Chemie-Academic Press 1965 3. Fisher, A. ]B. : Oxygen utilization and energy production. In: Biomedical Basis of Lung Function, p. 75-104 (ed. R. Crystal). New York: Marcel Dekker 1976 4. Frazier, M.E., Hodley, J.G., Andrews, T.K., Drucker, H.:Use of thermolysin for the dissociation of lung tissue into cellular components. Lab. Invest. 33, 231-238 (1975) 5. Gould, K.G. Jr., Clernents, A.J., Jones, A.L., Felts, J.M.: Dispersal of rabbit lung into individual viable cells. A new model for the study of lung metabolism. Science 178, 1209-1210 (1972) 6. Keyhanij E., Storey, B.T.: Energy conservation capacity andmorphologic integrity of rnitochondria in hypotonically treated rabbit epididyrnal spermatozoa. Biochem. Biophys. Acta 305, 557-569 (1975) 7. Kikkawa, Y. , Yoneda, K. : The type II epithelial Cell of the lung. I. Method of isolation. Lab. Invest. 30, 76 (1974) 8. Kikkawa, Y., Yoneda, K., Smith,--F., Packard, B., Suzuki, K.: The type II epithelial cells of the lung. If. Chemical composition and phospholipid synthesis. Lab. Invest. 32, 295-302 (1975) 9. Kuhn, C. III: Cylochemistry of pu~m~onary alveolar epithelial cells. Amer. J. Pathol. 53, 809-833 (1968) i0. Mason, R., Williams, M.C., Clements, J.A.:Isolation and identification of type II alveolar epithelial cells. Chest 67, 36S-37S (1975) 1 I. Phillips, If. J. : Dissociation of single cells from l---ungor kidney tissue with elastase. In Vitro 8, 101-105 (1972) 12. Sigma Technical Bullet~n No. 85: The histochemical demonstration of acid and alkaline phosphatase. Sigma Chemical Co., St. Louis, MO, revised 1971
t 65
13. 14.
15.
Sorokin, S. P. : A rnorphologic and cytochernical study on the great alveolar cell. J. Histochem. Cytochem. 14, 884-897 (1967) Wolfe, B.M.J., Rubinstein, D., Beck, J.C.:The metabolism of isolated pneurnonoeytes from rabbit lung. Canad. J. Bioehem. 46, 151 154 (1968) Yarborough, D.J., Myer, O.T., Dannenberg, A.M., Jr., Pearson, B.: Histochemistry of macrophage hydrolases. 3. Studies on B-galactosidase, 8-glueuronidase and arninopeptidase with indolyl and naphthyl substrates. J. Reticuloend. Soc. 4, 390-408 (1967)
Accepted May 23,
for publication: 1977
Aron B. Fisher, IVf.D. Department of Physiology/G4 University of Pennsylvania School of Medicine
Philadelphia, Pennsylvania 19174, U S A
