Protein Biosynthesis by the Pulmonary Alveolar Macrophage Comparison of Synthetic Activity of Suspended Cells and Cells on Surfaces'-•
CRAIG M. LEFFINGWELL and ROBERT B. LOW
SUMMARY _____________________ _____________________ ______________ An effort to optimize conditions for studying protein biosynthesis by the pulmonary alveolar macrophage in vitro has led to a comparative analysis of the activity of suspended and adherent cells. A number of differences were observed. (I) Suspended cells synthesized protein for only a limited period of time, after which they responded only partially to incubation in fresh medium. This was true even under reincubation conditions in which the cells were allowed to adhere to a surface. Adherent cells, however, synthesized protein during a longer period of time and were fully capable of responding to new medium within the time periods examined. (2) Analyses of the radioactive proteins synthesized using a dual-isotope technique suggested that, during a period of 2 hours, suspended cells synthesized relatively smaller quantities of high molecular weight proteins than adherent cells. (J) The administration of a phagocytic load (zymosan; particle to cell ratio, 10:1) inhibited by 20 per cent the incorporation of isotopic amino acid into protein during a period of 3 hours. The same phagocytic load, however, stimulated incorpora· tion by 20 per cent in adherent cells. (4)The rate of particle uptake measured using oil red 0-albumin complexes decreased by approximately 50 per cent in suspended cells preincubated for 2 hours, but was maintained in similarly preincubated adherent cells. It was concluded that pulmonary alveolar macrophages incubated adhered to a surface are more appropriate for meta· bolic studies than are pulmonary alveolar macrophages incubated in suspension.
Introduction Efforts in this laboratory to examine the protein biosynthetic capabilities of pulmonary alveolar macro phages (PAM) have necessitated an examination of the effects of in vitro incubation condi-
(Received in original form November 18, 1974 and in revised form june 4, 1975) 1 From the Department of Physiology and Biophysics, the University of Vermont, Burlington, Vermont 05401. 2 Supported by grant no. HL 14212 (SCOR) from the National Heart and Lung Institute. 3 Requests for reprints should be addressed to Robert B. Low, Ph. D., Department of Physiology and Biophysics, the University of Vermont, Burlington, Vermont 05401. A~!ERICAN
tions on the incorporation of radioactive amino acid into protein. Studies using noninduced PAM incubated in polypropylene test tubes with shaking at 37° C have been reported previously (1 ). The results indicated that the synthetic activity of cells incubated under those conditions was limited to approximately the first hour of incubation. The cells subsequently showed diminished incorporating ability, a finding in keeping with those of others (2, 3) who used bacille CalmetteGuerin-induced cells (BCG-cells). The present studies were undertaken ~o compare the capabilities of suspended cells with cells incubated under conditions in which the cells adhere to the surface of culture flasks. The results indicate that relative to suspended cells, adherent cells synthesize proteins for a longer peri-
REVIEW OF RESPIRATORY DISEASE, VOLUME 112, 1975
349
350
LEFFINGWELL AND LOW
od of time, show differences in the relative rates of incorporation of amino acid into different proteins and, in addition, respond differently to a phagocytic load. Materials and Methods The methods used were generally those reported previously (1). Briefly, PAM were lavaged from lungs of normal, adult male New Zealand rabbits using calcium- and magnesium-free phosphate-buffered saline. They were collected by centrifugation at 500 X gat room temperature and washed twice with the same medium before incubations were begun. Cell viability was determined by measuring eosin Y exclusion and lactic dehydrogenase (LDH) release from the cells. In vitro amino acid uptake into protein was measured in suspended cells by incubating them in polyprophylene tubes contaning I ml of Hanks medium buffered with tris (pH 7.4) (106 cells per ml) containing 19 nonradioactive amino acids plus radioactive leucine and antimicrobial drugs (HanksIris). Amino acid uptake by adherent cells was measured by preincubating 106 eels in 35- by 10-mm tissue culture dishes (Falcon no. 3001) to allow them to adhere, and subsequently incubating the cells in I ml oJ Hanks-tris medium. Experiments showed that cells not preincubated in the falcon flasks, but incubated immediately with radioactive medium, gave similar radioactive amino acid uptake curves. Hot trichloroacetic acid (TCA)-precipitable radioactivity was measured in incubations of cells in suspension, as described previously (1). A slightly modified procedure was used to count radioactivity from adherent cell incubations. After incubation, the medium in the flasks was poured off into an equal volume of cold 20 per cent TCA. One ml of 0.1 N sodium hydroxide was added to each flask, which was then shaken gently at room temperature for 5 min. The resultant wash fluid was poured off into an equal volume of cold 20 per cent TCA. All samples were then prepared for counting (1). Incorporation data were corrected for loss of cells during preincubation and wash experiments. The data from individual experiments are expressed as the mean ± I SD. Data based on more than one experiment are given as the mean ±I standard error of the mean (SE). Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of radioactive material from incubated cells was also used as described previously (!). Oxygen tension in incubation medium was determined immediately after incubation using an 0 2 monitor system (Model 53SA, Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio). Zymosan (Nutritional Biochemical Corp., Cleveland, Ohio) was used for experiments involving phagocytosis. It was prepared according to the method of Weissmann and associates (4), and was used in a particle-to-cell ratio of I 0: I. Measurements of the rate of particle uptake by
cells were made using oil red 0-albumin complexes (5). Uptake by suspended cells was measured in 1.0 ml-volumes in polypropylene tubes, using the same concentrations of cells and noncellular components as described by Stosscl and associates (5). Uptake by cells preincubated for varying times in flasks was accomplished by collecting the cells with a rubber policeman and subsequently reincubating them in suspension with oil reel 0 complexes as described above. The PAM were fixed for electron microscopy in 4 per cent glutaraldehyde buffered to pH 7.4 with sodium cacodylate. The cells were post-fixed after 2 hours in I per cent osmium tetroxide for I hour and subsequently dehydrated in a series of ethyl alcohols. The preparations were embedded in Epon® and sectioned at 35 to 50 nm for electron microscopy. The sections were studied on a Phillips 300 electron microscope. Results The time courses of the incorporation of radioactive leucine into protein by suspended and adherent cells are compared in figure I. The kinetics of incorporation during the first 60 min were closely similar. After that time, the suspended cell essentially ceased to incorporate amino acid into protein, in agreement with previous findings (1 ). Adherent cells, however, continued to incorporate amino acid, albeit at a slightly reduced rate relative to the first hour of incubation. The difference was reproducible: in 5 experiments, suspended cells incorporated only 8.8 ± 3.4 per cent more isotope into protein after 3 hours than they did after 2 hours. Adherent cells, however, incorporated 35.7 ± 4.6 per cent more isotope during the same time interval. In all cases, cells were greater than 90 per cent viable after a 3-hour incubation, as judged by the eosin Y test. In addition, cells incubated by both means released closely similar amounts of LDH (suspended cells, 9.9 ± 3.9 per cent; adherent cells, 8.8 ± 2.8 per cent). Finally, in all cases, less than 10 per cent of the radioactive protein synthesized by both groups of cells was released from them into the medium after 3 hours. These data suggest that the plasma membranes and protein export systems of the cells were not differentially affected by the incubated conditions. Measurements were made of Po 2 in fresh medium and medium used to incubate cells either in suspension or as adherent monolayers. After 2 or 3 hours of incubation, the Po 2 was the same in media used for suspended and adherent cells, in both cases being 10 ± 0.3 per cent less than the Po 2 in fresh medium.
PROTEI:\ BIOSYNTHESIS BY PUUIONARY ALVEOLAR MACROPHAGE
351
25
20
• = adherent o = suspended
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120
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Fig. I. Time course of incorporation of radioactive leucine into protein by pulmonary alveolar macrophages incubated in suspension and incubated after adherence to a surface. Macrophages isolated from a single rabbit were incubated at 37' C for the times shown in 1.0 ml of Hanks-tris medium (pH 7.4) at 37' C (I) containing the following additional components: 0.05 /LCi of DL-[14)-leucine (specific activity: 312 mCi per mmole;); 19 [12CJ-arnino acids (leucine excluded) at a concentration of I X J0-5 Af; 100 units of penicillin, 100 /Lg of streptomycin per ml, and 25 /Lg of Fungizone; 10 per cent heterologous rabbit serum made from blood collected by cardiac puncture and dialyzed at 2' C for 18 hours against 2 changes of 20 volumes of Hanks·tris medium; 106 cells (I). After incubation, suspended cells were precipitated by addition of 1 volume of icc· cold 20 per cent trichloroacetic acid (TCA), heated at 95' C for 10 min, and the precipitates were collected and washed with ice-cold 5 per cent TCA on Millipore filters of 0.45-l'm pore size (Milli· pore Corp., Redford, Mass.). Incubations in flasks were collected and precipitated with TCA as described in Methods, and were prepared for counting as above. Samples were counted in a Packard Model 3320 Liquid Scintillation Counter by adding to each in a vial, 0.7 ml of 88 per cent formic acid followed by 10 ml of a scintillation fluid containing 50 per cent toluene, 50 per cent methyl ccllosolve and 4 g of 2,5-bis-2· (5-tert-butyl benzoxazoly)·thiophene per liter (BROTPackard Instrument Company, Downers Grove, Ill.). In all cases, the individual time points were performed in triplicate, and the mean ± l SD are given. The results of an experiment to determine whether the replacement of old with fresh medium after varying incubation times would affect the ability of suspended and adherent PAM to TABLE 1 EFFECT OF MEDIUM RENEWAL ON INCORPORATION OF RADIOACTIVE LEUCINE INTO PROTEIN BY PULMONARY ALVEOLAR MACROPHAGES* Counts/min/1 o6 Cells Time
Suspended
Adherent
1 hour 2 hours 1 hour + 1 hour
23,348 ± 564 25,586 ± 2,456
20,170 ± 1,197 27,738 ± 756
with new medium
37,078 ±2,077 25,178 ± 1,843
36,105±1,552 38,636 ± 1,982
26,408 ± 1,201
51,519±1,840
4 hours 2 hours+ 2 hours with new medium
*Incubations were as described in legend to figure 1, except the isotope per incubation was 0.1 J.!Ci of D L[14c] -leucine (specific activity:312 mCi per mmole). The experiment was performed on cells from a single animal.
continue to incorporate radioactive leucine into protein as shown in table 1. Adherent cells responded almost fully to new medium at either I or 2 hours, the incorporation in new medium being, respectively, 91.5 and 85.7 per cent that expected of cells incubated for those time periods. Suspended cells, on the other hand, responded well to new medium at 1 hour (80 per cent of expected), but poorly with medium renewal at 2 hours (3.2 per cent of expected). It was next determined whether cells incubated in suspension for 120 min would be capable of incorporating radioactive leucine into protein if they were then reincubated in flasks under conditions that would allow them to adhere. The results given in table 2 show that this was not the case. Cells incubated for 2 hours in suspension showed little incorporating ability when reincubated either in suspension or adherent conditions. At the same time, adherent cells incubated for 120 min were just as active during the subsequent 60 min as were freshly incubated cells.
352
LEFFINGWELL AND !.OW
TABLE 2 RECOVERY OF PULMONARY ALVEOLAR MACROPHAGES FROM INCUBATION IN SUSPENSION* Preincubation None None Adherent, 2 hours Suspended, 2 hours Suspended, 2 hours
Incubation with Isotope 60 60 60 60 60
min min min min min
adherent suspended adherent suspended adherentt
Counts/min/106 Cells 13,299±1,160 874 16,039 ± 13,725±1,002 272 3,509 ± 241 2,805 ±
*Incubations were as described in legend to figure 1. tAfter incubation for 120 min in suspension, cells were incubated in flasks under conditions that would allow them to adhere during the incubation. The experiment was performed on cells from a single animal.
The distribution of radioactive protein in the cells preincubated in suspension and then under adherent conditions (table 2, line 5) indicated oue further important finding. After the 1-hour incubation with isotope, 46.8 per cent of the total radioactivity in protein was removed with the supernatant medium. Of this, 7.5 per cent of the total radioactivity remained in the supernate after it was centrifuged to remove cells (500 X g for 10 min), whereas the remainder was associated with pelleted cells. This suggested that almost one half of the cells preincubated in suspension for 2 hours were not capable of then adhering in the subsequent incubation period, whereas approximately 90 per cent of fresh cells were able to do so. It was not determined whether cells preincubated in suspension would recover if allowed to adhere during a time period longer than l hour. The dual-isotope technique of Dice and Schimke (6) was used in an effort to determine the molecular weight distribution of proteins synthesized by cells incubated by the 2 methods. Suspended cells were incubated with (14C]-leucine; adherent cells, with (3H]-leucine. After a 2-hour incubation, the cells were mixed and prepared for electrophoresis in polyacrylamide gels containing SDS. The results of the subsequent electrophoretic analysis are shown in figure 2. Shown in the top panel is a densitometric tracing of the Coomassie brilliant blue-stained proteins of washed PAM, demonstrating the wide molecular weight distribution of proteins of the cells. Mobility in SDS gels is determined by molecular weight (7). The (3H]- and [14C]-radioactivity profiles of the gel shown in the middle panel suggested that large molecular weight proteins contained relatively less [14C]-label than (3H]-label. This was confirmed when the ratio of (3H]- to [HC]-radioactivity was computed (bottom panel).
This experiment has been repeated a number of times with the same result. In an additional experiment, the isotopes were reversed; in this case, the isotope ratio increased along the length of the gel. Three experiments were also performed with only a 60-min incubation. Two of the 3 experiments showed the same trend as seen in the 120-min experiments, although the magnitude of the effect was smaller. The third 60-min experiment gave ambiguous results, with the isotope ratio more or less being the same along the length of the gel. The most reasonable interpretation of the data is that adherent cells were able to synthesize relatively larger amounts of high molecular weight proteins than were suspended cells. A possible alternative explanation is that the degradation of high molecular weight (labeled) proteins was selectively higher in suspended cells (see Discussion). The next question to arise was whether suspended and adherent PAM would respond differently to a phagocytic load in terms of their protein synthetic apparatus. In figure 3 are shown the results of an experiment in which cells were incubated for the times shown in the presence or absence of zymosan (particle: cell ratio, 10:1). Protein biosynthesis by suspended cells (test tube) was inhibited by the presence of zymosan. In the case of adhering cells, however, although there was slight inhibition at brief time intervals, at longer time periods the presence of zymosan stimulated the cells to incorporate more isotope into protein. The difference at 3 hours of approximately 20 per cent was reproducible (4 experiments, each with triplicate samples: P < 0.02). Electron microscopic evidence indicated that both suspended and adherent cells took up zymosan particles, although no effort was made to quantify the uptake using the electron micro-
PROTEI:-.! BIOSY:-.iTHESIS BY PUU!O:-IARY ALVEOLAR
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CRAIG M. LEFFINGWELL and ROBERT B. LOW
SUMMARY _____________________ _____________________ ______________ An effort to optimize conditions for studying protein biosynthesis by the pulmonary alveolar macrophage in vitro has led to a comparative analysis of the activity of suspended and adherent cells. A number of differences were observed. (I) Suspended cells synthesized protein for only a limited period of time, after which they responded only partially to incubation in fresh medium. This was true even under reincubation conditions in which the cells were allowed to adhere to a surface. Adherent cells, however, synthesized protein during a longer period of time and were fully capable of responding to new medium within the time periods examined. (2) Analyses of the radioactive proteins synthesized using a dual-isotope technique suggested that, during a period of 2 hours, suspended cells synthesized relatively smaller quantities of high molecular weight proteins than adherent cells. (J) The administration of a phagocytic load (zymosan; particle to cell ratio, 10:1) inhibited by 20 per cent the incorporation of isotopic amino acid into protein during a period of 3 hours. The same phagocytic load, however, stimulated incorpora· tion by 20 per cent in adherent cells. (4)The rate of particle uptake measured using oil red 0-albumin complexes decreased by approximately 50 per cent in suspended cells preincubated for 2 hours, but was maintained in similarly preincubated adherent cells. It was concluded that pulmonary alveolar macrophages incubated adhered to a surface are more appropriate for meta· bolic studies than are pulmonary alveolar macrophages incubated in suspension.
Introduction Efforts in this laboratory to examine the protein biosynthetic capabilities of pulmonary alveolar macro phages (PAM) have necessitated an examination of the effects of in vitro incubation condi-
(Received in original form November 18, 1974 and in revised form june 4, 1975) 1 From the Department of Physiology and Biophysics, the University of Vermont, Burlington, Vermont 05401. 2 Supported by grant no. HL 14212 (SCOR) from the National Heart and Lung Institute. 3 Requests for reprints should be addressed to Robert B. Low, Ph. D., Department of Physiology and Biophysics, the University of Vermont, Burlington, Vermont 05401. A~!ERICAN
tions on the incorporation of radioactive amino acid into protein. Studies using noninduced PAM incubated in polypropylene test tubes with shaking at 37° C have been reported previously (1 ). The results indicated that the synthetic activity of cells incubated under those conditions was limited to approximately the first hour of incubation. The cells subsequently showed diminished incorporating ability, a finding in keeping with those of others (2, 3) who used bacille CalmetteGuerin-induced cells (BCG-cells). The present studies were undertaken ~o compare the capabilities of suspended cells with cells incubated under conditions in which the cells adhere to the surface of culture flasks. The results indicate that relative to suspended cells, adherent cells synthesize proteins for a longer peri-
REVIEW OF RESPIRATORY DISEASE, VOLUME 112, 1975
349
350
LEFFINGWELL AND LOW
od of time, show differences in the relative rates of incorporation of amino acid into different proteins and, in addition, respond differently to a phagocytic load. Materials and Methods The methods used were generally those reported previously (1). Briefly, PAM were lavaged from lungs of normal, adult male New Zealand rabbits using calcium- and magnesium-free phosphate-buffered saline. They were collected by centrifugation at 500 X gat room temperature and washed twice with the same medium before incubations were begun. Cell viability was determined by measuring eosin Y exclusion and lactic dehydrogenase (LDH) release from the cells. In vitro amino acid uptake into protein was measured in suspended cells by incubating them in polyprophylene tubes contaning I ml of Hanks medium buffered with tris (pH 7.4) (106 cells per ml) containing 19 nonradioactive amino acids plus radioactive leucine and antimicrobial drugs (HanksIris). Amino acid uptake by adherent cells was measured by preincubating 106 eels in 35- by 10-mm tissue culture dishes (Falcon no. 3001) to allow them to adhere, and subsequently incubating the cells in I ml oJ Hanks-tris medium. Experiments showed that cells not preincubated in the falcon flasks, but incubated immediately with radioactive medium, gave similar radioactive amino acid uptake curves. Hot trichloroacetic acid (TCA)-precipitable radioactivity was measured in incubations of cells in suspension, as described previously (1). A slightly modified procedure was used to count radioactivity from adherent cell incubations. After incubation, the medium in the flasks was poured off into an equal volume of cold 20 per cent TCA. One ml of 0.1 N sodium hydroxide was added to each flask, which was then shaken gently at room temperature for 5 min. The resultant wash fluid was poured off into an equal volume of cold 20 per cent TCA. All samples were then prepared for counting (1). Incorporation data were corrected for loss of cells during preincubation and wash experiments. The data from individual experiments are expressed as the mean ± I SD. Data based on more than one experiment are given as the mean ±I standard error of the mean (SE). Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of radioactive material from incubated cells was also used as described previously (!). Oxygen tension in incubation medium was determined immediately after incubation using an 0 2 monitor system (Model 53SA, Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio). Zymosan (Nutritional Biochemical Corp., Cleveland, Ohio) was used for experiments involving phagocytosis. It was prepared according to the method of Weissmann and associates (4), and was used in a particle-to-cell ratio of I 0: I. Measurements of the rate of particle uptake by
cells were made using oil red 0-albumin complexes (5). Uptake by suspended cells was measured in 1.0 ml-volumes in polypropylene tubes, using the same concentrations of cells and noncellular components as described by Stosscl and associates (5). Uptake by cells preincubated for varying times in flasks was accomplished by collecting the cells with a rubber policeman and subsequently reincubating them in suspension with oil reel 0 complexes as described above. The PAM were fixed for electron microscopy in 4 per cent glutaraldehyde buffered to pH 7.4 with sodium cacodylate. The cells were post-fixed after 2 hours in I per cent osmium tetroxide for I hour and subsequently dehydrated in a series of ethyl alcohols. The preparations were embedded in Epon® and sectioned at 35 to 50 nm for electron microscopy. The sections were studied on a Phillips 300 electron microscope. Results The time courses of the incorporation of radioactive leucine into protein by suspended and adherent cells are compared in figure I. The kinetics of incorporation during the first 60 min were closely similar. After that time, the suspended cell essentially ceased to incorporate amino acid into protein, in agreement with previous findings (1 ). Adherent cells, however, continued to incorporate amino acid, albeit at a slightly reduced rate relative to the first hour of incubation. The difference was reproducible: in 5 experiments, suspended cells incorporated only 8.8 ± 3.4 per cent more isotope into protein after 3 hours than they did after 2 hours. Adherent cells, however, incorporated 35.7 ± 4.6 per cent more isotope during the same time interval. In all cases, cells were greater than 90 per cent viable after a 3-hour incubation, as judged by the eosin Y test. In addition, cells incubated by both means released closely similar amounts of LDH (suspended cells, 9.9 ± 3.9 per cent; adherent cells, 8.8 ± 2.8 per cent). Finally, in all cases, less than 10 per cent of the radioactive protein synthesized by both groups of cells was released from them into the medium after 3 hours. These data suggest that the plasma membranes and protein export systems of the cells were not differentially affected by the incubated conditions. Measurements were made of Po 2 in fresh medium and medium used to incubate cells either in suspension or as adherent monolayers. After 2 or 3 hours of incubation, the Po 2 was the same in media used for suspended and adherent cells, in both cases being 10 ± 0.3 per cent less than the Po 2 in fresh medium.
PROTEI:\ BIOSYNTHESIS BY PUUIONARY ALVEOLAR MACROPHAGE
351
25
20
• = adherent o = suspended
({)
--' --' w u 15
Q
""--~
Q X
10
::;; (L u 5
30
60
90
120
150
180
TIME (minutes)
Fig. I. Time course of incorporation of radioactive leucine into protein by pulmonary alveolar macrophages incubated in suspension and incubated after adherence to a surface. Macrophages isolated from a single rabbit were incubated at 37' C for the times shown in 1.0 ml of Hanks-tris medium (pH 7.4) at 37' C (I) containing the following additional components: 0.05 /LCi of DL-[14)-leucine (specific activity: 312 mCi per mmole;); 19 [12CJ-arnino acids (leucine excluded) at a concentration of I X J0-5 Af; 100 units of penicillin, 100 /Lg of streptomycin per ml, and 25 /Lg of Fungizone; 10 per cent heterologous rabbit serum made from blood collected by cardiac puncture and dialyzed at 2' C for 18 hours against 2 changes of 20 volumes of Hanks·tris medium; 106 cells (I). After incubation, suspended cells were precipitated by addition of 1 volume of icc· cold 20 per cent trichloroacetic acid (TCA), heated at 95' C for 10 min, and the precipitates were collected and washed with ice-cold 5 per cent TCA on Millipore filters of 0.45-l'm pore size (Milli· pore Corp., Redford, Mass.). Incubations in flasks were collected and precipitated with TCA as described in Methods, and were prepared for counting as above. Samples were counted in a Packard Model 3320 Liquid Scintillation Counter by adding to each in a vial, 0.7 ml of 88 per cent formic acid followed by 10 ml of a scintillation fluid containing 50 per cent toluene, 50 per cent methyl ccllosolve and 4 g of 2,5-bis-2· (5-tert-butyl benzoxazoly)·thiophene per liter (BROTPackard Instrument Company, Downers Grove, Ill.). In all cases, the individual time points were performed in triplicate, and the mean ± l SD are given. The results of an experiment to determine whether the replacement of old with fresh medium after varying incubation times would affect the ability of suspended and adherent PAM to TABLE 1 EFFECT OF MEDIUM RENEWAL ON INCORPORATION OF RADIOACTIVE LEUCINE INTO PROTEIN BY PULMONARY ALVEOLAR MACROPHAGES* Counts/min/1 o6 Cells Time
Suspended
Adherent
1 hour 2 hours 1 hour + 1 hour
23,348 ± 564 25,586 ± 2,456
20,170 ± 1,197 27,738 ± 756
with new medium
37,078 ±2,077 25,178 ± 1,843
36,105±1,552 38,636 ± 1,982
26,408 ± 1,201
51,519±1,840
4 hours 2 hours+ 2 hours with new medium
*Incubations were as described in legend to figure 1, except the isotope per incubation was 0.1 J.!Ci of D L[14c] -leucine (specific activity:312 mCi per mmole). The experiment was performed on cells from a single animal.
continue to incorporate radioactive leucine into protein as shown in table 1. Adherent cells responded almost fully to new medium at either I or 2 hours, the incorporation in new medium being, respectively, 91.5 and 85.7 per cent that expected of cells incubated for those time periods. Suspended cells, on the other hand, responded well to new medium at 1 hour (80 per cent of expected), but poorly with medium renewal at 2 hours (3.2 per cent of expected). It was next determined whether cells incubated in suspension for 120 min would be capable of incorporating radioactive leucine into protein if they were then reincubated in flasks under conditions that would allow them to adhere. The results given in table 2 show that this was not the case. Cells incubated for 2 hours in suspension showed little incorporating ability when reincubated either in suspension or adherent conditions. At the same time, adherent cells incubated for 120 min were just as active during the subsequent 60 min as were freshly incubated cells.
352
LEFFINGWELL AND !.OW
TABLE 2 RECOVERY OF PULMONARY ALVEOLAR MACROPHAGES FROM INCUBATION IN SUSPENSION* Preincubation None None Adherent, 2 hours Suspended, 2 hours Suspended, 2 hours
Incubation with Isotope 60 60 60 60 60
min min min min min
adherent suspended adherent suspended adherentt
Counts/min/106 Cells 13,299±1,160 874 16,039 ± 13,725±1,002 272 3,509 ± 241 2,805 ±
*Incubations were as described in legend to figure 1. tAfter incubation for 120 min in suspension, cells were incubated in flasks under conditions that would allow them to adhere during the incubation. The experiment was performed on cells from a single animal.
The distribution of radioactive protein in the cells preincubated in suspension and then under adherent conditions (table 2, line 5) indicated oue further important finding. After the 1-hour incubation with isotope, 46.8 per cent of the total radioactivity in protein was removed with the supernatant medium. Of this, 7.5 per cent of the total radioactivity remained in the supernate after it was centrifuged to remove cells (500 X g for 10 min), whereas the remainder was associated with pelleted cells. This suggested that almost one half of the cells preincubated in suspension for 2 hours were not capable of then adhering in the subsequent incubation period, whereas approximately 90 per cent of fresh cells were able to do so. It was not determined whether cells preincubated in suspension would recover if allowed to adhere during a time period longer than l hour. The dual-isotope technique of Dice and Schimke (6) was used in an effort to determine the molecular weight distribution of proteins synthesized by cells incubated by the 2 methods. Suspended cells were incubated with (14C]-leucine; adherent cells, with (3H]-leucine. After a 2-hour incubation, the cells were mixed and prepared for electrophoresis in polyacrylamide gels containing SDS. The results of the subsequent electrophoretic analysis are shown in figure 2. Shown in the top panel is a densitometric tracing of the Coomassie brilliant blue-stained proteins of washed PAM, demonstrating the wide molecular weight distribution of proteins of the cells. Mobility in SDS gels is determined by molecular weight (7). The (3H]- and [14C]-radioactivity profiles of the gel shown in the middle panel suggested that large molecular weight proteins contained relatively less [14C]-label than (3H]-label. This was confirmed when the ratio of (3H]- to [HC]-radioactivity was computed (bottom panel).
This experiment has been repeated a number of times with the same result. In an additional experiment, the isotopes were reversed; in this case, the isotope ratio increased along the length of the gel. Three experiments were also performed with only a 60-min incubation. Two of the 3 experiments showed the same trend as seen in the 120-min experiments, although the magnitude of the effect was smaller. The third 60-min experiment gave ambiguous results, with the isotope ratio more or less being the same along the length of the gel. The most reasonable interpretation of the data is that adherent cells were able to synthesize relatively larger amounts of high molecular weight proteins than were suspended cells. A possible alternative explanation is that the degradation of high molecular weight (labeled) proteins was selectively higher in suspended cells (see Discussion). The next question to arise was whether suspended and adherent PAM would respond differently to a phagocytic load in terms of their protein synthetic apparatus. In figure 3 are shown the results of an experiment in which cells were incubated for the times shown in the presence or absence of zymosan (particle: cell ratio, 10:1). Protein biosynthesis by suspended cells (test tube) was inhibited by the presence of zymosan. In the case of adhering cells, however, although there was slight inhibition at brief time intervals, at longer time periods the presence of zymosan stimulated the cells to incorporate more isotope into protein. The difference at 3 hours of approximately 20 per cent was reproducible (4 experiments, each with triplicate samples: P < 0.02). Electron microscopic evidence indicated that both suspended and adherent cells took up zymosan particles, although no effort was made to quantify the uptake using the electron micro-
PROTEI:-.! BIOSY:-.iTHESIS BY PUU!O:-IARY ALVEOLAR
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