EXPERIMENTAL
AND
MOLECULAR
Transformation
MICHAEL
PATHOLOGY
22,
142-150
of Alveolar Type Following Exposure
(1975)
2 Cells to Type to NO,ll*
1 Cells
J. EVANS, LINDA J. CABRAL, ROBERT J. STEPHENS, AND GUSTAVE FREEMAN Life
Sciences Division, Menlo Park, Received
Stanford California
Research 94025
Institute,
JuZy 11, 1974
This research was undertaken to study the fate of Type divided. To accomplish this, male rats were exposed to NO,
2 cells after they have to increase the number of dividing Type 2 cells. Dividing cells were labeled with ‘H-TdR and studied with autoradiographic techniques in the electron microscope for up to 14 days after labeling. The results show that initially most of the ‘H-TdR labeled cells were Type 2. However, by 2 days there was a decrease in frequency of labeled Type 2 cells and a large increase in labeled Type 1 cells. The new frequencies of labeled alveolar epithelial cells were stable from 2 through I4 days. This evidence supports the interpretation that Type 2 cells may transform into Type 1 cells. In addition, it was shown that under the conditions of this experiment: (1) the time for transformation was about 2 days, and (2) during this process an intermediate cell type was present.
INTRODUCTION The epithelium lining the walls of the alveoli is composed primarily of large, squamous Type 1 cells and smaller, cuboidal Type 2 cells. Type 1 cells cover most of the alveolar surface, and the Type 2 cells are dispersed throughout the alveoli between Type 1 cells. These cells are considered to be renewing cell populations. The process of cell renewa is basically a homeostatic mechanism for maintaining the integrity of a tissue (Bullough, 1965; Oehlert, 1973). In epithelial tissues, it involves replacement of a surface cells that is being sloughed off. Replacement is accomplished by division of progenitor cells, after which one or both sister cells may migrate and mature into the cell type that is to be replaced. Although this process is well understood in tissues such as epidermis and gastrointestina1 epithelium, a similar mechanism onIy recentIy has been described for pulmonary alveoli. Evans et al. (1972), using tritiated thymidine ( 3H-TdR) to label dividing cells, showed that Type 2 cells were the principal dividing cells in the alveolar epithelium of the rat following exposure to nitrogen dioxide (NOZ). Further studies described the kinetics of Type 2 cell division under these conditions and followed the SH-TdR labeled sister cells for 3 days 1 This Department
research was supported by PHS Grants of Health, Education and Welfare. 2 A portion of this research was presented before Colorado, June 1973. 142 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
ES the
00482
and
16th
Aspen
HL Lung
16330
from
Conference,
the
U.S.
Aspen,
TRANSFORMATION
OF TYPE 2 CELLS
FIG. 1. Average percentage of each cell type in the labeled alveolar epithelium. ubtained from data in Table I.
143
Points were
(Evans et al., 1973). Using light and electron microscopy, the authors observed a large increase in SH-TdR labeled Type 1 cells several days after division of the Type 2 cells. Their interpretation was that the Type 2 sister cells had transformed into Type 1 cells and thus were the progenitor cells of the alveolar epithelium. A similar result and interpretation were reported recently in a study of mice exposed to oxygen (Adamson and Bowden, 1974). Although it seemsclear that Type 2 cells may transform into Type 1 cells, the details of this process have not been described. The purpose of the present study was to determine the time for transformation of Type 2 to Type 1 cells, identify alveolar epithelial cells intermediate between Types 2 and 1, and determine whether the new Type 1 and Type 2 cell populations appeared stable. To accomplish this, rats were exposed to NOz, labeled with tritiated thymidine ( 3H-TdR), and studied for up to 14 days after labeling, using electron microscope autoradiography. MATERIALS
AND METHODS
One-month-old male Wistar rats weighing approximately 100 g were used in this study. A total of 14 rats were exposed to 15-17 ppm NOz for 48 hr, removed to ambient conditions, and injected intraperitoneally with 500 &i of SH-TdR (sp act 6.7 Ci/mmole). At intervals of 1 hr and 1, 2, 3, 4, 7, and 14 days after injection of SH-TdR, ,&herats were sacrificed. The lungs were fixed in situ with 2% glutaraldehyde in 0.1 M cacodylate buffer, removed, sliced, washed in 0.05 M Verona1 acetate buffer, posffixed in 1% 0~04 in Verona1 acetate buffer, dehydrated, and embedded in epoxy resin (Araldite) (Stephens and Evans, 1973). Some tissues were fixed only in 0~04 before dehydration and embedding. Samples were prepared for light and electron microscopy by previously described methods (Evans et aE., 1973), For light microscopy, 1-q sections were cut, coated with Ilford L-4 emulsion, exposed for 2-3 wk, developed, and stained with toluidine blue. For electron microscopy, pale-gold sections were cut, picked up on grids, double-stained with uranyl acetate and lead citrate, and
EVANS
144
FIG. 2. Labeled FIG. 3. Labeled
ET
AL.
Type 2 cell 4 days after an injection Type 1 celI 4 days after an injection
of ‘H-TdR of ‘H-TdR
(~11,600). (~8,600).
coated with carbon. The grids then were covered with Ilford L-4 emulsion, exposed for 6 wk, developed, and viewed with a Philips 200 electron microscope. The labeled alveolar epiihelial cell population was analyzed by scanning a
TRANSFORMATION
OF
TYPE
2 CELLS
FIG. 4. Labeled undetermined cell 1 day after an injection of BH-TdR. This more characteristic of a Type 2 cell (x9,000). FIG. 5. Labeled undetermined cell 1 day after an injection of *H-TdR. sidered more characteristic of a Type 1 cell (x9,000).
145
cell is considered This
cell
is con-
grid in the electron microscope and photographing each labeled cell in one section on the grid. Ten to 15 samplesfrom each rat were studied. All the labeled epithelial cells collected from an animal then were separated according to cell
146
FIG. inclusions
EVANS
6. Labeled (arrow)
Type similar
ET AL.
1 cell 2 days after an injection to those in Fig. 4 ( X11,600).
type and expressed as a following cell types were and S), and undetermined in the alveolar epithelium
of *H-TdR.
Note
the
cytoplasmic
percentage of the total labeled cell population. The involved: Type 2 cells (Fig. 2), Type 1 cells (Figs. 3 cells (Figs. 4 and 5). Undetermined cells were those that were not distinctly Type 1 or Type 2 cells.
TRANSFORMATION
OF TYPE 2 CELLS
14Y
TABLE I FREQUENCY
Time after %I-TdR
OF LABELED CELL TYPES IN THE ALWOLAR IDENTIFIED WITH THE ELECTRON MICROSCOPP
Total number labeled cells
‘% Labeled Type 2
EPITHELIUM
yO Labeled undetermined
TO Labeled Type 1
54 59
88.9
11.1 11.9
0
86.4
1 day
63 61
57.0 62.4
39.8 32.7
3.2 4.9
2 days
56 31
41.0 51.7
28.6 12.9
30.4 35.5
3 days
41 36
65.6 44.4
9.9 19.4
24.5 36.1
4 days
47 42
57.5 57.2
16.9 11.8
25.6 31.0
52.5 (72.8)
14.1 (9.0)
33.4 (18.2)
52.5 61.4
19.0 9.7
28.5 29.1
1 hr
7 days 14 days
&” 21 31
1.7
* Each line represents the data from one animal. b Not included in evaluation of data because of insufficient numbers of labeled cells.
RESULTS The pathologic observations of the tissue and degree of cell labeling were similar to those previously described (Evans et al., 1972; Evans et al., 1973; Stephens et al., 1972). After 48 hr of exposure to 15-17 ppm NOa, there were areas of cuboidal epithelium near the peripheral openings of the terminal bronchioles. However, after 3 days of recovery, the tissues appeared normal and remained so for up to 14 days. Labeled cells were distributed throughout the sections, although more were located near the openings of the terminal bronchioles. The Iabehng analysis of this study is presented in TabIe I and summarized in Fig. 1. At 1 hr after injection of 3H-TdR, most of the labeled cells had not divided (Cleaver, 1967; Thrasher, 1966; Evans et aE., 1973). At this time, an average of 87.8% were Type 2 cells, 0.8% were Type 1 cells, and 11.5% were undetermined. By the end of 1 day and thereafter, most of the labeled cells should have divided (Evans et al., 1973). At 1 day, 59.7% of the labeled cells were Type 2, 4.1% were Type 1, and 36.2% were undetermined. At 2 days, 46.4% were Type 2, 32.9% were Type 1, and 20.8% were undetermined. At 3 days, 55% were Type 2, 30.3% were Type 1, and 14.7% were undetermined. From 3-14 days, the percentages of labeled cell types remained about the same ( Table I ) . The morphology of the labeled Type 2 cells appeared similar at aII time intervals (Fig. 2). The size of the cells and the number of lamellar bodies were not determined. Labeled Type 1 cells appeared normal except for the presence of occasional inclusion bodies and irregular cell surfaces at 2 and 3 days (Fig. 6).
148
EVANS
ET AL.
After this time, they appeared normal (Fig. 3). The unidentified cells may have been Type 2, Type 1, or a cell intermediate between them. Electron microscopy showed that most of these tended to be cuboidal cells resting on the basement membrane (Figs. 4 and 5). They did not contaiu typical lamellar bodies but did have occasional inclusions and microvilli, similar to those observed in Type 1 cells at 2 and 3 days (Fig. 6). M’ost of these cells were rather characteristic of Type 2 cells (Fig, 4), although, a smaller proportion appeared to be spreading out over the basement membrane and thus resembled Type 1 cells more closely (Fig. 5). DISCUSSION In a previous study, the mechanism and kinetics of Type 2 cell division following exposure to NO2 were described (Evans et al., 1973). It was shown that as Type 2 cells divide, they move apart over the basement membrane so that after telophase, both sister cells remain on the basement membrane. Division of most of the labeled cells was shown to have occurred within 12 hr after injection of 3H-TdR. Another portion of this study presented data concerning the fate of Type 2 sister cells after division. It was shown with the light microscope at 1 hr through 3 days, and with the electron microscope at 1 hr and 2 days, that a large number of labeled Type 1 cells appear in the alveolar epithelium. From this and other information, it was concluded that Type 2 sister cells can transform into Type 1 cells. Adamson and Bowden (1974) report similar results in a recent article. Using mice exposed to 90% oxygen for 6 days and studying the survivors for 7 days during recovery, they showed with the light microscope a large increase in labeled Type 1 cells 3 days after an injection of 3H-TdR. Before this time, Type 2 cells were the main alveolar epithelial cells labeled. From these studies, they also conclude that Type 2 cells can transform into Type 1 cells. In the present study, information concerning the mechanism and kinetics of Type 2 transformation into Type 1 cells was ‘obtained. The first change observed was a decrease in the frequency of labeled Type 2 cells and an increase in labeled undetermined cells (Fig. l), followed by an increase in labeled Type 1 cells and a decrease in labeled undetermined cells. This sequence demonstrates the progression ~oflabel from Type 2 cells through an undetermined cell population and ending in the Type 1 cell population. That the frequency of labeled Type 2 cells decreases and then remains essentially the same from 1-14 days, despite changes in the other cell types, suggests that Type 2 cells destined to become Type 1 have begun the process of transformation before the end of 1 day. Also, under the conditions of this experiment, not all the Type 2 sister cells transform into Type 1 cells. For example, if each dividing Type 2 cell contributed one sister cell to the Type 1 cell population and the other remained as a Type 2 cell, the percentage of labeled Type 2 cells after 3 days (when labeled Type 1 cells appear) would be about one-half of the 1-l-n value, and the Type 1 cells would increase by a like amount. The actual decrease observed in Type 2 cells is 1~s than 5OoJ0,averaging about 30%. This indicates that not all Type 2 sister cells transform into Type 1 cells, thus allowing for a slight increase in the number of Type 2 cells in the tissue. In agreement with this, previous results
TRANSFORMATION
OF TYPE 2 CELLS
149
showed a slight overall increase in Type 2 cells following exposure to NOz (Evans et al., 1973). The wave of labeling passing through the undetermined cell population indicates the presence of a cell type intermediate between Type 2 and Type 1. The concept of intermediate cells transitional between Type 1 and Type 2 cells has been mentioned by several authors (Faulkner and Esterly, 1971; Greenberg et al., 1971). However, in these studies the cells in question appear to be more like Type 1 cells than intermediate cells (Evans et al., 1973). Recently, Adamson and Bowden (1974) stated that cuboidal cells in the alveolar epithelium that lacked lamellar bodies are cells intermediate between Type 2 and Type 1, although there was no labeling data to support this claim. In the present study the undetermined cell population consists of alveolar epithelial cells that are not distinctly Type 1 or Type 2 cells. In general, they are cuboidal cells containing several electron-dense inclusions but no lamellar bodies (Figs. 4 and 5). The electron-dense inclusions observed in labeled undetermined cells are similar to those present in labeled Type 1 cells at 2 and 3 days (Figs. 4-6). The presence of labeled, morphologically distinct cells in the undetermined population at 1 day and their decreased labeling coincidental with increased labeling of Type 1 cells suggest they are intermediate cells in the process of transforming from Type 2 to Type 1 cells. The sharp appearance of labeled Type 1 cells between 1 and 2 days after labeling and their relatively constant frequency thereafter indicate that the process of transformation from a Type 2 to a Type 1 cell requires about 2 days under the conditions of this experiment. Morphologically, the Type 1 cells at 2 and 3 days have an irregular surface and eleotron-dense inclusions (Evans et al., 1973). However, from the P14th day these disappear and the labeled Type 1 cells appear normal. In summary, the results from this and other studies indicate that Type 2 cells can divide and may transform into Type 1 cells (Evans et al., 1973; Adamson and Bowden, 1974). The time required for transformation is 2 days, after which time the labeled cell populations are stable for up to 14 days. Not all Type 2 sister cells transform into Type 1 cells, and there is a slight overall increase of Type 2 cells in the tissue. However, those that do transform are committed after the first day. The process of transformation involves an intermediate cell type that exists at 1 day. This cell is cuboidal and lacks lamellar bodies but does contain electron-dense inclusions. Presumably, this cell flattens out and becomes a Type 1 cell. Type 1 cells that appear at 2 and 3 days contain electron-dense inclusions; however, after the fourth day these are lost, and the cells appear normal thereafter. REFERENCES ADAMSON, I. Y. R., and BOWDEN, D. H. ( 1974). The type 2 cell as progenitor of alveolar epithelial regeneration. A cytodynamic study in mice after exposure to oxygen. Lab. Invest. 30, 35-42. BULLOUGH, W. S. ( 1963). Mitotic and functional homeostasis. Cancer Res. 25, 1683-1727. CLEAVER, J. E. ( 1967). “Thymidine Metabolism and Cell Kinetics.” North-Holland Publishing Co., Amsterdam. EVANS, M. J., STEPHENS, R. J., CABRAL, L. J., and FREEMAN, G. (1972). Cell renewal in the lungs of rats exposed to low levels of NOS Arch. En&on. Health 24, 180-188.
150
EVANS
ET AL.
EVANS, M. J., CABRAL, L. J., STEPHENS, R. J., and FREEMAN, G. (1973). Renewal of alveolar epithelium in the rat following exposure to NOa. Am. J. Pathol. 70, 175-198. FAULKNER, C. S., and ESTERLY, J. R. (1971). Ultrastructural changes in the alveolar epithelium in response to Freund’s Adjuvant. Am. J. Pathol. 64, 559-566. GREENBERG, S. D., GYORKEY, F., JENKINS, D. E., and GYORKEY, P. ( 1971). Alveolar epithelial cells following exposure to nitric acid. Arch. Environ. Health 22, 655-662. OEHLERT, W. ( 1973). Cell proliferation in carcinogenesis. Cell Tissue Kinet. 6, 325-335. STEPHENS, R. J., FREEMAN, G., and EVANS, M. J. (1972). Early response of lungs to low levels of nitrogen dioxide. Arch. Environ. Health 24, 160-179. STEPHENS, R. J., and EVANS, M. J. (1973). Selection and orientation of lung tissue for scanning and transmission electron microscopy. Environ. Rex 6, 52-59. THRASHER, J. D. ( 1966). Analysis of renewing epithelial cell populations. In Methods of Cell Physiology (D. M. Prescott, Ed.), pp. 323-357. Academic Press, New York.
AND
MOLECULAR
Transformation
MICHAEL
PATHOLOGY
22,
142-150
of Alveolar Type Following Exposure
(1975)
2 Cells to Type to NO,ll*
1 Cells
J. EVANS, LINDA J. CABRAL, ROBERT J. STEPHENS, AND GUSTAVE FREEMAN Life
Sciences Division, Menlo Park, Received
Stanford California
Research 94025
Institute,
JuZy 11, 1974
This research was undertaken to study the fate of Type divided. To accomplish this, male rats were exposed to NO,
2 cells after they have to increase the number of dividing Type 2 cells. Dividing cells were labeled with ‘H-TdR and studied with autoradiographic techniques in the electron microscope for up to 14 days after labeling. The results show that initially most of the ‘H-TdR labeled cells were Type 2. However, by 2 days there was a decrease in frequency of labeled Type 2 cells and a large increase in labeled Type 1 cells. The new frequencies of labeled alveolar epithelial cells were stable from 2 through I4 days. This evidence supports the interpretation that Type 2 cells may transform into Type 1 cells. In addition, it was shown that under the conditions of this experiment: (1) the time for transformation was about 2 days, and (2) during this process an intermediate cell type was present.
INTRODUCTION The epithelium lining the walls of the alveoli is composed primarily of large, squamous Type 1 cells and smaller, cuboidal Type 2 cells. Type 1 cells cover most of the alveolar surface, and the Type 2 cells are dispersed throughout the alveoli between Type 1 cells. These cells are considered to be renewing cell populations. The process of cell renewa is basically a homeostatic mechanism for maintaining the integrity of a tissue (Bullough, 1965; Oehlert, 1973). In epithelial tissues, it involves replacement of a surface cells that is being sloughed off. Replacement is accomplished by division of progenitor cells, after which one or both sister cells may migrate and mature into the cell type that is to be replaced. Although this process is well understood in tissues such as epidermis and gastrointestina1 epithelium, a similar mechanism onIy recentIy has been described for pulmonary alveoli. Evans et al. (1972), using tritiated thymidine ( 3H-TdR) to label dividing cells, showed that Type 2 cells were the principal dividing cells in the alveolar epithelium of the rat following exposure to nitrogen dioxide (NOZ). Further studies described the kinetics of Type 2 cell division under these conditions and followed the SH-TdR labeled sister cells for 3 days 1 This Department
research was supported by PHS Grants of Health, Education and Welfare. 2 A portion of this research was presented before Colorado, June 1973. 142 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
ES the
00482
and
16th
Aspen
HL Lung
16330
from
Conference,
the
U.S.
Aspen,
TRANSFORMATION
OF TYPE 2 CELLS
FIG. 1. Average percentage of each cell type in the labeled alveolar epithelium. ubtained from data in Table I.
143
Points were
(Evans et al., 1973). Using light and electron microscopy, the authors observed a large increase in SH-TdR labeled Type 1 cells several days after division of the Type 2 cells. Their interpretation was that the Type 2 sister cells had transformed into Type 1 cells and thus were the progenitor cells of the alveolar epithelium. A similar result and interpretation were reported recently in a study of mice exposed to oxygen (Adamson and Bowden, 1974). Although it seemsclear that Type 2 cells may transform into Type 1 cells, the details of this process have not been described. The purpose of the present study was to determine the time for transformation of Type 2 to Type 1 cells, identify alveolar epithelial cells intermediate between Types 2 and 1, and determine whether the new Type 1 and Type 2 cell populations appeared stable. To accomplish this, rats were exposed to NOz, labeled with tritiated thymidine ( 3H-TdR), and studied for up to 14 days after labeling, using electron microscope autoradiography. MATERIALS
AND METHODS
One-month-old male Wistar rats weighing approximately 100 g were used in this study. A total of 14 rats were exposed to 15-17 ppm NOz for 48 hr, removed to ambient conditions, and injected intraperitoneally with 500 &i of SH-TdR (sp act 6.7 Ci/mmole). At intervals of 1 hr and 1, 2, 3, 4, 7, and 14 days after injection of SH-TdR, ,&herats were sacrificed. The lungs were fixed in situ with 2% glutaraldehyde in 0.1 M cacodylate buffer, removed, sliced, washed in 0.05 M Verona1 acetate buffer, posffixed in 1% 0~04 in Verona1 acetate buffer, dehydrated, and embedded in epoxy resin (Araldite) (Stephens and Evans, 1973). Some tissues were fixed only in 0~04 before dehydration and embedding. Samples were prepared for light and electron microscopy by previously described methods (Evans et aE., 1973), For light microscopy, 1-q sections were cut, coated with Ilford L-4 emulsion, exposed for 2-3 wk, developed, and stained with toluidine blue. For electron microscopy, pale-gold sections were cut, picked up on grids, double-stained with uranyl acetate and lead citrate, and
EVANS
144
FIG. 2. Labeled FIG. 3. Labeled
ET
AL.
Type 2 cell 4 days after an injection Type 1 celI 4 days after an injection
of ‘H-TdR of ‘H-TdR
(~11,600). (~8,600).
coated with carbon. The grids then were covered with Ilford L-4 emulsion, exposed for 6 wk, developed, and viewed with a Philips 200 electron microscope. The labeled alveolar epiihelial cell population was analyzed by scanning a
TRANSFORMATION
OF
TYPE
2 CELLS
FIG. 4. Labeled undetermined cell 1 day after an injection of BH-TdR. This more characteristic of a Type 2 cell (x9,000). FIG. 5. Labeled undetermined cell 1 day after an injection of *H-TdR. sidered more characteristic of a Type 1 cell (x9,000).
145
cell is considered This
cell
is con-
grid in the electron microscope and photographing each labeled cell in one section on the grid. Ten to 15 samplesfrom each rat were studied. All the labeled epithelial cells collected from an animal then were separated according to cell
146
FIG. inclusions
EVANS
6. Labeled (arrow)
Type similar
ET AL.
1 cell 2 days after an injection to those in Fig. 4 ( X11,600).
type and expressed as a following cell types were and S), and undetermined in the alveolar epithelium
of *H-TdR.
Note
the
cytoplasmic
percentage of the total labeled cell population. The involved: Type 2 cells (Fig. 2), Type 1 cells (Figs. 3 cells (Figs. 4 and 5). Undetermined cells were those that were not distinctly Type 1 or Type 2 cells.
TRANSFORMATION
OF TYPE 2 CELLS
14Y
TABLE I FREQUENCY
Time after %I-TdR
OF LABELED CELL TYPES IN THE ALWOLAR IDENTIFIED WITH THE ELECTRON MICROSCOPP
Total number labeled cells
‘% Labeled Type 2
EPITHELIUM
yO Labeled undetermined
TO Labeled Type 1
54 59
88.9
11.1 11.9
0
86.4
1 day
63 61
57.0 62.4
39.8 32.7
3.2 4.9
2 days
56 31
41.0 51.7
28.6 12.9
30.4 35.5
3 days
41 36
65.6 44.4
9.9 19.4
24.5 36.1
4 days
47 42
57.5 57.2
16.9 11.8
25.6 31.0
52.5 (72.8)
14.1 (9.0)
33.4 (18.2)
52.5 61.4
19.0 9.7
28.5 29.1
1 hr
7 days 14 days
&” 21 31
1.7
* Each line represents the data from one animal. b Not included in evaluation of data because of insufficient numbers of labeled cells.
RESULTS The pathologic observations of the tissue and degree of cell labeling were similar to those previously described (Evans et al., 1972; Evans et al., 1973; Stephens et al., 1972). After 48 hr of exposure to 15-17 ppm NOa, there were areas of cuboidal epithelium near the peripheral openings of the terminal bronchioles. However, after 3 days of recovery, the tissues appeared normal and remained so for up to 14 days. Labeled cells were distributed throughout the sections, although more were located near the openings of the terminal bronchioles. The Iabehng analysis of this study is presented in TabIe I and summarized in Fig. 1. At 1 hr after injection of 3H-TdR, most of the labeled cells had not divided (Cleaver, 1967; Thrasher, 1966; Evans et aE., 1973). At this time, an average of 87.8% were Type 2 cells, 0.8% were Type 1 cells, and 11.5% were undetermined. By the end of 1 day and thereafter, most of the labeled cells should have divided (Evans et al., 1973). At 1 day, 59.7% of the labeled cells were Type 2, 4.1% were Type 1, and 36.2% were undetermined. At 2 days, 46.4% were Type 2, 32.9% were Type 1, and 20.8% were undetermined. At 3 days, 55% were Type 2, 30.3% were Type 1, and 14.7% were undetermined. From 3-14 days, the percentages of labeled cell types remained about the same ( Table I ) . The morphology of the labeled Type 2 cells appeared similar at aII time intervals (Fig. 2). The size of the cells and the number of lamellar bodies were not determined. Labeled Type 1 cells appeared normal except for the presence of occasional inclusion bodies and irregular cell surfaces at 2 and 3 days (Fig. 6).
148
EVANS
ET AL.
After this time, they appeared normal (Fig. 3). The unidentified cells may have been Type 2, Type 1, or a cell intermediate between them. Electron microscopy showed that most of these tended to be cuboidal cells resting on the basement membrane (Figs. 4 and 5). They did not contaiu typical lamellar bodies but did have occasional inclusions and microvilli, similar to those observed in Type 1 cells at 2 and 3 days (Fig. 6). M’ost of these cells were rather characteristic of Type 2 cells (Fig, 4), although, a smaller proportion appeared to be spreading out over the basement membrane and thus resembled Type 1 cells more closely (Fig. 5). DISCUSSION In a previous study, the mechanism and kinetics of Type 2 cell division following exposure to NO2 were described (Evans et al., 1973). It was shown that as Type 2 cells divide, they move apart over the basement membrane so that after telophase, both sister cells remain on the basement membrane. Division of most of the labeled cells was shown to have occurred within 12 hr after injection of 3H-TdR. Another portion of this study presented data concerning the fate of Type 2 sister cells after division. It was shown with the light microscope at 1 hr through 3 days, and with the electron microscope at 1 hr and 2 days, that a large number of labeled Type 1 cells appear in the alveolar epithelium. From this and other information, it was concluded that Type 2 sister cells can transform into Type 1 cells. Adamson and Bowden (1974) report similar results in a recent article. Using mice exposed to 90% oxygen for 6 days and studying the survivors for 7 days during recovery, they showed with the light microscope a large increase in labeled Type 1 cells 3 days after an injection of 3H-TdR. Before this time, Type 2 cells were the main alveolar epithelial cells labeled. From these studies, they also conclude that Type 2 cells can transform into Type 1 cells. In the present study, information concerning the mechanism and kinetics of Type 2 transformation into Type 1 cells was ‘obtained. The first change observed was a decrease in the frequency of labeled Type 2 cells and an increase in labeled undetermined cells (Fig. l), followed by an increase in labeled Type 1 cells and a decrease in labeled undetermined cells. This sequence demonstrates the progression ~oflabel from Type 2 cells through an undetermined cell population and ending in the Type 1 cell population. That the frequency of labeled Type 2 cells decreases and then remains essentially the same from 1-14 days, despite changes in the other cell types, suggests that Type 2 cells destined to become Type 1 have begun the process of transformation before the end of 1 day. Also, under the conditions of this experiment, not all the Type 2 sister cells transform into Type 1 cells. For example, if each dividing Type 2 cell contributed one sister cell to the Type 1 cell population and the other remained as a Type 2 cell, the percentage of labeled Type 2 cells after 3 days (when labeled Type 1 cells appear) would be about one-half of the 1-l-n value, and the Type 1 cells would increase by a like amount. The actual decrease observed in Type 2 cells is 1~s than 5OoJ0,averaging about 30%. This indicates that not all Type 2 sister cells transform into Type 1 cells, thus allowing for a slight increase in the number of Type 2 cells in the tissue. In agreement with this, previous results
TRANSFORMATION
OF TYPE 2 CELLS
149
showed a slight overall increase in Type 2 cells following exposure to NOz (Evans et al., 1973). The wave of labeling passing through the undetermined cell population indicates the presence of a cell type intermediate between Type 2 and Type 1. The concept of intermediate cells transitional between Type 1 and Type 2 cells has been mentioned by several authors (Faulkner and Esterly, 1971; Greenberg et al., 1971). However, in these studies the cells in question appear to be more like Type 1 cells than intermediate cells (Evans et al., 1973). Recently, Adamson and Bowden (1974) stated that cuboidal cells in the alveolar epithelium that lacked lamellar bodies are cells intermediate between Type 2 and Type 1, although there was no labeling data to support this claim. In the present study the undetermined cell population consists of alveolar epithelial cells that are not distinctly Type 1 or Type 2 cells. In general, they are cuboidal cells containing several electron-dense inclusions but no lamellar bodies (Figs. 4 and 5). The electron-dense inclusions observed in labeled undetermined cells are similar to those present in labeled Type 1 cells at 2 and 3 days (Figs. 4-6). The presence of labeled, morphologically distinct cells in the undetermined population at 1 day and their decreased labeling coincidental with increased labeling of Type 1 cells suggest they are intermediate cells in the process of transforming from Type 2 to Type 1 cells. The sharp appearance of labeled Type 1 cells between 1 and 2 days after labeling and their relatively constant frequency thereafter indicate that the process of transformation from a Type 2 to a Type 1 cell requires about 2 days under the conditions of this experiment. Morphologically, the Type 1 cells at 2 and 3 days have an irregular surface and eleotron-dense inclusions (Evans et al., 1973). However, from the P14th day these disappear and the labeled Type 1 cells appear normal. In summary, the results from this and other studies indicate that Type 2 cells can divide and may transform into Type 1 cells (Evans et al., 1973; Adamson and Bowden, 1974). The time required for transformation is 2 days, after which time the labeled cell populations are stable for up to 14 days. Not all Type 2 sister cells transform into Type 1 cells, and there is a slight overall increase of Type 2 cells in the tissue. However, those that do transform are committed after the first day. The process of transformation involves an intermediate cell type that exists at 1 day. This cell is cuboidal and lacks lamellar bodies but does contain electron-dense inclusions. Presumably, this cell flattens out and becomes a Type 1 cell. Type 1 cells that appear at 2 and 3 days contain electron-dense inclusions; however, after the fourth day these are lost, and the cells appear normal thereafter. REFERENCES ADAMSON, I. Y. R., and BOWDEN, D. H. ( 1974). The type 2 cell as progenitor of alveolar epithelial regeneration. A cytodynamic study in mice after exposure to oxygen. Lab. Invest. 30, 35-42. BULLOUGH, W. S. ( 1963). Mitotic and functional homeostasis. Cancer Res. 25, 1683-1727. CLEAVER, J. E. ( 1967). “Thymidine Metabolism and Cell Kinetics.” North-Holland Publishing Co., Amsterdam. EVANS, M. J., STEPHENS, R. J., CABRAL, L. J., and FREEMAN, G. (1972). Cell renewal in the lungs of rats exposed to low levels of NOS Arch. En&on. Health 24, 180-188.
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EVANS, M. J., CABRAL, L. J., STEPHENS, R. J., and FREEMAN, G. (1973). Renewal of alveolar epithelium in the rat following exposure to NOa. Am. J. Pathol. 70, 175-198. FAULKNER, C. S., and ESTERLY, J. R. (1971). Ultrastructural changes in the alveolar epithelium in response to Freund’s Adjuvant. Am. J. Pathol. 64, 559-566. GREENBERG, S. D., GYORKEY, F., JENKINS, D. E., and GYORKEY, P. ( 1971). Alveolar epithelial cells following exposure to nitric acid. Arch. Environ. Health 22, 655-662. OEHLERT, W. ( 1973). Cell proliferation in carcinogenesis. Cell Tissue Kinet. 6, 325-335. STEPHENS, R. J., FREEMAN, G., and EVANS, M. J. (1972). Early response of lungs to low levels of nitrogen dioxide. Arch. Environ. Health 24, 160-179. STEPHENS, R. J., and EVANS, M. J. (1973). Selection and orientation of lung tissue for scanning and transmission electron microscopy. Environ. Rex 6, 52-59. THRASHER, J. D. ( 1966). Analysis of renewing epithelial cell populations. In Methods of Cell Physiology (D. M. Prescott, Ed.), pp. 323-357. Academic Press, New York.
