Ciclo Celular Neumocitos

Cell cycle kinetics in the alveolar epithelium
BRUCE D. UHAL
Lung Cell Kinetics Laboratory, Pulmonary Division and Department of Medicine,
Michael Reese Hospital and University of Illinois at Chicago, Chicago, Illinois 60612
Uhal, Bruce D. Cell cycle kinetics in the alveolar epithelium. Am. J.

PhysioZ. 272 (Lung Cell. A&o,!. Physiol. 16): L1031-L1045, 1997.-The
type II alveolar epithelial cell has important metabolic and biosynthetic
functions but also serves as the stem cell of the alveolar epithelium. Much
of the evidence underlying this premise was obtained before 1980 and
provided the basis for a working model that has not been reconsidered for
more than fifteen years. With the exceptions to be discussed below, little
evidence has accumulated in the interim to suggest that the model
requires significant alteration. Important questions remain unanswered,
however, and some components of the model need to be supplemented,
particularly in light of recent investigations that have provided insights
not possible in earlier work. In particular, in vitro studies have suggested
that the relationship between the parent type II cell and its progeny may
not be as straightforward as originally thought. In addition, the rate of
epithelial cell loss was recognized long ago to be an important factor in
the regulation of this system, but its kinetics and mechanisms have
received little attention. These and other unresolved issues are critical to
our understanding of the homeostasis of the alveolar epithelium under
normal and pathological conditions.
lung injury; proliferation; apoptosis
THE TYPE II PNEUMOCYTE is a multifunctional epithelial type II cells accomplish alveolar repair is now known to
cell with important synthetic, secretory, and progenitor be inversely related to the development of pulmonary
capacities. The best-described function of this cell is the fibrosis (lls>, and uncontrolled proliferation of type II
synthesis and secretion of pulmonary surfactant, the cells may result in adenocarcinomas of the lung paren-
lipid-protein complex that prevents atelectasis at low chyma (100, 107). Thus the ability to therapeutically
lung volumes by lowering of the surface tension at the manipulate type II cell proliferation would constitute
air-alveolar interface (25, 36). The type II cell, referred an attractive strategy to improve the management of a
to also as the vacuolated alveolar cell or the granular variety of lung disorders. However, the careful design of
pneumocyte, functions in xenobiotic metabolism through such strategies will require extensive knowledge of the
the activity of cytochrome P-450 monoxygenases (51) cell cycle kinetics of these cells and the positive and
and in transepithelial ion movement driven by a vari- negative regulation of their proliferation under both
ety of interesting transport systems (22, 68, 120). In normal and pathological conditions.
recent years, the type II cell has been proposed to have The topic of cell cycle kinetics in the alveolar epithe-
immunomodulatory functions manifested through the lium was last reviewed by Kauffman (62) more than
activities of the surfactant-associated proteins (29, 70), fifteen years ago. That comprehensive work summa-
through modulation of the expression of adhesion mol- rized a series of autoradiographic and histological
ecules (6, 53), and through the regulated release of studies, beginning in the 1950s and continuing through
immunomodulatory cytokines (20,103). the 1970s which defined the anatomy and cytokinetics
In addition to its synthetic and secretory capacities, of the lung parenchyma. On the basis of the available
the type II pneumocyte is the stem cell of the alveolar data, an attempt was made to categorize AEC with
epithelium. After lung injury and during normal cell respect to other cell types according to classical defini-
turnover, type II cells divide and differentiate into type tions of cell kinetic behavior (73, 116). Since that
I alveolar epithelial cells (AEC), usually in a highly review, investigations of the AEC cycle have been
regulated fashion that restores normal tissue architec- performed almost exclusively in vitro; most recent
ture and function. This process is a critical component studies have employed primary cell isolates or pulmo-
of the response of the lung to a wide variety of inhaled nary cell lines to rank the potency of growth-promoting
toxicants as well as blood-borne xenobiotics that ex- activities or to begin identifying specific molecules and
hibit pulmonary toxicity (81). The efficiency with which regulatory pathways involved in the control of AEC
1040-0605/97 $5.00 Copyright o 1997 the American Physiological Society LlO31
Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (130.095.063.089) on January 7, 2019.
L1032 INVITED REVIEW
proliferation. The purpose of this manuscript is to

review the work that defined the cell cycle kinetics and
stem cell function of AEC, both in vivo and in vitro.
Older data will be reexamined in light of more recent
information, discrepancies will be discussed, and gaps
in our current knowledge will be noted. It is hoped that
this discussion will stimulate renewed interest and
lead to further investigation in this important aspect of
lung cell biology.
THE MODEL, CIRCA 1980
A drawing of the pulmonary alveoli is presented in k Type I Air Space

Fig. 1, top. In primates, the alveolar epithelium is
comprised of only two cell types, the type I and type II
pneumocytes, although a third epithelial cell termed
the “brush cell” has been described in the rat and dog
(114). In the normal lung, the type II cell resides
primarily in the corners of the alveoli, is cuboidal, and
appears by light microscopy to be vacuolated due to
numerous lamellar bodies, the storage organelles for
pulmonary surfactant. The squamous type I cell is
normally devoid of organelles, has extremely attenu-
ated cytoplasmic extensions, and may cover the walls of
several adjacent alveoli to comprise as many as 10 or fp*@:.,!.‘.‘T.
.0,..e-. . G1
more apical plates (115) or surfaces facing the lumen.
Although the two epithelial cell types are present in
similar numbers, type I cells are estimated to comprise
95% of the alveolar surface area (27). Excellent studies
Ll1 ....e-..‘m.-. .***-
,,..
.:,.....::;::,*.>
.-‘:::.‘,*.‘.+.;*.
.*
.....:.*:.
S
G2
of the alveolar anatomy have been published (27, 28,

114).
In the last review of this topic, the accepted model of
alveolar epithelial stem cell function was that depicted
in Fig. 1, bottom. The epithelium was conceptualized as
follows: the type II pneumocyte is the only stem cell of
both type II and type I AEC. If either cell type is lost,
the nearest type II cells are stimulated to proliferate
and, if necessary, to differentiate into squamous type I
cells, which are terminally differentiated and thus
incapable of division. However, only a fraction of the
Fig. 1. Sch .ematic of alveolar epithel ial cell origin in th .e adult lung,
daughter cells is believed to differentiate; the remain- 1980. Top: tYPeI alveolar epithelial cell is attenuated and has few
ing fraction is believed to retain the type II cell organelles and long cytoplasmic extensions to facilitate gas exchange
phenotype, thus replenishing the original stem cell with blood. The type II alveolar epithelial cell is cuboidal, metaboli-
population. Because no other cell type is known to be cally active, and contains storage organelles for pulmonary surfac-
tant. The two epithelial cell types are present in similar numbers, but
capable of giving rise to a type II cell (at least in the
the type I cell covers 95% of the alveolar surface area (27). Bottom:
adult lung), the daughter cell that retains the type II type II cell is the progenitor of both epithelial cell types. Division of a
phenotype must be capable of continued rounds of type II pneumocyte leads to daughter cells with distinctly different
division by reentering the cell cycle at an undetermined fates; one transforms into a type I cell, which is terminally differenti-
location. ated and incapable of division. The other retains the type II pheno-
type and replenishes the original stem cell population. No other stem
The requirement for continued rounds of division has cell contributes to alveolar epithelial population.
important implications regarding the overall capacity
of the epithelium for repair and for cell cycle kinetics,
both of which will be discussed. The factors that the present discussion will be limited to normal or
determine the fraction and locations of cells that go on preneoplastic cell kinetics in the normal or injured
to differentiate are the subject of intense investigation lung.
and likely include the composition of the underlying IDENTIFICATION OF THE STEM CELL AND ITS FATES
extracellular matrix (reviewed in Ref. 40). However,
this review will not cover AEC differentiation per se but By 1970, many investigators had noted greatly in-
will stress the quantitative aspects of the model and the creased numbers of cuboidal granular pneumocytes in
data upon which it is based. Considerable evidence the alveolar epithelium after lung injury induced by
suggests important roles for both AEC types in the oxygen (48, 54, 82) or during pneumonia (6, 19). The
repair of lung iniury and in fibrogenesis: for this reason, observation of primarily cuboidal epithelium in a wide

INVITED REVIEW LlO33
variety of lung injury models led to the suggestion that o--o TYPE 2
these cells might be involved somehow in repair of the W-0 TYPE 1
Q -0 UNDETERMINED
injury (19, 84). The first committment to the concept
that the type II pneumocyte was a stem cell was
published in 1969 by Kapanci et al. (55) after a careful
study of histological changes in the lungs of monkeys
exposed to 100% oxygen. The type I epithelium, which
was destroyed after 4 days of exposure to oxygen, was
replaced by cuboidal type II epithelium before the
recovery of functionally normal lung architecture upon
reexposure of the animals to normal room air. Impor-
tantly, the authors noted that, during the recovery
period, some of the cuboidal type II epithelial cells
exhibited thin cytoplasmic extentions, suggesting a
direct transformation of type II pneumocytes into type I
cells. This hypothesis was supported by the earlier
work of Bertalanffy and Leblond (12,13), who had used lhr Id 2d 36 4d Sd 6d 76 14 d
TIME AFTER INJECTION OF [~H]THYMIOINE
nuclear counting and the colchicine-induced meta-
phase arrest technique to study the cytodynamics of Fig. 2. Kinetics of alveolar epithelial cell transformation after lung
cell populations in the alveolar wall. Their work had injury by nitrogen dioxide. Data are the percentage distribution of
pulse-labeled cells in the alveolar epithelium with time after adminis-
demonstrated that the “granular” type II pneumocytes
tration of thymidine to rats exposed to 15-17 ppm NOZ. [Redrawn
had a much greater capacity for division and, interest- from Evans et al. (44).]
ingly, a shorter turnover time than did the “epithelial-
lining cells,” known today as type I cells. of the type II pneumocyte. It is important to note that
A more direct demonstration that type II cells trans- the data reflect only those cells that were both labeled
form into type I cells was obtained by the application of with tritiated thymidine and present in the epithelial
thymidine-labeling techniques coupled with electron layer. In lung sections obtained 1 h after labeling,
microscopy (EM). As discussed by Kauffman (62), the nearly all of the labeled cells in the epithelium (88%)
combination of the then newly refined autoradio- could be clearly identified by EM as type II, whereas
graphic techniques with the resolving power of EM ~1% of the labeled cells were identified as type I. About
permitted the positive identification of labeled cell 12% of the labeled cells were observed as- “undeter-
types within the heterogeneous lung parenchyma, a mined,” a term the authors used to describe cells that
task not easily accomplished in earlier studies relying were present in the epithelial layer but that were not
on light microscopy alone. Evans et al. (43, 45) were clearly recognizable as type I or type II. By 24 h after
among the first to use this improved approach to study pulse labeling, the number of labeled cells identified as
the alveolar epithelium; in work published in 1973, it type II had decreased to <60%, but the number ob-
was applied to lung sections obtained from adult rats served with indeterminate morphology had increased
exposed to 15-17 ppm NO2 for 48 h. The exposure was to almost 40%. By day 3, the indeterminate fraction
followed immediately by an intraperitoneal injection of had declined significantly, and the decrease was accom-
tritiated thymidine designed to achieve a “pulse” of panied by a reciprocal increase in the percentage of
available label. In lung sections obtained 1 h after labeled type I cells. Thereafter, the relative proportion
labeling, virtually all labeled cells in the alveoli were of labeled type II and type I epithelial cells remained
type II pneumocytes. By 24 h, however, labeled type I constant for up to 14 days after labeling.
cells began to appear, and, by 48 h after labeling, the A direct transition of type II cells to type I cells,
percentage of type I cells containing label had in- through an intermediate morph .ology reminiscent of
creased to nearly lo-fold that observed at 1 h. Over the both, is the most plausible interpretation of these data.
same time interval, the percentage of type II cells that The high percentage of labeled epithelial cells identifi-
were labeled fell to one-third the original value. As in able as type II (88%) immediately after labeling strongly
the earlier study by Kapanci et al. (55), EM revealed suggests that the type II, rather than some other cell, is
type II cells with morphological characteristics of type I the progenitor of all resulting epithelium. Although an
cells and vice versa; in the studies of Evans et al. earlier study by the same group had shown that
(43-45), however, these “transitional” cells often were nonepithelial cells in the alveolar wall also became
labeled with thymidine, lending additional support to labeled after NO2 exposure (45), it is extremely unlikely
the con.cept of a direct transition of the type II to the that some other unidentified stem cell could give rise to
type I cell associated with type II cell division. labeled type II cells in a span of only 1 h after the
Several years later, the same group published a injection of thymidine. Similarly, the possibility that
similar but more meticulous study (44) in which the the undetermined labeled cell population could have
time interval of data collection was lengthened and the been generated from a source other than type II cells
occurrence of transitional epithelial cells was more seems remote in light of the rapidity with which this
carefully monitored. Figure 2, taken from that report, population emerged (1 day), as well as its recurrent
is the most clear illustration of the progenitor function presence in locations and orientations consistent with

that of type II cells (43). In addition, the fact that the flattened morphology, and the average number of ex-
intermediate population was found to exhibit morpho- posed silver grains per nucleus was halved over the
logical characteristics of both type I and type II cells, 48-h interval. Again, the halving of nuclear grains and
but not of other cell types, argues against the possibil- the transition of label from cells of cuboidal to squa-
ity that a nonepithelial cell could give rise to type II mous morphology was consistent with a differentiation
cells. of type II cells to type I cells associated with cell
Similar studies of other models of lung injury or division. In a quantitative study of lung cell popula-
growth are less clear but nonetheless are consistent tions of mice exposed to urethan, Kauffman (60) noted
with the notion that type II cells give rise to type I cells. that both the type II and type I cell populations doubled
In a 1974 study of adult mice exposed to 90% oxygen, in the proliferative period immediately preceding the
Adamson and Bowden (1) also noted “intermediate” appearance of tumors. Although it was suggested that
cells exhibiting morphological characteristics of both these data were consistent with the stem cell function
type II and type I cells. These cells appeared immedi- of type II cells, that report by itself contributed little
ately after the point at which thymidine labeling of the additional evidence in support of that hypothesis.
alveolar epithelium became maximal, i.e., at 2 days Together, the studies discussed above supported the
after the termination of a 6-day exposure to oxygen. At concept that the type II pneumocyte is the sole progeni-
this same point in time, colchicine-induced mitoses tor of both AEC populations in the adult lung. As
were observed in the type II cell population, but neither discussed earlier, the rapidity with which type II cells
mitoses nor thymidine labeling were found in type I became labeled after a thymidine pulse (1 h; see Ref.
cells. By 3 days after exposure, the ratio of labeled type 44) argues against the presence of another unidentified
II to labeled type I cells had decreased from an initial progenitor cell for the epithelium. If such a population
value of 11:l to 1.4:1. This was accompanied by a existed, it likely would have been observed transform-
halving of the average number of exposed silver grains ing into type II cells in the careful EM studies of oxygen
per nucleus (in type II cells), which reached a value and NOa-induced lung injury (1, 43, 55). To date, such
equal to that observed in labeled type I cells. Together, an observation has not been reported.
these data supported the notion of a direct transition of In addition, related lines of evidence upheld the
type II cells to type I cells after oxygen-induced lung notion that type I cells must be derived from type II
injury. The decline of nuclear grain counts also sug- cells; one of these was the demonstration that type I
gested that this transition was associated with type II cells are incapable of division. Careful examinations of
cell division. the occurrence of mitotic figures in the alveolar wall
In the following year, the same authors investigated were unable to find evidence of mitosis in type I cells,
the pattern of lung cell labeling in the fetuses of despite the presence of abundant mitoses in neighbor-
pregnant rats injected with tritiated thymidine (2). The ing type II and other alveolar cells (1, 2, 12). This was
injections were administered 2 h before death ongesta- observed even after the administration of colchicine to
tional days 18-Z. Thymidine labeling of epithelial oxygen-injured animals to induce the accumulation of
cells was maximal at day 20, and over 50% of the mitotic nuclei (1). Indeed, before some of these works
labeled epithelial cells were cuboidal and vacuolated were published, Weibel (115) had described the ana-
and contained glycogen. Cells with a phenotype interme- tomic characteristics, which he referred to as “topologi-
diate between type II and type I were observed on this cal differentiation,” that intuitively would be expected
and subsequent days; mitotic figures were observed in to preclude the extremely attenuated type I cells from
type II and intermediate cells but not in squamous division. Another line of evidence came from the thymi-
epithelial cells. Because the animals were killed very dine labeling data itself. In short-term labeling experi-
shortly after the administration of thymidine, no signifi- ments (l-4 h), the type I cell population was not
cant labeling of type I cells was expected, nor was it labeled significantly, regardless of the lung injury model
observed. These data were consistent with the progeni- employed (1,44). However, in the longer pulse-labeling
tor function of type II cells in the fetal lung as well as in experiments in which thymidine labeling of type I cells
the adult. was observed (48 h-14 days), the labeling was associ-
Brody et al. (15) found similar evidence in studies of ated with the halving of nuclear grain counts (to values
compensatory growth of the lung after unilateral pneu- 50% of that in type II cells), consistent with the
monectomy in mice. After removal of the left lung, derivation of type I cells from the division of a parent
thymidine labeling of cells in the remaining lung tissue population (1, 15). Although a significant number of
became maximal at 6 days after lung resection, pro- thymidine-labeled type I cells were reported in studies
vided that the thymidine was administered immedi- of newborn rats (83), it has been speculated that this
ately before the animals were killed. In those animals, observation may have been erroneous due to technical
labeled cells in the alveolar wall were located primarily difficulties in distinguishing type I cells from capillary
in the alveolar corners and exhibited vacuoles, presum- endothelial cells in the preparations studied (62). Con-
ably lamellar bodies, when viewed by light microscopy. sidered together, studies of whole lung tissue in adult
In contrast, if the mice were injected with thymidine on animals overwhelmingly support the notion that the
day 6 but were not killed until 48 h later, labeled cells type I epithelium is derived from the type II cell.
were observed in the centers rather than the corners of This concept is supported also by more recent investi-
the alveolar wall. These labeled cells also had a more gations of primary isolates of type II pneumocytes. The

INVITED REVIEW L1035
first investigators to report the isolation of these cells

from the rat (67) noted that the cuboidal, lamellar Adult Rat
body-laden phenotype observed immediately after isola- Type II Cells
tion was transient. It was gradually replaced, over a
period of 3-10 days, by cells that were devoid of
organelles, flattened, and, at least under the micro-
scope, highly reminiscent of the morphology exhibited
by type I cells in vivo. On this basis, the authors
speculated that the cultured cells were undergoing a
FG d4 AG d4
phenotypic transition similar to that which occurs in DAY 4 \
the intact animal. This observation was confirmed detach on Day 4
/ I
many times in subsequent years (16, SS), leading to the
use of long-term cultures of primary type II cells as a
model of the type I phenotype in studies of ion transport
(21, 69), extracellular matrix metabolism (40, 90, 91), FG d8 AG d8 AG-FG
and other epithelial functions (98). Although this prac-
DAY 8
tice has been controversial (89), immunologic evidence
-*
r’lg. 3. In vitro evidence for transdifferentiation of alveolar epithelial
from two independent groups (32,38) has lent credence
to the underlying premise by demonstrating that pri- cells. Primary type II cells cultured on fibroblast feeder layer
overlying floating collagen gels (FG) retain type II phenotype, but
mary type II cells, with time in culture, eventually those overlying attached collagen gels (AG) differentiate into type I
become reactive to antibodies generated from type I cell cell-like phenotype (see Ref. 31). Differentiated cells on attached gels,
antigens. An earlier study of the lectin-binding profiles when subsequently detached (AG - FG), revert to type II-like
of primary AEC was consistent with these observations phenotype. d4, Day 4; d8, day 8. [Taken from Shannon et al. (95).]
(37). As more evidence of type I cell-specific functions is
found in long-term cultures of primary type II cells, the gels, the epithelial cells lost their lamellar inclusion
notion that cultured type II cells can differentiate into bodies and exhibited a flattened type I-like appearance;
type I, or at least type I-like, pneumocytes in vitro as this was accompanied by significantly reduced abun-
they do in vivo is gaining acceptance (86). dance of mRNAs for the surfactant apoproteins. When
However, the concept that cell division is required for the attached gels were detached and cultured for an
the differentiation of type II cells into type I cells is less additional 4 days, however, the epithelial cells regained
well supported. As discussed above, the fact that nuclear the ability to accumulate surfactant apoprotein mRNAs
grain counts in thymidine-labeled type I cells were and also regained lamellar inclusion bodies and cuboi-
one-half the value of those observed in type II cells after da1 morphology. In a later study employing similar
oxygen-induced injury (1) or pneumonectomy (15) cer- conditions of culture, Danto et al. (31) showed that the
tainly suggested that cell division had occurred. Fur- loss of type II cell characteristics that occurred on the
thermore, morphometric studies of type II cells after attached collagen gels was accompanied by increased
lung injury by butylated hydroxytoluene (101, 102) reactivity of the cells to antibodies raised against type I
suggested a link between mitosis and type II cell cell-specific antigens, supporting the notion that the
differentiation; in normal mice, the lamellar body area type II cells were differentiating into type I cells in this
per cell was found to decrease by ~80% between culture system. More importantly, when the attached
prophase and telophase (101). These data suggested a gels were subsequently detached as in the earlier study
cell cycle-dependent loss of lamellar bodies, specifically (95), the epithelial cells not only regained the type II
in mitosis. Both lines of evidence, however, were some- cell phenotype but also lost reactivity to the type I
what biased in that they included only those cells that cell-specific antibodies.
could be identified as mitotic or labeled with thymidine. These works suggest the possibility that the type I
The experimental designs thus precluded the analysis cell phenotype is plastic and is capable of reversion to
of quiescent cells, and so were incapable of examining the type II cell, at least in vitro. This interpretation of
the possibility that some type I cells might be derived the data relies on the assumption that cell turnover did
from unlabeled type II cells (or vice versa) through the not occur in the cell culture systems employed (31).
process termed “transdifferentiation” (l&92), i.e., with- However, the contribution of cell proliferation and/or
out undergoing division. death was not addressed experimentally in either
Recent studies suggest that AEC are in fact capable study. The data upon which this assumption was based,
of transdifferentiation, at least in vitro. In work pub- which suggest that primary type II cells are kinetically
lished in 1992, Shannon et al. (95) cultured primary immobile, will be discussed in KINETICS OF THE AEC
alveolar type II cells together with a fetal rat lung CYCLE; unfortunately, most of these data were derived
fibroblast feeder layer (see Fig. 3). The cells were from investigations of primary AEC cultured on sur-
cultured on rat tail collagen gels that were either faces other than fibroblast feeder layers and collagen
attached to the substratum, which promoted the loss of gels. For this reason, the alternative interpretation of
type II cell-differentiated characteristics, or on floating those experiments, i.e., selective turnover and amplifi-
collagen gels, which promoted the retention of those cation of pneumocyte subpopulations, was not strictly
characteristics. After 4 days on the attached collagen excluded (31, 95). Related observations were made in

an interesting investigation of the metabolism of ben- tion of tritiated thymidine and then were killed at
zo [alpyrene, in which Stoner et al. (105) cultured regular intervals for up to 12 h after injection. Figure
human lung explants for 25 days. On the 7th to 10th 4A, taken from that study, depicts the percentage of
days of culture, the alveolar epithelium was comprised mitotic figures labeled with thymidine within the type
primarily of type II cells rather than the expected II pneumocyte population. The schematic in Fig. 4B
normal mixture of squamous and cuboidal epithelium. displays the derivation of cell cycle parameters from a
This observation has been suggested to support the theoretically ideal cell population (94) analyzed by the
concept of reversion of type I cells to the type II same technique [the percent labeled mitosis (PLM)
phenotype (117); however, thymidine labeling of the method]. By analogy, the data clearly show that the
explants identified a significant percentage of labeled type II cell population had a duration of DNA synthesis
type II and bronchiolar cells, consistent with cell divi- (T,, or S phase duration) of -7.7 h and an approximate
sion. Unfortunately, mitoses in the epithelium were not duration of mitosis (actually G2 + 1/2M phases) of 1.8 h.
quantitated, and thus the origin of the cuboidal epithe- Although earlier studies had derived similar estimates
lium in the explants was not entirely clear. of the S phase duration in whole lung tissue (96, 97),
On the other hand, studies of fetal rat lung are these did not discriminate between cell types. It is
consistent with the possibility that the type I cell significant to note that the data of Evans et al. (Fig. 4A)
phenotype is plastic in vivo. Williams and Dobbs (117) could not provide a direct measure of the total cell cycle
showed that the reactivity of developing lung epithelial time (Tc in Fig. 4B) because the sampling was stopped
cells to a monoclonal antibody specific to type I cell at 12 h after the thymidine pulse, a time before that at
antigens occurred earlier in gestation than expected, which a second round of cell division could have begun.
significantly before the appearance of morphologically These data and the observed labeling index (LI = 32%)
mature type I epithelium. Conversely, the binding of a were, however, used subsequently by Kauffman (62) to
type II cell marker (Maclura pomifera lectin) also calculate an estimate of Tc (21-23 h) from the formula
occurred before the appearance of mature type II cells LI = TJT,.
but began after the appearance of the type I cell This estimate of the total Tc agreed well with that
marker. The latter observation argued against the obtained earlier for type II cells of the adult mouse
concept that mature type II cells are necessary precur- lung; in the previous year, Kauffman (59) had used
sors of type I cells in the fetal lung and led the authors
to speculate that the differentiation of the fetal alveolar
epithelium was a gradual process involving indetermi- A
nate cells of pluripotent capacity. Whether this is the
case in the adult lung is not yet known; the possibility
of type I cell reversion to type II cells has not been
assessed experimentally in an adult animal model but
neither is it precluded by any of the existing data.
Should this hypothesis be correct, the plasticity of the
type I cell phenotype would significantly affect the
overall capacity of the epithelium for repair, as well as
its kinetics.
KINETICS OF THE AEC CYCLE

-
Much of our knowledge of the cell cycle kinetics of 0 2 4 6 8 10 12 14
AEC, especially in vivo, came from the application of
what are now seldom-used techniques, such as nuclear HOURS AFTER INJECTION OF SH-TdR
counting and kinetic analyses of thymidine labeling.
Although in vivo labeling studies of lung tissue had
been performed before 1970, most of these did not
quantitate the epithelial cell cycle per se but were
concerned primarily with whole tissue kinetics and
turnover, topics that will be discussed in ALVEOLAR
EPITHELIAL TURNOVER IN VIVO. Nonetheless, by 1964
Bertalanffy (12) had used careful nuclear counting to T G2 + lt2M
gain an estimate of 50 min for the duration of mitosis in
Fig. 4. A: percent labeled mitosis (PLM) plot of type II pneumocytes
the alveolar walls. These measurements did not dis- after lung injury by nitrogen dioxide. Data are percentage of mitotic
criminate, however, between vacuolated (type II), non- type II cells labeled with thymidine vs. time after injection of label.
vacuolated (alveolar macrophage), and other alveolar Injection was given immediately after 48-h exposure of rats to 15-17
cells. One of the first direct measurements of the cell ppm NOz. 3H-TdR, tritiated thymidine. [Taken from Evans et al.
(43).] B: schematic of derivation of cell cycle phase durations from a
cycle of type II pneumocytes was reported in 1973 by
theoretically ideal PLM plot. Note that 2 rounds of cell division are
Evans et al. (43), who studied the alveolar epithelial required for measurement of the total cell cycle time (T,). [Taken from
response of rats exposed to 17 ppm NOa. After 48 h of Shackney and Ritch (94).] Ts, duration of S phase; TQ duration of Gz
exposure. the animals received an intraneritoneal iniec- chase.

grain-counting techniques to calculate an approximate duration of G2 phase + SM (1.5-1.9 h), the duration of
Tc of 21-24 h. In that study, normal mice and mice G1 phase (4.1-5.2 h), and Tc (12.3-15.5 h). Importantly,
treated with urethan were given intraperitoneal injec- sampling times were extended until the PLM plot
tions of radiolabeled thymidine, and counting was displayed a second maximum (see Fig. 4B), enabling
performed on type II cells within lung sections pre- the direct calculation of Tc and supporting the notion
pared by standard autoradiographic methods. The esti- that continuous cycling of the epithelium did occur in
mate of T, was calculated from the decline in the mean the fetal mouse model.
number of exposed silver grains per thymidine-labeled On the other hand, related studies of adult animal
nucleus (MGC); this index theoretically should de- models have produced no direct evidence of continuous
crease to 50% of its original value with each cell cycling by type II cells in the postnatal lung. Adamson
division (10). Two methods of calculation were used and and Bowden (1) and Brody et al. (15) both reported a
provided essentially identical estimates of Tc. The first halving of nuclear grain counts in type II cells within
was based on algorithms derived by Baserga et al. (10) the regenerating mouse lung. However, the decrease in
for similar studies of rapidly dividing Ehrlich tumor grain counts occurred at the expected early intervals
cells. In the second method, Tc was estimated as the after the administration of thymidine, but neither
time required for the MGC to decrease to two-thirds of group reported data at the longer sampling times
its original value. This approach, which had been necessary to document a second round of cell division.
applied to earlier analyses of bronchial epithelial cell In a study of butylated hydroxytoluene-induced lung
kinetics (58), relies on the assumption that the two injury in normal adult mice and beige adult mice (a
daughter cells resulting from each cell division have model of the Chediak-Higashi syndrome), Smith et al.
distinctly separate fates, i.e., that one daughter cell (102) showed that nuclear grain counts in type II cells
differentiates and does not divide again, whereas the remained constant for up to 4 days after the first cell
other immediately prepares for a second division. As division. That observation was contrary to the data of
shown in Fig. lB, this has been presumed to be the case Kauffman (59) discussed above and led the authors to
for type II pneumocytes in vivo. The notion that this conclude that sequential type II cell mitosis was not a
assumption was valid for type II cells was supported by significant occurrence in the normal adult mouse model.
the subsequent work of Evans et al. (44) but recently This disparity may be related to the theoretical limita-
has been brought into question by the in vitro work of tions and technical hardships inherent in autoradio-
Danto et al. (31) and Shannon et al. (Ref. 95 and see graphic grain-counting techniques, a topic that has
Fig. 3). Other assumptions underlying the two methods been reviewed (99).
just described are that 1) all labeled cells eventually However, more recent studies employing entirely
divide and 2) the role of cell death is negligible (10). The different methodology also failed to detect sequential
validity of these suppositions for the alveolar epithe- mitoses by type II cells in vivo. Continuously cycling
lium has not been explored rigorously. cell populations can be directly quantitated through
Other data in the report by Kauffman (59) were flow cytometric methods; an exemplary analysis of such
among the first to support the important premise that a population is shown in Fig. 5A. Proliferating adult rat
type II cells form a “continuously cycling” population. thymocytes in vivo were shown to complete S phase,
As discussed above (see Fig. 1, bottom), the daughter rapidly divide, and quickly enter a second S phase (8).
cell that retains the type II phenotype must be capable The second S phase is detected as the second maximum
of repeated rounds of division (continuous cycling), in a plot of time versus %S/B, i.e., the percentage of
provided that no other stem cell can give rise to type II cells that have incorporated bromodeoxyuridine (a thy-
cells. Consistent with that notion, Kauffman observed midine analog) and that exhibit S phase DNA content
that nuclear grain counts in type II cells fell to one- (for details of the technique, see Refs. 8, 39, 108). The
fourth their original value by 4 days after a thymidine timing and height of the second maximum afford
pulse, suggesting that more than one cell division had accurate measurements of the total Tc as well as the
occurred. Curiously, this was the case in normal adult fraction of daughter cells reentering the division cycle.
mice but not in those exposed to urethan to induce type In this example, -60% of the proliferating thymocytes
II cell proliferation. In the urethan-treated animals, entered a second S phase, the midpoint of which
the grain counts fell to only one-half their original occurred 10 h after the first S phase (a measure of T,).
value and remained at that level for up to 7 days after When the same analysis was applied to type II cells of
the administration of thymidine. The latter result was normal or pneumonectomized rats in vivo, no second
interpreted to indicate that urethan had prolonged the maximum of %S/B was observed for up to 30 h after
Tc of type II cells to a value longer than the interval of administration of the bromodeoxyuridine (Fig. 5B). If
study. That view was supported by reports that urethan the estimates of Tc obtained in earlier studies were
could inhibit both mitosis and S phase transit in vivo accurate (22 h, see above), a second maximum should
(26,57). In a later investigation of the fetal mouse lung have occurred at the 18- to 20-h interval of sampling.
(61), the same author used the PLM technique to show Details of the application of this method to AEC have
that Tc of peripheral lung epithelial cells increased with been discussed (108, 111, 113); one interpretation of
gestational age, consistent with findings in other fetal these results is the possibility that type II cells of the
tissues (77). The cell cycle phase durations, determined rat have a much longer T, than that suggested by
at 15-18 days gestation, were T, (6.3-7.9 h), the earlier estimates, most of which were derived from

INVITED REVIEW
tors had noted that primary isolates of type II cells did

not proliferate (as measured by cell number) without
experimental immortalization, particularly if the cells
were cultured at the relatively high plating densities
necessary for investigations of surfactant metabolism
or ion transport. Perhaps, for this reason, few kinetic
analyses have been performed on primary type II cells.
Furthermore, because the total cell number in these
preparations remains relatively constant with time,
many authors have assumed that primary type II cells
are kinetically static (31,95). The inability of these cells
to proliferate has been suggested to result from cell
cycle arrest in early S phase (24), possibly related to
unusually high rates of degradation of newly synthe-
sized DNA (23). On the other hand, several groups (46,
78, 106) have shown that primary type II cells are
capable of proliferation if they are cultured at sufi-
ciently low initial plating densities of at least lo-fold
lower than those in the works described above. More
recently, Kalina et al. (52) found that the capacity for
Time after BrdUrd injection (h) clonal growth in low-density culture was exhibited only
by a subpopulation of primary type II cells; it was
B speculated that this subgroup constitutes a small but
1 distinct cohort of committed stem cells within a much
larger and heterogeneous type II cell population.
Some of the kinetic data currently available support
the concept that type II cells are kinetically heteroge-
neous (see Table 1). In the intact animal and in culture,
60 AEC exhibit a relatively constant S phase duration of
7-9 h, regardless of the animal species, the lung injury
model, the density of cultured cells, or the method of
quantitation. In contrast, the duration of GJM phases
may be lengthened, especially in vitro at higher initial
5 /ioK 20 25 35
I Table 1. Qpe IIpneumocyte
cycle parameters
(TZP) cell
Hours
Ref.
Time after BrdU injection (hours) Model LI, % T, TG2YM Tc Method No.
Fig. 5. A: derivation of cell cycle parameters from flow cytometric In vivo

analyses of a continuously cycling population of adult rat thymocytes Fetal mouse, epithelium 35.0 7.3 1.0 14.5 TdRPLM 61
pulse labeled in vivo with bromodeoxyuridine (BrdUrd). Plot of 20.0 Calc
percentage of BrdUrd-positive cells exhibiting S phase DNA content Adult mouse 0.2 22.0 MGC 59
(PBUS) shows a second maximum as a fraction of the daughter cells Adult rat, NO2 repair 35.0 7.7 1.8 22.0 Calc 43
(-60%) enters a second S phase. This occurs at a time equal to Tc. Adult rat, normal 1.9 7.8 1.1 FACS 108
[Taken from Baron and Penit @).I B: flow cytometric analysis of type Adult rat, PNX 4.8 6.6 2.3 FACS 108
II pneumocytes pulse labeled with BrdUrd in vivo after left unilateral Adult rat, 02 repair 3.0 7.0 2.4 FACS UNP
pneumonectomy. Identical plot as that described in A (here %S/B) Type II cell adenoma 1.2 9.0 2.0 45.0 CL 41
yields no second maximum for up to 30 h after labeling. [From Uhal In vitro
(108); printed with permission by Wiley.] Primary rat
T2P, Oczy 0 2.3 13.8 11.9 FACS 108
T2P, Dczy 2 5.5 9.9 10.4 FACS 108
mouse lung models. Alternatively, continuous cycling of
Low density 7.7 7.9 2.1 FACS UNP
AEC may occur only when the need for cell replacement A549 cell line 18.9 7.3 FACS UNP
is more severe than that of the normal or slowly
regenerating rat lung models used in Fig. 5. Other LI, labeling index; T,, duration of DNA synthesis; T,, cell cycle
time; TdR PLM, percent thymidine-labeled metaphases; Calc, calcu-
interpretations will be discussed in ALVEOLAR EPITHE-
lation by formula Tc = T,/LI; MGC, mean grain count of thymidine
LIAL TURNOVER IN VIVO; in any case, the data supporting incorporation; FACS, flow cytometry and bromodeoxy uridine incorpo-
the concept of the type II cell as a continuously cycling ration; PNX, remaining lung after left unilateral pneumonectomy; 02
population in vivo are not overwhelmingly convincing. repair, 100% oxygen for 55 h + 96 h recovery in room air (from Ref.
17); CL, continuous labeling with tritiated thymidine; day 0, day of
Unfortunately, isolated AEC in culture have been of cell isolation; day 2, 2 days in primary culture; low density, primary
limited utility as models for kinetic studies. As dis- T2P, day 4 of culture at 1 X 104/cm2 on tissue culture plastic; UNP,
cussed in an earlier report (111), numerous investiga- unpublished data, B. D. Uhal.

plating densities. The accuracy of the delayed GZ/M rates and other common characteristics (42, 73). For
phase transit times obtained by flow cytometric deter- example, in the tongue and intestinal epithelia, rapid
mination of cellular DNA content (Ref. 108 and unpub- stem cell proliferation ensures immediate replacement
lished observation; Table 1) are supported by the of lost cells, and excess cells are desquamated from the
observation that binucleated cells accumulated in pri- apical surface. Kauffman (62) compared the known
mary cultures seeded at the same initial plating den- kinetic behavior of the alveolar epithelium with that of
sity (74). Curiously, type II cells that complete S phase other cell populations and concluded that type II cells
in vitro or in the regenerating lung display a wide range did not satisfy the criteria for designation as a renewal
of GB/M transit times (108), suggesting the existence of population. Foremost among these was the criterion
pneumocyte subpopulations that differ in susceptibility that the rate of cell proliferation (as determined by the
to G2/M phase arrest. To date, the mechanisms or daily mitotic rate) would be greater than that required
potential physiological significance of this behavior is to maintain daily growth of the tissue, leading to the
unknown. continuous removal of excess cells.
The number of total alveolar cells required for daily
growth of the rat lung (essentially zero for the adult
ALVEOLAR EPITHELIAL TURNOVER IN WO
human lung) had been determined by Enesco and
By 1953, careful application of the metaphase arrest Leblond (42), who estimated the daily rate of nuclear
technique had yielded estimates of the rate of turnover addition to the growing rat lung (0.47%/day) from the
of cells within the alveolar walls. Bertalanffy and increase in DNA content of the lung and the known
Leblond (13) had used light microscopy to discriminate DNA content per cell nucleus. Bertalanffy and Leblond
“vacuolated” (probable type II) and “nonvacuolated” (13) obtained a similar figure of O.l9%/day specifically
cells (probable macrophages and leukocytes) in the for the alveolar epithelium; this was calculated from
alveoli of adult rats and had quantitated the propor- the daily rate of gain in lung mass (0.91%) corrected for
tions of the various cell types present as well as the the fraction of that mass (21%) comprised by the
number of metaphases that accumulated in each at vacuolated alveolar cells (probable type II). In this and
regular intervals (6 h) after administration of colchi- a later study (12), the daily mitotic rate for the vacu-
tine. In this and a later report, Bertalanffy (12) com- olated alveolar cells was measured by the metaphase
piled the data obtained at 6-h intervals to derive a arrest technique to be 0.89%, significantly greater than
“daily mitotic rate,” i.e., the percentage of vacuolated the 0.19% required for tissue growth. On this basis, the
alveolar cells undergoing mitosis per day, of 3.4-3.7%. alveolar epithelium was classified as a renewal popula-
These data yielded estimates of 29-27 h, respectively, tion, with a daily mitotic rate -0.7%, or threefold, in
for the “renewal” or “turnover time,” the time required excess of that required for tissue growth. As discussed
for cell division by 100% of the vacuolated alveolar cell thereafter by Kauffman (62), however, the fraction of
population. The nonvacuolated cell population, which that excess that was required to offset the spontaneous
had a significantly higher daily mitotic rate, was calcu- death of type I or type II cells was unknown by 1980,
lated to have a turnover time of only 8 h (13). and it remains unknown to this day. The designation of
Those data were in good agreement with studies of the alveolar epithelium as a renewal population there-
the mouse lung that were published nearly fifteen fore remains tentative, at least in comparison with
years later. Bowden et al. (14) administered tritiated those renewal epithelia in which vast numbers of
thymidine to normal adult mice and quantitated the excess cells are continually shed.
decline in MGC (see KINETICS OF THE AEC CYCLE) over The idea that AEC also might be shed continuously
time in labeled nuclei within the alveolar walls. Al- was proposed by Bertalanffy and Leblond (13), who
though the authors did not discriminate between cell speculated that the “free alveolar cells” first noted by
types, the MGC plots displayed a bimodal decline, Macklin (76) might actually be excess AEC migrating
which was interpreted as evidence for two kinetically up the airways for excretion with tracheal secretions.
distinct cell populations with turnover times of 7 days This theory was supported by later studies of the
versus 28-35 days. It was postulated that the bimodal cellular fractions of the mucus layer overlying the
data reflected the mesenchymal and epithelial cell bronchial and tracheal epithelium (12); the mucus
populations, respectively. Together, these estimated layer contained numerous cells that were speculated to
turnover rates for the epithelia of the normal lung were reflect alveolar desquamation. However, little evidence
very different from those obtained from models of lung was available to support that contention, and positive
injury; an epithelial turnover time of only 3 days was identification of the free cells was not possible at the
obtained when the same experimental techniques were resolution of the light microscope. EM of bronchoalveo-
applied after exposure of mice to 90% oxygen (1). lar lavage samples, which were collected after experi-
The relatively long estimates of cell turnover in the mental lung injury by 90% oxygen (Ref. 1, see KINETICS
normal lung led to the contention that the alveolar OF THE AEC CYCLE), failed to detect thymidine-labeled
epithelium was one of the more slowly proliferating type II pneumocytes and argued against the occurrence
pulmonary cell populations (13, 14). That finding was of detectable shedding. On the other hand, EM of lung
in contrast to the epithelia of other organs, many of tissue injured by NO2 (43) or hyperoxia (55) revealed
which had been classified as “renewing” cell popula- dividing type II or transitional cells (intermediate
tions on the basis of much higher basal proliferative between type II and type I) that were partially covered

LlO40 INVITED REVIEW
by “flaps” of adjacent type I cells, suggesting that the that required for repair, as well as the mechanisms of
type I cells were in the process of sloughing off the its removal from the lung, are unknown at present.
basement membrane. More recently, significant num- The determination of whether an excess of type II cell
bers of type II pneumocytes were observed in bronchoal- proliferation does in fact exist requires knowledge of
veolar lavage samples from patients with acute onset of the rate of cell loss. Unfortunately, no kinetic studies to
the adult respiratory distress syndrome (104). It was date have considered the cell loss factor, or phi, as it
unclear, however, whether the cells were released by relates to the normal or injured alveolar epithelium in
underlying damage to the interstitium, by a disruption vivo. This may be due to the difficulty in making such
of the basement membrane, or whether they were measurements accurately in a cell population of rela-
simply shed as excess stem cells. Many of the lavage tively low abundance. Cell loss has been measured,
pneumocytes exhibited a morphology reminiscent of however, in epithelial-derived lung tumors. In a study
adenocarcinoma cells, including prominent nucleoli of urethan-induced adenocarcinomas in mice, Dyson
and nuclear membrane irregularities; this observation and Heppleston (41) used continuous labeling with
lends credence to the hypothesis that they were excess tritiated thymidine to show that the rate of cell loss was
stem cells rather than quiescent epithelia lost by 83-95% of the rate of cell birth (KB) in the tumor.
desquamation. Furthermore, because both of these indexes varied in
Although the premise that the alveolar epithelium is parallel with tumor age, it was proposed that the rates
continually shed remains controversial, two lines of of epithelial cell loss and birth were linked mechanisti-
reasoning support the notion that an “excess” of alveo- cally. Thaete and Malkinson (107) later demonstrated
lar stem cells can exist in both the normal and injured that urethan-induced tumors in mice are either solid or
lung. First, the estimates of the daily mitotic rate of papillary and that these tumor types are derived from
0.89% for the vacuolated epithelium (see above discus- type II pneumocytes or Clara cells, respectively. Al-
sion) depended on the detection of arrested metaphases though the work of Dyson and Heppleston (41) did not
within cells that clearly contained organelles (presum- distinguish tumor morphology, it is likely that some of
ably lamellar bodies) visible by light microscopy (12, the adenomata that they studied was of type II cell
13). Subsequent EM studies showed that type II cells origin. Regardless, the continuous labeling studies
undergoing mitosis, on average, lose -40% of their indicated the importance of the rate of cell loss in
determining the growth rate of peripheral lung carcino-
lamellar body volume in the interval between prophase
mas and underscore the importance of understanding
and metaphase and up to 90% by telophase (101). Thus
cell loss under non-neoplastic conditions as well.
the scoring of only those cells that contained vacuoles
As discussed above, little conclusive evidence is
may well have underestimated the number of arrested
available to suggest that shedding is an important
metaphases in the epithelium. Furthermore, the meta-
mechanism of AEC loss. An alternative fate for excess
phase arrest (stathmokinetic) technique employed in
cells, however, is programmed cell death, and recent
those measurements relies on the assumptions that 1) evidence suggests that apoptosis is an important fea-
all cells that reach metaphase are stopped by the ture of the normal and injured alveolar epithelium.
arresting agent, in this case colchicine, and 2) that cells Studies of epithelial-mesenchymal interaction have
arrested in metaphase are not lost to cell death (34). demonstrated that both primary type II cells and a
Although neither of these assumptions has been vali- human AEC line are capable of spontaneous and induc-
dated for the alveolar epithelium, significant loss of ible apoptosis in vitro (112); programmed cell death was
other cell types after metaphase arrest by colchicine or inducible by factors released from lung fibroblasts. The
other stathmokinetic agents is known to occur in vivo stimulatory fibroblasts were isolated from fibrotic lung
(5). For the se reasons, if the estimated daily mitotic specimens (88, 112), suggesting that regulated cell
rates of the type II epithelium were in error, they were death in the epithelium might be important in pulmo-
likely to have been underestimated. nary fibrosis. Consistent with that notion, fragmented
Second, a wide variety of insults to the lung can lead DNA was detected in significant numbers of AEC
to a hyperplastic response in which seemingly excess within thin sections of fibrotic human lung but not in
numbers of cuboidal type II cells (together with bron- sections of normal human lung (72).
chial cells) populate an epithelium devoid of type I cells In the liver, an increase in the percentage of apoptotic
(7, 56, 63, 93). This phenomenon has been termed the hepatocytes from a basal level of 0.1% to a value of
“cuboidal epithelium”, a common feature of the fibrotic 2-3% is sufficient to result in the loss of 25% of total
lung (31,93). Although the mechanisms that lead to the liver mass in as little as l-2 days (18). These data
cuboidal epithelium are poorly understood, it has been reflect the extremely rapid kinetics of the apoptotic
suggested that the cuboidal cells are stem cells that are process (mean duration 3 h) and show that a seemingly
rapidly proliferating in an attempt to repair the de- low incidence of apoptotic cells within a given cell
nuded epithelium but which are prevented from doing population can indicate considerable cell loss over time.
so by an inability to differentiate into type I cells (79). Consistent with that notion, the type II cell-like A549
In healing fibrotic lung tissue, some fraction of these human lung carcinoma cell line underwent significant
cells presumably goes on to differentiate into type I net decreases in total cell number and DNA content
cells to restore the normal epithelium. However, the upon induction of apoptosis (see Fig. 6), despite a low
percentage of the cuboidal stem cells that is in excess of abundance (~1%) of detectible apoptotic nuclei in the
which was speculated to contribute to the inability of

primary isolates of AEC to proliferate (23). Those
observations are consistent with the premise that
primary AEC manifest a relatively high basal rate of
apoptosis in vitro and suggest that programmed cell
death plays an important role in AEC kinetics. Consid-
ered together, the cell culture data and whole animal
studies imply that more attention should be paid to the
2 role of apoptosis and the rate of cell loss in the
E 1 A549 T AEC
homeostasis ofAEC in vitro and in vivo.
A NEW PERSPECTIVE, 1997
On the basis of the available data to date, the

following view is offered as a working model of alveolar
epithelial kinetics in the adult lung. As depicted in Fig.
NH FHPF N-R PA 7, nearly all type II cells in the normal lung are
TYPE OF MEDIUM quiescent (Go phase) and must traverse G1 phase and
Fig. 6. Steady-state cell number and DNA content of confluent
undergo balanced cell growth (33, 35, 110) before
cultures of alveolar epithelial cells (AEC) exposed to fibroblast- beginning DNA synthesis. Cell division leads to two
conditioned media. A549 human lung carcinoma cell line (left) and relatively undifferentiated daughter cells, both of which
primary AEC (right) were exposed for 20 h to serum-free media have the potential to rapidly revert to the type II cell
conditioned by fibroblasts isolated from normal [normal human (NH) phenotype or to differentiate into type I cells. Either
and normal rat (NR)] or fibrotic [human with idiopathic pulmonary
fibrosis (HIPF) and subjects treated with paraquat (PA)] human or
rat lung, respectively. Cell number and DNA content are expressed as
a percentage of that present at the beginning of incubation. Nos. e%
:-:,-.
-,:.
- e
..‘I.
above bars are the % change in DNA of cultures
NR-conditioned media. *P < 0.05 compared
exposed
with NH or NR media by
to NH- or
il _
cl.:.-:-.‘
,-_.
. GO
Student’s t-test. [Taken from Uhal et al. (112).]
?+ 1
stimulated cells (112). The A549 cell line is believed to
have originated from a type II cell-derived human lung f&3@ Gl
carcinoma (100); in mouse lung carcinomas, Dyson and

Heppleston (41) noted “isolated” karyorrhectic cells in
the apparent absence of necrosis. This observation led
the authors to suggest that the high rate of cell loss in
01 ----.
0
-.
.;... '.-I. .d
...::... ....*y,.,
--
*:...*...
.._.
..-.'-.--...-- .
,..-*..
.
. . a.
-....:.
I...
.
-:-
l .:
S
G2
the tumors (83-95% of KB) might be due to pro-

grammed cell death, which only a short time earlier
had been termed “apoptosis” by Kerr et al. (66). More
recently, Gaffney et al. (47) quantitated the apoptotic
indexes of a variety of human lung carcinomas (mean
values of 1.3% for adenocarcinomas) with in situ end
labeling of fragmented DNA, a method that correlated
well with light microscopic and ultrastructural
tions by EM.
observa- 1
When compared with the A549 cell line, primary
cultures of AEC underwent greater net loss of cell
number and total DNA upon stimulation of apoptosis
(see Fig. 6). This was the case particularly in cells
studied at day 4 of primary culture, a time at which
they manifest a type I cell-like phenotype (32). The
primary cells also exhibited a significantly higher inci- 1 1
dence of karyorrhectic cells under light microscopy; in
the absence of serum, ~3% of unstimulated primary 1 -cl@=@
c>
.--
...a.
-,.*.a:..
.-.**-*::;-;
.,:.:-
.-.-. LII+ apoptosis
AEC could be detected as apoptotic, and nearly 15%
were apoptotic after stimulation (112). Furthermore,
these values may have been underestimated by the Fig. 7. Alveolar epithelial stem cell function in the adult lung, 1997.
detection method employed, which relied on the obser- Type II pneumocyte is the sole progenitor of both alveolar epithelial
cell types. Cell division leads to 2 undifferentiated daughter cells that
vation of fragmented nuclei and thus did not include
can assume either mature phenotype. Tranformation of type II to
cells in the early stages of apoptosis (119). Earlier work type I cells is reversible, but reentry into the cell cycle requires
had shown that adult rat type II pneumocytes exhib- reversion to the type II phenotype. Both epithelial cell types have the
ited an unusually high basal rate of DNA degradation, potential for programmed cell death.

phenotype may transform to the other without cell subset constitutes a subpopulation of committed stem
division, although reentry into the cell cycle requires cells, such as those proposed by Kalina et al. (52), also is
reversion to the type II cell. Either differentiated unknown.
phenotype can be lost, not only by toxic ins ult and Although the phenomenon of continuous cycling
necrosis bu .t also through end .ogeno usly induced apopto- would be expected in pathological conditions involving
sis, the contribution of which to in vivo cell kinetics is type II cell hyperplasia, it was not observed in the
presently unknown. preneoplastic proliferative period induced by urethan,
The model of 1980 has been supplemented, but its which led to the speculation that the total T, was
basic structure is unchanged: no evidence has accumu- variable (59). In the light of more recent work, an
lated to refute the notion that the type II pneumocyte is alternative interpretation is the possibility that continu-
the sole precursor of all AEC in the adult lung. Al- ous cycling of type II cells might be invoked only when
though the type I cell may be capable of reversion to the the reserve of stem cells is depleted below some critical
type II in vivo (albeit the bulk of the evidence to date is level. If type I cells are indeed capable of reversion to
from in vitro studies), no evidence for division by type I the type II phenotype in vivo as they appear to be in
cells has been reported. As discussed by Witschi (llS), vitro (31, 95), that critical level would be determined
lung injuries that damage type I cells, such as butyl- not by the number of surviving type II cells but rather
ated hydroxytoluene injury (65), are generally repa- by the total number of viable AEC and the rate of type I
rable, whereas injuries that kill both type I and type II to type II transdifferentiation. Although the composi-
cells [such as that induced by paraquat (64)] are often tion of the underlying extracellular matrix (40, 90) is
fatal, presumably as a result of the destruction of the believed to be a very important determinant of the
only available stem cell population. Thus the bulk of phenotypic transition between type II and type I cells
the available data from animal models continues to (and vice versa), the kinetics of type I cell “reversion,”
support the premise that only the type II cell can the factors controlling it, and its relationship to AEC
function as the stem cell for alveolar repair. - On the turnover have yet to be evaluated.
other hand, infection of mice with the influenza virus One physiological context in which these issues are
PR8-A caused marked destruction of both type I and likely to be critical is in the pathogenesis of pulmonary
type II epithelium, but the subsequent repair of alveo- fibrosis. In human lung specimens, fibroblastic foci
lar lesions appeared to occur through proliferation of colocalize with regions of unrepaired or cuboidal epithe-
metaplastic bronchial epithelial cells rather than by lium (71), which suggests a mechanistic relationship
type II cell proliferation (75). Although bronchial epithe- between inefficient alveolar epithelial repair and the
lial cells have been observed together with type II proliferation of underlying fibroblasts. Studies of mouse
pneumocytes in the cuboidal epithelium of the fibrotic lung explants (4) or murine whole lung tissue (3)
human lung (63), the relative contributions of bronchial obtained after varying degrees of epithelial injury by
versus type II alveolar cells in the repair of human lung hyperoxic gas were among the first to support the
damage is largely unknown. contention that healthy reepithelialization was essen-
In addition, much is still to be learned about the cell tial for normal healing free of fibrosis. Subsequent
cycle kinetics of type II cells. The current model implies studies by Haschek and Witschi (50) and Haschek et al.
that the type II cell must form a continuously cycling (49) found that hyperoxia could potentiate the lung
population under non-neoplastic conditions, but data in damage induced by butylated hydroxytoluene and could
support of that concept are scant. For this reason, the induce fibrotic changes that persisted for up to one year.
point at which most daughter cells reenter the cell cycle This work clearly demonstrated that the severity of
is undetermined (Fig. 7). Recent work supports the fibrotic lesions in the mouse lung was inversely related
concept that most type II cells in the normal lung reside to the efficiency of alveolar repair. For these reasons, it
in a distinct quiescent metabolic state that is distin- is tempting to speculate that type II cell proliferation
guished by reduced size and low protein-to-DNA and and differentiation are likely to be critical factors in the
RNA-to-DNA ratios (110). These ratios are analogous to pathways leading to fibrogenesis in the human lung as
those found in the G,o subpopulation of lymphocytes well. As discussed above, the cuboidal epithelium of the
defined by Darzynkiewicz et al. (35); when G,o lympho- fibrotic lung is believed to be the result of type II cell
cytes progress further into the G1 phase of the cell cycle, hyperplasia in conjunction with faulty differentiation
they grow larger as they accu .mulate RNA and protein to type I cells (79). If the hypotheses discussed above
before DNA synthesis. Type II cells that are both are correct, continuous cycling of type II cells would
hypertrophic and of elevated RNA-to-DNA and protein- likely be observed in a cuboidal epithelium devoid of
to-DNA ratios emerge after lung injury by silica (80,87) type I cells but desperately attempting to replace them.
or during compensatory lung growth after pneumonec- Animal models of fibrotic lung injury may thus prove
tomy (110). These large cells are committed to DNA useful in future investigations of the interrelationships
synthesis (87,110) and are found in numbers as high as between cell proliferation, differentiation, and death in
30% of total recoverable type II cells. Continuously the alveolar epithelium.
cycling cell populations generally reenter the G1 phase
I gratefully acknowledge Ramona Jefferies for help with manu-
after mitosis (8), consistent with the hypothesis that script typing.
the hypertrophic type II cells might be a subset of This work was supported by National Heart, Lung, and Blood
cvcling pneumocvtes. However, whether or not this Institute Grant HL-45136.

Address for reprint requests: B. D. Uhal, Lung Cell Kinetics 23. Clement, A., N. Riedel, and J. S. Brody. [“Hlthymidine
Laboratory, Dept. of Medicine, Pulmonary Div., Michael Reese Hospi- incorporation does not correlate with growth state in cultured
tal, 2929 S. Ellis Ave., Rm. 405KND, Chicago, IL 60612. alveolar type II cells. Am. J. Respir. Cell MOL. BioZ. 3: 159-164,
1990.
REFERENCES 24. Clement, A., M. Steele, J. S. Brody, and N. Riedel. Growth-
related gene expression in type 2 alveolar epithelial cells. Am.
1. Adamson, I. Y. R., and D. H. Bowden. The type 2 cell as Rev. Respir. Dis. 142: S60-S62, 1990.
progenitor of alveolar epithelial regeneration. A cytodynamic 25. Clements, J. A. Surface tension of lung extracts. Proc. Sot.
study in mice after exposure to oxygen. Lab. Invest. 30: 35-42, Exp. BioZ. Med. 95: 170-172,1957.
1974. 26. Cornman, I. The properties of urethane considered in relation
2. Adamson, I. Y. R., and D. H. Bowden. Derivation of type 1 to its action on mitosis. Int. Reu. Cytol. 3: 113-130,1954.
and epithelium from type 2 cells in the developing rat lung. Lab. 27. Crapo, J. D., B. E. Barry, P. Gehr, M. Bachofen, and E. R.
Invest. 32: 736-745,1975. Weibel. Cell number and cell characteristics of the normal
3. Adamson, I. Y. R., and D. H. Bowden. Pulmonary injury and human lung. Am. Rev. Respir. Dis. 126: 332-337,1982.
repair. Organ culture studies of murine lung after oxygen. Arch. 28. Crapo, J. D., S. L.Young, E. K. Fram, K. E. Pinkerton, B. E.
Pathol. Lab. Med. 100: 640-643,1976. Hbarry, and R. 0. Crapo. Morphometric characteristics of
4. Adamson, I. Y. R., L. Young, and D. H. Bowden. Relation-
cells in the alveolar region of mammalian lungs. Am. Rev.
ship of alveolar epithelial injury and repair to the induction
Respir. Dis. 128: S542-S546,1983.
ofpulmonary fibrosis. Am. J. PathoZ. 130: 377-383,1988.
29. Crouch, E. C., A. Persson, G. L. Griffin, D. Chang, and
5. Aherne, W.A., and R. S. Camplejohn. On correcting the error
R. M. Senior. Interactions of pulmonary surfactant protein D
due to metaphase degeneration in stathmokinetic studies. Exp.
@P-D) with h uman blood leukocytes. Am. J. Respir. Cell MOL.
Cell Res. 74: 496-501,1972.
BioZ. 12: 410-415,1995.
6. Alley, M. R., and B. W. Matelow. Alveolar epitheliazation in
30. Crystal, R. G., V. J. Ferrans, and F. Basset. Biologic basis of
ovine pneumonia. J. Pathol. 103: 219-224,197l.
pulmonary fibrosis. In: The Lung: Scientific Foundations, edited
7. Aso, Y., K. Yoneda, and Y. Kikkawa. Morphologic and bio-
by R. G. Crystal and J. B. West, 1991, chapt. 7.8.1, p. 2031-
chemical study of pulmonary changes induced by bleomycin in
2046.
mice (Abstract). Lab. Invest. 35: 558, 1976.
31. Danto, S. I., J. M. Shannon, Z. Borok, S. M. Zabski, and
8. Baron, C., and C. Penit. Study of the thymocyte cell cycle by
E. D. Crandall. Reversible transdifferentiation of alveolar
bivariate analysis of incorporated bromodeoxyuridine and DNA
epithelial cells. Am. J. Respir. Cell MoZ. BioZ. 12: 497-502,1995.
content. Eur. J. Immunol. 20: 1231-1236,199O.
32. Danto, S. I., S. M. Zabski, and E. D. Crandall. Reactivity of
9. Barton, W. W., S. Wilcoxen, P. J. Christensen, and R.
alveolar epithelial cells in primary culture with type I cell
Paine. Disparate cytokine regulation of ICAM- in rat alveolar
monoclonal antibodies. Am. J. Respir. CeZZ MoZ. BioZ. 6: 296-
epithelial cells and pulmonary endothelial cells in vitro. Am. J.
306,1992.
PhysioZ. 269 (Lung Cell. MOL. Physiol. 13): L127-L135,1995.
33. Darzynkiewicz, Z., T. Sharpless, L. Staiano-Coico, and
10. Baserga, R., S. A. Tyler, and W. E. Klisieleski. The kinetics
M. R. Melamed. Subcompartments of the Gl phase of cell cycle
of growth of the Ehrlich tumor. Arch. Pathol. 76: g-13,1963.
detected by flow cytometry. Proc. NatZ. Acad. Sci. USA 77:
11. Beresford, W. A. Direct transdifferentiation: can cells change
their phenotype without dividing? Cell Differ. Dev. 29: 81-93, 6696-6699,198O.
1990. 34. Darzynkiewicz, Z., F. Traganos, and M. Kimmel. Assay of
12. Bertalanffy, F. D. Respiratory tissue: structure, histophysiol- cell cycle kinetics by multivariate flow cytometry using the
ogy, cytodynamics. II. New approach and interpretation. Int. principle of stathmokinesis. In: Techniques in CeZZ Cycle Analy-
Rev. Cytol. 17: 213-297,1964. sis, edited by J. W. Gray and Z. Darzynkiewicz. Clifton, NJ:
13. Bertalanffy, F. D., and C. P. Leblond. The continuous re- Humana, 1987, p. 291-336.
newal of the two types of alveolar cells in the lung of the ratl. 35. Darzynkiewicz, Z., F. Traganos, T. Sharpless, and M. R.
Anat. Rec. 115: 515-536,1953. Melamed. Lymphocyte stimulation. Proc. NatZ. Acad. Sci. USA
14. Bowden, D. J., E. Davies, and J. P. Wyatt. Cytodynamics of 73: 2881-2884,1976.
pulmonary alveolar cells in the mouse. Arch. PathoZ. 86: 867- 36. Dobbs, L. G. Pulmonary surfactant. Annu. Reu. Med. 40:
870,1968. 431-446,1989.
15. Brady, J. S., R. Burki, and N. Kaplan. Deoxyribonucleic acid 37. Dobbs, L. G., M. C. Williams, and A. E. Brandt. Changes in
synthesis in lung cells during compensatory lung growth after biochemical characteristics and pattern of lectin binding of
pneumonectomy.l-3. Am. Reu. Respir. Dis. 117: 307-316,1978. alveolar type II cells with time in culture. Biochim. Biophys.
16. Brody, J. S., and M. C. Williams. Pulmonary alveolar epithe- Acta 846: 55-66, 1985.
lial cell differentiation. Annu. Rev. Physiol. 54: 351-371,1992. 38. Dobbs, L. G., M. C. Williams, and R. Gonzalez. Monoclonal
17. Bui, K. C., S. Buckley, F. Wu, B. D. Uhal, I. Joshi, J. Liu, M. antibodies specific to apical surfaces of rat alveolar type I cells
Hussain, I. Makhoul, and D. Warburton. Induction of A and bind to surfaces of cultured, but not freshly isolated, type II
D-type cyclins and cdc2 kinase activity during recovery from cells. Biochim. Biophys. Acta 970: 146-156,198s.
short term hyperoxic lung injury. Am. J. Physiol. 268 (Lung 39. Dolbeare, F., H. Gratzner, M. G. Pallavicini, and J. W.
Cell. Mol. Physiol. 12): L625-L635,1995. Gray. Flow cytometric measurement of total DNA content and
18. Bursch, W., S. Patte, B. Putz, G. Barthel, and R. Schulte- incorporated bromodeoxyuridine. Proc. NatZ. Acad. Sci. USA
Hermann. Determination of the length of the histological 80: 5573-5577,1983.
stages of apoptosis in normal liver and altered hepatic foci of 40. Dunsmore, S. E., and D. E. Rannels. Extracellular matrix
rats. Carcinogenesis 11: 847-853, 1990. biology in the lung. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol.
19. Carrington, C. G., and T. J. Green. Granular pneumocytes in 14): L3-L27,1996.
early repair of diffuse alveolar injury. Arch. Intern. Med. 126: 41. Dyson, P., and A. G. Heppleston. Cell kinetics of urethane-
464-465,197O. induced murine pulmonary adenomata: II. The growth fraction
20. Chaucey, J. G., G. M. Peters, and R. H. Simon. Archidonic and cell loss factor. Br. J. Cancer 33: 105-111,1976.
acid metabolism by rat alveolar epithelial cells. Lab. Invest. 58: 42. Enesco, M., and C. P. Leblond. Cell proliferation in the
133-140,1988a. mammalian lung. J. Embryol. Exp. MorphoZ. 10: 530,1962.
21. Cheek, J. M., M. J. Evans, and E. D. Crandall. Type I 43. Evans, M. J., L. J. Cabral, R. J. Stephens, and F. Freeman.
cell-like morphology in tight alveolar epithelial monolayers. Renewal of alveolar epithelium in the rat following exposure to
Exp. CeZZ Res. 184: 375-387,1989. NOz.Am. J. Pathol. 70: 175-190,1973.
22. Cheek, J. M., K. J. Kim, and E. D. Crandall. Tight monolay- 44. Evans, M. J., L. J. Cabral, R. J. H. Stephens, and G.
ers of rat alveolar epithelial cells: bioelectric properties and Freeman. Transformation of alveolar type 2 cells to type 1 cells
active sodium transport. Am. J. Physiol. 256 (CeZZ Physiol. 25): following exposure to N021,2. Exp. MoZ. Pathol. 22: 142-150,
C688-C693.1989. 1975.

45. Evans, M. J., R. J. Stephens, L. J. Cabral, and F. Freeman. 67. Kikkawa, Y., and K. Yoneda. The type II epithelial cell of the
Cell renewal in the lungs of rats exposed to low levels of NOZ. lung. I. Method of isolation. Lab. Invest. 30: 76-82,1974.
Arch. Environ. Health 24: 180-188,1972. 68. Kim, K. J., J. Cheek, and E. D. Crandall. Contribution of
46. Everett, M. RI., R. J. King, M. B. Jones, and H. M. Martin. active Na+ and Cl- fluxes to net ion transport by alveolar
Lung fibroblasts from animals breathing 100% oxygen produce epithelium. Respir. Physiol. 85: 245-256, 1991.
growth factors for alveolar type II cells. Am. J. Physiol. 259 69. Kim, K. J., D. J. Suh, R. L. Lubman, S. I. Danto, Z. Borok,
(Lung Cell. Mol. Physiol. 3): L247-L254,1990. and E. D. Crandall. Studies on the mechanisms of active ion
47. Gaffney, E. F., A. J. O’Neill, and M. J. Staunton. In situ end fluxes across alveolar epithelial cell monolayers. J. Tissue CuZt.
labeling, light microscopic assessment and ultrastructure of Methods 14:187-194,1992.
apoptosis in lung carcinoma. J. CZin. Pathol. 48: 1017-1021, 70. Kremlev, S. G., and D. S. Phelps. Surfactant protein A
1995. stimulation of inflammatory cytokine and immunoglobulin
48. Gould, V E., R. Tosco, R. F. Wheels, N. S. Gould, and Y. production. Am. J. Physiol. 267 (Lung Cell. MOL. Physiol. 11):
Kapanci. Oxygen pneumonitis in man. Ultrastructural obser- L712-L719,1994.
vations on the development of alveolar lesions. Lab. Invest. 26: 71. Kuhn III, C., J. Boldt, T. E. King, Jr., E. Crouch, T. Vartio,
409-508,1972. and J. A. McDonald. An immunohistochemical study of archi-
49. Haschek, W. M., K. M. Reiser, A. J. P. Klein-Szanto, J. A. tectural remodeling and connective tissue synthisis in pulmo-
Last, and H. P. Witschi. Potentiation of butylated hydroxytolu- nary fibrosis l-3. Am. Rev. Respir Dis. 140: 1693-1703,1989.
ene induced lung damage by oxygen: cell kinetics and collagen 72. Kuwano, K., R. Kunitake, M. Kawasaki, Y. Nomoto, N.
metabolism. Am. Rev. Respir. Dis. 127: 28-34, 1983. Hagimoto, Y. Nakanishi, and N. Hara. p21 and p53 expres-
50. Haschek, WI M., and H. P. Witschi. Pulmonary fibrosis: a sion in association with DNA strand breaks in idiopathic
possible mechanism. Toxicol. Appl. Pharmacol. 51: 475-487, pulmonary fibrosis. Am. J. Respir Crit. Care Med. 154: 477-
1979. 483,1996.
51. Jones, K. G., J. F. Holland, G. L. Foureman, J. E. Bend, 73. Leblond, C. P. Classification of cell population on the basis of
and J. R. Fouts. Xenobiotic metabolism in Clara cells and their proliferative behavior, International Symposium on the
alveolar type II cells isolated from lungs of rats treated with Control of Cell Division and the Induction of Cancer. NatZ.
beta-naphthoflavone. J. Pharmacol. Exp. Ther. 225: 316-319, Cancer Inst. Monogr. 14: 119-150,1964.
1983. 74. Leslie, C. C., K. McCormick-Shannon, and R. J. Mason.
52. Kalina, M., S. Ricklis, and H. Blau. Pulmonary epithelial cell Heparin-binding growth factors stimulate DNA synthesis in rat
proliferaiton in primary culture of alveolar type II cells. Exp. alveolar type II cells. Am. J. Respir Cell Mol. Biol. 2: 99-106,
LungRes. 19:153-175,1993. 1990.
53. Kang, B., J. D. Crapo, C. D. Wegner, L. G. Letts, and L. 75. Loosli, C. G., S. F. Stinson, D. P. Ryan, M. S. Herweck, J. D.
Chang. Intercellular adhesion molecule-l expression on the Hardy, and R. Serebrin. The destruction of type II pneumo-
alveolar epithelium and its modification by pyperoxia. Am. J. cytes by by airborne influena PR8-A virus: its effect on surfac-
Respir Cell. Mol. Biol. 9: 350-355, 1993. tant and lecithin content of the pneumonic lesions of mice
54. Kapanci, U., R. Tosco, and J. Eggermann, et. al. Oxygen (Abstract). Chest 67: 7S, 1975.
pneumonitis in man. Light and electromicroscopic moephomet- 76. Macklin, C. C. The dust cells in the lungs of the albino mouse.
ric studies. Chest 62: 162-169, 1972. Lancet 260: 432-435,195l.
55. Kapanci, Y., E. R. Weibel, H. P. Kaplan, and F. R. Robinson. 77. Malamud, D. Differentiation and the cell cycle. In: The Cell
Pathogenesis and reversibility of the pulmonary lesions of Cycle and Cancer, edited by R. Baserga. New York: Dekker,
oxygen toxicity in monkeys. II. Ultrastructural and morphomet- 1971, p. 132-141.
ric studies. Lab. Invest. 20: 101-118, 1969. 78. Mason, R. J., C. C. Leslie, K. McCormick-Shannon, R. R.
56. Katzenstein, A. L., J. L. Myers, and M. T. Mazur. Acute Deterding, T. Nakamura, J. S. Rubin, and J. M. Shannon.
interstitial pneumonia A clinicopathologic, ultrastructural, and Hepatocyte growth factor is a growth factor for rat alveolar type
cell kinetic study. Am. J. Surg. Pathol. 10: 256-267, 1986. II cells. Am. J. Respir Cell Mol. Biol. 11: 561-567, 1994.
57. Kauffman, S. L. Cell proliferation in embryonic mouse neural 79. McDonald, J. A. Idiopathic pulmonary fibrosis: a paradigm for
tube following urethane exposure. Dev. BioZ. 20: 146-157,1969. lung injury and repair. Chest 99: 87S-93S, 1991.
58. Kauffman, S. L. Alterations in cell proliferation in mouse lung 80. Miller, B. E., and G. E. R. Hook. Isolation and characteriza-
following urethane exposure. II. Terminal bronchiolar epithe- tion of hypertrophic type II cells from the lungs of silica-treated
lium. Am. J. Pathol. 64: 531-538,197l. rats. Lab. Invest. 58: 565-575,1988.
Kauffman, S. L. Alterations in cell proliferation in mouse lung 81. Miller, B. E., and G. E. R. Hook. Hypertrophy and hyperpla-
following urethane exposure. III. Effects of chronic exposure on sia of alveolar type II cells in response to silica and other
type 2 alveolar epithelial cells. Am. J. Pathol. 68: 317-323, pulmonary toxicants. Environ. Health Perspect. 85: 15-23,
1972. 1990.
60. Kauffman, S. L. Kinetics of alveolar epithelial hyperplasia in 82. Nash, G., J. B. Blennerhassat, and J. Pontoppidan. Pulmo-
lungs of mice exposed to urethane. Lab. Invest. 30: 170-175, nary lesions associated with oxygen therapy and artificial
1974. ventilation. N. Engl. J. Med. 276: 368-374,1967.
61. Kauffman, S. L. Kinetics of pulmonary epithelial proliferation 83. Ohare, K. H., and P. L. Townes. Morphogenesis of albino rat
during prenatal growth of the mouse lung. Anat. Rec. 183: lung: an autoradiographic analysis of the embryological origin
393-404,1975. of the type I and II pulmonary epithelial cells. J. Morphol. 132:
62. Kauffman, S. L. Cell proliferation in the mammalian lung. Int. 69-75,197O.
Rev.Exp.PathoZ. 22:131-191,198O. 84. Omar, A. R. The characteristic cells of the lung and their
63. Kawanami, O., V. J. Ferrans, and R. G. Crystal. Structure of reaction to injury. Vet. BUZZ. 34: 371,1964.
alveolar epithelial cells in patients with fibrotic lung disorders. 85. Paine, R., A. Ben-Ze’Ev, S. R. Farmer, and and J. S. Brody.
Lab. Invest. 46: 39-53,1982. The pattern of cytokeratin synthesis is a marker of type 2 cell
64. Keeling, P. L., I. S. Pratt, W. N. Aldridge, and L. L. Smith. differentiation in adult and maturing fetal lung alveolar cell.
The enhancement of paraquat toxicity in rats by 85% oxygen: Dev.BioZ. 129:505-515,1988.
lethality and cell-specific lung damage. Br. J. Exp. Pathol. 62: 86. Paine, R., and R. A. Simon. Expanding the frontiers of lung
643-653,1981. biology through the creative use of alveolar epithelial cells in
65. Kehrer, J. P., and H. P. Witschi. Effects of drug metabolism culture. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14):
inhibitors on butylated hydroxytoluene induced pulmonary L484-L486,1996.
toxicity in mice. Toxicol. AppZ. Pharmacol. 53: 333-342,198O. 87. Panos, R. J., and R. J. Mason. Hypertrophic alveolar type II
Kerr, J. F. R., A. H. Wyllie, and A. R. Currie. Apoptosis. A cells isolated after silica-induced lung injury are progressing
basic biological phenomenon with wide-ranging implications in through the cell cycle and maintain a commitment to DNA
tissue kinetics (Abstract). Br J. Cancer 26: 239. 1972. synthesis in primary culture. Chest 99: 27S-28S, 1991.

Pardo, A., and M. Selman. Decreased collagenase production line. A model for cytokine networks in the lung. J. CZin. Inuest.
by fibroblasts derived from idiopathic pulmonary fibrosis. i&z- 86: 1945-1953,199o.
trix Suppl. 1: 417-448,1992. 104. Stanley, M. W., M. J. Henry-Stanley, K. J. Gajl-Peczalska,
89. Rannels, D. E. Type II cell differentiation in culture. Am. J. and P. B. Bitterman. Hyperplasia of type II pneumocytes in
Physiol. 270 (Lung Cell. Mol. Physiol. 14): L483, 1996. acute lung injury. Cytologic findings of sequential bronchoalveo-
90. Rannels, S. R., C. S. Fisher, L. J. Heuser, and D. E. lar lavage.Am. J. Clin. Pathol. 97: 669-677,1992.
Rannels. Culture of type II pneumocytes on a type II cell- Stoner, G. D., C. C. Harris, H. Authrup, B. F. Trump, E. W.
derived fibronectin-rich matrix. Am. J. PhysioZ. 253 (CeZZ PhysioZ. Kingsbury, and G. A. Myers. Explant culture of human
22): C759-C765,1987. peripheral lung. I. Metabolism of benzo[a]pyrene. Lab. Invest.
91. Rannels, S. R., R. N. Grove, and D. E. Rannels. Matrix- 38: 685-692,1978.
derived soluble components influence type II pneumocytes in
Tanswell, A. K., P. J. Byrne, R. N. Han, J. D. Edelson, and
primary culture. Am. J. Physiol. 256 (Cell Physiol. 25): C621-
V. K. M. Han. Limited division of low-density adult rat type II
C629,1989.
pneumocytes in serum-free culture. Am. J. Physiol. 260 (Lung
Selman, K., and F. C. Kafatos. Transdifferentiation in the
Cell. Mol. Physiol. 4): L395-L402,1991.
labial gland of the silk moth: is DNA synthesis required for
cellular metamorphosis? Cell. Differ. Deu. 3: 81-94,1974. 107. Thaete, L. G., and A. M. Malkinson. Cells of origin of primary
Selman, RI., M. Montano, C. Ramos, R. Barrios, and R. pulmonary neoplasms in mice: morphologic and histochemical
Perez-Tamayo. Experimental pulmonary fibrosis induced by studies. Exp. Lung Res. 17: 219-228,199l.
paraquat plus oxygen in rats: a morphologic and biochemical 108. Uhal, B. D. Flow cytometric study of the type II pneumocyte
sequential study. Exp. Mol. Pathol. 50: 147-166,1989. cell cycle in vivo and in vitro. Cytometry 15: 46-52,1994.
Shackney, S. E., and P. S. Ritch. Percent labeled mitosis 110. Uhal, B. D., and M. D. Etter. Type II pneumocyte hypertrophy
curve analysis. In: Techniques in Cell Cycle Analysis, edited by without activation of surfactant biosynthesis after partial pneu-
J. W. Gray and 2. Darzynkiewicz. Clifton, NJ: Humana, 1987, p. monectomy. Am. J. Physiol. 264 (Lung CeZZ. Mol. Physiol. 8):
31-45. L153-L159,1993.
Shannon, J. M., S. D. Jennings, and L. D. Nielsen. Modula- 111. Uhal, B. D., K. M. Flowers, and D. E. Rannels. Type II
tion of alveolar type II cell differentiated function in vitro. pneumocyte proliferation in vitro: problems and future direc-
Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L427-L436, tions. Am. J. Physiol. Suppl. (Oct.) 261: 110-117, 1991.
1992. 112. Uhal, B. D., I. Joshi,A. True, S. Mundle,A. Raza,A. Pardo,
96. Shorter, R. G., J. L. Titus, and M. B. Divertie. Cytodynamics and M. Selman. Fibroblasts isolated after fibrotic lung injury
in the respiratory tract of the rat. Thorax 21: 32-37,1966. induce apoptosis of alveolar epithelial cells in vitro. Am. J.
97. Simnett, J. D., and A. G. Heppleston. Cell renewal in the Physiol. 269 (Lung Cell. Mol. Physiol. 13): L819-L828, 1995.
mouse lung. The influence of sex, strain and age. Lab. Invest. 113. Uhal, B. D., and D. E. Rannels. DNA distribution analysis of
15: 1793-1801,1966. type II pneumocytes by laser flow cytometry: technical consider-
98. Simon, R. H., T. J. Gross, J. A. Edwards, and R. G. Sitrin. ations. Am. J. Physiol. 261 (Lung Cell. Mol. Physiol. 5): L296-
Fibrin degradation by rat pulmonary alveolar epithelial cells. L306,1991.
Am. J. Physiol. 262 (Lung CeZZ. Mol. Physiol. 6): L482-L488, 114. Weibel, E. R. Morphological basis of alveolar-capillary gas
1992.
exchange. Physiol. Rev. 53: 419-495,1973.
Simpson-Herren, L. Autoradiographic techniques for measure-
115. Weibel, E. R. A note on differentiation and diversibility of
ment of the labeling index. In: Techniques in CeZZ Cycle AnaZy-
alveolar. Chest 654: 195-215, 1974.
sis, edited by J. W. Gray and Z. Darzynkiewicz. Clifton, NJ:
116. Weiss, P., and J. L. Kavanau. A model of growth and growth
Humana, 1987, p. l-29.
Smith, B. T. Cell line A549: a model system for the study of control in mathematical terms. J. Gen. Physiol. 41: l-48, 1957.
alveolar type II cell function. Am. Rev. Respir. Dis. 115: 285- 117. Williams, M. C., and L. G. Dobbs. Expression of cell-specific
293,1977. markers for alveolar epithelium in fetal rat lung. Am. J. Respir.
Smith, L. J. The effect of type 2 cell mitosis on the surfactant Cell Mol. Biol. 2: 533-542, 1990.
system of injured mouse lungs. J. Lab. CZin. Med. 102: 434-443, 118. Witschi, H. Responses of the lung to toxic injury. Environ.
1983. Health Perspect. 85: 5-13,199O.
Smith, L. J., N. B. Kaplan, and J. Brody. Response of normal 119. Wyllie, A. H., J. F. R. Kerr, and A. R. Currie. Cell death: the
and beige mouse alveolar type 2 cells to lung injuryl-4. Am. significance of apoptosis. Int. Reu. CytoZ. 68: 251-306,198O.
Rev. Respir. Dis. 122: 947-957,198O. 120. Yue, G., R. L. Shoemaker, and S. Matalon. Regulation of
Standiford, T. J., S. L. Kunkel, M.A. Basha, S. W. Chensue, low-amiloride-affinity sodium channels in alveolar type II cells.
J. P. Lynch, G. B. Towes, J. Westwick, and R. M. Strieter. Am. J. Physiol. 267 (Lung CeZZ. Mol. Physiol. 11): L94-LlOO,
Interleukin-8 gene expression by a pulmonary epithelial cell 1994.

Ciclo Celular Neumocitos

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Ciclo Celular Neumocitos

Uploaded by

Copyright:

Available Formats

Cell cycle kinetics in the alveolar epithelium

Uhal, Bruce D. Cell cycle kinetics in the alveolar epithelium. Am. J.

proliferation. The purpose of this manuscript is to

THE MODEL, CIRCA 1980

A drawing of the pulmonary alveoli is presented in k Type I Air Space

of the alveolar anatomy have been published (27, 28,

Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (130.095.063.089) on January 7, 2019.

Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (130.095.063.089) on January 7, 2019.

Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (130.095.063.089) on January 7, 2019.

first investigators to report the isolation of these cells

Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (130.095.063.089) on January 7, 2019.

KINETICS OF THE AEC CYCLE

Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (130.095.063.089) on January 7, 2019.

Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (130.095.063.089) on January 7, 2019.

tors had noted that primary isolates of type II cells did

Fig. 5. A: derivation of cell cycle parameters from flow cytometric In vivo

Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (130.095.063.089) on January 7, 2019.

Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (130.095.063.089) on January 7, 2019.

which was speculated to contribute to the inability of

On the basis of the available data to date, the

Student’s t-test. [Taken from Uhal et al. (112).]

carcinoma (100); in mouse lung carcinomas, Dyson and

the tumors (83-95% of KB) might be due to pro-

Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (130.095.063.089) on January 7, 2019.

Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (130.095.063.089) on January 7, 2019.

Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (130.095.063.089) on January 7, 2019.

Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (130.095.063.089) on January 7, 2019.

Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (130.095.063.089) on January 7, 2019.

You might also like