Introduction
Intercellular communication between epithelial cells and fibroblasts
is important for cellular regulation in a variety of organ systems, particularly
during development (1,2). It has been shown that fibroblasts in the
lung or their secreted collagen fibers can influence epithelial cell growth
and differentiation, whereas the epithelium may control fibroblast growth
(3-5). In some cases, cell-cell interaction occurs through secreted
molecules (6,7), but there is also evidence that direct intercellular
contact between these different cell types may alter growth characteristics
or epithelial cell function (4,8).
These results have largely been obtained from studies of cells isolated
from normal, adult lungs or from developing lungs. Changes in this reciprocal
epithelial cell-fibroblast interaction may occur in injured lungs and so
may alter the reparative processes. The pulmonary epithelium is a common
target for a variety of environmental toxins as well as circulating drugs,
and severe injury to this cell is frequently associated with subsequent
fibro-blast proliferation (9). In considering the mechanisms underlying
pulmonary fibrosis after epithelial injury, most studies have focused on
cytokine production and fibroblast stimulation by macrophages in particular
(10). In the pulmonary re-sponse to bleomycin, for example, recovered
alveolar macrophages have been shown to secrete a fibroblast growth factor
and are also positive for transforming growth factor ß (TGF-ß),
which is associated with increased collagen production (11,12). However,
bleomycin induces epithelial injury (13,14), which may disrupt the
normal epithelial-fibroblast control system. It is not known whether this
change in the local microenvironment alters proliferation of either cell
type and induces abnormal repair, which is characterized by epithelial proliferation,
abnormal differentiation, and extensive underlying fibrosis.
In the present study, type 2 epithelial cells and fibroblasts were isolated
from rat lungs 10 days after administration of bleomycin, when epithelial
injury is maximal, and after 6 weeks when abnormal repair with fibrosis
occurs. This allowed us to determine if there are changes in either cell
type at these two stages of injury and repair, which could alter the proliferation
of the other cell type, either through a secreted molecule or by a cell-contact-related
process.
Materials and Methods
Male Sprague-Dawley rats (about 150 g) were used. In a trial series,
we injected animals intratracheally while they were under mild barbiturate
anesthesia with various doses from 1 to 15 U of bleomycin (kindly provided
by Bristol-Myers Squibb Co., Evansville, IN). Rats were killed by an intraperitoneal
barbiturate overdose at intervals to 12 weeks, and the lungs were processed
for histologic examination. Based on this trial, we chose a dose of 8 U/kg
body weight in 0.3 ml sterile water for further experiments. Two groups
of 12 rats were injected, then 1 set was killed at 10 days, the other at
6 weeks. We lavaged the lungs four times with 5 ml saline and counted inflammatory
cells by using a hemocytometer. After centrifugation to remove cells, we
assayed the lavage fluid for protein content. A sample of lung tissue was
taken to measure hydroxyproline (HYP), and the remainder was processed for
microscopy (15). Control groups received the same volume of water
and were killed at the same times. This experiment established that the
8 U/kg bleomycin induced acute lung injury at 10 days, followed by pulmonary
fibrosis at 6 weeks.
Cell Isolation
We isolated type 2 epithelial cells and fibroblasts from normal rats
and killed rats at 10 days and 6 weeks after intratracheal administration
of bleomycin or water. We isolated type 2 cells using the method of Dobbs
et al. (16). Briefly, the lungs were excised, and after perfusion,
4.3 U/ml elastase was instilled into the trachea. Subsequently, the lungs
were minced and filtered through successive nylon meshes down to 7 µm
to obtain a cell suspension that was incubated in serum-free media in petri
dishes coated with IgG. We collected nonadherent cells and spun them down;
viability by trypan blue was >90%. We resuspended the cells in Dulbecco's
modified Eagle's medium with 10% fetal bovine serum for counting by hemocytometer
and seeding into culture dishes. Cells were isolated from two rats, then
pooled for each experimental run. Previous experiments using special stains
and electron microscopy on cell pellets showed that cells prepared in this
way were >94% type 2 cells. We made a cytospin preparation of each starting
preparation to ensure purity; cells were only used when purity was >90%
with <2% macrophages present. There was no morphologic difference between
isolated cells from control or bleo-mycin-treated lungs.
We prepared fibroblasts from rats in the same group using a trypsin digestion
method as previously described and cultured the fibroblasts to confluence
(17). Cells were passaged once before using them in the experiments
described below. They stained >98% positive for vimentin and negatively
for factor VIII.
Culture System
Fibroblasts were seeded at a density of 2 x 105 per well on
a 24-well plastic culture dish and allowed to adhere for 1 hr. We seeded
epithelial cells at 4 x 105 per well into a well insert, which
had a filter as a base (Millicell-CM, Millipore Corp., Bedford, MA). These
filters were coated with 50 µg rat-tail collagen just before using
them to improve cell attachment. Other epithelial cells were seeded directly
onto a fibroblast layer and co-cultured. Using various combinations, we
studied 1) epithelial cells alone; 2) fibroblasts alone; 3) epithelial cells
exposed to secretions of underlying fibroblasts; 4) fibroblasts exposed
to secretions of overlying epithelial cells; and 5) co-cultures with epithelial
cells and fibroblasts in direct contact.
For both bleomycin groups, we cultured isolated epithelial cells and
fibroblasts under the five conditions outlined above. We carried out additional
experiments in which cells isolated from normal rats were cultured with
cells from bleomycin-exposed lungs; for example, normal type 2 cells were
cultured over fibroblasts from bleomycin-exposed lungs and vice versa.
Cell Growth
Cells were cultured as described for 1 day then changed to serum-free
conditions for a further day. In some experiments, we quantitated the growth
of each cell type by counting cell numbers after trypsinization of the wells
and filters separately, then by using a hemocytometer. In most experiments,
we measured DNA synthesis by adding tritiated thymidine (3HT)
at 0.1 µCi/ml for the final 4 hr of culture, when the cells were rinsed
well and scraped off for scintillation counting. The thymidine uptake (dpm)
was taken as the mean of four wells for each culture series. In addition,
from one additional well, we removed cells and made duplicate cytospin preparations
from each suspension. Slides were subsequently dipped in Kodak NTB2 emulsion
in the dark room under a red filter. After 2 weeks in total darkness, we
developed the slides and stained them with toluidine blue. For each time
period, we calculated the percentage of cells labeled after counting 500
cells per slide.
To quantitate DNA synthesis by the individual cell types in the co-cultures,
we stained cytospin preparations for cytokeratin and vimentin using standard
immuno-peroxidase methods (18), then we prepared slides for autoradiography.
This allowed us to count separately the thymidine-labeled nuclei of epithelial
cells and fibroblasts from the same culture dish. Similarly, slides of "pure"
epithelial cells were stained for vimentin so that any labeled, contaminating
fibroblasts could be eliminated from the cell counts.
Growth Factor Studies
A few additional experiments using cells from different rats were set
up to test the effects of selected, known growth factors and their antibodies
on epithelial cell proliferation. Agents used at various dose levels were
tumor necrosis factor (TNF-
),
platelet-derived growth factor (PDGF), TGF-, TGF-ß, epidermal growth factor (EGF), and their respective
antibodies (R and D Systems, Minneapolis, MN). Effects were quantitated
by counting thymidine-labeled cells in autoradiographs using the same experimental
outline described earlier.
Statistics
We used isolated cells from at least four different preparations for
each of the conditions studied. Means ± SEs were calculated for each
experimental group and compared to the corresponding control for significance
using the Student's t test.
Results
In vivo Studies
The intratracheal injection of water causes a mild inflammation response,
which in our experience ceases by 3-5 days. In this experiment, no change
in lung cells, lavage protein, or lung HYP over noninjected controls was
found at the times studied. The number of alveolar macrophages (AM) recovered
by bronchoalveolar lavage (BAL) from bleomycin-injected lungs was more than
double control values at 10 days and remained above normal at 6 weeks (Fig.
1). Few polymorphonuclear leukocytes (PMN) were recovered from normal lungs,
but signficantly higher numbers were present in BAL from bleomycin-injected
lungs. This inflammatory response was confirmed by measuring the protein
content of BAL fluid (Fig. 2). The protein level was greatly increased 10
days after bleomycin, and, although it fell subsequently, it was still above
control levels after 6 weeks. We indexed changes in lung collagen by the
HYP level, which was unchanged at 10 days after treatment but was significantly
increased after 6 weeks (Fig. 2).
Figure 1. Number
of cells (mean ± SE) lavaged from noninjected control and bleomycin
(BLM)-treated rats after 10 days and 6 weeks. (Open bars) Alveolar macrophages;
(shaded bars) polymorphonuclear leukocytes. *p < 0.01 compared
to water-injected control at the corresponding time.
Figure 2. Protein
content of bronchoalveolar lavage fluid and lung content of hydroxyproline
(HYP) in noninjected controls and in lungs at 10 days and 6 weeks after
bleomycin treatment (BLM). *p < 0.01 compared to water-injected
control at the corresponding time.
Lung morphology confirmed the general biochemical findings. At 10 days,
diffuse injury to the alveolar walls was seen, the tissue was edematous,
and the alveoli contained AM and PMN (Fig. 3A). By 6 weeks, fewer cells
were seen in alveolar spaces; the main change was in the pulmonary interstitium,
which was thicker than normal due to excess fibroblasts and collagen (Fig.
3B). Evidence of epithelial repair was seen, as some alveoli were lined
by cuboidal cells rather than the usual mixture of type 1 and 2 cells.
Figure 3. Lung
sections after bleomycin (X 550). (A) At 10 days, diffuse alveolar injury
is seen with interstitial edema. Many inflammatory cells are present in
the air spaces. (B) At 6 weeks, the interstitium is greatly thickened by
fibroblasts and collagen.
In vitro Studies: 10-Day Bleomycin Group
We studied growth characteristics of epithelial cells and fibroblasts
using 3HT uptake. Type 2 cells isolated from normal lung (N-EP)
or from lungs 10 days after bleomycin injection (B-EP) incorporated low
levels of 3HT (Fig. 4). When either set of EP cells was cultured
over fibroblasts from normal (N-FB) or bleomycin (B-FB)-treated rats in
the lower culture chamber, thymidine incorporation of EP cells significantly
increased (Fig. 4). Proliferation was confirmed by counting the number of
EP cells, which increased about 70% when EP were cultured over FB.
Figure 4. Incorporation
of tritiated thymidine (3HT) by epithelial cells (EP) from normal
(N) lungs or 10 days after bleomycin (B) treatment. The EP cells were cultured
alone or on a filter above fibroblasts (FB). *p < 0.05 compared
to corresponding EP cells alone.
When fibroblasts were grown alone, N-FB and B-FB incorporated 3HT
equally (Fig. 5). However, when fibroblasts were cultured in wells beneath
EP cells, DNA synthesis was significantly lower than in fibroblasts grown
alone. In particular, a secretion of B-EP depressed 3HT uptake
in B-FB to a greater extent (Fig. 5).
Figure 5. Incorporation
of 3HT by fibro-blasts (FB) from normal (N) lung or 10 days after
bleomycin (B) treatment. The FB were cultured alone or beneath epithelial
(EP) cells.*p < 0.05 compared to corresponding Fb cells alone;
p
< 0.05 compared to all other groups.
In vitro Studies: 6-Week Bleomycin Group
Epithelial cells isolated from this older group of rats, normal or treated,
showed a slightly lower level of 3HT uptake. However, in each
experimental group, N-EP and B-EP cells showed increased DNA synthesis when
cultured on inserts over N-FB or B-FB (Fig. 6). However, when using cells
derived from bleomycin-treated rats, a lower increase in 3HT
uptake by EP cells was observed compared to that seen when only normal cells
were used (N-EP, N-FB).
Figure 6. Epithelial
cells (EP) isolated from control lungs (N) and from lungs 6 weeks after
bleomycin (B) treatment. The EP were cultured alone or over fibroblasts
(FB). p < 0.05 compared to all other groups.
The 3HT uptake of normal fibroblasts was significantly reduced
when they were exposed to factors secreted by N-EP or B-EP (Fig. 7). Growth
of B-FB was suppressed by N-EP cells, but no effect was seen when B-FB were
cultured beneath B-EP (Fig. 7).
Figure 7. Fibroblasts
(FB) isolated from control lungs (N) and from lungs 6 weeks after bleomycin
(B) treatment. The FB were cultured alone or below epithelial cells (EP).
*p < 0.05 compared to corresponding FB cells alone.
Most of the experiments were carried out using 3HT uptake
(dpm) as the end point. Other experiments were also carried out using this
group of rats to determine cell counts to confirm cell division as opposed
to DNA synthesis only. We also used autoradiography to establish the identity
of cells in DNA synthesis. The results confirm that epithelial cells cultured
over, but separated from, fibroblasts show significantly increased labeling
and cell numbers compared to cells cultured alone (Table 1). This occurred
using normal cells, and to a lesser extent with cells from bleomycin-treated
lungs. The autoradiographic study also allowed quantitation of labeled cells
in co-cultures. When EP and FB were co-cultured, the stimulatory effect
on the EP population was lost. Total cell numbers in the co-cultures were
high due to fibroblast growth (data not shown), but only about 2.5-3% of
EP cells were labeled in autoradiographs, a value equal to that found when
EP cells were cultured alone (Table 1). This contrasts to an EP labeling
value of 7-12% when these cells were exposed to a fibroblast secretory product
.
In vitro Growth Factor Studies
In a preliminary set of studies to identify the fibroblast secretory
product responsible for epithelial growth stimulation, antibodies to the
well-known growth factors PDGF, EGF, TNF-, TGF-
and TGF-ß were added at various concentrations to the culture system.
The increase in 3HT incorporation by EP cells grown over fibro-blasts
was not blocked by antibodies to any of these growth factors (data not shown).
The antibodies were biologically active because lower growth was seen in
the underlying fibroblasts. One surprising result occurred using anti-TGF-ß:
it seemed to stimulate EP growth. This result was investigated more closely,
and results are shown in Table 2.
Addition of pure TGF-ß to the epithelial cells cultured over fibroblasts
lowered the percentage of labeled EP cells. When the anti-TGF-ß was
used, 3HT incorporation by EP cells alone, and in particular
those maintained over fibroblasts, was significantly increased (Table 2).
Discussion
Pulmonary fibrosis is a common sequel to severe or chronic injury to
the lung. Most research into the underlying mechanisms of fibroblast stimulation
has concentrated on cytokine release by macrophages because these cells
secrete a variety of hormonelike molecules that enhance fibroblast growth
and collagen production (10). This is also true of alveolar macrophages
recovered from fibrotic lungs injured by the antineoplastic drug bleomycin
(11). However, it has been previously shown that intravenously or
intratracheally injected bleomycin initially damages endothelial and epithelial
cells of the lung, and repair is abnormal leading to fibrosis (13).
Despite severe epithelial damage, the effects of bleomycin on the local
epithelial-fibroblast control system have not been investigated.
The model of intratracheal injection permits a lower dose to be instilled
directly to the lung and is a useful model of generating rapid fibrosis
(14). We chose a dose level of bleomycin to allow study of these
two cell types isolated from lungs at different stages of the injury-repair
cycle. At 10 days after injection, there is acute lung injury indexed by
the inflammatory response, a high level of alveolar protein, and morphologic
changes, all of which indicate diffuse alveolar injury, especially to type
1 cells. From these lungs, isolated type 2 cells, although probably injured
to some extent, were certainly viable and could be maintained in culture.
At 10-days in vivo, there was no change in collagen levels in the
lung. In contrast, 6 weeks after bleomycin treatment, fibrosis was evident
biochemically and by lung morphology. The acute inflammatory reaction had
subsided and the blood-air barrier was much less permeable. Previous studies
have shown that the peak of epithelial repair is over by this time (19),
when epithelial cells and fibroblasts were also isolated for in vitro
study.
Fibroblasts from each group, cultured alone, proliferated rapidly and
approximately equally as measured by cell counts and thymidine incorporation.
When normal fibroblasts were cultured in a lower chamber beneath epithelial
cells, their growth was slower and a significant decrease in fibroblast
DNA synthesis was observed. This was also true using cells from the 10-day
bleomycin group; fibro-blast growth was particularly suppressed when fibroblasts
were exposed to secreted products of epithelial cells from the same rats
(Fig. 5). When cells were prepared from the fibrotic lungs, epithelial cells
were still able to decrease 3HT uptake by normal fibroblasts,
but these cells had no effect on fibroblasts from bleomycin-treated rats.
This indicates that a product secreted by epithelial cells, which has some
fibroblast inhibitory effects on normal cells, is not secreted in fibrotic
lung in which excess fibroblast growth occurs. This suggests that in normal
cells, localized fibroblast control is mediated through the overlying epithelium.
However, the epithelial surface 6 weeks after bleomycin treatment is not
normal in vivo, and various cuboidal epithelial cell forms have been
described (19). These may be functionally deficient in producing
the fibroblast inhibitor as shown here and so may contribute to the development
of fibrosis.
The identity of the inhibitory molecule is not known. Although macro-phages
are a well-recognized source of cytokines, the low number present in our
epithelial cell preparation makes it more likely that the fibroblast inhibitory
molecule is produced by type 2 cells. One candidate is TGF-ß, which
has been shown in lung epithelial cells in bleomycin-treated lungs, though
it was associated more with areas of increased collagen deposition (12).
Another possible inhibitor is prostaglandin E2 (PGE2), which
is secreted by type 2 cells in culture (19) and is secreted in larger
amounts by these cells during injury such as exposure to silica (21).
Because PGE2 is associated with reduced fibroblast growth in
the lung, it may function at a local level in epithelial-fibroblast growth
control. It has been shown previously that alveolar epithelial cells secrete
a low-molecular-weight factor that inhibits proliferation of interstitial
cells (22). Evidence of epithelial control of fibroblast growth has
also been found in organ culture studies in which fibroblast growth is promoted
in the absence of epithelium and is controlled as the epithelial surface
is restored (23,24). The present results suggest that epithelial
cells are capable of fibroblast-growth control through diffusion of a secreted
factor, but in a fibrotic lung, this inhibitory effect is lost.
In a reciprocal interaction, epithelial cell proliferation appears to
be promoted by exposure to a diffusible product of lung fibroblasts. Type
2 cells isolated from control or treated rats incorporated a low level of
3HT and showed little division; as determined by autoradiography,
only about 2-3% of cells were labeled. However, when these cells were cultured
on a filter insert with fibroblasts in the lower chamber, all parameters
of cell growth were significantly increased, whether using cells from normal
or bleomycin-injected rats. This indicates that a fibroblast-derived factor
is capable of stimulating epithelial growth and so may accelerate repair.
It is interesting to note that this stimulation occurred through a secreted,
diffusing factor. When co-cultures were studied, fibroblast growth predominated,
but when autoradiographs were examined, epithelial cell labeling was low
and identical to levels seen in pure epithelial cultures. This suggests
that direct epithelial cell-fibroblast contact induces an inhibitor to epithelial
growth. Similar results were obtained earlier using fetal cells, whereby
direct cell-cell contact between these two cell types was associated with
reduced epithelial cell growth but enhanced type 2 cell differentiation
(4).
Pure type 2 cells proliferate poorly under normal culture conditions
and require addition of specific growth factors to stimulate cell division
(7,25). The identity of the fibroblast-derived factor that promotes
epithelial growth in the present study is not known; it could be one or
a combination of several fibroblast-derived growth factors that promote
lung cell proliferation (7). Fibroblast-conditioned media has been
shown to produce a competence factor for fetal lung epithelial cells (5),
and it has also been shown that lung fibroblasts produce a growth factor
for bronchial epithelial cells (26). In preliminary experiments to
identify the factor, we were unable to block the increase in epithelial
growth using antibodies to PDGF, TNF-, TGF-
or EGF. When TGF-ß was added to type 2 cells in culture over fibroblasts,
the usual growth increase was not seen, suggesting that TGF-ß may
act as an antagonist to the epithelial growth factor. This was supported
when the addition of anti-TGF-ß to the culture system resulted in
a significant increase in type 2 cell labeling. Lung fibroblasts are known
to secrete TGF-ß (27) and its regulation may play a key role
in both epithelial and fibroblast cell behavior during injury and repair.
This aspect of the epithelial-fibroblast interaction is now under more detailed
study.
The existence of a fibroblast-derived growth factor for epithelial cells
could be important in providing a stimulus to lung repair after type I cell
necrosis, when rapid regeneration of the epithelial surface by type 2 cell
proliferation is a key event in the restoration of normal lung structure
(23,28). Because type 1 cell injury is a common event when the lung
is exposed to various environmental toxins, the changes in cellular control
seen here after bleomycin may represent a general response to air- or bloodborne
agents that are capable of inducing fibrosis. Control mechanisms involving
epithelial cell-fibroblast interactions may be important in regulating normal
repair in a highly localized manner. This system may be effective in repairing
acute focal injury. It may be speculated that, in cases of more severe epithelial
injury, this localized control process is supplemented by more generalized
mechanisms of repair in which chronic inflammation, macrophage activation,
and general cytokine networks are involved.
