Cell size, cell cycle, and
-smooth muscle actin expression
by primary human lung fibroblasts
Bruce D.
Uhal1,
Carlos
Ramos2,
Iravati
Joshi1,
Antonio
Bifero1,
Annie
Pardo3, and
Moises
Selman2
1 Lung Cell Kinetics Laboratory and The
Cardiovascular Institute, Michael Reese Hospital, Chicago, Illinois
60616; and 3 Facultad de Ciencias, Universidad
Nacional Autonoma de Mexico, Coyoacan 04000; and
2 Instituto Nacional de Enfermedades
Respiratorias, Tlalpan 14080, Mexico
 |
ABSTRACT |
Primary human lung fibroblasts were separated
into small ( group I),
intermediate ( group II), and large
( group III) subpopulations by unit
gravity sedimentation (1 G). The three subsets retained differences in
cell size for up to 15 days of primary culture. Flow cytometric
(fluorescence-activated cell sorter) measurements of forward-angle
light scatter agreed well with fibroblast volume measured by image
analysis and confirmed the utility of forward-angle light scatter for
discriminating size subpopulations. Group
II fibroblasts accumulated most rapidly by 8 days of
culture and also contained the greatest proportion of S and
G2/M phase cells as determined by
fluorescence-activated cell sorter. Fibroblasts that were
immunoreactive with antibodies to
-smooth muscle actin (
-SMA)
were found only in group III. In situ
end labeling of fragmented DNA detected apoptotic cells in both
groups II and III, but double labeling for in situ
end labeling and
-SMA revealed apoptotic cells in both the
-SMA-positive and -negative populations. These results demonstrate
that primary human lung fibroblasts behave as predicted by classic
models of cell cycle progression and differentiation. However, they do
not support the hypothesis that the expression of
-actin is related
to apoptosis. We also describe a simple and reproducible method for the
high-yield isolation of human lung fibroblast subsets of differing
proliferative potential and phenotype.
lung cells; myofibroblast; apoptosis; proliferation; cell
heterogeneity
 |
INTRODUCTION |
LUNG FIBROBLAST SUBPOPULATIONS of functionally distinct
capacities have been isolated from rodent and human tissues. From the
mouse lung, two subpopulations have been identified by disparate expression of the allelic antigen Thy 1 (15, 16).
Subpopulations of human lung fibroblasts also have been discriminated
on the basis of expression of the receptor for the complement
subcomponent C1q (1). Fibroblastic foci in the lungs of patients with
interstitial lung disease contain fibroblasts of the subtype termed VA,
which was identified by expression of the intermediate filaments
vimentin and
-smooth muscle actin (
-SMA) (12). The VA
subpopulation is but one member of the myofibroblast phenotype, a
heterogeneous family of mesenchymal cells observed in a variety of
injured and/or repairing tissues (9, 18).
Investigations of primary isolates of these fibroblast subsets have
revealed differences in growth rate (17), collagen synthesis (5), and
responses to various cytokines (26). On this basis, it is believed that
the initial distribution and subsequent selection of these
subpopulations is likely a critical determinant in the pathogenesis
and/or resolution of pulmonary fibrosis (8). In vitro, these
fibroblast subpopulations exhibit reproducible patterns of morphology.
Fibroblast subsets of mouse lung, when cultured after separation by
fluorescence-activated cell sorter (FACS) analysis of Thy 1 expression,
displayed either a spindle-shaped morphology with lipid inclusion
bodies or a larger and more rounded morphology (15). Similarly, human
lung fibroblast subsets separated by C1q-receptor expression also
displayed two distinct morphologies: spindle-shaped cells with
elongated processes (high binding) or larger and more flattened cells
(1).
The large and flat fibroblast morphology was also observed in cultures
of microfilament-laden myofibroblasts isolated from experimental
granulating wounds inflicted on rats (23). In studies of mouse and
human lung cells in vitro (1, 17), the large and flat fibroblast
subsets were found to grow more slowly in culture than those with the
smaller spindle shape. Myofibroblasts isolated from connective tissue
stroma of human breast carcinomas or from associated granulating wounds
also grew more slowly than fibroblasts obtained from normal human
tissue (24). The finding of markers of apoptosis within myofibroblast
populations in vivo (7) has led to the speculation that fibroblast
differentiation to the myofibroblast phenotype might represent a
terminal pathway leading to apoptosis (6).
Together, these observations suggest an interdependence of lung
fibroblast phenotype and cell cycle progression. An understanding of
this relationship might provide new insights into lung fibroblast function as well as new tools for future investigations. To begin studying this topic, we hypothesized that a simple cell separation protocol based on differences in cell size would also discriminate fibroblast subsets of functionally distinct capacities, particularly with respect to growth kinetics. We describe here the application of
unit gravity sedimentation (1 G) as a cell separation method for human
fibroblasts isolated from normal lung tissue, and we report the
resolution of size subpopulations of high yield and disparate
proliferation kinetics. Initial evaluations of these subsets are
consistent with classic models of cell size and cell cycle progression
and suggest that expression of the myofibroblast phenotype is unrelated
to the commitment to apoptosis.
 |
METHODS |
Materials. Materials for
cell isolation and culture were purchased from sources described
elsewhere (22). Propidium iodide, trypsin, trypsin inhibitor,
avidin-rhodamine, and an FITC-conjugated monoclonal antibody to
-SMA
were obtained from Sigma (St. Louis, MO). Avidin-FITC, both FITC- and
rhodamine-conjugated anti-mouse IgGs, and DNase-free RNase, cytometry
grade, were purchased from Boehringer Mannheim (Indianapolis, IN).
Fluorescein-conjugated annexin V was obtained from PharMingen (San
Diego, CA). All other chemicals were of reagent grade.
Fibroblast isolation and culture.
Primary lung fibroblasts were isolated at the National Institute of
Respiratory Diseases (Tlalpan, Mexico) from a patient undergoing a
lobectomy for removal of a primary lung tumor (14). No morphological
evidence of disease was found in the tissue samples used for fibroblast
isolation. The cells were isolated by trypsin dispersion as described
earlier (14), and fibroblast strains were established in Dulbecco's modified Eagle's medium (or in Ham's F-12 medium) supplemented with
10% fetal bovine serum (FBS), 200 U/ml of penicillin, and 200 mg/ml of
streptomycin. All cells were cultured at 37°C in 95% air-5%
CO2 until early confluence. One
early-passage strain (N12, passage
10) was chosen arbitrarily for this study. Cell number was determined with the cell proliferation reagent WST-1 (Boehringer Mannheim), a tetrazolium salt cleaved by the mitochondria of viable cells to yield a soluble formazan chromophore. Relative cell
density was determined according to the instructions provided by the
manufacturer. In a pilot study, WST-1 absorbance was proportional to
cell number as determined by hemocytometer counts. In addition, no
significant differences were found in the WST-1 absorbances for small
( group I), intermediate
( group II), or large ( group III) fibroblasts assayed at equivalent cell numbers
predetermined by hemocytometer (data not shown). Thus the determination
of cell accumulation rates with WST-1 was unaffected by the differences in cell size between group I,
II, and
III fibroblasts.
Unit gravity sedimentation. Fibroblast
separation on the basis of cell size was conducted as described earlier
for type II alveolar epithelial cells (19, 20). Briefly, 5-10 × 106 fibroblasts were
trypsinized, washed, and resuspended in 50 ml of Ham's F-12 medium
buffered with HEPES and containing 2% FBS. The suspension was layered
over an eight-step discontinuous gradient of Ficoll (2-8%
wt /vol, 50 ml/step) in Ham's F-12 medium containing 2% FBS and
buffered with HEPES at pH 7.3, all in a 4°C cold room. The gradient
chamber was slowly lowered over a period of 20 min to the horizontal
position (15), where it remained for 60 min. The chamber was then
returned to a vertical position over 20 min, and 36 fractions of 15 ml
each were collected through a port in the chamber bottom. The cells
were either fixed immediately with 70% ethanol (22) or recovered for
subsequent culture in Ham's F-12 medium containing 10% FBS. Over five
separate experiments, as many as 20 × 106 cells or as few as 3 × 106 cells were separated, with no
change in light scatter or volume profiles of the resulting pooled
groups.
Flow cytometry. Flow cytometric
analyses were performed on a Partec CA-III flow cytometer equipped with
a 25-mW argon ion laser for excitation at 488 nm. Propidium iodide and
rhodamine (tetramethylrhodamine isothiocyanate) fluorescences were
acquired through a 610-nm long-pass filter and fluorescein (FITC)
fluorescence was acquired through an EM520 band-pass filter. After
standardization with fluorescent microspheres (Coulter, Hialeah, FL),
amplifier gains were not changed throughout an experiment. Preparation
of cells for DNA distribution and 5-bromo-2'-deoxyuridine
(BrdU) incorporation experiments was conducted as
described earlier (22), with cell fixation in 70% ethanol followed by
incubations with 4 N HCl and DNase-free RNase. For analysis of
apoptosis by in situ end labeling (ISEL) (25), the cells were labeled
with biotinylated dUTP. Depending on experimental requirements,
biotinylated DNA was detected with avidin-FITC or avidin-rhodamine
essentially as described by Gorczyca et al. (10).
For analyses of apoptosis by annexin V binding, the cells were
trypsinized from culture dishes and incubated for 1 h in suspension. Fluorescein-conjugated annexin V was added to the medium for 15 min at
the concentration recommended by the supplier, after which the cells
were washed and resuspended in PBS for immediate FACS analysis. For
immunocytochemistry, fibroblasts were fixed with ice-cold 70% ethanol
and stored at
20°C until assay. The cells were washed and
incubated for 1 h at 37°C with FITC-conjugated monoclonal
anti-human
-SMA antibody diluted 1:400 in 1% bovine serum albumin
in PBS, pH 7.3. The cells were washed and resuspended in
PBS for FACS analysis. Fluorescence and forward-angle light scatter
(FALS) data were acquired in linear or log scale as indicated in Figs.
2-5 and 8. Flow cytometric data were analyzed and quantitated with
WINMDI software (Scripps Institute, La Jolla, CA) with the Quandrant Statistics routine for compartmentation of bivariate histograms. When visual compartmentation of the histograms was not
possible, the histogram subtraction function of MULTI2D software (Phoenix Flow Systems, San Diego, CA) was used. Univariate DNA distribution data were compartmented into cell cycle phase fractions with the software MULTICYCLE (Phoenix Flow Systems); doublets were
eliminated from the compartmentation computations through software-resident curve-fitting algorithms based on 2N versus 4N peak
position.
Microscopy and image analysis.
Photomicroscopy was performed on an Olympus EMT-2
epifluorescence-phase-contrast microscope equipped with band-pass
filters for detection of FITC and rhodamine-propidium iodide,
respectively, and fitted with both color and gray-scale charge-coupled
device cameras. Cell volume was determined by automated measurement of
the ferret diameters of each of 50 cells/group with the image-analysis
program MOCHA (Jandel Scientific, San Rafael, CA). The ferret diameters
were converted to cell volumes by calculation. Quantitation of
fluorescence images was performed through intensity thresholding and
pixel summation algorithms resident in the MOCHA image-analysis
software.
 |
RESULTS |
Fibroblast size and light-scatter
profile. Unit gravity sedimentation (1 G) over an
eight-step gradient of Ficoll clearly resolved fibroblast subsets on
the basis of cell size. The most extreme differences in cell size were
observed between pooled gradient fractions
1-6 (Fig.
1A)
and
25-30
(Fig. 1B) immediately after removal
from the sedimentation chamber. The size difference was also evident
after 1 (Fig. 1, C and
D) and 15 days (Fig. 1, E and
F) of subsequent culture. At culture
day 1, the large cells were generally
of the "stellate" morphology and the small cells were primarily
"spindle" shaped; these phenotypes still differed in size on
culture day 15 (see
Fibroblast size and proliferation kinetics).

View larger version (102K):
[in this window]
[in a new window]
|
Fig. 1.
Phase-contrast microscopy of human lung fibroblast subpopulations
separated by unit gravity sedimentation (1 G). Relative size
differences were readily apparent in fibroblasts immediately after
pooling of Ficoll gradient fractions
1-6
(A) and
25-30
(B). Size gradient of the same
subsets was also evident after 1 (C
and D, respectively) and 15 (E and
F, respectively) days of primary
culture. Quantitation of cell volume in pooled gradient fractions is
reported in Fig. 3.
|
|
Comparison of pooled gradient fractions
1-6 and
25-30
also revealed the most extreme differences in light-scatter intensity. Figure 2 displays FALS profiles for
fractions
1-6 and
25-30
measured in conjunction with nuclear DNA content by propidium iodide
binding (22). Although heterogeneity of light-scatter intensity was evident in each sample, median FALS values were reproducible and easily
measured. The N12 fibroblast strain did not contain lipid inclusion
bodies detectable by phase-contrast microscopy or Oil Red O staining
(data not shown), and thus the light-scatter profiles of these cells
were not influenced by the presence of lipid-filled organelles. The
small bodies visible by light microscopy after time in culture (Fig. 1,
E and
F) were removed by washing and thus
were not intracellular.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 2.
Flow cytometric [fluorescence-activated cell sorter (FACS)]
analysis of light scatter and ploidy of human fibroblast subsets
separated by 1 G. Immediately after separation, cells in pooled Ficoll
gradient fractions
1-6
(A) and
25-30
(B) were fixed in ethanol and
subjected to FACS analysis of forward-angle light scatter (FALS) vs.
DNA content as previously described (19, 22).
1 and
2, diploid
(G0/G1
phase) and tetraploid (G2/M phase)
fibroblast populations, respectively, as detected by propidium iodide
fluorescence of RNase-treated cells. Quantitation of cell cycle phase
fractions is reported in Fig. 4 and text. Compare
x-axis position of
populations 1 and
2 relative to constant FALS marker at
channel 150 (dotted line).
|
|
Figure 3 plots the median FALS values for
each of five pooled gradient fractions as a function of cell volume
measured by static-image cytometry. With the exception of the smallest
group of fibroblasts (fractions
25-30), the
diameter of which approached that of bare nuclei (7.3 ± 0.6-µm
nuclear diameter vs. 8.44 ± 2.5-µm cell diameter), the median
FALS value correlated well with average cell volume
(r = 0.94). On the basis of this plot,
all subsequent measurements were made on either unfractionated cells or
Ficoll gradient fractions pooled into small ( group
I, fractions 25-30),
intermediate ( group II,
fractions
13-24), and
large ( group III,
fractions
1-12)
fibroblast subsets. In three experiments, the percentage of total cells
recovered in each of the three groups ranged from 23 to 28%
( group I), 38 to 42% ( group
II), and 30 to 39% ( group
III).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3.
Relationship of cell volume and FALS in ethanol-fixed human lung
fibroblasts. Immediately after separation, cells in pooled Ficoll
gradient fractions
1-6,
7-12,
13-18,
19-24,
and
25-30
were fixed in ethanol and subjected to FACS analysis of FALS (plotted
in log scale) as described in Fig. 2. Average cell volume was
determined by static image analysis of the same samples used for FACS
(see METHODS). Each point is mean ± SD of cell volume and median FALS channel for pooled fraction.
Line between fractions
19-24 and
1-6
is linear regression (r = 0.94). For
subsequent experiments, isolated fractions were combined into
groups I (small cells;
fractions
25-30),
II (medium cells;
fractions
13-24), and
III (large cells;
fractions
1-12).
|
|
Fibroblast size and proliferation
kinetics. DNA distribution data obtained by analyses of
propidium iodide binding (see Fig. 2) were compartmented by established
methods (22) to yield cell cycle phase fractions for each fibroblast
group. As shown in Fig. 4,
group II contained the highest
percentage of S and G2/M phase cells, roughly threefold higher than that of the unfractionated fibroblast strain. Groups I and
III exhibited cell cycle phase distributions of 82, 3, and 15% and 85, 4, and 11% for
G0/G1, S, and G2/M phases, respectively,
distributions essentially identical to the unfractionated N12 strain.
In addition, bivariate analysis of FALS and the thymidine analog BrdU
incorporated into the DNA of viable S phase cells (5) revealed that
BrdU incorporation was confined to fibroblasts of intermediate size
(Fig. 5). This was in contrast to BrdU
incorporation by a human lung epithelial cell line (Fig.
5B) and to primary alveolar type II
pneumocytes (19); in those cells, the analog was incorporated only by
cells of the highest relative size.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 4.
DNA distribution analysis of fibroblast subsets separated by 1 G. Ethanol-fixed fibroblasts were incubated with 5 µg/ml of
propidium iodide and DNase-free RNase for ploidy analysis by FACS
as described earlier (22). DNA distributions displayed are from
unfractionated (A) and
group II
(B) fibroblasts defined in Fig. 3.
Inset, percentage of cells in each
cell cycle phase. Groups I and
III exhibited percent distributions
nearly identical to unfractionated cells (see text). Experiment was
performed twice with similar results. For the 2 experiments, ranges of
cell cycle distributions were 81-86%
G0/G1,
4-7% S, and 10-12%
G2/M for unfractionated cells;
55-61%
G0/G1,
12-19% S, and 26-27%
G2/M for group
II; 82-84%
G0/G1,
4-6% S, and 10-12%
G2/M for group
I; and 80-85%
G0/G1,
3-7% S, and 8-13% G2/M
for group III.
|
|

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 5.
Relationship of human lung fibroblast size to incorporation of
5-bromo-2'-deoxyuridine (BrdU). Unfractionated primary N12 human
lung fibroblasts (A) and A549 human
lung carcinoma cell line (B) were
cultured to mid-log phase and were incubated for 1 h with 10 µM BrdU.
Cells were then harvested for FACS detection of incorporated BrdU (22)
vs. cell size measured as FALS. Note midrange FALS intensity of
BrdU-positive N12 fibroblasts (A,
top) and contrast that with A549
BrdU-positive subset of high-FALS intensity relative to unlabeled
population. Arrowheads, median FALS values for each group.
|
|
Consistent with these observations, group
II of the N12 human lung fibroblasts yielded the
highest rate of accumulation by 8 days of culture begun immediately
after separation by 1 G (Fig. 6). By
day 13 of culture, the rate of growth
of group I had surpassed that of
group II, but both
groups I and
II grew significantly faster than
group III. As shown in Fig. 6,
inset, cells of
groups I and
II increased in size by culture
day 13, but those of
group III did not. None of the three
fibroblast groups reached confluence by day
15 of culture (data not shown), and thus the slower
growth rates of group III and
eventually group II were unrelated to
density arrest.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 6.
Growth in primary culture of human lung fibroblasts separated by 1 G. Fibroblast groups I,
II, and
III defined in Fig. 3 were placed in
primary culture in presence of 10% fetal bovine serum for 15 days. At
the indicated days, cell number was estimated by WST-1 assay (see
METHODS). Each point is mean ± SD; where not visible, error bars fall within symbols.
Inset: cell volume measurements on
isolated groups
I-III on
days 0 and
13 of culture. Note increase in cell
size in groups I and
II but not in group
III. Significant difference compared with both other
groups on the same day of culture:
* P < 0.001;
** P < 0.05 (by ANOVA and
Student-Newman-Keuls test).
|
|
Fibroblast size, apoptosis, and
-actin
expression. To determine the relationship between
fibroblast phenotype, DNA fragmentation, and spontaneous apoptosis,
unfractionated N12 cells were harvested at mid-log phase and subjected
to ISEL of fragmented DNA (17). Microscopy of adherent cells (Fig.
7) revealed two populations of labeled
cells: fibroblasts with moderate (Fig. 7,
A and
B) and high (Fig. 7,
C and
D) intensity of ISEL labeling. Many
labeled cells displayed condensation of the labeled chromatin, nuclear fragmentation (Fig. 7D), and blebs
in the plasma membrane (Fig. 7C,
arrow), indicative of late apoptosis (21). Immunofluorescence detection
of
-SMA revealed both unlabeled cells (Fig.
7E, arrows) and cells
labeled with the monoclonal antibodies to varying degrees (Fig. 7F, same field as Fig.
7E).

View larger version (100K):
[in this window]
[in a new window]
|
Fig. 7.
In situ end labeling (ISEL) of fragmented DNA and immunofluorescence
detection of -smooth muscle actin ( -SMA) in primary human lung
fibroblasts. Shown are phase-contrast
(A,
C, and
E) and fluorescence
(B,
D, and
F) images of the same fields of
cells. N12 fibroblast strain was cultured to mid-log phase. Cells were
labeled by a modified ISEL protocol (25) or with FITC-conjugated
monoclonal antibodies to -SMA (see
METHODS). ISEL labeling of nuclei
was observed as negative, moderate (A
and B), or high intensity
(C and
D); some labeled cells displayed
chromatin condensation and nuclear fragmentation
(D) and blebbing of plasma membrane
(C, arrow), indicative of late
apoptosis (11, 21). Immunofluorescence detection of -SMA
(E and
F) revealed occasional heavily
labeled fibroblasts (F,
top left), moderate labeling
(F,
top), and many unlabeled fibroblasts
(E, arrows; compare with
F). See Figs. 8 and 9 for FACS and
imaging quantitations of ISEL and -SMA.
|
|
Flow cytometric analyses of FALS versus ISEL labeling (Fig.
8A)
resolved both the moderately and highly labeled subsets, which comprised 30.9 and 4.9%, respectively, of the total fibroblast population. In parallel experiments, labeling of viable fibroblast preparations with the sensitive apoptosis marker annexin V (Fig. 8B) also discriminated moderately
and highly labeled subsets, which were present in similar proportions.
Although the moderate ISEL subset was composed of both mid-FALS
( group II) and high-FALS ( group III) fibroblasts, the high-ISEL
and high-annexin V subsets were found to consist only of the large
group III cells. Similar analyses
(Fig. 8C) of FALS versus
immunoreactivity to
-SMA antibodies revealed that
-SMA expression
was also found only in group III fibroblasts.

View larger version (54K):
[in this window]
[in a new window]
|
Fig. 8.
Relationship of human lung fibroblast size to DNA fragmentation,
annexin V binding, and -SMA immunoreactivity.
A: unfractionated primary N12 human
lung fibroblasts were cultured to mid-log phase and harvested for FACS
detection of cell size, measured as FALS vs. fragmented DNA detected by
ISEL as described in Fig. 7. Note mid- to high-FALS profile of cells
with moderate ISEL labeling (M), and high-FALS profile of fibroblasts
with high ISEL labeling (H); compare FALS values of each to constant
FALS marker at channel 150 (dotted line). U, unlabeled fibroblast
population. M and H subsets comprised 30.9 and 4.9%, respectively, of
total fibroblast population. B:
unfractionated N12 cells in viable suspension were incubated with
fluorescein-conjugated annexin V and analyzed 1 h later (see
METHODS).
Populations M and
H comprised 21.1 and 3.2%,
respectively, of total fibroblasts. C:
N12 fibroblasts were cultured and harvested in the same way as in
A; cells were incubated with
FITC-conjugated monoclonal antibodies to -SMA (anti- -SMA; see
METHODS) and subjected to FACS
analysis of FALS vs. FITC fluorescence. Note high-FALS profile of cells
with positive -SMA labeling. Analysis was repeated 3 times with
similar results. D: control annexin
V-binding profile of viable suspension of A549 lung epithelial cells;
<5% displayed positive binding.
|
|
However, double labeling for ISEL and
-SMA (Fig.
9) revealed that the two
labels were not necessarily observed in the same cell.
-Actin-positive fibroblasts (Fig. 9, bracket) were found to be
either unlabeled or positively labeled by ISEL as were
-SMA-negative cells. Conversely, both groups of ISEL-labeled cells (moderate and
high; Fig. 9) were composed of either
-SMA-positive or -negative fibroblasts, as was the ISEL-negative population.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 9.
Bivariate analysis of DNA fragmentation and -SMA immunoreactivity in
primary human lung fibroblasts. N12 fibroblast strain was cultured as
described in Fig. 8. Adherent cells were subjected to double labeling
for ISEL and -SMA, each conducted as described in Fig. 7;
quantitation of both labels was performed by image analysis (see
methods). Bracket, -SMA-positive
(+) fibroblasts. M and H, same moderately and highly labeled cells,
respectively, detected by ISEL as in Fig. 8. Note that ISEL labeling is
found in both -SMA-negative and -positive fibroblast populations.
|
|
 |
DISCUSSION |
As discussed earlier by Bont et al. (2), unit gravity sedimentation (1 G) separates particles on the basis of size, independently of density.
This method therefore offered a theoretically feasible approach to
isolating the "large and flat" fibroblasts observed by our
laboratory and previously by other investigators (1, 15,
23). We found that the method easily resolved subsets of differing
volume (Fig. 2) and morphologies that were stable for up to 15 days in
culture. With the exception of the smallest fibroblast subset
(fractions
25-30), the
cells of which were only slightly larger than nuclei, cell volume
correlated well with FALS; this result confirms that FALS
will provide a reliable index of fibroblast size for future flow
cytometric studies of subpopulation dynamics.
The observation that group II
contained the greatest percentage of S and
G2/M phase cells (Fig. 4) and
virtually all BrdU-positive cells (Fig. 5) agrees with the model of
"balanced cell growth" discussed by Darzynkiewicz et al. (3).
In that concept of cell cycle progression, quiescent cells entering the
cell cycle must increase cellular RNA content (primarily ribosomal) and
protein content (and thus cell size) to a value above some threshold
level; reaching the threshold is a prerequisite to passage of the
"restriction point," believed to reside at the
G1/S phase border (4). Within group I fibroblasts, the small cell
size and paucity of S phase cells is consistent with the designation of
this subset as quiescent but capable of proliferation.
This interpretation is also supported by the growth curves in Fig. 6;
group I fibroblasts lagged behind
group II in their rate of accumulation
by 8 days of culture but surpassed both groups II and III by 13 days.
The latter observation suggests that within group
I competent fibroblasts entered the cell cycle between
8 and 13 days of culture, whereas many of the group
II cells were already in the cell cycle at the time of
1 G separation. In support of this view, the small
group I cells transformed into the
larger group II phenotype during the
8- to 13-day culture interval, indicating that balanced cell growth was
maintained after 1 G separation (Fig. 6,
inset).
Group III, the largest fibroblasts,
grew most slowly at all culture times (Fig. 6) and was among the two
groups ( groups II and
III) that contained ISEL-labeled
apoptotic cells (Figs. 7 and 8). These observations suggest that the
slow rate of growth of group III may
be the result of a high rate of spontaneous apoptosis relative to cell
division. Although cells labeled by ISEL were found in both
groups II and
III, group
II contained the highest proportion of S phase cells
(Figs. 4 and 5); these would be expected to offset cell death by
apoptosis, and the growth curves in Fig. 6 are consistent with this
interpretation. In this regard, it is interesting to note that the low
C1q-binding subset of human lung fibroblasts identified by Akamine et
al. (1) also displayed a poor rate of growth in culture despite
containing a higher percentage of cycling cells identified by flow
cytometry. This paradox might also be explained by a higher rate of
spontaneous apoptosis within this subpopulation, which exhibited the
same morphological characteristics (large and flat) as
group III of the present study. The
methods described here will offer an ideal approach to addressing this issue. These methods will also be useful in examining the relationship of lipid inclusion bodies (15) to fibroblast phenotype when coupled
with fluorescent lipophilic probes. The fibroblast strain studied here,
however, did not contain significant numbers of lipid inclusions to
permit such an analysis.
The presence of both immunoreactivity to
-actin antibodies and ISEL
labeling in group III suggested that
fibroblast apoptosis might be associated with the acquisition of the
myofibroblast phenotype. Such an association was suggested earlier (6)
and was supported by the finding of apoptosis in myofibroblast
populations within granulation tissue transforming to scar (7). Our
double-labeling data (Fig. 9) argue against such an association because
ISEL labeling was clearly observed in both
-actin-negative and
-positive fibroblasts in comparable proportions. In addition,
-actin
expression by primary isolates of rat lung mesenchymal cells has been
observed not only in large "lacy" cells but also in a group of
smaller, more tightly packed clones (13). Whether these discrepancies are due to species differences, cell culture conditions, or other factors awaits further investigation.
In summary, we describe a simple and reproducible unit gravity
sedimentation method for the isolation of human lung fibroblast subsets
in high yield on the basis of cell size. Kinetic and flow cytometric
analyses identified three size subsets corresponding to young quiescent
( group I), rapidly proliferating
( group II), and large slow-growing
( group III) groups. Under conditions of log-phase growth, only group III
exhibited expression of
-SMA, but double-labeling studies indicated
that both
-actin-positive and -negative cells were capable of
spontaneous apoptosis. These results suggest that
-actin expression
by lung fibroblasts does not necessarily precede or accompany a
commitment to apoptosis. The ease and high yield of the method
described will facilitate future investigation of the functions and
interrelationships of known fibroblast subsets.
 |
ACKNOWLEDGEMENTS |
We thank the Department of Medicine, University of Illinois at
Chicago, for administrative assistance in arranging the visit of
Research Scholar C. Ramos of the Instituto Nacional de Enfermedades Respiratorias de Mexico (Tlalpan).
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-45136 to B. D. Uhal; the Women's Board Endowment to the
Research and Education Foundation of the Michael Reese Medical Staff;
and the Universidad Nacional Autonoma de Mexico.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: B. D. Uhal, Cardiovascular Institute,
Michael Reese Hospital, 2929 S. Ellis Ave., Rm. 405KND, Chicago, IL
60616.
Received 14 April 1998; accepted in final form 10 August 1998.
 |
REFERENCES |
1.
Akamine, A.,
G. Raghu,
and
A. S. Narayanan.
Human lung fibroblast subpopulations with different Clq binding and functional properties.
Am. J. Respir. Cell Mol. Biol.
6:
382-389,
1992[Medline].
2.
Bont, W. S.,
J. E. DeVries,
M. Geel,
A. Van Dongen,
and
H. A. Loos.
Separation of human lymphocytes and monocytes by velocity sedimentation at unit gravity.
J. Immunol. Methods
29:
1-16,
1979[Medline].
3.
Darzynkiewicz, Z.,
H. Crissman,
F. Traganos,
and
J. Steinkamp.
Cell heterogeneity during the cell cycle.
J. Cell. Physiol.
113:
465-474,
1982[Medline].
4.
Darzynkiewicz, Z.,
T. Sharpless,
L. Staiano-Coico,
and
M. R. Melamed.
Subcompartments of the G1 phase of cell cycle detected by flow cytometry.
Proc. Natl. Acad. Sci. USA
77:
6696-6699,
1980[Abstract].
5.
Derdak, S.,
D. P. Penney,
P. Keng,
M. E. Felch,
D. Brown,
and
R. P. Phipps.
Differential collagen and fibronectin production by Thy 1+ and Thy 1
lung fibroblast subpopulations.
Am. J. Physiol.
263 (Lung Cell. Mol. Physiol. 7):
L283-L290,
1992[Abstract/Free Full Text].
6.
Desmouliere, A.,
and
G. Gabbiani.
The role of the myofibroblast in wound healing and fibrocontractive diseases.
In: The Molecular and Cellular Biology of Wound Repair. New York: Plenum, 1996, p. 391-423.
7.
Desmouliere, A.,
M. Redard,
L. Darby,
and
G. Gabbiani.
Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar.
Am. J. Pathol.
146:
56-66,
1995[Abstract].
8.
Fries, K. M.,
T. Blieden,
R. J. Looney,
G. D. Sempowski,
M. R. Silvera,
R. A. Willis,
and
R. P. Phipps.
Evidence of fibroblast heterogeneity and the role of fibroblast subpopulations in fibrosis.
Clin. Immunol. Immunopathol.
72:
283-292,
1994[Medline].
9.
Gabbiani, G.
The biology of the myofibroblast.
Kidney Int.
41:
530-532,
1992[Medline].
10.
Gorczyca, W.,
J. Gong,
and
Z. Darzynkiewicz.
Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays.
Cancer Res.
53:
1945-1951,
1993[Abstract].
11.
Grasl-Kraup, B.,
B. Ruttkay-Nedecky,
H. Koudelka,
K. Bukowska,
W. Bursch,
and
R. Schulte-Hermann.
In situ detection of fragmented DNA fails to discriminate among apoptosis, necrosis and autolytic cell death: a cautionary note.
Hepatology
21:
1465-1468,
1995[Medline].
12.
Kuhn, C.,
and
J. McDonald.
The roles of the myofibroblast in idiopathic pulmonary fibrosis.
Am. J. Pathol.
138:
1257-1265,
1991[Abstract].
13.
Mitchell, J.,
J. Woodcock-Mitchell,
L. Perry,
J. Zhao,
R. Low,
L. Baldor,
and
P. Absher.
In vitro expression of the
-smooth muscle actin isoform by rat lung mesenchymal cells: regulation by culture condition and transforming growth factor-
.
Am. J. Respir. Cell Mol. Biol.
9:
10-18,
1993[Medline].
14.
Pardo, A.,
and
M. Selman.
Decreased collagenase production by fibroblasts derived from idiopathic pulmonary fibrosis.
Matrix Suppl.
1:
417-448,
1992[Medline].
15.
Phipps, R. P.,
D. P. Penney,
P. Keng,
H. Quill,
A. Paxhia,
S. Derdak,
and
M. E. Felch.
Characterization of two major populations of lung fibroblasts: distinguishing morphology and discordant display of Thy 1 and class II MHC.
Am. J. Respir. Cell Mol. Biol.
1:
65-74,
1989[Medline].
16.
Reif, A. E.,
and
J. Allen.
The AKR thymic antigen and its distribution in leukemias and nervous tissues.
J. Exp. Med.
120:
413-433,
1964.
17.
Sempowski, G. D.,
M. P. Beckmann,
S. Derdak,
and
R. P. Phipps.
Subsets of murine lung fibroblasts express membrane-bound and soluble IL-4 receptors. Role of IL-4 in enhancing fibroblast proliferation and collagen synthesis.
J. Immunol.
152:
3606-3614,
1994[Abstract/Free Full Text].
18.
Skalli, W.,
T. Schurch,
R. Seemayer,
D. Lagace,
B. Montandon,
P. Gabbinai,
and
G. Gabbiani.
Myofibroblasts from diverse pathologic settings are heterogeneous in their content of actin isoforms and intermediate filament proteins.
Lab. Invest.
60:
275-285,
1989[Medline].
19.
Uhal, B. D.,
and
M. D. Etter.
Type II pneumocyte hypertrophy without activation of surfactant biosynthesis after partial pneumonectomy.
Am. J. Physiol.
264 (Lung Cell. Mol. Physiol. 8):
L153-L159,
1993[Abstract/Free Full Text].
20.
Uhal, B. D.,
G. D. Hess,
and
D. E. Rannels.
Density-independent isolation of type II pneumocytes after partial pneumonectomy.
Am. J. Physiol.
256 (Cell Physiol. 25):
C515-C521,
1989[Abstract/Free Full Text].
21.
Uhal, B. D.,
I. Joshi,
A. True,
S. Mundle,
A. Raza,
A. Pardo,
and
M. Selman.
Fibroblasts isolated after fibrotic lung injury induce apoptosis of alveolar epithelial cells in vitro.
Am. J. Physiol.
269 (Lung Cell. Mol. Physiol. 13):
L819-L828,
1995[Abstract/Free Full Text].
22.
Uhal, B. D.,
and
D. E. Rannels.
DNA distribution analysis of type II pneumocytes by laser flow cytometry: technical considerations.
Am. J. Physiol.
261 (Lung Cell. Mol. Physiol. 5):
L296-L306,
1991[Abstract/Free Full Text].
23.
Vande Berg, J. S.,
R. Rudolph,
and
M. Woodward.
Comparative growth dynamics and morphology between cultured myofibroblasts from granulating wounds and dermal fibroblasts.
Am. J. Pathol.
114:
187-200,
1984[Abstract].
24.
Vande Berg, J. S.,
R. Rudolph,
and
M. Woodward.
Growth dynamics of cultured myofibroblasts from human breast cancer and nonmalignant contracting tissues.
Plast. Reconstr. Surg.
73:
605-618,
1984[Medline].
25.
Wijsman, J. H.,
R. R. Jonker,
R. Keijzer,
C. J. H. Van De Velde,
C. J. Cornelisse,
and
J. H. Van Dierendonck.
A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA.
J. Histochem. Cytochem.
41:
7-12,
1993[Abstract/Free Full Text].
26.
Willis, R. A.,
A. K. Nussler,
K. M. Fries,
D. A. Geller,
and
R. P. Phipps.
Induction of nitric oxide synthase in subsets of murine pulmonary fibroblast: effect on fibroblast interleukin-6 production.
Clin. Immunol. Immunopathol.
71:
231-239,
1994[Medline].
Am J Physiol Lung Cell Mol Physiol 275(5):L998-L1005
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society