Alveolar epithelial cell death adjacent to underlying
myofibroblasts in advanced fibrotic human lung
Bruce D.
Uhal1,
Iravati
Joshi1,
W. Frank
Hughes2,
Carlos
Ramos3,
Annie
Pardo4, and
Moises
Selman3
1 The Cardiovascular Institute,
Michael Reese Hospital, Chicago 60616;
2 Department of Anatomy, Rush
Medical College, Chicago, Illinois 60612;
4 National Autonomous University
of Mexico, Coyoacan 04000; and
3 National Institute of
Respiratory Diseases, Tlalpan 14080, Mexico
 |
ABSTRACT |
Earlier work from this laboratory showed that
abnormal fibroblast phenotypes isolated from fibrotic human lung
produce factor(s) capable of inducing apoptosis and necrosis of
alveolar epithelial cells in vitro [B. D. Uhal, I. Joshi, A. True, S. Mundle, A. Raza, A. Pardo, and M. Selman. Am.
J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L819-L828, 1995]. To determine
whether epithelial cell death is associated with proximity to abnormal
fibroblasts in vivo, the spatial distribution of epithelial cell loss,
DNA fragmentation, and myofibroblasts was examined in the same tissue specimens used previously for fibroblast isolation. Paraffin sections of normal and fibrotic human lung were subjected to in situ end labeling (ISEL) of fragmented DNA and simultaneous immunolabeling of
-smooth muscle actin (
-SMA); replicate samples were subjected to
electron microscopy and detection of collagens by the picrosirius red
technique. Normal human lung exhibited very little labeling except for
positive
-SMA immunoreactivity of smooth muscle surrounding bronchi
and vessels. In contrast, fibrotic human lung exhibited moderate to
heavy ISEL of interstitial, cuboidal epithelial, and free alveolar
cells. ISEL of the alveolar epithelium was not distributed uniformly
but was most intense immediately adjacent to underlying foci of
-SMA-positive fibroblast-like interstitial cells. Both electron
microscopy and picrosirius red confirmed epithelial cell apoptosis,
necrosis, and cell loss adjacent to foci of collagen accumulation
surrounding fibroblast-like cells. These results demonstrate that the
cuboidal epithelium of the fibrotic lung contains dying as well as
proliferating cells and support the hypothesis that alveolar epithelial
cell death is induced by abnormal lung fibroblasts in vivo as it is in vitro.
cuboidalization; type II pneumocytes; programmed cell death; contractile interstitial cells
 |
INTRODUCTION |
THE CELLULAR AND MOLECULAR EVENTS leading to
fibrogenesis in the lung are believed to be intimately related to
alveolitis and alveolar epithelial repair processes (11).
Investigations of the damage caused by hyperoxic gas on murine whole
lung tissue (2) or explants derived from such tissue (3) were the first to suggest that repair of alveolar epithelial damage was critical for
healing without fibrosis. Subsequent studies (17, 18) of the ability of
oxygen to potentiate lung damage induced by butylated hydroxytoluene
supported the hypothesis that the severity of fibrogenesis was
inversely related to the efficiency of alveolar epithelial repair.
Consistent with that theory is the observation that foci of
accumulating fibroblasts in human lung tissue are found in close
proximity to unrepaired or abnormal alveolar epithelium (22).
In the fibrotic human lung and in animal models of pulmonary fibrosis,
the alveolar epithelium can be absent or metaplastic and often is
primarily cuboidal rather than the normal mixture of squamous type I
cells interspersed with cuboidal type II cells (11, 33). Electron
microscopy of human tissue suggests that the cuboidal cells are of both
alveolar and bronchiolar origin (21), but the relative importance of
alveolar versus bronchial cells in the genesis of new alveolar
epithelium has not been explored rigorously. For many years, it has
been believed that the cuboidal epithelium is composed of rapidly
proliferating cells that are attempting to repair the denuded
epithelium (11, 21, 26). It has been suggested that the high ratio of
cuboidal to squamous cells in the abnormal epithelium might be due to
an inability of dividing type II cells to differentiate into type I
cells, possibly as a result of altered composition or integrity of the underlying extracellular matrix (26).
Earlier work from this laboratory (38) demonstrated that fibroblasts
isolated from fibrotic human or rat lung produce factor(s) capable of
inducing apoptosis and necrosis of alveolar epithelial cells in vitro.
These observations suggested that apoptosis might be an important
mechanism of epithelial cell death in the fibrotic lung. Consistent
with that theory, apoptotic alveolar epithelial cells have been
recently observed in sections of fibrotic lung of both human and mouse
origin (15, 24). We hypothesized that the abnormal
fibroblast phenotypes that emerge in fibroblastic foci might constitute
an important source of factor(s) capable of inducing epithelial cell
apoptosis. In the lung, these phenotypes are known to include the
myofibroblast subtype termed "VA" that expresses the intermediate
filaments vimentin and
-smooth muscle actin (
-SMA) (23).
Accordingly, we further theorized that the cuboidal epithelium might
contain apoptotic and/or necrotic cells in addition to
proliferating cells, particularly in proximity to the VA fibroblast
subtype. To test this hypothesis, we examined the distribution of dying
cells within the cuboidal epithelium relative to underlying fibrotic
foci in the same biopsy samples that we used earlier for fibroblast
isolation (38). We report that the cuboidal epithelium of fibrotic
human lung contains apoptotic and necrotic cells in close proximity to
foci of
-actin-positive fibroblasts and collagen accumulation.
 |
METHODS |
Materials. Monoclonal
antibodies to
-SMA (clone 1A4) and biotinylated dUTP were obtained
from Sigma Immunochemicals (St. Louis, MO). DNA polymerase I was
obtained from Promega (Madison, WI). Solutions
A and B of the
Vectastain ABC Elite formulation were purchased from Vector
Laboratories (Burlingame, CA). All other materials were of reagent grade.
Tissue samples and handling. Human
lung tissue was obtained by open lung biopsies performed at the
National Institute of Respiratory Diseases (Tlalpan, Mexico). Lung
tissue was obtained from six patients with idiopathic pulmonary
fibrosis (IPF). All patients had clinical, functional, and radiological
features that fulfill the diagnostic criteria for an interstitial lung
disease (29, 32). Briefly, all had progressive dyspnea,
bilateral reticulonodular images on chest roentgenogram, restrictive
lung functional impairment with decreased lung volumes and compliance,
and hypoxemia at rest that worsened with exercise. Patients with IPF
had neither antecedents of any occupational or environmental exposure
nor other known cause of interstitial lung disease; samples from these
patients were the same as those designated human IPF
(HIPF) in the earlier study by Uhal et al. (38) of primary fibroblasts
isolated from fibrotic human and rat lung tissue. Normal lung tissue
was obtained from three individuals having a lobectomy for removal of a
primary lung tumor. No histopathological evidence of disease was found in those tissue samples. All tissue was fixed in 10% neutral-buffered Formalin for 16 h and embedded in paraffin. Sections were cut at
6.0-µm thickness and mounted on glass coverslips.
In situ end labeling and
immunohistochemistry. Tissue sections were
deparaffinized by being passed through xylene, 1:1 xylene-alcohol, 100% alcohol, and 70% alcohol for 10 min each. In situ end labeling (ISEL) of fragmented DNA was conducted by a modification of the method
of Mundle et al. (27). Briefly, ethanol was removed by rinsing in
distilled water for at least 10 min. The coverslips were then placed in
0.23% periodic acid (Sigma) for 30 min at 20°C. Samples were
rinsed once in water and three times in 0.15 M phosphate-buffered
saline (PBS) for 4 min each and were then incubated in saline-sodium
citrate solution (0.3 M NaCl and 30 mM sodium citrate in water, pH 7.0)
at 80°C for 20 min. After four rinses in PBS and four rinses in
buffer A (50 mM
Tris · HCl, 5 mM
MgCl2, 10 mM
-mercaptoethanol,
and 0.005% BSA in water, pH 7.5), the coverslips were incubated at
18°C for 2 h with ISEL solution (0.001 mM biotinylated dUTP, 20 U/ml of DNA polymerase I, and 0.01 mM each dATP, dCTP, and dGTP in
buffer A). Afterward, the sections
were rinsed thoroughly five times with buffer
A and three additional times in 0.5 M PBS. For
detections based on diaminobenzidine, the tissue was then incubated at
20°C with a solution consisting of 80 µl each of
solutions A (avidin
solution) and B (biotin-peroxidase solution) of the Vectastain Elite Kit in 3.84 ml of
buffer B (1% BSA and 0.5% Tween 20 in 0.5 M PBS). After 30 min, the sections were washed four times in PBS
and were then immersed for 10 min in a solution of 0.25 mg/ml of
diaminobenzidine in 0.05 M Tris · HCl, pH 7.5, containing 0.01% hydrogen peroxide. Alternatively, detection of
incorporated dUTP was achieved with a fast blue chromogen system. The
tissues were rinsed in distilled water three times and mounted under
Fluoromount solution (Southern Biotechnology Associates, Birmingham, AL).
For double labeling with ISEL and antibodies to
-SMA, fluorescein
(FITC)-conjugated monoclonal antibodies to
-actin were diluted 1:100
in ISEL solution and incubated with the tissue as described above for
single ISEL. Control samples, treated as described above but without
DNA polymerase I, gave no positive signal (data not shown).
Identification of collagen. Collagens
were localized through staining of lung sections by the picrosirius red
technique (30). Briefly, sections deparaffinized with xylene were
rehydrated through a descending series of ethanols to distilled water.
The hydrated sections were immersed in 0.2% aqueous phosphomolybdic
acid for 2 min, rinsed in distilled water, and then stained for 110 min with 0.1% sirius red F3BA (Pfaltz and Bauer, Stamford, CT) in saturated aqueous picric acid, pH 2.0. The sections were washed for 2 min in 0.01 N HCl, rinsed for 45 s in 70% ethanol, dehydrated in three
changes of absolute alcohol, cleared in xylene, and mounted in
Permount. Fibrotic foci were detected by polarization microscopy, under
which collagen fibers appear yellow-red (30) or white in gray-scale images.
Microscopy and image analysis. The
prepared sections were photographed under transmitted, epifluorescent,
or polarized light on an Olympus BH2 epifluorescence microscope fitted
with automatic photographic equipment. For electron microscopy,
portions of some biopsies were fixed in 1% gluteraldehyde in
cacodylate buffer. The tissues were osmicated and stained with uranyl
acetate, dehydrated with a graded series of alcohol solutions, and
embedded in Epon/Araldite. Thin sections were examined and photographed
on a Philips EM 300 instrument in the Electron Microscopy Facility,
Rush Medical College (Chicago, IL).
For image analysis of ISEL and
-SMA labeling, transmitted light and
fluorescence digital images were obtained by scanning color
photographic slides made with tungsten film. Scanned images were first
converted to 256-level gray scale, then to pseudocolor on the basis of
intensity thresholds derived by dividing each 256-level gray scale into
three equal levels of intensity (low, moderate, and high). Once
established, the same intensity thresholds were applied to all images
processed for colocalization studies (see Fig. 5).
For analyses of the relationship between epithelial cell death and
immunoreactivity to
-SMA (Table 1), ISEL
or
-SMA intensity thresholds were defined as described above. The
property of epithelial integrity was defined as the presence of
morphologically intact epithelium within 50 µm of any border of the
-SMA intensity threshold under evaluation. In the compilation of
data from a series of lung sections (Table 1), the scale for scoring
epithelial integrity was from zero to five marks (from least to
highest, respectively). A score of zero marks indicates complete
absence of epithelial cells overlying the stroma within 50 µm of the
indicated
-SMA threshold. A score of five marks reflects
morphologically intact epithelium within the entire scoring region.
The scale for scoring ISEL intensity also was from zero to five marks
(from least to highest, respectively). In the compiled intensity data,
a score of zero marks indicates that all epithelial cells within the
50-µm limit defined above display no ISEL. A score of five marks
indicates that all epithelial cells within the 50-µm limit display
the highest ISEL intensity. Regions in which both ISEL and
-SMA
labels exactly colocalized were scored at the highest ISEL intensity.
Processing of scanned images was performed with MOCHA image-analysis
software (Jandel Scientific, San Raphael, CA). The scoring of samples
from IPF patients was performed in a blinded fashion.
 |
RESULTS |
The histology of normal and fibrotic lung specimens is shown in Fig.
1,
A-C.
Tissue from patients with IPF showed patchy alveolar septal fibrosis
and interstitial inflammation consisting mostly of mononuclear cells
but also of neutrophils and eosinophils. A variable macrophage
accumulation was observed in the air spaces as was cuboidalization of
the alveolar epithelium. Biopsies lacked granulomas, vasculitis,
microorganisms, and inorganic material by polarized light microscopy
(4).

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Fig. 1.
Histology and in situ end labeling (ISEL) of biopsies of normal and
fibrotic lung tissue. A: hematoxylin
and eosin staining of normal lung tissue.
B and
C: hematoxylin and eosin
preparations of biopsies from 2 patients with idiopathic pulmonary
fibrosis (IPF). D: ISEL of fragmented
DNA in normal lung tissue. E:
ISEL of biopsy from same IPF patient as in
B; positive reaction is intense black
(actually blue generated by fast blue detection system). Bars, 50 µm;
B and
D are the same as
A.
|
|
ISEL of fragmented DNA in sections of normal human lung tissue revealed
very few labeled cells (Fig. 1D). In
contrast, sections prepared from lung tissue of patients with IPF (Fig.
1E) contained many labeled cells
within the interstitium, air spaces, and epithelium. Electron
microscopy of the alveolar epithelium revealed cells with morphological
characteristics of both apoptosis and necrosis (Fig.
2, B and
C); dying epithelial cells were
often observed in locations adjacent to fibrotic foci containing
numerous collagen bundles (Fig. 2B,
CB) interspersed with fibroblast-like interstitial cells (Fig.
2C, F).

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Fig. 2.
Electron microscopy of alveolar epithelial cells in fibrotic human
lung. A: alveolar epithelial cells
(AECs) distal to fibrotic foci have uniform cytoplasmic density and
abundant microvillae and mitochondria, which appear normal.
B: significant number of AECs near
fibrotic foci contain swollen cytosol characteristic of necrosis. Note
collapsed microvillae and collagen bundles (CB) immediately underlying
epithelial cell. C: many AECs adjacent
to fibrotic foci display characteristics of both necrosis and
apoptosis, such as basolateral swelling together with condensed and
marginated chromatin (arrow) and apical shrinkage (9, 36). Abundant
collagen bundles underlying these cells were interspersed with
fibroblast-like interstitial cells (F). All samples were from a patient
with IPF. Magnification, ×5,000.
|
|
Staining of lung sections by the picrosirius red technique (Fig.
3) confirmed the presence of collagen in
these foci and suggested a relationship between the locations of
fibrotic foci and epithelial cell death. The condensed nuclei of
apoptotic cells were found within the epithelium near regions of
moderate accumulation of collagen (Fig.
3C); the epithelium adjacent to
regions of very dense collagen accumulation was clearly injured and
often absent (Fig. 3E).

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Fig. 3.
Identification of collagen in normal and fibrotic human lung.
Deparaffinized biopsy sections were stained with picrosirius red
technique (30). B,
D, and
F: polarization microscopy of the same
fields in A,
C, and
E, respectively, under transmitted
light. Under polarized light, collagen appears white.
A and
B: normal human lung. Small amounts of
collagen are seen only in thickest parts of interstitium but not in
alveolar septa. Bar, 50 µm. C and
D: area of moderate accumulation of
collagen in thickened septum in biopsy from a patient with IPF. Note
condensed nucleus surrounded by halo in adjacent epithelium (C,
arrowhead). Bar, 20 µm. E and
F: area of severe accumulation of
collagen within septum of a separate IPF patient. Note
damaged or absent epithelium adjacent to most abundant collagen
(E, arrows). Bar, 20 µm.
|
|
To determine whether alveolar epithelial cell death was related to the
proximity of the affected epithelium to abnormal interstitial cells
within the fibrotic foci, double labeling of fibrotic lung specimens
was performed with ISEL and simultaneous immunolabeling of
-SMA. The
specificity of the antibodies to
-SMA was demonstrated first with
sections of normal human lung, in which immunoreactivity was found only
within the smooth muscle underlying vessels or bronchi (Fig.
4). In contrast, sections of fibrotic human
lung were immunoreactive to anti-
-SMA in numerous fibroblastic foci scattered throughout the parenchyma; one of these foci is displayed in
Fig. 5. The
-SMA labeling (Fig.
5B) was extensive in the
interstitium immediately underlying "sheets" of in situ
end-labeled cuboidal epithelium (Fig.
5A, arrowheads). In isolated areas
corresponding to the most intense
-SMA immunoreactivity (Fig.
5C, green areas), the adjacent
alveolar epithelium was absent or partially detached from the
underlying stroma (arrows).

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Fig. 4.
Specificity of antibodies to -smooth muscle actin ( -SMA).
A: phase-contrast micrograph of
deparaffinized section of normal human lung. Note large airway at
left.
B: fluorescence micrograph of same
field shown in A. Section was
immunolabeled with FITC-conjugated monoclonal antibodies to -SMA.
Note immunoreactivity of smooth muscle underlying airway wall.
Magnification, ×100.
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Fig. 5.
Double labeling of fibrotic human lung with ISEL and antibodies to
-SMA. A: in situ end-labeled lung
section from a patient with IPF. Positive labeling is brown color of
diaminobenzidine detection system. Note "sheets" of ISEL-positive
cuboidal epithelium (arrowheads). B:
fluorescence image of the same field shown in
A. Section also was immunostained with
FITC-conjugated monoclonal antibodies to -SMA during ISEL procedure
as described in METHODS.
C: composite pseudocolor image of ISEL
and -SMA labeling superimposed.
Inset: pseudocolor thresholds denote
light, moderate, and heavy intensity of each label, increasing from
left to
right. Pseudocolor black (far
right) denotes regions in which both
labels colocalized at highest intensity. Note ISEL of moderate
intensity (red) or, less frequently, of high intensity (blue) adjacent
to interstitial cells reacting moderately with -SMA (yellow). More
often, epithelium is absent (arrows) in areas adjacent to most intense
-SMA labeling (green). Bar, 50 µm. See Table 1 for evaluation of
relationship between ISEL and -SMA.
|
|
The pseudocolor image (Fig. 5C)
displays three intensity threshold levels for both ISEL (white, red,
and blue) and
-SMA labeling (white, yellow, and green), constructed
from images of each label analyzed individually. These thresholds were
used to obtain a semiquantitative evaluation of the relationship
between the two labels in a series of fibrotic lung samples. Table 1
displays the results of this analysis. Scoring of epithelial integrity, defined as the presence of intact epithelium within 50 µm of
-actin immunoreactivity, revealed that the most damaged or absent
epithelia were those in close proximity to interstitia that reacted
most intensely with
-SMA antibodies (
-SMA intensity level 3, green). The remaining epithelial cells were then scored on the basis of ISEL intensity as defined in the pseudocolor image; the most intense ISEL of epithelia was found adjacent to foci of the most intense
-SMA labeling. The epithelial layer adjacent to interstitia of light
or absent
-SMA immunoreactivity (intensity level 1, white) was of
higher integrity and relatively free of ISEL.
 |
DISCUSSION |
It is generally accepted that alveolitis and damage to the alveolar
epithelium are important antecedents of fibrogenesis in the lung. As
discussed by Crystal et al. (11), data supporting this concept come
from both animal models and studies of familial cases of IPF in which
the children of afflicted parents exhibit alveolitis well before
clinical evidence of the disease (7). In studies of intact animal
models, the experimental inhibition of alveolar repair in mice
previously injured with butylated hydroxytoluene led to an acceleration
of fibrogenesis (17, 18). Similarly, mouse lung explants with severe
epithelial damage induced by prior hyperoxic lung injury exhibited
marked interstitial cell proliferation and collagen deposition in
culture, but explants of less severely injured tissue did not (3). As
detailed by Witschi (39), these observations suggested that the
critical determinant in the pathway to fibrogenesis was not the
infiltrating inflammatory cells but rather the epithelial damage and
its repair. Alveolar epithelial repair normally occurs through the
proliferation and differentiation of type II alveolar epithelial cells,
a process that is believed to be regulated by factors produced by lung
fibroblasts (25, 28). The many important "antifibrotic" functions
of the intact alveolar epithelium have been discussed in detail by
Simon (35).
Cuboidalization of the alveolar epithelium is a common feature of the
fibrotic lung regardless of the animal species or the initiating
stimuli (11, 33, 35). Numerous investigations (1, 13, 19, 20) of animal
models of lung injury have documented a transient cuboidalization as
type II pneumocytes proliferate to replace damaged or lost type I
cells. Largely on the basis of these observations, it has been presumed
that epithelial cuboidalization in the fibrotic human lung is
indicative of sustained epithelial proliferation in the absence of
appropriate factors to signal the cessation of cell division and the
initiation of subsequent differentiation (26, 35). This interpretation, however, fails to consider other possible mechanisms underlying cuboidalization, such as reversion of type I cells into type II cells
by transdifferentiation (12, 34) or rapid loss of type I cells by
apoptosis (37, 38).
Apoptotic cells were observed in the bronchial and alveolar epithelia
of mice made fibrotic by intratracheal administration of bleomycin
(15); the apoptotic cells appeared 1 day after bleomycin administration
and reappeared during the progression of fibrosis at
days
7-14 after
administration. Although bleomycin is known to induce apoptosis in
isolated alveolar macrophages (16), it is not clear whether the agent
induces apoptosis directly on alveolar epithelial cells, and thus it is
unknown whether epithelial cell apoptosis is a common mechanism
underlying fibrogenesis in the mouse lung or an unrelated consequence
of direct damage by bleomycin. On the other hand, ligation of Fas
antigen with antibodies to Fas induced epithelial apoptosis and
pulmonary fibrosis in mice, suggesting involvement of apoptosis
in the pathogenesis of pulmonary fibrosis (14).
Our work suggests that the cuboidal epithelium of fibrotic human lung
contains dying as well as proliferating cells, at least during the late
stage of disease progression at which the tissue biopsies were
obtained. Although the study of samples from advanced fibrotic lung may
not yield information relevant to early events in the pathogenesis of
fibrosis, we contend that the contribution of epithelial cell death to
failure of epithelial repair likely deepens and extends the committment
to fibrogenesis. As discussed by Simon (35), intact alveolar epithelium
produces prostaglandins of the E series, known inhibitors of fibroblast
proliferation (6, 10), and protects underlying interstitial cells from exposure to growth factors released by intra-alveolar macrophages and
other inflammatory cells. For these reasons, failure of the epithelium
to repair may be equally detrimental in advanced fibrosis as well as in
the genesis of nascent fibrotic foci. In this study, the finding of
apoptotic and necrotic epithelial cells overlying fibroblastic foci in
the particular specimens examined is consistent with the earlier study
by Uhal et al. (38) of factors produced by fibroblasts isolated from
the same biopsy samples.
Our data also confirm the observations of Kuwano et al. (24) that DNA
fragmentation and apoptosis in the alveolar epithelium are prominent
features of fibrotic human lung tissue with no known prior exposure to
bleomycin or other potential xenobiotic inducers of apoptosis.
Apoptosis of mesenchymal cells is believed to constitute an important
and necessary component of the resolution of granulation tissue in the
lung (31); intuitively, apoptotic cells would be expected in a grossly
hypercellular tissue that is attempting to restore normal architecture.
The notion that excess alveolar epithelial cells are also eliminated by
programmed cell death is supported by the recent study of Bardales et
al. (5), who found that apoptotic alveolar epithelial cells were
relatively abundant in the lungs of patients with resolving acute lung
injury but not in tissue samples taken during the proliferative phase of chronic interstitial pneumonia. Those findings are in contrast to
the data of Hagimoto and colleagues (14, 15), which suggest a role for
apoptosis in the genesis of fibrosis as well as in its resolution.
Abnormal epithelial-mesenchymal interactions in lung injury were first
speculated to be important on the basis that unusual cell contacts
between fibroblasts and alveolar epithelial cells were observed in
butylated hydroxytoluene-injured mouse lung and in lung tissue from
patients with IPF (8). In the human lung, the predominant fibroblast
subtype that emerges in fibroblastic foci has been designated as the
subtype VA, which expresses the intermediate filaments vimentin and
-SMA and has been termed the myofibroblast (23). This subtype may be
the same as that identified by immunoreactivity to anti-pC1, which
recognizes the COOH-terminal polypeptide of human procollagen type I;
pC1-positive cells were found adjacent to regions of severe epithelial
shedding or cuboidalization in lung tissue from patients with IPF (22). In light of the earlier study (38) of fibroblasts derived
from the same specimens studied here, our findings of apoptotic
epithelial cells in proximity to
-SMA-positive interstitial cells in
situ suggests a role for the VA fibroblast subtype in the induction of
epithelial cell death in vivo. Characterization of the primary fibroblast strains and the proteins they secrete is currently underway.
In summary, electron microscopy and ISEL of fragmented DNA revealed
that the cuboidal epithelium of fibrotic human lung is composed of both
proliferating and dying cells. ISEL and simultaneous immunolabeling for
-SMA revealed that apoptotic and necrotic epithelial cells were
observed primarily in proximity to underlying foci of
-actin-positive interstitial cells. These results are consistent
with a role for the myofibroblast in the production of inducers of
epithelial cell apoptosis and necrosis. We speculate that this abnormal
epithelial-mesenchymal interaction contributes to the pathogenesis
and/or exacerbation of fibrotic lung disease by preventing
normal epithelial repair and thereby deepening the commitment to fibrogenesis.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood
Institute Grant HL-45136 to B. D. Uhal and by Instituto Nacional de
Enfermedades Respiratorias, (Tlalpan, Mexico).
 |
FOOTNOTES |
Address for reprint requests: B. D. Uhal, The Cardiovascular Institute,
Michael Reese Hospital, 2929 S. Ellis Ave., Rm. 405KND, Chicago, IL
60616.
Received 27 May 1997; accepted in final form 27 August 1998.
 |
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