Division of Respiratory, Cell and Molecular Biology, School of Medicine, University of Southampton, Southampton General Hospital, Southampton, SO16 6YD, United Kingdom
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ABSTRACT |
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Restitution of an epithelial layer
after environmental or biological damage is important to maintain the
normal function of the respiratory tract. We have investigated the role
of transforming growth factor (TGF)- isoforms in the repair of
layers of 16HBE 14o
bronchial epithelial-derived cells
after damage by multiple scoring. ELISA showed that both latent
TGF-
1 and TGF-
2 were converted to their active forms 2 h
after wounding. Time-lapse microscopy showed that the addition of
TGF-
1, but not TGF-
2, progressively increased the rate of
migration of damaged monolayers at concentrations down to 250 pg/ml.
This increase was blocked by addition of a neutralizing TGF-
1
antibody. Phase-contrast microscopy and inhibition of proliferation
with mitomycin C showed that proliferation was not required for
migration. These results demonstrate that conversion of latent to
active TGF-
1 and TGF-
2 during in vitro epithelial wound repair
occurs quickly and that TGF-
1 speeds epithelial repair. A faster
repair may be advantageous in preventing access of environmental agents
to the internal milieu of the lung although the production of active
TGF-
molecules may augment subepithelial fibrosis.
transforming growth factor-; 16HBE 14o
bronchial
epithelial cell; cell migration; wound healing; primary bronchial
epithelial cells
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INTRODUCTION |
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TRANSFORMING GROWTH
FACTOR (TGF)- is a multifunctional cytokine with significant
regulatory effects on extracellular matrix production from mesenchymal
cells in the lung (8, 12). Produced in an inactive form
tethered to a latency-associated peptide (LAP) by covalent bonding
(17), its signaling through TGF-
receptors requires
exposure of the active site of the ligand either through conformational
change or through cleavage of the LAP (17). The three
isoforms secreted by mammalian cells have been studied extensively in
connection with wound healing in skin where, in contrast to TGF-
3,
TGF-
1 and TGF-
2 provide a quicker wound repair but induce more
scarring (34).
The bronchial epithelium forms a barrier between the external and internal milieu of the respiratory tract and consists of ciliated and secretory columnar cells attached to a basal cell layer forming a pseudostratified epithelium. The intact epithelium prevents passive movement of environmental agents by the paracellular route, which is blocked by tight junctions (2). Damage to the epithelium involving loss of columnar epithelial cells can impact on the effectiveness of this barrier, allowing antigen access to the underlying bronchial tissue, as has been established in animal models (13). The restitution processes involved in the repair of damage to the bronchial epithelium are progressive and follow a number of characteristic and identifiable steps. After damage to the epithelial layer, epithelial cells abutting the wound edge dedifferentiate into a migratory phenotype to quickly cover the area of damage (15). The formation of a fibrin-fibrinogen gel rich in leukocytes at this point protects the underlying tissue until cell-cell contact has been reestablished. Finally, redifferentiation provides an intact fully functioning epithelium (16). Restitution to an intact undifferentiated epithelial barrier after damage to the airway can take as little as 1 h in the guinea pig (14), whereas the time taken in the human airway has not been determined.
Damage to the epithelium of the conducting airways has long been
recognized to be a characteristic feature of asthma (27), with the shed epithelial cells, predominately ciliated columnar cells
(24), found in bronchoalveolar lavage fluid and the
morphological appearance of a single monolayer of epithelial cells seen
in samples from bronchoscopy (24). Thus the proportion of
epithelial cells in a repairing, dedifferentiated phenotype would be
increased in asthmatic subjects. There is also an increase in the
"activation" of epithelial cells after damage associated with
increased release of mediators, including interleukin-5, interleukin-8,
and granulocyte-macrophage colony-stimulating factor, which may affect
the immunological responses in the surrounding tissue (5,
32). The increase in subbasement membrane thickening resulting
from the deposition of collagens I and III by myofibroblasts in
the lamina reticularis (31) is well established
and implicates TGF- as a potential mediator in the pathogenesis of asthma.
Given the increased proportion of repairing epithelial cells in asthma,
this study investigated the changes in TGF-1 and TGF-
2 isoforms
produced during the wound repair process and their effect on the speed
of movement of the monolayer.
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MATERIALS AND METHODS |
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Cell culture.
The 16HBE 14o bronchial epithelial cell line was a kind
gift from Dr. D. Gruenert (Cardiovascular Research Institute,
University of California; see Ref. 9). Stock cultures were
routinely maintained in MEM with Earle's salts (Life Technologies,
Paisley, UK); supplemented with 10% FCS (Life Technologies), 20 mM
L-glutamine, 10 U/ml penicillin G, 10 µg/ml streptomycin
sulfate, and 2.5 µg/ml amphotericin B (Life Technologies); and
incubated at 37°C in a 5% CO2 incubator. Experimental
cultures were grown free from antibiotics for a minimum of 24 h
before the experimental protocol. Epithelial cells were used with a
maximum range of 20 passages.
Primary cell culture. Primary epithelial cells were obtained from tissue after lung resection. Mucosal strips were dissected from bronchial samples, and excess matrix material was removed. Strips of mucosa (1-2 mm) were placed on 24-well plates with the epithelial surface downward. Epithelial cultures were maintained in LHC-9 medium (Life Technologies; see Ref. 21) with 2% Ultroser G serum replacement (Life Technologies). All fibroblast-contaminated cultures were discarded.
RT-PCR.
mRNA was extracted using Trizol (Life Technologies) following the
manufacturer's instructions and was stored at 20°C in diethyl pyrocarbonate-treated water until use. mRNA (1 µg) was reverse transcribed into cDNA with avian myeloblastosis virus reverse transcriptase (Promega, Southampton, UK).
ELISA.
After damage to epithelial monolayers by the cross-hatch method (see
above) and subsequent culture for 5 min and 2, 6, 24, 48, and 72 h, the supernatants were removed and stored at 20°C. TGF-
1 and
TGF-
2 were analyzed using a sandwich ELISA system (Promega) with
monoclonal capture and polyclonal detection antibodies and
visualization with tetramethylbenzidine according to the
manufacturer's instructions. Cross-reactivity between all three
isoforms was quantified, by the manufacturer, as <5% for both
TGF-
1 and TGF-
2 antibodies. After the reaction was stopped with 1 M phosphoric acid, the plate was read on an ELISA plate reader at 450 nm, and all values were corrected using a standard curve.
Time-lapse microscopy methods.
The 16HBE 14o cells were grown to confluence in 35-mm
petri dishes (Greiner) and were damaged with a single scrape from a pipette tip. All culture medium and damaged cells were removed, and the
wounded monolayer was washed one time in medium and changed to fresh
serum-containing medium containing 100 mM HEPES. The cells were
incubated at 37°C in a 5% CO2 incubator for 1 h
before time-lapse analysis. For cultures involving treatment with
TGF-
1 or TGF-
2 (R&D Systems, Abingdon, UK), 0.25, 2.5, or 25 ng/ml were added to the serum-containing medium immediately before
time-lapse analysis.
Time-lapse analysis. All images were converted into TIFF format and analyzed using Scion Image PC (Scion, Frederick, MD). The area of damage within a set 0.7 × 0.47-mm box was measured at each time, and the difference in area covered between time points was calculated, thus providing a measure of the area covered by the repairing epithelium. Because the length of the wound edge was the same in all experiments (~0.8 mm), the area covered during migration was used as an index of the linear rate of migration (mm2/h).
To measure the movement of individual cells within the epithelial sheet, the position of 20 wound edge cells was tracked through each frame. This was performed for control monolayers and 0.25 ng/ml TGF-Statistical analysis. Statistical analysis was performed using the SPSS statistical program version 10. Two independent sample t-tests were used to compare ELISA data between damaged and undamaged groups and time-lapse data between treated and untreated groups. Statistical significance was highlighted on individual data points.
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RESULTS |
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Epithelium and epithelial damage.
The 16HBE 14o cell line, an SV40 transformed bronchial
epithelial cell line, grew in a typical epithelial fashion forming a
fully confluent monolayer with a "cobblestone" appearance
(9). After confluence in 35-mm petri dishes, mechanical
damage in a cross-hatch pattern generated islands of epithelial cells,
with gaps between each island of 5-25 cell widths (Fig.
1). Cells from each island subsequently
migrated in all directions to cover the area of damage giving a high
proportion of cells in a migratory phase. Repair of small "wounds,"
five cell widths, commonly occurred within 6 h of damage, with
cell-cell contact being reestablished and confluence resumed ~24 h
after damage (Fig. 1). At this stage, the lines of damage could still
be seen where cell-cell borders overlapped (Fig. 1); however, over the
course of the next 24 h, the monolayer returned to a morphology
indistinguishable from undamaged cultures. Few mitotic figures were
seen in the islands of cells as they migrated to confluence. With the
exception of time-lapse analysis, this model of epithelial repair was
used for all subsequent experiments.
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TGF- expression profiles of 16HBE 14o
and primary
epithelial cells.
To confirm the similarity in the TGF-
profile between the 16HBE
14o
cell line and primary epithelial cells from lung
resection, RT-PCR for the three isoforms of TGF-
was performed (Fig.
2). Primary epithelial cells gave strong
PCR product bands for TGF-
1 and TGF-
2 that were broadly
equivalent to each other and showed a lower level of expression for
TGF-
3 (Fig. 2A). A similar expression profile was
observed in 16HBE 14o
cells (Fig. 2B). The
16HBE 14o
cell line was used for all subsequent
experiments.
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Production of active TGF- isoforms during epithelial repair.
Supernatants were taken from epithelial monolayers damaged by the
cross-hatch method at 5 min and 2, 6, 24, 48, and 72 h after addition of serum-containing medium. Although the overall amount of the
latent form of TGF-
1 declined over 72 h, a peak of latent TGF-
1 release was observed in the damaged cultures at 2 h
postwounding. This induction of latent TGF-
1 was mirrored by a
similar significant (P = 0.03) increase in the active
form of the molecule at the same time point compared with undamaged
control cultures, with the total percentage of TGF-
1 in the active
form peaking at 26% in damaged cultures (Fig.
3A).
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Migration of epithelial cells after exogenous addition of active
TGF- isoforms.
To measure the effect of TGF-
1 and TGF-
2 on the rate of cell
migration, 16HBE 14o
cells were tracked by time-lapse
microscopy after a single scrape wound to the confluent monolayer.
Images taken every hour were measured to determine the area covered by
the cells per hour. Because the wound edge in all samples was of equal
length (0.8 mm), the value of the area covered per unit time was
directly proportional to the mean migration rate per hour. Thus a
positive migratory rate indicates closure of the wound.
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Anti-TGF-1 blocks the increased migration induced by TGF-
.
To further support the role of TGF-
1 in the enhanced migratory rate,
a monoclonal antibody to the active form of TGF-
1 was used after
24 h of serum starvation to remove the exogenous active molecule.
After a single scrape damage to the serum-starved 16HBE 14o
cell monolayers, addition of 0.25 ng/ml TGF-
1
significantly enhanced the rate of migration at 2 and 3 h
(P = 0.05 and 0.038, respectively) compared with
control cultures without additional TGF-
1. Addition of a monoclonal
neutralizing antibody to the active form of TGF-
1 3 h after the
initial stimulus reduced the rate of migration such that it was not
significantly different from control cultures (Fig.
6).
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Relationship between cell migration and proliferation.
To establish that the enhanced rate of migration was not as a result of
increased cell proliferation, time-lapse microscopy was performed in
the presence of 1 µg/ml mitomycin C in cultures starved of serum for
24 h. This showed that, despite the inhibition of proliferation
induced by mitomycin C, there was an increase in the rate of migration
above the level of controls for the first 4 h of the 6-h
time-lapse experiment (Fig. 7).
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DISCUSSION |
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Repair of the bronchial epithelium after damage is an essential
feature of respiratory tract function. Through time-lapse analysis and
mechanical wounding of epithelial monolayers, this report highlights
the importance of the isoforms of TGF- for the repair of the
bronchial epithelium in vitro and shows that damage to the bronchial
epithelium can induce the activation of latent TGF-
.
Two models of epithelial repair were developed using the 16HBE
14o bronchial epithelial cell line. The cross-hatch
damage model was devised to generate a high proportion of cells in a
migratory phase, thus allowing the study of cells and cell products
during migration and later stages of the repair cycle
(20). In comparison, the time-lapse model generated
detailed measurements of the speed of migration over the initial 6-h
time period after damage. Thus the time-lapse system showed that an
enhanced migratory rate after TGF-
1 stimulation of the wounded
monolayer involved a progressive increase of speed over the first
4 h studied. The SV40-transformed 16HBE 14o
cell
line has been well characterized and has a number of the features of
primary human bronchial epithelial cells (9). Indeed, RT-PCR confirmed that both primary cells and 16HBE 14o
cells expressed mRNA for all three TGF-
isoforms, and the 16HBE 14o
cell line was used for all further experiments.
Few differences have previously been observed between TGF-1 and
TGF-
2 in terms of their expression in epithelial repair. Skin wound
models show that TGF-
1 and TGF-
2 show a similar pattern in terms
of kinetics of expression (1). In the present study, damage to a bronchial epithelial monolayer demonstrated a divergence between TGF-
1 and TGF-
2 in terms of TGF-
release and function in cell culture supernatants. In damaged cultures only, and in contrast
to the pattern for TGF-
1, latent TGF-
2 was detected in
progressively increasing concentrations at the later stages of the time
course, with this increase starting at a time point where resolution of
the wound had occurred. This is consistent with previous published
results using a similar model (37). Whether this reflects
an increased production of TGF-
2 at this stage or a decreased
breakdown and clearance of the molecule is unclear. However, time-lapse
measurements of the wound edge after addition of TGF-
2 to the
culture showed no detectable enhancement of migration, in contrast to
increased migration with additional TGF-
1. Therefore,
TGF-
2 appears to play no role in cell migration during wound repair
of bronchial epithelial cells.
Production of TGF- in most cell types is in the latent form, and
activation is necessary for production of the active form and for cell
signaling (17). In vitro, this can be performed by
acidification and then neutralization before ELISA measurement; however, in vivo, a number of putative activation mechanisms have been
identified, including plasmin and the urokinase plasminogen activator
system, the extracellular matrix molecule thrombospondin-1,
V
6-integrin (10, 26, 35),
and most recently a matrix metalloproteinase (MMP)-9/CD44 complex
(36).
Despite the differences in isoform synthesis and function detected by
ELISA, one similarity was evident. Damage to the bronchial epithelial
monolayer allowed the detection of active TGF-1 in the culture
supernatants at a peak of 2 h after wounding, and a similar
profile was seen for TGF-
2. Furthermore, TGF-
2 was only observed
in its active form during the early stages of the repair process. This
transient accumulation was only seen in cultures in which
serum-containing medium was used, indicating that latent TGF-
in the
serum was the substrate for activation. This was confirmed after damage
to the monolayer in the absence of serum, where the pattern of release
of both TGF-
1 and TGF-
1 was different from that in the presence
of serum. In particular, the peak in active TGF-
1 and TGF-
2
demonstrated in the presence of serum was absent after serum removal.
The use of serum-containing media in these systems reflects the in vivo
situation where immunocytochemistry has shown that the majority of
TGF-
present in the bronchial airway is found in the subepithelial
compartment (29), probably complexed with fibrillary
latent TGF-
binding proteins (11). Therefore, damage to
a bronchial epithelial monolayer enhances the ability of cells to
convert latent TGF-
1 and TGF-
2 into their active forms. This has
recently been shown graphically in a coculture system where damage to
guinea pig tracheal epithelial cells induced the loss of matrix-bound
TGF-
1 on the underlying human amniotic basement membrane
(25). The specific identity of these activation molecules
has not been elucidated fully and will likely differ between isoforms
and between organs.
V
6-Integrin, which is
upregulated at the wound edge of keratinocyte monolayers (18), has been shown to activate TGF-
1 in vitro,
binding through an RGD sequence on the TGF-
1 LAP, a sequence not
present on TGF-
2 LAP (26). Similarly, both MMP-9 and
CD44 have an increased expression during repair of the bronchial
epithelium (7, 22), with the complex of molecules at the
cell surface showing greater activation of TGF-
2 than TGF-
1.
The consequences of this concentration of active TGF-1 at the wound
edge were investigated using time-lapse microscopy. In line with
previous studies using dispersed cultures of mink lung epithelial cells
and cultures of primary rabbit tracheal cells, TGF-
1 on a wounded
human epithelial monolayer induced an increased migratory speed
(6, 38). However, the present study showed that the
concentration of TGF-
1 sufficient to establish an increased speed
could be reduced to 250 pg/ml. Furthermore, TGF-
2 at any concentration showed no change in migration different from untreated control cultures.
By the use of time lapse, we could observe that, in TGF-1-stimulated
wounded monolayers, migration speed progressively increased up to
4 h, thereafter plateauing at an enhanced level. Measurement of
the distance traveled during this time indicated that although some
cell spreading occurred, the increase in the migratory speed of the
epithelial layer was predominately the result of cell migration. Moreover, the total distance traveled by the monolayer (240 µm) was
>10-fold greater than the average cell diameter of the 16HBE 14o
cell line (~20 µm). The importance of continued
TGF-
1 stimulation during this enhanced but stable migration was
demonstrated by the neutralization of TGF-
1 in the system. After
initial stimulation with an optimal concentration of TGF-
1 (3 h), an
anti-TGF-
1 antibody reduced the rate of migration to the level of
cultures without addition of TGF-
1. Therefore, TGF-
1 but not
TGF-
2 can induce an increased migratory speed for the wounded
epithelial monolayer; furthermore, this increased speed was dependent
on the continued presence of active TGF-
1 in the system. This is, to
our knowledge, the first demonstration of a differential migration response to TGF-
isoforms in bronchial epithelial cells. However, an
enhanced migration to TGF-
1 has previously been shown in bovine aortic smooth muscle, and an enhanced migration to TGF-
2 has been
shown in bovine aortic endothelial cells (23). This
differential response has been linked to the relative expression of the
type III TGF-
receptor (33). Furthermore, the X-ray
crystal structure of the two isoforms reveals differences in a region
of the molecule implicated in receptor binding (19).
TGF-1 is recognized for its ability to inhibit cell proliferation in
most cell types (17); however, there was neither a reduction nor enhancement of proliferation in the current study. This
does not reflect a lack of responsiveness to TGF-
1, which has been
suggested to contribute to carcinogenesis (30), because application of TGF-
1 enhanced cell migration. Cell proliferation has
been shown to assist in the closure of chemically wounded primary
epithelial monolayers 48 h after damage at a site 160-400 µm distal to the wound edge. The use of time-lapse analysis of wounded monolayers after inhibition of proliferation with mitomycin C
in this study demonstrated that proliferation of the monolayer was not
responsible for the increased migration observed. In contrast, the
enhanced migration after inhibition of proliferation is consistent with
previous studies where induction of G1 arrest in mink lung epithelial cells increased the migratory response to TGF-
1
(38). In addition, by phase-contrast microscopy, few cells
could be observed in the process of division during the time course
(Fig. 1). Therefore, active TGF-
1 at a similar concentration to that produced in damaged cultures could enhance and thereafter sustain the
rate of migration of a wounded monolayer.
Bronchial epithelial repair is essential to normal airway function for
maintenance of the barrier between the external and internal milieu of
the lung after damage induced by inhaled pathogens, pollutants, or
allergen. It has been established that asthmatic airways contain more
evidence of epithelial damage than airways from nonasthmatic patients
(3), as indicated by the presence of creola bodies in
induced sputa and bronchial lavage samples (27).
Therefore, by inference, asthmatic airways contain a greater degree of
epithelial repair. It is clear from this study that bioactive TGF-1
can be released from its latent form after damage to an epithelial cell
monolayer and upregulation of activating molecules. A recent study has
shown that chemical and mechanical damage of 16HBE 14o
monolayers can induce myofibroblast proliferation (37),
partly through growth factors. The current study reinforces and
augments this by providing evidence that mechanical damage and repair
of the bronchial epithelial monolayer can induce the synthesis and activation of growth factors, in this case the TGF-
isoforms, and
that activation increases the speed of repair of the monolayer. This
corresponds with skin wound repair where blockade of the TGF-
isoforms TGF-
1 and TGF-
2 or injection of TGF-
3 in the wound
site prolongs the wound repair but induces the production of matrix and
alignment of fibers similar to normal skin (34). The
release of TGF-
after bronchial damage initiated by inhalation of
allergen (14) could explain the detection of increased
TGF-
in the bronchoalveolar lavage fluid of asthmatic patients, both basally and 24 h after allergen challenge (28).
In summary, this report highlights the divergence in function between
TGF-1 and TGF-
2 in terms of bronchial epithelial repair. Activation mechanisms that facilitate the conversion of latent to
active forms of TGF-
may be enhanced at an early stage in the repair
process. However, although active forms of both TGF-
2 and TGF-
1
are produced during wound repair, only TGF-
1 increases the speed of
epithelial repair.
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ACKNOWLEDGEMENTS |
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This work was supported by Medical Research Council Program Grant G8604034, Rhone-Poulenc Rorer, United Kingdom, and Hope Charity.
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FOOTNOTES |
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Address for reprint requests and other correspondence: W. J. Howat, Div. of Respiratory, Cell, and Molecular Biology, MP810, Level D, Centre Block, Southampton General Hospital, SO16 6YD United Kingdom (E-mail: W.J.Howat{at}soton.ac.uk).
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. Section 1734 solely to indicate this fact.
Received 10 June 2001; accepted in final form 14 August 2001.
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