Division of Pulmonary and Critical Care Medicine and Will Rogers Institute Pulmonary Research Center, University of Southern California, Los Angeles, California 90033
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ABSTRACT |
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We investigated expression
of the 3-integrin subunit by rat alveolar epithelial
cells (AECs) grown in primary culture as well as the effects of
monoclonal antibodies with blocking activity against the
3-integrin subunit on AEC monolayer formation.
3-Integrin subunit mRNA and protein were detectable in
AECs on day 1 and increased with time in culture.
3- and
1-integrin subunits coprecipitated in immunoprecipitation experiments with
3- and
1-subunit-specific antibodies, consistent with their
association as the
3
1-integrin receptor
at the cell membrane. Treatment with blocking anti-
3 monoclonal antibody from day 0 delayed development of
transepithelial resistance, reduced transepithelial resistance through
day 5 compared with that in untreated AECs, and resulted in
large subconfluent patches in monolayers viewed by scanning electron
microscopy on day 3. These data indicate that
3- and
1-integrin subunits are expressed
in AEC monolayers where they form the heterodimeric
3
1-integrin receptor at the cell
membrane. Blockade of the
3-integrin subunit inhibits
formation of confluent AEC monolayers. We conclude that the
3-integrin subunit modulates formation of AEC monolayers by virtue of the key role of the
3
1-integrin receptor in AEC adhesion.
alveolar epithelium; 1-integrin; cell adhesion; extracellular matrix; transepithelial resistance
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INTRODUCTION |
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THE PULMONARY ALVEOLAR AIR SACS are lined with a continuous layer of epithelial cells across which gas exchange occurs. The alveolar epithelium is composed of alveolar type I (AT1) and type II (AT2) cells that interact with the extracellular matrix (ECM; basement membrane) via cell surface receptors. This interaction influences many alveolar epithelial cell (AEC) functions, including cell adhesion and spreading, and modulates the repair of the alveolar epithelial barrier after lung injury.
The integrins are a family of cell surface proteins that mediate cell
adhesion and cell-cell interactions. Integrins are heterodimeric proteins that consist of one each of several distinct - and
-subunits that together determine the specificity of the molecule as
a receptor for different ECM proteins. Several different integrin
subunits have been identified in human and rat lungs and AECs, with
particular subunits thought to play a role in modulation of AEC
function in an ECM ligand-specific fashion (6,
7, 12, 19, 23, 24, 29, 38, 42,
46, 48). Current data also indicate that the
expression of different integrin receptors in the lung, and in alveolar
epithelium in particular, changes after lung injury or inflammation and
after neoplastic transformation (1, 2, 14, 20, 26, 33,
35-37, 39, 43).
The 3
1-integrin (VLA-3) is a
receptor for several known ligands, including laminin, fibronectin,
collagen type IV, epiligrin, and entactin/nidogen (2). It
is present in many epithelia, and its expression is required for
morphogenesis of both lung and kidney (27). The
3-integrin subunit and its
1-subunit companion are expressed in normal human alveolar epithelium in both AT1
and AT2 cells, where it is presumed that they form functional receptors
(46). The specific functions and substrates for the
3
1-integrin in the lung and alveolar
epithelia are largely unknown.
In this study, we investigated the expression and function of the
3
1-integrin in AEC monolayers. In this
model, primary cultured rat AT2 cells form confluent, electrically
resistive monolayers of cells that gradually acquire AT1 cell
phenotypic properties (4, 5, 9).
Our results indicate that AECs in culture express
3- and
1-integrins that form
3
1-integrin heterodimers based on
coprecipitation studies. Blockade of integrin binding with specific
anti-integrin monoclonal antibodies (MAbs) results in decreased AEC
adhesion and monolayer integrity, consistent with a role for this ECM
receptor in the formation of an intact epithelial barrier in situ.
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METHODS |
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Cell isolation and preparation of rat AEC monolayers. AT2 cells were isolated from adult male Sprague-Dawley rats by disaggregation with elastase (2.0-2.5 U/ml; Worthington Biochemical, Freehold, NJ), followed by differential adherence on IgG-coated bacteriological plates (16). The enriched AT2 cells were resuspended in a minimal defined serum-free medium (MDSF) consisting of Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mixture in a 1:1 ratio (Sigma, St. Louis, MO) supplemented with 1.25 mg/ml of bovine serum albumin (BSA; Collaborative Research, Becton Dickinson, Franklin Lakes, NJ), 10 mM HEPES, 0.1 mM nonessential amino acids, 2.0 mM glutamine, 100 U/ml of sodium penicillin G, and 100 µg/ml of streptomycin (5). Cells were plated onto tissue culture-treated polycarbonate (Nuclepore, Pleasanton, CA) filter cups (Transwell, Corning Costar, Cambridge, MA), 8-well chamber slides (Falcon, Becton Dickinson, Franklin Lakes, NJ), or 24-well tissue culture plastic dishes (Falcon, Becton Dickinson) at a density of 1.0 × 106 cells/cm2. Cultures were maintained in a humidified 5% CO2 incubator at 37°C. AT2 cell purity (>90%) was assessed by staining freshly isolated cells for lamellar bodies with tannic acid (32). Cell viability (>90%) was measured by trypan blue dye exclusion.
Media were changed, thereby removing nonadherent cells, on the second day after plating. Monolayers were subsequently fed every other day. Cells were maintained in MDSF or in MDSF supplemented with eitherA549 cells. A549 cells (a human adenocarcinoma-derived cell line) were obtained from the American Type Culture Collection and cultured in medium containing DMEM supplemented with 10% fetal bovine serum, 100 U/ml of sodium penicillin G, and 100 µg/ml of streptomycin. Cells were plated at a density of 2 × 106/cm2 on 100-mm tissue culture plastic dishes (Falcon, Becton Dickinson), grown until confluent in a humidified 5% CO2 incubator at 37°C, and solubilized directly into immunoprecipitation lysis buffer (see Western analysis and coprecipitation studies) for immunoprecipitation studies.
Immunofluorescence.
Two sets of fresh-frozen sections (4 µm) of adult rat lung were fixed
in 100% methanol at 20°C, blocked with PBS-3% BSA (pH 7.4) to
reduce nonspecific reactivity, reacted sequentially with anti-
3-integrin subunit polyclonal antibody (PAb) 1920P
(Chemicon, Temecula, CA) and FITC-labeled goat anti-rabbit secondary
Ab, and postfixed with 3.7% Formalin. The labeled sections were then viewed by differential interference contrast (DIC) optics and immunofluorescence (IF) with an Olympus microscope equipped with epifluorescence optics set at ×300 magnification. Monolayers
(day 7) grown on chamber slides maintained in MDSF were
rinsed with cold PBS, fixed with 100% methanol at
20°C for 10 min,
rinsed in PBS again, and treated with PBS-3% BSA. Monolayers were
reacted in situ with anti-
3-integrin subunit PAb 1920P.
After extensive washing, the monolayers were incubated with
FITC-labeled goat anti-rabbit secondary Ab and viewed by
epifluorescence at ×600 magnification.
Western analysis and coprecipitation studies.
SDS-PAGE was performed with the buffer system of Laemmli
(30), and immunoblotting (Western blotting) was performed
with procedures modified from Towbin et al.
(44). For detection of integrin subunits, AEC monolayers
were solubilized directly into 2% SDS sample buffer at 37°C for 15 min. Equal amounts of cell protein in sample buffer were resolved by
SDS-PAGE under reducing conditions for the detection of
1-integrin subunit or under nonreducing conditions for
the detection of
3-integrin subunit as previously described (22). Proteins were electrophoretically blotted
onto Immobilon-P (Millipore). The blots were blocked for 2 h with
5% nonfat dry milk in Tris-buffered saline (TBS; 20 mM Tris and 500 mM
NaCl, pH 7.5, and then incubated with primary PAb 1920P for detection
by immunoblot.
RNA isolation and Northern analysis.
Total RNA was isolated from AEC monolayers by the acid
guanidinium-phenol-chloroform method (10). Equal amounts
of RNA (5-20 µg) were denatured with formaldehyde,
size-fractionated by agarose gel electrophoresis under denaturing
conditions, and transferred to nylon membranes (Hybond N+, Amersham
Life Sciences, Cleveland, OH). RNA was immobilized by ultraviolet
cross-linking. Blots were prehybridized for 2 h at 65°C in 1 M
sodium phosphate buffer (pH 7), 7% SDS, and 1% BSA. Hybridization was
performed for 16 h at 65°C in the same buffer. Blots were probed
with an integrin subunit-specific oligonucleotide probe for the
3-subunit (Biognostik, Chemicon, Temecula, CA). Probes
were labeled with [
-32P]dCTP (Amersham) by the
random-primer method with the use of a commercially available kit
(Boehringer Mannheim, Indianapolis, IN). Blots were washed at high
stringency (0.5× saline-sodium citrate; 75 mM NaCl and 7.5 mM sodium
citrate, pH 7.0, with 0.1% SDS at 55°C) and visualized by
autoradiography. Differences in RNA loading were normalized with a
24-mer oligonucleotide probe rRNA end labeled with
[
-32P]ATP for 18S (31). Binding was
detected by autoradiography and quantified by densitometry.
Cell number.
AECs grown for 24 h in MDSF or MDSF supplemented with 5 µg/ml of
3-integrin blocking MAb CP11,
3-integrin
blocking MAb Ralph 3.2, or nonimmune mouse IgG in 24-well tissue
culture plastic plates were washed three times with ice-cold PBS,
trypsinized (0.05% trypsin) for 30 s, and resuspended in PBS. The
cell number was counted with a Coulter Counter (Coulter Electronics,
Hialeah, FL).
Nuclear staining of AEC monolayers with propidium iodide.
AEC monolayers grown in MDSF or MDSF supplemented with 5 µg/ml of
anti-3 MAb CP11 were fixed on day 3 for IF
(as described in Immunofluorescence). Fixed
monolayers were stained with propidium iodide (Molecular Probes,
Eugene, OR), a selective nuclear stain. Stained specimens were viewed
by epifluorescence microscopy at ×200 magnification.
Scanning electron microscopy.
AEC monolayers grown in MDSF or MDSF supplemented with 5 µg/ml of
anti-3 MAb CP11 were fixed on day 3 with
2.5% glutaraldehyde and 1.5% OsO4, dehydrated, and
critical-point dried. Specimens were viewed by scanning electron
microscopy (SEM) at ×400 magnification.
Chemicals. Except where otherwise indicated, cell culture media and all other chemicals were purchased from Sigma and were of the highest commercial quality available.
Statistical analysis. Results are expressed as means ± SE. Significance of differences (P < 0.05) was determined by ANOVA.
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RESULTS |
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Labeling of lung sections with anti-3 MAb.
Figure 1 shows corresponding DIC and
epifluorescence images indicating diffuse alveolar staining by the
anti-
3 PAb 1920P. The staining pattern, which is
similar to that shown for other anti-
3 Abs in human lung
(46), is consistent with staining of both AT1 and AT2
cells in situ.
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Immunofluorescence, Western blotting, and Northern blotting of
3-integrin in AEC monolayers.
AEC monolayers labeled with the anti-
3 PAb 1920P showed
staining on day 7 as illustrated in the epifluorescence
image shown in Fig.
2A. The same Ab labels
a single band corresponding to the expected molecular mass (145 kDa) of
the
3-integrin subunit on Western blot (Fig.
2B). AEC monolayers expressed progressively increasing
amounts of
3-integrin subunit protein as shown in this
representative Western blot (Fig. 2C). Protein abundance increased maximally by day 6 to ~3.5 times that observed
on day 1. Similarly, the 5-kb transcript corresponding to
the expected size of the
3-integrin subunit mRNA was
present on Northern blot from days 1 to 8, with a
relative increase in mRNA levels evident as a function of time in
culture.
3-Integrin subunit mRNA levels were maximal by
day 6 at ~4 times those observed on day 1 (Fig. 2D).
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Coprecipitation of integrin 3- and
1-subunits.
As illustrated in Fig. 3A,
immunoprecipitates of day 4 AEC lysates containing either
anti-
1-integrin subunit MAb 141720 (lane 2)
or anti-
3-integrin subunit PAb 1920P (lane 3)
were resolved by SDS-PAGE and immunoblotted with the
anti-
3-integrin subunit PAb. Positive 145-kDa bands are
seen for both
3 and
1 immunoprecipitates, indicating that both
3 and
1 Abs either
precipitate or coprecipitate the
3-integrin subunit.
Figure 3A, lane 1, shows day 8 AEC
lysate blotted as a positive control, demonstrating immunodetection of the same 145-kDa
3-integrin subunit in the
3 and
1 immunoprecipitates as in total
cell lysate. Immunoprecipitations with rat IgG (Figure 3A, lane
4) or in the absence of primary Ab (lane 5)
were negative for
3-integrin subunit.
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Immunoprecipitation of 3-integrin by
anti-
3-integrin blocking Abs.
As shown in Fig. 4, lysates of A549
cells (lanes 1 and 3) and day 4 rat
AEC lysates (lanes 2 and 4) were
immunoprecipitated with either anti-
3-integrin subunit
MAb CP11 (lanes 1 and 2) or Ralph 3.2 (lanes 3 and 4), and the precipitated proteins
were resolved by SDS-PAGE and immunoblotted with
anti-
3-integrin subunit PAb 1920P. Positive 145-kDa
bands are seen in Fig. 4, lanes 1 and 2,
indicating that MAb CP11 (an anti-human integrin Ab) precipitates both
human and rat
3-integrin subunits. A positive 145-kDa
band is also seen in Fig. 4, lane 4, but is absent in
lane 3, indicating that MAb Ralph 3.2 (an anti-rat integrin
Ab) precipitates rat, but not human,
3-integrin
subunits. These results indicate the potential for specific interaction
between each anti-integrin blocking Ab and the rat
3-integrin subunit in the functional studies described
below.
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Effect of anti-3 blocking MAb on AEC cell adhesion.
AECs grown in 24-well plates in MDSF or MDSF supplemented with
5 µg/ml of either anti-
3-integrin blocking
Ab CP11 or Ralph 3.2 or mouse IgG1 were gently rinsed with PBS at
24 h, and adherent cells were trypsinized off the plates and
counted with a Coulter Counter. As indicated in Fig.
5, the absolute number of
adherent cells per well was no different for MDSF versus MDSF
plus IgG1. In contrast, AECs grown in MDSF plus CP11 or Ralph 3.2 showed markedly reduced numbers of adherent cells per well. These
results indicate that either anti-
3-integrin blocking Ab
is capable of reducing cell adhesion at 24 h.
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Effect of anti-3 blocking MAb on AEC monolayer
formation: TER.
AEC monolayers grown in the absence of anti-
3-integrin
subunit blocking Ab formed confluent monolayers, with TER > 1,000
· cm2 on day 3, whereas AECs grown in
the presence of 5 µg/ml of Ab CP11 developed no measurable TER after
an equal amount of time in culture (Fig.
6). Intermediate concentrations of Ab (1 and 2 µg/ml) resulted in measurable but lower TER than no-Ab controls (data not shown). In separate experiments, AECs grown in the presence of 5 µg/ml of Ab Ralph 3.2 showed no measurable resistance on day 3, whereas TER was no different for monolayers treated
with 5 µg/ml of mouse IgG of the same subclass as the
anti-
3 blocking Ab (2.86 ± 0.12 k
· cm2) compared with that for untreated monolayers (3.13 ± 0.19 k
· cm2). TER
1,000
· cm2 (indicating development of confluent, electrically
tight monolayers) gradually develops on days 4-6 under
all conditions studied.
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Effect of anti-3 blocking MAb on AEC monolayer
formation: nuclear staining and SEM.
As shown in Fig.
7A,
fluorescence microscopy of fixed AEC monolayers (day 3)
grown in the absence of anti-
3 blocking Ab and stained
with the nuclear stain propidium iodide showed uniform distribution of
nuclei consistent with the development of confluence for monolayers. In
contrast, monolayers grown in the presence of 5 µg/ml of
anti-
3 blocking Ab CP11 showed large areas of the filter
without nuclei (i.e., without cells), probably due to the failure of cells to adhere to the substratum and/or cell sloughing before or during fixation (Fig. 7B). As shown in
Fig. 8A, control monolayers on day 3 appeared confluent by SEM, whereas those
grown in the presence of 5 µg/ml of anti-
3 blocking Ab
CP11 (Fig. 8B) showed areas of nonconfluence.
Monolayers grown in the presence of blocking Ab appeared to show cells
being sloughed off, leaving areas of bare filter.
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DISCUSSION |
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We demonstrated in this study that primary cultured AECs express
both integrin 3- and
1-subunits. Integrin
3-subunit mRNA and protein are detectable from day
1 and increase with time in culture through day 8,
coincident with the period during which the cells spread to form
confluent monolayers and undergo transdifferentiation from AT2 cells
toward the AT1 cell phenotype (4, 5).
Integrin
3- and
1-subunits can be
reciprocally coprecipitated, consistent with their association as the
3
1-integrin receptor at the cell membrane. AEC monolayer formation can be partially and transiently inhibited by anti-
3-subunit Abs previously shown to have
blocking activity as indicated by adhesion assays, bioelectric
measurements, and morphological assessment. Taken together, these data
demonstrate that cultured AECs express the
3
1-integrin receptor, a mediator of
epithelial cell adhesion, and indicate a role for this receptor in
mediating AEC adhesion.
Integrin 3-subunits have been previously identified
in adult rat and human alveolar epithelia as well as in fetal lung and isolated fetal distal lung epithelial cells (6,
7, 12, 19, 23,
24, 29, 42, 46,
48). Staining for the integrin
3-subunit is
abundant in evolving alveolar walls during the canalicular stage of
development (19-21 days gestation in the rat) where it colocalizes with laminin-5 (epiligrin), one of its ECM ligands (46). Antisera to both the integrin
3-subunit and its only known companion, the integrin
1-subunit, have been shown to label adult human alveolar
surfaces diffusely (46) in a pattern similar to that
observed with the anti-integrin
3-subunit PAb used in the current study (Fig. 1). Integrin
3-subunit is also
known to be expressed in some lung epithelium-derived cell lines (e.g., LM5 cells, an AT2-derived cell line), but it has not been previously identified in adult AECs grown in primary culture (29).
3-Integrin subunit mRNA is detectable in AECs as a 5-kb
transcript by Northern blot.
3-Integrin subunit protein
is detectable in AECs by IF (Fig. 2A) and as a 145-kDa
protein on Western blot (Fig. 2B). Both
3-integrin subunit mRNA and protein are minimally detectable in freshly isolated AT2 cells (data not shown), whereas mRNA
levels and protein abundance increase from day 1 through day 8 in culture, with the greatest increases occurring
during the first few days after plating (Fig. 2, C and
D). The most likely explanation for the relatively low
amount of this integrin subunit after cell isolation may be that
transcriptional downregulation and protein turnover of
3-integrin occur rapidly in the absence of a continuous
input of signal from an intact basement membrane. Conversely,
attachment of cells and secretion of basement membrane components
likely create positive feedback for further matrix secretion, integrin
expression, and cell adhesion (13, 15, 25). Although little is known about the specific
mechanisms by which
3-integrin expression is regulated
in any tissue, the recent description of the genomic organization of
the human and mouse
3-integrin subunit genes may
facilitate further efforts in this direction (21,
45).
It is also possible that the quantitative differences in
3-integrin subunit expression observed in the present
study reflect some degree of differential expression between AT1 and
AT2 cells. AT2 cells, especially those grown on permeable supports,
have been shown to transdifferentiate toward the AT1 cell phenotype on
the basis of morphology and expression of several distinct AT1 cell
markers, with a time course resembling that found for the increase in
3-integrin subunit expression (4,
5). AT1 and AT2 cells have been shown to reside on
basement membranes of somewhat different composition, consistent with a
role for different
3
1-integrin
receptor-matrix interactions in the transition from one differentiated
phenotype to the other (40). Alternatively,
3-integrin expression may be relatively greater in AT1
versus AT2 cells for the simple reason that AT1 cells have more
basolateral surface in contact with basement membrane than AT2 cells
and would need to express relatively more integrin (and other cell
surface) receptor than their AT2 cell counterparts to maintain a
similar membrane density of attachments. Notwithstanding the
demonstration of the labeling of AT2 cell basolateral membranes by an
anti-
3-integrin subunit Ab in situ in at least one
report (46), indicating that these cells express
3-integrin to some degree, further studies will be
necessary to distinguish among these possibilities.
3
1-Integrin receptors are expressed in a
variety of epithelial cells, including keratinocytes, and in smooth
muscle and connective tissue. Although many of the known integrin
subunits can associate with multiple molecular partners,
3-integrin subunits have only been shown to form
heterodimers with
1-integrin subunits to form
functioning integrin receptors. Conversely,
1-integrin subunits, which are expressed ubiquitously, can associate to form integrin receptors with at least 10 different
-subunits. In the current study, we have shown that the
3-integrin subunit
associates with its
1-integrin subunit partner in AECs
by demonstrating reciprocal coprecipitation (Fig. 3, A and
B). This does not preclude the association of
1-integrin subunits with other
-integrin subunits
such as the
5-integrin subunit shown to be present in AECs (38) or the currently less likely association of
3-integrin subunits with other as yet unidentified
-integrin subunits. The likely association of
1-integrin subunits with other
-integrin subunits is
supported by the fact that relatively little
3-integrin subunit appears to have been recovered by immunoprecipitation with an anti-
1-subunit Ab (Fig. 3A), whereas
the anti-
3- and anti-
1-subunit Abs
precipitate similar amounts of
1-subunit protein (Fig.
3B). This result may be a consequence of the fact that most
or all of the
3-integrin subunit present in heterodimers is paired with a
1-integrin subunit, whereas a
substantial fraction of the
1-integrin subunit present
in heterodimers is paired with an
-integrin subunit other than
3-integrin. The apparent lack of efficiency with which
the
1-integrin subunit coprecipitates the
3-integrin subunit may therefore be due, at least in
part, to the fact that much of the
-integrin subunit coprecipitated by the anti-
1-subunit Ab is actually some other
-subunit isoform that remains undetected on the immunoblot.
The 3
1-integrin receptor is best known as
a mediator of cell-matrix attachment. In this study, decreased adhesion
by AECs during monolayer formation in the presence of
3-integrin subunit blocking Abs (shown to specifically
associate with the
3-integrin subunit in rat AECs by
immunoprecipitation studies; Fig. 4) was demonstrated in several
different ways. First, AECs cultured in the presence of
anti-
3-integrin subunit Ab with blocking activity show
markedly fewer adherent cells at 24 h compared with those plated
with IgG or no Ab (Fig. 5). Second, AT2 cells plated from day
0 in the presence of blocking Ab show a delay in the development of electrically resistive monolayers (i.e., development of
TER > 1,000
· cm2) relative to untreated
controls (Fig. 6). Although control monolayers develop high electrical
resistance by day 3 as previously reported (9),
treated monolayers fail to show any measurable TER at the same point.
Third, epifluorescence images of propidium iodide-stained nuclei from
day 3 monolayers show large patches of bare polycarbonate filter in treated versus untreated AECs (Fig. 7). These bare areas could result either from failure of cells to attach (and secrete appropriate ECM components) or from suboptimal adhesion to the ECM
and/or adjacent AECs, with subsequent loss during fixation and
processing. The large defects created in the monolayers provide ample
explanation in either case for the lack of electrical resistance, which
depends on AEC confluence and the formation of functional tight
junctions between cells. Finally, SEM images of treated versus
untreated monolayers on day 3 confirm the lack (or loss) of
cell adhesion to the underlying substratum (Fig. 8). Although the
precise mechanism by which cell attachment is impaired cannot be
determined from these images, the sloughed appearance of some of the
cells on this and other similar images (data not shown) suggests that a
relative lack of adhesiveness rather than a complete inability to
attach to the substratum may account for the delay in development of
electrically resistive monolayers.
After day 3, the gradual development of TER > 1,000 · cm2 by AEC monolayers treated with the
3-integrin subunit blocking Ab can be explained in
several ways. As shown in Fig. 2, expression of the
3-integrin subunit, and presumably of the
3
1-integrin receptor, increases from
day 1 through day 8. ECM components serving as
integrin ligands are continuously secreted by the cells after attachment and promote further anchoring of the cells to the substratum (17). Therefore, part of the loss of effect of the
blocking Ab may simply be due to the stoichiometric increase in
3
1-integrin and ECM ligands relative to
the amount of Ab present. Alternatively, expression of other integrin
receptors capable of binding the same or additional matrix components
may gradually assume the functions of
3
1-integrin. Several other integrin
receptors, including
2
1,
5
1 and
V
3,
that collectively bind to a wide variety of matrix proteins have been
described in adult AECs (23, 24,
38). The lack of effect of blocking Ab when it is added after the development of TER in untreated monolayers (data not shown)
is compatible with either of these explanations and suggests that both
may be operative.
Little information is currently available concerning specific functions
of the 3
1-integrin receptor in adult
alveolar epithelium. Transforming growth factor-
(TGF-
) is shown
to be increased in alveolar lining fluid during inflammatory reactions
of the lung and to be present in AECs of developing lungs and
hyperplastic type II cells during repair. In 1997, Kim and Yamada
(25) suggested that growth factors such as TGF-
induce
an increase in ECM and enhance the ability of the cells to respond to
this increase. Surprisingly, Kumar et al. (29)
demonstrated downregulation of
3-integrin subunit
expression in the AT2-derived cell line LM5 after treatment with
TGF-
1, although expression of
6- and
1-integrin subunits (which form an integrin receptor
that binds laminin similar to
3
1-integrin
receptor) concurrently increased. Thus although growth factor-induced
regulation of
3-integrin could be shown to occur in this
study, a specific role for this integrin in lung inflammation and
repair could not. In contrast, the strong correlation between the loss
of
3-integrin expression and lung cell
dedifferentiation, tumorigenesis, and metastasis suggests that the
presence of this ECM receptor is at least one of many obligatory
components of a nonmalignant phenotype in AECs (1,
2, 14, 20, 26,
33, 36, 43).
The adhesive function of the 3
1-integrin
receptor in alveolar epithelium may be important during development as
well as for normal cell turnover and repair from injury in the adult. Several studies (6, 7, 12) have
demonstrated the presence of
3-integrin in fetal lung
epithelium, including developing alveolar epithelium. Fetal distal lung
epithelium expresses
3-integrin subunit that has been
shown to bind to laminin in vitro (7).
3-Integrin-deficient mice survive to birth but die
shortly thereafter due to severe defects in kidney and lung organ
formation (27). The latter appears to be due to a relative
failure of branching morphogenesis rather than from any abnormality of
the alveolar epithelium, although perinatal mortality of the
3-integrin-deficient mice precludes any evaluation of
the role of this integrin in adult AEC biology in this model. Further
insight into the role of the
3
1-integrin
receptor in alveolar epithelium during development and in the adult may
be possible due to the availability of mice deficient in multiple
integrin subunits and/or conditional knockouts that allow for normal
lung development through the perinatal period.
In addition to its role as an adhesion molecule in AECs,
3
1-integrin may serve a variety of
cellular functions as it does in other cell types.
3
1-Integrin has been shown to modulate the assembly of pericellular matrices via its effects on the rate of
ECM component secretion (47), increase matrix turnover by activation of matrix metalloproteases and phagocytosis of the matrix
(3, 11, 28), regulate epithelial
cell surface polarity (34), and effect cell
proliferation (18). Given its ability to activate multiple
cellular signaling pathways (41), it is likely that the
3
1-integrin has numerous effects on AEC
biology that remain to be identified.
In summary, we have identified the
3
1-integrin receptor in AECs grown in
primary culture. Inhibition of integrin-ECM interaction with an Ab with
blocking activity transiently reduced the ability of AECs to form
confluent, electrically resistive monolayers, indicating a role in
cell-substratum adhesion for this receptor. Further studies will be
necessary to define the involvement of the
3
1-integrin receptor in both fetal and
adult alveolar epithelial development, in lung injury and repair, and
in the pathogenesis of lung neoplasia.
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ACKNOWLEDGEMENTS |
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We thank Dr. Edward D. Crandall for thoughtful encouragement and support of this work. We note with appreciation the expert technical support of Martha Jean Foster and Susie Parra.
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FOOTNOTES |
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This work was supported in part by the American Heart Association and American Heart Association-Western States Affiliate; the American Lung Association; the Baxter Foundation; National Heart, Lung, and Blood Institute Research Grants HL-03609, HL-38578, HL-38621, HL-51928, and HL-62569; and the Hastings Foundation.
Address for reprint requests and other correspondence: R. L. Lubman, Division of Pulmonary and Critical Care Medicine, GNH 11900, Univ. of Southern California, 2025 Zonal Ave., Los Angeles, CA 90033 (E-mail: rlubman{at}hsc.usc.edu).
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.
Received 18 June 1999; accepted in final form 10 February 2000.
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