Will Rogers Institute Pulmonary Research Center and Division of Pulmonary and Critical Care Medicine, University of Southern California, Los Angeles, California 90033
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ABSTRACT |
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Despite a
presumptive role for type I (AT1) cells in alveolar epithelial
transport, specific Na transporters have not previously been localized
to these cells. To evaluate expression of Na transporters in AT1 cells,
double labeling immunofluorescence microscopy was utilized in whole
lung and in cytocentrifuged preparations of partially purified alveolar
epithelial cells (AEC). Expression of Na pump subunit isoforms and the
-subunit of the rat (r) epithelial Na channel (
-ENaC) was
evaluated in isolated AT1 cells identified by their immunoreactivity
with AT1 cell-specific antibody markers (VIIIB2 and/or
anti-aquaporin-5) and lack of reactivity with antibodies specific for
AT2 cells (anti-surfactant protein A) or leukocytes (anti-leukocyte
common antigen). Expression of the Na pump
1-subunit in
AEC was assessed in situ. Na pump subunit isoform and
-rENaC expression was also evaluated by RT-PCR in highly purified (~95%) AT1 cell preparations. Labeling of isolated AT1 cells with
anti-
1 and anti-
1 Na pump subunit and
anti-
-rENaC antibodies was detected, while reactivity with
anti-
2 Na pump subunit antibody was absent. AT1 cells in
situ were reactive with anti-
1 Na pump subunit antibody. Na pump
1- and
1- (but not
2-) subunits and
-rENaC were detected in highly
purified AT1 cells by RT-PCR. These data demonstrate that AT1 cells
express Na pump and Na channel proteins, supporting a role for AT1
cells in active transalveolar epithelial Na transport.
alveolar epithelium; sodium pumps; ion transport; immunofluorescence; sodium channel
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INTRODUCTION |
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THE ALVEOLAR EPITHELIUM IS comprised
of two morphologically distinct cell types, cuboidal type II (AT2)
cells and expansive type I (AT1) cells with long cytoplasmic processes
that cover most of the internal surface area of the lung (11,
18). Active Na transport across the alveolar epithelium
is believed to generate the osmotic gradient that drives alveolar fluid
clearance under both normal and pathological conditions. Studies in
isolated perfused lung (2, 17), alveolar epithelial cell
monolayers (10), intact animals (43), and
human lungs ex vivo (42) demonstrated that transalveolar
fluid transport is inhibitable by both amiloride and ouabain,
indicating that the predominant cellular pathways for vectorial Na
clearance are apical amiloride-sensitive Na channels and basolateral
ouabain-sensitive Na pumps (28, 49). Consistent with these
findings, specific Na transporters have been localized to AT2 cells
both in situ and in vitro. mRNA for all three subunits of the rat
epithelial Na channel, -,
-, and
-rENaC, have been identified
in freshly isolated AT2 cells (32) and in AT2 cells in
adult rat lung (16, 27). In addition, several isoforms of
the
- and
-subunits of Na-K-ATPase have been identified in freshly isolated AT2 cells (29, 33, 41, 48) and in AT2 cells in situ (19, 45).
In contrast to AT2 cells, which have been extensively investigated with
regard to their roles in both surfactant production and ion transport
in adult lung, the properties of AT1 cells are less well characterized.
This has been due, in large part, to difficulty in isolating highly
purified populations of AT1 cells and subsequently identifying them by
other than morphological means following isolation (1, 36,
53). Because AT2 cells acquire the morphological appearance and
phenotypic characteristics of AT1 cells over time, AT2 cells in culture
for several days have been used as a model of the alveolar epithelium
with which to study AT1 cell-like properties (9, 13). With
the use of this model, it has been demonstrated that alveolar
epithelial cells (AEC), after several days in culture, exhibit active
Na transport, which is inhibitable by both ouabain and amiloride (10). AEC also express abundant amounts of mRNA for all
three Na channel subunits and 1- and
1-Na
pump subunit mRNA and protein (3, 12). Given the extensive
surface area of the lung that is covered by AT1 cells, results of these
studies tend to support a role for AT1 cells in transalveolar Na
transport. However, specific Na transporters have not been successfully
localized to AT1 cells in situ, and the role of AT1 cells in
transepithelial Na transport remains presumptive.
We utilized colocalization immunofluorescence (IF) techniques to
evaluate expression of Na pump and Na channel subunits in isolated AT1
cells within partially purified populations of AEC and in whole lung.
Isolated AT1 cells were first identified by their reactivity with AT1
cell-specific antibodies [VIIIB2 and anti-aquaporin (AQP)-5] and
their lack of reactivity with AT2 cell [surfactant protein A
(SP-A)] or leukocyte [leukocyte common antigen (LCA)]
markers. The 1-,
2-, and
1-subunits of Na-K-ATPase and the
-subunit of the
rENaC (
-rENaC) were then localized by concurrent labeling with
either VIIIB2 or anti-AQP-5. In whole lung, expression of the
1-subunit in AT1 cells was assessed by concurrent
labeling with anti-AQP-5. Expression of Na pump subunit isoforms and
-rENaC was also evaluated in highly purified (~95%) AT1 cell
preparations by reverse transcriptase-polymerase chain reaction
(RT-PCR). The results of these studies demonstrate that AT1 cells
express Na pump and Na channel proteins, supporting a role for AT1
cells in active transalveolar epithelial Na transport.
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METHODS |
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Cell isolation and enrichment. AT1 cells were isolated using several modifications of previously described methods for AT2 and AT1 cell isolation (4, 5, 14, 15). Briefly, lungs from adult male Sprague-Dawley rats (250-300 g) were perfused via the pulmonary artery with RPMI 1640 containing 25 mM HEPES (solution A). Lungs were lavaged with phosphate-buffered saline (PBS; pH 7.2) containing 5 mM each of EDTA and EGTA and then filled with 10 ml of solution B (solution A with 10% dextran) containing elastase at 4.5 U/ml (Worthington, Freehold, NJ) at 37°C for a total of 40 min. Lung tissue was dissected away from the trachea and large airways and chopped in solution B containing 20% fetal bovine serum (FBS) and DNase (2 mg/ml). Lung fragments were agitated by end-over-end rotation and filtered sequentially through 100- and 20-µm Nitex mesh (Tetko, Elmsford, NY). Cells were resuspended in medium followed by panning on IgG-coated bacteriological plates for 30 min. Cytocentrifuged preparations of these partially purified AEC were processed for IF following fixation in 100% methanol for 10 min.
To further enrich for AT1 cells, partially purified cell suspensions were subjected to density gradient separation using a discontinuous iodixanol (OptiPrep; Nycomed Pharma, Oslo, Norway) gradient consisting of 2 ml each of Optiprep solutions of densities 1.068, 1.047, 1.033, and 1.012. Cells were centrifuged on the gradient at 600 g for 15 min. Cells between densities of 1.012 and 1.047 were collected, washed in Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mixture in a 1:1 ratio with 1% FBS, and resuspended in PBS-0.1% bovine serum albumin (BSA) at a concentration of 107 cells/ml. Immunoselection for AT1 cells was undertaken by incubating cells with the AT1 cell-specific monoclonal antibody (MAb) VIIIB2 for 30 min at 4°C. After being washed in RPMI with 1% FBS, cells were incubated with magnetic beads coated with a human anti-mouse IgG (CELLection Pan Mouse IgG kit; Dynal, Lake Success, NY) for 15 min at 4°C. Bound AT1 cells were selected by magnetic separation and resuspended in RPMI with 1% FBS. Beads were removed by incubating cells with DNase releasing buffer (Dynal) before processing for RT-PCR. Cell viability (>90%) was determined by trypan blue dye exclusion. Cell purity was assessed by IF using the AT1 cell-specific antibodies VIIIB2 or anti-AQP-5. All media and other chemicals were purchased from Sigma Chemical (St. Louis, MO) unless otherwise indicated and were of the highest commercial quality available.Cell-specific antibodies. VIIIB2 is a murine MAb that was generated in this laboratory that recognizes an epitope in the apical membrane of AT1 cells in situ that is not detectable in other adult rat lung cells or other tissues (13). By IF, VIIIB2 also labels AEC in primary culture that have undergone transdifferentiation toward the AT1 cell phenotype. The polyclonal rabbit anti-AQP-5 antibody (Alpha Diagnostic, San Antonio, TX) recognizes a 28-kDa protein present in AT1 cells. This antibody has been used for both immunofluorescent labeling (6) and immunoblotting (7) to detect AQP-5 in tissues and cells, respectively, and yields equivalent results to polyclonal antisera obtained from Landon S. King (Johns Hopkins Univ.) and Chemicon (Temecula, CA) that were raised to the same peptide. Polyclonal guinea pig or rabbit anti-SP-A antibodies [J. Whitsett (31) and J. Shannon (23, 46), University of Cincinnati] were used to identify AT2 cells. Although known to be expressed in some cell types other than AT2 cells (e.g., Clara cells), SP-A is not expressed by AT1 cells. Leukocytes were identified using a murine monoclonal anti-LCA antibody (Chemicon).
Na pump and Na channel antibodies.
The 1-subunit isoform of Na-K-ATPase was detected using
a murine MAb (6H or 464.4) obtained from M. Caplan, Yale University (24), although it is now also commercially available from
UBI (Lake Saranac, NY). This antibody has been used by many
investigators for detection of Na pumps in a wide variety of mammalian
cells and tissues by immunohistochemical and immunofluorescent
techniques (37) and can also detect Na pumps by
immunoblotting (50). We and others have previously used
this reagent to detect and quantify Na pump
1-subunits
in AEC by IF and Western blotting (3, 8, 12, 35, 55). The
1-subunit isoform of Na-K-ATPase was detected using a
murine MAb (IEC 1/48) obtained from A. Quaroni, Cornell
University (26). We have used it to identify Na pump
1-subunits in lung and AEC (12, 55). As
described by Zhang et al. (55), it can be used to
immunoprecipitate Na pump
1-subunits from rat lung and
other tissues. The presence of the
2-subunit isoform of
Na-K-ATPase was evaluated using a mouse MAb (McB2) obtained from K. Sweadner, Harvard University, and has also been used widely for
detection of Na pumps in many different cells and tissues by
immunoblotting and immunohistochemistry (47, 50, 51). The
-subunit of rENaC was detected using a rabbit polyclonal antibody
(1190-2) obtained from D. Benos, University of Alabama. It is an
anti-
-bENaC (bovine ENaC) antibody that recognizes
-rENaC as well
as
-bENaC but reacts negligibly with
- and
-rENaC and has been
used to detect cells expressing
-rENaC by ELISA, Western analysis
(20), and IF (22).
IF (isolated cells).
Fixed cytocentrifuged cell preparations were rinsed in PBS and treated
with PBS-3% BSA overnight to block nonspecific reactivity. Cells were
identified by double labeling with combinations of 1° antibodies
specific for AT1 cells (VIIIB2 and anti-AQP-5), AT2 cells (anti-SP-A),
or leukocytes (anti-LCA). In this set of experiments, specific
combinations of 1° MAb and polyclonal antibodies (PAb) used to
minimize cross-reactivity of 2° antibodies were VIIIB2 (MAb) and
anti-AQP-5 (PAb), VIIIB2 (MAb) and anti-SPA (PAb), and anti-AQP-5 (PAb)
and anti-LCA (MAb). Localization of the Na pump and Na channel subunits
to AT1 cells was determined by concurrent labeling of cells with either
VIIIB2 or anti-AQP-5 (used as the AT1 cell marker) and antibodies to
the 1-,
2-, or
1-subunits of Na-K-ATPase or the
-subunit of rENaC. For the reasons noted above
to avoid cross-reactivity of 2° antibodies, specific combinations of
1° antibodies used for this set of experiments were: anti-AQP-5 (PAb)
and anti-
1 (MAb), anti-AQP-5 (PAb) and
anti-
1 (MAb), anti-AQP-5 (PAb) and anti-
2
(MAb), and VIIIB2 (MAb) and anti-
-rENaC (PAb). Negative controls
included substitution of normal rabbit or guinea pig serum or rabbit
IgG for the PAbs, or a nonreacting mouse MAb MF20 (D. Fischman, Cornell
Univ.), which is reactive only with chicken myosin for the MAbs
(13). After being incubated with 1° antibodies or
negative controls, slides were washed with PBS, followed by incubation
with appropriate combinations of anti-mouse and anti-rabbit or
anti-guinea pig 2° antibodies linked to either rhodamine
isothiocyanate (red) or fluorescein isothiocyanate (FITC, green).
Slides were treated with Vectashield (Vector Laboratories, Burlingame,
CA) antifade mounting media and were viewed with an Olympus BX60
microscope equipped with epifluorescence optics.
IF (intact lung).
Normal rat lung was inflated and fixed in 4% paraformaldehyde. After
being embedded in paraffin, 2- to 5-µm sections were cut. After being
deparaffinized and rehydrated through graded alcohols, slides underwent
microwave antigen retrieval (Antigen Unmasking Solution; Vector) and
were double labeled using the anti-1-subunit of
Na-K-ATPase MAb and anti-AQP-5 PAb. Rabbit polyclonal anti-AQP-5
antibody was amplified using an avidin-biotin system with FITC
(Vector), and following an avidin-biotin block step, mouse monoclonal
anti-
1-antibody was amplified using an avidin-biotin
system with Texas red (Vector). Negative controls included substitution
of normal rabbit serum or rabbit IgG for the anti-AQP-5 PAb and MF20 or
MOPC21 (Sigma), an irrelevant mouse IgG1, for the
anti-
1-subunit antibody. Tissue sections were treated with Vectashield antifade mounting media. Images were captured using a
cooled charge-coupled device camera (Magnafire; Olympus, Melville,
NY) with a barrier filter equipped for simultaneous detection of Texas
red and FITC. Images were imported into Adobe Phostoshop (Adobe
Systems, Mountain View, CA) as TIFF files and printed in CMYK color
using a Hewlett-Packard Deskjet 970C.
RNA extraction and RT-PCR.
Total cellular RNA was extracted from highly purified (~95%) AT1
cell preparations immediately following cell isolation using an RNeasy
mini-kit for RNA extraction from small numbers of cells (Qiagen,
Valencia, CA). DNA was removed by on-column DNase treatment. RNA was
reverse transcribed using Moloney murine leukemia virus reverse
transcriptase (SuperScript II; Invitrogen Life Technologies, Carlsbad,
CA) in the presence of random primers, followed by amplification with
Taq polymerase using subunit-specific oligonucleotide
primers for the 1-,
2-, and
1-subunit isoforms of Na-K-ATPase and the
-subunit of
rENaC. Specific PCR primer pairs used for amplification were
1)
1-subunit: 5'-TCC-TCC-CTC-TTT-CCT-CCG-3'
(bases 99-116) and 5'-GCC-TCG-GCT-CAA-ATC-TGT-TC-3'
(complementary to bases 423-404); 2)
1-subunit: 5'-CAT-CTG-GAA-CTC-GGA-GAA-GAA-GG-3' (bases
504 to 526) and 5'-TTG-GGG-TCA-TTA-GGA-CGG-AAG-3' (complementary to bases 746 to 726); 3)
2-subunit:
5'-TGG-GGGTGG-CAA-GAA-GAA-AC-3' (bases 152 to 171) and
5'-CAT-AGG-CTA-AGA-AGC-AGA-GAA-GCG-3' (complementary to bases 438 to
415); and 4)
-rENaC:
5'-TCA-CTT-CAG-CAC-ATC-TTC-CCC-AGC-G-3' (bases 2211 to 2235) and
5'-GTA-CCT-GCC-TAC-CCG-TCC-CAA-GTG-G-3' (complementary to bases 3062 to
3038). Samples were amplified for 27-30 cycles, and PCR
products were separated by agarose gel electrophoresis. Assuming that
all the contaminating cells were AT2 cells, parallel experiments were
performed using RNA from the number of AT2 cells equal to the number of
contaminating cells present in the AT1 cell preparations, followed by
PCR amplification within the linear range (27-30 cycles). Ethidium
bromide-stained gels were visualized with ultraviolet light.
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RESULTS |
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Characterization of isolated alveolar cells. Labeling of cytocentrifuged preparations using combinations of 1° antibodies specific for AT1 cells (VIIIB2), AT2 cells (anti-SP-A antibody), or leukocytes (anti-LCA antibody) indicates that the partially purified alveolar cell populations are comprised of approximately equal numbers of these three cell types. Immunomagnetic selection to further enrich for AT1 cells yielded ~500,000 AT1 cells per rat, with purity ranging from 70-98% and >90% viability. Preparations of ~95% purity were used for RT-PCR analysis.
Identification of AT1 cells by double labeling IF.
Cytocentrifuged cell preparations were concurrently labeled
with VIIIB2 and anti-AQP-5. As shown in Fig.
1, all cells that are reactive with
VIIIB2 are also reactive with anti-AQP-5. Furthermore, all cells not
reactive with VIIIB2 are also not reactive with anti-AQP-5. In
contrast, cells reactive with VIIIB2 do not label with anti-SP-A
antibody (which identifies AT2, but not AT1, cells in whole lung in
situ) and vice versa (Fig. 2). Similarly,
cells that are reactive with anti-AQP-5 are also not labeled with
anti-SP-A, and double labeling with anti-LCA and either anti-AQP-5 or
anti-SP-A indicates that neither AT1 nor AT2 cells are reactive with
anti-LCA (data not shown). No reactivity was observed when normal
rabbit or guinea pig serum (or IgG) was substituted for the polyclonal anti-AQP-5 and anti-SP-A antibodies, respectively, or when MF20 was
substituted for VIIIB2 or anti-LCA antibodies. Colocalization of both
AT1 cell markers to the same cells, together with the absence of
labeling of these same cells with either anti-SP-A or anti-LCA,
confirms that these AT1 cell-specific antibodies can be used
successfully to identify AT1 cells within partially purified
populations of alveolar cells.
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Identification of Na pump subunits by IF in isolated alveolar
cells.
Localization of the 1-,
1-, and
2-subunits was performed by concurrent labeling of cells
with the AT1 cell-specific anti-AQP-5 PAb and the Na pump subunit
isoform MAbs. As shown in Fig. 3, AT1
cells, identified by reactivity with anti-AQP-5, also label strongly
with 6H, an antibody specific for the
1-subunit of
Na-K-ATPase. Similarly, AT1 cells are strongly reactive with IEC 1/48
antibody to the
1-subunit of Na-K-ATPase (Fig.
4). Other cells in
the field (presumably AT2 cells) are also labeled with the
anti-
1 and anti-
1 Na pump subunit
antibodies, but not with anti-AQP-5. No reactivity was observed when
normal rabbit serum (or IgG) or MF20 were substituted for either the
polyclonal rabbit anti-AQP-5 or the monoclonal anti-Na pump subunit
antibodies, respectively. Neither AT1 cells nor any other cells are
labeled with the anti-
2 Na pump subunit antibody McB2
(Fig. 5). Rat brain, used as a positive control for
2, was strongly reactive with the
anti-
2-subunit antibody. Double labeling with anti-SP-A
and Na pump subunit antibodies confirms that AT2 cells within these
partially purified populations also express the
1 and
1 Na pump subunit isoforms (data not shown).
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Identification of rENaC -subunit by IF in isolated alveolar
cells.
Localization of the
-subunit of rENaC was performed by concurrent
labeling with the AT1 cell-specific MAb VIIIB2 and the anti-
-rENaC
subunit PAb. As shown in Fig. 6, AT1
cells, identified by their reactivity with VIIIB2, are also reactive
with anti-
-rENaC. Other cells in the field not reactive with VIIIB2
(presumably AT2 cells) are also reactive with anti-
-rENaC. No
reactivity was observed when MF20 or normal rabbit serum (or IgG) was
substituted for either the monoclonal VIIIB2 or polyclonal rabbit
anti-
-rENaC antibodies, respectively.
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Identification of 1 Na pump subunit in AT1 cells in
whole lung.
Sections of whole rat lung were double labeled with anti-AQP-5 PAb and
mouse anti-
1 Na pump subunit MAb. AT1 cells are
identified by labeling of the apical cell surface with anti-AQP-5
(green) (Fig. 7). As shown in Fig.
7A, linear staining (red) localizes the
1-subunit of Na-K-ATPase to the basolateral surface of
AQP-5-expressing AT1 cells. Substitution of mouse IgG for the
anti-
1 MAb reveals no reactivity with AT1 cells (Fig.
7B). Figure 7C demonstrates that relative signal
intensity for the
1-subunit is much greater on the
basolateral membrane of AQP-5-negative AT2 cells than AT1 cells,
perhaps accounting for the inability to detect AT1 cell Na pumps in
situ in previous reports.
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Identification of Na pump and Na channel subunits by RT-PCR in
isolated AT1 cells.
RT-PCR of highly purified AT1 cell RNA demonstrates appropriately sized
bands for the 1- and
1-subunits of
Na-K- ATPase and the
-subunit of rENaC (Fig.
8). Within the linear range of amplification, a relatively faint band is seen using RNA from a number
of AT2 cells equal to the number of contaminating cells in the AT1 cell
preparations (Fig. 8). Consistent with the IF data reported above in
isolated cells, the
2 Na pump subunit is not detected in
AT1 cells, although a strong band is seen with the PCR-amplified
2-subunit plasmid. No bands are seen following reverse
transcription without PCR.
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DISCUSSION |
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We evaluated expression of Na pump and Na channel subunits in
isolated AT1 cells and in AT1 cells in situ. Utilizing a panel of
cell-specific antibodies to identify AT1 cells within partially purified populations of freshly isolated distal lung cells, we demonstrated that isolated cells labeled by VIIIB2 are also reactive with anti-AQP-5. Furthermore, cells identified by these AT1
cell-specific antibodies do not react with anti-SP-A or anti-LCA,
demonstrating the success of this approach for identification of AT1
cells within mixed alveolar cell populations. Using a strategy of
concurrent labeling and immunofluorescent detection, we localized Na
channel and Na pump subunits to isolated cells that are reactive with the AT1 cell-specific antibodies. Expression of the 1-
and
1-subunits, but not the
2-subunit
isoforms of Na-K-ATPase, and the
-subunit of rENaC was demonstrated
in isolated AT1 cells. Consistent with these IF results, transcripts
for the Na pump
1- and
1- (but not
2-) subunits and
-rENaC were also detected in highly
purified AT1 cells by RT-PCR. Concurrent labeling with anti-AQP-5 in
whole lung demonstrates expression of the
1 Na pump
subunit by AT1 cells in situ. These findings represent the first
successful demonstration that AT1 cells express Na transport proteins,
suggesting a role for AT1 cells in Na transport across alveolar epithelium.
Until recently, AT1 cells have largely been characterized by their morphological appearance. Because AT1 cell morphology changes markedly following cell isolation, definitive identification of isolated AT1 cells has been problematic. The advent of a number of antibody probes that react specifically with AT1 cells in the adult rat lung in situ has provided nonmorphological means to identify AT1 cells in vivo and, as methods for isolation of AT1 cells, have been developed, within populations of isolated lung cells. VIIIB2 is a murine MAb generated in our laboratory that recognizes an epitope in the apical membrane of AT1 cells that is not detected in other rat lung or nonlung cells (13). The polyclonal anti-AQP-5 antibody recognizes a 28-kDa water channel present in the adult in salivary and lacrimal tissues and in lung (7, 25, 38). In the alveolar epithelium, AQP-5 is expressed only on the apical surface of AT1 cells (30). Using these antibodies for labeling of cytocentrifuged preparations of isolated alveolar cells, colocalization of these antibodies that are specific for AT1 cells in situ is seen. Furthermore, cells that are reactive with anti-SP-A (AT2 cells) or anti-LCA (leukocytes) are not reactive with the AT1 cell-specific antibodies, consistent with their identity as AT1 cells.
Several methods have been described for isolation of AT1 cells.
Approaches have included digestion using different combinations of
enzymes in conjunction with density gradient separation
(53), centrifugal elutriation (1), and/or
selective adhesion. However, results have been variable, and routine
isolation of large numbers of viable AT1 cells to high levels of purity
has been problematic. In part, this may reflect the inability to easily
detach these large cells, with their expansive cytoplasmic processes,
from the underlying basement membrane. Most recently, using elastase at
4.5 U/ml, together with subsequent magnetic selection with cell-specific antibodies, isolation of AT1 cells with purity ranging from 60-86% has been reported (15). Perhaps as a
consequence of the large surface over which they are spread, and to
release AT1 cells from the basement membrane, elastase was used at more than twice the concentration usually required for AT2 cell isolation. In addition, to allow easy passage of the larger AT1 cells, filtration steps were performed using Nitex mesh of relatively large pore size (20 µm). Using a modification of this approach, we successfully isolated
mixed cell populations comprised of approximately equal numbers of AT1
cells, AT2 cells, and leukocytes. We demonstrated that using a strategy
in which cells are concurrently labeled with AT1 cell-specific
antibodies, proteins of interest can successfully be localized to
individual AT1 cells. Nevertheless, since this level of purity is not
adequate for analysis of mRNA in AT1 cells, immunomagnetic selection
was undertaken using the AT1 cell-specific MAb VIIIB2 in conjunction
with a secondary antibody conjugated to magnetic beads. This approach
yielded AT1 cells of sufficient purity (95%) for analysis of mRNA
expression by RT-PCR. However, immunoselection with MAb VIIIB2
precluded the concurrent use of Na pump subunit MAbs for colocalization
in highly purified AT1 cell populations, making it preferable to
perform the double labeling IF experiments with partially purified cell preparations.
Na-K-ATPase is a heterodimeric protein consisting of an - and a
-subunit. The Na pump subunits occur in one of several distinct isoforms having characteristic tissue distribution (34).
There is general agreement that the predominant isoforms expressed in lung are
1- and
1-isoforms, which have
been localized to both AT2 cells in situ (19, 45) and
isolated AT2 cells (29, 33, 41, 48). Although low levels
of the
2-subunit isoform have been detected in whole
lung (34, 54), the precise distribution, if any, of this
isoform within cells of the alveolar epithelium has not been resolved
(3, 40). Although it is generally accepted that AT2 cells
possess functional Na pumps, for reasons delineated further below,
previous attempts to localize antigenic Na pumps to AT1 cells in situ
have been unsuccessful to date (19, 45). The IF and RT-PCR
data in this study in freshly isolated cells indicate that the
1- and
1-subunits, but not the
2-subunit, of Na-K-ATPase are in fact expressed by AT1
cells, while expression of the
1-subunit was also
confirmed in AT1 cells in situ. Although we demonstrate here expression
of the
1 and
1 Na pump subunits in AT1
cells, further studies are needed to delineate their functional role in
these cells.
mRNA for all three subunits of the rENaC have been identified in adult
rat lung (27, 39, 52), in freshly isolated rat AT2 cells
(32, 49), and in AEC after several days in culture (3, 12). In situ hybridization demonstrates diffuse
labeling of the distal alveolar surface for predominantly the - and
-subunits of rENaC (16, 27, 52), although none of these
subunits has been detected to date by immunohistochemistry in distal
rat lung (39). The pattern of labeling of the alveolar
surface for
-rENaC by in situ hybridization has been interpreted to
be consistent with labeling primarily of AT2 cells, but the diffuse
nature of the signal makes it impossible to conclude that AT1 cells do
not express
-rENaC. We demonstrate in the current study that the
-subunit of rENaC is expressed by freshly isolated AT1 cells. Consistent with our findings in isolated cells, a recent preliminary study by Johnson et al. (21) reported that all three Na
channel subunits are present by IF in AT1 cells in whole lung. Although we did not evaluate rENaC function in this study, further work will be
needed to determine its functional properties in these cells.
The large surface area occupied by AT1 cells in the lung, in
conjunction with observations that alveolar fluid clearance is both
ouabain sensitive and amiloride inhibitable, has always made it seem
unlikely that AT1 cells do not express Na pumps and Na channels.
Failure to detect antigenic Na pumps in AT1 cells in situ in previous
studies is thought to perhaps reflect the fact that Na pumps in AT1
cells are present at lower antigenic density than in AT2 cells (due
perhaps to their larger surface area) at a level below the sensitivity
of detection with the methods previously employed (45).
Lower antigenic density in AT1 cells than in AT2 cells is supported by
our observations that although the 1-subunit is detected
in AT1 cells in situ in the current study, relative signal intensity is
far stronger in AT2 cells than in adjacent AT1 cells in whole lung
(Fig. 7C). Increased sensitivity for detection of Na pump
subunits in isolated AT1 cells compared with whole lung may reflect, in
part, differences in the methods of fixation used for isolated cells
compared with whole lung. Reduction of antibody binding by fixation
(especially with aldehyde fixatives) was suggested by Schneeberger and
McCarthy (45) as a possible explanation for the patchy
detection of Na pumps in AT2 cells in whole lung and for failure to
detect antigenic Na pumps at all in AT1 cells. In addition, following
isolation, the cells round up, probably resulting in a higher antigenic
density per apparent membrane surface area than is present in situ.
Induction of Na transporter proteins during the isolation procedure,
while possible, seems an unlikely explanation given that the cells in the partially purified preparations undergo very few manipulations in
the short time before fixation. In any event, given that Na pump
subunits in AT1 cells may be present at a lower antigenic density than
in AT2 cells, availability of different antibodies in conjunction with
the use of antigen retrieval and signal amplification approaches likely
resulted in increased sensitivity, allowing us to identify the
antigenic signal in AT1 cells in situ.
Besides the recent demonstration that AT1 cells are highly permeable to water, suggesting a role for AT1 cells in water transport across alveolar epithelium (15), and a presumptive role in gas exchange, knowledge of AT1 cell function and identification of the genes and proteins expressed by these cells has been limited (44). Characterization of AT1 cell function and patterns of gene expression have been hindered by the difficulty in isolating purified populations of AT1 cells and in identifying isolated AT1 cells as well as AT1 cells in situ definitively by nonmorphological means. In this study, using complementary approaches in isolated cells and whole lung, we successfully localized both Na pump and Na channel subunits for the first time to AT1 cells, implicating AT1 cells in active Na transport across the alveolar epithelium.
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ACKNOWLEDGEMENTS |
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This work was supported in part by the American Lung Association, the American Heart Association, National Heart, Lung, and Blood Institute Research Grants HL-03609, HL-38578, HL-38621, HL-38658, HL-51928, HL-64365, and HL-62569, the Baxter Foundation, and the Hastings Foundation.
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FOOTNOTES |
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E. D. Crandall is Hastings Professor and Norris Chair of Medicine.
Address for reprint requests and other correspondence: Z. Borok, Division of Pulmonary and Critical Care Medicine, Univ. of Southern California, IRD 602, 2020 Zonal Ave., Los Angeles, CA 90033 (E-mail: zborok{at}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. Section 1734 solely to indicate this fact.
10.1152/ajplung.00130.2000
Received 21 April 2000; accepted in final form 27 November 2001.
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