Identification and isolation of mouse type II cells on the basis of intrinsic expression of enhanced green fluorescent protein
Jason M. Roper,1
Rhonda J. Staversky,2
Jacob N. Finkelstein,2
Peter C. Keng,3 and
Michael A. O'Reilly2
Departments of 1Environmental Medicine,
2Pediatrics, and 3Radiation
Oncology, School of Medicine and Dentistry, University of Rochester,
Rochester, New York 14642
Submitted 4 February 2003
; accepted in final form 7 May 2003
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ABSTRACT
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The unique morphology and cell-specific expression of surfactant genes have
been used to identify and isolate alveolar type II epithelial cells. Because
these attributes can change during lung injury, a novel method was developed
for detecting and isolating mouse type II cells on the basis of transgenic
expression of enhanced green fluorescence protein (EGFP). A line of transgenic
mice was created in which EGFP was targeted to type II cells under control of
the human surfactant protein (SP)-C promoter. Green fluorescent cells that
colocalized by immunostaining with endogenous pro-SP-C were scattered
throughout the parenchyma. EGFP was not detected in Clara cell secretory
protein-expressing airway epithelial cells or other nonlung tissues. Pro-SP-C
immunostaining diminished in lungs exposed to hyperoxia, consistent with
decreased expression and secretion of intracellular precursor protein. In
contrast, type II cells could still be identified by their intrinsic green
fluorescence, because EGFP is not secreted. Type II cells could also be
purified from single-cell suspensions of lung homogenates using
fluorescence-activated cell sorting. Less than 1% of presorted cells exhibited
green fluorescence compared with >95% of the sorted population. As expected
for type II cells, ultrastructural analysis revealed that the sorted cells
contained numerous lamellar bodies. SP-A, SP-B, and SP-C mRNAs were detected
in the sorted population, but T1
and CD31 (platelet endothelial cell
adhesion molecule) were not, indicating enrichment of type II epithelial
cells. This method will be invaluable for detecting and isolating mouse type
II cells under a variety of experimental conditions.
fluorescence-activated cell sorting; surfactant proteins; transgenic
ALVEOLAR TYPE II epithelial cells are critical for normal lung
development, homeostasis, and repair after injury. Type II cells produce
pulmonary surfactant lipids and proteins required for reducing alveolar
surface tension (10,
29,
30). As essential progenitors
for type I epithelial cells, they are also critical for normal alveolar
development and tissue remodeling after injury
(1,
2). Type II cell hypertrophy
and hyperplasia are often associated with chronic lung disease
(17). Because type II cells
express vascular endothelial growth factor, they may also regulate
vascularization (21). Given
their importance, it is no surprise that a large number of different animal
models have been used to study the type II cell phenotype in vivo and ex vivo.
The ability to investigate organogenesis and disease progression by
overexpressing and deleting genes in mice, particularly genes expressed by
type II cells, has recently favored the use of mice in pulmonary research
(27).
Although mice are advantageous for manipulating genes, they have not been
useful for isolating type II cells for ex vivo study. In contrast, rat and
rabbit type II cells have successfully been isolated using velocity
centrifugation through a gradient of albumin
(8,
10). Isolation of mouse type
II cells by this method has been less successful, because airway Clara cells,
which are extremely abundant in mice, frequently contaminate the preparations
(7,
18). Relatively pure
populations of mouse type II cells have been obtained by dispase with agarose
instillation and laser-flow cytometry, but it was necessary to label cells
with lipid-soluble phosphine fluorescent dye
(14). High yields have also
been obtained when flow sorting was replaced with panning on plates containing
anti-CD32 and anti-CD45 (5,
29). Although epithelial cells
are readily obtained with this method, we found that type II cell purity
varied between preparations (unpublished observations). Given that the
strength of the mouse model is its ability to be genetically manipulated, it
would be advantageous to develop new methods by which type II cells could be
routinely identified and isolated from genetically defined strains under
different experimental conditions.
Green fluorescent protein (GFP) from the jellyfish Aequorea
victoria fluoresces brightly when exposed to ultraviolet or blue light
(34). The active chromophore
is encoded within the primary translation product and does not require
enzymatic digestion for activity. A mutant form of GFP has been generated with
a red-shifted peak that fluoresces 35 times more intensely than wild-type GFP.
This enhanced GFP (EGFP) has been introduced into mammalian cells in vitro and
in vivo (9,
13,
24,
32,
37). Fluorescence microscopy
and fluorescence-activated cell sorting (FACS) have been used to visualize and
purify EGFP-expressing cells from transgenic mouse thymus, bone marrow, and
cardiac myocytes (9,
24,
37). On the basis of these
studies, we generated a line of mice in which EGFP was expressed in type II
epithelial cells under control of the human surfactant protein (SP)-C
promoter. This promoter targets genes specifically to the respiratory
epithelium (11,
12). Using these mice, we
demonstrate that type II cells may be visualized in real time and isolated on
the basis of their endogenous green fluorescence.
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MATERIALS AND METHODS
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Generation and identification of transgenic mice. The EGFP
open-reading frame was excised from pEGFP plasmid (Clontech, Palo Alto, CA)
using SalI and EcoRI enzymes and ligated directly into a
pUC18 vector containing the 3.7-kb human SP-C gene promoter kindly provided by
Dr. Jeffrey Whitsett (35). The
promoter sequences comprised -3683 to +21 of the human SP-C gene. The EGFP
cDNA was followed by a simian virus 40 (SV40) small t intron and a 0.4-kb
sequence containing a poly(A) addition signal with stop codons in all reading
frames. The SV40 sequence does not encode viral proteins. The expression
cassette of 4.8 kb was excised with SstI, gel purified onto
diethylaminoethyl membrane, and microinjected (5 ng/µl) into the pronuclei
of C57Bl/6J mice at the University of Rochester's Transgenic Animal Facility.
The University Committee on Animal Resources of the University of Rochester
approved the generation and handling of the mice.
Tail snips were obtained from potential founders and their progeny at
weaning and used to isolate genomic DNA
(19). DNA (10 µg) was
digested with EcoRI and separated on 0.8% agarose gels. DNA was
transferred to Nytran and hybridized with the EGFP cDNA radiolabeled with
32P (3,000 Ci/mmol; New England Nuclear, Boston, MA). Hybridized
blots were washed in 1% bovine serum albumin, 40 mM sodium phosphate, and 2 mM
EDTA before visualization on a PhosphorImage screen (Molecular Dynamics). Copy
number was determined by comparing transgene DNA band intensity with genomic
equivalents of pEGFP plasmid.
Analysis of transgene expression in whole lungs. Adult (8-12 wk
old) mice were exposed to room air or >95% oxygen (hyperoxia) for 72 h
(25). Animals were euthanized
by injection with pentobarbital sodium (100 mg/kg ip). Lungs were inflation
fixed through the trachea for 15 min with 10% neutral buffered formalin at 10
cm of pressure. Lungs were removed and further fixed for 12 h in the same
fixative. Individual lobes were removed and embedded in optimum cutting
temperature compound (Sakura Finetek, Torrance, CA) before they were frozen in
liquid nitrogen. Frozen sections (6 µm) were prepared from these lobes
using a cryostat and stored at -80°C. Slides were equilibrated to room
temperature and washed free of optimum cutting temperature compound using
Tris-buffered saline before coverslips were applied, and the slides were
visualized. Additional lobes were dehydrated in graded alcohol, cleared in
xylene, and embedded in paraffin. Paraffin sections (4-µm) were
immunostained with rabbit anti-Clara cell secretory protein (CCSP) serum
(28) or rabbit anti-pro-SP-C
serum (Chemicon International, Temecula, CA). Paraffin-embedded tissue
sections were deparaffinized with xylene and rehydrated through graded ethanol
and water. Sections for CCSP immunostaining were incubated overnight in
primary antibody and detected with Texas red dye-conjugated donkey anti-rabbit
IgG (Jackson Labs, West Grove, PA). Sections for pro-SP-C immunostaining were
subjected to antigen retrieval (AR) by boiling in 50 mM Tris, pH 9.5,
incubated overnight in primary antibody, subjected to tyramide signal
amplification (TSA) using a TSA-biotin system, and detected with
streptavidin-Texas red (New England Nuclear). EGFP was detected with anti-EGFP
serum (Clontech) and 3,3'-diaminobenzidine precipitation using
previously described methods
(25) or in sections subjected
to AR with an FITC-conjugated goat anti-EGFP antibody (Novus Biologicals,
Littleton, CO). Sections were immersed in 4',6-diamidino-2-phenylindole,
and fluorescence was visualized with a fluorescence microscope (model E800,
Nikon, Melville, NY). Images were captured with a digital camera (SPOT-RT,
Diagnostic Instruments, Sterling Heights, MI).
Protein expression was also determined by Western blot analysis. Tissues
were homogenized in lysis buffer containing protease inhibitors
(25). Proteins were separated
on 12% polyacrylamide gels and transferred to polyvinylpyrolidine membrane.
Membranes were blocked overnight at 4°C in 5% nonfat dry milk before
incubation in anti-EGFP serum (Clontech) or
-actin serum (Sigma
Chemical, St. Louis, MO). Immune complexes were detected by chemiluminescence
(Amersham, Arlington Heights, IL) and visualized by exposure to Kodak Bio-Max
film.
Isolation of type II cells by FACS. Mice were euthanized by
injection with pentobarbital sodium (100 mg/kg ip). The renal vessels of the
left kidney were isolated and lacerated to reduce blood volume. The pulmonary
capillaries were perfused with 10 ml of sterile saline to remove erythrocytes.
The lungs were dissected free of the thoracic cavity and rinsed in sterile
saline. Two different methods were used to dissociate epithelial cells from
the lung. One method involved dissociating the lungs with surgical scissors
and chopping them into a fine paste using an automatic tissue chopper. Minced
tissues were incubated in 10 ml of protease cocktail containing 0.5 mg/ml
collagenase, 0.5 mg/ml pronase, 0.4 mg/ml DNase, and 0.006% sodium bicarbonate
in Hanks' balanced salt solution for 1 h at 37°C. Dissociated tissue was
then filtered through a sterile 100-µm nylon cell strainer, and the
single-cell suspension was pelleted at 300 g and 4°C. Pellets
were resuspended in a suitable volume of PBS, and cell counts were obtained
using a Coulter counter. A second method involved instillation of perfused
lungs with dispase and then with low-melt agarose before preparation of
single-cell suspensions (5).
Cell suspensions were successively filtered through 100- and 40-µm cell
strainers and finally through 25-µm nylon gauze. Single-cell suspensions
were then pelleted by centrifugation at 300 g for 5 min at 4°C.
Cells obtained from transgenic mice (10 per experiment) were pooled and
resuspended in 10 ml of DMEM with 0.5% FBS and 25 mM HEPES. Cells obtained
from nontransgenic mice (1 per experiment) were resuspended in 2 ml of medium.
Presort cell counts were obtained using a Coulter counter.
Green fluorescent (EGFP) type II cells from dissociated lung tissues were
isolated using a cell sorter (B-D FACSVantage SE, Becton Dickinson
Immunocytometer Systems, Palo Alto, CA). A two-step sorting procedure was used
in our study to isolate the small population of EGFP-expressing cells with
high purity and yield. Single-cell suspensions were sorted first with the
"enrich" mode to collect every EGFP-positive cell. This procedure
resulted in enrichment of the EGFP-positive cells from
1% to 26%. The
enriched cell populations were sorted again with the "normal-R"
mode to achieve final purity of
95%. Cellular EGFP was excited by an
argon ion laser emitted at the wavelength of 488 nm, and the fluorescence was
collected after a 530 ± 30 nm band-pass filter. A two-parameter sorting
window (forward light scattering and EGFP fluorescent intensity) was used to
define the EGFP-positive cell populations. The cells were sorted through a
flow chamber with an 80-µm nozzle tip under 12 psi sheath fluid pressure.
The sorted cells were collected into 15-ml conical tubes filled with sterile
media for morphological and biochemical assays. On average,
2.5 h were
required to prepare a single-cell suspension of 1.5 x 108
cells from 10 mice. The enrich-mode sort was carried out at a rate of 10,000
cells/s, and completion requires
4.5 h. The normal-R mode sort occurred
at a rate of 1,000 cells/s, and completion requires
1 h.
Isolated cells were cytospun onto glass slides, fixed in a 2.5%
phosphate-buffered glutaraldehyde, pH 7.4, and post-fixed in a 1.0%
phosphate-buffered osmium tetroxide. The slides were passed through a graded
series of ethanol to 100% and infiltrated with liquid Spurr epoxy resin and
embedded on the glass surface with inverted capsular molds containing fresh
resin. After polymerization at 70°C, the hardened capsules were then
popped off the glass slide by dipping into liquid nitrogen. The
"popped-off" capsules were examined with a light microscope to
determine the area to be trimmed and thin sectioned with a diamond knife onto
200-mesh copper grids. The grids were contrasted with aqueous uranyl acetate
for 10 min and lead citrate for 15 min, examined, and photographed with a
transmission electron microscope (model 7100, Hitachi).
Genotypic analysis by RT-PCR. Gene expression was determined by
RT-PCR using total RNA isolated by phase-lock gel microcentrifuge tubes (5
Prime-3 Prime, Boulder, CO)
(25). cDNA templates were
synthesized at 42°C for 30 min using RT and oligo(dT) primers
(Perkin-Elmer, Foster City, CA). All products were amplified with 15 µM
primers and cycling 35 times at 94°C for 1 min, 51°C (SP-C), 57°C
(SP-A and T1-
) or 60°C (EGFP, SP-B, and aquaporin-5) for 1 min, and
72°C for 1 min. Primers for EGFP were
5'-ATGGTGAGCAAGGGCGAGGAGCTG-3' and
5'-CTTGTACAGCTCGTCCATGCCGAG-3', which amplified a 716-bp product.
Primers for SP-A were 5'-AAAGGGGGCTTCCAGGGTTTCCAGC-3' and
5'-ATTCCTCGGGGCAGCAATGTGG-3', which amplified a 224-bp product.
Primers for SP-B were 5'-TGTCTACCTGCCCCTGGTTATTG-3' and
5'-GCATCCTCAGTGGAACATCGG-3', which amplified a 471-bp product.
Primers for SP-C were 5'-CATGAAGATGGCTCCAGA-3' and
5'-TTTGTGATAGGATCCCCC-3', which amplified a 400-bp product.
Primers for aquaporin-5 were 5'-GGAGGTGTGTTCAGTTGCCTTC-3' and
5'-CTCAGCGAGGAGGGGAAAAG-3', which amplified a 684-bp product.
Primers for T1
were 5'-TACTGGCAAGGCACCTCTGG-3' and
5'-TCTGCGTTTCATCCCCTGC-3', which amplified a 200-bp product.
Primers for CD31 (platelet endothelial cell adhesion molecule) were
5'-AAGCGAAGGATAGATAAGACCTC-3' and
5'-CAGGATGGAAATCACAACTTCA-3', which amplified a 995-bp
product.
Statistical analysis. Where appropriate, values are means ±
SD. Group means were compared by ANOVA using Fisher's procedure post hoc
analysis, and P < 0.05 was considered significant.
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RESULTS
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Identification and characterization of transgenic mice. The EGFP
cDNA was inserted into unique SalI and EcoRI sites located
between the 3.7-kb human SP-C gene promoter and the 0.4-kb simian virus small
t intron and poly(A) signal (Fig.
1A). The small t intron and poly(A) signal enhance RNA
stability and do not encode viral oncogenic proteins. The 4.8-kb transgene was
excised free of plasmid sequences using SstI and injected into
C57Bl/6J pronuclei. Three founder mice were identified by Southern blot
analysis from a total of 76 mice screened. Two male and one female founder
mice were identified with 3-40 copies of the transgene integrated. All three
mice were independently mated to establish germ-line transmission. Although
plugs were observed when founder 9 was mated, females never became
pregnant. After unsuccessfully breeding for >1 yr, founder 9
unexpectedly died and was destroyed before transgene expression could be
ascertained. Germ-line transmission of the transgene was successful in the
remaining two founders. The transgene was detected in
20% of progeny from
founder 28a, indicating that her germ cells were mosaic for the
transgene. Her F1 progeny transmitted the transgene in typical Mendelian
fashion. Unfortunately, EGFP mRNA and protein were not detected, so this line
was not examined further. Founder 67 transmitted the transgene in
50% of his progeny, with typical Mendelian inheritance by the second
generation. Recently, homozygous (transgenic on both chromosomes) mice have
been obtained. Transgenic haplozygous (transgenic on one chromosome with no
complementary allele) progeny contained approximately three to five copies of
the transgene integrated as head-tail concatamers
(Fig. 1B). Because
EGFP mRNA was detected in their lungs (data not shown), these haplozygous mice
were studied further.

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Fig. 1. Transgene construction and identification of mice. A: human
surfactant protein-C (hSP-C) promoter from -3.7 to +21 kb was inserted into
pUC 18, where PstI (P) is at +21. The 0.7-kb enhanced green
fluorescent protein (EGFP) coding region was inserted into unique
SalI (S) and EcoRI (E) sites located just 3' of the
PstI site. The entire insert of 4.8 kb, including the simian virus 40
(SV40) poly(A), was excised free of pUC through unique SstI sites,
purified, and injected. B: genomic DNA (10 µg) from tail snips was
digested with EcoRI, separated by size, and hybridized with
32P-labeled EGFP cDNA. Lanes 1, 2, 3, and 4
contain 1-, 5-, 10-, and 50-genome copies of EGFP plasmid DNA, respectively,
as a positive control. Remaining lanes contain DNA from transgenic and
nontransgenic mice. Larger size of the transgene DNA (arrow) reflects addition
of the hSP-C promoter and SV40 3'-untranslated region.
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EGFP expression in transgenic mice did not affect fecundity, Mendelian
inheritance, or health of the mice. Nor have signs of respiratory distress or
abnormal histopathology been observed in older mice. EGFP expression was
assessed in a number of tissues by Western analysis. EGFP was readily detected
in lungs of transgenic, but not nontransgenic, littermates
(Fig. 2). As predicted for the
lung-specific expression of the SP-C promoter, EGFP was also not detected in
heart, intestine, kidney, liver, or brain of transgenic mice. The expression
of
-actin confirmed that all lanes contained protein.
Several methods were used to detect cells expressing EGFP.
Immunohistochemistry was initially used to localize EGFP expression in
transgenic and nontransgenic lungs (Fig. 3,
A and B). EGFP-positive cells were restricted to
cells in corners of alveoli, rather than along alveolar walls. Under
higher-power resolution, they exhibited cuboidal and granular morphology,
consistent with the type II cell phenotype (inset,
Fig. 3A). Staining was
not detected in airway epithelium, fibroblasts underlying airway, endothelial
cells surrounding blood vessels, or any cells of the nontransgenic lung. This
pattern of expression was confirmed by visualization of intrinsic green
fluorescence in frozen sections prepared from lungs of trangenic and
nontransgenic mice (Fig. 3, C and
D). To confirm EGFP expression in type II cells, tissue
sections were immunostained with anti-pro-SP-C and visualized using TSA and
streptavidin-Texas red conjugate. Sections were then stained with
FITC-conjugated anti-EGFP serum, because endogenous EGFP fluorescence is
extinguished by the AR method required for pro-SP-C staining
(Fig. 3, E and
F). Cells with green and red fluorescence were readily
detected, indicating that EGFP-expressing cells also expressed pro-SP-C.
Although all EGFP-positive cells expressed pro-SP-C, all type II cells did not
express detectable levels of EGFP. EGFP cells also colocalized with
pro-SP-B-expressing cells (data not shown). Because the human SP-C promoter
can target transgenes to airway Clara cells, EGFP expression was also compared
with CCSP expression. Intrinsic green fluorescence was used instead of EGFP
immunostaining, because, unlike pro-SP-C, CCSP staining does not require AR.
Red CCSP fluorescence was observed in terminal airway epithelial cells that
did not overlap with intrinsic green fluorescence
(Fig. 3, G and
H). These findings indicate that, in this particular line
of mice, EGFP expression is restricted to type II epithelial cells with little
to no expression in bronchiolar epithelium.

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Fig. 3. Type II epithelial cells express EGFP. EGFP was localized by
immunocytochemistry in paraffin-embedded sections prepared from transgenic
(A) and nontransgenic (B) lungs. Brown EGFP-positive cells
(arrows in A) are observed among methyl green-counter-stained blue
nuclei. Frozen sections were prepared from transgenic (C) and
nontransgenic (D) lungs, stained with
4',6-diamidino-2-phenylindole, and visualized by epifluorescence. Bright
green fluorescent cells (arrows in C) are observed among blue
fluorescent nuclei. EGFP- and pro-SP-C-expressing cells were colocalized in
transgenic mice (E and F). Inset in E is
enlarged in F. FITC-EGFP-positive green cells consistently overlapped
with pro-surfactant protein (SP)-C red cells (arrows in F).
G and H: intrinsic EGFP fluorescence (solid arrow in
H) does not overlap with Clara cell secretory protein
(red)-expressing airway epithelial cells (dashed arrow in H).
Inset in G is enlarged in H.
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EGFP fluorescence is retained during lung injury. SP expression is
markedly altered when lungs are injured. For example, hyperoxia (>95%
oxygen) stimulates SP-B mRNA and protein expression in bronchiolar epithelium
while diminishing expression in alveolar type II cells
(16,
36). Although the mechanism is
unknown, decreased protein expression in type II cells is associated with mRNA
loss and, presumably, failure to maintain intracellular levels of precursor
protein that is secreted along with surfactant lipids. Because EGFP is not
secreted, we hypothesized that it would be retained during hyperoxia, thereby
permitting identification of type II cells when SP expression is lost.
Transgenic mice were exposed to room air or >95% oxygen for 72 h, and
pro-SP-C and EGFP immunostaining were compared
(Fig. 4). Green EGFP-positive
type II cells were detected in lungs exposed to room air as a yellow-green
color because of their colocalization with red pro-SP-C immunostaining.
Although EGFP-positive cells were readily detected in hyperoxic lungs, red
pro-SP-C immunostaining was markedly diminished throughout the parenchyma.
Compared with room air-exposed tissues, the EGFP-positive cells exhibited a
bright green fluorescence due to the lack of red pro-SP-C staining. Thus EGFP
protein expression is retained during oxygen-induced lung injury and can be
used to identify type II cells that have diminished SP expression.

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Fig. 4. Expression of EGFP and pro-SP-C in lungs exposed to room air and hyperoxia.
Transgenic lungs exposed to room air (A) or hyperoxia (B)
were immunostained for EGFP and pro-SP-C. FITCEGFP green fluorescence
colocalized with red pro-SP-C fluorescence in room air-exposed lungs (arrows
in A). In hyperoxic lungs, FITCEGFP fluorescent cells were detected
(arrows in B) when red pro-SP-C fluorescence diminished.
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Isolation of type II epithelial cells on the basis of green
fluorescence. Previous studies have shown that intact cells expressing
EGFP can be isolated by FACS when excited under ultraviolet light
(9,
24,
37). Two different protease
cocktails were used to prepare single-cell suspensions that were visualized by
fluorescence microscopy. A rare fluorescent cell was detected when lungs were
dissociated with pronase and collagenase (data not shown). This suggested that
these proteases did not effectively remove type II cells from their matrix. In
contrast, brightly fluorescent cells were frequently seen when dispase and
agarose were instilled. This confirmed previous studies showing that this
method efficiently dissociates epithelial cells from lung tissue
(5,
14). Because all cells have
low intrinsic fluorescence, especially conducting airway epithelial and red
blood cells, the overall fluorescence was initially determined by FACS using
dissociated cells from a nontransgenic lung
(Fig. 5). After we set an
upper-limit gate that considered 0.053 ± 0.075% (M2 region in
Fig. 5) of the cells highly
fluorescent, cells from a transgenic lung were sorted using the normal-R mode
and a 1.5-drop sort envelope. We discovered that a long sorting time was
required and very few EGFP-positive cells were collected. Furthermore, overall
purity varied between experiments because of contaminating red blood cells. To
improve the purity and recovery of sorted cells, a two-step sorting procedure
was established. Cells were first sorted using the enrich mode to ensure that
every EGFP-positive cell was collected. Assessment of the fluorescence
intensity showed that the first-step sorting increased the percentage of
EGFP-positive cells from 1.12 ± 0.32% (M2 region in
Fig. 5B) to 26.55
± 1.07% (M2 region in Fig.
5C). The smaller fluorescent peaks in
Fig. 5C comprised
mostly red blood cells, which were discarded. Enriched EGFP-positive cells
were resorted using the normal-R mode and a three-drop sort envelope under
more stringent conditions (M3 = 20.79 ± 1.83%). As expected for the
heterocellular nature of lung tissue, forward light scatter revealed that the
presorted population exhibited a wide variation in cell size
(Fig. 6). Although multiple
cell sizes were present in the discarded EGFP-negative population, the
EGFP-positive population exhibited a uniform cell size consistent with
enrichment of a single cell type.

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Fig. 5. Graphs showing fluorescence intensity vs. cell number during sorting
procedure. A: fluorescence intensity was assessed in cells collected
from nontransgenic lungs and a positive gate M2 was set for cells exhibiting
high fluorescence. B: fluorescence intensity was then assessed in
cells collected from a transgenic lung, and cells under gate M2 were
collected. C: several highly fluorescent populations were detected
when collected cells were reanalyzed. Cells with highest fluorescence were
resorted and collected under gate M3. Blood cells comprised most of the
population under gate M1 and were discarded. Each histogram represents
50,000 events.
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Fig. 6. Graphs showing forward scatter (FS) light intensity vs. cell number.
Forward light scatter was measured in presorted (A), postsorted
EGFP-negative (B), and postsorted EGFP-positive (C) cells.
Each histogram represents 10,000 events examined within gate B
(line).
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Pre- and postsorted cells were visualized by fluorescence microscopy. An
occasional brightly fluorescent green cell was observed in the presorted
population (Fig. 7). Dispase
and agarose were used to obtain
2,000,000 cells per lung, of which 2.5
± 0.71% (n = 3) were highly fluorescent. The higher percentage
of fluorescent cells counted by eye than gated by FACS (
1%, region M2 in
Fig. 5B) represents
the more stringent criteria used by the laser in the cell sorter. In contrast,
94 ± 6.8% (n = 3) of the postsorted cells were highly
fluorescent as assessed by fluorescence microscopy. Approximately 50-100,000
fluorescent cells were obtained per lung, which could vary depending on the
rate at which cells passed through the FACS. Ultrastructural analysis of
sorted cells revealed that they contained lamellar bodies, characteristic of
type II epithelial cells (Fig. 7,
C and D).

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Fig. 7. Fluorescence-activated cell sorting of cells expressing EGFP. Cytospin of
presorted (A) and sorted (B) cells stained with
4',6-diamidino-2-phenylindole display green EGFP fluorescence and blue
nuclei. Arrow in B points to a contaminating nonfluorescent cell in
the sorted population. This photograph was derived from an experiment in which
>98% of the isolated cells were fluorescent. Ultrastructural analyses
confirmed that the sorted cells exhibited morphological characteristics of
alveolar type II cells, including the presence of lamellar bodies (C
and D). Magnification: x1,500 (C) and x6,000
(D).
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RT-PCR was used to confirm enrichment of type II epithelial cells in the
sorted population. With the exception of EGFP, primers were designed to bridge
introns so that amplification of genomic DNA could be distinguished from RNA.
Amplification of RNA-derived products was also confirmed by failure to amplify
EGFP when RT was omitted from the reaction
(Fig. 8). SP-A, SP-B, SP-C,
aquaporin-5, T1
, CD31, and CCSP were detected in RNA isolated from
whole lung. As expected, SP-A, SP-B, and SP-C were detected in the sorted
cells, consistent with their expression by type II cells. The type I
epithelial cell gene T1
and the endothelial cell gene CD31 (platelet
endothelial cell adhesion molecule) were not detected in the sorted cells.
Interestingly, aquaporin-5 was detected in some experiments, but only when
most of the RT-PCR product was loaded onto the gel (not shown). Because
aquaporin-5 was not always detected, it is likely to be due to the presence of
a contaminating cell type or occasionally expressed by type II cells. Clara
cells are most likely the predominant contaminant, because CCSP was faintly
detected in the sorted population, even though EGFP did not colocalize with
cells expressing CCSP.

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Fig. 8. Sorted cells express surfactant genes. Total RNA was prepared from intact
lung (top) or sorted cells (bottom) and analyzed by RT-PCR
for expression of EGFP, SP-A, SP-B, SP-C, aquaporin-5 (AQ-5), T1 , CD31,
and Clara cell secretory protein (CCSP). Reverse transcriptase was omitted (-)
in some samples to ensure that products were amplified from RNA. PECAM,
platelet endothelial cell adhesion molecule.
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DISCUSSION
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This study established that transgenic expression of EGFP could be used to
identify or isolate alveolar type II epithelial cells from mice. In one
example of its utility, type II cells were identified in hyperoxic lungs by
their green fluorescence, even though endogenous SP expression had diminished.
In another example, type II cells were purified on the basis of their
intrinsic green fluorescence. Purity exceeded 90% and could be increased by
narrowing the gate used to select the cells or slowing the rate at which cells
passed through the sorter. Each mouse lung yielded
2,000,000 cells from
which 50-100,000 fluorescent type II cells were obtained. Although transgene
expression was not ascertained in the discarded cells, nearly all
EGFP-expressing cells are calculated to have been positively selected for.
Higher yields per mouse will require the generation of additional lines with a
greater percentage of type II cells expressing EGFP. Even with these
limitations, this study established that type II cells can be tracked during
lung injury or isolated by FACS simply on the basis of transgenic expression
of EGFP.
The human SP-C/EGFP mice were developed with the intention that the
intrinsic nature of EGFP-mediated fluorescence might allow one to observe and
isolate type II cells in real time. Taking advantage of the targeting
capabilities of the human SP-C promoter and the knowledge that other lines of
mice have been generated that express EGFP, we successfully generated a line
of mice from which fluorescent type II cells can be identified and isolated.
Surprisingly, EGFP expression recapitulated endogenous SP-C expression, in
that it was restricted to the parenchyma. Previous studies revealed that the
human promoter drives ectopic expression of chloramphenicol acetyltransferase
(CAT) in bronchiolar epithelium and in alveolar type II cells
(12). A detailed deletion
analysis of the promoter revealed that ectopic bronchiolar expression was
dependent on sequences between -1910 and -215, with alveolar expression being
dependent on more proximal sequences
(11). Although the proximal
region provided appropriate expression to type II cells, the proportion of
type II cells expressing CAT varied between individual lines of mice. As
pointed out by Glasser et al.
(11), variation in transgene
expression has also been reported for the
-globin promoter. On the basis
of studies with the
-globin promoter, Glasser et al. speculated that the
chromatin environment around the integration site influences the proportion of
type II cells that express the SP-C/CAT transgene. Such a scenario would be
consistent with the relatively low proportion of type II cells that express
EGFP in our line of mice. In the same manner, the integration site may have
blocked activity of the distal elements responsible for the ectopic expression
typically seen in bronchiolar epithelium. The Southern blot analysis in
Fig. 1 indicates that the
SP-C/EGFP transgene did not undergo a gross deletion or inversion, which might
explain why bronchiolar expression is not detected. In some ways, the lack of
bronchiolar expression in this line of mice is a benefit for those interested
in exclusively tracking type II cells.
Even though high levels of EGFP can be toxic, it is unlikely to explain why
only some type II cells express the protein. First, lung pathology is normal,
and the mice appear to be unaffected by the transgene. Second, propidium
iodide exclusion revealed that viability of the isolated cells was 94.2
± 0.9% (n = 3). Third, transgenic mice have been created in
which EGFP is expressed in the lung and other tissues under control of the
chicken
-actin promoter and cytomegalovirus enhancer
(24). Furthermore, EGFP has
recently been expressed throughout the lung or in specific epithelial
populations depending on when Cre-mediated recombination occurred during
embryogenesis (26). Although
it is highly speculative, EGFP may be expressed only in a subpopulation of
type II cells that are distinguished by factors other than expression of
surfactant genes. Evidence that supports this hypothesis is as follows: weak
expression of aquaporin-5 was detected in some experiments, even though
T1
was not. Interestingly, aquaporin-5 was faintly detected in one of
the sorting experiments where 98% of the sorted cells expressed EGFP. The fact
that T1
has never been detected in the sorted population suggests that
type I cells were successfully excluded. Normally, alveolar type II cells do
not express aquaporin-5 (22).
However, aquaporin-5 has been observed in hyperplastic type II cells as a
result of conditional expression of fibroblast growth factor-7, indicating
that type II cells have the capacity to express this gene
(33). The finding that
aquaporin-5 is occasionally detected in the sorted cells suggests that some or
all type II cells may express low levels of this gene. Genotypic analysis of
these cells using microarray technology may someday reveal whether
subpopulations of type II cells exist.
A number of different methods have been used previously to isolate type II
cells. One method involved purification by velocity sedimentation
(8,
10,
18). Although type II cell
purity approximated
80% in rats and rabbits, yields from mice rarely reach
65%. Alternatively, type II cells have been isolated by flow cytometry on the
basis of their size, shape, and lipid content using phosphine fluorescence
(14,
20). Purity was improved by
dissociating lungs with dispase and gating against macrophages labeled with
fluorescent lectins. With these methods,
1,000,000-2,000,000 type II
cells per mouse were obtained at >90% purity. The present study also used
dispase and FACS to purify mouse type II cells. Unlike previous flow studies
that used fluorescent dyes to label lipids, type II cells were selected on the
basis of their expression of EGFP. Even though yields were considerably lower
than those from other published methods, type II cell purity was extremely
high. Indeed, only 1 of
100 cells examined by electron microscopy did not
contain lamellar bodies, a key characteristic of type II cells. Moreover, RNA
was purified and used successfully to genotype enrichment of genes expressed
by type II cells. Expression of the CCSP message in the sorted population
indicates that Clara cells, which did not express EGFP, may contaminate the
sorted population. Increasing the sorting stringency will reduce this
population, albeit at the expense of reducing the yield. To our knowledge,
this is the first time that type II cells were not isolated on the basis of
predetermined characteristics of cell shape, size, or lipid content. As such,
a uniform population of type II cells was purified solely on the basis of
their ability to express a single gene.
In addition to isolating cells, this line of mice can also be used to
follow type II cells in real time. As shown in
Fig. 4, EGFP may be used to
identify type II cells during hyperoxia when SP expression diminishes. We have
recently been able to isolate fluorescent type II cells from oxygen-exposed
lungs for studies on molecular oxidative damage. Thus these isolation methods
will be an invaluable new tool for distinguishing and isolating type II cells
from animals exposed to a number of different inhaled pollutants. EGFP
expression may also be followed in real time as cultured type II cells
differentiate into type I cells
(3,
4). This process is thought to
mimic the normal differentiation events that occur during embryogenesis or
remodeling of the injured lung
(1,
2). It will be interesting to
determine whether EGFP fluorescence can be maintained or restored by altering
culturing conditions that allow differentiation of type I-like cells to a type
II phenotype (6,
30,
31). Branching morphogenesis
has been studied in explant cultures. This process is thought to involve
inductive interactions between mesenchyme and epithelium
(15). A previous study
followed ureteric bud development in three dimensions using EGFP-mediated
fluorescence and confocal microscopy
(32). Similar studies may now
be applied to lung branching morphogenesis. EGFP fluorescence may also be used
to follow SP-C transcriptional activity under various experimental conditions,
such as during organogenesis or type II cell injury
(21,
23). By first backcrossing the
present line of mice onto genetically modified strains, one can also study the
role of specific genes in type II cell function. Clearly, the use of EGFP as a
noninvasive marker will enhance our ability to study type II cells under a
variety of experimental conditions.
In summary, we have shown that mouse type II cells can be identified and
isolated on the basis of transgenic expression of EGFP. As we identify new
cell type-specific promoters and develop new isoforms of GFP that fluoresce at
different wavelengths, our ability to track multiple cell types at the same
time should increase. Indeed, the present method for isolating EGFP-expressing
type II cells should be amenable to isolating other cell types as new lines of
intrinsically fluorescent mice become available. Until that time, the present
isolation procedures used with this line of mice provides an exciting new
opportunity to investigate mouse type II epithelial cells.
 |
DISCLOSURES
|
---|
This work was funded in part by National Heart, Lung, and Blood Institute
Grants HL-58774 and HL-67392 (M. A. O'Reilly). J. M. Roper was supported by
National Institutes of Health Training Grants ES-07026 and HL-66988. The Flow
Cytometry and Animal Inhalation Facilities are supported in part by National
Institute of Environmental Health Sciences Center Grant ES-01247.
 |
ACKNOWLEDGMENTS
|
---|
We thank Rick Watkins for advice on fluorescent microscopy and Barry Stripp
for the rabbit anti-CCSP antibody. The University of Rochester's Transgenic
Mouse Facility generated the transgenic mice, and the Electron Microscopy Core
performed the ultrastructural analyses on isolated cells.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: M. A. O'Reilly, Dept.
of Pediatrics, Box 850, School of Medicine and Dentistry, University of
Rochester, 601 Elmwood Ave., Rochester, NY 14642 (E-mail:
michael_oreilly{at}urmc.rochester.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.
 |
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