From the Departments of Internal Medicine, Howard Hughes Medical
Institute, Physiology and Biophysics, University of Iowa College of
Medicine, Iowa City, Iowa 52242, the Division of
Immunologic and Infectious Diseases, Children's Hospital of
Philadelphia, Philadelphia, Pennsylvania 19104, and the ¶ Dana
Farber Cancer Institute, Boston, Massachusetts 02115
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ABSTRACT |
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Recent identification of two receptors for the
adenovirus fiber protein, coxsackie B and adenovirus type 2 and 5 receptor (CAR), and the major histocompatibility complex (MHC) Class I The mechanism of infection by type 2 and type 5 adenovirus has
been extensively studied. However, most of the knowledge on adenoviral
infection has been obtained from studies done on immortalized cell
lines. The first steps in adenovirus infection are thought to involve
primarily two proteins in the capsid, fiber and penton base (1-3). The
fiber protein is important for binding to a high affinity fiber
receptor. In a human oral epidermoid carcinoma cell line (KB cells),
A549 cells, and HeLa cells, this receptor is thought to be present in
the range of 3,000-10,000 receptors/cell (4-6). NIH 3T3 cells, which
are resistant to adenovirus infection, have less than 100 receptors/cell (7). After binding to the fiber receptor, penton base
interaction with We and others (15-22) have found infection of ciliated airway
epithelia by adenovirus to be inefficient. In an in vitro
model of human airway epithelia, we found that unlike infections of HeLa cells adenovirus infection of ciliated airway epithelia was quite
limited and that which did occur was not blocked by co-incubation with
soluble fiber protein (15). In a second set of experiments, we measured
fiber-mediated binding of adenovirus and found that fiber protein had
no effect on binding to differentiated airway epithelia. Based on these
studies, we concluded that ciliated airway epithelia lack high affinity
fiber receptor activity.
Two separate groups have recently cloned receptors that bind adenovirus
fiber and may be involved in the pathogenesis of adenovirus infection.
Bergelson et al. (23) identified a fiber receptor, the
coxsackie B and adenovirus type 2 and 5 receptor
(CAR).1 When transfected with
CAR cDNA, non-permissive Chinese hamster cells became susceptible
to adenovirus attachment and infection. The results from Bergelson
et al. (23) are in concordance with Mayr and Freimuth (24)
who found that human chromosome 21 DNA-containing CAR sequence allows
Chinese hamster cells to bind fiber. Hong et al. (25) used a
phage display hexapeptide library to identify mimotopes of the fiber
protein receptor. One of these mimotopes was homologous to the core
motif of the MHC-I Human Airway Epithelia--
Airway epithelial cells were
obtained from surgical polypectomies or from trachea and bronchi of
lungs removed for organ donation from non-CF and CF patients as
indicated. Cells were isolated by enzyme digestion as described
previously (26). Freshly isolated cells were seeded at a density of
5 × 105 cells/cm2 onto collagen-coated,
0.6-cm2 diameter millicell polycarbonate filters (Millipore
Corp., Bedford, MA). The cells were maintained at 37 °C in a
humidified atmosphere of 5% CO2 and air. Twenty four hours
after plating, the mucosal media was removed, and the cells were
allowed to grow at the air-liquid interface (27, 28). The culture media
consisted of a 1:1 mix of Dulbecco's modified Eagle's medium/Ham's
F12, 5% Ultraser G (Biosepra SA, Cedex, France), 100 units/ml
penicillin, 100 µg/ml streptomycin, 1% nonessential amino acids, and
0.12 units/ml insulin. Epithelia were tested for transepithelial
resistance and for morphology by scanning electron microscopy.
Recombinant Adenoviruses--
Recombinant adenovirus vectors
expressing Viral Infection and Binding Assays--
Epithelia were allowed
to reach confluence and develop a transepithelial electrical resistance
(Rt), indicating the development of tight junctions and an intact
barrier. All epithelia had values of Rt>500 Ribonuclease Protection Assay--
To obtain a quantitative
assessment of CAR message in human airway epithelia, we used the
ribonuclease protection assay. Total RNA was isolated from primary
airway epithelia using RNA-STAT 60 (Tel-Test Corp., Friendswood, TX)
according to the manufacturer's instructions. [32P]UTP
randomly labeled antisense RNA probes for CAR and Analysis of CAR and MHC by Immunocytochemistry--
To evaluate
CAR expression at the apical and basolateral membrane of differentiated
airway epithelia, unpermeabilized ciliated airway epithelia were
incubated for 3 h with 1:60 dilution of an anti-CAR monoclonal
antibody (RmcB) applied either to the apical or to the basolateral side
at 4 °C. After careful rinsing, the epithelia were incubated with a
1:200 dilution of FITC-labeled anti-mouse antibody (Jackson
ImmunoResearch) for 1 h. Non-immune ascites fluid and secondary
antibody alone were used as negative controls. Staining with RmcB was
detected on HeLa cells and CAR cDNA-electroporated Chinese hamster
cells but not on CFTR cDNA-electroporated cells (data not shown).
To evaluate MHC-I Measurement of Measurement of GFP Expression--
To detect GFP expression, we
dissociated the epithelia with 0.05% trypsin and 0.53 mM
EDTA. Fluorescence from 50,000 individual cells was analyzed using
fluorescence-activated cell analysis (FACScan, Lysys II software,
Becton Dickinson, San Jose, CA). The percentage of cells positive for
GFP was assessed by determining the percent of highly fluorescent cells
in each group and subtracting the fluorescence of the control cells. To
assess the GFP expression of immature, basal non-ciliated epithelial
cells, aliquots of the cell suspension were fixed with 1.8%
formaldehyde and 2% glutaraldehyde for 5 min. The cells were spun at
1000 rpm and resuspended in PBS at a density of 107
cells/ml. The cell suspension was incubated with 1:100 monoclonal anti-CK-14 antibody or no antibody for 2 h. The cells were
centrifuged and resuspended with a 1:50 Texas Red anti-mouse IgG for
1 h. GFP and Texas Red fluorescence (CK-14) from 10,000 individual cells were analyzed using fluorescence-activated cell analysis (16). To
assess the level of CAR expression by GFP-positive cells, aliquots of
the cell suspension were incubated with RmcB antibody (1:60 dilution)
for 2 h. The cells were centrifuged and resuspended with a
1:200 dilution Texas Red anti-mouse IgG (Jackson ImmunoResearch)
for 1 h. GFP and Texas Red (CAR) fluorescence from 10,000 individual cells were analyzed using fluorescence-activated cell analysis.
Measurement of Transepithelial Electrical
Properties--
Epithelia were mounted in modified Ussing chambers
(Jim's Instruments, Iowa City, IA) as described previously (33).
Epithelia were bathed on the submucosal surface with a Ringer's
solution containing, in mM, 135 NaCl, 2.4 KH2PO4 0.6 KH2PO2 1.2 CaCl2, 1.2 MgCl2, 10 Hepes (titrated to pH 7.4 with NaOH), and 10 dextrose. The mucosal solution was identical with
the exception that NaCl was replaced with 135 mM sodium
gluconate. Amiloride (10 µM) was added to the mucosal
solution to inhibit Na+ channels and transepithelial
Na+ transport. The cAMP agonists, 10 µM
forskolin and 100 µM isobutylmethylxanthine, were added
to the mucosal and submucosal solutions to stimulate transepithelial
Cl Binding of Fluorescent Virus by Confocal Microscopy--
Airway
epithelia were treated with EMEM, H2O, or 8 mM
EGTA in EMEM as described above. Immediately after treatment, 50 m.o.i. of Cy3-labeled Ad2/ Expression of CAR Message by Ciliated Airway Epithelia--
We
investigated the abundance of CAR mRNA in airways by using primary
cultures of human airway epithelia grown on permeable filter supports.
We previously showed that 3 days after seeding, the airway epithelia
showed a fiber-dependent infection by adenovirus (15, 16).
Fourteen days after seeding most of the cells in the epithelia were
covered with cilia, and these epithelia were resistant to adenovirus
infection. To evaluate the contribution of CAR to adenovirus infection,
we studied CAR mRNA in poorly differentiated (3 days after seeding)
or well differentiated (14 days after seeding) airway epithelia. Fig.
1 shows that both poorly differentiated
and well differentiated airway epithelia expressed similar levels of
CAR mRNA. These data indicate that ciliated airway epithelia
express CAR message at similar levels to HeLa cells and poorly
differentiated airway epithelia. There are at least two possible
explanations as follows: first, CAR is expressed in differentiated
epithelia, but the protein is present only on the basolateral membrane;
second, only a few cells express CAR, and vectors applied to the lumen
do not have access to those cells.
CAR and MHC Immunocytochemistry--
We first evaluated the
possibility that CAR is not expressed at the apical membrane of
differentiated airway epithelia by immunocytochemistry. No staining was
observed when the antibody was added to the apical side (Fig.
2A). However, CAR staining was
detected on the surface of most cells when the antibody was added to
the basolateral side. Furthermore, a small population of cells showed
very high levels of CAR staining (Fig. 2A). When no primary
or non-immune ascites fluid was used, no stain was visible (data not
shown). To more quantitatively analyze CAR expression, we used
fluorescence-activated cell scanning (FACS) to determine the
number of cells expressing CAR and the level of CAR expression by each
cell. Fig. 2B shows examples of histograms obtained from cells stained with anti-CAR antibody. The shift in fluorescence suggests that most cells express CAR. In addition, the histogram shows
that a subpopulation of the epithelial cells was highly fluorescent.
These highly fluorescent cells may represent basal cells, since we
found a similar number of cells were positive for cytokeratin 14 (CK-14) expression, a basal cell marker (16) (data not shown). Because
both anti-CAR and anti-CK-14 were mouse monoclonals, studies to
evaluate co-localization of CK-14 and CAR by FACS were not
possible.
We also studied the cellular distribution of MHC-I Adenovirus Infection of Airway Epithelia Is Polar--
We
investigated the ability of adenovirus to infect human airway epithelia
through the basolateral side by a fiber-dependent mechanism. Since the adenovirus diameter is only 70-130 nm (3), it
should be able to pass freely through the Millipore filters (0.4 µm
pores) used to grow the airway epithelia. To test this hypothesis, we
turned the epithelia upside down and carefully applied 50 m.o.i.
of Ad2/
To estimate the number of cells achieving gene transfer via the
basolateral side, we applied Ad2/GFP to the basolateral surface for 30 min, and after 48 h we assayed GFP expression by FACS. Fig.
5A shows that 41.3 ± 4.4% of the cells were positive for GFP expression when the virus was
applied via the basolateral side compared with 1.3 ± 0.2% when
it was applied via the apical side. Fig. 5B shows a
fluorescence photomicrograph of epithelia infected with Ad2/GFP via the
apical and basolateral sides. Many cells showed moderate GFP
fluorescence, and a small population of cells had a higher intensity of
fluorescence (arrows). We hypothesized that depending on the
level of CAR expression, some cells will be more prone to infection. To
evaluate this possibility further, we analyzed cells by FACS. Fig.
6A shows two different
populations of GFP-positive cells, one with moderate levels of
fluorescence and one with very high levels of fluorescence. To
characterize further the population of cells expressing high and
moderate levels of GFP, we used an anti-CAR antibody or an anti-CK14
antibody to assess for expression of CAR and CK-14 (a basal cell
marker). Many of the cells with the highest GFP fluorescence also
showed high levels of anti-CAR fluorescence (top right
quadrant in Fig. 6B) and high levels of anti-CK-14
fluorescence (top right quadrant in Fig. 6C). The
data suggest that a subpopulation of cells, probably basal cells, may
express higher levels of CAR and therefore are more prone to adenovirus
infection. However, some cells highly positive for GFP expressed low
levels of CK14 and CAR suggesting that other receptors (perhaps MHC-1)
may also be involved in adenovirus binding and infection. Moreover,
most of the cells expressing moderate levels of GFP expressed low
levels of CAR (bottom left quadrant in Fig. 6B)
suggesting that low levels of CAR may limit the degree of adenovirus
infection.
Adenovirus-mediated Gene Transfer of CFTR through the Basolateral
Side--
For CFTR to mediate transepithelial Cl The Basolateral Surface Is Accessible to Virus in Airway Epithelia
following Disruption of the Tight Junctions--
Chelation of
extracellular Ca2+ with 8 mM EGTA can
reversibly increase the permeability of tracheal epithelia (36, 37). Also, a brief apical application of H2O has been reported
to transiently increase permeability of airway epithelia to
macromolecules and DNA (38). We tested the effect of exposing well
differentiated human airway epithelia to EGTA or to H2O in
an attempt to disrupt the tight junctions, improve access of adenovirus
to the basolateral surface of cells, and improve adenovirus-mediated
gene transfer. Fig. 8 shows that both 8 mM EGTA and H2O added to the apical surface for
30 min significantly decreased Rt, indicating disruption of the tight
junctions. The effect was fully reversible with time, with Rt returning
to base-line values within 12 h.
We used fluorescently labeled adenovirus to test the hypothesis that
disruption of the tight junctions with H2O or 8 mM EGTA would allow adenovirus to reach the basolateral
surface of the airway epithelial cells. Fig.
9, A, C, and E,
shows 180-µm thick X-Z projections of confocal images
taken of control epithelia and epithelia treated with H2O
or EGTA prior to addition of the fluorescently labeled adenovirus. The
location of cells is shown by staining for F-actin. The experiments
were performed at 4 °C to avoid endocytosis of vector. Control
epithelia showed no detectable apical surface binding or virus between
the cells. In contrast, epithelia pretreated with H2O or 8 mM EGTA showed large amounts of virus within the epithelia
but not at the apical surface. Fig. 9, B, D, and
F, shows a single X-Z confocal section (0.35 µm). The fluorescently labeled adenovirus can be seen in the
intercellular spaces of the epithelia pretreated with H2O
or 8 mM EGTA. Thus, disruption of the tight junctions
increased adenovirus access to the basolateral surface of airway
epithelia where CAR and MHC Class I are expressed.
Transient Disruption of Tight Junctions Enhances Gene Transfer by
Apical Adenovirus--
We tested the hypothesis that disrupting the
tight junctions of differentiated epithelia would increase
adenovirus-mediated gene transfer. The apical surfaces of airway
epithelia were pretreated for 30 min with either H2O or 8 mM EGTA followed by a 30-min incubation with 50 m.o.i.
of Ad2/
We also tested the hypothesis that disrupting the tight junctions of
well differentiated epithelia would increase adenovirus-mediated CFTR
gene transfer to CF airway epithelia. We observed a significant correction of total Cl Our earlier work showed that ciliated airway epithelia are
relatively resistant to adenovirus infection and adenovirus-mediated gene transfer because they lack high affinity fiber receptor activity on the apical surface (15, 16). Our current work confirms those
findings and provides a molecular explanation, the two cellular receptors for fiber, CAR and MHC Class I, were not present on the
apical membrane of well differentiated ciliated airway epithelia. Lack
of fiber binding appears to be the rate-limiting step to explain the
inefficient adenovirus-mediated gene transfer to the airway epithelia
seen in vitro (16, 17, 21) and in vivo (19, 39,
40). Our results are also in agreement with a recent report by Pickles
et al. (41) suggesting that adenovirus binding and CAR
expression are limited to the basolateral surface of human airway
epithelia. However, Pickles et al. (41) found that in contrast to human, adenovirus internalization was rate-limiting for
adenovirus infection to the apical membrane of rat airway epithelia.
Despite their absence on the apical membrane, both receptors were
expressed on the basolateral membrane. Our data showing that excess
fiber knob inhibits basolateral gene transfer suggests that fiber
receptor is responsible, at least in part, for the efficiency of gene
transfer from the basolateral surface. However, it is still not clear
if one or both of the two fiber receptors are required for adenovirus
binding and infection of airway epithelia. Our data cannot rule out the
requirement for MHC Class I (25) since we found it uniformly expressed
in the basolateral membrane of most cells. However, our data also
suggest that basal cells may express higher levels of CAR and may be
more susceptible to adenovirus-mediated gene transfer. Experiments
using recombinant expression and knock-out or antisense down-regulation
of these receptors will be required to assess their relative
contribution to fiber binding and adenovirus internalization.
Some viruses infect epithelia via the basolateral side (35, 42, 43),
the apical side (44-48), or both sides (49, 50). The sidedness of
infection may even vary with maturation of the epithelia. Rossen
et al. (51) found that porcine-transmissible gastroenteritis
virus infected equally from the basolateral side and the apical side of
a porcine epithelial cell line (LLC-PK1) initially after plating.
However, several days after seeding, infection became restricted to the
apical membrane. These results are similar to our original observation
that the apical side of poorly differentiated airway epithelia was
infected by adenovirus in a fiber-dependent manner (16).
The sidedness of infection for the same virus may also vary in
different epithelia. Topp et al. (43) found that herpes
simplex virus preferentially infected Madin-Darby canine kidney cells
via the basolateral side, whereas it infected a retinal cell epithelia
preferentially via the apical side. These observations underscore the
importance of studying the interaction of viruses with a model relevant
to the pathophysiology of the virus.
At present it is difficult to conceive of a way to increase CAR or MHC
Class I expression on the apical surface of airway cells. Obviously,
overexpression by gene transfer is not a practical possibility.
However, it might be possible to bypass the fiber receptor by changing
the capsid proteins to bind to a different receptor (52-56).
Alternatively, if vector could be delivered from the basolateral side,
or if the vector was able to gain access through the tight junctions to
the basolateral membrane, then it would be possible to target most
cells of the airway epithelia. At present it is not clear how this
might be done in a way that is safe in vivo.
Understanding the mechanism of adenovirus entry into human airway
epithelia is required to understand the biology of adenovirus and its
normal mechanism of infection. Given the epidemiologic data suggesting
the frequency of adenovirus infection (3), the relative inefficiency of
adenovirus infection is striking. It is possible that wild-type
infection requires either a large initial inoculum or damage to the
integrity of the epithelia. Subsequent dissemination of the infection
could then be due to replication of wild-type adenovirus and spread via
the basolateral side. The current models of adenovirus pathogenesis
rely on a murine model (Sigmodon hispidus) and a large viral
inoculum (>108 plaque-forming units) (57). Further studies
of the pathogenesis of wild-type adenovirus in a relevant model may
yield important insights into the prevention of wild-type infection
and/or improvements in the utility of adenovirus for gene transfer.
-2 domain allows the molecular basis of adenoviral infection to be
investigated. Earlier work has shown that human airway epithelia are
resistant to infection by adenovirus. Therefore, we examined the
expression and localization of CAR and MHC Class I in an in vitro model of well differentiated, ciliated human airway
epithelia. We found that airway epithelia express CAR and MHC Class I. However, neither receptor was present in the apical membrane; instead, both were polarized to the basolateral membrane. These findings explain
the relative resistance to adenovirus infection from the apical
surface. In contrast, when the virus was applied to the basolateral
surface, gene transfer was much more efficient because of an
interaction of adenovirus fiber with its receptors. In addition, when
the integrity of the tight junctions was transiently disrupted, apically applied adenovirus gained access to the basolateral surface and enhanced gene transfer. These data suggest that the receptors required for efficient infection are not available on the apical surface, and interventions that allow access to the basolateral space
where fiber receptors are located increase gene transfer efficiency.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
V
3 and
V
5 integrins facilitates internalization
via receptor-mediated endocytosis (2, 8, 9). The acidic pH the virus
encounters in the endosome may trigger a conformational change that
releases the virus into the cytoplasm (10-12) and allows the
adenovirus capsid to travel to the nucleus (2, 13). Then viral proteins
and DNA bind to the nuclear pore complex, capsid disassembly continues, and DNA enters the nucleus accompanied by DNA-associated protein 7 (1,
2, 14). These studies have concluded that a high affinity fiber
receptor is required for binding and infection and that an
V
integrin acts as a co-receptor.
-2 consensus region (25). In a lymphoblastoid
cell line they found that expression of MHC-I resulted in increased
fiber protein binding and adenovirus-mediated gene transfer as compared
with cells that lack expression of MHC-I. The discovery of CAR and
MHC-I
-2 domain allowed us to investigate the molecular basis for
our earlier finding of a lack of fiber receptor activity on the apical
membrane of human airway epithelia and the limited efficiency of
adenovirus infection.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase, Ad2/
Gal-2, and CFTR, Ad2/CFTR-16, were
prepared as described previously (29) by the University of Iowa Gene
Transfer Vector Core at titers of ~1010 infectious
units/ml. Recombinant adenovirus vectors expressing green fluorescent
protein (GFP) (30, 31), Ad2/GFP, and the purified fiber knob protein
used for competition experiments (32) were a gift of Dr. Sam Wadsworth
(Genzyme, Framingham, MA).
·cm2.
Fourteen days after seeding, 50 µl containing 50 m.o.i. of the recombinant viruses in phosphate-buffered saline (PBS) were added to
the apical surface. The particle to infectious unit ratio ranged between 24 and 34, and the cell number in a 0.6-cm diameter well differentiated airway epithelium is approximately 750,000. Following the indicated incubation time, the viral suspension was removed, and
the monolayers were rinsed twice with PBS. After infection, the
epithelia were incubated at 37 °C for an additional 30-72 h.
Transepithelial resistance (Rt) was measured with an ohm meter (EVOMTM, World Precision Instrument Inc., Sarasota, FL) before infection, and Rt was not altered by application of virus. To disrupt
the tight junctions, we pretreated the apical surface of the epithelia
for 30 min with 400 µl of either Eagle's modified essential media
(EMEM), deionized H2O, or 8 mM EGTA in EMEM
(EGTA) at 37 °C. To evaluate virus association with cells,
adenovirus was covalently labeled with the carbocyanine dye, Cy3
(Amersham Pharmacia Biotech) (13). The labeling procedure decreased the infectious units/particle ratio by 5-35%.
-actin were
transcribed from linearized plasmid DNA using T3 polymerase for CAR and
Sp6 polymerase for
-actin. Antisense probes were hybridized with 10 µg of total RNA from primary airway epithelia using a ribonuclease
protection assay II kit (Ambion, Austin, TX). The RNA-RNA hybrids were
digested with a 1/200 dilution of RNase A/T1 provided to yield a
216-nucleotide CAR-protected fragment and a 127-nucleotide
-actin-protected fragment. Protected fragments were separated on a
5% denaturing polyacrylamide gel and transferred to a nylon membrane.
Visualization and quantitation of CAR and
-actin was achieved by
exposing the membrane to a phosphoscreen and scanning with a STORM
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Quantitation of the
CAR message was normalized to
-actin message and expressed as a
ratio of CAR/
-actin.
-2 domain expression at the apical and basolateral
membrane of differentiated airway epithelia, we used a similar protocol
with 1:100 dilution of anti-human MHC-I antibody (ICN
Immunobiologicals, Costa Mesa, CA). The epithelia were mounted on
slides in Gelmount (Biomedia Corp., Foster City, CA) and examined using
epifluorescence with a Leica photomicroscope.
-Galactosidase Activity--
We measured total
-galactosidase activity using a commercially available method
(Galacto-LightTM, Tropix, Inc., Bedford, MA). Briefly,
after rinsing with PBS, cells were removed from filters by incubation
with 120 µl of lysis buffer (25 mM Tris phosphate, pH
7.8, 2 mM dithiothreitol; 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic
acid; 10% glycerol; and 1% Triton X-100) for 15 min. Light emission
was quantified in a luminometer (Analytical Luminescence Laboratory,
San Diego).
current through CFTR Cl
channels. To
assess total Cl
current, we then added 100 µM bumetanide to the submucosal solution and measured the
change in current.
Gal-2 in 100 µl of PBS were added to the
apical surface of epithelia at 4 °C. After 20 min the virus was
removed, and epithelia were rinsed twice with PBS. Cultures were fixed with 4% paraformaldehyde and permeabilized with Triton X-100 at 20 °C for 10 min and then rinsed 3 times with PBS. The cells were stained with BODIPY 650/665-labeled phalloidin (Molecular Probes, Eugene, OR) for 10 min, rinsed, and then mounted on glass slides with
Vectashield (Vector Laboratories, Burlingame, CA). Optical sections of
fluorescent samples were obtained using a Bio-Rad MRC-1024 equipped
with a Kr/Ar laser at × 60 magnification. Data was reconstructed
in X-Z series and projected with varying thickness to
display a cross-section of the epithelia.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Determination of CAR expression by
ribonuclease protection assay on poorly differentiated
(P.D., 3 days) and well differentiated
(W.D., 14 days) airway epithelia. A,
protected fragments correspond to message for CAR and -actin as
indicated. B, relative abundance of CAR compared with
-actin for five different experiments. Data are mean ± S.E.;
p = 0.15.
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Fig. 2.
Expression of CAR in well differentiated
human airway epithelia. A, immunocytochemistry of
unpermeabilized epithelia incubated with monoclonal anti-CAR Ab and an
FITC-labeled secondary antibody applied to either the apical or
basolateral side. Top panel shows absence of CAR staining on
the apical membrane. Bottom panel shows a low intensity
staining on most cells, with higher intensity staining of a small
proportion of cells. The bar represents 50 µm.
B, FACS analysis for CAR expression. Histogram shows the
fluorescent intensity of 10,000 cells stained in suspension for CAR
with anti-CAR antibody (RmcB) followed by Texas Red-labeled secondary
antibody. Dashed histogram shows the control cells stained
only with Texas Red-labeled secondary antibody.
-2 domain on well
differentiated human airway epithelia. Epithelia were incubated with an
FITC-labeled anti-human MHC-I antibody applied either to the apical or
basolateral surface. As with anti-CAR antibodies, no staining was
observed when the antibody was added to the apical side (Fig.
3). However, membrane staining was
observed throughout the epithelium when the antibody was added to the
basolateral side (Fig. 3). No staining was seen with the FITC-labeled
secondary antibody alone or when mouse cells were used (data not
shown). These results suggest that the adenovirus fiber receptors, CAR and MHC-I
-2 domain receptor, are polarized to the basolateral plasma membrane of the airway epithelia. In addition, they suggest that
a small population of cells, probably basal cells, express higher
levels of CAR. This polarity may account for the relative resistance to
adenovirus infection when vector is applied to the apical membrane.
Furthermore, the results predict that infection from the basolateral
side should be more efficient and fiber-dependent.
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Fig. 3.
Expression of MHC Class I in well
differentiated human airway epithelia evaluated by
immunocytochemistry. Unpermeabilized epithelia were incubated with
an FITC-labeled monoclonal anti-MHC Class I antibody applied to either
the apical or basolateral side. Top panel shows absence of
MHC Class I staining on the apical membrane. Bottom panel
shows high intensity staining on all cells. The bar
represents 50 µm.
Gal-2 in a volume of 25 µl to the bottom of the Millipore
filter. After 30 min, the epithelia were rinsed thoroughly. Fig.
4 shows that airway epithelia expressed
-galactosidase activity at levels 2 logs greater when the virus was
applied from the basolateral side than when it was placed on the apical
surface. In contrast to apical infection, infection from the
basolateral surface was blocked by addition of 70 µg/ml fiber
knob.
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Fig. 4.
Effect of polarity of infection on
adenovirus-mediated transgene expression by human airway
epithelia. Well differentiated airway epithelia were exposed to
50 m.o.i. Ad2/ Gal-2 for 30 min either from the apical or
basolateral side (BL). Vector was then removed, and the
epithelia were cultured for 2 additional days before analysis. Control
epithelia received no virus. Data are
-galactosidase activity/mg of
protein under control conditions (open bars) or in the
presence of 70 µg/ml fiber knob (shaded bars). Data are
mean ± S.E., n = 14. Asterisk
indicates p < 0.01 between basolateral and apical
viral application. ¶ indicates p < 0.01 between
basolateral expression and fiber knob competition.
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Fig. 5.
Effect of polarity of infection on
adenovirus-mediated GFP expression by human airway epithelia.
A, well differentiated airway epithelia were exposed to
50 m.o.i. Ad2/GFP for 30 min from either the apical or basolateral
(BL) side. Vector was then removed, and the epithelia were
cultured for 2 additional days before FACS analysis. Data are the
percentage of dissociated airway epithelia cells that were positive by
FACS analysis for GFP. Ad2/ Gal-2 and non-infected cells were used as
negative controls. Data are mean ± S.E.; n = 4. Asterisk indicates p < 0.01 between
basolateral and apical viral application. B, fluorescent
photomicrograph of epithelia infected with Ad2/GFP from the apical and
basolateral side. Arrows indicate a subpopulation of
GFP-positive cells that are higher in fluorescence. The bar
represents 50 µm.
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Fig. 6.
Effect of polarity of infection on
adenovirus-mediated GFP expression by human airway cells. Well
differentiated airway epithelia were exposed to 50 m.o.i. Ad2/GFP
for 30 min from the basolateral side. Vector was then removed, and the
epithelia were cultured for 1 additional day before FACS scan analysis.
A, histogram of fluorescence intensity of 10,000 cells
exposed to virus from the basolateral surface; dashed histogram shows
control cells. Arrows and dotted lines indicate
two subpopulations of cells with moderate and very high fluorescence
from GFP. B, dot plot analysis of 10,000 cells exposed from
the basolateral surface to the Ad2/GFP virus and analyzed by FACS for
GFP fluorescence and for fluorescence from anti-CAR antibody followed
by Texas Red-labeled secondary antibody. x axis represents
the GFP fluorescence intensity and the y axis represents the
Texas Red (CAR) fluorescence intensity. Dotted vertical
lines separates populations of cells that express high and
moderate levels of GFP fluorescence. Dotted horizontal line
separates cells that express high and low levels of CAR. C,
dot plot analysis of 10,000 cells exposed from the basolateral surface
to the Ad2/GFP virus and stained in suspension for CK-14 with an
anti-CK-14 antibody followed by Texas Red-labeled secondary antibody.
x axis represents the GFP fluorescence intensity, and the
y axis represents the Texas Red (CK-14) fluorescence
intensity. Dotted vertical lines separate the populations of
cells that express high and moderate levels of GFP fluorescence.
Dotted horizontal line separates the cells that express
CK-14.
transport it must be present in the apical membrane of cells that are
in contact with the lumen. Thus, basal cells cannot contribute to
Cl
transport across the apical membrane. Therefore we
asked if delivery of adenovirus expressing CFTR via the basolateral
side would correct the Cl
transport defect in well
differentiated CF epithelia. Finally, whereas the relationship between
-galactosidase expression on measured
-galactosidase activity is
linear, the relationship between expression of CFTR and transepithelial
Cl
transport is not (33). Fig.
7 shows that 48 h after 50 m.o.i. of Ad2/CFTR-16 were added to the basolateral side of CF
epithelia for 30 min, total Cl
current
(
IscBum) increased into the range observed in normal epithelia (34). No correction was observed when the virus was added to
the apical side. We cannot exclude the possibility that cell division
and differentiation of basal cells resulted in adenovirus-mediated expression of CFTR in columnar cells; however, the low rate of cell
division (35) and the short time between adenovirus infection and the
expression assay make this less likely. These data suggest that
columnar non-basal airway epithelia can be infected with adenovirus via
the basolateral side and can express sufficient amounts of CFTR to
correct the CF Cl
transport defect.
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Fig. 7.
Effect of polarity of infection on
adenovirus-mediated CFTR expression by CF human airway epithelia.
CF airway epithelia were infected with 50 m.o.i. of Ad2/CFTR-16
for 30 min either from the apical or basolateral (BL) side.
Forty eight hours later the epithelia were studied in Ussing chambers.
Data are mean ± S.E. change in current after the addition of
bumetanide to cAMP-stimulated, amiloride-treated epithelia
(IscBum). Asterisk indicates p 0.01, n = 6.
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Fig. 8.
Effect of apical application of
H2O or 8 mM EGTA on transepithelial resistance
(Rt) in well differentiated human airway epithelia. The apical
surface was treated for 30 min (solid bar) with either 400 µl of H2O or 400 µl of an EMEM solution containing 8 mM EGTA and then rinsed with EMEM. Rt was measured at the
times indicated. Data are mean ± S.E., n = 9 in
each group.
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Fig. 9.
Effect of disrupting tight junctions on
adenovirus binding to well differentiated human airway epithelia.
A, C, and E are 180-µm thick X-Z
projections of confocal images taken of control epithelia and epithelia
treated with H2O or EGTA prior to addition of the
fluorescently labeled adenovirus. B, D, and
F show a single vertical axis X-Z section (0.35 µm) of control epithelia and epithelia treated with H2O
or EGTA prior to addition of the fluorescently labeled adenovirus. The
individual epithelial cells stained with an F-actin stain (pseudo-color
green) can be visualized on B, D, and
F. Scale bar = 25 µm.
Gal-2. Fig. 10 shows that
these treatments increased
-galactosidase expression 8-9-fold as
compared with epithelia pretreated with EMEM. Furthermore, the increase
in
-galactosidase expression could be blocked by excess fiber knob
protein. Moreover, if Ad2/
Gal-2 was applied 12 h after
treatment at a time when Rt had returned to basal values,
-galactosidase expression was not significantly different from that
in control epithelia (data not shown).
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Fig. 10.
Effect of disruption of tight junctions on
adenovirus-mediated -galactosidase expression
in human airway epithelia. Well differentiated airway epithelia
pretreated with EMEM (control), H2O, or EGTA were exposed
to 50 m.o.i. Ad2/
Gal-2 for 30 min from the apical side. Vector
was then removed, and the epithelia were cultured for 48 h before
analysis. Data are
-galactosidase activity/mg of protein under
control conditions (open bars) or in the presence of 70 µg/ml fiber knob (shaded bars). Data are mean ± S.E.
of
-galactosidase activity/mg of protein. Asterisk
indicates p < 0.01 compared with EMEM control,
n = 6.
current (
IscBum) in
CF airway epithelia transfected with 50 m.o.i. of Ad2/CFTR-16
after treatment with H2O or 8 mM EGTA (Fig. 11). No correction in
IscBum was seen when the virus was added to the apical
surface of epithelia for 30 min. We had previously shown that
adenovirus-mediated gene transfer to airway epithelia can correct the
CF defect if the incubation time with the virus was increased (16);
therefore, we also applied Ad2/CFTR-16 to the apical surface for
24 h. We also found that treatment of airway epithelia with
H2O or 8 mM EGTA alone (without adding
Ad2/CFTR-16) did not affect the
IscBum defect of CF
airway epithelia (n = 3, data not shown). Fig. 6 shows
that there was more correction of Cl
transport in
epithelia treated with H2O or 8 mM EGTA for 30 min than with a 24-h exposure to virus alone. These data indicate that
disruption of the tight junctions before apical application of the
virus significantly improved adenovirus infection and
adenovirus-mediated expression of CFTR. However, the efficiency of
adenovirus-mediated gene transfer was slightly lower with
H2O or 8 mM EGTA pretreatment than with
application of virus to the basolateral side.
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Fig. 11.
Effect of disruption of tight junctions on
adenovirus-mediated CFTR expression in CF airway epithelia.
Control CF airway epithelia and CF epithelia pretreated with
H2O or EGTA were infected with 50 m.o.i. of
Ad2/CFTR-16 applied to the apical surface for 30 min. Forty eight hours
later the epithelia were studied in Ussing chambers. Data are mean ± S.E. change in current after the addition of bumetanide
(IscBum) in cAMP-stimulated, amiloride-treated CF
epithelia. Asterisk indicates p < 0.01, n = 6.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Pary Weber, Phil Karp, Thomas Moninger, Janice Launspach, Theresa Mayhew, and Christine McLennan for excellent assistance. We especially appreciate the help of Dr. Mary Schroth for the CF tissue. We thank Dr. Sam Wadsworth, Genzyme (Framingham, MA), for the gift of wild-type adenovirus and Ad2/GFP and fiber knob. We appreciate the support of the University of Iowa Gene Transfer Vector Core (supported in part by the Roy J. Carver Charitable Trust).
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FOOTNOTES |
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* This work was supported by the NHLBI and the NIAID of the National Institutes of Health, the Cystic Fibrosis Foundation, and the Roy J. Carver Charitable Trust.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.
§ Established Investigator of the American Heart Association.
Investigator of the Howard Hughes Medical Institute.
** Fellow of the Roy J. Carver Charitable Trust. To whom correspondence should be addressed: University of Iowa College of Medicine, 500 EMRB, Iowa City, IA 52242. Tel.: 319-353-5511; Fax: 319-335-7623; E-mail: Joseph-Zabner{at}uiowa.edu.
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ABBREVIATIONS |
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The abbreviations used are: CAR, coxsackie B and adenovirus type 2 and 5 receptor; MHC, major histocompatibility complex; GFP, green fluorescent protein; m.o.i., multiplicity of infection; PBS, phosphate-buffered saline; Rt, resistance; EMEM, Eagle's modified essential media; FITC, fluorescein isothiocyanate; CF, cystic fibrosis; FACS, fluorescence-activated cell scanning; CFTR, cystic fibrosis conductance transmembrane regulator.
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REFERENCES |
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