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1998, 72(7):6014. J. Virol.
J. Hart, Scott H. Randell and Richard C. Boucher
Raymond J. Pickles, Douglas McCarty, Hirotoshi Matsui, Pádraig
Responsible for Inefficient Gene Transfer
Well-Differentiated Airway Epithelium Is
Limited Entry of Adenovirus Vectors into
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JOURNAL OF VIROLOGY,
0022-538X/98/$04.0010July 1998, p. 6014–6023 Vol. 72, No. 7
Copyright © 1998, American Society for Microbiology. All Rights Reserved.
Limited Entry of Adenovirus Vectors into Well-Differentiated
Airway Epithelium Is Responsible for Inefficient Gene Transfer
RAYMOND J. PICKLES,
1
* DOUGLAS MCCARTY,
2
HIROTOSHI MATSUI,
1
PA
´DRAIG J. HART,
1
SCOTT H. RANDELL,
1
AND RICHARD C. BOUCHER
1
CF/Pulmonary Research and Treatment Center
1
and Gene Therapy Center,
2
University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248
Received 29 December 1997/Accepted 23 March 1998
Investigations of the efficiency and safety of human adenovirus vector (AdV)-mediated gene transfer in the
airways of patients with cystic fibrosis (CF) in vivo have demonstrated little success in correcting the CF
bioelectrical functional defect, reflecting the inefficiency of AdV-mediated gene transfer to the epithelial cells
that line the airway luminal surface. In this study, we demonstrate that low AdV-mediated gene transfer
efficiency to well-differentiated (WD) cultured airway epithelial cells is due to three distinct steps in the apical
membrane of the airway epithelial cells: (i) the absence of specific adenovirus fiber-knob protein attachment
receptors; (ii) the absence of a
v
b
3/5
integrins, reported to partially mediate the internalization of AdV into the
cell cytoplasm; and (iii) the low rate of apical plasma membrane uptake pathways of WD airway epithelial cells.
Attempts to increase gene transfer efficiency by increasing nonspecific attachment of AdV were unsuccessful,
reflecting the inability of the attached vector to enter (penetrate) WD cells via nonspecific entry paths.
Strategies to improve the efficiency of AdV for the treatment of CF lung disease will require methods to increase
the attachment of AdV to and promote its internalization into the WD respiratory epithelium.
Successful gene therapy for cystic fibrosis lung disease re-
quires efficient in vivo gene transfer to airway epithelia (2). We
have previously reported that the efficiency of adenovirus vec-
tor (AdV)-mediated gene transfer to poorly differentiated
(PD) airway epithelial cells in vitro is high whereas the effi-
ciency of gene transfer to well-differentiated (WD) ciliated
airway epithelium in vivo is low (18, 25). We have speculated
that the lower efficiency observed in vivo is due to the absence
of an early step in the vector-cell interaction (25). Ad is
thought to enter cells by a two-step process: (i) initial attach-
ment of the viral fiber-knob protein to high-affinity receptors,
for which a candidate has recently been identified (the human
coxsackievirus B and Ad2 and Ad5 receptor [hCAR]) (1, 29),
and (ii) translocation of the virus into the cell cytoplasm via
coated-pit internalization processes, mediated in part by an
interaction of the viral penton base with a
v
b
3/5
integrins (32).
The target cell type for cystic fibrosis lung gene therapy is the
WD ciliated columnar airway epithelial cell, but the interac-
tions of AdV with this cell type in vivo have not been compre-
hensively studied. Inefficient AdV-mediated gene transfer to a
bronchial xenograft model of human ciliated airway epithelia
has been reported to reflect the absence of the a
v
b
3/5
integrins
from the luminal membrane of the epithelium (14). Similarly,
luminal a
v
b
3/5
integrin expression was reported to correlate
with the efficiency of AdV-mediated gene transfer to murine
airway epithelium (13). However, the exact role of a
v
b
3/5
in-
tegrins in determining AdV-mediated gene transfer efficiency
to the respiratory epithelium is not known and may not alone
account for decrements in efficacy. Mutants of AdV that lack
penton base RGD sequences (normally required for a
v
b
3/5
integrin interactions) are able to efficiently transduce human
epithelial cells, although the rate of internalization is reduced
(12). In a b
5
-knockout mouse model, airway epithelial cells
lacking the b
5
integrin subunit had the same susceptibility to
AdV-mediated gene transfer as did wild-type airway cells (17),
again suggesting that a
v
b
3/5
integrins may be facilitative rather
than necessary for efficient vector entry into the cell cytoplasm.
In this study, we set out to identify the rate-limiting steps
responsible for limiting the uptake of AdV into the respiratory
airway epithelium to determine which step(s) is responsible for
the inefficiency of AdV-mediated gene transfer to this tissue.
For these studies, we have used human and rat airway epithe-
lial cell culture models that can generate cultures with PD and
WD phenotypes from common progenitor cells. With this sys-
tem, we systematically compared AdV entry into and attach-
ment to PD and WD cells and related the findings to the
differences in gene transfer efficiencies.
MATERIALS AND METHODS
Virus preparations. Human Ad5 vector (AdV), with deletions of the E1a,E1b,
and E3 genes, contained the Escherichia coli lacZ gene under the control of the
cytomegalovirus promoter (9). [
35
S]methionine-radiolabeled AdVCMVlacZ
(
35
S-AdV) was prepared as described previously (16) with modification for 293
cells by harvesting at 48 h postinfection. The adenovirus particle-to-infectious-
unit ratio was routinely 100:1 and was determined by plaque assay on either 293
or 911 cells (similar values were obtained for the two cell lines). Serial dilutions
of virus in Dulbecco’s modified Eagle medium (DMEM-H) were incubated with
50% confluent cell cultures in six-well plates at a volume of 1 ml per well (fluid
height, 0.1 cm). The cultures were incubated for1hat37°C without agitation.
The medium was removed and replaced with 3 ml of agar overlay (13
DMEM-H, 10% fetal bovine serum [FBS], and 1% SeaPlaque LGT agarose).
The cultures were fed with 1 ml of overlay (containing 2% FBS) every 3 days
until the plaques were counted at 6 days postinfection for 911 cells or 10 to 12
days postinfection for 293 cells. The specific activity of the radiolabeled vector
was approximately 4 310
25
cpm per particle. To directly visualize AdV, Cy3
FluoroLink (Amersham Life Science Inc., Arlington Heights, Ill.) was conju-
gated to the AdV capsid coat by incubation of 10
12
particles of AdVlacZ as
described previously to form CyAdV (23). This process reduced the AdV titer by
less than 10-fold (23). CyAdV was characterized by assessment of fluorescent
attachment and transgene expression with HeLa and CHO K1 cell lines in the
absence and presence of competing purified fiber-knob protein. The Ad5 fiber-
knob protein was produced by expressing pBEVafibre (a gift from Robert Ger-
ard, Katholieke Universtiet Leuven, Louvain, Belgium) in E. coli TG-1 cells, and
purified fiber-knob protein was obtained exactly as described previously (19).
Cell culture. Human tracheobronchial epithelial cells were derived from
non-CF airway specimens and were cultured by procedures similar to those
* Corresponding author. Mailing address: CF/Pulmonary Research
and Treatment Center, UNC School of Medicine, University of North
Carolina at Chapel Hill, Chapel Hill, NC 27599-7248. Phone: (919)
966-7044. Fax: (919) 966-7524. E-mail: branston@med.unc.edu.
6014
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described by Gray et al. (15). Portions of the lower trachea and mainstem
bronchi representing excess donor tissue were obtained at the time of lung
transplantation under institutional review board-approved protocols. Epithelial
cells were removed from the specimens by protease XIV digestion as described
previously (34), and 10
6
cells were plated per 100-mm tissue culture dish in
modified LHC9 medium (22). The modifications were increased epidermal
growth factor concentration to 25 ng/ml, adjustment of the retinoic acid concen-
tration to 5310
28
M, and supplementation with 0.5 mg of bovine serum albumin
per ml and 0.8% bovine pituitary extract. At approximately 75% confluence, the
cells were harvested with trypsin and passage 1 cells were plated at a density of
3.33 310
5
cells on Transwell-Col inserts (diameter, 24 mm; pore size, 0.4 mm;
Corning-Costar, Cambridge, Mass.) in modified medium. The medium is similar
to the supplemented LHC9, except that a 50:50 mixture of LHC Basal (Biofluids
Inc., Rockville, Md.) and DMEM-H was used as the base, amphotericin and
gentamicin were omitted, and the epidermal growth factor concentration was
reduced to 0.5 ng/ml. After 4 to 6 days, the cells became confluent and were used
as PD cultures. For the production of WD cultures, confluent cultures were
maintained with an air/liquid interface for another 25 to 30 days.
Rat tracheal epithelial cells were isolated from pathogen-free male F344 rats
(200 g), and 2 310
5
cells were plated on permeable Transwell-Col matrix
supports (as above) by the method of Kaartinen et al. (21). After 5 days of
culture, the cells became confluent and were used as PD cultures. For the
production of rat WD cultures, after the cells became confluent the apical
surfaces of the cultures were given an air/liquid interface for at least 19 days.
HeLa cells (American Type Culture Collection) were plated on Transwell-Col
supports (as above), grown to confluence, and maintained in Eagle’s minimum
essential medium supplemented with nonessential amino acids and 10% FBS.
HeLa cells expressing the tetracycline-sensitive wild-type and K44a mutant form
of dynamin were kind gifts from Sandra Schmid (Scripps Research Institute, La
Jolla, Calif.) (7) and were initially maintained in DMEM–10% FBS–400 mgof
gentamicin per ml–200 ng of puromycin per ml–1 mg of tetracycline per ml. To
induce dynamin overexpression, the cells were cultured in the absence of tetra-
cycline for 2 days before being exposed to AdV.
Expression, entry, and attachment studies. Analyses of expression, entry, and
attachment were performed within a single batch of cells, on the same day, with
identical reagents. For the lacZ expression studies, the luminal surfaces of cul-
tures were exposed to 10
10
particles of AdV (multiplicity of infection, ;100) at
37°C for 6 h, unbound virus was removed from the cells by three washes in
ice-cold medium, and the cells were returned to 37°C before gene expression
analyses 48 h after the initial exposure to AdV. b-Galactosidase (b-gal) enzyme
activity was assessed as described previously (18).
Internalization of radiolabeled AdV was assessed as follows. After exposure of
cultures to AdV (10
10
particles) for6hat37°C, the cultures were transferred to
4°C and washed three times in ice-cold medium. The cells were then rinsed with
an acid-salt wash (0.2 N acetic acid, 0.5 M NaCl [pH 2.5]) at 4°C and exposed for
1 h to protease (0.25% pronase XIV with 0.0025% DNase in serum-free culture
medium at 4°C) to remove extracellular bound vector. This method effectively
removed more than 95% of the vector attached to the cell surface at 4°C, as
determined by assessing removal-resistant counts after this treatment. After
further washing in ice-cold medium, the cells were solubilized in 1% sodium
dodecyl sulfate (SDS)–0.3 N NaOH and the counts were assessed by liquid
scintillation counting. For attachment studies, cultures maintained at 4°C were
exposed to
35
S-AdV (10
10
particles) for 6 h. The cultures were then washed three
times in ice-cold medium, and the cells were solubilized and subjected to liquid
scintillation counting. For the studies with purified fiber-knob protein and RGD
peptides, the cultures were preexposed to fiber-knob protein (10 mg/ml), RGD
peptide (4.0 mg/ml; Gibco BRL, Bethesda, Md.) or cyclical RGD peptide (0.4
mg/ml; Immunodynamics, La Jolla, Calif.) for2hat4°Cbefore the addition of
AdVfor6hat4°C. The cultures were then washed three times in ice-cold
medium and either immediately solubilized as above for scintillation counting or
maintained at 37°C for a further 48 h before being subjected to expression
analyses.
For both species, only fully confluent cultures were exposed to AdV to ensure
a standard surface area and to avoid nonspecific binding of AdV to the matrix
support. The transepithelial resistance (R
t
) values of the cultures at the time of
AdV exposure were as follows: human PD and WD cultures, 1,076 695 (n548)
and 1,342 653 (n536) Vzcm
2
, respectively; rat PD and WD cultures, 223 6
5 and 2,653 675 Vzcm
2
, respectively (n512 for both). Radioactive counts per
minute and b-gal activity were standardized with respect to the nominal sur-
face area of the culture surface, since we consider the apical surface area of
cells exposed to vector to be the most appropriate denominator, allowing
direct comparison to the epithelium in vivo. The b-gal activity was measured
as microunits per square centimeter of epithelium, where1Uofenzyme will
hydrolyze 1 mmol of o-nitrophenyl-b-D-galactopyranoside per min at pH 7.3 and
37°C.
For attachment and expression studies with HeLa cell mutants, cells were
plated onto six-well plates in tetracycline-deficient medium to induce wild-type
or mutant dynamin expression. After 2 days in culture, the cells were cooled to
4°C and exposed to 10
10
particles of AdVlacZ per ml for 2 h. After three washes
with ice-cold medium the cells were prepared for liquid scintillation counting as
above or placed at 37°C for 48 h until used for expression analyses. For each
stage of the experiment, parallel cultures were used to determine cell numbers.
Generation of hCAR-expressing cell lines. To generate stable expression of
hCAR in cell lines, hCAR cDNA was placed in a Moloney murine leukemia
virus-based retroviral vector. The hCAR cDNA was a gift from Jeffrey Bergelson
(Dana-Farber Cancer Institute, Boston, Mass.). Briefly, hCAR cDNA was cloned
into the LxPin retroviral vector (24a) containing a poliovirus internal ribosome
entry site and a neomycin resistance gene. The resulting plasmid was packaged
by transient transfection of PA317 cells and pseudotyped with vesicular stoma-
titis virus glycoprotein G.
Functional analyses of the retroviral construct was performed by introducing
hCAR into a cell line that exhibits reduced susceptibility to AdV transduction.
Chinese hamster ovary (CHO K1; American Type Culture Collection) cells were
infected with retroviral vectors containing hCAR-Neo (CAR-CHO) or Neo
alone (CHO) as a control, and stably expressing cell lines were selected by
standard methods (4). To test the functional expression of hCAR, cells were
grown in 12-well culture dishes until confluent,
35
S-AdVlacZ was exposed to the
cultures (10
10
particles/ml for2hat4°C) in the absence or presence of excess
purified fiber-knob protein (10 mg/ml), and attachment and expression were
assessed as above.
HeLa, CHO, and CAR-CHO cells were exposed to monoclonal antibody
(MAb) RmcB (a hybridoma cell culture supernatant generated against hCAR
[20], a gift from Jeffrey Bergelson) as follows. Cells grown on glass coverslips
were cooled to 4°C and blocked with 3% bovine serum albumin. After incubation
with RmcB, the cells were washed and exposed to fluorescein isothiocyanate-
conjugated goat anti-mouse immunoglobulin G1 (IgG1) (Jackson ImmunoRe-
search Labs Inc., West Grove, Pa.) in the presence of CyAdV (10
10
particles).
The cells were washed in phosphate-buffered saline and fixed in paraformalde-
hyde (4%), mounted with Vectashield containing 49,6-diamidino-2-phenylindole
(DAPI) (Vector Laboratories Inc.), and viewed by conventional fluorescence
microscopy. Controls consisted of untransfected CHO cells or CHO cells ex-
pressing neo alone. In addition, the absence of primary antibody or the use of
irrelevant isotyped MAb (anti-bromodeoxyuridine; Boehringer-Mannheim
Corp., Indianapolis, Ind.) were used as controls. Localization of hCAR in human
airway cultures was performed as follows. Human PD and WD cultures were
fixed and permeabilized in paraformaldehyde (4%) and Triton X-100 (0.2%),
respectively. The cultures were then exposed to RmcB (or anti-bromodeoxyuri-
dine as a control) and, after being washed, exposed to Texas Red-conjugated
goat anti-mouse IgG1 (Jackson ImmunoResearch Labs Inc.). After being
washed, the cultures were fixed in paraformaldehyde and viewed by confocal
fluorescence microscopy, and XZ sections were generated.
Electron and confocal microscopy. For the transmission electron microscopy
studies, the luminal surfaces of human cultures were exposed to AdV (10
10
particles) for6hat4°C, washed as above, and fixed in 1.5% glutaraldehyde
overnight before being processed for electron microscopy by standard methods
(28). For experiments with fluorescent microspheres, the luminal surfaces of
human cultures were exposed to 100-nm fluorescent microspheres (0.02% in
medium; Molecular Probes) for6hat37°C and then washed three times with
medium. En face fluorescent images were taken with a conventional inverted
fluorescence microscope (Leica DM IRB). Images perpendicular to the cell layer
were captured by XZ sectioning with a confocal microscope (Leica TCS/4D), and
images were generated with the Metamorph image analysis system (Universal
Image Co.).
Immunoprecipitation and Western analyses. Apical or basolateral membranes
of human and rat PD and WD cultures were exposed to sulfosuccinimidobiotin
(0.5 mg/ml; Pierce Chemical Co.) for 30 min at 4°C and then the cells were
washed in ice-cold phosphate-buffered saline containing protease inhibitors (2
mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1 mM [each] leupeptin-
pepstatin) and solubilized in lysis buffer (150 mM NaCl, 1% Nonidet P-40, 0.5%
deoxycholate, 0.1% SDS, 50 mM Tris-HCl [pH 7.5] with protease inhibitors).
Before immunoprecipitation of integrins, the cell lysates were precleared with
normal rabbit serum and protein G beads (Pierce Chemical Co.). The a
v
b
3/5
integrins were immunoprecipitated by rabbit anti-human/rat a
v
b
3/5
integrin poly-
clonal antibody (838 [3], a kind gift of S. Albelda, University of Pennsylvania,
Philadelphia) or a rabbit anti-human vitronectin receptor polyclonal antibody
(AB1904; Chemicon International Inc.). Precipitates were subjected to SDS-
polyacrylamide gel electrophoresis (14% Tris-glycine gel under reduced condi-
tions) and transferred to polyvinylidene difluoride, and biotinylated membrane
proteins were detected with streptavidin-conjugated peroxidase and visualized by
enhanced chemiluminescence.
Inulin uptake studies. To measure the nonspecific uptake capacity of cells,
confluent PD human primary airway epithelial cells were generated on tissue
culture plastic and WD cultures were generated as described above. The luminal
surfaces of the cultures were exposed to freshly prepared [
3
H]inulin (35 mg/ml,
4 cpm/nl; Amersham Life Sciences Inc.) at either 4 or 37°C for 6 h. The cultures
were then returned to 4°C, washed five times with ice-cold medium containing
excess inulin (1 mg/ml), and solubilized for liquid scintillation counting. To
measure cell uptake of [
3
H]inulin, cell-associated counts at 37°C were subtracted
from those at 4°C as previously described (31). The volume of fluid uptake into
the cells was calculated based on the known counts per minute of the applied
solution.
Statistics. Statistical analysis was performed by Student’s ttest, and P,0.05
was considered significant.
VOL. 72, 1998 ADENOVIRUS-MEDIATED GENE TRANSFER TO AIRWAY EPITHELIUM 6015
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RESULTS
Analyses of gene transfer, vector entry, and attachment with
models of respiratory epithelium. (i) Human cultures. Modi-
fications of a cell culture system reported by Gray et al. (15)
generate from human tracheobronchial airway epithelial cells
polarized cultures with PD and WD cellular phenotypes from
the same patient (Fig. 1A). The morphology of WD cultures
resembles the pseudostratified ciliated epithelium exhibited by
human cartilaginous airway in vivo. Exposure of the apical
surfaces of human WD and PD cultures to AdVlacZ resulted
in significantly less b-gal expression in WD cultures than in PD
cultures (Fig. 1B). These results demonstrate that human WD
and PD airway epithelial cells are resistant and susceptible,
respectively, to AdV-mediated gene transfer, recapitulating
the phenomenon observed in vivo for human and rodent car-
tilaginous airway epithelia (18).
We have previously shown that the reduced efficiency of
gene transfer to WD cells compared to PD cells is not due to
a specific interaction of the transgene promoter with these cell
types or to the proliferative status of the cells at the time of
transduction (25). In the present study, we tested the hypoth-
esis that early steps in the vector-cell interaction determine the
gene transfer efficiency. Using radiolabeled AdV, we measured
the penetration of AdV into cells and determined which
step(s) is rate limiting for efficient AdV-mediated gene trans-
fer to human airway epithelial cells.
To determine if WD and PD cultures internalized different
amounts of AdV, both culture types were exposed to
35
S-AdV
and the quantity of internalized AdV was determined by mea-
suring the cell-associated counts that were resistant to removal
from the external cell surface by acid and protease treatment.
Figure 1C shows that the internalization of AdV into WD
cultures was markedly reduced compared to the amount inter-
nalized into PD cultures. To investigate whether the differ-
ences in entry reflect the degree of attachment of AdV, the
apical surfaces of human WD and PD cultures were exposed to
35
S-AdV at 4°C to measure cellular attachment of vector in the
absence of internalization. These studies showed that the sur-
face of WD cultures bound markedly less AdV than the PD
cultures (Fig. 1D). Collectively, these results suggest that the
reduction in gene transfer to WD cultures results from a re-
duced internalization of AdV into this cell type, which may be
related to the absolute amount of AdV attached to the luminal
surface of the cultures.
We confirmed this conclusion with a second human culture
system, containing cellular islands that exhibit a WD pheno-
type in the center and a PD phenotype at the edges (24). These
cultures were exposed to fluorescence-labeled AdV (CyAdV)
at 37°C and viewed by fluorescence microscopy after being
washed. At 6 h after exposure, CyAdV was routinely associated
with PD cells at the periphery and only rarely associated with
individual cells within the WD regions (Fig. 2A). This cellular
distribution of CyAdV localization is paralleled by the cellular
distribution of lacZ expression in the same cultures 24 h later,
assayed by 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside
(X-Gal) histochemistry (Fig. 2B). Therefore, by direct visual-
ization of vector, AdV entry into PD cells far exceeds that into
WD cells.
To investigate whether the differences in attachment and
internalization with WD and PD culture types are specific to
AdV, the uptake of fluorescent microspheres that approximate
the size of the vector (100 nm) into PD and WD cultures was
examined. Incubation of cultures with microspheres for6hat
37°C resulted in a large quantity of microspheres associated
with PD but not WD cultures, as shown by conventional fluo-
rescence microscopy (Fig. 2C and E, respectively). To deter-
mine the cellular localization of the microspheres, confocal
microscopy-generated XZ sections revealed that they were
both attached and internalized only into PD cultures (Fig. 2D).
These data suggest that PD cultures can internalize nonspe-
cifically attached particles and that the apical membrane of PD
cultures can undergo nonspecific uptake processes. In contrast,
microspheres did not enter WD cultures (Fig. 2F), due to
reduced nonspecific attachment and possibly to reduced up-
take into the cells.
To determine whether AdV attachment to PD and WD
cultures is a specific fiber-knob-mediated interaction, trans-
gene expression and AdV attachment were measured in the
presence of excess purified fiber-knob protein. Experiments
with HeLa cells showed that purified fiber-knob protein was
capable of inhibiting transgene expression by more than 99%
and AdV attachment by ;75% (Fig. 3), indicating that specific
fiber-knob binding to HeLa cells accounted for the subsequent
transgene expression. Excess fiber-knob protein inhibited
;70% of transgene expression in PD cultures and completely
inhibited the low level of expression in WD cultures, suggest-
FIG. 1. Interaction of AdV with human airway epithelial cell cultures. (A)
Representative histological cross-sections of human airway cells after 5 days in
culture showing PD epithelial cells and after .25 days in culture showing WD
pseudostratified ciliated epithelial cells. Hematoxylin and eosin counterstain;
magnification, 3215. (B to D) Comparative analyses of lacZ gene expression in
PD and WD cultures 48 h after exposure to AdVlacZ (6 h at 37°C) (B), inter-
nalization of AdV into PD and WD cultures after exposure to
35
S-AdVlacZ (6 h
at 37°C) (C), and attachment of AdV to PD and WD cultures after exposure to
35
S-AdVlacZ (6 h at 4°C) (D). Only the apical surfaces of cultures were exposed
to AdV (10
10
particles/ml). b-Gal activity and counts per minute (CPM) were
measured per square centimeter of epithelial surface area. Values shown are
mean 6standard error (SE) (n53). The results shown are representative of a
total of three different experiments.
6016 PICKLES ET AL. J. VIROL.
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ing that the predominant route of AdV entry into both culture
types is via fiber-knob receptor-mediated endocytosis. In the
PD but not the WD cultures, however, a significant portion of
transgene expression was not blocked by excess fiber-knob
protein. This non-fiber-knob-protein-mediated expression may
be due to nonspecific uptake processes, as described above
(Fig. 2). In contrast to transgene expression, AdV attachment
to both PD and WD cultures was not inhibited by fiber-knob
protein, suggesting that we cannot detect fiber receptor-spe-
cific attachment given the large component of “nonspecific”
attachment in both culture types.
To investigate the mechanism of nonspecific attachment of
AdV, we visually assessed the binding of AdV to PD and WD
cultures by transmission electron microscopy (TEM). The ma-
jority of AdV attached to the human PD culture surface was
associated with the abundant glycocalyx present on the mi-
crovilli (Fig. 4A), suggesting that the glycocalyx itself was bind-
ing AdV by a fiber-knob-independent mechanism. Assessment
of the binding of AdV to human WD cultures by TEM (Fig.
4B) showed reduced levels of glycocalyx on the WD cultures
and little AdV associated with the cell surface. Therefore, we
speculate that the great nonspecific attachment of AdV to PD
cultures reflects the relative mass of glycocalyx on these cells.
The recent identification of hCAR as a putative specific
AdV attachment receptor (1, 29) prompted investigation of the
localization of this receptor in human airway epithelial cul-
tures. Retrovirus-mediated overexpression of hCAR cDNA in
CHO cells (normally resistant to AdV-mediated gene transfer)
leads to increased specific AdV attachment and increased spe-
cific AdV-mediated gene transfer to levels above that observed
for HeLa cells (Fig. 5A). Immunofluorescence detection of
hCAR was performed with MAb RmcB on HeLa, CHO, and
CAR-CHO cells. RmcB detected hCAR on HeLa and CAR-
CHO cells but not on control CHO cells (Fig. 5B, panels I, III,
and II, respectively). In addition, coincubation of the cells with
RmcB and CyAdV showed a similar distribution for the recep-
tor and ligand, respectively, indicating that increased hCAR
expression led to increased AdV attachment. These results
show that hCAR mediates AdV attachment and AdV-medi-
ated gene transfer in these cell lines and that RmcB can detect
the presence of hCAR.
To determine whether hCAR is expressed in human airway
epithelia, human PD and WD cultures were permeabilized and
probed for hCAR expression with RmcB. In permeabilized
human PD cultures, hCAR immunoreactivity was detected on
the surfaces of all of the epithelial cells (Fig. 6A, panel I)
whereas in permeabilized WD cultures hCAR was restricted to
the basolateral membranes of the columnar cells (panel II).
Control cultures probed with an irrelevant MAb showed little
immunoreactivity above autofluorescence (panels III and IV).
These results parallel the earlier findings of the AdV attach-
ment and expression profiles for these culture types.
FIG. 2. Direct visualization of specific and nonspecific cellular uptake path-
ways with human cultures expressing both PD and WD cellular phenotypes. (A)
Exposure of cellular islands to CyAdV (10
10
particles/ml for6hat37°C) (red)
resulted in association of CyAdV with PD cells at the periphery of the islands
(arrow) and only rarely with individual cells within the WD regions. (B) The
cellular distribution of CyAdV is paralleled by the cellular distribution of lacZ
expression (arrow) in the same cultures 24 h later. (C and E) To assess whether
uptake into PD cultures was restricted to specific AdV uptake, the apical surfaces
of confluent cultures were exposed to fluorescent microspheres (;10
10
spheres/ml for6hat37°C), resulting in a large quantity of microspheres (red)
associated with PD (C) but not WD (E) cultures viewed en face. (D and F) To
determine the cellular localization of microspheres, confocal microscopy-gener-
ated XZ sections revealed that microspheres (red) were both attached to and
internalized into PD cultures (D) but not WD cultures (F). For panels D and F,
the cells were counterstained with calcein. Magnifications, 348 (A and B), 324
(C and E), and 3240 (D and F).
FIG. 3. Specificity of AdV interactions with human PD and WD airway
cultures. Inhibition of AdV-mediated gene transfer (top) and AdV attachment
(bottom) to cells preincubated with purified fiber-knob protein (10 mg/ml)
(1knob). Fiber-knob protein produced only a partial inhibition of AdV-medi-
ated gene transfer to PD cultures but inhibited the small amount of gene transfer
to WD cultures. In contrast to HeLa cells, fiber-knob protein did not inhibit AdV
attachment to either PD or WD cultures. AdV represents cultures exposed to
AdV but not preincubated with fiber-knob protein. Values shown represent
mean 6SE of ndeterminations, where nis shown in parentheses.
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The basolateral distribution of both hCAR and a
v
b
3/5
inte-
grins (see below and reference 14) in WD cells suggests that
these cultures may be more susceptible to AdV gene transfer if
access to the basolateral membrane was feasible. To test this
notion, AdV was applied to either the basolateral or apical
surface of human WD cultures. These studies showed that the
gene transfer efficiency is enhanced when the basolateral sur-
face rather than the apical surface is exposed to AdV (Fig. 6B).
(ii) Rat cultures. Preliminary experiments suggested that PD
airway epithelial cells derived from the rat tracheal epithelium
exhibited less nonspecific AdV attachment than human PD
cells (26). We therefore compared this well-characterized sys-
tem (Fig. 7A) with our human studies. In agreement with the
human model and in vivo studies, WD cultures were resistant
and PD cultures were susceptible to AdV-mediated gene trans-
fer (Fig. 7B). In contrast to human airway epithelia, the at-
tachment of AdV to PD and WD cultures was similar (Fig.
7D). Because of the lower total attachment, specific fiber-knob
inhibitable attachment could be detected and accounted for
approximately 50% of the total attachment (rat PD, 648 6125
and 320 673 cpm per cm
2
in the absence and presence,
respectively, of fiber-knob protein [n59 for each]; rat WD,
449 649 and 224 660 cpm per cm
2
in the absence and
presence, respectively, of fiber-knob protein [n59 for each]).
Thus, the rat model allows a comparison of AdV internaliza-
tion when specific and nonspecific AdV attachment was similar
on both culture types. This comparison shows that the amount
of AdV internalized into WD cells is far smaller than that
internalized in PD cells, which correlates with the gene transfer
efficiencies (Fig. 7C). These data indicate that the rate-limiting
step for efficient gene transfer to the rat WD cultures is the
internalization of AdV.
Mechanisms of internalization. (i) a
v
b
3/5
integrin localiza-
tion and interactions with AdV. AdV interactions with fiber-
knob receptors and/or a
v
b
3/5
integrins are thought to initiate
endosomogenesis via a mechanism involving receptor cluster-
ing. To test the hypothesis that reduced gene transfer in WD
cultures compared to PD cultures reflects the absence of a
v
b
3/5
integrin “initiation” of coated-pit endosomogenesis, we mea-
sured the distribution of a
v
b
3/5
integrins in the apical and
basolateral regions of PD and WD cultures. For human cul-
tures (Fig. 8A, panel i) and rat cultures (data not shown), the
a
v
b
3/5
integrins are distributed predominantly in the basolat-
eral membranes of both PD and WD cultures. The a
v
b
3/5
integrins are absent from the WD apical membranes of both
species and were expressed at low levels in the apical mem-
branes of the PD cultures, a finding in agreement with that of
Goldman and Wilson (14).
Whereas these data on integrin distribution correlate with
the high and low levels of gene transfer to the respective
culture types, attempts to demonstrate the functional impor-
tance of integrins in AdV-mediated gene transfer were unsuc-
cessful. In our models of airway epithelial cells, neither cyclical
RGD (cRGD) peptide (0.4 mg/ml) nor RGD peptide (4 mg/
ml) significantly altered the level of transgene expression in
human or rat PD cultures, respectively (Fig. 8A, panel ii). AdV
attachment to human or rat cultures was also not altered by
cRGD or RGD peptides, respectively (results not shown).
These data suggest that mediation of endosomogenesis and
endosomolysis leading to internalization of AdV into PD cul-
tures may occur by mechanisms other than a
v
b
3/5
integrin
interactions, suggesting that the absence of a
v
b
3/5
integrins
from the apical membrane of WD cells may not entirely ac-
count for the resistance of these cells to gene transfer.
FIG. 4. Investigation of nonspecific attachment of AdV to human PD and WD airway cells. The apical surfaces of human cultures were exposed to AdV (10
10
particles/ml for6hat4°C), and tissues were processed for TEM to assess AdV attachment. (A) With human PD cultures, AdV (arrows) was associated with cellular
glycocalyx-like structures on the apical membrane (inset). (B) With WD cultures, in agreement with the attachment studies, little AdV was associated with the apical
surface. Magnifications, 37,000 (A and B) and 20,000 (inset).
6018 PICKLES ET AL. J. VIROL.
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(ii) Dynamin-mutant cells deficient in receptor-mediated
endocytosis. The data from rat WD cultures emphasize the
importance of entry across the apical membrane as a limiting
variable for gene transfer. Whereas there are multiple modes
of entry across the cellular membrane (see below), morpho-
logical studies have identified coated-pit vesicles as an impor-
tant path for AdV entry (11, 30). We initiated studies to func-
tionally test the importance of this path for AdV-mediated
gene transfer with HeLa cells and HeLa cell mutants that have
reduced coated-pit-mediated endocytosis. Dynamin is respon-
sible for “pinching off” endocytotic invaginations formed dur-
ing receptor-mediated endocytosis (7). While overexpression
of wild-type (Wt) dynamin in HeLa cells does not affect endo-
cytotic processes (7), overexpression of the K44a dynamin mu-
tant selectively and reproducibly reduces receptor-mediated
endocytosis. Overexpression of either Wt or K44a dynamin did
not affect AdV attachment to the cells (Fig. 8B, panel i), but
b-gal expression was significantly reduced in K44a dynamin-
FIG. 5. Expression of hCAR mediates AdV attachment and gene expression. (A) Specific AdV attachment (i) and gene expression (ii) were measured for the HeLa,
CHO, and CAR-CHO cell lines by incubating cells in the absence (solid bars) or presence (open bars) of purified fiber-knob protein (10 mg/ml) for1hat4°Cbefore
adding
35
S-AdV (10
10
particles/ml for2hat4°C). Values shown represent the mean 6SE (n56 and 3 for solid and open bars, respectively). (B) Representative
immunofluorescent detection of hCAR with HeLa (i), CHO (ii), and CAR-CHO (iii) cell lines exposed at 4°C to anti-hCAR MAb with fluorescein isothiocyanate-
conjugated goat anti-mouse IgG (green) and CyAdV (red). Cell nuclei are counterstained with DAPI (blue). Localization of hCAR and CyAdV was restricted to HeLa
and CAR-CHO cells; there was no localization of hCAR and little CyAdV associated with CHO cells. Magnification, 3250.
FIG. 6. Distribution of endogenous hCAR in human PD and WD cultures. (A) Human PD (i and iii) and WD (ii and iv) cultures after permeabilization were
exposed to either RmcB MAb (i and ii) or an irrelevant isotyped MAb (iii and iv), and binding was detected with goat anti-mouse IgG-Texas Red. For PD cultures,
hCAR was detected on all surfaces of epithelial cells, whereas WD cultures show a basolateral distribution of hCAR. Magnification, 3100. (B) Exposure of AdV to
apical versus basolateral surfaces of human WD cultures. AdV (10
10
particles/ml) was applied to the respective surfaces for6hat37°C, and gene expression was
measured 48 h later. The values shown represent mean 6SE (n53).
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expressing cells compared to Wt dynamin-expressing cells
(panel ii). These findings show that the predominant route of
entry of AdV into HeLa cells occurs via receptor-mediated
endocytosis and that cells expressing identical amounts of AdV
attachment/internalization receptors can display a reduced
gene transfer efficiency reflecting a reduced plasma membrane
uptake pathway process.
Relationship between attachment and expression in PD and
WD cultures. Increased efficiency of AdV-mediated gene
transfer to cells in vitro has been achieved by the manipulation
of the AdV capsid coat to increase cellular attachment (10, 33).
To determine if increased attachment of AdV to human PD
and WD cultures leads to enhanced gene transfer efficiency,
increasing concentrations of AdV were applied to both culture
types. As shown in Fig. 9A, panel i, exposure of PD cultures to
a 10-fold increase in AdV concentration resulted in significant
enhancement of both the amount of AdV attached to the
cultures and transgene expression. These data suggest that in
this culture type, similar in morphology to many cell types
grown in vitro, the absolute amount of AdV attachment di-
rectly correlates with the subsequent level of gene transfer. For
WD cultures (panel ii) exposed to AdV concentrations similar
to or up to 1,000-fold higher than those to which PD cultures
were exposed, the level of gene transfer was not enhanced even
though the absolute amount of AdV attachment was increased
to levels associated with significant gene transfer in the PD
cultures. These data clearly show that increased AdV attach-
ment to WD cultures failed to overcome the inefficient gene
transfer.
The direct relationship between the quantity of attached
vector and expression in PD cells suggests that once attach-
ment has occurred, entry can be achieved by constitutive non-
specific pathways, e.g., pinocytosis and phagocytosis. In con-
trast, although vector can nonspecifically attach to WD cells,
the absence of expression suggests that few or no constitutive
or nonspecific entry pathways exist in the apical surface of this
cell type. To test the capacity for constitutive (unstimulated)
uptake of luminal solutions by airway epithelia in different
states of differentiation, we measured the uptake of a fluid-
FIG. 7. Interaction of AdV with rat airway epithelial cell cultures. (A) Rep-
resentative histological cross-sections of rat airway cells after 5 days in culture
showing PD epithelial cells and after .19 days in culture showing WD
pseudostratified mucociliary epithelial cells. Hematoxylin and eosin counter-
stain; magnification, 3220. (B to D) Comparative analyses of lacZ gene expres-
sion in PD and WD cultures 48 h after exposure to AdVlacZ (6 h at 37°C) (B),
internalization of AdV into PD and WD cultures after exposure to
35
S-AdVlacZ
(6 h at 37°C) (C), and attachment of AdV to PD and WD cultures after exposure
to
35
S-AdVlacZ (6 h at 4°C) (D). Only the apical surfaces of cultures were
exposed to AdV (10
10
particles/ml). b-Gal activity and counts per minute (CPM)
were measured per square centimeter of epithelial surface area. Values shown
are mean 6SE (n.8).
FIG. 8. a
v
b
3/5
integrin localization in human airway cultures and entry of
AdV mediated by dynamin-associated uptake pathways. (A) Panel i shows im-
munoprecipitates of biotinylated human a
v
b
3/5
integrins with a rabbit anti-hu-
man a
v
b
3/5
integrin polyclonal antibody. Apical (Ap) or basolateral (Bl) mem-
branes of either PD (lanes 1, 2, and 5) or WD (lanes 3, 4, and 6) cultures were
biotinylated and, after immunoprecipitation, probed with streptavidin conju-
gated to horseradish peroxidase for detection. Lanes 5 and 6 show immunopre-
cipitation with a rabbit IgG control. The arrowhead shows approximately 120
kDa. The experiment shown is representative of four experiments. Panel ii shows
lack of inhibition of gene transfer with a
v
b
3/5
integrin-interacting peptides. Nei-
ther cRGD peptide (0.4 mg/ml) with human cultures (HBE) nor RGD peptide
(4 mg/ml) with rat cultures (RTE) significantly altered the level of transgene
expression in the respective cultures after exposure to AdVlacZ (10
10
particles/
ml). Values shown represent mean 6SE (n54 and 3 for cultures in the absence
[closed bars] and presence [open bars] of RGD peptides, respectively). (B)
Comparison of AdV attachment (i) and AdV-mediated gene transfer (ii) to
HeLa cells overexpressing either Wt or mutant (K44a) dynamin. Cells were
exposed to AdVlacZ (10
10
particles/ml) for2hat4°C, and either attachment was
measured immediately or expression was measured 24 h later. Values shown
represent mean 6SE (n56 for each).
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phase marker and correlated this parameter with gene transfer
efficiency. Human cells grown on tissue culture plastic (i.e., PD
cells) internalized measurable amounts of [
3
H]inulin, whereas
WD cells did not (Fig. 9B, panel i). These differences were
paralleled by the differences in gene transfer (panel ii). These
findings suggest that a relative reduction in the constitutive
activity of the aggregate apical membrane entry pathways in
WD cells contributes to the resistance of these cells to gene
transfer.
DISCUSSION
The WD ciliated airway epithelium is the target tissue for
AdV-mediated gene transfer approaches for the treatment of
the pulmonary manifestations of CF. However, AdV are inef-
ficient gene transfer vectors for the respiratory epithelium in
vivo (18, 25, 35, 36) and high AdV doses delivered to the lung
are inflammatory (5, 6, 27), limiting the usefulness of the cur-
rently available vectors. Therefore, efforts are being directed at
increasing the efficiency of AdV-mediated gene transfer in the
hope that lower, less inflammatory doses can be administered
to provide effective gene transfer to the airway epithelium.
A strategy to identify the mechanisms that account for the
low in vivo gene transfer efficiency of AdV has emanated from
studies that demonstrate that WD epithelial cells of human
and rodent airways are resistant to AdV gene transfer whereas
injured or PD airway epithelial cells are efficiently transduced
(8, 25, 36). Since quantitative studies of the interactions of
AdV with the airway epithelium in vivo are difficult and prone
to considerable variation, we have used cell culture models that
reproduce (i) the WD (ciliated) and PD cellular phenotypes
and (ii) the relative resistance of WD cells and permissiveness
of PD cells to AdV-mediated gene transfer as observed in vivo
(Fig. 1 and 7).
Our analyses of AdV interactions with human airway epi-
thelial cells show that decreased gene transfer efficiency in WD
cultures compared to PD cultures is due to limited entry of
AdV across the apical membrane of WD cultures, which may
reflect as many as three independent steps: (i) reduced specific
AdV attachment to the apical surface of WD cells; (ii) the
absence of a
v
b
3/5
integrins at the apical surface of WD cells;
and (iii) a reduced rate of AdV internalization across the
apical membrane of WD cells.
Using radiolabeled virus, we demonstrate that the specific
attachment of AdV to human WD cultures is reduced com-
pared to attachment to PD cultures (Fig. 1, 3, and 4) and that
differences in specific attachment mirror differences in AdV-
mediated gene transfer efficiency (Fig. 1 and 3). In support of
these functional attachment data, immunofluorescence studies
with the recently identified specific AdV fiber-knob attachment
receptor (hCAR [1, 29]) demonstrated that hCAR is not ex-
pressed at the luminal surface of human WD cultures but is
expressed on all surfaces of PD cells. The distribution of hCAR
and the attachment data correlate with the relative degree of
AdV-mediated transgene expression in the respective cultures
(Fig. 6). These findings are consistent with those from a study
recently reported by Zabner et al. (35) with a similar model of
human WD airway epithelial cells, which concluded that a
determining factor for inefficient gene transfer is the lack of
high-affinity fiber receptors on the apical surface of WD cells.
FIG. 9. Increased AdV attachment leads to increased gene transfer in cells with active nonspecific uptake pathways. (A) Effect on AdV attachment and gene transfer
in human PD (i) and WD (ii) cultures with exposure to different numbers of AdV particles. Values shown represent mean 6SE (n.3). (B) Panel i shows measurement
of nonspecific fluid-phase uptake pathways in human PD and WD cells with [
3
H]inulin exposed to the luminal surface of the cultures at 37°C for 6 h. Values shown
represent mean 6SE (n.5). Panel ii shows gene expression in parallel cultures exposed to AdVlacZ (10
10
particles/ml) for6hat37°C, with enzyme activity measured
24 h later. Values shown represent mean 6SE (n.8).
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While the results of the present study are consistent with
those of Zabner et al. (35), additional steps may also determine
the efficiency of AdV-mediated gene transfer. It has been re-
ported that fiber-knob receptors and/or a
v
b
3/5
integrins medi-
ate endosomogenesis by a mechanism involving receptor-me-
diated clustering of clathrin-rich membrane regions (11, 30).
The apical membrane of WD epithelia may have a generally
low capacity for performing endosomogenesis because of a low
expression of fiber-knob/a
v
b
3/5
integrin receptors (Fig. 6 and
8A) (14, 35). However, the a
v
b
3/5
integrins do not appear to be
necessary for efficient gene transfer to PD cultures since RGD
peptides failed to reduce gene expression in these culture types
(Fig. 8A), a finding that is supported by studies with RGD-
mutated AdV (12) and efficient gene transfer to airway epi-
thelial cells derived from a b
5
integrin knockout mouse model
(17). These observations cast doubt on the absolute require-
ment of these integrins for gene transfer in WD cells.
With respect to the importance of apical membrane inter-
nalization capacity, we observed that internalization of AdV
into human WD cultures is lower than into human PD cultures
but that differences in attachment efficiency precluded an anal-
ysis of the direct effects of entry rates on this parameter. How-
ever, an indication that absolute rates of entry (endocytosis)
are important for gene transfer efficiency came from studies
with rat airway cultures. In contrast to human cultures, AdV
attachment is similar in rat PD and WD cells and does not
reflect the differences in gene transfer efficiency (Fig. 7). How-
ever, the quantity of AdV internalized into WD rat cultures is
greatly reduced compared to the quantity internalized into the
PD cultures. The rates of internalization of AdV directly cor-
relate with the relative gene transfer efficiencies observed with
the two cellular phenotypes (Fig. 7), indicating that the inter-
nalization process for the entry of AdV into WD cells is rate
limiting.
PD airway epithelial cells are susceptible to AdV-mediated
gene transfer via specific hCAR-mediated pathways. However,
PD cells also appear transducible via nonspecific attachment
and nonspecific uptake processes. Both human and rat PD
cultures, but not WD cells, appear capable of internalizing
nonspecifically attached AdV via nonspecific mechanisms such
as pinocytosis (Fig. 9B). This observation is important to the
design of targeted vectors that attempt to increase gene trans-
fer efficiency based on the assumption that attachment alone is
the rate-limiting step to efficient gene transfer (10, 33). Retar-
geted vectors attached via nonspecific interactions or to non-
internalizing receptors will probably depend on nonspecific
uptake pathways to enter cells; while this approach is useful for
PD cells in vitro, increasing attachment to WD cultures which
do not exhibit these cellular entry pathways does not increase
gene transfer efficiency (Fig. 9A). In addition, nonspecific entry
pathways that do not use dynamin-associated coated pits may
not efficiently allow the productive entry of AdV into cells (Fig.
8B).
In summary, human WD cultures are resistant to AdV-
mediated gene transfer because of decreased specific attach-
ment sites and reduced nonspecific entry paths that internalize
a fraction of a large vector load typical of Ad CF gene therapy
protocols. To circumvent the inefficiency of AdV-mediated
gene transfer to the respiratory epithelium, AdV will require
retargeting to receptor types that both undergo endocytosis via
coated-pit mechanisms and are present in sufficient numbers
on the airway epithelial luminal surface.
ACKNOWLEDGMENTS
We gratefully thank R. J. Samulski for helpful discussions during the
preparation of the manuscript and Steven Albelda, Jeffrey Bergelson,
Robert Gerard, John Olsen, Sandra Schmid, and James Yankaskas for
generous gifts of reagents and human tissue.
This work was supported by NIH grant SCOR CF HL42384 and
Cystic Fibrosis Foundation (CFF) grant S880. R.J.P. was in receipt of
a CFF Research Fellowship.
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