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Journal of Virology, October 2000, p. 9617-9628, Vol. 74, No. 20
Thomas E. Starzl Transplantation
Institute,1 Department of
Surgery,2 Department of Molecular
Genetics and Biochemistry,4 and
Department of Dermatology and the University of Pittsburgh
Cancer Institute,3 University of Pittsburgh
Medical Center, Pittsburgh, Pennsylvania 15213-2582
Received 5 May 2000/Accepted 12 July 2000
Recombinant adenovirus (rAd) infection is one of the most effective
and frequently employed methods to transduce dendritic cells (DC).
Contradictory results have been reported recently concerning the
influence of rAd on the differentiation and activation of DC. In this
report, we show that, as a result of rAd infection, mouse bone
marrow-derived immature DC upregulate expression of major
histocompatibility complex class I and II antigens, costimulatory molecules (CD40, CD80, and CD86), and the adhesion molecule CD54 (ICAM-1). rAd-transduced DC exhibited increased allostimulatory capacity and levels of interleukin-6 (IL-6), IL-12p40, IL-15, gamma
interferon, and tumor necrosis factor alpha mRNAs, without effects on
other immunoregulatory cytokine transcripts such as IL-10 or IL-12p35.
These effects were not related to specific transgenic sequences or to
rAd genome transcription. The rAd effect correlated with a rapid
increase (1 h) in the NF- As professional antigen-presenting
cells (APC), dendritic cells (DC) exhibit the unique ability to
stimulate both naive and memory T lymphocytes and play a critical role
in central and peripheral T-cell tolerance (3, 4, 34, 55,
58). Their potential to determine the balance between immunity
and tolerance makes DC targets for the therapeutic manipulation of
immune responses against tumor cells or microorganisms or for the
control of undesired immune reactions against allo- or autoantigens. In
this respect, gene transfer approaches have been explored in an effort
to potentiate the adjuvant (12, 29) or tolerogenic
properties of DC (30, 35, 57). Recombinant adenovirus (rAd)
has been demonstrated to be one of the most effective vehicles to
deliver foreign DNA into DC (1, 15, 16, 29, 41, 59, 71).
However, a fundamental problem with the use of replication-deficient
rAd is that they generate the rapid development of natural killer (NK)
cell and cytotoxic T-lymphocyte (CTL) responses that eliminate rAd-infected cells and induce neutralizing antibodies (Abs) that "limit" readministration of the same rAd serotype
(65-67). The immunogenicity of rAd is a particular drawback
when long-term transgene expression is required or when transduced DC
are employed to generate antigen-specific tolerance for therapy of
graft rejection or autoimmune diseases (26, 30, 35, 65-67).
Although the mechanistic basis of rAd immunogenicity is unknown,
evidence has accumulated that this phenomenon is associated with the
expression of transgene and viral proteins (17, 26, 53, 67)
and the active participation of DC (52). Thus, human DC
infected with E1a There is evidence that DC-virus interaction affects DC activity in
different ways. Thus, whereas influenza virus infection augments DC
stimulatory activity, measles or human immunodeficiency viruses silence
the host immune response through negative effects on DC function
(6). With respect to rAd, different groups have recently
reported contradictory effects on DC (25, 27, 46, 59, 71).
Zhong et al. (71) and Tillman et al. (59)
concluded that rAd is a nonperturbing vector for DC. By contrast, Rea
et al. (46) demonstrated that rAd infection activates human DC.
NF- In the present study, we investigated the effects of rAd on mouse DC,
both in vitro and in vivo. We show that DC infected with rAd exhibited
increased expression of major histocompatibility complex (MHC),
adhesion, and costimulatory molecules and enhanced allostimulatory
function. After rAd infection, increased levels of IL-6, IL-12p40,
IL-15, gamma interferon (IFN- Experimental animals.
Ten- to twelve-week-old C57BL/10 (B10;
H2Kb IAb IE Reagents.
Mouse recombinant granulocyte-macrophage
colony-stimulating factor (GM-CSF) was a gift from the Schering-Plough
Research Institute (Kenilworth, N.J.), and mouse rIL-4 was purchased
from R&D (Minneapolis, Minn.). The NF- Generation of BM DC.
The method was modified from one
described earlier (22,37). Briefly, bone-marrow (BM) cells
from femurs and tibias of B10 mice were depleted of red blood cells by
lysis with ammonium chloride. NK cells, granulocytes, erythroid
precursors, and T and B cells were killed by labeling with a cocktail
of monoclonal Abs (MAbs; anti-NK-1.1, anti-Gr-1, anti-TER-119/erythroid
cells, anti-CD4, anti-CD8, and anti-CD45R/B220; all MAbs were from
PharMingen, San Diego, Calif.), followed by incubation with
low-toxicity rabbit complement (Cedarlane) BM cells were cultured in
RPMI 1640 (Life Technologies, Grand Island, N.Y.) with 10%
heat-inactivated fetal calf serum (Life Technologies), glutamine,
nonessential amino acids, sodium pyruvate, HEPES, 2-mercaptoethanol,
and penicillin-streptomycin, supplemented with 1,000 U of murine
rGM-CSF and 1,000 U of murine rIL-4 per ml. Culture medium was replaced
at day 3. At day 5, nonadherent cells were removed, and fresh medium
with cytokines was added. Two days later (day 7) approximately 60 to
90% of the new population of nonadherent cells was CD11c+
BM DC, with a predominance of immature DC (CD11c+
CD86
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Recombinant Adenovirus Induces Maturation of
Dendritic Cells via an NF-
B-Dependent Pathway
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
B-DNA binding activity detected by
electrophoretic mobility shift assays. rAd-induced DC maturation was
blocked by the proteasome inhibitor
N
-p-tosyl-L-lysine chloromethyl
ketone (TLCK) or by infection with rAd-IB, an rAd-encoding the
dominant-negative form of IB. In vivo studies showed that after
intravenous administration, rAds were rapidly entrapped in the spleen
by marginal zone DC that mobilized to T-cell areas, a phenomenon
suggesting that rAd also induced DC differentiation in vivo.
These findings may explain the immunogenicity of rAd and the
difficulties in inducing long-term antigen-specific T-cell hyporesponsiveness with rAd-transduced DC.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
rAd induce the generation of
virus-specific CTLs in vitro (52). Moreover, DC infected ex
vivo with rAd-encoding cDNAs unrelated to immunostimulatory
molecules (e.g., reporter genes) exhibit a higher T-cell stimulatory
ability compared to non-transduced DC (27, 30, 35).
B is an evolutionarily conserved family of regulatory,
DNA-binding transcription factors that are resident in the cytoplasm in
various homo- and heterodimeric associations (2, 28, 38). NF-B factors play a crucial role during generation and
maturation of myeloid DC (9, 10, 19, 43, 45, 48, 64).
NF-B is sequestered in the cytoplasm by the inhibitory
B proteins, IB and IB
.
Signal transduction results in the phosphorylation and subsequent
ubiquitination of IB, which serves to target
IB protein for degradation by the proteasome. As a result
of IB proteolysis, the NF-B dimers are
liberated for nuclear translocation, bind to specific DNA sequence
elements (B motifs), and regulate target gene
transcription by interactions with the general transcription machinery.
Recent progress has dissected the signaling pathway by which tumor
necrosis factor alpha (TNF-), interleukin-1 (IL-1), and bacterial lipopolysaccharide (LPS) activate the nuclear
translocation of NF-B. After cytokine-receptor
engagement, the FADD/MORT/MyD family proteins are recruited to the
cytoplasmic tail of the receptor; subsequently, FADD/MORT/MyD proteins
interact with and activate either the TRAF2 or TRAF6 proteins.
Activated TRAF binds to and activates the NF-B inducing
kinase complex, which in turn phosphorylates and activates the
inhibitory B kinase complex. Activated inhibitory kinase
complex can directly phosphorylate serines 32 and 36 of IB, which targets IB for proteolysis,
thereby releasing NF-B dimers to enter the nucleus
(2, 28, 38).
), and TNF- mRNAs were observed,
whereas transcription of IL-12p35, IL-10, or transforming growth factor
1 (TGF-1) remained unaffected, or even decreased. DC
activation after rAd infection was not associated with any particular
transgenic sequence and was independent of viral transcription.
However, the viral effect on DC correlated with rapid nuclear
translocation of NF-B, which was blocked either by the proteasome
inhibitor N-p-tosyl-L-lysine
chloromethyl ketone (TLCK) or by an rAd encoding the mutant form of
IB, both inhibitors (by different mechanisms) of NF-B nuclear
translocation. Our in vivo studies reveal that splenic DC in the
marginal zone were among the first targets of rAd infection after
intravenous (i.v.) administration. In agreement with the in vitro
results, rAd induced rapid mobilization of immature DC from the
marginal zone into the T-cell-dependent areas, an observation
indicative of DC activation in vivo.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) and
C3H/He (C3H; H2Kk IAk
IEk) mice were purchased from The Jackson Laboratory
(Bar Harbor, Maine). Animals were maintained in the
specific-pathogen-free facility of the University of Pittsburgh Medical Center.
B inhibitor TLCK and
7-amino-actinomycin D (7-ADD) were purchased from Calbiochem (San
Diego, Calif.).
cells) (Fig. 1A). In
experiments in which the whole population of BM cells (day 7) was
infected with rAd, flow cytometric analysis was performed exclusively
on the CD11c+ DC subset (CD11c is a DC-restricted marker in
the mouse). In other cases, CD11c+ CD86 and
CD11c+ CD86+ BM DC were isolated by flow
sorting (purity, 90 to 94%). For molecular analysis,
CD11c+ BM DC were positively selected by incubating BM
cells cultured in GM-CSF plus IL-4 (day 7) with a bead-conjugated
hamster anti-mouse CD11c MAb (Miltenyi Biotec, Auburn, Calif.),
followed by passage through a paramagnetic column (Miltenyi) (DC
purity, 90 to 93%). Because of the variability in the percentage of DC
and in the immature/mature DC ratio in each experiment, each had its
own nontreated control. Results were compared only within and not between experiments.
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FIG. 1.
(A) BM DC generated in GM-CSF plus IL-4 (day 7) as
described in Materials and Methods, labeled, and analyzed by flow
cytometry. Two populations of DC were detected: CD11c+
CD86 immature DC (upper left quadrant) and
CD11c+ CD86+ DC (upper right quadrant). (B)
Allostimulatory activity of gamma-irradiated, purified immature
CD11c+ CD86 (
) or mature
CD11c+ CD86+ B10 DC (
), assessed using naive
C3H (H2k) splenic T cells as
responders in a 3-day MLR. BM DC were propagated as described in
Materials and Methods, harvested at day 7, purified by flow sorting,
and set up at various concentrations with 2 × 105
responder T cells. [3H]thymidine (TdR) was added 18 h before harvesting. The MLR stimulatory activity of freshly-isolated
B10 (
) or C3H (
) bulk spleen cells is also shown. The results are
expressed as mean counts per minute ± 1 SD, and are
representative of at least three separate experiments. (C) Transduction
efficiency of immature (CD11c+ CD86) and
mature (CD11c+ CD86+) DC purified by sorting
and infected with rAd-EGFP (at an MOI of 100). Transgene expression was
evaluated 24 h later by flow cytometry. (D) Transduction
efficiency of DC infected for 2 days with increasing MOIs of rAds and
detected by flow cytometry. In panels C and D the transgene expression
was evaluated exclusively on CD11c+ cells. Dead cells were
excluded by 7-ADD staining. No positive cells were detected in
noninfected controls or in cells infected with rAd-Control (data not
shown). The data are representative of three independent experiments.
rAd vectors.
E1 E3 deleted rAds
included (i) Add1434 (rAd-Control), encoding no transgene or
transpromoter, kindly provided by A. Shaked (Department of Surgery,
University of Pennsylvania) (44); (ii) rAd-EGFP, encoding
the enhanced variant of the Aequorea victoria green
fluorescent protein (EGFP) cDNA; (iii) rAd-LacZ, encoding the
Escherichia coli -galactosidase cDNA; (iv)
rAd-Luc, encoding the luciferase cDNA; and (v) rAd-IB,
encoding the cDNA for the mutant form of IB, kindly
provided by D. A. Brenner (University of North Carolina)
(24). All transgenes were under the control of the
hCMV-MIEp. rAds were scaled up and purified by a
1,1,2-trichloro-trifluoroethane extraction procedure (Sigma), followed
by two CsCl gradients (Life Technologies). CsCl was removed from viral
bands by dialysis against 2 liters of buffer (Tris, 10 mM;
MgCl2, 1 mM; NaC1, 135 mM [pH 7.5]). The final dialysis
was performed against the same buffer containing 10% glycerol. Virus
stocks were tested for the presence of replication-competent Ad as
described elsewhere (18) and preserved at 
80°C. Viral titers were
measured by standard endpoint dilution assay using 293 cells. All Ad
preparations were analyzed for endotoxin (LPS) by using the
Limulus amebocyte lysate test (E-TOXATE; Sigma) according to
the manufacturer's protocol. The detection limit of the test was 0.015 endotoxin units/1010 PFU/ml. All experiments were done with
viral aliquots that tested negative for endotoxin.
Flow cytometry.
For triple color labeling, BM DC were
blocked with normal goat serum (1:10, 10 min at 4°C) and then
incubated (1h at 4°C) with (i) biotin-conjugated hamster anti-mouse
CD11c MAb (HL3), (ii) phycoerythrin (PE)-conjugated rat anti-mouse CD86
MAb (GL1), and (iii) one of the following fluorescein isothiocyanate
(FITC)-conjugated MAbs: mouse anti-H2Kb
(AF6-88.5), mouse anti-IAb chain (25-9-17), hamster
anti-mouse CD40 (HM40-3), hamster anti-mouse CD80 (16-10A1), rat
anti-mouse CD11b (M1/70), or hamster anti-mouse CD54 (3E2). Incubation
with primary MAb was followed by treatment with Cy-Chrome-conjugated
streptavidin (PharMingen) at 1:2,000 for 30 min at 4°C. After
staining, cells were washed and fixed in 2% paraformaldehyde. For
double immunostaining of rAd-infected BM DC, cells were blocked with
normal goat serum as described above and then incubated (1 h at 4°C)
with (i) PE anti-mouse CD11c MAb and (ii) one of the following
FITC-conjugated MAbs: anti-H2Kb, anti-IAb chain, anti-mouse CD40, anti-mouse CD80, anti-mouse CD86, anti-mouse CD11b, or anti-mouse CD54. After staining, cells were washed and incubated with 7-ADD to exclude dead cells. Appropriate
fluorochrome-conjugated species- and isotype-matched, irrelevant MAbs
were used as negative controls. All MAbs were purchased from
PharMingen. Cytometric analysis was performed using an EPICS Elite flow
cytometer (Coulter, Hialeah, Fla.).
RPA.
RNA was isolated using a total RNA Isolation Kit
(PharMingen) from 5 × 106 CD11c+ BM DC
highly purified by positive selection with bead-anti-CD11c MAb,
followed by passage through a paramagnetic column as described above.
RNA was dissolved in RNase-free water and stored at 80°C until use.
The RNase protection assay (RPA) for cytokine mRNAs was performed using
the RiboQuant Multi-Probe RPA System (PharMingen). Two different
multiprobe template kits containing cDNAs encoding mouse
IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p35, IL-12p40, IL-13,
IL-15, IFN-, IFN-, TNF-, TGF-1, TGF-2, TGF-3,
macrophage migration inhibition factor, and the housekeeping genes L32
and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as templates for the T7 polymerase-directed synthesis of
[-32P]UTP labeled antisense RNA probes. Hybridization
(16 h at 56°C) of 5 µg of each target mRNA with the antisense RNA
probes sets was followed by RNase and proteinase K treatment,
phenol-chloroform extraction, and ammonium acetate precipitation of
protected RNA duplexes. In each RPA, the corresponding antisense RNA
probe set (3 × 103 cpm) was included as the molecular
weight standard and to examine probe set integrity. Mouse RNA
(PharMingen) (positive control) and RNA degradation controls were
included. Yeast tRNA served as negative control. Samples were
electrophoresed on acrylamide-urea sequencing gels. Dried gels were
exposed on Fujifilm X-ray film, with two intensifying screens, at
80°C. Quantification of the bands was performed by densitometry
(Personal Densitometers 1; Molecular Dynamics, Sunnyvale, Calif.),
followed by assessment using ImageQuantNT software (Molecular
Dynamics). The signals from specific mRNAs were normalized to signals
from housekeeping genes (L32 and GADPH) run on each lane to adjust for
loading differences.
Cytokine quantitation.
Enzyme-linked immunosorbent assay
(ELISA) kits (OptEIA; PharMingen) were used to quantify murine IL-12
p40/70 and IFN- in 48-h supernatants of rAd-transduced DC. The
detection limits for IL-12 and IFN- were 6.25 and 15.6 pg/ml, respectively.
Allostimulatory activity (MLR).
B10 BM DC (day 7) were
purified by sorting into CD11c+ CD86
(immature) DC and CD11c+ CD86+ (mature) DC.
Both subpopulations were employed as stimulators in 72-h primary mixed
leukocyte reactions (MLR) using nylon wool column purified naive
allogeneic (C3H) splenic T cells as responders (30,35). In
experiments with rAd-infected DC, CD11c+ BM DC infected
with increasing doses of virus and purified by positive selection with
paramagnetic columns were employed as stimulators. As controls,
nontransduced B10 BM DC, or B10 splenocytes, or C3H splenocytes were
used as stimulators.
Preparation of nuclear protein fractions and electrophoretic
mobility shift assays (EMSAs).
BM DC (day 7) were purified by
labeling with bead anti-CD11c MAb, followed by passage through
paramagnetic columns, and infected with rAd-Control or rAd-IB
(multiplicity of infection [MOI] of 100) for 1, 3, or 18 h. To
prepare nuclear extracts, BM DC were pelleted, resuspended in 0.5 ml of
buffer A (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl,
and 0.5% Nonidet P-40 [NP-40]) and disrupted by gently pipetting.
Nuclei were washed in buffer B (buffer A without NP-40), and nuclear
proteins were extracted with 20 mM HEPES (pH 7.9), 10% glycerol, 1.5 mM MgCl2, 420 mM KCl, and 0.2 mM EDTA. All steps were
performed on ice or at 4°C. All buffers contained 1 to 2 µg each of
aprotinin, benzamidine, chymostatin, leupeptin, and pepstatin per ml;
0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, and 0.1 µM sodium vanadate. Protein concentrations were measured by using the
Bio-Rad protein assay with bovine serum albumin as a standard. The
NF-B binding sequence from the immunoglobulin light chain
enhancer (5' AGT TGA GGG GAC TTT CCC AGG C 3') was used as a
probe. DNA probes were end labeled with [-32P]ATP
using T4 polynucleotide kinase (Boehringer Mannheim) and purified on
Sephadex G-50 columns (Sigma) equilibrated with TEN (10 mM Tris, 1 mM
EDTA, 100 mM NaCl) as described by the manufacturer (Sigma). A portion
(5 µl [1 µg]) of each sample of nuclear protein was incubated
with ~105 cpm of 32P-labeled consensus
NF-B oligonucleotide (0.5 ng) for 45 min at room temperature. The
nuclear proteins and NF-B probe were incubated in buffer (10 mM
Tris, pH 7.5; 10% glycerol; 0.2% NP-40). Poly(dI-dC) (Boehringer
Mannheim) was included as nonspecific competitor DNA. Protein-DNA
complexes were resolved on 4% nondenaturing polyacrylamide gels in
running buffer (450 mM Tris borate, 1 µM EDTA; pH 8.0). After
electrophoresis, gels were dried and subjected to autoradiography.
Antibody supershift experiments included the addition of 1 µl of
anti-p50 or anti-p65 polyclonal antibodies (Santa Cruz Biotechnology,
Santa Cruz, Calif.).
Immunofluorescence staining of tissue sections.
Spleen
samples were embedded in Tissue-Tek OCT (Miles Laboratories, Inc.,
Elkhart, Ind.), snap frozen in isopentane-liquid nitrogen, and stored
at 80°C until further use. To preserve the tissue localization of
EGFP, spleens from animals injected with rAd-EGFP were fixed in 2%
paraformaldehyde (2 h at 4°C) and cryopreserved in 30%
sucrose-phosphate-buffered saline (PBS) overnight, and snap frozen.
Cryostat sections (8 µm) were air dried, fixed in acetone (10 min at
4°C), blocked with normal goat serum, and incubated with PE-labeled
anti-mouse CD11c MAb and FITC-labeled anti-mouse CD3
MAb (145-2C11;
PharMingen), for 1 h at room temperature. Sections were fixed in
2% paraformaldehyde and mounted with glycerol-PBS (pH 7.0). Slides
were examined with a Zeiss Axiovert 135 microscope equipped with
appropriate filters. Fluorescence was visualized using a cooled
charge-coupled-device camera (Photometrics CH250; Photometrics, Tucson,
Ariz.). Signals from different fluorochromes were acquired
independently, and final montages were edited using the Adobe Photoshop
software program (Adobe Systems, Mountain View, Calif.).
Statistical analysis. Results are expressed as means ± one standard deviation (SD). Comparisons between different means were performed by analysis of variance, followed by the Student Newman-Keuls test. Comparison between two means was performed by the Student t test. A P value of <0.05 was considered significant.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Immature and mature BM DC are infected by rAd.
BM DC generated
in vitro with GM-CSF plus IL-4 showed at day 7 a mixed population
of CD11c+ CD86 (69 ± 11%) and
CD11c+ CD86+ (30 ± 8%) DC (Fig. 1A).
Triple labeling of the CD11c+ CD86 cells,
followed by flow cytometric analysis, revealed surface markers of
immature myeloid DC (MHC class Ilo [MHC-Ilo],
MHC-IIlo, CD40/lo, CD80lo,
CD11bhi, and CD54lo). These cells induced only
minimal levels of allogeneic T-cell proliferation in vitro (Fig. 1B).
In contrast, CD11c+ CD86+ cells exhibited the
phenotype of mature DC (MHC-Ihi, MHC-IIhi,
CD40+, CD80hi, CD11blo, and
CD54hi) and triggered a potent allogeneic T-cell response
(Fig. 1B).
and CD11c+
CD86+ DC, and infected with rAd-EGFP (MOI = 100). At
24 h after infection, the expression of EGFP was analyzed by flow
cytometry. Figure 1C shows that similar percentages of
CD86 and CD86+ BM DC were infected by
rAd-EGFP.
The efficiency of rAd-mediated gene delivery to BM DC (day 7) was
evaluated after 24 h of infection with rAd-EGFP. Figure 1D
illustrates the expression of EGFP detected by flow cytometry on live
(7-ADD) CD11c+ DC. Maximum infectivity was
achieved at an MOI of 500. No positivity was found in noninfected
cells or in cells infected with rAd-Control (data not shown). The
mortality of rAd-transduced BM DC (evaluated by 7-ADD labeling and flow
cytometry) did not increase significantly (P > 0.05)
24 to 48 h after infection, when MOIs of from 10 to 500 were used
(not shown).
rAd increases the expression of MHC antigen, costimulatory
molecules, ICAM-1 (CD54), and levels of cytokine mRNAs in BM DC.
The effect of rAd on the cell surface phenotype of BM DC (day 7) was
studied 48 h (day 9) after infection with rAd-Control. Nonadherent
cells were double labeled, and the specific markers were analyzed by
flow cytometry exclusively in live DC (gate: CD11c+,
7-ADD). Infection with rAd caused an increase in the
intensity of expression (as measured by the mean fluorescence channel
[MFC]) and in the percentage DC positive for MHC-II, CD40, CD80, and
CD86 (Fig. 2). For those markers such as
MHC-I and CD54 (ICAM-1) that were present on all CD11c+ BM
DC, there was increased intensity of expression (MFC) (Fig. 2). Most
changes in the DC phenotype were detected at MOIs of 10 to 100. Consistent with an rAd-associated DC activation-differentiation process, expression of CD11b (an integrin downregulated upon myeloid DC
maturation [49]) decreased after rAd infection (Fig.
2). As a control, the intensity of expression of CD11c, a molecule expressed at the same level by immature and mature DC (Fig. 1A), remained stable (Fig. 2).
|
, and
TNF- and decreased the expression of TGF-1 mRNA. The increase in
IL-12p40 and IFN- mRNAs was correlated with the levels of protein
detected by ELISA in the supernatants 48 h after infection (Fig.
3C) and by intracellular staining of CD11c+ BM DC by flow
cytometry (data not shown). No changes were detected in the level of
IL-12 p35 mRNA (detected as an extremely weak band in some of the
samples; data not shown) or in the low levels of TGF-2 and -3
mRNAs. It was not possible to detect IL-2, IL-3, IL-4, IL-5, IL-9,
IL-10, IL-13, or IFN- transcripts in any of the noninfected of
rAd-Control transduced samples. Similar data were obtained after
CD11c+ BM DC infection with rAd-EGFP (not shown).
|
Infection with rAd increases the allogeneic stimulatory capacity of
DC.
The allostimulatory activity of DC following rAd infection was
tested in BM DC cultures (day 7) transduced with increasing MOIs of
rAd-Control. After 24 h, CD11c+ BM DC were isolated by
positive selection with paramagnetic columns, irradiated, and used as
stimulators of allogeneic (C3H) naive T cells in 72-h MLR.
CD11c+ BM DC infected with increasing MOIs of rAd-Control
showed an increased capacity to stimulate allogeneic splenic T cells, a phenomenon consistent with the phenotypic changes observed after rAd
infection. The relatively low allostimulatory capacity of noninfected
BM DC shown in Fig. 4 was due to the high
proportion of immature DC (85%) in the sample.
|
, 100; 
, 500), assessed at various
concentrations with 2 × 105 responder splenic naive
C3H (H-2k) T cells in 3-day MLR
experiments. BM DC (day 7) were propagated as described in Materials
and Methods, treated with rAd for 24 h, and purified by labeling
with bead anti-CD11c MAb and passage through a paramagnetic column.
[3H]TdR was added 18 h before harvesting. The MLR
stimulatory activity of freshly isolated allogeneic (B10 [Effect of transgene sequences and rAd transcription on BM DC
activation.
To investigate whether the changes observed in BM DC
were associated with transgenic DNA sequences carried by the virus, we studied (by flow cytometry) the expression of MHC-II and CD86 in the
live DC (gate: CD11c+, 7-ADD) after infection
with rAds with distinct transgenes (rAd-LacZ or rAd-Luc) or without
transgene (rAd-Control). The fact that, by 48 h after
transduction, the three rAds had upregulated MHC-II and CD86 in
CD11c+ DC (Fig. 5A) suggested
that the viral effect might be caused by the nontransgenic rAd
genome and/or by viral proteins.
|
)
upregulated CD86 and MHC-II (Fig. 5C) to the same extent as DC
transduced with non-UV-irradiated rAd at the same MOI. Therefore, these
results suggest that rAd transcription is not associated with DC activation.
DC maturation induced by rAd is associated with NF-B
activation.
Because many of the surface molecules and cytokines
whose expression was increased by rAd-transduced DC have been shown to be upregulated by NF-B, we sought to investigate the involvement of
NF-B in rAd-induced DC maturation, using both pharmacologic and
genetic inhibition strategies. The peptide TLCK is an inhibitor of
proteosome-mediated degradation of the IB protein and has been shown
to inhibit the expression of NF-B-dependent gene expression (20). We observed a dose-dependent inhibition of the
expression of CD86 and MHC-II induced by rAd-Control at an MOI of 100 on DC (gate: CD11+, 7-ADD) (Fig. 6A). Maximal
inhibition by TLCK was observed at a concentration of 100 mM. These
data are consistent with a role for NF-B in rAd-induced DC
maturation. Since TLCK is a nonspecific inhibitor of trypsin-like
proteases, we sought to directly address the role of NF-B in
rAd-induced DC maturation by utilizing rAd engineered to express a
mutant IB protein that replaces serines 32 and 36 with alanine. The
mutant IB protein is not phosphorylated or degraded in response to
cytokines and, when overexpressed, functions as a dominant-negative
inhibitor of NF-B-dependent gene transcription (24).
Expression of CD86 and MHC-II was analyzed by flow cytometry 24 h
after rAd-IB infection. Inclusion of the mutant IB transgene prevented the increase in CD86 and MHC-II expression induced by rAd in
BM DC (Fig. 6B). These data clearly
demonstrate an important role for NF-B in the upregulation of CD86
and MHC-II in DC after rAd infection.
|
2 of negative cells
when isotype controls were used. The percentage of positive cells and
the MFC (in parentheses) are shown. Nontransduced DC (NT) and NT DC
treated with TLCK (TLCK) and cultured under similar conditions were
included as controls. (B) Transduction with rAd-IrAd increase the nuclear DNA binding activity of NF-B in BM
DC.
Since expression of the mutant IB transgene prevented the
upregulation of CD86 and MHC-II in DC after rAd infection, we sought to
address directly the possibility that rAd transduction could activate
nuclear NF-B DNA binding activity in DC by employing the EMSA.
Figure 7 shows EMSA results using nuclear
protein extracts from purified CD11c+ DC at various times
after rAd infection. Notice that there was an increase in the nuclear
NF-B-DNA binding complex as early as 1 h postinfection,
suggesting that rAd is a direct activator of NF-B signal
transduction. A more pronounced increase in NF-B DNA binding
activity was observed at 3 and 18 h postinfection. Importantly,
the increase in NF-B DNA binding activity observed at 3 and 18 h postinfection was blocked if the rAd encoded the dominant-negative
IB mutant transgene. Antibody supershift EMSAs indicated that the
p50 NF-B and, to a lesser extent, the p65 NF-B protein were the
predominant NF-B proteins activated by rAd infection in DC.
Antibodies to the p300/CBP protein were included as a negative control
and did not affect the NF-B DNA complex as expected. These data
demonstrate that rAd increased nuclear p50 and p65 NF-B DNA binding
as early as 1 h postinfection, which suggests that rAd is a
direct activator of NF-B in this system. Furthermore, expression of
the dominant-negative mutant IB protein interfered with the
rAd-induced nuclear NF-B DNA binding activity at 3 and 18 h
postinfection, while preventing the rAd-induced expression of CD86 and
MHC-II (Fig. 6B). These data provide strong evidence for the
involvement of NF-B in the regulation of CD86 and MHC-II gene
expression during the maturation of DC induced by rAd.
|
i.v injection of rAd preferentially infects splenic marginal zone
DC and induces mobilization of DC into T-cell areas.
DC in the
marginal zone of spleen resemble immature DC, whereas interdigitating
DC in the T-cell area (periarteriolar lymphoid sheath [PALS]) are
mature APC (23, 56, 63). It has been demonstrated in the
mouse that i.v. administration of either LPS or Toxoplasma
gondii antigen activates marginal DC and elicits their
mobilization into the PALS (14, 47). We used this in vivo
model to test whether rAd could trigger the maturation and trafficking
of marginal zone splenic DC. rAd-EGFP was administered via the tail
vein (1.5 × 109 PFU/mouse), and the distribution of
DC was analyzed in spleen sections 6, 24, and 48 h after virus
injection. As soon as 6 h after rAd administration, EGFP
expression was detected exclusively in the marginal zone (Fig. 8A to
C). Staining with a PE-anti-CD11c MAb
confirmed that marginal zone DC were targets of rAd infection (Fig.
8C). CD11c EGFP+ cells were also detected. At
24 and 48 h following rAd-EGFP administration, CD11c+
EGFP+ DC were detected in the PALS (Fig. 8D), an
observation suggesting that rAd initially infected marginal zone DC
that later migrated to the PALS. However, this approach allowed us to
trace only the movement of those DC that expressed detectable levels of
intact EGFP and that may have corresponded to a subset of the entire population of DC influenced by the viral effect. To analyze the mobilization of the entire splenic DC population into T-cell areas, animals were injected i.v. with rAd without reporter gene
(rAd-Control/1.5 × 109 PFU/mouse), and tissue
sections from different time points were double labeled with PE
anti-CD11c MAb for DC and FITC anti-CD3 MAb for T cells. After
administration of PBS or in noninjected animals, CD11c+ DC
were clearly distributed in the marginal zone and intermingled with T
cells in PALS (Fig. 9A). At 6 and 24 h after virus administration, the number of marginal zone DC decreased,
and the population of DC in the PALS increased substantially (Fig. 9B
and C). After 48 h, the marginal zones repopulated with
CD11c+ DC (Fig. 9D).
|
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
rAd is one of the most effective viral vehicles for delivery of exogenous DNA to a variety of nonreplicating cells, included DC (1, 15, 16, 25, 29, 71). However, the molecular basis of its strong immunogenicity is not well understood (65-67). Information concerning the effect(s) of rAd on DC, the APC that will determine the outcome of the primary immune response, is still contradictory (25, 27, 29, 46, 59, 71). In this study, we demonstrated that BM DC infected with rAd encoding distinct transgenes upregulated the expression of MHC antigens and costimulatory (CD40, CD80, and CD86) and adhesion (CD54) molecules. As a result, rAd-infected DC became potent stimulators of allogeneic T cells. These rAd effects on DC correlated with the viral input and were even detected at MOIs ranging from 10 to 100, viral doses commonly used for ex vivo DC transduction in gene therapy protocols. Our results agree with those reported recently by Rea et al. (46) using human DC but contrast with those of Zhong et al. (71) and Tillman et al. (59), who concluded that human DC maturation was not a function of rAd infection. As pointed out by Rea et al., in the system used by Zhong et al, immature DC already expressed the maturation marker CD83, a fact that makes it difficult to discriminate between the spontaneous maturation of DC and that induced by rAd (46, 71). The low efficiency of transduction of human DC reported by Tillman et al. after rAd-EGFP infection at an MOI of 100 (<20% compared with ~80% [Fig. 1D] in the present work) may have diluted the effect of the Ad on the DC population (59). Differences in species (mouse versus human DC) may also account for the discrepancies.
We demonstrated that rAd infection increased the transcript levels of
IL-6, IL-12p40, IL-15, IFN-, and TNF- genes. These data confirm
and extend the recent report by Rea et al. (46), in which
they showed that rAd increased the stimulatory capacity of human DC but
without inducing polarization into Th1-driving APC. Production of the
IL-12p40/35 heterodimer by DC is the key signal during the early steps
of antigen presentation that drives differentiation of naive Th0 cells
into Th1 lymphocytes (60). The fact that, in our system, rAd
increased exclusively the transcription of IL-12p40 mRNA, without
affecting basal levels of IL-12p35 transcripts, allows us to explain at
a molecular level, the lack of Th1 polarization of rAd-infected DC
reported previously (46).
Different pieces of evidence prompted us to investigate whether
activation of the transcriptional factor NF-B was involved in
rAd-induced DC maturation: (i) components of hCMV, Epstein-Barr virus,
herpes simplex virus, and human T-cell leukemia virus type 1 can
activate NF-B (2, 40); (ii) specific NF-B DNA binding sequences (B motifs) are known to be present in the promoters of
several of the genes that are upregulated by DC after rAd infection (2, 38, 51, 69, 70); and (iii) the NF-B pathway is involved in the development and activation of myeloid DC (9, 10,
19, 43, 45, 48, 64). Our data indicate that there was rapid
NF-B activation in nuclei of DC within 1 h of rAd infection, probably as a consequence of virus- cell receptor interaction and/or
internalization. The early NF-B activation increased during the
following 18 h. The late NF-B activation suggests that viral gene transcription might have been responsible for NF-B nuclear translocation, an unlikely mechanism based on the fact that both UV-irradiated and nonirradiated rAd exerted a similar effect on the BM
DC. Another, more plausible explanation may be that, as a consequence
of the early (within 1 h) NF-B nuclear translocation, DC
upregulated the transcription of certain proinflammatory cytokines (i.e., TNF-) that, in turn, may have induced a second phase of NF-B activation (32).
There is evidence that rAd can activate NF-B in other cell
types, such as human vascular smooth muscle cells (11) and mouse hepatocytes (33). The mechanism(s) employed by rAd to activate NF-B
is unknown. In our experiments the effect did not depend on viral
transcription. Consistent with these results, McCoy et al.
(39) demonstrated that in vivo administration of
UV-inactivated or incomplete Ad particles induced a strong inflammatory
response in the respiratory tract. Borgland et al. reported recently
that the chemokine induction occurring within 24 h of rAd
infection in mice is mediated through capsid-dependent activation of
NF-B (7, 42). Taken together, these observations suggest
that some of the viral components (DNA or proteins) are responsible for
NF-B activation.
In order for rAd to be adsorbed and then internalized by the host cell,
the fiber knob must interact with the coxsackievirus and Ad receptor
(CAR) and the penton base with v3 or
v5 integrins (5, 62). The
M2 integrin has been reported as the
receptor for the penton protein of Ad2 in human hematopoietic cells
(21). DC do not express CAR but do exhibit moderate levels
of v3, v5,
and M2 integrins (46, 59).
Both immature and mature DC are infectable by rAd in humans and mice
(46, 59). It is likely that interaction between the Ad fiber
and the integrins expressed on the DC surface may activate the NF-B
pathway (50, 68). It has been reported that the
v5-dependent cell migration in carcinoma
cell lines and the v3-induced survival in
endothelial cells are phenomena mediated though activation of NF-B
(50, 68). Penton capsid binding to cell surface integrins
induces an enzymatic cascade that phosphorylates the mitogen-activated protein kinase and the phosphoinositide-3-OH kinase (8,
31). Whether these kinases, when activated after rAd
infection, are involved in the phosphorylation and subsequent
degradation of IB in DC is still unknown. Disruption of endosomes by
internalized Ad particles, with the subsequent release of proteolytic
enzymes and viral DNA into the cytosol, has been also proposed as a
possible mechanism of NF-B activation (11). Unmethylated CpG
bacterial DNA sequences have been demonstrated to activate NF-B and
trigger maturation of DC (54). However, in the present work,
the induction of DC differentiation by CpG sequences present in
transgenic DNA of rAd seems unlikely, since rAd encoding no transgene,
or vectors encoding different transgenic cDNAs lacking CpG
motifs, produced the same effect in DC.
We demonstrated that, within 6 h of systemic administration of
rAd-EGFP, there was exclusive expression of the transgene in the
splenic marginal zone, a region populated mainly by immature DC, B
cells, and specialized macrophages (23, 56, 63). A considerable proportion of EGFP+ cells in the marginal zone
were also positive for the DC marker CD11c. Immature DC of the marginal
zone are able to differentiate and migrate to the PALS after i.v.
administration of LPS or soluble T. gondii antigen
(14,47). Using a similar in vivo model, we demonstrated
that, by 6 and 24 h after rAd i.v. injection, splenic DC rapidly
mobilized to the T-cell-dependent areas, a phenomenon accompanied by a
proportional decrease in the DC number in the marginal zone.
Interestingly, not all mobilized DC expressed the transgene. This
observation suggests that rAds might induce DC differentiation by
different mechanisms: first, and as we demonstrated here in vitro, by
direct stimulation of infected DC through NF-B activation; and
second, by induction of proinflammatory cytokines in DC or other
neighboring cells that, in turn, trigger the maturation of
nontransduced DC.
In conclusion, the data presented herein strongly suggest that rAd
accelerates the differentiation of immature mouse DC, a phenomenon that
is linked to activation of the transcription factor NF-B. The
enhancement of the stimulatory ability of DC induced by rAd may be the
basis of the strong immune response elicited against rAd. It may
explain the success of therapies based on rAd-transduced DC against
neoplasms and infectious agents. However, the rAd effect on DC can be
an obstacle when rAd-transduced DC are required to promote tolerance to
treat autoimmune disease or to avoid graft rejection. The fast NF-B
activation induced by rAd in different cell types might explain the
systemic inflammatory response and the activation of innate immunity
reported recently as side effects of rAd in human clinical trials
(36).
| |
ACKNOWLEDGMENTS |
|---|
We thank Nancy Zurowski and Bridget Colvin for skillful
assistance with cell culture techniques, A. Shaked (Department of Surgery, University of Pennsylvania) for the rAd Add1434, D. A. Brenner (University of North Carolina) and A. Gambotto (University of
Pittsburgh) for the rAd-IB, and Cipriano Almonte and Simon Watkins
for image processing. We thank Schering-Plough Research Institute,
Kenilworth, N.J., for gifts of cytokines.
This study was supported by National Institutes of Health grants DK 49745 and AI 41011 (to A.W.T.) and by an NRSA grant (F32-GM-19877) from the National Institutes of Health (to R.W.G.).
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Surgery, University of Pittsburgh Medical Center, E1504 Biomedical Science Tower, 200 Lothrop St., Pittsburgh, PA 15213. Phone: (412) 624-6627. Fax: (412) 624-1172. E-mail: morelli{at}imap.pitt.edu.
| |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Virology, October 2000, p. 9617-9628, Vol. 74, No. 20
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