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IMMUNOBIOLOGY
From the Regulation of Cell Growth Laboratory, the
Laboratory of Experimental Immunology, and the Mouse Cancer Genetics
Program, National Cancer Institute at Frederick, MD, and the Center for
Surgery Research, Cleveland Clinic Foundation, OH.
To determine whether infection by a model virus is capable of
initiating dendritic cell (DC) differentiation, human CD14+
peripheral blood monocytes were infected with replication-defective type 5 adenovirus. Under serum-free conditions, this resulted in differentiation of a majority of cells toward a DC phenotype within
36 to 48 hours, without the need for cytokine-induced
predifferentiation. Infection induced DC morphology and altered the
expression of surface markers, including loss of CD14, de novo
induction of CD83 and CD25, and strongly augmented expression of CD86,
CD80, CD40, and HLA-DR and HLA class I molecules.
Differentiated cells maintained immunophenotype without loss of
viability for at least 2 days after removal of the differentiation
agent and cytokines. A greatly enhanced capacity to stimulate
T-lymphocyte alloproliferation and increased expression of the
DC-associated transcription factor RelB were observed. Virus without
transgene was found to induce changes similar to transgene-expressing
viruses. RelB up-regulation and DC immunophenotype were sensitive to
the antioxidant N-acetylcysteine, suggesting a critical
role for nuclear factor Dendritic cells (DCs) are potent antigen-presenting
cells (APCs) that sensitize T lymphocytes to alloantigens and nominal antigens.1,2 Because of their role in priming T-cell
responses, DCs are a focus of efforts to develop immunotherapeutic
strategies aimed at treating malignancies and chronic infections. DCs
can be derived in vitro from CD14+ monocytes with the use
of granulocyte-macrophage colony-stimulating factor (GM-CSF) and
interleukin-4 (IL-4).3 Cells so treated down-regulate CD14
expression and differentiate into immature DCs. If tumor necrosis
factor- Because of our previous success in inducing rapid DC
differentiation under serum-free conditions using LPS, a compound
likely to signal Gram-negative bacterial infection in vivo, we have now turned our attention to other classes of pathogens, in this case a
modified type 5 adenovirus. As a group, adenoviruses have a relatively broad tissue tropism, and replication-defective deletion mutants were initially considered to have a relatively low pathogenic potential.10 Because of these perceived features,
recombinant, replication-defective adenoviruses have become workhorses
for the introduction of transgenes into a variety of cultured cell types for investigational purposes, as well as vehicles for recombinant vaccines and, perhaps most importantly, a delivery system for transgenes in human gene therapy settings.
Adenovirus has been previously shown to infect CD14+
monocytes cultured in GM-CSF11 as well as immature
monocyte-derived DCs.12 It has been reported that
adenovirus infection of immature DCs under tested conditions was
completely nonperturbing: adenovirus infection did not cause any
apparent activation or further maturation of DCs.13-15
However, work by others suggests that adenovirus infection of immature
DCs can promote varying degrees of activation.16-18
In the studies reported here, we demonstrate that under serum-free
conditions, replication-defective type 5 adenovirus initiates the rapid
differentiation of CD83+ DCs from
CD83 Replication-defective recombinant adenoviruses
Monoclonal antibody and fluorescence-activated cell
sorting analysis
Human peripheral blood monocyte and allogenic mixed lymphocyte reaction cultures Human CD14+ peripheral blood monocytes (92% to 94% purity) were obtained from healthy donors by leukapheresis and countercurrent centrifugal elutriation according to National Institutes of Health guidelines for human subjects and were either cultured immediately or cryopreserved as described previously.22 Lymphocyte-rich fractions were also collected and cryopreserved. Monocytes were plated at a density of 2.5 × 106 cells per well in 24-well tissue-culture plates (Costar, Corning, NY) in 2 mL per well macrophage serum-free medium (M -SFM) (Life Technologies,
Gaithersburg, MD) supplemented with 50 ng/mL recombinant human GM-CSF
(Immunex, Seattle, WA), which is necessary for maintaining cell
viability, as described previously.21 This combination of
M-SFM and GM-CSF, which constitutes basal culture medium for all
monocytes and DCs used in these studies, is henceforth referred to in
the text simply as SFM/G. Where indicated, cultures were supplemented
with additional agents prior to the addition of adenovirus or other
differentiation-inducing stimuli. These agents included N-acetylcysteine (NAC) (Sigma, St Louis, MO), a
TNF- -neutralizing mAb 5N,23 or polymyxin B
(Sigma). For allosensitization studies, T cells, purified from
lymphocyte-rich elutriation fractions by means of T-cell isolation
columns (R&D Systems, Minneapolis, MN), were plated in 96-well plates
in RPMI medium as described previously.8 Gamma-irradiated
(20 Gy) monocytes or monocyte-derived DCs were added at varying T
cell-to-APC ratios and cocultured for 96 hours at 37°C in 5%
CO2. Cells were then pulsed with 1 µCi (.038 MBq) per well [3H]-thymidine
([3H]-TdR) (3.2 TBq/mmol) (Amersham
Pharmacia Biotech, Arlington Heights, IL) and harvested 18 hours later.
The [3H]-TdR incorporation was assessed by liquid
scintillation spectroscopy.
Induction of DC differentiation with LPS or replication-defective adenovirus Monocytes cultured overnight in SFM/G were treated with 50 ng/mL Escherichia coli O26:B6 LPS (Sigma) for 3 additional days, a treatment previously shown to induce rapid DC differentiation in about half of the cells.8 For viral infections of freshly purified monocytes, cells were plated in 200 µL SFM/G. For monocytes cultured in 2 mL SFM/G for 24 to 48 hours prior to infection, supernatants were carefully removed until the culture volume was 200 µL. Adenovirus suspensions were added to the cells and gently mixed. Infection was allowed to proceed for 2 hours at 37°C; then, fresh SFM/G was added to bring the cultures to 2 mL per well. Infected cells were typically cultured an additional 36 to 48 hours to achieve peak adenovirus-induced differentiation. Since maximal adenovirus-induced differentiation could usually be completed 1 day earlier than with LPS treatment, this staggered introduction of differentiating agents synchronized the development of DCs after 4 total days of culture, the time when most immunophenotypical, morphological, molecular, and functional properties were evaluated.Microscopy Monocytes treated with LPS, infected with adenovirus, or left untreated, as described above, were harvested by careful removal of medium, gentle washing of plates with Ca2+ Mg2+-free phosphate-buffered saline, and 20 minutes' incubation with enzyme-free dissociation buffer (Life Sciences, Gaithersburg, MD) to remove residual adherent cells. Cytospin preparations were made onto glass slides, stained with Wright solution, and subjected to light photomicroscopy, as described previously.8RNAse protection assay The multiprobe RNAse protection assay (Pharmingen, San Diego, CA) was performed according to the manufacturer's directions, with the following modifications:Hybridization. 33P-uridine 5' triphosphate (70 to 80 µCi [2.6 to 3 MBq] per reaction) was used to synthesize the probe, and 0.5 to 1.0 × 106 cpm was added to each hybridization reaction. After synthesis and addition of yeast transfer RNA (tRNA) and EDTA (final volume, 50 µL), the reaction mixture was placed on a G25 microspin column (Amersham Pharmacia Biotech), and the probe was purified by centrifugation for 2 minutes at 3000 rpm in a microcentrifuge. RNAse inactivation.
A master cocktail was prepared containing (per RNA sample) 200 µL
RNAse inactivation reagent (Ambion, Austin, TX), 50 µL ethanol, 5 µg yeast tRNA, and 1 µL Ambion GycoBlue coprecipitate. Then, 250 µL aliquots of this cocktail were pipetted into 1.5-mL
microcentrifuge tubes, and the individual RNAse-treated samples were
added to each tube. After mixing, the samples were kept at
Preparation of whole-cell extracts and immunoblotting Cells were pelleted and lysed in a buffer containing 10 mM Tris (pH 7.4), 220 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 5 µM ZnCl2, 1% Triton X-100, and protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN) supplemented with additional protease inhibitor2-macroglobulin (1.25 U/mL) (Roche Molecular Biochemicals). The extract was incubated on ice
for 10 minutes and centrifuged at 15 000g for 15 minutes at
4°C; the supernatant constituted the whole-cell extract. Protein
concentrations were measured by using the Bio-Rad (Hercules, CA)
protein assay with the use of a bovine serum albumin standard. Then, 5 to 10 µg protein were resolved by 10% Tricine sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (Invitrogen, Carlsbad, CA)
and transferred to Immobilon-P (Millipore, Bedford, MA). The membrane
was probed with anti-RelB antiserum 1319 and antiactin mAb (Chemicon,
Temecula, CA).8 Immunoreactive proteins were
revealed with an enhanced chemiluminescent system (Amersham
Pharmacia Biotech).
Cytokine and chemokine quantitation Enzyme-linked immunosorbent assay kits (R&D Systems) were used to quantify several cytokines and chemokines in 24- to 48-hour culture supernatants of adenovirus-transduced monocytes. Detection limits were as follows: for IL-6, IL-1, and IL-1 , 8 pg/mL; RANTES and
TNF-, 16 pg/mL; for IL-10, 47 pg/mL; for IL-12 p40/70, 15 pg/mL; and
for macrophage inflammatory protein-1 (MIP-1) and MIP-1, 62 pg/mL. Assays were performed by the Lymphokine Testing Laboratory,
SAIC, National Cancer Institute at Frederick, MD.
Infection of human CD14+ monocytes with adenoviral vectors under serum-free conditions results in the rapid acquisition of DC immunophenotype Starting populations of human monocytes were 92% to 94% CD33+, CD11c+, CD14+, and negative for the DC activation marker, CD83 (Figure 1A-B). As reported previously,8,21 these cells were negative for the costimulatory molecule CD80 and low/negative for CD40, but showed moderate levels of CD86, HLA-DR, and HLA class I antigens (Figure 1B).
Dose-response studies indicated that maximum infectivity with adenovirus-GFP (as assessed by transgene expression), coupled with maximal induction of DC phenotype (without significant loss of viability), was achieved at an MOI of 100 to 200 (data not shown). These results are consistent with reports of others using immature DCs,13,16-18 and an MOI of 200 was therefore used in all our subsequent experiments. Infection with a GFP-encoding adenovirus in this dose range for 48 to
96 hours resulted in dramatic changes in surface immunophenotype consistent with differentiation of monocytes into DCs. Infected cells
decreased CD14 expression while strongly up-regulating surface CD80,
CD86, CD40, and HLA-DR. HLA class I levels rose slightly above those
seen in uncultured cells (Figure 1B). Markers of activated DCs, CD83
(Figure 1B) and CD25 (Figure 1C), were also highly induced in
adenovirus-infected cultures. Similar results were obtained regardless
of whether monocytes were cultured in SFM/G for only 2 hours or for 1 to 2 days prior to infection. It is clear that the observed
differentiation, seen in 20 separate experiments with 14 different
donors, was triggered by adenovirus and not by the GM-CSF-containing
SFM/G. When monocytes were cultured for 96 hours in SFM/G in the
absence of virus, most cells increased expression of CD80 and CD40, but
they showed decreased expression of CD86 and HLA class I. Most
importantly, they remained CD14+ and CD83 One characteristic of fully differentiated cells is the ability to maintain phenotype after removal of the differentiating agent(s) and cytokines. To test the adenovirus-infected cells, they were washed following 48 hours exposure to virus and were cultured for an additional 48 hours in SFM (without GM-CSF). No change in immunophenotype and no loss of viability were observed in the differentiated cells (Figure 1B). We also found that adenovirus-induced differentiation is not
attributable to the expression of a transgene: virus without transgene
induced profound alterations in immunophenotype similar to those seen
with adenovirus-GFP and adenovirus-Luc (Figures 2 and 7). The possibility that endotoxin
contamination makes a major contribution to adenovirus-mediated DC
differentiation has been minimized, since heat inactivation of virus
(which does not affect LPS activity) strongly inhibited
adenovirus-induced DC immunophenotype (Figure 2). In addition,
polymyxin B, an inactivator of LPS, did not significantly affect
adenovirus-induced DC differentiation (Figure 2).
Since in previous studies8 we showed that rapid DC
differentiation induced by LPS or TNF- Infection with adenovirus in SFM/G had no major effect on cell viability. In most experiments, viability ranged from 80% to 90%, whether measured at 48, 72, or 96 hours after infection. In summary, infection of human monocytes with adenovirus vectors under serum-free conditions results in the rapid (48-hour) and virtually complete differentiation of the cells into DCs, without need for cytokine-induced predifferentiation. Adenovirus-infected monocytes acquire DC morphology Virus infection also dramatically affected the appearance and behavior of the cells in culture. Cells cultured in SFM/G were moderately adherent in culture, with little tendency to aggregate. In contrast, adenovirus-infected cells formed large, loosely adherent clusters (Figure 3), which are typical of DCs matured in vitro.5,24 Judging from expression of GFP as determined by FACS, at least 70% of the monocytes were virus infected (Figure 3). GFP expression was also apparent by Western blot analysis (data not shown) and fluorescent microscopy. The FACS-based comparison of GFP expression with CD83 expression shows that even the cells that expressed little or no GFP had elevated CD83 expression. Since there are many more noninfectious than infectious (ie, GFP-expressing) particles in the virus preparation (as determined on the 293 adenovirus propagation line), the result may mean that viral infectivity is not required to trigger differentiation of the cells. Alternatively, the result may indicate that the differentiating effects of infectious adenovirus may not be direct. Instead, the effects may stem from the induced secretion of a soluble factor(s), a point to which we will return later.
When fixed and stained cells were microscopically examined at higher
magnifications, morphological changes to individual cells induced by
adenovirus infection became apparent. Cells from adenovirus-infected cultures displayed elaborate cellular processes consistent with DCs
(Figure 4B), very similar to what was
seen when LPS was used to drive differentiation in parallel (Figure
4C). In contrast, cells cultured for the same length of time in SFM/G
displayed the unremarkable morphology of monocytes (Figure 4A),
demonstrating that culture without adenovirus infection did not cause
any apparent differentiation of cells.
Virus-infected cells acquire enhanced allostimulatory activity We tested the ability of virus-infected cells to stimulate T lymphocytes at low APC numbers in the allogenic mixed lymphocyte reaction (MLR), since DCs have been shown to be extremely efficient APCs in this assay. As expected, uncultured monocytes had a very poor allostimulatory capacity (Figure 5). As reported previously,8 monocytes cultured in SFM/G displayed a somewhat greater allosensitizing capacity, probably owing to the culture-enhanced levels of CD80 and CD40. Stimulation of proliferation by these cells, however, was reduced to nearly background levels at APC-to-T-cell ratios of 1:15 625. In contrast, adenovirus-GFP-infected monocytes were highly allostimulatory compared with other groups, with the capacity to induce considerable proliferation of allogenic T cells even at a 1:15 625 (stimulator-to-responder) ratio, which corresponds to a mere 13 cells added per well (Figure 5). Cells differentiated with adenovirus containing no transgene showed similar, albeit slightly lower, APC capacity (not shown). DCs differentiated with LPS also showed enhanced capacity to induce allogenic T-cell proliferation, but appeared generally inferior to adenovirus-derived cells. As expected, heat-inactivated adenovirus showed a much inhibited capacity to enhance MLR activity of cultured cells (Figure 5), consistent with the generally modest immunophenotypical modulations (Figure 2). We conclude that infection of normal CD14+ monocytes with adenovirus vectors yields cells with enhanced allostimulatory capacity even when very small numbers of APCs are added to T-cell coculture, a functional capacity characteristic of DCs.
Adenovirus infection induced expression of RelB The nuclear factor (NF)- B family member RelB is associated
with, and essential for, normal development of myeloid
DCs.25-30 To test whether infection of monocytes with
adenovirus vectors resulted in enhanced RelB expression, immunoblots
from lysates of infected cells were probed with an antibody specific
for RelB (Figure 6A). Very little RelB
was detected in control cells cultured in SFM/G (lane 1), but the
protein was highly induced in both LPS- and adenovirus-treated cells
(lanes 2-4). Thus, adenovirus infection causes the induction of the
transcription factor RelB, which is associated with DC
maturation.
Cytokine and chemokine profiles of adenovirus-infected cells DCs synthesize a large number of cytokines and chemokines that exert profound autocrine and paracrine effects. To test whether monocytes infected with adenovirus vectors resemble DCs in their pattern of cytokine/chemokine expression, we performed RNAse protection assays, using total RNA isolated 24 hours after infection. The results showed that infection with adenovirus vectors was nearly as efficient as LPS treatment in inducing expression of cytokines and chemokines typical of DCs.31-33 Both adenovirus infection and LPS treatment induced high levels of messenger RNA (mRNA) encoding IL-1,
as well as appreciable increases of mRNA for IL-1, IL-6, IL-10, and
IL-12 p40 (Figure 6B). Adenovirus also induced low-level expression of
IL-12 p35 mRNA, which was not seen in LPS-treated cells. Control cells
cultured in SFM/G expressed none of these mRNAs at detectable levels.
We also monitored the secretion of several cytokine proteins. Whereas
LPS induced secretion of low to moderate levels of IL-10, IL-12, and
IL-1 Chemokine genes up-regulated by both virus and LPS include RANTES,
MIP-1
Adenovirus-induced differentiation is inhibited by NAC and by
neutralization of TNF- .38-41 We previously showed that much of the
DC-differentiating effect of LPS on monocytes was a result of induced
secretion of TNF-.8 Therefore, the possibility that
adenovirus vector-infected monocytes might produce TNF- was
investigated. We found that TNF- was readily detectable in culture
supernatants at 24 hours after infection, although at a much lower
level (> 10 × ) than seen in LPS-treated cultures.
We then investigated whether virus-induced differentiation of some or
all of the cells was dependent on the presence of TNF-
Our previous study showed that NAC, an antioxidant and a
nonspecific inhibitor of NF-
Our previous studies showed that human CD14+ monocytes
cultured in serum-free medium can rapidly differentiate into DCs upon treatment with TNF- Both TNF- Transcription factors belonging to the NF- Currently, published reports regarding the effect of adenovirus
infection on the maturation of DCs are somewhat conflicting. Some
studies indicate that adenovirus infection of immature DCs is
completely nonperturbing and does not initiate further activation or
maturation of DCs in the absence of other agents.13-15,50
In contrast, others have convincingly demonstrated maturation-inducing effects of adenovirus infection for both immature human and murine DCs.16-18 We clearly show that adenovirus infection under
serum-free conditions induces actual differentiation of
CD14+CD83 Our results have several important implications. First, our results show that serum-free culture can be used for simultaneous adenovirus-mediated gene transfer and rapid induction of differentiation of DCs from monocytes. This method speeds and simplifies the process of introducing target proteins to APCs for DC-based vaccine strategies and thus represents an appreciable advance. Second, the vigorous response of monocytes to replication-defective adenovirus offers an additional cautionary statement on the use of such vectors in gene therapy trials where the purpose is to install transgenes into human tissues while provoking minimal inflammatory responses. The proinflammatory cytokine/chemokine response to adenovirus that we have described in vitro is consistent with in vivo murine studies, reported by others,36,41,51 that were likewise independent of transgene expression and viral replication.36,51 The strong proinflammatory response induced by adenovirus vectors suggest that the in vivo response of monocytes (and presumably other cell types) to infection with adenovirus could have potentially negative consequences in human gene therapy settings, unless these qualities can be engineered out of the virus. Finally, these studies suggest that CD14+ monocytes have an
innate capacity to sense the presence of viruses in vitro and respond by rapidly differentiating into potent DCs. There is good reason to
suppose that this can also happen in vivo. Respiratory epithelial cells, when infected with a variety of viruses that cause respiratory tract infections, secrete not only GM-CSF, but also considerable amounts of RANTES and MIP-1 Several questions regarding the adenovirus-induced differentiation of
DCs from monocytes remain to be resolved and form the focus of our
ongoing studies. For instance, the precise mechanism by which monocytes
are signaled to the presence of adenovirus is not clear. Since the
virus we used is competent to enter human monocytes and transcribe the
inserted sequences but is defective for replication, and since
psoralen/UV- inactivation had no affect on adenovirus-mediated
differentiation,17 it is likely that the signal(s) is
delivered prior to the transcription of any virus-encoded products. It
is also not clear whether the absence of IL-12 secretion by the
apparently fully differentiated infected cells represents an
appropriate physiologic response directed at the generation of robust
Th2/antibody-mediated (ie, virus-neutralizing) immunity or is a viral
subversion of the immune response that limits Th1-type cell-mediated
immunity (which could enhance viral survival). Since RANTES and
MIP-1 The studies reported here clearly demonstrate that a model virus is capable of initiating actual and rapid differentiation of DCs directly from CD14+ monocytes, lending additional credibility to the notion that monocytes represent a bona fide precursor pool for DCs that can be quickly mobilized by infectious agents. The elucidation of the mechanisms by which this occurs and the interplay between virus and host that modulates the character of the developing immune response will be of critical value to our developing understanding of host-virus relationships.
We wish to express our gratitude to Drs Susan Leitman, E. J. Read, Harvey Klein, Thomas Trischmann, and Charles Carter of the Cell Processing Section of the Transfusion Medicine Branch at the National Cancer Institute, National Institutes of Health, Bethesda, MD, for their generosity and technical support.
Submitted May 4, 2001; accepted September 20, 2001.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Nancy R. Rice, National Cancer Institute at Frederick, PO Box B, Frederick, MD, 21702-1201; e-mail: ricen{at}ncifcrf.gov.
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Top
Abstract
Introduction
CD14+ monocytes. The present studies
differ from previous work because adenovirus is shown to act very
rapidly upon native CD14+ monocytes, rather than upon
immature, CD14
70°C for 30 minutes, followed by centrifugation for 15 minutes at
14 000 rpm at room temperature. The supernatants were poured off; a
sterile cotton swab was used to remove excess liquid from the tube; and the pellet was resuspended in 3 µL Pharmingen sample buffer.
) indicates uncultured
monocytes; (
), monocytes cultured 72 hours in SFM/G; (
),
monocytes cultured overnight in SFM/G, infected with adenovirus-GFP,
and incubated for 48 hours; (
), monocytes that received similar
treatment with heat-inactivated (HI) adenovirus-GFP; (
), monocytes
that were cultured overnight in SFM/G and treated with LPS for 48 hours.
