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, and Calcium Ionophore Under Serum-Free Conditions Promote Rapid Dendritic Cell-Like Differentiation in CD14+ Monocytes Through Distinct Pathways That Activate NF-
B
*
Division of Basic Sciences, Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, MD 21702;
Medicine Branch, National Cancer Institute, Bethesda, MD 20814; and
The Center for Surgery Research, Cleveland Clinic Foundation, Cleveland, OH 44195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,
or calcium ionophore (CI). Within 48–96 h, depending on the inducing
agent, the cells acquire many immunophenotypical, morphological,
functional, and molecular properties of DC. However, there are
significant differences in the signaling pathways used by these agents,
because 1) LPS-induced, but not CI-induced, DC differentiation required
TNF- production; and 2) cyclosporin A inhibited differentiation
induced by CI, but not that induced by LPS. Nevertheless, all three
inducing agents activated members of the NF-
B family of
transcription factors, including RelB, suggesting that despite
differences in upstream elements, the signaling pathways all involve
NF-B. In this report we also demonstrate and offer an explanation
for two observed forms of the RelB protein and show that RelB can be
induced in myeloid cells, either directly or indirectly, through a
calcium-dependent and cyclosporin A-sensitive
pathway. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(4). Upon
receiving such signals, DC enter a terminal differentiation/activation
program, during which they up-regulate activation, MHC, and T cell
costimulatory molecules and quickly leave peripheral tissues,
presumably taking with them an antigenic sample of their local
environment. DC then enter the afferent lymphatics and migrate to the T
cell-dependent areas of the draining lymph nodes. Here they form
associations with Ag-specific T lymphocytes, thereby initiating T
cell-dependent immune responses. Transcription factors of the
Rel/NF-B family, including p50, p52, and RelB, are expressed by and
play an important role in the biology of DCs (5).
DCs derived from CD14+ monocytes are often
obtained in vitro first by culturing these cells in FCS- or human
serum-containing medium supplemented with a combination of GM-CSF and
IL-4 (6). Monocytes cultured under these conditions for 1
wk or more down-regulate surface CD14 and enter a developmental state
comparable to that of immature, tissue-resident DC. To promote the
attainment of full DC maturity, additional agents, such as bacterial
LPS, TNF-, and monocyte-conditioned medium, have been used
successfully (7, 8, 9). We have recently demonstrated that
Ca2+-mobilizing agents can also induce the
differentiation of DC from CD14+ monocytes
(10). This Ca2+-dependent
differentiation process is extremely rapid (1–2 days) and is sensitive
to calcineurin antagonists, including cyclosporin A (CsA)
(11).
Our primary interest is elucidation of the signaling pathways involved in the differentiation of DC from CD14+ precursors. Such studies would be facilitated if the differentiation period of DC could be cut from 1–2 wk to only a few days. Another inducement for the discovery of rapid DC culture methods is the potential for use of DC in immunotherapy. Cell-based therapies aimed at treating malignancies or infections demand that time in culture be kept to a practical minimum. In fact, ideal culture systems would be not only rapid, but free of animal or human serum, because issues of safety and consistency are paramount. For these reasons, we have developed a compositionally simplified serum-free culture system that can rapidly (4 days or less) yield mature DC from human CD14+ monocytes using a variety of agents to drive differentiation.
In the studies presented here we show that under serum-free conditions,
LPS, TNF-, and calcium ionophore (CI) A23187 are each capable of
inducing rapid DC differentiation in human monocytes. Most
interestingly, we show that CI treatment leads to the induction of
NF-B proteins, particularly RelB, in myeloid cells. Despite the
Ca2+ dependence and CsA sensitivity of CI-induced
differentiation, we found no evidence of NF-AT involvement in this
pathway. Although LPS, TNF-, and A23187 all induced DC
differentiation, there must be significant differences in the signaling
pathways used, because 1) TNF- production is required for
LPS-induced, but not for A23187-induced, differentiation; and 2) CsA
blocks differentiation induced by A23187, but not that induced by LPS.
However, in all cases expression of the DC activation marker CD83 was
associated with enhanced levels of nuclear NF-B. Thus, despite
upstream differences, the signaling pathways that induce DC
differentiation each activate NF-B.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The methods employed and the mAb specific for human CD80, CD86, CD14, CD83, CD40, and HLA-DR as well as isotype-matched control mAbs were identical with those used in previously published studies (11). A gate (R1) was employed in all FACS analysis (except FACS separation experiments) to include all viable cells based on lack of propidium iodide staining. The instrumentation employed in flow cytometry studies was a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA) running CellQuest analysis software. For separation of CD14+CD83- from CD14-CD83+ cells, LPS-treated monocytes were double stained with FITC-anti-CD14 and PE-anti-CD83, washed, and resuspended in PBS with 2% FCS at 5 x 106 cells/ml. Cells were sorted using a Becton Dickinson FACSvantage SE high speed fluorescence-activated cell sorter. The resulting populations were confirmed by back-analysis to be >98% pure.
Culture of human peripheral blood monocytes, T cells, and cell lines
Human CD14+ peripheral blood monocytes
(92–95% purity) were obtained from 16 healthy donors by leukapheresis
and elutriation according to National Institutes of Health guidelines
for human subjects, and either cultured immediately or cryopreserved as
described previously (10). Lymphocyte-rich fractions were
also collected, and T cells for allosensitization studies were purified
using T cell isolation columns (R&D Systems, Minneapolis, MN).
Monocytes were plated at a density of 2.5 x
106 cells/well in 24-well tissue culture plates
(Costar, Corning, NY) in 2 ml/well macrophage serum-free medium
(M
-SFM; Life Technologies, Gaithersburg, MD) supplemented with 50
ng/ml recombinant human GM-CSF (Immunex, Seattle, WA), as described
previously (11). Cells cultured overnight were induced to
differentiate by addition of varying doses of LPS derived from
Escherichia coli strain O26:B6 (Sigma, St. Louis, MO),
100–200 U/ml TNF- (PeproTech, Rocky Hill, NJ), or 188–225 ng/ml CI
A23187 (Sigma). Because DC differentiation was achieved more rapidly
with CI than with either LPS or TNF- (24 h vs up to 72 h), in
experiments aimed at immunophenotypical characterization, cell
morphology, and function, CI addition was delayed for 2 days so that at
the end of a 96-h total culture period all treatment groups would
be developmentally synchronized. Signaling pathway inhibitors included
CsA (Sandoz, Basel, Switzerland), N-acetyl cysteine (NAC;
Sigma), and a TNF--neutralizing mAb 5N (12). The human
promyelocytic leukemia line, HL-60 (CCL 240), was obtained from
American Type Culture Collection (Manassas, VA) and maintained in RPMI
medium with 10% FCS (HyClone, Logan, UT) as described previously
(13).
Measurement of TNF- in monocyte culture supernatants
Monocytes cultured overnight in M-SFM supplemented with
GM-CSF were treated with 50 ng/ml LPS or 188 ng/ml A23187. After
24 h, culture supernatants were removed and assayed by ELISA (R&D
Systems) for the presence of TNF- by the Lymphokine Testing
Laboratory, Frederick Cancer Research and Development Center, National
Cancer Institute (Frederick, MD). The limit of detection of this assay
is 15 pg/ml TNF-.
Antisera
Rabbit antisera that recognize human cRel (serum 265), human p50 (serum 1141), and NF-AT (serum 796) have been described previously (14, 15). RelB antisera were raised against the following peptides: 1319, NH2-CREAAFGGGLLSPGPEAT, located at the C terminus of human RelB; and 1393, NH2-LRSGPASGPSVPTGRC, located at the N terminus of human RelB (16). The cysteine residues were added to facilitate coupling of the peptides to hemocyanin.
Oligonucleotides
For the EMSA we used oligonucleotides containing binding sites
for NF-B (5'-AGT TGA GGG GAC TTT CCC AGG C-3'; Promega, Madison,
WI), octamer-1 (5'-TGT CGA ATG CAA ATC ACT AGA A-3'; Promega), and
NF-AT (5'-ATA AAA TTT TCC AAT GTA AA-3'). The double-stranded
oligonucleotides were labeled to high specific activity with
[
-32P]ATP (6000 Ci/mmol; Amersham Pharmacia
Biotech, Piscataway, NJ) and T4 polynucleotide kinase (Roche Molecular
Biochemicals, Indianapolis, IN).
Preparation of cellular extracts and EMSA
Cells were rinsed and resuspended in ice-cold hypotonic buffer
(25 mM Tris-HCl (pH 7.4), 1 mM MgCl2, 5 mM KCl,
and protease inhibitor mixture (Roche Molecular Biochemicals)) at a
concentration of 1–2 x 107 cells/ml,
incubated on ice for 15 min, and lysed with an equal volume of
hypotonic buffer containing 0.3% Nonidet P-40, additional protease
inhibitor 2-macroglobulin (2 U/ml; Roche
Molecular Biochemicals), and phosphatase inhibitors (1 mM sodium
vanadate and 1 µM okadaic acid). The lysate was centrifuged at
500 x g for 5 min; the supernatant constitutes the
cytoplasmic fraction. The pelleted nuclei were extracted with 30–50
µl of a solution containing 10 mM Tris-HCl (pH 7), 220 mM NaCl, 30 mM
sodium pyrophosphate, 50 mM NaF, 5 µM ZnCl2,
0.05% Nonidet P-40, and inhibitors at 4°C for 20 min with agitation.
The extract was centrifuged at 15,000 x g for 15 min
at 4°C; the supernatant constitutes the nuclear extract.
For EMSA, the binding reaction mixture was 10 mM HEPES (pH 7.5), 80 mM KCl, 1 mM EDTA, 1 mM EGTA, 6% glycerol, 0.5 µg of polydeoxyinosinic-deoxycytidylic acid (poly(dI-dC)), 0.5 µg of sonicated double-stranded salmon sperm DNA, 32P-labeled oligonucleotide (1 x 105 to 3 x 105 cpm), and nuclear extract (2–3 µg of protein) in a total volume of 20 µl. The mixture was incubated at room temperature for 15 min. For supershift analysis, the reaction mixture minus 32P-labeled DNA was preincubated for 15 min on ice with 1 µl of a 1:3 dilution of antiserum. The 32P-labeled oligonucleotide was then added, and the product was analyzed on 6% DNA retardation gels (Invitrogen/NOVEX, Carlsbad, CA).
Immunoprecipitation and immunoblotting
Extracts were incubated overnight with antiserum and protein A-Sepharose (Amersham Pharmacia Biotech) after dilution with TNT buffer (20 mM Tris-HCl (pH 7.5), 200 mM NaCl, and 1% Triton X-100). Washed precipitates were resolved by 10% Tricine SDS-PAGE (NOVEX) and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Immunoreactive proteins were revealed with an enhanced chemiluminescent system (Amersham Pharmacia Biotech).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and A23187 each rapidly
induce mature DC immunophenotype in a majority of CD14+
monocytes
We previously showed that A23187 induced rapid differentiation of
DC from monocytes under serum-free conditions (11) and
began these studies by testing the differentiating effects of LPS and
TNF- under identical conditions. As expected, uncultured, elutriated
monocytes almost uniformly expressed surface CD14, CD86, and HLA-DR,
but were largely low/negative for the expression of CD83, CD80, and
CD40 (Fig. 1
). To maintain the viability
of these cells in M-SFM, the addition of GM-CSF proved necessary.
Culture for 96 h under these conditions usually led to a loss of
surface CD86 and to a slight to moderate down-regulation of CD14
expression. Moderate up-regulation of CD80 and CD40 was also observed,
but there was little effect on HLA-DR expression, and no effect on the
expression of the DC activation marker CD83. Markedly different results
were obtained when Escherichia coli O26:B6 LPS at 50 ng/ml
was included for the final 72 h of culture (Fig. 1). Between 50
and 80% of the cells from all donors lost surface CD14, and these
cells showed marked up-regulation of CD83. Greatly enhanced expression
of CD80, CD86, CD40, and HLA-DR was also observed compared with that of
controls treated with GM-CSF only. Dose-response studies indicated that
monocytes cultured in M-SFM were very sensitive to LPS (data not
shown). Plateau responses were seen in the dose range of 10–100 ng/ml,
although a response was still evident at concentrations as low as 0.1
ng/ml LPS. In two-color analysis, it was clear that the
subpopulation of cells that became negative for the monocyte/macrophage
marker CD14 were always the highest expressers of CD80, HLA-DR, and the
DC activation marker CD83, consistent with this population’s identity
as DC (not shown).
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likewise displayed dramatic effects (Fig. 1
). In most donors,
TNF- caused a loss of CD14 expression that was much more uniform
than that induced by LPS. A majority of cells expressed CD83, and
marked up-regulation of CD80, CD86, CD40, and HLA-DR was also observed.
The effects of A23187 were the most rapid and complete. After only 24-h
treatment, there was near total down-regulation of CD14 and uniform
up-regulation of CD83 expression. As with LPS and TNF-, A23187 also
enhanced the expression of CD80, CD86, and HLA-DR, but the CD40
increase was sometimes slightly less than that seen with the other
treatments. Results from five separate experiments showed that
differentiation treatments did not cause an unacceptable loss of
viability over the 96-h culture period. Relative to undifferentiated
cells, viabilities were 99 ± 3% for LPS treatment, 79 ±
7% for TNF- treatment, and 74 ± 4% for CI treatment. Thus,
LPS, TNF-, and A23187 are each capable of inducing rapid conversion
of CD14+ peripheral blood monocytes into cells
with a DC-like immunophenotype under serum-free conditions. Human AB serum has an inhibitory effect on DC differentiation
Most methods using cytokines for deriving mature DC from
CD14+ monocytic precursors use serum-containing
medium and require 1–2 wk of culture. We tested whether the more rapid
kinetics we observed were due to some positive factor(s) supplied by
the M-SFM or to an inhibitory factor(s) found in serum. DC
differentiation was induced by TNF- or LPS in elutriated monocytes
as described above, except that some groups were cultured in M-SFM
supplemented with 5% human AB serum. The starting (uncultured)
population was 92% positive for surface CD14 and low/negative for CD83
(Fig. 2, population defined by R2), and
only about 1% of the cells had a
CD14-CD83+ mature DC
phenotype (defined by R3). Treatment with either LPS or TNF- in
M-SFM resulted in 71% of the cells converting to the
CD14-CD83+ phenotype.
However, inclusion of 5% serum resulted in decrease of the
CD14-CD83+ cells from 71
to 7% (LPS treatment) and from 71 to 1% (TNF- treatment).
Viability remained very high in all cultures, ranging from 79 to 91%
for the LPS- and TNF--treated samples. Similar inhibitory effects
were observed in three separate lots of human serum from two different
suppliers. Suppression of rapid differentiation was also seen when
FCS was used instead of human serum (data not shown). These
results suggest that under our experimental conditions the absence of
serum facilitates rapid DC differentiation induced by LPS or
TNF-.
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-, and A23187-treated monocytes acquire DC morphology
To examine the morphological changes induced by various
treatments, CD14+ peripheral blood monocytes were
cultured in M-SFM plus GM-CSF with or without LPS, TNF-, or
A23187 as described above. At the end of the 96-h culture period, cells
from all treatment groups were harvested, and cytospin preparations
were made onto glass slides and Wright stained. Dendritic morphology,
including the characteristic cellular processes, was virtually absent
on cells cultured in M-SFM supplemented only with GM-CSF (Fig. 3A). In contrast, such
processes were clearly apparent on a majority of cells treated with LPS
(Fig. 3B), TNF- (Fig. 3C), and A23187 (Fig. 3D).
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-, and A23187-treated monocytes acquire enhanced
allostimulatory capacity
We next tested uncultured and cultured cells for their capacity to
stimulate T lymphocytes in the allogenic MLR. Uncultured cryopreserved
monocytes (Fig. 4) demonstrated a
relatively poor capacity to stimulate T cells, with appreciable
proliferation only induced at APC:T cell ratios of 1:25 or less. In
contrast, cells cultured in M-SFM with GM-CSF alone exhibited
markedly enhanced allosensitizing capacity. Although these cells lack
many features characteristic of DCs, this enhanced capacity to
allosensitize T cells may rest on the demonstrated induction of
costimulatory molecules such as CD80 and CD40 (Fig. 1). Nevertheless,
the most efficient cells at stimulating T cell proliferation in the
allogenic MLR were those cells with activated DC characteristics: LPS-,
TNF--, and A23187-treated monocytes. As expected, combinations of
autologous T cells and APC resulted in almost no
[3H]thymidine incorporation. These results show
that in the absence of serum, human monocytes can acquire enhanced APC
function consistent with DC.
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neutralizing Ab inhibits acquisition of DC immunophenotype
in monocytes treated with TNF- or LPS, but not A23187
Having established that activated DC characteristics can be
rapidly induced in normal human monocytes under serum-free conditions,
we turned our attention to the signaling pathways involved in this
process. Because TNF- secretion is a characteristic response of
monocytes to LPS (17, 18), and because TNF- has a
differentiating effect on monocyte-derived DC (7, 9), we
asked whether LPS induces DC differentiation in monocytes wholly or in
part through the induced secretion of TNF-. We found that monocytes
cultured in M-SFM with GM-CSF and LPS produced high levels of
TNF- (typically on the order of 30–40 ng/ml in 24 h).
A23187-treated cells also produced TNF-, but at a level almost
200-fold lower (200 pg/ml) than that induced by LPS. We then asked
whether DC differentiation could be inhibited by TNF--neutralizing
Ab. We found that, as expected, the Ab completely inhibited the
acquisition of DC immunophenotype in cells treated with TNF- (Fig. 5). Up-regulation of CD83, CD80, and
HLA-DR was abrogated, as was down-regulation of CD14. When cells were
treated with LPS, the Ab also had a dampening effect. Although the
inhibition was not as complete as that with TNF-, a nonetheless
marked reversal of CD14 loss as well as suppressed up-regulation of
CD83, CD80, and HLA-DR were evident, suggesting an important
contribution from TNF- in the LPS-mediated conversion of monocytes
to activated DC under the conditions tested. In contrast, with
A23187-treated cells the TNF--neutralizing Ab had little or no
effect on CD14 loss or on CD83, CD80, and HLA-DR up-regulation. This
suggests that TNF- is not required for A23187-induced DC
differentiation, and that A23187 acts either downstream of TNF receptor
1 (TNFR1) in the TNF signaling pathway or through a different pathway
altogether.
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B through pathways differentially sensitive to CsA
Previous studies have implicated NF-B transcription factors in
the process of DC differentiation (19, 20, 21). To test
whether NF-B is activated under serum-free conditions, monocytes
were treated with LPS or A23187 for 24 h, and nuclear proteins
were analyzed by EMSA using an NF-B consensus oligonucleotide.
Extracts from cells cultured in M-SFM plus GM-CSF had a low level of
nuclear DNA-binding activity (Fig. 6A, lane 1). In
contrast, treatment of the cells with LPS (lane 2) or
A23187 (lane 5) for 24 h resulted in a high
level of nuclear NF-B DNA-binding activity, with LPS clearly the
more potent inducer of NF-B. TNF- treatment also resulted in
elevated NF-B DNA binding (data not shown).
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B DNA-binding activity (Fig. 6A, lane
7). However, after treatment of cells with A23187 for 24 h, a
high level of DNA-binding activity was detected in nuclear extracts
(lane 9). Thus, all treatments (LPS, TNF-, A23187)
that led to acquisition of DC phenotype in either monocytes or HL-60
cells also resulted in induction of nuclear NF-B DNA-binding
activity.
To gain insight into the mechanism of activation of NF-B by LPS and
A23187, we tested the effect of CsA, which inhibits the
calcium/calmodulin-dependent phosphatase calcineurin. We found that the
pretreatment of monocytes with CsA blocked A23187-induced NF-B DNA
binding activity (Fig. 6A), nuclear expression of cRel and
RelB proteins (Fig. 6B), and surface expression of the DC
activation marker, CD83 (Fig. 6C, compare lanes 5
and 6 in all panels). Similar results were obtained
with HL-60 cells (compare lanes 9 and 8 in all
panels). However, CsA had no detectable inhibitory effect on
LPS-induced differentiation (Fig. 6, compare lanes 2 and
4). In contrast, induction by LPS of nuclear NF-B and
surface expression of CD83 were efficiently blocked by NAC, a potent
inhibitor of NF-B activation (Fig. 6, lanes 2 and
3). (The effect of NAC on either A23187-treated monocytes or
HL-60 cells could not be assessed due to severe combined toxicity of
NAC and A23187.) Thus, in all cases the expression of CD83, a marker
for DC differentiation, was associated with the expression of nuclear
NF-B. These data are consistent with the hypothesis that NF-B
activity is closely associated with the differentiation process.
Furthermore, the differential sensitivity of LPS- and A23187-treated
cells to CsA offers strong evidence that there are differences in the
upstream signaling pathways activated by these inducers despite common
activation of NF-B proteins. Also of considerable interest is our
observation that RelB (Fig. 6B), which is known to be
associated with DC differentiation, is induced not only by LPS and
TNF- (not shown) but also by A23187, a
Ca2+-mobilizing agent.
The purified CD83+/CD14- population from
LPS-treated monocytes expresses high levels of nuclear NF-B,
including RelB
As shown above, LPS treatment of CD14+ human
monocytes leads to the loss of CD14 expression and the concomitant
expression of the DC activation marker CD83 in a sizable fraction, but
not all, of the cells. To determine whether nuclear NF-B is
associated preferentially with the DC-like population, LPS-treated
human monocytes were separated by FACS into
CD83+/CD14- and
CD83-/CD14+ subpopulations
(Fig. 7A), and nuclear
extracts were analyzed by EMSA using the NF-B consensus
oligonucleotide. Although both subpopulations of these cells had
demonstrable nuclear NF-B DNA-binding activity (Fig. 7B,
lanes 1 and 3), the
CD83+/CD14- fraction
showed by far the higher level (lane 3). The
specificity of DNA binding was confirmed by supershift analysis with
anti-p50 serum (lanes 2 and 4).
Nuclear extracts from the two subpopulations were also analyzed by
Western blot (Fig. 7D). As expected, nuclear RelB was
predominantly detected in the
CD83+/CD14- fraction
(compare lanes 1 and 2). DNA binding of the
ubiquitously expressed transcription factor OCT-1 was identical in the
two subpopulations, demonstrating the integrity of the nuclear extracts
(Fig. 7C). These studies show that the cells responding to
LPS under serum-free conditions by acquiring a DC characteristics also,
as a group, displayed a higher level of NF-B activation and nuclear
RelB expression compared with cells retaining the monocyte/macrophage
immunophenotype.
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As shown in Figs. 6B and 7D, anti-RelB
serum 1319 detected two proteins of very similar size in both human
monocytes and HL-60 cells. Inspection of the human RelB cDNA sequence
(16) revealed that the 5'-most in-frame ATG lies in a
suboptimal context for translation initiation (22), while
the second in-frame ATG is located in a better context. If translation
can begin at either ATG, two forms of the protein are predicted,
differing in size by 17 aa. Antiserum 1319, which was raised against a
peptide at the C terminus of the protein, would detect both forms. To
test the prediction, we used anti-RelB serum 1393, raised against a
15-residue peptide starting at the first ATG. This serum would be
expected to detect only the longer of the two proteins.
We tested antisera 1319 and 1393 on lysates of cells transiently
transfected with human RelB as well as on lysates of LPS-treated human
monocytes. In both cases immunoprecipitation with serum 1319 resulted
in two bands, while precipitation with serum 1393 yielded only the
upper band (Fig. 8, lanes 2,
3, 5, and 6). Competition with cognate
peptide demonstrated the specificity of the recognition
(lanes 1 and 4). These results are
completely consistent with the prediction of two possible initiation
codons in human RelB mRNA, and they indicate that differentiating human
monocytes express at least two forms of the RelB protein.
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The transcription factor NF-AT has been found in variety of cell types, including T cells, B cells, mast cells, and NK cells. NF-AT is activated in these cells by agents that increase intracellular Ca2+ flux. The increased Ca2+ activates the phosphatase calcineurin, which results in dephosphorylation and nuclear translocation of pre-existing cytoplasmic NF-AT and up-regulation of NF-AT synthesis (23, 24). Because CI promotes DC differentiation in monocytes through a calcineurin antagonist-sensitive pathway, we assessed the possible role of NF-AT in this process.
To minimize contamination with lymphocytes, elutriated monocytes were
further purified by positive selection for CD14.
CD14+ cells were cultured with or without A23187
for 5 or 24 h, and nuclear and cytoplasmic extracts were tested by
EMSA for the ability to bind an NF-AT probe. No NF-AT DNA-binding
activity was observed in either nuclear or cytoplasmic extracts from
these cells (Fig. 9A,
lanes 1–6). As a positive control, we tested whole cell
extracts from human lymphocytes, which, as expected, contained a high
level of NF-AT DNA-binding activity (lane 7). A
pan-NF-AT antiserum blocked this binding completely, demonstrating the
specificity of the interaction (lane 8). Integrity of
the monocyte nuclear extracts was confirmed by EMSA with an OCT-1
binding site (Fig. 9B). Finally, we were unable to detect
NF-AT protein in the treated monocytes by immunoblot analysis (data not
shown). Thus, we found no evidence of NF-AT involvement in
A23187-induced DC differentiation of monocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our demonstration of similar rapid differentiation induced by agents
known to signal infection or inflammation lends additional credibility
to the idea that monocytes indeed contribute to the generation of
mature, activated DC in vivo. Our studies clearly show that under
serum-free conditions both TNF- and bacterial LPS can promote rapid
DC differentiation in a majority of CD14+ human
peripheral blood monocytes. The treated cells exhibited
immunophenotypical, morphological, and functional characteristics of DC
while maintaining good viability. Thus, treatment under serum-free
conditions may offer a useful alternative to the more typical regimen
involving longer culture in medium with serum and IL-4. Given the
current interest in culturing DC for therapeutic purposes, both the
speed of the differentiation process and the elimination of serum may
offer considerable advantages. In fact, when we supplemented our
M-SFM base medium with 5% human AB serum, rapid DC differentiation
in response to LPS and TNF- was not observed. This suggests that
human serum may actually contain components that retard the generation
of DC from monocytes under the conditions tested.
Differentiation of DC under serum-free conditions can be driven not
only by LPS and cytokines, but also, as we showed previously
(11), by CI. In the present studies we demonstrated that
the CI-induced differentiation process differs in several respects from
that induced by LPS. First, LPS, but not CI, induced high levels of
TNF- secretion. Second, TNF- played a major role in LPS-induced
differentiation, for TNF--neutralizing Ab significantly inhibited
this process. In contrast, neutralizing Ab had no effect on CI-induced
differentiation. This suggests that either CI acts downstream of TNFR1
in the TNF signaling pathway, or it activates a separate pathway
altogether. Third, CI-induced differentiation was strongly blocked by
CsA, but this drug had no effect on LPS-induced differentiation. This
experiment offers evidence that the phosphatase calcineurin plays a
vital role in the former pathway, but is not required in the
latter.
Regarding the role of TNF- in LPS-induced differentiation of DC, our
results suggest two possibilities. The first is that TNF- is
primarily responsible for the differentiation process, and beyond
stimulating TNF- production, LPS has no additional role to play.
This possibility is consistent with the fact that TNF- by itself
induces DC differentiation, and TNF--neutralizing Ab effectively
inhibits LPS-induced differentiation. Alternatively, LPS may activate
discrete pathways that modify DC differentiation, resulting in DC with
functional capacities different from those of cells differentiated with
TNF- alone. This possibility is consistent with the
observation that in the absence of TNF, LPS can activate the
expression of various genes, including some cytokine genes, in
monocytes (27), macrophages (28), and
TNF--deficient mice (29).
With respect to signaling pathways involved in the differentiation
process, we found an association between DC differentiation and
activation of NF-B. All three inducing agents, LPS, TNF, and CI,
resulted in nuclear NF-B, and treatment that blocked the appearance
of nuclear NF-B also inhibited differentiation. Because it is well
known that both TNF- and LPS activate NF-B in many cell types,
these results are not surprising. In fact, the involvement of NF-B,
and especially RelB, in the process of LPS-induced DC differentiation
has been reported previously (30, 31, 32). As in other
cell types, signaling most likely proceeds through a Toll-like
receptor (for LPS) or TNFR p55 (for TNF) through pathways that lead to
the activation of the IB kinases and hence to NF-B (33, 34).
However, our observation that CI also activates NF-B in the absence
of any apparent costimulator was unexpected. There are many reports
showing that Ca2+ synergizes with various other
agents or treatments in activation of NF-B, but we are unaware of
any previous experiment showing that CI by itself can induce NF-B.
We were unable to exclude the possibility that GM-CSF supplies
additional signals necessary for CI-induced activation of NF-B
because this cytokine was essential for maintaining cell viability. It
also remains a possibility that CI acts indirectly, through the induced
secretion of biological agents that, in turn, act in an autocrine
fashion to promote differentiation. However, thapsigargin, a compound
that causes a rapid efflux of Ca2+ from the
endoplasmic reticulum, has been shown, as a single agent, to activate
NF-B in HeLa cells (35), normal renal tubular
epithelial cells (36), and pancreatic lobules
(37). This activation is inhibited by pyrrolidine
dithiocarbamate and therefore requires the presence of reactive oxygen
intermediates. We tried to test whether reactive oxygen intermediates
were necessary for ionophore-induced NF-B activation under our
conditions, but unfortunately both pyrrolidine dithiocarbamate and NAC
were very toxic for monocytes and HL-60 cells when used in combination
with ionophore. Therefore, beyond the fact that it is CsA sensitive
(and thus likely to involve calcineurin), the mechanism of
ionophore-induced NF-B activation and DC differentiation remains to
be defined.
What is the role of NF-B in the DC differentiation process? Many of
the genes up-regulated in DCs contain functional NF-B binding sites
in their regulatory regions. These include the genes for IL-1,
IL-1ß, IL-6, IL-8, IL-12 (p40), macrophage inflammatory protein-1,
macrophage inflammatory protein-1ß, CCR5, CD80, MHC class I (H-2Kb),
MHC class II (HLA-B7), CD54, Fas ligand, Fas, RANTES, and, of course,
TNF- (5, 38). In addition, the CD86 gene has a required
NF-B binding site (39), and the human CD83 gene has a
potential NF-B binding site whose functional significance has not
yet been reported (40). Thus, expression of many genes
important for DC function is likely to involve NF-B. Expression of
the pro-survival Bcl-XL gene is also up-regulated in DC and is
influenced by NF-B (41, 42), consistent with the
anti-apoptotic function of RelA that has been demonstrated in many
cell types. It is interesting, in light of the importance of RelB to DC
function (see below), that RelB is also capable of inhibiting apoptosis
in at least one model system (our unpublished observations).
Targeted disruption of the RelB gene in mice has revealed that it plays
a unique and critical role in DC differentiation and/or function
(21, 43). In wild-type mice, RelB is expressed largely in
the thymus, lymph nodes, and spleen, and its localization in those
organs correlates with that of DC. RelB-/- mice
produce no apparent mature DC of myeloid origin, and bone marrow
chimeras (RelB -/- bone marrow into lethally
irradiated wild type host) have shown that this is due to a direct
effect of RelB on stem cell development (21, 44).
Consistent with this finding, we and others have shown that RelB is
up-regulated and activated during differentiation of DC in vitro from
both mouse and human precursors (19, 30, 45, 46). In this
report we demonstrated two forms of the RelB protein. Whether these two
forms differ in functional properties is a subject of our current
studies. Most interestingly, we showed that RelB can be induced in
myeloid cells either directly or indirectly through a
calcium-dependent, CsA-sensitive pathway. In contrast, NF-B/RelB
activation induced by LPS or TNF- was not sensitive to CsA,
indicating that at least two different signaling pathways are available
for the induction of NF-B proteins and DC differentiation, each with
distinct upstream components.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nancy R. Rice, Frederick Cancer Research and Development Center, National Cancer Institute, P.O. Box B, Frederick, MD 21702-1201.
3 Abbreviations used in this paper: DC, dendritic cell; CI, calcium ionophore; CsA, cyclosporin A; M-SFM, macrophage serum-free medium; NAC, N-acetyl cysteine; poly(dI-dC), polydeoxyinosinic-deoxycytidylic acid; TNFR1, TNF receptor 1.
Received for publication May 1, 2000. Accepted for publication July 14, 2000.
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References |
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,
Isotype-matched control Ab staining; 
, marker-specific Ab. Results
are representative of six experiments with different donors.
and 
) or monocytes
cultured in M
and •), GM-CSF
plus LPS (
and 
), GM-CSF plus TNF-
and 
), or GM-CSF
plus A23187 (
2 x
106 cells each). B, Nuclear extracts (3 µg
of protein) from both subpopulations were analyzed by EMSA using a
32P-oligonucleotide containing the NF-
