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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 277-285
IMMUNOBIOLOGY
Differential expression of Rel/NF- B and octamer factors is a
hallmark of the generation and maturation of dendritic cells
M. Neumann,
H.-W. Fries,
C. Scheicher,
P. Keikavoussi,
A. Kolb-Mäurer,
E.-B. Bröcker,
E. Serfling, and
E. Kämpgen
From the Institute of Pathology, Department of Molecular Pathology,
and the University Hospital, Department of Dermatology, University of
Würzburg, 97080 Würzburg, Germany.
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Abstract |
A key feature of maturation of dendritic cells is the
down-regulation of antigen-processing and up-regulation of
immunostimulatory capacities. To study the differential expression of
transcription factors in this process, we investigated the nuclear
translocation and DNA binding of Rel/NF- B and octamer factors
during in vitro generation and maturation of dendritic cells compared
with macrophage development. RelB was the only factor strongly
up-regulated during the generation of both immature dendritic cells and
macrophages. Cytokine-induced maturation of dendritic
cells resulted in an increase in nuclear RelB, p50, p52, and especially
c-Rel, whereas cytokine-treated macrophages responded poorly. This
up-regulation of NF-B factors did not correlate with lower
levels of cytosolic NF-B inhibitors, the IBs. One IB, Bcl-3,
was strongly expressed only in mature dendritic cells. Furthermore,
generation and maturation of dendritic cells led to a continuous
down-regulation of the octamer factor Oct-2, whereas monocytes and
macrophages displayed high Oct-2 levels. A similar pattern of
maturation-induced changes in transcription factor levels was found in
cultured murine epidermal Langerhans cells, suggesting a general
physiological significance of these findings. Finally, this pattern of
differential activation of Rel and octamer factors appears to be
suitable in determining the maturation stage of dendritic cells
generated by treatment with different cytokine combinations in vitro.
(Blood. 2000;95:277-285)
© 2000 by The American Society of Hematology.
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Introduction |
Dendritic cells constitute a system of
antigen-presenting cells that control immunity by interacting with
lymphocytes.1 Classical myeloid-derived dendritic cells are
specialized in several ways to specifically activate primary T cell
responses.2 Immature dendritic cells, such as epidermal
Langerhans cells, develop from hematopoietic precursors and reside as
sentinels at body surfaces and interstitial spaces. They are equipped
to capture antigens and to produce immunogenic major histocompatibility
complex [MHC]-peptide complexes. In the presence of
maturation-inducing stimuli, such as inflammatory cytokines, dendritic
cells develop into potent T cell stimulators by up-regulating adhesion
and costimulatory molecules. Furthermore, they migrate into secondary
lymphoid organs to select and stimulate rare antigen-specific T cells.
This scenario of cascadelike changes in functional capacities was first
elucidated with the use of Langerhans cells as a model
system,3 but was later recognized as a principle feature of
dendritic cells derived from different organs.4
Distinct alterations in gene expression of dendritic cells are a
prerequisite for the maturation process. However, little is known about
dendritic cell-specific gene regulation and signal transduction. The
family of Rel/NF-B transcription factors plays a pivotal role in the
regulation of immunological processes, as demonstrated by the phenotype
of several Rel/IB-deficient mice.5-8 RelB-deficient mice
show a multifocal and mixed infiltration of inflammatory cells in
several tissues and impaired cellular immunity. These animals also
exhibit a dramatically reduced number of dendritic cells in the
thymus,5,6 recently found to be due to a selective loss of
myeloid-related, but not of lymphoid-related, dendritic cells.9 In a few studies, high expression of Rel proteins
was detected mainly in mature dendritic cells.10-13 In the
developing mouse, the highest RelB levels are found in interdigitating
dendritic cells of thymic medulla and the deep cortex of lymph
nodes.14 In addition, RelB expression correlates with the
activation of their antigen-presenting capacity, a property of mature
dendritic cells.11
NF-B is a dimer composed of virtually any of the 5 mammalian Rel
proteins (p65/RelA, c-Rel, RelB, p50/NF-B1, and p52/NF-B2), which
are structurally characterized by the Rel homology
domain.15-17 The Rel homology domain mediates important
functions, such as specific DNA binding, nuclear translocation, and
protein-protein interactions. The nuclear translocation and activity of
NF-B factors is controlled by a family of cytoplasmic inhibitory
proteins, the IBs.18,19 The IBs interact with NF-B
dimers, thereby blocking their nuclear translocation. The individual
IBs differ in their affinities for various NF-B dimers and in
their inducible degradation.20 Many external stimuli lead
to a site-specific phosphorylation of IBs followed by their
proteolytic degradation and release of active NF-B, which is then
translocated into the nucleus.21,22
The genes controlled by NF-B factors encode proteins of principal
importance for the immune system, such as MHC I and II molecules,
cytokines and their receptors (eg, interleukin[IL]-2 and the
 -chain of the IL-2R), or cell adhesion molecules, such as the
intercellular cell adhesion molecule 1.15,19 Most of these
proteins are also expressed in dendritic cells and markedly up-regulated when dendritic cells switch from an antigen-capturing to
an antigen-presenting mode.23
Octamer factors are another class of transcription factors that play a
central role in the immune system. The octamer motif was originally
identified as a regulatory promoter element necessary for B
cell-specific expression of immunoglobulin (Ig)
genes.24,25 Oct-1 and Oct-2 are the 2 most prominent
octamer factors. Whereas Oct-1 is expressed ubiquitously, Oct-2
expression is restricted largely to B lymphocytes.24-26
Oct-2 appears to be involved in the late phases of B cell development
and the proliferation of mature B cells.27,28 However,
nothing is known about its role in the development of macrophages or
maturation of dendritic cells.
To elucidate the expression of Rel/NF-B and octamer factors during
the generation and maturation of dendritic cells, we studied the
nuclear appearance and DNA binding of these factors in human and murine
dendritic cells and macrophages. We demonstrate that in both
monocyte-derived dendritic cells and macrophages, the expression of
Rel/NF-B and octamer transcription factors is differentially regulated and that expression patterns are found specific for the
developmental states of dendritic cells and macrophages.
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Materials and methods |
Cell culture and preparation of human dendritic cells, macrophages,
and murine Langerhans cells
Human dendritic cells were prepared from peripheral blood monocytes
according to Romani et al.29 For maturation of dendritic cells, cells were cultured either in monocyte-conditioned
medium29 or in a cytokine cocktail of IL-1 (500U/ml),
IL-6, tumor necrosis factor[TNF]-, IL-1 (all 1000U/ml;
Strathmann Biotech, Hannover, Germany) and PGE2
(10-8M; Sigma, Deisenhofen, Germany)30 for 3 days. The yield of dendritic cells was normally between 5% and 10% of
the initially processed PBMC, with an average purity
higher than 80%. For preparation of macrophages, CD14[+]
cells were isolated by adherence of monocytes
(5 × 107/10cm2) to cell culture dishes
in R0 medium (RPMI 1640/ 2mM L-glutamine/ 50 µg/ml
gentamicin) containing 1% autologous plasma. The nonadherent cells
were removed by 2 × gentle washing with warm PBS.
Adherent monocytes were cultured for 7 days in X-VIVO-15 medium (BioWhittaker, Walkersville, MD) supplemented with
10% fetal calf serum, 2 mM L-glutamine, 50 µg/ml
gentamicin, 100 U/ml granulocyte-macrophage-stimulating factor (GM-CSF)
and 10 U/ml M-CSF. The adherent cells were collected and
analyzed by flow cytometry for the expression of
Mph-specific markers (CD14, C36, CD64, CD68) and the
absence of dendritic cell markers (CD1a, CD83). Epidermal cells were
prepared from the ear skin of mice according to Schuler and
Koch.31 Fresh Langerhans cells (day 0) were prepared from
epidermal cells by mismatched panning31 with -class II antibodies (Abs) ( I-E: 14-4-4S and I-A: MK-D6, both from
American Type Culture Collection). Alternatively,
trypsinized epidermal cells were first cultured in R0
medium with 10% FCS and with 1000U/ml muGM-CSF for 1 day prior to
mismatched panning of Langerhans cells or cultured for 3 days and then
enriched by density centrifugation with the use of a dense bovine serum
albumin-gradient. The purity of each Langerhans cell preparation was
between 80% and 95% as assessed by fluorescence-activated cell
sorter staining.
Antibodies for immunodetection and electrophoretic mobility
shift assays (EMSAs)
The following polyclonal antibodies were used for immunodetection:
-p65-NF-B (Santa Cruz Biotechnology, Santa Cruz, CA, sc-109),
-c-Rel (sc-070), -RelB (sc-226), -p50-NF-B (Dr. N. Rice,
NCI, Frederick, MD, Ab 1141), -p52-NF-B (Upstate
Biotechnology, Lake Placid, NY, Ab 06-413), -B (sc-371),
-IB (sc-945), -IB (Dr. N. Rice; Ab 891 & 1775),
-Bcl-3 (sc-185), -Oct-1 (Dr. T. Wirth, Würzburg, Germany)
and -Oct-2 (sc-233). For supershift EMSAs, 2 different antibodies
were used: -p50-NF-B (sc-114) and -Oct-1 (sc-232).
Flow cytometry
Flow cytometry was used to assess the differentiation of monocyte
precursors into dendritic cells or macrophages. Usually, 105 cells in 100 µl PBS/1% bovine serum albumin were
incubated with 1 µg primary antibody for 30 minutes on ice, washed
once, and then incubated with the fluorescein isothiocyanate (FITC)-
or phosphatidylethanolamine (PE)-coupled secondary antibody under the
same conditions. The antibodies used were CD1a (OKT6), -HLA class
II (L243 and 9.3F10, all American Type Culture Collection, Rockville,
MD); CD14, CD25, CD64, CD86 (IT2.2) (all Pharmingen, Hamburg, Germany);
CD68 (KP1, Dako, Glostrup, Denmark); CD83 (Immunotech, Hamburg, Germany); CD115 (3-4A4-E4, Calbiochem, Bad Soden, Germany); CD36 (mAb 89, Serotec/Camon, Wiesbaden, Germany); and FITC- or PE-conjugated -mouse or - rat Ig (Dianova, Hamburg, Germany). The stained cells were analyzed on a
florescence-activated cell scan (Becton Dickinson, Heidelberg, Germany).
Preparation of protein extracts and immunoblotting
Nuclear and cytoplasmic protein extracts from all cells were
prepared according to Schreiber et al33 with slight
modifications as described.34 Protein concentrations were
determined by a Bradford assay.35 For immunoblot assays, 6 to 10 µg of protein extract were separated on 8% or 10% sodium
dodecyl sulfate-polyacrylamide gels and electrophoretically
transferred to nitrocellulose membranes. Equal loading was confirmed by
Ponceau S staining (Sigma, St. Louis, MO). The purity of
the nuclear protein extracts was controlled by immunoblotting with the
use of the p50-NF-B-specific antibody also detecting the p105
precursor exclusively located in the cytoplasm. The cytoplasmic
extracts were checked for contaminating nuclear proteins by
immunoblotting with the use of the Oct-2-specific antibody. Oct-2 is
found only in the nucleus.
EMSAs and supershift EMSAs
For each EMSA, 2 to 4 µg nuclear protein extracts were incubated
with 6000 cpm (equivalent to approximately 0.25 ng) of a 32P-labeled oligonucleotide probe. The reaction mixtures
contained 1 µg poly (dIdC) per 4 µg nuclear protein
as nonspecific competitor. As a control for specificity of DNA binding,
a 50 mol/L excess of an unlabeled B-specific probe was added to
nuclear proteins. The samples were incubated for 30 minutes on ice and
fractionated on a nondenaturing 6% polyacrylamide gel at 200 V/15 cm.
One µl of each of the Rel- and octamer-specific antibodies was added in supershift EMSA. The following oligonucleotide probes were used for
EMSAs: Consensus NF-B binding site derived from B enhancer element of the IL-2 promoter ('TCEdA > C')36:
5'-GACCAAGAGGGATTTCACCCCTAAATC-3'. Consensus octamer site from the immunoglobulin heavy chain
enhancer37: 5'-AGCAGAAATGCAAATTATACCCG-3'
| |
Results |
Differentiation of monocytes into dendritic cells and
macrophages
To identify molecular events involved in dendritic cell
differentiation, we used the established GM-CSF/IL-4 protocol for generation of immature dendritic cells from monocytes (Figure 1A). This
resulted in a population of nonadherent, highly veiled, strongly CD1a
and class II positive and CD14, CD64 negative cells after a
7-day culture (Figure 1B).
Treatment of these cells for 3 days with
monocyte-conditioned medium induced the typical phenotype of mature
dendritic cells characterized by up-regulated CD83, CD25
(IL-2R-chain), and the costimulatory molecule CD86 (Figure 4C).
In addition, monocyte-conditioned medium decreased, by a factor of 5 to 10, the number of dendritic cells required to stimulate maximal T cell proliferation in the mixed leukocyte reaction (data not
shown). Alternatively, culture of monocytes in X-VIVO-15 medium with
low doses of GM-CSF and M-CSF resulted in the generation of adherent CD1a , CD14+, and
CD64+ macrophages (Figure 1D) with low T cell stimulatory
capacity (data not shown). Treatment of these cells with
monocyte-conditioned medium led to enhanced expression of the Mph
marker CD68 (Figure 1E), but not of costimulatory
molecules or CD83.
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| Fig 1.
Phenotypes of human monocytes and of human dendritic
cells and macrophages generated in vitro.
The expression of cell surface proteins characteristic
for dendritic cells (CD1a, MHC class II, CD83, CD115) and macrophages
(CD14, CD36, CD64, CD68), and dendritic cell maturation (CD86, CD25)
were investigated by (A) fluorescence-activated cell sorter analysis of
monocytes, (B) immature dendritic cells, (C) mature dendritic cells,
(D) macrophages (on day 7), and (E) monocyte-conditioned
medium-treated macrophages. Cells were generated in vitro and
florescence-activated cell sorter analyses performed as described in
Materials and Methods. Dot plots: Forward scatter on the x-axis and
side scatter on the y-axis. Histograms: Fluorescence intensity in a
logarithmic scale (x-axis) was blotted against cell numbers (y-axis).
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Generation of immature dendritic cells and macrophages leads
to the specific accumulation of nuclear RelB
We first wanted to know whether the concentrations of nuclear Rel
proteins change during the generation of immature dendritic cells from
monocytes. In immunoblot assays, using nuclear protein extracts from
monocyte precursors (day 0; Figure 2, lane
1) and immature dendritic cells (day 7; Figure 2, lane 2) the
expression of p65/RelA, c-Rel, p50/NF-B1 and p52/NF-B2 was found
to be similar in monocytes and immature dendritic cells. In contrast, a
striking increase in the expression of nuclear RelB was observed in
immature dendritic cells. A similar increase in the nuclear concentrations of RelB was also detected in macrophages. The pattern of
other nuclear Rel/NF-B proteins was also very similar, if not
identical to that found in immature dendritic cells (Figure 2,
lanes 4-6).
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| Fig 2.
Nuclear Rel proteins are differentially expressed during
generation and maturation of human dendritic cells and development of
macrophages in vitro.
Immunoblots were performed with nuclear proteins from monocytes (day 0, lanes 1 and 4), immature dendritic cells (day 7, lane 2), mature
dendritic cells (day 10, lane 3), differentiated macrophages (day 7, lane 5), and macrophages treated with monocyte-conditioned medium for
another 3 days (day 10, lane 6). Proteins were separated on an 8%
sodium dodecyl sulfate-polyacrylamide gel and electrophoretically
transferred to a nitrocellulose membrane.
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Maturation of dendritic cells by cytokines results in a
specific nuclear accumulation of c-Rel
Monocyte-conditioned medium-induced maturation of immature
dendritic cells resulted in nuclear accumulation of p50, p52, and RelB
detected by immunoblot analysis. Again, p65 remained unchanged, whereas
the strongest up-regulation was observed for c-Rel (Figure 2, lanes 2 and 3). In contrast, treatment of macrophages with monocyte-conditioned
medium resulted in only a moderate increase in nuclear p50, p52, RelB,
and c-Rel levels (Figure 2, lanes 5 and 6).
Increased nuclear translocation of Rel proteins during the
generation of dendritic cells and macrophages also results in an
increase in DNA binding
To determine whether the enhanced nuclear concentration of Rel
proteins also results in an enhanced DNA binding, we studied the DNA
binding of nuclear Rel proteins from dendritic cells and macrophages by
EMSAs and supershift EMSAs. A strong increase in DNA binding activity
was observed after induction of dendritic cell maturation (Figure
3A, lane 3), resulting in 2 major
DNA/protein bands. Both consisted of 2 complexes. The slower migrating
band consisted of p65/RelA-containing heterodimers (complex I) and RelB-containing heterodimers (complex II) as detected by supershift EMSAs (Figure 3B). Complex I is composed of p65/p50 and p65/c-Rel heterodimers (Figure 3B; eg, see lanes 2, 3, and 5). The
`classical' NF-B complex, p65/p50, is best seen after supershift
with the -c-Rel-specific antibody (Figure 3B, lane 9). The
second, slightly faster, migrating complex visible after c-Rel
supershifting represents the RelB/p50 heterodimer (complex II) (Figure
3B, lane 9). Although no supershift signal was detected with the use of
a RelB-specific antibody (Figure 3B, lanes 10 and 16), complex II
disappeared after addition of -RelB, clearly indicating the presence
of RelB. This is best recognizable by a comparison of lane 9 with lane 10 of figure 3B. The RelB/p50 dimer of lane 9 was no longer visible as
a distinct band in lane 10. Here only the p65 heterodimers with a
slightly lower electrophoretic mobility were detectable. RelB forms
heterodimers not only with p50 (Figure 3B, lanes 11 and 17) but also
with c-Rel (Figure 3B, lanes 9 and 15). It should be noted that complex
II is visible only in immature (day 7) and mature dendritic cells (day
10) but not in peripheral blood monocytes (day 0) (Figure 3A; compare
lane 1 with lanes 2 and 3). A specific supershift signal was observed
in dendritic cells with the use of the c-Rel-specific antibody (Figure
3B, lanes 3, 9 and 15). This signal distinctly increased during
dendritic cell maturation (Figure 3B; compare lanes 9 and 15). The band
with the highest electrophoretic mobility consisted of p50/p50
homodimers (complex III) and of a complex of unknown composition
(marked by an asterisk in Figure 3).
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| Fig 3.
Increased nuclear translocation of Rel proteins during in
vitro generation of human dendritic cells and macrophages results in an
increase in B-specific DNA binding activity.
(A) EMSAs performed with nuclear proteins from monocytes (day 0, lanes
1 and 4), immature dendritic cells (day 7, lane 2), and mature
dendritic cells (day 10, lane 3). As a control for specificity of DNA
binding, an excess of an unlabeled B-specific probe (C, lane 4) was
added to nuclear proteins from monocytes. The 3 detectable
B-specific DNA binding complexes are designated I through III (see
below). The asterisk indicates a complex of unknown composition. The
signals of free probes were cut off. (B) Supershift EMSAs were
performed with nuclear proteins from monocytes (day 0, lanes 1-6),
immature dendritic cells (day 7, lanes 7-12), and mature dendritic
cells (day 10, lanes 13-19). For supershift EMSAs, 1 µl of each of
the Rel protein-specific antibodies was added to the extracts as
indicated (lanes 2-6, 8-12, and 14-18). As a control for the
specificity of supershifts, 1 µl of normal rabbit serum (NRS) was
added to nuclear extracts from mature dendritic cell (lane 19). The 3 indicated B-specific DNA complexes are composed of p50 homodimers
(III) and RelB/p50 and RelB/c-Rel heterodimers (II). Complex I consists
of 2 NF-B complexes: p65/p50 and p65/c-Rel heterodimers. The
asterisk indicates a complex of unknown composition. (C) EMSAs using
nuclear proteins from monocytes (day 0, lane 1), differentiated
macrophages (lane 2), and macrophages treated with
monocyte-conditioned medium for another 3 days (Mph+,
lane 3). The specificity of DNA binding was controlled by
addition of an excess of unlabeled B-specific probe to nuclear
extracts from monocytes (C, lane 4). (D) Supershift EMSAs using nuclear
proteins from macrophages (lanes 1-6) and macrophages treated with
monocyte-conditioned medium for another 3 days (Mph+, lanes
7-13).
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The differentiation of macrophages was also accompanied by a moderate
up-regulation of B-specific DNA binding activity (Figure 3C, lanes 1 and 2), and a further increase was observed after monocyte-conditioned
medium treatment (Figure 3C, lanes 2 and 3). As in mature dendritic
cells, a RelB-containing binding complex (II) was detected in fully
differentiated macrophages (Figure 3C, lanes 2 and 3; Figure 3D lanes 4 and 10). The patterns of supershifts performed with Mph
extracts revealed a composition of 4 complexes, very
similar to those in mature dendritic cells (compare Figures 3B and 3D).
However, a c-Rel-specific supershift signal was observed only after
longer exposures of the films, indicating a reduced binding activity of
c-Rel in macrophages compared with dendritic cells.
Maturation of dendritic cells correlates with an increase in
IB, IB, and Bcl-3
The inducible degradation of IBs is crucial for NF-B
activation. Therefore, we investigated whether the strong increase in
the nuclear concentrations of Rel proteins during dendritic cell
maturation might be mediated by a sustained degradation of one or
several IBs. However, immunoblot analysis of cytoplasmic proteins
showed that this is not the case. The concentrations of IBs either
remained constant (IB) or were increased
(IB Iß, and Bcl-3) during the maturation of
dendritic cells (Figure 4, lanes 1-3). In
particular, this was observed for Bcl-3, whose expression was strongly
up-regulated in mature dendritic cells. IB, detected as 2 isoforms,38 was found to be poorly expressed in monocytes
(Figure 4, lane 1), but more strongly expressed in immature and mature
dendritic cells (Figure 4, lanes 2 and 3).
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| Fig 4.
The concentration of Bcl-3 is strongly increased in
mature dendritic cells.
Immunoblots with cytoplasmic proteins from monocytes (day 0, lanes 1 and 4), immature dendritic cells (day 7, lane 2), mature dendritic
cells (day 10, lane 3), macrophages (day 7, lane 5), and macrophages
treated with monocyte-conditioned medium for another 3 days (day 10, lane 6).
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In vitro-generated macrophages showed a small decrease in IB and
IB compared with monocytes (Figure 4, lanes 4 and 5), while IB levels were slightly up-regulated following
monocyte-conditioned medium treatment (Figure 4, lane 6). IB was
relatively weakly expressed in macrophages and was up-regulated upon
monocyte-conditioned medium treatment. In contrast to the strong
induction of Bcl-3 in mature dendritic cells, monocyte-conditioned
medium treatment did not induce Bcl-3 in macrophages (Figure 4, lane 6).
Interestingly, immunoblot analysis of cytoplasmic protein extracts
performed in parallel showed that the total cellular concentration of
Rel proteins was up-regulated (data not shown) during generation of
dendritic cells and macrophages. Thus the increase of active, nuclear
Rel protein is due principally to an enhanced expression of these
transcription factors and not to a sustained degradation of IBs.
Differential expression of Rel proteins during maturation
of epidermal mouse Langerhans cells corresponds to the expression
pattern in human dendritic cells
To confirm the data obtained for the human dendritic cells generated
and maturated in vitro, we analyzed mouse epidermal Langerhans cells
representing the "gold standard" of a dendritic cell maturation system. Freshly prepared murine Langerhans cells correspond in their
differentiation stage to immature human dendritic cells. Immunoblot
analyses using highly enriched, freshly explanted Langerhans cells
showed a strong expression of RelB, p65, and p50, but not c-Rel (Figure
5A, lane 1). This pattern corresponds to
that of immature human dendritic cells. Maturation of Langerhans cells by GM-CSF induced strong c-Rel expression, a gradual increase in the
concentrations of RelB and p50, and a slight increase in p65, very
similar to the increase in Rel proteins during human dendritic cell
maturation (Figure 5A, lane 3). In contrast to human dendritic cell, we
were unable to detect p52 at any stage of murine Langerhans cell
maturation. However, the pattern of B-specific DNA binding complexes
in Langerhans cells at defined stages of maturation was comparable to
those obtained for human dendritic cells (Figures 5B and C; compare
with Figures 3A and B). Separate control experiments using normal
rabbit serum were also performed (data not shown). No unspecific
supershift signals were observed in these experiments.
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| Fig 5.
Changes of nuclear Rel proteins during maturation of
epidermal mouse Langerhans cells correspond to those in human dendritic
cells.
(A) Immunoblots of nuclear proteins from freshly explanted Langerhans
cells (immature phenotype, day 0, lane 1), Langerhans cells cultured
for 24 hours (intermediate phenotype, day 1, lane 2), and maturated
Langerhans cells (mature phenotype, day 3, lane 3). (B) EMSAs performed
with nuclear proteins. The protein extracts were prepared from freshly
explanted Langerhans cells (day 0, lane 1), Langerhans cells cultured
for 24 hours (day 1, lane 2), and mature Langerhans cells cultured for
3 days (day 3, lane 3). As a control for DNA binding specificity, an
excess of unlabeled B-specific probe was added to nuclear extracts
from mature Langerhans cells (C, lane 4). The 3 detectable
B-specific DNA binding complexes are designated I-III (see below).
The asterisk indicates an additional complex of unknown composition.
(C) Supershift EMSAs with nuclear proteins from freshly explanted
Langerhans cells (day 0, lanes 1-5), Langerhans cells cultured for 24 hours (day 1, lanes 6-10), and mature Langerhans cells after 3 days in
culture (day 3, lanes 11-15). For supershift EMSAs, 1 µl of each of
the Rel protein-specific antibodies was added to the extracts as
indicated. The 3 detectable B-specific DNA complexes are composed of
p50 homodimers (III), RelB/p50 heterodimers (II), and p65/p50 and,
probably, p65/c-Rel heterodimers (I).
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Octamer factors are also differentially expressed during generation
and maturation of human dendritic cells
In addition to Rel/NF-B factors, we investigated several other
transcription factors, such as AP-1, NF-AT, and octamer factors, for
their expression during the generation and maturation of dendritic cells and differentiation of macrophages. In contrast to AP-1 and NF-AT
factors, whose concentrations remained unchanged during these processes
(data not shown), the octamer factors displayed a differential
expression. While the concentration of Oct-1 remained almost constant,
a strong, gradual down-regulation of Oct-2, which is highly expressed
in monocytes, was observed in dendritic cells (Figure
6, lanes 1-3). This down-regulation started
as early as day 3 after GM-CSF/IL-4 treatment of monocytes (data not
shown). All different isoforms of Oct-239 were found to be
down-regulated (Figure 6, lanes 2 and 3). The maturation of dendritic
cells resulted in an almost complete loss of Oct-2 (Figure 6, lane 3).
This loss of Oct-2 expression is accompanied by a down-regulation of
the cell surface molecule CD36 (see Figure 1). The CD36 gene has been shown to be mainly controlled by Oct-2 in murine B cells and some Mph
cell lines.40
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| Fig 6.
Down-regulation of Oct-2 expression during in vitro
differentiation of human dendritic cells but not macrophages.
Immunoblots with nuclear proteins from monocytes (day 0, lanes 1 and
4), immature dendritic cells (day 7, lane 2), mature dendritic cells
(day 10, lane 3), macrophages (day 7, lane 5), and macrophages treated
with monocyte-conditioned medium for another 3 days (day 10, lane
6).
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In contrast to the profound down-regulation of Oct-2 during dendritic
cell differentiation, Oct-2 was only slightly down-regulated during Mph
development (Figure 6, lane 5), and following
monocyte-conditioned medium treatment, Oct-2 concentrations even
appeared to be up-regulated again (Figure 6, lane 6). Thus, the
expression pattern of Oct-2 completely differs during the
differentation to dendritic cell and macrophages. Furthermore, contrary
to in vitro-generated human dendritic cells, no correlation between
Oct-2 and CD36 expression was found during the development of human macrophages.
The gradual loss of Oct-2 expression during dendritic cell
differentation was also detected at the level of DNA binding (Figure 7). Four prominent DNA/protein complexes
were detected in EMSAs with the use of an octamer binding site. As
shown in supershift EMSAs, the slowest migrating complex I contains
Oct-1 (Figure 7B, lanes 2, 5 and 8). The formation of this complex
increases slightly during dendritic cell maturation (Figures 7A and 7B) as well as differentiation of macrophages (Figures 7C and 7D). Although
only complex III reacted with the Oct-2-specific antibody used in
these supershift assays, the generation of complexes II and III (and of
a further, less prominent complex that can be seen best after
supershifting Oct-1 as in lane 2 of Figure 7B, marked by a circle) is
probably due to the binding of different Oct-2 isoforms. The synthesis
of multiple Oct-2 isoforms is known to be due to alternative
splicing.39 The formation of both Oct-2 complexes
disappeared during the generation and maturation of dendritic cells. A
fourth prominent complex migrating faster than complex III (indicated
by an asterisk in Figure 7) is probably due to an unspecific binding
since its formation is suppressed by both an excess of unlabeled
octamer- and B-specific binding probes (see Figures 7A
and C, lane 4; and Figure 7C, lane 5). In contradiction to the
sustained expression of Oct-2, the intensity Oct-2-specific binding
complexes (Figure 7C, lane 1-3) decreases during Mph differentiation,
indicating a less efficient DNA binding capacity of Oct-2 in these
cells. However, contrary to mature dendritic cells, Oct-2 complexes can
clearly be recognized in supershift EMSAs using nuclear proteins from
macrophages cultured for 10 days in vitro (Figure 7D, lanes 5-7).
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| Fig 7.
Different octamer-specific DNA binding complexes in human
dendritic cells and macrophages.
(A) EMSAs with nuclear proteins from monocytes (day 0, lanes 1 and 4),
immature dendritic cells (day 7, lane 2), and mature dendritic cells
(day 10, lane 3). As a control for specificity of DNA binding, an
excess of an unlabeled octamer binding probe was added to nuclear
proteins from monocytes (C1, lane 4). The 3 detectable octamer-specific
complexes are designated I to III (see below). The asterisk indicates a
prominent unspecific band, and the circle marks another unshiftable
complex, probably containing Oct-2. As shown by supershift EMSAs (see
B), the 3 detectable octamer-specific DNA complexes are composed of
Oct-1 (complex I) and different isoforms of Oct-2 (complexes II and
III). The signals of the free probe are cut off. (B) Supershift EMSAs
with nuclear proteins from monocytes (day 0, lanes 1-3), immature
dendritic cells (day 7, lanes 4-6), and mature dendritic cells (day 10, lanes 7-10). For supershift assays, 1 µl of each of the
octamer-factor-specific antibodies was added as indicated (lanes
2 + 3, 5 + 6, and 8 + 9). As a control for specificity of the
supershifts, 1 µl of NRS was added to nuclear protein from mature
dendritic cells (lane 10). (C) EMSAs with nuclear proteins from
monocytes (day 0, lane 1), macrophages (lane 2), and macrophages
treated with monocyte-conditioned medium for another 3 days (Mph+,
lane 3). The specificity of DNA binding was controlled by
addition of an excess of an octamer-specific probe (C1, lane 4) and of
a B-specific probe (C2, lane 5) to nuclear extracts from Mph+ cells.
(D) Supershift EMSAs with nuclear proteins from
macrophages (lanes 1-3) and macrophages treated with
monocyte-conditioned medium for another 3 days (Mph+, lanes
4-7).
|
|
In nuclear protein extracts from freshly prepared mouse Langerhans
cells only small amounts of Oct-1 and Oct-2 could be detected by
immunoblotting (Figure 8 A, lane 1).
Maturation of Langerhans cells resulted in an increase in
concentrations of Oct-1 but not Oct-2 (Figure 8A, lane 3). According to
the down-regulation of Oct-2 expression in immature dendritic cells, no
Oct-2-specific DNA binding complex could be found in Langerhans cells
(Figures 8B and 8C). The only detectable specific complex consisted of Oct-1 (complex I), which increased during Langerhans cell maturation (Figures 8B, lanes 1-3, and 8C lanes 2, 5, and 8). Thus, the pattern of
Oct-1 and Oct-2 expression found for human dendritic cells is also
typical for murine Langerhans cells.
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[in a new window]
| Fig 8.
Octamer factor expression in mouse epidermal Langerhans
cells.
(A) Immunoblots with nuclear proteins from freshly explanted Langerhans
cells (immature phenotype, day 0, lane 1), Langerhans cells cultured
for 24 hours (intermediate phenotype, day 1, lane 2), and maturated
Langerhans cells (mature phenotype, day 3, lane 3). (B) EMSAs with
nuclear proteins from freshly explanted Langerhans cells (day 0, lane
1), Langerhans cells cultured for 24 hours (day 1, lane 2), and mature
Langerhans cells cultured for 3 days (day 3, lane 3). As a control for
specificity of DNA binding, an excess of unlabeled octamer probe was
added to nuclear extract from mature Langerhans cells (C, lane 4). The
octamer-specific complex is designated I (see below). The asterisk and
the circle indicate prominent unspecific complexes. The signals of the
free probe are cut off. (C) Supershift EMSAs performed with nuclear
extracts from freshly explanted Langerhans cells (day 0, lanes 1-3),
Langerhans cell cultured for 24 hours (day 1, lanes 4-6), and maturated
Langerhans cells after 3 days in culture (day 3, lanes 7-9). For
supershift EMSA, 1 µl of each of the octamer-factor-specific
antibodies was added as indicated (lanes 2 + 3, 5 + 6, and
8 + 9). The detectable octamer-specific DNA complex is composed of
Oct-1 (I).
|
|
Differential expression of Rel and octamer factors reflects the
maturation stage of dendritic cells generated after treatment with
various stimuli
For maturation of dendritic cells in vitro, several protocols have
been applied, including different cytokines and/or maturation-promoting media. To determine the maturation status of human dendritic cells generated by different stimuli, we used the expression of Rel and
octamer factors as a marker. In these experiments, the dendritic cells
were grown in media containing human plasma instead of fetal calf serum
because fetal calf serum often contains
components, such as lipopolysaccharide, that induce
dendritic cell maturation. The expresssion of Rel and octamer proteins
in mature dendritic cells generated by monocyte-conditioned medium
treatment (Figure 9, lane 1) was compared
with that in dendritic cells whose maturation was induced by a defined
cytokine cocktail consisting of IL-1 and , IL-6, TNF- and
PGE2 (Figure 9, lane 2). Further, these monocyte-conditioned medium-treated dendric cells were compared to
dendritic cells treated only with TNF- (Figure 9, lane 3), and to
dendritic cells arrested at the immature state by culture in
GM-CSF/IL-4-containing medium (Figure 9, lane 4). The
patterns of transcription factors in mature dendritic cells generated
with the cytokine cocktail were almost identical to those from
dendritic cells generated by monocyte-conditioned medium (Figure 9,
lanes 1 and 2). In contrast, TNF- alone was unable to induce full
dendritic cell maturation. Compared with mature dendritic cells
generated by monocyte-conditioned medium, TNF- induced only a minor
up-regulation of RelB, p50, and c-Rel typical for mature dendritic
cells (Figure 9, lane 3). In addition, significant levels of Oct-2,
typical for immature dendritic cells, were detected in dendritic cells treated with TNF-. Dendritic cells arrested at the immature stage showed a continuous expression of Oct-2 and a poor expression of Rel
proteins (Figure 9, lane 4), which was below the Rel protein levels
found in immature dendritic cells (Figure 2, lane 2). The low levels of
nuclear Rel proteins in maturation-arrested dendritic cells might be
due both to a decreased expression and to an enhanced degradation of
Rel proteins induced by cell death. This conclusion is supported by an
increased apoptosis observed in these cells (data not shown).
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[in a new window]
| Fig 9.
Patterns of nuclear Rel/NF-B and octamer factors
detected in dendritic cells after treatment with various stimuli.
Immunoblots of nuclear proteins from immature dendritic cells treated
with various stimuli for 3 days. These stimuli either preserve the
immature state of dendritic cells (GM-CSF/IL-4) or induce their
maturation (all other stimuli). The stimuli used were
monocyte-conditioned medium (lane 1); a cytokine cocktail consisting of
IL-1, IL-1, IL-6, TNF-, and PGE2 (lane 2);
TNF- (lane 3); and GM-CSF/IL-4 (lane 4).
|
|
| |
Discussion |
Dendritic cells have the unique capacity to switch from
antigen-processing to antigen-presenting cells capable of stimulating naive T cells. This process involves specific alterations in gene expression reflecting the differential activation of specific transcription factors. Mice deficient for RelB, a member of the Rel/NF-B factor family, showed a severe impairment of dendritic cell
function.5,6 This prompted us to study the expression of
Rel/NF-B factors during (1) the generation of immature dendritic cells derived from human monocytes and (2) the cytokine-induced maturation of human and mouse dendritic cells.
During the generation of dendritic cells from monocytes, RelB was the
only nuclear protein found to be strongly up-regulated, emphasizing a
role for RelB during early development of dendritic cells.12 However, we also found a comparable up-regulation
of RelB in monocyte-derived macrophages, indicating that up-regulation of RelB is not a property specific to immature dendritic cells. Furthermore, since RelB-deficient mice still possess immature dendritic
cells, such as epidermal Langerhans cells, RelB might be replaceable by
other transcription factors in the early stages of dendritic cell
development. Interestingly, RelB-deficient mice miss mature dendritic
cells in lymphoid organs.5,6 This finding indicates a
predominant role of Rel/NF-B factors in the maturation but not the
generation of immature dendritic cells. Such a role has been also
suggested by another study using a dendritic cell-like murine cell
line, demonstrating that NF-B activation is an important signaling
pathway involved in dendritic cell maturation.13
We have shown that all Rel proteins, except p65, were strongly
expressed during cytokine-induced human dendritic cell maturation, indicating that the up-regulation of B-dependent gene expression is
pivotal for this process. Surprisingly, c-Rel, which has not been
implicated in dendritic cell maturation so far, was found to be
drastically up-regulated. This suggests an important role for c-Rel in
dendritic cell function, although no obvious defect in cellular immune
responses has been reported in c-Rel-deficient mice.41 A
similar pattern of increased Rel protein concentrations was also found
in mature mouse epidermal Langerhans cells, whereas cytokine-treated
macrophages showed only a moderate, or no, increase in Rel factor
concentrations. Therefore, the up-regulation of c-Rel, RelB, and p50
expression reflects a specific response of immature dendritic cells
toward maturation-inducing cytokines and marks a distinct biological
difference to macrophages, a further cell type of the myelo-monocytic lineage.
The increased expression of nuclear NF-B factors observed in mature
dendritic cells seems to be due to enhanced transcription of Rel genes.
Instead of distinctly lower steady-state levels of IB proteins, we
reproducibly detected increased levels of IB and in immature
and mature dendritic cells as well as in macrophages, especially after
monocyte-conditioned medium treatment. This can be explained by the
NF-B-mediated control of IB gene expression.15
A strong increase in Bcl-3 concentrations was detected in mature
dendritic cells, but not in macrophages. Bcl-3 not only is able to
function as an NF-B-inhibitor, but also exerts (after formation of
ternary complexes with p50 and p52) transactivating properties.8,15 Interestingly, Bcl-3-deficient mice
display severe defects in T cell-mediated immune responses. The
cellular basis of this phenotype is unknown.8 Our
observation of selective Bcl-3 up-regulation in mature dendritic cells
suggests this factor to be specifically involved in dendritic cell
maturation, and a disturbance of this process, as in Bcl-3-deficient
mice, might contribute to the observed defects in T cell responses.
In addition to the Rel/NF-B factors, the activity of octamer factors
seems also to be involved in the differentiation of dendritic cells. A
strong down-regulation of Oct-2 expression starts rapidly after the
onset of monocyte differentiation to immature dendritic cells. Cells
cultured for 3 days in the presence of GM-CSF/IL-4 exhibit already much
lower levels of Oct-2 than monocytes (data not shown). In contrast,
monocyte-derived macrophages show only a moderate down-regulation of
Oct-2, indicating a molecular hallmark distinguishing dendritic cells
from macrophages.
The decrease in Oct-2 concentration during dendritic cell
differentiation correlates with a loss of CD36 expression. CD36 was
recently identified as an important receptor for the uptake of
apoptotic vesicles and virally infected apoptotic cells by dendritic
cells.42,43 The CD36 gene is known to be an Oct-2-specific target gene in murine B cells and some Mph cell
lines.40 Our studies suggest that in human dendritic cells
the expression of the CD36 gene might also be controlled by Oct-2.
However, this assumption is based solely on the observation that Oct-2
expression declines with a comparable kinetics as the expression of
CD36 during the development and maturation of dendritic cells. In
contrast, no such correlation was notable in macrophages. Therefore,
direct experimental evidence is needed to determine whether in human dendritic cells the cd36 gene is indeed a candidate for an
Oct-2-dependent regulation.
The specific expression patterns of Rel and octamer proteins during
dendritic cell differentation might be a useful molecular marker to
determine the maturation state of dendritic cells generated in vitro.
In clinical trials, monocyte-conditioned medium is frequently used to
induce dendritic cell maturation.29 However, in addition to
several cytokines, this supernatant contains other unknown and possibly
inhibitory components. Thus, variations in its maturation-inducing potency are commonly observed. Alternatively, a cocktail consisting of
TNF-, IL-1, IL-6, and PGE2 has been proposed as a
substitute for monocyte-conditioned medium.30 Others
described TNF- to be sufficient to induce dendritic cell
maturation,44 but this requires the presence of fetal calf
serum.29,30 Identical expression patterns of
Rel and octamer factors were observed in dendritic cells grown in media
containing either monocyte-conditioned medium or a defined cytokine
cocktail, indicating that this cocktail can replace
monocyte-conditioned medium. In contrast, dendritic cells treated with
TNF- alone displayed a factor pattern typical for an intermediate
state of dendritic cell maturation characterized by a lower CD83 and
CD25 expression, when compared with monocyte-conditioned medium-treated dendritic cells (data not shown). Thus, the expression patterns of Rel and octamer factors are useful additional markers for
determining the maturation state of dendritic cells.
| |
Acknowledgments |
The authors thank C. Staskewitz and C. Kurzmann for excellent technical
assistance and Drs N. R. Rice and T. Wirth for gifts of reagents. We
are indebted to Drs A. McLellan, A. Schimpl, F. Weih, A. Wilisch-Neumann, and T. Wirth for critical reading of the manuscript.
| |
Footnotes |
Submitted June 21, 1999; accepted September 6, 1999.
Supported by a grant from the German Ministry of Education and Research
(01GE9602/4 and IZKF Würzburg to E.K.) and grants from the
Deutsche Forschungsgemeinschaft, SFB 465 (to E.K., H.W.F. and E.S.),
and NE 608/1-2 (to M.N.). M.N. is also supported by a fellowship from
the habilitation program of the Deutsche Forschungsgemeinschaft (NE
608/2-1).
The first 2 authors contributed equally to this work.
Reprints: M. Neumann, Institute of Pathology,
Department of Molecular Pathology, University of Würzburg,
Josef-Schneider-Strasse 2, D-97080 Würzburg, Germany.
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.
| |
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Top
Abstract
Introduction