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INTRODUCTION |
Dendritic cells (DCs)1
are highly specialized antigen-presenting cells that have an essential
role in the initiation and control of the cytotoxic T cell response. As
"professional" antigen-presenting cells, they are specialized to
take up, process, and present soluble antigens in complexes with either
class I or class II MHC molecules (1, 2). They are present in most
tissues in a relatively immature state, but in the presence of
inflammatory signals, they rapidly take up foreign antigens and undergo
maturation into potent antigen-presenting cells that migrate to
lymphoid organs where they initiate an immune response. Their
phenotypic and functional characteristics are intimately linked to
their stage of maturation. However, the specific genes whose expression
mediates differentiation of pluripotent progenitors to DCs are largely
undefined. The generation of large numbers of DCs has become feasible
through the in vitro culturing of progenitors using
exogenous hematopoietic cytokines to support their growth,
differentiation, and maturation (3, 4). Human myeloid DCs can be
generated from various sources, including blood, bone marrow, and
CD34+ stem cells. Monocytes from peripheral blood have
served as a ready source for generating myeloid DCs in vitro
following incubation with granulocyte-macrophage colony-stimulating
factor (GM-CSF) and interleukin-4 (IL-4) for use in immunotherapy
(4-6). Thus, DCs have become accessible for detailed molecular and
cell biological analysis and for clinical applications.
Microarrays and proteomics technologies for identifying the
mRNA and protein constituents of living organisms and determining
their pattern of expression are emerging (7-9). Few studies have been
undertaken that simultaneously analyzed cell populations at both RNA
and protein levels. Potential sources of discordance between RNA and
protein levels include translational control and altered protein
stability. Additionally, proteomic analysis may uncover
post-translational modifications that are not predictable at the RNA
level. Here we used in vitro cultures of circulating
CD14+ monocytes treated with GM-CSF and IL-4 followed by
treatment with TNF-
, in order to analyze systematically gene
expression during DC differentiation and maturation, using both
oligonucleotide microarrays and proteomics.
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EXPERIMENTAL PROCEDURES |
Generation of DCs from Peripheral Blood--
Generation of DCs
was performed as described previously (10). Peripheral blood
mononuclear cells (PBMCs) were obtained from leukapheresis specimens of
normal donors after Ficoll-Paque density gradient separation. PBMCs
were washed twice in Hank's balanced salt solution (Life Technologies,
Inc.) and were resuspended in XVIVO-15 medium (BioWhittaker,
Walkersville, MD). PBMCs were incubated with anti-CD14 monoclonal
antibody coated with microbeads (Miltenyi Biotec, Auburn, CA), and
CD14+ monocytes were isolated by passing the PBMCs through
a magnetic cell separation system (Miltenyi Biotec). CD14+
monocytes (2 × 107-4 × 107) were
cultured in XVIVO-15 medium containing GM-CSF (100 ng/ml) and IL-4 (50 ng/ml). After 7 days of culture, fresh XVIVO-15 medium containing
GM-CSF (100 ng/ml) and IL-4 (50 ng/ml) plus TNF-
(10 ng/ml) was
added to the cells for 7 additional days. All cytokines were purchased
from PeproTech (Rocky Hill, NJ).
Cell Surface Antigen Analysis--
The analysis of cell surface
antigens was performed by direct immunofluorescence (FACScan, Becton
Dickinson, Mountain View, CA). Cells were washed twice with culture
cell medium and incubated for 30 min on ice with each test monoclonal
antibody diluted to the optimal concentration for immunostaining.
Labeled cells were then washed, fixed in 1% paraformaldehyde, and
analyzed for fluorescence. Data analysis was based on examination of
10,000 cells/sample. Staining was performed with the following FITC-
and phycoerythrin (PE)-labeled monoclonal antibodies: FITC-CD1a,
FITC-CD14, FITC-HLA-DR, FITC-CD83, FITC-mouse IgG1, FITC-mouse IgG2
(all from PharMingen, San Diego, CA); PE-CD86 (Coulter/Immunotech,
Miami, FL); and PE-mouse IgG1 (Becton Dickinson). Primary antibodies
were compared with the appropriate isotype-matched controls.
Preparation of cRNA and Gene Chip Hybridization--
Total RNA
was isolated using Trizol reagent (Life Technologies, Inc.) and used to
generate cRNA probes. Preparation of cRNA, hybridization, and scanning
of the HuGeneFL arrays were performed according to the manufacturer's
protocol (Affymetrix, Santa Clara, CA). Briefly, 5 µg of the RNA was
converted into double-stranded cDNA by reverse transcription using
a cDNA synthesis kit (SuperScript Choice, Life Technologies, Inc.)
with an oligo(dT)24 primer containing a T7 RNA polymerase
promoter site added 3' of the poly(T) (Genset, La Jolla, CA). After
second-strand synthesis, labeled cRNA was generated from the cDNA
sample by an in vitro transcription reaction supplemented
with biotin-11-CTP and biotin-16-UTP (Enzo, Farmingdale, NY). The
labeled cRNA was purified by using RNeasy spin columns (Qiagen,
Valencia, CA). Fifteen micrograms of each cRNA was fragmented at
94 °C for 35 min in fragmentation buffer (40 mM Tris
acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate) and then used to prepare 300 µl of
hybridization mixture (100 mM MES, 0.1 mg/ml herring sperm
DNA (Promega), 1 M sodium chloride, 10 mM Tris,
pH 7.6, 0.005% Triton X-100) containing a mixture of control cRNAs for
comparison of hybridization efficiency between arrays and for relative
quantitation of measured transcript levels. Before hybridization, the
cRNA samples were heated at 94 °C for 5 min, equilibrated at
45 °C for 5 min, and clarified by centrifugation (14,000 × g) at room temperature for 5 min. Aliquots of each sample (10 µg of cRNA in 200 µl of the master mix) were hybridized to HuGeneFL Arrays at 45 °C for 16 h in a rotisserie oven set at 60 rpm then washed with non stringent wash buffer (6 × saline/sodium phosphate/EDTA) at 25 °C, followed by stringent wash buffer (100 mM MES (pH 6.7), 0.1 M NaCl, 0.01% Tween 20) at 50 °C,
stained with streptavidin-phycoerythrin (Molecular Probes), washed
again with 6 × saline/sodium phosphate/EDTA, stained with
biotinylated anti-streptavidin lgG, followed by a second staining with
streptavidin-phycoerythrin, and a third washing with 6 × saline/sodium phosphate/EDTA. The arrays were scanned using the
GeneArray scanner (Affymetrix). Data analysis was performed using
GeneChip 4.0 software. The software includes algorithms that determine
whether a gene is absent or present and whether the expression level of
a gene in an experimental sample is significantly increased or
decreased relative to a control sample. To assess differences in gene
expression, we selected genes based on a sort score value equal or
greater than 2. The sort score is calculated by Affymetrix software by
using a combination of actual values of the average differences.
Two-dimensional Polyacrylamide Gel Electrophoresis--
The
procedure followed was as described previously (11). Cells were
solubilized in 200 µl of lysis buffer containing 9.5 M
urea (Bio-Rad), 2% Nonidet P-40, 2% carrier ampholytes pH 4-8 (Gallard/Schlessinger, Carle Place, NY), 2%
-mercaptoethanol, and
10 mM phenylmethanesulfonyl fluoride. Aliquots containing ~5 × 106 cells were applied onto isofocusing gels.
Isoelectric focusing was conducted using pH 4-8 carrier ampholytes at
700 V for 16 h, followed by 1000 V for an additional 2 h. The
first-dimension gel was loaded onto the second-dimension gel, after
equilibration in 125 mM Tris, pH 6.8, 10% glycerol, 2%
SDS, 1% dithiothreitol, and bromphenol blue. For the second-dimension
separation, a gradient of 11-14% of acrylamide (Serva, Crescent
Chemical, Hauppauge, NY) was used. Proteins were transferred to an
Immobilon-P polyvinylidene difluoride membrane (Millipore, Bedford, MA)
or visualized by silver staining of the gels (11, 12). Phosphoproteins
were visualized by phosphorimaging technology.
Radioactive Labeling and Heat Shock
Treatment--
[32P]Orthophosphate labeling was
performed by preincubating the cells for 2 h with 200 µCi/ml
32PO
(Amersham Pharmacia
Biotech) in phosphate-free culture medium (Life Technologies, Inc.).
Heat shock treatment was then performed by incubating the cells for 1 h at 42-44 °C as described previously (12, 13).
Western Blotting--
Following transfer, membranes were
incubated for 2 h in blocking buffer containing 5% milk in 10 mM Tris-HCl, pH 7.5, 2.5 mM EDTA, pH 8, 50 mM NaCl. The membranes were incubated for 2 h with
antibodies against calreticulin, SPA-600 (StressGen, Victoria, Canada),
T-19 (Santa Cruz Biotechnology, Santa Cruz, CA), vimentin, V9 (Santa
Cruz Biotechnology), or FABP5 (kindly provided by Professor Celis,
University of Aarhus, Denmark) at a dilution 1:1,000,000, 1:10,000,
1:100,000, and 1:1000, respectively. The membranes were then incubated
for 1 h with horseradish peroxidase-conjugated anti-rabbit
(Amersham Pharmacia Biotech) or anti-goat (Sigma) IgG antibodies, at a
dilution 1:1000. Immunodetection was accomplished by enhanced
chemiluminescence (ECL) (Amersham Pharmacia Biotech) followed by
autoradiography on hyperfilm MP (Amersham Pharmacia Biotech).
In-gel Enzymatic Digestion--
The two-dimensional gels were
silver-stained by successive incubations in 0.02% sodium thiosulfate
for 2 min, 0.1% silver nitrate for 40 min, and 0.014% formaldehyde
plus 2% sodium carbonate. The proteins of interest were excised from
the two-dimensional gels and destained for 5 min in 15 mM
potassium ferricyanide and 50 mM sodium thiosulfate as
described (14). Following 3 washes with water, the gel pieces were
dehydrated in 100% acetonitrile for 5 min and dried for 30 min in
a vacuum centrifuge. Digestion was performed by addition of 100 ng of
trypsin (Promega, Madison, WI) in 200 mM ammonium
bicarbonate or by the addition of 100 ng of the endoproteinase Glu-C
(Promega, Madison, WI) in 100 mM ammonium bicarbonate. The
Lys-C digestion was performed with 500 ng of the endoproteinase Lys-C
(Roche Molecular Biochemicals) in 100 mM Tris-HCl, pH 9. Following enzymatic digestion overnight at 37 °C, the peptides were
extracted twice with 50 µl of 60% acetonitrile, 1% trifluoroacetic
acid. After removal of acetonitrile by centrifugation in a vacuum
centrifuge, the peptides were concentrated by using pipette tips C18
(Millipore, Bedford, MA).
Mass Spectrometry--
Analyses were performed primarily using a
Perspective Biosystem matrix-assisted laser desorption ionization-time
of flight (MALDI-TOF) Voyager-DE mass spectrometer (Framingham, MA),
operated in delayed extraction mode. Peptide mixtures were analyzed
using a saturated solution of
-cyano-4-hydroxycinnamic acid (Sigma) in acetone containing 1% trifluoroacetic acid (Sigma). Peptides were
selected in the mass range of 800-4000 Da. Spectra were calibrated using calibration mixture 2 of the Sequazyme peptide mass standards kit
(Perspective Biosystems, Framingham, MA). The search program MS-Fit,
developed by the University of California at San Francisco, was
used for searches in the data base NCBI. Search parameters were as
follows: maximum allowed peptide mass error of 400 ppm, consideration
of one incomplete cleavage per peptide, and pH range between 4 and 8. MALDI-TOF mass spectrometry was also used for molecular weight
determination as described (15). In some cases, the amino acid sequence
of some peptides of interest was determined by electrospray
ionization-mass spectrometry analysis.
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RESULTS |
Surface Phenotype of CD14+ Monocytes-derived
DCs--
Differentiation of CD14+ blood monocytes into
mature dendritic cells can be induced in vitro by treatment
with a combination of GM-CSF, IL-4, and TNF-
(10, 16). We isolated
CD14+ cells from PBMCs obtained from leukapheresis
specimens of healthy donors. Adherent CD14+ monocytes were
cultured for 7 days in the presence of GM-CSF (100 ng/ml) and IL-4 (50 ng/ml) followed by 7 additional days in the presence of GM-CSF (100 ng/ml), IL-4 (50 ng/ml), and TNF-
(10 ng/ml). The differentiation
stage of the cells was determined by two criteria, morphology and cell
surface expression of specific markers. At day 7, the DCs displayed
phenotypic and morphologic characteristics of immature DCs. The cells
expressed CD1a, the costimulatory molecule CD86 (50 and 52% positive
cells, respectively), high levels of MHC class II antigens, while being
negative for the monocyte marker CD14, as determined by
fluorescence-activated cell sorter analysis. Following further culture
in the presence of TNF-
for 7 days, almost all the cells exhibited
high levels of HLA-DR, CD86, and CD83, which represent markers of
mature DC (Fig. 1) (16-19). Development
of the dendrite/neurite morphology was progressively more prevalent and
pronounced after addition of TNF-
(data not shown). Furthermore,
up-regulation of CD83 expression presented the same temporal
kinetics as the morphologic changes.

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Fig. 1.
Surface phenotype of DCs derived from
CD14+ monocytes. CD14+ blood monocytes
were cultured in XVIVO-15 serum-free medium containing GM-CSF (100 ng/ml) and IL-4 (50 ng/ml) for 7 days (A) or for an
additional 7-day period after the addition of TNF- (10 ng/ml)
(B). Cells were washed and then incubated with various
antibodies as indicated. The data are shown as histograms
depicting the number of cells (y axis) exhibiting various
fluorescence intensities (x axis). The black
histograms represent the isotype-matched control, and the
white histograms represent staining with specific
antibodies.
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Analysis of Overall Gene Expression in CD14+ Monocytes
and Their Derived DCs by Oligonucleotide Arrays--
Three independent
differentiation experiments were performed, and RNA transcript levels
for different genes were determined at day 1 (CD14+
monocytes) and after 7 days of GM-CSF/IL-4 treatment (immature DCs) and
14 days of GM-CSF/IL-4 plus TNF-
treatment (mature DCs), using
oligonucleotide arrays. Transcripts for ~40% of the 6,300 unique
genes assessed were detected in all the cell populations tested. We
identified a subset of genes that differed in their expression levels
during DC differentiation and maturation, by 2.5-fold or greater, in
all three experiments. The 255 genes identified are presented in
Tables I and II, for up-regulated and
down-regulated genes, respectively. The
number of genes whose expression decreased upon DC differentiation
and/or maturation was as large as the number of genes whose expression
increased. In addition, comparison of overall gene expression between
immature and mature DCs showed only few differences. Genes known to be
differentially expressed during DC differentiation changed their
expression accordingly in our analysis. This group included the
monocytic marker CD14, CD163, and C5a anaphylatoxin receptor (CD88),
which were strongly down-regulated, and the cell surface proteins CD1a,
CD1b, CD1c, CD36, CD59, CD83, CD86, and CCR7, which were up-regulated
with DC differentiation and maturation. Up-regulation of Fc-
RII and Fc-
RII, of several genes encoding for MHC class II, and of genes encoding for the secreted proteins TARC (CCR4 ligand), MCP-4, and the
macrophage-derived chemokine was also observed.
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Table I
Up-regulated mRNAs in monocyte-derived DCs
RNA from CD14+ monocytes (D1), immature (D7), and mature (D14)
DCs were hybridized onto Affymetrix oligonucleotide arrays and
quantified as indicated under "Experimental Procedures." For each
gene, the fold change was calculated by Affymetrix software; NC
indicates no change; the ~ indicates fold change calculation for
which the smaller value is replaced by an estimate of the minimum value
for detectable transcripts. PPA- is peroxisome
proliferator-activated receptor- .
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Table II
Down-regulated mRNAs in monocyte-derived DCs
RNA from CD14+ monocytes (D1), immature (D7), and mature (D14)
DCs were hybridized onto Affymetrix oligonucleotide arrays and
quantified as indicated under "Experimental Procedures." For each
gene, the fold change was calculated by Affymetrix software; NC
indicates no change; the ~ indicates fold change calculation for
which the smaller value is replaced by an estimate of the minimum value
for detectable transcripts.
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Most of the 255 genes we have uncovered were not previously known to be
expressed differentially in DCs. Novel changes included differential
expression of many cell surface molecules related to cell adhesion,
such as E-cadherin, galectin 2, CD11a/LFA-1
, ninjurin-1, macmarcks,
syndecan 2, CD44E, and presenilin 1. Transcript levels for genes
encoding for several secreted proteins increased as the cells
differentiated. These genes include the growth factor BPGF1, TGF-
,
CSF-1, semaphorin E, activin
A subunit, and the macrophage chemoattractant osteopontin. In contrast, expression of the
chemokines belonging to the IL-8 superfamily (IL-8, CTAPIII, MIP-2
,
MIP-2
, platelet factor 4 (PF4), and ENA-78) was decreased. We also
observed a decrease of neuromedin B, PEDF, and PBEF mRNAs. Expression of mRNAs encoding for proteins localized in the nuclear compartment or involved in signaling has been poorly described in DCs.
Our results demonstrate that expression of the interferon regulatory
factor 4, C/EBPa, MRG1, peroxisome proliferator-activated receptor-
, TRIP7, SLA, Rap1GAP, cAMP-dependent protein
kinase, IP3 protein kinase B, cyclophilin C, and cyclins A1, D2, G2,
and H genes, was increased. Expression of interferon regulatory factor 47A, TAL2, NAP-2, epidermal growth factor response factor 2, CtBP, IEX-1, SAP49, HRH1, I
B
, Fyb, Net, and cyclophilin F genes is decreased. Finally, the regulation of a large group of genes encoding for lipid-binding proteins or enzymes involved in lipid metabolism was
observed. Levels of acyl-CoA thioester hydrolase, 15-lipoxygenase, lysosomal acid lipase, lipoprotein lipase, lysophospholipase homolog (HU-K5), FABP3, FABP4, FABP5, apolipoproteins C-I and E, and
3-oxoacyl-CoA thiolase mRNAs were increased. Concomitantly, a
strong decrease of MRP8 and MRP14 mRNAs was observed.
Proteomic Profiling of Monocyte-derived DCs--
To identify
protein changes during the differentiation and maturation of the
monocyte-derived DCs, total proteins were extracted from
CD14+ monocytes at day 1 of culture, after 7 days of
GM-CSF/IL-4 treatment (immature DCs), and after 14 days of GM-CSF/IL-4
plus TNF-
treatment (matures DCs), as for microarray analysis.
Proteins were separated by two-dimensional gel electrophoresis, and
following silver staining, the gels were digitized. Two-dimensional
protein patterns were matched by computer analysis. In this study, 900 protein spots were matched and quantitated. Whereas the overall
two-dimensional patterns of CD14+ monocytes and immature
and mature DCs were largely similar, numerous protein changes were
reproducibly detected. As for the microarray analysis, we selected
protein spots whose intensities changed in all experiments by 2.5-fold
or greater during DC differentiation or maturation. A set of 37 proteins was identified. Fig. 2 shows the
position of the 37 regulated proteins (25 up- and 12 down- regulated)
in a two-dimensional pattern of immature DCs. As observed by microarray
analysis, most changes occurred during the first 7 days of culture,
whereas only few additional changes were observed between 7 and 14 days
in culture.

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Fig. 2.
Two-dimensional profiles of DCs.
A, up-regulated (black arrows) and down-regulated
(white arrows) protein spots are reported on a
representative two-dimensional gel corresponding to protein expression
profile of immature DCs. These results are representative of three
independent experiments. B, close-up sections of
silver-stained two-dimensional gels from CD14+ monocytes
(left panel) and mature DCs (right panel),
corresponding to based sections in A, are shown for
comparison.
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Identification of Differentially Expressed Proteins--
In
order to identify the proteins of interest, additional two-dimensional
gels were produced with the same cellular extracts and silver-stained
as described under "Experimental Procedures." The 37 proteins of
interest were then excised from the gels, digested with trypsin, and
subsequently analyzed by MALDI-TOF mass spectrometry. The
resulting spectra were used to identify the proteins, using the
MS-FIT search program. Of the 37 spots excised from the gels, 18 were
identified without ambiguity, consisting of 11 up-regulated and 7 down-regulated proteins (Table III).
Specific antibodies confirmed the identification based on mass
spectrometry for all proteins analyzed by Western blotting (see Table
III). The proteins identified were members of specific families
including chaperones, Ca2+-binding proteins, fatty-acid
binding proteins, and structural proteins. Expression of three
members of the fatty-acid binding protein (FABP) family, FABP4, FABP5,
and acyl-CoA-binding protein, was highly increased after 7 days of
culture. The increased protein and RNA levels for these genes were
concordant (Table I). Concomitant with the up-regulation of FABP4
and FABP5, we observed a strong down-regulation of two members of the
S100 family, the myeloid-related proteins MRP14 and MRP8.
Interestingly, it has been recently shown that the heterodimer
MRP8/MRP14, designated fatty acid p34 (FA-p34), exerts a fatty acid
binding activity (20-22). MRP14 and MRP8 down-regulation was
progressive upon DC differentiation and maturation, leading to a 9- and
12-fold decrease in spot intensities, respectively. Again, the results
obtained for these two genes at both the RNA and protein levels were
highly concordant (Table II).
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Table III
Identification by mass spectrometry of the regulated protein-spots
during DC differentiation
Analysis by MALDI-TOF-MS of the tryptic peptide profiles of the
protein-spots followed by search in the NCBI database.
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There were discrepancies between the protein and gene expression
data for vimentin and hsp27 that were previously shown to be induced at
the mRNA level during DC differentiation (25). Close analysis of
the microarray hybridization data showed saturation level intensities
for these genes resulting from their high level expression. Therefore,
the discordance between mRNA and protein levels observed in our
data for these genes most likely reflects their high expression level,
reaching saturation at the RNA hybridization level using microarrays
but not at the protein level using two-dimensional gels. Vimentin and
hsp27 proteins can be resolved into several isoforms on two-dimensional
gels. Therefore, we wished to analyze the expression of these isoforms
in DCs, by Western blotting using specific antibodies. Four vimentin
spots, including two (spots 278 and 279) previously identified by mass
spectrometry and two additional spots (spots 237 and 327), were
revealed by Western blotting using a specific antibody against vimentin
(Fig. 3). All four spots increased in
intensity with DC differentiation (see Fig. 2). There was no detectable
differential expression of the four isoforms of vimentin during DC
differentiation. Hsp27 was resolved by two-dimensional polyacrylamide
gel electrophoresis into four isoforms, a nonphosphorylated (hsp27A;
pI = 6.6) and three phosphorylated forms (hsp27B, -C, and -D;
pI = 6.2, 5.7 and 5.5, respectively), as described previously (12,
13). Spot 574 was identified by mass spectrometry as corresponding to
the unphosphorylated hsp27A isoform, and spot 570 was identified as the
phosphorylated form hsp27B. Expression of both hsp27A (spot 574) and
hsp27B (spot 570) was increased during DC differentiation, whereas the
hyperphosphorylated forms hsp27C and hsp27D remained undetectable (Fig.
4A). Heat shock treatment of
either CD14+ monocytes or DCs, preincubated with
[32P]orthophosphate, resulted in the induction of all
phosphorylated forms of hsp27 (hsp27B, -C, and -D) (Fig.
4B). A decrease in the unphosphorylated form hsp27A in
response to heat shock treatment correlated with an increase in
hsp27-phosphorylated forms, as determined by silver staining (data not
shown). These results suggest that the increase in hsp27 expression
observed during DC differentiation was not due to a stress response of
the cells but was specific to their differentiation stage and that
phosphorylation of hsp27 was not modulated during DC
differentiation.

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Fig. 3.
Identification of four vimentin
isoforms. Close-up sections of silver-stained two-dimensional gel
of DCs (left panel) and of a Western blot using a specific
monoclonal antibody V9 against vimentin (right panel).
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Fig. 4.
hsp27 regulation during DC differentiation
and heat shock. A, close-up sections of silver-stained
two-dimensional gels from CD14+ monocytes and DCs showing
an increase in the unphosphorylated form hsp27A and a minor increase in
the phosphorylated form hsp27B during DC differentiation. B,
CD14+ monocytes untreated or treated for 7 days in the
presence of GM-CSF and IL-4 were labeled for 2 h with
[32P]orthophosphate and subjected (HS) or not
(C) to a heat shock treatment of 45 min at 42 °C.
Two-dimensional electrophoresis and autoradiography showed that all the
phosphorylated forms hsp27B, -C, and -D were induced in both cell
types.
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Identification of a Novel Calreticulin Isoform--
The
calreticulin protein was found to be down-regulated with DC
differentiation in our two-dimensional gel analysis, whereas the
corresponding transcript was unchanged at the RNA level by microarray
analysis. Hybridization data for calreticulin transcript did not show
any saturation. Interestingly, a protein (spot 412) with an estimated
molecular mass of 32 kDa and pI of 4.1 was found to be induced
in immature DCs (Fig. 2). After enzymatic digestions using trypsin or
endoproteinase Lys-C and analysis of the resulting peptides by
MALDI-TOF mass spectrometry, the peptide masses were consistent with
those of peptides derived from calreticulin, a protein with a mass of
48 kDa and a pI of 4.3. Calreticulin is a chaperone protein localized
in the endoplasmic reticulum (23, 24), and no forms of calreticulin
with a mass of 32 kDa have been reported previously. Interestingly, the
peptides obtained from the tryptic and Lys-C digestions matched only
with the C-terminal portion of calreticulin (Table
IV). Additional enzymatic digestions using the endoproteinase Glu-C were performed. Peptides that exhibited high intensities after Lys-C or Glu-C digestions were further analyzed
by electrospray ionization-mass spectrometry in order to obtain the
sequence of these peptides. Two peptides were identified as
LIVRPDNTYEVK and LIVRPDNTYE. These two peptides, obtained after different digestions, contained the same N-terminal end and therefore correspond to the N-terminal end of the protein. The molecular mass and
pI values of the novel calreticulin form were calculated as 28825.69 Da
and 4.07, respectively, in agreement with the mass determined by
MALDI-TOF (29 kDa) and with the mass/pI estimated based on migration in
two-dimensional gels. Altogether, these results indicated that the
protein in spot 412 is a cleavage product of calreticulin,
corresponding to the C-terminal end (amino acids 157-400) (Fig.
5A). We designated this newly
identified form of calreticulin as Crt32. Identification of spot 412 as
the C-terminal portion of calreticulin was further confirmed by Western
blotting using two specific antibodies against calreticulin, SPA-600
and T-19 antibodies, produced against a C-terminal and an N-terminal peptide, respectively. Spot 412 was revealed by SPA-600 antibody (Fig.
5B) but not by T-19 antibody (data not shown). Three
additional spots (413, 414, and 415), present only in DCs, were also
recognized by the antibody produced against the C-terminal region of
calreticulin. These three isoforms remain to be characterized.
Concomitant with the increase in Crt32 levels, full-length calreticulin
(spots 138 and 182) was decreased during DC differentiation and
maturation. These data suggested that calreticulin is most likely
cleaved during DC differentiation yielding Crt32. Therefore, whereas
microarray analysis did not show any changes involving calreticulin,
proteomic analysis allowed the detection of a post-translational
modification of calreticulin occurring during DC differentiation.
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Table IV
Assignment of peptide masses to the human calreticulin sequence
Analysis by MALDI-TOF-MS of the peptide masses followed by search in
the NCBI database.
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Fig. 5.
Characterization of the calreticulin isoform,
Crt32. A, schematic representation of the full-length
and the Crt32 fragment of calreticulin. Calreticulin is divided into
three domains as follows: the N-domain, the P-domain, and the C-domain.
The protein contains an N-terminal signal sequence (17 amino acids)
represented by a black box. The P-domain is a site of
chaperone activity and high affinity Ca2+ binding. The
C-domain is a site of high capacity Ca2+ binding and
contains the KDEL endoplasmic reticulum retrieval signal. The
calreticulin product Crt32 (amino acids 157-400) contains the
P-domain and C-domain. B, close-up sections of
silver-stained two-dimensional gels from DCs (left panel)
and of a Western blot using a specific polyclonal antibody
SPA-600 directed against the C-terminal region of the calreticulin
(right panel).
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DISCUSSION |
DCs are professional antigen-presenting cells that are critically
involved in the initiation of a primary immune response (1, 2). DCs
acquire their function with differentiation that occurs through a
programmed expression of specific proteins. In order to develop a
better understanding of DC differentiation, we utilized two
complementary approaches to identify specific genes regulated during DC
differentiation and maturation. One approach relies on the quantitative
analysis of mRNAs by oligonucleotide microarrays. The other
approach relies on quantitative analysis and identification of proteins
by proteomics. Indeed, proteins represent the most functional
compartment of a cell, and the information obtained at the protein
level cannot simply be predicted from examining expression at the RNA
level. The proteomics approach is also appropriate to identify
post-translational modifications, which may regulate protein function.
A systematic analysis of genes that are differentially expressed with
monocyte-derived DC differentiation using SAGE has been reported
recently (25). Most of the genes described as differentially expressed
were also found to be differentially expressed in our study. In
addition, we have uncovered a large number of additional genes. We
identified close to 4% of the genes and proteins analyzed as regulated
during DC differentiation. The regulated genes were in major part
related to cell adhesion and motility, growth control, regulation of
the immune response and antigen presentation, and lipid metabolism. These include all genes previously reported to be modulated during DC
differentiation. A large number of additional genes not previously reported in DCs have been identified in this study.
Immature DCs traffic from the blood to tissues where they take up and
process antigens. DCs subsequently migrate to the draining lymphoid
organs where they are converted to mature DCs with up-regulation of
co-stimulatory and HLA molecules, resulting in priming of naive T
cells. Interestingly, we identified a large number of genes encoding
for proteins involved in cell adhesion and motility that are regulated
during DC differentiation. Expression of galectin 2, CD11a/LFA-1
,
ninjurin 1, macmarcks, syndecan 2, CD44E, and presenilin 1 was
down-regulated. Expression of secreted proteins involved in cell
motility, autotaxin-t and semaphorin E, reported to play a role in axon
guidance in the nervous system (26), was up-regulated. Up-regulation of
the cytoskeleton-related proteins, the macrophage capping protein, and
vimentin, all involved in cell motility (27, 28), were also observed
during DC differentiation. Therefore, the concomitant decrease in
expression of integrins and cell adhesion molecules, the increase in
expression of genes involved in cell motility, and regulated expression
of enzymes such as
1-antitrypsin and macrophage
metalloelastase (HME) likely have an effect on the enhanced migration
properties of DCs compared with their precursors. HME belongs to the
family of related matrix-degrading enzymes that are important in tissue
remodeling and repair during development and inflammation (29).
Differentiation of DCs was accompanied by differential expression of
genes involved in the immune response. Noticeable was the up-regulation
of genes encoding anti-inflammatory proteins such as cyclophilin C and
TSG-6 (30, 31) with a concomitant decrease in the production of
pro-inflammatory cytokines. Several genes encoding pro-inflammatory
cytokines and their receptors, such as prointerleukin-1
, TNF-
,
CD163, C5a anaphylatoxin receptor, IL-6 receptor, and TNF receptor,
were down-regulated. A noticeable change was the down-regulation of a
set of chemokines belonging to the IL-8 superfamily such as CTAPIII,
MIP2-
, MIP2-
, ENA78, PF4, and IL-8. It has been reported that
these chemokines are pro-inflammatory cytokines that act as potent
neutrophil chemoattractants and activators (32). Interestingly, these
chemokines that were coordinately down-regulated have been co-localized
to the same genomic region (33). Osteopontin, a key cytokine involved
in T lymphocyte activation (34), was up-regulated. The maturation of
DCs was accompanied by the up-regulation of Mac-2-binding protein. Mac-2-binding protein is an adhesion molecule with a potent immune stimulatory activity. Indeed, it has been demonstrated that
Mac-2-binding protein stimulates host defense systems, such as NK and
LAK cell activities and induces the secretion of IL-2 (35).
Up-regulation of TGF-
was also observed during DC maturation.
An important function of DCs is antigen uptake, processing, and
presentation. As expected, mRNAs for Fc-
RII and Fc-
RII as
well as for several MHC class II genes were up-regulated. A marked
increase in macrophage mannose receptor (MRC1) RNA was observed. MRC1
is involved in the capture of antigens by immature DCs and in their
delivery to MHC class II compartments (36). Several proteins known for
their chaperone activity including hsp73, hsp27, and calreticulin were
also regulated during DC differentiation. An emerging hypothesis is
that heat shock proteins participate in antigen processing and
presentation and play a central role in the activation of T lymphocytes
by DCs (37-39). hsp70 targets immature DC precursors to enhance
antigen uptake (40). We observed an up-regulation of hsp73 protein,
related to the hsp70 family (41), during DC differentiation. hsp73 has
been recently reported to bind specifically to the cell surface of
monocytic and dendritic cell lines and to be internalized spontaneously
by receptor-mediated endocytosis (38). In addition, the murine hsp73
has been recently reported to accumulate in exosomes from immature DCs
(42). The role of hsp27 up-regulation during DC differentiation is less clear. It has been reported that an increase in cellular levels of
hsp27 promotes a resistance of monocytes to apoptotic cell death (43,
44). Increased hsp27 expression in DCs may therefore have a protective
role against cytotoxicity. In contrast to hsp27 and hsp73, the cognate
chaperone protein calreticulin was down-regulated during DC
differentiation due to post-translational modification. In addition,
whereas the expression of hsp27 and hsp73 was maximal in immature DCs,
calreticulin was mostly down-regulated during DC maturation.
Calreticulin participates in the assembly of MHC class I with peptide
and
2-microglobulin in the endoplasmic reticulum, a
process required for the presentation of antigenic peptides to
cytotoxic T lymphocytes at the cell surface (23, 24). In addition, it has been reported recently that calreticulin elicits tumor- and peptide-specific immunity (45). Calreticulin displays in vivo peptide binding activity and can elicit
cytotoxic T lymphocyte responses against bound peptides (46).
Proteomic analysis of DCs allowed us to identify a truncated form of
calreticulin, present only in DCs. We designated this novel form of
calreticulin as Crt32. This form contains the P-domain, a site of
chaperone activity, the C-domain, which contains the endoplasmic
reticulum retrieval sequence, but lacks the N-domain. A calreticulin
fragment corresponding to the N-domain has been recently purified from
the supernatant of an Epstein-Barr virus-immortalized cell line. This
fragment, named vasostatin, is an angiogenesis inhibitor that exerts
antitumor effects in vivo (47, 48). Therefore, even though
the C-terminal end of vasostatin has not been characterized precisely,
Crt32 most likely corresponds to the complementary part of vasostatin, following cleavage of calreticulin. A decrease in levels of the cognate
form of calreticulin and an increase in Crt32 levels may be relevant to
DC function, and the precise function(s) of Crt32 in mature DCs is
currently under investigation.
This study suggests a role for genes involved in lipid metabolism in DC
function. Several genes encoding enzymes or proteins involved in the
production, uptake, transport, and solubilization of cholesterol and
fatty acids were up-regulated in DCs. This group includes
apolipoprotein E, apolipoprotein C-I, ABCG1, lysosomal acid lipase, and
lipoprotein lipase. The fatty acids are translocated from the
extracellular environment to the cytoplasm by the fatty-acid translocase (FAT/CD36) and then solubilized and transported by FABPs to
the site where they are metabolized (49). We observed marked
up-regulation of CD36 as well as of the lipid-binding proteins FABP3,
FABP4, FABP5, CRABPII, and acyl-CoA-binding protein. Up-regulation of
FABPs was concomitant with a strong down-regulation of the S100
proteins, MRP8 and MRP14. Interestingly, MRP8 and MRP14 are expressed
by myeloid cells during inflammatory reactions, and it has been
reported that MRP-8/MRP-14 heterodimer (FA-p34) has a fatty acid
binding activity and specifically binds (poly)unsaturated fatty acids
(20-22). In contrast, FABP4 and FABP5 bind saturated or
(mono)unsaturated fatty acids with a high affinity. Long chain fatty
acids and acyl-CoA esters affect a large number of cell functions
during cell growth and differentiation, including signal transduction,
gene regulation, ion channel activities, and membrane fusion (49, 50).
In this context, we observed an up-regulation of 15-lipoxygenase that
promotes the formation of lipoxins that are modulators of leukocyte
recruitment (51-53).
The oligonucleotide array and proteomics analyses undertaken in this
study have uncovered novel genes and proteins with potential roles in
DC function, differentiation and/or maturation. Microarray analysis has
identified important changes in genes involved in cell adhesion and
motility, immune response, growth control, as well as in lipid
metabolism. Following the simultaneous analysis of several thousand
genes at the mRNA level, the challenge is to utilize efficiently
this information to develop a better understanding of DC function. This
study also demonstrates that a proteomics approach may provide
information that could not be obtained at the RNA level, due either to
poor correlation between mRNA and protein levels or due to
post-translational modifications that may result in several isoforms
generated from one mRNA, as in the case of calreticulin in our
study. Genes and proteins identified to be expressed selectively in DCs
may provide further understanding of the biological function of DCs in
host defense system and of the mechanisms of antigen processing and presentation.