Profiling Changes in Gene Expression during Differentiation and Maturation of Monocyte-derived Dendritic Cells Using Both Oligonucleotide Microarrays and Proteomics*

Dendritic cells (DCs) are antigen-presenting cells that play a major role in initiating primary immune responses. We have utilized two independent approaches, DNA microarrays and proteomics, to analyze the expression profile of human CD14+ blood monocytes and their derived DCs. Analysis of gene expression changes at the RNA level using oligonucleotide microarrays complementary to 6300 human genes showed that ∼40% of the genes were expressed in DCs. A total of 255 genes (4%) were found to be regulated during DC differentiation or maturation. Most of these genes were not previously associated with DCs and included genes encoding secreted proteins as well as genes involved in cell adhesion, signaling, and lipid metabolism. Protein analysis of the same cell populations was done using two-dimensional gel electrophoresis. A total of 900 distinct protein spots were included, and 4% of them exhibited quantitative changes during DC differentiation and maturation. Differentially expressed proteins were identified by mass spectrometry and found to represent proteins with Ca2+ binding, fatty acid binding, or chaperone activities as well as proteins involved in cell motility. In addition, proteomic analysis provided an assessment of post-translational modifications. The chaperone protein, calreticulin, was found to undergo cleavage, yielding a novel form. The combined oligonucleotide microarray and proteomic approaches have uncovered novel genes associated with DC differentiation and maturation and has allowed analysis of post-translational modifications of specific proteins as part of these processes.

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 ϩ 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)(8)(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 ϩ 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.
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 FITCand phycoerythrin (PE)-labeled monoclonal antibodies: FITC-CD1a, FITC-CD14, FITC-HLA-DR, FITC-CD83, FITC-mouse IgG1, FITCmouse 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 g 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 3Ј 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 l 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 ϫ g) at room temperature for 5 min. Aliquots of each sample (10 g of cRNA in 200 l 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 ϫ 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 ϫ saline/sodium phosphate/EDTA, stained with biotinylated anti-streptavidin lgG, followed by a second staining with streptavidin-phycoerythrin, and a third washing with 6 ϫ 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.
In-gel Enzymatic Digestion-The two-dimensional gels were silverstained 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 twodimensional 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 l 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 Ϫ2 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.

RESULTS
Surface Phenotype of CD14 ϩ Monocytes-derived DCs-Differentiation of CD14 ϩ 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 ϩ cells from PBMCs obtained from leukapheresis specimens of healthy donors. Adherent CD14 ϩ 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 fluorescenceactivated 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). Further-more, up-regulation of CD83 expression presented the same temporal kinetics as the morphologic changes.
Analysis of Overall Gene Expression in CD14 ϩ 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 ϩ 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 ϳ40% 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.
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,  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 ϩ 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 ϩ 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 twodimensional 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.
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, Ca 2ϩ -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).
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 ϭ 6.6) and three phosphorylated forms (hsp27B, -C, and -D; pI ϭ 6.2, 5.7 and 5.5, respectively), as DEDEEDEEDKEEDEEEDVPGQAKDEL 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 ϩ monocytes or DCs, preincubated with [ 32 P]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. 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 ionizationmass 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 Cterminal 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. 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 matu- 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 Ca 2ϩ binding. The Cdomain is a site of high capacity Ca 2ϩ 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 silverstained 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). ration. 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 downregulated. 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 dem-onstrated 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)(38)(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 receptormediated 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 15lipoxygenase that promotes the formation of lipoxins that are modulators of leukocyte recruitment (51)(52)(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.