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Originally published online as doi:10.1189/jlb.0307194 on June 26, 2007

Published online before print June 26, 2007
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(Journal of Leukocyte Biology. 2007;82:710-720.)
© 2007 by Society for Leukocyte Biology

Gene expression profiling during differentiation of human monocytes to macrophages or dendritic cells

Anne Lehtonen*,1,2, Helena Ahlfors{dagger},1, Ville Veckman*, Minja Miettinen*, Riitta Lahesmaa{dagger},1 and Ilkka Julkunen*,1,3

* Department of Viral Diseases and Immunology, National Public Health Institute, Helsinki, Finland; and
{dagger} Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland

3 Correspondence: Department of Viral Diseases and Immunology, National Public Health Institute, Mannerheimintie 166, FI-00300 Helsinki, Finland. E-mail: ilkka.julkunen{at}ktl.fi


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ABSTRACT
 
Macrophages and dendritic cells (DC) are APC, which regulate innate and adaptive immune responses. Macrophages function locally mainly, maintaining inflammatory responses in tissues, whereas DC take up microbes, mature, and migrate to local lymph nodes to present microbial antigens to naïve T cells to elicit microbe-specific immune responses. Blood monocytes can be differentiated in vitro to macrophages or DC by GM-CSF or GM-CSF + IL-4, respectively. In the present study, we performed global gene expression analyses using Affymetrix HG-U133A Gene Chip oligonucleotide arrays during macrophage and DC differentiation. During the differentiation process, 340 and 350 genes were up-regulated, and 190 and 240 genes were down-regulated in macrophages and DC, respectively. There were also more that 200 genes, which were expressed differentially in fully differentiated macrophages and DC. Macrophage-specific genes include, e.g., CD14, CD163, C5R1, and Fc{gamma}R1A, and several cell surface adhesion molecules, cytokine receptors, WNT5A and its receptor of the Frizzled family FZD2, fibronectin, and Fc{epsilon}R1A were identified as DC-specific. Our results reveal significant differences in gene expression profiles between macrophages and DC, and these differences can partially explain the functional differences between these two important cell types.

Key Words: microarray • GM-CSF • IL-4 • WNT5A


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INTRODUCTION
 
Macrophages and dendritic cells (DC) are APC, which regulate innate and adaptive immune responses during microbial infections [1 2 3 4 5 ]. Macrophages recognize, phagocytose, and destroy infectious agents and present microbe-specific peptides to lymphocytes. Interaction of macrophages with microbes leads to the activation of intracellular signal transduction pathways, followed by enhanced expression of cytokines and other molecules involved in an inflammatory response. Macrophages act locally mainly, by destroying microbial pathogens and maintaining inflammatory responses within the site of infection. DC reside in tissues in an immature state, on alert for possible invading pathogens. Like macrophages, DC also recognize and phagocytose microbes, but upon contact with a microbe, they undergo a maturation process, which leads to enhanced expression of cell surface adhesion molecules, production of cytokines, and reduced capacity to endocytose foreign particles [1 , 6 ]. Mature DC leave the peripheral tissues, migrate to local lymph nodes, and present antigens to naïve T cells, thus initiating the development of adaptive immune responses. Peripheral blood-derived monocytes can be differentiated in vitro to macrophages or DC, depending on the presence of differentiation-initiating cytokines, namely GM-CSF and GM-CSF plus IL-4, respectively. Humans also have other types of DC, plasmacytoid (pDC) and myeloid (mDC), which are found in blood in a differentiated but immature form [7 ].

Differentiation of monocytes from the common myeloid progenitor takes place in the bone marrow and is orchestrated by sequential expression and action of a specific set of transcription factors, such as PU.1, C/EBPß, acute myeloid leukemia, and IFN regulatory factor 8 (IRF8) [8 , 9 ]. Monocytes, which leave the bone marrow and enter the circulation, are already mature cells, capable of, e.g., phagocytosing microbes and secreting cytokines, but these functions are potentiated by further differentiation into macrophages or DC in peripheral tissues. Monocytes may differentiate into macrophages in tissues, but controversy still exists about whether monocytes actually differentiate into functional DC in vivo. Human and mouse macrophages and DC have been broadly studied by microarray analyses using various infection models and have been found to respond in cell type-specific ways to different pathogens or their components (reviewed in ref. [10 ]), reflecting their roles as primary instigators of immune responses and effectors in local infections and inflammation. The basal state of these cells has not been analyzed so far, and the molecular determinants regulating macrophage and DC responses have remained elusive. In the present work, we have analyzed, by microarray technology, the global changes in mRNA expression patterns during differentiation of human primary blood monocytes into macrophages or DC.

Our results showed that more that 1000 genes were up-regulated, and nearly 500 genes were down-regulated rapidly (within 24 h) after the initiation of the monocyte differentiation program to macrophages or DC. During the differentiation program, hundreds of genes also showed stable differences in their gene expression profile, leading to more than 200 genes, whose expression was significantly different in 7-day macrophages and DC. Examples of these genes include high expression of, e.g., cell surface adhesion molecules, cytokine receptors, WNT5A and its receptor of the Frizzled family FZD2, fibronectin (FN), and Fc{epsilon}R1A in DC and CD14, CD163, C5R1, and Fc{gamma}R1A in macrophages. Our study shows that cell types sharing a common origin of differentiation and having overlapping, functional profiles display surprisingly different patterns of gene expression.


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MATERIALS AND METHODS
 
Cell culture
Monocytes were purified from freshly collected, leukocyte-rich buffy coats obtained from healthy blood donors (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland). Human PBMC were isolated by a density gradient centrifugation over a Ficoll-Paque gradient (Amersham-Pharmacia Biotech, Uppsala, Sweden), as described previously. Mononuclear cells were collected, and monocytes were purified further as described previously [11 ]. Briefly, mononuclear cells were centrifuged over a Percoll gradient (Amersham-Pharmacia Biotech). Next, T and B cells were depleted by using anti-CD3 and anti-CD19 magnetic beads (Dynal, Oslo, Norway). Monocytes were adhered onto plastic six-well plates (Falcon, Becton Dickinson, Franklin Lakes, NJ, USA) for 1 h at 37°C in RPMI-1640 medium without FCS (2.5x106 cells/well). After incubation, nonadherent cells were removed, and the wells were washed with PBS. Monocytes were allowed to differentiate into macrophages in macrophage serum-free medium supplemented with antibiotics and recombinant human (rh)GM-CSF (10 ng/ml, Nordic Biosite, Täby, Sweden). Immature, monocyte-derived DC were generated using RPMI-1640 medium plus 10% FCS, rhGM-CSF (10 ng/ml), and rhIL-4 (20 ng/ml, R&D Biosystems, Abingdon, UK). In vitro-differentiated cell populations were devoid of CD3+ and CD19+ cells. pDC were isolated from PBMC by positive selection using blood DC antigen-4 (BDCA-4)-conjugated, paramagnetic beads (Miltenyi Biotec, Gladbach, Germany). To enhance cell purity, the labeled pDC were separated twice through MACS LS columns. Otherwise, the protocol recommended by the manufacturer was followed. The purity of isolated CD123/BDCA-2, double-positive cells was over 95% constantly, and no CD11c expression was detected. mDC were isolated from PBMC by using the BDCA-1 DC isolation kit, according to the manufacturer’s instructions (Miltenyi Biotec). Briefly, CD19+ cells were removed, first by positive selection, after which BDCA-1+ DC were positively selected. Again, the BDCA-1+ cells were run twice through an LS column to enhance purity. Isolated BDCA-1+ cells were over 95% CD11c+.

Flow cytometry
The expression of cell surface proteins was analyzed by flow cytometry. One-, 3-, and 7-day cells were collected and washed with PBS, and unspecific binding of antibodies was blocked by incubating cells in 10% FCS-PBS. Next, the cells were stained with fluorescence label-conjugated anti-CD1b (BD Biosciences, San Jose, CA, USA), anti-CD14 (Dako A/S, Glostrup, Denmark), or anti-DC-specific ICAM-grabbing nonintegrin (DC-SIGN; BD Biosciences) antibodies and their respective isotype control antibodies (Caltag Laboratories, S. San Francisco, CA, USA). The expression of cell surface proteins was analyzed with FACScan flow cytometer and CellQuest software (BD Biosciences).

Affymetrix oligonucleotide microarray analyses
For the Affymetrix sample preparations (Affymetrix, Santa Clara, CA, USA), 20 µg total cellular RNA, pooled from four different donors, was used as the starting material. The sample preparation was performed according to the instructions and recommendations provided by the manufacturer. The samples were hybridized by the Finnish DNA Microarray Centre to HG-U133A arrays containing ~22,000 probes covering ~13,000 human genes. Two biological repeats with RNA from eight donors in total (two pools of four different donors) were performed for 3- and 7-day microarray experiments (two sets of five arrays). Kinetic data for 3, 6, and 24 h were studied from one culture (a pool of four donors). The data were analyzed with GeneChip Microarray Suite software, Version 5 (MAS5, Affymetrix), and filtered according to recommendations of the manufacturer. Briefly, the probe sets were excluded if the detection call for target and reference were absent, if the change call gave no change in comparative analysis, or if the signal:log ratio between target and reference were between –1 and 1. Gene expression was considered up-regulated if the signal:log ratio between the reference and the target samples was higher than one (greater than twofold increase) and if the target sample were "present." Similarly, a gene was defined as down-regulated if the signal:log ratio were >–1 (greater than twofold decrease) and if the reference sample were present. Genes were considered as differentially expressed when they presented a consistent change in two separate biological repeats (the two different 3- and 7-day cultures) or in the case of the immediate differentiation response, consistent regulation at all of the time-points (3, 6, and 24 h culture). All of the genes, which fulfilled these criteria in at least one of the comparisons and one of the time-points, were selected for further analysis, where the expression of the genes was explored in parallel in different conditions without fold-change threshold. Microsoft Access and Excel for Windows software were used for data analysis and processing. Visualization was performed using Eisen’s Treeview [12 ]. The gene annotations were obtained from the NetAffx database [13 ]. The microarray data were also analyzed through the use of Ingenuity Pathways Analysis (Ingenuity Systems, Redwood City, CA, USA). The significance is expressed as a P value, which is calculated by comparing the number of user-specified genes of interest participating in a given pathway, relative to the total number of occurrences of these genes in all pathway annotations, stored in the Ingenuity Pathways Knowledge Base (right-tailed Fisher’s Exact test).

Quantitative RT-PCR (qRT-PCR) analysis
For qRT-PCR analyses, total RNA was isolated from macrophages or DC derived from two to four donors using the Qiagen Rneasy Midi kit. RNA from different donors was pooled before purification. The primers used in the TaqMan analyses, corresponding to C3 (Hs00163811_m1), TCF7L2 (Hs00181036_m1), Fc{gamma}R1A (Hs02340030_m1), Fc{epsilon}R1A (Hs00758599_m1), TGFa (Hs00177401_m1), suppressor of cytokine signaling 1 (SOCS1; Hs00705164_s1), FZD2 (Hs00361432_s1), and WNT5A (Hs0018013_m1) genes, were obtained from Applied Biosystems (Foster City, CA, USA).

DNA affinity binding and Western blot analysis
Monocytes were isolated and differentiated into macrophages or DC for 3 or 7 days. Cells were collected, and samples were treated as described [11 ]. Both strands of the DNA elements containing a NFAT/AP-1-binding site of the human IL-2 gene promoter (proximal –135 and distal –280 [14 , 15 ]) were synthesized with BamHI overhangs as spacers, with 5'-biotinylation of the upper strand oligonucleotide (DNA Technology, Aarhus, Denmark). The oligonucleotides were annealed in 0.5 M NaCl and incubated with streptavidin-agarose beads (Neutravidin, Pierce, Rockford, IL) at +4°C for 2 h, in a ratio to yield maximum saturation of the beads with the biotinylated oligonucleotide. Samples were incubated with agarose beads, saturated with the oligonucleotide for 2 h at +4°C. After washing, the bound proteins were released in SDS sample buffer, and equal aliquots were subjected to SDS-PAGE and Western blotting.

For direct Western blot analyses, cells were lysed, and 30 µg protein aliquots were separated on 10% SDS-PAGE (8% for detection of FN) using the Laemmli buffer system. Proteins separated on gels were transferred onto Immobilon-P membranes (Millipore, Bedford, MA, USA). Binding of primary and secondary antibodies was performed in PBS (pH 7.4) containing 5% nonfat milk for 1 h at room temperature. Primary antibodies used in immunoblotting were anti-Wnt5A (sc-23698), anti-FN (sc-9068), and anti-NFATc1 (sc-7294; all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). HRP-conjugated, anti-guinea pig (P0141, Dako A/S), anti-goat (P0449, Dako A/S), and anti-rabbit (P0448; Dako A/S) Igs were used as secondary antibodies. The protein bands were visualized on Hyper-Max film using the ECL system (Amersham Biosciences, Piscataway, NJ, USA).


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RESULTS
 
Experimental setting for studying gene expression profiles of differentiating macrophages and DC
In humans, peripheral blood monocytes can function as precursors for macrophages and DC in vitro when the cells are treated with GM-CSF or GM-CSF plus IL-4, respectively. We set out to study the differentiation process in two separate time-frames: an immediate response of monocytes to differentiating cytokines (0, 3, 6, and 24 h time-points) and a stable response leading to fully differentiated macrophages or DC (3 and 7 days with untreated, 1-day monocytes as the reference sample). Already at early time-points (3 and 6 h), after the initiation of the cytokine-driven differentiation process, the expression of a large number of genes was up- or down-regulated. We considered changes in the gene expression to be significant and reproducible when they were detected in two consecutive time-points (3 and 6 h or 6 and 24 h; Table 1 ). Even with these criteria, the number of regulated genes remained high (Table 1) .


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  		Table 1. Number of Genes Up- or Down-Regulated During the Initiation of Monocyte Differentiation to Macrophages or DC

Monocytes responded rapidly and robustly to differentiation-inducing cytokines
GM-CSF is a growth factor, which activates several signal transduction pathways in hematopoietic cells. IL-4 is a pleiotropic cytokine with a broad range of cell type-specific effects. Accordingly, monocytes stimulated with GM-CSF or GM-CSF + IL-4 display a robust, transcriptional response and changes in the expression of a large set of genes. At the 3 + 6-h time-points, 526 and 529 genes were up-regulated, and 380 and 489 genes were down-regulated in macrophages and DC, respectively (Table 1) . At 6 + 24 h time-points, the corresponding figures were 972 and 1051 for up-regulated and 453 and 441 for down-regulated genes, respectively. Comparison of the constant gene expression profiles (in all three time-points) in cells directed for macrophage or DC differentiation revealed that 17 genes were expressed at a higher level in macrophage-driven than in DC-driven cells (Table 1 ; Fig. 1 ). FGFR1, TKTL1, and ADFP (Fig. 1 ; three right-most colums, marked with red) were among the genes, which were up-regulated specifically in the macrophage differentiation lineage. In total, 22 genes were expressed at a higher level in the DC lineage and included, e.g., FN1, Wnt5A, MAOA, TGM2, KIAA0992, CTNS, and CCL22 (Fig. 1 ; three right-most columns, marked with green). It is interesting that the expression of the monocyte/macrophage marker CD14 gene was down-regulated in GM-CSF- and GM-CSF + IL-4-stimulated cells as compared with untreated monocytes (Fig. 1) .


Figure 1
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  		Figure 1. Genes differentially regulated in cytokine-stimulated monocytes, which were isolated and stimulated with GM-CSF or GM-CSF and IL-4 for 3, 6, and 24 h or left untreated. Total RNA was isolated, and gene expression profiles were determined using Affymetrix oligonucleotide microarrays. In comparisons, 3 h DC versus monocyte (ms), 6 h DC versus ms, and 24 h DC versus ms, as well as 3 h macrophage (Mf) versus ms, 6 h Mf versus ms, and 24 Mf versus ms, green color stands for decreased and red color for increased gene expression among indicated comparisons. In comparisons made among 3 h Mf and DC, 6 h Mf and DC, and 24 h Mf and DC, green color denotes higher expression levels in DC, and red color marks higher expression in macrophages.

Differentiation-specific gene expression patterns in macrophages and DC
GM-CSF- or GM-CSF + IL-4 stimulated monocytes showed differences in their gene expression pattern, already at 24 h after cytokine stimulation. However, as the full differentiation of macrophages and DC in vitro takes, ca., 6–7 days, we also carried out gene expression profiling of 3- and 7-day-differentiated macrophages and DC. As in the short time-point experiments, the expression of hundreds of genes was changed at 3 and 7 days (Fig. 2 ). To limit the number of genes to be studied further, we focused on genes, which showed stable changes in their expression at 3- and 7-day time-points. However, a complete list of all cytokine-regulated genes is available as supplementary data (Supplementary Tables 1, a–c).


Figure 2
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  		Figure 2. Experimental set-up and numbers of regulated genes during macrophage and DC differentiation. The arrows between the spheres indicate the cell types whose gene expression profiles were compared. Numbers of differentially expressed genes are indicated in the boxes, where arrows indicate up- or down-regulation of genes. The changes in the expression of a given gene were considered significant when there was a greater than or equal to twofold increase or decrease in the mRNA expression pattern in two separate experiments. Mo, Monocyte.

Altogether, 342 genes were up-regulated, and 194 were down-regulated during macrophage differentiation (Fig. 2) . Correspondingly, 354 and 236 genes were up- and down-regulated, respectively, when monocytes differentiated into DC. The majority of these genes was regulated similarly in both cell types, which is expected, as GM-CSF is a differentiation factor for macrophages and DC. In addition, another 76 genes were expressed at a higher level in macrophages, and 120 genes were expressed more strongly in DC (Fig. 2) . These genes included several known cell-surface molecules, such as CD14, Fc{gamma}R1A, and CD163 in macrophages and CD1a, CD1e, Fc{epsilon}R1A/R2, CD1c, CD1b, LY75 (DEC-205), and CD209 (DC-SIGN) in DC (Fig. 3 ). HLA Class II genes were also expressed at higher levels in DC compared with macrophages, reflecting a high antigen-presenting capacity of DC. TNFRSF11A (receptor activator of NF-{kappa}B), encoding for an important costimulatory molecule regulating DC–T cell interactions, was also induced strongly during DC differentiation. Receptors for cytokines and some other ligands, whose expression was induced differentially, included CSF3R in macrophages and FZD2, IL21R, and IL10RA in DC (Fig. 3) . Several cytokines and extracellular ligands also showed a cell type-specific expression pattern: WNT5A, chemokines CCL17 and CCL22, and TGFA were strongly up-regulated during DC differentiation, and the expression of GDF15/MIC-1 was induced almost exclusively in macrophages. DC-specific genes linked to transcription included IRF4, NR4A3, NFIL3, SMAD1, and two Ets-TF family members EHF and ETV5. Basic helix-loop-helix (BHLH)B3, minichromosome maintenance mutant 2 (MCM2), MCM6, and NR1H3 genes were expressed at a higher level in macrophages (Fig. 3) .


Figure 3
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  		Figure 3. Stable gene expression changes during macrophage and DC differentiation from monocytes. After isolation, monocytes were differentiated to macrophages or DC with GM-CSF or GM-CSF + IL-4, respectively, for 3 or 7 days. Total cellular RNA was extracted, and gene expression profiles were determined by hybridizing to Affymetrix arrays. Red and green denote increased and decreased gene expression, respectively.

It is of note that often differences in the gene expression levels between DC and macrophages resulted from a specific down-regulation of gene expression in one cell type over the other. For example, the expression of C3, a central component of the complement cascade, was down-regulated in DC and macrophages compared with monocytes, but its expression remained at a higher level in macrophages than in DC (Fig. 3) , and their expression in macrophages remained at a comparable level with that seen in monocytes, thus leading to a macrophage-specific expression pattern of these genes.

We also analyzed the data with Ingenuity Pathways Analysis software, which analyzes microarray data and detects expression patterns of genes whose expression is linked to specific signaling pathways. Macrophages and DC were analyzed separately. For both cell types, data from 24 h and 7-day time-points were first proportioned to the monocyte data, and then, the expression of canonical signaling pathway-related genes was compared between different cells types. The results of this pathway analysis are depicted in Figure 4 . Expression of genes related to antigen presentation, NF-{kappa}B signaling, and IL-4 signaling pathways was up-regulated specifically in DC. Genes linked to IL-6 signaling and apoptosis were up-regulated in both cell types. Death receptor signaling pathway genes were up-regulated specifically in macrophages. Chemokine signaling, p38 MAPK signaling, and VEGF signaling-related genes were down-modulated in both cell types. These results further suggest that differentiation of macrophages and DC leads to the acquisition of different signaling capacities in the two cell types.


Figure 4
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  		Figure 4. Expression analysis of signaling pathway-linked genes. The microarray data were analyzed by Ingenuity Pathways Analysis Software. The x-axis displays selected canonical pathways, and the y-axis displays the –(log) significance. The significance is expressed as a P value, which is calculated using the right-tailed Fisher’s Exact test. The threshold line denotes the cutoff for significance, which equalizes P = 0.05. EGF, Epidermal growth factor; IGF-1, insulin growth factor 1; PDGF, platelet-derived growth factor; PPAR, perixisome proliferator-activated receptor; VEGF, vascular EGF.

Cell surface expression of known lineage markers for macrophages and DC
Several genes encoding known cell surface marker molecules for macrophages and DC, such as CD14 and CD163 for macrophages and CD1a-c, CD1e, DC-SIGN (CD209), and DC-lysosome-associated membrane protein (CD208) for DC, were found to be expressed at 7 days of differentiation. To validate mRNA expression data and the phenotype of differentiated cells, we performed flow cytometric analyses to detect cell surface expression of these marker proteins. Nonfixed monocytes and 3- and 7-day macrophages and DC were stained with anti-CD14-, -CD1b-, or DC-SIGN-specific antibodies, followed by flow cytometric analyses of the expression of these molecules on the surface of the cells. CD14 expression was high in monocytes and 3- and 7-day macrophages, whereas 3-day DC showed weak and 7-day DC almost no CD14 expression (Fig. 5A ). CD1b was expressed in all cell types, but the highest expression was seen in 3- and 7-day DC. Monocytes and 7-day macrophages were devoid of DC-SIGN expression, whereas 3- and 7-day DC displayed high expression levels. Three-day macrophages, however, were transiently positive for this DC-specific marker. In conclusion, the protein expression of CD14, CD1b, and DC-SIGN correlated well with the relative mRNA expression pattern detected by microarray.


Figure 5
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  		Figure 5. Expression of cell-surface marker proteins and mRNA of a selected set of genes during macrophage and DC differentiation. Purified monocytes were differentiated to macrophages or DC with GM-CSF or GM-CSF + IL-4, respectively, for 3 or 7 days. (A) Cells from different blood donors were analyzed separately for cell-surface expression of CD14, CD1b, and DC-SIGN. Flow cytometric profiles of one representative individual are shown (solid black profiles). Isotype controls are indicated as white profiles. (B) Real-time quantitative PCR analyses of expression of genes representing high expression in monocytes, macrophages, or DC. Note the differences in scale of the y-axis. Data are representative of two individual experiments.

Validation of the Affymetrix expression data by qRT-PCR analyses
To further validate the data obtained from the Affymetrix gene expression array, we chose representative genes for further analyses with qRT-PCR (TaqMan) based on their differential expression in macrophages and DC. Fc{gamma}R1A, C3, and TCF7L2 were expressed preferentially in macrophages, and the expression of WNT5A, FZD2, SOCS-1, TGFA, and Fc{epsilon}R1A was higher in DC. The expression patterns of all of these genes were fully concordant between the Affymetrix and qRT-PCR results (Fig. 5B) .

Wnt5A mRNA expression in different DC subtypes
As described above, the Wnt5A gene was found to be expressed in high levels in monocyte-derived DC. In humans, in addition to monocyte-derived DC, there are at least two other DC subtypes, namely pDC and mDC, respectively, which can be isolated directly from blood. To study the basal expression and the role of DC differentiation-specific cytokines on Wnt5A expression, freshly isolated pDC and mDC were left unstimulated or stimulated with GM-CSF, IL-4, or their combination for 4 h, and WNT5A mRNA expression was analyzed by qRT-PCR. The basal WNT5A mRNA expression was low in pDC, and it was readily detectable in mDC and 7-day-differentiated, monocyte-derived DC obtained from the same blood donors (Fig. 6 ). pDC and mDC responded to IL-4 stimulation by a fourfold increase in WNT5A mRNA expression, leading to clearly higher expression levels compared with those seen in monocyte-derived DC. The results suggest that WNT5A is a mDC-specific (mDC and monocyte-derived DC) gene, but its expression can also be induced in pDC by IL-4. This indicates that the WNT5A gene is likely to be under a direct regulation by Stat6 and other IL-4-stimulated TFs.


Figure 6
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  		Figure 6. Wnt5A expression is induced in DC subtypes by IL-4 stimulation. pDC and mDC were isolated directly from peripheral blood with magnetic cell sorting and stimulated with GM-CSF, IL-4, or GM-CSF + IL-4 for 4 h. Expression of Wnt5A was analyzed by quantitative real-time PCR. Monocyte-derived, 7-day-differentiated DC were included in the analysis as a reference sample. Data are representative of two individual experiments.

WNT5A and FN proteins are expressed specifically in DC
As shown above, DC, but not macrophages, were found to express high levels of WNT5A and FN mRNAs. As WNT5A and FN proteins associate with or take part in the formation of extracellular matrix (ECM), respectively, we also studied their expression during macrophage and DC differentiation. FN expression was detected in monocytes, but macrophages were completely devoid of FN protein expression (Fig. 7 ). In contrast, FN expression was strong in 3-day DC, and its expression increased even further in 7-day DC. WNT5A expression was detected in monocytes and 3-day-differentiated macrophages and DC. However, in 7-day macrophages, WNT5A expression was barely detectable, and DC continued to express this protein in high levels (Fig. 7) . Wnt proteins are known to associate with proteins of the ECM. However, although the expression of WNT5A and FN was high in 7-day DC, we could not detect direct association of WNT5A with FN by coimmunoprecipitation (data not shown).


Figure 7
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  		Figure 7. Wnt5A and FN proteins are expressed specifically in the DC lineage. Monocytes were isolated and differentiated to macrophages or DC with GM-CSF or GM-CSF + IL-4, respectively, for 3 or 7 days. Whole-cell lysates were prepared, and proteins were separated on 8% (for FN) and 10% (for Wnt5A) SDS-PAGE. Expression of Wnt5A and FN proteins was analyzed by Western blotting with specific antibodies. Data are representative of two individual experiments.

Constitutive NFAT DNA-binding activity is detected in DCs but not in macrophages
Instead of the canonical Wnt/ß-catenin pathway, the FZD2 receptor can activate cellular signaling via intracellular Ca2+ influx [16 ], resulting in the activation and DNA binding of NFAT. To study whether the basal activation of NFAT was different in 7-day-differentiated macrophages and DC, we carried out oligonucleotide-binding experiments using proximal (–135) and distal (–280) NFAT-binding sites of the human IL-2 gene promoter [14 , 15 ]. The constitutive binding of NFATc1 to the IL-2 promoter NFAT sites was high in DC extracts, and the corresponding binding of NFATc1 in macrophage cell extracts was clearly weaker (Fig. 8A ). To study whether the constitutively high NFAT DNA-binding activity was mediated by G-protein-coupled receptors such as FZD2, we used PT to block the activity of such receptors. PT reduced the basal NFAT DNA-binding activity in DC but not in macrophages (Fig. 8B) , suggesting that PT-sensitive receptors, including FZD2, as a result of its cell type-specific expression, may be involved in constitutive NFAT activation in human monocyte-derived DC. CsA (1 µg/ml) treatment of DC, but not that of macrophages, also reduced the constitutive, NFAT-binding activity to the same extent as PT, suggesting that the calcineurin-dependent pathway is involved in NFAT activation in monocyte-derived DC.


Figure 8
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  		Figure 8. Constitutive NFAT DNA-binding activity, which is sensitive to Ca-influx inhibition is detected in DC. Purified monocytes were differentiated to macrophages or DC as described. (A) Whole-cell lysates were prepared and subjected to DNA pull-down using oligonucleotides corresponding to the proximal and distal NFAT-binding elements found in the IL-2 promoter. Interacting proteins were analyzed by SDS-PAGE and Western blotting with NFAT-specific antibodies. (B) Cells were treated with pertussis toxin (PT; 10 and 100 ng/ml) or cyclosporin A (CsA; 1 µg/ml) for 1 h. Whole-cell lysates were prepared and analyzed as described above for interaction with the proximal NFAT-binding element. Data are representative of two individual experiments.


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DISCUSSION
 
Macrophages and DC are the key players in the interface between innate and adaptive immune responses. Their functions in inflammatory conditions differ in many aspects, and the molecular basis for this difference has been the focus of intense investigation [17 18 19 20 21 22 23 24 ]. Previously, the main focus has been on analyzing the gene expression changes occurring in these cells during microbial infections. Our study provides a global analysis of human macrophage and DC gene expression profiles during their differentiation from monocytes, providing an important reference point for further studies focusing on modulating macrophage activity in chronic inflammatory conditions or in vitro production of DC for immunotherapeutic purposes.

Our gene expression profiling revealed that ~1000 genes (~5% of analyzed genes) were regulated after the initiation of the macrophage or DC differentiation program. It was of interest that also, nearly 500 genes were down-regulated in both cell types (Table 1) . During the actual differentiation process (7-day differentiation; Fig. 2 ), stable gene expression changes were less frequent, but yet, there were ~350 genes up-regulated and 190 or 240 genes down-regulated in macrophages and DC, respectively. Our DC microarray data are well in line with a previous study [25 ], where an exceptionally large proportion of genes was also found to be up- or down-regulated during DC differentiation. There are no previous studies comparing gene expression profiles between differentiating macrophages and DC.

Macrophage and DC differentiation and that of hematopoietic cells in general are known to be regulated by TFs directing the sequential expression of more cell type-specific genes [8 , 9 , 26 ]. In our microarray analysis, 216 genes were found to be expressed differentially between macrophages and DC; 25 of them (13%) encode proteins classified as TFs. The list includes IRF4, SMAD1, C/EBPß, and other leucine zipper family TFs, several nuclear receptor family members, and four members of the Ets-family, forkhead family, and bHLH family TFs. More specifically, in humans, IRF4 expression is enhanced specifically during DC differentiation [27 ]. Similarly, IRF4 knockout mice show impaired DC differentiation and functional deficiencies [28 , 29 ], possibly as a result of importance of IRF4 in TLR signaling. In our data, C/EBPß expression was down-modulated in DC compared with macrophages, and this protein has a well-established role in macrophage biology. MafB is considered as a macrophage-specific TF, but it is surprising that MafB mRNA was expressed in DC at a higher level (Fig. 3) . It has been suggested that relative expression levels of PU.1 and MafB proteins regulate macrophage/DC differentiation in such a way that high PU.1 levels favor DC differentiation by inhibiting MafB expression and activity [30 ]. However, PU.1 mRNA expression does not vary significantly between macrophages and DC [27 ], suggesting that other mechanisms, apart from the mere expression of PU.1, may be involved. The functions of some of the differentially regulated TFs have been linked to other hematopoietic cell lineages, such as B cell lymphoma 6 and musculin/activated B cell factor-1 to B cells [31 , 32 ] and Sry-related high mobility group box 4 to T cells [33 ]. For the majority of these TFs, however, there are no previous reports indicating a role for them in myeloid cell differentiation or hematopoietic cell differentiation in general. Some TFs, such as ARID5B, have remained characterized relatively poorly. This suggests that many TFs may have broader functions than anticipated previously, depending on cell type and available protein interaction partners. It is interesting that many of these TFs have been reported to function as repressors of gene expression, suggesting that fine-tuning of gene expression during terminal differentiation may be mediated by selective inhibition of gene expression rather than by positive regulation.

The Wnt signaling pathway [34 ] has been investigated intensely in the context of embryogenesis and cancer [35 ]. Considerably less is known about the role of this pathway in the normal differentiation of human hematopoietic cells, although Wnt signaling pathway plays a role in lymphopoiesis and stem cell proliferation [36 ]. WNT5A is expressed in adult human bone marrow CD34+ Lin– primitive progenitor cells [37 ], and treatment of progenitor cells with WNT5A-conditioned medium enhances their repopulating capacity in a xenotransplantation model [38 ]. We found that the expression of WNT5A, a member of the nontransforming group of Wnts, was up-regulated specifically during monocyte-derived DC differentiation and that blood-derived DC subtypes, pDC and mDC, also showed enhanced expression of WNT5A mRNA after IL-4 stimulation. In contrast, monocytes and macrophages did not express WNT5A mRNA or protein. Such a specific expression pattern in DC subtypes suggests that Wnts in general and WNT5A in particular may be an essential factor for DC development and functions. It is interesting that in an avian model of hematopoietic cell differentiation, WNT5A was found to inhibit macrophage colony formation and differentiation of monocytes to macrophages [39 ]. In a recent report by Blumenthal et al. [40 ], it was also shown that WNT5A expression is induced via TLR2 in Mycobacteriun tuberculosis-infected human macrophages, and enhanced WNT5A expression was required for IL-12 production in macrophages and subsequent IFN-{gamma} production in T cells. It will also be of great interest to study whether cytokines or inflammatory stimuli can induce WNT5A expression further in DC and macrophages.

In the canonical signaling pathway, WNT5A binds to FZD4 or FZD5 and stabilizes ß-catenin to induce the expression of T cell factor/lymphoid enhancer factor-regulated target genes. Alternatively, WNT5A can use the FZD2 receptor to activate the intracellular Ca2+ pathway [16 ] or the orphan tyrosine kinase receptor Ror2 [41 ]. FZD2 mRNA expression was also found to be induced strongly during DC differentiation, suggesting that a functional ligand–receptor pair may be expressed during DC differentiation. We also showed that constitutive activation of the TF NFAT can be seen in DC but not in macrophages (Fig. 7) . NFAT activity is stimulated when the intracellular Ca2+ level is increased. An autocrine loop of WNT5A/FZD2 could thus be involved in constitutive NFAT activity in DC. NFAT activity is required for the expression of IL-2. It is of interest that Streptococcus pyogenes-stimulated, monocyte-derived DC are capable of producing IL-2 [11 ], a cytokine considered to be T cell-specific. The constitutively active Wnt signaling pathway could provide sufficient NFAT activity for IL-2 production in microbe-stimulated DC.

Cytokine signaling via the JAK/STAT pathway, activated by IL-4 and GM-CSF, is modulated by specific protein inhibitors of the SOCS family. In our study, basal SOCS-1 expression was detected almost exclusively in DC but not in macrophages. SOCS-1 expression in mouse DC has been reported to be regulated by IL-4 [42 ]. Studies in mice with SOCS-1-deficient DC and macrophages have demonstrated an important role for SOCS-1 in preventing spontaneous hyperactivation of DC and ensuing systemic autoimmunity [43 ]. Similarly, silencing SOCS-1 expression potentiates antigen presentation by DC and leads to enhanced anti-tumor immunity in an experimental mouse tumor model [44 ]. It is interesting that SOCS-1 expression in DC seems to be linked to differentiation and maturation status of the cells, whereas in macrophages, it is inducible by proinflammatory cytokines.

FN, an important component of ECM, was expressed almost exclusively in monocytes and DC. FN is also found in human plasma in relatively high levels. In a study evaluating the effect of principal plasma proteins on monocyte-derived DC maturation, FN was not found to induce DC maturation [45 ]. In view of our results, this can be expected, as FN is produced by DC themselves. It is interesting that IL-4-activated macrophages express FN mRNA and protein readily [46 ], suggesting that although macrophages do not express FN basally, they can be induced to do so in inflammatory conditions.

In the present study, we observed that a great number of gene regulatory changes are associated with cytokine-induced differentiation of human monocytes to macrophages or DC. In addition, we observed that more that 200 genes were expressed differentially between differentiated macrophages and DC (immature DC). A large proportion of these genes encode cell surface molecules and receptors, which regulate DC or macrophage interactions with other cells, microbes, or their components and soluble mediators such as cytokines and Igs. An interesting group of molecules, which were expressed differentially in monocyte-derived macrophages and DC, was certain TFs, which in the end, may regulate cell type-specific gene expression in macrophages and DC. Our gene expression profiling of human macrophages and DC provides a good background for further functional analyses, which ultimately, will give us a full picture of the molecular determinants regulating the functional differences of these important cell types.


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ACKNOWLEDGEMENTS
 
The Medical Research Council of the Academy of Finland, the National Technology Agency of Finland, Turku University Hospital Fund, and the Sigrid Juselius Foundation supported this study. We thank Mari Aaltonen, Hanna Valtonen, and Johanna Lahtinen for expert technical assistance. We also thank Miina Miller at the Finnish DNA Microarray Center for her help with microarray analysis.


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FOOTNOTES
 
1 These authors contributed equally to this work. Back

2 Current address: Genome-Scale Biology Program, Biomedicum Helsinki, FI-00014, University of Helsinki, Finland. Back

Received March 30, 2007; revised May 23, 2007; accepted May 27, 2007.


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