Proteomic data are scarce in relation to DC biology, and where available they only focus on moDCs 141516. Others have focused on gene expression, as well as obtaining some proteomic data, in monocytes and moDCs 171819. The present study compared the whole cell proteome of immature mDCs, moDCs and KG-1 cells. Clearly, a major challenge in proteomic studies of DCs is obtaining enough protein for performing two-dimensional electrophoresis. This limitation was partly overcome by using a large volume of blood (approximately 120 ml) for cell separation. We also pooled whole cell lysates of DCs from seven individuals to obtain sufficient quantities of protein and to eliminate inter-individual variations. We found that peripheral blood mDCs have six- and five-fold lower protein content per cell than moDCs and KG-1 cells, respectively (data not shown). Unfortunately, the low numbers of mDCs in peripheral blood (approximately 1% of PBMCs), together with their lower protein content, meant that, despite pooling samples, we were able to run only duplicate gels for mDCs.

Figures 2 and 3 show three representative two-dimensional gel images of the different cell types. Gel images were analyzed using PDQuest software and all images were normalized before any comparisons between gels were made. The total number of spots in the gels were 661, 619, and 770 for mDCs, moDCs and KG-1 cells, respectively. To analyze the comparability of gels, the densities of spots matched in all three gels were plotted and a correlation coefficient value was calculated. The proteome of mDCs showed different levels of similarity compared with those of moDCs and KG-1 cells (correlation coefficient 0.68 and 0.62, respectively) (Figure 4). Duplicate gels of mDCs were reproducible (correlation coefficient >0.90), as were triplicate gels of moDC and KG-1 cells. Figure 5 shows an overlay of Gaussian images of mDCs, moDCs and KG-1 cells.

Thirty-five spots were up-regulated more than four-fold in mDCs compared with the DC models, and fifty were down-regulated by the same amount (Table 1). A number of differentially expressed proteins, which appeared to be more than two-fold different in intensity (either up- or down-regulated) in the DC models compared to mDCs, were excised from the gels and subjected to trypsin digestion and MALDI-TOF (matrix-assisted laser desorption/ionisation-time of flight) mass spectrometric analysis; Table 2 shows the corresponding protein data. The factor of difference was calculated by dividing the intensity of the protein spot in mDCs by that of the corresponding spot in moDCs or KG-1 cells. Eighteen spots were successfully identified using MALDI-TOF mass spectrometry. These proteins are known to be involved in a wide spectrum of biological processes, including functions related to cell integrity and metabolism (Table 2).

The majority of the proteins that showed higher levels of expression in mDCs are known to be involved in cell growth and maintenance, including FGG, ubiquinol cytochrome c reductase, glutathione S transferase, nuclease isoform sm3 and annexin A1. Some of these differentially expressed proteins also appear to be involved in DC maturation. Pereira et al. 15 have shown higher expression of FGG in the proteome of immature moDCs compared to mature moDCs. Furthermore, fascin and actin, which showed substantially lower expression (8- and 16-fold, respectively) in mDCs compared with both DC models, are known to play important roles in maintaining cell structure and in the formation of immunological synapses between DCs and T cells 202122. Al-Alwan et al. 21 have previously shown that increased fascin expression correlates with DC maturation state, and recent work supports this, suggesting that fascin is a mature DC marker 23. This, together with our data on FGG expression, suggests that, at least in their resting state, mDCs have a less mature phenotype compared to moDCs and KG-1 cells.

To confirm the proteomics data, we used a capture ELISA to assess the relative expression of five proteins that had an induction factor greater than two, namely actin related protein 2/3 complex 2 (ARPC2), phosphoglucomutase 1 (PGM1), fascin, FGG and carbonic anhydrase 2 (CAH2) (Table 2). The pattern obtained was in general agreement with the proteomes obtained for each of the three cell types. Thus, as expected, ARPC2, PGM1, fascin and CAH2 were found to be lower in mDCs compared to the two models, whereas FGG was higher (Figure 6).

We compared the three cell types by studying their surface phenotypes. Immature cells were cultured in the presence of lipopolysaccharide (LPS) for 24 h to produce a mature cell type. The cell markers used for characterization were CD11c, CD40, CD62L, CD80, CD83, CD86, CD206, CD209, HLA DR and Toll-like receptor (TLR)-4, which have all been reported to be found on dendritic cells 424.

As with our proteomic data, cell surface marker expression suggested that immature mDCs expressed lower levels of the usual DC maturation markers compared with both moDCs and KG-1 cells. The mDC models, moDcs and KG-1 cells, expressed significantly higher levels of CD11c, CD40, CD80, CD83 and CD209 than mDCs (Figure 7). However, mDCs showed significant up-regulation of the classic DC maturation markers CD40, CD80, CD83 and CD86 after 24 h stimulation with LPS; levels of these markers were more than ten-fold higher in mature compared to immature cells. The mDC models also showed up-regulation of these markers, but to a lesser extent (more than three-fold). The expression of cell surface markers on mature KG-1 cells was lower than on both mDCs and moDCs, with CD11c, CD40, CD80 and CD86 being expressed at significantly (p < 0.05) lower levels than on mDCs (Figure 7).

Myeloid DCs showed a more mature phenotype after stimulation with LPS (as shown by higher expression of CD40, CD80, CD83 and CD86) compared with moDCs and KG-1 cells. Interestingly, the mannose receptor (CD206), which has important functions in endocytosis, antigen recognition and binding and MHC class II presentation 252627, was only detectable on moDCs and was down-regulated by 4-fold after stimulation with LPS for 24 h; only negligible levels were found on mDCs and KG-1 cells. This would, therefore, suggest that in in vitro assays, moDCs could bind and internalize certain antigens, particularly glycoproteins, more efficiently. These findings are in keeping with those of Hajas et al. 13 showing that moDCs express much higher levels of CD206 than KG-1 cells and they could internalize antigens relatively more efficiently. The expression of DC-SIGN (DC-specific intercellular adhesion molecule-3-grabbing non-integrin or CD209) was low on all three cell types, but significantly higher on immature moDCs and KG-1 cells compared with mDCs.

Our finding of negligible levels of TLR4 on all three cell types is somewhat different from those of others 2829 who found no expression of TLR4 on mDCs, but did show expression on moDCs. However, there are studies showing TLR4 expression by both mDCs and moDCs, but not on pDCs 30. This discrepancy in data could have been caused by the use of different monoclonal antibodies and experimental conditions.

Peripheral blood mDCs were found to express significantly higher levels of key inflammatory (IL-1β, IL-6 and IL-8) and regulatory (IL-10) cytokines, compared to moDCs and KG-1 cells. Levels of IL-1β, IL-6, IL-8 and IL-10 were dose dependent, and following 24 h culture with either 50 or 100 pg/ml LPS were significantly higher in mDCs than in moDC and KG-1 cells (Figure 8). The IL-6, IL-10 and IL-12 data are at variance with a previous study 12, but this may be due to the use of different stimuli (for example, intact Escherichia coli rather than LPS), culture conditions and cytokine detection method by the authors. This pattern of cytokine production clearly makes mature mDCs more efficient in the cross-talk with T cells 3132 and other cells of the innate immune system (for example, natural killer cells), as well as in exerting inflammatory and/or regulatory effects mediated through cytokine production. This again is in line with our proteomic data suggesting that mDCs have a less mature phenotype, at least in their resting state, compared to the two DC models, moDCs and KG-1 cells 33.

Having demonstrated that unstimulated moDCs have a more mature phenotype than freshly isolated mDCs, as shown by proteomics (for example, lower FGG and higher fascin) and surface marker expression (higher CD83), we then proceeded to assess the endocytic and T cell stimulatory abilities of the DCs using dextran uptake and autologous mixed leukocyte reaction, respectively. moDCs were found to be better in endocytosis (Figure 9) and T cell activation (Figure 10) compared to mDCs, and this is in keeping with their more advanced maturation status. Others have shown 12 that, upon stimulation, both mDCs and moDCs are equally efficient in autologous T cell activation, which is in agreement with our finding that mDCs acquire a fully mature phenotype after LPS stimulation (Figure 7).

Heparinized whole blood from healthy volunteers (obtained with prior consent and Ethical Committee approval) was used for separation of PBMCs on a Histopaque density gradient (HISTOPAQUE-1077, Sigma, Poole, UK). CD1c⁺ peripheral DCs were isolated using the CD1c dendritic cell isolation kit from Miltenyi Biotech (Bisley, UK). Briefly, this involved depleting PBMCs of CD19⁺ B cells followed by positive selection of CD1c⁺ cells. CD14⁺ monocytes were isolated by positive selection from the CD1c^- cell fraction, and immature CD1a⁺CD83^- moDCs were generated as previously described 24. Briefly, this involved culturing CD14⁺ monocytes in the presence of IL-4 (250 IU/ml; R&D systems, Oxford, UK) and GM-CSF (50 ng/ml; R&D systems) for six days. Cells were cultured at 1 × 10⁶/ml in RPMI 1640 medium (Sigma) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin (Gibco Life Technologies, Paisley, UK) and 10% (v/v) fetal calf serum (FCS; Harlan Sera-Lab, Loughborough, UK) at 37°C in 5% CO₂. On day 3, cultured cells were fed with fresh medium containing relevant cytokines.

The human monocytic cell line KG1 was purchased from ECACC (Salisbury, UK). Cells were maintained at 0.35 × 10⁶/ml in Iscoves modified Dulbecco's medium (Sigma) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin (Gibco Life Technologies) and 10% (v/v) FCS (Harlan Sera-Lab) at 37°C in 5% CO₂. Cells were stimulated with 10 ng/ml PMA and 100 ng/ml ionomycin (both from Sigma) for 24 h, as described previously 6.

Immature peripheral mDCs, immature moDCs and DC-like KG1 cells were cultured in 48-well culture plates at 0.25 × 10⁶ cells/ml. Four conditions were set up in parallel, stimulating cells with 0, 10, 50 or 100 pg/ml LPS (Sigma). After 24 h, 250 μl of supernatant was collected and frozen at -80°C, and cells were harvested for cell surface marker staining.

Immature mDCs, moDCs and KG1 cells were generated as described earlier. Cells were harvested and washed three times in 1 ml of 0.05% PBS/Tween blocking buffer (300 g for 5 minutes). Cell pellets were resuspended in 500 μl carbonate buffer (pH 8.6, 7.6 mM Na₂CO₃, 142 mM NaHCO₃) and sonicated for 5 minutes. The sonicated cells were labeled by incubation with 20 μg digoxigenin (Roche, Basel, Switzerland) for 1 h at room temperature. The remaining free digoxiginin was neutralized with 150 mM Tris followed by dialysis against PBS (pH 7.2) overnight. The protein concentration for each cell type was measured at 280 nm using a Nanodrop (Agilent Technologies, Berkshire, UK). In the ELISA, anti-PGM1 (Abnova, Taipei, Taiwan), anti-fascin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), anti-CAH2 (Abnova), anti-FGG (Abnova) and anti-APRC2 (Abnova) antibodies (10 μl/ml) were diluted in carbonate buffer and plated onto a Nunc Immobilizer™ Amino 96-well plate n amino-reactive 96-well (Nunc, Roskilde, Denmark). Plates were incubated for 2 h at room temperature with shaking at 200 rpm. Liquid was removed from the plates and the plates were washed three times with PBS/Tween. The plate was incubated with 100 μl of each cell suspension (10 μg/ml total protein) for 2 h at room temperature. The plate was then washed 3 times with PBS/Tween and incubated with peroxidase-conjugated polyclonal anti-dioxigenin Fab fragment (Roche) at 1:5,000 in 1% BSA in PBS/Tween at 100 μl per well. Plates were incubated at room temperature for 1 h and washed 3 times as above. ABTS^® Peroxidase Substrate (100 μl at 5 mg/ml) (Roche) was added to each well and 30 minutes later the absorbance was measured at 405 nm.

For the analysis of the endocytic activity of the three cell types, 1 × 10⁵ cells were incubated with FITC-dextran (40,000 MW; Sigma) for 1 h at 37°C. As a control, 1 × 10⁵ cells were cooled to 4°C prior to incubation with dextran at 4°C for 1 h. Cells were washed three times and immediately analyzed on a FACS EPICS Altra cytometer.

Human PBMCs were obtained as described above. Un-touched T cells were then purified by negative selection (Pan T cell isolation kit, Miltenyi Biotech) to a purity of >95%. Cells were resuspended in RPMI 1640 medium (Sigma) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin (Gibco Life Technologies) and 10% (v/v) FCS (Harlan Sera-Lab). Autologous T cells (1 × 10⁵) were cultured in the absence (medium alone) or presence of 1 × 10⁴ irradiated (3,000 rad ¹³⁷Cs) immature mDCs and moDCs in 96-well U-bottomed microplates (Nunc) in 200 μl of medium per well. Cells were cultured for 72 h followed by an 18 h pulse with 1 μCi (0.037 MBq) of [³H]thymidine (Amersham Life Science, Buckingham, UK). Cells were transferred to a Unifilter-96 plate GF/C using a cell harvester and [³H]thymidine incorporation was measured in scintillation fluid (Microscint O) using a sinctillation counter (Canberra Packard Limited, Pangbourne, UK). All determinations were carried out in quadruplicate.

Cell surface marker expression was analyzed using WinMDI version 2.8 36 and 7,000 live cells were gated for each analysis. The cytokine bead array data were analyzed using the BD CBA software (Becton Dickinson).

The paired Student t-test was used to compare surface marker expression and cytokine expression between mDCs, moDCs and KG-1 cells. Data with p values of less than 0.05 or 0.01 were taken to be significant.