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Originally published In Press as doi:10.1074/mcp.M500262-MCP200 on January 19, 2006.
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Molecular & Cellular Proteomics 5:726-736, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

High Expression of Antioxidant Proteins in Dendritic Cells

Possible Implications in Atherosclerosis*,S

Aymeric Rivollier{ddagger}, Laure Perrin-Cocon{ddagger}, Sylvie Luche§, Hélène Diemer, Jean-Marc Strub, Daniel Hanau||, Alain van Dorsselaer, Vincent Lotteau{ddagger}, Chantal Rabourdin-Combe{ddagger}, Thierry Rabilloud§,** and Christine Servet-Delprat{ddagger}

From the {ddagger} INSERM U503, Université Lyon 1, IFR128-Biosciences Gerland, 21 avenue Tony Garnier, 69 007 Lyon cedex 07, § Commissariat à l’Energie Atomique (CEA), Laboratoire d’Immunochimie, DRDC/ICH, INSERM U548, CEA-Grenoble, 17 rue des martyrs, F-38054 Grenoble cedex 9, Laboratoire de Spectrométrie de Masse Bio-Organique, UMR CNRS 7509, Ecole de Chimie, Polymères et Matériaux, 25 rue Becquerel, 67008 Strasbourg cedex, and || Biologie des Cellules Dendritiques Humaines, INSERM U725, Etablissement Français du Sang-Alsace, 10 rue Spielmann, 67065 Strasbourg cedex, France


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DCs) display the unique ability to activate naive T cells and to initiate primary T cell responses revealed in DC-T cell alloreactions. DCs frequently operate under stress conditions. Oxidative stress enhances the production of inflammatory cytokines by DCs. We performed a proteomic analysis to see which major changes occur, at the protein expression level, during DC differentiation and maturation. Comparative two-dimensional gel analysis of the monocyte, immature DC, and mature DC stages was performed. Manganese superoxide dismutase (Mn-SOD) reached 0.7% of the gel-displayed proteins at the mature DC stage. This important amount of Mn-SOD is a primary antioxidant defense system against superoxide radicals, but its product, H2O2, is also deleterious for cells. Peroxiredoxin (Prx) enzymes play an important role in eliminating such peroxide. Prx1 expression level continuously increased during DC differentiation and maturation, whereas Prx6 continuously decreased, and Prx2 peaked at the immature DC stage. As a consequence, DCs were more resistant than monocytes to apoptosis induced by high amounts of oxidized low density lipoproteins containing toxic organic peroxides and hydrogen peroxide. Furthermore DC-stimulated T cells produced high levels of receptor activator of nuclear factor {kappa}B ligand, a chemotactic and survival factor for monocytes and DCs. This study provides insights into the original ability of DCs to express very high levels of antioxidant enzymes such as Mn-SOD and Prx1, to detoxify oxidized low density lipoproteins, and to induce high levels of receptor activator of nuclear factor {kappa}B ligand by the T cells they activate and further emphasizes the role that DCs might play in atherosclerosis, a pathology recognized as a chronic inflammatory disorder.

Dendritic cells (DCs)1 are the most potent antigen-presenting cells in the body, and their unique ability to stimulate a primary T cell response places them at the center of the immune response (1). Immature DCs differentiate from bone marrow progenitors or from monocytes and then either stay in the blood stream or migrate into the peripheral tissues. Immature DCs, such as Langerhans cells in the skin, survey incoming pathogens. They are equipped with receptors to become activated when exposed to pathogen-associated molecular patterns (2). Their capacity to recognize pathogens and become activated therefore represents the first critical event in the initiation of the immune response. An encounter with a pathogen leads to DC maturation and migration through lymphatic vessels to T cell areas of secondary lymphoid organs. Antigen presentation by DCs activates specific naive T cells to express CD40 ligand (CD154) (3), which, in turn, activates DCs, achieving their terminal differentiation, as assessed by the up-regulation of MHC I and II molecules and of co-stimulatory molecules CD80/CD86 and by the production of cytokines, such as IL-12 and IL-1{alpha}/ß, which all participate in T cell stimulation (3–545) and in the development of adaptive immunity (4, 6). Mature DCs modulate T cell responses through the secretion of various cytokines such as IL-12, promoting a Th1-type cellular immune response (7), or IL-4 following thymic stromal lymphopoietin activation, promoting a Th2-type humoral immune response (8). Finally a DC apoptosis program can be triggered at the end of the maturation process so that mature DCs do not produce an overstimulation of the immune system (9). DCs frequently operate under stress conditions induced by tissue damage, infectious pathogens, or inflammatory reactions. Oxidative stress enhances the production of inflammatory cytokines by DCs (10).

Atherosclerosis is considered as a chronic inflammatory disease of the arterial vasculature initiated by endothelial cell damage and implicating monocytes, DCs, and macrophages that operate under intense oxidative stress conditions. Indeed the accumulation of oxidized low density lipoproteins (oxLDLs), generated from native LDL trapped in the subendothelial space of the arterial wall, is a main feature of the disease and plays a key role in its progression, leading to the formation of vascular lesions (11, 12). This accumulation of oxLDLs induces the activation of macrophages and elicits an oxidative burst (13), generating toxic components such as the superoxide anions derived from the NADPH oxidase activity. Lipid peroxides and hydrogen peroxide directly contained in oxLDL are also toxic per se (14). Due to their pro-oxidant actions, superoxide anions resulting from macrophage activation by oxLDL as well as lipid peroxides contained in the oxLDLs are able to induce apoptosis in resident monocytes and macrophages (14–161516). This apoptosis is a common feature within early atherosclerotic lesions (17). Superoxide anions can be reduced into H2O and O2 by combination of the actions of cellular superoxide dismutase (SOD) and peroxiredoxin (Prx), catalase, or other peroxidases. Thus, it is hypothesized that monocytes and macrophages undergo apoptosis when their antioxidant enzymatic protection systems (SOD and Prx) are overflowed. Contrary to monocytes and macrophages, the expression pattern of oxidative stress response proteins in DCs is still unknown. Although both immature and mature DCs have been observed in the atherosclerotic plaques in close association with activated T cells (18) and in para-aortic and jugulodigastric lymph nodes attached to atherosclerotic arterial wall (19), their exact role in atherosclerosis progression is still poorly understood. These studies, however, suggest that vascular DCs may be implicated in the local induction of immune and autoimmune reactions (20, 21).

Besides their toxic action on diverse cell types, oxLDLs also increase monocyte adhesion to the endothelial cell layer as well as their transmigration (22–242324) toward adjacent tissues. Finally oxLDLs trigger the production of proinflammatory cytokines such as monocyte chemoattractant protein-1, macrophage colony-stimulating factor, and granulocyte macrophage colony-stimulating factor (GM-CSF) by endothelial cells and stimulate monocyte differentiation into DCs (25).

Here we investigated the relationship between oxidative stress protein expression profiles and DC differentiation and maturation. We performed a proteomic analysis of both immature and mature DCs derived from monocytes in the presence of IL-4 and GM-CSF and compared it with the monocyte proteome. This comparative proteomic approach revealed the original ability of DCs to express high levels of antioxidant enzymes such as Mn-SOD and Prx. These high expression levels of antioxidant enzymes confer a great capacity to DCs to resist apoptosis induced by oxLDLs, mimicking the oxidative stress microenvironment of atherosclerosis lesions. Moreover when co-cultured with T cells, DCs produce themselves, and induce T cells to produce, high levels of receptor activator of nuclear factor {kappa}B ligand (RANKL), a chemotactic factor for monocytes and a key survival factor for DCs (26–282728). Altogether these in vitro results suggest that DCs play a crucial role in the atherosclerosis pathogenesis and in the maintenance of its chronicity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Reagents
Monocytes and T cells were purified (29) from the adult blood of healthy volunteer donors (Etablissement français du sang, Lyon Gerland, France). Monocyte-derived DCs were generated in vitro as described previously (29). Briefly monocytes were seeded at 106 cells/ml and maintained in RPMI 1640 medium (Invitrogen) supplemented with 10 mM Hepes, 2 mM L-glutamine, 40 µg/ml gentamicin (Invitrogen), 10% heat-inactivated FCS (Roche Applied Science), 50 ng/ml human recombinant GM-CSF, and 500 units/ml human recombinant IL-4. After 6 days of culture, more than 95% of the cells were DCs as assessed by CD1a labeling. Recombinant human GM-CSF and IL-4 were purchased from PeproTech (Rocky Hill, NJ). For maturation, immature DCs were plated at 106 cells/ml and were stimulated with 1 µg/ml LPS or with 105/ml irradiated (7000 rads) fibroblastic CD40L- or control CD32-transfected L cells (both kindly provided by Schering-Plough Laboratory for Immunological Research, Dardilly, France) for 24 h. For oxLDL cultures, 10% lipoprotein-deficient FCS (Sigma) was used. T cell activation was performed at 106 cells/ml with 1 µg/ml anti-CD3 (HIT3a murine mAb) and 10 µg/ml anti-CD28 (CD28.2 murine mAb) from Pharmingen. For proteomic studies, cells were harvested by centrifugation, rinsed in PBS, and resuspended in homogeneization buffer (0.25 M sucrose, 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA). A buffer volume approximately equal to the packed cell volume was used. The suspension was transferred to a polyallomer ultracentrifuge tube, and the cells were lysed by the addition of 4 volumes (respective to the suspension volume) of 8.75 M urea, 2.5 M thiourea, 25 mM spermine base, and 50 mM DTT. After 1 h at room temperature, the extracts were ultracentrifuged (30 min at 200,000 x g). The supernatant was collected, and the protein concentration was determined by a Bradford assay using bovine serum albumin as a standard. Carrier ampholytes (0.4% final concentration) were added, and the protein extracts were stored at –20 °C. Human Prx2 was purified from freshly isolated human red blood cell by ion-exchange chromatography and gel filtration as described previously (30).

LDL Preparation
LDL (1.025 ≤ density ≤ 1.055 g/ml) was isolated from human plasma of normolipidemic healthy individuals by ultracentrifugation as described previously (25). The protein content of LDL was estimated by Coomassie Protein MicroAssay procedure (Pierce), and its lipid composition was determined using cholesterol RTU, triglyceride enzymatic PAP 150, and phospholipid enzymatic PAP 150 kits from bioMérieux (Marcy l’Etoile, France).

LDL Oxidation
LDL concentration was adjusted to 1 mg/ml protein by dilution in PBS and dialyzed at 4 °C against PBS to eliminate EDTA. Cu2+-mediated oxidation was conducted at 37 °C for 24 h by dialysis against 5 µM CuSO4 in PBS. The reaction was stopped by addition of 40 µM butylated hydroxytoluene and extensive dialysis at 4 °C against PBS containing 100 µM diethylenediamine pentaacetic acid.

Flow Cytometry
Cell suspensions were labeled according to standard procedures using the following monoclonal antibodies: CD1a-PE, CD14-PE, CD25-PE, CD80-FITC, CD83-PE, CD86-PE, MHC I (HLA-ABC-FITC), MHC II (HLA-DR-FITC), or an isotype control (Beckman Coulter). 0.5 µg/ml propidium iodide were added to detect apoptotic cells. Immunostaining was performed in 1% BSA and 3% human serum in PBS and then quantified on a FACSCalibur (BD Biosciences).

Immunofluorescence Staining
Cells cultured on glass coverslips were first fixed for 10 min with 3.7% formaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS for 7 min. After preincubation for 20 min in normal human serum with 10% PBS, cells were incubated with anti-RANKL (catalog number sc-9073, rabbit polyclonal, Santa Cruz Biotechnology) and anti-CD3 (UCHT1 mAb, Beckman Coulter) antibodies. Coverslips were then treated with the appropriate conjugated secondary antibodies (donkey anti-rabbit or donkey anti-mouse antibodies, Jackson Immunoresearch, West Grove, PA). Primary and secondary antibodies were applied for 60 min in a humidified chamber. Between each step, coverslips were washed three times for 5 min in PBS buffer. Observations were performed by epifluorescence using a Zeiss Axioplan microscope.

Allogeneic T Cell Stimulation
DCs were cultured in various numbers (10–105), for 7 days, in the presence of a constant number of T cells (105 cells/well) purified from the blood of another donor (allogeneic) as described previously (31). [3H]Thymidine incorporation was measured after a 12-h pulse with 1 µCi of [3H]thymidine/well using a Top Count NXT counter (PerkinElmer Life Sciences).

Western Blots
Cells were lysed in a buffer containing 200 mM NaCl, 40 mM Tris-HCl, pH 8.0, 1% Nonidet P-40, 2 mM EDTA, 1 mM PMSF, 1 mM NaF, 10 µg/ml aprotinin, and a mixture of protease inhibitors (protease inhibitor set III, Calbiochem) for 15 min at 4 °C. Insoluble materials were removed by centrifugation at 10,000 x g for 10 min. Proteins from cell lysates were separated by SDS-PAGE using NuPAGE 4–12% bis-Tris gels (Invitrogen) and then transferred to Immobilon-P membranes (Millipore, Bedford, MA). Membranes were blocked using 5% BSA in TBS-T (20 mM Tris, pH 7.6, 130 mM NaCl, and 0.1% Tween 20) and incubated for 1 h with a specific anti-Mn-SOD antibody (catalog number 06-984, Upstate Cell Signaling Solutions, Charlottesville, VA). Immunoreactive bands were visualized by using a secondary goat anti-rabbit horseradish peroxidase-conjugated antibody (Jackson Immunoresearch) and chemiluminescence (ECL Western blotting substrate kit, Pierce). The membranes were not stripped before reblotting with anti-actin antibody (catalog number A-2066, Sigma).

Two-dimensional Electrophoresis
Two-dimensional electrophoresis was performed with immobilized pH gradients for isoelectric focusing. Home-made linear pH 4–8 or 4–12 gradients were used (32) and prepared according to published procedures (33). IPG strips were cut with a paper cutter and rehydrated in 7 M urea, 2 M thiourea, 4% CHAPS, and 0.4% carrier ampholytes (pH 3–10 range) containing either 20 mM DTT (pH 4–8 gradients) or 5 mM Tris cyanoethyl phosphine (purchased from Molecular Probes, for pH 4–12 gradients) (34). The protein sample was mixed with the rehydration solution in the case of pH 4–8 gradients or cup-loaded at the anode for pH 4–12 gradients. Isoelectric focusing was carried out for a total of 60,000 V-h. After focusing, the strips were equilibrated for 2 x 10 min in 6 M urea, 2% SDS, and 125 mM Tris-HCl, pH 7.5, containing either 50 mM DTT (first equilibration step) or 150 mM iodoacetamide (second equilibration step). The equilibrated strip was loaded on the top of a 10 or 11% polyacrylamide gel and resolved by SDS-PAGE at 12 watts/gel using the Tris-taurine system (35).

After migration, the gels were stained either with silver nitrate for 2D gels with a pH 4–8 gradient (36) or with ammoniacal silver for 2D gels with a pH 4–12 gradient (37). Quantitative gel analysis was performed on the silver-stained gels with Melanie II software (Genebio, Geneva, Switzerland). The experiments were performed in triplicate, starting with different cell batches. Several gels were made for each culture to select gels with very close detection signal levels for quantitative analysis. This allowed us to keep the gel analysis parameters constant for better reproducibility. As a matter of fact, the total spot intensity in the analyzed gels ranged from 531,400 to 662,750 arbitrary absorbance units with a mean of 600,290 units, i.e. a maximum deviation of ±11%.

Mass Spectrometry
In-gel Digestion—
Excised gel slice rinsing was performed by the Massprep (Micromass, Manchester, UK) as described previously (38). Gel pieces were completely dried with a SpeedVac before digestion. The dried gel volume was evaluated, and 3 volumes of 12.5 ng/µl trypsin (Promega, Madison, WI) freshly diluted in 25 mM NH4HCO3 were added. The digestion was performed at 35 °C overnight. Then the gel pieces were centrifuged for 5 min in a SpeedVac, and 5 µl of 35% H2O, 60% acetonitrile, 5% HCOOH were added to extracted peptides. The mixture was sonicated for 5 min and centrifuged for 5 min. The supernatant was recovered, and the procedure was repeated once.

MALDI-TOF MS Analysis—
Mass measurements were carried out on an UltraflexTM MALDI-TOF/TOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany). This instrument was used at a maximum accelerating potential of 20 kV and was operated in reflector positive mode. Sample preparation was performed with the dried droplet method using a mixture of 0.5 µl of sample with 0.5 µl of matrix solution. The matrix solution was prepared from a saturated solution of {alpha}-cyano-4-hydroxycinnamic acid in H2O, 50% acetonitrile diluted three times. Internal calibration was performed with tryptic peptides resulting from autodigestion of trypsin (monoisotopic masses at m/z = 842.51, m/z = 1045.564, and m/z = 2211.105).

MS Data Analysis—
Monoisotopic peptide masses were assigned and used for database searches using the search engines MASCOT (Matrix Science, London, UK) (39) and Aldente (www.expasy.org). All proteins present in Swiss-Prot were used without any pI and Mr restrictions. The peptide mass error was limited to 90 ppm, and one possible missed cleavage was accepted.

MS/MS Data Analysis—
LC-MS/MS analysis of the digested proteins was performed using a CapLC capillary LC system (Micromass) coupled to a hybrid quadrupole orthogonal acceleration time-of-flight tandem mass spectrometer (Q-TOF II, Micromass). The LC-MS union was made with a PicoTip (New Objective, Woburn, MA) fitted on a ZSPRAY (Micromass) interface. Chromatographic separations were conducted on a reversed-phase capillary column (Pepmap C18, 75-µm inner diameter, 15-cm length, LC Packings) with a 200 nl/min flow. The gradient profile used consisted of a linear gradient from 95% A (H2O, 0.05% HCOOH) to 45% B (acetonitrile, 0.05% HCOOH) in 35 min followed by a linear gradient to 95% B in 1 min. Mass data acquisitions were piloted by MassLynx software (Micromass) using automatic switching between MS and MS/MS modes. The internal parameters of Q-TOF II were set as follows. The electrospray capillary voltage was set to 3.0 kV, the cone voltage was set to 30 V, and the source temperature was set to 80 °C. The MS survey scan was m/z 300–1500 with a scan time of 1 s and an interscan time of 0.1 s. When the intensity of a peak rose above a threshold of eight counts, tandem mass spectra were acquired. Normalized collision energies for peptide fragmentation were set using the charge state recognition files for +1, +2, and +3 peptide ions. The scan range for MS/MS acquisition was from m/z 50 to 1500 with a scan time of 1 s and an interscan time of 0.1 s. Fragmentation was performed using argon as the collision gas and with a collision energy profile optimized for various mass ranges of precursor ions. Mass data collected during a nano-LC-MS/MS analysis were processed and converted into a .PKL file to be submitted to the search software MASCOT (Matrix Science).

Statistics
Statistical comparisons were made using the Student’s two tailed t test. All results are representative of at least three experiments and expressed as means ± S.D. of at least three replicates.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Human monocyte-derived DCs are classically CD1a+ and CD14–, opposite of monocytes. The immature DC phenotype was attested by intermediate surface expression levels of HLA-ABC and HLA-DR and by negative or low expression of CD25, CD83, and co-stimulatory molecules CD80 and CD86 (Fig. 1 A). Following stimulation by the bacterially derived danger signal LPS or by the T cell-derived signal CD40 ligand, DC phenotype displayed the typical inductions of CD25 and CD83 and enhancements of CD80, CD86, HLA-ABC, and HLA-DR surface expressions. DCs differed from monocytes in their function because they elicited allogeneic T cell responses (Fig. 1B).


Figure 1
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  		FIG. 1. Phenotype and function of human monocytes and DCs. A, phenotype analysis of peripheral blood monocytes (dashed line), monocyte-derived immature DCs (thick line), and CD40L-activated mature DCs (bold line) by cytofluorometry. Cells were stained with antibodies against CD1a, CD14, CD25, CD80, CD83, CD86, HLA-ABC, and HLA-DR or related isotype control antibodies (gray histogram). Data are representative of more than 10 experiments. B, T cell proliferation measured by thymidine incorporation, at the indicated time points, in co-culture of allogeneic T cells with either DCs (circles) or monocytes (squares). DC:T cell ratio was 1:100 cells. The triplicate experiment is representative of more than 10 experiments. Mo, monocytes; imm DC, immature DCs; mat DC, mature DCs.

 
Comparative 2D gel analysis of the monocyte, immature DC, and mature DC stages is shown on Figs. 2 and 3. The most striking and reproducible differences indicated by arrows on the figures, as pulled from a quantitative gel image analysis, were further analyzed by mass spectrometry to determine the nature of these differentially expressed proteins. Gels with close detection sensitivity were selected so that the detection parameters for image analysis could be kept constant and lead to close quantitative values (see "Materials and Methods"). It is striking to note that there are relatively few reproducible differences especially between immature and mature dendritic cells. This step was our initial main focus as the monocyte-immature dendritic cell transition had been investigated before (40). These few differences point to well known functions of dendritic cells, such as antigen presentation (HLA class II) or cytokine production (IL1). They also point to cytoskeletal remodeling (gelsolin), which is obvious when taking into account the morphological changes between monocytes and immature and mature dendritic cells. However, one of the most striking differences is Mn-SOD. This protein has been described previously as heavily induced during the monocyte-immature DC transition (40), and it is rather surprising to see that despite the high levels reached at this stage, a further induction was observed during DC maturation. As Mn-SOD is an oxidative stress response protein whose expression is often modulated during proapoptotic conditions (41) or during cancer transformation (42), this oriented us to focus our study on oxidative stress response proteins. This was further reinforced by the fact that Prx1, which is also an important oxidative stress response protein, and Trx1, which plays an important role in several cellular redox processes, were also shown to be induced at both stages similar to Mn-SOD.


Figure 2
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  		FIG. 2. 2D electrophoresis of whole cell extracts (acidic and neutral proteins). Whole cell extracts (120 µg of protein) prepared from monocytes and immature and mature dendritic cells were analyzed by 2D electrophoresis. The pH gradient in the first dimension ranged from 4 to 8, thereby separating the acidic and neutral cellular proteins only. The second dimension was a 10% gel using a pH 8 gel and the taurine system. The oxidative stress response proteins identified on the gels are shown by arrows. The boxed zone is the one shown in Fig. 4.

 

Figure 3
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  		FIG. 3. 2D electrophoresis of whole cell extracts (basic proteins). The same analysis as in Fig. 2 was performed, but a pH 4–12 gradient was used in the first dimension. The second dimension was an 11% gel using a pH 8.2 gel and the taurine system. This allowed analysis of the basic proteins. Silver staining was with ammoniacal silver.

 
An additional benefit of the proteomic approach in the study of peroxiredoxins is that this technology allows access to both native and oxidized forms of the proteins (43, 44). From previously mentioned studies, we knew the positions of the various peroxiredoxin spots on the gels. However, we reassessed their positions on our DC gels by mass spectrometry to further secure our identifications. Sequence coverages were always above 50% (data not shown), thereby providing safe protein identifications. The identification of the peroxiredoxins and of Mn-SOD, however, was also secured by MS/MS analysis (see supplemental material).

The quantitative results obtained on oxidative stress proteins are summarized in Table I. Although they were identified on the gels as very minor spots (indicated by arrowheads), the oxidized forms of Prx1, Prx2, and Prx6 are not mentioned in Table I as they did not show any quantitative variation along DC differentiation and maturation processes. Expressions of the various proteins belonging to the Prx family are divergent and depend on the differentiation and maturation states. Some Prxs decreased moderately but steadily during the differentiation and maturation process (Prx6 and Prx4), whereas others increased steadily (Prx1), and others showed a bell-shaped expression curve (Prx2), peaking at the immature DC stage. The low but steady expression level of the mitochondrial Prx3 is surprising as it is a mitochondrial oxidative stress response protein (as is Mn-SOD). However, Mn-SOD was expressed at much higher levels and also showed a clear induction in our biological system. Finally results similar to those obtained with CD40 ligand were also obtained upon DC maturation with LPS (data not shown). To confirm those results, we used two different strategies. One was a co-migration strategy (Fig. 4). Pure Prx2 purified from human red blood cells (30) was added in increasing amounts to monocytes extracts, and the spiked extracts were separated by two-dimensional electrophoresis and submitted to image analysis. The results obtained showed a 2-fold difference between the amount of Prx2 that should be added to mimic the intensity observed in DCs (e.g. 20 ng for immature DCs) and the amount that was deduced from the quantitative image measurements (theoretically 40 ng). This can be accounted for by postulating that half of the loaded proteins are lost in the pH 4–8 two-dimensional analysis either from proteins lying outside the separation space (in pI and Mr) or from proteins lost because of poor solubility (e.g. membrane proteins). Such a quantitative yield has been described already (45). The second strategy was Western blotting, and it was used for Mn-SOD (Fig. 5). SDS-PAGE gels were used to secure against any artifact arising from 2D electrophoresis. The results clearly show an induction of the protein as observed from 2D gels. However, the induction factors observed by blotting were clearly inferior to those observed by 2D gel electrophoresis in accordance with the known non-linear behavior of Western blotting (46).


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  		TABLE I Quantitative measurements of the oxidative stress response proteins

The values are expressed in ppm of the total spot volume as calculated by the Melanie software. Due to the precision of the measurement (typically ±10%, but indicated on the table) they have been rounded (to the closest decennial). Study of the variations in expression of the antioxidant proteins between the various cellular stages showed significant variations (p < 0.05) except for Prx3 (no variation), Prx4 between immature and mature DCs (p < 0.5), and Prx6 between monocytes and immature DCs (p < 0.1).

 

Figure 4
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  		FIG. 4. Co-migration of Prx2 with monocyte extracts. Only the Prx2 zone is shown in this figure. Increasing amounts of chromatographically purified Prx2 (30) were added to a monocyte extract, and the spiked extracts were separated by two-dimensional electrophoresis using a linear pH 4–8 gradient. A, starting monocyte extract. B, monocyte + 10 ng of Prx2. C, monocyte + 20 ng of Prx2. D, monocyte + 50 ng of Prx2. E, immature dendritic cells. F, mature dendritic cells. Arrows point out Prx2 spots in the different conditions.

 

Figure 5
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  		FIG. 5. Western blotting of Mn-SOD. Western blot analysis of Mn-SOD expression in monocytes, immature monocyte-derived DCs (Imm DC), and LPS-matured monocyte-derived DCs (Mat DC). Immunoblot analysis was performed with anti-Mn-SOD antibody (catalog number 06-984, Upstate Cell Signaling Solutions) and anti-actin (catalog number A-2066, Sigma) antibody as loading control. Data are representative of two experiments.

 
Due to their high rate of antioxidant enzymes, we postulated that DCs are more resistant to oxidative environments than are monocytes. In human advanced atherosclerosis, the atherosclerotic lesions (plaques) constitute an extensive oxidative environment. Indeed oxLDLs accumulating in these lesions trigger the production of superoxide and peroxide by macrophages but also directly exert a toxic activity toward different cell types because of the lipid peroxides and hydrogen peroxide they contain. In vitro experiments have revealed previously that oxLDLs are toxic and apoptosis-inducing for macrophages and smooth muscle cells (15, 47). We thus studied and compared the survival capacities of both DCs and monocytes exposed to high concentration of oxLDLs. Cell death was measured after 24 h of incubation by intracellular incorporation of propidium iodide. The number of apoptotic monocytes following oxLDL treatment dramatically increased in the presence of oxLDLs from 12 to 76% (Fig. 6A). A preliminary dose-response study showed that monocytes were as potent as DCs to detoxify oxLDLs until a 50 µg/ml concentration (30–35% death). When oxLDL concentration exceeded this 50 µg/ml threshold, more than 87.3% of monocytes underwent apoptosis, whereas immature DCs and mature DCs were less affected (47.8 and 67.3% death, respectively) (Fig. 6B). More detailed investigations, working with 75 µg/ml oxLDL doses, demonstrated that immature DCs and, to a lower extent, mature DCs were more resistant than monocytes to the apoptosis induced by high doses of oxLDLs (Fig. 6C). Thus the survival of immature DCs in the oxidative stress environment generated by oxLDLs in vitro is better than monocyte survival. This finding can be correlated to their respective expression levels of Mn-SOD and Prx.


Figure 6
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  		FIG. 6. Apoptosis in human monocytes and DCs untreated or treated with oxLDL. Apoptosis was quantified by cytofluorometry after incorporation of propidium iodide. A, monocytes were cultured for 24 h with or without 75 µg/ml oxLDL. B, monocytes (diamonds), immature monocyte-derived DCs (squares), and CD40L-activated mature DCs were cultured for 24 h with increasing doses of oxLDL. C, mortality of monocytes, immature monocyte-derived DCs, and CD40L-activated mature DCs was measured after 24-h culture in the presence (black bars) or in the absence (white bars) of 75 µg/ml oxLDL. Means and S.D. of three experiments are shown. FSC, forward scattering.

 
The enhanced capacity of immature DCs to survive in an oxLDL-enriched environment, compared with monocytes, as well as the presence of DC-associated T cells in atherosclerosis lesions led us to investigate the production of RANKL in DC/T cell co-cultures. RANKL is the ligand of the TNF receptor family member RANK expressed on T cells activated by anti-CD3 and anti-CD28 antibodies (48) and on memory T cells (27). It is a crucial survival factor for DCs and a chemotactic factor for monocytes (26, 28, 49). Moreover expression of RANK and RANKL has been reported in atherosclerotic plaques (50). Immunofluorescent staining was used to study the expression of RANKL on T cells co-cultured with monocytes or DCs. T cells co-cultured with monocytes weakly expressed RANKL, and monocytes did not express RANKL themselves. In contrast, T cells co-cultured for 5 days with DCs strongly expressed RANKL on their surface. Interestingly DCs also displayed RANKL expression (Fig. 7). Consequently in addition to Mn-SOD and Prx overexpression, DC-induced RANKL expression may also be a key parameter accounting for a role of DCs in atherosclerosis pathogenesis.


Figure 7
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  		FIG. 7. RANKL expression in co-cultures of T cells with either monocytes or DCs. Shown are RANKL (green) and CD3 (red) stainings of T cells after 5 days of culture with monocytes (A), immature DCs (B), and anti-CD3 plus anti-CD28 antibodies (C). Stars indicate monocytes (A) or DCs (B). T cells were distinguished from other cells based on CD3 expression and Hoechst nuclei staining (not shown).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Because of their outstanding interest in immunology, gene expression in DCs has been studied in detail both at the monocyte-immature DC transition (40, 51) and at the immature-mature DC transition (52, 53). However, proteomic studies complementing the latter transcriptomic studies on DC maturation are required. We focused our study on a major protein induction observed in proteomics: Mn-SOD reached (by combining the two major Mn-SOD spots) 0.7% of the gel-displayed proteins at the mature DC stage. This important amount oriented us to study the oxidative stress response pathways. Mn-SOD is up-regulated by a variety of proinflammatory mediators, such as TNF-{alpha}, LPS, IL-1b, and interferon-{gamma} (54, 55). Therefore, the observed induction of Mn-SOD protein can be correlated with its well documented role as an antiapoptotic protein (41, 56). The main activity of Mn-SOD enzyme is to reduce anion superoxides into hydrogen peroxides. Together with glutathione peroxidase and catalase, Prx enzymes play an important role in eliminating peroxides (57), which are produced by numerous pathways, including the dismutation of superoxide. For example, Prx1 and Prx2 are involved in the removal of H2O2 in thyroid cells and can protect these cells from undergoing apoptosis (58). Only a moderate increase of DC apoptosis was observed in the presence of H2O2 (59) probably due to high Prx expression in DCs. Strikingly H2O2 rather seems to be an activating signal for DCs as it stimulates their production of IL-8 and TNF-{alpha} in a dose-dependent manner (10).

The high amount of antioxidant enzymes awards DCs the ability to survive in a highly oxidant environment. This also correlates with the antiapoptotic effects observed upon DC maturation (60). Recent studies have demonstrated that oxLDLs promote DC differentiation from monocytes (25), whereas oxLDLs induce apoptosis of human macrophages, a main feature in the first steps of atherogenesis (61). As oxLDLs produce both organic peroxides and hydrogen peroxide (14), it was interesting to study the changes in Prx expression during DC differentiation and maturation. In this case, proteomic study is especially well suited as the native and oxidatively inactivated forms of Prx can be separated on 2D gels (43, 44). In the case of DC differentiation and maturation, we did not notice any changes in these oxidized forms, which were present at very low amounts. However, interesting quantitative findings were made on the normal forms of Prx. First of all, the relative amounts of the various Prxs are very different. Mitochondrial Prx3 was always expressed at low levels. However, among the cytosolic Prxs (Prx1, Prx2, and Prx6), Prx2 was expressed at a much lower level than was Prx1 or Prx6 in monocytes (350 versus above 2000 ppm), although this is not the case in Jurkat cells (where Prx6 is almost undetectable (43)) or in HeLa cells (where Prx2 is expressed at much higher levels (44)). Prx2 counteracts the nuclear factor {kappa}B pathway (57), which is required for DC maturation (60). It was therefore not surprising to find low levels of Prx2 in mature DCs and, more generally, low levels of Prx2 in the monocyte-DC lineage compared with other hematopoietic lineages (62).

It is generally believed that all mammalian cytosolic peroxiredoxins have similar substrate specificity and are thus able to destroy both hydrogen peroxide and organic peroxides (63). In terms of peroxide destruction, the total cytosolic peroxiredoxin amount (i.e. Prx1 + Prx2 + Prx6) increases from 4800 ppm at the monocyte stage to 7500 ppm at the immature DC stage and to 9300 ppm at the mature DCs. Although Prx6 also exhibits other different actions, which may alter this simplistic scheme, our results suggest that the Prx defense line is induced steadily during DC differentiation and maturation. This overall increase, however, masks divergent quantitative changes in the cytosolic peroxiredoxins upon DC differentiation and maturation; this is the most surprising part of our data. Prx1 continuously increased during this process (close to 3-fold), whereas Prx6 continuously decreased (2-fold) and Prx2 peaks at the immature DC stage.

An interesting trend is provided by the dual function of Prx6, which is at the same time a peroxiredoxin and a phospholipase A2 (PLA2) (64). Thus, a decrease in Prx6 amount also means a decrease in PLA2 activity. Easily oxidized polyunsaturated fatty acids are often found at the 2-position in phospholipids and are therefore liberated by PLA2 activity. Inhibition of PLA2 can be of physiological interest because it prevents the rise of lysophosphatidylcholine levels and diminishes the death-inducing effects of oxLDLs on monocytes (65). Apart from the context of oxidized lipids, it must be kept in mind that Prx6 is also an activator of NADPH oxidase (66), which is active in DCs and especially mature ones (67). All these factors may explain the reorientation from Prx6 to Prx1 that occurs during DC differentiation and maturation.

In a more general frame, our data concerning monocyte and DC survival in an oxLDL-enriched stress environment cannot be directly correlated to the global cellular expression levels of the antioxidant enzymes Prxs. Indeed mature DCs, which have the highest content of Prxs, are more sensitive to oxLDL-induced apoptosis than are immature DCs. Cell survival to oxLDL-induced death correlates rather well with the cytosolic amount of Prx-2. This is rather surprising as the various mammalian Prxs are known to be able to reduce the same scope of peroxides in vitro (63). However, it must be kept in mind that Prxs interact with multiple and different partners (68). These other interactions may alter their operational efficiency either by altering their catalytic efficiency or by segregating some types of Prxs away from the peroxide substrates generated by oxLDLs, especially lipid hydroperoxides. This may explain why resistance to oxLDLs correlates with the expression of one particular Prx and not with the total Prx amount.

Concerning the role of DCs in atherosclerosis, it is now accepted that mature DCs, which are known to be present in advanced plaques (69), contribute to plaque destabilization especially through T cell activation and CD40-CD40L interactions play a key role in atherosclerosis progression (70). Besides being implicated in the induction of local immune or autoimmune responses, our results indicate, that when interacting with T cells, DCs produce the cytokine RANKL themselves and induce its production by T cells. RANKL, also called osteoprotegerin ligand or TNF-related activation-induced cytokine, is a chemotactic factor for monocytes, together with monocyte chemoattractant protein-1 produced by endothelial cells, following oxLDL stimulation in atherosclerosis. RANKL expression has been detected in advanced calcified lesions of atherosclerosis (50). Thus DC- and T cell-derived RANKL might also enhance the recruitment of monocytes from blood toward the atherosclerosis lesions.

RANKL also plays important roles in DC homeostasis. RANKL-RANK interaction has been shown to sustain DC survival by inducing the antiapoptotic gene Bcl-xL (28). Skin CD1a+ DCs express RANK but lack RANKL and are short lived. However, they can be rescued from cell death either by recombinant soluble RANKL or by RANKL+ DCs generated in vitro from CD34+ progenitors (28). In addition to enhancing DC survival, RANKL induces the expression of proinflammatory cytokines (IL-6 and IL-1) and T cell growth and differentiation factors (IL-12 and IL-15) by DCs in vitro (27). RANKL also provides co-stimulation required for efficient CD4+ T cell priming during viral infection in the absence of CD40L/CD40 (49, 71). These data further suggest that, besides recruiting more monocytes to the atherogenic plaque, RANKL produced locally may also increase DC lifespan in the plaque and amplify their functions, then contributing to the progression of atherosclerosis.

In conclusion, DC recruitment, maturation, and survival may be critical factors for the progression of atherosclerosis. (i) DC recruitment into the vascular wall is increased by atherogenic stimuli such as oxLDLs, TNF-{alpha}, and hypoxia (72). (ii) DC maturation is induced by different atherogenic stimuli such as superoxide, oxLDLs, lysophosphatidylcholine, nicotine, angiotensin II, atrial natriuretic peptide, and TNF-{alpha} (7–108910, 73, 74). (iii) DC survival in the atherosclerotic plaque environment is a third important factor enabling these professional antigen-presenting cells to induce a more important antigen-specific T cell activation. The high antioxidant enzyme expression levels, the better resistance to oxLDL-induced apoptosis, and the production of RANKL upon DC-T cell interactions that we highlighted in this work are three elements that further implicate DCs in the pathogenesis of atherosclerosis and emphasize the role they may play in the amplification of the chronic inflammation together with hypercholesterolemia and oxidative stress. This role of DCs in atherosclerosis progression was recently further supported by observations indicating that oxLDLs and platelet-activating factor contained in the lesions locally activate DCs but inhibit their migration to draining lymph nodes (75). This activated DC sequestration, in addition to enhanced DC survival, may further aggravate the local inflammation.


  FOOTNOTES
 
Received, August 15, 2005, and in revised form, December 23, 2005.

Published, MCP Papers in Press, January 19, 2006, DOI 10.1074/mcp.M500262-MCP200

Supported by CNRS.

1 The abbreviations used are: DC, dendritic cell; LPS, lipopolysaccharide; MHC, major histocompatibility complex; Mn-SOD, manganese superoxide dismutase; LDL, low density lipoprotein; oxLDL, oxidized low density lipoprotein; Prx, peroxiredoxin; RANK, receptor activator of nuclear factor {kappa}B; RANKL, receptor activator of nuclear factor {kappa}B ligand; IL, interleukin; GM-CSF, granulocyte macrophage colony-stimulating factor; mAb, monoclonal antibody; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; 2D, two-dimensional; TNF, tumor necrosis factor; PLA2, phospholipase A2. Back

* This work was supported in part by grants from INSERM, Université Claude Bernard Lyon, and CNRS; emergence project of Rhone-Alpes Region; Association pour la Recherche sur le Cancer Grants 4800 and 3637; Ligue contre le Cancer Ardèche, Drôme, and Rhône; and Ministère de l’ÉEducation Nationale, de la Recherche et de la Technologie Grant ACI 8BC05H. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

** To whom correspondence should be addressed: DRDC/ICH, INSERM U 548, CEA-Grenoble, 17 rue des martyrs, F-38054 Grenoble cedex 9, France. Tel.: 33-4-38-78-32-12; Fax: 33-4-38-78-98-03; E-mail: Thierry.Rabilloud{at}cea.fr


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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