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Originally published online as doi:10.1189/jlb.0205109 on April 13, 2006

Published online before print April 13, 2006
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(Journal of Leukocyte Biology. 2006;80:45-58.)
© 2006 by Society for Leukocyte Biology

Mixed Langerhans cell and interstitial/dermal dendritic cell subsets emanating from monocytes in Th2-mediated inflammatory conditions respond differently to proinflammatory stimuli

Nicolas Bechetoille*,{dagger}, Valérie André{dagger}, Jenny Valladeau*, Eric Perrier{dagger} and Colette Dezutter-Dambuyant*,1

* INSERM Unit 346, EA No. 37-32, Human Skin and Immunity, Claude Bernard University Lyon 1, Edouard Herriot Hospital, Cedex, France; and
{dagger} COLETICA, Lyon, France

1 Correspondence: INSERM Unit 346, Human Skin and Immunity, EA No. 37-32, Claude Bernard University Lyon 1, Edouard Herriot Hospital, 69437 Lyon Cedex 03, France. E-mail: dezutter{at}lyon.inserm.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The skin harbors two dendritic cell (DC) subsets, Langerhans cells (LC) and interstitial/dermal DC (IDDC), which traffic to lymph nodes after inflammation and ultraviolet stress. To demonstrate that monocytes may act as DC precursors for skin DC in postinflammatory recolonization, we generated LC and IDDC from monocytes by using cytokines related to the T helper cell type 2 environment [granulocyte macrophage-colony stimulating factor/transforming growth factor-ß/interleukin-13/tumor necrosis factor {alpha} (GM-CSF/TGF-ß/IL-13/TNF-{alpha})]. In this study, skin DC [LC as Langerin/CD207+ cells and IDDC as DC-specific intercellular adhesion molecule-grabbing nonintegrin (SIGN)/CD209+ cells] displayed desynchronized programs along their differentiation, activation/maturation processes in response to stimuli characteristics of a proinflammatory context. First, we demonstrate that monocytes are able to diverge simultaneously along two distinct pathways toward Langerin+-LC-type DC and DC-SIGN+-IDDC. Second, as TGF-ß is known to antagonize the TNF-{alpha}-induced maturation process of DC, we showed that IDDC did not mature and acquired a low CC chemokine receptor 7 (CCR7) receptor expression even when stimulated with prolonged incubation with TNF-{alpha}. It is striking that the LC subset is able to express a high level of CCR7 expression and the maturation marker DC-lysosome-associated membrane protein (DC-LAMP). Third, mixed LC and IDDC subsets secrete IL-10 and IL-12 when stimulated by CD40 ligand and lipopolysaccharide (LPS) but not after prolonged incubation with TNF-{alpha}. In contrast, LPS was a better activator of IL-10 secretion than the CD40 ligand for GM-CSF/IL-4-generated DC and for GM-CSF/TGF-ß/IL-13-generated LC and IDDC populations. To summarize, the phenotypic/migratory maturation status of LC may be more easily enhanced by stimuli mimicking a proinflammatory situation, and IDDC are more resistant. Moreover, our culture system provided a means of studying cross-talk between two skin DC outside of their respective skin compartment.

Key Words: skin • differentiation • maturation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) are professional antigen-presenting cells, which play a critical role in the initiation of primary immune responses [1 , 2 ]. They differentiate from their precursors into so-called immature DC (iDC), which are present in most tissues in a sentinel position. The best-characterized iDC are Langerhans cells (LC), which are located in the epidermis of skin, where they form a cellular network that constitutes the first immunological barrier against environmental insults and pathogens [3 ]. Epidermal LC have a high capacity of internalizing exogenous antigens and responding to "danger signals" [3 ]. During the initiation of an immune response, epidermal LC migrate from the skin to draining lymph nodes to present processed antigens to T cells and induce antigen-specific T cell immunity [4 5 6 ]. In the skin, the dermis harbors another DC population, interstitial/dermal DC (IDDC), which should play an important role in the regulation of skin immune responses [7 ]. At present, the relationship between IDDC and epidermal LC is still unclear, and their respective/interactive contributions to skin immune responses to environmental stimuli are still not defined clearly.

Epidermal LC are characterized by intracellular structures formed by double membrane joints—Birbeck granules [8 ]. Unlike other members of the DC family, epidermal LC express CD1a, E-cadherin, cutaneous lymphocyte-associated protein (CLA) and CC chemokine receptor 6 (CCR6) [9 10 11 12 13 ]. Epidermal LC also specifically express the lectin Langerin/CD207, an endocytic receptor that may be implicated in Birbeck granule formation [14 , 15 ]. In contrast, IDDC are poorly characterized. Several subsets of IDDC have been described, showing a phenotypic heterogeneity, which may reflect different steps of maturation but also different origins [7 , 16 17 18 ]. Finally, a C-type lectin, DC-specific intercellular adhesion molecule-grabbing nonintegrin (SIGN)/CD209, is expressed exclusively on IDDC and consequently represents a useful marker for these IDDC [18 , 19 ].

LC and IDDC can be generated in vitro from CD34+ hematopoietic progenitor cells [20 21 22 23 24 ] or CD14+ circulating monocytes [25 26 27 28 29 30 31 ]. In vitro, Ito et al. [32 ] have also shown that a CD1a+/CD11c+ subset of peripheral blood DC may represent a direct precursor of LC. Although combinations of hematopoietic growth factors, including granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-3 (IL-3), tumor necrosis factor {alpha} (TNF-{alpha}), IL-4, and IL-13, stimulate the in vitro development of interstitial DC, the presence of transforming growth factor-ß1 (TGF-ß1) in the cytokine milieu appears to be essential for the development of characteristics of LC [25 , 33 34 35 36 37 ]. However, Mohamadzadeh et al. [26 ] have generated LC-type DC (i.e., E-cadherin and Langerin), which have some characteristics of mature DC [mDC; i.e., DC-lysosome-associated membrane protein (LAMP)/CD208 but no intracellular Birbeck granules] from CD14+ monocytes cultured in the presence of GM-CSF plus IL-15 without TGF-ß1 [26 ].

Although the mechanism by which LC and IDDC enter draining lymph nodes is rather well-characterized, no mechanism by which skin DC precursors populate the skin or the identity of these precursors has been well-determined. First, with regard to epidermal "colonization" of LC, it has been postulated that bone marrow-derived, myeloid CLA+ LC precursors travel via peripheral blood through the dermis to the overlying, clinically normal human epidermis [38 ]. Using a skin explant model, Larregina et al. [39 ] described a population of CD14+ cells that differ from peripheral blood monocytes and that may represent direct precursors of LC. Macrophage inhibitory protein-3{alpha} (MIP-3{alpha})/liver and activation-regulated chemokine (LARC)/Exodus-1/CC chemokine ligand 20 (CCL20) is a constitutive chemokine released by keratinocytes, which is a key to the attraction of LC precursors to the epidermis [40 ]. In inflamed human skin, where a transient epidermal depletion of LC was observed, the mechanisms of LC repopulation by the recruitment of precursor cells from the circulating blood or from the dermis or alternatively, from migration from the surrounding normal epidermis into the inflamed area, are also not clear. MIP-3{alpha}, secreted in excess by keratinocytes after inflammatory stimuli or T cell signal, has also been shown to play a major role in the recruitment of LC precursors at sites of inflammation [41 ]. Second, little is known about the origin of IDDC and their potential precursors. In a model of transendothelial circulation, monocytes and CD14+CD34+ leukocytes differentiated into interstitial-type DC [42 , 43 ]. Previously, Vanbervliet et al. [44 ] demonstrated that CD34+ hematopoietic progenitor cell-derived CD14+ precursors of interstitial DC sequentially express CCR2 and CCR6 and consequently, can migrate into inflamed human skin, where chemokine monocyte chemoattractant protein-4 and MIP-3{alpha} are secreted heavily by keratinocytes.

Therefore, in this study, we examined the simultaneous differentiation of CD14+ peripheral blood monocytes via two distinct pathways toward LC-type DC and IDDC by using the combined cytokine cocktail composed of GM-CSF, TGF-ß, IL-13, and TNF-{alpha}. We highlighted a distinct, exclusive, and independent differentiation/maturation program of both skin DC subsets in response to soluble proinflammatory stimuli.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Culture media, antibodies, and cytokines
The medium used for cell culture experiments was RPMI 1640, supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% heat-inactivated fetal calf serum (FCS) Myoclone (all from Gibco-BRL, Grand Island, NY), hereafter called complete medium. Mouse monoclonal antibodies (mAb) used were: fluorescein isothiocyanate (FITC)-conjugated anti-CD1a [Clone NA1/34, immunoglobulin G2a (IgG2a), Dako, Glostrup, Denmark], FITC-conjugated anti-CD11b (Clone Bear1, IgG1, Immunotech, Marseille, France), phycoerythrin (PE)-conjugated anti-CD11c (Clone BU15, IgG1, Immunotech), Texas red tandem conjugated (TC) anti-CD14 (Clone TÜK-4, IgG1, Caltag Laboratories, Burlingame, CA), PE/FITC-conjugated anti-CD14 (Clone RMO52, IgG2a, Immunotech), PE-conjugated anti-CD16 (Clone 3G8, IgG1, Immunotech), FITC-conjugated anti-CD64 (Clone 22, IgG1, Immunotech), TC anti-CD68 (Clone KP1, IgG1, Dako), TC anti-CD83 (Clone HB15, IgG2b, Caltag Laboratories), FITC-conjugated anti-CD86 (Clone BU63, IgG1, Caltag Laboratories), PE-conjugated anti-CD207/Langerin (Clone DCGM4, IgG1, Immunotech), PE-conjugated anti-CD208/DC-LAMP (Clone 104.G4, IgG1, Immunotech), TC anti-human leukocyte antigen (HLA)-DR (Clone TÜ36, IgG2b, Caltag Laboratories), PE-conjugated anti-CCR6 (Clone 53103.111, IgG2b, R&D Systems, Minneapolis, MN), PE-conjugated anti-CCR7 (Clone 150503, IgG2a, R&D Systems), anti-CD80 (Clone 104, IgG1, Immunotech), anti-CD208/DC-LAMP (Clone 104.G4, IgG1, Immunotech), anti-CD209/DC-SIGN (Clone 1B10, IgG1, kindly provided by Ali Amara, Institut Pasteur, Paris, France), anti-E-cadherin (Clone HECD-1, IgG1, Takara, Shiga, Japan), and anti-CCR7 (Clone 2H4, IgM, Becton Dickinson, San Jose, CA). Rabbit polyclonal antibody anti-factor XIII-A was purchased from Nordic Immunological Laboratories (Tilburg, Netherlands). Recombinant human cytokines (rh)IL-13, rhTGF-ß1, and rhTNF-{alpha} were purchased from R&D Systems. rhGM-CSF was kindly provided by S. Sealand and S. Lebecque S. (Schering-Plough, Kenilworth, NJ). Chemokines rhMIP-3{alpha}/LARC/CCL20 and rhMIP-3ß/ELC/CCL19 were purchased from R&D Systems.

Murine fibroblast cell lines transfected with human CD40 ligand or CD32 were used as controls and were kindly provided by Schering-Plough. Lysine-fixable FITC-dextran (molecular weight=40,000) was purchased from Molecular Probes, Inc. (Eugene, OR).

In vitro generation of monocyte-derived LC/IDDC and flow cytometry analysis
CD14+ monocytes were isolated as described previously by Geissmann et al. [25 ]. Briefly, fresh CD14+ monocytes were isolated from peripheral blood mononuclear cells of healthy volunteers by the standard Ficoll-Paque method and immediately separated by negative magnetic depletion using hapten-conjugated CD3, CD7, CD19, CD45RA, CD56, and anti-IgE antibodies [monocyte isolation kit, magnetic cell sorter (MACS), Miltenyi Biotec, Bergisch Gladbach, Germany] and a magnetic cell separator (Midi MACS), according to the manufacturer’s instructions, routinely resulting in >95% pure CD14+ cells. Purified CD14+ monocytes were cultured in six-well tissue-culture plates (Costar Corp., Cambridge, MA) for 6 days in complete medium supplemented at Day 0 with 200 ng/mL GM-CSF, 10 ng/mL TGF-ß1, and 10 ng/mL IL-13. At Days 2 and 4, fresh medium supplemented with GM-CSF and TGF-ß1 (but not IL-13) was added. At Day 6, monocyte-derived DC were or were not stimulated with 200 U/mL TNF-{alpha} for 6, 12, 18, 24, and 48 h.

Phenotype analysis was carried out by flow cytometry. Briefly, 1 x 105 cells were incubated for 30 min at 4°C with affinity-purified mouse mAb at the appropriate concentration or with isotype control at the same concentration. Cells were washed and for indirect staining, incubated for 30 min at 4°C with FITC-conjugated goat F(ab')2 anti-mouse Ig (Immunotech).

For double-staining, cells were stained successively with anti-CD208/DC-LAMP, anti-CD209/DC-SIGN, or anti-CCR7 antibodies and then with FITC-conjugated goat F(ab')2 anti-mouse Ig and PE-conjugated anti-CD207/Langerin, PE-conjugated anti-CD208/DC-LAMP, or PE-conjugated anti-CCR7 antibodies. Intracellular staining was carried out using the "Fix and Perm" cell permeabilization kit (Caltag Laboratories), according to the manufacturer’s instructions. Aliquots of 5 x 104 were then analyzed with a FACScan using CELLQUEST-Pro software (Becton Dickinson, Le Pont de Claix, France).

Characterization of differentiation/activation/maturation states of in vitro generation of monocyte-derived LC/IDDC
FITC-dextran uptake procedure
FITC-dextran uptake of monocyte-derived DC was assessed as described previously [45 ]. Briefly, 1.5 x 105 cells were resuspended in internalization buffer (phosphate-buffered saline-2% heat-inactivated FCS) and incubated with FITC-dextran at a final concentration of 1 mg/mL for 15 min at 37°C. As a control, a portion of DC suspension was incubated with FITC-dextran at 4°C. Cells were washed four times with cold internalization buffer and were analyzed by flow cytometry (Becton Dickinson, France).

Chemotaxis assay
Cell migration was assessed as described previously [41 ]. Briefly, rhMIP-3{alpha}/LARC/CCL20 and rhMIP-3ß/ELC/CCL19 were diluted at a final concentration of 1 µg/mL and were added to 12-well plates (Costar Corp.). A total of 2 x 105 cells was added to Transwell inserts with a standard 5-µm pore polycarbonate filter. Plates were incubated for 2 h at 37°C. After removal of the Transwell inserts, cells were counted. Each assay was performed in triplicate, and the results are expressed as the mean number of cells having migrated in the lower well compartment.

CD40 ligation procedure
As control, fibroblastic L cells, transfected with CD40 ligand or CD32, were irradiated at 80 Gy and added to cultures of monocyte-derived DC in a proportion of 1:10 for 40 h.

Quantitation of cytokine secretion by enzyme-linked immunosorbent assay (ELISA)
Supernatants were stored at –70°C until cytokines were measured. Production of IL-10 and IL-12p70 was measured in duplicate using ELISA Quantikine kits (R&D Systems) according to the manufacturer’s instructions. Sensitivity of IL-10 and IL-12 detection was, respectively, 3.5 pg/mL and 0.5 pg/mL.

Mixed lymphocyte/DC reaction
Allogeneic T cells were isolated from lymphocyte pellets obtained after Percoll gradient by rosetting with sheep red blood cells as described previously [46 ]. The T cell population contained more than 95% CD3-positive cells, as assessed by flow cytometry. DC were resuspended in RPMI 1640 with 10% human AB serum and added in triplicate at various concentrations to 105 allogeneic T cells/well in round-bottom, 96-well tissue-culture plates (Falcon, Oxnard, CA). Triplicate cultures were maintained for 5 days at 37°C. T cell proliferation was measured by pulsing the cells with 1 µCi [3H] methylthymidine (Amersham France SA, Les Ulis) for the final 18 h of culture. Cells were then harvested, and incorporated thymidine was quantitated in a direct ß-counter (Matrix 96, Packard Instruments, Meriden, CT).

Immunogold labeling in transmission electron microscopy
Langerin expression was investigated by immunogold labeling. Briefly, 1 x 105 cells were incubated for 1 h at 4°C with anti-Langerin mAb (DCGM4, IgG1, Immunotech) at the dilution 1:100 or with isotype control. Then, cells were washed and incubated for 1 h at 4°C with 5 nm gold granule-conjugated goat F(ab')2 anti-mouse Ig (Amersham France SA). After washing, cells were fixed with 2% glutaraldehyde in cacodylate buffer for 18 h. After rinsing in cacodylate buffer with sucrose for 12 h, the cells were processed for transmission electron microscopy. Cells were postfixed with an aqueous solution of 1% osmium tetroxide in cacodylate buffer with sucrose and embedded in epoxy medium after dehydration through a graded series of ethanol. Ultrathin sections were stained with lead citrate and uranyl acetate and examined with a JEOL 1200EX electron microscope (CMEABG, Lyon University, France).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
GM-CSF/TGF-ß1/IL-13-generated DC composed of two DC subsets displaying LC precursor and immature interstitial/dermal DC phenotypes
The cytokines GM-CSF and IL-13 have been described to skew the differentiation of CD14+ monocytes toward DC [29 30 31 ]. Furthermore, CD14+ monocytes cultured in the presence of GM-CSF, TGF-ß1, and IL-4 can differentiate into LC-type DC [25 ]. As IL-4 and IL-13 have been evolved by gene duplication and have consequently shown many structural and biological similarities [47 ], we investigated whether the combination of the cytokines GM-CSF, TGF-ß1, and IL-13 induces CD14+ monocytes to become LC-type DC.

Purified CD14+ monocytes through negative selection on magnetic columns expressed characteristic monocyte markers: CD14+high/CD11b+high, CD14+high/CD11c+high, and CD14+high/CD64+high (Fig. 1A ). A gradient of expression from a low level up to a high level of expression was noted for CD16 and HLA-DR (Fig. 1A) .


Figure 1
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  		Figure 1. Phenotype analysis, internalization, and migratory capacity of monocyte-derived DC generated with GM-CSF, TGF-ß1, and IL-13. (A) Freshly purified CD14+ monocytes were analyzed for the expression of characteristic monocyte markers by fluorescein-activated cell sorter (FACS) analysis (n=4 experiments). Freshly isolated CD14+ monocytes were cultured for 6 days in the presence of GM-CSF, TGF-ß1, and IL-13. (B and D) Cells were then harvested, and expression of indicated markers was determined by FACS analysis (n=12 experiments). Filled histograms represent antigen staining, and open histograms represent isotype-matched controls. Percentage of positive cells and mean fluorescence intensity (MFI) are mentioned in dot plots. (C) Cells were cultured for an additional 40 h without or with CD40 ligand-transfected fibroblasts, and then expression of maturation markers and capacity to internalize FITC-dextran were determined by FACS analysis (n=4 experiments). Filled histograms represent antigen staining, and open histograms represent isotype-matched controls. For dextran internalization, 1.5 x 105 cells were incubated in the presence of FITC-dextran at 37°C (filled histograms) for 15 min. No uptake was detected at 4°C (open histograms). All flow cytometry analyses were performed without gating using a FACScan with CellQuest Pro software (Becton Dickinson, Le Pont de Claix, France). (E) Cells were tested for their capacity to migrate in response to MIP-3{alpha}/CCL20. Migration assays were performed by seeding 2 x 105 cells in TranswellTM inserts of 5 µm for 2 h at 37°C. Each assay was performed in triplicate (mean±SD), and results are expressed as the index of migrating cells (ratio of chemokine:medium; n=4 experiments). FSC, Forward-scatter.

 
CD14+ monocytes, cultured for 6 days in the presence of GM-CSF, TGF-ß1, and IL-13, generated large numbers of dendritic-shaped cells, as revealed by Giemsa staining (data not shown). GM-CSF, TGF-ß1, and IL-13 gave rise to cells with a DC phenotype. Expression of the monocyte marker CD14 and the lysosomal marker CD68 was still observed on these cells, which expressed CD1a antigen and major histocompatibility complex (MHC)-class II molecules at the surface (Fig. 1B) . At Day 6 of culture, the generated DC were immature with low or no expression of CD80, CD83, CDw197/CCR7, and CD208/DC-LAMP (Fig. 1C) . Upon ligation with CD40 ligand for 40 h, these DC showed increased surface expression of HLA-DR, CD80, and CD83 and acquired CCR7/CDw197 and DC-LAMP/CD208 expression, consistent with maturation of DC (Fig. 1C) . As the capacity of internalization is a hallmark of iDC, we investigated whether GM-CSF/TGF-ß1/IL-13-generated DC were able to take up FITC-dextran after only 15 min of incubation, as described previously by Caux et al. [48 ]. As expected, the generated DC efficiently captured soluble FITC-dextran but lost this capacity upon maturation (Fig. 1C) .

Although several studies have shown that TGF-ß1 is essential for the development of LC [25 , 33 34 35 36 37 ], the DC generated with GM-CSF/TGF-ß1/IL-13 expressed no Langerin at the intracellular or surface level (Fig. 1D) . However, a subset of these DC expressed the two characteristic markers of epidermal LC: E-cadherin, an adhesion molecule involved in the binding to keratinocytes [9 ], and CCR6, the receptor for MIP-3{alpha}/LARC/CCL20/Exodus-1 [13 ]. These two LC-specific markers (E-cadherin and CCR6) were observed after 6 days of culture of the CD14+ monocytes in the presence of GM-CSF/TGF-ß1/IL-13 (Fig. 1D) . Furthermore, we determined whether the CCR6 chemokine receptor was functional by testing the capacity of GM-CSF/TGF-ß1/IL-13-generated DC to migrate in response to MIP-3{alpha} in TranswellTM inserts. As illustrated in Figure 1E , the cells migrated in response to MIP-3{alpha}, proving the functionality of CCR6. We also analyzed whether a subpopulation could express markers of dermal DC—DC-SIGN and factor XIIIa, two specific markers of dermal DC. We observed that a subset of GM-CSF/TGF-ß1/IL-13-generated DC was related to IDDC (Fig. 1D) .

The exclusive effect of differentiation by TNF-{alpha} on the generated LC-type DC and IDDC subsets is time-dependent
TNF-{alpha} is a proinflammatory cytokine, which is used widely to provoke full DC differentiation of LC-type DC [49 ]. We therefore investigated whether TNF-{alpha} could enhance the LC differentiation by analyzing Langerin expression on GM-CSF/TGF-ß1/IL-13-generated DC (Days 3 and 6 and in the presence of TNF-{alpha} for 18 h and 48 h).

After 3 days of culture in the presence of cytokines GM-CSF, TGF-ß1, and IL-13, CD14+ monocytes acquired DC-SIGN expression independently of the presence of TNF-{alpha} (Fig. 2A ). DC-SIGN expression was maintained until Day 8 of the culture (Fig. 2A) . We observed that incubation of TNF-{alpha} for 18 h strongly induced surface expression of Langerin (42±20% of Langerin+ cells) on only one subset of DC (Fig. 2A) , and the chemokine receptor CCR6 was disappearing (Fig. 2B) . It was noted that on Day 6 of the monocyte culture in the presence of GM-CSF, TGF-ß1, and IL-13, the DC-SIGN+ cells did not express CCR6 (Fig. 2B) . As CCR6 expression was not found on the DC-SIGN+ cells, the chemokine receptor CCR6 may be restricted to the Langerin– LC precursor pool on Day 6 of the culture. As the direct isolation of CCR6+-labeled cells by affinity column selection led to a high cell mortality of purified CCR6+ cells (data not shown), we had to purify the CCR6+ cells (without mAb labeling) by using their capacity to be chemoattracted by the specific ligand to CCR6, i.e., the chemokine MIP-3{alpha}. To demonstrate that CCR6+ cells can progressively lose MIP-3{alpha} receptor in favor of Langerin expression while they are differentiating, we isolated CCR6+ cells by using a chemotaxis assay. Day 6-generated CCR6+ cells (20±10%) were chemoattracted by MIP-3{alpha}, then cultured in the presence of TNF-{alpha} (48 h), and analyzed further for cytoplasmic Langerin expression. As expected, all the cultured MIP-3{alpha}-chemoattracted cells expressed Langerin (Fig. 2C) . Therefore, on Day 6 of the culture, the CCR6+ cells may be considered as Langerin– LC precursors.


Figure 2
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  		Figure 2. Expression of Langerin, DC-SIGN, and CCR6 on GM-CSF/TGF-ß1/IL-13-generated DC in time-course of culture. Freshly isolated CD14+ monocytes were cultured for 6 days in the presence of GM-CSF, TGF-ß1, and IL-13. Cells were then recovered and reseeded in the presence of GM-CSF, TGF-ß1, and TNF-{alpha} for 18 h and 48 h. At indicated time-points, cells were harvested, and expression of indicated markers was determined by FACS analysis. (A) Expression of Langerin and DC-SIGN on fresh CD14+ monocytes and on generated DC in time-course of culture, i.e., Days 3 and 6, Day 6 + 18 h TNF-{alpha}, and Day 6 + 48 h TNF-{alpha}. (B) Expression of CCR6 and DC-SIGN was analyzed on the same cells. Percentage of positive cells is mentioned in dot plots. Results are representative of four experiments. (C) Six day-generated DC were analyzed for CCR6 expression. The chemoattracted CCR6+ cells, in response to MIP-3{alpha}, were cultured further for 2 days in the presence of GM-CSF, TGF-ß1, and TNF-{alpha} and analyzed for Langerin expression by FACS. Filled histograms represent antigen staining, and open histograms represent isotype-matched controls. These cytometry graphs are representative of the four experiments performed. Results were stated statistically by expressing mean ± SD.

 
A prolonged stimulation by TNF-{alpha} up to 48 h induced a down-regulation of Langerin surface expression (Fig. 2A) , as it was described previously by Valladeau et al. [14 ]. The localization changes of Langerin epitopes recognized by DCGM4 mAb, from surface to intracellular levels, seem to be attributed to a activation/maturation process of LC-type DC. To investigate more precisely the effect of TNF-{alpha} on the differentiation of the LC-type DC, we performed a kinetics study to clarify the effect of TNF-{alpha} on DC populations generated in the presence of GM-CSF, TGF-ß, and IL-13, i.e., LC-type DC and IDDC. Langerin epitopes were recognized early, after 6 h of TNF-{alpha} incubation at the intracellular level and then were progressively translocated to the cell surface, and the intracellular intensity decreased (Fig. 3 ). The Langerin surface expression was optimum after 18 h of TNF-{alpha} incubation and then was down-regulated progressively, and the Langerin gradually increased at the intracellular level up to 48 h incubation with TNF-{alpha} (Fig. 3) . In contrast, and as we have already shown in Figure 2A , surface DC-SIGN expression (35±8% of DC-SIGN+ cells) was not affected by the addition of TNF-{alpha} from a short (6 h) to a prolonged (48 h) incubation period (Fig. 3) , as described in iDC or mDC [19 ]. These results are schematized in Figure 3 and revealed that Langerin expression at the surface versus cytoplasmic level was modulated by TNF-{alpha} in time-dependent manner, and in contrast, DC-SIGN expression was TNF-{alpha}-independent.


Figure 3
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  		Figure 3. Analysis of Langerin and DC-SIGN expression on GM-CSF/TGF-ß1/IL-13-generated DC during TNF-{alpha} stimulation. Freshly isolated CD14+ monocytes were cultured for 6 days in the presence of GM-CSF, TGF-ß1, and IL-13. Cells were then recovered and reseeded in the presence of GM-CSF, TGF-ß1, and TNF-{alpha} for 6–48. At indicated time-points, i.e., 6, 12, 18, 24, and 48 h, TNF-{alpha} cells were harvested, and expression of indicated markers was determined by FACS analysis. Percentage of positive cells and MFI are mentioned in dot plots. Results are representative of three experiments.

 
As Langerin and DC-SIGN markers are expressed on subsets of GM-CSF/TGF-ß1/IL-13-generated DC after TNF-{alpha} stimulation (18 h), we used double-staining experiments to control that these two populations were distinct and consequently, to demonstrate that our culture model was able to generate distinct and exclusive populations of skin DC from CD14+ monocytes. By means of IL-13, after incubation with TNF-{alpha} for 18 h, two populations characterized by the exclusive expression of Langerin and DC-SIGN emerged independently (Fig. 4 ). In accordance with Arrighi et al. [50 ], IL-4 generated a double-positive subpopulation expressing surface Langerin and DC-SIGN (9±5% in the presence of IL-4 vs. 0.5±2% in the presence of IL-13). As a low percentage of double-positive DC-SIGN+/Langerin+ cells was found in the presence of IL-13, our DC generation model reinforces the role that may play IL-13 in the exclusive emergence of lectin expression, allowing the generation of skin DC subpopulations—the Langerin+ LC and the DC-SIGN+ dermal DC in a unique suspension.


Figure 4
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  		Figure 4. Expression of Langerin and DC-SIGN on DC generated with GM-CSF/TGF-ß1 plus IL-13 or IL-4. Freshly isolated CD14+ monocytes were cultured for 6 days in the presence of GM-CSF and TGF-ß1, plus IL-13 or IL-4. Cells were then recovered and reseeded in the presence of GM-CSF, TGF-ß1, and TNF-{alpha} for 18 h. The expression of Langerin and DC-SIGN was determined by double staining and analyzed by FACS. Quadrant setting was performed according to the reactivity of isotype-matched control mAb. Percentage of positive cells is mentioned in dot plots. Data are representative of four experiments.

 
As LC are characterized by their intracytoplasmic organelles, Birbeck granules, we also performed electron microscopy studies. Typical Birbeck granules, i.e., tennis racket-like structures with rod-like profiles, electron-dense central lamella, and rounded ends (Fig. 5A ), were identified by electron microscopy in 30–35% of DC stimulated by a short incubation (18 h) of TNF-{alpha} in three experiments. Long rod-like structures were observed forming from the plasma membranes (Fig. 5B) , whereas short and typical short rod-like Birbeck granules (Fig. 5C) were found in the cytoplasm, mainly nearby the Golgi apparatus. By incubation with DCGM4 mAb plus 5 nm gold particle-conjugated goat IgG anti-mouse IgG, cross-linked gold particle-Birbeck granules were induced from plasma membrane (Fig. 5D 5E) and internalized into the Langerin+ LC cytoplasm (Fig. 5F) . The induction of Birbeck granules occurred along the labeling procedure and only in the presence of DCGM4 mAb (1 h incubation at 4°C) + 5 nm gold particle-conjugated goat IgG anti-mouse IgG (1 h incubation at 4°C), without the necessity of further incubation at 37°C. As Valladeau et al. [15 ] already demonstrated on epidermal LC, the cross-linkage of Langerin molecules recognized by DCGM4 mAb induced typical Birbeck granule formation from a Langerin+ cell membrane.


Figure 5
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  		Figure 5. Electron microscopy analysis of GM-CSF/TGF-ß1/IL-13-generated DC after short TNF-{alpha} stimulation. Freshly isolated CD14+ monocytes were cultured for 6 days in the presence of GM-CSF, TGF-ß1, and IL-13. Cells were then seeded in the presence of GM-CSF, TGF-ß1, and TNF-{alpha} for 18 h. Cells were then harvested, processed for standard electron microscopy analysis or for immunogold labeling, and then analyzed by electron microscopy as described in Materials and Methods. Ultrathin sections were examined on a JEOL 1200 EX electron microscope. Results are representative of three experiments. (A) Typical tennis racket-like Birbeck granules (BG) were found gathered underneath the plasma membrane (black arrows). (B) Long Birbeck granules (black arrow) were also seen forming from plasma membrane. (C) Some short and isolated Birbeck granules (black arrows) were found in the cytoplasm near the Golgi apparatus. (D–E) Plasma membrane displayed 5 nm gold granule (gg) binding (dotted arrow). Some 5 nm gold granules are observed in the round part of Birbeck granules, formed from plasma membrane or internalized in the cytoplasm (F, dotted arrow). Observation is representative of three experiments. Original bar, 200 nm.

 
Taken together, our results showed that TNF-{alpha} delivers a signal-inducing terminal differentiation of LC precursors into Langerin+ LC for the first 18 h in the context of Langerin acquisition and transfer to plasma membrane. The earlier acquisition of DC-SIGN expression occurred in the absence of TNF-{alpha} (on Day 3 of the culture). In conclusion, Langerin expression at the surface versus cytoplasmic level is modulated in TNF-{alpha} in a time-dependent manner in contrast to DC-SIGN expression. Furthermore, CCR6 expression was only found on cells capable to differentiate into Langerin+ cells, suggesting that a Langerin– LC precursor pool is restricted to a CCR6+ cell population.

Desynchronized process of activation/maturation by TNF-{alpha} on the generated LC-type DC and IDDC subsets
We have showed above that TNF-{alpha} delivers a signal-inducing LC differentiation in the first 18 h of incubation. For further time lapse, it was well-known that TNF-{alpha} acts as a proinflammatory cytokine by activating and maturing conventional DC. Thus, we investigated whether TNF-{alpha} may deliver a similar activation/maturation signal to our model of generated DC, which mimic skin DC subpopulations, the Langerin+ LC, and the DC-SIGN+ dermal DC. To this end, we explored whether the hallmarks that are encountered on activated DC (HLA-DR, T cell costimulatory molecules, and CCR7) and mDC (CD83 and DC-LAMP) may be found in our skin DC model for a long-time incubation with TNF-{alpha} (48 h). As expected, we observed no expression increase of membrane HLA-DR, CD80, CD86, and CD83 and no acquisition of DC-LAMP expression on Day 6-generated DC maintained for 18 h in the presence of TNF-{alpha} (Fig. 6 ). In contrast, 48-h incubation in the presence of TNF-{alpha} leads to an activated/mDC phenotype, as proven by acquisition of higher expression of membrane HLA-DR (increase of 1.5-fold of percentage of positive cells and increase of 7.5-fold of MFI) and T cell costimulatory molecules (twofold increase of the percentage of CD80+ and CD86+ cells, ninefold increase of CD80 antigen density, and twofold increase of CD86 antigen density). The DC maturation CD83 antigen only showed a 1.6-fold increase of percentage of positive cells, whereas the acquisition of the maturation marker DC-LAMP antigen was noted. In this context, it is noteworthy that the acquisition of DC-LAMP+ expression is restricted to a low proportion of GM-CSF/TGF-ß1/IL-13-generated DC (Fig. 6) .


Figure 6
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  		Figure 6. Expression of activation/maturation markers on GM-CSF/TGF-ß1/IL-13-generated DC after TNF-{alpha} stimulation. Freshly isolated CD14+ monocytes were cultured for 6 days in the presence of GM-CSF, TGF-ß1, and IL-13. Cells were then recovered and reseeded in the presence of GM-CSF, TGF-ß1, and TNF-{alpha} for 18 h and 48 h. At indicated time-points, Day 6, Day 6 + 18 h TNF-{alpha}, and Day 6 + 48 h TNF-{alpha}, cells were harvested, and expression of indicated markers was determined by FACS analysis. Percentage of positive cells and MFI are mentioned in dot plots. Results are representative of four experiments.

 
To know whether GM-CSF/TGF-ß1/IL-13-generated DC populations, i.e., Langerin+ subset and DC-SIGN+ subset, followed a synchronized activation/maturation program, we looked for the expression of CD80, CD83, CD86, and CCR7 after 48 h incubation with TNF-{alpha} in both subsets. We observed a lower expression of the activation marker CCR7 on DC-SIGN+ subset (MFI value, 10) only and not on the Langerin+ subset (MFI value, 43) in comparison with the total GM-CSF/TGF-ß1/IL-13-generated DC population (MFI value, 37; Fig. 7A ). All other activation/maturation DC markers (CD80, CD83, CD86) did not differ in Langerin+ and DC-SIGN+ subsets (Fig. 7A) . We confirmed the functionality of CCR7 by means of the chemotaxis assay in response to MIP-3ß. After a short incubation with TNF-{alpha} (18 h), GM-CSF/TGF-ß1/IL-13-generated DC exhibited a statistically nonsignificant migratory response to MIP-3ß in comparison with negative control (Fig. 7B) . As expected, after incubation with TNF-{alpha} for 48 h, the DC acquired a strong capacity to migrate toward MIP-3ß (Fig. 7B) , which is consistent with DC maturation. The increase in the number of migrating cells after TNF-{alpha} stimulation (48 h) might be obviously a result of the acquisition of CCR7 expression into the Langerin+ subset (Fig. 7A) .


Figure 7
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  		Figure 7. Dual expression of activation/maturation markers and migratory response to MIP-3ß by Langerin+ cells and DC-SIGN+ cells after TNF-{alpha} stimulation. Freshly isolated CD14+ monocytes were cultured for 6 days in the presence of GM-CSF, TGF-ß1, and IL-13. Cells were then recovered and reseeded in the presence of GM-CSF, TGF-ß1, and TNF-{alpha} for 18 h and 48 h. (A) Cells were double-labeled for expression of Langerin or DC-SIGN and other DC activation markers. The expression of DC activation markers was analyzed on cells gated on Langerin expression or DC-SIGN expression. Filled histograms represent antigen staining, and open histograms represent isotype-matched controls. For CCR7 marker, percentage of positive cells and MFI are mentioned (n=4 experiments). (B) Cells were tested for their capacity to migrate in response to MIP-3ß/CCL19. Migration assays were performed by seeding 2 x 105 cells in a TranswellTM insert of 5 µm for 2 h at 37°C. Each assay was performed in triplicate (mean±SD), and results are expressed as the migration index (ratio of chemokine:medium; n=4 experiments).

 
To further investigate the maturation program between Langerin+ subset and DC-SIGN+ subset, we studied the acquisition of DC-LAMP expression in both subsets after 18 h and 48 h incubation with TNF-{alpha}. After TNF-{alpha} stimulation for 18 h, Langerin+ and DC-SIGN+ cells were not mDC, as demonstrated by the lack of expression of DC-LAMP (Fig. 8A ). Conversely, prolonged exposure of TNF-{alpha} (48 h) induces maturation of about half of the Langerin+ population (56±7% of Langerin+/DC-LAMP+ cells), as illustrated by the induction of DC-LAMP expression (Fig. 8B) . In contrast, a low proportion of the DC-SIGN+ population acquired DC-LAMP expression (12±8% of DC-SIGN+/DC-LAMP+ cells) after stimulation by TNF-{alpha} for 48 h. The monocytes cultured with GM-CSF, IL-13, and TGF-ß1 for 6 days and with TNF-{alpha} ± TGF-ß1 for 2 more days contained a similar proportion of DC-SIGN+/DC-LAMP– DC (0.3±1% with TGF-ß1 vs. 0.5±2% without TGF-ß1) and DC-SIGN+/CCR7+ DC (40±10% with TGF-ß1 vs. 42±9% without TGF-ß1). The DC activation phenotype of DC-SIGN+ cells was not antagonized by the presence or not of TGF-ß1 (Fig. 9 ). Thus, prolonged exposure to TNF-{alpha} (48 h) enables a part of the Langerin+ population to mature and to acquire DC-LAMP expression.


Figure 8
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  		Figure 8. Expression of DC-LAMP on Langerin+ cells and DC-SIGN+ cells after TNF-{alpha} stimulation. Freshly isolated CD14+ monocytes were cultured for 6 days in the presence of GM-CSF, TGF-ß1, and IL-13. Cells were then recovered and reseeded in the presence of GM-CSF, TGF-ß1, and TNF-{alpha} for 18 h and 48 h. At indicated time-points, (A) 18-h TNF-{alpha} and (B) 48-h TNF-{alpha}, cells were double-stained as the following: Langerin/DC-SIGN, Langerin/DC-LAMP, and DC-SIGN/DC-LAMP. Quadrant parameters were determined using the appropriate isotype controls. Percentage of positive cells is mentioned in dot plots. Data are representative of four experiments.

 

Figure 9
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  		Figure 9. Expression of DC-LAMP and CCR7 on DC-SIGN+ cells after TNF-{alpha} stimulation in the presence or not of TGF-ß1. Freshly isolated CD14+ monocytes were cultured for 6 days in the presence of GM-CSF, TGF-ß1, and IL-13. Cells were then recovered and reseeded in the presence of GM-CSF/TNF-{alpha}, with or without TGF-ß1 for 18 h and 48 h. Cells were harvested, and expression of indicated markers was determined by FACS analysis. Percentage of positive cells is mentioned in dot plots. Results are representative of four experiments.

 
Allostimulatory activity is a functional hallmark exhibited by mDC. This activity is highly promoted by CD40 ligation. We therefore compared the allostimulatory properties of GM-CSF/TGF-ß1/IL-13-generated DC submitted to a series of short and prolonged incubations with TNF-{alpha} or CD40 ligation. As expected, the monocytes cultured with GM-CSF, IL-13, and TGF-ß1 for 6 days were inefficient in stimulating naïve, allogeneic T cell proliferation (Fig. 10 ). The cells cultured for 18 h with TNF-{alpha} displayed a weak allostimulatory capacity, which was promoted moderately and strongly, respectively, after TNF-{alpha} stimulation (for 48 h) and CD40/CD40 ligand interaction (for 40 h).


Figure 10
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  		Figure 10. Allostimulatory properties of GM-CSF/TGF-ß1/IL-13-generated DC after TNF-{alpha} stimulation and interaction with CD40 ligand. Freshly isolated CD14+ monocytes were cultured for 6 days in the presence of GM-CSF, TGF-ß1, and IL-13. Cells were then recovered and reseeded in the presence of GM-CSF or TGF-ß1 plus TNF-{alpha} (for 18 h and 48 h) or plated for 40 h on CD40 ligand-transfected fibroblasts. Cells were then harvested, and graded numbers were added to 105 allogeneic T cells. T cell proliferation was assessed by [3H] thymidine incorporation for the final 18 h of culture. Results are expressed as the mean counts per minute (cpm) ± SD from triplicate wells. T cells alone gave less than 30 cpm. Data are representative of four experiments.

 
Other proinflammatory stimuli may act as decisive signals in the production of IL-12 versus IL-10
Then, we investigated the ability of the GM-CSF/TGF-ß1/IL-13-generated DC in the antigen-specific T cell priming by comparison with IL-10 and IL-12 production after maturation-inducing stimuli [51 ]. In this context, we compared the production of culture supernatants from GM-CSF/TGF-ß1/IL-13-generated DC and the DC generated from GM-CSF/IL-4-cultured monocytes, with or without TNF-{alpha} (18 h and 48 h incubations), lipopolysaccharide (LPS), and CD40 ligand additions.

GM-CSF/IL-4-derived DC constitutively produced a low amount of IL-10 (77±16 pg/mL) in the absence of stimulation, and GM-CSF/TGF-ß1/IL-13-generated DC only produce IL-10 after stimulation (Fig. 11A ). A low increase of IL-10 production was observed in GM-CSF/TGF-ß1/IL-13-generated DC after 48 h of incubation with TNF-{alpha} (23±6 pg/mL). After addition of LPS and CD40 ligand, no difference between GM-CSF/TGF-ß1/IL-13-generated DC and GM-CSF/IL-4-generated DC was observed in IL-10 production. As expected, LPS markedly increased IL-10 production. We noted that GM-CSF/IL-4-generated DC produced bioactive IL-12p70 when stimulated by short (8±4 pg/mL) or prolonged (15±2 pg/mL) incubation with TNF-{alpha}, LPS (58±4 pg/mL), or CD40 ligand (61±3 pg/mL; Fig. 11B ). In contrast, GM-CSF/TGF-ß1/IL-13-generated DC only produced IL-12p70 when activated with LPS (10±3 pg/mL) or CD40 ligand (48±3 pg/mL). It is striking that the amount of IL-12 produced was sixfold lower when GM-CSF/TGF-ß1/IL-13-generated DC were stimulated with LPS.


Figure 11
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  		Figure 11. Secretion of IL-10 and IL-12 by GM-CSF/TGF-ß1/IL-13-generated DC in response to TNF-{alpha}, LPS, and CD40 ligand. Freshly isolated CD14+ monocytes were cultured for 6 days in the presence of GM-CSF, TGF-ß1, and IL-13. Cells were then recovered and reseeded in the presence of GM-CSF or TGF-ß1 plus TNF-{alpha} (for 18 h and 48 h), LPS (48 h), or CD40 ligand (40 h). As control, "conventional" DC generated from GM-CSF/IL-4-cultured monocytes (solid bars) were used. Supernatants were collected and then analyzed for (A) IL-10 and (B) IL-12p70 production using high-sensitive ELISA techniques. Each assay was performed in triplicate (mean±SD), and results are expressed as pg/mL quantified amounts of secreted cytokines (n=4 experiments).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our experiments report the findings that in skin postinflammatory conditions, monocytes may act as DC precursors for skin DC subsets, i.e., LC and IDDC. Monocyte recruitment from blood, differentiation, and activation/maturation of these monocyte-derived DC is regulated by the balance of different soluble, proinflammatory, and environmental stimuli.

Many studies have shown LC to be capable of inducing tolerance, as LC may induce tolerance to self-antigens as they traffic to lymph nodes in the steady state [51 ]. This current paradigm was also established for other interstitial DC in their steady status [52 ]. Studies of the biology of DC are focused nowadays on efforts to engineer immune responses and to better understand the immunopathology of skin diseases. In this context, current studies demonstrate the active participation of keratinocytes (antimicrobial peptides) and DC in skin innate immune responses. However, the biological mechanisms by which the still-enigmatic precursors of skin DC populate the skin are poorly understood in steady and pathological conditions. Nevertheless, in atopic dermatitis and inflamed skin after ultraviolet (UV) irradiation, blood-borne CCR2+ DC precursors and dermal CD36+ cells are known to be actively recruited to the skin [44 , 53 ]. Thus, in similar pathological conditions, we have investigated whether a predominant T helper cell type 2 (Th2) microenvironment (i.e., IL-13) could support and regulate the skin re-colonization of LC and IDDC derived from CD14+ blood monocytes.

Several precursors for the LC lineage have been described in human peripheral blood: CD34+ progenitors expressing the skin-homing receptor CLA need to be treated with GM-CSF/TNF-{alpha} [38 ]; the CD1a+/CD11c+/CD14– subset of human blood DC needs to be treated with GM-CSF/TGF-ß/IL-4 [32 ]; and CD14+ monocytes need to treated with GM-CSF/TGF-ß/IL-4 [25 ] or GM-CSF/TGF-ß/IL-13 [54 ]. In the skin, dermal CD14+ cells that already express Langerin are also able to acquire features of LC when cultured with TGF-ß [39 ]. Although previous studies in vitro have shown that commitment to LC differentiation was already established at the level of circulating DC precursors early on during ontogeny [38 ], later studies strongly suggested that LC pathways could depend on the skin cytokine environment once LC precursor cells have entered the conjunctive tissue of the skin. Moreover, human monocytes represent putative dermal DC precursors, as when cultured in the presence of GM-CSF and IL-4, they differentiate into DC that are phenotypically similar to dermal DC [19 , 27 , 28 , 55 ]. In this study, we used a cytokine cocktail composed of GM-CSF, TGF-ß, IL-13, and TNF-{alpha} to mimic a suitable physiological skin environment in which the Th2 cytokine profile predominates. In vivo, all of these cytokines are expected to be present into skin environment and consequently, to act simultaneously on recruited monocytes. In this context, our data demonstrated that monocytes can diverge simultaneously along two pathways in vitro, i.e., LC-type DC and IDDC.

Indeed, our study described for the first time the in vitro generation from monocytes of two distinct DC subsets exclusively expressing Langerin (specific marker of LC) and DC-SIGN (specific marker of dermal DC). The monocyte suspension used to generate the Langerin+ subset and DC-SIGN+ subset constitutes a homogeneous population expressing characteristic and typical markers of peripheral blood CD14+ monocytes such as CD11b+high, CD11c+high, CD64+high, CD16+weak, and HLA-DR+weak. None of these monocyte markers allowed us to distinguish a peculiar monocyte subpopulation skewing toward a conventional DC or LC differentiation pathway. In this context, it is impossible to conclude whether Langerin+ cell and DC-SIGN+ cell subsets share a common CD14+ progenitor or derive from heterogeneous and distinct monocyte subpopulations.

Described previously as a proinflammatory cytokine known to induce DC maturation and full differentiation of LC-type DC in the presence of IL-4 [49 ], we showed that TNF-{alpha} favors the early emergence of Langerin expression in the presence of the cytokine cocktail GM-CSF, TGF-ß, and IL-13. It is interesting that we also demonstrated that a short incubation of TNF-{alpha} (less than 18 h) induced acquisition of Langerin. In contrast, membrane expression of DC-SIGN in the dermal-type DC population was TNF-{alpha}-independent. Dermal macrophages that are known to secrete TNF-{alpha} could be implicated in the early LC differentiation of monocytes before entering the epidermis. It is well-known that TNF-{alpha}, which is keratinocyte-derived cytokine, appeared to be more crucial for terminal differentiation of LC-type DC by inducing expression of specific markers such as Langerin and Birbeck granules in the epidermal environment. Our results differ from those of Geissmann et al. [49 ], who showed that TNF-{alpha} only strongly increased the percentage of Langerin+ LC-type DC. This difference may be explained by the use of IL-13 in our experiment, whereas Geissmann et al. [49 ] generated LC-type DC in the presence of IL-4 by using a longer incubation with TNF-{alpha}. It is also noteworthy that the differentiation of monocytes seems to diverge preferentially into the LC pathway rather than into the pathway of dermal DC, probably by the addition of exogenous TGF-ß, as described previously by Geissmann et al. [25 ] with CD14+ monocytes and Caux et al. [34 ] with a CD34+ hematopoietic progenitor-derived CD14+ subset. The wide range of percentage of generated Langerin+ cells may only reflect the differences that reside in the monocyte populations of healthy donors. Such a suggestion cannot be clarified really, as no experimental data concerning the LC progenitors in the blood CD14+ monocytes are known. Furthermore, Langerin and DC-SIGN expression was found mutually exclusive in the presence of IL-13 and not in the presence of IL-4, which is known to favor the production of some DC expressing Langerin and DC-SIGN simultaneously [50 ]. Such a striking finding may reside in the efficiency of their receptors in response to the respective ligand (IL-4 vs. IL-13), although both receptors share a common {alpha} chain (IL-4R{alpha}).

Our results strongly underlined that the exogeneous level of TNF-{alpha} was critical for LC differentiation and the maturation/migration steps of both types of skin DC. Indeed, a short incubation with TNF-{alpha} was mandatory to generate "genuine" LC-type DC without induction of the maturation of LC-type and IDDC, as revealed by the absence of DC-LAMP expression. When incubated for a prolonged period (from 18 h to 48 h), a strong maturation signal was delivered by TNF-{alpha}. Indeed, we strikingly demonstrated that IDDC were not sensitive to the maturing effect of TNF-{alpha}, whereas 56 ± 7% of Langerin+ LC-type DC acquired the maturation marker DC-LAMP. Moreover, the LC-type DC population acquired higher CCR7 receptor expression than the IDDC subset. Thus, our data confirm the current concept that TNF-{alpha}, depending on its concentration, acts on differentiation versus migration/activation of the LC subset. In addition, we revealed the notion that LC may constitute a heterogeneous cell subset with regard to maturation (DC-LAMP+ vs. DC-LAMP–) and expression (CCR7+ vs. CCR7–) of the CCR7 receptor. This result may be correlated to a previous study that described two distinct epidermal LC subsets, which expressed respectively high and low levels of membrane HLA-DR antigens [56 ]. However, it is still unclear whether the CCR7+ LC subset was involved in the peripheral tolerance and/or the initiation of a specific immune response. Conversely, IDDC, which did not mature, could potentially induce tolerance. These observations are consistent with the work of Nair and co-workers [57 ], which underscores that acquisition of migratory capacity by DC cannot be interpreted as a hallmark of a complete maturation process correlated to an enhanced immunostimulatory capacity.

We found that mixed skin DC populations secreted only a low level of IL-10 and did not produce significant amounts of IL-12 when stimulated by prolonged incubation with TNF-{alpha}. Thus, LC-type DC and IDDC seem to express rather a Th2 cytokine-like profile than a Th1 profile known to be implicated in cell-mediated immunity. It is interesting that LC-type DC and IDDC, which expressed a high level of membrane MHC-class II molecules, displayed a semi-mature phenotype and consequently, were potentially able to elicit the CD4+/CD25+ TReg cell generation, as discussed previously by Lutz and Schuler [51 ]. Furthermore, it was shown that exposure of DC to TNF-{alpha} could promote development of tolerance-inducing cells, although the phenotype of DC does not correlate with iDC in terms of surface marker expression [58 ]. In contrast, maturation signals such as LPS and CD40 ligand induced IL-12 secretion and increased IL-10 secretion in mixed skin DC populations. Thus, our results are consistent with large differences previously observed between TNF-{alpha} and LPS as maturation signals for DC [59 ]. In addition, our results confirmed that skin DC mature poorly in response to noncognate signals such as TNF-{alpha}, and activation via bacterial components such LPS or CD40 ligand cognate signal induced their full maturation as assessed by IL-12 secretion and subsequently, their ability to elicit specific immune responses. However, the precise identification of the subset(s) secreting IL-10 and/or IL-12 remains to be elucidated.

Taken together, in skin inflammatory environments such as in atopic dermatitis and after UV irradiation, where TNF-{alpha} and IL-1 are produced at relatively high levels, we showed that maturation/migration pathways of both skin DC subsets are distinct, which would support the view that epidermal LC and dermal DC could participate differently but complementarily in eliciting skin immune responses. In line with this, Bacci et al. [60 ] demonstrated in a mouse model that the selective participation of LC or dermal DC in promoting contact hypersensitivity depends on the dose of hapten applied. Indeed, the contact hypersensitivity reaction seems to be mediated, respectively, by LC and dermal DC after application of low and high doses of hapten. It is noteworthy that our results are in perfect accordance with the observations of Bacci et al. [60 ], as LC-type DC are more susceptible to be activated/matured at lower doses of TNF-{alpha} than IDDC. The responsiveness of LC to bacterial components is much weaker than that of dermal DC [61 , 62 ]. Indeed, Takeuchi et al. [61 ] demonstrated that the hyporesponsiveness to bacteria observed in LC may have some correlation with down-regulated Toll-like receptors (TLR) such as TLR2 and TLR4. These results, apparently in contradiction to ours, may be a result of the respective proportion of different monocyte-derived DC subsets obtained with various experimental protocols (IL-4 vs. IL-13, with/without TGF-ß and TNF-{alpha}). It is interesting that our culture system provides the possibility of simultaneously generating a known proportion of both types of skin DC and then allowing their challenge by a bacterial component in a comparative manner.

In summary, LC, which are located in the epidermis, are unique DC, as they differ from dermal DC in their life cycle [53 ] and in their way of maturation and migration [49 , 61 ]. Previous studies suggest that LC are the primary targets of exogenous pathogens, which may cross the epithelial barrier. Furthermore, their epithelial environment may amplify their activation/maturation by the release of proinflammatory cytokines, i.e., TNF-{alpha}, IL-1, and ß-defensins. In this study, we confirmed that human monocytes could be potential precursors of skin DC, and we further postulated that during inflammatory skin events, such as after UV irradiation or in atopic dermatitis, in which the Th2 cytokine environment predominates, the recruited monocytes could differentiate simultaneously into LC and IDDC to repopulate the skin. Under these particular inflammatory conditions, we demonstrated the existence of desynchronized maturation programs within the LC subset and especially between LC and IDDC. As LC are more sensitive to phenotypic maturation in response to activation signals, they may be a more potent vaccine tool than monocyte-derived DC in the preparation of antigen-pulsed DC in antitumoral immunotherapy. The great originality of our culture system consists in the possibility of studying the relationship and potential cross-talk between both types of skin DC. Such cross-talk was evoked recently in a study of skin DC homeostasis, where the system TNF-related activation-induced cytokine/receptor activator of nuclear factor-{kappa}B is expressed distinctly on both types of skin iDC [63 ]. Such interactions between LC and IDDC need to be clarified in certain infectious pathologies where both types of skin DC elicit particular immune responses to bacterial or viral pathogens.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from "Agence Nationale de la Recherche Technique" (ANRT). The authors thank Dr. Pierre Garrone (Schering-Plough) for providing us with GM-CSF and Mrs. Jane Mitchell for reviewing the English version of the manuscript.

Received February 23, 2005; revised February 3, 2006; accepted March 6, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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