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(Journal of Leukocyte Biology. 2008;84:143-151.)
© 2008 by Society for Leukocyte Biology

Transcriptional profiling of human skin-resident Langerhans cells and CD1a+ dermal dendritic cells: differential activation states suggest distinct functions

Saskia J. A. M. Santegoets*,{dagger}, Susan Gibbs{ddagger}, Kim Kroeze{ddagger}, Rieneke van de Ven*, Rik J. Scheper*,1, Carl A. Borrebaeck§, Tanja D. de Gruijl{dagger} and Malin Lindstedt§

Departments of
* Pathology,
{dagger} Medical Oncology, and
{ddagger} Dermatology, VU University Medical Center, Amsterdam, The Netherlands; and
§ Department of Immunotechnology, Lund University, Lund, Sweden

1Correspondence: VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. E-mail: rj.scheper{at}vumc.nl


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ABSTRACT
 
In human skin, two main populations of dendritic cells (DC) can be discriminated: dermal DC (DDC) and epidermal Langerhans cells (LC). Although extensively studied, most of the knowledge about DDC and LC phenotype and function is obtained from studying DDC and LC cultured in vitro or DDC and LC migrated from skin explants. These studies have left the exact relationship between steady-state human LC and DDC unclear: in particular, whether CD1a+ DDC represent migrated LC or whether they constitute a separate subset. To gain further insight in the kinship between skin-resident CD1a+ DDC and LC, we analyzed CD1a+ DDC and LC, isolated from steady-state skin samples, by high-density microarray analysis. Results show that the CD1a+ DDC specifically express markers associated with DDC phenotype, such as the macrophage mannose receptor, DC-specific ICAM-grabbing nonintegrin, the scavenger receptor CD36, coagulation factor XIIIa, and chemokine receptor CCR5, whereas LC specifically express Langerin, membrane ATPase (CD39), and CCR6, all hallmarks of the LC lineage. In addition, under steady-state conditions, both DC subsets display a strikingly different activation status, indicative of distinct functional properties. CD1a+ DDC exhibit a more activated, proinflammatory, migratory, and T cell-stimulatory profile, as compared with LC, whereas LC mainly express molecules involved in cell adhesion and DC retention in the epidermis. In conclusion, transcriptional profiling is consistent with the notion that CD1a+ DDC and LC represent two distinct DC subsets but also that under steady-state conditions, CD1a+ DDC and epidermal LC represent opposites of the DC activation spectrum.

Key Words: microarray • DC lineage


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INTRODUCTION
 
Dendritic cells (DC) are professional APC with the unique ability to initiate and maintain primary immune responses. As key sensors of danger, immature DC reside in peripheral tissues, such as the skin and mucosal sites. DC are specialized in antigen capture and constantly sample the environment for antigens, local inflammation, and pathogens [1 2 3 4 ]. Upon recognition of danger signals, immature DC undergo a process called DC maturation, resulting in the inhibition of antigen uptake, as well as in a switch in chemokine receptor expression and subsequent migration of the DC to the secondary lymphoid organs, where they present the antigen to T cells. In the human skin, two main populations of DC can be discriminated: Langerhans cells (LC), which can be found in the epidermis [5 , 6 ], and dermal DC (DDC), which are located in the dermis. In vitro differentiation studies from CD34+ precursor cells suggest that DDC and LC originate from a common myeloid DC precursor and have several features in common. These include the expression of high levels of MHC class I and class II molecules, costimulatory and adhesion molecules, as well as the expression of certain leukocyte/myeloid markers such as CD45RO, CD13, and CD33 and a lack of CD3, CD19, CD20, CD16, and CD56 lineage markers, as reviewed by Larregina and Falo Jr. [7 ]. However, both DC subsets also exhibit specific "DDC" and "LC" characteristics. LC are characterized by the expression of the C-type lectin Langerin, which is responsible for the formation of Birbeck granules, a typical hallmark for the LC lineage [8 , 9 ]. In addition, LC express E-cadherin [10 ], membrane adenosine triphosphatase (ATPase), and CCR6 [11 ], whereas DDC do not. On the other hand, DDC can be distinguished from LC by the expression of certain C-type lectins, such as macrophage mannose receptor (MMR) and DC-specific ICAM-grabbing nonintegrin (SIGN) [12 , 13 ], as well as by the expression the scavenger receptor CD36 [14 ] and the expression of coagulation factor XIIIa (FXIIIa) [15 , 16 ]. Furthermore, DDC can also express the monocyte/macrophage marker CD14 [5 , 6 , 17 ].

Although it has been described that DDC and LC are professional APC capable of inducing primary immune responses in vitro and in vivo, functional differences between the DC subsets have also been reported. The observed difference in C-type lectin expression indicates that both DC subsets may recognize and react to different spectra of pathogens [18 ]. Besides that, in vitro-generated DDC have been described to more efficiently drive the differentiation of naïve B cells into IgM-secreting plasma cells, and LC have been described to be more potent in vitro stimulators of cytotoxic T cells [19 ], as well as more potent inducers of Th1 responses as a result of their inability to produce IL-10 upon CD40 ligation [6 ]. However, compared with the abundant in vitro data, data clarifying in vivo functions of human LC are scarce. Recently, immunogenic properties of LC in vivo have been questioned based on murine studies. Rather than directly activating immune effector cells, LC were reported to function as transporters of antigen, carrying antigen from skin to lymph nodes (LN) and transferring the antigenic cargo to LN-resident DC for actual antigen presentation and CTL priming [20 21 22 ].

It must be emphasized that most of our current knowledge about DDC and LC phenotype and function was obtained through the study of DDC and LC, cultured in vitro from CD34+ hematopoietic progenitor cell or blood-derived monocytes [5 , 19 , 23 , 24 ]. Analyzing DC migrated from skin explants or directly isolated from epidermal and dermal cell suspensions revealed that the skin DC population is quite heterogeneous. Based on the expression of CD1a and CD14, different DC populations could be identified: Besides epidermal LC (CD1ahigh/Langerin+/Birbeck granule+), three distinct DDC subsets could be discerned, i.e., CD1a+/CD14– DDC, CD1a–/CD14+ DDC, and CD1a–/CD14– DDC [25 , 26 ]. Although studied extensively, the relationship between human LC and the various DDC subsets remains unclear and controversial, and CD14+-to-CD1a+ and CD1a+-to-CD14+ (trans-)differentiation events having been reported [17 , 27 ]. Larregina and co-workers [7 , 28 ] recently defined CD1a+ skin-emigrated DC as LC and CD1a–/CD14– skin-emigrated DC as DDC, whereas Angel and co-workers [26 ] suggested CD1a+ DDC to be distinct from migrating LC, as concluded from intermediate levels of CD1a and the absence of Langerin expression. Notably, activated LC have been demonstrated to down-regulate CD1a and Langerin, making it difficult to distinguish skin migratory LC from skin-resident DDC [14 , 29 ].

Microarray technology has made it possible to study the expression levels of thousands of genes in parallel, with only relatively small amounts of material. By performing global transcriptional profiling of skin-derived CD1a+ DDC and CD1a+ LC, using high-density microarray analysis and extracting the differentially expressed genes, we aimed to elucidate whether CD1a+ DDC and CD1a+ LC, obtained from resting, noninflamed skin, are two truly separate DC subsets or whether they represent a functional continuum of one subset. Besides that, we also studied transcript levels of genes that might be related to their in vivo function.

Our data demonstrate a remarkable difference in maturation status under steady-state conditions between CD1a+ DDC and LC, with CD1a+ DDC displaying a more activated, proinflammatory, and migratory profile and LC exhibiting a more quiescent profile, expressing genes involved in cell adhesion and DC retention in the epidermis. Based on the gene expression profiles obtained, they nevertheless adhere to the previously proposed, classic definitions of the DDC and LC subset phenotypes, suggesting that CD1a+ DDC and LC consist of two distinct DC subsets and that CD1a+ DDC do not merely represent one end of the functional continuum of LC.


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MATERIALS AND METHODS
 
Isolation of DDC and LC from skin
Human skin specimens were obtained from healthy donors undergoing corrective breast or abdominal plastic surgery after informed consent. Thick slices (3 mm) of skin containing the epidermis and the dermis were cut by use of a dermatome. Slices of skin were cut in pieces of 1 cm2 and incubated with 2.4 U/ml Dispase II (Roche Diagnostics, Mannheim, Germany) for 30–60 min at 37°C. The epidermis and dermis were separated with tweezers and washed with PBS. To isolate LC, the epidermal sheets were incubated with PBS with 0.05% trypsin (Invitrogen Life Technologies, Carlsbad, CA, USA) for 10 min at 37°C, and a single-cell suspension was prepared by pushing the tissue through 100 µm pore nylon cell strainers (Falcon) with a plunger of a 2-ml syringe. Epidermal cell suspension was enriched for LC by density centrifugation over Lymphoprep (Nycomed AS, Oslo, Norway) and CD1a-guided MACS (Miltenyi Biotec, Bergisch Gladbach, Germany). To isolate DDC, the dermis was incubated with PBS containing 0.48 U/ml Dispase and 6 mg/ml Collagenase A (Boehringer Mannheim, Mannheim, Germany) at 37°C for 2 h, after which, a single-cell suspension was prepared by pushing through 100 µm pore nylon cell strainers with a plunger of a 2-ml syringe. Cell suspension was enriched for DDC by CD1a-guided MACS.

Antibodies and flow cytometry
PE- or FITC-labeled antibodies directed against human CD83, Langerin (Immunotech, Marseille, France), CD1a, CD86, and DC-SIGN (all from BD Biosciences, Mountain view, CA, USA) were used for flow cytometric analysis. Antibody staining was performed in PBS supplemented with 0.1% BSA and 0.02% natrium-azide for 30 min at 4°C. Stained cells were analyzed on a FACSCalibur (BD Biosciences) using CellQuest software.

Preparation of cRNA and gene chip hybridization
RNA isolation and gene chip hybridization were performed as described [30 ]. Briefly, cell pellets of skin-isolated DDC and LC from three different donors were dissolved in TRIzol reagent (Invitrogen Life Technologies) and stored at –20°C. After chloroform extraction, total RNA was precipitated in isopropanol, rinsed with 70% ethanol, lyophilized, and dissolved in 10 µl distilled water. Fragmentation, hybridization, and scanning of the Human Genome U133 Plus 2.0 arrays were performed according to the manufacturer’s protocol (Affymetrix, Santa Clara, CA, USA). The preparation of labeled cRNA was performed according to the Two-Cycle Eukaryotic Target Labeling assay protocol, using the GeneChip Expression 3' amplification two-cycle labeling and control reagents kit (Affymetrix). Briefly, cDNA was generated from total RNA (20–150 ng) using SuperScript II (Invitrogen Life Technologies) and a T7-oligo(dT) promoter primer (Affymetrix). After a second-strand cDNA synthesis, cDNA was converted to cRNA by an in vitro transcription reaction (MEGAscript T7 kit, Ambion, Foster City, CA, USA). Thereafter, the cRNA was purified using a RNeasy Mini kit (Qiagen, Hilden, Germany), and the yield was controlled with a spectrophotometer. A second cycle of cDNA synthesis was performed, followed by the same cleanup as above and a second in vitro transcription reaction cycle with biotin-labeled ribonucleotides and T7 RNA polymerase. Labeled cRNA was purified, using a RNeasy Mini kit (Qiagen), quality-controlled with an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and denatured at 94°C before hybridization. The samples were hybridized to the Human Genome U133 Plus 2.0 array at 45°C for 16 h by rotation (60 rpm) in an oven. The arrays were then washed, stained with streptavidin-PE (Invitrogen Molecular Probes, Eugene, OR, USA), washed again, and scanned with a GeneArray scanner (Affymetrix).

Microarray data analysis
The fluorescence intensity was analyzed using the GeneChip Operating software 1.1 (Affymetrix) and scaled to a target value of 100. Further data analysis was performed with GeneSpring 7.1 software (Agilent Technologies). For clustering, the samples were normalized per gene, which makes the median value for each gene across the samples equal to 1. A gene and condition tree clustering was performed on the LC and DC samples to distinguish replicate similarities. The tree-clustering algorithm, based on Pearson correlation, was used on genes denoted P (present) in DDC or LC (three replicates) with a signal intensity above 200 to eliminate borderline expression and displaying a fold change in mean expression level of ±2 between the two populations, giving a total of 1480 genes. The expression of selected genes was significantly different (P<0.05) as determined by one-way ANOVA.


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RESULTS
 
Phenotyping CD1a+ DDC and LC by flow cytometry
Dermal and epidermal cell suspensions were analyzed for the presence of DC by flow cytometry. As shown in Figure 1A , CD1a+ DC could be detected in dermal (Fig. 1A , left, upper panels) and epidermal cell suspensions (Fig. 1A , left, lower panels). Next, DC were isolated from the dermal and epidermal suspensions on the basis of the pan-skin DC marker CD1a. Purity of CD1a+ dermis-derived DC (hereafter referred to as CD1a+ DDC) and CD1a+ epidermis-derived LC (hereafter referred to as LC) was more than 90%. As shown in Figure 1A , CD1a+ DDC exhibit DDC characteristics, expressing intermediate levels of CD1a and no Langerin (Fig. 1A , right, upper panels), whereas skin LC exhibit LC characteristics, expressing high levels of CD1a and Langerin (Fig. 1A , right, lower panels). The isolated LC displayed an immature phenotype, as indicated by the absence of CD83 expression, whereas the CD1a+ DDC expressed CD83, indicative of a mature phenotype (Fig. 1A and 1B) . Importantly, this differential expression of CD83 was also observed for CD1a+ cells in freshly prepared dermal and epidermal cell suspensions (see Fig. 1A ), suggesting that CD83 expression was not induced by the isolation procedure. Moreover, exposure of the epidermal suspensions to the (longer) enzymatic digestion procedure, used for dissociation of the dermal suspensions, did not result in LC maturation (data not shown), illustrating that the observed difference in CD83 expression was not the result of a prolonged incubation time. Furthermore, both DC subsets exhibit similar levels of the costimulatory and adhesion molecules CD86 and HLA-DR (Fig. 1C) .


Figure 1
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  		Figure 1. Phenotype and morphology of skin-derived CD1a+ DDC and LC. Skin DDC and LC, obtained from healthy human skin specimens, were analyzed by flow cytometry for the expression of CD1a and CD83 prior to CD1a-guided MACS (A, left) and for the expression of CD1a, Langerin, and DC-SIGN (A, right), CD83 (B), and CD86 and HLA-DR (C) after CD1a-guided MACS. (A) Skin DDC expressed intermediate levels of CD1a and no Langerin (A, right, upper panels), whereas skin LC expressed high levels of CD1a and Langerin (A, right, lower panels). (B) CD83 expression of skin DDC and LC is depicted as the mean fluorescence intensity (MF) ± SEM of three independent experiments. (C) Photographs of skin DDC and LC were taken from cytocentrifuge preparations (cytospins, 400x original magnification). Flow cytometric analysis of MACS-isolated CD1a+ DDC and LC. Open histograms, Isotype-matched controls; closed histograms, the marker as indicated above. Mean fluorescence indices are listed in the upper right corners. Data shown are representative of three independent experiments.

Phenotyping CD1a+ DDC and LC by transcriptional analysis
To make an extensive transcriptional comparison of CD1a+ DDC and LC, RNA isolated from each DC subset was hybridized to Affymetrix Human Genome U133 Plus 2.0 arrays containing >54,000 probe sets and covering 38,500 human genes. mRNA expression profiles of CD1a+ DDC and LC were assessed from three individual donors. To determine whether the isolated CD1a+ DDC and LC indeed represented true DDC and LC, both DC subsets were first subjected to a global transcriptional analysis, and markers associated with DDC and LC biology were extracted. For each subset, the intensity signals for selected marker genes that were expressed (denoted present) and had an intensity level of >200 were assessed. As shown in Table 1 , the transcriptional patterns of CD1a+ DDC and LC fully support previous reports about DDC and LC phenotype definitions [31 , 32 ]. CD1a+ DDC express the C-type lectins MMR and DC-SIGN, the scavenger receptor CD36, coagulation FXIIIa, and the chemokine receptor CCR5 but do not express markers associated with the LC phenotype such as Langerin, membrane ATPase (CD39), and CCR6, and skin LC express the C-type lectin Langerin, membrane ATPase, and the chemokine receptor CCR6 (Table 1) but do not express MMR, DC-SIGN, CD36, or FXIIIa (Table 1) . Based on this panel of DC subset-defining markers (Table 1) , we thus conclude that CD1a+ DDC adhere to the previously proposed definition of the DDC phenotype and are not likely to represent migratory LC, as also described by others [26 ]. In particular, the observation that CD1a+ DDC express DDC-defining markers associated with an immature phenotype such as DC-SIGN and MMR argues against the possibility of migrating LC adopting a skin DDC transcriptional profile under the influence of environmental (i.e., dermal) factors, as "de novo" expression of such a marker is not consistent with the degree of maturation that might be expected in migrating LC. Moreover, it seems unlikely that the relatively brief period of time it takes for migrating LC to traverse the dermis would allow for the profound shift in a transcriptional profile from a LC to a DDC signature, as displayed in Table 1 , with such clear-cut "on" and "off" signals for a wide array of LC- and DDC-specific transcripts.


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  		Table 1. Differential Characteristics of CD1a+ DDC and LC

LC display a nonimmunogenic, nonmigratory phenotype under steady-state conditions
To gain insight into functional differences between CD1a+ DDC and LC, genes that were differentially expressed (twofold change in expression level, P<0.05) were further clustered into groups, as illustrated by a heat map in Figure 2 . Seven groups of differentially expressed genes are presented according to function: chemokine/chemokine receptor, IL/IL-R, TNF/TNFR family, adhesion, immune response, endo/exocytosis, and "others." In addition, to further characterize both DC subsets, a panel of genes was selected based on their specific known function in DC biology (Tables 2 3 4 5 6 7 ). This comparative analysis revealed that LC, residing in the epidermis under steady-state conditions, exhibit a nonstimulatory phenotype. As illustrated by Figure 2 , LC display a nonmigratory profile, expressing molecules involved in cell adhesion such as E-cadherin, ICAM-3, and epidermal surface antigen or involved in DC retention such as junctional adhesion molecules and CD47 but lacking expression of molecules involved in DC migration such as CCR7 (Table 3) , as also described by others [10 , 33 34 35 36 ]. Indeed, ligation of CD47, also known as integrin-associated protein, has been demonstrated to regulate LC maturation and migration, resulting in the suppression of LC function, inhibition of T cell priming, and the subsequent inhibition of the establishment of an immune response [37 ]. Yu and co-workers [37 ] suggested that the firm adhesion between LC and keratinocytes, resulting in suppression of DC migration, might be CD47-mediated. Indeed, it has been shown that CD47-expressing cells can firmly adhere to other CD47-expressing cells without the need for interaction with CD47 ligands, such as signal regulatory protein-1{alpha} or thrombospondin-1 [37 , 38 ]. Furthermore, these data support findings described previously that LC trafficking is not only controlled at the level of chemokine/chemokine receptor expression but also at the level of cell adhesion [31 , 39 ]. In addition, LC express relatively low levels of costimulatory and adhesion molecules such as CD80, CD40, and CD54 (Table 2) or proinflammatory cytokines and cytokine receptors (Table 4) and do not express T cell stimulation molecules such as 4-1BB and CD30L, indicating that under steady-state conditions, LC are poor T cell stimulators (Table 6) . The non-T cell stimulatory profile of LC is further illustrated by the expression of CD43 (also known as leukosialin), a glycoprotein that is only expressed on immature DC and implicated in the inhibition of nonspecific T cell contacts [40 , 41 ]. In addition, under steady-state conditions, LC do not exhibit proper B cell stimulatory capacity, as illustrated by the absence of BAFF, a TNF family member, which is known to play an important role as a costimulator of B lymphocyte proliferation and function [42 ]. Of note, the observation that LC isolated from steady-state skin are quiescent and sedentary does not exclude the findings described previously of the potential of (a fraction of) LC to mature and migrate to regional LN under steady-state conditions [43 44 45 ]. However, the discrete nature of the DDC and LC signatures found for the respective CD1a+ DDC and LC populations (Table 1) suggests that such a population of migrating LC could only account for a small fraction of the dermis-derived CD1a+ DC.


Figure 2
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  		Figure 2. Hierarchical clustering of differentially expressed genes in skin DDC and skin LC. Subset-selective transcriptional profiles in freshly isolated skin CD1a+ DDC (n=3, left column) and LC (n=3, right column) were identified by filtering (described in Materials and Methods) and sorted according to their gene ontogeny into various groups: chemokine/chemokine receptor, IL/IL-R, TNF/TNFR, adhesion, immune response, endo/exocytosis, and others. Color changes, within a row, indicate expression levels relative to the median of the sample population. As the samples are normalized to a median value of 1, the color bar range of 5 (red) to –5 (green) represents high and low expression levels, respectively. FC, fold change.


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  		Table 2. Relative Expression Levels of Costimulatory, Adhesion Molecules, and Integrins


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  		Table 3. Relative Expression Levels of Chemokines and Chemokine Receptors


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  		Table 4. Relative Expression Levels of Cytokines and Cytokine Receptors


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  		Table 5. Relative Expression Levels of C-Type Lectins, FcRs, and Scavenger Receptors


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  		Table 6. Relative Expression Levels of TNF/TNFR Family


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  		Table 7. Relative Expression Levels of Transcription Factors

CD1a+ DDC display an activated and migratory phenotype under steady-state conditions
In stark contrast to LC, CD1a+ DDC exhibit an activated phenotype, expressing higher levels of costimulatory and adhesion molecules (e.g., CD54, CD80, CD86, CD40; see Table 2 ), proinflammatory cytokines (such as IL-6, IL-8, IL-15, and IL-16), cytokine receptors (IL-1R, IL-4R, IL-7R, and IL-27Ra; see Table 4 ), PGE2 receptors EP2 and EP4, and TNF family members, capable of providing T and B cell stimulation such as 4-1BB, CD30L, and BAFF (Table 6) . This activated phenotype is also illustrated by the observation that CD1a+ DDC express chemokines that are involved in directing leukocyte migration, such as MDC (CCL22), MIP-1 {alpha} (also known as CCL3), MIP-1β (CCL4), Gro-{alpha} (CXCL1), and SDF-{alpha} (CXCL12). In addition, CD1a+ DDC expressed chemokine receptors that are involved in LN homing such as CCR7 and CXCR4 (Table 3) .

The more activated, immunostimulatory phenotype of CD1a+ DDC as compared with LC was also confirmed by the expression CD44, C3aR1, and C5aR1. CD44 has been described to be up-regulated on skin DDC and LC upon activation and to be involved in emigration from the skin and in adhesion to the T cell zones of the LN, thereby showing its importance for the ability of DC to induce primary T cell responses within the LN [46 ]. As shown in Figure 2 , CD44 is significantly up-regulated on CD1a+ DDC compared with skin LC, indicating that indeed CD1a+ DDC exhibit a more activated phenotype capable of T cell binding in the LN. This more activated phenotype was further illustrated by the observation that CD1a+ DDC, but not LC, expressed the complement receptors C3aR1 and C5aR1, both of which are involved in DC homing to inflammatory sites. This is in line with findings from others, demonstrating that freshly isolated CD83+ skin DC expressed C3aR1 and C5aR1 [47 ], whereas C5aR1 was not observed in the majority of immature skin LC [48 ]. Moreover, the observation reported previously that C5aR1 was expressed on a small number of skin LC that were located in the proximity of the basal membrane and exhibited a more activated and migratory phenotype further supports the apparent association between complement receptor expression and maturation induction [48 ]. Interestingly, the difference in CD83 protein expression between DDC and LC could not be confirmed by mRNA expression levels, suggesting differences between the transcriptional and translational levels (Table 2) .

Mature CD1a+ DDC express immunosuppressive factors: maintenance of tolerance in the steady-state
The finding that even in the absence of apparent danger signals, CD1a+ DDC display a fully mature and T cell-stimulatory phenotype may seem counterintuitive. It should, however, be noted that although CD1a+ DDC exhibit an activated and migratory phenotype under steady-state conditions, they also display immunosuppressive features. As demonstrated in Table 8 , under steady-state conditions, CD1a+ DDC express relatively high levels of IL-10 and indoleamine 2,3-dioxygenase (IDO) transcripts. The anti-inflammatory cytokine IL-10 has been described to negatively regulate the immune response via the induction of T cell tolerance [49 ] or the development of regulatory T cells [50 , 51 ], and IDO has been described to convey immunosuppressive effects by degrading the essential amino acid tryptophan, thereby down-regulating T cell functions [52 , 53 ]. The observation that the activated CD1a+ DDC also display immunosuppressive features under steady-state conditions indicates that the activated phenotype does not automatically equal T cell activation and that without additional danger signals, CD1a+ DDC may be mainly involved in the maintenance of tolerance. In keeping with this notion,we described previously CD1a+ DC in skin-draining LN to all display a CD83+ mature phenotype, even in the steady-tate [54 ].


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  		Table 8. Relative Expression Levels of Immune-Modulatory Factors


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DISCUSSION
 
In conclusion, this extensive comparative analysis of CD1a+ DDC and LC by means of transcriptional profiling was the first of its kind and generated valuable information about the in vivo phenotype and function of both DC subsets. The obtained gene expression profiles clearly demonstrate that the two isolated DC populations adhere to the previously proposed definitions of the DDC and LC phenotype, suggesting them to constitute separate subsets. Furthermore, CD1a+ DDC and LC show a remarkable difference in functional maturation status under steady-state conditions, suggesting that steady-state, epidermis-derived LC are quiescent and sedentary, whereas steady-state, dermis-derived CD1a+ DDC are continuously carrying antigen to LN and capable of stimulating T cells, as also described recently by Angel et al. [26 ] in humans and Kissenpfennig et al. [55 ] in mice. As LC have been described to be capable of migrating under steady-state conditions, we cannot exclude that a fraction of the CD1a+ DDC is indeed migrating LC, thereby contributing to the more activated profile of CD1a+ DDC. Yet, the clear-cut and discrete manner in which dermis-derived CD1a+ DC express markers associated with DDC phenotype and not markers associated with LC phenotype (Table 1) indicates that such migrating LC can only account for a small fraction of this DC population. Of note, the expression of transcripts of suppressive factors such as IL-10 and IDO suggests that T cell stimulation by mature CD1a+ DDC in the steady-state may result in the induction of tolerance.


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ACKNOWLEDGEMENTS
 
This work was supported by a grant from the European Commission (LSHB-CT-2005-018681) as part of the Integrated Project, "Novel Testing Strategies for In Vitro Assessment of Allergens (Sens-it-iv)."

Received November 14, 2007; revised February 28, 2008; accepted March 18, 2008.


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