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Originally published online as doi:10.1189/jlb.0204087 on June 14, 2004

Published online before print June 14, 2004
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(Journal of Leukocyte Biology. 2004;76:657-666.)
© 2004 by Society for Leukocyte Biology

Autocrine IL-10 partially prevents differentiation of neonatal dendritic epidermal leukocytes into Langerhans cells

Souyet Chang-Rodriguez*, Rupert Ecker{dagger}, Georg Stingl* and Adelheid Elbe-Bürger*,1

* Department of Dermatology, Division of Immunology, Allergy and Infectious Diseases, Medical University of Vienna, Austria; and
{dagger} Competence Center for Bio Molecular Therapeutics, Vienna, Austria

1 Correspondence: Department of Dermatology, DIAID, Medical University of Vienna, Brunner Str. 59, A-1235 Vienna, Austria. E-mail: adelheid.elbe-buerger{at}meduniwien.ac.at


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To test whether reduced immune responsiveness in early life may be related to the immaturity of neonatal antigen-presenting cells, we comparatively assessed the phenotypic and functional characteristics of dendritic epidermal leukocytes (DEL) and epidermal Langerhans cells (LC) in newborn (NB) and adult mice, respectively. We report that purified, 3-day-cultured DEL do not acquire the morphology and phenotype typical of LC and are significantly weaker stimulators of naive, allogeneic CD4+ and CD8+ T cells than LC. Freshly isolated DEL are twice as efficient as LC in the uptake of fluorescein isothiocyanate-conjugated tracers but are not able to present these to antigen-specific T cell hybridomas. To clarify the underlying cause, cytokine expression of NB and adult epidermal cells (EC) was examined. We found that DEL express considerable amounts of interleukin (IL)-10, that IL-10 in NB EC supernatants partially inhibits LC maturation, and that DEL-enriched EC from IL-10–/– mice induce stronger primary T cell responses compared with those from IL-10+/+ mice. We conclude that IL-10 is one of the factors preventing maturation and differentiation of DEL into immunocompetent LC in intrauterine life and is at least partly responsible for the poor immune responsiveness of neonates.

Key Words: antigen presentation/processing • cytokines • cytokine receptors • dendritic cells • skin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neonates often respond poorly to conventional vaccines or microbial infections [1 ]. Immaturity of the immune system has been considered to play a role. The mechanisms underlying this immaturity are still unclear. Several studies have shown that neonatal T cells exhibit reduced functionality relative to adult T cells such as lower production of interleukin (IL)-2, poorer proliferation in response to anti-CD3 stimulation, bias toward T helper cell type 2 (Th2) responses, lower levels of CD40 ligand upon activation, and impaired development of Th1 memory effector function [2 3 4 5 ]. Lower numbers of T cells in neonatal versus adult spleen may be another critical component of reduced neonatal responsiveness [6 ]. In addition, CD8 responses are reduced in numbers in neonates as compared with adults [7 8 9 ]. However, using appropriate stimulatory conditions, neonatal T cells can mount adult-like responses [10 11 12 13 14 15 16 17 ]. This led to the hypothesis that immune responsiveness in early life could be a result of the immaturity of dendritic cells (DC), the most potent antigen-presenting cell (APC), unique in their ability to efficiently prime and polarize naive CD4 and CD8 T cells [18 ]. Indeed, human cord blood-derived DC produce much less IL-12(p70) and express lower costimulatory molecules as compared with adult DC, which may account for the Th2 bias of the otherwise reduced T cell response [19 , 20 ]. The impaired IL-12 synthesis by neonatal DC is apparently directly related to a repressed IL-12(p35) gene expression [21 ]. Similarly, newborn (NB) mouse DC from epidermis, spleen, and lymph nodes are poor stimulators of antigen-specific T cell proliferation [22 23 24 ].

In this study, we have made an attempt to purify and culture dendritic epidermal leukocytes (DEL) from NB mice to investigate their maturation capacity and function and to explore the mechanisms that keep DEL in an immature state. We found that DEL do not acquire the morphology, phenotype [major histocompatibility complex (MHC) class IIlow/neg, CD205–, CD25–], and function typical of in vitro-cultured Langerhans cells (LC) and that autocrine IL-10 partially precludes their maturation and differentiation into immunocompetent LC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
BALB/c (H-2d), C3H/HeN (C3H, H-2k), and C57BL/6 (H-2b) mice (Charles River Wiga GmbH, Sulzfeld, Germany) were bred and maintained at the animal facility of the Department of Dermatology, Medical University of Vienna, Austria. Mice deficient for IL-10 (IL-10–/–) and corresponding wild-type mice (IL-10+/+) on a C57BL/6 genetic background were kindly provided by M. Mähler and A. Bleich (Institute for Laboratory Animal Science and Central Animal Facility, Medical School Hannover, Germany).

Reagents
RPMI-1640 medium was supplemented with 10% heat-inactivated fetal calf serum (FCS; PAA Laboratories GmbH, Linz, Austria), 25 mM HEPES, 10 µg/ml gentamycin, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol, and 1x antibiotic-antimycotic solution (all from Gibco Life Technologies, Inc., Grand Island, NY). The following unlabeled fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, and allophycocyanin (APC)-conjugated monoclonal antibodies (mAb) were used: 2C11 (anti-CD3), GK1.5 (anti-CD4), 53–6.72 (anti-CD8), M1/70 (anti-CD11b), 2.4G2 (anti-CD16/CD32), M1/69, and J11d (anti-CD24), 3C7 and 7D4 (anti-CD25), 3/23 (anti-CD40), 30-F11 (anti-CD45), 3E2 (anti-CD54), 16-10A1 (anti-CD80), GL1 (anti-CD86), G7 (anti-CD90, Thy-1), M5/114.15.2 (anti-I-Ab,d,q and I-Ed,k), JES5-16E3 (anti-IL-10), 1B1.3a [anti-IL-10 receptor (IL-10R)] (all from PharMingen, San Diego, CA); F7D5 (anti-CD90, Thy1.2, Biosource International, Camarillo, CA); and ECCD-2 (anti-E-cadherin, Zymed, South San Francisco, CA). Hybridomas 10-2-16 (I-Ak, TIB93), 14-4-4S (I-Ed,k, HB32), M5/114.15.2 (anti-I-Ab,d,q and I-Ed,k, TIB120), M1/9 (anti-CD45, TIB122), N418 (anti-CD11c, HB224), NLDC-145 (anti-DEC-205), F4/80 (anti-macrophage, HB198), IM7.8.1 (anti-CD44), and RA3-3A1/6.1 (anti-CD45R/B220, TIB146) were obtained from American Type Culture Collection (Manassas, VA). Second-step reagents were polyclonal FITC-labeled F(ab')2 goat anti-rat immunoglobulin G (IgG; H+L, Immunotech, Marseille, France), FITC-conjugated goat anti-hamster IgG (H+L, Caltag, South San Francisco, CA), streptavidin-PE (SAv-PE; PharMingen), and SAv Texas RedTM (Amersham Pharmacia Biotech, Vienna, Austria). Irrelevant, isotype-matched mAb were used as negative controls. FITC-ovalbumin (OVA) and FITC-dextran (40 kDa) were purchased from Molecular Probes (Leiden, The Netherlands), and OVA was obtained from Sigma Chemical Co. (St. Louis, MO). The peptides OVA265–280 (I-Ab), OVA257–264 (SIINFEKL, H2-Kb) [25 , 26 ], and mouse tyrosinase-related protein-2181–188 (mTRP-2; H-2Kb) [27 ] were synthesized at InterCell AG (Clinical Immunology Department, Campus Vienna Biocenter, Austria) [28 ].

Epidermal sheets and cell suspensions
Epidermal sheets from adult (6-week) and neonatal [NB, 2, 3, and 7 days postpartum (pp)] mice were prepared and subjected to immunofluorescence staining [29 ]. Immunostained sheets were analyzed using a confocal laser-scanning microscope (CLM 510, Carl Zeiss Jena, Germany). For all fluorochromes, the optical thickness was adjusted to 3 µm. Airy units were as follows: FITC, 3.96; PE, 3.52; APC, 2.81. Epidermal cell (EC) suspensions from trunk skin of mice at various age-groups were prepared essentially as described [30 ]. EC were used for flow cytometry analyses and antigen-processing assays or were cultured (1.5x106 cells/ml) at 37°C. After 72 h, nonadherent EC were harvested, and dead cells were largely eliminated by density gradient centrifugation (Lympholyte-M, Cedarlane Laboratories, Hornby, Ontario, Canada). The resulting cell suspensions were analyzed by flow cytometry, further enriched, or used for functional studies.

Purification and culture of NB DEL and LC
NB DEL and LC were purified using the mismatched panning technique [31 ]. Briefly, pre-enrichment of DEL and LC was achieved by removing keratinocytes and dendritic epidermal T cells by treatment with anti-Thy-1 mAb (clones G7 and F7D5) for 30 min at 4°C, followed by Low-Tox-M-rabbit C' (Cedarlane Laboratories) for 40 min at 37°C (=pre-enriched EC). After the removal of dead cells by density gradient centrifugation, EC were treated for 30 min at 4°C with culture supernatants with 14-4-4S (anti-I-Ek,d, mouse IgG2a) in combination with 10-2-16 (anti-I-Ak, mouse IgG2b) or M1/9 (anti-CD45, rat IgG2b). After extensive washes, cells were transferred for 30 min at room temperature into petri dishes coated with goat anti-Ig antibodies, which were mismatched; i.e., for panning of cells treated with mouse mAb, goat anti-rat Ig was used, and for panning of DEL and LC treated with rat mAb, goat anti-mouse Ig was used. Nonadherent cells were gently rinsed off the dish with prewarmed phosphate-buffered saline (PBS). Adherent DEL and LC were released by adding excess amounts of {gamma} globulin matched to the antibody bound to the petri dishes. The purity of DEL and LC was verified by flow cytometry and was consistently ≥80%. These cells were used for functional assays or further cultured in 96-well microtiter plates (5x104 per well) for 3 days in culture medium alone or with granulocyte macrophage-colony stimulating factor (GM-CSF), a survival factor for LC [32 33 34 ]. At the end of the culture period, the cells were analyzed by flow cytometry or used for functional studies.

Endocytosis assay
Pre-enriched EC from NB and adult mice were incubated for 10–60 min at 37°C or 4°C (to assess nonspecific FITC signal) in culture medium containing FITC-OVA/dextran (1 mg/ml). After three washes in PBS supplemented with 5% FCS and 0.1% NaN3, cells were stained with PE-conjugated M5/114 mAb or the isotype control (30 min, 4°C). Endocytosis was assessed by flow cytometry analysis of gated MHC class II+ cells.

Antigen-presentation assay
Antigen-presentation capacity of neonatal DEL and adult LC from C57BL/6 and C3H mice was measured using MHC-restricted T cell hybridomas 2D5 (CD4+, specific for I-Ab-restricted OVA265–280), 4B10 (CD8+, specific for H-2Kb-restricted OVA257–264), and E8 (CD4+, specific for I-Ek-restricted OVA). The activation of hybridoma cells was determined by measuring IL-2 production by enzyme-linked immunosorbent assay (ELISA; Endogen, Woburn, MA). Pre-enriched EC or purified neonatal DEL and LC (1x106 cells/ml) were cultured with/without OVA and the OVA peptide265–280 for 24 h or with/without OVA peptide257–264 (SIINFEKL) and its control peptide mTRP-2 for 2 h in 24-well culture plates at concentrations specified in the respective figure. Thereafter, nonadherent cells were harvested, washed, and adjusted to equal numbers of DEL and LC as determined by flow cytometry. OVA or peptide-pulsed, and for control purposes, unpulsed, neonatal DEL and LC (5x103/well) were cultured with hybridoma cells (1x105 per well) or alone in 96-well flat-bottom microtiter plates. Supernatants were harvested after 24 h and stored at –20°C until use.

Flow cytometric analysis
For two-color analysis, cells (5x105 per sample) were resuspended in cold PBS/1% FCS/0.1% NaN3 and serially incubated with FITC-conjugated mAb directed against selected mouse antigens and PE-conjugated anti-CD45 or anti-MHC class II mAb (30 min, 4°C). For the detection of E-cadherin molecules, EC were prepared as described [35 ]. Specificity of staining was confirmed using isotype-matched control mAb. Fluorescence was measured using a FACScan® flow cytometer, and data were analyzed with Cell QuestTM software (both from Becton Dickinson, San Jose, CA). Dead cells were excluded by 7-amino-actinomycin D (Sigma Chemical Co.) uptake.

Mixed lymphocyte reactions (MLR)
CD4+ and CD8+ T cells were prepared from mesenteric lymph nodes essentially as described [30 ]. EC suspensions of different age groups were cultured for 3 days. Nonadherent cells were harvested, and dead cells were eliminated (98–99%) by density gradient centrifugation (Lympholyte-M). Adjusted numbers of neonatal DEL and LC (1x103/well) were cocultured with allogeneic CD4+ and CD8+ T cells (1x105/well) or alone in 96-well round-bottom culture plates (Costar, Corning, NY). At the indicated time-points, cells were pulsed with 37 KBq/well (3H)-thymidine (TdR) for the last 8 h of culture, and incorporation of the radionucleotide was measured using scintillation spectroscopy (Wallac Oy, Turku, Finland). In certain experiments, highly purified NB DEL and LC were used. All cultures were performed in triplicates, and results are expressed as mean cpm ± SD.

Assessment of cytokine production in EC cultures
For the assessment of cytokine production, freshly prepared EC from NB and adult C3H mice were cultured at a density of 2 x 106/ml [GM-CSF, tumor necrosis factor {alpha} (TNF-{alpha})] or 3 x 106 cells/ml (IL-10) in 24-well plates (Costar). In certain experiments, purified DEL (>92%) and LC (>94%) were incubated in 96-well culture plates (2.5x105/ml per 200 µl in each well). At selected time-points, supernatants were collected and stored at –20°C until use. Cytokine concentrations were determined by ELISA according to the manufacturer’s instructions for GM-CSF, IL-10 (Endogen), and TNF-{alpha} (Genzyme, Cambridge, MA). In certain experiments, 24 h supernatants from NB and adult EC cultures (7x106 cells/ml) were added to adult and NB EC suspensions, respectively. As a control, each EC population was cultured alone. In specificity control experiments, 0.012 µg/ml neutralizing anti-IL-10 antibody (BioSource International) or isotype control was added to the cultures from day 0 onward. After 3 days, nonadherent EC were enriched for APC by density gradient centrifugation, stained, and analyzed for marker expression by flow cytometry.

Quantification of CD45+/IL-10+ neonatal DEL
Data acquisition and image cytometry were done using an automated fluorescence-confocal laser-scanning microscope and the analysis software TissueQuest, which permitted recognition of individual cells by a specific identification strategy [36 ]. In brief, 40 randomly selected fields under 40x object lenses (one field was 0.2375 mm2) were scanned. Target cells were labeled as described above. Specificity of the staining procedure and accuracy of instrument settings were cross-checked by isotype-matched negative controls. Cellular subpopulations were defined, and the thresholds for statistical evaluation were set according to the negative controls.

Statistical analyses
The Student‘s t-test was used to analyze the results, and a P value <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phenotypic characterization of NB DEL
Under nonpathologic conditions, LC are the only cells that constitutively express MHC class II molecules in the adult epidermis. Accordingly, in epidermal sheets and EC suspensions of adult C3H mice, one part of CD45+ cells exhibited a pronounced anti-MHC class II reactivity, thus qualifying as LC, and CD45+, MHC class II– cells, represent dendritic epidermal T cells (Fig. 1A 1B 1C 1D ). In contrast, the majority of CD45+ dendritic leukocytes in NB epidermis did not express MHC class II antigens (Fig. 1E 1F 1G 1K) . Some areas contained MHC class II+ cells appearing as single cells or as doublets (Fig. 1I) , and a few areas contained these cells at higher densities (see Fig. 7A ). When compared with LC, freshly isolated NB DEL expressed higher levels of CD45, H-2K, F4/80, and CD32, similar levels of H-2D, CD11b, CD44, e-cadherin, and CD54, but significantly less MHC class II (I-A and I-E) and CD24 molecules (Fig. 1K and 1L) . Like LC, DEL failed to express CD3, CD90, CD40, CD80, and CD86 (data not shown). Similar results were obtained in C57BL/6 mice (data not shown).



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  		Figure 1. NB DEL are MHC class II–. Epidermal sheets from adult and NB C3H mice were stained for CD45 (A, E, H) and MHC class II (B, F, I). Merged panels represent superimpositions of red and green staining. Colocalizing cells appear in yellow. Note the absence of MHC class II staining on the majority of CD45+ DEL in situ (F) and the weak expression of these molecules on most of the CD45+ cells upon isolation (K, box). Occasionally, MHC class II+ cells were observed in the NB epidermis (I, K, circle). Freshly isolated EC (5x104) from adult and NB mice were acquired and examined for surface expression of the indicated molecules by flow cytometry. Data are shown as dot blot (D, K) and as geometric mean fluorescence intensity (MFI) ± SD (L) on gated CD45+ cells (n=4). MFI of corresponding isotype-matched control mAb was subtracted from each value. Original scale bars, 20 µm. ***, P < 0.001, versus LC; **, P < 0.01, versus LC; *, P < 0.05, versus LC.

 


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  		Figure 7. Altered surface molecule and stimulatory capacity of NB IL-10–/– DEL. Epidermal sheets from neonatal IL-10+/+ (A) and IL-10–/– mice (B) were stained for MHC class II. Note that the intensity of MHC class II expression on DEL is higher in IL-10–/– compared with IL-10+/+ mice. (C) Three-day-cultured EC suspensions from adult (IL-10+/+) and NB (IL-10+/+, IL-10–/–) mice were examined for surface expression of the indicated molecules by flow cytometry. The levels of surface molecule expression are represented as MFI. (D) The MLR was performed in the same way as described in Figure 3 . Proliferative responses were determined on day 4 of culture. Original scale bars, 20 µm. ***, P < 0.001, versus DEL-enriched EC (IL-10+/+).

 
NB DEL capture antigen more efficiently than LC but are defective in presenting antigen
To compare the antigen-uptake capacity of NB DEL and LC, their endocytic activity was assessed by determining the internalization of FITC-conjugated tracers. At all time-points investigated, NB DEL were twice as efficient as LC in the uptake of FITC-conjugated proteins, indicating that NB DEL are functionally active DC (Fig. 2A and 2B ). In line with this are data by Bellette et al. [37 ], showing that neonatal LC are also more efficient in antigen uptake compared with adult LC. Although Bellette et al. [37 ] have shown that neonatal LC preferentially use a wortmannin-sensitive, fluid-phase pathway to internalize exogenous antigen, they have not investigated whether this finding may be of relevance for the function of these cells. We studied the antigen-presentation capacity of NB DEL and for comparison, LC, using OVA-specific T cell hybridomas. Pre-enriched EC from NB or adult C57BL/6 mice were pulsed with OVA protein and OVA peptide265–280 for 24 h and OVA peptide257–264 for 2 h. Pulsed cells were then cocultured with the appropriate T cell hybridomas, and their activation was measured as IL-2 secretion. Whereas LC were most efficient in the presentation of OVA protein and peptides, NB DEL presented neither OVA protein nor MHC class I- and II-restricted peptides efficiently (Fig. 2C and 2D) . To ensure that the impairment seen in NB DEL was not a result of a function of the response of a particular T cell hybridoma and a specific mouse strain, the ability of NB DEL to present antigen was examined using another T cell hybridoma (E8) and another mouse strain (C3H). Results obtained showed that NB DEL were not able to present OVA antigen (data not shown). To exclude exogenous inhibitory effects (e.g., factors secreted from keratinocytes) on the antigen-presenting capacity of neonatal DEL, they were highly purified, pulsed with OVA protein, and cocultured with the OVA-specific T cell hybridoma E8. Again, NB DEL were defective in presenting antigen. However, when DEL were isolated from 3-day-old mice, they presented antigen almost as effectively as LC (Fig. 2E) .



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  		Figure 2. NB DEL fail to present antigen. Pre-enriched EC from adult and NB C3H mice were incubated with 1 mg/ml FITC-OVA (A) or FITC-dextran (B). Then cells were stained with PE-labeled anti-MHC class II mAb or isotype control before fluorescein-activated cell sorter acquisition. For each time-point, the surface binding of the reagents at 4°C was measured and subtracted from 37°C values and presented as MFI of internalized FITC. (C, D) Pre-enriched EC from adult and NB C57BL/6 mice were cultured (1x106 cells/ml) with/without OVA protein (2 mg/ml) and the OVA peptide265–280 (5 µM) for 24 h (C) or with/without OVA peptide257–264 (1, 5 µM; D) and its control peptide mTRP-2 (data not shown) for 2 h in 24-well culture plates. Thereafter, cells were harvested, washed, and adjusted to equal numbers of NB DEL and LC as determined by flow cytometry. OVA protein or peptide-pulsed, and for control purposes, unpulsed, DEL and LC (5x103/well) were cultured with the appropriate hybridoma cells (105/well) or alone in 96-well flat-bottom microtiter plates. Supernatants were harvested, and IL-2 production was determined by ELISA. Cells pulsed with the control peptide mTRP-2 failed to stimulate hybridoma cells. (E) Highly purified neonatal DEL and LC (≥90%) from C3H mice were pulsed with OVA protein in the presence of GM-CSF (200 U/ml), washed, and cocultured with E8 hybridoma cells as described. Hybridoma cells failed to produce IL-2 when cultured alone or with unpulsed DEL/LC (data not shown). One of at least two experiments is shown.

 
To investigate whether NB DEL are capable of inducing a primary T cell response, NB DEL- and for comparison, LC-enriched EC were tested for their ability to stimulate naive T cells. A marked defect in the ability of NB DEL-enriched EC to stimulate CD4+ and CD8+ T cell proliferation was evident when the APC were derived from NB mice (Fig. 3A ). The ability of NB DEL to present antigen improved with age. When neonatal DEL-enriched EC were taken 3 and 6 days pp, ≥47% and ≥75% of the adult response was observed, respectively (Fig. 3A and data not shown). To distinguish whether the failure to stimulate T cell proliferation results from a functional deficiency of all EC or only a given subpopulation, purified NB DEL were tested as described above. Repeated experiments showed that on a per-cell basis, DEL were able to stimulate only 10% and 20% of naive CD4+ and CD8+ T cell proliferation, respectively, of that elicited by LC from adult mice (Fig. 3B) . The low T cell response does not reflect a demise of DEL during culture (data not shown). Addition of IL-2 only partially improved the T cell response to NB DEL (data not shown). Collectively, these data imply that the deficit in antigen presentation of NB DEL may be a problem of presentation and not in uptake of antigen.



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  		Figure 3. NB DEL are greatly impaired in their capacity to activate naive, allogeneic T cells. (A) EC were isolated from adult and neonatal mice and cultured for 3 days. Then, equal numbers of viable LC/neonatal DEL (103/well) were cocultured with naive, allogeneic CD4+ and CD8+ T cells (105/well) in 96-well round-bottom microtiter plates. T cell proliferation was determined after 4 days by measuring the incorporation of (3H)-TdR. Error bars represent SD of triplicate cultures. LC/DEL-enriched EC and CD4+and CD8+ T cells did not proliferate when cultured with medium (<1000 cpm). (B) The allogeneic MLR with 3-day-cultured [in the presence of GM-CSF (200 U/ml)], purified LC (98.4%) and NB DEL (93.4%) was performed as described (A). Shown is one representative of three independent experiments with similar results.

 
NB DEL do not acquire the morphology and phenotype of LC in culture
As NB DEL are functionally deficient in their ability to effectively present antigen to T cells, we tested whether differences in surface expression of molecules relevant for T cell activation may be responsible for this phenomenon. Purified NB DEL and for comparison, LC, were cultured with GM-CSF. After 3 days, the morphologic and phenotypic analysis of NB DEL and LC revealed major differences (Fig. 4A and 4B ). During culture, LC became highly dendritic and formed numerous homotypic clusters, and NB DEL developed short dendrites and created only few small-sized clusters. When DEL were isolated 2–3 days after birth, their morphology was comparable with LC (data not shown). Phenotypically, cultured NB DEL and LC exhibited similar intensity in surface expression of CD40, F4/80, and CD11b and were CD3–. However, NB DEL expressed considerably less MHC class II (I-A and I-E), CD54, CD80, and CD86 and failed to express CD205 and CD25 when compared with LC (Fig. 4 , Exp. 1 and 2). As costimulatory molecules are normally up-regulated during culture, this indicated that DEL have functional deficiencies in up-regulating those molecules required for fully professional APC activity.



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  		Figure 4. NB DEL do not acquire the morphology and phenotype of LC in culture. Highly enriched LC (A) and NB DEL (B) were cultured for 3 days with medium supplemented with GM-CSF (200 U/ml). LC were highly dendritic and formed numerous homotypic clusters, and DEL had short dendrites and created only few small-sized clusters. These cells were stained with the indicated markers in Exp. 1 and 2, and analyzed by flow cytometry. Solid lines in the dot plots denote the position of the cut-off channel fixed with the isotype controls. Shown are two representative experiments of five.

 
IL-10 in EC supernatants from NB mice prevents LC maturation in vitro
To examine whether a special epidermal cytokine milieu may be responsible for the distinct phenotype of neonatal DEL, we comparatively assessed the cytokine pattern of NB and adult cultures by testing EC supernatants by ELISA. We have chosen cytokines that are important for LC survival/maturation and the cytokine IL-10, which is known for its immunosuppressive properties and which plays a dominant role in several immune reactions including regulatory mechanisms in the skin [38 , 39 ]. While comparable amounts of GM-CSF were measured in NB and adult EC culture supernatants during the entire culture period of 72 h, the levels of TNF-{alpha} and IL-10 in the supernatants of NB-EC cultures far exceeded those secreted by EC from adult animals (Fig. 5A 5B 5C ). To assess whether the different cytokine secretion pattern may have any modulating effect on the phenotypic maturation of LC and DEL, EC from NB and adult mice were cultured with 24 h EC supernatants from adult and NB mice, respectively, and analyzed after 3 days for surface marker expression. While EC supernatants from adult mice failed to modulate the expression of cell-surface molecules on NB DEL (data not shown), EC supernatants from NB mice markedly prevented (range, 41–47%) the up-regulation of CD86 and MHC class II molecules on adult LC (Fig. 5D) . To determine whether the inhibitory effect of NB EC supernatants on the up-regulation of these molecules is mediated by IL-10, attempts were made to neutralize the effects of this cytokine. Although addition of a neutralizing polyclonal anti-IL-10 antibody to the cultures significantly reversed the inhibitory effect of NB EC supernatant onto the phenotypic changes of LC, addition of a nonspecific isotype control had no consequences on the inhibitory effect of the EC supernatant (Fig. 5E) . These data suggest that IL-10 is at least partly responsible for the observed inhibition and imply that EC isolated from NB mice may secrete other immunosuppressive factors active in the cultures.



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  		Figure 5. NB EC secrete IL-10, which has an inhibitory effect on LC marker up-regulation in vitro. (A–C) EC from adult and NB C3H mice were cultured as described in Materials and Methods. Supernatants, harvested at 24, 48, and 72 h, were screened for the presence of GM-CSF (A), TNF-{alpha} (B), and IL-10 (C) by ELISA. Data are expressed in pg/ml as mean concentration ± SD of duplicates (GM-CSF, IL-10) or triplicates (TNF-{alpha}). Data shown are representative of two experiments. (D) EC (1x105/ml) from adult mice were cultured with 24 h EC supernatants (SP) generated from EC cultures (7x106 cells/ml) of NB mice. After 72 h, EC were examined for expression of the indicated molecules by flow cytometry. Cells (5x104 per sample) were acquired. Percent inhibition of MFI of surface molecules on LC is shown. (E) Culture of cells and analysis were performed in the same way as in D in the presence of a neutralizing anti-IL-10 antibody or isotype control. The levels of surface molecule expression are represented as MFI. LC cultured in medium alone indicate 100% expression. Data are represented as mean calculated from three independent experiments. **, P < 0.01, versus control; *, P < 0.05, versus control.

 
DEL are the primary source of IL-10 in the neonatal epidermis
As the epidermis is composed of heterogeneous cell populations, we wanted to determine which EC type is the main IL-10 producer in situ. Thus, epidermal sheets from NB and for comparison, adult mice were double-stained for IL-10 and selected surface antigens. We found that the majority (≥68%) of CD45+ DEL and some MHC class II+ DEL was strongly anti-IL-10-reactive (Fig. 6A and 6B , and data not shown). Occasionally we observed a faint IL-10 reactivity of CD45–, MHC class II– EC in NB skin, presumably keratinocytes, and this was judged to be specific, as the isotype controls never showed this staining pattern. In contrast, complete absence of IL-10 binding was observed in adult epidermis. To confirm specific reactivity of the IL-10 antibody, the epidermis from NB IL-10-deficient mice was used. Complete absence of IL-10 binding was observed in DEL and keratinocytes from IL-10–/– mice (Fig. 6A) . Kinetics experiments revealed that DEL show IL-10 expression until day 7 after birth, although in decreasing intensity (data not shown). IL-10 exerts its effects via a specific receptor (reviewed in ref. [40 ]). We therefore tested, by examining the expression of IL-10R, whether DEL could respond to IL-10. In concordance with the IL-10 staining pattern, fetal and NB CD45+ and MHC class II+ DEL expressed moderate levels of IL-10R, and these levels decreased with age (Fig. 6C , and data not shown). To examine whether NB DEL can produce biologically active IL-10 protein, supernatants from NB DEL and for comparison, LC cultures were tested for IL-10 bioactivity. Concentrations up to 2.4 ng/ml IL-10 were detected in NB DEL culture supernatants, whereas no IL-10 protein was detectable in supernatants from LC cultures (Fig. 6D) . Thus, DEL proved to be the predominant source of IL-10 in the neonatal skin.



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  		Figure 6. NB DEL express IL-10 and the IL-10R and secrete high amounts of IL-10 protein. (A) Epidermal sheets from adult and NB (IL-10+/+, IL-10–/–) mice were stained for CD45 and IL-10. While CD45+ DEL from IL-10+/+ mice expressed high levels of IL-10, LC from IL-10+/+ and CD45+ DEL from IL-10–/– failed to do so. Some NB DEL are stuffed with IL-10 (inset). (B) Dot plots show the mean relative intensity of IL-10 on CD45+ cells in epidermal sheet preparations. From each sheet, a total area of 9.5 mm2 was screened, and the numbers of CD45+/IL-10+ cells were evaluated. The cut-off values (solid lines) were determined using isotype-matched control antibodies. Based on these cut-off values, the relative number of positive and negative events was calculated. (C) Kinetics of IL-10R expression on fetal and neonatal DEL. EC from mice of various age groups were analyzed for surface-marker expression by flow cytometry. EC (5x104 per sample) were acquired, and MFI of IL-10R expression on gated CD45+ cells is shown. Data are represented as mean ± SD from three independent experiments. (D) Purified LC and NB DEL from C3H mice were cultured in the presence of GM-CSF (200 U/ml), and supernatants were screened for the presence of IL-10 by ELISA. Data are represented as mean ± SD from five experiments. Original scale bars, 20 µm. ***, P < 0.001, versus LC.

 
The deletion of the IL-10 gene enhances the stimulatory capacity of NB DEL
Reasoning that the low expression of MHC class II by NB DEL may be a result of the effect of IL-10, we evaluated the MHC class II expression pattern in IL-10–/– mice. We found that the intensity of MHC class II expression on neonatal DEL was higher in IL-10–/– compared with IL-10+/+ mice (Fig. 7A and 7B ). In line with that, 3-day-cultured NB DEL from IL-10–/– mice displayed higher membrane levels of MHC class II, CD40, and CD86 when compared with NB DEL from IL-10+/+ mice, although adult expression levels were not reached (Fig. 7C) . Consequently, the ability of NB DEL-enriched EC from IL-10–/– mice to stimulate allogeneic, naive T cells was greatly enhanced when compared with NB DEL from IL-10+/+ mice (Fig. 7D) . These data show that IL-10 production by DEL themselves influences their differentiation and their T cell stimulatory capacity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We and others [22 , 41 42 43 44 45 46 ] have previously reported that the fetal and NB murine epidermis contains ADPase+, MHC class II–, CD205–, CD207–, CD90–, CD3– dendritic leukocytes. In this study, we have purified these cells from NB epidermis and performed phenotypic and functional studies. Furthermore, we have investigated which factors maintain them in an immature state. Our data show for the first time that upon culture with GM-CSF, purified DEL do not express markers that are typically expressed by LC, DEL capture antigen more efficiently than LC but are markedly impaired in their antigen-presenting function, and autocrine IL-10 secretion partly inhibits the differentiation and maturation of DEL into LC.

Compared with LC, freshly isolated NB DEL expressed significantly higher levels of CD45, MHC class I, F4/80, and CD32; identical levels of CD11b, CD44, e-cadherin, and CD54; and much less MHC class II and CD24 molecules. Similar to LC, highly purified NB DEL matured in the presence of GM-CSF in vitro but never achieved the LC phenotype. NB DEL moderately up-regulated MHC class II and costimulatory molecules and failed to express CD205 and CD25. Reasoning that this distinct phenotype of DEL may be a consequence of an altered cytokine milieu in neonatal epidermis, we compared the cytokine pattern of NB and adult cultures by testing EC supernatants for the presence of selected cytokines. We found that neonatal EC produced significantly more TNF-{alpha} than adult EC and that neonatal but not adult EC secrete high levels of IL-10. We show for the first time that neonatal DEL but not keratinocytes are the main source for IL-10 production and that they express the IL-10R. Constitutive expression of IL-10R has been observed on murine bone marrow-derived DC [47 , 48 ] and on human peripheral blood mononuclear cell-derived DC [49 ], suggesting that IL-10 and IL-10R constitute a general modulatory loop for the regulation of DC differentiation and function. In fact, DC functions are tightly regulated in that protective immune responses are elicited, and unwanted immune responses are prevented. IL-10 is an important immunoregulatory cytokine, which inhibits the production of IL-12 and the expression of costimulatory molecules by various types of DC, including LC, which correlate with its ability to inhibit primary alloantigen-specific T cell responses and to induce a state of alloantigen-specific or peptide-specific anergy in primed and naive CD4+ and CD8+ T cells (reviewed in ref. [40 ]). Recently, it has been shown that IL-10 renders DC incapable of up-regulating cathepsin S and cathepsin B activity and attenuates the level of both enzymes. Suppressed cathepsin S and cathepsin B activity delays MHC class II-sodium dodecyl sulfate stable dimer formation and impairs antigen degradation. Accordingly, IL-10 reduces the number of MHC class II peptide complexes accessible to antigen-specific T cell receptor [50 ]. We found that NB DEL internalized protein antigens much more efficiently than LC. However, despite an increased endocytic activity, NB DEL were greatly impaired in their capacity to up-regulate MHC class II and costimulatory molecules in culture and to stimulate proliferative T cell immune responses. Our further findings that IL-10 in NB EC suspensions partly inhibits up-regulation of MHC class II and CD86 expression on LC, that 3-day-cultured NB DEL from IL-10–/– mice displayed higher surface levels of MHC class II and costimulatory molecules compared with NB DEL from IL-10+/+ mice, and that DEL-enriched EC from NB IL-10–/– mice are superior to DEL-enriched EC from NB IL-10+/+ mice in their ability to stimulate naive, allogeneic T cells indicate that autocrine IL-10 secretion in neonatal DEL partly inhibits their differentiation/maturation and function. This is supported by data showing that IL-10 modifies the maturation process of DC precursors and the differentiation of LC in that overexpression of the IL-10 gene in the epidermis of mice alters the density of LC and reduces MHC class II, CD80, and CD86 expression on these cells and their ability to stimulate allogeneic T cell proliferation [51 , 52 ].

In contrast to neonatal DEL, LC do not produce IL-10 (ref. [53 ] and this study). Similarly, LC differentiated from monocytes or CD34+ progenitors in the presence of transforming growth factor-ß and DC generated from CD1a+ progenitors [54 ] fail to synthesize IL-10 [55 , 56 ], whereas certain DC subsets producing IL-10 have been identified from Peyer’s patches, liver, lung, and peripheral blood [57 58 59 60 ]. These findings suggest that IL-10 production is a property of some DC subsets and according to our findings, may occur only during a certain time-period during their development.

The ability of IL-10 to inhibit induction and effector function of T cell-mediated and inflammatory immune responses led to numerous studies of its potential use in bone marrow and organ transplantation. With regard to skin, transplantation of untreated neonatal or ultraviolet light B-irradiated adult skin had tolerogenic effects [61 , 62 ]. Although it has been shown that tolerogenicity of neonatal skin grafts derives in part from natural expression of IL-10 by the graft, another possible contribution to tolerogenicity may be the inability of NB DEL to up-regulate MHC class II and costimulatory molecules. Indeed, we have provided evidence in our study that the low expression of MHC class II and costimulatory molecules by NB DEL is partly attributable to autocrine IL-10 secretion. Furthermore, we have demonstrated that although a few DEL start to express MHC class II around birth, they retain immature characteristics until days 5–7 after birth (expression of low levels of IL-10, IL-10R, DEC-205, and costimulatory molecules), which is also reflected in their functional incompetence. These data correlate with transplantation studies, demonstrating that grafts from neonates up to 1 week enjoy a significant survival advantage [63 ]. Additional contributions to tolerogenicity may be made by other cytokines expressed by the neonatal skin. We have found that culture supernatants of neonatal EC contain high levels of TNF-{alpha}. This cytokine is a key player for the migration capacity of LC from sites of antigen capture to the draining lymph nodes where they initiate an adaptive immune response [64 , 65 ]. However, evidence has been provided from skin organ culture experiments that high concentrations of TNF-{alpha} can partly inhibit LC migration [66 ]. Furthermore, it has been shown that autocrine IL-10 impairs the migration of antigen-bearing DC to the lymph nodes [47 ] and that IL-10 transgenic mice have fewer MHC class II+ hapten-bearing cells in regional lymph nodes after hapten painting of the skin compared with controls [52 ]. In analogy to these findings, higher amounts of TNF-{alpha} in neonatal epidermis and autocrine IL-10 secretion may hinder DEL emigration out of the skin. Recent findings indicate that neonatal DEL are indeed inefficient in transporting antigen to the draining lymph nodes [45 ]. Whether TNF-{alpha} and/or IL-10 contribute to this phenomenon remains to be clarified.

Collectively, the results of our study provide evidence that autocrine IL-10 and IL-10R expression by DEL in the fetal and neonatal period serves as a relevant, modulatory loop for the regulation of LC development, with important consequences on the outcome of the immune response.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant of the Austrian Science Fund (P14243-MED) to A. E-B. We thank J. Kapp (Department of Ophthalmology, Winship Cancer Institute, Atlanta, GA) and A. Egyed (InterCell Biotechnologies Inc., Vienna, Austria) for the gift of T cell hybridomas and helpful discussions and S. Olt and W. Hötzenecker (Department of Dermatology, Medical University of Vienna, Austria) for excellent technical help.

Received February 12, 2004; revised April 8, 2004; accepted May 11, 2004.


   REFERENCES
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