Published online before print December 8, 2004
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* Department of Dermatology, Division of Immunology, Allergy and Infectious Diseases, Medical University of Vienna, Austria;
Novartis Institutes for Biomedical Research, Vienna, Austria;
Department of Dermatology, Eberhard-Karls-University of Tübingen, Germany; and
Schering-Plough, Laboratory for Immunological Research, Dardilly, France
1 Correspondence: Department of Dermatology, Division of Immunology, Allergy and Infectious Diseases, Medical University of Vienna, Lazarettgasse 19, A-1090 Vienna, Austria. E-mail: adelheid.elbe-buerger{at}meduniwien.ac.at
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: transplantation • epidermis • leukocytes • cell cycle • cytokines • apoptosis • grafts
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Like all hematopoietic cells, LC originate in the bone marrow [4 , 5 ]. Recent studies in adult mice indicate that under steady-state conditions, LC are derived from radio-resistant, long-lived, proliferating, local precursors and that rapid recruitment of bone marrow-derived LC precursors occurs during skin inflammation [6 , 7 ]. Migratory LC have been found in skin-draining, lymphatic vessels under steady-state conditions, which suggests that there is a constant turnover of LC even in the absence of inflammatory signals [6 , 8 , 9 ]. Although we are beginning to understand the mechanisms that regulate the migration of LC and their precursors, far less is known about their ontogeny. In mice, initial colonization of the epidermis with a considerable number of ADPase+major histocompatibility complex (MHC) class II– dendritic epidermal leukocytes (DEL), which are considered to be LC precursors, takes place at approximately fetal day 16 (d16) [10 ]. A few dendritic ADPase+ MHC class II+ cells appear around birth, but a dramatic numerical increment of these cells occurs after birth [10 11 12 ]. Recently, we and others have reported that fetal and newborn DEL do not express markers, typically expressed by LC (e.g., CD205 and CD207) [13 14 15 ] and that DEL capture antigen more efficiently than LC but are markedly impaired in their antigen-presenting function [15 ]. Here, we analyzed whether DEL represent a subpopulation, which is present only in fetal/neonatal life and subsequently undergoes apoptosis in situ, or indeed represent LC precursors. Our results show that fetal and newborn epidermis does not contain apoptotic leukocytes and that DEL are the actual LC precursors.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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, Grand Island, NY). The following unlabeled fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, and allophycocyanin (APC)-conjugated monoclonal antibodies (mAb) were used: 30-F11 (anti-CD45), KH74 (anti-I-Ab), AMS-32.1 (anti-I-Ad), 11-5.2 (I-Ak), 17-3-3 (anti-I-Ek), and anticyclin B1 (GNS-1; all from PharMingen, San Diego, CA); 10-2.16 (anti-I-Ak, TIB 93) and NLDC-145 (anti-CD205; from the American Type Culture Collection, Manassas, VA); and 929F3 (anti-CD207; raised at Schering-Plough, Laboratory for Immunological Research, Dardilly, France). Second-step reagents were FITC goat F(ab')2 anti-rat immunoglobulin (Ig; Zymed, South San Francisco, CA); R-PE-conjugated goat anti-rat Ig-specific polyclonal antibody and streptavidin-PE (PharMingen); and Texas red-streptavidin (Amersham Pharmacia Biotech, Vienna, Austria). Irrelevant, isotype-matched mAb were used as negative controls.
Assessment of apoptotic leukocytes in the epidermis by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)
To monitor apoptosis in situ, epidermal sheets from selected age groups {fetal [E19], newborn [
10 h postpartum (pp)], 3 days pp, and adult [6 weeks]} were prepared as described previously [16
], cut into small pieces (
3 mm2), fixed with 4% paraformaldehyde (30 min, room temperature), and then washed three times in phosphate-buffered saline (PBS). Sheets were permeabilized with 0.1% Triton X-100 (Sigma Chemical Co., St. Louis, MO) in 0.1% sodium citrate for 1 h, extensively washed, and afterward, were subjected to FITC-TUNEL labeling according to the manufacturer’s instructions (Roche Molecular Biochemicals, Mannheim, Germany). For the identification of DEL and LC, epidermal sheets were counterstained with APC-labeled mAb [anti-CD45 (clone 30F11), biotinylated anti-MHC class II (clone KH74)] for 1 h at 37°C. As a positive control, sheets were first incubated with 1500 U/ml DNase I (1 h, 37°C) to induce DNA breaks in the nucleus and were then subjected to double-staining procedures. Immunostained sheets were analyzed with a confocal laser-scanning microscope 510 (Carl Zeiss, Jena, Germany).
Epidermal and dermal cell suspensions and purification of subpopulations
For the preparation of skin cell suspensions, fetal and neonatal mice were decapitated, submerged for 5 min in a solution of 10% Betaisodona (Mundipharma, Vienna, Austria) and for 2 min in 70% ethanol, washed (5 min, PBS), and dried. Adult mice were killed by CO2, shaved, and disinfected as described for neonatal skin. Full-thickness trunk skin was then excised and placed on a sterile petri dish (dermal side up), and the subcutaneous fat was scraped off. Skin was cut into small pieces (20x8 mm), placed in cell-culture dishes (150x25 mm, Corning, NY), and floated dermal side down on a 1% trypsin-PBS solution (Merck, Darmstadt, Germany) for 40 min at 37°C. Epidermal sheets were peeled off from the underlying dermis, and both tissues were vigorously agitated in a shaking water bath (15 min, 37°C) in FCS-supplemented RPMI 1640 (Gibco Life Technologies) containing 0.025% DNase (Sigma Chemical Co.). The resulting cell suspension was stained and analyzed by confocal laser-scanning microscopy and flow cytometry or further used for enrichment procedures. Recently, we adapted a method to highly enrich DEL [15
] and dermal leukocytes (data not shown). Using this technique, the purity of the two cell populations from newborn C3H mice was consistently 
90% as determined by flow cytometry. One part of the cells was stained for cytoplasmic and surface marker expression, placed onto adhesion slides, and analyzed by a confocal laser-scanning microscope. Remaining cells were cultured in 96-well round-bottom microtiter plates (1x103/well) in serum-free medium X-VIVO 15 (BioWhittaker, Walkersville, MD) containing 2 mM L-glutamine and 1x antibiotic-antimycotic solution (Gibco Life Technologies). Cultures were supplemented with previously optimized concentrations of the following factors and cytokines: granulocyte macrophage-colony stimulating factor (GM-CSF; 100 ng/ml, Novartis Institutes for Biomedical Research, Vienna, Austria), stem cell factor (SCF; 20 ng/ml, PeproTech EC Ltd., London, UK), fetal liver tyrosine kinase 3 ligand (Flt3L; 100 ng/ml, Immunex Corp., Seattle, WA), and transforming growth factor-ß1 (TGF-ß1; 0.5 ng/ml, Biotec AG, Hannover, Germany). Culture medium was exchanged every second day. After 2 weeks, all cells were harvested and stained for fluorescence analyses (see below). Stained cells were placed on each reaction field of an adhesion slide (Bio-Rad, Richmond, CA) and incubated in a humidified chamber (30 min, room temperature) to allow their sedimentation. Nonattached cells were rinsed off with PBS, and the slides were embedded in fluorescence-mounting fluid (Dako, Vienna, Austria) and analyzed with a confocal laser-scanning microscope. The same procedure was performed for double-stainings of lymph node cells and highly purified LC and DEL.
Immunofluorescence analysis of cells by flow cytometry and confocal laser-scanning microscopy
Cells (1x106/sample) were resuspended in cold PBS/1% FCS/0.1% NaN3 and serially incubated with biotinylated- and FITC-conjugated mAb directed against selected mouse antigens (30 min, 4°C). To block nonspecific binding of mAb, cells were incubated with mouse/goat serum [5% (v/v) in PBS, 20 min, 4°C] prior to the staining procedures. Specificity of staining was confirmed using isotype-matched, control mAb. Dead cells were excluded by 7-amino-actinomycin D (1 µg/ml, Sigma Chemical Co.) uptake. For the detection of cytoplasmic CD207, cells were fixed and permeabilized with a Cytofix/Cytoperm solution (20 min, 4°C, PharMingen), washed in a buffer containing saponin (Perm/Wash buffer, PharMingen), then incubated with the nonconjugated mAb anti-CD207 diluted in Perm/Wash buffer (30 min, 4°C), exposed to FITC-goat anti-rat, and counterstained with a biotinylated, anti-MHC class II mAb (30 min, 4°C) followed by strepavidin-PE. For the detection of cytoplasmatic cyclin B1 in MHC class II+ epidermal cells, cells were stained with the PE-labeled anti-MHC class mAb 11-5.2 (30 min, 4°C), washed, and fixed in 75% ethanol (2 h, –20°C). Ethanol was removed by centrifugation, aspirated, and washed once with wash buffer. The cells were permeabilized with 0.25% Triton X-100 in wash buffer (5 min, 4°C). After three additional washing steps, cells were stained with the FITC-conjugated anticyclin B1 mAb (30 min, 4°C), washed, and resuspended in a propidium iodide (Sigma Chemical Co.) solution for simultaneous analysis of DNA cell-cycle and cyclin B1 expression. Fluorescence was measured with a FACScan flow cytometer, and data were analyzed with Cell Quest software (both from Becton Dickinson, Mountain View, CA). For confocal laser-scanning microscope analyses, cells were incubated with nonconjugated mAb anti-CD207, diluted in Perm/Wash buffer (30 min, 4°C), exposed to an R-PE-conjugated goat anti-rat Ig polyclonal antibody (PharMingen), and counterstained with FITC-labeled anti-CD45 or anti-MHC class II (clone 10-2.16) mAb.
Transplantation studies
Six-week-old female mice (C3H/B6/F1, SCID) were anesthetized intraperitoneally with 100 µl Ketalar (ketamine-hydrochloride; 2 ml of a 50 mg/ml solution, Parke-Davis GesmbH, Vienna, Austria) and Rompun (xylazine-hydrochloride; 800 µl of a 2% solution, Bayer Austria, Vienna; plus 5.2 ml Aqua dest.). The transplantation area on the back was carefully shaved. The graft bed was prepared by excising a full-thickness skin specimen in a size approximately equal to that of the fetal skin grafts (15x15 mm). Full-thickness fetal (E18) skin grafts from female B6 mice consisting of almost the entire integument were freed of subcutaneous fat, and the graft was then placed into the graft bed of adult recipient mice, fixed with surgical glue, and covered with a polyvinyl bandage [17
]. For each transplantation experiment (n=10), we have used one fetal mouse/adult recipient. Fifteen mice were transplanted per experiment. Experiments with successfully engrafted mice were performed at selected time-points after transplantation. Epidermal and dermal sheets from the grafts of three to five transplanted mice were stained for each time-point.
Immunofluorescence and quantitative analysis of epidermal and dermal cell subpopulations
Double-labeling of sheets prepared from grafts before and after transplantation of fetal d18 skin (B6; I-Ab) onto adult (C3H/B6/F1; I-AkI-EkI-Ab) mice
Epidermal and dermal sheets were prepared as described [16
] and incubated with appropriately diluted FITC-conjugated mouse anti-mouse MHC class II mAb (I-Ab, clone KH74; I-Ek, clone 17-3-3) for 16 h at 4°C. After several washes, sheets were counterstained for 1 h at 37°C with APC-labeled anti-CD45 (clone 30F11) mAb or biotinylated mouse anti-mouse MHC class II (I-Ab; clone KH74) mAb and then reacted with Texas red-streptavidin (1 h, 37°C). In some experiments, sheets were incubated with an appropriately diluted FITC-conjugated rat anti-mouse CD45 (clone 30F11) mAb for 16 h at 4°C (data not shown).
Double-labeling of sheets prepared from grafts after transplantation of fetal d18 skin (B6; I-Ab) onto adult SCID (I-AdI-Ed) mice
In the first series of experiments, sheets were incubated with an appropriately diluted FITC-conjugated mouse anti-mouse MHC class II (I-Ad; clone AMS-32.1) mAb (16 h, 4°C). After several washes, sheets were counterstained (1 h, 37°C) with a biotinylated mouse anti-mouse MHC class II (I-Ab; clone KH74) mAb and then reacted with Texas red–streptavidin (1 h, 37°C). In the second series of experiments, sheets were first incubated with the purified rat anti-mouse CD205 mAb (16 h, 4°C), counterstained with a FITC-labeled goat F(ab')2 anti-rat Ig, and then reacted with biotinylated mouse anti-mouse MHC class II (I-Ab; clone KH74) and Texas red-streptavidin (1 h, 37°C). In a third series of experiments, sheets were incubated with purified rat anti-mouse CD207 mAb for 16 h at 4°C and FITC-labeled goat F(ab')2 anti-rat Ig and counterstained with biotinylated mouse anti-mouse MHC class II (I-Ab; clone KH74) mAb and Texas red-streptavidin (1 h, 37°C).
Immunolabeled cells in epidermal sheets were enumerated at 400x magnification using a rectangular grid with 40x oil objective in a conventional immunofluorescence microscope (Leitz Diaplan, Wetzlar, Germany). Forty fields were randomly chosen, and the density of positive cells was determined and expressed as the number of cells (±SD) per mm2 of skin surface.
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Figure 1. DEL do not undergo apoptosis in situ. Epidermal sheets (one sheet/mouse, n=3) from C57BL/6 mice were prepared at the indicated time-points and subjected to the TUNEL technique. For the identification of DEL and LC, sheets were counterstained with APC-labeled anti-CD45 and anti-MHC class II mAb. To induce DNA breaks in the nucleus (=Pos. control), epidermal sheets were incubated with 1500 U/ml DNase I and then subjected to double-labeling. Immunostained sheets were analyzed using a confocal laser-scanning microscope. Results are representative of three independent experiments for each time-point. Original scale bars: 20 µm.
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Figure 2. Generation of LC from DEL in vitro. EC suspensions from newborn and adult C3H mice were treated with anti-Thy-1 and complement and subsequently enriched for DEL and LC, respectively, as described in Materials and Methods. One portion of the cells was immediately stained for the indicated markers and analyzed using a confocal laser-scanning microscope (A); the other portion was seeded at 1 x 103 cells/200 µl in 96-well round-bottom tissue-culture plates in serum-free medium in the presence of GM-CSF, SCF, Flt3L, and TGF-ß1. (B) Clusters of dendritically shaped cells first appeared after 7 days of culture. Several dividing cells (inset) were observed. After 2 weeks, total cells were harvested from the suspension culture and analyzed for their anti-CD45, -MHC class II, and -CD207 immunoreactivity (C, D). Isotype-matched control antibodies consistently gave negative results (data not shown). CD45+ cells consisted of highly dendritic and round cells with a fuzzy surface. Cytoplasmic anti-CD207 reactivity was detected in both cell types. The dendrites are only faintly visualized because of the cytoplasmic (and predominantly perinuclear) location of CD207. Shown is one representative of five independent experiments with similar results. Original scale bars: 20 µm.
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Figure 3. DEL are dividing in situ. Immunostaining for cyclin B1 and subsequent analysis by flow cytometry were performed as described in Materials and Methods. Cells (2x104) were acquired per sample, and bars represent the mean ± SD of three experiments for each time-point. 20–35% of all MHC class II+ cells in neonatal epidermis express the cyclin B1 protein, indicative of G2/M phases of the cell cycle. There is a sharp decline of cyclin B1+MHC class II+ cells in adult epidermis. NB, Newborn.
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2 months). Similar data were obtained when we transplanted d18 fetal C3H (I-Ek) mouse skin onto adult C57BL/6 SCID (I-Ab) mice. Thus, examination of epidermal sheets from the grafts 3 days after transplantation revealed a few leukocytes of donor (I-Ek: 56 cells/mm2) and recipient (I-Ab: 24 cells/mm2) origin. One to 2 weeks post-grafting, the density of both MHC class II populations, considerably increased (I-Ek: 1594 cells/mm2; I-Ab: 1523 cells/mm2). From these results, we can conclude that the fetal skin contains LC precursors, expressing their potential in vivo in adult mice.
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Figure 4. Transient appearance of donor-type dendritic MHC class II+ cells in d18 fetal skin (B6; I-Ab) specimens grafted onto adult C3H/B6/F1 (I-AkI-EkI-Ab) recipients. (A) At the time of transplantation (day 0), epidermal and dermal sheets from fetal mice on d18 of gestation (E18) were exposed to FITC-anti-MHC class II (I-Ab)/APC-anti-CD45 double-labeling. The epidermis contained a considerable number of CD45+ DC, which failed to express MHC class II antigens. In contrast, the dermis harbored abundant numbers of round-shaped CD45+ cells, most of which coexpress MHC class II molecules. (B) Three days after transplantation (day 3), epidermal and dermal sheets were exposed to FITC-anti-MHC II (I-Ek)/biotinylated anti-MHC II (I-Ab)-Texas red-streptavidin double-labeling. Confocal microscopy revealed few scattered recipient-type (I-EkI-Ab) but no donor-type MHC class II+ (I-Ab), single-positive cells in the epidermis. In the dermis, most of the MHC class II+ cells were double-positive. Occasionally, donor-type (I-Ab), single-positive cells were visible (insets, arrows). (C) Seven days after transplantation (day 7), we encountered donor (I-Ab) cells (arrows) with one or two dendrites and recipient double-MHC class II+ (I-AbI-Ek) cells in the epidermis. (D) Four weeks after transplantation (day 28), only cells of recipient origin were visible in the epidermis and the dermis. One representative of five experiments is shown. Original scale bars: 20 µm.
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Figure 5. Appearance and persistence of donor LC in d18 fetal skin (B6) grafted onto adult C.B-17-SCID (I-AdI-Ed) mice. (A) Fourteen days after transplantation (day 14), epidermal sheets were exposed to FITC-anti-MHC class II (I-Ad)/biotinylated anti-MHC class II (I-Ab)-Texas red–streptavidin double-labeling. Two exclusive populations of donor and recipient cells were observed. (B) Twenty-eight days after transplantation (day 28), we found changes in donor cells, as evidenced by an increase in cellularity and development of a polygonal cell body with several, slender dendrites protruding from its surface. When epidermal sheets were stained with FITC-anti-CD207/biotinylated anti-MHC class II (I-Ab)-Texas red-strepavidin or FITC-anti-CD205/biotinylated anti-MHC class II (I-Ab)-Texas red-streptavidin, we found that donor cells (I-Ab) have matured into LC given by reactivity with anti-CD205 and anti-CD207 mAb. One representative of five experiments is shown. Original scale bars: 20 µm.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumor necrosis factor 
(TNF-) is known to initiate apoptosis in multiple cell types including keratinocytes [25
, 26
]. As neonatal epidermal cells produce significantly more TNF- than adult epidermal cells [15
], we reasoned that DEL may undergo apoptosis and are subsequently replaced by LC precursors from the bone marrow in the neonatal period. To test this hypothesis, fetal and neonatal epidermis was screened for the presence of apoptotic leukocytes. At no time-point of investigation do DEL undergo apoptotic cell death. To test whether DEL can give rise to cells with LC features, we have used a defined, serum-free culture system, described previously for the generation of human LC from CD34+ precursors [19
]. In the DEL cultures, supplemented with GM-CSF, SCF, Flt3L, and TGF-ß1, LC outgrowth could already be detected morphologically. Colonies were composed of underlying cells that loosely adhered to plastic and attached to overlying clustered cells, most of which expressed MHC class II. In all of these colonies, the majority of the cells was CD207+. Serum-free culture of DEL thus allows the outgrowth of cells with LC features. Morphologically, these colonies resemble previously described DC clusters generated from murine bone marrow [27
] and are reminiscent of mixed monocyte-DC clusters obtained in semisolid, serum-containing cultures of human CD34+ progenitors [28
]. In our cultures, differentiation into LC only occurred when DEL were highly enriched, the culture medium was replaced every second day, and TGF-ß1 was present. The influence of TGF-ß1 on LC development has been studied in recent years by several investigators [29
, 30
]. In vitro studies have demonstrated that TGF-ß combined with other hematopoietic growth factors can induce LC from various hematopoietic progenitor/precursor cells [19
, 31
32
33
34
35
], monocytes [36
], peripheral blood CD1a+CD11c+ [37
], and dermal CD14+ cells [38
]. Previous studies also showed that SCF and Flt3L potently synergize with GM-CSF to promote the expansion of DC [19
, 39
, 40
]. The amplification efficiency by these factors in our cultures remains to be investigated.
An important result of this study was the demonstration that 20–35% of all MHC class II+ neonatal DEL were in G2/M phase of the cell cycle, indicating a high turning over of LC precursors in neonatal epidermis. Our data demonstrating that approximately 5% of LC in adult epidermis is in the cell cycle are consistent with results by other investigators, showing that LC divide in the skin [6 , 41 42 43 44 45 ]. Thus, our results concur with the supposition that the younger the organism is, the more proliferative potential it will display. Similar observations have been reported for epidermal stem cells [46 ].
Our observation that DEL can give rise to LC in vitro seems to contrast with our previous studies, which showed that highly purified, newborn DEL, cultured in the presence of GM-CSF alone, do not acquire LC morphology, phenotype, or function and that autocrine interleukin (IL)-10 secretion partly inhibits the differentiation and maturation of DEL into LC [15 ]. A critical difference from our former work is that we have modified our culture conditions. In this study, we have used a serum-free medium and a different cytokine cocktail. The mechanism(s) accounting for neutralization of the suppressive effect of IL-10 in our cultures are not known. A possible explanation is that as a result of continual medium change, the usually high amounts of IL-10 were low and biologically ineffective in our cultures. As a result of the constant medium replacement, the activity of other suppressive factors may also be diminished. Finally, one or more of the cytokines added to the cultures may have a dominant effect over IL-10 and allow DEL to undergo differentiation.
We have shown that transplantation of fetal skin onto adult immunocompetent F1 mice leads to the appearance of dendritic MHC class II+ donor cells, which are often found in clusters. However, already after 2–3 weeks, these cells completely disappeared from the epidermis and were replaced by dendritic MHC class II+ recipient cells. The nature and fate of the MHC class II+ donor cells that disappeared from the epidermis require further investigation. Reasoning that in SCID mice, emigration of MHC class II+ donor cells from the epidermis would be diminished substantially, as their lymph nodes are very small, and induction of proinflammatory cytokines (as a result of surgical trauma associated with grafting) in SCID mice is less severe compared with F1 mice, we transplanted fetal skin onto adult SCID mice. Similar to the previous model, MHC class II+ donor cells emerged in the epidermis. Further characterization of their nature revealed that they acquired CD205 and CD207 molecules, thus qualifying them as LC. In timed kinetics experiments, we found that some LC of graft origin remained detectable in the epidermis for more than 2 months. The latter observation is in accordance with previous findings, which showed in different transplantation models, that donor LC persist for long periods in the epidermis of the grafts and are not completely replaced by recipient LC in long-tolerated grafts [47
48
49
50
51
]. In our system where no circulating LC precursor cells are available for repopulation, the quantitative increase of donor LC in the grafts soon after transplantation strongly suggests that they are able to proliferate in situ. Our observation that DEL have strong proliferative capacity in vitro and in vivo is in favor of this hypothesis. Using different techniques, other investigators have also shown that LC undergo cell division in situ [6
, 41
42
43
44
45
]. Our further findings that dermal MHC class II+ donor cells preferentially emigrate from the skin to regional lymph nodes and not to the epidermis and that fetal dermal cells, highly enriched for CD45 (98%) and cultured under conditions where DEL gave rise to LC, never differentiated into LC (data not shown) strongly suggest that donor LC are derived from skin precursors, presumably from epidermal precursors. Whether recipient LC in our grafts are recruited from the surrounding skin or from blood precursors is not clear. Holzmann et al. [7
] have recently shown that newly immigrating cells into tape-stripped epidermis are MHC class II-positive and -negative, express CC chemokine receptor 6, and do not express CD207. As in our transplantation model, we cannot investigate the phenotype of cells entering the grafts until they are MHC class II+, other mouse models have to be used to address this point.
The failure of DEL to differentiate into LC in the fetal skin is incompletely understood. In a previous work, we have shown that IL-10 partially inhibits their differentiation and demonstrate here that factors such as TGF-ß1 are necessary for this process. In line with the latter observation are studies showing reduced expression of TGF-ß receptors and their ligands in fetal skin compared with adults [52 ]. In addition, so far, unknown suppressive factors may be present in fetal skin, which disappear when the skin differentiates.
We have reported that IL-10 precludes LC precursors from acquiring their potential as initiators of adaptive immunity [15 ]. It is interesting that it has recently been shown that IL-10 enhances the ability of DC to act as initiators of innate immunity in response to Toll-like receptor signaling [53 ]. Whether LC precursors have a similar function in the neonatal period remains to be investigated. In addition, it has recently been described that neonatal skin in mice and humans expresses increased levels of antimicrobial peptides, which may provide a compensatory, innate defense mechanism during the establishment of cellular immune-response capacity in the newborn period [54 ].
Collectively, we have shown that a pool of hematopoietic precursors seeds the skin during embryonic life and can give rise to LC. In light of the data presented here and according to a previously published study [6 ], it is therefore tempting to speculate that these precursors are able to compensate for the low LC loss that occurs during steady-state turnover and minor injuries. We conclude that understanding the mechanism of LC development might be crucial to our knowledge of the immune status of the newborn mouse and for the design of new vaccine strategies in humans.
Received October 13, 2004; revised November 10, 2004; accepted November 11, 2004.
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