Adequate numbers and functional maturity are needed for leukocytes to exhibit a protective role in host defense. During intrauterine life, the skin immune system has to acquire these prerequisites to protect the newborn from infection in the hostile external environment after birth. We investigated the quantitative, phenotypic, and functional development of skin leukocytes and analyzed the factors controlling their proliferation and trafficking during skin development. We show that CD45+ leukocytes are scattered in embryonic human skin and that their numbers continuously increase as the developing skin generates an environment that promotes proliferation of skin resident leukocytes as well as the influx of leukocytes from the circulation. We also found that CD45+HLA-DRhighCD1c+ dendritic cells (DCs) are already present in the epidermis and dermis at 9 wk estimated gestational age (EGA) and that transforming growth factor β1 production precedes Langerin and CD1a expression on CD45+CD1c+ Langerhans cell (LC) precursors. Functionally, embryonic antigen-presenting cells (APCs) are able to phagocytose antigen, to up-regulate costimulatory molecules upon culture, and to efficiently stimulate T cells in a mixed lymphocyte reaction. Collectively, our data provide insight into skin DC biology and the mechanisms through which skin DCs presumably populate the skin during development.

Multipotent mesenchymal stromal cells (MSCs) are found in a variety of adult tissues including human dermis. These MSCs are morphologically similar to bone marrow–derived MSCs, but are of unclear phenotype. To shed light on the characteristics of human dermal MSCs, this study was designed to identify and isolate dermal MSCs by a specific marker expression profile, and subsequently rate their mesenchymal differentiation potential. Immunohistochemical staining showed that MSC markers CD73/CD90/CD105, as well as CD271 and SSEA-4, are expressed on dermal cells in situ. Flow cytometric analysis revealed a phenotype similar to bone marrow–derived MSCs. Human dermal cells isolated by plastic adherence had a lower differentiation capacity as compared with bone marrow–derived MSCs. To distinguish dermal MSCs from differentiated fibroblasts, we immunoselected CD271+ and SSEA-4+ cells from adherent dermal cells and investigated their mesenchymal differentiation capacity. This revealed that cells with increased adipogenic, osteogenic, and chondrogenic potential were enriched in the dermal CD271+ population. The differentiation potential of dermal SSEA-4+ cells, in contrast, appeared to be limited to adipogenesis. These results indicate that specific cell populations with variable mesenchymal differentiation potential can be isolated from human dermis. Moreover, we identified three different subsets of dermal mesenchymal progenitor cells.

The adult human skin harbors a variety of leukocytes providing immune surveillance and host defense, but knowledge about their ontogeny is scarce. In this study we investigated the number and phenotype of leukocytes in prenatal human skin (dermal dendritic cells (DDCs), macrophages, T cells (including FoxP3 + regulatory T cells), and mast cells) to unravel their derivation and to get a clue as to their putative function in utero . By flow cytometry and immunofluorescence, we found a distinction between CD206 + CD1c + CD11c + DDCs and CD206 + CD209 + CD1c − skin macrophages by 9 weeks estimated gestational age (EGA). T cells appear at the end of the first trimester, expressing CD3 intracytoplasmatically. During midgestation, CD3 + FoxP3 − and CD3 + FoxP3 + cells can exclusively be found in the dermis. Similarly, other leukocytes such as CD117 + (c-kit) mast cells were not identified before 12–14 weeks EGA and only slowly acquire a mature phenotype during gestation. Our data show at which time point during gestation antigen-presenting cells, T cells, and mast cells populate the human dermis and provide a step forward to a better understanding of the development of the human skin immune system.

IntroductionTwo in vitro systems for the generation of human dendritic cells (DCs) are widely used for basic and clinically oriented research: monocyte-derived DCs (moDCs) and hematopoietic progenitor cell (HPC)-derived DCs. In the first system, granulocytemacrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4) are added to peripheral blood monocytes to generate moDCs. 1 This system is most widely applied for generating human DCs. In the second model, CD34 ϩ HPCs are stimulated in transforming growth factor ␤1 (TGF-␤1)-supplemented serumfree cultures (containing basic DC cytokines GM-CSF, tumor necrosis factor ␣ [TNF␣], stem cell factor [SCF] with or without Flt3L) for generating Langerhans cells (LCs), a DC subtype that resides within epithelial tissue. 2,3 Exclusion of TGF-␤1 from this latter culture system abrogates LC differentiation and de-represses a program of monocyte generation from CD34 ϩ cells. 3 When these latter HPC cultures are initiated in the presence of serum without TGF-␤1, 2 pathways of CD1a ϩ DCs are simultaneously generated, ie LCs and non-LC interstitial/dermal-type DCs. 4 These populations arise from 2 separate precursor pathways at culture day 5. A CD1a ϩ CD14 Ϫ precursor gives rise to LCs, whereas a CD1a Ϫ CD14 ϩ precursor can give rise to non-LC DCs, which share many features with moDCs generated from peripheral blood monocytes. 4,5 This CD1a Ϫ CD14 ϩ intermediate population can be further subdivided into 2 functional subsets based on CD11b (CD11b ϩ CD14 ϩ versus CD11b Ϫ CD14 ϩ monocytic cells). CD11b ϩ CD14 ϩ cells can be induced by GM-CSF plus IL-4 to develop into intDC/moDCs or by M-CSF to macrophage differentiation. Conversely, CD11b Ϫ CD14 ϩ intermediates, which represent early monocytic cells, retain LC differentiation capacity in the presence of TGF-␤1. 6 Therefore, a CD14 ϩ monocytic precursor can be induced to develop along 2 separate pathways in vitro depending on IL-4 (moDCs) versus TGF-␤1 (LCs) cytokine signals.IL-4 versus TGF-␤1 antagonize each other's function to induce moDCs versus LCs. Neutralizing anti-TGF-␤1 mAb represses LC generation in favor of CD14 ϩ monocyte generation. 7 Similarly, the addition of IL-4 to TGF-␤1-supplemented LC generation cultures of CD34 ϩ cells represses LC differentiation in favor of inducing non-LC DC differentiation. 7 Conversely, the addition of TGF-␤1 to GM-CSF plus IL-4-containing moDC cultures of peripheral blood monocytes polarizes these cells toward an LC-like phenotype. 8 In line with this, TGF-␤1 in the epidermal microenvironment is critical for LC differentiation in vivo. [9][10][11] Functional differences between moDCs versus LCs are increasingly recognized. However, very little is known about the molecular mechanisms controlling the lineage commitment of these cells to develop into different DC subsets (ie, LCs and non-LC moDCs).We recently performed mechanistic studies on the involvement of family members of the nuclear hormone receptor system, ie, retinoid X receptor-alpha (RXR␣) and vitamin D 3 receptor (VDR) i...

Atopic dermatitis arises primarily in early infancy. In these patients, corticosteroids are used especially with great caution because of their side effects. Calcineurin inhibitors such as pimecrolimus (PIM) could be useful, but safety concerns have been raised in particular because of the lack of knowledge about their effects on the developing skin immune system. This study was designed to investigate the impact of PIM and corticosteroids on epidermal cells (EC) in infants and newborn mice. We found that the percentage of unfractionated viable infant ECs was significantly decreased in the presence of beta-methasone-17-valerate (BMV) but not PIM. Exposure of unfractionated infant ECs to BMV but not to PIM and vehicle control caused a significant inhibition of the upregulation of CD86 molecules on Langerhans cells (LC). The release of cytokines by LCs and ECs, cultured in the presence of BMV and PIM, was not significantly reduced compared with controls. Topical corticosteroid but not PIM application onto newborn mice induced apoptosis in some LC precursors. Our data suggest that similar to the situation in adult skin, corticosteroids may impair LC maturation as well as viability of ECs in infants, effects not seen with PIM.