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Phenotypic characterization of leukocytes in prenatal human dermis.

Schuster C, Vaculik C, Prior M, Fiala C, Mildner M, Eppel W, Stingl G, Elbe-Bürger A - J. Invest. Dermatol. (2012)

Bottom Line: 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).During midgestation, CD3(+)FoxP3(-) and CD3(+)FoxP3(+) cells can exclusively be found in the dermis.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria.

ABSTRACT
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.

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Mast cells appear during the first trimester but are not fully mature at midgestation. (a) Flow cytometric analysis of freshly isolated single cell suspensions of embryonic, fetal, and adult skin revealed the presence of immature mast cells in fetal skin. Shown are representative dot plots of five experiments per group. (b) Graphs show the increase in mast cell numbers of developing skin analyzed by flow cytometry. Five specimens were investigated per age group. Bars represent the mean of investigated groups. (c) The size, granularity, and numbers of CD45+CD117+ cells (red dots) and CD45+CD3+ cells (green dots) between fetal and adult skin are compared. (d, e) Toluidin blue (d) and chymase (e) stainings were performed on cryostat sections of embryonic, fetal, and adult skin (left panel). Arrows denote toluidine blue-expressing cells in fetal dermis. Bar=100 μm. The numbers of metachromatic cells (d, right panel) and chymase+ cells (e, right panel) were determined and bars represent the mean of investigated groups (n=5–6 per group). EGA, estimated gestational age; FCS, forward scatter; SSC, side scatter.
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fig6: Mast cells appear during the first trimester but are not fully mature at midgestation. (a) Flow cytometric analysis of freshly isolated single cell suspensions of embryonic, fetal, and adult skin revealed the presence of immature mast cells in fetal skin. Shown are representative dot plots of five experiments per group. (b) Graphs show the increase in mast cell numbers of developing skin analyzed by flow cytometry. Five specimens were investigated per age group. Bars represent the mean of investigated groups. (c) The size, granularity, and numbers of CD45+CD117+ cells (red dots) and CD45+CD3+ cells (green dots) between fetal and adult skin are compared. (d, e) Toluidin blue (d) and chymase (e) stainings were performed on cryostat sections of embryonic, fetal, and adult skin (left panel). Arrows denote toluidine blue-expressing cells in fetal dermis. Bar=100 μm. The numbers of metachromatic cells (d, right panel) and chymase+ cells (e, right panel) were determined and bars represent the mean of investigated groups (n=5–6 per group). EGA, estimated gestational age; FCS, forward scatter; SSC, side scatter.

Mentions: By flow cytometry, CD45+CD117+ mast cells are not detectable in human prenatal skin until 11 weeks EGA (Figure 6a and b). Coinciding with the beginning of bone marrow hematopoiesis a minute, though distinct, population of mast cells can be first identified at 12–14 weeks EGA. The frequency of CD45+CD117+ cells increases considerably during the second trimester, occasionally reaching adult-like levels (Figure 6a and b). A comparison of the scatter profiles of fetal and adult CD45+CD117+ skin mast cells reveals a substantially weaker granularity of fetal than adult mast cells (Figure 6c, red dots), indicating immaturity of fetal mast cells due to the lack of specific granules. By contrast, CD45+CD3+ T cells in fetal and adult skin have a similar forward and side scatter profile, but significantly differ in numbers (Figure 6c, green dots). In line with the suspected mast cell immaturity, we could not detect mast cells by their metachromatic staining with toluidin blue up to 14 weeks EGA (Figure 6d). Toluidin blue–expressing cells were first identified in the second trimester, but their numbers were approximately 8-fold lower than in adult skin. Similar to adult skin, these cells are preferentially found around vessels and in vicinity to skin appendages. Mature, chymase-expressing mast cells are rarely found in fetal skin (Figure 6e), but can be easily identified in adult skin. The ratio of toluidin blue/chymase-positive cells changes from 6.4:1 in fetal skin to 0.9:1 in adult skin.


Phenotypic characterization of leukocytes in prenatal human dermis.

Schuster C, Vaculik C, Prior M, Fiala C, Mildner M, Eppel W, Stingl G, Elbe-Bürger A - J. Invest. Dermatol. (2012)

Mast cells appear during the first trimester but are not fully mature at midgestation. (a) Flow cytometric analysis of freshly isolated single cell suspensions of embryonic, fetal, and adult skin revealed the presence of immature mast cells in fetal skin. Shown are representative dot plots of five experiments per group. (b) Graphs show the increase in mast cell numbers of developing skin analyzed by flow cytometry. Five specimens were investigated per age group. Bars represent the mean of investigated groups. (c) The size, granularity, and numbers of CD45+CD117+ cells (red dots) and CD45+CD3+ cells (green dots) between fetal and adult skin are compared. (d, e) Toluidin blue (d) and chymase (e) stainings were performed on cryostat sections of embryonic, fetal, and adult skin (left panel). Arrows denote toluidine blue-expressing cells in fetal dermis. Bar=100 μm. The numbers of metachromatic cells (d, right panel) and chymase+ cells (e, right panel) were determined and bars represent the mean of investigated groups (n=5–6 per group). EGA, estimated gestational age; FCS, forward scatter; SSC, side scatter.
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fig6: Mast cells appear during the first trimester but are not fully mature at midgestation. (a) Flow cytometric analysis of freshly isolated single cell suspensions of embryonic, fetal, and adult skin revealed the presence of immature mast cells in fetal skin. Shown are representative dot plots of five experiments per group. (b) Graphs show the increase in mast cell numbers of developing skin analyzed by flow cytometry. Five specimens were investigated per age group. Bars represent the mean of investigated groups. (c) The size, granularity, and numbers of CD45+CD117+ cells (red dots) and CD45+CD3+ cells (green dots) between fetal and adult skin are compared. (d, e) Toluidin blue (d) and chymase (e) stainings were performed on cryostat sections of embryonic, fetal, and adult skin (left panel). Arrows denote toluidine blue-expressing cells in fetal dermis. Bar=100 μm. The numbers of metachromatic cells (d, right panel) and chymase+ cells (e, right panel) were determined and bars represent the mean of investigated groups (n=5–6 per group). EGA, estimated gestational age; FCS, forward scatter; SSC, side scatter.
Mentions: By flow cytometry, CD45+CD117+ mast cells are not detectable in human prenatal skin until 11 weeks EGA (Figure 6a and b). Coinciding with the beginning of bone marrow hematopoiesis a minute, though distinct, population of mast cells can be first identified at 12–14 weeks EGA. The frequency of CD45+CD117+ cells increases considerably during the second trimester, occasionally reaching adult-like levels (Figure 6a and b). A comparison of the scatter profiles of fetal and adult CD45+CD117+ skin mast cells reveals a substantially weaker granularity of fetal than adult mast cells (Figure 6c, red dots), indicating immaturity of fetal mast cells due to the lack of specific granules. By contrast, CD45+CD3+ T cells in fetal and adult skin have a similar forward and side scatter profile, but significantly differ in numbers (Figure 6c, green dots). In line with the suspected mast cell immaturity, we could not detect mast cells by their metachromatic staining with toluidin blue up to 14 weeks EGA (Figure 6d). Toluidin blue–expressing cells were first identified in the second trimester, but their numbers were approximately 8-fold lower than in adult skin. Similar to adult skin, these cells are preferentially found around vessels and in vicinity to skin appendages. Mature, chymase-expressing mast cells are rarely found in fetal skin (Figure 6e), but can be easily identified in adult skin. The ratio of toluidin blue/chymase-positive cells changes from 6.4:1 in fetal skin to 0.9:1 in adult skin.

Bottom Line: 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).During midgestation, CD3(+)FoxP3(-) and CD3(+)FoxP3(+) cells can exclusively be found in the dermis.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria.

ABSTRACT
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.

Show MeSH