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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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Skin T cells can be identified at midgestation. (a) Multiparameter flow cytometric analysis was performed after incubation with mAbs against the cell surface markers indicated. Gates in dot plots were set according to isotype-matched control staining. Dead cells were excluded by 7-amino-actinomycin-D uptake. Dot plots are representative of 5–8 experiments. (b) Immunofluorescence double staining was performed on cryostat sections of embryonic, fetal, and adult skin. One of at least three experiments per group is shown. Arrows denote CD45+CD3+ T cells. (c) Graphs show an increase of T-cell numbers in developing prenatal skin analyzed by flow cytometry (n=5–8). (d) Single cell suspensions of embryonic, fetal, and adult skin were cultured for 48 hours, supernatants were harvested, and CCL27 levels were analyzed by ELISA. Bars represent the mean. (e) Immunohistochemical staining was performed on cryostat sections of fetal and adult skin, and CCL27 binding was visualized using amino-ethyl-carbazole. Bars=50 μm. EGA, estimated gestational age.
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fig4: Skin T cells can be identified at midgestation. (a) Multiparameter flow cytometric analysis was performed after incubation with mAbs against the cell surface markers indicated. Gates in dot plots were set according to isotype-matched control staining. Dead cells were excluded by 7-amino-actinomycin-D uptake. Dot plots are representative of 5–8 experiments. (b) Immunofluorescence double staining was performed on cryostat sections of embryonic, fetal, and adult skin. One of at least three experiments per group is shown. Arrows denote CD45+CD3+ T cells. (c) Graphs show an increase of T-cell numbers in developing prenatal skin analyzed by flow cytometry (n=5–8). (d) Single cell suspensions of embryonic, fetal, and adult skin were cultured for 48 hours, supernatants were harvested, and CCL27 levels were analyzed by ELISA. Bars represent the mean. (e) Immunohistochemical staining was performed on cryostat sections of fetal and adult skin, and CCL27 binding was visualized using amino-ethyl-carbazole. Bars=50 μm. EGA, estimated gestational age.

Mentions: CD45+CD3+ T cells were not detectable in skin during the first trimester in six out of eight samples (Figure 4a-c), whereas two samples (11 and 14 weeks EGA) show a minute population, comprising 0.01% of total skin single cell suspensions. In these embryonic samples, CD3 was rarely found by immunofluorescence and seemed to be expressed only intracellulary (Figure 4b). During midgestation, a moderate increase in T cells takes place, but numbers are approximately 15- to 20-fold lower than in adult skin. CD3+CD45+ cells are exclusively found in the dermis during skin development.


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)

Skin T cells can be identified at midgestation. (a) Multiparameter flow cytometric analysis was performed after incubation with mAbs against the cell surface markers indicated. Gates in dot plots were set according to isotype-matched control staining. Dead cells were excluded by 7-amino-actinomycin-D uptake. Dot plots are representative of 5–8 experiments. (b) Immunofluorescence double staining was performed on cryostat sections of embryonic, fetal, and adult skin. One of at least three experiments per group is shown. Arrows denote CD45+CD3+ T cells. (c) Graphs show an increase of T-cell numbers in developing prenatal skin analyzed by flow cytometry (n=5–8). (d) Single cell suspensions of embryonic, fetal, and adult skin were cultured for 48 hours, supernatants were harvested, and CCL27 levels were analyzed by ELISA. Bars represent the mean. (e) Immunohistochemical staining was performed on cryostat sections of fetal and adult skin, and CCL27 binding was visualized using amino-ethyl-carbazole. Bars=50 μm. EGA, estimated gestational age.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3472563&req=5

fig4: Skin T cells can be identified at midgestation. (a) Multiparameter flow cytometric analysis was performed after incubation with mAbs against the cell surface markers indicated. Gates in dot plots were set according to isotype-matched control staining. Dead cells were excluded by 7-amino-actinomycin-D uptake. Dot plots are representative of 5–8 experiments. (b) Immunofluorescence double staining was performed on cryostat sections of embryonic, fetal, and adult skin. One of at least three experiments per group is shown. Arrows denote CD45+CD3+ T cells. (c) Graphs show an increase of T-cell numbers in developing prenatal skin analyzed by flow cytometry (n=5–8). (d) Single cell suspensions of embryonic, fetal, and adult skin were cultured for 48 hours, supernatants were harvested, and CCL27 levels were analyzed by ELISA. Bars represent the mean. (e) Immunohistochemical staining was performed on cryostat sections of fetal and adult skin, and CCL27 binding was visualized using amino-ethyl-carbazole. Bars=50 μm. EGA, estimated gestational age.
Mentions: CD45+CD3+ T cells were not detectable in skin during the first trimester in six out of eight samples (Figure 4a-c), whereas two samples (11 and 14 weeks EGA) show a minute population, comprising 0.01% of total skin single cell suspensions. In these embryonic samples, CD3 was rarely found by immunofluorescence and seemed to be expressed only intracellulary (Figure 4b). During midgestation, a moderate increase in T cells takes place, but numbers are approximately 15- to 20-fold lower than in adult skin. CD3+CD45+ cells are exclusively found in the dermis during skin development.

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