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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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CD11c+ leukocytes are present throughout skin development. (a, b) Multiparameter flow cytometry of freshly isolated single cell suspensions of embryonic, fetal, and adult skin was performed by 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 3–6 experiments. (b) CD45+HLA-DRhigh (R1), CD45+HLA-DRweak (R2), and CD45+HLA-DR− (R3) cells from the indicated age groups are compared with regard to expression of CD11c and CD1c (lower panels). EGA, estimated gestational age.
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fig3: CD11c+ leukocytes are present throughout skin development. (a, b) Multiparameter flow cytometry of freshly isolated single cell suspensions of embryonic, fetal, and adult skin was performed by 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 3–6 experiments. (b) CD45+HLA-DRhigh (R1), CD45+HLA-DRweak (R2), and CD45+HLA-DR− (R3) cells from the indicated age groups are compared with regard to expression of CD11c and CD1c (lower panels). EGA, estimated gestational age.

Mentions: To assess whether macrophages in prenatal human dermis express important lineage molecules, skin of defined gestational age groups and, in parallel, of adults was immunostained for CD45, CD206, and CD209. Similar to adult skin, CD45+CD206+CD209+ cells (Figure 1, arrows), most probably macrophages, as well as CD45+CD206+CD209− cells (Figure 1, arrrowheads) are identified already in embryonic dermis. The staining intensity of CD206 and CD209 in embryonic skin is mostly comparable to adult skin, yet some cells show only weak expression (compare Figure 2, left column, arrowhead), implying that the upregulation of these markers occurs in the skin. Of note, CD206 staining of linear structures, most likely blood vessels, is observed occasionally in prenatal but not in adult skin (Figure 1, middle column, double arrow). Epidermal CD45+ cells at all age groups investigated lack CD206 and CD209. To evaluate whether CD1c and CD209 are mutually exclusive, triple immunofluorescence staining was performed. We found virtually nonoverlapping populations of CD206+CD1c+ DDCs (Figure 2, arrowhead) and CD206+CD209+ macrophages (arrow) already in prenatal dermis, similar to what has been reported in adult dermis, with the exception that CD1c+ cells more often lack or weakly express CD206. Statistical analysis revealed that the frequency of CD1c+ leukocytes significantly increases with gestational age (Figure 2b) and that CD209+ cells outnumber CD1c+ cells between 9 and 14 weeks EGA, reaching adult-like ratios already by midgestation (Figure 2c). By flow cytometry, we found that in all age groups investigated CD11c+ cells are detectable and that CD11c expression is restricted to CD45+ leukocytes (Figure 3a). In depth analysis of HLA-DRhighCD45+ cells reveals that CD1c+CD11c+ cells are already present in the developing skin. The HLA-DRhighCD45+ population contains a similar percentage of CD1c−CD11c+ cells but, in contrast to adult skin, a higher percentage of CD1c−CD11c− cells (Figure 3b, R1). In addition, HLA-DRlow/negCD45+ cells express CD11c to a similar extent at all time points investigated (Figure 3b, R2 and R3).


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)

CD11c+ leukocytes are present throughout skin development. (a, b) Multiparameter flow cytometry of freshly isolated single cell suspensions of embryonic, fetal, and adult skin was performed by 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 3–6 experiments. (b) CD45+HLA-DRhigh (R1), CD45+HLA-DRweak (R2), and CD45+HLA-DR− (R3) cells from the indicated age groups are compared with regard to expression of CD11c and CD1c (lower panels). EGA, estimated gestational age.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472563&req=5

fig3: CD11c+ leukocytes are present throughout skin development. (a, b) Multiparameter flow cytometry of freshly isolated single cell suspensions of embryonic, fetal, and adult skin was performed by 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 3–6 experiments. (b) CD45+HLA-DRhigh (R1), CD45+HLA-DRweak (R2), and CD45+HLA-DR− (R3) cells from the indicated age groups are compared with regard to expression of CD11c and CD1c (lower panels). EGA, estimated gestational age.
Mentions: To assess whether macrophages in prenatal human dermis express important lineage molecules, skin of defined gestational age groups and, in parallel, of adults was immunostained for CD45, CD206, and CD209. Similar to adult skin, CD45+CD206+CD209+ cells (Figure 1, arrows), most probably macrophages, as well as CD45+CD206+CD209− cells (Figure 1, arrrowheads) are identified already in embryonic dermis. The staining intensity of CD206 and CD209 in embryonic skin is mostly comparable to adult skin, yet some cells show only weak expression (compare Figure 2, left column, arrowhead), implying that the upregulation of these markers occurs in the skin. Of note, CD206 staining of linear structures, most likely blood vessels, is observed occasionally in prenatal but not in adult skin (Figure 1, middle column, double arrow). Epidermal CD45+ cells at all age groups investigated lack CD206 and CD209. To evaluate whether CD1c and CD209 are mutually exclusive, triple immunofluorescence staining was performed. We found virtually nonoverlapping populations of CD206+CD1c+ DDCs (Figure 2, arrowhead) and CD206+CD209+ macrophages (arrow) already in prenatal dermis, similar to what has been reported in adult dermis, with the exception that CD1c+ cells more often lack or weakly express CD206. Statistical analysis revealed that the frequency of CD1c+ leukocytes significantly increases with gestational age (Figure 2b) and that CD209+ cells outnumber CD1c+ cells between 9 and 14 weeks EGA, reaching adult-like ratios already by midgestation (Figure 2c). By flow cytometry, we found that in all age groups investigated CD11c+ cells are detectable and that CD11c expression is restricted to CD45+ leukocytes (Figure 3a). In depth analysis of HLA-DRhighCD45+ cells reveals that CD1c+CD11c+ cells are already present in the developing skin. The HLA-DRhighCD45+ population contains a similar percentage of CD1c−CD11c+ cells but, in contrast to adult skin, a higher percentage of CD1c−CD11c− cells (Figure 3b, R1). In addition, HLA-DRlow/negCD45+ cells express CD11c to a similar extent at all time points investigated (Figure 3b, R2 and R3).

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