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HLA-DR+ leukocytes acquire CD1 antigens in embryonic and fetal human skin and contain functional antigen-presenting cells.

Schuster C, Vaculik C, Fiala C, Meindl S, Brandt O, Imhof M, Stingl G, Eppel W, Elbe-Bürger A - J. Exp. Med. (2009)

Bottom Line: We also found that CD45(+)HLA-DR(high)CD1c(+) dendritic cells (DCs) are already present in the epidermis and dermis at 9 wk estimated gestational age (EGA) and that transforming growth factor beta1 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, DIAID, 1090 Vienna, Austria.

ABSTRACT
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-DR(high)CD1c(+) dendritic cells (DCs) are already present in the epidermis and dermis at 9 wk estimated gestational age (EGA) and that transforming growth factor beta1 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.

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Proliferating CD45+ cells contribute to the increase of leukocyte numbers in developing skin. Dot graphs show the increase of relative (A) and absolute (B) numbers of CD45+ leukocytes in developing skin analyzed by flow cytometry and immunofluorescence, respectively. (C) The percentage of Ki67+CD45+ cells among total CD45+ skin cells during different stages of development was determined. 9-14 wk EGA: A, n = 18; B, n = 9; C, n = 7. 18–24 wk EGA: A and B, n = 9 each; C, n = 7. Adult: A–C, n = 7 each. Bars represent the mean of investigated groups. (D) Immunofluorescence triple labeling identified proliferating CD45+HLA-DR+Ki67+ cells (arrows) in all investigated age groups. Alexa Fluor 488–labeled Laminin5 mAb visualizes the dermo-epidermal junction. Shown is one representative experiment of three independent experiments per group with similar results. Bars, 50 μm.
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fig1: Proliferating CD45+ cells contribute to the increase of leukocyte numbers in developing skin. Dot graphs show the increase of relative (A) and absolute (B) numbers of CD45+ leukocytes in developing skin analyzed by flow cytometry and immunofluorescence, respectively. (C) The percentage of Ki67+CD45+ cells among total CD45+ skin cells during different stages of development was determined. 9-14 wk EGA: A, n = 18; B, n = 9; C, n = 7. 18–24 wk EGA: A and B, n = 9 each; C, n = 7. Adult: A–C, n = 7 each. Bars represent the mean of investigated groups. (D) Immunofluorescence triple labeling identified proliferating CD45+HLA-DR+Ki67+ cells (arrows) in all investigated age groups. Alexa Fluor 488–labeled Laminin5 mAb visualizes the dermo-epidermal junction. Shown is one representative experiment of three independent experiments per group with similar results. Bars, 50 μm.

Mentions: During intrauterine life, the skin immune system has to acquire the prerequisites to protect the newborn from infection in the hostile environment after birth, which includes the formation of a leukocyte network. To explore the kinetics of leukocyte numbers in developing and adult human skin, we enzymatically digested whole skin and analyzed the percentage of CD45+ leukocytes using flow cytometry. We found that relative leukocyte numbers among all skin cells in developing skin increase from 1.13% in embryonic skin (9–14 wk EGA) to 2.97% in fetal skin (18–24 wk EGA), which is significantly lower than in adult skin (11.59%; Fig. 1 A). To exclude skin digestion's influence on the quantity of released leukocytes, we analyzed in parallel the development of the absolute leukocyte numbers by counting CD45+ cells in skin sections. This confirmed that leukocyte numbers are significantly lower in embryonic and fetal skin than in adult skin (Fig. 1 B). To test whether the increase in leukocyte numbers in developing skin is caused by proliferating leukocytes, skin sections were counterstained with the nuclear proliferation marker Ki67. We found that 17.0% of CD45+ cells in embryonic skin, but only 3.7% in fetal skin and 0.9% in adult skin, expresses Ki67 (Fig. 1 C). Triple immunofluorescence staining of skin sections revealed that Ki67 is found in CD45+HLA-DR+ leukocytes in both the dermis and the epidermis (Fig. 1 D, arrows; and not depicted) and in dermal CD45+HLA-DR− leukocytes (not depicted) in all developmental stages as well as in adult skin. CD45−Ki67+ cells represent, among others, proliferating keratinocytes, fibroblasts and endothelial cells. We next asked whether the high rate of proliferating leukocytes in embryonic and fetal skin could be explained by higher concentrations of proliferation-inducing cytokines. Indeed, analysis of skin cell culture supernatants from embryonic, fetal, and adult single cells revealed that GM-CSF, SCF, and IL-6 concentrations are five- to sevenfold higher in supernatants of embryonic compared with adult skin (Fig. 2 A). No obvious differences in M-CSF concentrations among all investigated age groups were observed (Fig. 2 A), and IL-3 was not detectable (not depicted). In addition to the local proliferation of skin-resident leukocytes, their numerical increase may also be caused by the attraction of leukocytes from the circulation. As it is impossible to directly study the influx of leukocytes into skin in humans, we measured surrogate markers for leukocyte immigration such as chemokine levels. We found that MCP-1 (CCL2) concentrations are 80-fold higher and RANTES (CCL5) levels are twofold higher in supernatants from embryonic skin than from adult skin (Fig. 2 B). In contrast to MCP-1, RANTES reaches adult-like levels already at 18–24 wk EGA. We observed no significant differences in CCL20 and MDC (CCL22) concentrations among all age groups investigated (unpublished data).


HLA-DR+ leukocytes acquire CD1 antigens in embryonic and fetal human skin and contain functional antigen-presenting cells.

Schuster C, Vaculik C, Fiala C, Meindl S, Brandt O, Imhof M, Stingl G, Eppel W, Elbe-Bürger A - J. Exp. Med. (2009)

Proliferating CD45+ cells contribute to the increase of leukocyte numbers in developing skin. Dot graphs show the increase of relative (A) and absolute (B) numbers of CD45+ leukocytes in developing skin analyzed by flow cytometry and immunofluorescence, respectively. (C) The percentage of Ki67+CD45+ cells among total CD45+ skin cells during different stages of development was determined. 9-14 wk EGA: A, n = 18; B, n = 9; C, n = 7. 18–24 wk EGA: A and B, n = 9 each; C, n = 7. Adult: A–C, n = 7 each. Bars represent the mean of investigated groups. (D) Immunofluorescence triple labeling identified proliferating CD45+HLA-DR+Ki67+ cells (arrows) in all investigated age groups. Alexa Fluor 488–labeled Laminin5 mAb visualizes the dermo-epidermal junction. Shown is one representative experiment of three independent experiments per group with similar results. Bars, 50 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2626673&req=5

fig1: Proliferating CD45+ cells contribute to the increase of leukocyte numbers in developing skin. Dot graphs show the increase of relative (A) and absolute (B) numbers of CD45+ leukocytes in developing skin analyzed by flow cytometry and immunofluorescence, respectively. (C) The percentage of Ki67+CD45+ cells among total CD45+ skin cells during different stages of development was determined. 9-14 wk EGA: A, n = 18; B, n = 9; C, n = 7. 18–24 wk EGA: A and B, n = 9 each; C, n = 7. Adult: A–C, n = 7 each. Bars represent the mean of investigated groups. (D) Immunofluorescence triple labeling identified proliferating CD45+HLA-DR+Ki67+ cells (arrows) in all investigated age groups. Alexa Fluor 488–labeled Laminin5 mAb visualizes the dermo-epidermal junction. Shown is one representative experiment of three independent experiments per group with similar results. Bars, 50 μm.
Mentions: During intrauterine life, the skin immune system has to acquire the prerequisites to protect the newborn from infection in the hostile environment after birth, which includes the formation of a leukocyte network. To explore the kinetics of leukocyte numbers in developing and adult human skin, we enzymatically digested whole skin and analyzed the percentage of CD45+ leukocytes using flow cytometry. We found that relative leukocyte numbers among all skin cells in developing skin increase from 1.13% in embryonic skin (9–14 wk EGA) to 2.97% in fetal skin (18–24 wk EGA), which is significantly lower than in adult skin (11.59%; Fig. 1 A). To exclude skin digestion's influence on the quantity of released leukocytes, we analyzed in parallel the development of the absolute leukocyte numbers by counting CD45+ cells in skin sections. This confirmed that leukocyte numbers are significantly lower in embryonic and fetal skin than in adult skin (Fig. 1 B). To test whether the increase in leukocyte numbers in developing skin is caused by proliferating leukocytes, skin sections were counterstained with the nuclear proliferation marker Ki67. We found that 17.0% of CD45+ cells in embryonic skin, but only 3.7% in fetal skin and 0.9% in adult skin, expresses Ki67 (Fig. 1 C). Triple immunofluorescence staining of skin sections revealed that Ki67 is found in CD45+HLA-DR+ leukocytes in both the dermis and the epidermis (Fig. 1 D, arrows; and not depicted) and in dermal CD45+HLA-DR− leukocytes (not depicted) in all developmental stages as well as in adult skin. CD45−Ki67+ cells represent, among others, proliferating keratinocytes, fibroblasts and endothelial cells. We next asked whether the high rate of proliferating leukocytes in embryonic and fetal skin could be explained by higher concentrations of proliferation-inducing cytokines. Indeed, analysis of skin cell culture supernatants from embryonic, fetal, and adult single cells revealed that GM-CSF, SCF, and IL-6 concentrations are five- to sevenfold higher in supernatants of embryonic compared with adult skin (Fig. 2 A). No obvious differences in M-CSF concentrations among all investigated age groups were observed (Fig. 2 A), and IL-3 was not detectable (not depicted). In addition to the local proliferation of skin-resident leukocytes, their numerical increase may also be caused by the attraction of leukocytes from the circulation. As it is impossible to directly study the influx of leukocytes into skin in humans, we measured surrogate markers for leukocyte immigration such as chemokine levels. We found that MCP-1 (CCL2) concentrations are 80-fold higher and RANTES (CCL5) levels are twofold higher in supernatants from embryonic skin than from adult skin (Fig. 2 B). In contrast to MCP-1, RANTES reaches adult-like levels already at 18–24 wk EGA. We observed no significant differences in CCL20 and MDC (CCL22) concentrations among all age groups investigated (unpublished data).

Bottom Line: We also found that CD45(+)HLA-DR(high)CD1c(+) dendritic cells (DCs) are already present in the epidermis and dermis at 9 wk estimated gestational age (EGA) and that transforming growth factor beta1 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, DIAID, 1090 Vienna, Austria.

ABSTRACT
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-DR(high)CD1c(+) dendritic cells (DCs) are already present in the epidermis and dermis at 9 wk estimated gestational age (EGA) and that transforming growth factor beta1 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.

Show MeSH
Related in: MedlinePlus