Limits...
Tracking and quantification of dendritic cell migration and antigen trafficking between the skin and lymph nodes.

Tomura M, Hata A, Matsuoka S, Shand FH, Nakanishi Y, Ikebuchi R, Ueha S, Tsutsui H, Inaba K, Matsushima K, Miyawaki A, Kabashima K, Watanabe T, Kanagawa O - Sci Rep (2014)

Bottom Line: Tape stripping (mechanical injury) induced a long-lasting four-fold increase in CD103(-)DDC migration to the dLN and accelerated the trafficking of exogenous protein antigens by these cells.Both stresses increased the turnover of CD103(-)DDCs within the dLN, causing these cells to die within one day of arrival.Therefore, CD103(-)DDCs act as sentinels against skin invasion that respond with increased cellular migration and antigen trafficking from the skin to the dLNs.

View Article: PubMed Central - PubMed

Affiliation: 1] Center for Innovation in Immunoregulative Technology and Therapeutics, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Kyoto 606-8501, Japan [2] Laboratory for Autoimmune Regulation, Research Center for Allergy and Immunology, RIKEN, 1-7-22 Suehiro-cho, Tsurumi, Yokohama City, Kanagawa 230-0045, Japan [3] Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, 7-3- 1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

ABSTRACT
Skin-derived dendritic cells (DCs) play a crucial role in the maintenance of immune homeostasis due to their role in antigen trafficking from the skin to the draining lymph nodes (dLNs). To quantify the spatiotemporal regulation of skin-derived DCs in vivo, we generated knock-in mice expressing the photoconvertible fluorescent protein KikGR. By exposing the skin or dLN of these mice to violet light, we were able to label and track the migration and turnover of endogenous skin-derived DCs. Langerhans cells and CD103(+)DCs, including Langerin(+)CD103(+)dermal DCs (DDCs), remained in the dLN for 4-4.5 days after migration from the skin, while CD103(-)DDCs persisted for only two days. Application of a skin irritant (chemical stress) induced a transient >10-fold increase in CD103(-)DDC migration from the skin to the dLN. Tape stripping (mechanical injury) induced a long-lasting four-fold increase in CD103(-)DDC migration to the dLN and accelerated the trafficking of exogenous protein antigens by these cells. Both stresses increased the turnover of CD103(-)DDCs within the dLN, causing these cells to die within one day of arrival. Therefore, CD103(-)DDCs act as sentinels against skin invasion that respond with increased cellular migration and antigen trafficking from the skin to the dLNs.

Show MeSH

Related in: MedlinePlus

Skin irritant painting promotes the migration and turnover of CD103–DDCs.(A–F) The clipped abdominal skin of KikGR mice was photoconverted by exposure to violet light, then the same region was painted with a skin irritant (acetone:dibutylphthalate mixture) (A). At the time points indicated, cells isolated from the axillary dLNs were stained with fluorochrome-conjugated mAbs for analysis by flow cytometry. (B) Graph shows total cell number and KikGR-red cell number of skin-derived DCs (see Supplementary Fig. S2A for gating strategy). (C) Fold increases relative to the steady state (S) of KikGR-red skin-derived DCs in the dLN after skin photoconversion. (D) Sections of axillary LNs from KikGR-BM→WT chimeric mice 24 h after skin photoconversion and irritant painting were examined under a confocal microscope. Scale bars, 40 μm. (E) Graph shows total cell number and KikGR-red cell number of CD103–DDCs, CD103+DCs, and CD326+DCs (see Supplementary Fig. S3 for gating strategy). (F) Fold increases relative to the steady state (S) of KikGR-red CD103–DDCs in the dLN after skin photoconversion. Percentage decreases relative to the peak KikGR-red CD103–DDC number on day 1 are indicated by blue arrows. At least four samples were analyzed for each time point. Cell numbers within each population were calculated by multiplying total cell number by percentages as determined by flow cytometry, and are presented as mean ± SE. (G and H) The clipped abdominal skin of KikGR mice was painted with a skin irritant on day zero, before photoconversion of the axillary dLNs on day one and flow cytometry analysis of the dLNs on day two (G). (H) Graphs shows the proportions (mean ± SE; n = 3) of CD103–DDCs in the dLN that were KikGR-red and KikGR-green. Data are representative of three independent experiments. Illustration created by M.T. using Adobe Photoshop software.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4129424&req=5

f4: Skin irritant painting promotes the migration and turnover of CD103–DDCs.(A–F) The clipped abdominal skin of KikGR mice was photoconverted by exposure to violet light, then the same region was painted with a skin irritant (acetone:dibutylphthalate mixture) (A). At the time points indicated, cells isolated from the axillary dLNs were stained with fluorochrome-conjugated mAbs for analysis by flow cytometry. (B) Graph shows total cell number and KikGR-red cell number of skin-derived DCs (see Supplementary Fig. S2A for gating strategy). (C) Fold increases relative to the steady state (S) of KikGR-red skin-derived DCs in the dLN after skin photoconversion. (D) Sections of axillary LNs from KikGR-BM→WT chimeric mice 24 h after skin photoconversion and irritant painting were examined under a confocal microscope. Scale bars, 40 μm. (E) Graph shows total cell number and KikGR-red cell number of CD103–DDCs, CD103+DCs, and CD326+DCs (see Supplementary Fig. S3 for gating strategy). (F) Fold increases relative to the steady state (S) of KikGR-red CD103–DDCs in the dLN after skin photoconversion. Percentage decreases relative to the peak KikGR-red CD103–DDC number on day 1 are indicated by blue arrows. At least four samples were analyzed for each time point. Cell numbers within each population were calculated by multiplying total cell number by percentages as determined by flow cytometry, and are presented as mean ± SE. (G and H) The clipped abdominal skin of KikGR mice was painted with a skin irritant on day zero, before photoconversion of the axillary dLNs on day one and flow cytometry analysis of the dLNs on day two (G). (H) Graphs shows the proportions (mean ± SE; n = 3) of CD103–DDCs in the dLN that were KikGR-red and KikGR-green. Data are representative of three independent experiments. Illustration created by M.T. using Adobe Photoshop software.

Mentions: Previous studies have reported that the appearance of labeled skin-derived Langerin−DDCs (including CD103−DDCs) in the dLN precedes the appearance of Langerin+ DCs following the application of a fluorescent dye and a chemical stressor to the skin31724. We therefore attempted to quantify changes in skin-derived DC dynamics after inducing skin inflammation in KikGR mice. The abdominal skin of KikGR mice was photoconverted, painted with a skin irritant, and the appearance of KikGR-red cells in an axillary dLN was analyzed (Fig. 4A). One day after photoconversion and painting, the number of KikGR-red skin-derived DCs in the dLN had increased more than 12-fold (Fig. 4B and “d1” in Fig. 4C) compared to the number of KikGR-red cells present in the steady state (“S” in Fig. 4C), and large numbers of KikGR-red cells with DC-like morphology were observed in the dLN (Fig. 4D). The increase in skin-derived DCs in the dLN following skin irritant painting was largely the result of an increase in KikGR-red CD103−DDCs (Fig. 4 E and F). Intriguingly, however, in contrast to the findings of previous skin irritant painting studies31724, only very small increases in the number of cells from other subsets (including Langerin+ DCs) were detected in the dLN during the later phases after painting (on days three and four) (Fig. 4E).


Tracking and quantification of dendritic cell migration and antigen trafficking between the skin and lymph nodes.

Tomura M, Hata A, Matsuoka S, Shand FH, Nakanishi Y, Ikebuchi R, Ueha S, Tsutsui H, Inaba K, Matsushima K, Miyawaki A, Kabashima K, Watanabe T, Kanagawa O - Sci Rep (2014)

Skin irritant painting promotes the migration and turnover of CD103–DDCs.(A–F) The clipped abdominal skin of KikGR mice was photoconverted by exposure to violet light, then the same region was painted with a skin irritant (acetone:dibutylphthalate mixture) (A). At the time points indicated, cells isolated from the axillary dLNs were stained with fluorochrome-conjugated mAbs for analysis by flow cytometry. (B) Graph shows total cell number and KikGR-red cell number of skin-derived DCs (see Supplementary Fig. S2A for gating strategy). (C) Fold increases relative to the steady state (S) of KikGR-red skin-derived DCs in the dLN after skin photoconversion. (D) Sections of axillary LNs from KikGR-BM→WT chimeric mice 24 h after skin photoconversion and irritant painting were examined under a confocal microscope. Scale bars, 40 μm. (E) Graph shows total cell number and KikGR-red cell number of CD103–DDCs, CD103+DCs, and CD326+DCs (see Supplementary Fig. S3 for gating strategy). (F) Fold increases relative to the steady state (S) of KikGR-red CD103–DDCs in the dLN after skin photoconversion. Percentage decreases relative to the peak KikGR-red CD103–DDC number on day 1 are indicated by blue arrows. At least four samples were analyzed for each time point. Cell numbers within each population were calculated by multiplying total cell number by percentages as determined by flow cytometry, and are presented as mean ± SE. (G and H) The clipped abdominal skin of KikGR mice was painted with a skin irritant on day zero, before photoconversion of the axillary dLNs on day one and flow cytometry analysis of the dLNs on day two (G). (H) Graphs shows the proportions (mean ± SE; n = 3) of CD103–DDCs in the dLN that were KikGR-red and KikGR-green. Data are representative of three independent experiments. Illustration created by M.T. using Adobe Photoshop software.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4129424&req=5

f4: Skin irritant painting promotes the migration and turnover of CD103–DDCs.(A–F) The clipped abdominal skin of KikGR mice was photoconverted by exposure to violet light, then the same region was painted with a skin irritant (acetone:dibutylphthalate mixture) (A). At the time points indicated, cells isolated from the axillary dLNs were stained with fluorochrome-conjugated mAbs for analysis by flow cytometry. (B) Graph shows total cell number and KikGR-red cell number of skin-derived DCs (see Supplementary Fig. S2A for gating strategy). (C) Fold increases relative to the steady state (S) of KikGR-red skin-derived DCs in the dLN after skin photoconversion. (D) Sections of axillary LNs from KikGR-BM→WT chimeric mice 24 h after skin photoconversion and irritant painting were examined under a confocal microscope. Scale bars, 40 μm. (E) Graph shows total cell number and KikGR-red cell number of CD103–DDCs, CD103+DCs, and CD326+DCs (see Supplementary Fig. S3 for gating strategy). (F) Fold increases relative to the steady state (S) of KikGR-red CD103–DDCs in the dLN after skin photoconversion. Percentage decreases relative to the peak KikGR-red CD103–DDC number on day 1 are indicated by blue arrows. At least four samples were analyzed for each time point. Cell numbers within each population were calculated by multiplying total cell number by percentages as determined by flow cytometry, and are presented as mean ± SE. (G and H) The clipped abdominal skin of KikGR mice was painted with a skin irritant on day zero, before photoconversion of the axillary dLNs on day one and flow cytometry analysis of the dLNs on day two (G). (H) Graphs shows the proportions (mean ± SE; n = 3) of CD103–DDCs in the dLN that were KikGR-red and KikGR-green. Data are representative of three independent experiments. Illustration created by M.T. using Adobe Photoshop software.
Mentions: Previous studies have reported that the appearance of labeled skin-derived Langerin−DDCs (including CD103−DDCs) in the dLN precedes the appearance of Langerin+ DCs following the application of a fluorescent dye and a chemical stressor to the skin31724. We therefore attempted to quantify changes in skin-derived DC dynamics after inducing skin inflammation in KikGR mice. The abdominal skin of KikGR mice was photoconverted, painted with a skin irritant, and the appearance of KikGR-red cells in an axillary dLN was analyzed (Fig. 4A). One day after photoconversion and painting, the number of KikGR-red skin-derived DCs in the dLN had increased more than 12-fold (Fig. 4B and “d1” in Fig. 4C) compared to the number of KikGR-red cells present in the steady state (“S” in Fig. 4C), and large numbers of KikGR-red cells with DC-like morphology were observed in the dLN (Fig. 4D). The increase in skin-derived DCs in the dLN following skin irritant painting was largely the result of an increase in KikGR-red CD103−DDCs (Fig. 4 E and F). Intriguingly, however, in contrast to the findings of previous skin irritant painting studies31724, only very small increases in the number of cells from other subsets (including Langerin+ DCs) were detected in the dLN during the later phases after painting (on days three and four) (Fig. 4E).

Bottom Line: Tape stripping (mechanical injury) induced a long-lasting four-fold increase in CD103(-)DDC migration to the dLN and accelerated the trafficking of exogenous protein antigens by these cells.Both stresses increased the turnover of CD103(-)DDCs within the dLN, causing these cells to die within one day of arrival.Therefore, CD103(-)DDCs act as sentinels against skin invasion that respond with increased cellular migration and antigen trafficking from the skin to the dLNs.

View Article: PubMed Central - PubMed

Affiliation: 1] Center for Innovation in Immunoregulative Technology and Therapeutics, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Kyoto 606-8501, Japan [2] Laboratory for Autoimmune Regulation, Research Center for Allergy and Immunology, RIKEN, 1-7-22 Suehiro-cho, Tsurumi, Yokohama City, Kanagawa 230-0045, Japan [3] Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, 7-3- 1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

ABSTRACT
Skin-derived dendritic cells (DCs) play a crucial role in the maintenance of immune homeostasis due to their role in antigen trafficking from the skin to the draining lymph nodes (dLNs). To quantify the spatiotemporal regulation of skin-derived DCs in vivo, we generated knock-in mice expressing the photoconvertible fluorescent protein KikGR. By exposing the skin or dLN of these mice to violet light, we were able to label and track the migration and turnover of endogenous skin-derived DCs. Langerhans cells and CD103(+)DCs, including Langerin(+)CD103(+)dermal DCs (DDCs), remained in the dLN for 4-4.5 days after migration from the skin, while CD103(-)DDCs persisted for only two days. Application of a skin irritant (chemical stress) induced a transient >10-fold increase in CD103(-)DDC migration from the skin to the dLN. Tape stripping (mechanical injury) induced a long-lasting four-fold increase in CD103(-)DDC migration to the dLN and accelerated the trafficking of exogenous protein antigens by these cells. Both stresses increased the turnover of CD103(-)DDCs within the dLN, causing these cells to die within one day of arrival. Therefore, CD103(-)DDCs act as sentinels against skin invasion that respond with increased cellular migration and antigen trafficking from the skin to the dLNs.

Show MeSH
Related in: MedlinePlus