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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.

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Each skin-derived DC subset displays distinct migration kinetics in its migration from the skin to the dLN.(A) The clipped abdominal skin of KikGR mice was photoconverted by exposure to violet light before examination under a stereoscopic fluorescence microscope. Unclipped areas of skin remained black because the photoconversion light could not reach them. (B) The skin and axillary dLNs were resected immediately after skin photoconversion, or from non-photoconverted mice, and the cells from these tissues isolated for analysis by flow cytometry. (C) Following skin photoconversion, cells from the dLNs were isolated and stained for analysis of skin-derived DCs by flow cytometry (see Supplementary Fig. S2A for gating strategy). Data represent the proportion of skin-derived DCs labeled KikGR-red. (D) Sections of dLNs collected from KikGR-BM→WT mice 24 h after skin photoconversion were observed under a confocal microscope. Scale bars, 40 μm. (E) Flow cytometry plots showing KikGR-green and KikGR-red cells within each DC subpopulation in the dLN 24 h after skin photoconversion. (F) Flow cytometry plot showing KikGR-green and KikGR-red DCs (CD11c+ cells) within the dLN of CCR7–/– KikGR mice 24 h after skin photoconversion. (G) Total skin-derived DCs from the dLN of KikGR mice after skin photoconversion (as in panel C) were further gated into CD103–DDCs and CD103+DCs (see Supplementary Fig. S3 for gating strategy). (H) Following skin photoconversion, cells from the dLN of WT-BM→KikGR chimeric mice were isolated and stained for analysis of LCs by flow cytometry (see Supplementary Fig. S4 for gating strategy). Data in G and H represent the proportion of each subpopulation labeled KikGR-red. (I) After skin photoconversion, single-cell suspensions from the epidermis and dermis were analyzed by flow cytometry as described in Supplementary Fig. S2B. Percentages on flow cytometry dot plots (B, E and F) indicate the proportion of KikGR-red cells within each subpopulation. At least four samples from each time point were analyzed. Data represent mean ± SE (C, G, H, and I) and are representative of three independent experiments.
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f1: Each skin-derived DC subset displays distinct migration kinetics in its migration from the skin to the dLN.(A) The clipped abdominal skin of KikGR mice was photoconverted by exposure to violet light before examination under a stereoscopic fluorescence microscope. Unclipped areas of skin remained black because the photoconversion light could not reach them. (B) The skin and axillary dLNs were resected immediately after skin photoconversion, or from non-photoconverted mice, and the cells from these tissues isolated for analysis by flow cytometry. (C) Following skin photoconversion, cells from the dLNs were isolated and stained for analysis of skin-derived DCs by flow cytometry (see Supplementary Fig. S2A for gating strategy). Data represent the proportion of skin-derived DCs labeled KikGR-red. (D) Sections of dLNs collected from KikGR-BM→WT mice 24 h after skin photoconversion were observed under a confocal microscope. Scale bars, 40 μm. (E) Flow cytometry plots showing KikGR-green and KikGR-red cells within each DC subpopulation in the dLN 24 h after skin photoconversion. (F) Flow cytometry plot showing KikGR-green and KikGR-red DCs (CD11c+ cells) within the dLN of CCR7–/– KikGR mice 24 h after skin photoconversion. (G) Total skin-derived DCs from the dLN of KikGR mice after skin photoconversion (as in panel C) were further gated into CD103–DDCs and CD103+DCs (see Supplementary Fig. S3 for gating strategy). (H) Following skin photoconversion, cells from the dLN of WT-BM→KikGR chimeric mice were isolated and stained for analysis of LCs by flow cytometry (see Supplementary Fig. S4 for gating strategy). Data in G and H represent the proportion of each subpopulation labeled KikGR-red. (I) After skin photoconversion, single-cell suspensions from the epidermis and dermis were analyzed by flow cytometry as described in Supplementary Fig. S2B. Percentages on flow cytometry dot plots (B, E and F) indicate the proportion of KikGR-red cells within each subpopulation. At least four samples from each time point were analyzed. Data represent mean ± SE (C, G, H, and I) and are representative of three independent experiments.

Mentions: To track DC migration from the skin to the dLN, the abdominal hair of KikGR mice was clipped, and the skin was exposed to violet light (436 nm) to induce photoconversion of the KikGR protein from green (KikGR-green) to red (KikGR-red)(Fig. 1A). Unlike shorter wavelenths, exposure to violet light in this manner does not cause inflammation or other immunomodulatory effects303235. Although 100% of the cells in violet light-exposed skin were KikGR-red immediately after photoconversion, cells in non-photoconverted skin and in the axillary dLNs remained KikGR-green (Fig. 1B).


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)

Each skin-derived DC subset displays distinct migration kinetics in its migration from the skin to the dLN.(A) The clipped abdominal skin of KikGR mice was photoconverted by exposure to violet light before examination under a stereoscopic fluorescence microscope. Unclipped areas of skin remained black because the photoconversion light could not reach them. (B) The skin and axillary dLNs were resected immediately after skin photoconversion, or from non-photoconverted mice, and the cells from these tissues isolated for analysis by flow cytometry. (C) Following skin photoconversion, cells from the dLNs were isolated and stained for analysis of skin-derived DCs by flow cytometry (see Supplementary Fig. S2A for gating strategy). Data represent the proportion of skin-derived DCs labeled KikGR-red. (D) Sections of dLNs collected from KikGR-BM→WT mice 24 h after skin photoconversion were observed under a confocal microscope. Scale bars, 40 μm. (E) Flow cytometry plots showing KikGR-green and KikGR-red cells within each DC subpopulation in the dLN 24 h after skin photoconversion. (F) Flow cytometry plot showing KikGR-green and KikGR-red DCs (CD11c+ cells) within the dLN of CCR7–/– KikGR mice 24 h after skin photoconversion. (G) Total skin-derived DCs from the dLN of KikGR mice after skin photoconversion (as in panel C) were further gated into CD103–DDCs and CD103+DCs (see Supplementary Fig. S3 for gating strategy). (H) Following skin photoconversion, cells from the dLN of WT-BM→KikGR chimeric mice were isolated and stained for analysis of LCs by flow cytometry (see Supplementary Fig. S4 for gating strategy). Data in G and H represent the proportion of each subpopulation labeled KikGR-red. (I) After skin photoconversion, single-cell suspensions from the epidermis and dermis were analyzed by flow cytometry as described in Supplementary Fig. S2B. Percentages on flow cytometry dot plots (B, E and F) indicate the proportion of KikGR-red cells within each subpopulation. At least four samples from each time point were analyzed. Data represent mean ± SE (C, G, H, and I) and are representative of three independent experiments.
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Related In: Results  -  Collection

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f1: Each skin-derived DC subset displays distinct migration kinetics in its migration from the skin to the dLN.(A) The clipped abdominal skin of KikGR mice was photoconverted by exposure to violet light before examination under a stereoscopic fluorescence microscope. Unclipped areas of skin remained black because the photoconversion light could not reach them. (B) The skin and axillary dLNs were resected immediately after skin photoconversion, or from non-photoconverted mice, and the cells from these tissues isolated for analysis by flow cytometry. (C) Following skin photoconversion, cells from the dLNs were isolated and stained for analysis of skin-derived DCs by flow cytometry (see Supplementary Fig. S2A for gating strategy). Data represent the proportion of skin-derived DCs labeled KikGR-red. (D) Sections of dLNs collected from KikGR-BM→WT mice 24 h after skin photoconversion were observed under a confocal microscope. Scale bars, 40 μm. (E) Flow cytometry plots showing KikGR-green and KikGR-red cells within each DC subpopulation in the dLN 24 h after skin photoconversion. (F) Flow cytometry plot showing KikGR-green and KikGR-red DCs (CD11c+ cells) within the dLN of CCR7–/– KikGR mice 24 h after skin photoconversion. (G) Total skin-derived DCs from the dLN of KikGR mice after skin photoconversion (as in panel C) were further gated into CD103–DDCs and CD103+DCs (see Supplementary Fig. S3 for gating strategy). (H) Following skin photoconversion, cells from the dLN of WT-BM→KikGR chimeric mice were isolated and stained for analysis of LCs by flow cytometry (see Supplementary Fig. S4 for gating strategy). Data in G and H represent the proportion of each subpopulation labeled KikGR-red. (I) After skin photoconversion, single-cell suspensions from the epidermis and dermis were analyzed by flow cytometry as described in Supplementary Fig. S2B. Percentages on flow cytometry dot plots (B, E and F) indicate the proportion of KikGR-red cells within each subpopulation. At least four samples from each time point were analyzed. Data represent mean ± SE (C, G, H, and I) and are representative of three independent experiments.
Mentions: To track DC migration from the skin to the dLN, the abdominal hair of KikGR mice was clipped, and the skin was exposed to violet light (436 nm) to induce photoconversion of the KikGR protein from green (KikGR-green) to red (KikGR-red)(Fig. 1A). Unlike shorter wavelenths, exposure to violet light in this manner does not cause inflammation or other immunomodulatory effects303235. Although 100% of the cells in violet light-exposed skin were KikGR-red immediately after photoconversion, cells in non-photoconverted skin and in the axillary dLNs remained KikGR-green (Fig. 1B).

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