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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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Tape stripping promotes exogenous protein trafficking, migration to the dLN, and the rapid loss of CD103–DDCs from the dLN.(A) The dorsal skin of KikGR mice was clipped, subjected to tape stripping (some mice only), painted with Alexa647-conjugated OVA (A647-OVA) and photoconverted according to the timeline shown for each treatment group. (B–D) At the time points indicated, cells isolated from the brachial dLNs were stained with fluorochrome-conjugated mAbs for analysis of total skin-derived DCs, CD103–DDCs, CD103+DCs, and CD326+DCs by flow cytometry (see Supplementary Fig. S2A and Supplementary Fig. S3 for gating strategy). Cell numbers within each population were calculated by multiplying total cell numbers by percentages as determined by flow cytometry, and are presented as mean ± SE (B and C). Flow cytometry dot plots showing KikGR-green vs. KikGR-red cells within the CD103–DDC population (upper plots) and CD326 vs. A647-OVA within the KikGR-red CD103–DDC population (lower plots) (D). At least four samples were analyzed for each time point. Data are representative of two independent experiments. (E) Flow cytometry dot plot showing KikGR-green vs. KikGR-red cells within the CD103–DDC population 24 h after tape stripping without OVA painting. Percentages on flow cytometry dot plots indicate the proportion of KikGR-red or A647-OVA+ cells within each subpopulation. Data are representative of two independent experiments. Illustration created by M.T. using Adobe Photoshop software.
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f5: Tape stripping promotes exogenous protein trafficking, migration to the dLN, and the rapid loss of CD103–DDCs from the dLN.(A) The dorsal skin of KikGR mice was clipped, subjected to tape stripping (some mice only), painted with Alexa647-conjugated OVA (A647-OVA) and photoconverted according to the timeline shown for each treatment group. (B–D) At the time points indicated, cells isolated from the brachial dLNs were stained with fluorochrome-conjugated mAbs for analysis of total skin-derived DCs, CD103–DDCs, CD103+DCs, and CD326+DCs by flow cytometry (see Supplementary Fig. S2A and Supplementary Fig. S3 for gating strategy). Cell numbers within each population were calculated by multiplying total cell numbers by percentages as determined by flow cytometry, and are presented as mean ± SE (B and C). Flow cytometry dot plots showing KikGR-green vs. KikGR-red cells within the CD103–DDC population (upper plots) and CD326 vs. A647-OVA within the KikGR-red CD103–DDC population (lower plots) (D). At least four samples were analyzed for each time point. Data are representative of two independent experiments. (E) Flow cytometry dot plot showing KikGR-green vs. KikGR-red cells within the CD103–DDC population 24 h after tape stripping without OVA painting. Percentages on flow cytometry dot plots indicate the proportion of KikGR-red or A647-OVA+ cells within each subpopulation. Data are representative of two independent experiments. Illustration created by M.T. using Adobe Photoshop software.

Mentions: Mechanical injury to the skin, such as tape stripping, which removes the stratum corneum, disrupts the permeability of the epithelial barrier and induces the maturation and emigration of LCs from the epidermis40. We therefore examined changes in the migration patterns of skin-derived DCs and exogenous protein trafficking following tape stripping. As depicted in Fig. 5A, the dorsal skin of KikGR mice was subjected to tape stripping and painting with Alexa647-obalbumin (OVA) in PBS, then photoconverted either immediately (d0PC group), after one day (d1PC group), or after two days (d2PC group), with analysis of the dLN one day after photoconversion in each case. Following tape stripping and protein painting, numbers of skin-derived DCs (gray column) and Alexa647+ skin-derived DCs (blue column) in the dLN were elevated on days 1–3 (d0PC, d1PC, and d2PC groups) compared to the no tape stripping control group (no-TS-d0PC group) (Fig. 5B). The increase in skin-derived DCs in the dLN was made up primarily of KikGR-red CD103−DDCs (Fig. 5C). In the d0PC group, almost half of KikGR-red cells consisted of Alexa647+ (i.e. exogenous protein-carrying) cells (Fig. 5 C and D), and the majority (92%) of KikGR-red Alexa647+ cells were CD103−DDCs (Fig. 5C). These results clearly indicate that exogenous protein is carried from the skin to the dLN by CD103−DDCs. In addition, the number of Alexa647+CD103−DDCs was 6- to 10-fold higher in the d0PC group than at the other time points, suggesting that the trafficking of exogenous protein to the dLN is greatest on the first day after tape stripping (Fig. 5 C and D).


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)

Tape stripping promotes exogenous protein trafficking, migration to the dLN, and the rapid loss of CD103–DDCs from the dLN.(A) The dorsal skin of KikGR mice was clipped, subjected to tape stripping (some mice only), painted with Alexa647-conjugated OVA (A647-OVA) and photoconverted according to the timeline shown for each treatment group. (B–D) At the time points indicated, cells isolated from the brachial dLNs were stained with fluorochrome-conjugated mAbs for analysis of total skin-derived DCs, CD103–DDCs, CD103+DCs, and CD326+DCs by flow cytometry (see Supplementary Fig. S2A and Supplementary Fig. S3 for gating strategy). Cell numbers within each population were calculated by multiplying total cell numbers by percentages as determined by flow cytometry, and are presented as mean ± SE (B and C). Flow cytometry dot plots showing KikGR-green vs. KikGR-red cells within the CD103–DDC population (upper plots) and CD326 vs. A647-OVA within the KikGR-red CD103–DDC population (lower plots) (D). At least four samples were analyzed for each time point. Data are representative of two independent experiments. (E) Flow cytometry dot plot showing KikGR-green vs. KikGR-red cells within the CD103–DDC population 24 h after tape stripping without OVA painting. Percentages on flow cytometry dot plots indicate the proportion of KikGR-red or A647-OVA+ cells within each subpopulation. Data are representative of two independent experiments. Illustration created by M.T. using Adobe Photoshop software.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Tape stripping promotes exogenous protein trafficking, migration to the dLN, and the rapid loss of CD103–DDCs from the dLN.(A) The dorsal skin of KikGR mice was clipped, subjected to tape stripping (some mice only), painted with Alexa647-conjugated OVA (A647-OVA) and photoconverted according to the timeline shown for each treatment group. (B–D) At the time points indicated, cells isolated from the brachial dLNs were stained with fluorochrome-conjugated mAbs for analysis of total skin-derived DCs, CD103–DDCs, CD103+DCs, and CD326+DCs by flow cytometry (see Supplementary Fig. S2A and Supplementary Fig. S3 for gating strategy). Cell numbers within each population were calculated by multiplying total cell numbers by percentages as determined by flow cytometry, and are presented as mean ± SE (B and C). Flow cytometry dot plots showing KikGR-green vs. KikGR-red cells within the CD103–DDC population (upper plots) and CD326 vs. A647-OVA within the KikGR-red CD103–DDC population (lower plots) (D). At least four samples were analyzed for each time point. Data are representative of two independent experiments. (E) Flow cytometry dot plot showing KikGR-green vs. KikGR-red cells within the CD103–DDC population 24 h after tape stripping without OVA painting. Percentages on flow cytometry dot plots indicate the proportion of KikGR-red or A647-OVA+ cells within each subpopulation. Data are representative of two independent experiments. Illustration created by M.T. using Adobe Photoshop software.
Mentions: Mechanical injury to the skin, such as tape stripping, which removes the stratum corneum, disrupts the permeability of the epithelial barrier and induces the maturation and emigration of LCs from the epidermis40. We therefore examined changes in the migration patterns of skin-derived DCs and exogenous protein trafficking following tape stripping. As depicted in Fig. 5A, the dorsal skin of KikGR mice was subjected to tape stripping and painting with Alexa647-obalbumin (OVA) in PBS, then photoconverted either immediately (d0PC group), after one day (d1PC group), or after two days (d2PC group), with analysis of the dLN one day after photoconversion in each case. Following tape stripping and protein painting, numbers of skin-derived DCs (gray column) and Alexa647+ skin-derived DCs (blue column) in the dLN were elevated on days 1–3 (d0PC, d1PC, and d2PC groups) compared to the no tape stripping control group (no-TS-d0PC group) (Fig. 5B). The increase in skin-derived DCs in the dLN was made up primarily of KikGR-red CD103−DDCs (Fig. 5C). In the d0PC group, almost half of KikGR-red cells consisted of Alexa647+ (i.e. exogenous protein-carrying) cells (Fig. 5 C and D), and the majority (92%) of KikGR-red Alexa647+ cells were CD103−DDCs (Fig. 5C). These results clearly indicate that exogenous protein is carried from the skin to the dLN by CD103−DDCs. In addition, the number of Alexa647+CD103−DDCs was 6- to 10-fold higher in the d0PC group than at the other time points, suggesting that the trafficking of exogenous protein to the dLN is greatest on the first day after tape stripping (Fig. 5 C and D).

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