Semaphorins guide the entry of dendritic cells into the lymphatics by activating myosin II

Journal name:
Nature Immunology
Volume:
11,
Pages:
594–600
Year published:
DOI:
doi:10.1038/ni.1885
Received
Accepted
Published online

Abstract

The recirculation of leukocytes is essential for proper immune responses. However, the molecular mechanisms that regulate the entry of leukocytes into the lymphatics remain unclear. Here we show that plexin-A1, a principal receptor component for class III and class VI semaphorins, was crucially involved in the entry of dendritic cells (DCs) into the lymphatics. Additionally, we show that the semaphorin Sema3A, but not Sema6C or Sema6D, was required for DC transmigration and that Sema3A produced by the lymphatics promoted actomyosin contraction at the trailing edge of migrating DCs. Our findings not only demonstrate that semaphorin signals are involved in DC trafficking but also identify a previously unknown mechanism that induces actomyosin contraction as these cells pass through narrow gaps.

At a glance

Figures

  1. Plxna1-/- mice show impaired T cell responses due to defects in the migration of DCs into the lymph nodes.
    Figure 1: Plxna1−/− mice show impaired T cell responses due to defects in the migration of DCs into the lymph nodes.

    (a) CFSE dilution by CD4+ OT-II T cells intravenously transferred into wild-type (WT) or Plxna1−/− mice given subcutaneous injection of OVA peptides in complete Freund's adjuvant into the footpads, assessed as antigen-specific T cell responses in the draining lymph nodes (black lines) and nondraining lymph nodes (red lines). Data are representative of three independent experiments. (b) Two-photon microscopy of CMTMR-labeled wild-type and Plxna1−/− bone marrow–derived DCs (BMDCs; orange) injected into the footpads of wild-type recipient mice that also received CMFDA-labeled CD4+ OT-II T cells (green), showing DC trafficking into popliteal lymph nodes at 24 h after injection. Original magnification, ×40. Results are representative of two experiments. (c) Trafficking of CFSE-labeled wild-type or Plxna1−/− DCs into the popliteal lymph nodes of wild-type recipient mice after foodpad injection, calculated according to the following equation: (% input cells) = (total cells) × (% CFSE+ cells) / (input cells). *P < 0.01 and **P < 0.001 (Mann-Whitney U-test). Data are representative of three experiments (mean and s.d.). (d) Absolute number of endogenous DCs isolated from the brachial lymph nodes of wild-type and Plxna1−/− mice at 24 or 48 h after epicutaneous administration of FITC–isomer I to the shoulder skin. Data are representative of three experiments.

  2. Uptake of FITC-dextran and responses to chemokines are not affected in Plxna1-/- DCs.
    Figure 2: Uptake of FITC-dextran and responses to chemokines are not affected in Plxna1−/− DCs.

    (a) Uptake of FITC-dextran by wild-type and Plxna1−/− BMDCs at 37 °C for 30 min. Control, cells cultured on ice with FITC-dextran; max, maximum. (b) Chemotaxis of wild-type and Plxna1−/− DCs toward gradients of CCL19, CCL21 or CXCL12 in Transwell systems (pore size, 5 μm). (c) Directional sensing of wild-type and Plxna1−/− DCs in response to a CCL21 gradient in a Zigmond chamber, assessed as DC position after 60 min relative to original position and presented as the percentage of cells that ended within a 30° arc facing the CCL21 source. (d) Expression of CCR7 and CXCR4 in wild-type and Plxna1−/− DCs. Data are representative of three experiments (error bars (b), s.d.).

  3. Impaired transmigration of Plxna1-/- DCs across the lymphatics.
    Figure 3: Impaired transmigration of Plxna1−/− DCs across the lymphatics.

    (a) Confocal z-stack imaging (top) of CFSE-labeled wild-type and Plxna1−/− BMDCs (green) injected intradermally into the ear tissues of oxazolone-sensitized mice, assessed in whole mounts stained 24 h after transfer with biotinylated anti–LYVE-1 and streptavidin-indocarbocyanine (red). Scale bars, 50 μm. Below, quantification of retained DCs in the fields above; each symbol represents an individual field, and red circles indicate the mean. *P < 0.001 (Mann-Whitney U-test). (b) Transmigration of wild-type and Plxna1−/− BMDCs across a lymphatic endothelial cell monolayer, assessed by time-lapse video microscopy as interactions recorded every 30 s. Yellow dotted lines indicate junctions of endothelial cells; white arrows indicate DCs in contact with lymphatic endothelial cells; red arrows (top) indicate the transmigration process of wild-type DCs. Scale bars, 50 μm. (c) Confocal microscopy (left) of wild-type and Plxna1−/− CFSE-labeled DCs added to endothelial cell monolayers, incubated for 45 min, fixed and then stained with Alexa Fluor 546–conjugated phalloidin; images were obtained with an optical section separation (z-interval) of 0.22 μm. Right, quantification of DC transmigration, presented as transmigrated DCs relative to total DCs. *P < 0.001 (Student's t-test). (d) Chemotaxis of wild-type or Plxna1−/− DC across Transwell inserts (pore size, 5 μm) layered with lymphatic endothelial cell, in response to a CCL21 gradient. *P < 0.001 (Student's t-test). Data are representative of three experiments (mean and s.d. in c,d).

  4. Sema3A-NRP1-plexin-A1 interactions are responsible for DC trafficking.
    Figure 4: Sema3A–NRP1–plexin-A1 interactions are responsible for DC trafficking.

    (a) Trafficking of wild-type DCs in the lymphatics after adoptive transfer into wild-type, Sema3a−/−, Sema6c−/− or Sema6d−/− recipient mice. *P < 0.01 (Mann-Whitney U-test). Data are pooled from three independent experiments (standard error ± 95% confidence interval). (b) Trafficking of wild-type and Nrp1sema− knock-in (KI) DCs into the lymphatics after adoptive transfer into wild-type recipients (left), and chemotaxis of wild-type and Nrp1sema− knock-in DCs through Transwell inserts (pore size, 5 μm) layered with lymphatic endothelial cells, in response to a CCL21 gradient (right). *P < 0.05 (Mann-Whitney U-test; left) and **P < 0.01 (Student's t-test; right). Data are representative of three experiments (mean and s.d.). (c) In vitro proliferation of CD4+ T cells in response to keyhole limpet hemocyanin (KLH) after immunization of wild-type, Sema3a−/−, Nrp1sema− knock-in, Sema6c−/− and Sema6d−/− mice with keyhole limpet hemocyanin in complete Freund's adjuvant. *P < 0.01 and **P < 0.001 (Student's t-test). Data are representative of three experiments (mean ± s.d.).

  5. Sema3A acts on the rear side of DCs.
    Figure 5: Sema3A acts on the rear side of DCs.

    (a) Chemotaxis of DCs in the presence of human immunoglobulin G (IgG) or recombinant Sema3A protein in the lower (left) or upper (right) chamber of a Transwell system, with CCL21 present (CCL21) or absent (−) in the lower chamber. *P < 0.01 (Student's t-test). Data are representative of three experiments (mean and s.d.). (b) DC speed in a two-dimensional DC chemotaxis assay with Sema3A or human IgG added to the opposite side of CCL21, assessing the frequency distribution (bars) and cumulative frequency distribution (lines) of the instantaneous speed. P < 0.001 (Mann-Whitney U-test). Data are representative of three independent experiments. (c) Confocal time-lapse video microscopy of BMDCs expressing green fluorescent protein–labeled plexin-A1 (plexin-A1–GFP), treated with lipopolysaccharide, suspended in type I collagen gels and placed into a Zigmond chamber with chemokine gradients; DC locomotion was examined at 1-minute intervals (time (in minutes:seconds), bottom left corner). Scale bar, 10 μm. Results are representative of three experiments. (d) Confocal z-stack imaging (left) of the localization and intensity of plexin-A1 (anti–plexin-A1 plus indocarbocyanine-labeled anti–rabbit IgG; red) and F-actin (Alexa Fluor 488–conjugated phalloidin; green) in DCs. Scale bar, 10 μm. Right, frequency of cells with no colocalization of signals. Data are representative of three independent experiments.

  6. Sema3A induces phosphorylation of MLC and promotes actomyosin contraction.
    Figure 6: Sema3A induces phosphorylation of MLC and promotes actomyosin contraction.

    (a) Confocal z-stack imaging (left) of DCs on fibronectin-coated coverslips treated with human IgG or recombinant Sema3A protein fused with human immunoglobulin Fc portion (Sema3A-Fc) and stained with antibody to phosphorylated MLC (pMLC) plus indocarbocyanine-labeled anti–rabbit IgG (bottom row); eight z-stack images with an optical section separation (z interval) of 0.36 μm were projected onto one single image. DIC (top row), differential interference contrast image. Scale bars, 10 μm. Right, frequency distribution (bars) and cumulative frequency distribution (lines) of the average intensity of dendrite regions in DCs stimulated with human IgG or Sema3A-Fc. P < 0.001 (Mann-Whitney U-test). Data are representative of three independent experiments. (b) Speed (left) and frequency distribution (bars, right) or cumulative frequency distribution (lines, right) of the instantaneous speed (right) of a single DC in response to chemokines in the presence of human IgG or Sema3A-Fc in type I collagen matrices, analyzed by time-lapse microscopy and MetaMorph software. Each symbol (left) represents an individual cell; red horizontal lines indicate the mean. *P < 0.001 (Mann-Whitney U-test). Data are representative of three independent experiments. (c) Chemotaxis of wild-type or Plxna1−/− DCs in response to CCL21 in the presence of human IgG or Sema3A-Fc; DCs were added to the upper chamber of a Transwell system with type I collagen. NS, not significant; *P < 0.05 (overall difference, one-way analysis of variance (ANOVA); post-hoc multiple comparisons, Tukey's test). Data are representative of three experiments (mean and s.d.). (d) Chemotaxis of DCs left untreated (No Tx) or treated for 30 min at 37 °C with 50 μM blebbistatin or 30 μM Y-27632 and then added to the upper chamber of a Transwell system (pore size, 5 μm) layered with type I collagen (3 mg/ml; left) and a monolayer of human dermal lymphatic microvascular endothelial cells (right), in response to CCL21 in the presence of human IgG or Sema3A-Fc in the upper chamber. *P < 0.05 and **P < 0.01 (overall difference, one-way ANOVA; post-hoc multiple comparisons, Tukey's test). Data are representative of three experiments (mean and s.d.).

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Author information

Affiliations

  1. Department of Immunopathology, Research Institute for Microbial Diseases, Osaka University, Suita, Japan.

    • Hyota Takamatsu,
    • Noriko Takegahara,
    • Yukinobu Nakagawa,
    • Tatsusada Okuno,
    • Sujin Kang,
    • Satoshi Nojima,
    • Toshihiko Toyofuku &
    • Atsushi Kumanogoh
  2. World Premier International Immunology Frontier Research Center, Osaka University, Suita, Japan.

    • Hyota Takamatsu,
    • Noriko Takegahara,
    • Yukinobu Nakagawa,
    • Tatsusada Okuno,
    • Masayuki Mizui,
    • Sujin Kang,
    • Satoshi Nojima,
    • Toshihiko Toyofuku,
    • Hitoshi Kikutani &
    • Atsushi Kumanogoh
  3. Department of Dermatology, Osaka University Graduate School of Medicine, Suita, Japan.

    • Yukinobu Nakagawa &
    • Ichiro Katayama
  4. Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, RIKEN, Wako, Japan.

    • Michio Tomura
  5. Department of Biochemistry, Cancer Research Institute, Sapporo Medical University School of Medicine, Sapporo, Japan.

    • Masahiko Taniguchi
  6. Institute of Developmental Genetics, Helmholtz Center Munich, Neuherberg, Germany.

    • Roland H Friedel
  7. Department of Developmental Biology, Stanford University, Stanford, California, USA.

    • Helen Rayburn
  8. Division of Research, Genentech, South San Francisco, California, USA.

    • Marc Tessier-Lavigne
  9. Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.

    • Yutaka Yoshida
  10. Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Japan.

    • Masayuki Mizui &
    • Hitoshi Kikutani
  11. Department of Pathology, Osaka University Graduate School of Medicine, Suita, Japan.

    • Satoshi Nojima
  12. Department of Pathology, Hyogo College of Medicine, Nishinomiya, Japan.

    • Tohru Tsujimura
  13. Department of Neurology, Osaka University Graduate School of Medicine, Suita, Japan.

    • Yuji Nakatsuji

Contributions

A.K. and H.T. designed the study and wrote the manuscript; H.T. did most of the experiments and analyzed the data with Y. Nakagawa, T.O., M.M., S.K. and S.N.; N.T. produced Sema6d−/− mice, recombinant Sema6D protein and antibody to Sema6D (anti-Sema6D); M. Tomura did two-photon microscopic experiments; M. Tanaguchi produced Sema3a−/− mice; R.H.F., H.R. and M.T.-L. produced Sema6c−/− mice; Y.Y. produced anti-plexin-A1; T. Tsujimura did histological analyses; and Y. Nakatsuji, I.K., T. Toyofuku and H.K. provided collaborative suggestions.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Text and Figures (2M)

    Supplementary Figures 1–7 and Supplementary Methods

Movies

  1. Supplementary Video 1 (2M)

    Normal to sense direction in response to chemokines in plexin-A1−/− DCs. DCs from wild-type or plexin-A1−/− mice were allowed to adhere to fibronectin-coated cover-slips and placed in a CCL21 gradient in a Zigmond chamber. DC trafficking was recorded every 30 sec by a confocal time-lapse video microscope.

  2. Supplementary Video 2 (4M)

    Plexin-A1−/− DCs exhibit impaired transmigration across a lymphatic EC-monolayer. BMDCs derived from wild-type or plexin-A1−/− mice were added to lymphatic EC monolayers, and interactions between DCs and the lymphatic ECs were recorded every 30 sec by a time-lapse video microscope. The yellow dotted lines show the cellular junctions of the ECs. White arrows indicate DCs that were contacting the lymphatic ECs.

  3. Supplementary Video 3 (980K)

    Impaired transmigration in plexin-A1−/− DCs. CFSE-labeled DCs derived from wild-type or plexin-A1−/− mice were added to lymphatic EC monolayers, incubated for 45 min, fixed, and then stained with Alexa 546-conjugated phalloidin. Confocal microscope images were obtained with an optical section separation (Z-interval) of 0.22 μm. Twelve Z-stack images were reconstituted into a 3Dimage using Imaris 3D software. Wild-type DCs penetrated from the apical to basal sides, but plexin-A1−/− DCs could not reach the basal side.

  4. Supplementary Video 4 (980K)

    Sema3A acts on the rear side of migrating DCs. Two-dimensional DC chemotaxis assays using EZ-TAXIScan were performed, in which recombinant Sema3A or human IgG was applied to the opposite side of CCL21. DC migration was recorded every 30 sec by a time-lapse video microscope.

  5. Supplementary Video 5 (192K)

    Plexin-A1 localizes to the back of migrating DCs. Plexin-A1-EGFP fusion protein-expressed BMDCs treated with LPS were suspended in type I collagen (3 mg/ml) containing 2% FCS and then placed on one side of the Zigmond chamber to cover the stage with gel. After gel was polymerized, CCL21was added to the other side. DC locomotion was examined at 1-min intervals by a confocal time-lapse video microscope.

  6. Supplementary Video 6 (1M)

    Sema3A enhances the velocity of DC migration in 3D-collagen matrices. BMDCs treated with LPS for 12 h were suspended in type I collagen (3 mg/ml) containing 2% FCS with either a human IgG or Sema3A-Fc and then placed on one side of the Zigmond chamber to cover the stage with gel. The cells were incubated at 37C for 30 min to polymerize the matrix, and then RPMI containing 0.5% BSA with CCL21 (5 μg/ml) was added to the other chamber. After a 20-min incubation, DC locomotion was examined at 1-min intervals by a confocal time-lapse video microscope.

Additional data