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

Nature Immunology volume 11, pages 594–600 (2010)Cite this article

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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.

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Figure 1: Plxna1−/− mice show impaired T cell responses due to defects in the migration of DCs into the lymph nodes.
Figure 2: Uptake of FITC-dextran and responses to chemokines are not affected in Plxna1−/− DCs.
Figure 3: Impaired transmigration of Plxna1−/− DCs across the lymphatics.
Figure 4: Sema3A–NRP1–plexin-A1 interactions are responsible for DC trafficking.
Figure 5: Sema3A acts on the rear side of DCs.
Figure 6: Sema3A induces phosphorylation of MLC and promotes actomyosin contraction.

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Acknowledgements

We thank D.D. Ginty and A.L. Kolodkin (Howard Hughes Medical Institute) for neuropilin-1-mutant mice; W.R. Heath (University of Melbourne) for OT-II-transgenic mice; and T. Yazawa for technical support. Supported by the Japan Society for the Promotion of Science (Research Fellowships for Young Scientists to H.T.), the US National Institutes of Health (R01NS065048 to Y.Y.), the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Ministry of Health, Labour and Welfare, the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (A.K.), the Target Protein Research Program of the Japan Science and Technology Agency (T.T. and A.K.), the Uehara Memorial Foundation (A.K.) and the Takeda Scientific Foundation (T.T., N.T. and A.K.).

Author information

Authors and 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

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  1. Hyota Takamatsu
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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.

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Correspondence to Atsushi Kumanogoh.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Methods (PDF 1548 kb)

Supplementary Video 1

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. (MOV 1612 kb)

Supplementary Video 2

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. (MOV 4486 kb)

Supplementary Video 3

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. (MOV 979 kb)

Supplementary Video 4

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. (MOV 977 kb)

Supplementary Video 5

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. (MOV 191 kb)

Supplementary Video 6

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. (MOV 1449 kb)

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Takamatsu, H., Takegahara, N., Nakagawa, Y. et al. Semaphorins guide the entry of dendritic cells into the lymphatics by activating myosin II. Nat Immunol 11, 594–600 (2010). https://doi.org/10.1038/ni.1885

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