Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo

Nature volume 498, pages 371–375 (2013)Cite this article

Subjects

Abstract

Neutrophil recruitment from blood to extravascular sites of sterile or infectious tissue damage is a hallmark of early innate immune responses, and the molecular events leading to cell exit from the bloodstream have been well defined1,2. Once outside the vessel, individual neutrophils often show extremely coordinated chemotaxis and cluster formation reminiscent of the swarming behaviour of insects3,4,5,6,7,8,9,10,11. The molecular players that direct this response at the single-cell and population levels within the complexity of an inflamed tissue are unknown. Using two-photon intravital microscopy in mouse models of sterile injury and infection, we show a critical role for intercellular signal relay among neutrophils mediated by the lipid leukotriene B4, which acutely amplifies local cell death signals to enhance the radius of highly directed interstitial neutrophil recruitment. Integrin receptors are dispensable for long-distance migration12, but have a previously unappreciated role in maintaining dense cellular clusters when congregating neutrophils rearrange the collagenous fibre network of the dermis to form a collagen-free zone at the wound centre. In this newly formed environment, integrins, in concert with neutrophil-derived leukotriene B4 and other chemoattractants, promote local neutrophil interaction while forming a tight wound seal. This wound seal has borders that cease to grow in kinetic concert with late recruitment of monocytes and macrophages at the edge of the displaced collagen fibres. Together, these data provide an initial molecular map of the factors that contribute to neutrophil swarming in the extravascular space of a damaged tissue. They reveal how local events are propagated over large-range distances, and how auto-signalling produces coordinated, self-organized neutrophil-swarming behaviour that isolates the wound or infectious site from surrounding viable tissue.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Neutrophil extravascular swarming dynamics.
Figure 2: LTB4 promotes neutrophil recruitment from distant sites.
Figure 3: Integrin and GPCR signalling at the neutrophil cluster.
Figure 4: LTB4 requirement for swarming in infected lymph nodes.

Similar content being viewed by others

References

  1. Nathan, C. Points of control in inflammation. Nature 420, 846–852 (2002)

    Article  CAS  ADS  PubMed  Google Scholar 

  2. Ley, K., Laudanna, C., Cybulsky, M. I. & Nourshargh, S. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nature Rev. Immunol. 7, 678–689 (2007)

    Article  CAS  Google Scholar 

  3. Chtanova, T. et al. Dynamics of neutrophil migration in lymph nodes during infection. Immunity 29, 487–496 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ng, L. G. et al. Visualizing the neutrophil response to sterile tissue injury in mouse dermis reveals a three-phase cascade of events. J. Invest. Dermatol. 131, 2058–2068 (2011)

    Article  CAS  PubMed  Google Scholar 

  5. Peters, N. C. et al. In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies. Science 321, 970–974 (2008)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  6. Bruns, S. et al. Production of extracellular traps against Aspergillus fumigatus in vitro and in infected lung tissue is dependent on invading neutrophils and influenced by hydrophobin RodA. PLoS Pathog. 6, e1000873 (2010)

    Article  PubMed  PubMed Central  Google Scholar 

  7. Yipp, B. G. et al. Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo. Nature Med. 81, 1386–1393 (2012)

    Article  Google Scholar 

  8. Liese, J., Rooijakkers, S. H., van Strijp, J. A., Novick, R. P. & Dustin, M. L. Intravital two-photon microscopy of host–pathogen interactions in a mouse model of Staphylococcus aureus skin abscess formation. Cell Microbiol. http://dx.doi.org/10.1111/cmi.12085 (2012)

  9. Harvie, E. A., Green, J. M., Neely, M. N. & Huttenlocher, A. Innate immune response to Streptococcus iniae infection in zebrafish larvae. Infect. Immun. 81, 110–121 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kreisel, D. et al. In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation. Proc. Natl Acad. Sci. USA 107, 18073–18078 (2010)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  11. Nakasone, E. S. et al. Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance. Cancer Cell 21, 488–503 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lämmermann, T. et al. Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature 453, 51–55 (2008)

    Article  ADS  PubMed  Google Scholar 

  13. McDonald, B. et al. Intravascular danger signals guide neutrophils to sites of sterile inflammation. Science 330, 362–366 (2010)

    Article  CAS  ADS  PubMed  Google Scholar 

  14. Guggenberger, C., Wolz, C., Morrissey, J. A. & Heesemann, J. Two distinct coagulase-dependent barriers protect Staphylococcus aureus from neutrophils in a three dimensional in vitro infection model. PLoS Pathog. 8, e1002434 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. McDonald, B. & Kubes, P. Cellular and molecular choreography of neutrophil recruitment to sites of sterile inflammation. J. Mol. Med. 89, 1079–1088 (2011)

    Article  CAS  PubMed  Google Scholar 

  16. Sánchez-Madrid, F. & del Pozo, M. A. Leukocyte polarization in cell migration and immune interactions. EMBO J. 18, 501–511 (1999)

    Article  PubMed  PubMed Central  Google Scholar 

  17. Cho, H. et al. The loss of RGS protein-Gαi2 interactions results in markedly impaired mouse neutrophil trafficking to inflammatory sites. Mol. Cell. Biol. 32, 4561–4571 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kim, N. D., Chou, R. C., Seung, E., Tager, A. M. & Luster, A. D. A unique requirement for the leukotriene B4 receptor BLT1 for neutrophil recruitment in inflammatory arthritis. J. Exp. Med. 203, 829–835 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Chen, M. et al. Neutrophil-derived leukotriene B4 is required for inflammatory arthritis. J. Exp. Med. 203, 837–842 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Oyoshi, M. K. et al. Leukotriene B4-driven neutrophil recruitment to the skin is essential for allergic skin inflammation. Immunity 37, 747–758 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chou, R. C. et al. Lipid-cytokine-chemokine cascade drives neutrophil recruitment in a murine model of inflammatory arthritis. Immunity 33, 266–278 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sadik, C. D., Kim, N. D., Iwakura, Y. & Luster, A. D. Neutrophils orchestrate their own recruitment in murine arthritis through C5aR and FcγR signaling. Proc. Natl Acad. Sci. USA 109, E3177–E3185 (2012)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  23. Afonso, P. V. et al. LTB4 is a signal-relay molecule during neutrophil chemotaxis. Dev. Cell 22, 1079–1091 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. DiPersio, J. F., Billing, P., Williams, R. & Gasson, J. C. Human granulocyte-macrophage colony-stimulating factor and other cytokines prime human neutrophils for enhanced arachidonic acid release and leukotriene B4 synthesis. J. Immunol. 140, 4315–4322 (1988)

    CAS  PubMed  Google Scholar 

  25. Malawista, S. E., de Boisfleury Chevance, A., van Damme, J. & Serhan, C. N. Tonic inhibition of chemotaxis in human plasma. Proc. Natl Acad. Sci. USA 105, 17949–17954 (2008)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  26. Palmblad, J. et al. Leukotriene B4 is a potent and stereospecific stimulator of neutrophil chemotaxis and adherence. Blood 58, 658–661 (1981)

    CAS  PubMed  Google Scholar 

  27. Ford-Hutchinson, A. W., Bray, M. A., Doig, M. V., Shipley, M. E. & Smith, M. J. Leukotriene B, a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 286, 264–265 (1980)

    Article  CAS  ADS  PubMed  Google Scholar 

  28. Calderwood, D. A. & Ginsberg, M. H. Talin forges the links between integrins and actin. Nature Cell Biol. 5, 694–697 (2003)

    Article  CAS  PubMed  Google Scholar 

  29. Kastenmüller, W., Torabi-Parizi, P., Subramanian, N., Lämmermann, T. & Germain, R. N. A spatially-organized multicellular innate immune response in lymph nodes limits systemic pathogen spread. Cell 150, 1235–1248 (2012)

    Article  PubMed  PubMed Central  Google Scholar 

  30. Rudolph, U. et al. Ulcerative colitis and adenocarcinoma of the colon in Gαi2-deficient mice. Nature Genet. 10, 143–150 (1995)

    Article  CAS  PubMed  Google Scholar 

  31. Pero, R. S. et al. Gαi2-mediated signaling events in the endothelium are involved in controlling leukocyte extravasation. Proc. Natl Acad. Sci. USA 104, 4371–4376 (2007)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  32. Potocnik, A. J., Brakebusch, C. & Fassler, R. Fetal and adult hematopoietic stem cells require beta1 integrin function for colonizing fetal liver, spleen, and bone marrow. Immunity 12, 653–663 (2000)

    Article  CAS  PubMed  Google Scholar 

  33. Petrich, B. G. et al. Talin is required for integrin-mediated platelet function in hemostasis and thrombosis. J. Exp. Med. 204, 3103–3111 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Gao, J. L. et al. Impaired host defense, hematopoiesis, granulomatous inflammation and type 1-type 2 cytokine balance in mice lacking CC chemokine receptor 1. J. Exp. Med. 185, 1959–1968 (1997)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Gao, J. L., Lee, E. J. & Murphy, P. M. Impaired antibacterial host defense in mice lacking the N-formylpeptide receptor. J. Exp. Med. 189, 657–662 (1999)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Chen, K. et al. A critical role for the G protein-coupled receptor mFPR2 in airway inflammation and immune responses. J. Immunol. 184, 3331–3335 (2010)

    Article  CAS  PubMed  Google Scholar 

  37. Radin, J. N. et al. β-Arrestin 1 participates in platelet-activating factor receptor-mediated endocytosis of Streptococcus pneumoniae. Infect. Immun. 73, 7827–7835 (2005)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  38. Adachi, O. et al. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9, 143–150 (1998)

    Article  CAS  PubMed  Google Scholar 

  39. Riedl, J. et al. Lifeact mice for studying F-actin dynamics. Nature Methods 7, 168–169 (2010)

    Article  CAS  PubMed  Google Scholar 

  40. Faust, N., Varas, F., Kelly, L. M., Heck, S. & Graf, T. Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. Blood 96, 719–726 (2000)

    CAS  PubMed  Google Scholar 

  41. Glaccum, M. B. et al. Phenotypic and functional characterization of mice that lack the type I receptor for IL-1. J. Immunol. 159, 3364–3371 (1997)

    CAS  PubMed  Google Scholar 

  42. Gaiser, M. R. et al. Cancer-associated epithelial cell adhesion molecule (EpCAM; CD326) enables epidermal Langerhans cell motility and migration in vivo. Proc. Natl Acad. Sci. USA 109, E889–E897 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Li, J. L. et al. Intravital multiphoton imaging of immune responses in the mouse ear skin. Nature Protocols 7, 221–234 (2012)

    Article  CAS  PubMed  Google Scholar 

  44. Davies, D. G. et al. The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280, 295–298 (1998)

    Article  CAS  ADS  PubMed  Google Scholar 

  45. Boxio, R., Bossenmeyer-Pourie, C., Steinckwich, N., Dournon, C. & Nusse, O. Mouse bone marrow contains large numbers of functionally competent neutrophils. J. Leukoc. Biol. 75, 604–611 (2004)

    Article  CAS  PubMed  Google Scholar 

  46. Woodfin, A. et al. The junctional adhesion molecule JAM-C regulates polarized transendothelial migration of neutrophils in vivo. Nature Immunol. 12, 761–769 (2011)

    Article  CAS  Google Scholar 

  47. Kühn, R., Schwenk, F., Aguet, M. & Rajewsky, K. Inducible gene targeting in mice. Science 269, 1427–1429 (1995)

    Article  ADS  PubMed  Google Scholar 

  48. Oh, H., Siano, B. & Diamond, S. Neutrophil isolation protocol. J. Vis. Exp. 17, 745 (2008)

    Google Scholar 

  49. Pflicke, H. & Sixt, M. Preformed portals facilitate dendritic cell entry into afferent lymphatic vessels. J. Exp. Med. 206, 2925–2935 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank L. Birnbaumer, R. Fässler, E. Tuomanen, P. M. Murphy, S. Akira, S. Monkley, D. Critchley, R. Wedlich-Söldner and M. Sixt for providing mice for this study, J.G. Egen and J. Tang for assistance with imaging, M. Parsek for providing P. aeruginosa–GFP, J.H. Kehrl, P.M. Murphy, R. Varma and members of the Germain laboratory for discussions. This work was supported by the Intramural Research Program of National Institute of Allergy and Infectious Diseases and National Cancer Institute, National Institutes of Health. T.L. was supported by a Human Frontiers Science Program Long-Term Fellowship. W.K. is presently a member of the Deutsche Forschungsgemeinschaft (DFG)-funded excellence cluster ImmunoSensation, Bonn, Germany.

Author information

Authors and Affiliations

  1. Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, 20892-0421, Maryland, USA

    Tim Lämmermann, Bastian R. Angermann, Wolfgang Kastenmüller & Ronald N. Germain

  2. Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, 20892-4256, Maryland, USA

    Philippe V. Afonso & Carole A. Parent

  3. Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, 21702-1201, Maryland, USA

    Ji Ming Wang

  4. Institutes of Molecular Medicine and Experimental Immunology (IMMEI), University of Bonn, 53105 Bonn, Germany,

    Wolfgang Kastenmüller

Authors
  1. Tim Lämmermann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Philippe V. Afonso
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bastian R. Angermann
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ji Ming Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wolfgang Kastenmüller
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Carole A. Parent
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ronald N. Germain
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.L. and R.N.G. designed the experiments, interpreted the data and wrote the paper. P.V.A., J.M.W. and C.A.P. contributed to data interpretation and experimental design, as well as the development of the final version of the paper. T.L. performed all skin-imaging experiments. W.K. performed intravital imaging of infected lymph nodes. B.R.A. developed the software for graphical display of imaging data. T.L., B.R.A. and P.V.A. conducted quantitative analysis of the data.

Corresponding authors

Correspondence to Tim Lämmermann or Ronald N. Germain.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-16, a Supplementary Table, Supplementary Notes 1-2 and additional references. (PDF 12491 kb)

Differential kinetics of neutrophil and monocyte directed migration towards a focal tissue damage site

Three hours after induction of a 15 s ear skin trauma, endogenous blood-circulating inflammatory cells had entered into the extravascular space of the ear dermis of a DsRed+/+Cx3cr1gfp/gfpTyrc-2J/c-2J mouse before laser-induced focal tissue damage was induced (center). This representative video shows the immediate response of fast-migrating neutrophils (small red cells) towards a focal tissue damage site, while monocytes (green) migrate with slower speeds and follow the developing neutrophil cluster with delay. Accumulating, small DsRed-positive cells were identified as Ly6G-positive neutrophils in Supplementary Fig. 3a differing from other static stromal elements (vessels, hair follicles, skin-resident cells) that are also pseudo-colored in red. Graphic analysis of this video is presented in Fig. 1b and Supplementary Fig. 3a. Similar neutrophil and monocyte kinetics were observed in DsRed+/+Cx3cr1gfp/+Tyrc-2J/c-2J mice. Two-photon intravital microscopy (x, y, z = 246µm, 246µm, 26µm; merge of z-stack), time-lapse over 2 h (10 frames/s). (MOV 10371 kb)

Biphasic neutrophil swarming response to focal tissue damage

Bone marrow-derived neutrophils from C57BL/6 mice were CMFDA-labeled and injected intradermally into the ventral ear skin of a Tyrc-2J/c-2J mouse 3 h before laser-induced focal tissue damage. This representative video shows the biphasic chemotactic response of neutrophils (pseudo-colored in green) sensing the focal tissue damage (autofluorescence, green) (left) within the fibrous collagenous connective tissue of the ear skin dermis (visualized by second harmonic generation, white) (right). Graphic analysis of this video is presented in Fig. 1c. Two-photon intravital microscopy (x, y, z = 322µm, 351µm, 16µm; merge of z-stack), time-lapse over 49 min 30 s (10 frames/s). (MOV 4553 kb)

Cell death/lysis of few neutrophils leads to amplified recruitment of neutrophils from distant sites

Neutrophils from C57BL/6 mice were CMFDA-labeled and injected together with propidium iodide (PI) intradermally into the ventral ear skin of a Tyrc-2J/c-2J mouse 2-4 h before laser-induced focal tissue damage. This video shows a single early-recruited neutrophil at the damage site that loses its intracellular dye (green) while its nucleus becomes PI-positive (red) as an indicator of cell death/lysis. Exactly at that time, neutrophils from distant sites increase in speed and directionality and migrate towards the dying neutrophil (left; with motion paths over the last 5 min as white dragon tails, right). At the beginning of the video, some skin-resident cells are PI-positive as a consequence of the intradermal injection and/or unspecific dye uptake. Graphic analysis of this video is presented in Fig. 2a and Supplementary Fig. 4. Two-photon intravital microscopy (x, y, z = 116µm, 155µm, 22µm; merge of z-stack), time-lapse over 21 min 30 s (10 frames/s). (MOV 1557 kb)

Cxcr2-/-, Fpr1-/-, and Fpr2-/- neutrophils show unaltered interstitial chemotaxis to sites of focal tissue damage

Various GPCR-deficient (Cxcr2-/-, upper row; Fpr2-/-, middle row; Fpr1-/-, lower row) and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of Tyrc-2J/c-2J mice 2-4 h before laser-induced focal tissue damage. This video shows representative experiments of GPCR-deficient (pseudo-colored in yellow) and control neutrophils (pseudo-colored in turquois) migrating side-by-side towards a damage site (left panels) with motion paths over the last 5 min as dragon tails in the corresponding pseudo-color (WT, middle; KO, right panels). Graphic analysis of several experiments is presented in Supplementary Fig. 8 and did not reveal any differences between knockout and control neutrophil dynamics. Two-photon intravital microscopy (x, y, z = 105µm, 147µm, 14-22µm; merge of z-stack), time-lapse over 29 min 30 s (10 frames/s). (MOV 2681 kb)

Leukotriene B4 optimizes neutrophil interstitial recruitment to sites of focal damage

Ltb4r1-/- and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a Tyrc-2J/c-2J mouse 3 h before laser-induced, 25 µm-sized focal tissue damage. This representative video shows Ltb4r1-/- (pseudo-colored in yellow) and control neutrophils (pseudo-colored in turquois) migrating side-by-side towards the damage site (left) with motion paths over the last 10 min as dragon tails in the corresponding pseudo-color (middle and right). Graphic analysis of several experiments is presented in Fig. 2b and Supplementary Fig. 9 and revealed impaired recruitment of Ltb4r1-/- neutrophils with both chemotactic index and velocity reduced at later time points. Two-photon intravital microscopy (x, y, z = 167µm, 209µm, 18µm; merge of z-stack), time-lapse over 30 min (10 frames/s). (MOV 2640 kb)

Leukotriene B4 optimizes neutrophil interstitial recruitment from distant tissue sites

Ltb4r1-/- and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a Tyrc-2J/c-2J mouse 3 h before laser-induced, 10 µm-sized focal tissue damage. This representative video shows Ltb4r1-/- (pseudo-colored in yellow) and control neutrophils (pseudo-colored in turquois) migrating side-by-side towards the damage site (left) with motion paths over the last 10 min as dragon tails in the corresponding pseudo-color (middle and right). Graphic analysis of this experiment is presented in Fig. 2c and Supplementary Fig. 10 and revealed that Ltb4r1-/- neutrophils are poorly recruited from distant tissue sites. After the experiment, non-motile neutrophils responded with directed migration to a second laser damage (that was set closer to these cells) confirming viability and responsiveness of these cells (not shown). Two-photon intravital microscopy (x, y, z = 167µm, 302µm, 20µm; merge of z-stack), time-lapse over 30 min (10 frames/s). (MOV 2713 kb)

Neutrophil-derived LTB4 optimizes neutrophil interstitial recruitment to sites of focal damage

Ltb4r1-/- and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a leukotriene-deficient Alox5-/-Tyrc-2J/c-2J mouse 3 h before laser-induced focal tissue damage. This representative video shows Ltb4r1-/- (pseudo-colored in yellow) and control neutrophils (pseudo-colored in turquois) migrating side-by-side towards the damage site (left) with motion paths over the last 10 min as dragon tails in the corresponding pseudo-color (middle and right). Graphic analysis of several experiments is presented in Fig. 2d and Supplementary Fig. 11 and revealed that neutrophil-derived LTB4 improves the interstitial recruitment response of the neutrophil population. Two-photon intravital microscopy (x, y, z = 171µm, 165µm, 16µm; merge of z-stack), time-lapse over 34 min 30 s (10 frames/s). (MOV 3084 kb)

Interstitial neutrophil recruitment does not require high-affinity integrin-mediated adhesion

Talin-deficient (Tln1-/-) and control neutrophils were differentially dye labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of Tyrc-2J/c-2J mice 2-3 h before laser-induced focal tissue damage was performed. This video shows two representative experiments of Tln1-/- and control neutrophils migrating side-by-side towards a damage site. In upper panels, Tln1-/- neutrophils were labeled with CMTPX (pseudo-colored in red) and control neutrophils with CMFDA (pseudo-colored in green). In lower panels, cell dyes were switched. Cell migration in relation to the fibrous collagenous connective tissue of the ear skin dermis was visualized by second harmonic generation (white; middle column) and motion paths over the last 10 min are presented as yellow dragon tails. Graphic analysis of several experiments is presented in Supplementary Fig. 13 and shows that active integrins are dispensable for interstitial neutrophil recruitment. Two-photon intravital microscopy (x, y, z = 149µm, 159µm, 16-20µm; merge of z-stack), time-lapse over 26 min 30 s (10 frames/s). (MOV 2428 kb)

Congregating neutrophils displace fibrous collagen bundles from the wound center

This video shows the collagenous fiber network (visualized by second harmonic generation) as neutrophils accumulate at the focal damage site in the dermis of a LysMgfp/+Tyrc-2J/c-2J mouse (Fig. 3a). Fiber bundles are displaced over time in the x-y-axis. Some disappearing fibers are also displaced in the z-axis out of the imaging volume (not shown). Two-photon intravital microscopy (x, y, z = 122µm, 49µm, 30µm; merge of z-stack), time-lapse over 32 min 30 s (10 frames/s). (MOV 1498 kb)

Neutrophils migrate in the collagen-free center in close contact with each other

15 s ear skin trauma was induced in a DsRed+/+Tyrc-2J/c-2J mouse (for endogenous blood-circulating neutrophils to enter the extravascular space), 1 h later bone marrow-derived neutrophils from a Lifeact-GFP transgenic mouse were injected intradermally into the ventral ear skin followed 2 h later by laser-induced focal tissue damage. This representative video shows a zoom into the collagen-free wound center where both injected Lifeact-GFP neutrophils (pseudo-colored in green) and endogenous DsRed-positive neutrophils (pseudo-colored in red) migrate in close contact with each other. Lifeact-GFP outlines the actin cortex of neutrophils to demarcate the cell borders of injected individual cells. Two-photon intravital microscopy (x, y, z = 78µm, 78µm, 3µm; merge of z-stack), time-lapse over 34 min 28 s (10 frames/s). (MOV 12302 kb)

Active integrins are essential for sustained access to the neutrophil cluster in the collagen-free wound center

Talin-deficient (Tln1-/-) and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a Tyrc-2J/c-2J mouse 3 h before laser-induced focal tissue damage. This representative video shows Tln1-/- (pseudo-colored in green) and control neutrophils (pseudo-colored in red) accumulating at the damage site (left). Neutrophil movement in relation to collagen displacement is visualized by second harmonic generation (pseudo-colored in white) (middle, right). Lower panels show zoom-in on the margin of collagen displacement. The analysis of the clustering response is presented in Fig. 3b,c. Two-photon intravital microscopy (x, y, z = 368µm, 368µm, 10µm; merge of z-stack), time-lapse over 29 min 30 s (10 frames/s). (MOV 2618 kb)

β2 integrins are essential for access of the neutrophil cluster in the collagen-free wound center

Itgb2-/- and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a Tyrc-2J/c-2J mouse 3 h before laser-induced focal tissue damage. This representative video shows Itgb2-/- (pseudo-colored in green) and control neutrophils (pseudo-colored in red) accumulating at the damage site (left). Neutrophil movement in relation to collagen displacement is visualized by second harmonic generation (pseudo-colored in white) (middle, right). The analysis of the clustering response is presented in Fig. 3c and Supplementary Fig. 15a. Two-photon intravital microscopy (x, y, z = 368µm, 368µm, 12µm; merge of z-stack), time-lapse over 29 min 30 s (10 frames/s). (MOV 2646 kb)

Gαi2-, not Gαi3-dependent signals control neutrophil clustering

Gnai isoform-deficient and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a Tyrc-2J/c-2J mouse 3 h before laser-induced focal tissue damage. This video shows representative experiments of Gnai2-/- (upper panels) or Gnai3-/- (lower panels) (both pseudo-colored in green) and control neutrophils (pseudo-colored in red) accumulating at the damage site. Neutrophil movement in relation to collagen displacement is visualized by second harmonic generation (pseudo-colored in white) (middle, right). The analysis of the clustering response is presented in Fig. 3f and Supplementary Fig. 15b. Two-photon intravital microscopy (x, y, z = 185µm, 185µm, 14µm; merge of z-stack), time-lapse over 56 min 30 s (10 frames/s). (MOV 4956 kb)

Neutrophil-derived LTB4 controls neutrophil clustering

Ltb4r1-/- and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a Tyrc-2J/c-2J mouse (upper panels) or leukotriene-deficient Alox5-/-Tyrc-2J/c-2J mouse (lower panels) 3 h before laser-induced focal tissue damage. This video shows representative experiments of Ltb4r1-/- (pseudo-colored in green) and control neutrophils (pseudo-colored in red) accumulating at the damage site of the respective host. Neutrophil movement in relation to collagen displacement is visualized by second harmonic generation (pseudo-colored in white) (middle and right). The analysis of the clustering response is presented in Fig. 3d-f and Supplementary Fig. 15c and 16a. Two-photon intravital microscopy (x, y, z = 185µm, 185µm, 14µm; merge of z-stack), time-lapse over 30 min (10 frames/s). (MOV 2657 kb)

LTB4 requirement for swarming in infected lymph nodes

Mice were infected with P. aeruginosa-GFP in the footpad before 2P-IVM was performed on the draining popliteal lymph nodes when comparable neutrophil numbers were present in the subcapsular sinus at indicated times after infection (WT: 3 h, Ltb4r1-/-: 4.5 h). Neutrophil-GFP signal is pseudo-colored (heat map) to indicate neutrophil clusters (white) in WT-LysMgfp/+ mice (left) and Ltb4r1-/-LysMgfp/+ mice (right). The analysis of the clustering response is presented in Fig. 4a-c. Time-lapse over 39 min 20 s (10 frames/s). (MOV 2568 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lämmermann, T., Afonso, P., Angermann, B. et al. Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo . Nature 498, 371–375 (2013). https://doi.org/10.1038/nature12175

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature12175

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing