Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo (original) (raw)

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
  2. Philippe V. Afonso
  3. Bastian R. Angermann
  4. Ji Ming Wang
  5. Wolfgang Kastenmüller
  6. Carole A. Parent
  7. Ronald N. Germain

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 toTim Lämmermann or Ronald N. Germain.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information (download PDF )

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 (download MOV )

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 (download MOV )

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 (download MOV )

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 (download MOV )

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 (download MOV )

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 (download MOV )

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 (download MOV )

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 (download MOV )

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 (download MOV )

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 (download MOV )

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 (download MOV )

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 (download MOV )

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 (download MOV )

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 (download MOV )

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 (download MOV )

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

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