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. 2013 Jun 20;498(7454):371-5.
doi: 10.1038/nature12175. Epub 2013 May 26.

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

Tim Lämmermann  1 , Philippe V Afonso, Bastian R Angermann, Ji Ming Wang, Wolfgang Kastenmüller, Carole A Parent, Ronald N Germain
Affiliations

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

Tim Lämmermann et al. Nature. .

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 defined. Once outside the vessel, individual neutrophils often show extremely coordinated chemotaxis and cluster formation reminiscent of the swarming behaviour of insects. 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 migration, 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.

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Figures

Figure 1
Figure 1. Neutrophil extravascular swarming dynamics
2P-IVM on intact ear dermis of anesthetized mice. Interstitial cell recruitment towards focal damage (blue dotted circle) was recorded. a, b, Time-lapse sequence of endogenous innate immune cell dynamics in DsRed+/−Lyz2gfp/+Tyrc-2J/c-2J mice (myelomonocytic cells in green-yellow, stroma in red) (a) and DsRed+/+Cx3cr1gfp/gfp Tyrc-2J/c-2J mice (macrophages/monocytes in green, neutrophils and stroma in red). Cell tracks over the last 10 min (n=4) (b, bottom). Scale bars, 50 µm. Time, h:min. c, Distance-time plot (DTP) of intradermal (i.d.) injected bone marrow neutrophils; individual cell-migration paths towards the damage site are each highlighted with instantaneous chemotactic index (colour) and velocity (opacity). Representative experiment of n=169.
Figure 2
Figure 2. LTB4 promotes neutrophil recruitment from distant sites
a, 2P-IVM images of a single neutrophil becoming propidium iodide-positive (arrow) at 13 min and its correlation to neutrophil-amplified chemotaxis (white tracks). DTP analysis for migration paths coloured as in Fig. 1. b, Comparative analysis of interstitial recruitment after i.d. co-injection of Ltb4r1−/− and wild-type (WT) neutrophils into Tyrc-2J/c-2J mice. DTP of one representative experiment (n=8). c, Time-course of migration tracks towards 10 µm-damage. Time, h:min. Scale bars, 20 µm (a), 50 µm (c). Track durations, 5 min (a), 10 min (c). d, Comparative analysis of interstitial recruitment after i.d. co-injection of Ltb4r1−/− knockout (KO) and wild-type neutrophils into Alox5−/− Tyrc-2J/c-2J mice. Radial velocity-time plots with regression lines showing the recruitment dynamics for three individual experiments (of n=7). n (in graph) indicates the number of analyzed tracks. Each dot represents the instantaneous radial velocity for one cell at that time point.
Figure 3
Figure 3. Integrin and GPCR signaling at the neutrophil cluster
a, After focal damage (blue dotted circle), congregating neutrophils rearrange collagenous fibers (visualized by collagen second harmonic generation, SHG). Time, h:min. Scale bar, 50 µm. b–f, Comparative analysis of neutrophil clustering after i.d. co-injection of gene-deficient and wild-type neutrophils into Tyrc-2J/c-2J or Alox5−/− Tyrc-2J/c-2J mice. b, d, 2P-IVM images at the endpoint of the clustering response. Scale bars, 50 µm. c, f, Accumulation index as quantitative parameter for neutrophil entry into the collagen-free wound center. Each dot represents analysis of one damage site. Median in red. ns, non-significant (Mann Whitney U-test). (e) Time-course of neutrophil accumulation in the wound center. Three representative experiments are presented for each gene-deficiency.
Figure 4
Figure 4. LTB4 requirement for swarming in infected lymph nodes
Mice were infected with P. aeruginosa-GFP in the footpad of mice and 2P-IVM was then performed on the draining popliteal lymph nodes at the time indicated. a, Time-lapse sequence of infected subcapsular sinuses of wild-type Lyz2gfp/+ (top) and Ltb4r1−/−Lyz2gfp/+ (bottom) mice. Analysis was performed when comparable neutrophil numbers were in the sinus (wild-type: 3 h, Ltb4r1−/−: 4.5 h). Neutrophil-GFP signal is pseudo-coloured (heat map) to indicate neutrophil clusters (white). Time, h:min. Scale bars, 100 µm. b, c, Quantification of cluster diameter (b) and persistence (c), data pooled from three independent experiments. b, ***: P<0.001 (Student’s t-test), c, **: P<0.01 (Mann-Whitney U-test). Red bars, mean (b), median (c).
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