Neutrophils scan for activated platelets to initiate inflammation

Abstract

Immune and inflammatory responses require leukocytes to migrate within and through the vasculature, a process that is facilitated by their capacity to switch to a polarized morphology with an asymmetric distribution of receptors. We report that neutrophil polarization within activated venules served to organize a protruding domain that engaged activated platelets present in the bloodstream. The selectin ligand PSGL-1 transduced signals emanating from these interactions, resulting in the redistribution of receptors that drive neutrophil migration. Consequently, neutrophils unable to polarize or to transduce signals through PSGL-1 displayed aberrant crawling, and blockade of this domain protected mice against thromboinflammatory injury. These results reveal that recruited neutrophils scan for activated platelets, and they suggest that the neutrophils' bipolarity allows the integration of signals present at both the endothelium and the circulation before inflammation proceeds.

Figure 1. Neutrophils recruited to inflamed venules interact with activated platelets via protruding PSGL-1 clusters

(A) Micrographs of polarized neutrophils interacting with platelets (red; yellow arrowheads) through the leading edge or the CD62L-labeled uropod (blue). (B) Quantification of total or domain-specific platelet interactions in wild-type mice or mice deficient in P-selectin (Selp^–/–), PSGL-1 (Selplg^–/–) or Mac-1 (Itgam^–/–); n=5-8 mice, 38-133 interactions. (C) In vivo receptor distribution on polarized wild-type neutrophils. (D) Examples of luminal and lateral projections from 3D reconstructions of polarized Dock2-GFP neutrophils (see also Movie S4). (E) Frequency of neutrophils extending PSGL-1 clusters into the lumen (Lu), laterally (La) or between the cell body and the endothelium (En). n= 6 mice, 251 cells. (F) 3D reconstructions of an inflamed vessel showing the distribution of PSGL-1 clusters (see Movie S5). (G) Representative micrographs of neutrophils interacting with non-activated (arrow) or activated P-selectin+ platelets (arrowhead); and quantification of interactions of each domain with P-selectin+ or JON/A+ platelets. n=3-4 mice, 66-116 interactions. Scale bars, 10 μm. Bars show mean ± SEM. *, p<0.05; ***, p<0.001, one-way ANOVA analysis with Tukey's post-hoc test.

Figure 2. PSGL-1 controls intravascular motility and the distribution of Mac-1 and CXCR2

(A) Tracks of crawling neutrophils within inflamed vessels in untreated wild-type mice, mice deficient in PSGL-1 (Selplg^–/–) or Mac-1 (Itgam^–/–), and mice depleted of platelets using anti-platelet serum or treated with a PSGL-1-blocking antibody. (B) Quantification of the crawling displacements and instantaneous velocities of neutrophils in the same groups as (A); n=50-56 cells, 4-9 mice. (C) Tracks of neutrophils with conditional deletion of Cdc42 or expressing a mutant form of PSGL-1 that lacks the cytoplasmic tail (PSGL-1^ΔCyt), and (D) quantification of the displacement per minute and instantaneous velocities of adhered neutrophils. n=50-55 cells, 3-5 mice. (E) Representative micrographs and quantification (F) of the in vivo distribution of CXCR2 and Mac-1 in polarized neutrophils from wild-type and PSGL- 1-deficient mice; n=17-19 cells, 3 mice. Scale bar, 10 μm. Data show mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001; ANOVA with Tukey's multigroup test (B) or unpaired t-test (F).

Figure 3. PSGL-1 at the uropod becomes a preferred docking site for platelets during pathological inflammation

(A) Survival curves of Balb/c mice treated with LPS alone or LPS plus anti-MHC-I to induce ALI. Neutrophils were depleted using anti-Ly6G and platelets using anti-platelet serum prior to induction of ALI. n=5-20 mice. (B) Left: representative micrographs of inflamed venules during ALI Asterisk indicates platelets interacting with the uropod of neutrophils. Right: quantification of platelet interactions with the leading edge or uropod in control (LPS only) and ALI-induced mice. Scale bar, 10 μm. n=3-4 mice, 32-73 interactions. (C) Frequency of interactions with the leading edge or uropod in TNFα-treated or ALI-induced mice, and distribution of interactions in WT mice, and mice deficient in PSGL-1 (Selplg^–/–) or Mac-1 (Itgam^–/–). n=3-5 mice, 23-137 interactions. (D) Frequency of interactions with the leading edge or uropod during sepsis in WT mice, and mice deficient in PSGL-1 or Mac-1. n= 3- 4 mice, 32-56 interactions. Bars show mean ± SEM. *, p<0.05; ***, p<0.001 as determined by ANOVA analysis with Tukey's multigroup test.

Figure 4. PSGL-1-mediated interactions trigger vascular injury

(A) Survival curves of Balb/c mice treated with LPS alone or LPS + anti-MHC-I to induce ALI. Absence of Mac-1 or inhibition of PSGL-1 protect from death; n=5-19 mice. (B) Representative axial slices of the thorax of Balb/c mice at different times after induction of ALI. White signal in the pulmonary space identifies edema, which is quantified in (C); n=7-8 mice per group. (D) Quantification of hepatic injury as levels of AST and ALT transaminases in plasma of the indicated group of mice 24h after treatment with LPS; n=7-11 mice. (E) Representative brain sections of wild-type mice 24h after inducing ischemia showing vessels at increasing magnifications, and intravascular neutrophil (Ly6G, green)-platelet (CD41, red) aggregates. Scale bars, 10 μm. (F) Percentages of infarcted hemispheres 24h after arterial occlusion in control wild-type mice (WT), mice deficient in Mac-1 (Itgam^–/–) and WT mice after blocking PSGL-1. Images are representative brain sections stained with TTC showing the extent of ischemia as white areas with a red outline; n=5- 8 mice. Bars show mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001, ANOVA analysis with Tukey's multigroup test.