CD31 signaling promotes the detachment at the uropod of extravasating neutrophils allowing their migration to sites of inflammation
- Université Paris Cité and Université Sorbonne Paris Nord, INSERM, Laboratory for Vascular Translational Science (LVTS), France
- Centre for Immunobiology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, United Kingdom
- Université Paris Cité, INSERM, Paris Cardiovascular Research Center (PARCC), France
- Cell and Tissue Imaging Platform, INSERM, CNRS, ERL8252, Centre de Recherche sur l’Inflammation (CRI), France
- Department of Pharmacy, School of Medicine and Surgery, University of Naples Federico II, Italy
- Department of Cardiology and of Physiology, Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpitaux Universitaires Paris Nord Val-de-Seine, Site Bichat, France
Figures
Figure 1
Pecam1-/- neutrophils exhibit efficient mobilization in the circulation but fail to reach the inflammatory site after crossing the vessel wall.
(A) Absolute number of Ly6G+ cells harvested in the peritoneal cavity at different time points after the induction of sterile peritonitis in WT or Pecam1-/- mice. (B) Blood neutrophil levels at the indicated time points. (C) Concentration of SDF-1, CD62P, CXCL1, and PTX-3 in the plasma and cell-free peritoneal fluid of Pecam1-/- and WT mice at 4 hr after PBS or IL-1β injection. Data are presented as mean ± SE. n = 3/5 per condition. The data represent three independent experiments. Statistical significance is indicated relative to the previous time point. Mann–Whitney test: *p<0.05, **p<0.01, ***p<0.001. (D) Confocal micrographs of whole-mount omentum showing extravasating Ly6G+ cells (clone 1A8; green) from a post-capillary venule with CD31 expression (polyclonal, R&D #AF3628; white). Samples were also stained with a monoclonal antibody against phosphorylated CD31 tyrosine 713 (pY713, clone EPR8079; red staining). Inset shows colocalization of pY713 and Ly6G. Scale bar 50 µm. (E) Inverse correlation between pY713 staining intensity of each Ly6G+ cell and the distance from the vessel (Spearman correlation and exponential one-phase decay regression with 95% confidence interval). (F) Quantification of neutrophil distance from the closest vessel in WT and Pecam1-/- mice 4 hr after IL-1β injection. n = 153 and n = 89 cells for WT and Pecam1-/- mice, respectively. Unpaired nonparametric Mann–Whitney test: ***p<0.001. (G) Ly6G+ cells (green) accumulate around the outer edge of post-capillary venules (phalloidin staining, white) in the omentum of Pecam1-/- mice. Scale bar 50 µm. (H) Percentage of neutrophils attached to the outer part of the vessels (gray) and percentage of neutrophils released into the extravascular space (black) in WT and Pecam1-/- mice.
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Figure 1—source data 1
Quantification of total number of neutrophils in WT and Pecam1-/- mice after IL-1β challenging (Figure 1A).
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Figure 1—source data 2
Fraction of neutrophils in the peripheral blood of WT and Pecam1-/- mice after IL-1β challenging (Figure 1B).
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Figure 1—source data 3
Quantification of inflammatory cytokines in the plasma or peritoneal fluid of WT and Pecam1-/- mice 4 hr after IL-1β challenging (Figure 1C).
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Figure 1—source data 4
Spearman correlation between the distance of each neutrophil from the closest capillary venule and its CD31 phospho(p)-Tyrosine(Y)–713 level (Figure 1E).
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Figure 1—source data 5
Distance (µm) of neutrophils from the closest capillary venule in the omentum of WT and Pecam1-/- mice upon IL-1β challenge (n = 3/group) (Figure 1F).
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Figure 1—source data 6
Fraction of neutrophils attached (<5 µm) or released (>5 µm) from the closest capillary venule in the omentum of WT and Pecam1-/- mice upon IL-1β challenge (n = 3/group) (Figure 1H).
- https://cdn.elifesciences.org/articles/84752/elife-84752-fig1-data6-v2.xlsx
Figure 2 with 1 supplement
Dynamic involvement of CD31 in integrin molecular complexes at the neutrophil uropod.
(A) Experimental strategy of shotgun proteomic analysis used to identify CD31 partners in human neutrophils under resting or fMLP-activated conditions. (B–D) Venn diagrams (B) and dot plots (C) illustrating the proteins identified by mass spectrometry upon CD31 co-immunoprecipitation in resting and fMLP-activated human neutrophils. (D) Ingenuity analysis of the top canonical pathways enriched in the CD31 interactome. The x-axis represents the statistical score, while the y-axis represents the corresponding canonical pathways. Blue color indicates negative Z scores (pathway suppression), red color indicates positive Z scores (pathway activation), and white color indicates a neutral Z score. (E) Ingenuity network analysis of the CD31 interactome under resting (light blue) or activated conditions (red), with unchanged partners depicted in gray. (F) Timeframe images from live video microscopy showing neutrophils stained with fluorophore-coupled monoclonal antibodies against CD11b (light blue) and CD31 (red) moving on a fibronectin-coated surface. (G) Cell motion over time is visualized by a time color-coded superposition of frames. (H) IL-8-stimulated primary human neutrophils migrating on recombinant human ICAM-1 stained for CD31 (clone WM59; green) and phosphorylated CD31 ITIM tyrosine 713 (pY713, clone EPR8079; red). (I) Staining for SHIP-1 (clone P1C1; red) or (J) total CD11a (clone TS2/4; white) and CD11a opened/active conformation (clone NKI-L16, red) in IL-8-stimulated primary human neutrophils. Representative 3D reconstructions of top and side views are shown at the bottom of each panel. Quantification of mean fluorescence intensities (MFIs) for the indicated staining along the cell axis is also presented (mean ± SE [SE bars in gray color]; n > 10 cells/staining; statistical comparisons between the first 20 frontal and last 20 posterior measurement points). *p<0.05, **p<0.01, ***p<0.001 (Mann–Whitney test).
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Figure 2—source data 1
Protein pulled down with CD31 and identified by mass spectrometry (Figure 2C).
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Figure 2—source data 2
Quantification of mean fluorescence intensities (MFIs) of CD31 (clone WM59) and CD31 pY713 (clone EPR8079) along the cell axis (Figure 2H).
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Figure 2—source data 3
Quantification of mean fluorescence intensities (MFIs) of CD31 (clone WM59) and SHIP-1 (clone P1C1) along the cell axis (Figure 2I).
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Figure 2—source data 4
Quantification of mean fluorescence intensities (MFIs) of CD31 (clone WM59), total CD11a (clone TS2/4), and opened CD11a (clone NKI-L16) along the cell axis (Figure 2J).
- https://cdn.elifesciences.org/articles/84752/elife-84752-fig2-data4-v2.xlsx
Figure 2—figure supplement 1
Orbitrap data analysis for the identification of CD31 pathways in human neutrophils.
(A, B) KEGG functional annotation pathways enriched in human neutrophil CD31 interactome under resting (A) or fMLP-activated (B) conditions. (C) Regulation of actin cytoskeleton KEGG pathway showing the proteins that were found to be associated with CD31 under resting condition (light blue), fMLP-activated condition (red) or found in both (gray).
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Figure 2—figure supplement 1—source data 1
KEGG functional annotation pathways enriched in human neutrophil CD31 interactome under resting conditions.
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Figure 2—figure supplement 1—source data 2
KEGG functional annotation pathways enriched in human neutrophil CD31 interactome under fMLP-activated conditions.
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Figure 3 with 1 supplement
Phenotype of Pecam1-/- and ITIM-less CD31 neutrophils on laminin in vitro.
(A–G) Confocal microscopy phenotypic characterization of purified WT or Pecam1-/- neutrophils placed on immobilized recombinant mouse laminin α4 and recombinant mouse CXCL1. (A) Representative confocal images and (B, C) quantification of talin (polyclonal, ab71333; red) and F-actin (Phalloidin, green) staining in WT mice (B) and Pecam1-/- mice (C). (D) Quantification of the uropod lengths of WT and Pecam1-/- neutrophils calculated as the distance from the backside of the nucleus to the end of the cellular tail. (E) Neutrophil total area. Data comes from three independent experiments, *p<0.05, **p<0.01, ***p<0.001 (unpaired Student’s t-test). (F, G) Quantification of CD44 (clone IM7; red) and SHIP1 (clone PC1C; green) staining in WT mice (F) and Pecam1-/- mice (G). (H, I) Quantification of talin (red) and F-actin (Phalloidin, green) staining in Pecam1ITIM+/+ mice (H) and Pecam1ITIM-/- (I). Quantification of talin/phalloidin ratio at the front (J) and back (K) edge of analyzed cells. Quantification of mean fluorescence intensities (MFIs) of the indicated staining along the cell axis (mean ±SE [SE bars in gray color]; n > 8 cells/staining; statistical comparisons between the 20 first frontal and 20 last posterior measurement points are shown). *p<0.05, **p<0.01, ***p<0.001 (Mann–Whitney test).
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Figure 3—source data 1
Quantification of mean fluorescence intensities (MFIs) of polymerized F-actin (phalloidin) and Talin (polyclonal ab71333) along the cell axis of WT neutrophils (Figure 3B).
- https://cdn.elifesciences.org/articles/84752/elife-84752-fig3-data1-v2.xlsx
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Figure 3—source data 2
Quantification of mean fluorescence intensities (MFIs) of polymerized F-actin (phalloidin) and Talin (polyclonal ab71333) along the cell axis of Pecam1-/- neutrophils (Figure 3C).
- https://cdn.elifesciences.org/articles/84752/elife-84752-fig3-data2-v2.xlsx
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Figure 3—source data 3
Uropod length (µm) of WT and Pecam1-/- neutrophils migrating on laminin α4-coated surfaces (Figure 3D).
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Figure 3—source data 4
Cell area (µm2) of WT and Pecam1-/- neutrophils migrating on laminin α4-coated surfaces (Figure 3E).
- https://cdn.elifesciences.org/articles/84752/elife-84752-fig3-data4-v2.xlsx
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Figure 3—source data 5
Quantification of mean fluorescence intensities (MFIs) of CD44 (clone IM7) and SHIP-1 (clone PC1C) along the cell axis of WT neutrophils (Figure 3F).
- https://cdn.elifesciences.org/articles/84752/elife-84752-fig3-data5-v2.xlsx
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Figure 3—source data 6
Quantification of mean fluorescence intensities (MFIs) of CD44 (clone IM7) and SHIP-1 (clone PC1C) along the cell axis of Pecam1-/- neutrophils (Figure 3G).
- https://cdn.elifesciences.org/articles/84752/elife-84752-fig3-data6-v2.xlsx
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Figure 3—source data 7
Quantification of mean fluorescence intensities (MFIs) of polymerized F-actin (phalloidin) and Talin (polyclonal ab71333) along the cell axis of WT Pecam1ITIM-/- neutrophils (Figure 3H).
- https://cdn.elifesciences.org/articles/84752/elife-84752-fig3-data7-v2.xlsx
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Figure 3—source data 8
Quantification of mean fluorescence intensities (MFIs) of polymerized F-actin (phalloidin) and Talin (polyclonal ab71333) along the cell axis of Pecam1ITIM-/- neutrophils (Figure 3I).
- https://cdn.elifesciences.org/articles/84752/elife-84752-fig3-data8-v2.xlsx
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Figure 3—source data 9
Quantification of polymerized F-actin (phalloidin) and Talin (polyclonal ab71333) ratio at the leading edge of WT and Pecam1ITIM-/- neutrophils (Figure 3J).
- https://cdn.elifesciences.org/articles/84752/elife-84752-fig3-data9-v2.xlsx
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Figure 3—source data 10
Quantification of polymerized F-actin (phalloidin) and Talin (polyclonal ab71333) ratio at the uropod of WT and Pecam1ITIM-/- neutrophils (Figure 3K).
- https://cdn.elifesciences.org/articles/84752/elife-84752-fig3-data10-v2.xlsx
Figure 3—figure supplement 1
Generation of CD31 exons 13–14 floxed, ITIM-less mice.
(A) Schematic representation of the experimental setup used for generating CD31 exons 13–14 floxed mice (Pecam1ITIM-/-). (B, C) RT-QPCR analysis showing the expression of the different CD31 exons in the spleen (B) and lungs (C) coming from WT or Pecam1ITIM-/- mice. (D) Western blot of whole lung lysate coming from WT, conventional Pecam1-/- and Pecam1ITIM-/- mice. Membranes were stained with an antibody recognizing an epitope located within the extracellular CD31 domain (sc-28188) or with an antibody recognizing an epitope located within the intracellular CD31 C-terminal tail (sc-1506-R). Membranes were also stained for GAPDH as an internal protein loading control. Note that the decrease of the molecular weight of Pecam1ITIM-/- mice is consistent with the loss of the ITIM-bearing portion of the cytoplasmic tail. (E) Hematological parameters and blood formula of WT and Pecam1ITIM-/- mice. (F) Representative confocal micrographs showing DAPI (blue staining), CD31 (red staining) extracellular (upper panels), and intracellular (lower panels) expression in the lymph nodes of WT Pecam1ITIM+/+, heterozygous Pecam1ITIM+/- and homozygous Pecam1ITIM-/- mice. Scale bar represents 50 µm.
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Figure 3—figure supplement 1—source data 1
RT-QPCR analysis showing the expression of the different CD31 exons in the spleen of WT or Pecam1ITIM-/- mice (Figure 3—figure supplement 1B).
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Figure 3—figure supplement 1—source data 2
Western blot of CD31 intracellular and extracellular staining of WT, Pecam1-/-, and Pecam1ITIM-/- mice (Figure 3—figure supplement 1D).
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Figure 3—figure supplement 1—source data 3
RT-QPCR analysis showing the expression of the different CD31 exons in the lungs of WT or Pecam1ITIM-/- mice (Figure 3—figure supplement 1C).
- https://cdn.elifesciences.org/articles/84752/elife-84752-fig3-figsupp1-data3-v2.xlsx
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Figure 3—figure supplement 1—source data 4
Hematological parameters and blood formula of WT and Pecam1ITIM-/- mice (Figure 3—figure supplement 1E).
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Figure 4 with 1 supplement
Effect of CD31 engagement on neutrophil detachment from the vessel wall and recruitment in vivo.
In vivo multiphoton intravital microscopy of Lyz2-GFP+ mice ear pinna intradermally injected with IL-1β with or without the CD31 agonist (Video 5). (A) Post-capillary venules at 1 hr after IL-1β injection in Lyz2-GFP+ mice show GFP+ neutrophils (in green) that are extravasating (white arrows). Blue color represents the collagen fibers imaged with the secondary harmonic generation (SHG). Scale bar 20 µm. (B) Cell locomotion during time is visualized by time color-coded superposition of frames. Scale bar 50 µm. (C) Quantification of detachment speed of individual neutrophils from the vessel wall. Data are expressed as mean showing all points. ***p<0.001 (Mann–Whitney test). n = 34–90 cells pulled from two mice per condition. (D) Extravasated leukocytes at 3 hr after IL-1β injection. Scale bar 40 µm. (E) Time color-coded superposition of frames at 3 hr after IL-1β injection. Scale bar 40 µm. (F) Quantification of migratory speed at 3 hr after IL-1β injection. (G) Morphological parameters (length and circularity) of leukocytes at 3 hr after IL-1β injection. Data are expressed as mean showing all points. **p<0.01, ***p<0.001 (Mann–Whitney test). n = 60 cells pulled from two mice per condition. (H) Track plots of Lyz2-GFP+ cells at 3 hr after IL-1β injection treated with vehicle (left) and treated with the CD31agonist (right) normalized to the starting position.
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Figure 4—source data 1
Detachment speed of neutrophils egressing from the post-capillary venules 1 hr after IL-1β challenge in the ear pinna of Lyz2-GFP mice treated or not with CD31 agonist (Figure 4C).
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Figure 4—source data 2
Migratory speed of neutrophils 3 hr after IL-1β challenge in the ear pinna of Lyz2-GFP mice treated or not with CD31 agonist (Figure 4F).
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Figure 4—source data 3
Morphological parameters of neutrophils 3 hr after IL-1β challenge in the ear pinna of Lyz2-GFP mice treated or not with CD31 agonist (Figure 4G).
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Figure 4—source data 4
Track plots of Lyz2-GFP+ cells at 3 hr after IL-1β injection treated with vehicle (Figure 4H, left).
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Figure 4—source data 5
Track plots of Lyz2-GFP+ cells at 3 hr after IL-1β injection treated with CD31 agonist (Figure 4H, right).
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Figure 4—figure supplement 1
CD31 agonist peptide is able to sustain CD31 functionality in human and murine neutrophils.
(A) Western blot of total cellular lysate coming from purified primary human neutrophils pre-treated or not with incremental doses of the CD31agonist challenged with or without fMLP. A monoclonal antibody against phosphorylated CD31 tyrosine-713 (pY713, clone EPR8079) was used to reveal ITIM phosphorylation on the blots normalized on the GAPDH content in each condition. (B) TIRF or epifluorescence (EPI) analysis of CD31 molecular clusters (clone MBC78.2-A488, green) and CD31agonist (rhodamine-conjugated, red) membrane distribution in resting or fMLP-stimulated human neutrophils. N = 4 per time point, **p<0.01 and *p<0.05, one-way Brown–Forsythe and Welch ANOVA test with Dunnett correction for multiple comparisons. (C) Dynamic binding of the CD31agonist (FITC-conjugated) on human neutrophil under basal and activated conditions analyzed at the indicated time points by flow cytometry. Median fluorescent intensities (MFIs) were reported as a fold change from the unstimulated, baseline condition (time 0, not stimulated). (D) Representative contour plot (left) and quantification (right) of CD31agonist binding on mouse neutrophils. In order to assess the specificity of the CD31agonist binding for its target, WT and Pecam1-/- neutrophils purified from the bone marrow were incubated with a FITC-conjugated version of the CD31 agonist and challenged with fMLP or vehicle (resting condition). FITC fluorescence in the granulocyte gate (FSC/SSC) was used to evaluate the binding of the CD31 agonist on neutrophil’s membrane. Fluorescence intensity is expressed as the fold-change from the resting condition. (E) Representative histograms (left) and quantification (right) of actin polymerization upon fMLP challenging the presence or absence of CD31 agonist. At the indicated time point, neutrophils were fixed with 2% PFA, stained with phalloidin fluorescent probe, and analyzed by flow cytometry. Data are representative of at least two independent experiments (n = 3 per group) giving similar results. Data are expressed as mean ± SEM. **p<0.01 and *p<0.05, Two-way ANOVA test with Šidák correction for multiple comparisons.
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Figure 4—figure supplement 1—source data 1
Dynamic binding of the CD31 agonist (FITC-conjugated) on human neutrophil under basal and activated conditions analyzed at the indicated time points by flow cytometry (Figure 4—figure supplement 1C).
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Figure 4—figure supplement 1—source data 2
Western blot of phospho-ITIM CD31 in human neutrophils treated with fMLP with incremental doses of CD31 agonist (Figure 4—figure supplement 1A).
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Figure 4—figure supplement 1—source data 3
Quantification of CD31 agonist binding on WT and Pecam1-/- neutrophils (Figure 4—figure supplement 1D).
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Figure 4—figure supplement 1—source data 4
Quantification of actin polymerization (Phalloidin staining) upon fMLP challenging the presence or absence of CD31 agonist (Figure 4—figure supplement 1E).
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Figure 5
Restoring Pecam1-/- neutrophil motility in vivo through pharmacological interventions.
(A) Experimental design. WT or Pecam1-/- mice (4–6/group) were injected intraperitoneally (i.p) with IL-1β to induce the peritonitis. After 2 hr, mice were injected i.p. with vehicle or RGD peptides. At 4 hr, mice were sacrificed, and absolute peritoneal neutrophil number was assessed. (B) Representative dot plots showing harvested cellular exudate at 4 hr in the indicated conditions. Among CD45+ cells, neutrophils were identified as CD11b+ and Ly6G+ by flow cytometry. (C) Absolute numbers of extravasated neutrophils at 4 hr in the indicated conditions. (D) Confocal micrographs of whole-mount omentum showing extravasating Ly6G+ cells (green) from a post-capillary venule (Phalloidin, white) in Pecam1-/- mice. Scale bar 50 µm. (E) Quantification of Pecam1-/- neutrophil distance from the closest post-capillary vessels in vehicle or RGD-treated mice. (F) Experimental design. WT (4–5/group) were injected i.p with or without IL-1β to induce the peritonitis. After 2 hr, mice were injected i.p. with vehicle or CD31agonist. At 4 hr, mice were sacrificed, and absolute peritoneal neutrophil number was assessed. (G) Representative dot plots and (H) absolute numbers of extravasated neutrophils at 4 hr in the indicated conditions. Data are expressed as mean ± SD. Mann–Whitney test *p<0.05, **p<0.01, ***p<0.001. (I) Experimental setup. WT or Pecam1ITIM-/- neutrophils were purified from the bone marrow, differentially stained with CTV or CFSE, and mixed in 1:1 ratio. Cells were adoptively transferred into WT recipient. After 24 hr, recipient mice were challenged with intraperitoneal injection of IL-1β, and transmigrated neutrophils were analyzed in the peritoneal wash after 4 hr. Neutrophils were identified as live, singlets, CD45+, CD11b+, and Ly6G+ cells. (J, K) Representative FACS plots (left) and proportion (right) of WT and Pecam1ITIM-/- neutrophils in the blood before (J) and 4 hr after (K) IL-1β challenge. (L) Representative FACS plots (left) and proportion (right) of WT and Pecam1ITIM-/- neutrophils in the peritoneal wash 4 hr after IL-1β challenge. N = 7, **p<0.01 (Wilcoxon matched-pairs signed-rank test).
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Figure 5—source data 1
Absolute numbers of extravasated neutrophils at 4 hr in WT and Pecam1-/- mice treated or not with RGD peptides (Figure 5C).
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Figure 5—source data 2
Quantification of Pecam1-/- neutrophil distance from the closest post-capillary vessels in vehicle or RGD-treated mice (Figure 5E).
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Figure 5—source data 3
Absolute numbers of extravasated neutrophils at 4 hr in the indicated conditions (Figure 5G).
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Figure 5—source data 4
Proportion of transferred WT or Pecam1ITIM-/- neutrophils in the blood before IL-1β challenge (Figure 5J).
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Figure 5—source data 5
Proportion of transferred WT or Pecam1ITIM-/- neutrophils in the blood 4 hr after IL-1β challenge (Figure 5K).
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Figure 5—source data 6
Proportion of transferred WT or Pecam1ITIM-/- neutrophils in the peritoneal fluid 4 hr after IL-1β challenge (Figure 5L).
- https://cdn.elifesciences.org/articles/84752/elife-84752-fig5-data6-v2.xlsx
Author response image 1
Videos
Video 1
Live video microscopy of purified human neutrophils stained with anti CD31 and anti CD11b fluorescent antibody moving on fibronectin-coated surface.
Timeframe images from live video microscopy of neutrophils stained with fluorophore-coupled monoclonal antibodies directed against CD11b (light blue) and CD31 (red) moving onto a fibronectin-coated surface.
Video 2
Behavior of Lyz2-GFP+ neutrophils in the ear pinna after 1 hr of IL-1β challenge.
Example of post-capillary venule at 1 hr after IL-1β injection in Lyz2-GFP+ mice showing neutrophils (in green) that are extravasating. Blue color represents the collagen fibers imaged with the secondary harmonic generation (SHG). Scale bar represents 50 µm.
Video 3
Behavior of CD31agonist-treated Lyz2-GFP+ neutrophils in the ear pinna after 1 hr of IL-1β challenge.
Example of post-capillary venule at 1 hr after IL-1β injection in Lyz2-GFP+ mice injected with CD31 agonist. Scale bar represents 50 µm.
Video 4
Behavior of CD31agonist-treated Lyz2-GFP+ neutrophils in the ear pinna after 3 hr of IL-1β challenge.
Example of migrating Lyz2-GFP+ neutrophils (in green) 3 hr after IL-1β injection. Scale bar represents 100 µm.
Video 5
Behavior of Lyz2-GFP+ neutrophils in the ear pinna after 3 hr of IL-1β challenge.
Example of migrating Lyz2-GFP+ neutrophils (in green) 3 hr after IL-1β injection. Scale bar represents 100 µm.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-mouse CD31, rat monoclonal (clone 390) | BD Biosciences | Cat# 558736, RRID:AB_397095 | 1:100 |
| Antibody | Anti-mouse CD31, rabbit monoclonal (clone SP38) | Abcam | Cat# ab231436 | 1:100 |
| Antibody | Anti-human CD31, mouse monoclonal (clone MBC78.2) | Merk | Cat# MABF2034 | 1:100 |
| Antibody | APC anti-human CD11b, mouse monoclonal (clone D12) | BD Biosciences | Cat# 561015, RRID:AB_10561676 | 1:100 |
| Antibody | Alexa Fluor 647 anti-human CD11a, mouse monoclonal (clone NKI-L16) | Thermo Fisher | Cat# MUB0364P | 1:100 |
| Antibody | PE anti-human CD11a, mouse monoclonal (clone TS2/4) | BioLegend | Cat# 350605, RRID:AB_10660819 | 1:100 |
| Antibody | Alexa Fluor 647 anti-human SHIP-1, mouse monoclonal (clone P1C1-A5) | BioLegend | Cat# 656608, RRID:AB_2563145 | 1:100 |
| Antibody | Alexa Fluor 488 anti-human CD31, mouse monoclonal (clone WM59) | BioLegend | Cat# 303109, RRID:AB_493075 | 1:100 |
| Antibody | FITC anti-mouse CD45, rat monoclonal (clone 30-F11) | BD Biosciences | Cat# 553080, RRID:AB_394610 | 1:100 |
| Antibody | PE anti-mouse Ly6G, rat monoclonal (clone 1A8) | BD Biosciences | Cat# 551461, RRID:AB_394208 | 1:100 |
| Antibody | APC anti-mouse CD11b, rat monoclonal (clone M1/70) | BD Biosciences | Cat# 553312, RRID:AB_398535 | 1:100 |
| Antibody | Alexa Fluor 647 AffiniPure anti-rat IgG, (H+L), donkey polyclonal | Jackson ImmunoResearch | Cat# 712-605-153, RRID:AB_2340694 | 1:500 |
| Antibody | Alexa Fluor 594 AffiniPure anti-goat IgG (H+L), donkey polyclonal | Jackson ImmunoResearch | Cat# 705-585-147, RRID:AB_2340433 | 1:500 |
| Antibody | Alexa Fluor 488 AffiniPure anti-rabbit IgG (H+L), donkey polyclonal | Jackson ImmunoResearch | Cat# 711-545-152, RRID:AB_2313584 | 1:500 |
| Antibody | Anti-CD31 (phospho Y713) antibody, rabbit monoclonal (clone EPR8079(2)) | Abcam | Cat# ab180175 | 1:100 |
| Antibody | Anti-mouse/rat CD31, goat polyclonal | R&D Systems | Cat# AF3628, RRID:AB_2161028 | 1:100 |
| Antibody | Anti-mouse/human Talin 1, rabbit polyclonal | Abcam | ab71333, RRID:AB_2204002 | 1:100 |
| Antibody | Anti-mouse SHIP-1, mouse monoclonal (clone P1C1-A5) | BioLegend | Cat# 656601, RRID:AB_2562400 | 1:100 |
| Antibody | Alexa Fluor(R) 488 anti-mouse/human CD44, rat monoclonal (clone IM7) | BioLegend | Cat# 103016, RRID:AB_493679 | 1:100 |
| Antibody | Anti-human GAPDH, rabbit polyclonal | Sigma-Aldrich | Cat# G9545, RRID:AB_796208 | 1:1000 |
| Antibody | Anti-rabbit IgG, HRP-conjugate antibody, giat polyclonal | Millipore | Cat# 12-348, RRID:AB_390191 | 1:2000 |
| Peptide, recombinant protein | Recombinant human IL-8/CXCL8 protein | R&D Systems | Cat# 208-IL-050 | 10 ng/ml |
| Peptide, recombinant protein | Recombinant human ICAM-1 | R&D Systems | Cat# ADP4 | 20 µg/ml |
| Peptide, recombinant protein | Recombinant mouse laminin a4 | R&D Systems | Cat# 3837-A4 | 20 µg/ml |
| Peptide, recombinant protein | Recombinant mouse CXCL1 | R&D Systems | Cat# 453-KC | 10 ng/ml |
| Peptide, recombinant protein | Recombinant mouse IL-1 beta | R&D Systems | Cat# 401ML-005 | 40 ng/ml |
| Peptide, recombinant protein | RGD peptide | Tocris | Cat# 3498 | 10 mg/kg |
| Peptide, recombinant protein | CD31 agonist (P8RI) | PMID:29957231 | N/A | 2.5 mg/kg |
| Peptide, recombinant protein | N-Formyl-Met-Leu-Phe | Tocris | Cat# 1921 | 1 µM |
| Chemical compound, drug | Viobility 405/520 fixable dye | Miltenyi Biotec | Cat# 130-109-816 | 1:100 |
| Chemical compound, drug | Alexa Fluor 647 Phalloidin | Thermo Fisher Scientific | Cat# A22287, RRID:AB_2620155 | 150 nM |
| Chemical compound, drug | DAPI | Thermo Fisher Scientific | Cat# D1306; RRID:AB_2629482 | 5 µM |
| Chemical compound, drug | Percoll | Sigma | Cat#P1644 | |
| Commercial assay or kit | ProLong Gold Antifade Mountant | Invitrogen | Cat# P36930 | |
| Commercial assay or kit | CellTrace Violet Cell Proliferation Kit | Thermo Fisher Scientific | Cat# C34571 | |
| Commercial assay or kit | CellTrace CFSE Cell Proliferation Kit | Thermo Fisher Scientific | Cat# C34554 | |
| Commercial assay or kit | EasySep Mouse Neutrophil Enrichment Kit | Stem Cell Technologies | Cat# 19762 | |
| Commercial assay or kit | Mouse CXCL1/KC DuoSet ELISA | R&D Systems | Cat# DY453-05 | |
| Commercial assay or kit | Bio-Plex Pro Magnetic COOH Beads 26 | Bio-Rad | Cat# MC10026-01 | |
| Commercial assay or kit | Bio-Plex Amine Coupling Kit | Bio-Rad | Cat# 171406001 | |
| Commercial assay or kit | Pierce BCA Protein Assay Kit | Thermo Scientific | Cat# 23225 | |
| Commercial assay or kit | 10% Mini-PROTEAN TGX Precast Protein Gels, 10-well, 50 µl | Bio-Rad | Cat# 4561034 | |
| Commercial assay or kit | Trans-Blot Turbo Mini 0.2 µm Nitrocellulose Transfer Packs | Bio-Rad | Cat# 1704158 | |
| Commercial assay or kit | Clarity Western ECL substrate kit | Bio-Rad | Cat# 1705060S | |
| Strain, strain background (Mus musculus) | C57BL/6, C57BL/6 | Charles River | Internal colony | |
| Strain, strain background (M. musculus) | Pecam1-/-, C57BL/6 | PMID:10072554 | Internal colony | |
| Strain, strain background (M. musculus) | Lyz2-eGFP, C57BL/6 | PMID:10887140 | Internal colony | |
| Strain, strain background (M. musculus) | Pecam1ITIM-/-, C57BL/6 | Generated for this work | Internal colony | |
| Software, algorithm | FlowJo V10 | FlowJo | https://www.flowjo.com/ | |
| Software, algorithm | Mascot | Matrix Sciences | http://www.matrixscience.com | |
| Software, algorithm | Enrichr | PMID:27141961 | https://maayanlab.cloud/Enrichr/ | |
| Software, algorithm | Prism 9 | GraphPad Software | https://www.graphpad.com/scientific-software/prism | |
| Software, algorithm | BD FACSDiva V8 | BD Biosciences | https://www.bdbiosciences.com/en-us/products/software/instrument-software/bd-facsdiva-software | |
| Software, algorithm | Leica Application Suite X (LAS X) | Leica Microsystems | https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/ | |
| Software, algorithm | Ingenuity Pathway Analysis (IPA) | QIAGEN | https://www.qiagen.com | |
| Software, algorithm | Fiji-ImageJ | ImageJ | https://imagej.net/Fiji/ | |
| Other | FACS LSR II | BD Biosciences | N/A | Flow cytometer |
| Other | LTQ Velos Orbitrap | Thermo Fisher Scientific | N/A | Mass spectrometer |
| Other | Zeiss LSM7 MP | Zeiss | N/A | Fluorescent microscope |
| Other | AxioObserver | Zeiss | N/A | Fluorescent microscope |
| Other | Trans-Blot Turbo Transfer System | Bio Rad | N/A | Western blot transfer system |
| Other | Zeiss TIRF 3 system | Zeiss | N/A | TIRF microscope |
| Other | Vet ABC | scil | N/A | Hemocytometer |
Additional files
-
Supplementary file 1
List of proteins identified by Orbitrap co-immunoprecipitate with CD31 in resting and fMLP-treated human neutrophils.
- https://cdn.elifesciences.org/articles/84752/elife-84752-supp1-v2.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/84752/elife-84752-mdarchecklist1-v2.docx
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CD31 signaling promotes the detachment at the uropod of extravasating neutrophils allowing their migration to sites of inflammation
eLife 12:e84752.
https://doi.org/10.7554/eLife.84752
