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Endothelial signaling by neutrophil-released oncostatin M enhances P-selectin–dependent inflammation and thrombosis

Hendra Setiadi, Tadayuki Yago, Zhenghui Liu and Rodger P. McEver

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    Adhesion of human or mouse neutrophils to P-selectin triggers release of OSM. (A-F) Representative flow cytometric data of intracellular OSM expression in permeabilized CD16b+ human neutrophils, CD14-high human monocytes, CD3+ human lymphocytes, Ly6G+ mouse neutrophils, M-CSFR+ mouse monocytes, or CD3+ mouse T lymphocytes. The indicated cell population was defined by its specific surface marker and by light scatter. (G) Measurement of OSM in medium or in lysates of human neutrophils after incubation with immobilized human serum albumin (HSA) or human platelet-derived P-selectin with or without shear on a rotary shaker, in the presence or absence of blocking anti-human PSGL-1 mAb PL1 or nonblocking anti–PSGL-1 mAb PL2. (H) Measurement of OSM in medium or in lysates of neutrophils from WT or Selplg−/− mice after incubation with immobilized mouse CD45-IgM or mouse P-selectin–IgM with or without shear on a rotary shaker. The data in panels A-F are representative of 3 experiments. The data in panels G-H represent the mean ± standard deviation (SD) from 3 experiments. *P < .05; **P < .01.

  • Figure 2.

    Interactions of neutrophil-derived OSM with gp130-containing receptors on endothelial cells enhance P-selectindependent rolling in mouse postcapillary venules. (A) Rolling flux fraction of leukocytes in postcapillary venules of trauma-stimulated cremaster muscle from WT mice with or without injection of the indicated mAb. (B) Representative flow cytometric data of intracellular OSM expression in Ly6G+ neutrophils from WT mice, Osm−/− mice, irradiated Osm−/− mice transplanted with bone marrow from WT mice, or irradiated WT mice transplanted with bone marrow from Osm−/− mice. (C-D) Rolling flux fraction and rolling velocity of leukocytes in postcapillary venules of trauma-stimulated cremaster muscle from the indicated genotype. (E) Representative fluorescent images of injected labeled neutrophils rolling in postcapillary venules of trauma-stimulated cremaster muscle of Osm−/− mice. Venular endothelial cells were labeled with Alexa Fluor 488–conjugated anti-CD31 mAb, outlined by the blue dashed line. The images depict distances rolled by the circled cells in a 2-second interval. The arrow indicates the path of each rolling cell. Top, Injected WT neutrophils (red); middle, injected Osm−/− neutrophils (blue); bottom, injected 1:1 mixture of WT (red) and Osm−/− (blue) neutrophils. Scale bar, 10 μm. (F-G) Rolling flux fraction and rolling velocity of injected labeled cells. (H, left) Representative images of Fluoresbrite Red microspheres coated with anti-gp130 mAb or isotype control IgG adhering to endothelial cells in trauma-stimulated venules of cremaster muscle from tamoxifen-treated WT or gp130flox/floxVECad-Cre-ERT2 mice. To block rolling of leukocytes, which also express gp130, anti-mouse PSGL-1 mAb 4RA10 was injected 1 hour before exteriorization of the cremaster muscle. (H, right) Quantification of the covered area of adherent microspheres with digital image analysis software. The data represent the mean ± standard error of the mean (SEM) from 10 venules from 5 mice. Scale bar, 10 μm. (I-J) Rolling flux fraction and rolling velocity of leukocytes in postcapillary venules of trauma-stimulated cremaster muscle from tamoxifen-treated WT or gp130flox/floxVECad-Cre-ERT2 mice, with or without preinjection of blocking anti–P-selectin mAb. The data in panels B and H are representative of 5 experiments. The data in panels A,C-D,F,I-J represent the mean ± SEM from 10 to 20 venules from 4 to 8 mice in each group. **P < .01.

  • Figure 3.

    Interactions of neutrophil-derived OSM with gp130-containing receptors enhance P-selectindependent rolling on stimulated mouse and human endothelial cells in vitro. (A) Number of mouse neutrophils rolling on MLECs 30 minutes after A23187 stimulation in the presence of the indicated mAb. (B) Mean rolling velocity of mouse neutrophils on MLECs 30 minutes after A23187 stimulation in the presence of the indicated antibody. (C) Number of human neutrophils rolling on HUVECs at the indicated time before or after adding histamine (time = 0), in the presence of the indicated antibody. (D) Mean rolling velocity of neutrophils on HUVECs 5 minutes after adding histamine, in the presence of the indicated antibody. (E) Number of neutrophils rolling on HUVECs at the indicated time before or after adding thrombin (time = 0), in the presence of the indicated antibody. (F) Mean rolling velocity of neutrophils on HUVECs 5 minutes after adding thrombin, in the presence of the indicated antibody. (G) Number of neutrophils rolling on immobilized purified P-selectin at the indicated time after initiation of perfusion, in the presence or absence of histamine, thrombin, and the indicated antibody. The data represent the mean ± SD from 3 experiments. *P < .05; **P < .01.

  • Figure 4.

    OSM increases clustering of P-selectin in clathrin-coated pits of human endothelial cells. (A) Density of P-selectin molecules on the surface of HUVECs 10 minutes after adding control buffer or the indicated agonist. (B) Internalization rate of P-selectin on HUVECs 10 minutes after adding buffer with or without or OSM. (C) Internalization rate of P-selectin on HUVECs 10 minutes after adding histamine-containing buffer with or without OSM. (D) Representative confocal immunofluorescence images of HUVECs 10 minutes after treatment with histamine, OSM, or both agonists. The sections were fixed, permeabilized, and incubated with 4′,6-diamidino-2-phenylindole (DAPI) (blue) to visualize cell nuclei and with anti–P-selectin antibody (green) to visualize the distribution of P-selectin. Scale bar, 20 μm. (E) Quantification of the areas of P-selectin puncta. (F) Representative confocal immunofluorescence images of HUVECs 10 minutes after treatment with histamine or with histamine plus OSM. The sections were fixed, permeabilized, and incubated with anti–P-selectin antibody (green) and anti–α-adaptin antibody (red). Merged images revealed partial colocalization of α-adaptin with P-selectin (yellow). Scale bar, 20 μm. (G) Quantification of colocalization of P-selectin with α-adaptin. (H) Number of fixed human neutrophils, in isotonic or hypertonic medium with or without OSM, rolling on HUVECs at the indicated time. (I) Number of fixed human neutrophils, in isotonic or hypertonic medium with or without OSM, rolling on HUVECs at the indicated time after addition of histamine. (J) Number of fixed human neutrophils, in isotonic or hypertonic medium with or without OSM, rolling on immobilized purified P-selectin at the indicated time. The data in panels A-C,E,G represent the mean ± SEM from 3 to 8 experiments. The data in panels D and F are representative of 4 experiments. The data in panels H-J represent the mean ± SD from 3 experiments. *P < .05; **P < .01.

  • Figure 5.

    Neutrophil-derived OSM increases P-selectindependent thrombosis in flow-restricted veins. (A-B) Quantification of endothelial surface area covered with firmly adherent Ly6G+ neutrophils or M-CSFR+ monocytes using spinning-disk intravital microscopy, 3 hours after ligation of the IVC in mice of the indicated genotype. (C-D) Kinetics of thrombus development (frequency) and thrombus size (area) in the indicated genotype, measured by ultrasonography at the indicated times after ligation. (E) Thrombus weight in the indicated genotype 24 hours after ligation. Each symbol represents an individual thrombus. Horizontal red bars represent median values. (F-G) Number of Ly6G+ neutrophils or M-CSFR+ monocytes per thrombus 24 hours after ligation in the indicated genotype, as measured by flow cytometry. (H-I) Normalized thrombus weight per Ly6G+ neutrophil or M-CSFR+ monocyte. (J) Western blot of thrombus lysates probed with antibodies to fibrin, Ly6G, and citrullinated histone. (K) Western blot of thrombus lysates probed with antibodies to fibrin, M-CSFR, and tissue factor. The Aα, Bβ, and γ chains of fibrin are marked. The data in panels A-I represent the mean ± SEM from 5 to 12 mice in each group. The data in panels J-K are representative of 3 experiments. *P < .05; **P < .01.

  • Figure 6.

    Signaling through gp130-containing receptors on endothelial cells increases P-selectindependent thrombosis in flow-restricted veins. (A) Representative images of gp130 expression (red) in the IVC of tamoxifen-treated WT or gp130flox/floxVECad-Cre-ERT2 mice obtained with spinning-disk intravital microscopy. Fluoresbrite red microspheres coated with anti-gp130 mAb or isotype control mAb were injected IV into tamoxifen-treated WT or gp130flox/floxVECad-Cre-ERT2 mice 20 minutes before exposing the IVC. Top, IVC of tamoxifen-treated WT mouse injected with isotype control mAb-coated beads; middle, IVC of tamoxifen-treated WT mouse injected with anti-gp130 mAb-coated beads; bottom, IVC of tamoxifen-treated gp130flox/floxVECad-Cre-ERT2 mouse injected with anti-gp130 mAb-coated beads. The graph at right indicates quantification of the covered area of adherent microspheres with digital image-analysis software. The data represent the mean ± SEM from 5 mice. Scale bar, 10 μm. (B-C) Quantification of endothelial surface area covered with firmly adherent Ly6G+ neutrophils or M-CSFR+ monocytes, 3 hours after ligation of the IVC. (D-E) Kinetics of thrombus development (frequency) and thrombus size (area) in mice of the indicated genotype, measured by ultrasonography at the indicated times after ligation. (F) Thrombus weight in the indicated genotype 24 hours after ligation. Each symbol represents an individual thrombus. Horizontal red bars represent median values. (G-H) Number of Ly6G+ neutrophils or M-CSFR+ monocytes per thrombus 24 hours after ligation in the indicated genotype, as measured by flow cytometry. (I-J) Normalized thrombus weight per Ly6G+ neutrophil or M-CSFR+ monocyte. The data represent the mean ± SEM from 5 to 12 mice in each group. *P < .05; **P < .01.

  • Figure 7.

    Neutrophil-derived OSM triggers endothelial gp130 signaling that enhances P-selectindependent leukocyte adhesion and thrombosis. Neutrophils rolling on P-selectin release OSM (A) that triggers gp130 signaling in endothelial cells (B). Signaling through gp130 augments clustering of P-selectin in clathrin-coated pits, which enhances rolling of neutrophils and monocytes and facilitates integrin-dependent arrest (B-C). In flow-restricted veins, the increased leukocyte adhesion promotes thrombosis (D). See “Discussion” for details.