Figure 6. Extravasating leukocytes... | Rockefeller University Press

Figure 6.

$Figure 6. Extravasating leukocytes deposit CD18-enriched microparticles at the subendothelial layer during the stretched uropod retraction. (A) Microparticle formation from the uropods of elongated leukocytes was visualized in the CXCL2-stimulated venules of CD18-mCFP (top) and LysM-GFP mice (bottom). The circles indicate extravasating leukocytes. A series of representative time-lapse images are shown among at least five microparticle formation videos from the MP-IVM analysis of the cremaster venules from CD18-mCFP and LysM-GFP mice. See corresponding Videos 13 and 14. (B) Microparticles were generated from human neutrophils during the in vitro transmigration through a TNF-stimulated HUVEC monolayer. The circle indicates a transmigrating neutrophil through the HUVEC monolayer. A series of representative time-lapse images are shown among three independent microparticle formation videos of in vitro migration. See corresponding Video 15. (A and B) Arrows indicate microparticles. (C) Microparticles formed during the transmigration of primary human T cells and neutrophils toward 0.2 µg chemokines (CXCL12 for T cells and CXCL2 for neutrophils) through a TNF-stimulated HUVEC layer grown on basement membrane (BM)–coated transwells (dashed lines; middle and right). After incubation at 37°C for 1 h, the cells were labeled with anti-CD18 antibodies (CBR LFA1/2 and TS1/18) and Alexa Fluor 488–anti–mouse IgG antibody after fixation. Three-dimensional images were taken using multiphoton microscopy. Representative still images are shown among three microparticle formation videos of the in vitro transwell chemotaxis of human T cells and neutrophils. A schematic description of the assay is shown (left). See corresponding Video 16. (D) Microparticles (MP) derived from transmigrating human T cells and neutrophils were counted. Cell numbers were counted from three independent experiments described in C. The results are expressed as the mean ± SEM. (E) Microparticle formation from the uropods of the elongated granulocytes was visualized in the CXCL2-stimulated venules of VLA-3–cKO mouse by i.v. injection of Alexa Fluor 488–anti-Gr1 antibody. The circle indicates an extravasating leukocyte. The arrow shows the formation of a microparticle. A series of representative time-lapse images are shown from three microparticle formation videos from the MP-IVM analysis of the cremaster venules of VLA-3–cKO mice. See corresponding Video 17. (F) Scanning EM was performed to detect submicrometer-sized LFA-1–expressing microparticles. Microparticles were isolated from CXCL2-stimulated cremaster tissue by collagenase digestion and centrifuge as described in Materials and methods. Magnetic beads conjugated with anti–rat IgG were used to collect CD11a antibody (M17/4)–bound particles from the lower molecular weight fraction than normal cells. Scanning EM of magnetic beads bound to LFA-1–positive particles revealed that there are a large number of LFA-1–expressing microparticles, whose sizes are in submicrometer ranges. Bars: (A and E) 30 µm; (B and C) 10 µm; (F) 1 µm.$

Extravasating leukocytes deposit CD18-enriched microparticles at the subendothelial layer during the stretched uropod retraction. (A) Microparticle formation from the uropods of elongated leukocytes was visualized in the CXCL2-stimulated venules of CD18-mCFP (top) and LysM-GFP mice (bottom). The circles indicate extravasating leukocytes. A series of representative time-lapse images are shown among at least five microparticle formation videos from the MP-IVM analysis of the cremaster venules from CD18-mCFP and LysM-GFP mice. See corresponding Videos 13 and 14. (B) Microparticles were generated from human neutrophils during the in vitro transmigration through a TNF-stimulated HUVEC monolayer. The circle indicates a transmigrating neutrophil through the HUVEC monolayer. A series of representative time-lapse images are shown among three independent microparticle formation videos of in vitro migration. See corresponding Video 15. (A and B) Arrows indicate microparticles. (C) Microparticles formed during the transmigration of primary human T cells and neutrophils toward 0.2 µg chemokines (CXCL12 for T cells and CXCL2 for neutrophils) through a TNF-stimulated HUVEC layer grown on basement membrane (BM)–coated transwells (dashed lines; middle and right). After incubation at 37°C for 1 h, the cells were labeled with anti-CD18 antibodies (CBR LFA1/2 and TS1/18) and Alexa Fluor 488–anti–mouse IgG antibody after fixation. Three-dimensional images were taken using multiphoton microscopy. Representative still images are shown among three microparticle formation videos of the in vitro transwell chemotaxis of human T cells and neutrophils. A schematic description of the assay is shown (left). See corresponding Video 16. (D) Microparticles (MP) derived from transmigrating human T cells and neutrophils were counted. Cell numbers were counted from three independent experiments described in C. The results are expressed as the mean ± SEM. (E) Microparticle formation from the uropods of the elongated granulocytes was visualized in the CXCL2-stimulated venules of VLA-3–cKO mouse by i.v. injection of Alexa Fluor 488–anti-Gr1 antibody. The circle indicates an extravasating leukocyte. The arrow shows the formation of a microparticle. A series of representative time-lapse images are shown from three microparticle formation videos from the MP-IVM analysis of the cremaster venules of VLA-3–cKO mice. See corresponding Video 17. (F) Scanning EM was performed to detect submicrometer-sized LFA-1–expressing microparticles. Microparticles were isolated from CXCL2-stimulated cremaster tissue by collagenase digestion and centrifuge as described in Materials and methods. Magnetic beads conjugated with anti–rat IgG were used to collect CD11a antibody (M17/4)–bound particles from the lower molecular weight fraction than normal cells. Scanning EM of magnetic beads bound to LFA-1–positive particles revealed that there are a large number of LFA-1–expressing microparticles, whose sizes are in submicrometer ranges. Bars: (A and E) 30 µm; (B and C) 10 µm; (F) 1 µm.