Figure 4.
Figure 4. Leukocyte elongation is mediated by LFA-1 at the tail. (A) A scanning EM image of an extravasating leukocyte from the endothelium of a CXCL2-stimulated cremaster from a CD18-mCFP mouse is shown. A representative image is shown from two independent scanning EM experiments. (B) Two representative transmission EM images are shown together with corresponding cartoons that indicate endothelial cells (EC) in red and neutrophils in gray. Among 50 randomly selected neutrophil images from two independent experiments that showed perivascular elongation, 100% of these cells laid the uropod attached to the basolateral side of the endothelial cell layer but not its apical side. Transmission EM was independently performed two times using the CXCL2-stimulated cremasters of CD18-mCFP mice. Interst., interstitium; Lu., lumen. (C) CFP signal in an elongated CD18-mCFP is shown in rainbow colors. Texas red dextran was i.v. injected to indicate the blood vessel (red), the cremaster muscle of CD18-mCFP mice was stimulated with CXCL2, and then the tissue was fixed. The arrow indicates that CD18 is localized at the leukocyte uropod. Three-dimensional images of CFP and Texas red were taken using MP-IVM, and the images were processed as extended images using the Volocity software. Bars: (A and B) 1 µm; (C) 10 µm. (D) Antibody diffusion into the perivascular space through i.v. injection. Mice were immunized in one ear with 5 µg Keyhole Limpet Hemocyanin emulsified in complete Freund’s adjuvant. 4 d later, 50 µg PE-conjugated anti–mouse CD11a antibody (M17/4) or CD11b antibody (M1/70) was i.v. injected. After 30 min, the mice were sacrificed, and the leukocytes were extracted from the ear by collagenase digestion of the tissue. In vivo labeling of intradermal leukocytes was then evaluated by flow cytometry. The plot represents the CD45+CD4+ lymphocytes from anti-CD11a– or anti-CD11b–treated mice (black lines; gray closed histograms indicate PBS-treated mice). (E–H) The cell lengths (E) and extravascular retention times (F) of CD18-mCFP cells were measured in the CXCL2-stimulated cremaster venules of CD18-mCFP mice in the presence of the IgG isotype control or CD11a (M17/4) or CD11b (M1/70) blocking antibodies. The cell lengths (G) and extravascular retention times (H) of the granulocytes labeled with Alexa Fluor 488–anti-Gr1 antibody were measured in WT and CD11a KO mice. The cells were visualized using MP-IVM. The leukocytes located in the interstitium and the intravascular area have been excluded from our analysis. The cell length and the extravascular retention time were measured from images of at least three venules stimulated for >30 min in the cremasters of two CXCL2-stimulated mice. *, P < 0.05; **, P < 0.0001. See corresponding Videos 7–9. (I) The interstitial migration velocities of CD18-mCFP cells were analyzed in CXCL2-stimulated cremaster tissues from CD18-mCFP mice in the presence of IgG isotype control or CD11a or CD11b blocking antibody using MP-IVM. The velocities were measured from cremaster tissues of two CXCL2-stimulated mice per condition. (E, G, and I) Horizontal lines indicate the mean.

Leukocyte elongation is mediated by LFA-1 at the tail. (A) A scanning EM image of an extravasating leukocyte from the endothelium of a CXCL2-stimulated cremaster from a CD18-mCFP mouse is shown. A representative image is shown from two independent scanning EM experiments. (B) Two representative transmission EM images are shown together with corresponding cartoons that indicate endothelial cells (EC) in red and neutrophils in gray. Among 50 randomly selected neutrophil images from two independent experiments that showed perivascular elongation, 100% of these cells laid the uropod attached to the basolateral side of the endothelial cell layer but not its apical side. Transmission EM was independently performed two times using the CXCL2-stimulated cremasters of CD18-mCFP mice. Interst., interstitium; Lu., lumen. (C) CFP signal in an elongated CD18-mCFP is shown in rainbow colors. Texas red dextran was i.v. injected to indicate the blood vessel (red), the cremaster muscle of CD18-mCFP mice was stimulated with CXCL2, and then the tissue was fixed. The arrow indicates that CD18 is localized at the leukocyte uropod. Three-dimensional images of CFP and Texas red were taken using MP-IVM, and the images were processed as extended images using the Volocity software. Bars: (A and B) 1 µm; (C) 10 µm. (D) Antibody diffusion into the perivascular space through i.v. injection. Mice were immunized in one ear with 5 µg Keyhole Limpet Hemocyanin emulsified in complete Freund’s adjuvant. 4 d later, 50 µg PE-conjugated anti–mouse CD11a antibody (M17/4) or CD11b antibody (M1/70) was i.v. injected. After 30 min, the mice were sacrificed, and the leukocytes were extracted from the ear by collagenase digestion of the tissue. In vivo labeling of intradermal leukocytes was then evaluated by flow cytometry. The plot represents the CD45+CD4+ lymphocytes from anti-CD11a– or anti-CD11b–treated mice (black lines; gray closed histograms indicate PBS-treated mice). (E–H) The cell lengths (E) and extravascular retention times (F) of CD18-mCFP cells were measured in the CXCL2-stimulated cremaster venules of CD18-mCFP mice in the presence of the IgG isotype control or CD11a (M17/4) or CD11b (M1/70) blocking antibodies. The cell lengths (G) and extravascular retention times (H) of the granulocytes labeled with Alexa Fluor 488–anti-Gr1 antibody were measured in WT and CD11a KO mice. The cells were visualized using MP-IVM. The leukocytes located in the interstitium and the intravascular area have been excluded from our analysis. The cell length and the extravascular retention time were measured from images of at least three venules stimulated for >30 min in the cremasters of two CXCL2-stimulated mice. *, P < 0.05; **, P < 0.0001. See corresponding Videos 7–9. (I) The interstitial migration velocities of CD18-mCFP cells were analyzed in CXCL2-stimulated cremaster tissues from CD18-mCFP mice in the presence of IgG isotype control or CD11a or CD11b blocking antibody using MP-IVM. The velocities were measured from cremaster tissues of two CXCL2-stimulated mice per condition. (E, G, and I) Horizontal lines indicate the mean.

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