Figure 5.
Figure 5. Leukocyte elongation is maintained by VLA-3 integrin–mediated penetration through the endothelial basement membrane at the leading edge. (A–C) The extravasating LysM-GFP cells or Alexa Fluor 488–anti-Gr1 antibody–stained granulocytes were tracked after stimulation with CXCL2 in mice treated with the control (A) or VLA-3 blocking peptide (B) and in VLA-3–cKO mice (C). The cell lengths (top) were measured at each time point during migration. The velocities of the head and tail (bottom) of migrating cells were measured by tracking the positions of the leading edge and uropod at each time point with Volocity software and plotted at the indicated time points. A representative dataset of the cell length and velocity profiles was paired among three independent analyses of each condition. See corresponding Videos 10–12. (D–F) The correlations between the angle from the venule and the cell length during extravasation are shown for control (D), VLA-3 blocking peptide–treated (E), and VLA-3–cKO (F) mice. The cell lengths and the angles from each mouse were analyzed using MP-IVM. The maximum angle of the venule and the longest length of each cell were measured during granulocyte extravasation in MP-IVM. The angles and the cell lengths of extravasating cells were measured from at least five stimulated cremaster venules of three mice. (G) Cell lengths were measured at each time point during extravascular retention in control, VLA-3 blocking peptide–treated, and VLA-3–cKO mice from videos taken with MP-IVM. The data are additional representatives of cell length profiles in A–C. (H) Extravascular retention times were measured in the control peptide treated–, Ela-Cre, VLA-3 blocking peptide–treated, and VLA-3–cKO mice from videos taken with MP-IVM. Extravasating LysM-GFP cells in the cremaster venules of LysM-GFP mice were tracked for the control and VLA-3 blocking peptide treatments. The LXY2 peptide was superfused onto the exteriorized cremaster muscle for VLA-3 integrin blocking. For the analysis of Ela-Cre and VLA-3–cKO mice, Alexa Fluor 488–anti-Gr1 antibody was i.v. injected to visualize the granulocytes. The extravascular retention time was measured from at least five stimulated cremaster venules from three mice per condition.

Leukocyte elongation is maintained by VLA-3 integrin–mediated penetration through the endothelial basement membrane at the leading edge. (A–C) The extravasating LysM-GFP cells or Alexa Fluor 488–anti-Gr1 antibody–stained granulocytes were tracked after stimulation with CXCL2 in mice treated with the control (A) or VLA-3 blocking peptide (B) and in VLA-3–cKO mice (C). The cell lengths (top) were measured at each time point during migration. The velocities of the head and tail (bottom) of migrating cells were measured by tracking the positions of the leading edge and uropod at each time point with Volocity software and plotted at the indicated time points. A representative dataset of the cell length and velocity profiles was paired among three independent analyses of each condition. See corresponding Videos 10–12. (D–F) The correlations between the angle from the venule and the cell length during extravasation are shown for control (D), VLA-3 blocking peptide–treated (E), and VLA-3–cKO (F) mice. The cell lengths and the angles from each mouse were analyzed using MP-IVM. The maximum angle of the venule and the longest length of each cell were measured during granulocyte extravasation in MP-IVM. The angles and the cell lengths of extravasating cells were measured from at least five stimulated cremaster venules of three mice. (G) Cell lengths were measured at each time point during extravascular retention in control, VLA-3 blocking peptide–treated, and VLA-3–cKO mice from videos taken with MP-IVM. The data are additional representatives of cell length profiles in A–C. (H) Extravascular retention times were measured in the control peptide treated–, Ela-Cre, VLA-3 blocking peptide–treated, and VLA-3–cKO mice from videos taken with MP-IVM. Extravasating LysM-GFP cells in the cremaster venules of LysM-GFP mice were tracked for the control and VLA-3 blocking peptide treatments. The LXY2 peptide was superfused onto the exteriorized cremaster muscle for VLA-3 integrin blocking. For the analysis of Ela-Cre and VLA-3–cKO mice, Alexa Fluor 488–anti-Gr1 antibody was i.v. injected to visualize the granulocytes. The extravascular retention time was measured from at least five stimulated cremaster venules from three mice per condition.

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