Figure 6.
Impaired adhesion strength reduces migration of ERM-deficient neutrophils into extravascular tissues. (A) Distribution of PSGL-1 (red), CD44 (blue), or integrin αMβ2 (red) on neutrophils crawling in venules of the cremaster muscle of control or DKO mice superfused with WKYMVm peptide, visualized by spinning disk confocal microscopy. The data are representative of 3 experiments. Bar, 10 μm. Quantitative data are presented in supplemental Figure 7. Representative videos are shown in supplemental Videos 1-3. (B) Schematic of peptide gradient created by inserting a diffusible Matrigel plug impregnated with WKYMVm adjacent to the cremaster muscle. (C) Images of adherent neutrophils from control (green) or DKO (red) mice at the indicated times after IV injection into WT mice. Venular endothelial cells were stained with anti-CD31 mAb (blue). The orientation of the WKYMVm peptide gradient is marked. The images are representative of 4 independent experiments. Bar, 50 µm. A representative video is shown in supplemental Video 4. (D) Quantification of adherent luminal and extravascular neutrophils at the indicated time points after injection. The data represent the mean ± SEM of 44 to 269 neutrophils counted in 3 to 5 venules from each of 4 independent experiments. *P < .05 for luminal control relative to DKO neutrophils at the same time point. #P < .05 for extravascular control relative to DKO neutrophils at the same time point. (E) Luminal and extravascular crawling velocities of cells 2 hours after injection. The data represent the mean ± SEM from 20 to 30 neutrophils from 4 independent experiments. (F) Migration plots of extravascular neutrophils 2 hours after injection. The starting point of each track was normalized to zero for the x- and y-axis. Positive y-axis values represent movement toward the WKYMVm chemoattractant source. The plots are representative of 3 independent experiments. (G) Chemotactic index of extravascular neutrophils from panel F. The data represent the mean ± SEM from 20 to 30 neutrophils from 3 independent experiments. A representative video is shown in supplemental Video 5. (H) Competitive recruitment of IV injected neutrophils from control mice labeled with green (PKH67) dye and from control or DKO mice labeled with red (PKH26) dye into the peritoneum of WT mice 2 hours after thioglycollate challenge. Data are plotted as the ratio of PKH26-labeled control or DKO neutrophils relative to PKH67-labeled control neutrophils. The data represent the mean ± SEM from 4 recipient mice in each genotype group.

Impaired adhesion strength reduces migration of ERM-deficient neutrophils into extravascular tissues. (A) Distribution of PSGL-1 (red), CD44 (blue), or integrin αMβ2 (red) on neutrophils crawling in venules of the cremaster muscle of control or DKO mice superfused with WKYMVm peptide, visualized by spinning disk confocal microscopy. The data are representative of 3 experiments. Bar, 10 μm. Quantitative data are presented in supplemental Figure 7. Representative videos are shown in supplemental Videos 1-3. (B) Schematic of peptide gradient created by inserting a diffusible Matrigel plug impregnated with WKYMVm adjacent to the cremaster muscle. (C) Images of adherent neutrophils from control (green) or DKO (red) mice at the indicated times after IV injection into WT mice. Venular endothelial cells were stained with anti-CD31 mAb (blue). The orientation of the WKYMVm peptide gradient is marked. The images are representative of 4 independent experiments. Bar, 50 µm. A representative video is shown in supplemental Video 4. (D) Quantification of adherent luminal and extravascular neutrophils at the indicated time points after injection. The data represent the mean ± SEM of 44 to 269 neutrophils counted in 3 to 5 venules from each of 4 independent experiments. *P < .05 for luminal control relative to DKO neutrophils at the same time point. #P < .05 for extravascular control relative to DKO neutrophils at the same time point. (E) Luminal and extravascular crawling velocities of cells 2 hours after injection. The data represent the mean ± SEM from 20 to 30 neutrophils from 4 independent experiments. (F) Migration plots of extravascular neutrophils 2 hours after injection. The starting point of each track was normalized to zero for the x- and y-axis. Positive y-axis values represent movement toward the WKYMVm chemoattractant source. The plots are representative of 3 independent experiments. (G) Chemotactic index of extravascular neutrophils from panel F. The data represent the mean ± SEM from 20 to 30 neutrophils from 3 independent experiments. A representative video is shown in supplemental Video 5. (H) Competitive recruitment of IV injected neutrophils from control mice labeled with green (PKH67) dye and from control or DKO mice labeled with red (PKH26) dye into the peritoneum of WT mice 2 hours after thioglycollate challenge. Data are plotted as the ratio of PKH26-labeled control or DKO neutrophils relative to PKH67-labeled control neutrophils. The data represent the mean ± SEM from 4 recipient mice in each genotype group.

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