American Society of Hematology

Figure 2

$Figure 2. ESL-1 cooperates with PSGL-1 in all stages of neutrophil recruitment during inflammation. Analyses were performed in mice transplanted with BM from WT, ESL-1−/−, PSGL-1−/−, or DKO mice together with competing WT-DsRed BM cells. (A) Flow cytometric analyses of soluble E-selectin binding to Ly6GHI blood neutrophils. Histograms show overlays of E-selectin binding to experimental neutrophils (empty histograms) and WT-DsRed competitors (dark gray histograms). Binding in the presence of EDTA was used as a negative control (light gray histograms). (B) Quantification of E-selectin binding, as measured by the mean fluorescence intensities, in all groups. Values are represented as ratios relative to internal WT-DsRed competitor cells. There were 7 to 9 mice per group from 3 independent experiments. (C) Intravital microscopy analysis of neutrophil rolling within inflamed cremasteric venules. Near-simultaneous acquisition in 2 channels discriminates experimental (bright field) and WT-DsRed (red) neutrophils. The bar graph shows rolling flux fractions represented as ratios relative to internal WT-DsRed competitor cells. There were 22 to 58 venules in 5 to 7 mice per group. (D) Representative micrographs of adherent neutrophils from the same groups shown in (C). White dotted lines demarcate adherent nonfluorescent cells. Nonfluorescent structures in the bottom panel are erythrocytes bound to WT-DsRed cells. Bar graph shows ratios of adherent fractions relative to WT-DsRed competitors. There were 26 to 58 venules in 5 to 8 mice per group. (E) Neutrophil extravasation after 8 hours of thioglycollate-induced peritonitis. Histograms show percentages of mutant (gray histograms) and WT-DsRed (red histograms) neutrophils in blood before extravasation (top panels), and in peritoneal exudates (bottom panels). (F) Ratios of mutant relative to WT-DsRed neutrophils in blood vs the peritoneum, thereby representing extravasation efficiencies. There were 5 to 14 mice per group from 2 independent experiments. Data are shown as mean ± SEM and was analyzed by 1-way ANOVA and the Tukey multiple comparison test.$

ESL-1 cooperates with PSGL-1 in all stages of neutrophil recruitment during inflammation. Analyses were performed in mice transplanted with BM from WT, ESL-1^−/−, PSGL-1^−/−, or DKO mice together with competing WT-DsRed BM cells. (A) Flow cytometric analyses of soluble E-selectin binding to Ly6G^HI blood neutrophils. Histograms show overlays of E-selectin binding to experimental neutrophils (empty histograms) and WT-DsRed competitors (dark gray histograms). Binding in the presence of EDTA was used as a negative control (light gray histograms). (B) Quantification of E-selectin binding, as measured by the mean fluorescence intensities, in all groups. Values are represented as ratios relative to internal WT-DsRed competitor cells. There were 7 to 9 mice per group from 3 independent experiments. (C) Intravital microscopy analysis of neutrophil rolling within inflamed cremasteric venules. Near-simultaneous acquisition in 2 channels discriminates experimental (bright field) and WT-DsRed (red) neutrophils. The bar graph shows rolling flux fractions represented as ratios relative to internal WT-DsRed competitor cells. There were 22 to 58 venules in 5 to 7 mice per group. (D) Representative micrographs of adherent neutrophils from the same groups shown in (C). White dotted lines demarcate adherent nonfluorescent cells. Nonfluorescent structures in the bottom panel are erythrocytes bound to WT-DsRed cells. Bar graph shows ratios of adherent fractions relative to WT-DsRed competitors. There were 26 to 58 venules in 5 to 8 mice per group. (E) Neutrophil extravasation after 8 hours of thioglycollate-induced peritonitis. Histograms show percentages of mutant (gray histograms) and WT-DsRed (red histograms) neutrophils in blood before extravasation (top panels), and in peritoneal exudates (bottom panels). (F) Ratios of mutant relative to WT-DsRed neutrophils in blood vs the peritoneum, thereby representing extravasation efficiencies. There were 5 to 14 mice per group from 2 independent experiments. Data are shown as mean ± SEM and was analyzed by 1-way ANOVA and the Tukey multiple comparison test.

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