American Society of Hematology

Figure 3

$Figure 3. ESL-1 mediates neutrophil recruitment in the absence of PSGL-1, but is dispensable for integrin-mediated slow rolling. Analyses in panels A-D were performed in WT mice transplanted with BM cells from PSGL-1−/−, DsRed+, and DKO donor mice. (A) Flow cytometric analyses of soluble E-selectin binding to Ly6GHI blood neutrophils. Histograms show overlays of E-selectin binding to DKO vs PSGL-1−/− DsRed+ neutrophils present in the blood of the same mice. Binding in the presence of EDTA was used as a negative control. E-selectin binding of WT neutrophils is included as a reference. Bar graphs at right show quantification of E-selectin–binding intensities of DKO and PSGL-1−/− DsRed+ neutrophils. Data are from 4 mice and was analyzed using the paired t test. (B) Rolling-flux fractions of DKO and PSGL-1−/− DsRed+ neutrophils in inflamed cremastric venules. There were 27 venules from 5 mice. (C) Representative micrograph of adherent neutrophils in an inflamed venule during the adhesion phase. Dotted lines demarcate a nonfluorescent DKO cell. Bar graph at right shows quantification of the adherent fractions. There were 27 venules from 5 mice. (D) Neutrophil extravasation in thioglycollate-induced peritonitis. Bar graph represents the relative frequencies of DKO and PSGL-1−/− DsRed+ neutrophils in blood vs the peritoneum, thereby representing extravasation efficiencies. There were 4 mice from 2 independent experiments. Data were compared using a paired t test. (E) Cumulative frequency histograms of rolling velocities of mutant neutrophils obtained from analyses of chimeric mice reconstituted with BM cells from WT, ESL-1−/−, PSGL-1−/−, or DKO mice. Dotted lines indicate velocities of the median. The bar graph represents mean rolling velocities. There were 37 to 82 cells per group from 5 to 7 mice per group, from 2 independent sets of experiments. Data were analyzed using a 1-way ANOVA with the Tukey multiple comparison test. (F) Rolling velocities of leukocytes in autoperfused flow chambers coated with E-selectin alone or in combination with ICAM-1. Data are from 3 to 4 individual mice per group and was analyzed using the 2-tailed Student t test. Data are shown as mean ± SEM.$

ESL-1 mediates neutrophil recruitment in the absence of PSGL-1, but is dispensable for integrin-mediated slow rolling. Analyses in panels A-D were performed in WT mice transplanted with BM cells from PSGL-1^−/−, DsRed+, and DKO donor mice. (A) Flow cytometric analyses of soluble E-selectin binding to Ly6G^HI blood neutrophils. Histograms show overlays of E-selectin binding to DKO vs PSGL-1^−/− DsRed+ neutrophils present in the blood of the same mice. Binding in the presence of EDTA was used as a negative control. E-selectin binding of WT neutrophils is included as a reference. Bar graphs at right show quantification of E-selectin–binding intensities of DKO and PSGL-1^−/− DsRed+ neutrophils. Data are from 4 mice and was analyzed using the paired t test. (B) Rolling-flux fractions of DKO and PSGL-1^−/− DsRed+ neutrophils in inflamed cremastric venules. There were 27 venules from 5 mice. (C) Representative micrograph of adherent neutrophils in an inflamed venule during the adhesion phase. Dotted lines demarcate a nonfluorescent DKO cell. Bar graph at right shows quantification of the adherent fractions. There were 27 venules from 5 mice. (D) Neutrophil extravasation in thioglycollate-induced peritonitis. Bar graph represents the relative frequencies of DKO and PSGL-1^−/− DsRed+ neutrophils in blood vs the peritoneum, thereby representing extravasation efficiencies. There were 4 mice from 2 independent experiments. Data were compared using a paired t test. (E) Cumulative frequency histograms of rolling velocities of mutant neutrophils obtained from analyses of chimeric mice reconstituted with BM cells from WT, ESL-1^−/−, PSGL-1^−/−, or DKO mice. Dotted lines indicate velocities of the median. The bar graph represents mean rolling velocities. There were 37 to 82 cells per group from 5 to 7 mice per group, from 2 independent sets of experiments. Data were analyzed using a 1-way ANOVA with the Tukey multiple comparison test. (F) Rolling velocities of leukocytes in autoperfused flow chambers coated with E-selectin alone or in combination with ICAM-1. Data are from 3 to 4 individual mice per group and was analyzed using the 2-tailed Student t test. Data are shown as mean ± SEM.

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