American Society of Hematology

Figure 4

$Figure 4. Leukocyte rolling and recruitment during inflammation in St3gal4-deficient, St3gal6-deficient, and double-deficient mice. (A) Leukocyte rolling in the autoperfused ex vivo microflow chamber. Microflow chambers were coated with recombinant murine (rm) P-selectin or rm E-selectin and perfused for 10 minutes with whole blood through a catheter connected to the mouse carotid artery (at least 5 chambers per group). Using an upright microscope, the number of rolling leukocytes calculated by rollers/(WBC count × 0.001) was assessed at 10 minutes. All values are shown as mean ± SEM (n = 6-12). *P < .05. (B) Effect of sialidase treatment on neutrophil rolling in the flow chamber. Isolated wild-type neutrophils were either left untreated for 1 hour (n = 15) or treated for 1 hour with Arthrobacter ureafaciens (AU) sialidase (n = 11) or with Streptococcus pneumonia (SP) sialidase (n = 14). Thereafter, neutrophils were perfused through flow chambers coated either with P- or E-selectin at a wall shear stress of 1 dyne/cm2. All values are shown as mean ± SEM (*P < .05). (C) Leukocyte rolling in TNF-α–treated cremaster muscle venules in vivo. Leukocyte rolling flux fraction (%) was assessed for each group. Blocking of E-selectin and P-selectin were performed by systemic injection of E-selectin–blocking mAb 9A9 and P-selectin blocking mAb RB40.34, respectively. All values are depicted as mean ± SEM (n = 3-6). *P < .05. (D) Leukocyte rolling velocities in TNF-α–treated cremaster muscle venules in vivo. Cumulative distribution of leukocyte rolling velocities are shown for no treatment (left), treatment with E-selectin–blocking mAb 9A9 (middle), and treatment with P-selectin–blocking mAb RB40.34 (right). Average rolling velocities are indicated as mean ± SEM with 3-6 mice per group. *P < .05 versus wild-type. (E) Total peritoneal leukocytes were collected and neutrophil and macrophage numbers analyzed by flow cytometry before (0 hours) or 2, 4, or 24 hours after IP injection of 3% thioglycollate in PBS to induce acute peritoneal inflammation. All values are presented as mean ± SEM (n = 6). ***P < .001; **P < .01; *P < .05.$

Leukocyte rolling and recruitment during inflammation in St3gal4-deficient, St3gal6-deficient, and double-deficient mice. (A) Leukocyte rolling in the autoperfused ex vivo microflow chamber. Microflow chambers were coated with recombinant murine (rm) P-selectin or rm E-selectin and perfused for 10 minutes with whole blood through a catheter connected to the mouse carotid artery (at least 5 chambers per group). Using an upright microscope, the number of rolling leukocytes calculated by rollers/(WBC count × 0.001) was assessed at 10 minutes. All values are shown as mean ± SEM (n = 6-12). *P < .05. (B) Effect of sialidase treatment on neutrophil rolling in the flow chamber. Isolated wild-type neutrophils were either left untreated for 1 hour (n = 15) or treated for 1 hour with Arthrobacter ureafaciens (AU) sialidase (n = 11) or with Streptococcus pneumonia (SP) sialidase (n = 14). Thereafter, neutrophils were perfused through flow chambers coated either with P- or E-selectin at a wall shear stress of 1 dyne/cm². All values are shown as mean ± SEM (*P < .05). (C) Leukocyte rolling in TNF-α–treated cremaster muscle venules in vivo. Leukocyte rolling flux fraction (%) was assessed for each group. Blocking of E-selectin and P-selectin were performed by systemic injection of E-selectin–blocking mAb 9A9 and P-selectin blocking mAb RB40.34, respectively. All values are depicted as mean ± SEM (n = 3-6). *P < .05. (D) Leukocyte rolling velocities in TNF-α–treated cremaster muscle venules in vivo. Cumulative distribution of leukocyte rolling velocities are shown for no treatment (left), treatment with E-selectin–blocking mAb 9A9 (middle), and treatment with P-selectin–blocking mAb RB40.34 (right). Average rolling velocities are indicated as mean ± SEM with 3-6 mice per group. *P < .05 versus wild-type. (E) Total peritoneal leukocytes were collected and neutrophil and macrophage numbers analyzed by flow cytometry before (0 hours) or 2, 4, or 24 hours after IP injection of 3% thioglycollate in PBS to induce acute peritoneal inflammation. All values are presented as mean ± SEM (n = 6). ***P < .001; **P < .01; *P < .05.

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