![Figure 7. Effect of 4F-GalNAc on selectin binding and leukocyte recruitment to peritoneum. BMCs were cultured in media containing granulocyte colony-stimulating factor and IL-3, and 25μM 4F-GalNAc, 25μM GalNAc, or vehicle control (0.125% DMSO). (A) After 2 days in culture, 2 cell populations (regions R1 and R2) stained positive for nuclear dye LDS-751 in the flow cytometer. Cells in R1 were neutrophils (Gr-1+1A8+). (B) PSGL-1 and CD45 antigen expression, and P-selectin IgG binding to Gr-1+ cells were measured. 4F-GalNAc, but not GalNAc or vehicle control, reduced P-selectin fusion protein binding to cells. Data are mean ± SEM with respect to vehicle control. *P < .05 with respect to all other treatments. (C) BMCs cultured for 2 days with 25μM 4F-GalNAc or vehicle control were labeled with either DiL or DiO dyes. Mixed-cell populations just before injection into C57BL/6 recipient mice contained approximately equal numbers of DiL-labeled vehicle control cells [(Vehicle)DiO] and DiO-labeled 4F-GalNAc–treated cells [(4F-GalNAc)DiL] or vice versa. (D) Peritoneal lavage sample from mice obtained 5 hours after intraperitoneal injection of thioglycollate shows substantial accumulation of neutrophils in the peritoneum. These cells were positive for LDS-751, Gr-1, and 1A8. DiO/DiL-labeled cells were not injected in this experiment. (E) Granulocytes from the peritoneal lavage of animals, which were tail-vein injected with mixed DiO + DiL BMCs just before induction of peritonitis. Number of DiL-labeled cells (vehicle control) exceeds DiO-labeled cells (4F-GalNAc–treated). (F) Ratio of 4F-GalNAc to vehicle control cells in infusion and peritoneum lavage sample. Data are mean ± SEM for 4 animals injected with DiL-labeled 4F-GalNAc–treated cells along with paired DiO-labeled vehicle controls. Four animals were injected with DiO/DiL labeling switched. 4F-GalNAc treatment reduced cell migration into peritoneum irrespective of whether these cells were labeled with DiO or DiL. *P < .05 with respect to infusion sample. (G) A total of 100 mg/kg/day 4F-GalNAc or vehicle control was injected into mice for 4 days before induction of peritonitis. Total leukocyte (Total), neutrophil (Neut), eosinophil (Eos), and macrophage (Mac) counts were determined in peritoneal lavage 5 hours after intraperitoneal injection of thioglycollate. Data are mean ± SEM for 4 animals for each treatment. *P < .05 with respect to vehicle control.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/115/6/10.1182_blood-2009-07-231480/4/zh89990948290007.jpeg?Expires=1724269433&Signature=zRl2vpIqHN5u-K27ERudgX~3pN9WbgpbGTF2n4XD4~V8GkmBxO1-~DMh66UQqMeFJiRfQ0fT14jCInANycr~VFvJdrzRFghVevm5d0opiOU3QeHDIAc3ivxsyr-xBjSxTA9djcmDYJWMXWczIIq1YIqx3ZkbZoNdXJQU0BtKe06LKIH0wi4a6YypBwGpW9ji-0wIA92zG3VhiPnVwVZwbrHNCA~NakEVR2HXjywHbcLVkP1u9Tk5p6ASesYLIuBaSJw1hnVIXpDrjd4fwyxe-UeGXxNXI80hC3N-L0K5UVYAmveBxukuA9DZRXBv7DbRaGnpqa7lIm2ErzKUB5cm-w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Effect of 4F-GalNAc on selectin binding and leukocyte recruitment to peritoneum. BMCs were cultured in media containing granulocyte colony-stimulating factor and IL-3, and 25μM 4F-GalNAc, 25μM GalNAc, or vehicle control (0.125% DMSO). (A) After 2 days in culture, 2 cell populations (regions R1 and R2) stained positive for nuclear dye LDS-751 in the flow cytometer. Cells in R1 were neutrophils (Gr-1+1A8+). (B) PSGL-1 and CD45 antigen expression, and P-selectin IgG binding to Gr-1+ cells were measured. 4F-GalNAc, but not GalNAc or vehicle control, reduced P-selectin fusion protein binding to cells. Data are mean ± SEM with respect to vehicle control. *P < .05 with respect to all other treatments. (C) BMCs cultured for 2 days with 25μM 4F-GalNAc or vehicle control were labeled with either DiL or DiO dyes. Mixed-cell populations just before injection into C57BL/6 recipient mice contained approximately equal numbers of DiL-labeled vehicle control cells [(Vehicle)DiO] and DiO-labeled 4F-GalNAc–treated cells [(4F-GalNAc)DiL] or vice versa. (D) Peritoneal lavage sample from mice obtained 5 hours after intraperitoneal injection of thioglycollate shows substantial accumulation of neutrophils in the peritoneum. These cells were positive for LDS-751, Gr-1, and 1A8. DiO/DiL-labeled cells were not injected in this experiment. (E) Granulocytes from the peritoneal lavage of animals, which were tail-vein injected with mixed DiO + DiL BMCs just before induction of peritonitis. Number of DiL-labeled cells (vehicle control) exceeds DiO-labeled cells (4F-GalNAc–treated). (F) Ratio of 4F-GalNAc to vehicle control cells in infusion and peritoneum lavage sample. Data are mean ± SEM for 4 animals injected with DiL-labeled 4F-GalNAc–treated cells along with paired DiO-labeled vehicle controls. Four animals were injected with DiO/DiL labeling switched. 4F-GalNAc treatment reduced cell migration into peritoneum irrespective of whether these cells were labeled with DiO or DiL. *P < .05 with respect to infusion sample. (G) A total of 100 mg/kg/day 4F-GalNAc or vehicle control was injected into mice for 4 days before induction of peritonitis. Total leukocyte (Total), neutrophil (Neut), eosinophil (Eos), and macrophage (Mac) counts were determined in peritoneal lavage 5 hours after intraperitoneal injection of thioglycollate. Data are mean ± SEM for 4 animals for each treatment. *P < .05 with respect to vehicle control.
