Adhesion to E-selectin promotes growth inhibition and apoptosis of human and murine hematopoietic progenitor cells independent of PSGL-1 by Ingrid G. Winkler, Karen R. Snapp, Paul J. Simmons, and Jean-Pierre Lévesque Blood Volume 103(5):1685-1692 March 1, 2004 ©2004 by American Society of Hematology

Human CD34+ HPCs adhere to P- and E-selectins resulting in growth inhibition and apoptosis. Human CD34+ HPCs adhere to P- and E-selectins resulting in growth inhibition and apoptosis. (A) Dose-dependent adhesion of CD34+ HPCs to microwells coated with increasing concentrations of CD14-Fc control or E- and P-selectin–Fc. (B) BM CD34+ proliferation at day 7 of culture in the presence of 36GS on microwells coated with increasing concentrations of CD14-Fc (○), E-selectin-Fc (▴) or P-selectin-Fc (♦). Cells were seeded at 104/mL. (C) Apoptosis of BM CD34+ cells at day 7 of culture in the presence of 36GS on microwells coated with increasing concentrations of CD14-Fc, E-selectin–Fc, or P-selectin–Fc. (D) Proliferation of CD34+ cells isolated from steady-state BM, mobilized peripheral blood (PBPC), or umbilical cord blood (UCB) on microwells coated with BSA (10 μg/mL), P-selectin (1800 sites/μm2), or E-selectin (550 sites/μm2) alone or in combination (P+E). Cells (104/mL) were seeded at day 0 in the presence of 36GS and counted on day 7. *indicates statistically different (P < .05) from BSA-treated control group. (E) Apoptosis of CD34+ cells isolated from steady-state BM, mobilized peripheral blood, or umbilical cord blood on microwells coated as described for panel D with BSA, P- or E-selectin alone, or in combination. Cells were grown in the presence of 36GS and TUNEL assays performed on day 7. (A-C) Data are expressed as a mean ± SD of one representative experiment. (D-E) Data are expressed as mean ± SEM for 3 independent experiments on BM, peripheral blood, or umbilical cord blood CD34+ HPCs. * indicates statistically different (P < .05) from BSA control groups. Ingrid G. Winkler et al. Blood 2004;103:1685-1692 ©2004 by American Society of Hematology

Differing adhesion of human CD34+ cells to P- and E-selectin in the presence of function-blocking antibody to PSGL-1 or the proteases O-sialoglycoprotein endopeptidase, neutrophil elastase, and cathepsin G. CD34+ HPCs were preincubated with either isotype c... Differing adhesion of human CD34+ cells to P- and E-selectin in the presence of function-blocking antibody to PSGL-1 or the proteases O-sialoglycoprotein endopeptidase, neutrophil elastase, and cathepsin G. CD34+ HPCs were preincubated with either isotype control mAb, KPL1 mAb, O-sialoglycoprotein endopeptidase (OSG), or a combination of neutrophil elastase and cathepsin G (NE+CG) and added to microwells coated with 10 μg/mL BSA, P-selectin (1800 sites/μm2), or E-selectin (550 sites/μm2). Results are expressed as mean ± SD of one representative experiment from 3 made in triplicate. *indicates statistically different (P < .05) from isotype-treated control groups. Ingrid G. Winkler et al. Blood 2004;103:1685-1692 ©2004 by American Society of Hematology

K562 cells stably transfected with FucT-VII exhibit E-selectin-mediated growth inhibition and apoptosis in the absence of PSGL-1. K562 cells stably transfected with FucT-VII exhibit E-selectin-mediated growth inhibition and apoptosis in the absence of PSGL-1. (A) Adhesion of K562 alone or transfected with PSGL-1 (KP cells), FucT-VII (KF cells), or both (KPF cells) to microwells coated with P-selectin (1800 sites/μm2) or E-selectin (550 sites/μm2). (B) Proliferation of K562, KP, KF, and KPF cells grown in serum-deprived medium on microwells coated with BSA, P-selectin, or E-selectin. Cells were seeded at 104/mL and counted after 5 days incubation. (C) Apoptosis of K562, KP, KF, and KPF cells grown in serum-deprived medium on microwells coated with 10μg/mL BSA, P-selectin, or E-selectin. TUNEL assay was performed after 5 days incubation. *indicates statistically different (P < .05) from BSA control groups. Results are expressed as mean ± SEM of 3 independent experiments made in triplicate. Ingrid G. Winkler et al. Blood 2004;103:1685-1692 ©2004 by American Society of Hematology

E-selectin inhibits proliferation and induces apoptosis of mouse HPCs E-selectin inhibits proliferation and induces apoptosis of mouse HPCs. (A) Adhesion of murine BM lineageneg Sca-1+ c-KIT+ (LSK) cells to immobilized BSA, rmu P-selectin (1800 sites/μm2), or rmu E-selectin (550 sites/μm2). E-selectin inhibits proliferation and induces apoptosis of mouse HPCs. (A) Adhesion of murine BM lineageneg Sca-1+ c-KIT+ (LSK) cells to immobilized BSA, rmu P-selectin (1800 sites/μm2), or rmu E-selectin (550 sites/μm2). (B) Proliferation of murine BM LSK cells grown in the presence of cytokine combinations 11FTS, 36GS, or 3611FTS on microwells coated with BSA, P-selectin, or E-selectin. Cells were seeded at 104/mL and counted after 7 days incubation in serum-deprived medium. (C) Apoptosis of murine BM LSK cells cultured 7 days in the presence of 11FTS, 36GS, or 3611FTS on microwells coated with BSA, P-selectin, or E-selectin. (D) Pre–colony-forming cell assay. Bone marrow LSK cells were cultured in the presence of 11FTS, 36GS, or 3611FTS on microwells coated with BSA, P-selectin, or E-selectin. After 7 days incubation, cells were transferred to fresh noncoated wells and cultured an additional 7 days before colony assays were performed. The results are expressed as the number of colonies derived from a single original LSK cell. The clonogenic efficiencies (expressed as a percentage) of the cells put into methylcellulose are indicated above each column. *Statistically different (P < .05) from BSA treated control groups. Results are expressed as mean ± SEM of 3 (D) or 5 (A-C) independent experiments made in triplicate. Ingrid G. Winkler et al. Blood 2004;103:1685-1692 ©2004 by American Society of Hematology

E-selectin inhibits proliferation and induces apoptosis of HPCs from PSGL-1–/– mice. E-selectin inhibits proliferation and induces apoptosis of HPCs from PSGL-1–/– mice. Bone marrow lineageneg Sca-1+ c-KIT+ (LSK) sorted cells from PSGL-1–/– and wild-type control mice were cultured for 5 days in the presence of 6 cytokines (3611FTS) on wells coated with either BSA (□), rmu P-selectin–Fc at 1800 sites/μm2 (▦), or rmu E-selectin–Fc at 550 sites/μm2 (▪). Concentrations of viable cells and percentage of apoptotic cells are indicated in panels A and B, respectively. Mean values ± SD of 2 experiments made in triplicate. *Statistically different (P < .05) from BSA control groups. Ingrid G. Winkler et al. Blood 2004;103:1685-1692 ©2004 by American Society of Hematology