PMC

FACS sorted Ly6G^pos neutrophil and Ly6G^neg non neutrophil BM cells were co-cultured either with MC3T3 pre-osteoblast cells or differentiated OB in presence of G-CSF for 3 days. (A) MC3T3 cells apoptosis and (B) differentiated OB apoptosis was determined by flow cytometry after Annexin V staining (X ± SEM; N=3 experiment). (C) Annexin V expression on BM CD45⁻ CD31⁻ Ter119⁻ osteolineage cell from the G-CSF treated control or neutropenic mice (X ± SEM; N=5 mice in each group). *P< 0.05 compared to vehicle; † P< 0.05 compared to isotype plus G-CSF; ¶ P > 0.05 compared to vehicle.

(A) Effect of ROS production blockade on G-CSF induced HSPC mobilization. Mice were treated with G-CSF or G-CSF plus NAC and LSK cell mobilization in PB was determined by flow-cytometry. (B) ROS production in G-CSF treated mice BM neutrophil and non-neutrophil cells. (X ± SEM; N= 4 mice). (C) Effect of ROS production blockade on G-CSF mediated oeteolineage cell apoptosis. Mice were treated with G-CSF alone or G-CSF plus NAC and Annexin V expression in CD45⁻CD31⁻Ter119⁻ BM cells was determined by flow cytometry. † P< 0.05 compared to G-CSF; *P< 0.05 compared to Ly6G^pos vehicle (PBS); ¶ P< 0.05 compared to PBS treated Ly6G^neg cells.

(A) Effect of Ly6G^posCD115⁻ neutrophil or Ly6G^neg non-neutrophil cells on SDF-1 production by MC3T3 cells, a pre-osteoblast and (B) by differentiated OB. FACS sorted Ly6G^pos neutrophil and Ly6G^neg non neutrophil BM cells were co-cultured with pre-osteoblast or with differentiated OB in presence of G-CSF for 3 days and SDF-1 expression in MCET3 cells was determined by Q-RT-PCR and SDF-1 levels in culture supernatant was measured in by ELISA (X ± SEM; N=3 experiments). (C) FACS sorted Ly6G^pos BM neutrophils were co-cultured at different ration with pre-osteoblast cells in presence of G-CSF for 3 days and SDF-1 was measured in culture supernatant by ELISA (X ± SEM; N=2 experiment). *P< 0.05 compared to vehicle; † P< 0.05 compared to isotype plus G-CSF; § P > 0.05 compared to isotype plus G-CSF; ¶ P > 0.05 compared to vehicle.

Mice were treated with single dose of anti-Gr1 antibody or IgG isotype control antibody on day 1 of a 3 day G-CSF mobilization. (A) Ly6G⁺ CD115⁻ neutrophils in BM and manual differential neutrophil counts (200 cells/slide) on Wright-Geimsa-stained cytospins (insert) (B) SLAM LSK and LSK cells in the PB of IgG isotype plus G-CSF or anti-Gr1 plus G-CSF treated mice. (C) Spearman’s rank correlation between the number of BM neutrophils and PB LSK cells. (D) SLAM LSK and LSK in the BM of G-CSF treated control or neutropenic mice. (E) Competitive transplantation of repopulating HSC using equal volumes of PB from G-CSF treated control or neutropenic mice (CD45.2 C57BL/6) in combination 2 × 10⁵ BM competitor cells from CD45.1 BoyJ mice. Donor chimerism was measured by flow cytometry in peripheral blood at 6 months after transplantation. (F) Donor derived SLAM LSK cell number in BM of transplanted mice at 6 month (X ± SEM; N= ≥ 5 mice per group). *P< 0.05 compared to vehicle; † P< 0.05 compared to isotype plus G-CSF; § P > 0.05 compared to isotype plus G-CSF.

Mice were treated with G-CSF for 3 days and neutrophils were depleted by single injection of anti-Gr1 antibody on day 1 of the G-CSF regime. (A) Left: Representative photomicrographs show endosteal OB after H&E staining and graph show average OB counts per 400× field and Right: Bone associated cells were recovered from the femurs and tibiae of mice by flushing and treating with collagenase type 1 and CD45⁻ CD31⁻ Ter119⁻ gated cell population quantitated by flow cytometry. (B) MPC enriched Sca-1⁺Alcam⁻ cells and OB enriched Sca-1⁻Alcam⁺ cells in the collagenase treated BM fraction was determined by flow cytometry (X ± SEM; N=3 per group in each of 2 experiment). (C) SDF-1 and VCAM-1 m-RNA expression relative to β-actin in CD45⁻ CD31⁻ Ter119⁻ enriched BM cells harvested from G-CSF treated control or neutropenic mice (X ± SEM; N=4 per group) (D) SDF-1, and SCF in the BM extracellular fluid of G-CSF treated control or neutrophil depleted mice as measured by ELISA (X ± SEM; N=3 per group in each of 2 experiment). (E) Effect of anti-Ly6G antibody plus G-CSF administration on BM Ly6G⁺ CD115⁻ neutrophil and Ly6G⁻ CD115⁺ F4/80⁺ macrophage and on (F) CD45⁻ CD31⁻ Ter119⁻ osteolineage cells in the BM, analyzed by flow cytometry (X ± SEM; N=4 per group). *P< 0.05 compared to vehicle; † P< 0.05 compared to isotype plus G-CSF; § P > 0.05 compared to isotype plus G-CSF; ¶ P > 0.05 compared to vehicle.