Analysis of CD14^hi cells generated from band-stage neutrophils. (A) Neutrophils from G-CSF–mobilized donors were stimulated with proinflammatory cytokines (GM-CSF 10 ng/mL, TNFα 25 ng/mL, IL-1β 10 ng/mL). At day 5, CD14^hi cells were FACS-sorted and cell morphology was assessed (bar = 20 µm). Data are representative of 4 independent experiments. (B) Cell morphology and cell size were analyzed by May-Grünwald-Giemsa staining (days 2-7; bar = 10 µm) or FACS (day 5; CD14⁺ or LF⁺ cell subsets were gated and analyzed for forward scatter intensity). (C) Day 5–stimulated neutrophils were gated as indicated and analyzed for the expression of informative marker molecules. Data are representative of 3 experiments. (D) CD15⁺CD54^– neutrophils exhibit increased CD14^hi cell differentiation potential. Freshly isolated neutrophils from G-CSF–mobilized donors were FACS-sorted for CD15 and CD54. CD15⁺CD54⁺ and CD15⁺CD54^– neutrophils were stimulated with the cytokines GM-CSF (10 ng/mL), TNFα (25 ng/mL), and IL-1β (10 ng/mL) for 5 days and analyzed by FACS for CD14 and LF. Bars represent mean ± SD (percentages of phenotypically defined cells; n = 3). (E) Neutrophils from G-CSF–mobilized donors were stimulated as in (A) for 5 days. Phospho-MKK6 and MKK6 expression levels were assessed by western blot comparing proteins from equal number of cells. FACS-sorted CD14^hi vs CD14^– cells were separately analyzed.