G-CSF selectively reduces levels of surface CXCR4 in myeloid lineage cells. (A) Flow cytometric analysis of surface CXCR4 expression in bone marrow Gr1⁺ myeloid cells. Unfractionated bone marrow cell populations were incubated (1 × 10⁶/mL) at 37°C for 6 hours in medium alone or with G-CSF at concentrations of 0.1, 10, and 100 ng/mL. Cells were double stained for Gr1 (APC-labeled) and CXCR4 (FITC-labeled 2B11 antibody) or with APC- and FITC-labeled control antibodies. Gr1⁺ cells (R1 gate) represented 40.1% of total nucleated cells. (B) Analysis of CXCR4 expression in CD19⁺ B-lineage (top panel) and CD3e⁺ T-lineage (bottom panel) cells. Unfractionated bone marrow cell populations were incubated at 37°C for 6 hours with or without G-CSF (100 ng/mL). Cells were double stained for CD19 (APC-labeled) and CXCR4 (FITC-labeled) or CD3e (APC-labeled) and CXCR4 (FITC-labeled) and gated on CD19⁺ cells (44% of total nucleated cells) or CD3e⁺ (5.2% of total nucleated cells) cells. (C) Expression of CXCR4 in Gr1⁺ cells as a function of culture time (0-18 hours) in medium alone or with G-CSF (100 ng/mL). The results are expressed as mean fluorescence intensity (MFI) of CXCR4 detected on Gr1⁺ cells (mean ± SEM of 3 experiments). (D) Time-dependent reduction of surface CXCR4 in Gr1⁺ cells induced by G-CSF (100 ng/mL). Unfractionated bone marrow cell populations were incubated at 37°C for 18 hours with or without G-CSF (100 ng/mL). The results are expressed as the mean (± SEM of 5 experiments) percent reduction of surface CXCR4 induced by G-CSF compared with medium alone. *P < .05 medium versus G-CSF. (E) G-CSF reduces surface CXCR4 in precultured bone marrow cells. Unfractionated bone marrow cell populations were preincubated (37°C, 18 hours) in medium alone to achieve high-level surface CXCR4 expression, and further incubated for 18 hours with or without G-CSF (100 ng/mL). The results reflect levels of surface CXCR4 expression (expressed as MFI) in Gr1⁺ cells cultured with or without G-CSF after the preculture (mean ± SEM of 3 experiments). (F) Unfractionated bone marrow cell populations were incubated (37°C) for 18 hours with or without G-CSF (100 ng/mL), or for 40 minutes with or without SDF-1α (400 ng/mL) to achieve a reduction of surface CXCR4. After washing (PBS containing 50 mM glycine), the cells were incubated for 2 hours at 37°C in medium containing cycloheximide (100 μg/mL). The results reflect surface CXCR4 expression in Gr1⁺ myeloid cells (representative experiment of 3 performed).