Analysis of the relative contribution of G-CSF and soluble mediators induced by G-CSF to the reduction of surface CXCR4 on bone marrow myeloid cells. Unfractionated bone marrow cell populations were preincubated (1 × 10⁶/mL) at 37°C for 18 hours in medium alone or with G-CSF (100 ng/mL). (A) Cell-free supernatants from these cultures were preincubated (1 hour, 37°C) with rabbit neutralizing antibody against human (final concentration, 5 μg/mL) and rat neutralizing antibodies against mouse (final concentration, 3 μg/mL) G-CSF or with PBS only, and then added undiluted (1 mL) to freshly isolated bone marrow cell populations (1 × 10⁶ cells); the mixture was further incubated (6 hours, 37°C). Levels of CXCR4 expression in Gr1⁺ cells were measured by flow cytometry. The results are expressed as MFI (± SE) of triplicate determinations. *P < .05 (medium versus G-CSF). (B) Detection of G-CSF bioactivity in culture supernatants from bone marrow cell populations incubated with or without neutralizing antibodies against mouse and human G-CSF (as described under “Preparation of murine and human bone marrow cells”). G-CSF bioactivity was measured by ³H thymidine incorporation in murine NFS-60 cells, and the results are expressed as mean (± SE) of triplicate cultures. *P < .05. (C-D) Neutrophil elastase reduces levels of surface CXCR4 in bone marrow myeloid (C) and lymphoid (D) cells. Unfractionated bone marrow cell populations were incubated (1 × 10⁶/mL in DMEM medium with 0.2% BSA for 4 hours at 37°C) with medium alone, leukocyte elastase (enzyme, 40 μg/mL), and with leukocyte elastase plus leukocyte elastase inhibitor III (10 μM). Levels of surface CXCR4 were measured by flow cytometry on Gr1⁺ cells and lymphoid cells. (E) Dose dependency of leukocyte elastase–induced reduction of surface CXCR4 on Gr1⁺ cells and lymphoid cells. Unfractionated bone marrow cell populations were incubated with leukocyte elastase (5-100 μg/mL) for 2 hours at 37°C. The results are expressed as percent CXCR4 expression compared with untreated cells (MFI of elastase-treated cells/MFI of untreated cells × 100). (F) Surface CXCR4 expression in the presence of specific enzyme inhibitors. Unfractionated bone marrow cell populations were preincubated (2 hours, 37°C) in medium only (DMEM with 0.2% BSA); or with leukocyte elastase inhibitor III (LEI 10 μM); cathepsin-G (CGI, 10 μM); LEI plus CGI (10 μM each); the CD26/dipeptdyl peptidase inhibitor AB192 (50 μM); and the metalloproteinase inhibitor BB94 (10 μM). Subsequently, cells were incubated with or without G-CSF (100 ng/mL, 4 hours, 37°C). Surface CXCR4 was measured on Gr1⁺ cells by flow cytometry. The results are expressed as percent MFI compared with cells incubated in medium alone without inhibitors.