GM-CSF enhances neutrophil cytokine responses to influenza virus and specific TLR ligands. (A) MIP-1β was measured from supernatants of human neutrophils (10⁶ cells) incubated with or without GM-CSF (100 U/mL) for 30 minutes, then stimulated with Pam₂CSK₄ (10 ng/mL), poly (I:C) (50 μg/mL), LPS (10 ng/mL), R-848 (3 μM), CpG (6 μg/mL), or influenza virus (X31, 100 000 HA). Error bars here and elsewhere represent standard deviation (SD) of triplicates. Data shown are from 1 representative experiment; similar results were obtained in 6 independent experiments using different donors. (B) IL-8 was measured in the same cell supernatants as in panel A. (C) Intracellular cytokine staining for IL-8 was performed on human neutrophils treated with G-CSF (100 U/mL) and GM-CSF (100 U/mL). Cells were stimulated with Pam₂CSK₄ (100 ng/mL), CpG (10 μg/mL), R-848 (10 μM), or influenza virus (X31, 100 000 HA), treated with brefeldin A, and stained with antibodies to CD16, CD3, CD19, and HLA-DR. Cells were permeabilized then stained with antibody to IL-8 (solid line) or isotype control (gray histogram). Gates were set for CD16⁺, CD3⁻, CD19⁻, and HLA-DR⁻ cells.