STAT3-deficient neutrophils have impaired CXCR2-mediated chemotaxis. (A) Bone marrow neutrophils from wild-type (WT, □) or STAT3-deficient (KO, ▪) mice were examined for MIP-2–dependent chemotaxis in migration assays with 3-μm Transwells (n = 6 per genotype) (P = .065 comparing WT with KO). Average values from at least 3 independent experiments are shown. (B) Neutrophils were derived from lin^– progenitors in G-CSF–containing medium ex vivo. At day 12, neutrophils were tested for MIP-2–dependent chemotaxis (n = 5 for WT, □;n = 4 for KO, ▪) (P = .006 comparing WT with KO). Average values from at least 3 independent experiments are shown. (C) Bone marrow neutrophils were tested for chemotaxis toward KC (n = 5 for WT, □;n = 4 for KO, ▪)(P = .028 comparing WT with KO). Average values from at least 3 independent experiments are shown. (D) Chemotaxis toward fMLP was tested with bone marrow neutrophils as indicated (WT, □; KO, ▪; n = 4 per genotype) (P = .710 comparing WT with KO). Average values from at least 3 independent experiments are shown. (E) Bone marrow cells from wild-type or STAT3-deficient mice were stained with antibodies for Gr-1 and CXCR2 and examined by flow cytometry. The histogram shows CXCR2 cell-surface expression in the Gr-1⁺ population from WT (open gray) and KO (open black) mice relative to isotype control (solid black). Results are representative of 3 independent experiments. (F) Bone marrow cells were stained with PE-conjugated Gr-1 antibody and then stimulated with MIP-2 or left untreated for the times indicated. Cells were fixed and stained with phalloidin-FITC and analyzed by flow cytometry. Relative F-actin levels were determined as described in “Chemotaxis and actin polymerization assays” and plotted against MIP-2 treatment time points (n = 5 per genotype) (P = .049, F-actin at 30 seconds comparing WT with KO). Average values from 4 independent experiments are shown. Error bars represent SEM in all panels.