STAT3-dependent regulation of neutrophil migration and CXCR2-mediated Raf/MEK/ERK signaling. (A) Peripheral blood neutrophil numbers were determined for WT (□) and STAT3-deficient (knockout [KO], ■) mice treated with MIP-2 (50 μg/kg) or BSA carrier for 30 minutes. Neutrophil amounts in MIP-2–treated mice were normalized to BSA-treated controls of the appropriate genotype and relative levels are shown (n = at least 4 for WT and KO). (B) Absolute neutrophil numbers in total spleen, blood, and bone marrow of BSA- or MIP-2–treated mice are shown (n = at least 3 for WT and KO for each condition). (C) CXCR2 expression within the Gr-1⁺ bone marrow population of BSA- and MIP-2–treated WT (black line) and STAT3-deficient (KO, gray line) mice is shown, relative to isotype controls (dashed black line). Results are representative of 3 independent experiments. (D) Activation of c-Raf, MEK1/2, and ERK1/2 in MIP-2–treated (100 ng/mL) or unstimulated Gr-1⁺ cells was analyzed by immunoblotting and quantified by densitometry. Results are representative of at least 3 independent experiments. (E) MIP-2–responsive chemotaxis of bone marrow neutrophils from WT mice was examined in the absence of or after, 30 minutes pretreatment with the MEK1/2 inhibitor U0126 (10μM or 50μM as indicated, n = 5 for each condition). Average values from 3 independent experiments are shown. (F) WT bone marrow neutrophils were stimulated with MIP-2 (100 ng/mL for 5 minutes) in the presence or absence of U0126 (10μM or 50μM as indicated). ERK1/2 activation was assessed by immunoblotting with whole-cell lysates; results are representative of 3 independent experiments. Error bars represent SEM; *P < .05, **P < .01, ***P < .001.