The inhibitory effect of G-CSF is mediated by activation of STAT3 signaling and the subsequent suppression of MIP-2–elicited cell signaling. (A) G-CSF treatment does not alter the KC and MIP-2 serum levels in mice i.p. injected with E. coli. Mice were i.p. injected with 2 ×10⁶ CFU E. coli and/or i.v. injected with 2 µg G-CSF. Data shown are representative of three experiments (n = 5 mice). (B) Mouse BM neutrophils were stimulated with MIP-2, G-CSF, or G-CSF + MIP-2 for the indicated times. Phosphorylated STAT3 (P-STAT3), ERK1/2 (P-ERK1/2), and AKT (P-AKT) levels were analyzed by Western blotting. Data shown are representative of four independent experiments. (C) Relative amounts of phosphorylated Akt, Erk, and STAT3 were quantified with ImageJ software (National Institutes of Health; Subramanian et al., 2007). All samples were normalized to total actin. Basal level refers to phosphorylated protein levels at time 0 min. Data are representative of four independent experiments. Student’s t test was used. (D) The inhibitory effort of G-CSF– on MIP-2–elicited signaling was rescued by 0.5 µg/ml JAK inhibitor INCB018424. (E) JAK inhibitor INCB018424 blocks G-CSF–elicited STAT3 phosphorylation. (F) Treatment with INCB018424 reverses G-CSF–induced reduction of Erk phosphorylation in cells stimulated with MIP-2. Data are representative of four independent experiments. (G–K) The inhibitory effect of G-CSF on MIP-2–elicited neutrophil chemotaxis could be rescued by JAK inhibitor INCB018424. (G) Chemotaxis of mouse neutrophils in response to MIP-2 (also see Videos 6 and 10). (H) Tracks of migrating neutrophils. (I–K) Neutrophil chemotaxis was analyzed for migration speed (I), directionality (J), and upward directionality (K) as described in Fig. 5 (C–E). Data are representative of three experiments (n = 20 cells). All data are represented as means ± SD. *, P < 0.01 versus neutrophils treated with G-CSF but not INCB018424.