FIGURE 6.

CCL7 is induced during anti-IgE sensitization, and its absence reduces Ag-induced Akt phosphorylation. Mature BMMCs (>4 wk of culture) from wild-type mice were sensitized or not with anti-DNP IgE (100 ng/ml), and levels of CCL7 mRNA and protein were quantified at different time points. (A) Total RNA from sensitized cells was isolated, and Ccl7 expression was examined using real-time PCR. (B) Supernatants of culture medium were collected, and protein levels were analyzed by ELISA. (C) BMMCs were stimulated or not with different concentrations of ionophore for 20 min, and β-hexosaminidase activity was determined as a measure of degranulation. Data are mean ± SEM and are representative of four separate experiments. (D) A total of 1 × 10⁶ BMMCs from wild-type and CCL7^−/− mice was incubated overnight in medium containing anti-DNP IgE (100 ng/ml) and treated with 10 ng/ml Ag for 5 min. To examine the phosphorylation of Akt, Western blotting was performed on 30 μg of total protein using Abs to total Akt, phospho-Akt (Ser⁴⁷³), and β-actin (loading control). Relative densitometric analysis is presented. Data are mean ± SEM and are representative of three separate experiments. *p < 0.05, **p < 0.01, ANOVA. ns, not significant.