IRp60 inhibits eotaxin-induced eosinophil transmigration and IL-5/GMCSF-induced eosinophil survival and activation. Eosinophils were incubated with either anti-IRp60 or control antibodies (isotype, anti-p58.2), followed by sheep anti-mouse F(ab′)₂ to cross-link IRp60 receptors. The cells were washed and used in 3 different assays: (A) transmigration, (B) survival, and (C) mediator release. (A) Transmigration was assessed in a microwell dual-chamber system in response to recombinant human eotaxin (1-100 ng/mL) for 90 minutes (37°C, 5% CO₂). After incubation, the cells that migrated to the lower chamber were evaluated by FACS analysis obtained by acquiring events for 60 seconds. Data are expressed as percent inhibition of eotaxin-treated cells ± SD; n = 4. (B) Eosinophil survival was evaluated by incubating them with various concentrations of IL-5/GM-CSF (5-50 ng/mL) for the indicated time points. Thereafter, the cells were assessed for apoptosis using annexin V-PI staining (n = 4; ^*P < .05; ^**P < .005). (C) Eosinophil mediator release was evaluated by incubating the cells with various concentrations of IL-5/GM-CSF (5-50 ng/mL) for 18 to 20 hours. Cytokine release in the cell supernatants was measured by using the FlowMultiplex array. Data are expressed as pg/mL of each cytokine ± SD (n = 5; ^*P < .05; ^**P < .005).