Production and release of biologically active RA by IL-3–stimulated basophils. In vitro reporter assay. RA-responsive F9-RARE-lacZ reporter cells were incubated for 24 hours without basophils or cocultured with basophils (± Ba), which were left untreated or stimulated with 10 ng/mL IL-3, as indicated. Reporter cells were harvested and assayed for β-galactosidase activity, which relates to the amounts of RA released by basophils. (A) Reporter cells were incubated with or without basophils in the presence of rising concentrations of retinal or retinol and (B) with or without 10 nM retinal or 30 nM retinol, in the presence or absence of RAR antagonist AGN193109 (AGN, 100 nM). (Inset) As a control, β-galactosidase activity in reporter cells as a function of exogenous RA concentration and its inhibition by AGN193109 (100 nM) is shown. (C) The amount of RA released is proportional to basophil numbers. Different numbers of basophils were cultured for 24 hours with IL-3, and then coincubated with reporter cells in the absence or presence of retinoids (10 nM retinal or 30 nM retinal) for additional 24 hours. Mean values (± SD) of triplicates of representative experiments are shown. Data in the different panels are from separate experiments with basophils from different donors.