OBL differentiation is inhibited by IL-3. (A) Primary mouse OBL cells were cultured for 10 days in osteogenic media with or without the addition of IL-3 (0.01 to 10 ng/mL), TNF-α (1 ng/mL), or IL-6 (1 ng/mL). ALP activity was measured as described in “Materials and methods.” ALP activity was decreased in a dose-dependant manner by the presence of IL-3. TNF-α and IL-6 are positive and negative controls, respectively. ALP activity in the untreated control ranged from 40 to 70 mU per 10⁴ cells plated. Inhibition by IL-3 ranged from 50% to 80% of control. Results represent the mean ± standard deviation for 4 wells for each concentration and are representative of 3 independent experiments. ^*P < .05. (B) Primary human BM adherent cells were cultured for 10 days (CFU-F) or 21 days (CFU-OB) in osteogenic media and treated with IL-3 (0.02 to 20 ng/mL). Alkaline phosphatase and Alizarin Red staining was performed after that culture period, respectively, and the colonies were counted and quantified. Results represent the mean ± standard deviation of the number of CFU-Fs and CFU-OBs in 3 independent experiments. ^*P < .05. (C) Cells were treated for 10 days with osteogenic media supplemented with BMP-2 (50 ng/mL) with or without IL-3, and ALP activity was measured as described in “Materials and methods.” BMP-2 alone stimulated ALP expression and was blocked by IL-3. IL-6 is a negative control. (D) Primary murine OBL cells were cultured for 3 weeks in osteogenic media with or without the addition of IL-3 and then stained for mineral deposition by the von Kossa method as described in “Materials and methods.” Mineral deposition was inhibited in response to IL-3. Shown is a representative well from each concentration. (E) The culture plate from panel D was scanned and quantified for relative amount stained using Adobe Photoshop. Quantification of von Kossa staining shows a dose-dependent decrease in amount stained in response to IL-3. (F) IL-3 increases the capacity of TNF-α to inhibit OBL differentiation. Primary murine OBL cultures were treated with osteogenic media and increasing amounts of TNF-α (0.01 to 10 ng/mL) without (▪) or with (▧) IL-3 (100 pg/mL). IL-6 treatment with () or without (▧) IL-3 is a negative control. ALP activity was determined as described in “Materials and methods.” In all panels, data are represented as average ± standard deviation for 4 wells for each concentration and are representative of 3 independent experiments. ^*P < .05.