Effect of iron on IFN-γR2 chain internalization. (A) ST4 cells were cultured at 37°C in complete medium (▪ and ▪) or in serum-free medium (□ and ○). After 24 hours of culture, 10 μM FeSO4 was added (▪ and ○); aliquots were recovered at different time points and IFN-γR2 surface expression was evaluated by flow cytometric analysis. Results are expressed as percentage of positive cells calculated by subtracting nonspecific fluorescence detected with isotype-matched control IgG1 from specific fluorescence. One of 3 independently performed experiments is shown. (B) Total RNA was extracted from ST4 T cells cultured for 24 hours in complete or serum-free medium, with or without 10 μM DFO or 10 μM FeSO4, and IFN-γR2 and GAPDH (housekeeping gene) mRNAs were analyzed. (C) IFN-γR2 surface expression was evaluated in ST4 cells cultured in serum-free medium (left), serum-free medium plus 10 μM FeSO4 for 1 hour (middle), serum-free medium plus 10 μM FeSO4 for 1 hour, washed 3 times, and then treated for 1 hour with 10 μM DFO (right). Open histograms represent background of mouse IgG. Negative control and gray histograms represent the expression of IFN-γR2. (D) ST4 T cells cultured for 24 hours with or without serum, or without serum plus 10 μM FeSO4, were recovered and incubated with anti–IFN-γR2 mAb at 4°C (□) or 37°C (▪). After 1 hour, cells were permeabilized and stained with PE-conjugated anti–mouse Ig. Mean of IFN-γR2 internalized fluorescence was calculated by subtracting the mean of internalized fluorescence obtained with isotype-matched mAb from that obtained with specific anti–IFN-γR2 mAb.
