Fig 4.

Human Fe-Tf inhibits the effect of HFE on iron uptake but not release. (A) Human Fe-Tf was titrated into cultures of HFE-positive (open squares) or HFE-negative (filled circles) HeLa-HFE cells. With no added Tf, HFE-negative cells contain more ferritin than HFE-positive cells, but as human Fe-Tf increases toward 100 μg/ml (1.33 μM), the difference between the ferritin expression in HFE-positive and -negative cells disappears. Points plotted are mean fluorescence of 25,000 cells. (B) U937 cells grown in media containing 670 nM (50 μg/ml) human Fe-Tf express the same level of ferritin in the presence (green line histogram) or absence (gray filled histogram) of added 300 nM WT HFE. (C and D) The effect of HFE on iron uptake is dose-dependent: 400 nM HFE reduces the uptake of iron by U937 cells exposed to 26.6 nM ⁵⁹Fe-Tf (C) but does not have a detectable effect on cells exposed to 2 μM ⁵⁹Fe-Tf (D). (E–G) By contrast Fe-Tf does not prevent the effect of HFE on THP-1 cells. HFE expressed by HFE-vacc infection still reduces TfR1 (green histogram, E) and soluble HFE raises ferritin (green line, G) in THP-1 cells grown in media containing 4 μM (300 μg/ml) Fe-Tf. (H) Unlabeled Fe-Tf (133 nM) slightly enhanced the inhibitory effect of HFE (at given doses) on iron release over 20 min from THP-1 cells. Controls: Expression of H2-Kb by vacc-Kb infection did not alter TfR1 (F) or ferritin expression (not shown) in THP-1 cells cultured in media containing 4 μM Fe-Tf. U937 cells (B) and THP-1 cells (G) responded to DFO (red) and FAC (blue) as in Fig. 1.