Fig 3.

Effects of mutations in HFE on iron handling by U937 cells, THP-1 cells, and ex vivo macrophages. (A) Log-phase U937 (left two columns) and stationary phase THP-1 (right two columns) cells were exposed to 300 nM WT HFE, S43C HFE, V78A HFE, H41D HFE, R44E HFE, or W141A HFE soluble proteins for 16 h, and TfR1 (first and third columns) and ferritin expression (second and fourth columns) were measured (green line histograms) relative to untreated cells (gray filled histogram). Controls: Cells exposed to 50 μM DFO (red) or 250 μg/ml FAC (blue) are also shown. Staining with isotype control antibodies are shown as filled purple histograms. (B and C) Effect of mutations in HFE on iron release by THP-1 cells; cells were loaded with iron as in Fig. 2D and allowed to export iron in the presence of WT or mutant HFE proteins at 300 nM for the times shown (B) or for 30 min (C). Error bars in B indicate the range of values in the triplicate samples for each condition; each point in C represents ⁵⁹Fe released by one aliquot of 10⁶ cells. (D) Effect of HFE on iron release by ex vivo macrophages (homozygous WT HFE). (E) Effect of HFE on iron release by ex vivo macrophages from a treated HH patient (homozygous C260Y). (F) Summary of percent inhibition of iron release by WT HFE from macrophages from controls (open bars) and treated HH patients (black bars). The HFE genotype of each individual is shown. Error bars represent the range of inhibition when multiple experiments were performed on cells from the same individual (each experiment was performed on triplicate wells). (Inset) Adherent macrophages stained with anti-CD68 antibody (red histogram) or isotype control (purple). (G) Location of amino acids on HFE (α1 and α2 domains depicted by green ribbons) required for inhibition of iron release (red spheres). The HFE-contacting regions of TfR1 are shown by white strands. The figure was derived from the crystal structure of HFE:TfR1 (24).