Iron overload is common in patients undergoing allogeneic hematopoietic cell transplantation (HCT), but the mechanisms leading to overload are unknown. Here, we determined iron levels and the expression of iron regulatory proteins in the liver and gut of nonobese diabeticsevere combined immunodeficient (NOD/ SCID) mice that underwent transplantation with syngeneic (histocompatible) or allogeneic (histoincompatible) T lymphocytes. Infusion of histoincompatible T cells resulted in a significant rise in serum iron levels and liver iron content.
IntroductionIron overload is common in patients undergoing hematopoietic cell transplantation (HCT). 1-3 Many of these patients develop iron overload even without heavy red cell transfusion support; the mechanisms are largely unknown. High-dose conditioning preceding HCT may result in hyperferremia and increased non-transferrinbound iron, in association with the cessation of erythropoietic activity. 4,5 In many patients hyperferremia persists after HCT, 1,2 suggesting aberrant iron release into the circulation.The major regulator of iron release into the circulation is hepcidin (Hamp), secreted mainly by the liver. 6 Hepcidin binds to the iron transporter ferroportin 1 (Fpn), expressed on enterocytes and macrophages, which delivers iron from inside the cell to the circulation. 7 Hepcidin binding results in internalization and cytoplasmic degradation of ferroportin. As both the intestinal tract and liver are targets of graft-versus-host disease (GVHD), 2,8,9 we hypothesized that one effect of allogeneic transplantation may be direct or indirect interference by T lymphocytes with the expression or function of iron regulatory proteins in liver and gut, thereby contributing to iron overload. Here we characterized Hamp and Fpn expression and dietary iron uptake in a murine model of histoincompatible allogeneic T-lymphocyte transplantation.
MethodsFemale NOD/LtSz-scid/scid (NOD/SCID [H-2d]), C57BL/6J (H-2b), and BALB/cJ mice (H-2d) were purchased from The Jackson Laboratory (Bar Harbor, ME). NOD/SCID mice were maintained in a pathogen-free environment and kept on normal chow (iron content ϳ35 ppm) or chow with low (Յ 1 ppm) or high (30 000 ppm) iron content (Test Diets, Richmond, IN), for 14 to 28 days before transplantation. Histocompatible Mice were euthanized at 7 or 14 days after transplantation (3-6 weeks after initiating a particular diet) by CO 2 inhalation. Blood was collected via cardiac puncture and total serum iron was measured. Hepatic iron content was determined using a colorimetric assay as described. 10 Tissue for histology was fixed in 10% formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin and with Perl Prussian blue to detect iron deposition.Hepatocyte suspensions were generated by mincing and straining liver tissue through a 75-m mesh. Enterocytes were obtained by scraping the mucosa off the inverted duodenum and mincing and straining through a 75-m mesh. Total RNA was isolated and cDNA synthesized using the MACS One-Step cDNA Sy...