Transferrin fails to provide protection against Fas-induced hepatic injury in mice with deletion of functional transferrin-receptor type 2

Abstract

We reported previously that Fas-induced hepatic failure in normal mice was attenuated or prevented by exogenous transferrin (Tf), particularly apoTf. Here we show in C57BL6J/129 mice with genetic inactivation of transferrin receptor 2 (TfR2(Y245X)), that Fas-induced hepatotoxicity (apoptosis; rise in plasma aspartate aminotransferase (AST) levels) was comparable to that in wild-type mice, but was not modified by pretreatment with Tf. Rises in plasma AST were preceded by a decline in serum iron levels. AST elevations and iron declines were more profound in female than in male mice. Female mice also showed higher baseline levels of Bcl-xL in hepatocytes, which declined significantly upon treatment with agonistic anti-Fas antibody. These data confirm the cytoprotective function of Tf, and show a novel property of TfR2. Both apoptotic Fas responses and cytoprotective effects of Tf were associated with significant shifts in plasma iron levels, which quantitatively differed between male and female mice.

Figure 1. Changes in aspartate-aminotransferase (AST) and total iron (Fe) levels in blood plasma and Bcl-xL expression in liver lysates of C57BL6 mice exposed to agonistic anti-Fas antibody (aFas)

Mice were given i.p. aFas at sublethal doses of 0.08 µg/g body weight. Blood was sampled at 0. 6, 12, 24, 48 hours and 7 days after aFas. Shown are the means ± SE of AST (U/L) (A) and iron (µg/dL) (B). AST levels (at 24 hours) were significantly higher in female than in male mice (p<0.05). Iron levels declined by 6 hours and were increased by 48 hours in aFas-treated mice in comparison to controls (“bleeding”= blood sampling only); however, differences reached significance only in female mice (*p<0.05 and **p<0.002, respectively). C) Expression of Bxl-xL was determined in liver cell lysates obtained from mice sacrificed at 0 or 6 hours after aFas injection (C57BL6 mice; 5 per group) by Western blot and analyzed with Image J. Female mice expressed Bcl-xL at higher levels than male mice and showed significant decreases in Bcl-xL protein following treatment with aFas; very little change was noted in male mice.

Figure 2. Blood plasma iron levels and hepatic iron deposition in TfR2 mutant mice

(A) Blood samples were obtained from 2–3 months old mice (five to eleven animals/group), and iron was determined by QuantiChrom Iron Assay Kit. Shown are the means ± SE. (B) Female mice, 2 months old, were killed, liver tissue was fixed in 10% formalin, and paraffin-embedded sections were stained with Prussian Blue (x40). Shown are examples of liver sections from wild type (WT) mice and mice heterozygous (HT) or homozygous (HO) for the TfR2 mutation. Only HO mice showed severe hepatic iron loading [9].

Figure 3. Serum aspartate aminotransferase (AST) and plasma iron (Fe) levels in wild type (WT) mice and in mice heterozygous (HT) or homozygous (HO) for the TfR2 mutation

Mice were injected i.p. with sublethal doses of anti-Fas MAB (aFas, Jo2, 0.08 µg/g body weight) and treated with 0.25 mL saline or human ApoTf, 0.1 mg/0.25 mL/mouse at 48, 24, 1 hour before, and 1 hour after aFas injection. Additional mice received no treatment (“bleeding” = blood sampling only). Blood for AST and Fe determination was collected at 0, 6, 12, 24, 48 hours and 7 days after aFas. Results are given as mean ± SE, plotted over time. A) wild type (WT) female mice (*p<0.02, **p<0.01 versus control); B) WT male mice (*p<0.05, **p<0.01 versus control); C) heterozygous (HT) female mice; D) HT male mice; E) homozygous (HO) female mice; F) HO male mice. AST levels remained in the normal range after injection of saline or Tf without aFas, and changes in iron levels in mice injected with Tf only paralleled those seen in controls (not shown). G) Mice were treated as in A–F, but iron levels were followed for 7 days (168 hours). Fluctuations were more extensive in female than in male mice, (two male WT “bleeding” controls died due to technical problems). Arrows indicate time of injection of aFas. *p<0.01 – saline [109±2 µg/dL] versus ApoTf [194±8 µg/dL], **p<0.01 – saline [68±2 µg/dL] versus ApoTf [118±9 µg/dL].

Figure 4. Proposed model

Left panel depicts events in an unmodified hepatocyte exposed to agonistic anti-Fas antibody (aFas). The middle panel shows an unmodified hepatocyte exposed to aFas in the presence of exogenous transferrin (Tf). The arrow-less line between intracellular iron (Fe) and extracelluar Fe indicates absence of significant unidirectional shifts. The right panel shows a hepatocyte lacking functional transferrin receptor 2 (TfR2) exposed to aFas in the presence of exogenous Tf; in contrast to the middle panel, no protection is provided by Tf (see text for additional abbreviations).