Haptoglobin modifies the hemochromatosis phenotype in mice

Classic hereditary hemochromatosis (HH) is a common genetic disorder of iron metabolism characterized by a gradual but progressive expansion of the plasma iron compartment. Affected patients develop toxic iron overload and tissue damage including liver cirrhosis, hepatocarcinoma, and heart disease. Most patients with HH are homozygous for a missense mutation (C282Y) that disrupts the conformation of HFE, an atypical major histocompatibility class I molecule.¹  Similar to human patients, mice lacking the Hfe protein or producing a mutated protein analogous to the human C282Y protein develop increased hepatic iron levels and elevated transferrin saturation.^2,3  Whereas C282Y is among the most prevalent human mutations, the clinical penetrance of the disease is low, suggesting that the HFE C282Y mutation is a necessary but not sufficient cause of clinical HH.⁴  Variable penetrance may be due to epigenetic, environmental, and genetic factors.⁵  Also, in mice there is a marked difference in hepatic iron loading between C57BL/6 and DBA/2 Hfe^-/- strains, indicating that other genes modify the murine HH phenotype.^6,7  Several candidate modifier genes have been proposed in mice and humans,^8-10  including haptoglobin (Hp). Hp is the plasma protein with the highest binding affinity for hemoglobin (K_d ≈ 1 pM).¹¹  It binds free hemoglobin and delivers it to the reticuloendothelial system by CD163 receptor-mediated endocytosis.¹²  Thus, Hp is believed to reduce loss of hemoglobin through the glomeruli and to allow heme-iron recycling.

Hp is synthesized as a single polypeptide, which is cleaved into α- and β-chains. The mammalian isoform Hp1-1 is a homodimer of 2 Hp molecules linked by a single disulfide bond through their respective α-chains. In humans, a variant with a longer α-chain is also present. Individuals homozygous for the longer allele show a multimeric Hp phenotype designated Hp2-2. Hp2-1 refers to the molecular phenotype (both Hp dimers and multimers) seen in humans heterozygous for the 2 variant alleles. Complexes of hemoglobin and multimeric Hp (the 2-2 phenotype) exhibit higher functional affinity for CD163 than do complexes of hemoglobin and dimeric Hp (the 1-1 phenotype).

In healthy men, the Hp2-2 type is associated with higher serum iron and ferritin levels than the Hp2-1 and Hp1-1 types. Moreover, in healthy men carrying the Hp2-2 type, a fraction of Hp-hemoglobin complexes is shunted into monocyte-macrophages, resulting in partial iron retention.^13,14 

Hp was recently proposed to be a genetic modifier of HFE-associated hemochromatosis, as the Hp2-2 type was overrepresented in hemochromatotic patients and iron loading was more pronounced in patients carrying Hp2-2.¹⁵  However, this finding has been challenged by other studies.^16,17  To gain insight into the role of Hp as modifier gene in HH, we used Hfe and Hp double-null mice. Here, we show that Hfe and Hp compound mutant mice accumulate significantly less hepatic iron than Hfe-null mice, thus demonstrating that Hp-mediated heme-iron recovery may contribute significantly to iron loading in HH.

Hfe-null and Hp-null mice were described previously.^2,18  Hfe^-/- and Hp^-/- mice on 129S6/SvEvTac and C57BL/6J genetic backgrounds, respectively, were bred and the resulting double heterozygous progeny were intercrossed to generate Hfe^?/+Hp^?/+, Hfe^?/+Hp^-/-, Hfe^-/-Hp^?/+, and Hfe^-/-Hp^-/- mice. Animals were genotyped by polymerase chain reaction (PCR) as previously described.^18,19  All mice used in this study were F₂ males of C57BL/6J × 129S6/SvEvTac hybrid strain background of 1, 2, or 3 months of age. Mice were fed with a standard diet that contains 0.02% iron (4RF25; Mucedola, Milano, Italy).

Tissue iron content was determined with the colorimetric method using BPS (4,7-diphenyl-1,10-phenantroline disulfonic acid) as chromogen.²⁰  Results were expressed as μg iron/g dry tissue weight.

Tissues were fixed in 10% formalin for 48 hours and embedded in paraffin. Microtome sections were stained with the Perls reaction followed by DAB (methanol 3,3′ diamino-benzidine; Boehringer Mannheim, Mannheim, Germany) development.²¹  Tissue sections were analyzed on an Olympus BX41 microscope (objective Olympus Plan 20 ×/0.40) equipped with an Olympus DP50 camera for image acquisition (acquisition software: Image-Pro Plus). Images were processed with Adobe Photoshop 7.0.

Results were expressed as mean ± SEM. Statistical analyses were performed using the one-way analysis of variance (ANOVA). A P value of less than .05 was regarded as significant.

To investigate the contribution of Hp-mediated heme-iron recovery to liver iron overload in HH, we generated Hfe and Hp compound null mice by intercrossing Hfe-null and Hp-null mice on 129S6/SvEvTac and C57BL/6J genetic backgrounds, respectively.

Firstly we verified that the “low-iron” phenotype typically displayed by the C57BL/6J strain was not dominant over the “high-iron” phenotype of the 129S6/SvEvTac strain by comparing liver iron content in 129S6/SvEvTac Hfe^-/- × C57BL/6J Hfe^+/+ F₁ mice and 129S6/SvEvTac Hfe^+/- mice. This analysis showed that liver iron was not significantly different in 129S6/SvEvTac-C57BL/6J Hfe^+/- and 129S6/SvEvTac Hfe^+/- mice (212.74 ± 26.15 μg/g dry tissue weight vs 273.79 ± 42.01 μg/g dry tissue weight; n = 8, P = .24), thus excluding a dominant effect of the C57BL/6J strain on the 129S6/SvEvTac strain.

Mice used in this study were F₂ males of C57BL/6J × 129S6/SvEvTac hybrid strain background of 1, 2, and 3 months of age. As no differences in iron parameters between Hfe^+/- and Hfe^+/+ mice were detectable, heterozygous animals were combined with wild type.

Liver iron content was similar in wild-type and Hp-null mice and strongly increased in Hfe-null mice as expected. In Hfe and Hp compound null mice of 1 and 2 months of age, we observed reductions in hepatic iron of 35% and 31%, respectively, compared with age-matched Hfe-null mice (P < .01). Perls staining of liver sections showed a significant reduction in periportal iron deposits in HfeHp double-null mice compared with Hfe-deficient mice (Figure 1).

On the other hand, no differences in liver iron deposits were detectable between wild-type and Hp-null mice (Figure 1). This was probably due to the activation of compensatory systems to maintain normal hepatic iron content in Hp-deficient mice, including increased iron absorption and/or accelerated iron turnover in reticuloendothelial cells. The same mechanisms would not be activated in Hfe-null mice that are unable to “sense” iron stores.

Serum iron levels and transferrin saturation were significantly higher in Hfe-null mice than in wild-type controls, as previously reported by Zhou et al.²  On the other hand, no significant differences in both serum iron and transferrin saturation were detectable between Hfe-null and HfeHp-null mice or between wild-type and Hp-null mice (not shown).

Interestingly, higher kidney iron content was found in the HfeHp double-null mice compared with Hfe-null mice. The difference in kidney iron between Hfe-null and HfeHp-null mice was weak at 1 and 2 months of age and strongly significant at 3 months of age. Perls staining of kidney sections showed that in HfeHp double-null mice, iron accumulated in proximal tubular cells (Figure 2). The difference in renal iron between Hfe-null and HfeHp-null mice was likely caused by the lack of Hp, as the same difference was detectable between wild-type and Hp-null mice (Figure 2).

These data indicate a diversion of hemoglobin-derived iron to the kidney rather than to the hepatic stores in Hfe and Hp compound mice and are in agreement with our recent results showing a role of Hp in modulating renal iron deposits.²² 

Our results strongly support the conclusion that Hp is a modifier of HH in mice. Data reported by Bensaid et al²³  showed that the region encompassing the Hp locus on mouse chromosome 8 is associated with a possible modifier gene for iron loading. We speculate that that modifier might be Hp; however, we cannot rule out the extremely unlikely possibility that Hp^-/- mice carry a linked, allelic variant of another gene that corresponds to the human modifier on chromosome 8.

Moreover, data obtained in HfeHp-null mice support the hypothesis that, in humans, different capacities for heme-iron recovery associated with different Hp types could modify the rate of liver iron accumulation in HH. Undoubtedly, the rate of progression of hepatic iron loading and the potential for toxicity and organ disease in human HH will also depend on other genetic and environmental factors⁵  unrelated to Hp status.

We wish to thank D. Chiabrando, S. Marro, N. Morello, and R. Pierini for help with some experiments.