A role for ferritin in the antioxidant system in coffee cell cultures

Iron (Fe) is an essential nutrient for plants, but it can generate oxidative stress at high concentrations. In this study, Coffea arabica L. cell suspension cultures were exposed to excess Fe (60 and 240 mu M) to investigate changes in the gene expression of ferritin and antioxidant enzymes. Iron content accumulated during cell growth, and Western blot analysis showed an increase of ferritin in cells treated with Fe. The expression of two ferritin genes retrieved from the Brazilian coffee EST database was studied. CaFER1, but not CaFER2, transcripts were induced by Fe exposure. Phylogenetic analysis revealed that CaFER1 is not similar to CaFER2 or to any ferritin that has been characterised in detail. The increase in ferritin gene expression was accompanied by an increase in the activity of antioxidant enzymes. Superoxide dismutase, guaiacol peroxidase, catalase, and glutathione reductase activities increased in cells grown in the presence of excess Fe, especially at 60 mu M, while the activity of glutathione S-transferase decreased. These data suggest that Fe induces oxidative stress in coffee cell suspension cultures and that ferritin participates in the antioxidant system to protect cells against oxidative damage. Thus, cellular Fe concentrations must be finely regulated to avoid cellular damage most likely caused by increased oxidative stress induced by Fe. However, transcriptional analyses indicate that ferritin genes are differentially controlled, as only CaFER1 expression was responsive to Fe treatment.

Immunosuppressive effects of melanoma-derived heavy-chain ferritin are dependent on stimulation of IL-10 production

Cultured melanoma cells release soluble factors that influence immune responses. Screening of a cDNA library with anti-sera from a melanoma patient identified an immunoreactive plaque, which encoded heavy-chain ferritin (H-ferritin), Previous studies have drawn attention to the immunosuppressive effects of this molecule and prompted further studies on its biochemical and functional properties in human melanoma, These studies demonstrated, firstly, that H-ferritin appeared to be secreted by melanoma cells, as shown by immunoprecipitation of a 21.5 kDa band from supernatants. It was also detected in extracts of melanoma cells by Western blotting as 43 and 64 kDa dimers and trimers of the 21.5 kDa fraction. Secondly, flow-cytometric analysis of H- and light-chain ferritin (L-ferritin) expression on melanoma showed a wide variation in L-ferritin expression and consequently of the ratio of H- to L-ferritin expression. Suppression of mitogenic responses of lymphocytes to anti-CD3 showed a correlation with the ratio of H- to L-ferritin in the supernatants and was specific for H-ferritin, as shown by inhibition studies with a monoclonal antibody (MAb) against H-ferritin, Similar results were obtained with H- and L-ferritin from other sources. Suppression of mitogenic responses of lymphocytes to anti-CD3 by H-ferritin was inhibited using a MAb against IL-IO, which suggested that the immunosuppressive effect of H-ferritin was mediated by IL-IO, Assays of cytokine production from anti-CD3-stimulated lymphocytes showed that H-ferritin markedly increased production of IL-10 and IFN-gamma and had only slight effects on IL-2 and IL-4 production, Our results suggest that melanoma cells may be a major source of H-ferritin and that production of the latter may account for some of the immunosuppressive effects of melanoma, (C) 2001 Wiley-Liss. Inc.

Heavy chain ferritin activates regulatory T cells by induction of changes in dendritic cells

Heavy chain ferritin (H-ferritin) Is a component of the Iron-binding protein, ferritin. We have previously shown that H-ferritin Inhibits anti-CD3-stimulated lymphocyte proliferation and that this was due to Increased production of Interleukin-10 (IL-10). In the present study we have shown that Induction of IL-10 production was due to effects of H-ferritin on adherent antigen-presenting cells (APCs) In blood and monocyte-derived dendritic cells (MoDCs). IL-10 was produced by a subpopulation of CD4 T cells, which expressed the CD25 component of the IL-2 receptor and the CTLA-4 receptor characteristic of regulatory T cells. The changes Induced In MoDCs were compared with those Induced by CD40L and their significance tested by Inhibition with monoclonal antibodies. These studies Indicated that H-ferritin Induced relatively greater expression of CD86 and B7-H1 on MoDCs and that monoclonal antibodies against their receptors, CTLA-4 and programmed death receptor-1 (PD-1), Inhibited IL-10 production from the regulatory T cells. H-ferritin did not appear to Induce direct production of the cytokines IL-2, IL-4, IL-6, IL-10, IL-12, or Interferon-gamma from the DCs. These results are consistent with the thesis that H-ferritin Induces B7-H1 and CD86 (B7-2) on APCs, which In turn Induce IL-10 production from regulatory T cells. This is possibly one mechanism by which melanoma cells may Induce changes In APCs In the vicinity of the tumor and result in suppression of Immune responses by induction of regulatory T cells. (C) 2002 by The American Society of Hematology.

Association of increased levels of heavy-chain ferritin with increased CD4(+) CD25(+) regulatory T-Cell levels in patients with melanoma

We have shown previously that melanoma cells in culture release heavy-chain ferritin (H-Ferritin) into supernatants and that this is responsible for the suppression of responses of peripheral blood lymphocytes stimulated by anti-CD3. These effects were mediated by activation of regulatory T cells to produce interleukin (IL)-10. In the present study, we examined whether a similar relation might exist between levels of H-Ferritin and activation of regulatory T cells in patients with melanoma. Ferritin levels were evaluated by ELISA and regulatory T-cell numbers were assessed by three-color flow cytometry to identify CD4(+) CD25(+) CD69(-) T cells. CD69 positive cells were excluded to avoid inclusion of normal activated CD4, CD25 expressing T cells. Measurements of H- and light-chain (L)-Ferritin by ELISA revealed that H- but not L-Ferritin was elevated in the circulation of melanoma patients. In addition, these studies revealed a marked increase in the number of CD4+ CD25+ CD69- T cells in such patients, compared with age-matched controls. The ratio of H-Ferritin:L-Ferritin correlated with the levels of regulatory T cells consistent with a causal relation between unbound H-Ferritin levels and the activation of regulatory T cells. H-Ferritin or regulatory T cells did not, however, correlate with the stage of the melanoma. These results provide evidence for the importance of H-Ferritin in the induction of regulatory T cells in patients with melanoma and provide additional insight into the suppression of immune responses in such patients.

Serum Ferritin Level Remains a Reliable Marker of Bone Marrow Iron Stores Evaluated by Histomorphometry in Hemodialysis Patients

Background and objectives: As well as being a marker of body iron stores, serum ferritin (sFerritin) has also been shown to be a marker of inflammation in hemodialysis (HD) patients. The aim of this study was to analyze whether sFerritin is a reliable marker of the iron stores present in bone marrow of HD patients. Design: Histomorphometric analysis of stored transiliac bone biopsies was used to assess iron stores by determining the number of iron-stained cells per square millimeter of bone marrow. Results: In 96 patients, the laboratory parameters were hemoglobin = 11.3 +/- 1.6 g/dl, hematocrit = 34.3 +/- 5%, sFerritin 609 +/- 305 ng/ml, transferrin saturation = 32.7 +/- 22.5%, and C-reactive protein (CRP) = 0.9 +/- 1.4 mg/dl. sFerritin correlated significantly with CRP, bone marrow iron, and time on HD treatment W = 0.006, 0.001, and 0.048, respectively). The independent determinants of sFerritin were CRP (beta-coef = 0.26; 95% CI = 24.6 to 132.3) and bone marrow iron (beta-coef = 0.32; 95% CI = 0.54 to 2.09). Bone marrow iron was higher in patients with sFerritin >500 ng/ml than in those with sFerritin :5500 ng/ml. In the group of patients with sFerritin :5500 ng/ml, the independent determinant of sFerritin was bone marrow iron (beta-coef = 0.48, 95% CI = 0.48 to 1.78), but in the group of patients with sFerritin >500 ng/ml, no independent determinant of sFerritin was found. Conclusions: sFerritin adequately reflects iron stores in bone marrow of HD patients.

Lead, hemoglobin, zinc protoporphyrin and ferritin concentrations in children

OBJECTIVE: To assess the relationship of blood lead and hemoglobin, zinc protoporphyrin, and ferritin concentrations in children. METHODS: A cross-sectional study was carried out in 136 anemic and non-anemic children from two rural villages near a lead smelter in Adrianópolis, Southern Brazil, from July to September 2001. Hemoglobin electrophoresis was performed to exclude children with hemoglobin variants and thalassemia syndromes associated with anemia. Lead was determined by atomic absorption spectrophotometry; hemoglobin by automated cell counting; zinc protoporphyrin by hematofluorometry; ferritin by chemiluminescence. Student's t-test, Mann-Whitney test, and the c² test were used to assess the significance of the differences between the variables investigated in anemic and non-anemic children. Stepwise multivariate linear regression analysis was performed using two models for anemic and non-anemic children respectively. RESULTS: Lead was negatively associated to hemoglobin (p<0.017) in the first model, and in the second model lead was positively associated to zinc protoporphyrin (p<0.004) after controlling for ferritin, age, sex, and per capita income. There was an inverse association between hemoglobin and blood lead in anemic children. It was not possible to confirm if anemic children had iron deficiency anemia or subclinical infection, considering that the majority (90.4%) had normal ferritin. CONCLUSIONS: The study detected a relationship between anemia and elevated blood lead concentrations. Further epidemiological studies are necessary to investigate the impact of iron nutritional interventions as an attempt to decrease blood lead in children.

Functional and structural studies of a mini-ferritin protein

Dissertação para obtenção do Grau de Mestre em Bioquímica

Erarbeitung von Richtwerten für das Ferritin im Serum durch Auswertung der in einer Laborarztpraxis gemessenen Konzentrationen männlicher und weiblicher Personen im Alter von 0 bis 20 Jahren : Vergleich mit den verfügbaren Referenzbereichen

Magdeburg, Univ. Med. Fak., Diss., 2011

The impact of iron supplementation efficiency in female blood donors with a decreased ferritin level and no anaemia. Rationale and design of a randomised controlled trial: a study protocol.

ABSTRACT: BACKGROUND: There is no recommendation to screen ferritin level in blood donors, even though several studies have noted the high prevalence of iron deficiency after blood donation, particularly among menstruating females. Furthermore, some clinical trials have shown that non-anaemic women with unexplained fatigue may benefit from iron supplementation. Our objective is to determine the clinical effect of iron supplementation on fatigue in female blood donors without anaemia, but with a mean serum ferritin </= 30 ng/ml. METHODS/DESIGN: In a double blind randomised controlled trial, we will measure blood count and ferritin level of women under age 50 yr, who donate blood to the University Hospital of Lausanne Blood Transfusion Department, at the time of the donation and after 1 week. One hundred and forty donors with a ferritin level </= 30 ng/ml and haemoglobin level >/= 120 g/l (non-anaemic) a week after the donation will be included in the study and randomised. A one-month course of oral ferrous sulphate (80 mg/day of elemental iron) will be introduced vs. placebo. Self-reported fatigue will be measured using a visual analogue scale. Secondary outcomes are: score of fatigue (Fatigue Severity Scale), maximal aerobic power (Chester Step Test), quality of life (SF-12), and mood disorders (Prime-MD). Haemoglobin and ferritin concentration will be monitored before and after the intervention. DISCUSSION: Iron deficiency is a potential problem for all blood donors, especially menstruating women. To our knowledge, no other intervention study has yet evaluated the impact of iron supplementation on subjective symptoms after a blood donation. TRIAL REGISTRATION: NCT00689793.

Conditional deletion of ferritin h in mice reduces B and T lymphocyte populations.

The immune system and iron availability are intimately linked as appropriate iron supply is needed for cell proliferation, while excess iron, as observed in hemochromatosis, may reduce subsets of lymphocytes. We have tested the effects of a ferritin H gene deletion on lymphocytes. Mx-Cre mediated conditional deletion of ferritin H in bone marrow reduced the number of mature B cells and peripheral T cells in all lymphoid organs. FACS analysis showed an increase in the labile iron pool, enhanced reactive oxygen species formation and mitochondrial depolarization. The findings were confirmed by a B-cell specific deletion using Fth(lox/lox) ; CD19-Cre mice. Mature B cells were strongly under-represented in bone marrow and spleen of the deleted mice, whereas pre-B and immature B cells were not affected. Bone marrow B cells showed increased proliferation as judged by the number of cells in S and G2/M phase as well as BrdU incorporation. Upon in vitro culture with B-cell activating factor of the tumor necrosis factor family (BAFF), ferritin H-deleted spleen B cells showed lower survival rates than wild type cells. This was partially reversed with iron-chelator deferiprone. The loss of T cells was also confirmed by a T cell-specific deletion in Fth(lox/lox) ;CD4-Cre mice. Our data show that ferritin H is required for B and T cell survival by actively reducing the labile iron pool. They further suggest that natural B and T cell maturation is influenced by intracellular iron levels and possibly deregulated in iron excess or deprivation.

Construction and analysis of a new conditional ferritin H knockout mouse strain/

Résumé Le fer joue un rôle important dans la plupart des fonctions biologiques mais sa présence excessive provoque la production de molécules réactives d'oxygène (ROS) qui peuvent contribuer à diverses maladies. La protéine de stockage du fer, la ferritine H, capte l'excès en fer et le stocke sous forme non-toxique, ce qui empêche des dommages potentiels. La délétion de la ferritine H dans des souris knock-out a été essayée antérieurement, mais ces souris mouraient au stade précoce du développement embryonnaire. Pour étudier l'importance du fer, et en particulier son stockage dans la ferritine, et pour pouvoir mieux comprendre les fonctions de la ferritine H, nous avons créé un modèle de souris knock-out conditionnelles de la ferritine H, selon le système classique de Cre-LoxP. Le premier exon et la région du promoteur du gène de la ferritine H ont été entourés de sites loxP. La mortalité embryonnaire provoquée par la délétion constitutive du gène de la ferritine H a été confirmée en croisant nos souris avec des souris exprimant nestin-Cre1. En croisant nos souris avec des souris transgéniques Mx-Cre, nous avons observé que l'induction de Cre par injection de polyI-polyC provoque la délétion presque complète de la ferritine H dans le foie (> 99%) et la rate (> 88%). Ces tissus ont également perdu une grande partie de leur réserve de fer. Cette observation apporte pour la première fois la preuve in vivo que la ferritine H est indispensable pour le stockage du fer, que les fonctions de la ferritine H et de la ferritine L ne sont pas équivalentes, et que la ferritine L ne peut pas assumer seule la fonction de stockage du fer. Dans le foie des souris knock-out, l'expression de l'ARN messager de l'hepcidine a été induite après 10 jours. En même temps, l'expression de l'ARN messager des gènes codant pour des protéines de l'absorption de fer (DMT1, ferroportin, Dcytb1 et hephaestin) a été réprimée mais dans le duodénum seulement. L'expression d'hepcidine est inversément corrélée avec celle des gènes liés à l'absorption de fer. Cette observation corrobore des études antérieures. Mais, en plus, elle montre également que cette répression se produit seulement dans l'intestin. Nous pouvons ainsi tirer la conclusion suivante : ou bien l'hepcidine a un récepteur spécifique dans le duodénum ou bien les gènes liés à l'absorption de fer dans le duodénum ont un facteur spécifique de transcription sensible à l'hepcidine. Aucune répression de DMT1 et de ferroportin n'a été observée dans les macrophages de la rate après l'induction d'hepcidine. La délétion de ferritine H a entraîné une augmentation du taux de mortalité des cellules hépatiques, ainsi que des altérations dans l'architecture normale du tissu de la rate. Vu par l'immunohistologie, le nombre de lymphocytes B et T était réduit dans la rate, tendant à démontrer que la ferritine H et l'homéostase du fer jouent un rôle dans l'immunité. En conclusion, le modèle de souris knock-out conditionnelles de la ferritine H nous fournit un outil précieux pour l'étude in vivo du rôle joué par la ferritine dans l'homéostase du fer, dans les dommages créés par les ROS, ainsi que dans l'apoptose et l'immunité. Summary Iron plays an important role in most biological functions. However, excess of iron results in production of reactive oxygen species (ROS) which could substantially contribute to pathology of various diseases. Ferritin H scavenges excess of iron and stores it in non-toxic form and potentially prevents the damage. Fenitin H targeting in mice has been attempted before, however, straight knockout was lethal in early embryonic stage. To study the role of iron and its storage protein ferritin and to further elucidate ferritin H functions, we aimed at creating a conditional ferritin H knockout mouse model by classical Cre-LoxP system. First exon along with promoter region of the ferritin H gene was foxed. Embryonic lethality of the constitutive ferritin H deletion was confirmed by crossing the foxed mice with mice expressing nestin Cre-1 as transgene. Almost complete deletion was observed in liver (> 99%) and spleen (>88%) upon induction of Cre by injecting polyI-polyC in Fth Lox/Lox; MxCre mice. These tissues also lost substantial fraction of their iron stores. This provides first in vivo evidence that ferritin H is required for iron storage, ferritin H and L functions are not redundant and that ferritin L cannot perform iron storage function alone. Hepcidin mRNA expression was induced after 10 days in the livers of deleted mice and, simultaneously, mRNA expression of iron absorption related genes (DMT 1, ferroportin, Dcytb1 and hephaestin) was repressed in duodenum only. Hepcidin expression is inversely correlated with that of duodenal iron absorption related genes. This is in agreement with previous studies. However, we also show that this repression happens only in intestine. This leads to the conclusion that either hepcidin has a specific receptor in duodenum or the iron absorption related genes have duodenum specific transcription factor that is responsive to hepcidin. No repression of DMT1 and ferroportin was observed in spleen macrophages upon hepcidin induction. Ferritin H deletion showed increased cell death in liver and disruption of normal architecture of spleen. B lymphocytes were reduced in spleen on immunohistology which point towards a role of ferritin H and iron homeostasis in immunity. In conclusion, ferritin H conditional knockout mouse model provides us with an invaluable tool to study the in vivo role of ferritin H in iron homeostasis, ROS mediated damage, apoptosis and immunity.

Light-induced retinal degeneration correlates with changes in iron metabolism gene expression, ferritin level, and aging.

PURPOSE: Retinal degeneration is associated with iron accumulation in several rodent models in which iron-regulating proteins are impaired. Oxidative stress is catalyzed by unbound iron. METHODS: The role of the heavy chain of ferritin, which sequesters iron, in regulating the thickness of the photoreceptor nuclear layer in the 4- and 16-month-old wild-type H ferritin (HFt(+/+)) and heterozygous H ferritin (HFt(+/-)) mice was investigated, before and 12 days after exposure to 13,000-lux light for 24 hours. The regulation of gene expression of the various proteins involved in iron homeostasis, such as transferrin, transferrin receptor, hephaestin, ferroportin, iron regulatory proteins 1 and 2, hepcidin, ceruloplasmin, and heme-oxygenase 1, was analyzed by quantitative (q)RT-PCR during exposure (2, 12, and 24 hours) and 24 hours after 1 day of exposure in the 4-month-old HFt(+/+) and HFt(+/-) mouse retinas. RESULTS: Retinal degeneration in the 4-month-old HFt(+/-) mice was more extensive than in the HFt(+/+) mice. Yet, it was more extensive in both of the 16-month-old mouse groups, revealing the combined effect of age and excessive light. Injury caused by excessive light modified the temporal gene expression of iron-regulating proteins similarly in the HFt(+/-) and HFt(+/+) mice. CONCLUSIONS: Loss of one allele of H ferritin appears to increase light-induced degeneration. This study highlighted that oxidative stress related to light-induced injury is associated with major changes in gene expression of iron metabolism proteins.

Effect of iron supplementation on fatigue in nonanemic menstruating women with low ferritin: a randomized controlled trial.

BACKGROUND: The true benefit of iron supplementation for nonanemic menstruating women with fatigue is unknown. We studied the effect of oral iron therapy on fatigue and quality of life, as well as on hemoglobin, ferritin and soluble transferrin receptor levels, in nonanemic iron-deficient women with unexplained fatigue. METHODS: We performed a multicentre, parallel, randomized controlled, closed-label, observer-blinded trial. We recruited from the practices of 44 primary care physicians in France from March to July 2006. We randomly assigned 198 women aged 18-53 years who complained of fatigue and who had a ferritin level of less than 50 ug/L and hemoglobin greater than 12.0 g/dL to receive either oral ferrous sulfate (80 mg of elemental iron daily; n = 102) or placebo (n = 96) for 12 weeks. The primary outcome was fatigue as measured on the Current and Past Psychological Scale. Biological markers were measured at 6 and 12 weeks. RESULTS: The mean score on the Current and Past Psychological Scale for fatigue decreased by 47.7% in the iron group and by 28.8% in the placebo group (difference -18.9%, 95% CI -34.5 to -3.2; p = 0.02), but there were no significant effects on quality of life (p = 0.2), depression (p = 0.97) or anxiety (p = 0.5). Compared with placebo, iron supplementation increased hemoglobin (0.32 g/dL; p = 0.002) and ferritin (11.4 μg/L; p < 0.001) and decreased soluble transferrin receptor (-0.54 mg/L; p < 0.001) at 12 weeks. INTERPRETATION: Iron supplementation should be considered for women with unexplained fatigue who have ferritin levels below 50 μg/L. We suggest assessing the efficiency using blood markers after six weeks of treatment. Trial registration no. EudraCT 2006-000478-56.

Eliminating heterophilic antibody interference for ferritin detection using Olympus F(ab')2 based reagent.

Interferences with the Olympus immunoturbidimetric assay for ferritin have been reported because the antibodies used in the immunoassay are derived from rabbits. Rabbits are familiar pets known to be a risk factor for developing heterophilic (or interfering) antibodies. This report shows how the current Olympus Ferritin assay has been improved to eliminate the interference from heterophilic antibodies.

Serum ferritin levels are associated with a distinct phenotype of chronic hepatitis C poorly responding to pegylated interferon-alpha and ribavirin therapy.

Elevated serum ferritin levels may reflect a systemic inflammatory state as well as increased iron storage, both of which may contribute to an unfavorable outcome of chronic hepatitis C (CHC). We therefore performed a comprehensive analysis of the role of serum ferritin and its genetic determinants in the pathogenesis and treatment of CHC. To this end, serum ferritin levels at baseline of therapy with pegylated interferon-alpha and ribavirin or before biopsy were correlated with clinical and histological features of chronic hepatitis C virus (HCV) infection, including necroinflammatory activity (N = 970), fibrosis (N = 980), steatosis (N = 886), and response to treatment (N = 876). The association between high serum ferritin levels (> median) and the endpoints was assessed by logistic regression. Moreover, a candidate gene as well as a genome-wide association study of serum ferritin were performed. We found that serum ferritin ≥ the sex-specific median was one of the strongest pretreatment predictors of treatment failure (univariate P < 0.0001, odds ratio [OR] = 0.45, 95% confidence interval [CI] = 0.34-0.60). This association remained highly significant in a multivariate analysis (P = 0.0002, OR = 0.35, 95% CI = 0.20-0.61), with an OR comparable to that of interleukin (IL)28B genotype. When patients with the unfavorable IL28B genotypes were stratified according to high versus low ferritin levels, SVR rates differed by > 30% in both HCV genotype 1- and genotype 3-infected patients (P < 0.001). Serum ferritin levels were also independently associated with severe liver fibrosis (P < 0.0001, OR = 2.67, 95% CI = 1.68-4.25) and steatosis (P = 0.002, OR = 2.29, 95% CI = 1.35-3.91), but not with necroinflammatory activity (P = 0.3). Genetic variations had only a limited impact on serum ferritin levels. Conclusion: In patients with CHC, elevated serum ferritin levels are independently associated with advanced liver fibrosis, hepatic steatosis, and poor response to interferon-alpha-based therapy.