Abraham M. Konijn

Ferritin synthesis in inflammation. I. Pathogenesis of impaired iron release.

Summary. Plasma iron turnover (PIT) and ferritin synthesis in the liver and spleen were studied in rats within the first 24 h of inflammation produced by turpentine injection. Comparison of the sequential changes in PIT and ferritin synthesis showed that alterations in ferritin synthesis preceded the changes in plasma iron exchange throughout the study. Thus, after 4 h inflammation ferritin synthesis was twice normal whereas plasma iron and PIT were still unchanged. Conversely, maximal reduction in plasma iron occurred after 12 h inflammation, at a time when ferritin synthesis had already declined to normal rates. These correlations seem to indicate that, in analogy with other acute phase reacting proteins, increased ferritin synthesis is a primary nonspecific response which is part of a general pattern of the systemic effects of inflammation. This increase in ferritin synthesis is assumed to be responsible for the diversion of labile iron into ferritin stores, and its reduced availability for release from tissues.

Suppressive Effect of Ferritin on in Vitro Lymphocyte Function

Summary. This study describes the effect of ferritin on lymphocyte function in vitro. Peripheral blood lymphocytes isolated from normal donors were incubated with purified human splenic ferritin, and the mitogenic effect of phytohaemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and mixed lymphocyte reaction (MLR) were assessed by the uptake of 3H‐thymidine (3H‐TdR). Ferritin (0·25–5·0 μg/ml culture) caused a marked suppression of PHA and Con A blastogenesis but had no suppressive effect on PWM‐induced transformation. Maximal suppression was obtained at a ferritin concentration of 1 μg/ml and this was not enhanced by increasing ferritin concentrations. Ferritin also reduced the Con A capping phenomenon in normal lymphocytes from 22% to 6%, suppressed the MLR reaction but had no effect on the ability of normal lymphocytes to form E, EA and EAC rosettes or on in vitro lymphocyte cytoxicity against the K‐562 cell line. Visual proof of the suppressive effect of ferritin on mitogen induced blastogenesis was provided by scanning electron microscopy, and direct evidence for the ability of lymphocytes to bind ferritin was obtained from studies with radioiodine labelled ferritin. The above findings indicate that ferritin suppresses certain parameters of T‐lymphocyte function in vitro. The relation of the present findings to recognized abnormalities of T‐cell function encountered in certain neoplastic disorders associated with high serum ferritin levels is at present unknown.

Iron Chelators for Thalassaemia

The iron-chelating compound desferrioxamine (DFO) was discovered accidentally, as a byproduct of antibiotic research by scientists in the Swiss Federal Institute of Technology in Zurich and Ciba in Basel. Its development into a useful clinical drug was not by design, but thanks to natural curiosity and ingenuity, as described by Keberle (1992). Although DFO has been available for treating transfusional iron overload from the early 1960s, the era of modern and effective iron-chelating therapy started only 20 years ago with the introduction of subcutaneous DFO infusions by portable pumps. Today, long-term DFO therapy is an integral part of the management of thalassaemia and other transfusion-dependent anaemias, with a major impact on wellbeing and survival. In this Review we will discuss the abnormalities in iron metabolism associated with thalassaemia major and the chelatable iron pools representing the targets of iron-chelating therapy, examine the results of longterm DFO therapy in thalassaemia, and, finally, review the development of new, orally effective, iron chelators which may be considered for present or future use.

Ferritin synthesis in inflammation. II. Mechanism of increased ferritin synthesis.

Summary. The mechanism of increased ferritin synthesis in inflammation was studied in rat livers 0—48 h after turpentine injection. A subcellular protein synthesizing system was employed in which the respective roles of cell sap factors and polysomes from normal and treated animals could be studied. Two waves of increased ferritin synthesis were found, an early wave with peak activity at 6 h of inflammation, and a second wave starting at about 24 h. The early wave of enhanced ferritin synthesis was associated with increased activity of cell sap factors. In contrast, the late enhancement of ferritin synthesis was characterized by increased polysomal activity as well as increased cell sap activity. These observations suggest a post‐transcriptional control mechanism for the early phase of enhanced ferritin synthesis in inflammation, and a transcriptional as well as post‐transcriptional control for the late phase of enhanced ferritin synthesis.

Objectives and Mechanism of Iron Chelation Therapy

Abstract: Prevention of cardiac mortality is the most important beneficial effect of iron chelation therapy. Unfortunately, compliance with the rigorous requirements of daily subcutaneous deferoxamine (DFO) infusions is still a serious limiting factor in treatment success. The development of orally effective iron chelators such as deferiprone and ICL670 is intended to improve compliance. Although total iron excretion with deferiprone is somewhat less than with DFO, deferiprone may have a better cardioprotective effect than DFO due to deferiprones ability to penetrate cell membranes. Recent clinical studies indicate that oral ICL670 treatment is well tolerated and is as effective as parenteral DFO used at the standard dose of 40 mg/kg of body weight/day. Thus, for the patient with transfusional iron overload in whom results of DFO treatment are unsatisfactory, several orally effective agents are now available to avoid serious organ damage. Finally, combined chelation treatment is emerging as a reasonable alternative to chelator monotherapy. Combining a weak chelator that has a better ability to penetrate cells with a stronger chelator that penetrates cells poorly but has a more efficient urinary excretion may result in improved therapeutic effect through iron shuttling between the two compounds. The efficacy of combined chelation treatment is additive and offers an increased likelihood of success in patients previously failing DFO or deferiprone monotherapy.

Macrophages function as a ferritin iron source for cultured human erythroid precursors

Iron is essential for the survival as well as the proliferation and maturation of developing erythroid precursors (EP) into hemoglobin‐containing red blood cells. The transferrin–transferrin receptor pathway is the main route for erythroid iron uptake. Using a two‐phase culture system, we have previously shown that placental ferritin as well as macrophages derived from peripheral blood monocytes could partially replace transferrin and support EP growth in a transferrin‐free medium. We now demonstrate that in the absence of transferrin, ferritin synthesized and secreted by macrophages can serve as an iron source for EP. Macrophages trigger an increase in both the cytosolic and the mitochondrial labile iron pools, in heme and in hemoglobin synthesis, along with a decrease in surface transferrin receptors. Inhibiting macrophage exocytosis, binding extracellular ferritin with specific antibodies, inhibiting EP receptor‐mediated endocytosis or acidification of EP lysosomes, all resulted in a decreased EP growth when co‐cultured with macrophages under transferrin‐free conditions. The results suggest that iron taken up by macrophages is incorporated mainly into their ferritin, which is subsequently secreted by exocytosis. Nearby EP are able to take up this ferritin probably through clathrin‐dependent, receptor‐mediated endocytosis into endosomes, which following acidification and proteolysis release the iron from the ferritin, making it available for regulatory and synthetic purposes. Thus, macrophages support EP development under transferrin‐free conditions by delivering essential iron in the form of metabolizable ferritin. J. Cell. Biochem. 103: 1211–1218, 2008.

Differential effect of isolated placental isoferritins on in vitro T‐lymphocyte function

Summary The effect of isoferritins isolated from human term placenta on certain T‐lymphocyte parameters was studied in vitro using normal human lymphocytes. These isoferritins differed in ion exchange affinity, isoelectric point, and subunit composition. Only the acidic isoferritins caused a marked suppression of phytohaem‐agglutinin (PHA) blastogenesis and the most acidic isoferritin (‘Acid I’) was suppressive at a concentration as low as 0.25 μg/ml. All four isoferritins suppressed concanavalin A (Con A) blastogenesis in a similar concentration dependent manner, with maximum effect at an isoferritin concentration of 1 μg/ml.

A rapid and sensitive ELISA for serum ferritin employing a fluorogenic substrate

A fluorescent enzyme-linked immunosorbent assay is described for the rapid measurement of serum ferritin. Increased sensitivity was achieved by using 4-methyl-umbelliferyl-beta-D-galactopyranoside as the substrate for beta-galactosidase coupled to the purified antiferritin antibody. Further enhancement of the specific antigen-antibody reaction was attained by the addition of 4% polyethylene glycol 6000 to the antiferritin-beta-galactosidase conjugate. The procedure is performed in microELISA plates. These modifications of the method permit the measurement of serum ferritin at concentrations ranging from 0.25 to 50 microgram/liter with a coefficient of variation of 8% or less. The entire procedure is performed at ambient temperature and is completed within one working day. The cost of the assay is less than 10% of the immunoradiometric assay for serum ferritin.

Roles of ferritin and iron in ischemic preconditioning of the heart

Iron and copper play major roles in biological systems, catalyzing free radical production and consequently causing damage. The relatively high levels of these metals, which are mobilized into the coronary flow following prolonged ischemia, have been incriminated as key players in reperfusion injury to the heart. In the present communication we investigated other roles of iron — providing protection to the ischemic heart via preconditioning (PC).

Cardioprotective effect of α-tocopherol, ascorbate, deferoxamine, and deferiprone: Mitochondrial function in cultured, iron-loaded heart cells

Because mitochondrial inner membrane respiratory complexes are important targets of iron toxicity, we used iron-loaded rat heart cells in culture to study the beneficial effect on mitochondrial enzymes of the iron chelators deferoxamine (DFO) and deferiprone (L1) and of antioxidants and reducing agents (ascorbate and alpha-tocopherol). Reduced nicotinamide adenine dinucleotide-cytochrome c oxidoreductase (complex I-III) and succinate dehydrogenase were the most-sensitive indicators of iron toxicity and cardioprotective effect. Although at concentrations below 0.3 mmol/L the iron-mobilizing effect of L1 was less than that of DFO, both were equally effective in protecting or restoring mitochondrial respiratory enzyme activity. At 1.0 mmol/L, L1 toxicity was manifested in respiratory enzyme inhibition, whereas DFO had no such effect. Ascorbate (0.057 to 5.7 mmol/L) had a mild cardioprotective effect at the highest concentration only, in association with decreased cellular iron uptake. By contrast, alpha-tocopherol (0.023 mmol/L) completely inhibited mitochondrial iron toxicity without affecting iron uptake or release, and irrespective of whether it was used before, during, or after in vitro iron loading. These observations illustrate the usefulness and limitations of iron chelators and other agents used for preventing iron toxicity to the heart and other vital organs, and they underline the need for exploring in more detail the effects of these agents in the clinical setting.