Hematopoietic progenitor cell transplantation toxicities in multiple myeloma patients with bisphosphonate-induced osteonecrosis of the jaw: a longitudinal cohort study.

PURPOSE: There is no information regarding the toxicity associated with autologous hematopoietic progenitor cell transplantation (AHPCT) in patients with multiple myeloma (MM) who have bisphosphonate-induced osteonecrosis of the jaw (ONJ). There is also limited information regarding long-term outcome of these patients. METHODS: In this retrospective cohort study, we compared the toxicity after AHPCT in MM patients with and without ONJ. We also analyzed the response rate and overall survival of this population of patients. RESULTS: During the study period, 176 patients underwent AHPCT at our institution for MM. Ten patients with ONJ prior to AHPCT were matched to 40 control patients without ONJ. The incidence and severity of transplantation-associated toxicities were similar in both groups, including mucositis, 50 % in patients with ONJ vs. 68 % in controls (p = 0.889) and febrile days, median 1 vs. 3 days, respectively (p = 0.524). Myeloid engraftment and hospital length of stay were also similar between patients with ONJ and controls. There were significantly more complete remissions in patients with ONJ than in control patients (45 % vs. 15 %, p = 0.0336), but survival between the groups was not significantly different (log-rank p = 0.0818). CONCLUSIONS: We conclude that the incidence and severity of transplantation-associated toxicities are similar in MM patients with and without ONJ. Long-term survival was also similar between both groups.

Deficiency in the divalent metal transporter 1 increases bleomycin-induced lung injury.

Exposure to bleomycin can result in an inflammatory lung injury. The biological effect of this anti-neoplastic agent is dependent on its coordination of iron with subsequent oxidant generation. In lung cells, divalent metal transporter 1 (DMT1) can participate in metal transport resulting in control of an oxidative stress and tissue damage. We tested the postulate that metal import by DMT1 would participate in preventing lung injury after exposure to bleomycin. Microcytic anemia (mk/mk) mice defective in DMT1 and wild-type mice were exposed to either bleomycin or saline via intratracheal instillation and the resultant lung injury was compared. Twenty-four h after instillation, the number of neutrophils and protein concentrations after bleomycin exposure were significantly elevated in the mk/mk mice relative to the wild-type mice. Similarly, levels of a pro-inflammatory mediator were significantly increased in the mk/mk mice relative to wild-type mice following bleomycin instillation. Relative to wild-type mice, mk/mk mice demonstrated lower non-heme iron concentrations in the lung, liver, spleen, and splenic, peritoneal, and liver macrophages. In contrast, levels of this metal were elevated in alveolar macrophages from mk/mk mice. We conclude that DMT1 participates in the inflammatory lung injury after bleomycin with mk/mk mice having increased inflammation and damage following exposure. This finding supports the hypothesis that DMT1 takes part in iron detoxification and homeostasis in the lung.

The department of veterans affairs nutritional status classification scheme allows for rapid assessment of nutritional status prior to autologous peripheral blood stem cell transplantation and identifies patients at high risk of transplant-related complications.

The nutritional assessment of patients prior to autologous peripheral blood stem cell transplantation (APBSCT) is labor intensive. A simple method of nutritional assessment prior to APSCT would be extremely helpful, especially if this method could identify patients at high risk of transplant-related complications. The Department of Veterans Affairs (VA) developed a Nutritional Status Classification Scheme (NSCS) to identify nutritionally compromised inpatients rapidly and reliably. The objective of this study was to determine if the use of the VA-NSCS could be utilized as a tool for the evaluation of patients prior to APBSCT and to determine if this tool could be used to identify patients at high risk of transplant-related complications. The nutritional status of 128 patients who underwent APBSCT was assessed by a registered dietician, utilizing the VA-NSCS, upon admission to the hospital and prior to conditioning regimen. Patients with moderately compromised nutritional status pretransplantation experienced a higher incidence of infections, longer duration of diarrhea, and longer length of hospital stay when compared to patients with normal or mildly compromised nutritional status. Our study demonstrates that the VA-NSCS, a simple and inexpensive tool to assess nutritional status, was useful in determining the pretransplant nutritional status of patients with lymphogenous malignancies who underwent APBSCT. In addition, this method was able to identify patients at a higher risk of posttransplant complications. Future studies should be undertaken to determine the optimal method for the nutritional assessment of autologous stem cell transplant candidates.

The dentate nucleus in Friedreich's ataxia: the role of iron-responsive proteins.

Frataxin deficiency in Friedreich's ataxia (FRDA) causes cardiac, endocrine, and nervous system manifestations. Frataxin is a mitochondrial protein, and adequate amounts are essential for cellular iron homeostasis. The main histological lesion in the brain of FRDA patients is neuronal atrophy and a peculiar proliferation of synaptic terminals in the dentate nucleus termed grumose degeneration. This cerebellar nucleus may be especially susceptible to FRDA because it contains abundant iron. We examined total iron and selected iron-responsive proteins in the dentate nucleus of nine patients with FRDA and nine normal controls by biochemical and microscopic techniques. Total iron (1.53 +/- 0.53 mumol/g wet weight) and ferritin (206.9 +/- 46.6 mug/g wet weight) in FRDA did not significantly differ from normal controls (iron: 1.78 +/- 0.88 mumol/g; ferritin: 210.9 +/- 9.0 mug/g) but Western blots exhibited a shift to light ferritin subunits. Immunocytochemistry of the dentate nucleus revealed loss of juxtaneuronal ferritin-containing oligodendroglia and prominent ferritin immunoreactivity in microglia and astrocytes. Mitochondrial ferritin was not detectable by immunocytochemistry. Stains for the divalent metal transporter 1 confirmed neuronal loss while endothelial cells reacting with antibodies to transferrin receptor 1 protein showed crowding of blood vessels due to collapse of the normal neuropil. Regions of grumose degeneration were strongly reactive for ferroportin. Purkinje cell bodies, their dendrites and axons, were also ferroportin-positive, and it is likely that grumose degeneration is the morphological manifestation of mitochondrial iron dysmetabolism in the terminals of corticonuclear fibers. Neuronal loss in the dentate nucleus is the likely result of trans-synaptic degeneration.

Hepcidin expression and iron transport in alveolar macrophages.

Alveolar macrophages express many proteins important in iron homeostasis, including the iron importer divalent metal transport 1 (DMT1) and the iron exporter ferroportin 1 (FPN1) that likely participate in lung defense. We found the iron regulatory hormone hepcidin (HAMP) is also produced by alveolar macrophages. In mouse alveolar macrophages, HAMP mRNA was detected at a low level when not stimulated but at a high level when exposed to lipopolysaccharide (LPS). LPS also affected the mRNA levels of the iron transporters, with DMT1 being upregulated and FPN1 downregulated. However, iron had no effect on HAMP expression but was able to upregulate both DMT1 and FPN1 in alveolar macrophages. IL-1 and IL-6, which are important in HAMP augmentation in hepatocytes, also did not affect HAMP expression in alveolar macrophages. In fact, the LPS-induced alterations in the expression of HAMP as well as DMT1 and FPN1 were preserved in the alveolar macrophages isolated from IL-1 receptor or IL-6-deficient mice. When alveolar macrophages were loaded with transferrin-bound (55)Fe, the subsequent release of (55)Fe was inhibited significantly by LPS. In addition, treatment of these cells with either LPS or HAMP caused the diminishment of the surface FPN1. These findings are consistent with the current model that HAMP production leads to a decreased iron efflux. Our studies suggest that iron mobilization by alveolar macrophages can be affected by iron and LPS via several pathways, including HAMP-mediated degradation of FPN1, and that these cells may use unique regulatory mechanisms to cope with iron imbalance in the lung.

Regulation of hepcidin and ferroportin expression by lipopolysaccharide in splenic macrophages.

Acute and chronic inflammatory states are associated with many changes in intracellular iron metabolism including sequestration of iron in the mononuclear-phagocyte system (MPS) and a decline in serum iron. Previous work in rodent models of acute inflammation has demonstrated inflammation-induced downregulation of intestinal and MPS iron exporter, ferroportin 1, mRNA and protein. In addition, these models have also demonstrated hepatic induction of mRNA of the small 25 amino acid peptide hepcidin. Hepcidin has been hypothesized to be the mediator of iron- and inflammation-induced changes in iron metabolism. The molecular details of the connection between iron metabolism, hepcidin and inflammation have become clearer with the recent finding of hepcidin-induced internalization and degradation of FPN1. The work presented here demonstrates that the lipopolysaccharide-induced splenic macrophage FPN1 mRNA downregulation is not dependent upon the action of a single cytokine such as IL-6, IL-1 or TNF-alpha because mice deficient in these pathways downregulate FPN1 normally. Furthermore, hepcidin is also synthesized in the spleen of normal mice and induced by lipopolysaccharide. Additionally, in vitro, splenic adherent cells produce hepcidin in response to lipopolysaccharide in an IL-6-dependent manner. There appear to be both probable transcriptional and post-transcriptional control of FPN1 expression by lipopolysaccharide-induced inflammation. The former effect is on mRNA expression and is independent of hepcidin, whereas the latter is IL-6- and hepcidin-dependent.

Functional consequences of ferroportin 1 mutations.

The cellular iron exporter ferroportin 1 is expressed in both the duodenum and in cells of the mononuclear phagocyte system. Expression of ferroportin 1 protein on the cell surface is regulated by the interaction of ferroportin 1 with hepcidin. Hepcidin treatment of cells results in internalization and lysosomal degradation of cell surface ferroportin 1. Recently, ferroportin 1 mutations leading to hemochromatosis (HFE4) have been identified. HFE4 differs from classical hemochromatosis in that there is a greater amount of macrophage iron sequestration. The data presented here demonstrate that HFE4 mutations are heterogeneous in their effects on protein function. Some mutations result in loss of function with partial protein sequestration in the ER. Others are indistinguishable from native ferroportin 1 and have a similar ability to deplete transfected cells of iron as evidenced by activation of the iron-response proteins and cellular ferritin depletion. Significantly, all mutants appear to be unresponsive to hepcidin and do not demonstrate the expected internalization on exposure to hepcidin. The clinical phenotypes observed in patients may be secondary to cell-type-specific defects in hepcidin-mediated inhibition of ferroportin 1 expression.

Apical location of ferroportin 1 in airway epithelia and its role in iron detoxification in the lung.

Ferroportin 1 (FPN1; aka MTP1, IREG1, and SLC40A1), which was originally identified as a basolateral iron transporter crucial for nutritional iron absorption in the intestine, is expressed in airway epithelia and upregulated when these cells are exposed to iron. Using immunofluorescence labeling and confocal microscopic imaging techniques, we demonstrate that in human and rodent lungs, FPN1 localizes subcellularly to the apical but not basolateral membrane of the airway epithelial cells. The role of airway epithelial cells in iron mobilization in the lung was studied in an in vitro model of the polarized airway epithelium. Normal human bronchial epithelial cells, grown on membrane supports until differentiated, were exposed to iron, and the efficiency and direction of iron transportation were studied. We found that these cells can efficiently take up iron across the apical but not basolateral surface in a concentration-dependent manner. Most of the iron taken up by the cells is then released into the medium within 8 h in the form of less reactive protein-bound complexes including ferritin and transferrin. Interestingly, iron release also occurred across the apical but not basolateral membrane. Our findings indicate that FPN1, depending on its subcellular location, could have distinct functions in iron homeostasis in different cells and tissues. Although it is responsible for exporting nutrient iron from enterocytes to the circulation in the intestine, it could play a role in iron detoxification in airway epithelial cells in the lung.

Copper-induced ferroportin-1 expression in J774 macrophages is associated with increased iron efflux.

Copper is known to play a role in iron recycling from macrophages. To examine whether cellular copper status affects expression of the iron exporter ferroportin-1 (FPN1), J774 macrophage cells were exposed to 10-100 microM CuSO(4) for up to 20 h. Copper treatment significantly increased FPN1 mRNA in a dose- and time-dependent manner. After 20 h, 100 microM CuSO(4) up-regulated FPN1 transcript levels approximately 13-fold compared to untreated controls. Induction was detected 8 h after copper treatment was initiated and markedly increased thereafter. A corresponding increase in FPN1 protein levels was observed upon copper treatment. Induction of J774 cell FPN1 expression by copper was also associated with a dose-dependent increase in (59)Fe release after erythrophagocytosis of labeled red blood cells. Thus, a previously uncharacterized role for copper in the regulation of macrophage iron recycling is suggested by the induction of FPN1 gene expression and iron efflux by this metal.

The role of the iron responsive element in the control of ferroportin1/IREG1/MTP1 gene expression.

BACKGROUND/AIMS: MTP1/Ferroportin1/IREG1, the product of the SLC40A1 gene, is a main iron export protein in mammals. However, the way this gene is regulated by iron is still unclear. The aim of this study was to investigate the functional role of genomic SLC40A1 elements in response to iron. METHODS: Vectors containing either reverse similar 2.6 kb 5' flanking region or deletion constructs, including one devoid of an iron responsive element (SLC40A1-DeltaIRE-Luc), were analyzed by luciferase reporter gene in transfected HepG2, CaCO2 and U937 cells. Expression of iron genes and activity of the iron regulatory protein were also studied. RESULTS: Iron increased and desferrioxamine decreased luciferase activity in all the cell types using both the full-length construct and the promoter deletion constructs, in the absence of changes in SLC40A1 or luciferase mRNA levels. To test the role of the SLC40A1 5' untranslated region, we first demonstrated that wild type and not SLC40A1-DeltaIRE-Luc could bind iron regulatory protein. Then, in cells transfected with SLC40A1-DeltaIRE-Luc, we found that, in spite of iron regulatory protein activation, the response to iron manipulation was lost. CONCLUSIONS: We demonstrate that the iron responsive element in the SLC40A1 gene is functional and that it controls gene expression through the cytoplasmic iron regulatory protein system.

Iron loading and erythrophagocytosis increase ferroportin 1 (FPN1) expression in J774 macrophages.

The expression of ferroportin1 (FPN1) in reticuloendothelial macrophages supports the hypothesis that this iron-export protein participates in iron recycling from senescent erythrocytes. To gain insight into FPN1's role in macrophage iron metabolism, we examined the effect of iron status and erythrophagocytosis on FPN1 expression in J774 macrophages. Northern analysis indicated that FPN1 mRNA levels decreased with iron depletion and increased on iron loading. The iron-induced induction of FPN1 mRNA was blocked by actinomycin D, suggesting that transcriptional control was responsible for this effect. After erythrophagocytosis, FPN1 mRNA levels were also up-regulated, increasing 8-fold after 4 hours and returning to basal levels by 16 hours. Western analysis indicated corresponding increases in FPN1 protein levels, with maximal induction after 10 hours. Iron chelation suppressed FPN1 mRNA and protein induction after erythrophagocytosis, suggesting that FPN1 induction results from erythrocyte-derived iron. Comparative Northern analyses of iron-related genes after erythrophagocytosis revealed a 16-fold increase in FPN1 levels after 3 hours, a 10-fold increase in heme oxygenase-1 (HO-1) after 3 hours, a 2-fold increase in natural resistance macrophage-associated protein 1 (Nramp1) levels after 6 hours, but no change in divalent metal ion transporter 1 (DMT1) levels. The rapid and strong induction of FPN1 expression after erythrophagocytosis suggests that FPN1 plays a role in iron recycling.

Haptoglobin reduces lung injury associated with exposure to blood.

The biological functions of the acute- phase protein haptoglobin (Hp) may be related to its ability to bind hemoglobin (Hb) or to modulate immune response. Hp is expressed at a high level in lung cells, yet its protective role(s) in the lung is not known. With the use of transgenic mice overexpressing Hp in alveolar macrophages, we demonstrated that Hp diminished Hb-induced lung injury when the lung was exposed to whole blood. In transgenic mouse lungs, Hb was more efficiently removed, and the induction of stress- responsive heme oxygenase-1 gene was significantly lower when compared with wild-type mice. At 24 h after blood treatment, the ferritin level that serves as an index for intracellular iron content was also lower in alveolar macrophages in transgenic mice than in wild-type mice. We propose that an Hp-mediated Hb catabolism process exists in alveolar macrophages. This process is likely coupled to an iron mobilization pathway and may be an efficient mechanism to reduce oxidative damage associated with hemolysis.

Iron increases expression of iron-export protein MTP1 in lung cells.

Accumulation of reactive iron in acute and chronic lung disease suggests that iron-driven free radical formation could contribute to tissue injury. Safe transport and sequestration of this metal is likely to be of importance in lung defense. We provide evidence for the expression and iron-induced upregulation of the metal transporter protein-1 (MTP1) genes in human and rodent lung cells at both the protein and mRNA levels. In human bronchial epithelial cells, a 3.8-fold increase in mRNA level and a 2.4-fold increase in protein level of MTP1 were observed after iron exposure. In freshly isolated human macrophages, as much as an 18-fold increase in the MTP1 protein level was detected after incubation with an iron compound. The elevation in expression of MTP1 gene was also demonstrated in iron-instilled rat lungs and in hypotransferrinemic mouse lungs. This is similar to our previous findings with divalent metal transporter-1 (DMT1), an iron transporter that is required for iron uptake and intracellular iron trafficking. These studies suggest the presence of iron mobilization and/or detoxification pathways in the lung that are crucial for iron homeostasis and lung defense.

Regulation of reticuloendothelial iron transporter MTP1 (Slc11a3) by inflammation.

Acute and chronic inflammation cause many changes in total body iron metabolism including the sequestration of iron in phagocytic cells of the reticuloendothelial system. This change in iron metabolism contributes to the development of the anemia of inflammation. MTP1, the duodenal enterocyte basolateral iron exporter, is also expressed in the cells of the reticuloendothelial system (RES) and is likely to be involved in iron recycling of these cells. In this study, we use a lipopolysaccharide model of the acute inflammation in the mouse and demonstrate that MTP1 expression in RES cells of the spleen, liver, and bone marrow is down-regulated by inflammation. The down-regulation of splenic expression of MTP1 by inflammation was also observed in a Leishmania donovani model of chronic infection. The response of MTP1 to lipopolysaccharide (LPS) requires signaling through the LPS receptor, Toll-like receptor 4 (TLR4). In mice lacking TLR4, MTP1 expression is not altered in response to LPS. In addition, mice lacking tumor necrosis factor-receptor 1a respond appropriately to LPS with down-regulation of MTP1, despite hyporesponsiveness to tumor necrosis factor-alpha signaling, suggesting that this cytokine may not be required for the LPS effect. We hypothesize that the iron sequestration in the RES system that accompanies inflammation is because of down-regulation of MTP1.

Role of the ferroportin iron-responsive element in iron and nitric oxide dependent gene regulation.

The newly described iron transporter, ferroportin (MTP1, IREG1), is expressed in a variety of tissues including the duodenum and cells of the mononuclear phagocyte system (MPS). In the MPS, ferroportin is hypothesized to be a major exporter of iron scavenged from senescent erythrocytes. Changes in iron metabolism, including the sequestration of iron in the MPS, are characteristic of both acute and chronic inflammation and these conditions induce changes in ferroportin expression. In a mouse model of acute inflammation, LPS administration is associated with reduced MPS ferroportin protein and mRNA expression. In addition, the ferroportin 5' UTR also has an iron-responsive element that binds to the iron-response proteins, but whether there is a role for this IRE in inflammation induced regulation of ferroportin has been unclear. A luciferase reporter gene under the control of the mouse ferroportin promoter and 5' UTR was used to determine if this 5' UTR conferred IRE-dependent regulation on this reporter gene. Stimulation of reporter gene transfected RAW 264.7 cells (a mouse macrophage cell line) with LPS resulted in IRE-dependent inhibition of luciferase production. Inhibitors of nitric oxide synthase abrogated the IRE-dependent effect of LPS. In addition, direct treatment of RAW 264.7 and with NO donor S-nitroso-N-acetylpenicillamine resulted in IRE-dependent down-regulation of luciferase expression. The effect of NO was consistent with IRP1/IRE mediated translation block. There are most likely both inflammation-mediated transcriptional and post-transcriptional (IRE-dependent) mechanisms for inhibiting ferroportin expression in MPS cells.