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From the Centro di Studio sulla Patologia Cellulare CNR, Istituto di Patologia Generale and Cattedra di Gastroenterologia I, Università degli Studi, IRCCS Ospedale Maggiore, Milano; and Dipartimento di Medicina Interna, Università di Modena, Italy.
In genetic hemochromatosis (GH), excess iron is deposited in parenchymal cells, whereas little iron is found in reticuloendothelial (RE) cells until the later stages of the disease. As iron absorption is inversely related to RE cells stores, a failure of RE to retain iron has been proposed as the basic defect in GH. In RE cells of GH subjects, we examined the activity of iron regulatory protein (IRP), a reliable indicator of the elusive regulatory labile iron pool, which modulates cellular iron homeostasis through control of ferritin (Ft) and transferrin receptor gene expression. RNA-bandshift assays showed a significant increase in IRP activity in monocytes from 16 patients with untreated GH compared with 28 control subjects (1.5-fold) and five patients with secondary hemochromatosis (SH) with similar iron burden (fourfold). In 17 phlebotomy-treated GH patients, IRP activity did not differ from that of control subjects. In both GH and SH monocyte-macrophages, Ft content increased by twofold and the L subunit-rich isoferritin profile was unchanged as compared with controls. IRP activity was still upregulated in vitro in monocyte-derived macrophages of GH subjects but, following manipulations of iron levels, was modulated normally. Therefore, the sustained activity of monocyte IRP found in vivo in monocytes of GH patients is not due to an inherent defect of its control, but is rather the expression of a critical abnormality of iron metabolism, eg, a paradoxical contraction of the regulatory iron pool. By preventing Ft mRNA translation, high IRP activity in monocytes may represent a molecular mechanism contributing to the inadequate Ft accumulation and insufficient RE iron storage in GH.
GENETIC HEMOCHROMATOSIS (GH) is a very common autosomal recessive disease of iron metabolism. Although the location of the gene on chromosome 6 in linkage with the HLA locus is well established,1 the gene product has not yet been identified. Recently, mutations in a sequence encoding a major histocompatibility complex (MHC) class I-like protein have been reported in a large majority of GH patients, making this gene, HLA-H, a very strong candidate for the GH gene.2 However, the function of the HLA-H gene product is still unknown. Despite intensive investigations by various groups over the past decades, the cellular location of the defect is still controversial. In fact, the liver, duodenum, and reticuloendothelial (RE) system have all been proposed as the primary sites of the metabolic abnormality.3
The RE system, in particular, plays a central role in iron metabolism, as it is responsible, through processing hemoglobin-iron from senescent erythrocytes, for iron supply to peripheral tissues, including the bone marrow.4 One peculiarity of GH is that excess iron is deposited mainly in parenchymal cells with relatively low iron stores in RE cells until late in the disease.5,6 As intestinal iron absorption is inversely related to RE cell iron deposits, an iron-handling defect of RE cells might be responsible for both excess iron deposition in parenchymal cells and lack of feed-back regulation of duodenal iron uptake.7 In GH, several studies on different aspects of RE cell iron metabolism have been performed using a variety of technical approaches,8-18 but conclusive evidence for a specific defect is still lacking.
Recently, considerable progress has been made in the molecular characterization of cellular iron metabolism. The metabolic status of iron in the cell is reflected in a regulatory pool of low molecular weight iron, the labile iron pool. As a consequence, the iron regulatory proteins (IRPs), the cytoplasmic sensors of the labile iron pool, are now regarded as the main regulators of cellular iron homeostasis.19-21 In addition to IRP-1 (formerly referred to as IRE-BP, FRP, IRF or IRP), a second IRP (IRP-2) has been characterized in rodent22,23 and human cells.24 IRPs control cellular iron storage and uptake by binding to specific RNA motifs called iron responsive elements (IRE) in untranslated regions of mRNAs for the H and L subunits of ferritin (Ft), which sequesters iron, and of transferrin receptor (TfR), which mediates uptake of the metal. IRP binding prevents translation of Ft mRNAs and increases TfR mRNA stability. When iron in the labile pool is scarce, IRPs bind to IRE and coordinately inhibit Ft synthesis and increase that of TfR, thus providing the cell with readily available iron. Conversely, in conditions of iron abundance, IRPs do not bind IRE and iron sequestration exceeds iron uptake. In iron replete cells, IRP-1 bears a 4Fe-4S cluster and represents the cytoplasmic homologue of mitochondrial aconitase; reversible disassembly of the cluster converts IRP-1 to its RNA-binding form. This posttranslational interconversion between the two forms constitutes the basis for the regulation of IRP-1 activity by iron. IRP-2 binds to IRE with an affinity and a specificity similar to those of IRP-1, but lacks aconitase activity and is not activated by reducing agents.25 Furthermore, its response to iron follows different pathways: de novo synthesis after iron starvation and degradation by the proteasome following iron repletion.26-28 Moreover, it has recently been demonstrated that molecules other than iron such as nitric oxide produced during inflammation29-31 and reactive oxygen species generated under conditions of oxidative stress31-34 can also modulate activity of IRPs.
We reasoned that, whatever the biochemical lesion of GH might be, assaying IRP activity in circulating monocytes and monocyte-derived macrophages was the only way to gain precise insights into the status of the regulatory labile iron pool and, as a consequence, the molecular mechanisms responsible for the suggested impairment of iron accumulation in RE cells of patients with GH.
Reagents
Subjects
Biochemical Evaluation Serum iron, total iron binding capacity, transferrin saturation index, and HLA typing were determined by standard techniques as previously reported.35 Serum Ft was measured by an enzyme immunoassay. Hepatic iron stores were evaluated and graded microscopically (0-4) by two independent observers and chemically by atomic absorption spectrophotometry as described previously.35Isolation of Monocytes Monocytes were purified as described by Colotta et al.37 Briefly, buffy coats prepared from heparinized venous blood were diluted with saline, layered on Ficoll-Paque, and centrifuged at 440g for 30 minutes. Mononuclear cells were then washed twice with saline and resuspended in RPMI 1640 medium containing 2 mmol/L glutamine, antibiotics and 10% fetal calf serum, adjusted to 285 mOsm. A total of 5 mL of the cellular suspension was layered over an equal volume of a solution composed of RPMI 1640 medium (54%) and 285 mOsm Percoll (46%) and centrifuged at 550g for 30 minutes. Lymphocytes at the bottom of the tube were resuspended in saline, pelletted in aliquots, and stored at -80°C. Monocytes banding at the interface were collected, washed twice with saline, and examined under the microscope to check yield, purity, and viability. A total of 10 to 15 × 106 cells/100 mL blood were recovered consisting of 95% to 98% monocytes, as shown by analysis of specific markers.37 Cell viability, measured by trypan blue dye exclusion test, was greater than 90%. The cells were either pelletted and stored at -80°C in aliquots or cultured (see below). Monocytes of patients with anemia and transfusional siderosis were separated from 50 mL of blood by the use of magnetic beads coated with antihuman CD14 antibody.38 Purity and viability of monocytes were similar to those reported above.Culture of Monocytes Monocytes were resuspended in RPMI 1640 medium containing 20% autologous or heat-inactivated human serum and cultured in 5% CO2 at 37°C.39 On different culture days, both adherent and nonadherent cells were harvested, pelletted, and stored at -80°C. As previously observed,39 both cell populations terminally differentiated to macrophages. Iron overload was obtained by culturing cells continuously in the presence of 50 µg/mL ferric ammonium citrate and iron deprivation by culture in the presence of 50 µmol/L desferrioxamine for 16 hours.In Vitro RNA Transcription The pSPT-fer plasmid containing the IRE of the human ferritin H chain40 was linearized with BamHI and transcribed in vitro with T7 RNA polymerase in the presence of 100 µCi of ( -32P) UTP (800 Ci/mmol). Probe-specific activity was determined by trichloroacetic acid precipitation of the reaction products and was routinely 1 × 106 dpm/ng.
RNA-Protein Gel Retardation Assay Cells were lysed in the buffer described by Leibold and Munro,41 the lysate was centrifuged at 16,000g for 5 minutes, and the supernatant was used for RNA-protein band-shift assays. Samples containing 2 µg protein, as determined using the Bio Rad protein assay kit, were incubated with a molar excess of IRE probe, digested with RNase T1, and treated with heparin as described previously.42 After separation on 6% nondenaturing polyacrylamide gels, RNA-protein complexes were visualized by autoradiography. For quantitation of IRP activity, radioactivity of bands excised from dried gels was determined by liquid scintillation counting and was converted to pmol/mg protein taking into account the specific activity and molecular mass (15.7 kD) of the probe and assuming a 1:1 molar ratio between IRP and RNA.43Determination of Ft Content The intracellular concentration of Ft was determined in the cytoplasmic extracts used for bandshift assays using a radioimmunoassay kit (Magic-Fer; Ciba Corning, Cassina de Pecchi, Milano, Italy) based on an antihuman liver Ft antibody. The subunit composition of Ft was measured by immunoassays based on specific anti-H and anti-L Ft subunit monoclonal antibodies.44Statistical Analysis The values shown in Table 1 are expressed as means ± standard deviation (SD). Significant differences were evaluated with Student's t-test using the Stat View 4.0 program (Abacus Concept Inc, Berkeley, CA).
IRP Activity in Circulating Monocytes To assess whether alterations of the molecular control of iron metabolism are present in RE cells from GH subjects, we examined IRP activity in monocytes from a large series of patients with untreated or treated GH in comparison to that of control subjects and patients with SH. A representative bandshift assay of IRP activity is shown in Fig 1 and quantitation of IRP activity in monocytes of all the subjects examined is reported in Table 1. Human IRPs are of similar charge and cannot be distinguished by bandshift assay, thus precluding analysis of the individual activities of the two forms. In this report, we use the term IRP activity to mean a combination of the two IRPs, although IRP-1 is probably the major contributor to the detected activity. Although some variability of IRP activity was found within all the groups examined, evaluation of a large number of subjects showed that untreated GH patients presented the highest amount of active IRP with a significant difference from control subjects (Table 1). IRP activity after phlebotomy returned to that seen in controls. Interestingly, in subjects with secondary iron overload with a tissue iron burden comparable to that of GH patients (Table 1), IRP binding activity was significantly decreased. Incubation of the extracts in the presence of 2% 2-mercaptoethanol to measure total cellular IRP increased the binding activity in all of the samples and almost blunted the differences between the various subjects (Fig 1). The remaining differences in total IRP activity are possibly accounted for by the contribution of IRP-2, which is not sensitive to 2-mercaptoethanol. Regarding total IRP activity, similar results were found throughout all the experiments performed in both monocytes and monocyte-derived macrophages (see below). With the limitations due to the influence of IRP-2 on quantitation of total activity (see above), the percentage of spontaneous IRP activity was 18% in normal subjects. As internal control to the experiments, lymphocyte IRP activity was also measured in selected cases, and a typical example is shown in Fig 2. No significant differences of IRP activity were found in lymphocytes from individual patients with GH when compared with IRP activity of lymphocytes from control subjects.
Ft Content in Circulating Monocytes
IRP Activity in Monocyte-Derived Macrophages As tissutal RE cells might represent the site of the metabolic defect in GH,15,45 monocytes from control subjects, patients with treated or untreated GH and with SH were cultured and allowed to mature to macrophages for several days, as reported.37,39 In agreement with a previous study,46 IRP activity of cells from controls and from patients with GH or SH increased with monocyte differentiation (Fig 4). In RE cells of untreated GH patients, IRP activity increased to a lesser extent, but still remained at a higher level than that of cells of other subjects.
The basic defect of GH is still elusive. The role of the product of the newly described candidate gene for GH in the derangement of iron metabolism in GH is still unknown.2 Using biochemical and molecular biology approaches, it has been shown that expression of the major iron-related genes in GH is normal in the liver,48-50 whereas duodenal abnormalities have been reported, including reduced accumulation of Ft and upregulation of TfR.51-53 Low accumulation of Ft is not due to a defective control of Ft synthesis, but to low expression of the corresponding mRNA and sustained activity of IRP.35,54 Thus, if the duodenal labile iron pool in GH is reduced, as IRP activity indicates, excessive transfer/release of iron to the bloodstream may represent the underlying cause.55 The cytoplasmic proteins from the whole duodenal tissue specimen used in a previous study35 derive from a variety of cell types, including both absorptive and nonabsorptive cells. Among the latter, macrophages of the lamina propria are especially important for iron metabolism, as they may be implicated in the control of iron absorption.16 In the absence of a convenient experimental system for studying human intestinal macrophages, circulating monocytes, the precursors of tissue macrophages, or monocyte-derived macrophages, can be reliably used as a suitable model for studying generalized disorders of the RE system.8,10
Submitted June 6, 1996;
accepted October 25, 1996.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hearly marked
``advertisment'' in accordance with 18 U.S.C. section 1734 solely to
indicate this fact. We are grateful to S. Levi for determination of ferritin content, F. Colotta and N. Polentarutti for help and advice in setting-up the procedure for monocyte isolation and culture, L. Kuhn for the generous gift of the pSPTFer plasmid, and F. Ravagnani for providing buffy coats of control subjects.
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Abstract
Introduction
Abstract
), 2 patients with SH caused by sporadic PTC and 
-thalassemia intermedia (
), 5 untreated (
), and 6 treated unrelated GH patients (
) were allowed to differentiate in vitro. Lysates were prepared from cells freshly isolated (day 0) or cultured for 3 and 6 days and IRP activity was measured by RNA-bandshift assay as described in the legend to Fig 1. Values were calculated as described in Materials and Methods and are expressed as means with SD. The inset shows a representative bandshift analysis of samples from a control (A) and an untreated GH patient (B) at the various time points. IRP activity increased with differentiation to macrophages in all the populations examined, maintaining the differences observed in freshly isolated monocytes.
) or in the presence of either 50 µg/mL ferric ammonium citrate (
). Iron loading was achieved by continuous culture in the presence of the iron salt and iron deprivation by adding desferrioxamine 16 hours before cell harvest. Lysates were prepared from cells freshly isolated (day 0) or cultured for 3 and 6 days, and IRP activity was measured by RNA-bandshift assay as described in the legend to Fig 1. Values were calculated as described in Materials and Methods and are expressed as means with SD. IRP activity was repressed by iron loading and upregulated by iron chelation at all stages of differentiation in RE cells of both controls and GH patients.
Interferon modulates human monocyte/macrophage transferrin receptor expression.
Blood
71:1590,
1988
