Density Of Transferrin Receptors Research Articles

Brain Iron Deficiency Changes the Stoichiometry of Adenosine Receptor Subtypes in Cortico-Striatal Terminals: Implications for Restless Legs Syndrome.

Brain iron deficiency (BID) constitutes a primary pathophysiological mechanism in restless legs syndrome (RLS). BID in rodents has been widely used as an animal model of RLS, since it recapitulates key neurochemical changes reported in RLS patients and shows an RLS-like behavioral phenotype. Previous studies with the BID-rodent model of RLS demonstrated increased sensitivity of cortical pyramidal cells to release glutamate from their striatal nerve terminals driving striatal circuits, a correlative finding of the cortical motor hyperexcitability of RLS patients. It was also found that BID in rodents leads to changes in the adenosinergic system, a downregulation of the inhibitory adenosine A1 receptors (A1Rs) and upregulation of the excitatory adenosine A2A receptors (A2ARs). It was then hypothesized, but not proven, that the BID-induced increased sensitivity of cortico-striatal glutamatergic terminals could be induced by a change in A1R/A2AR stoichiometry in favor of A2ARs. Here, we used a newly developed FACS-based synaptometric analysis to compare the relative abundance on A1Rs and A2ARs in cortico-striatal and thalamo-striatal glutamatergic terminals (labeled with vesicular glutamate transporters VGLUT1 and VGLUT2, respectively) of control and BID rats. It could be demonstrated that BID (determined by measuring transferrin receptor density in the brain) is associated with a selective decrease in the A1R/A2AR ratio in VGLUT1 positive-striatal terminals.

Friedreich's Ataxia, No Changes in Mitochondrial Labile Iron in Human Lymphoblasts and Fibroblasts: A DECREASE IN ANTIOXIDATIVE CAPACITY?

Friedreich's ataxia (FRDA) is caused by low expression of frataxin, a small mitochondrial protein. Studies with both yeast and mammals have suggested that decreased frataxin levels lead to elevated intramitochondrial concentrations of labile (chelatable) iron, and consequently to oxidative mitochondrial damage. Here, we used the mitochondrion-selective fluorescent iron indicator/chelator rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzylester (RPA) to determine the mitochondrial chelatable iron of FRDA patient lymphoblast and fibroblast cell lines, in comparison with age- and sex-matched control cells. No alteration in the concentration of mitochondrial chelatable iron could be observed in patient cells, despite strongly decreased frataxin levels. Uptake studies with (55)Fe-transferrin and iron loading with ferric ammonium citrate revealed no significant differences in transferrin receptor density and iron responsive protein/iron regulatory element binding activity between patients and controls. However, sensitivity to H(2)O(2) was significantly increased in patient cells, and H(2)O(2) toxicity could be completely inhibited by the ubiquitously distributing iron chelator 2,2'-dipyridyl, but not by the mitochondrion-selective chelator RPA. Our data strongly suggest that frataxin deficiency does not affect the mitochondrial labile iron pool or other parameters of cellular iron metabolism and suggest a decreased antioxidative defense against extramitochondrial iron-derived radicals in patient cells. These results challenge current concepts favoring the use of mitochondrion-specific iron chelators and antioxidants to treat FRDA.

Clinical Significance of Apoptosis Is Independent from Proliferation in Acute Myeloid Leukemia (AML).

Multidrug resistance and recurrent disease are key problems in the variable response of AML pts to treatment. Tumor cells in a high proliferative state have a high density of transferrin receptors, as demonstrated in breast cancer (Yang DC, 2001) and in adult T-cell leukemia/lymphoma (Moura IC, 2004). On the other hand, defects in apoptotic pathways such as higher levels of bcl-2 and Mcl-1 were reported in “poor risk” AML pts. Current availability of antisense oligonucleotides targeted both to the transferrin receptor genes and bcl-2 incited us to evaluate the impact of proliferative and/or apoptotic pathways on AML prognosis. Therefore, a large series of 325 pts, affected by de novo AML, except FAB M3, median age 55 years, treated with intensive chemotherapy regimens, were studied. The aims of our research were: 1) to correlate bax/bcl-2 ratio with the proliferation levels, determined by the transferrin receptor (CD71) and 2) to demonstrate that the clinical significance of spontaneous apoptosis is independent from proliferation. CD71, bcl-2 and bax proteins were determined by multicolor flow cytometry. CD71 was evaluated as mean fluorescence intensity (MFI) and bax/bcl-2 ratio was obtained by dividing MFI bax/MFI bcl-2. One hundred-seventy five pts (53.8%) were bax/bcl-2 ratio positive and 204/324 (63%) were CD71 positive, respectively. There was a close correlation between higher CD71 expression and Ki-67 positive staining by flow cytometry (r=0.86), confirming that transferrin receptor overexpression is really linked to increased cellular proliferation in AML. No significant correlation was found between a higher bax/bcl-2 ratio and a lower CD71 MFI (p=0.16), confirming that an apoptosis resistant protein profile may have variable proliferation levels. A significant lower complete remission (CR) rate was found in pts with lower bax/bcl-2 ratio (43% vs 72%, p<0.00001) or higher CD71 (47% vs 74%, p=0.00001). Overall survival (OS) was significantly shorter either in pts with lower bax/bcl-2 ratio (1% vs 15% at 4 years; p=0.00001) or higher CD71 MFI (5% vs 104% at 5 years; p=0.002).Noteworthy, higher bax/bcl-2 ratio plus lower CD71 MFI identified an AML subset at better prognosis with regard to CR (91% vs 32%; p<0.00001) and OS (31% vs 0% at 2 years; p<0.00001). In order to confirm the independent prognostic value of bax/bcl-2 ratio from proliferation levels, we investigated the CD71 + (203 pts) and the CD71- (121 pts) AML subgroups. As a matter of fact, a lower CR rate was found in patients with lower bax/bcl-2 ratio either within CD71+ (34% vs 61%, p=0.0009) or CD71- (60% vs 90%, p=0.0006) pts. Also a lower bax/bcl-2 ratio was associated with a shorter OS both within CD71+ (2% vs 10% at 4 years, p=0.008) and within CD71- (0% vs 24% at 3.5 years, p=0.00008) pts. The independent prognostic value of bax/bcl-2 ratio was confirmed in multivariate analysis with regard to CR (p=0.00007) and OS (p=0.00003). The lack of significant correlation between bax/bcl-2 ratio and CD71 MFI confirms the independence of apoptosis from proliferation pathways. Furthermore, the capacity of bax/bcl-2 ratio of clearly identifying patients at different prognosis within the CD71+ and CD71- subgroups implies that apoptosis has an intrinsic more relevant clinical significance as compared to proliferation. That has to be taken in account when strategies are to be planned in order to resolve both chemoresistance and relapse in AML.

Intracellular iron regulates iron absorption and IRP activity in intestinal epithelial (Caco-2) cells.

In vertebrates, body Fe homeostasis is maintained through the regulation of its intestinal absorption. In addition, because Fe is both essential and toxic, intracellular Fe levels are tightly regulated. Consequently, intestinal epithelial cells are in the unique position of being responsible simultaneously for the regulation of body Fe absorption and the regulation of their intracellular Fe levels to remain viable. We tested the hypothesis that the regulation of transepithelial Fe transport and the regulation of intracellular Fe levels are sensitive to a common effector. To this end, we used a recently developed protocol to obtain cultured intestinal epithelial cells with defined intracellular Fe concentrations. In these cells we tested Fe absorption and Fe regulatory protein (IRP) activities. We found that transepithelial Fe transport was inversely related to 20-200 microM intracellular Fe and that Caco-2 cells expressed Fe regulatory protein-1 and Fe regulatory protein-2 activities. Fe regulatory protein-1 activity, Fe regulatory protein-2 mass, transferrin receptor density, and ferritin levels were regulated by intracellular Fe in the same range (20-200 microM) that affected transepithelial Fe transport. These results suggest that a common Fe-responsive factor regulates both intracellular Fe levels and Fe absorption by Caco-2 cells.

Autoradiography of transferrin receptors in the human brain

Transferrin is the major protein concerned with iron transport in the serum and may provide a route by which iron enters the brain. This study was designed to show the optimal binding conditions for in vitro transferrin receptor autoradiography and to show the regional distribution of transferrin receptors in the human brain. Optimal binding conditions were: 120 min incubation with 5.0 nM 125I-transferrin at 37°C. Transferrin receptors degrade quickly even with storage at −70°C, therefore binding studies should be performed within 7 days post mortem. Transferrin receptors were widely distributed in the human brain, with high density in the neocortex, moderate densities in the putamen and caudate nuclei, and very low densities in the globus pallidus and substantia nigra. Therefore transferrin receptor density shows a mismatch with the known distribution of iron in the human brain. The presence and distribution of transferrin receptors in the human brain are important because they may provide a route to deliver lipophobic substances across the blood-brain barrier by binding them to antibodies raised against transferrin receptors.

Expression of lactoferrin receptors is increased in the mesencephalon of patients with Parkinson disease.

The degeneration of nigral dopaminergic neurons in Parkinson disease is believed to be associated with oxidative stress. Since iron levels are increased in the substantia nigra of parkinsonian patients and this metal catalyzes the formation of free radicals, it may be involved in the mechanisms of nerve cell death. The cause of nigral iron increase is not understood. Iron acquisition by neurons may occur from iron-transferrin complexes with a direct interaction with specific membrane receptors, but recent results have shown a low density of transferrin receptors in the substantia nigra. To investigate whether neuronal death in Parkinson disease may be associated with changes in a pathway supplementary to that of transferrin, lactoferrin (lactotransferrin) receptor expression was studied in the mesencephalon. In this report we present evidence from immunohistochemical staining of postmortem human brain tissue that lactoferrin receptors are localized on neurons (perikarya, dendrites, axons), cerebral microvasculature, and, in some cases, glial cells. In parkinsonian patients, lactoferrin receptor immunoreactivity on neurons and microvessels was increased and more pronounced in those regions of the mesencephalon where the loss of dopaminergic neurons is severe. Moreover, in the substantia nigra, the intensity of immunoreactivity on neurons and microvessels was higher for patients with higher nigral dopaminergic loss. These data suggest that lactoferrin receptors on vulnerable neurons may increase intraneuronal iron levels and contribute to the degeneration of nigral dopaminergic neurons in Parkinson disease.

Radiotoxicity of 67-Gallium on myeloid leukemic blasts

Promising clinical results are obtained with radiolabeled antibodies in leukemia patients. 67Gallium ( 67Ga) is a radionuclide that accumulates in many malignant tissues without need for a monoclonal antibody. For this reason, the use of 67Ga as a therapeutic agent is appealing. In the present we study, we report data about the radiotoxicity of 67Ga on peripheral blast cells of 23 patients with acute myelogenous leukemia (AML) in vitro. Isolated blast cells were incubated for 4 days with 0.74 MBq/ml (20 μCi/ml), 1.48 MBq/ml (40 μCi/ml) or 2.96 MBq/ml (80 μCi/ml) 67Ga. Compared with non-irradiated control cells proliferation during incubation was almost abolished. Clonogenic survival was measured by a colony forming unit assay (CFU-assay). In 13 of the 23 patients (56%) sufficient colony growth was observed for evaluation. The mean clonogenic survival of blasts after incubation with 0.74 MBq/ml, 1.48 MBq/ml and 2.96 MBq/ml 67Ga was 22.5, 11.3 and 3.5%, respectively. In some cases colony growth was completely abolished after incubation with only 0.74 MBq/ml 67Ga. No correlation was found between cellular 67Ga-uptake, (micro)dosimetry and transferrin receptor density (CD-71) via which 67Ga enters the cell. In vitro the blasts received a dose of >10 Gy in 9 of the 2.96 MBq/ml, in 3 of the 1.48 MBq/ml and in 2 of the 0.74 MBq/ml incubations. In one patient, even a radiation dose > 40 Gy was reached. Low dose rate irradiation is known to arrest cells in G2/M-phase of the cell cycle, but no such arrest was observed during incubation with 67Ga. Thus, 67Ga induces clonogenic cell death in leukemic blasts. Cellular uptake of 67Ga in vitro varies between patients and can be very high in some patients. The easy availability, low costs and absence of immunological problems warrant further investigation of the therapeutic potential of 67Ga in refractory or relapsed AML patients.

Transferrin receptors in the Parkinsonian midbrain

Several hypotheses have been put forward to explain the pathogenesis of Parkinson's disease (PD) and recently it has been suggested that alterations in iron homeostasis may be implicated. Because of the central role of the transferrin receptor in providing access of iron to cells, we have studied the distribution and density of transferrin receptors using [3H]-transferrin ([3H]-Tf) binding and tritium film autoradiography in the normal and PD midbrain. High levels of [3H]-Tf binding were found in the dorsal raphé, oculomotor nucleus and periaqueductal grey whilst lower levels of [3H]-Tf binding were found in the tegmentum, red nucleus and substantia nigra. Significant reductions in binding were found in the substantia nigra, red nucleus and oculomotor nucleus in PD, the reductions in [3H]-Tf binding being similar to the loss of nigral neurons in PD. The data suggest that the increased iron content of surviving nigral neurons may reflect a compensatory metabolic response rather than abnormal transferrin receptor expression.

Distribution of 125I-ferrotransferrin binding sites in the mesencephalon of control subjects and patients with Parkinson's disease.

Iron is abnormally accumulated in the substantia nigra pars compacta of patients with Parkinson's disease (PD). Because neuronal and glial iron uptake seems to be mediated by the binding of ferrotransferrin to a specific high-affinity receptor on the cell surface, the number of transferrin receptors could be altered in this disease. The regional distribution of specific binding sites for human 125I-diferric transferrin has been studied in the mesencephalon, on cryostat-cut sections from autopsy brains of control subjects and parkinsonian patients by in vitro autoradiography. Densities of binding sites were highest in the central gray substance (approximately 10 fmol/mg of tissue equivalent), intermediate in the catecholaminergic cell group A8, superior colliculus, and ventral tegmental area, and almost nonexistent in the substantia nigra. The density of 125I-transferrin binding sites was not significantly different between parkinsonian and control brains in any region analyzed. These results show that in the mesencephalon the regional density of transferrin binding sites is lowest in the dopaminergic cell groups, which are the most vulnerable to PD, and suggest that iron does not accumulate through an increased density of transferrin receptors at the level of the substantia nigra.

The role of transferrin in heme transport

Porphyrin accumulation by proliferating cells, e.g., those associated with cancers or wounds, tends to correlate with increased transferrin receptor density. To determine whether transferrin might be implicated in porphyrin transport, fluorescence and absorption spectroscopy were used to study the interaction of porphyrins with transferrin. A single high-affinity binding site for heme and other porphyrins (Kd ∼ 20–25 nM) was detected by fluorescence spectroscopy. Difference spectroscopy revealed three additional heme-binding sites. These sites were distinct from the iron-binding sites: 1) Apotransferrin and diferric transferrin bound porphyrins with equal affinity; 2) 59Fe was not displaced from transferrin by porphyrins. Murine erythroleukemia cells incubated with [ 59Fe]hemin-[ 125I]transferrin internalized both labels concomitantly. Accumulation of [ 59Fe]hemin could be blocked by a 100-fold excess of diferric transferrin but not by apotransferrin. These results indicate that cells can internalize exogenous heme, and possibly porphyrins, bound to transferrin via its receptor.

Brain iron homeostasis

The anatomical and cellular distribution of non-haem iron, ferritin, transferrin, and the transferrin receptor have been studied in postmortem human brain and these studies, together with data on the uptake and transport of labeled iron, by the rat brain, have been used to elucidate the role of iron and other metal ions in certain neurological disorders. High levels of non-haem iron, mainly in the form of ferritin, are found in the extrapyramidal system, associated predominantly with glial cells. In contrast to non-haem iron, the density of transferrin receptors is highest in cortical and brainstem structures and appears to relate to the iron requirement of neurones for mitochondiral respiratory activity. Transferrin is synthesized within the brain by oligodendrocytes and the choroid plexus, and is present in neurones, consistent with receptor mediated uptake. The uptake of iron into the brain appears to be by a two-stage process involving initial deposition of iron in the brain capillary endothelium by serum transferrin, and subsequent transfer of iron to brain-derived transferrin and transport within the brain to sites with a high transferrin receptor density. A second, as yet unidentified mechanism, may be involved in the transfer of iron from neurones possessing transferrin receptors to sites of storage in glial cells in the extrapyramidal system. The distribution of iron and the transferrin receptor may be of relevance to iron-induced free radical formation and selective neuronal vulnerability in neurodegenerative disorders.

Transferrin receptors of rat and human brain and cerebral microvessels and their status in Alzheimer's disease

We studied the regional distribution of specific [ 125I]transferrin binding to transferrin receptors in the brains and cerebral microvessels of humans and rats. We also assessed transferrin receptors in subjects with Alzheimer's disease. Human diferric [ 125I]transferrin bound to regional brain and cerebral microvessels with high affinity (dissociation constants of 1–10 nM), and the maximal binding densities ranged from 30 to 90 pmol/mg protein in the brain and were several-fold higher in cerebral microvessels. In Alzheimer's disease, transferrin receptor densities were significantly reduced in the hippocampus and the temporal and occipital cortex but were unchanged in the frontal and parietal cortex and the cerebellum. Although [ 125I]transferrin binding was higher in cerebral microvessels from subjects with Alzheimer's disease than in those of age-matched controls, this difference did not attain statistical significance. These results suggest that transferrin receptor density was decreased in some cortical areas including the hippocampus in Alzheimer's disease but relatively unchanged in cerebral microvessels.

Transferrin receptor expression in myelin deficient (md) rats.

The question of iron regulation in the brain is the subject of increasing interest as the evidence continues to accumulate that a loss of brain iron homeostasis plays a significant role in some neurodegenerative diseases. Most cells acquire iron through a specific receptor mediated process involving transferrin, the iron mobilization protein. It appears that in the brain, endothelial cells, neurons, and oligodendrocytes express the transferrin receptor. This study uses a strain of rats (myelin deficient, md) in which oligodendrocytes fail to mature, and examines the consequences of this genetic defect on the expression of the transferrin receptor in the brain. The affinity of transferrin for its receptor is similar between the cerebral cortex and cerebellum in both the normal and myelin deficient rats (Kd = 7.8-10.6 nM). The transferrin receptor density is normally 2-3 times higher in the cerebellum than in the cerebral cortex. In the myelin deficient rat strain, the density of the transferrin receptor is decreased in both the cerebrum (56%) and cerebellum (70%) compared to the littermate control animals. Because oligodendrocytes are the only cell type affected in this mutant, the results suggest that these cells are responsible for a considerable amount of the transferrin receptors that are expressed in the brain (excluding the endothelial cell contributions). These observations are consistent with the existing literature stating that oligodendrocytes are responsible for the majority of transferrin and transferrin mRNA which is expressed in the brain, and support the working hypothesis that imbalances in brain iron homeostasis, particularly during development, are associated with myelin disorders.

Distribution and number of transferrin receptors in Parkinson's disease and in MPTP-treated mice

Transferrin is a glycoprotein that functions primarily to deliver iron to the cell. Recent studies suggest that the transferrin receptor mediates the intracellular delivery and transport of iron bound to transferrin in the CNS. Iron-catalyzed free radical generation has been proposed as a possible cause of nigral cell death in Parkinson's disease. Our hypothesis is that abnormal iron handling by the transferrin receptor may contribute to the formation of free radical species which catalyze the lipid peroxidation of nigral cell membranes. We have assessed the number of transferrin receptors on membrane fractions prepared from the human striatum from control subjects and patients with Parkinson's disease. Equilibrium-binding studies demonstrated a reversible, saturable, and high-affinity transferrin binding site ( K D = 3 nM) in human brain membranes. Regional binding assays indicate that the number of transferrin receptors in the putamen was reduced significantly in Parkinson's disease. The density of transferrin receptors was unaltered in membranes prepared from the caudate nuclei and the globus pallidus. To address the possibility that transferrin receptors are located on dopaminergic terminals, we have examined the distribution and number of transferrin receptors in the striatum of MPTP-treated mice using in vitro autoradiographic methods. In these experiments, the loss of dopaminergic terminals in the striatum was visualized by differential [ 3H]mazindol uptake site autoradiography. A marked reduction in the density of both transferrin receptors and [ 3H]mazindol binding sites was observed in the mouse striatum 7 days post-MPTP treatment. Quantitative densitometric analysis demonstrated a recovery in [ 125I]ferrotransferrin binding in the striata of mice sacrificed at later survival times. The time-course for the recovery of striatal transferrin receptors preceded the rise in [ 3H]mazindol binding sites. Taken together, these observations suggest that transferrin receptors may be located, in part, on dopaminergic terminals. The recovery of [ 125I]ferrotransferrin binding in the striatum may reflect early sprouting of dopaminergic fibers. Alternatively, transferrin receptors may be up-regulated in surviving dopaminergic terminals as a cellular adaptation to increased metabolic activity.

Transferrin-polycation-mediated introduction of DNA into human leukemic cells: stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels.

We have subverted a receptor-mediated endocytosis event to transport genes into human leukemic cells. By coupling the natural iron-delivery protein transferrin to the DNA-binding polycations polylysine or protamine, we have created protein conjugates that bind nucleic acids and carry them into the cell during the normal transferrin cycle [Wagner, E., Zenke, M., Cotten, M., Beug, H. & Birnstiel, M. L. (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414]. We demonstrate here that this procedure is useful for a human leukemic cell line. We enhanced the rate of gene delivery by (i) increasing the transferrin receptor density through treatment of the cells with the cell-permeable iron chelator desferrioxamine, (ii) interfering with the synthesis of heme with succinyl acetone treatment, or (iii) stimulating the degradation of heme with cobalt chloride treatment. Consistent with gene delivery as an endocytosis event, we show that the subsequent expression in K-562 cells of a gene included in the transported DNA depends upon the cellular presence of the lysosomotropic agent chloroquine. By contrast, monensin blocks "transferrinfection," as does incubation of the cells at 18 degrees C.

Immunohistochemical localization of epithelial cells expressing transferrin receptors in normal and diseased human dento-gingival junction.

The rate of cell proliferation, which is associated with the density of transferrin receptors on the cell surface, may have an effect on the homeostasis of epithelial cell populations in the dento‐gingival junction. In addition to moderate labeling of basal cells, intensive labeling of transferrin receptors was found on suprabasal cells of sulcular epithelium towards the gingival margin of healthy tissues. In contrast, biopsies from diseased tissue exhibited increased labeling towards the base of the pocket. This distribution of labeling suggests that the distinct suprabasal population of cells expressing the transferrin receptors is associated with the process of pocket formation. The density of transferrin receptors decreased towards the lamina propria of gingiva and towards the apical termination of the junctional epithelium. In samples from periodontal pockets, the number of receptors was high eveo in the germinative cell layers. Labeled cells were also demonstrated in crevicular washings. The washing method together with the demonstration of transferrin receptors may provide a new approach for the study of epithelial cell homeostasis in the dento‐gingival tissues.

Transferrin and the Growth-Promoting Effect of Nerves

In addition to its role in the activity of specialized proteins such as hemoglobin and myoglobin, iron is required as a cofactor in several important enzymes common to most animal cells. One such enzyme, ribonucleotide reductase, which regulates the production of deoxyribonucleotides during DNA synthesis, requires a continuous supply of iron to maintain its activity throughout the process of DNA replication. The mechanism by which animal cells normally acquire iron involves receptor-mediated uptake of iron-loaded transferrin, followed by release of apotransferrin. The density of transferrin receptors on the cell surface is greatly increased in rapidly dividing normal and neoplastic cells. Various mitogens and certain organogenic tissue interactions have been shown to induce the appearance of transferrin receptors, signalling the onset of DNA replication. Interference with this process of iron delivery causes the rapid arrest of cell cycling, frequently during the S phase itself, which underscores the importance of iron for DNA replication. Although most circulating transferrin is synthesized in the liver and embryonic yolk sac, smaller quantities are produced in several other embryonic organs and certain other adult tissues. It has been suggested that local synthesis and/or release of transferrin supplies the iron required by rapidly growing cells in situations where the cells do not have ready access to adequate amounts of plasma transferrin due to incomplete development of the vasculature or the presence of blood-tissue barriers (Ekblom and Thesleff, 1985; Meek and Adamson, 1985). Oligodendrocytes and Schwann cells have been shown to synthesize and/or contain high concentrations of transferrin and these cells therefore may constitute a local source of this factor for neurons, whose growth and survival in vitro require transferrin. Transferrin in central and peripheral nervous tissues may be significant for the trophic or growth-promoting effect neurons exert on cells of certain tissues. Transferrin duplicates the activity of neural tissue or neural extracts on growth and development of cultured skeletal myoblasts from chick embryos and on proliferation of mesenchymal cells in blastemas from regenerating amphibian limbs, two systems that have been widely used in investigations of the growth-promoting influence of nerves. Moreover, removal of active transferrin from neural extracts, either with antibodies to transferrin or chelation of the iron, inhibits reversibly the effect of the extract in these developing systems. While the physiological significance of the extract in these developing systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Macrophage cytotoxicity: interleukin 1 as a mediator of tumor cytostasis.

Purified macrophage interleukin 1 (IL 1) induced a concentration-dependent inhibition of the proliferation of two commonly used tumor cell target lines, the human myeloid K562 and the murine T lymphoma Eb. In contrast, mastocytoma-derived P815 cells were not inhibited. The cytostatic action of IL 1 was not associated with direct cytotoxicity and was only partially reversible. PGE or interferon did not appear to mediate these effects. IL 1 treatment of the multipotential K562 cells revealed no morphologic evidence for the induction of specific differentiation. FACS analysis of IL 1-treated K562 cells showed a rapid decrease in transferrin receptor density, and a more delayed, but highly significant, increase in HLA-A,B,C antigen density. These findings provide one explanation for the frequently reported macrophage cytostatic actions against tumor cells, and indicate as well that IL 1, like interferon, may enhance the expression of Class I MHC antigens. These observations further extend the range of IL 1 actions and underscore the fundamental and direct role of this monokine in macrophage antitumor activity.

Transferrin receptors in rat brain: neuropeptide-like pattern and relationship to iron distribution.

We have characterized and visualized the binding of 125I-labeled transferrin to sections of rat brain. This saturable, reversible, high-affinity (Kd = 1 X 10(-9) M) binding site appears indistinguishable from transferrin receptors previously characterized in other tissues. Moreover, a monoclonal antibody raised to rat lymphocyte transferrin receptors could immunoprecipitate recovered intact transferrin solubilized from labeled brain slices, indicating that labeling was to the same molecular entity previously characterized as the transferrin receptor. The pattern of transferrin receptor distribution visualized in brain with both 125I-labeled transferrin and an anti-transferrin receptor monoclonal antibody are almost indistinguishable but differ from the pattern of iron distribution. Iron-rich brain areas generally receive neuronal projections from areas with abundant transferrin receptors, suggesting that iron may be transported neuronally. However, many brain areas with a high density of transferrin receptors appear unrelated to iron uptake and neuronal transport and form a receptor distribution pattern similar to that of other known neuropeptides. This "neuropeptide-like" distribution pattern suggests that transferrin may have neuromodulatory, perhaps behavioral, function in brain.

Chromatography of erythroblasts on immobilized transferrin.

Affinity chromatography with immobilized transferrin has been used to effect a partial purification of erythroblasts and reticulocytes from bone marrow. Human transferrin was covalently attached to Sepharose CL-2B, Bio-Gel A-1.5, A-0.5, P-20, P-6, P-4, and P-2, and Sephadex G-75. Nucleated erythroid cells bound to the greatest degree to the polyacrylamide P-6 bead, whereas reticulocytes had the greatest affinity for the Sepharose CL-2B bead. The polyacrylamide P-6 bead has a greater density of surface groups than the Sepharose bead, and this correlates to the greater density of transferrin receptors on erythroblasts. The beads were used in a column which contained a central stirring bar with multiple crosspieces. The stirrer provided a method to uniformly agitate the beads to facilitate elution of the cells bound to the matrix. Approximately 1-2 x 10(8) bone marrow cells could be applied to a 25-ml matrix bed. This procedure resulted in a considerable resolution of cell types and gave a high yield of reticulocytes and erythroblasts from rat and rabbit bone marrow.