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• W2070771807 abstract "The reticuloendothelial system has a central role in erythropoiesis and iron homeostasis. An important function of reticuloendothelial macrophages is phagocytosis of senescent red blood cells. The iron liberated from heme is recycled for delivery to erythrocyte precursors for a new round of hemoglobin synthesis. The molecular mechanism by which recycled iron is released from macrophages remains unresolved. We have investigated the mechanism of macrophage iron efflux, focusing on the role of ceruloplasmin (Cp), a copper protein with a potent ferroxidase activity that converts Fe2+ to Fe3+ in the presence of molecular oxygen. As shown by others, Cp markedly increased iron binding to apotransferrin at acidic pH; however, the physiological significance of this finding is uncertain because little stimulation was observed at neutral pH. Introduction of a hypoxic atmosphere resulted in marked Cp-stimulated binding of iron to apotransferrin at physiological pH. The role of Cp in cellular iron release was examined in U937 monocytic cells induced to differentiate to the macrophage lineage. Cp added at its normal plasma concentration increased the rate of 55Fe release from U937 cells by about 250%. The stimulation was absolutely dependent on the presence of apotransferrin and hypoxia. Cp-stimulated iron release was confirmed in mouse peritoneal macrophages. Stimulation of iron release required an intracellular “labile iron pool” that was rapidly depleted in the presence of Cp and apotransferrin. Ferroxidase-mediated loading of iron into apotransferrin was critical for iron release because ferroxidase-deficient Cp was inactive and because holotransferrin could not substitute for apotransferrin. The extracellular iron concentration was critical as shown by inhibition of iron release by exogenous free iron, and marked enhancement of release by an iron chelator. Together these data show that Cp stimulates iron release from macrophages under hypoxic conditions by a ferroxidase-dependent mechanism, possibly involving generation of a negative iron gradient. The reticuloendothelial system has a central role in erythropoiesis and iron homeostasis. An important function of reticuloendothelial macrophages is phagocytosis of senescent red blood cells. The iron liberated from heme is recycled for delivery to erythrocyte precursors for a new round of hemoglobin synthesis. The molecular mechanism by which recycled iron is released from macrophages remains unresolved. We have investigated the mechanism of macrophage iron efflux, focusing on the role of ceruloplasmin (Cp), a copper protein with a potent ferroxidase activity that converts Fe2+ to Fe3+ in the presence of molecular oxygen. As shown by others, Cp markedly increased iron binding to apotransferrin at acidic pH; however, the physiological significance of this finding is uncertain because little stimulation was observed at neutral pH. Introduction of a hypoxic atmosphere resulted in marked Cp-stimulated binding of iron to apotransferrin at physiological pH. The role of Cp in cellular iron release was examined in U937 monocytic cells induced to differentiate to the macrophage lineage. Cp added at its normal plasma concentration increased the rate of 55Fe release from U937 cells by about 250%. The stimulation was absolutely dependent on the presence of apotransferrin and hypoxia. Cp-stimulated iron release was confirmed in mouse peritoneal macrophages. Stimulation of iron release required an intracellular “labile iron pool” that was rapidly depleted in the presence of Cp and apotransferrin. Ferroxidase-mediated loading of iron into apotransferrin was critical for iron release because ferroxidase-deficient Cp was inactive and because holotransferrin could not substitute for apotransferrin. The extracellular iron concentration was critical as shown by inhibition of iron release by exogenous free iron, and marked enhancement of release by an iron chelator. Together these data show that Cp stimulates iron release from macrophages under hypoxic conditions by a ferroxidase-dependent mechanism, possibly involving generation of a negative iron gradient. Reticuloendothelial macrophages have a central role in regulating normal iron homeostasis (1Knutson M. Wessling-Resnick M. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 61-88Crossref PubMed Scopus (253) Google Scholar). Macrophages in bone marrow, liver, and spleen recognize and phagocytose senescent or damaged erythrocytes, and the heme iron is processed and returned to circulation for reutilization by red cell precursors during erythropoiesis (2Noyes W.D. Bothwell T.H. Finch C.A. Br. J. Haematol. 1960; 6: 43-55Crossref PubMed Scopus (64) Google Scholar). Macrophages may also utilize iron to generate toxic reactive oxygen species as a defense mechanism against pathogens (3Alford C.E. King Jr., T.E. Campbell P.A. J. Exp. Med. 1991; 174: 459-466Crossref PubMed Scopus (109) Google Scholar). Macrophage iron release appears to be tightly regulated. The relative amount of iron released, compared with the amount shunted for storage as ferritin, depends on iron status and is increased during iron deficiency. The rate of macrophage iron release is also regulated by, or at least coupled to, the rate of erythropoiesis. The reticuloendothelial system accumulates iron during several pathological states including chronic inflammation and renal failure, indicating an inability of the macrophage to release iron normally.Understanding the regulation of macrophage iron release has been an area of intense investigation during the past three decades. Several fundamental issues remain unresolved including the intracellular form of released iron, its mechanism of transport through the plasma membrane, the form of the emerging iron, and the mechanism of iron delivery to apotransferrin (4Brittenham G.M. Hoffman R. Benz E.J. Shattil S.J. Furie B. Cohen H.J. Silberstien L.E. McGlave P. Hematology: Basic Principles and Practice. Harcourt, New York2000: 397-428Google Scholar). A mechanism for the terminal step in the export process was suggested by Osaki et al. (5Osaki S. Johnson D.A. Frieden E. J. Biol. Chem. 1966; 241: 2746-2751Abstract Full Text PDF PubMed Google Scholar). They showed that ceruloplasmin (Cp, 1The abbreviations used are: Cp, ceruloplasmin; FPN1, ferroportin-1; LIP, labile iron pool; NTA, nitrilotriacetic acid; PBS, phosphate-buffered saline; PMA, phorbol myristoyl acetate; MOPS, 4-morpholinepropanesulfonic acid.1The abbreviations used are: Cp, ceruloplasmin; FPN1, ferroportin-1; LIP, labile iron pool; NTA, nitrilotriacetic acid; PBS, phosphate-buffered saline; PMA, phorbol myristoyl acetate; MOPS, 4-morpholinepropanesulfonic acid. EC 1.16.3.1) has a potent ferroxidase activity that catalyzed the oxidation of Fe2+ to Fe3+ at the expense of O2, and accelerated the binding of iron by apotransferrin. They proposed that this process generated a steep, negative free-iron concentration gradient that increased iron efflux from the cell (5Osaki S. Johnson D.A. Frieden E. J. Biol. Chem. 1966; 241: 2746-2751Abstract Full Text PDF PubMed Google Scholar, 6Osaki S. Johnson D.A. Frieden E. J. Biol. Chem. 1971; 246: 3018-3023Abstract Full Text PDF PubMed Google Scholar). The finding of massive tissue iron deposits in hereditary Cp deficiency patients (7Harris Z.L. Takahashi Y. Miyajima H. Serizawa M. MacGillivray R.T. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2539-2543Crossref PubMed Scopus (508) Google Scholar, 8Yoshida K. Furihata K. Takeda S. Nakamura A. Yamamoto K. Morita H. Hiyamuta S. Ikeda S. Shimizu N. Yanagisawa N. Nat. Genet. 1995; 9: 267-272Crossref PubMed Scopus (421) Google Scholar), and ferrokinetic studies in mice with a targeted disruption in the Cp gene, support an important role of Cp in tissue iron release (9Harris Z.L. Durley A.P. Man T.K. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10812-10817Crossref PubMed Scopus (488) Google Scholar).Arguments have been raised against this proposed mechanism of iron release. The rapid autoxidation of iron under physiological conditions, particularly in the presence of apotransferrin (which itself may have ferroxidase activity), may eliminate the need for Cp-dependent catalysis to oxidize and load iron into apotransferrin (10Rydén L. Lontie R. Copper Proteins and Copper Enzymes. Vol. III. CRC Press, Boca Raton, FL1984: 37-100Google Scholar, 11Bates G.W. Workman Jr., E.F. Schlabach M.R. Biochem. Biophys. Res. Commun. 1973; 50: 84-90Crossref PubMed Scopus (36) Google Scholar, 12Mareschal J.C. Rama R. Crichton R.R. FEBS Lett. 1980; 110: 268-270Crossref PubMed Scopus (10) Google Scholar). In fact, the rate of iron binding by apotransferrin is too fast to measure by usual kinetic techniques at pH 7, thus in vitro studies of Cp-mediated iron loading into apotransferrin are done under acidic conditions that slow the reaction, generally at pH 5.5 to 6.0 (13Johnson D.A. Osaki S. Frieden E. Clin. Chem. 1967; 13: 142-150Crossref PubMed Scopus (103) Google Scholar). It is also difficult to reconcile the gradient mechanism with the inverse relationship between plasma Cp concentration and transferrin iron saturation, for example, newborns have very high transferrin iron saturation and low Cp, whereas their mothers have the opposite condition (14Shokeir M.H. Clin. Biochem. 1972; 5: 115-120Crossref PubMed Scopus (6) Google Scholar). Likewise, the marked elevation of plasma Cp during the anemia of inflammation, when reticuloendothelial cell iron stores are elevated, remains paradoxical (15Roeser H.P. Lee G.R. Cartwright G.E. Proc. Soc. Exp. Biol. Med. 1973; 142: 1155-1158Crossref PubMed Scopus (7) Google Scholar). The requirement for apotransferrin in macrophage iron release in vitro is also controversial; several investigators have shown a lack of stimulation by apotransferrin (16Esparza I. Brock J.H. Br. J. Haematol. 1981; 49: 603-614Crossref PubMed Scopus (44) Google Scholar, 17Saito K. Nishisato T. Grasso J.A. Aisen P. Br. J. Haematol. 1986; 62: 275-286Crossref PubMed Scopus (56) Google Scholar), whereas others have found a stimulatory effect (18Rama R. Sánchez J. Octave J.-N. Biochim. Biophys. Acta. 1988; 968: 51-58Crossref PubMed Scopus (20) Google Scholar). In fact, under some circumstances, Cp may enhance iron uptake by cells, possibly by a trivalent cation transporter on the cell surface (19Mukhopadhyay C.K. Attieh Z.K. Fox P.L. Science. 1998; 279: 714-717Crossref PubMed Scopus (178) Google Scholar, 20Attieh Z.K. Mukhopadhyay C.K. Seshadri V. Tripoulas N.A. Fox P.L. J. Biol. Chem. 1999; 274: 1116-1123Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 21Qian Z.M. Tsoi Y.K. Ke Y. Wong M.S. Exp. Brain Res. 2001; 140: 369-374Crossref PubMed Scopus (24) Google Scholar).The role of plasma Cp in iron release by macrophages has not been investigated experimentally, and studies from non-macrophage cells are inconsistent. Young et al. (22Young S.P. Fahmy M. Golding S. FEBS Lett. 1997; 411: 93-96Crossref PubMed Scopus (56) Google Scholar) reported a 40% stimulation of iron release from human hepatocarcinoma HepG2 cells by Cp in the presence of apotransferrin. The mechanism of stimulation was not specifically investigated in detail, and in view of the comparable stimulation of iron release by either apo- or holotransferrin, an important role of iron loading to transferrin is unlikely. In other studies, Cp increased iron release from iron-loaded HepG2 cells (23Richardson D.R. J. Lab. Clin. Med. 1999; 134: 454-465Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) and from central nervous system astrocytes (24Jeong S.Y. David S. J. Biol. Chem. 2003; 278: 27144-27148Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar), but the mechanism underlying the stimulation was unclear because it occurred even in the absence of apotransferrin. Interestingly, astrocyte Cp is glycosylphosphatidylinositol-linked suggesting that both free and membrane-bound forms of Cp are involved in iron transport processes. Three other laboratories reported that Cp did not increase iron release (even in the presence of apotransferrin) from HepG2 or erythroleukemic K562 cells (19Mukhopadhyay C.K. Attieh Z.K. Fox P.L. Science. 1998; 279: 714-717Crossref PubMed Scopus (178) Google Scholar, 20Attieh Z.K. Mukhopadhyay C.K. Seshadri V. Tripoulas N.A. Fox P.L. J. Biol. Chem. 1999; 274: 1116-1123Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), placental BeWo cells (25Danzeisen R. Ponnambalam S. Lea R.G. Page K. Gambling L. McArdle H.J. Placenta. 2000; 21: 805-812Crossref PubMed Scopus (43) Google Scholar), or glioblastoma BT325 cells (21Qian Z.M. Tsoi Y.K. Ke Y. Wong M.S. Exp. Brain Res. 2001; 140: 369-374Crossref PubMed Scopus (24) Google Scholar).In this report we rigorously investigate the requirements for Cp stimulation of iron binding to apotransferrin and iron release from myeloid cells. We show that Cp markedly enhances iron release from macrophages only in the presence of apotransferrin and only under hypoxic conditions. Regarding the mechanism of Cp-stimulated macrophage iron release, we show that the iron source is the intracellular labile iron pool (LIP), that Cp ferroxidase activity is required, and that development of a negative iron gradient is an essential feature of iron release from these cells.EXPERIMENTAL PROCEDURESMaterials—Purified human Cp was obtained from Vital Products (Boynton Beach, FL) and Calbiochem (La Jolla, CA) and homogeneity was verified by an absorbance ratio (610 nm/280 nm) greater than 0.04. Integrity of the protein was established by SDS-PAGE and Coomassie stain; intact, 132-kDa protein was the predominant form, and the primary degradation product was the 115-kDa protein present in human serum (26Ehrenwald E. Chisolm G.M. Fox P.L. J. Clin. Invest. 1994; 93: 1493-1501Crossref PubMed Scopus (238) Google Scholar). Cp mass was determined by nephelometry by the Reference Laboratory at the Cleveland Clinic Foundation, and metal content was determined by inductively coupled plasma mass spectroscopy. Cp contained 6.3 ± 0.3 copper atoms per molecule, and the iron content was below the instrument detection level (i.e. <0.1 atoms per molecule). Human apotransferrin and human holotransferrin were obtained from Sigma, and the mass determined nephelometrically. The iron content of apo- and holotransferrin was 0.060 ± 0.002 and 2.03 ± 0.02 atoms per molecule, respectively; the copper content of the transferrin preparations was negligible (i.e. <0.001 atoms per molecule). RPMI 1640 medium (Invitrogen, Carlsbad, CA) was selected for cell experiments because it does not contain iron, copper, or ascorbic acid, a metal ion reducing agent (27Freshney R.I. Culture of Animal Cells: A Manual of Basic Technique. Wiley-Liss, New York2000Google Scholar). Reduced glutathione, nitrilotriacetic acid (NTA), ferrous ammonium sulfate, ferric chloride, bathophenanthroline disulfonic acid, phorbol myristoyl acetate (PMA), desferrioxamine, and all other reagents were from Sigma. 55FeCl3 (9.1 mm, 20 mCi/mg) was from PerkinElmer Life Sciences. A 55Fe-NTA solution was prepared by incubating 55FeCl3 (9.1 mm) and NTA (45.5 mm) for 1 h. The resulting 55Fe-NTA solution was mixed at a 1:9 molar ratio with unlabeled iron-NTA (prepared similarly) in serum-free medium containing ascorbate (100 μm) so that the final concentration of 55Fe-NTA was 10 μm. Iron solutions were freshly prepared before each experiment.Determination of Iron Binding by Apotransferrin—Iron binding to apotransferrin was measured by a modification of the method of Osaki et al. (5Osaki S. Johnson D.A. Frieden E. J. Biol. Chem. 1966; 241: 2746-2751Abstract Full Text PDF PubMed Google Scholar). Ferrous ammonium sulfate was dissolved in glycine (0.1 mm) buffer at pH 3.0. Apotransferrin and Cp were dissolved in RPMI 1640 medium (without phenol red dye) containing 20 mm Hepes at pH 7.4. Apotransferrin was used at 55 μm, the upper limit of the normal range in adult serum, as described previously (5Osaki S. Johnson D.A. Frieden E. J. Biol. Chem. 1966; 241: 2746-2751Abstract Full Text PDF PubMed Google Scholar, 13Johnson D.A. Osaki S. Frieden E. Clin. Chem. 1967; 13: 142-150Crossref PubMed Scopus (103) Google Scholar). For most experiments, the solutions were brought to 1% O2, 5% CO2, and 94% N2 by bubbling with a mixture of N2, CO2, and 21% O2 + 79% N2 at the appropriate ratio using a 3-channel gas controller and mixer. In some experiments the O2 or CO2 concentration was varied independently. The solutions were added to a plastic cuvette in a final volume of 1 ml. The cuvette was sealed with parafilm and transferrin-bound iron was measured as A 460 nm at 2-s intervals. The data were fitted to a hyperbola by non-linear least square optimization, and the initial rate was determined from the fitted parameters.Determination of 55Fe Release from U937 Cells—U937 cells were grown to a density of about 1 × 106 cells/ml in RPMI 1640 medium containing 10% fetal bovine serum. The cells were centrifuged at 1,000 × g for 5 min, the supernatant was aspirated, and the pellet washed with phosphate-buffered saline (PBS). The cells (7 × 105 cells/well in a 12-well plate) were differentiated toward the macrophage lineage by incubation for 16 h with PMA (15 ng/ml). To load the cells with iron, the media and non-adherent cells were aspirated, and the adherent cells were washed and incubated in serum-free RPMI 1640 medium for 24 h. The cells were incubated with 55Fe-NTA (10 μm) in the same medium containing ascorbate (100 μm) for 3 h (or in some experiments, 24 h) in a hypoxia chamber (Pro-Ox, Reming, Redfield, NY). The chamber was maintained at 37 °C with an atmosphere regulated to contain 1% O2 using an oxygen controller (Pro-Ox model 110, Reming), and with the remainder occupied by a mixture of 5% CO2 and 95% N2. Cell viability after 3 and 24 h of hypoxia and normoxia was determined by trypan blue dye exclusion; no significant differences in viability were observed and cell viability was greater than 95% under all conditions. The medium was aspirated and the cells were washed with ice-cold PBS containing 100 μm EDTA to remove iron non-specifically bound to the cell surface, and twice with ice-cold PBS. 55Fe-NTA uptake was measured in triplicate wells by lysis in 20% Nonidet P-40 followed by liquid scintillation counting. To measure 55Fe release, test reagents (apotransferrin, Cp) were dissolved in serum-free RPMI 1640 medium containing 20 mm Hepes at pH 7.4, and the solutions exhaustively bubbled with gases at the appropriate O2 and CO2 concentrations before addition (0.5 ml) to cells. The cells were placed in the hypoxia chamber for a 15-min iron release interval (unless otherwise noted). The medium was collected, centrifuged to remove suspended cells and cell debris, and the supernatant was collected and counted by liquid scintillation.Preparation of Mouse Peritoneal Macrophages—C57BL/6J mice were injected with 0.5 ml of thioglycollate medium into the peritoneum. After 72 h, the mice were sacrificed and the peritoneum was injected with 10 ml of ice-cold PBS. The peritoneal lavage was removed and the cells were centrifuged (1,000 rpm, 10 min, 4 °C) and resuspended (50,000 cells per well in a 12-well plate) in RPMI 1640 with 10% fetal bovine serum supplemented with l-glutamine and penicillin/streptomycin. Non-adherent cells were removed by rinsing with PBS.Determination of the Intracellular Labile Iron Pool by Quenching of Calcein Fluorescence—The intracellular LIP was determined by an assay based on quenching of calcein fluorescence by bound iron (28Epsztejn S. Kakhlon O. Glickstein H. Breuer W. Cabantchik Z.I. Anal. Biochem. 1997; 248: 31-40Crossref PubMed Scopus (324) Google Scholar). PMA-treated U937 cells were iron-loaded by incubating for 3 or 24 h with unlabeled Fe-NTA (10 μm) and ascorbate (100 μm) in serum-free RPMI 1640 medium. The entire experiment was done in a hypoxic atmosphere consisting of 1% O2, 5% CO2, and 94% N2. The cells were washed with ice-cold PBS containing EDTA (100 μm) and then twice with ice-cold PBS. Fresh RPMI 1640 medium was replaced and the iron-loaded cells were permitted to release iron for 15 min in the presence of various test agents. The cultures were then washed and incubated with calcein (0.5 μm) in serum-free RPMI 1640 medium (without phenol red) for 10 min. The cells were washed and observed in an inverted fluorescent microscope, and the mean cellular fluorescence intensity (488 nm excitation/517 nm emission) in 15 or more cells was quantitated (Image Pro Plus). To determine the LIP size before the iron release period, one set of wells was examined immediately after calcein treatment. To provide a no-iron control, one set of wells for each treatment condition was incubated for 30 min with desferrioxamine (100 μm) to chelate all free and calcein-bound iron. The difference in intensity between a given cell treatment and cells treated with desferrioxamine gives a relative measure of the LIP.Preparation of Apo-Cp by Reductive Copper Chelation and Reconstitution of Holo-Cp—Apo-Cp was prepared by removal of copper by complexing with cyanide under reducing conditions as described by Musci et al. (29Musci G. Di Marco S. Bellenchi G.C. Calabrese L. J. Biol. Chem. 1996; 271: 1972-1978Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Cp was dialyzed at 4 °C against sodium acetate buffer (100 mm, pH 5.9) containing ascorbate (10 mm) under anaerobic conditions achieved by continuous bubbling with N2 gas. Dialysis was continued until the “blue” coppers were reduced as shown by complete sample decolorization. Potassium cyanide (50 mm) was added and the dialysis continued for 5 h. The copper-cyanide complex and excess cyanide were removed by dialysis against sodium acetate (100 mm) containing cysteine (1 mm), and then by an overnight dialysis against buffer containing KCl (150 mm) and MOPS (50 mm, pH 7.0). Holo-Cp was reconstituted from apo-Cp as described (29Musci G. Di Marco S. Bellenchi G.C. Calabrese L. J. Biol. Chem. 1996; 271: 1972-1978Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). A Cu2+-reduced glutathione complex was prepared immediately before use by addition of copper sulfate (0.1 mm) to reduced glutathione (0.3 mm) in phosphate buffer (0.1 m, pH 7.0) containing ascorbate (100 μm). A 7-fold molar excess of the Cu2+-reduced glutathione complex was added to apo-Cp in KCl (150 mm), MgCl2 (5 mm), and MOPS (50 mm, pH 7.0) for 4 h. Unbound copper was removed by overnight dialysis against PBS.RESULTSCp Increases Iron Binding to Apotransferrin under Hypoxic Conditions—Previous studies have shown that Cp stimulated iron binding to apotransferrin under non-physiological, acidic conditions (pH 5.5 to 6.0) (5Osaki S. Johnson D.A. Frieden E. J. Biol. Chem. 1966; 241: 2746-2751Abstract Full Text PDF PubMed Google Scholar, 13Johnson D.A. Osaki S. Frieden E. Clin. Chem. 1967; 13: 142-150Crossref PubMed Scopus (103) Google Scholar). We verified this observation and found that Cp increased the initial rate of binding of ferrous iron to apotransferrin in acetate-buffered solution at pH 6.0 by about 10-fold (Fig. 1A). A similar experiment over a range of pH showed that the stimulation of iron binding to apotransferrin by Cp decreased as the pH increased, and the stimulation at pH 7.4 was less than 20% (Fig. 1B). Essentially identical results were observed when acetate buffer was replaced with RPMI 1640 culture medium containing 20 mm Hepes (not shown). The decrease in the apparent stimulatory activity of Cp was most likely because of the high rate of iron autoxidation at physiological pH.To minimize iron autoxidation, and to replicate tissue conditions, iron binding to apotransferrin was measured at pH 7.4 under conditions of varying degrees of hypoxia. The gas phase included 5% CO2 because maximal iron binding to apotransferrin requires HCO3- anion, which serves as a bridging ligand between the protein and the iron, and completes the coordination requirements of the metal ion (30Aisen P. Aasa R. Malmström B.G. Vänngård T. J. Biol. Chem. 1967; 242: 2484-2490Abstract Full Text PDF PubMed Google Scholar). The rate of iron binding to apotransferrin was essentially linear with respect to O2 concentration in the absence of Cp (Fig. 2A). In contrast, the rate of iron binding to apotransferrin in the presence of Cp increased dramatically as the O2 level was increased from 0 to 1% of the total applied gas. The rate of iron binding did not increase further even at an atmospheric O2 level of 21%. The net stimulation of iron binding to apotransferrin by Cp was about 10–15-fold at low O2 (1% or less), whereas the stimulation was 10–20% under normoxic conditions. To determine whether HCO3- anion influenced the stimulatory activity of Cp, an experiment was done in which the CO2 level was varied from 0 to 5% while the O2 level was maintained at 1%. Inclusion of 20 mm Hepes in the medium maintained the pH to within 0.05 units at all CO2 levels. Cp-independent and -dependent iron binding to apotransferrin both had an absolute requirement for CO2. Cp-dependent iron binding was halfmaximal at about 2% CO2 and near-maximal at about 5% CO2 (Fig. 2B). The dependence of iron binding on Cp concentration was determined under optimal conditions, i.e. 1% O2, 5% CO2, and pH 7.4. The initial rate of iron binding to apotransferrin increased almost linearly as Cp was increased up to its physiological plasma concentration of 300 μg/ml (Fig. 2C).Fig. 2Influence of O2 and CO2 levels on Cp-stimulated binding of iron to apotransferrin. A, ferrous ammonium sulfate (60 μm) was added to apotransferrin (55 μm) in the presence (•) or absence (○) of Cp (120 μg/ml) in phenol red-free, RPMI 1640 medium at pH 7.4. The O2 level in the applied gas was varied from 0 to 21% while maintaining CO2 at a constant 5% level, the remainder was made up with N2. The initial rate of iron binding to apotransferrin was measured as described in the legend to Fig. 1. B, iron binding to apotransferrin was measured as in A except that CO2 levels were varied while maintaining a constant 1% level of O2, the remainder was N2. C, iron loading into apotransferrin was measured as a function of Cp concentration in 1% O2, 5% CO2, and 94% N2 as described in A.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cp Enhances Iron Release from Monocytic Cells—We investigated the influence of Cp on iron release from monocytic cells using the human promonocytic U937 cell line pretreated with PMA to induce differentiation toward a macrophage phenotype. Under hypoxic conditions, and in the presence of apotransferrin, a linear increase in cellular iron release was observed up to 300 μg/ml Cp, the mean plasma concentration in healthy adults (Fig. 3A). At its physiological plasma concentration, Cp stimulated iron release by almost 2.5-fold, and by about 3.5-fold at a concentration of 600 μg/ml, the extreme pathophysiological plasma concentration during a severe acute phase response. The small deviation from linearity when the concentration was increased to 600 μg/ml was probably not because of saturation of any Cp-mediated process (for example, binding to cell surface receptors or apotransferrin), but rather because of the fact that even in the short, 5-min release period used here, about 85% of the pre-loaded 55Fe-NTA was released from the cells (Fig. 3B). A control experiment showed that apotransferrin was required for the stimulation of iron release because Cp by itself had no significant influence on release from cells (Fig. 3B).Fig. 3Stimulation of iron release from U937 monocytic cells by Cp. A, U937 cells (1 × 106 cells per well) in RPMI 1640 medium were incubated with PMA (15 ng/ml) for 16 h to induce differentiation toward the macrophage phenotype. The cells were iron-loaded by incubation with 55Fe-NTA (10 nmol) for 3 h in a hypoxic atmosphere maintained at 1% O2, 5% CO2, and 94% N2. Cellular iron release was measured as a function of Cp concentration (○) in the presence of apotransferrin (55 μm) and in the same hypoxic atmosphere. After a 5-min release interval, the medium was collected (cells centrifuged for 5 min at 2,000 rpm) and 55Fe determined by liquid scintillation counting and expressed as picomole/well. B, total 55Fe taken up by the cells during the uptake phase is indicated (open bar). As controls, iron release was measured in the presence of medium alone or in the presence of Cp (300 μg/ml) in the absence of apotransferrin (striped bars). C, iron release from U937 cells was measured after a 15-min release interval as a function of O2 level balanced by a gas mixture containing 5% CO2 and 95% N2 in the presence of apotransferrin (55 μm), and in the presence (•) or absence (○) of Cp (120 μg/ml). Iron release was measured as in A.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We investigated the effect of O2 on basal and Cp-stimulated iron release from U937 cells. In the absence of Cp, iron release was slow, about 20 pmol/well, increasing gradually to more than 50 pmol/well at atmospheric O2 (Fig. 3C). Cp markedly stimulated iron release, particularly at low O2 levels. At 0.1 or 1% O2, Cp (at 120 μg/ml) increased cellular iron release by about 50 to 75% (Fig. 3C). The rate of iron release in the presence of Cp was maximal at 1% O2, and did not change as O2 was increased to 21%. These results were very nearly parallel to the Cp-mediated stimulation of iron binding to ap" @default.
• W2070771807 created "2016-06-24" @default.
• W2070771807 creator A5005301360 @default.
• W2070771807 creator A5017688815 @default.
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• W2070771807 date "2003-11-01" @default.
• W2070771807 modified "2023-10-07" @default.
• W2070771807 title "Role of Ceruloplasmin in Macrophage Iron Efflux during Hypoxia" @default.
• W2070771807 cites W1489844805 @default.
• W2070771807 cites W1508143080 @default.
• W2070771807 cites W1526578321 @default.
• W2070771807 cites W1565446694 @default.
• W2070771807 cites W1567706026 @default.
• W2070771807 cites W1597924995 @default.
• W2070771807 cites W1602373035 @default.
• W2070771807 cites W1618001767 @default.
• W2070771807 cites W1965106723 @default.
• W2070771807 cites W1967855521 @default.
• W2070771807 cites W1981569438 @default.
• W2070771807 cites W1987352903 @default.
• W2070771807 cites W1987873761 @default.
• W2070771807 cites W1991047577 @default.
• W2070771807 cites W1993916376 @default.
• W2070771807 cites W2001442318 @default.
• W2070771807 cites W2005687534 @default.
• W2070771807 cites W2006210932 @default.
• W2070771807 cites W2007919022 @default.
• W2070771807 cites W2011537701 @default.
• W2070771807 cites W2016736821 @default.
• W2070771807 cites W2029443758 @default.
• W2070771807 cites W2030992731 @default.
• W2070771807 cites W2031265586 @default.
• W2070771807 cites W2032352201 @default.
• W2070771807 cites W2036615604 @default.
• W2070771807 cites W2039953837 @default.
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• W2070771807 cites W2044318447 @default.
• W2070771807 cites W2054895606 @default.
• W2070771807 cites W2060349323 @default.
• W2070771807 cites W2063617352 @default.
• W2070771807 cites W2067672885 @default.
• W2070771807 cites W2076136219 @default.
• W2070771807 cites W2081635255 @default.
• W2070771807 cites W2095571987 @default.
• W2070771807 cites W2104657893 @default.
• W2070771807 cites W2132544275 @default.
• W2070771807 cites W2132863560 @default.
• W2070771807 cites W2142411057 @default.
• W2070771807 cites W2143900678 @default.
• W2070771807 cites W2149472502 @default.
• W2070771807 cites W2159316975 @default.
• W2070771807 cites W2161359965 @default.
• W2070771807 cites W2163235960 @default.
• W2070771807 cites W2164208660 @default.
• W2070771807 cites W2166257949 @default.
• W2070771807 cites W2171525399 @default.
• W2070771807 cites W2292717771 @default.
• W2070771807 cites W2322889308 @default.
• W2070771807 cites W2419121323 @default.
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