59Fe Release Research Articles

A Nitric Oxide Storage and Transport System That Protects Activated Macrophages from Endogenous Nitric Oxide Cytotoxicity

Nitric oxide (NO) is integral to macrophage cytotoxicity against tumors due to its ability to induce iron release from cancer cells. However, the mechanism for how activated macrophages protect themselves from endogenous NO remains unknown. We previously demonstrated by using tumor cells that glutathione S-transferase P1 (GSTP1) sequesters NO as dinitrosyl-dithiol iron complexes (DNICs) and inhibits NO-mediated iron release from cells via the transporter multidrug resistance protein 1 (MRP1/ABCC1). These prior studies also showed that MRP1 and GSTP1 protect tumor cells against NO cytotoxicity, which parallels their roles in defending cancer cells from cytotoxic drugs. Considering this, and because GSTP1 and MRP1 are up-regulated during macrophage activation, this investigation examined whether this NO storage/transport system protects macrophages against endogenous NO cytotoxicity in two well characterized macrophage cell types (J774 and RAW 264.7). MRP1 expression markedly increased upon macrophage activation, and the role of MRP1 in NO-induced 59Fe release was demonstrated by Mrp1 siRNA and the MRP1 inhibitor, MK571, which inhibited NO-mediated iron efflux. Furthermore, Mrp1 silencing increased DNIC accumulation in macrophages, indicating a role for MRP1 in transporting DNICs out of cells. In addition, macrophage 59Fe release was enhanced by silencing Gstp1, suggesting GSTP1 was responsible for DNIC binding/storage. Viability studies demonstrated that GSTP1 and MRP1 protect activated macrophages from NO cytotoxicity. This was confirmed by silencing nuclear factor-erythroid 2-related factor 2 (Nrf2), which decreased MRP1 and GSTP1 expression, concomitant with reduced 59Fe release and macrophage survival. Together, these results demonstrate a mechanism by which macrophages protect themselves against NO cytotoxicity.

Nitric Oxide Storage and Transport in Cells Are Mediated by Glutathione S-Transferase P1-1 and Multidrug Resistance Protein 1 via Dinitrosyl Iron Complexes

Nitrogen monoxide (NO) plays a role in the cytotoxic mechanisms of activated macrophages against tumor cells by inducing iron release. We showed that NO-mediated iron efflux from cells required glutathione (GSH) (Watts, R. N., and Richardson, D. R. (2001) J. Biol. Chem. 276, 4724-4732) and that the GSH-conjugate transporter, multidrug resistance-associated protein 1 (MRP1), mediates this release potentially as a dinitrosyl-dithiol iron complex (DNIC; Watts, R. N., Hawkins, C., Ponka, P., and Richardson, D. R. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 7670-7675). Recently, glutathione S-transferase P1-1 (GST P1-1) was shown to bind DNICs as dinitrosyl-diglutathionyl iron complexes. Considering this and that GSTs and MRP1 form an integrated detoxification unit with chemotherapeutics, we assessed whether these proteins coordinately regulate storage and transport of DNICs as long lived NO intermediates. Cells transfected with GSTP1 (but not GSTA1 or GSTM1) significantly decreased NO-mediated 59Fe release from cells. This NO-mediated 59Fe efflux and the effect of GST P1-1 on preventing this were observed with NO-generating agents and also in cells transfected with inducible nitric oxide synthase. Notably, 59Fe accumulated in cells within GST P1-1-containing fractions, indicating an alteration in intracellular 59Fe distribution. Furthermore, electron paramagnetic resonance studies showed that MCF7-VP cells transfected with GSTP1 contain significantly greater levels of a unique DNIC signal. These investigations indicate that GST P1-1 acts to sequester NO as DNICs, reducing their transport out of the cell by MRP1. Cell proliferation studies demonstrated the importance of the combined effect of GST P1-1 and MRP1 in protecting cells from the cytotoxic effects of NO. Thus, the DNIC storage function of GST P1-1 and ability of MRP1 to efflux DNICs are vital in protection against NO cytotoxicity.

Abstract C40: Understanding the mechanism of action of novel iron chelators on drug-resistant neoplastic cells.

Abstract Introduction: Deprivation of cellular iron (Fe) may represent a novel and effective anti-cancer strategy1. Our laboratory has developed iron chelators with marked and selective anti-tumor activity against a variety of tumor types in vitro and in vivo. One of our leading compounds, Dp44mT, demonstrated potent anti-tumor activity against drug-resistant cell lines1,2 over-expressing P-gp and MRP1. Moreover, these compounds possess high activity at releasing cellular 59Fe3. Aims: To further understand the mechanism of chelator-mediated cellular 59Fe release we investigated:The difference in iron efflux between drug-resistant MCF7-VP (MRP1 hyper-expressing) cells and their wild-type counterparts (MCF7);Whether chelator-mediated Fe release was MRP1 and/or GSH-dependent and;If chelator-mediated cellular Fe release was temperature-dependent. Results: These studies demonstrated that MRP1 over-expressing MCF7-VP cells showed reduced chelator-mediated59Fe release compared to MCF-7 cells (P<0.001). However, this was not due to functional MRP1 and was not GSH-dependent, as neither MRP1 inhibition (using MK571) nor GSH-depletion (using BSO) had any effect on chelator-mediated 59Fe efflux. We found 59Fe efflux was shown to be temperature-dependent, as chelator-mediated 59Fe efflux was reduced at 4°C compared to 37°C. This suggested chelator-mediated Fe release by chelators was an energy dependent process. Interestingly, MCF7-VP cells showed higher 59Fe uptake from 59Fe-transferrin than wild-type cells. This was due to marked transferrin receptor-1 expression in MCF7-VP compared to MCF7 cells. Additionally, the only known Fe efflux pump, ferroportin1, was down-regulated in MCF7-VP cells. This latter observation was intriguing as chelator-mediated Fe efflux was depressed in this cell type. These studies indicate that chelator-mediated 59Fe release is temperature-dependent and MRP-independent. Further studies will examine the role of MRP1 in Fe uptake and metabolism.

Vesicular transport of Fe and interaction with other metal ions in polarized Caco2 Cell monolayers

Two aspects of the mechanisms by which iron is absorbed by the intestine were studied in the Caco2 cell model, using 59Fe(II)-ascorbate. Data showing the importance of vesicular processes and cycling of apotransferrin (apoTf) to uptake and overall transport of Caco2 cell monolayers (or basolateral 59Fe release) were obtained by comparing effects of: a) adding apoTf to the basal chamber; b) adding vesicular transport inhibitors; or c) cooling to 4 degrees C. These showed that apoTf may be involved in as much as half of Fe transfer across the basolateral membrane, and that vesicular processes may also play a role in non-apoTf-dependent Fe transport. Studies were initiated to examine potential interactions of other metal ions with Fe(II) via DMT1. Kinetic data showed a single, saturable process for uptake of Fe(II) that was pH dependent and had a Km of 7 microM. An excess of Mn(II) and Cu(I) over Fe(II) of 200: 1 (microM: microM) in 1 mM ascorbate markedly inhibited Fe uptake. The kinetics were not competitive. Km increased and Vmax decreased. We conclude that vesicular transport, involving endo- and exocytosis at both ends of the enterocyte, is a fundamental aspect of intestinal iron absorption and that DMT1 may function as a transporter not just for divalent but also for monovalent metal ions.

Overexpression of hemochromatosis protein, HFE, alters transferrin recycling process in human hepatoma cells

HFE is a MHC class 1-like protein that is mutated in hereditary hemochromatosis. In order to elucidate the role of HFE protein on cellular iron metabolism, functional studies were carried out in human hepatoma cells (HLF) overexpressing a fusion gene of HFE and green fluorescent protein (GFP). The expression of HFE-GFP was found to be localized on cell membrane and perinuclear compartment by fluorescent microscopy. By co-immunoprecipitation and Western blotting, HFE-GFP protein formed a complex with endogenous transferrin receptor and β 2-microglobulin, suggesting that this fusion protein has the function of HFE reported previously. We then examined the 59Fe uptake and release, and internalization and recycling of 125I-labeled transferrin in order to elucidate the functional roles of HFE in the cell system. In the transfectants, HFE protein decreased the rate of transferrin receptor-dependent iron ( 59Fe) uptake by the cells, but did not change the rate of iron release, indicating that HFE protein decreased the rate of iron influx. Scatchard analysis of transferrin binding to HFE-transfected cells showed an elevation of the dissociation constant from 1.9 to 4.3 nM transferrin, indicating that HFE protein decreased the affinity of transferrin receptor for transferrin, while the number of transferrin receptors decreased from 1.5×10 5/cell to 1.2×10 5/cell. In addition, the rate of transferrin recycling, especially return from endosome to surface, was decreased in the HFE-transfected cells by pulse-chase study with 125I-labeled transferrin. Our results strongly suggest an additional role of HFE on transferrin receptor recycling in addition to the decrease of receptor affinity, resulting in the reduced cellular iron.

Role of ceruloplasmin and ascorbate in cellular iron release

The process of iron (Fe) release from cells plays an important role in health and disease, although the mechanisms responsible remain unclear. In this study we have examined the process of Fe efflux from HepG2 cells, including the possible roles of Cp and ascorbate in this process. Recently, it has been suggested that Cp plays no role in Fe release but can increase Fe uptake by Fe-deficient HepG2 cells (Mukhopadhyay et al. Science 1998;279:714–717). However, this latter study used a nonphysiologically relevant Fe complex (iron 59-NTA) to label cells with 59Fe at a nonphysiologic temperature (25°C) and Cp concentration (<100 μg/mL). Because of these problems, the experiments have been repeated by maintaining physiologic conditions and labeling cells with the physiologic Fe donor diferric Tf. When cells were labeled at 37°C with 59Fe-Tf in the presence of a physiologically relevant Cp concentration (300 μg/mL), this latter protein had no effect on the uptake of 59Fe in control cells or in cells depleted of Fe by using desferrioxamine. In addition, when Fe-replete or Fe-depleted cells were incubated with 59Fe-NTA at 25°C or 37°C, Cp had no effect on 59Fe uptake compared with the control. When the effect of Cp (10–500 μg/mL) on 59Fe release was examined in cells prelabeled with 59Fe-Tf, a concentration-dependent increase in 59Fe efflux was observed, whereas BSA had no effect. However, in contrast to membrane-permeable Fe chelators that caused a marked increase in Fe release, the effect of Cp on Fe efflux was less impressive. To further investigate the mechanism of 59Fe mobilization, we compared 59Fe efflux among HepG2 cells, SK-Mel-28 melanoma cells, and SK-N-MC neuroblastoma cells. These studies demonstrated that 59Fe release was dependent on the incubation time with 59Fe-Tf, the cell line, and the reincubation temperature. Although 59Fe mobilization from cells was markedly temperature dependent, a range of metabolic inhibitors did not affect 59Fe release. Additional experiments showed that physiologic concentrations of ascorbate reduced 59Fe efflux, whereas glutathione had no effect. This study provides further evidence that Cp is involved in Fe mobilization but does not appear to affect Fe uptake from Tf or NTA.

Transferrin Receptor-Dependent and -Independent Iron Transport in Gallium-Resistant Human Lymphoid Leukemic Cells

AbstractRecent studies showed that gallium and iron uptake are decreased in gallium-resistant (R) CCRF-CEM cells; however, the mechanisms involved were not fully elucidated. In the present study, we compared the cellular uptake of 59Fe-transferrin (Tf) and59Fe-pyridoxal isonicotinoyl hydrazone (PIH) to determine whether the decrease in iron uptake by R cells is caused by changes in Tf receptor (TfR)-dependent or TfR-independent iron uptake. We found that both 59Fe-Tf and 59Fe-PIH uptake were decreased in R cells. The uptake of 59Fe-Tf but not59Fe-PIH could be blocked by an anti-TfR monoclonal antibody. After 59Fe-Tf uptake, R cells released greater amounts of 59Fe than gallium-sensitive (S) cells. However, after 59Fe-PIH uptake 59Fe release from S and R cells was similar. 125I-Tf exocytosis was greater in R cells. At confluency, S and R cells expressed equivalent amounts of TfR; however, at 24 and 48 hours in culture, TfR expression was lower in R cells. Our study suggests that the decrease in Tf-Fe uptake by R cells is caused by a combination of enhanced iron efflux from cells and decreased TfR-mediated iron transport into cells. Furthermore, because TfR-dependent and -independent iron uptake is decreased in R cells, both uptake systems may be controlled at some level by similar regulatory signal(s).

Transferrin Receptor-Dependent and -Independent Iron Transport in Gallium-Resistant Human Lymphoid Leukemic Cells

Recent studies showed that gallium and iron uptake are decreased in gallium-resistant (R) CCRF-CEM cells; however, the mechanisms involved were not fully elucidated. In the present study, we compared the cellular uptake of 59Fe-transferrin (Tf) and59Fe-pyridoxal isonicotinoyl hydrazone (PIH) to determine whether the decrease in iron uptake by R cells is caused by changes in Tf receptor (TfR)-dependent or TfR-independent iron uptake. We found that both 59Fe-Tf and 59Fe-PIH uptake were decreased in R cells. The uptake of 59Fe-Tf but not59Fe-PIH could be blocked by an anti-TfR monoclonal antibody. After 59Fe-Tf uptake, R cells released greater amounts of 59Fe than gallium-sensitive (S) cells. However, after 59Fe-PIH uptake 59Fe release from S and R cells was similar. 125I-Tf exocytosis was greater in R cells. At confluency, S and R cells expressed equivalent amounts of TfR; however, at 24 and 48 hours in culture, TfR expression was lower in R cells. Our study suggests that the decrease in Tf-Fe uptake by R cells is caused by a combination of enhanced iron efflux from cells and decreased TfR-mediated iron transport into cells. Furthermore, because TfR-dependent and -independent iron uptake is decreased in R cells, both uptake systems may be controlled at some level by similar regulatory signal(s).

Mobilization of iron from neoplastic cells by some iron chelators is an energy-dependent process

Iron (Fe) chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class may be useful agents to treat Fe overload disease and also cancer. These ligands possess high activity at mobilizing 59Fe from neoplastic cells, and the present study has been designed to examine whether their marked activity may be related to an energy-dependent transport process across the cell membrane. Initial experiments examined the release of 59Fe from SK-N-MC neuroblastoma (NB) cells prelabelled for 3 h at 37°C with 59Fe-transferrin (1.25 μM) and then reincubated in the presence and absence of the chelators for 3 h at 4°C or 37°C. Prelabelled cells released 4–5% of total cellular 59Fe when reincubated in minimum essential medium at 4°C or 37°C. When the chelators desferrioxamine (DFO; 0.1 mM) or PIH (0.1 mM) were reincubated with labelled cells at 4°C, they mobilized only 4–5% of cellular 59Fe, whereas as 37°C, these ligands mobilized 21% and 48% of cell 59Fe, respectively. The lipophilic PIH analogue, 311 (2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone; 0.1 mM), which exhibits high anti-proliferative activity, released 10% and 53% of cellular 59Fe when reincubated with prelabelled cells at 4°C and 37°C, respectively. Almost identical results were obtained using the SK-Mel-28 melanoma cell line. These data suggest that perhaps temperature-dependent mechanisms are essential for 59Fe mobilization from these cells. Interestingly, the metabolic inhibitors, 2,4-dinitrophenol, oligomycin, rotenone, and sodium azide, markedly decreased 59Fe mobilization mediated by PIH, but had either no effect or much less effect on 59Fe release by 311. Considering that an ATP-dependent process was involved in 59Fe release by PIH, further studies examined 4 widely used inhibitors of the multi-drug efflux pump P-glycoprotein (P-gp). All of these inhibitors, namely, verapamil (Ver), cyclosporin A (CsA), reserpine (Res) and quinine (Qui), decreased 59Fe mobilization by PIH but had little or no effect on 59Fe release mediated by analogue 311. Further, both CsA and Ver increased the proportion of ethanol-soluble 59Fe within cells in the presence of PIH, suggesting inhibited transport of the 59Fe complex from the cell. However, when PIH-mediated 59Fe release was compared between a well characterized Chinese hamster ovary cell line (CH RB30) expressing high levels of P-gp and the relevant control cell line (AuxB1), no appreciable difference in the kinetics of 59Fe release were found. In contrast, it was intriguing that the CH RB30 cells released more 59Fe into control medium (i.e., without PIH) than the AuxB1 control line (16.7% compared to 5.9%, respectively). In summary, the results suggest that a temperature- and energy-dependent process was involved in the efflux of the PIH- 59Fe complex from the cells. In contrast, 59Fe release mediated by 311 was temperature-dependent but not energy-dependent, and could occur by simple diffusion or passive transport. Further studies investigating the membrane transport of Fe chelators may be useful in designing regimes that potentiate their anti-neoplastic effects.© 1997 Elsevier Science B.V. All rights reserved.

The Potential of Iron Chelators of the Pyridoxal Isonicotinoyl Hydrazone Class as Effective Antiproliferative Agents II: The Mechanism of Action of Ligands Derived From Salicylaldehyde Benzoyl Hydrazone and 2-Hydroxy-1-Naphthylaldehyde Benzoyl Hydrazone

AbstractWe have recently screened 36 analogues of the lipophilic iron (Fe) chelator, pyridoxal isonicotinoyl hydrazone (PIH), for their antiproliferative effect (Richardson et al, Blood 86:4295, 1995). Of these compounds, 1 chelator derived from salicylaldehyde benzoyl hydrazone (206) and 4 ligands derived from 2-hydroxy-1-naphthylaldehyde benzoyl hydrazone (308, 309, 311, and 315) showed pronounced antiproliferative activity, being far more effective than desferrioxamine (DFO). The present study was designed to investigate in detail the mechanism of action of these PIH analogues in a variety of neoplastic cell lines. This investigation showed that the analogues were far more active than DFO at inhibiting cellular proliferation and 3H-thymidine, 3H-leucine, and 3H-uridine incorporation. Additional experiments showed that, in contrast to DFO, the 5 analogues were potent at preventing 59Fe uptake from transferrin (Tf ) and increasing 59Fe release from cells at concentrations as low as 10 μmol/L. Examination of the distribution of 59Fe in neoplastic cells using native polyacrylamide gel electrophoresis (PAGE)/59Fe-autoradiography showed that most of the 59Fe taken up from Tf was incorporated into ferritin, although 3 other previously unrecognized components (bands A, B, and C) were also identified. Band C comigrated with 59Fe-citrate and was chelated on incubation of neuroblastoma cells with DFO, PIH, or the PIH analogues, with this compartment being the main intracellular target of these ligands. Further work showed that the effects of the chelators at inducing characteristics consistent with apoptosis or necrosis were cell line-specific, and while DFO increased the percentage of cells in the Go/G1 phases in all cell types, the effect of analogue 311 on the cell cycle was variable depending on the cell line. This study provides further evidence for the potential use of these Fe chelators as anticancer agents.

Intermediate steps in cellular iron uptake from transferrin. II. A cytoplasmic pool of iron is released from cultured cells via temperature-dependent mechanical wounding.

A previous study described a cytoplasmic, transferrin (Tf)-free, iron (Fe) pool that was detected only when cells were mechanically detached from the culture substratum at 4 degrees C, after initial incubation with 59Fe-125I-Tf at 37 degrees C (Richardson and Baker, 1992a). The release of this internalized 59Fe could be markedly reduced if the cells were treated with proteases or incubated at 37 degrees C prior to detachment. The present study was designed to characterize this Fe pool and understand the mechanism of its release. The results show that cellular 59Fe release increased linearly as a function of preincubation time with 59Fe-Tf subsequent to mechanical detachment at 4 degrees C using a spatula. These data suggest that the 59Fe release was largely composed of end product(s) and was not an "intermediate Fe pool." When the Fe(II) chelator, dipyridyl (DP), was incubated with 59Fe-Tf and the cells, it prevented the accumulation of 59Fe that was released following mechanical detachment at 4 degrees C. Other chelators had much less effect on the proportion of 59Fe released. Examination of the 59Fe released showed that after a 4-h preincubation with 59Fe-Tf, approximately 50% of the 59Fe was present in ferritin. These data indicate that mechanical detachment of cells at 4 degrees C resulted in membrane disruptions that allow the release of high M(r), molecules. Moreover, electron microscopy studies showed that detachment of cells from the substratum at 4 degrees C resulted in pronounced membrane damage. In contrast, when cells were detached at 37 degrees C, or at 4 degrees C after treatment with pronase, membrane damage was minimal or not apparent. These results may imply that temperature-dependent processes prevent the release of intracellular contents on membrane wounding, or alternatively, prevent wounding at 37 degrees C. The evidence also indicates that caution is required when interpreting data from experiments where cells have been mechanically detached at 4 degrees C.

Nitrogen monoxide decreases iron uptake from transferrin but does not mobilise iron from prelabelled neoplastic cells

The effect of congeners of nitrogen monoxide (NO) on iron (Fe) uptake from 59Fe 125I-transferrin (Tf) and release of 59Fe from prelabelled cells have been investigated in SK-MEL-28 human melanoma cells, human K562 cells and mouse MDW-4 cells. These studies have been initiated as it has been suggested that the tumoricidal effects of NO may be mediated by its acting to release Fe from cells (Hibbs et al., 1984 Biochem. Biophys. Res. Commun. 123, 716–723; Hibbs et al., 1988 Biochem. Biophys. Res. Commun. 157, 87–94). The nitrosonium ion (NO +) generator, sodium nitroprusside (SNP), decreased 59Fe uptake by melanoma cells to 57% of the control without decreasing 125ITf uptake after a 4-h incubationwith 59Fe 125-Tf (1.25 μM). Longer incubations up to 24 h decreased 59Fe uptake and also 125ITf uptake. Two breakdown products of SNP, ferricyanide and cyanide, had no effect on 59Fe uptake. In addition, photolysis of the SNP solution prevented the inhibition of 59Fe uptake, suggesting that NO was the active agent. Two nitric oxide (NO · ) producing agents, 3-morpholinosydnonimine (SIN), and S-nitroso- N-acetylpenicillamine (SNAP), also decreased 59Fe uptake from 59Fe 125ITf. Superoxide dismutase increased efficacy of SIN, and the NO-scavenger, oxyhaemoglobin, prevented the inhibition of 59Fe uptake mediated by SNAP, again suggesting that NO was the active agent. Furthermore, dialysis studies demonstrated that none of the NO-generating agents could remove 59Fe from 59Fe 125ITf, suggesting that the decrease in cellular Fe uptake observed was not due to NO releasing Fe from the Fe-binding sites of Tf. Despite the ability of NO-producing agents at inhibiting 59Fe uptake by cells, they could not remove significant amounts of 59Fe from melanoma cells prelabelled with either 59Fe-citrate or 59Fe 125 ITf. Similar data were obtained using K562 and MDW-4 cells. Interestingly, the NO + generating agent, SNP, had no effect on [ 3H]thymidine uptake. However, when SNP was converted to an NO · generator by the addition of 1 mM ascorbate, its effect was similar to the NO · generator, SNAP, markedly reducing [ 3H]thymidine incorporation to 33% of the control value. The addition of unlabelled diferric Tf (0.625 μM) to SNAp ameliorated its inhibitory effect on cellular [ 3H]thymidine uptake, suggesting that the interaction of NO · with Fe was of importance in the inhibition observed. The results are discussed in the context of the cytostatic potential of NO via its binding to Fe.

Effects of chelators on iron uptake and release by the brain in the rat

The iron chelators desferrioxamine (DFO), pyridoxal isonicotinoyl hydrazone (PIH), 2,2'-bipyridine, diethylenetriamine penta-acetic acid (DTPA) and 1,2 dimethyl-3-hydroxy pyrid-4-one (CP20) were analysed for their ability to change 59Fe uptake and release from the brain of 15- and 63-day rats either during or after intravenous injection of 59Fe-125I-transferrin. DTPA was the only chelator unable to significantly reduce iron uptake into the brain of 15-day rats. This indicates that iron is not released from transferrin at the luminal surface of brain capillary endothelial cells. CP20 was able to reduce iron uptake in the brain by 85% compared to 28% with DFO. Only CP20 was able to significantly reduce brain iron uptake in 63 day rats. Once 59Fe had entered the brain no chelator used was able to mediate its release. All of the chelators except CP20 had similar effects on femur iron uptake as they did on brain uptake, suggesting similar iron uptake mechanisms. It is concluded that during the passage of transferrin-bound iron into the brain the iron is released from transferrin within endothelial cells after endocytosis of transferrin.

59Fe release from alveolar macrophages by macromolecular forms of chelating agents

Pro-drug forms of DTPA (diethylenetriaminepentaacetic acid), prepared by immobilising DTPA on various polysaccharides, polyvinylalcohol and silica, have proved to be of little value for mobilising intracellular 59Fe from alveolar macrophages radiolabelled by an in vivo process.

The effect of gamma-irradiation on the adherent capacity and iron metabolism of alveolar macrophages in mice and rats.

The effect of external gamma-irradiation on the survival of alveolar macrophages (AM) and on the extracellular release of 59Fe from AM-ingested [59Fe]iron hydroxide colloid was investigated in vitro using cells from C3H mice and Wistar rats. 59Fe release from mouse AM was enhanced by irradiation in a dose-dependent fashion, but irradiation up to 112 Gy did not affect the release from rat AM. Cell survival of AM, measured by direct counting of cell nuclei of adherent AM or vital staining with crystal violet, decreased dose dependently in mice and rats, but mouse AM were more radiosensitive than rat AM.

The Effect of g-Irradiation on the Adherent Capacity and Iron Metabolism of Alveolar Macrophages in Mice and Rats

The effect of external gamma-irradiation on the survival of alveolar macrophages (AM) and on the extracellular release of 59Fe from AM-ingested [59Fe]iron hydroxide colloid was investigated in vitro using cells from C3H mice and Wistar rats. 59Fe release from mouse AM was enhanced by irradiation in a dose-dependent fashion, but irradiation up to 112 Gy did not affect the release from rat AM. Cell survival of AM, measured by direct counting of cell nuclei of adherent AM or vital staining with crystal violet, decreased dose dependently in mice and rats, but mouse AM were more radiosensitive than rat AM.

Difference between C3H Mice and Wistar Rats in the Effect of External .GAMMA.-Irradiation on 59Fe Release from Alveolar Macrophage-Ingested 59Fe-Iron Hydroxide Colloid.

The effect of external gamma-irradiation on 59Fe release from alveolar macrophages (AMs) that had ingested 59Fe-iron hydroxide colloid was determined in C3H mice and Wistar rats with an in vitro assay system. The release of 59Fe from AM was increased by irradiation in mice but not in rats. A significant increase in 59Fe release was seen in mouse AM cultured for 72 hrs after irradiation at a dose of more than 25 Gy; but, graded doses of gamma-irradiation up to 100 Gy had no effect on 59Fe release from rat AMs. This difference between mice and rats may be attributable to the magnitude of death of or damage to AM because the survival of AMs assessed by vital staining with crystal violet decreased much more in mice than in rats.

The effect of external .GAMMA.-irradiation on 59Fe release in vitro from alveolar macrophages previously having ingested 59Fe-iron hydroxide colloid.

The effect of external gamma-irradiation on the digestive function of rat alveolar macrophages (AM) was studied by using the in vitro assay system, where the 59Fe release from AM engulfing 59Fe-iron hydroxide colloid was measured as an indicator of their digestive capability. Graded doses of gamma-irradiation up to 100 Gy had no effect on the extracellular release of 59Fe in AM cultured at 4 and 37 degrees C for 8-72 hrs postirradiation. When 10 mM Ca-DTPA was added to the culture medium, the 59Fe release was slightly depressed by irradiation at a dose of 30 Gy. It is apparent from these results that gamma-irradiation had no effect on the 59Fe transfer between cell and medium, and little effect on the catabolism of 59Fe-colloid in rat AM.

Factors Involved in the Regulation of Iron Transport through Reticuloendothelial Cells

The effects of various maneuvers on the handling of 59Fe-labeled heat-damaged red cells (59Fe HDRC) by the reticuloendothelial system were studied in rats. Raising the saturation of transferrin with oral carbonyl iron had little effect on splenic release of 59Fe but markedly inhibited hepatic release. Splenic 59Fe release was, however, inhibited by the prior administration of unlabeled HDRC or by the combination of carbonyl iron and unlabeled HDRC. When carbonyl iron was administered with unlabeled free hemoglobin, the pattern of 59Fe distribution was the same as that observed when carbonyl iron was given alone. 59Fe ferritin was identified in the serum after the administration of 59Fe HDRC but the size of the fraction was not affected by raising the saturation of transferrin. Sizing column analyses of tissue extracts from the spleen at various times after the administration of 59Fe HDRC revealed a progressive shift from hemoglobin to ferritin, with only small amounts present in a small molecular weight fraction. The small molecular weight fraction was greater in hepatic extracts, with the difference being marked in animals that had received prior carbonyl iron. The increased hepatic retention of 59Fe associated with a raised saturation of transferrin was reduced by a hydrophobic ferrous chelator (2,2'-bipyridine), a hydrophilic ferric chelator (desferrioxamine), and an extracellular hydrophilic ferric chelator (diethylene-triaminepentacetic acid). Transmembrane iron transport did not seem to be a rate-limiting factor in iron release, since no differences in 59Fe membrane fractions were noted in the different experimental settings. These findings are consistent with a model in which RE cells release iron from catabolized red cells at a relatively constant rate. When the saturation of transferrin is raised, a significant proportion of the iron is transported from the spleen to the liver either in small molecular weight complexes or in ferritin. Although a saturated transferrin had no effect on the release of iron from reticuloendothelial cells, prior loading with HDRC conditions them to release less iron.

L-Arginine independent macrophage tumor cytotoxicity

We have investigated the role of L-arginine in macrophage tumor cytotoxicity in coculture. L929, EMT-6, MCA-26, and P815 targets were all susceptible to cytolysis by activated macrophages when cocultured in medium containing L-arginine. When cocultured in arginine-free medium, these targets displayed comparable or even higher levels of lysis. L1210 targets were lytically resistant under either condition. However, 59Fe release from this target did reflect strong dependence on the presence of arginine. The structural analogue, N G-monomethyl-L-arginine, was an effective inhibitor of iron-release from L1210 targets cocultured with activated macrophages, whereas it had minimal inhibitory effects on release of 51Cr from cocultured L929 cells. These results suggest that the L-arginine requiring cytotoxic pathway of activated macrophage is independent of major effector mechanisms involved in tumor cell lysis.