Role of Iron in Nramp1-Mediated Inhibition of Mycobacterial Growth

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BRUCE S. ZWILLING,1,2* DONALD E. KUHN,1 LISA WIKOFF,1 DAVID BROWN,1. AND WILLIAM LAFUSE2. Departments of Microbiology1 and Medical ...
INFECTION AND IMMUNITY, Mar. 1999, p. 1386–1392 0019-9567/99/$04.0010 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 67, No. 3

Role of Iron in Nramp1-Mediated Inhibition of Mycobacterial Growth BRUCE S. ZWILLING,1,2* DONALD E. KUHN,1 LISA WIKOFF,1 DAVID BROWN,1 2 AND WILLIAM LAFUSE Departments of Microbiology1 and Medical Microbiology and Immunology,2 The Ohio State University, Columbus, Ohio 43210 Received 8 October 1998/Accepted 1 December 1998

Innate resistance to mycobacterial growth is mediated by a gene, Nramp1. We have previously reported that Nramp1 mRNA from macrophages of Mycobacterium bovis BCG-resistant (Bcgr) mice is more stable than Nramp1 mRNA from macrophages of BCG-susceptible (Bcgs) mice. Based on these observations and on reports that show that the closely related Nramp2 gene is a metal ion transporter, we evaluated the effect of iron on the growth of Mycobacterium avium within macrophages as well as on the stability of Nramp1 mRNA. The addition of iron to macrophages from Bcgs mice resulted in a stimulation of mycobacterial growth. In contrast, iron increased the capacity of macrophages from Bcgr mice to control the growth of M. avium. When we treated recombinant gamma interferon (IFN-g)-activated macrophages with iron, we found that iron abrogated the growth inhibitory effect of IFN-g-activated macrophages from Bcgs mice but that it did not affect the capacity of macrophages from Bcgr mice to control microbial growth. A more detailed examination of the effect of iron on microbial growth showed that the addition of small quantities of iron to resident macrophages from Bcgr mice stimulated antimicrobial activity within a very narrow dose range. The effect of iron on the growth inhibitory activity of macrophages from Bcgr mice was abrogated by the addition of catalase or mannitol to the culture medium. These results are consistent with an Fe(II)-mediated stimulation of the Fenton/Haber-Weiss reaction and hydroxyl radical-mediated inhibition of mycobacterial growth. The Bcg gene confers resistance to mycobacterial growth during the early nonimmune phase of infection (7, 19, 22, 23). Typing of recombinant inbred mouse strains for resistance and susceptibility to Mycobacterium bovis (strain BCG) as well as Mycobacterium avium and other mycobacterial species, combined with linkage analyses and dissection of a 30-centimorgan segment on murine chromosome 1, led to the cloning of the cDNA for the Bcg gene, designated Nramp1 (natural resistance-associated macrophage protein) (8, 33, 46). Sequence analysis of Nramp1 revealed a 1.4-kb open reading frame encoding a 484-amino-acid transmembrane protein with structural features common to eukaryotic transporters (17, 26, 43, 45). Susceptibility to mycobacterial growth in macrophages is associated with a glycine to aspartic acid substitution at position 169 within the fourth transmembrane domain of Nramp1 (5, 6, 21). However, the mechanism of Nramp1-mediated resistance to mycobacterial infection and the functional defect in Nramp1 mutants are still unknown. Previous studies in our laboratory have shown that a variety of mRNA species, including Nramp1, from macrophages of BCG-susceptible (Bcgs) mice are less stable than mRNAs from macrophages of BCG-resistant (Bcgr) mice (11, 12). The reduced stability of mRNA from macrophages of mice expressing the Nramp1Asp169-susceptible allele was also observed in mRNA species induced by recombinant gamma interferon (rIFN-g). mRNA stability is controlled by differences in sequences that facilitate protein binding (29, 40, 42), and this phenomenon promotes protection of mRNA from nucleases. Furthermore, protein binding to some mRNAs is controlled by Fe levels (29, 31, 42). For example, the transferrin receptor

mRNA is stabilized by the binding of a cytoplasmic aconitase (iron response element binding protein) to stem loop structures in the 39 untranslated region of the transferrin receptor mRNA when intracellular iron is low. An increase in intracellular iron results in the binding of iron to the aconitase, releasing it from the mRNA, and the subsequent degradation of transferrin receptor mRNA. Additionally, iron serves as an important trace element for the generation of reactive intermediates of oxygen and nitrogen (15, 30, 31, 48, 50) and is an important nutrient for the growth of mycobacteria (20, 51). Since iron affects mRNA stability and mycobacterial growth, we evaluated the effect of iron on the capacity of macrophages to inhibit the growth of M. avium and on the stability of Nramp1 mRNA. We found that iron stimulated the capacity of macrophages from mice expressing the Nramp1Gly169resistant allele to limit the growth of M. avium. In contrast, iron stimulated mycobacterial growth in macrophages from mice expressing Nramp1Asp169. The antimycobacterial activity of IFN-g-activated macrophages from mice expressing Nramp1Asp169 was abrogated by iron, but iron did not affect the capacity of IFN-g-activated macrophages from mice expressing Nramp1Gly169 to control the growth of the mycobacteria. The effect of Fe on the growth inhibitory effects of macrophages from these Bcgr mice occurred over a very narrow dose range and was abrogated by the addition of hydroxyl radical scavengers. These data are consistent with a model in which Nramp1 acts to decrease cellular iron levels by transporting iron into phagosomes, where it serves as a catalyst for the Fenton/ Haber-Weiss reaction to generate hydroxyl radicals and at the same time stabilizes mRNA.

* Corresponding author. Mailing address: Department of Microbiology, College of Biological Sciences, 484 West 12th Ave., Columbus, Ohio 43210. Phone: (614) 292-3310. Fax: (614) 292-8120. E-mail: [email protected].

Mice. Male BALB/c Bcgs (Nramp1Asp169) mice were obtained from the Charles River Laboratory at 6 weeks of age. The animals were housed in groups of five in microisolation cages and given food and water ad libitum. Male BALB/c

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VOL. 67, 1999 congenic C.D2Idhb-Ityr-Pep-3b mice, which are Bcgr (Nramp1Gly169), were initially provided by Michael Potter (NCI) and bred in our facilities (11). Reagents. M. avium (ATCC 35713) was obtained from the American Type Culture Collection (Manassas, Va.) and cultured in our laboratory as previously described (11). rIFN-g was obtained from Gibco BRL (Grand Island, N.Y.). Actinomycin D, desferrioxamine, ferric ammonium sulfate, NG-monomethyl-Larginine (MMLA), superoxide dismutase (SOD), mannitol, catalase, and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, Mo.). The cDNA probe for Nramp1 was produced by reverse transcriptase PCR as previously described by us (12). The b-actin cDNA probe was obtained by screening a macrophage cDNA library with actin-specific oligonucleotides. [32P]dCTP (specific activity, 3,000 Ci/mmol) was purchased from Amersham (Arlington Heights, Ill.). 55Fe-labeled ferric chloride (specific activity, 17 mCi/mg) was obtained from NEN (Boston, Mass.). Isolation of macrophages. Resident peritoneal macrophages or splenic macrophages from both Bcgr and Bcgs mice were enriched by overnight adherence to plastic culture dishes (Falcon, Franklin Lakes, N.J.) by using Iscove’s modified Dulbecco’s medium (IMDM) (Gibco/BRL, Gaithersburg, Md.) supplemented with 20% defined fetal bovine serum (FBS) (HyClone, Logan, Utah) containing less than 0.03 endotoxin unit (EU) of endotoxin/ml. Nonadherent cells were removed by gentle washing with Hanks balanced salt solution (Gibco/BRL). The splenic macrophages were removed by using a cell scraper as previously described by us (11) and cultured again in 35-mm-diameter culture dishes, at a concentration of 5 3 106 macrophages per dish, in IMDM containing 20% FBS. Following the second incubation for 16 h at 37°C, the purified splenic macrophages (as determined by both differential and nonspecific esterase staining) were then washed with Hanks balanced salt solution, and the medium was replenished with IMDM containing 100 U of rIFN-g/ml and subsequently treated as described. Macrophage cultures were incubated at 37°C in 5% CO2 for the times indicated for each experiment. Peritoneal macrophages were obtained by lavage and purified by culturing directly in 96-well plates. Macrophage-mediated mycobacterial growth inhibition. The growth inhibitory capacity of the macrophage populations was assessed as previously described by us (11). Briefly, macrophages were added to 96-well microtiter plates so that a concentration of 2 3 105 macrophages per well was attained. The purified macrophages (.90% macrophages as determined by nonspecific esterase staining) were then infected with 8 3 105 CFU of M. avium suspended in IMDM without serum and antibiotics and incubated overnight to allow for phagocytosis. The cultures were then washed to remove unphagocytized bacteria and incubated for 5 days to allow the intracellular growth of the bacteria. The macrophages were then lysed, and bacteria were pulsed overnight by incubating in media containing a mixture (1:1) of 7H9 (Difco) and IMDM with [3H]uracil (5 mCi/ml) (Amersham, Chicago, Ill.) (specific activity, 40 to 60 Ci/mmol) and 0.2% saponin. The bacteria were harvested onto glass fiber filter strips with a PHD cell harvester (Cambridge Technology, Inc., Watertown, Mass.). Radioactivity incorporated by the bacteria was quantitated by liquid scintillation spectrometry. To induce increased antimycobacterial activity of the macrophages, the cells were treated with 100 U of rIFN-g/ml (Gibco) in IMDM for 24 h prior to infection with M. avium. Iron was added to the macrophage cultures following removal of unphagocytized bacteria. In other experiments, resident macrophages were treated with iron but were not treated with rIFN-g. Isolation of RNA and Northern blot analysis. Splenic macrophages from Bcgr and Bcgs mice were stimulated with 100 U of rIFN-g/ml in the presence or absence of 100 mM desferrioxamine or 50 mM ferric ammonium sulfate. After 20 h the macrophage cultures were treated with 20 mg of actinomycin D or medium over a 48-h course prior to extraction and isolation of total RNA. After treatment, the macrophage monolayers were washed with ice-cold phosphatebuffered saline (PBS) (Gibco/BRL) and incubated on ice for 20 min. The PBS was removed and replaced with lysing buffer containing 8 M guanidine hydrochloride, 0.3 M sodium acetate, and 10% sarcosyl. Total RNA was extracted by the procedure as modified by Evans and Kamdar (16). Equal amounts of RNA isolated from each treatment were size fractionated by gel electrophoresis (1.5% formaldehyde agarose), and RNAs were transferred to a Hybond-N1 membrane (Amersham) by capillary blotting. A separate lane containing a 0.24- to 9.5-kb RNA marker ladder (Gibco/BRL) was stained following electrophoresis with ethidium bromide and used to determine RNA size. Northern hybridization was carried out by using the protocol as previously described by us (12). Gel purified insert cDNAs were 32P labeled by random priming. Blots initially hybridized to Nramp1 cDNA were stripped with 0.1% sodium dodecyl sulfate and rehybridized to a 32P-labeled b-actin cDNA probe. Autoradiographs were quantified by using the UVP ImageStore 5000 gel documentation program (San Gabriel, Calif.) and NIH Image version 1.58. Iron transport into M. avium-containing phagosomes. In order to determine if iron was differentially taken up by phagosomes from macrophages expressing wild-type or mutant Nramp1, we used the RAW264.7 macrophage cell line that had been stably transfected with Nramp1Gly169 (resistant) or Nramp1Asp169 (susceptible) alleles. The cell lines were kindly provided by Jenefer Blackwell (5). The cells were labeled with 55Fe by first growing the cells for 24 h in IMDM without FBS and then growing them in complete medium supplemented with 5 mM 55Fe-labeled iron citrate for 24 h to label endogenous Fe pools. The amounts of 55Fe taken up by the cells did not differ. Mycobacteria were added at a bacteria to macrophage ratio of 10/1 for 1 h at 37°C. The cells were then washed two times

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FIG. 1. Effect of iron on the growth of M. avium in macrophages from Bcgr and Bcgs mice. Resident peritoneal macrophages were infected with M. avium and then treated with ferric ammonium sulfate. After 5 days of culture, the macrophages were lysed and the bacteria were labeled with [3H]uracil. The radioactivity incorporated by the bacteria is an indication of the number of metabolically active bacteria remaining in the culture. The data are expressed as the percentages of inhibition of growth compared to growth of cultures of macrophages not treated with iron. The amount of radioactivity (mean 6 standard deviation) incorporated by M. avium from Bcgr macrophages was 18,627 6 2,899 cpm, while that incorporated by M. avium from macrophages from Bcgs mice was 31,095 6 2,602 cpm. There were no differences noted in the numbers of bacteria phagocytized by the macrophages at the beginning of the experiment. The effect of iron on the growth of the bacteria in macrophages from Bcgs mice was significant as determined by analysis of variance (ANOVA). Similarly, the effect of iron on the inhibition of mycobacterial growth in macrophages from Bcgr mice was also significant as determined by ANOVA. Thus, Fe stimulated the growth of the mycobacteria in macrophages from Bcgs mice. In contrast, at low levels iron suppressed the growth of the mycobacteria in macrophages from Bcgr mice.

with PBS to remove unphagocytized bacteria and then incubated in complete IMDM at 37°C for an additional hour. The cells were removed from monolayers by using a cell scraper, and the phagosomes were isolated by a modification of the method described by Sturgill-Koszycki et al. (25, 44). Briefly, the cells were suspended in lysis buffer containing 250 mM sucrose, 20 mM HEPES (pH 7.0), 0.5 M EGTA, and 0.1% gelatin. Lysis of the cells was accomplished by repeated passage of the cells through a 21-gauge needle until at least 95% of the cells were lysed. The resulting solution was diluted threefold with PBS and centrifuged at 200 3 g for 5 min to remove unbroken cells and nuclei. The supernatant was filtered through a 5-mm-pore-size Nucleopore filter (diameter, 25 mm; Corning, Corning, N.Y.), and the filter was rinsed with an equal volume of PBS. The filtrate was layered on top of a 50 to 12% sucrose step gradient and centrifuged at 800 3 g for 45 min. The fraction at the 50 to 12% sucrose interface was removed and diluted threefold with PBS. This fraction was then layered onto a 15% Ficoll (low-molecular-weight) cushion, and the sample was centrifuged at 1,300 3 g for 45 min. The M. avium-containing phagosomes were removed and resuspended in IMDM, and the amount of radioactivity incorporated into the phagosomes was determined. Equal amounts of phagosomes were isolated from the Nramp1Gly169- or Nramp1Asp169-transfected cell lines. The data are expressed as picomoles of iron per milligram of protein based on the amount of 55Fe added. This value includes the bacteria within the phagosome. However, in preliminary experiments we found that the amounts of bacteria taken up by the cell lines did not differ under the conditions we used to infect the cells.

RESULTS Iron stimulates the antimycobacterial activity of resident macrophages. The results of experiments performed to determine the effect of iron on mycobacterial growth are presented in Fig. 1. The addition of Fe to cultures of infected macrophages from Bcgr mice resulted in an inhibition of mycobacterial growth. The addition of 0.05 mM iron to cultures of macrophages from Bcgr mice resulted in the greatest level of growth inhibition. In contrast to the inhibitory effect of iron on mycobacterial growth in macrophages from Bcgr mice, increasing concentrations of iron resulted in increased growth of M.

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FIG. 2. The addition of iron to cultures of macrophages infected with M. avium results in the inhibition of mycobacterial growth. For comparison with macrophages treated with rIFN-g, resident peritoneal macrophages were treated with 100 U of rIFN-g/ml overnight prior to infection with M. avium. Other cultures were infected with M. avium and treated with iron but not treated with rIFN-g. After 5 days, the macrophages were lysed and the bacteria were labeled with [3H]uracil. The amount of radioactivity is an indication of the amount of metabolically active bacteria in the cultures. The data are expressed as the percentages of inhibition of bacterial growth compared to growth of cultures not treated with rIFN-g or iron. The effect of rIFN-g treatment was significant, as was the effect of iron, on mycobacterial growth as determined by analysis of variance.

avium in macrophages from Bcgs mice. The stimulatory effect of iron was observed at concentrations of Fe of 5 mM or higher. Results shown in Fig. 2 indicate that iron stimulated the antimycobacterial activity of the macrophages from Bcgr mice within a very narrow dose range. Maximal inhibition was obtained at 0.05 mM Fe. The inhibitory capacity of the cells was comparable to that attained following treatment of the macrophages with 100 U of rIFN-g in the absence of added Fe. Iron abrogates the antimycobacterial activity of IFN-g-activated macrophages from Bcgs mice. In contrast to the stimulatory effect of Fe on the inhibition of mycobacterial growth of resident macrophages from Bcgr mice, iron did not affect the capacity of rIFN-g-activated macrophages to control mycobacterial growth (Fig. 3). At the same time, however, Fe stimulated the growth of M. avium in rIFN-g-activated macrophages from Bcgs mice. IFN-g lowers intracellular iron levels. Preliminary reports by Hentze and Kuhn (30) and others (31) showed that IFN-g treatment lowered intracellular iron levels in macrophages from Bcgr mice. The results presented in Table 1 show that macrophages from Bcgr mice contain 344 pg of intracellular iron per mg of cell protein; this concentration is similar to that found in macrophages from susceptible mice (291 pg/mg). Treatment of the cells with rIFN-g lowered the intracellular iron content of macrophages from Bcgr mice to 200 pg/mg, while the intracellular iron levels of macrophages from Bcgs mice remained unaffected. Inhibitors of hydroxyl radical generation prevent increased antimicrobial activity induced by iron. Iron catalyzes the generation of hydroxyl radicals via the Fenton/Haber-Weiss reaction. The addition of the hydroxyl radical inhibitors catalase, mannitol, and DMSO resulted in an amelioration of the growth inhibitory capacity of macrophages from Bcgr mice that had been treated with iron (Fig. 4). The addition of neither SOD nor MMLA affected the growth inhibitory activity induced by iron.

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FIG. 3. Iron differentially affects the capacity of IFN-g-activated macrophages from Bcgr or Bcgs mice to inhibit the growth of mycobacteria. Macrophages were activated by treatment with 100 U of rIFN-g/ml prior to infection with M. avium. The radioactivity incorporated by the bacteria was determined after 5 days in culture. The data are expressed as the percentages of inhibition of mycobacterial growth based on the growth of the mycobacteria in macrophages not treated with rIFN-g. The mycobacteria isolated from macrophages of Bcgs mice that had not been treated with rIFN-g incorporated 42,025 6 2,561 cpm (mean 6 standard deviation), while those isolated from macrophages of Bcgr mice incorporated 31,689 6 2,882 cpm. The effect of iron on the growth of M. avium in macrophages from Bcgs mice was significant. Iron did not affect the capacity of the rIFN-g-treated macrophages from Bcgr mice to inhibit the growth of the bacterium.

Manipulation of intracellular iron affects Nramp1 mRNA stability. We have already reported that Nramp1 mRNA expression is inducible with rIFN-g (11) and that Nramp1 mRNA in macrophages from Bcgr mice is more stable than that in macrophages from Bcgs mice (12). As shown in Table 1, treatment with rIFN-g also lowered cellular iron content in macrophages from Bcgr mice. We tested the possibility that the decrease in intracellular iron is causally related to the enhanced mRNA stability by artificially altering cellular iron levels by using desferrioxamine, an iron chelator, or by adding iron. The results, shown in Fig. 5, show that chelation of iron with desferrioxamine resulted in increased half-life of Nramp1 mRNA in macrophages from Bcgs mice. The half-life of Nramp1 mRNA increased from 6 to 16 h in the presence of desferrioxamine, as contrasted to an increase of less than 2 h observed in macrophages from Bcgr mice. To determine if excess iron affected the expression of Nramp1 mRNA, rIFNg-stimulated macrophages from Bcgr and Bcgs mice were incubated in the presence or absence of ferric ammonium sulfate, TABLE 1. The effect of IFN-g on the iron content of macrophages from Bcgr and Bcgs micea Group

Untreated IFN-g

Iron content (mg of Fe/mg of cell protein) Bcgr mice

Bcgs mice

344 6 16 200 6 4b

291 6 16 261 6 16

a Resident peritoneal macrophages were treated overnight with 100 U of rIFN-g, washed, and digested with 6 N nitric acid to release bound iron (41). The iron was reduced by adding ascorbic acid, and ferrizone reagent was added. The reagent was allowed to react with the reduced iron and the optical density at 562 nm was determined. The amount of iron was determined by using an iron standard. b Significantly different from untreated macrophages (P , 0.05) as determined by Student’s t test. The iron contents of macrophages from Bcgr and Bcgs mice not treated with IFN-g were not different.

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FIG. 4. Inhibitors of hydroxyl radical generation prevent the increased antimycobacterial activity induced by iron. Resident peritoneal macrophages from Bcgr mice were infected with M. avium and cultured with 0.05 mM Fe in the presence or absence of the following: SOD, 1 mg/ml; MMLA, 5 mM; mannitol, 50 mM; catalase, 2 mg/ml; and DMSO, 150 mM. Several concentrations were used for each of the inhibitors; the effect of the optimal concentration is shown. The effects of mannitol, catalase, and DMSO were significant at a P of ,0.05 or lower. The effect of SOD or MMLA was not significant.

added to artificially raise iron levels. The results presented in Fig. 5 also show that incubation of macrophages from Bcgr mice with iron resulted in a decrease in Nramp1 mRNA halflife from 20 h to about 16 h, while that for Nramp1 from macrophages from Bcgs mice decreased from about 10 to 5 h. Thus, manipulation of intracellular iron levels had a greater effect on mRNA stability in macrophages from Bcgs mice. Iron transport by phagosomes isolated from M. avium-infected RAW264.7 macrophage cell lines transfected with Nramp1Gly169 allele differs from that by phagosomes isolated from lines transfected with the Nramp1Asp169 allele. The results of this investigation suggest that iron is transported into phagosomes and stimulates hydroxyl radical formation. To test this model, we measured iron transport into phagolysosomes from M. avium-infected cells. The results, presented in Fig. 6, show that phagosomes isolated from cells transfected with the Nramp1Gly169 (resistance) allele and infected with M. avium imported more iron then did phagosomes isolated from infected cells transfected with the Nramp1Asp169 (susceptibility) allele. We did not observe any differences in the amounts of iron imported by the intact cells, nor did we observe that iron was exported differently by the cell lines or by the phagosomes (32a). DISCUSSION The results of this investigation show that macrophages from Bcgr mice and Bcgs mice respond differently to iron. Iron stimulated the growth inhibitory capacity of macrophages from resistant mice. In contrast, iron stimulated mycobacterial growth in macrophages from susceptible mice. Our observation that treatment of macrophages from Bcgr mice with inhibitors of hydroxyl radical generation prevented the enhancing effect of iron on the growth inhibitory capacity of the macrophages strongly suggests that the presence of iron stimulates hydroxy radical formation via the Fe-catalyzed Fenton/HaberWeiss reactions (28, 32, 35, 37). The generation of OH z from H2O2 (Fenton’s reagent) requires the availability of Fe21. Our results (Fig. 6) suggest that the Nramp1 gene product regulates this transport. The iron-catalyzed interaction of H2O2 and O2z2 is the Haber-Weiss reaction, and the Fe31 formed by Fenton’s reagent is reduced by O2z2. When levels of Fe21 are

FIG. 5. Manipulation of intracellular iron affects mRNA stability. Splenic macrophages from Bcgr or Bcgs mice were stimulated with 100 U of rIFN-g/ml in the presence of 100 mM desferrioxamine (Desferox) or 50 mM ferric ammonium sulfate. After 20 h, the macrophage cultures were treated with 20 mg of actinomycin D (Act D) or medium for 2, 4, 8, 16, 32, or 48 h prior to extraction and isolation of total RNA. Control cultures (lane 1) were treated with rIFN-g only. The corresponding decay curve of Nramp1 mRNA expression, representing the percentages of mRNA remaining following actinomycin D treatment, is derived from densitometric analysis of Northern blots. ■, Mutant (Bcgs) plus desferrioxamine or Fe; h, mutant (Bcgs) without desferrioxamine or Fe; Œ, wild-type (Bcgr) plus desferrioxamine or Fe; ‚, wild-type (Bcgr) without desferrioxamine or Fe.

sufficient it can react directly with H2O2 to form OHz. The reactions are inhibited by catalase and mannitol but are not affected by SOD and may further require the interaction with iodine (32). This possibility is currently being explored by us. The lack of effect of MMLA, a nonmetabolizable form of L-arginine and thus an inhibitor of nitric oxide (NO) genera-

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FIG. 6. Iron transport by phagosomes isolated from the RAW264.7 macrophage cell lines, transfected with Nramp1Gly169 (resistance) or Nramp1Asp169 (susceptibility) alleles and infected with M. avium. Macrophage cell lines were labeled with 55Fe-labeled iron citrate prior to infection with M. avium. Phagosomes were isolated following lysis of the cells and differential centrifugation on a sucrose gradient. The radioactivity incorporated into the phagosomes is adjusted for total protein, which includes bacterial protein, and the data are expressed as picomoles of Fe per milligram of protein. The number of moles of Fe was calculated based on the specific activity of the isotope and does not take into account endogenous Fe levels.

tion, in preventing the Fe-mediated growth inhibition reinforces our previous observation that inhibition of arginine metabolism does not affect Nramp1-mediated control of mycobacterial growth (11). At the same time, as we have previously reported, the addition of MMLA to cultures of IFN-gactivated macrophages inhibited their capacity to limit the growth of M. avium. Thus, the antimycobacterial activity mediated by rIFN-g-activated macrophages and the growth inhibitory capacity mediated by Nramp1 are different. The stimulatory effect of iron on the growth inhibitory capacity of macrophages from Bcgr mice occurred over a very narrow dose range. The inability of high concentrations of iron to stimulate the growth of mycobacteria in IFN-g-activated macrophages may indicate that iron transport into the phagosome was limited and that only limited quantities of iron were available to stimulate the growth of the microorganism. Alternatively, it is possible that the increased production of NO by the rIFN-g-stimulated cells and the formation of peroxynitrite ion contributed to the increased capacity to limit the growth of the bacterium, even in the presence of increasing concentrations of iron in vitro (15, 32, 48, 50, 51). Nitric oxide has been reported to cause an efflux of nonheme iron from infected cells (47, 49), and the addition of MMLA to these cultures reduces their growth inhibitory capacity (11). We have reported, in preliminary studies, that the level of intracellular iron in macrophages from Bcgr mice decreases following treatment of the cells with rIFN-g (52). The results presented here confirm our earlier report and that by Atkinson and Barton (4). The mechanisms that account for the differences in iron content remain unknown, especially since whole cells were used to make these measurements and Nramp1 is expressed in phagosomal membranes and not in the plasma membrane (25). We do not think that iron flux is responsible for the changes in iron content because iron does not appear to be differentially taken up by the cells but is taken up only by phagosomes where Nramp1 is expressed (32a). However, the formation of NOz induces the loss of iron in part by an attack

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on iron-sulfur centers with associated inhibition of cell metabolism and growth (28, 32, 35, 37, 47, 49). While the generation of these reactive nitrogen intermediates does not appear to play an important role in Nramp1-mediated growth inhibition, it is possible that this mechanism may account for the loss of iron from the IFN-g-treated macrophages from Bcgr mice, which have been reported to produce more NO than macrophages from Bcgs mice (5, 36). What is the direction of Fe transport? Blackwell and Searle have suggested that iron is transported from the phagosome to the cytoplasm (9). This possibility would account for the scavenger function of macrophages, i.e., the removal of effete erythrocytes by phagocytosis and the recycling of iron. Removal of iron would limit its availability to microorganisms, thus accounting for the role of Nramp1 in limiting mycobacterial growth. This possibility points to a rather passive role for Nramp1. The results described by de Chastellier et al. (14) instead suggest a more active role for Nramp1. They showed, by ultrastructural analysis, that macrophages expressing the wild-type Nramp1 allele contained more-damaged bacilli, especially at a very early stage of infection. This observation argues instead for a more active role for Nramp1 than just the limitation of iron. Our results suggest that limited quantities of iron transported into the phagosome catalyze the generation of antimicrobial hydroxyl radicals via the Fenton/Haber-Weiss reactions. The observation that limited quantities of iron result in increased antimicrobial activity is analogous to that reported by Alford et al. (2), who showed that differences in the iron content of macrophages correlated with differences in antilisterial activity. High iron levels were synonymous with increased Listeria growth, while lower, but sufficient, levels of iron resulted in increased listericidal activity. Transferrin receptor-mediated iron uptake could also result in higher levels of iron within the phagosome. However, a reduction in the net uptake of iron occurs in infected host cells following the down regulation of transferrin receptors that occurs following infection (47) or upon interaction with macrophage-activating cytokines (13, 27, 39). This, together with the generation of NO, serves to lower the availability of iron. The transport of redox-active iron into phagosomes by Nramp1 may be required to supply sufficient catalyst for the Fenton/ Haber-Weiss reactions when the availability of cellular iron is limited. The observation that chelation of iron results in increased stability of Nramp1 mRNA supports the observations of Atkinson et al. (3), who showed that chelation of iron resulted in an increased expression of Nramp1 protein in cells that have been treated with rIFN-g. Changes in mRNA stability can have profound effects on the quantity of translated proteins (34, 41). Thus, the differences we have observed in the stability of Nramp1 mRNA and in the stability of mRNAs of other IFNg-activated genes can significantly alter the levels of antimicrobial effector molecules and cytokines produced by the cells. The results of this investigation show that changes in cytoplasmic Fe alter the stability of Nramp1 mRNA as well as other mRNAs (unpublished observations). The increased stability of mRNA from macrophages from Bcgr mice may, in part, be the result of the decreased levels of Fe associated with activation (13, 27, 39) as well as the transport of iron from the cytoplasm to the phagosome. This could account for the many pleiotropic effects of the Nramp1 gene, even though some of the differences that have been reported are the result of different genes whose induction requires different signal transduction pathways. One important mechanism by which Fe regulates mRNA stability is by regulating the binding of an iron response protein

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to a consensus iron response element sequence in the 39 untranslated regions of some mRNAs (e.g., transferrin receptor) (39). It has been proposed by Blackwell and Searle that Nramp1 mRNA stability may be similarly regulated, based on the observation that the consensus sequence for the iron response element is found in the 39 untranslated region of rat Nramp2 mRNA (9). However, we have not been able to identify this sequence in the 39 untranslated region of mouse Nramp1 mRNA (unpublished observations). One mechanism that could account for iron-regulated mRNA stability is the observation that iron regulates protein kinase C (PKC) mRNA transcription (1). Our group (10) and others (38) have reported that the PKC activity of macrophages from Bcgr mice is greater than that of macrophages from Bcgs mice. The Nramp1 protein contains three putative PKC phosphorylation sites (6, 46). In preliminary studies we have found that inhibition of PKC inhibits the induction of stable mRNA by macrophages from Bcgr mice. Thus, PKC activity, iron, mRNA stability, and the function of Nramp1 are linked. The role of iron in regulating PKC and the effects of PKC inhibitors on Nramp1-mediated iron transport and antimicrobial activity are currently being explored by us. The recent identification of the murine gene controlling microcytic anemia as Nramp2 and the demonstration that Nramp2 is a metal ion transporter, which transports Fe21, Zn21, Mn21, Co21, Cd21, Cu21, Ni21, and Pb21, provides further support that the members of this gene family function as metal ion transporters (17, 18, 24, 26). We propose that the role of Nramp1 is to transport iron from the cytoplasm into the maturing phagolysosome. This results in a lowering of biologically active cytoplasmic iron, which is sufficient to stabilize mRNA. The increased stability of mRNAs alone can account for the pleiotropic effects that have been attributed to Nramp1. The influx of iron into the phagosome, under conditions in which cellular iron may be limited, serves as a catalyst for the Fenton/Haber-Weiss reaction, which results in the limitation of mycobacterial growth. ACKNOWLEDGMENTS This work is supported by grants HL59795, AI42901, and MH54966 from the National Institutes of Health. REFERENCES 1. Alcantara, O., L. Obeid, Y. Hannun, P. Ponka, and D. H. Boldt. 1994. Regulation of protein kinase C (PKC) expression by iron: PKC-beta and PKC-alpha gene expression and role of the 59 flanking region of the PKC beta gene in the response to ferric transferrin. Blood 84:3510–3517. 2. Alford, C. E., T. E. King, and P. A. Campbell. 1991. Role of transferrin, transferrin receptors and iron in macrophage listericidal activity. J. Exp. Med. 174:459–466. 3. Atkinson, P. G., J. M. Blackwell, and C. H. Barton. 1997. Nramp1 locus encodes a 65 kDa interferon-gamma inducible protein in murine macrophages. Biochem. J. 325:779–786. 4. Atkinson, P. G. P., and C. H. Barton. 1998. Ectopic expression of Nramp1 in COS-1 cells modulates iron accumulation. FEBS Lett. 425:239–242. 5. Barton, C. H., S. H. Whitehead, and J. M. Blackwell. 1995. Nramp transfection transfers Ity/Lsh/Bcg related pleiotropic effects on macrophage activation: influence on oxidative burst and nitric oxide pathways. Mol. Med. 1:267–279. 6. Barton, C. H., J. K. White, T. I. A. Roach, and J. M. Blackwell. 1994. NH2-terminal sequencing of macrophage expressed natural resistance associated macrophage protein (Nramp1) encodes a proline/serine rich putative Src homology 3 binding domain. J. Exp. Med. 179:1683–1687. 7. Blackwell, J. M., T. I. A. Roach, S. E. Atkinson, J. W. Ajioka, C. H. Barton, and M. A. Shaw. 1991. Genetic regulation of macrophage priming/activation: the Lsh gene story. Immunol. Lett. 30:241–248. 8. Blackwell, J. M., C. H. Barton, J. D. White, S. Searle, A.-M. Baker, H. Williams, and M.-A. Shaw. 1995. Genomic organization and sequence of the human NRAMP gene: identification and mapping of a promoter region polymorphism. Mol. Med. 1:194–205. 9. Blackwell, J. M., and S. Searle. Understanding the multiple functions of Nramp1. Res. Immunol., in press.

1391

10. Brown, D. H., M. Faris, M. Hilburger, and B. S. Zwilling. 1994. The induction of persistence of I-A expression by macrophages from Bcgr mice occurs via a protein kinase C dependent pathway. J. Immunol. 152:1323–1331. 11. Brown, D. H., W. P. LaFuse, and B. S. Zwilling. 1995. Cytokine-mediated activation of macrophages from Mycobacterium bovis BCG-resistant and -susceptible mice: differential effects of corticosterone on antimycobacterial activity and expression of the Bcg gene (candidate Nramp). Infect. Immun. 63:2983–2988. 12. Brown, D. H., W. P. Lafuse, and B. S. Zwilling. 1997. Stabilized expression of mRNA is associated with mycobacterial resistance controlled by Nramp1. Infect. Immun. 65:597–603. 13. Byrd, T. F., and M. A. Horwitz. 1989. Interferon-gamma activated human monocytes down regulate transferrin receptors and inhibit the intracellular multiplication of Legionella pneumophila by limiting the availability of iron. J. Clin. Investig. 83:1457–1465. 14. de Chastellier, C., C. Fre´hel, C. Offredo, and E. Skamene. 1993. Implication of phagosome-lysosome fusion in restriction of Mycobacterium avium growth in bone marrow macrophages from genetically resistant mice. Infect. Immun. 61:3775–3784. 15. Drapier, J. C., H. Hirling, J. Wietzerbin, P. Kaldy, and L. C. Kuhn. 1993. Biosynthesis of nitric oxide activates iron regulatory factor in macrophages. EMBO J. 12:3643–3650. 16. Evans, R., and S. J. Kamdar. 1990. Stability of RNA isolated from macrophages depends on the removal of an RNA degrading activity early in the extraction procedure. BioTechniques 8:357–360. 17. Fleming, M. D., C. C. Trenor, M. A. Su, D. Foernzler, D. R. Beier, W. F. Dietrich, and N. C. Andrews. 1997. Microcytic anemia mice have a mutation in Nramp2, a candidate iron transporter gene. Nat. Genet. 16:383–386. 18. Fleming, M. D., M. A. Romano, M. A. Su, L. M. Garrick, M. D. Garrick, and N. C. Andrews. 1998. Nramp2 is mutated in the anemic Belgrade (b) rat: evidence of a role for Nramp2 in endosomal iron transport. Proc. Natl. Acad. Sci. USA 95:1148–1153. 19. Forget, A., E. Skamene, P. Gros, A.-C. Miailhe, and R. Turcotte. 1981. Differences in response among inbred mouse strains to infection with small doses of Mycobacterium bovis BCG. Infect. Immun. 32:42–47. 20. Gobin, J., and M. A. Horwitz. 1996. Exochelins of Mycobacterium tuberculosis remove iron from human iron binding proteins and donate iron to mycobactins in the M. tuberculosis cell wall. J. Exp. Med. 183:1527–1532. 21. Govoni, G., S. Vidal, S. Gauthier, E. Skamene, D. Malo, and P. Gros. 1996. The Bcg/Ity/Lsh locus: genetic transfer of resistance to infections in C57BL/6J mice transgenic for the Nramp1Gly169 allele. Infect. Immun. 64: 2923–2929. 22. Gros, P., E. Skamene, and A. Forget. 1981. Genetic control of natural resistance to Mycobacterium bovis (BCG) in mice. J. Immunol. 127:2417– 2421. 23. Gros, P., E. Skamene, and A. Forget. 1983. Cellular mechanisms of genetically controlled host resistance to Mycobacterium bovis (BCG). J. Immunol. 131:1966–1972. 24. Gruenheid, S., M. Cellier, S. Vidal, and P. Gros. 1995. Identification and characterization of a second mouse Nramp gene. Genomics 25:514–525. 25. Gruenherd, S. E., M. Pinner, M. Desjardins, and P. Gros. 1997. Natural resistance to infection with intracellular pathogens: the Nramp1 protein is recruited to the membrane of the phagosome. J. Exp. Med. 185:717–730. 26. Gunshin, H., B. Mackenzie, U. V. Berger, Y. Gunshin, M. F. Romero, W. F. Boron, S. Nussberger, J. L. Gollan, and M. A. Hediger. 1997. Cloning and characterization of a mammalian proton coupled metal-ion transporter. Nature 388:482–488. 27. Hamilton, T. A., J. E. Weiel, and D. O. Adams. 1984. Expression of transferrin receptor in murine peritoneal macrophages is modulated in the different stages of activation. J. Immunol. 132:2285–2290. 28. Henle, E. S., and S. Linn. 1997. Formation, prevention and repair of DNA damage by iron/hydrogen peroxide. J. Biol. Chem. 272:19095–19098. 29. Hentze, M. W. 1991. Determinants and regulation of cytoplasmic mRNA stability in eukaryotic cells. Biochim. Biophys. Acta 1090:281–292. 30. Hentze, M. W., and L. C. Kuhn. 1996. Molecular control of vertebrate iron metabolism: mRNA based regulatory circuits operated by iron, nitric oxide and oxidative stress. Proc. Natl. Acad. Sci. USA 93:8175–8182. 31. Klausner, R., T. A. Rouault, and J. B. Harford. 1993. Regulating the fate of mRNA: the control of cellular iron metabolism. Cell 72:19–28. 32. Klebanoff, S. J. 1992. Oxygen metabolites from phagocytes, p. 541–588. In J. I. Gallin, I. M. Goldstein, and R. Snyderman (ed.), Inflammation: basic principals and clinical correlations, 2nd ed. Raven Press, Ltd., New York, N.Y. 32a.Kuhn et al. Submitted for publication. 33. Malo, D., K. Vogan, S. Vidal, J. Hu, M. Cellier, E. Schurr, A. Fuks, N. Bumstead, K. Morgan, and P. Gros. 1994. Haplotype mapping and sequence analysis of the mouse Nramp gene predict susceptibility to infection with intracellular parasites. Genomics 23:51–61. 34. Malter, J. S. 1998. Posttranscriptional regulation of mRNA’s important in T cell function. Adv. Immunol. 68:1–49. 35. McCord, J. M. 1998. Iron, free radicals and oxidative injury. Semin. Hematol. 35:5–12.

1392

ZWILLING ET AL.

36. Nathan, C. 1995. Natural resistance and nitric oxide. Cell 82:873–876. 37. Olakanmi, O., S. E. McGowan, M. B. Hayek, and B. E. Britigan. 1993. Iron sequestration decreases the potential for extracellular hydroxyl radical formation. J. Clin. Investig. 91:889–899. 38. Olivier, M., P. Cok, J. Desanitis, Z. Hel, W. Wojciechowski, N. E. Reiner, E. Skamene, and D. Radzioch. 1998. Phenotypic difference between Bcg(r) and Bcg(s) macrophages is related to differences in protein kinase C dependent signalling. Eur. J. Biochem. 251:734–743. 39. Pantopoulos, K., and M. W. Hentze. 1995. Nitric oxide signaling to iron regulatory protein: direct control of ferritin mRNA translation and transferrin receptor mRNA stability in transfected fibroblasts. Proc. Natl. Acad. Sci. USA 92:1267–1271. 40. Peltz, S. W., C. F. Higgins, and A. Jacobsen. 1991. Turnover of mRNA in prokaryotes and lower eukaryotes. Curr. Opin. Genet. Dev. 2:739–747. 41. Rajagopalan, L. E., and J. S. Malter. 1997. Regulation of eukaryotic messenger RNA turnover. Prog. Nucleic Acid Res. Mol. Biol. 56:257–286. 42. Sachs, A. B. 1993. Messenger RNA degradation in eukaryotes. Cell 74:413– 421. 43. Stearman, R., D. S. Yuan, Y. Yamaguchi-Iwai, R. D. Klausner, and A. Dancis. 1996. A permease oxidase complex involved in high affinity iron uptake in yeast. Science 271:1552–1557. 44. Sturgill-Koszycki, S., U. E. Schaible, and D. G. Russell. 1996. Mycobacterium containing phagosomes are accessible to early endosomes and reflect a

Editor: S. H. E. Kaufmann

INFECT. IMMUN. transitional state in normal phagosome biogenesis. EMBO J. 15:6960–6968. 45. Supek, F., L. Supekova, H. Nelson, and N. Nelson. 1996. A yeast manganese transporter related to the macrophage protein conferring resistance to mycobacteria. Proc. Natl. Acad. Sci. USA 93:5105–5110. 46. Vidal, S. D., D. Malo, K. Vogan, E. Skamene, and P. Gros. 1993. Natural resistance to infection with intracellular parasites: isolation of a candidate for Bcg. Cell 73:469–485. 47. Weinberg, E. D. 1992. Iron depletion: a defense against intracellular infection and neoplasia. Life Sci. 50:1289–1297. 48. Weiss, G., B. Goosen, W. Doppler, D. Fuchs, K. Pantopoulos, G. WernerFelmayer, H. Wachter, and M. W. Hentze. 1993. Translational regulation via iron-responsive elements by the nitric oxide NO synthase pathway. EMBO J. 12:3651–3657. 49. Weiss, G., H. Wachter, and D. Fuchs. 1995. Linkage of cell mediated immunity to iron metabolism. Immunol. Today 16:495–500. 50. Weiss, G., C. Bogdan, and M. W. Hentze. 1997. Pathways for the regulation of macrophage iron metabolism by the anti-inflammatory cytokines IL-4 and IL-13. J. Immunol. 158:420–425. 51. Wheeler, P. A., and C. Ratledge. 1994. Metabolism of Mycobacterium tuberculosis, p. 353–385. In B. R. Bloom (ed.), Tuberculosis: pathogenesis, protection, and control. ASM Press, Washington, D.C. 52. Zwilling, B. S., W. Lafuse, and D. H. Brown. 1996. Mycobacterial resistance, mRNA stabilization, intracellular iron and the function of Nramp1. J. Leukoc. Biol. 60(Suppl.):28. (Abstract 93.)