Effect of Oxygen on Induction of the Cystine Transporter by Bacterial Lipopolysaccharide in Mouse Peritoneal Macrophages

Amino acid transport in mouse peritoneal macrophages is mediated by several membrane carriers with different substrate specificity and sensitivity to environmental stimuli. We reported previously that transport activities of cystine and arginine in the macrophages were induced markedly by low concentrations of bacterial lipopolysaccharide (LPS). It is known that a variety of macrophage functions are affected by ambient oxygen tension. In this study, we have investigated the effects of oxygen on the induction of amino acid transport activity by LPS and found that the induction of cystine, but not arginine, transport activity was dependent on the ambient oxygen tension. When the macrophages were cultured with 2% O2 in the presence of 1 ng/ml LPS, induction of cystine transport activity was reduced by ∼70% compared with cells cultured under normoxic conditions. In macrophages, transport of cystine is mediated by a Na+-independent anionic amino acid transporter named system xc−. System xc− is composed of two protein components, xCT and 4F2hc , and the expression of xCT was closely correlated with system xc−activity. A putative NF-κB binding site was found in the 5′-flanking region of the xCT gene, but the enhanced expression of xCT by LPS and oxygen was not mediated by NF-κB binding. An increase in intracellular GSH in macrophages paralleled induction of xCT, but not γ-glutamylcysteine synthetase. These results suggest the importance of system xc− in antioxidant defense in macrophages exposed to LPS and oxidative stress.AB037650AB037661 Amino acid transport in mouse peritoneal macrophages is mediated by several membrane carriers with different substrate specificity and sensitivity to environmental stimuli. We reported previously that transport activities of cystine and arginine in the macrophages were induced markedly by low concentrations of bacterial lipopolysaccharide (LPS). It is known that a variety of macrophage functions are affected by ambient oxygen tension. In this study, we have investigated the effects of oxygen on the induction of amino acid transport activity by LPS and found that the induction of cystine, but not arginine, transport activity was dependent on the ambient oxygen tension. When the macrophages were cultured with 2% O2 in the presence of 1 ng/ml LPS, induction of cystine transport activity was reduced by ∼70% compared with cells cultured under normoxic conditions. In macrophages, transport of cystine is mediated by a Na+-independent anionic amino acid transporter named system xc−. System xc− is composed of two protein components, xCT and 4F2hc , and the expression of xCT was closely correlated with system xc−activity. A putative NF-κB binding site was found in the 5′-flanking region of the xCT gene, but the enhanced expression of xCT by LPS and oxygen was not mediated by NF-κB binding. An increase in intracellular GSH in macrophages paralleled induction of xCT, but not γ-glutamylcysteine synthetase. These results suggest the importance of system xc− in antioxidant defense in macrophages exposed to LPS and oxidative stress. AB037650AB037661 lipopolysaccharide 4F2 heavy chain γ-glutamylcysteine synthetase electrophile response element Transport of amino acids across the plasma membrane of mammalian cells is mediated by several systems with different substrate specificity (1Christensen H.N. Physiol. Rev. 1990; 70: 43-77Crossref PubMed Scopus (971) Google Scholar). We have previously characterized anionic, neutral, and cationic amino acid transport systems in mouse peritoneal macrophages, which are known to be activated by a variety of factors such as bacterial lipopolysaccharide (LPS)1 and cytokines (2Watanabe H. Bannai S. J. Exp. Med. 1987; 165: 628-640Crossref PubMed Scopus (137) Google Scholar, 3Sato H. Watanabe H. Ishii T. Bannai S. J. Biol. Chem. 1987; 262: 13015-13019Abstract Full Text PDF PubMed Google Scholar, 4Sato H. Ishii T. Sugita Y. Bannai S. Biochim. Biophys. Acta. 1991; 1069: 46-52Crossref PubMed Scopus (30) Google Scholar). Macrophages exhibit cytoprotective functions including antigen presentation and microbicidal or tumoricidal activity. The major anionic amino acid transport system in macrophages is system xc−, which mediates the exchange of an anionic form of cystine and glutamate across the plasma membrane (2Watanabe H. Bannai S. J. Exp. Med. 1987; 165: 628-640Crossref PubMed Scopus (137) Google Scholar). The transport system for neutral amino acids in macrophages seems to be unique, transporting most neutral substrates such as serine, alanine, and leucine via a Na+-independent mechanism (3Sato H. Watanabe H. Ishii T. Bannai S. J. Biol. Chem. 1987; 262: 13015-13019Abstract Full Text PDF PubMed Google Scholar). For cationic amino acids, a system y+-like cationic amino acid transport system mediates the uptake of arginine, lysine, and ornithine, although the system in macrophages has slightly different characteristics from those of the typical system y+(4Sato H. Ishii T. Sugita Y. Bannai S. Biochim. Biophys. Acta. 1991; 1069: 46-52Crossref PubMed Scopus (30) Google Scholar). We have found that a very low concentration of LPS markedly enhances the activities of cystine and arginine transport in mouse peritoneal macrophages (5Sato H. Fujiwara K. Sagara J. Bannai S. Biochem. J. 1995; 310: 547-551Crossref PubMed Scopus (105) Google Scholar, 6Sato H. Fujiwara M. Bannai S. J. Leukoc. Biol. 1992; 52: 161-164Crossref PubMed Scopus (49) Google Scholar). The enhanced uptake of cystine results from the induction of system xc− activity and increased influx of arginine is because of the induction of the typical system y+ activity. The induction of cystine transport may contribute to the maintenance of intracellular GSH levels because cystine taken up by the cells is reduced to cysteine, a rate-limiting precursor for GSH synthesis (7Bannai S. Tateishi N. J. Membrane Biol. 1986; 89: 1-8Crossref PubMed Scopus (310) Google Scholar). In contrast, the induction of the arginine transport activity is a key event for production of nitric oxide involved in microbicidal and tumoricidal processes in activated macrophages (8Bogle R.G. Baydoun A.R. Pearson J.D. Mann G.E. Biochem. J. 1992; 284: 15-18Crossref PubMed Scopus (213) Google Scholar, 9Shibazaki T. Fujiwara M. Sato H. Fujiwara K. Abe K. Bannai S. Biochim. Biophys. Acta. 1996; 1311: 150-154Crossref PubMed Scopus (19) Google Scholar). A cDNA encoding a protein with properties consistent with system y+ in LPS-activated mouse macrophages has been identified and named mCAT-2B (10Closs E.I. Lyons C.R. Kelly C. Cunningham J.M. J. Biol. Chem. 1993; 268: 20796-20800Abstract Full Text PDF PubMed Google Scholar). Recently, we reported that system xc− is composed of two proteins and have cloned cDNAs for these proteins, namely 4F2hc (heavy chain of the surface antigen 4F2, also named CD98) and xCT (11Sato H. Tamba M. Ishii T. Bannai S. J. Biol. Chem. 1999; 274: 11455-11458Abstract Full Text Full Text PDF PubMed Scopus (802) Google Scholar). Coexpression of the ubiquitous transmembrane protein 4F2hc with xCT (and cDNAs of some other amino acid transporters) has been shown to induce amino acid transport activity in Xenopus oocytes (12Mastroberardine L. Spindler B. Pfeiffer R. Skelly P.J. Loffing J. Shoemaker C.B. Verrey F. Nature. 1998; 395: 288-291Crossref PubMed Scopus (478) Google Scholar, 13Kanai Y. Segawa H. Miyamoto K. Uchino H. Takeda E. Endou H. J. Biol. Chem. 1998; 273: 23629-23632Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, 14Torrents D. Estevez R. Pineda M. Fernandez E. Lloberas J. Shi Y-B. Zorzano A. Palacı́n M. J. Biol. Chem. 1998; 273: 32437-32445Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 15Deves R. Boyd C.A.R. J. Membrane Biol. 2000; 173: 165-173Crossref PubMed Scopus (149) Google Scholar). Recent studies have shown that in macrophages, ambient oxygen tension alters the morphology, expression of cell surface markers, viability, phagocytosis, metabolic activity, and release of cytokines (16Lewis J.S. Lee J.A. Underwood J.C.E. Harris A.L. Lewis C.E. J. Leukoc. Biol. 1999; 66: 889-900Crossref PubMed Scopus (324) Google Scholar). It is likely that antioxidant systems such as intracellular GSH are affected by ambient oxygen tension and/or oxidative stress in these cells activated by LPS. In the present study, we have investigated the effects of oxygen on the induction of cystine and arginine transport activity in the macrophages caused by treatment with LPS. We have established that induction of cystine transport by LPS is dependent on ambient oxygen levels, whereas LPS-induced activation of arginine transport via system y+ was unaffected. l-14CCystine,l-14Carginine, andl-14Cserine were obtained from PerkinElmer Life Sciences. Thioglycolate broth (Brewer's formula) and Bacto LPS (Salmonella typhosa 0901) were from Difco Laboratories, Detroit, MI. Fetal bovine serum was obtained from BioWhittaker, Walkersville, MD, and the lot which contains less than 0.3 ng/ml endotoxin was used. Monobromobimane was purchased from Molecular Probes, Inc., OR. Macrophages were collected by peritoneal lavage from female C57BL/6 mice weighing 20–25 g who had previously received 4 days prior an intraperitoneal injection of 2 ml of 4% thioglycolate broth. The lavage medium was RPMI 1640 containing 10 units/ml heparin. The cells were washed twice with RPMI 1640 and plated at 1 × 106 cells/35-mm diameter culture dish containing RPMI 1640, 10% fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin. Cells were incubated at 37 °C in 5% CO2, 95% air, and after 1 h the medium was replaced to remove nonadherent cells. For hypoxic conditions, cells were cultured in a gas-tight chamber flushed with 95% N2 and 5% CO2, and the required concentrations of O2were measured using an oxygen meter. Amino acid uptake was measured using techniques described previously (17Bannai S. Kitamura E. J. Biol. Chem. 1980; 255: 2372-2376Abstract Full Text PDF PubMed Google Scholar). Cells were rinsed three times in warmed PBSG (10 mm phosphate-buffered saline (137 mm NaCl, 3 mm KCl), pH 7.4, containing 0.01% CaCl2, 0.01% MgCl2·6H2O, and 0.1% glucose) and then incubated in 0.5 ml of the warmed uptake medium at 37 °C for specified time periods. The uptake medium was PBSG with a labeled amino acid (0.1 μCi/0.5 ml). Uptake was terminated by rapidly rinsing the culture dishes three times with ice-cold phosphate-buffered saline, and radioactivity associated with cell extracts was determined as described previously (17Bannai S. Kitamura E. J. Biol. Chem. 1980; 255: 2372-2376Abstract Full Text PDF PubMed Google Scholar). Amino acid uptake was determined under conditions approaching initial rates of uptake, i.e. measuring uptakes for cystine, serine, and arginine at 120, 30, and 15 s, respectively. For each of the amino acids, uptake increased linearly during the specified incubation interval. Intracellular GSH was extracted with 5% trichloroacetic acid and then treated with ether to remove the acid. The GSH content in the aqueous layer was measured using an enzymatic method described previously, which is based on the catalytic action of GSH in the reduction of 5,5′-dithiobis(2-nitrobenzoic acid) by the GSH reductase system (18Tietze F. Anal. Biochem. 1969; 27: 502-522Crossref PubMed Scopus (5617) Google Scholar). The GSH extracted from the cells was mostly reduced GSH, and the content of the oxidized form, GSSG, was negligible throughout the experiments in this study. The efflux of GSH was measured as follows. The cells were rinsed three times with PBSG and incubated with 0.5 ml of PBSG at 37 °C for 1 h. Then GSH in the PBSG was quantified by the method described above. The cysteine content in the cells was determined by the method of Cotgreave and Moldéus (19Cotgreave I.A. Moldéus P. J. Biochem. Biophys. Methods. 1986; 13: 231-249Crossref PubMed Scopus (233) Google Scholar) with a slight modification (20Sagara J. Miura K. Bannai S. J. Neurochem. 1993; 61: 1667-1671Crossref PubMed Scopus (136) Google Scholar). The cells were rinsed three times with PBSG and incubated in the dark at room temperature for 10 min with 100 μl of 8 mmmonobromobimane in 50 mm N-ethylmorpholine, pH 8 and 100 μl of 50 mm phosphate-buffered saline containing 0.01% CaCl2, 0.01% MgCl2·6H2O, and 0.1% glucose. Then 10 μl of 100% trichloroacetic acid was added. The protein precipitate was removed by centrifugation at 3000 × g for 5 min and aliquots were analyzed for cysteine-bimane adduct by HPLC. The HPLC separation was achieved on a steel column (4.6 × 100 mm) packed with 3-μm octadodecylsilica reversed-phase material. The fluorescence at 480 nm was monitored with excitation at 394 nm. The elution was performed with 9% (v/v) acetonitrile in 0.25% (v/v) acetic acid, pH 3.7 for 8 min, and then with 75% (v/v) acetonitrile in water for 5 min. The was 1 throughout the The mouse cDNAs for xCT, and were as The were labeled using and system of was and as described previously (11Sato H. Tamba M. Ishii T. Bannai S. J. Biol. Chem. 1999; 274: 11455-11458Abstract Full Text Full Text PDF PubMed Scopus (802) Google Scholar). was using mouse xCT cDNA as a The were and of the that 1 and the 5′-flanking region of the was The site was determined using a system for of cDNA and System the The extracts were by a based on the method of J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). cells × were washed with phosphate-buffered saline and collected by The cell were in ml of (10 mm pH 10 2 mm mm and incubated for 15 min on The cells were for after of then and the was in μl of mm pH 50 mm mm 10% a incubation on by a proteins were from the were performed as described previously using to the NF-κB site N. J. Biol. Med. 1998; PubMed Scopus (49) Google Scholar). The were incubated with 3 of protein under the binding conditions (10 mm pH 100 mm 5 mm 1 mm 1 mm 2 of and 5% in a of The were at 4 °C for 15 min and were on The were analyzed by The activity of cystine transport was measured in mouse peritoneal macrophages cultured under oxygen in the or presence of LPS transport activity was induced by LPS in an oxygen reduced the activity of cystine other amino acid in a we investigated the of oxygen tension on arginine and transport activity arginine transport was induced markedly by the low concentrations of LPS, as reported previously H. Fujiwara M. Bannai S. J. Leukoc. Biol. 1992; 52: 161-164Crossref PubMed Scopus (49) Google the induction of transport was by LPS oxygen tension had on the activity of of oxygen on the uptake of arginine and by macrophages incubated with LPS. Macrophages were incubated under for 1 and the medium was replaced by medium with or 1 ng/ml LPS. The cells were incubated under or for and the rates of uptake of mm andl-14Cserine were the The expression of xCT and 4F2hc by LPS was investigated in macrophages cultured under and 2% O2 shown in under expression of xCT was not in the cells in the of LPS, but three xCT and from cells treated with 1 ng/ml LPS were These may or a of (11Sato H. Tamba M. Ishii T. Bannai S. J. Biol. Chem. 1999; 274: 11455-11458Abstract Full Text Full Text PDF PubMed Scopus (802) Google Scholar). The expression of these was in cells treated with ng/ml LPS. These results are consistent with the that the induction of the activity of system xc− is in macrophages treated with 1 ng/ml (5Sato H. Fujiwara K. Sagara J. Bannai S. Biochem. J. 1995; 310: 547-551Crossref PubMed Scopus (105) Google Scholar). The expression of xCT in cells treated with 1 ng/ml LPS under was than that in the cells treated with the of LPS under The expression of xCT in the cells treated with ng/ml under was 3 the expression of xCT and 4F2hc in cells cultured with 1 ng/ml LPS under and The expression of xCT was not in the macrophages, but expression increased levels after h of culture under of xCT in cells cultured under was increased a time although expression was than that in cells cultured under The expression of 4F2hc also increased during culture with LPS, but in oxygen tension had negligible effects on These results suggest that the activity of cystine transport in the cells cultured with LPS under is caused in by the of xCT We have the xCT from the mouse and analyzed the of 5′-flanking shown in are several putative binding and a putative NF-κB binding We have investigated the effects of oxygen on the binding activity of NF-κB by LPS. The binding activity of NF-κB low in the macrophages treated with 1 ng/ml LPS, and a increase was the cells were treated with ng/ml LPS not the NF-κB binding activity. The of the NF-κB binding was with those of xCT that the induction of xCT by LPS and oxygen is not mediated by of NF-κB in macrophages cultured with LPS under and were incubated under for 1 and the medium was replaced by medium containing or ng/ml LPS. The cells were incubated for 1 h under or and the extracts were The was performed as described under We have previously that intracellular GSH levels are increased in the macrophages cultured with LPS (5Sato H. Fujiwara K. Sagara J. Bannai S. Biochem. J. 1995; 310: 547-551Crossref PubMed Scopus (105) Google Scholar). It is known that γ-glutamylcysteine synthetase the rate-limiting in GSH We have investigated the of LPS and oxygen on the expression of in these cells. the expression of for and xCT in the cells cultured with or LPS under normoxic or hypoxic conditions. is an which with GSH or The expression of was induced markedly by but by LPS. shown in the intracellular GSH in cells cultured under for h was than the in cells cultured under These results are consistent with the results that the cystine transport activity in the cells treated with LPS is under In with GSH levels in cells treated LPS under the GSH was in cells treated with LPS. contrast, the GSH in cells cultured under for h with which for system xc− and were increased GSH levels in macrophages incubated with and LPS under hypoxic and normoxic conditions. Macrophages were incubated under for 1 and the intracellular GSH was For other the medium was replaced by medium containing 100 or 1 ng/ml LPS (LPS). The cells were incubated under or for 3 and and intracellular GSH levels were the It is of importance to the intracellular cysteine, which is a rate-limiting substrate for GSH in response to induction of cystine transport activity. We investigated the intracellular cysteine levels in the cells cultured with or LPS under or The intracellular cysteine paralleled the activity of system xc−, the that the induction of system xc− activity is for increased It be that LPS and/or oxygen the efflux of GSH from the and this for increased intracellular as shown in the of efflux of GSH was in the cells cultured with LPS than that in the cells cultured LPS. The of the efflux the intracellular GSH because the efflux of GSH is and on the intracellular GSH levels J. N. Bannai S. J. Neurochem. 1996; 66: PubMed Scopus Google Scholar). is that the GSH efflux system is affected by LPS and/or the present that the in intracellular GSH levels caused by LPS is dependent on the induction of xCT but not contrast, the increased intracellular GSH levels caused by are to an induction of xCT and It is that GSH levels in the cells treated with for 4 h. initial of intracellular GSH levels seems to be the of an enzymatic GSH and by PubMed Scopus Google of LPS on GSH efflux in macrophages incubated under hypoxic and normoxic conditions. Macrophages were incubated under for 1 and the medium was replaced by medium containing 1 ng/ml LPS (LPS). The cells were incubated under or for and efflux of GSH was measured as described under the In a variety of of cells in the intracellular GSH is by the activity of system xc− (7Bannai S. Tateishi N. J. Membrane Biol. 1986; 89: 1-8Crossref PubMed Scopus (310) Google Scholar). is known to be the rate-limiting in GSH synthesis and also GSH The is induced by stress including the M. J. PubMed Scopus (49) Google Scholar). also the activity of system xc− (5Sato H. Fujiwara K. Sagara J. Bannai S. Biochem. J. 1995; 310: 547-551Crossref PubMed Scopus (105) Google Scholar). the increase in intracellular GSH caused by is most likely caused by the induction of and system xc−. In the present study, we have that LPS and oxygen induce the expression of xCT The intracellular cysteine was increased with the increase in cystine transport activity. The increase in the intracellular GSH by LPS the increase in intracellular The increase in GSH in the macrophages exposed to LPS is dependent on the induced activity of system xc−, not the activity of The results of the present that the induced activity of system xc− by LPS is enhanced by It is likely that the activity of system xc− is low in macrophages in because oxygen tension in the is J. Physiol. PubMed Scopus Google Scholar). these cells are activated by the activity of system and oxygen or oxygen enhances the GSH the oxygen or oxidative is likely that GSH rapidly in the macrophages exposed to and oxidative stress in a such as The response of system xc− activity to LPS and oxygen may be to the macrophages, which are the Recently, H. A.R. J. Physiol. 1999; PubMed Google Scholar) have that of bovine cells to a increase in cystine transport activity and that the increased cystine transport activity is mediated by system xc−. It be that the induction of xCT in macrophages by LPS is mediated by by the cells. as we previously T. Fujiwara M. Sato H. Fujiwara K. Abe K. Bannai S. Biochim. Biophys. Acta. 1996; 1311: 150-154Crossref PubMed Scopus (19) Google production by the macrophages incubated with 1 ng/ml LPS was the activity of system xc− by 1 ng/ml LPS. the of in the induction of xCT under the conditions in the present is We have identified several binding in the 5′-flanking region of the xCT also in the 5′-flanking region of the 4F2hc T. C.B. J.M. Biol. PubMed Scopus Google Scholar). These may be involved in the of the expression of xCT and induction of system by is very low in with LPS under (5Sato H. Fujiwara K. Sagara J. Bannai S. Biochem. J. 1995; 310: 547-551Crossref PubMed Scopus (105) Google a for in the of xCT by LPS. We have also found that is a putative NF-κB binding site in the 5′-flanking NF-κB a in the of involved in defense and expression of oxygen have been as the involved in the activation of NF-κB by and R. P. J. 1991; PubMed Scopus Google Scholar). Recent studies have that the LPS is mediated by the 4 in the presence of and NF-κB in macrophages K. T. H. H. T. Takeda K. S. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). NF-κB is of the factors involved in the induction of xCT by LPS and in the presence of LPS, the activation of NF-κB from the macrophages cultured under to be to that from the cells cultured under These results are with those of the uptake of In NF-κB was activated by 1 ng/ml LPS, whereas induction of system xc− activity was activated by 1 ng/ml (5Sato H. Fujiwara K. Sagara J. Bannai S. Biochem. J. 1995; 310: 547-551Crossref PubMed Scopus (105) Google Scholar). In contrast, NF-κB was activated by ng/ml LPS, expression of xCT in cells treated with ng/ml LPS was than that in the cells treated with 1 ng/ml LPS. These results suggest that other of the involved in the induction of xCT by LPS and although is not at present which is involved in the induction of xCT by LPS and using mice have that expression of is by activity is induced by oxidative stress and K. T. S. Ishii T. K. Y. T. N. K. M. Y. Biochem. Biophys. PubMed Scopus Google Scholar). is to to the electrophile response element in the 5′-flanking region of these and to the activity of The for contains the in 5′-flanking and this region is for expression of by the J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We have found that the mouse also contains an in 5′-flanking region that expression is by the in a to that for Recently, we have shown that in peritoneal macrophages from LPS the activity of system xc−, whereas not T. K. S. Sato H. T. Y. Bannai S. M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). that the by LPS is by a different mechanism from expression of xCT seems to be an system for of from LPS, because the expression is by a concentration of LPS and is We J. K. for the cDNA for mouse