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Originally published online as doi:10.1189/jlb.0606385 on January 2, 2007

Published online before print January 2, 2007
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(Journal of Leukocyte Biology. 2007;81:974-982.)
© 2007 by Society for Leukocyte Biology

Expression and function of cystine/glutamate transporter in neutrophils

Yuki Sakakura*,{dagger}, Hideyo Sato{ddagger},1, Ayako Shiiya*, Michiko Tamba*, Jun-ichi Sagara§, Manabu Matsuda*, Naomichi Okamura*, Nobuo Makino{dagger} and Shiro Bannai*

* Department of Biochemistry, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan;
{ddagger} Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata, Japan; and Centers for
§ Medical Science and
{dagger} Humanity and Health Sciences, Ibaraki Prefectural University of Health Sciences, Ami, Ibaraki, Japan

1 Correspondence: Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan. E-mail: shideyo{at}tds1.tr.yamagata-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reactive oxygen species (ROS) produced by neutrophils are essential in the host defense against infections but may be harmful to neutrophils themselves. Glutathione (GSH) plays a pivotal role in protecting cells against ROS-mediated oxidant injury. Cystine/glutamate transporter, designated as system xc– and consisting of two proteins, xCT and 4F2hc, is important to maintain GSH levels in mammalian-cultured cells. In the present paper, we have investigated system xc– in neutrophils. In human peripheral blood neutrophils, neither the activity of system xc– nor xCT mRNA was detected. The activity was induced, and xCT mRNA was expressed when they were cultured in vitro. The mRNA expression was much enhanced in the presence of opsonized zymosan or PMA. In contrast, mouse peritoneal exudate neutrophils, immediately after preparation, exhibited system xc– activity and expressed xCT mRNA. The activity and the expression were heightened further when they were cultured. Peritoneal exudate cells (mostly neutrophils) from xCT-deficient (xCT–/–) mice had lower cysteine content than those from the wild-type mice. GSH levels in the xCT–/–cells decreased rapidly when they were cultured, whereas those in the wild-type cells were maintained during the culture. Apoptosis induced in culture was enhanced in the xCT–/–cells compared with the wild-type cells. These results suggest that system xc– plays an important role in neutrophils when they are activated, and their GSH consumption is accelerated.

Key Words: glutathione • redox • superoxide • apoptosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils are short-lived polymorphonuclear leukocytes, which play an important role in immune responses to bacteria, yeast, and fungi. When the microorganisms invade a living body, neutrophils migrate into the site of inflammation to kill the microorganisms by reactive oxygen species (ROS), including superoxide anion produced by NADPH oxidase, hydrogen peroxide by superoxide dismutase (SOD), and hypochlorous acid by myeloperoxidase [1 ]. As neutrophils generate a large amount of ROS, they might need antioxidants to protect themselves against oxidative injury while performing their antimicrobial or inflammatory activities. The lifespan of the neutrophils is regulated by endogenous factors and extracellular stimuli, which in turn, determine the length of an inflammatory process. Neutrophil apoptosis occurs at the site of inflammation and is instigated by the production of ROS [2 , 3 ]. Spontaneous neutrophil apoptosis can be accelerated by oxidative stress and thiol depletion [4 , 5 ].

Glutathione (GSH) provides a major intracellular defense against ROS from the inflammatory production and is consumed in the reaction with ROS [6 7 8 9 ]. Cysteine is a rate-limiting precursor in GSH synthesis in cultured cells, and its availability is a regulatory factor for GSH synthesis [10 ]. However, cysteine autooxidizes easily to cystine in extracellular fluid. We have found a Na+-independent cystine/glutamate exchange transport system, designated as system xc–, in various cultured cells such as human fibroblasts and mouse peritoneal macrophages [11 , 12 ]. Cystine taken up by the cells via this system is reduced rapidly to cysteine. Thus, the GSH level in the cells is regulated by system xc– activity. System xc– is composed of two protein components, xCT and 4F2hc, and the transport activity is thought to be mediated by xCT [13 ]. The constitutive expression of the xCT gene was found in the brain but not in lung, liver, and kidney [13 ]. The activity of system xc– is highly inducible. In cultured cells, it is induced by various stimuli, including electrophilic agents such as diethyl maleate [14 ], oxygen [15 ], and bacterial LPS [16 ]. We have demonstrated that the induction of xCT by diethyl maleate is regulated by the antioxidant response element, also referred to as the electrophile response element, located in the 5'-flanking region of the xCT gene [14 ]. The transcription factor Nrf2 binds to this element to enhance the transcription of the xCT gene. Exposure of the cells to electrophilic agents or ROS results in the translocation of Nrf2 to nuclei and the transcriptional activation of genes having an antioxidant response element [17 ]. Thus, the induction of the activity of system xc– is thought to be an important protective mechanism against the oxidative stress.

In this paper, we investigate system xc– in neutrophils from blood and peritoneal exudate. Recently, we have described the generation and initial characterization of xCT gene-deficient (xCT–/–) mice and found that they are apparently healthy for at least several months after birth [18 ]. However, it has been shown that system xc– contributes to maintaining the plasma cystine/cysteine redox balance and GSH level in vivo. We examine here the peritoneal exudate cells (PEC), mostly neutrophils, prepared from xCT–/– mice to clarify a possible role of system xc– in neutrophils.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
L-[14C]Cystine was obtained from PerkinElmer Life and Analytical Sciences Inc. (Wellesley, MA, USA). PMA and zymosan A were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Mono-Poly resolving medium was purchased from Dainippon Pharmaceutical Co. (Osaka, Japan). Ficoll-Paque and Percoll were from Amersham Pharmacia (Uppsala, Sweden).

Animals
C57BL/6J mice used for wild-type were purchased from Charles River Laboratories Japan Inc. (Yokohama). xCT–/– mice of 129/Svj-C57BL/6J mixed genetic background were as described previously [18 ]. In some experiments, littermate controls, designated xCT+/+, were used as wild-type. The Institutional Review Board of University of Tsukuba (Japan) approved all animal studies.

Isolation of human peripheral blood neutrophils
Fresh heparinized blood was obtained from healthy volunteers after they had given informed consent. Neutrophils were isolated using Mono-Poly resolving medium by following the manufacturer’s instructions. The isolated fraction contained more than 90% neutrophils, as estimated by Giemsa stains. The neutrophils were washed with PBS [10 mM PBS (137 mM NaCl, 3 mM KCl), pH 7.4] and resuspended at 1 x 106 cells/ml in RPMI-1640 medium supplemented with 10% FBS, 50 units/ml penicillin, and 50 µg/ml streptomycin. The cells were used immediately for the experiments or incubated in polypropylene tubes at 37°C in 5% CO2 in air for 2–10 h.

Isolation of mouse blood neutrophils
Heparinized blood was obtained by cardiac puncture under pentobarbital anesthesia, and neutrophils were purified by the methods of Mizuno et al. [19 ]. Blood was mixed with an equal volume of prewarmed plasma gel, which was prepared by dissolving 3 g gelatin, 0.7 g NaCl, and 0.2 g CaCl2 in 100 ml distilled water. The mixture was allowed to stand until erythrocytes settled. The leukocyte-rich supernatant was transferred to another tube, centrifuged, and washed with MEM. The pellet was suspended in MEM, overlaid onto the same volume of Ficoll-Paque, and centrifuged at 800 g for 10 min at 4°C. The pellet contains neutrophils and remaining erythrocytes, which were lysed with 0.144 M NH4Cl, 17 mM Tris-HCl, pH 7.2, and washed with PBS. The average purity of neutrophils was over 93%, as estimated by Giemsa stains. The cells were collected and used immediately for the experiments.

Preparation of mouse PEC and purification of neutrophils from the PEC
Mouse PEC were collected by peritoneal lavage from mice 16 h after the i.p. injection of 2 ml 4% thioglycollate broth. The lavage medium was RPMI 1640 containing 10 U/ml heparin. The cells obtained from two to three mice were collected into a tube, washed with RPMI 1640, and suspended at 1 x 106 cells/ml in RPMI 1640 containing 10% FBS, 50 units/ml penicillin, and 50 µg/ml streptomycin. The percentage of neutrophils in the PEC was ~70%. The cells were used immediately or incubated at 37°C in 5% CO2 in air for various periods of time indicated.

Neutrophil purification from PEC was performed by Percoll density gradient [20 ]. Nine volumes of Percoll was mixed with 1 vol 10x concentrated Ca++-, Mg++-free HBSS. The solution was diluted further with single-strength Ca++-, Mg++-free HBSS supplemented with 10 mM HEPES to 81%, 70%, 55%, and 50% Percoll. These solutions were of 1.1002, 1.0871, 1.0693, and 1.0643 g/ml density, respectively. Each solution was layered into a 15-ml centrifuge tube successively, 3, 2, 2, and 2 ml with Pasteur pipettes. PEC were diluted in 3 ml 45% Percoll solution (1.0575 g/ml density) and layered on the top of the gradient. Tubes were centrifuged at 1600 g for 30 min at 10°C. The cell bands formed between the 55% and 81% layer were harvested, and the cells were washed and suspended at 1 x 106 cells/ml in RPMI 1640 containing 10% FBS, 50 units/ml penicillin, and 50 µg/ml streptomycin. More than 92% of the cells were neutrophils. They were used immediately or incubated at 37°C in 5% CO2 in air for 6–10 h.

Stimulus
To stimulate oxidative metabolism, opsonized zymosan or PMA was used. Opsonized zymosan was prepared by treating zymosan A with fresh mouse serum for 1 h at 37°C, and it was added to the cells at 50 µg per ml cell suspension.

Uptake of cystine
The activity of cystine transport was measured as described previously [12 ]. Briefly, the cells were collected by centrifugation, washed with PBS containing 0.01% CaCl2, 0.01% MgCl2·6H2O, and 0.1% glucose (PBSG), and suspended at 2 x 106 cells per 0.125 ml in prewarmed PBSG. Uptake was started by addition of 0.025 ml PBSG containing L-[14C]cystine (0.3 mM and 0.1 µCi/0.025 ml) to 0.125 ml of the cell suspension, which was incubated at 37°C for specified time periods with shaking. Then 0.1 ml of the suspension was removed and layered on a mixture (0.2 ml) of mineral oil and dibutyl phthalate (15:85 by volume) in microtubes. Cells were separated rapidly from the medium by centrifugation for 10 s, and the radioactivity in the cell pellet was measured. Cystine uptake was determined under conditions approaching initial rates of uptake, i.e., measuring uptake for cystine at 2 min, and the uptake of cystine increased linearly with time during this incubation.

RT-PCR analysis
Cells were collected by centrifugation, and total RNA was isolated using Isogen (Nippongene, Toyama, Japan) by following the manufacturer’s instructions. The first-strand cDNA was synthesized using the Superscript II first-strand synthesis system for RT-PCR (Invitrogen Corp., Carlsbad, CA, USA). The cDNA product was amplified using the primer sets 5'-GCTCATTACAGCTGTGGG-3' and 5'- CCAATGGTGACAATGGCC-3' for xCT and 5'-GACCCCTTCATTGACCT-3' and 5'-CCACCACCCTGTTGCTGT-3' for GAPDH.

Northern blot analysis
The RNA probes for mouse xCT and {gamma}-glutamylcysteine synthetase ({gamma}GCS) heavy subunit were digoxigenin (DIG)-labeled by transcription from the linearized plasmid using RNA-labeling mix (Roche Diagnostics, Basel, Switzerland) and T3/T7 RNA polymerase (Stratagene, La Jolla, CA, USA). Total RNA (3 µg) isolated from the cells as described above was electrophoresed on a 1% agarose gel in the presence of 2.2 M formaldehyde, transferred onto a positively charged nylon membrane, and hybridized with the DIG-labeled RNA probes in DIG Easy Hyb (Roche Diagnostics) for 16 h at 68°C. The membrane was washed twice for 5 min at room temperature with 1x SSC, 0.1% SDS, and then washed twice for 15 min at 68°C with 0.1x SSC, 0.1% SDS.

Measurement of intracellular cysteine and GSH
The cysteine content in the cells was determined by the method of Cotgreave and Moldéus [21 ] with a slight modification. The cells were rinsedthree times with 20 mM HEPES-buffered saline (137 mM NaCl, 3 mM KCl), pH 7.4, containing 0.01% CaCl2, 0.01% MgCl2·6H2O, and 0.1% glucose, and incubated in the dark at room temperature for 10 min with 100 µl 8 mM monobromobimane in 50 mM N-ethylmorpholine, pH 8, and 100 µl 50 mM HEPES saline containing 0.01% CaCl2, 0.01% MgCl2·6H2O, and 0.1% glucose. Then, 10 µl 100% trichloroacetic acid was added. The protein precipitate was removed by centrifugation at 3000 g for 5 min, and a cysteine-bimane adduct in the supernatant was analyzed by HPLC, and HPLC separation was achieved on a steel column (4.6x100 mm) packed with 3 µm octadodecylsilica reversed-phase material. The fluorescence at 480 nm was monitored with the excitation at 394 nm. The elution was performed with 9% (vol/vol) acetonitrile in 0.25% (vol/vol) acetic acid, pH 3.7, for 8 min. The flow rate was 1 ml/min throughout the process.

Total GSH [GSH+oxidized GSH (GSSG)] was extracted with 5% trichloroacetic acid and measured using the enzymatic method [22 ], which is based on the catalytic action of GSH in the reduction of 5,5'-dithiobis(2-nitrobenzoic acid) by the GSH reductase system. Total GSH extracted from the cells was mostly GSH, and the content of GSSG was negligibly low throughout the experiments in this study.

Assessment of apoptosis
The activity of caspase 3 in lysate of 1 x 106 cells was measured using Caspase-3/CPP32 fluorometric assay kit (BioVision, Mountain View, CA, USA). The activity of caspase 3 was expressed as the mean fluorescence intensity per 1 x 106 cells. Assessment of the percentage of cells showing morphology of apoptosis was performed with the cytospin preparations stained with Giemsa [23 ].

Assay for superoxide generation
Superoxide-generating activity was evaluated by cytochrome c reduction assay, and the assay mixtures consisted of 1 x 106 cells/ml, 20 µM cytochrome c, and 0.2 µM PMA, with or without 20 µg/ml SOD in a total volume of 1 ml HBSS. After incubation at 37°C for 10 min, the reactions were terminated by cooling the samples on ice. The samples were centrifuged at 10,000 g for 5 min, and the absorbance at 550 nm of the supernatant was measured. SOD-inhibitable cytochrome c reduction was measured from the absorbance difference at 550 nm, and the amount of superoxide was calculated using an absorption coefficient of 21,000 M–1 cm–1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
System xc– in human peripheral blood neutrophils
The system xc– activity is characterized by cystine transport into the cell, which is inhibitable by the extracellular glutamate. We investigated the activity of cystine transport in freshly isolated human peripheral blood neutrophils immediately after preparation and those cultured for 10 h. As shown in Figure 1A , the activity of cystine transport was weak in the freshly isolated cells and was not inhibited by glutamate, indicating that the weak cystine transport is not mediated by system xc–. In the cells cultured for 10 h, the activity of cystine transport was raised slightly, and the raised portion was inhibited by glutamate. The activity of system xc– seems to be induced during the culture in these cells. The expression of xCT mRNA in human peripheral blood neutrophils was examined by RT-PCR (Fig. 1B) . xCT mRNA in the freshly isolated cells was not detected. However, xCT mRNA was expressed in a time-dependent manner in the cells cultured for 2–8 h. When the neutrophils were activated by opsonized zymosan or by PMA, xCT mRNA expression was enhanced much. Activity of cystine transport in the cells treated with zymosan and PMA could not be measured, as the zymosan particle interfered with the transport assay, and the cells were aggregated and loosely adhered to the culture vessels in the presence of PMA.


Figure 1
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  		Figure 1. System xc– in human peripheral blood neutrophils. (A) The activity of cystine uptake in human peripheral blood neutrophils. Rates of uptake of 0.05 mM L-[14C]cystine were measured in neutrophils cultured for 0 h or for 10 h. Open and solid bars represent the rates of uptake of cystine measured in the absence or presence of 2.5 mM glutamate, respectively. Data represent the means ± SD (n=5). *, P < 0.05 (relative to 0 h in culture in the absence of glutamate). (B) Time-dependent expression of xCT mRNA in human peripheral blood neutrophils, which were cultured for 0–8 h in the absence (Cont) or presence of 50 µg/ml opsonized zymosan (Zym) or 1 µM PMA, and xCT mRNA was detected by RT-PCR in a semiquantitative manner. The figure shows the ethidium bromide-stained agarose eletrophoresis of the PCR products using the primer sets corresponding to the sequence of xCT (upper) and GAPDH (lower). Results shown are representative of three independent experiments.

 
System xc– in mouse neutrophils
The activity of cystine transport was measured in mouse PEC, in which the percentage of neutrophils was ~70%, and in the purified neutrophils from the PEC (Fig. 2A ). In freshly prepared PEC, immediately after preparation, the rate of uptake of cystine was one order of magnitude higher than in the human peripheral blood neutrophils (0.087 vs. 0.0092 nmol/mg protein) and inhibited strongly by glutamate. This means that the mouse PEC in vivo have a significant system xc– activity. When the PEC were cultured for 10 h, the activity increased. We also investigated the activity of system xc– in purified neutrophils from PEC. The activity of system xc– in purified neutrophils was significantly higher than in PEC, indicating that the cells other than neutrophils in PEC have little, if any, activity of system xc– in vivo. Then, the expression of xCT mRNA was investigated by Northern blot analysis. As shown in Figure 2B , the expression of xCT mRNA was detected in the freshly prepared PEC and enhanced by culturing for 6 h. In these cells, xCT mRNA was induced further by culturing the cells with opsonized zymosan or with PMA for 6 h. Conversely, xCT mRNA was not detected in mouse blood neutrophils. The results suggest that neutrophils express xCT mRNA and acquire system xc– activity after they are elicited into the peritoneal cavity.


Figure 2
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  		Figure 2. System xc– in mouse blood neutrophils and peritoneal exudate neutrophils. (A) The activity of cystine uptake in mouse PEC and purified neutrophils from PEC. Rates of uptake of 0.05 mM L-[14C]cystine were measured in the cells cultured for 0 h or for 10 h. Open and solid bars represent the rates of uptake of cystine measured in the absence or presence of 2.5 mM glutamate, respectively. Data represent the means ± SD (n=4–6). *, P < 0.01 (relative to 0 h in culture in the absence of glutamate). (B) Expression of xCT mRNA in mouse blood neutrophils, PEC, and purified neutrophils from PEC. Cells were cultured in the absence (–) or presence of 50 µg/ml opsonized zymosan (+Zym) or 1 µM PMA (+PMA) for 0 h or for 6 h, and total RNA was isolated. Northern blot analysis was performed using the DIG-labeled RNA probe for mouse xCT cDNA. Blots shown are for xCT mRNA of 12 kb and are representative of three independent determinations.

 
Cysteine and GSH levels in neutrophils derived from xCT–/– mice
Recently, we have generated the xCT–/– mice [18 ]. To investigate the role of xCT in neutrophils, we have prepared the PEC from wild-type and xCT–/– mice. There was no significant difference in the yield of PEC and the percentage of neutrophils in PEC between wild-type [both from C57BL/6J and from littermate controls (xCT+/+)] and xCT–/– mice. Glutamate-inhibitable cystine transport activity, i.e., system xc– activity, was not detected in the PEC from xCT–/– mice, and xCT mRNA was not detected by Northern blot analysis, regardless of culturing with zymosan (Fig. 3 ). The results demonstrate clearly the deficiency of system xc– in the PEC from xCT–/– mice. There was no difference in the system xc– activity between PEC from wild-type C57BL/6J mice and from wild-type littermates of xCT–/– mice (data not shown). Intracellular cysteine content could be a rate-limiting factor for the GSH synthesis, and in many types of cultured cells, it depends on the activity of cystine transport via system xc– [10 ]. Thus, we have investigated the intracellular cysteine levels in the PEC derived from wild-type and xCT–/– mice (Fig. 4 ). In the freshly prepared cells from wild-type mice, intracellular cysteine was 1.3 ± 0.18 nmol/mg protein, and it was decreased by culturing for 8 h. However, when the cells were cultured with zymosan or PMA, intracellular cysteine did not decrease in the presence of PMA and rather, increased in the presence of zymosan, probably as xCT is induced by these stimuli. In the freshly prepared cells from xCT–/– mice, intracellular cysteine was 0.5 ± 0.17 nmol/mg protein and decreased severely by culturing, regardless of the presence of zymosan or PMA. It is noteworthy that the intracellular cysteine in the freshly prepared PEC from xCT–/– mice was significantly lower than that from wild-type mice. Figure 5 demonstrates the GSH levels in the PEC derived from wild-type and xCT–/– mice. In the cells from wild-type mice, GSH levels decreased slightly at 1 h and increased after that. In the presence of zymosan or PMA, GSH levels increased more rapidly. It is likely that the increase in GSH levels was a result of the induction of system xc–. Conversely, in the cells from xCT–/– mice, the initial level of intracellular GSH was similar to that in the cells from wild-type mice, but GSH levels decreased rapidly in a time-dependent manner.


Figure 3
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  		Figure 3. Deficiency of system xc– in PEC prepared from xCT–/– mice. (A) The activity of cystine uptake in PEC from wild-type and xCT–/– mice. Rates of uptake of 0.05 mM L-[14C]cystine were measured in the freshly prepared cells. Open and solid bars represent the rates of cystine uptake measured in the absence or presence of 2.5 mM glutamate, respectively. Data represent the means ± SD (n=4). *, P < 0.01 (relative to wild-type in the absence of glutamate). (B) Expression of xCT mRNA in PEC from wild-type and xCT–/– mice. Cells were cultured in the absence (–) or presence of 50 µg/ml opsonized zymosan (+Zym) for 0 h or for 6 h, and total RNA was isolated. Northern blot analysis was performed using the DIG-labeled RNA probe for xCT cDNA. Blots shown are for xCT mRNA of 12 kb and are representative of three independent determinations.

 

Figure 4
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  		Figure 4. Intracellular cysteine levels in PEC prepared from wild-type (A) and xCT–/– (B) mice. PEC were cultured for 0 h or for 8 h in the absence (open bars) or presence of 50 µg/ml opsonized zymosan (solid bars) or 1 µM PMA (hatched bars), and intracellular cysteine levels were measured by HPLC. Data represent the means ± SD (n=4–7). *, P < 0.01 (relative to 0 h in culture), and #, P < 0.01 (relative to 8 h in culture in the absence of the stimulant).

 

Figure 5
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  		Figure 5. Changes in intracellular GSH levels during culture in PEC prepared from wild-type (A) and xCT–/– (B) mice. PEC were cultured in the absence ({circ}) or presence of 50 µg/ml opsonized zymosan ({blacksquare}) or 1 µM PMA ({blacktriangleup}), and after the time indicated, the intracellular GSH levels were measured. Data represent the means ± SD (n=4–6). *, P< 0.01 (relative to 0 h in culture), and #, P < 0.05 (relative to the same hour in culture in the absence of the stimulant).

 
Although the initial level of intracellular cysteine in the cells from xCT–/– mice was lower than that of wild-type, there was no difference in GSH levels between these cells. {gamma}GCS is a rate-limiting enzyme for GSH synthesis, and its expression was shown to be down-regulated by cysteine in hepatocytes [24 ]. We have investigated the expression of {gamma}GCS mRNA in the PEC from wild-type and xCT–/– mice by Northern blot analysis (Fig. 6 ). The expression of {gamma}GCS mRNA was detected in the freshly prepared PEC, slightly increased by culturing for 6 h, and greatly enhanced when the cells were activated by opsonized zymosan or PMA. In any of the cases examined, {gamma}GCS mRNA expression is much higher in the cells from xCT–/– than those from wild-type.


Figure 6
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  		Figure 6. Expression of {gamma}GCS mRNA in PEC prepared from wild-type and xCT–/– mice. PEC were cultured in the absence (–) or presence of 50 µg/ml opsonized zymosan (+Zym) or 1 µM PMA (+PMA) for 0 h or for 6 h, and total RNA was isolated. Northern blot analysis was performed using the DIG-labeled RNA probe for mouse {gamma}GCS cDNA. Blots shown are representative of three independent determinations.

 
Apoptosis and superoxide production in neutrophils derived from xCT–/– mice
To determine the effect of xCT gene deficiency on the neutrophil apoptosis, caspase 3 activity was analyzed in the PEC prepared from littermate wild-type (xCT+/+) and xCT–/– mice (Fig. 7 ). The activity of caspase 3 was measured by the cleavage of the fluorogenic substrate Asp-Glu-Val-Asp-amino-trifluoromethylcoumarin in a fluorometric assay. In the freshly prepared cells, no significant difference in the activity was observed in wild-type and xCT–/– cells. However, when the cells were cultured with and without zymosan for 6 h, the activity of caspase 3 was increased and significantly higher in xCT–/– cells than in wild-type cells. In the presence of BSO, an inhibitor of GSH synthesis, the activity of caspase 3 in wild-type cells increased and reached the level close to that in xCT–/– cells. These findings show that apoptosis is more accelerated in xCT–/– cells than in wild-type cells during the culture in vitro, probably as a result of the decrease in GSH. The percentage of cells undergoing apoptosis was examined. The proportion of cells showing typical features of apoptosis by Giemsa stains was 7.4% and 9.0% in freshly prepared PEC from xCT+/+ and xCT–/– mice, respectively, with no significant difference, and 25.3% and 30.0% in 6 h-cultured PEC from xCT+/+ and xCT–/– mice, respectively, with significant difference (P<0.05).


Figure 7
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  		Figure 7. Caspase 3 activity in PEC prepared from xCT+/+ and xCT–/– mice. PEC from xCT+/+ (open bars) and xCT–/– (solid bars) mice were cultured for 0 h or for 6 h in the absence or presence of 50 µg/ml opsonized zymosan (+Zym) or 0.1 mM buthionine sulfoximine (+BSO). Caspase 3 activity in 1 x 106 cells was measured. Data represent the means ± SD (n=6). *, P < 0.05 (relative to the corresponding xCT+/+), and #, P < 0.05 (relative to 6 h in culture of the same cell type without additives).

 
Finally, we measured the PMA-stimulated superoxide generation by the PEC prepared from littermate wild-type (xCT+/+) and xCT–/– mice (Fig. 8 ). There is no significant difference in the superoxide generation by freshly prepared PEC derived from wild-type and xCT–/–. When the cells were cultured for 6 h, the superoxide generation was reduced, but the generation by xCT–/– cells was more reduced compared with wild-type cells. In the presence of BSO, the amounts of superoxide generated by wild-type cells was decreased further and became similar to those by xCT–/– cells, suggesting that the depletion of GSH in xCT–/– cells in culture accounts for their deteriorated generation of superoxide compared with wild-type cells. BSO had no effect on the superoxide generation by xCT–/– cells, most probably, as they are deprived of cysteine.


Figure 8
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  		Figure 8. Generation of superoxide by PEC prepared from xCT+/+ and xCT–/– mice. PEC from xCT+/+ (open bars) and xCT–/– (solid bars) mice were cultured for 0 h or for 6 h in the absence (–) or presence of 0.1 mM BSO (+BSO), and superoxide generation was determined. Data represent the means ± SD (n=6). *, P < 0.01 (relative to the corresponding xCT+/+), and #, P < 0.05 (relative to 6 h in culture of the same cell type without BSO).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we have investigated the activity of system xc– and expression of xCT mRNA in blood neutrophils and peritoneal exudate neutrophils. In freshly isolated human peripheral blood neutrophils, neither the activity of system xc– nor the expression of xCT mRNA was detected. The activity was induced significantly when the neutrophils were cultured. In addition, xCT mRNA was detected in the cultured neutrophils, particularly in the presence of opsonized zymosan or PMA. It should be noted that cells in routine culture, i.e., under atmospheric oxygen, are exposed to oxidative stress more heavily than those in vivo. We also showed that xCT mRNA was not detected in freshly isolated mouse blood neutrophils. However, in freshly prepared mouse PEC and neutrophils purified from the PEC, the activity of system xc– and the expression of xCT mRNA were detected without culturing them. In the present experiments, PEC were taken from mice 16 h after i.p. injection of thioglycollate broth, and the percentage for neutrophils in the PEC was ~70%, which is close to that reported by Nathan et al. [25 ]. Taken together, the results suggest that the activity of system xc– is induced when the neutrophils migrate from the blood vessel into the site of inflammation, where they may be exposed to stronger oxidative stress than in blood.

GSH, a natural thiol antioxidant, maintains the reduced state in the cells and protects against oxidative damage of the cell. GSH is synthesized by two consecutive ATP-dependent reactions. {gamma}GCS catalyzes the first and rate-limiting step, forming {gamma}-glutamylcysteine from glutamate and cysteine. This is followed by the GSH synthetase-catalyzed reaction, which binds glycine to {gamma}-glutamylcysteine, forming GSH. Protective action of GSH is based on the oxidation of its cysteine residue with the formation of GSSG, which is catalytically reduced back to the thiol form by GSH reductase or effluxed from the cells. There are many reports that activation of neutrophil causes a decrease in their GSH concentration [6 7 8 9 ]. In the activated neutrophils, GSH is consumed rapidly, because of their production of ROS. Respiratory burst markedly accelerates NADPH consumption, as NADPH participates in the reduction of oxygen to superoxide by NADPH oxidase. The reduction of GSSG to GSH by GSH reductase also requires NADPH. Thus, deficiency in NADPH may limit the reduction of GSSG in the activated neutrophils and lead to depletion of GSH, as GSSG is extruded actively from the cells and degraded there. Cysteine is a rate-limiting precursor in GSH synthesis [10 ]. The increased activity of cystine transport via system xc– shown in the present study augments the supply of precursor cysteine for GSH synthesis in the neutrophils. We have also shown that {gamma}GCS is induced by culturing PEC with or without zymosan or PMA (Fig. 6) . Induction of {gamma}GCS by PMA has been shown in HepG2 cells [26 ]. Both genes encoding xCT and {gamma}GCS heavy subunit have an antioxidant response element, and their expression is up-regulated by oxidant stress such as hydrogen peroxide [14 , 27 ]. It is highly likely that neutrophils compensate the loss of GSH by the induction of these proteins to combat against self-produced oxidant. Correlated expression of xCT and {gamma}GCS has been shown in a wide variety of cancer cell lines [28 ].

We have examined cysteine and GSH contents in PEC derived from wild-type and xCT–/– mice. As shown in Figure 4 , the cysteine level in the freshly prepared PEC from xCT–/– cells was significantly lower than that from the wild-type cells, indicating that the activity of system xc– contributes at least in part to the regulation of the cysteine level in PEC in vivo. In contrast, the GSH level in freshly prepared PEC from xCT–/– mice was almost the same as that from wild-type mice (Fig. 5) . Thus, the question arises why the GSH levels in xCT–/– cells and in wild-type cells are similar despite the difference in their cysteine levels. As shown in Figure 6 , expression of {gamma}GCS was higher in xCT–/– cells than in wild-type cells. Kwon and Stipanuk [24 ] described that the expression of {gamma}GCS is regulated by cysteine; {gamma}GCS mRNA and {gamma}GCS activity decrease by cysteine supplementation. Higher expression of {gamma}GCS in xCT–/– cells may result from lower cysteine level in these cells. In GSH synthesis in xCT–/– cells, enhanced {gamma}GCS activity may compensate for the decrease of the intracellular cysteine, and consequently, the GSH level in xCT–/– cells becomes almost the same as in wild-type cells. It is noteworthy that {gamma}GCS was induced potently when xCT–/– PEC were cultured with zymosan or PMA (Fig. 6) . Presumably, the transcription of the {gamma}GCS gene is enhanced in these cells, partly by the depletion of cysteine and partly by the strong, oxidative stress as a result of GSH depletion. Figure 5 shows that the GSH level in wild-type cells, although slightly decreased early several hours in culture, increased thereafter, particularly in the presence of zymosan or PMA. This increase is accounted for by the induction of xCT and {gamma}GCS in these cells. Conversely, the GSH level in xCT–/– cells decreased constantly during culture as a result of the severe depletion of cysteine (Fig. 4B) .

Apoptosis of neutrophils is induced when they are cultured in vitro. Coxon et al. [23 ] examined apoptosis of mouse peritoneal exudate neutrophils in culture. Short-term culture of these cells leads to the time-related morphological changes of nuclei representing the apoptotic state; 37% cells are apoptotic at 8 h in culture. In the present experiments, apoptosis was more enhanced in the cultured xCT–/– cells than in the cultured wild-type cells (Fig. 7) , suggesting that the activity of system xc– contributes to preventing neutrophil apoptosis. Caspases represent a central mechanism mediating apoptosis of neutrophils [29 ]. Freshly isolated neutrophils express pro-caspase 3, which is cleaved and activated on phagocytosis. Fas ligation is an important trigger of this caspase cascade, and Fas antibody-induced apoptosis is mediated through caspase 3 [30 ]. When the neutrophil GSH is elevated by adding GSH or N-acetylcysteine, Fas-mediated apoptosis is inhibited [31 ]. Conversely, GSH depletion by diethyl maleate induces the caspase 3-dependent apoptosis in neutrophils [4 , 5 ]. In the present experiment, the inhibition of GSH synthesis by BSO enhanced the apoptosis in wild-type cells (Fig. 7) . It is likely that compared with wild-type cells, the enhanced apoptosis in xCT–/– cells in culture results from the decreased GSH.

Freshly prepared PEC from xCT–/– and wild-type mice exhibit almost the same capacity to generate superoxide in response to PMA (Fig. 8) . The result suggests that system xc– does not contribute to superoxide generation in peritoneal exudate neutrophils in vivo. No difference in GSH contents in cells from xCT–/– and wild-type mice, immediately after preparation, (Fig. 5) , may support this view. However, when the cells were cultured, superoxide generation by xCT–/– cells and by wild-type cells was reduced, and the extent was more in the former cells. The difference between the two types of cells could be explained by the depletion of GSH in xCT–/– cells, as the GSH synthesis inhibitor (BSO) reduces superoxide generation by wild-type cells but not xCT–/– cells. Akard et al. [32 ] have shown that N-ethylmaleimide, the covalent, thiol-modifying reagent, strongly inhibits the activation of NADPH oxidase by inactivating the cytosolic cofactor required for the enzyme activation, leading to the reduction of superoxide generation by neutrophils. The present results suggest that the activation of NADPH oxidase is affected by system xc– through the alteration of the GSH level in the cytosol.

Chronic GSH deficiency has been suspected as a causative factor in a number of pathologies, including diabetes mellitus [33 ] and AIDS [34 ]. Immunological functions such as resistance to infection may be impaired by GSH depletion [35 ]. Recently, xCT has been identified with a receptor mediating Kaposi’s sarcoma-associated herpesvirus fusion entry [36 ]. Redox conditions have substantial influences during infection of this virus. We have reported redox imbalance and decreased plasma GSH level in xCT–/– mice [18 ]. The present study suggests that xCT–/– mice provide insight into the mechanisms underlying inflammatory diseases involving neutrophils.


   ACKNOWLEDGEMENTS
 
This work was supported in part by a Grant-in-Aid in Scientific Research (16590239) from Monbukagakusho, Japan. We express our gratitude to Dr. Motoka Masuda for her help in collecting blood samples.

Received June 9, 2006; revised November 2, 2006; accepted December 1, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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