The Phagocytosis-Associated Respiratory
Burst in Human Monocytes Is Associated
with Increased Uptake of Glutathione
This information is current as
of June 17, 2017.
Tamas Seres, Roy G. Knickelbein, Joseph B. Warshaw and
Richard B. Johnston, Jr.
J Immunol 2000; 165:3333-3340; ;
doi: 10.4049/jimmunol.165.6.3333
http://www.jimmunol.org/content/165/6/3333
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References
The Phagocytosis-Associated Respiratory Burst in Human
Monocytes Is Associated with Increased Uptake of
Glutathione1
Tamas Seres,* Roy G. Knickelbein,* Joseph B. Warshaw,* and Richard B. Johnston, Jr.2*†
E
limination of invading micro-organisms by neutrophils,
monocytes, and macrophages depends heavily on the generation of reactive oxygen species during the phagocytosis-associated respiratory burst. The NADPH oxidase that mediates this process assembles in the plasma membrane, and toxic
oxidants are released to the inside and outside of the cell (1). Thus,
the oxidants created in this process, particularly superoxide anion
(O2⫺), hydrogen peroxide (H2O2), hypochlorous acid, and hydroxyl radical, carry the potential to damage the phagocytes themselves as well as other cells at sites of inflammation (2).
The intracellular reducing potential supplied by the thiol tripeptide glutathione (L-␥-glutamyl-L-cysteinylglycine (GSH)3) is
believed to play an essential role in protecting cells against oxidant
damage (3). We have published data indicating that GSH also
serves as the key component in the respiratory burst-driven formation of cellular protein-mixed disulfides, a process termed
S-thiolation (4 –7). This reversible process can modify enzyme
function and could protect protein sulfhydryl groups from irreversible oxidative damage (4, 5). The importance of GSH to cell in*Department of Pediatrics, Yale University School of Medicine, New Haven, CT
06520; and †Department of Pediatrics, University of Colorado School of Medicine
and National Jewish Medical and Research Center, Denver, CO 80206
tegrity is emphasized by experiments in which inhibition of GSH
synthesis by L-buthionine-(S,R)-sulfoximine in adult mice or newborn rats led to mithochondrial degeneration, multiorgan failure,
and death (3, 8).
Most cell types can metabolize extracellular GSH by an ectoenzyme, ␥-glutamyl transpeptidase (GGT), which transfers glutamate to an amino acid acceptor. Cysteinylglycine is then hydrolyzed, and the amino acids are internalized independently (9, 10).
The rate of transport and the intracellular availability of cysteine/
cystine appear to control the rate of GSH synthesis (9 –12). Transport of these amino acids is accomplished by well-characterized
amino acid transporters (13, 14). More recently, distinct transport
systems for GSH have been described on the canalicular and sinusoidal membranes of rat and human liver, and analysis of cell
lines and tissues suggests that a transport mechanism also exists on
certain other cell types (15–18).
The studies reported here were framed on the hypothesis that
stimulation of the phagocytic respiratory burst induces increased
uptake of cysteine, cystine, or GSH, individually or in combination. The results indicate that activation of the respiratory burst in
human monocytes or exposure of the cells to H2O2 decreases the
uptake of cysteine, does not affect cystine uptake, and induces a
prominent increase in uptake of GSH.
Received for publication December 28, 1999. Accepted for publication July 5, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Materials and Methods
1
Mononuclear leukocytes were separated from heparinized blood of normal
donors using lymphocyte separation medium (Organon Teknika, Durham,
NC). Monocytes were washed and resuspended in DMEM (Life Technologies, Grand Island, NY), and 4 ⫻ 106 cells were added to each 35-mmdiameter tissue culture dish. The cells were allowed to adhere for 2 h at
37°C in a humidified chamber containing 5% CO2 (4). The adherent cell
population contained at least 82– 89% monocytes by microscopic examination after staining with Wright-Giemsa or esterase stains (Sigma, St.
Louis, MO); other cells appeared to be lymphocytes. More than 98% of the
This work was supported by Grants AI24782 and P50HL46488 from the National
Institutes of Health.
2
Address correspondence and reprint requests to Dr. Richard B. Johnston, Jr., National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206.
E-mail address: [email protected]
Abbreviations used in this paper: GSH, L-␥-glutamyl-L-cysteinylglycine, glutathione; GGT, ␥-glutamyl transpeptidase; OZ, opsonized zymosan; Vmax, maximum velocity; GSSG, glutathione disulfide; SOD, superoxide dismutase; DPI, diphenyleneiodonium; DTE, dithioerythritol.
3
Copyright © 2000 by The American Association of Immunologists
Preparation of monocytes and lymphocytes
0022-1767/00/$02.00
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During the phagocytic respiratory burst, oxygen is converted to potent cytotoxic oxidants. Monocytes and macrophages are
potentially long-lived, and we have hypothesized that protective mechanisms against oxidant stress are varied and fully expressed
in these cells. We report here that the respiratory burst in monocytes is accompanied by an increase in the uptake of [35S]glutathione ([35S]GSH) after 20 –30 min to levels up to 10-fold greater than those at baseline. By 30 min, 49% of the cell-associated
radioactivity was in the cytosol, 41% was in membrane, and 10% was associated with the nuclear fraction. GSH uptake was
inhibited by catalase, which removes hydrogen peroxide (H2O2), and micromolar H2O2 stimulated GSH uptake effectively in
monocytes and also lymphocytes. Oxidation of GSH to glutathione disulfide with H2O2 and glutathione peroxidase prevented
uptake. Acivicin, which inhibits GSH breakdown by ␥-glutamyl transpeptidase (GGT), had no effect on the enhanced uptake seen
during the respiratory burst. Uptake of cysteine or cystine, possible products of GGT activity, stayed the same or decreased during
the respiratory burst. These results suggest that a GGT-independent mechanism is responsible for the enhanced GSH uptake seen
during the respiratory burst. We describe here a sodium-independent, methionine-inhibitable transport system with a Km (8.5
␮M) for GSH approximating the plasma GSH concentration. These results suggest that monocytes have a specific GSH transporter
that is triggered by the release of H2O2 during the respiratory burst and that induces the uptake of GSH into the cell. Such a mechanism
has the potential to protect the phagocyte against oxidant damage. The Journal of Immunology, 2000, 165: 3333–3340.
3334
INCREASED GSH UPTAKE IN MONOCYTE RESPIRATORY BURST
cells were viable as determined by trypan blue exclusion. Lymphocyte
separation medium, DMEM, and other incubation solutions were free of
bacterial LPS (⬍0.01 ng/ml), as tested by the Limulus amebocyte lysate
assay (E-toxate, Sigma).
To isolate lymphocytes, cells nonadherent following 2-h incubation in
DMEM were transferred to new dishes and allowed to adhere during a
second 2-h incubation. The final nonadherent cell population contained
⬎95% lymphocytes by microscopic examination of Wright-Giemsastained smears. PMA-stimulated superoxide anion release by these lymphocyte preparations was negligible.
In control experiments we studied the effect on GSH uptake of inhibiting the PMA-stimulated respiratory burst with diphenyleneiodonium
(DPI) (21, 22). DPI (ICN, Costa Mesa, CA) dissolved in DMSO (21) was
added in a volume of 2 ␮l at a final concentration of 0.1–2 ␮M. After
preincubation with the cells in Na⫹-HEPES buffer for 10 min, PMA (500
ng/ml) was added for an additional 10 min (O2⫺ release) or 30 min (GSH
uptake). DMSO alone (2 ␮l) had no effect on GSH uptake or O2⫺ release.
Monocyte viability (methylene blue exclusion) was 99% in buffer and 98%
after incubation for 30 min in 2 ␮M DPI.
Subcellular localization of cell-associated radioactivity
Amino acid and GSH uptake
Respiratory burst
Release of O2⫺ from monocytes was measured as superoxide dismutase
(SOD)-inhibitable reduction of ferricytochrome c, corrected for the protein
content (4, 20). The cells were stimulated by PMA (500 ng/ml) or OZ
(1 mg/ml). Values in the absence of stimulus were subtracted from those
obtained after stimulation. Study of the respiratory burst and of amino
acid/GSH transport were performed in parallel using cells from the same
preparation. In other experiments PMA-stimulated release of O2⫺ and uptake of L-[35S]GSH were studied in the presence of 50 ␮g/ml SOD or 2000
U/ml catalase.
We studied the subcellular localization of the respiratory burst-stimulated
cell-associated 35S-labeled thiol by separating the cells into membrane,
nuclear, and cytosolic fractions. After incubation of the cells with
35
L-[ S]GSH and stimulation with PMA as described, the monocytes were
scraped from the dishes and sonicated for 60 s (six 10-s bursts) on ice. An
aliquot was taken to determine total counts per minute. Cell debris and
nuclear material were pelleted from the remaining homogenate, using an
Eppendorf centrifuge at 1,000 ⫻ g for 10 min. The supernatant was saved,
and the pellet was further fractionated using Opti Prep, as described in the
manufacturer’s booklet (Nycomed Pharma, Oslo, Norway). To isolate nuclei, the 1,000 ⫻ g pellet was homogenized in diluent A (8% (w/v) sucrose,
25 mM KCl, 5 mM MgCl2, and 20 mM Tris, pH 7.8), and a second centrifugation was performed at 1000 ⫻ g for 10 min. The pellet was layered
on the 25/30/35% (w/v) discontinuous gradient of Opti Prep and centrifuged at 10,000 ⫻ g for 20 min. The nuclear material was harvested from
a distinct band at the 30/35% interface. The initial supernatant, saved after
the first centrifugation at 1,000 ⫻ g, was centrifuged at 100,000 ⫻ g for 30
min, and the radioactivity of the membrane fraction (pellet) was measured
by directly resuspending it in scintillation fluid. The radioactivity remaining in the supernatant represented the nonmembrane-bound cytosolic thiol.
This supernatant was treated with 10% (final concentration) TCA, then
centrifuged at 14,000 ⫻ g for 10 min to precipitate soluble proteins and any
protein-bound thiol. Radioactivity in the TCA supernatant represented free
thiol.
Each cell fraction was studied to verify the success of the separation
procedure. We found 93% (mean; n ⫽ 3) of the total activity of GGT in the
membrane fraction, consistent with the known localization of this enzyme
(19, 23). In contrast, 99% (mean; n ⫽ 3) of the total activity of glucose6-phosphate dehydrogenase (19) was located in the cytosolic fraction.
Release of radioactivity from monocytes
Monocytes were incubated with L-35S-labeled GSH with or without stimulation with PMA for 30 min as described above. The reaction was stopped
by removing the medium and washing the cells four times. The cells were
then incubated for 10 min at 37°C in HEPES buffer with or without 1 mM
dithioerythritol (DTE) to release plasma membrane protein-bound radioactivity by reducing disulfide bonds. Radioactivity released into the extracellular medium and that which remained associated with the washed adherent cells was determined. The uptake and release of GSH was presented
as picomoles per milligram of protein, as described above.
FIGURE 1. Cystine (Cys2), cysteine (Cys) and GSH uptake in human
monocytes during the respiratory burst. Uptake of 35S-labeled Cys2, Cys,
or GSH was determined over 30 min in the presence of Na⫹-HEPES buffer
with and without PMA as a stimulus for the respiratory burst. When Cys
uptake was studied, 1 mM GSH was added to the reaction mixture to
prevent auto-oxidation. Data points represent the mean ⫾ SEM of duplicate measurements in each of three separate cell preparations (n ⫽ 3). PMA
induced a significant reduction in uptake of Cys (p ⬍ 0.01) and an increase
in uptake of GSH (p ⬍ 0.002).
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Immediately before uptake studies, the plates were removed from the CO2
incubator, and DMEM was replaced with Na⫹-HEPES buffer containing
130 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.0 mM MgSO4, 1.2 mM
KH2PO4, 20 mM HEPES, and 1.0 mM glucose, pH 7.4. For Na⫹-free
conditions, NaCl was replaced with an equal concentration of choline chloride. (In this paper we refer to the choline buffer as Na⫹-free HEPES
buffer.) Uptake was initiated by replacing the above solution with 1.0 ml of
HEPES buffer containing 0.9 ␮Ci of L-[35S]cystine (0.8 nM) with 1 ␮M
cold cystine, 0.9 ␮Ci L-[35S]cysteine (0.8 nM) with 1 ␮M cold cysteine in
the presence of 1 mM GSH as a reducing agent, or 0.9 ␮Ci L-[35S]GSH
(2.1 nM) in the presence of 1 ␮M cold GSH, as we previously described
(14). The radioactivity of the mixture of radioactive and cold thiols was
measured by liquid scintillation counting, and the specific activity of the
thiol was calculated as counts per minute per picomole. PMA (500 ng/ml)
or opsonized zymosan (OZ; 1 mg/ml) to stimulate the respiratory burst,
H2O2, or buffer control was present in the radiolabeled thiol mix that was
added to the cells.
The reaction was allowed to proceed at 37°C, then was terminated by
removing medium with suction and washing four times with ice-cold unlabeled HEPES buffer. Cells were lysed by adding 1 ml of a solution of 0.1
N NaOH, 2% Na2CO3, and 0.02% sodium-potassium tartrate. After incubation for 3 h, aliquots were taken for protein analysis (Lowry method) and
determination of radioactivity using a liquid scintillation counter. Cellassociated thiol (picomoles per milligram of protein) was calculated on the
basis of the radioactivity (counts per minute) in the cells, the specific activity of the thiol (counts per minute per picomoles of thiol), and the protein
content of the reaction mixture (milligrams).
The cell-associated radioactivity measured in these experiments might
be a combination of uptake, extracellular binding, and association with any
nonwashable extracellular fluid volume. Therefore, control experiments
were performed at 4°C to inhibit uptake. Cell-associated radioactivity at
4°C, consisting of extracellular binding and trapping, which was negligible, was subtracted from the cell-associated radioactivity measured at
37°C. In this paper we refer to the accumulation of cell-associated thiol as
uptake, except when classic transport methodology was used.
Acivicin (500 ␮M; 10-min preincubation) was used for estimating the
extent of radiolabeled thiol uptake mediated by GGT, which cleaves GSH
to glutamate and cysteinylglycine (19). Experiments were performed in
Na⫹-HEPES and Na⫹-free HEPES buffers; results were equivalent.
Characterization of cysteine and cystine uptake was performed using
inhibitors selective for each transporter system. ASC, L, XAG, and xc were
inhibited using 1 mM serine, 1 mM 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, 400 ␮M L-aspartic acid-␤-hydroxamate, or 200 ␮M quisqualate, respectively (14). All chemicals were obtained from Sigma.
Michaelis constants (Km) were calculated from the Lineweaver-Burk
plot of GSH uptake determined at five different concentrations (5–500
␮M). Thirty- and 5-min GSH uptakes were determined in unstimulated or
PMA-stimulated cells, respectively, and the maximum velocity (Vmax; picomoles per milligram per minute) was calculated.
Methionine (1–100 ␮M), bromsulfophtalein (1–100 ␮M), and GSH disulfide (GSSG) (10 –500 ␮M) were studied as expected inhibitors of GSH
uptake. The Ki, which is the concentration of inhibitor that doubles the
apparent Km (or decreases the affinity) of the substrate, was determined
using the Dixon plot (14).
The Journal of Immunology
3335
FIGURE 2. Relationship between the release of superoxide anion and
uptake of GSH during the respiratory burst. A, Monocytes cultured in Na⫹HEPES buffer were stimulated with PMA, and the uptake of 35S-labeled
GSH and release of O2⫺ were measured at the indicated times after addition of PMA. B, Rate of the release of O2⫺ and rate of GSH uptake expressed as a function of the time after addition of PMA. C, Effect of preincubation for 10 min with varying concentrations of DPI on PMAstimulated release of O2⫺ and uptake of GSH. Data points in A, B, and C
represent the mean ⫾ SEM (n ⫽ 3).
Data presentation
All data shown are the mean ⫾ SEM of n experiments, each performed in
duplicate or triplicate. Statistical significance was determined using Student’s t test unless otherwise specified.
Results
Thiol uptake in human monocytes during the respiratory burst
Under basal culture conditions, in Na⫹-HEPES buffer uptake of
cysteine over 30 min was 4.3-fold higher than that of cystine and
10.3-fold higher than that of GSH (Fig. 1). The combined cystine
and cysteine uptake represented 93% of the total thiol taken up in
these experiments. In the presence of PMA cysteine uptake decreased by 70%, whereas cystine uptake was not significantly altered. In contrast, GSH uptake was increased 3.1-fold (Fig. 1).
Total thiol uptake decreased during the respiratory burst because
of the marked decrease in cysteine uptake.
The relationship between the PMA-induced respiratory burst
and GSH uptake was studied as a function of time after addition of
PMA (Fig. 2A). During the first 20 min total superoxide release
and total GSH uptake increased together; after 20 min GSH uptake
gradually declined. In these experiments GSH uptake increased
⬃12-fold over baseline during the respiratory burst. When the data
were expressed as rates (Fig. 2B), a close relationship was shown
between superoxide release and GSH uptake in the presence of
PMA. Both rates peaked by 5 min after addition of PMA and
declined rapidly thereafter.
The relationship between the PMA-stimulated respiratory burst
and GSH uptake was also studied after inhibition of the respiratory
burst by preincubation of the monocytes for 10 min with DPI (21,
22). Release of O2⫺ and uptake of GSH were inhibited to an equivalent extent over a range of DPI concentrations of 0.2–2 ␮M (Fig.
2C). Preincubation for 10 min with 1 ␮M DPI had no effect on the
increased GSH uptake stimulated by 0.5 mM H2O2 (without DPI
58 ⫾ 6 pmol/mg; with DPI 59 ⫾ 11 pmol/mg; mean ⫾ SEM; n ⫽ 3).
Sodium-independent uptake of thiols during the respiratory burst
and in unstimulated monocytes
Both Na⫹-dependent and Na⫹-independent mechanisms have
been described for cystine, cysteine, and GSH uptake. To explore
these mechanisms in monocytes, uptake was measured in HEPES
buffer in which sodium chloride was replaced with choline chloride. The effect of stimulating the respiratory burst with PMA was
similar to the effect in Na⫹-HEPES buffer; there was a decrease in
uptake of cysteine (44%), a minimal effect on cystine uptake, and
a stimulation of GSH uptake by 4.9-fold during the respiratory
burst. Almost all (93%) the GSH uptake was sodium independent
(Fig. 3, compared with Fig. 1). Under basal (unstimulated) conditions, about 60% of GSH uptake was Na⫹ independent.
Transporters for cysteine, cystine, or both can be differentiated
by their substrate and inhibitor specificity and whether Na⫹ is
necessary for transport activity. We explored cystine and cysteine
uptake (5 min, 37°C) in monocytes under basal culture conditions
by determining transport in the presence of these selective inhibitors (14). Cystine transport was predominantly (72%) Na⫹ independent (Figs. 1 and 3). This transport was inhibited 43 ⫾ 13%
(mean ⫾ SEM; n ⫽ 4) by quisqualate, a specific inhibitor of system xc, a glutamate/cystine exchanger. Sodium-dependent cystine
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FIGURE 3. Cystine (Cys2), cysteine (Cys), and GSH uptake in human
monocytes during the respiratory burst in sodium-free buffer. Uptake of
35
S-labeled Cys2, Cys, or GSH was determined over 30 min in Na⫹-free
HEPES buffer with and without PMA. When Cys uptake was studied,
1 mM GSH was added to the reaction mixture to prevent auto-oxidation.
Data points represent the mean ⫾ SEM (n ⫽ 3). PMA induced a significant
reduction in the uptake of Cys (p ⬍ 0.04) and a significant increase in the
uptake of GSH (p ⬍ 0.001); the reduction in Cys2 uptake was not
significant.
3336
INCREASED GSH UPTAKE IN MONOCYTE RESPIRATORY BURST
FIGURE 4. Relationship between release of superoxide anion and uptake of GSH during the respiratory burst induced by OZ. Monocytes cultured in sodium-free HEPES buffer were stimulated by OZ (1 mg/ml), and
release of O2⫺ and uptake of [35S]GSH were measured at the indicated
time points. GSH uptake in the absence of OZ is labeled as a control. Data
points represent the mean ⫾ SEM (n ⫽ 3).
Characterization of Na⫹-independent GSH uptake
The effect of the respiratory burst on uptake of GSH in Na⫹-free
buffer was studied during phagocytosis of a serum-opsonized particle (OZ) (20). The relationship between the OZ-stimulated respiratory burst and GSH uptake as a function of time (Fig. 4) was
similar to that shown with PMA as stimulus in Na⫹-HEPES buffer
(Fig. 2A). The characteristically slower rate of O2⫺ release with
OZ compared with that using PMA (20) was associated with a
longer interval before decline in GSH uptake (30 min with OZ,
Fig. 4; 20 min with PMA, Fig. 2A). In these experiments GSH
FIGURE 5. Effect of SOD or catalase on the uptake of GSH during
stimulation with PMA. The uptake of L-[35S]GSH was measured in Na⫹free HEPES buffer in the presence or the absence (control) of PMA at 37°C
for 30 min. The effect of PMA was also measured in the presence of SOD
(50 ␮g/ml) or catalase (2000 u/ml). Results represent the mean ⫾ SEM
(n ⫽ 3). Catalase significantly inhibited the PMA-induced increase in GSH
uptake ( p ⬍ 0.001).
uptake during the respiratory burst was 10.2 times higher than
uptake under basal conditions (control).
During the respiratory burst, superoxide anion is converted to
the more stable and membrane-permeable oxidant, H2O2. Uptake
of GSH during the PMA-induced respiratory burst was consistently (but not statistically significantly) further enhanced by SOD,
which accelerates H2O2 production (Fig. 5). In contrast, catalase,
which removes H2O2, reduced GSH transport during the respiratory burst almost to the level of the control (no PMA).
These results suggested that among the oxygen derivatives of
the respiratory burst, H2O2 plays a particularly important role in
inducing GSH uptake. In support of this conclusion, H2O2 in micromolar concentrations induced a concentration-dependent enhancement of GSH transport to levels over 7 times greater than
those at baseline (Fig. 6). In experiments not shown the time
course of this effect was similar to that seen with the PMA-induced
respiratory burst, with GSH uptake reaching a maximum after 15
min of exposure, then decreasing slowly (by ⬃15%) by 30 min
(n ⫽ 3). The highly reactive H2O2 derivative sodium hypochlorite
(NaOCl; Sigma; 0.05 mM) did not enhance GSH uptake significantly during a 30-min incubation: NaOCl, 35.1 ⫾ 6.5 pmol/mg;
0.05 mM H2O2, 214 ⫾ 28 pmol/mg; control, 27.5 ⫾ 6.6 pmol/mg
(n ⫽ 3).
Because the GSH used for uptake studies was radiolabeled on
the cysteinyl moiety, it was necessary to demonstrate that we were
measuring the uptake of intact GSH and not cysteinylglycine or
FIGURE 7. The role of GGT in GSH uptake. Uptake of [35S]GSH was
studied in Na⫹-free HEPES buffer at 30 min in either the absence (control)
or the presence of PMA, with or without a 10-min preincubation with 0.5
mM acivicin, an inhibitor of GGT. Data points represent the mean ⫾ SEM
(n ⫽ 3).
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
transport was inhibited 83 ⫾ 12% (n ⫽ 4) by L-aspartic acid-␤hydroxamate, a specific inhibitor of system XAG, which also transports aspartate and glutamate.
Cysteine transport was only 24% Na⫹ independent (Figs. 1 and
3). Most (82 ⫾ 2%; n ⫽ 4) of the Na⫹-dependent transport was
inhibited by serine, implicating system ASC. The Na⫹-independent cysteine transport was inhibited by 2-aminobicyclo-[2.2.1]heptane-2-carboxylic acid (46 ⫾ 3%), indicating the participation
of system L, a transporter that carries a number of amino acids,
including cysteine (14).
FIGURE 6. Dose-response effect of H2O2 on sodium-independent GSH
uptake. The uptake of L-[35S]GSH (2.1 nM in the presence of 1 ␮M cold
GSH) was measured in the presence of the indicated concentrations of
H2O2 at 37°C for 30 min. Results represent the mean ⫾ SEM (n ⫽ 3).
The Journal of Immunology
3337
Table II. Effect of H2O2 and transport inhibitors on GSH uptake in
human monocytesa
H2O2 (–)
H2O2 (⫹)
Km (␮M)
GSH
6.1 ⫾ 0.3
8.5 ⫾ 2.8
Vmax (pmol/mg/min)
GSH
3.8 ⫾ 1.8
42 ⫾ 9.0
KI (␮M)
cysteine released by the action of GGT, then dipeptidase. Therefore, cells were preincubated for 10 min with acivicin, an irreversible inhibitor of GGT, before exposure to PMA. As shown in Fig.
7, acivicin preincubation resulted in a modest (14%) decrease in
GSH uptake in unstimulated cells, but no change in the enhancement associated with the respiratory burst, suggesting that GSH is
being transported in intact form, and that GGT is not involved in
the increased GSH uptake seen during the respiratory burst.
We explored the possibility that H2O2 stimulated GSH uptake
by oxidizing GSH to GSSG, which, in turn, was taken up preferentially. When uptake was studied in the presence of glutathione
peroxidase (1 U/ml) and H2O2 (10 ␮M; 30 min) to convert GSH
to GSSG, uptake of [35S]GSH was less than uptake at baseline (no
stimulus or H2O2 added; 9 ⫾ 1 pmol/mg protein vs baseline, 20 ⫾
3 pmol/mg; mean ⫾ SEM; n ⫽ 3). In these experiments, H2O2
alone enhanced uptake of GSH, as shown in Fig. 6, and glutathione
peroxidase alone had no appreciable effect. A similar effect (mean
70% reduction below baseline; n ⫽ 3) was noted when the GSH
was preincubated with the strong oxidant diamide (50 ␮M).
Distribution of GSH taken up by human monocytes during
oxidant stress
We explored the cellular distribution of radioactivity in monocytes
after a 30-min incubation in Na⫹-free HEPES buffer with
Table I. Estimation of thiol bound to the outside surface of the plasma
membrane using disulfide reducing agent DTEa
Buffer
DTE
111.8 ⫾ 45.5
–
–
53.6 ⫾ 12.3
18.5 ⫾ 4.5
77.5 ⫾ 1.5
a
Results are means ⫾ SEM for transport studied in at least three different monocyte preparations. Uptake of GSH was studied in the absence of Na⫹. Uptake was
determined for at least five different substrate concentrations. Km was calculated from
Lineweaver-Burk plots, and Ki was calculated by using the Dixon plot. BSP,
Bromsulfophtalein.
[35S]GSH under basal conditions or stimulated with PMA (Fig. 8).
In unstimulated cells, most (78%) of the cell-associated radioactivity was in the cytosol, with 18% being in the membrane and 4%
in the nuclear fraction (n ⫽ 3). After stimulation with PMA, there
was an almost 6-fold increase in total radioactivity. The cellular
distribution of radioactivity was different from that under basal
conditions, with equivalent levels being achieved in the cytosol
and membrane fractions (49 and 41% of the total, respectively) and
a smaller amount localizing in the nuclear fraction (10%; Fig. 8).
Thiol in the cytosol under basal conditions and after PMA stimulation was further separated into TCA-soluble and -precipitable
fractions; the respiratory burst increased the level of cytosolic free
GSH by 2.1-fold and cytosolic protein-bound GSH by 3.4-fold
(mean; n ⫽ 3), in agreement with our previous studies showing
avid binding of GSH to cytosolic proteins during the respiratory
burst (4 –7).
In an attempt to probe further the differentiation of internalized
thiol from extracellular binding, we allowed GSH to associate with
the cell for 30 min in basal or PMA-stimulated conditions. After
washing the cells as usual, the ability of 1 mM DTE to reduce
disulfide bonds and release radioactivity into the supernatant during a 10-min incubation was taken as a measure of extracellularly
bound GSH (Table I). The total cell-associated radioactivity before
the incubation with buffer or DTE was increased about 7-fold during the respiratory burst. There was only a minor (3%) difference
in the amount of radioactivity released into the supernatant of stimulated cells by buffer compared with DTE, in agreement with the
concept that cell-associated radioactivity represents internalized
thiol.
Table I as well as Figs. 2A and 4 show that there was gradual
efflux of radiolabel after peak uptake had occurred. Thus, uptake
(accumulation of radiolabel) is a net value, although efflux appears
to be small during the initial 30 – 40 min.
Kinetics of GSH uptake
Thiol Content (pmol/mg)
Unstimulated
GSSG
Methionine
BSP
PMA stimulated
Cell
Supernatant
Cell
Supernatant
31.1 ⫾ 4.0
19.6 ⫾ 0.1
10.8 ⫾ 0.4
15.7 ⫾ 0.7
242.1 ⫾ 4.2
234.4 ⫾ 7.4
41.6 ⫾ 0.9
49.6 ⫾ 0.4
a
PMA was used to stimulate the respiratory burst in monocytes for 30 min in the
presence of radiolabeled GSH, as described in Materials and Methods. After washing
four times, the cells were exposed to HEPES buffer with or without DTE, 1 mM, for
10 min at 37°C, and radioactivity was measured in the supernatant and in adherent
cells. Results are mean ⫾ SEM, n ⫽ 3.
If activation of a transport protein were responsible for the increase
in GSH entering the cell during the respiratory burst or exposure to
H2O2, then uptake might be expected to follow Michaelis-Menten
kinetics. Consistent with this premise, uptake was found to be a
saturable event, with a similar high affinity (Km) for GSH in both
the presence and the absence of added H2O2 (Table II). The Km
demonstrated was in the range of the concentration of GSH in
plasma under physiologic conditions (15, 24, 25). In contrast, the
Vmax was increased 11-fold in the presence of H2O2.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 8. Uptake and distribution of GSH in monocytes during the
respiratory burst. Monocytes were preincubated with L-[35S]GSH in Na⫹free HEPES buffer with or without PMA for 30 min. The reaction was
stopped by scraping the monocytes from the dishes and sonicating them in
Eppendorf tubes for 60 s (six 10-s bursts) on ice. An aliquot was taken to
determine total counts per minute. The cell debris and the nuclear material
were centrifuged at 1,000 ⫻ g for 10 min. The supernatant was separated
into the membrane fraction, obtained as the pellet after centrifugation at
100,000 ⫻ g for 30 min, and the supernatant, which represented the cytosolic fraction. Nucleus-associated GSH was separated from the cell debris as described in Materials and Methods. Results represent the mean ⫾
SEM of three experiments performed in duplicate. Measured radioactivity
was significantly higher in each of the three fractions from cells exposed to
PMA ( p ⬍ 0.005 to p ⬍ 0.05).
3338
INCREASED GSH UPTAKE IN MONOCYTE RESPIRATORY BURST
The effects of compounds known to inhibit various GSH transporters in hepatocytes (15, 16) were also investigated, and Ki was
determined by analyzing the data using a Dixon plot. Methionine
inhibited hydrogen peroxide-stimulated GSH transport at a concentration close to the Km; concentrations severalfold higher were
required for inhibition of GSH transport by GSSG or bromsulfophtalein (Table II).
GSH uptake in lymphocytes
The role of H2O2 or the respiratory burst induced by PMA in GSH
uptake was compared in human monocytes and lymphocytes (Fig.
9). PMA did not induce superoxide anion release in our lymphocyte preparations; therefore, the direct effect of PMA on GSH uptake could be evaluated. Hydrogen peroxide caused a significant
increase in GSH uptake in both purified monocytes ( p ⬍ 0.002)
and purified lymphocytes ( p ⬍ 0.02). In contrast, the respiratory
burst induced by PMA caused a 4-fold increase in GSH uptake in
monocytes, but PMA had no effect on GSH uptake by lymphocytes.
Discussion
During the process of phagocytosis professional phagocytes undergo a vigorous respiratory burst in which they consume oxygen
and convert it enzymatically to O2⫺, which interacts to form H2O2,
then hypochlorous acid, hydroxyl radical, and other potent oxidants. It has been recognized that this process is accompanied by
activation of the hexose monophosphate shunt and oxidation of
GSH to GSSG through the action of GSH peroxidase and H2O2
(26). GSSG is cycled back to GSH by glutathione reductase. Data
from a variety of experimental systems indicate that the redox
cycling of GSH plays an important role in protecting the cell
against oxidant damage (26, 27). We report here that the respiratory burst in human monocytes is also accompanied by increased
uptake of GSH into the cell, up to 10-fold over baseline.
Virtually all animal cells synthesize GSH (3). Most of the
body’s GSH is intracellular, ⬎99% remaining in the reduced form
in the absence of oxidant stress, in concentrations of 0.5–10 mM
(3, 28). Total GSH in plasma has been reported to be 6 –33 ␮M
(15, 24, 25), 60% as reduced GSH (28). Synthesis is achieved
using the constituent amino acids (Glu, Cys, and Gly) supplied in
part by the breakdown of plasma GSH by the ectoenzymes GGT
and dipeptidase (9, 19). The key sulfhydryl amino acid cysteine
and, in greater concentration, its oxidized form cystine also appear
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FIGURE 9. GSH uptake in monocytes and lymphocytes in the presence
of H2O2 or PMA. Uptake of 35S-labeled GSH was measured in purified
monocytes or lymphocytes in the presence or the absence of H2O2 (50 ␮M)
or of PMA as a stimulus for the respiratory burst at 37°C for 30 min. Data
points represent the mean ⫾ SEM (n ⫽ 4). H2O2 (but not PMA) induced
a significant increase in uptake of GSH by lymphocytes (p ⬍ 0.001).
in plasma and are available for transport (24, 25). Cysteine and
cystine transport has been shown to be essential for GSH synthesis
in lymphocytes, macrophages, and other cells (29, 30).
We report here that under basal conditions human monocytes
take up cysteine primarily by a Na⫹-dependent system (Fig. 1 vs
Fig. 3). This system appears to be system ASC, based on the defining inhibition studies (9, 14) as used by us previously with alveolar type II cells (14). System L appeared to play a lesser role in
monocyte uptake of cysteine. In contrast, transport of cystine was
predominantly (72%) Na⫹ independent and mediated at least in
part by system xc. In the the presence of Na⫹, cystine was transported primarily by system XAG. This finding supports the conclusion of Rimaniol et al. (31) that human mononuclear phagocytes possess a system XAG cystine/glutamate transporter. They
have proposed that this transporter might clear neuroexcitatory
glutamate in the brain. The changes induced by the respiratory
burst in uptake of all three thiols were similar in the presence and
the absence of Na⫹ (Figs. 1 and 3).
Our data suggest that the respiratory burst- and H2O2-induced
increase seen in uptake of GSH, which was 35S-radiolabeled in the
Cys moiety, was due primarily to transport of intact GSH. Preincubation of the cells with acivicin, which inhibits GSH breakdown
by ␥-glutamyl transpeptidase (19), had no effect on the H2O2induced GSH uptake (Fig. 7). Uptake of Cys and Cys2, possible
thiol products of GSH breakdown, was decreased or did not appreciably change during the respiratory burst (Figs. 1 and 3). Oxidation of [35S]GSH to [35S]GSSG by preincubation with H2O2/
GSH peroxidase or diamide reduced uptake of the thiol to 30 –50%
of the baseline (no H2O2) value, in agreement with the higher Km
for GSSG than for GSH found on kinetic analysis (Table II). In
these experiments we cannot rule out a direct effect of the diamide
on the cell, but the facts that [35S]GSH uptake was inhibited by the
strong oxidant diamide and was not increased by hypochlorous
acid support the conclusion that the H2O2-induced cell-associated
radioactivity is due primarily to uptake, not oxidant-stimulated
mixed disulfide formation with plasma membrane ectoproteins. In
addition, incubation of the PMA-stimulated cells with DTE, which
would release externally bound thiols, elicited only a few more
counts per minute than did buffer alone (Table I).
Uptake of GSH against a concentration gradient would be expected to require active transport. The data reported here support
the existence of a Na⫹-independent GSH transporter on human
monocytes. A transport mechanism for GSH has been described in
several cell types, including human platelets and canalicular and
sinusoidal membranes of rat and human liver (15–18, 32–35).
GSH transport in various mammalian cell lines can be bidirectional, at least under certain conditions (33, 35). The Na⫹-independent activity, redox regulation, and inhibition by methionine
and bromsulfophthalein that we found with monocyte GSH transport are characteristic of the liver sinusoidal GSH transporter (32,
35, 36), but the Km of this hepatic transporter is in the millimolar
range, as would be appropriate for its mediating GSH efflux from
the liver into the bloodstream. We found monocytes to have a
transport mechanism with a considerably higher affinity for GSH
(Km ⫽ 6 –9 ␮M), which was close to the GSH concentration in
plasma. A high affinity transporter for GSH has also been described in human platelets (34).
The rate of the respiratory burst-associated uptake of GSH
closely approximated the rate of O2⫺ release; both peaked at 5 min
(Fig. 2B); accumulated GSH uptake after addition of H2O2 peaked
at 15 min. SOD, which removes O2⫺ by catalyzing its dismutation
into H2O2, increased GSH uptake slightly, whereas catalase reduced the stimulatory effect of PMA by ⬎80% (Fig. 5). H2O2,
which can cross cell plasma membranes relatively efficiently (37),
The Journal of Immunology
timing of increased GSH concentration might also be important to
protecting and maintaining these regulatory systems, and thus cell
function.
Acknowledgments
We thank Dr. Nazzareno Ballatori, University of Rochester School of Medicine, for constructive criticism.
References
1. Babior, B. M. 1999. NADPH oxidase: an update. Blood 93:1464.
2. Henson, P. M., and R. B. Johnston, Jr. 1987. Tissue injury in inflammation:
oxidants, proteinases, and cationic proteins. J. Clin. Invest. 79:669.
3. Meister, A. 1994. Glutathione-ascorbic acid antioxidant system in animals.
J. Biol. Chem. 269:9397.
4. Seres, T., V. Ravichandran, T. Moriguchi, K. Rokutan, J. A. Thomas, and
R. B. Johnston, Jr. 1996. Protein S-thiolation and dethiolation during the respiratory burst in human monocytes: a reversible post-translational modification
with potential for buffering the effects of oxidant stress. J. Immunol. 156:1973.
5. Ravichandran, V., T. Seres, T. Moriguchi, J. A. Thomas, and R. B. Johnston, Jr.
1994. S-thiolation of glyceraldehyde-3-phosphate dehydrogenase induced by the
phagocytosis-associated respiratory burst in blood monocytes. J. Biol. Chem.
269:25010.
6. Rokutan, K., J. A. Thomas, and R. B. Johnston, Jr. 1991. Phagocytosis and
stimulation of the respiratory burst by phorbol diester initiate S-thiolation of
specific proteins in macrophages. J. Immunol. 147:260.
7. Chai, Y. C., S. S. Ashraf, K. Rokutan, R. B. Johnston, Jr., and J. A. Thomas.
1994. S-thiolation of individual human neutrophil proteins including actin by
stimulation of the respiratory burst: evidence against a role for glutathione disulfide. Arch. Biochem. Biophys. 310:273.
8. Martensson, J., A. Jain, E. Stole, W. Frayer, P. A. M. Auld, and A. Meister. 1991.
Inhibition of glutathione synthesis in the newborn rat: a model for endogenously
produced oxidative stress. Proc. Natl. Acad. Sci. USA 88:9360.
9. Deneke, S. M., and B. L. Fanburg. 1989. Regulation of cellular glutathione.
Am. J. Physiol. 257:L163.
10. van Klaveren, R. J., M. Demedts, and B. Nemery. 1997. Cellular glutathione
turnover in vitro, with emphasis on type II pneumocytes. Eur. Respir. J 10:1392.
11. Bannai, S., H. Sato, T. Ishii, and Y. Sugita. 1989. Induction of cystine transport
activity in human fibroblasts by oxygen. J. Biol. Chem. 264:18480.
12. Sato, H., K. Kuriyama-Matsumura, R. C. M. Siow, T. Ishii, S. Bannai, and
G. E. Mann. 1998. Induction of cystine transport via system xc⫺ and maintenance
of intracellular glutathione levels in pancreatic acinar and islet cell lines. Biochim.
Biophys. Acta 1414:85.
13. Bannai, S. 1984. Transport of cystine and cysteine in mammalian cells. Biochim.
Biophys. Acta 779:289.
14. Knickelbein, R. G., T. Seres, G. Lam, R. B. Johnston, Jr., and J. B. Warshaw.
1997. Characterization of multiple cysteine and cystine transporters in rat alveolar type II cells. Am. J. Physiol. 273:L1147.
15. Ookhtens, M., and N. Kaplowitz. 1998. Role of the liver in interorgan homeostasis of glutathione and cyst(e)ine. Semin. Liver. Dis. 18:313.
16. Ballatori, N., and J. F. Rebbeor. 1998. Roles of MRP2 and oatp1 in hepatocellular
export of reduced glutathione. Semin. Liver. Dis. 18:377.
17. Konig, J., D. Rost, Y. Cui, and D. Keppler. 1999. Characterization of the human
multidrug resistance protein isoform MRP3 localized to the basolateral hepatocyte membrane. Hepatology 29:1156.
18. Paulusma, C. C., M. A. van Geer, R. Evers, M. Heijn, R. Ottenhoff, P. Borst, and
R. P. J. Oude Elferink. 1999. Canalicular multispecific organic anion transporter/
multidrug resistance protein 2 mediates low-affinity transport of reduced glutathione. Biochem. J. 338:393.
19. Knickelbein, R. G., D. H. Ingbar, T. Seres, K. Snow, R. B. Johnston, Jr.,
O. Fayemi, F. Gumkowski, J. D. Jamieson, and J. B. Warshaw. 1996. Hyperoxia
enhances expression of ␥-glutamyl transpeptidase and increases protein S-glutathiolation in rat lung. Am. J. Physiol. 270:L115.
20. Johnston, R. B., Jr., C. A. Godzik, and Z. A. Cohn. 1978. Increased superoxide
anion production by immunologically activated and chemically elicited macrophages. J. Exp. Med. 148:115.
21. O’Donnell, V. B., D. G. Tew, O. T. G. Jones, and P. I. England. 1993. Studies on
the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase. Biochem. J. 290: 41.
22. Jankowski, A., and S. Grinstein. 1999. A noninvasive fluorometric procedure for
measurement of membrane potential: quantification of the NADPH oxidase-induced depolarization in activated neutrophils. J. Biol. Chem. 274: 26098.
23. Grisk, O., U. Kuster, and S. Ansorge. 1993. The activity of ␮-glutamyl transpeptidase (␥-GT) in populations of mononuclear cells from human peripheral blood.
Biol. Chem. Hoppe-Seyler 374:287.
24. Andersson, A., A. Lindgren, M. Arnadottir, H. Prytz, and B. Hultberg. 1999.
Thiols as a measure of plasma redox status in healthy subjects and in patients with
renal or liver failure. Clin. Chem. 45:1084.
25. Mansoor, M. A., A. M. Svardal, and P. M. Ueland. 1992. Determination of the in
vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in
human plasma. Anal. Biochem. 200:218.
26. Roos, D., R. S. Weening, and A. A. Voetman. 1980. Protection of human neutrophils against oxidative damage. Agents Actions 10:528.
27. Roos, D., R. S. Weening, S. R. Wyss, and H. E. Aebi. 1980. Protection of human
neutrophils by endogenous catalase: studies with cells from catalase-deficient
individuals. J. Clin. Invest. 65:1515.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
stimulated rapid and vigorous GSH uptake when added to the outside of the cells in micromolar concentrations. Added hypochlorous acid had a negligible stimulatory effect. Thus, respiratory
burst-associated GSH uptake appears to be driven primarily by
H2O2.
Small amounts of radiolabeled GSH probably entered in PMAinduced pinocytic vesicles and OZ-induced phagocytic vacuoles,
but most of the GSH uptake induced by PMA was inhibited by
catalase, which removes H2O2 (Fig. 5), and by DPI, which inhibits
the respiratory burst (Fig. 2C). DPI did not inhibit the increased
GSH uptake induced by H2O2. In addition, PMA stimulated selective uptake of GSH, not cysteine, cystine, or GSSG.
The mechanism by which H2O2 drives such a profound and
rapid uptake of GSH remains to be defined. Rapid modulation of
signal transduction is a likely explanation. The phagocytic respiratory burst and other systems of oxidant stress have been related
to modification of signaling in a variety of systems (38 – 44). H2O2
in particular has been reported to inhibit tyrosine phosphatase activity (39 – 41), to stimulate tyrosine phosphorylation (42, 43), and
to stimulate the activity of mitogen-activated protein kinase (42)
and protein kinase C (44). H2O2 has also been reported to influence
the activation of transcription factors, including NF-␬B and AP-1
in mammalian cells (42, 45) and OxyR in Escherichia coli (46),
and to activate RBC KCl cotransport (47).
It is not clear what survival advantage might be offered by this
rapid uptake of GSH and by the shift from uptake primarily of
cysteine/cystine to uptake of GSH. We reported previously that
stimulation of the respiratory burst in human monocytes induced a
rapid decrease in intracellular GSH, with a nadir at 10 min of PMA
stimulation; GSSG increased, with a peak at 5 min (4). The decrease at 10 min was 2.3 nmol/mg protein. The Vmax for GSH
uptake (42 pmol/mg/min; Table II) suggests that about 20% of the
GSH decline in the first 10 min could be offset by rapid GSH
uptake.
Stimulation of the respiratory burst for 30 min increased thiol
labeling by 2.7-fold in the cytosol, 10-fold in the membrane fraction, and 10-fold in the nuclear fraction (Fig. 8). The GSH taken up
during the respiratory burst might interact in these cell compartments to form mixed disulfides (the process of S-thiolation) between a variety of cellular proteins and GSH, a process that we
have demonstrated with human neutrophils and monocytes and
mouse macrophages (4 –7). S-thiolation of a protein can significantly modify its function (5), and the covalent disulfide bonds
formed in this process are reversed enzymatically as the respiratory burst subsides (4). Thus, S-thiolation represents a form of
redox buffering that could protect proteins against oxidant denaturation and modulate cellular metabolic events during phagocytosis. For example, GSH has been shown to play a protective role
in maintaining the functional integrity of nuclear and mitochondrial DNA (48) and in control of apoptosis (49 –51). It seems possible that the amount of GSH taken up during the respiratory burst,
although small relative to the total intracellular GSH concentration, could appear at the right place in the cell at the right time to
protect specific monocyte proteins against oxidant damage.
In addition to protecting against oxidant damage, GSH has been
reported to play a central role in regulating a large number of
biologic systems that are fundamental to the immune response,
inflammation, and host defense. These include activation in T cells
of NF-␬B, IL-2-dependent functions including cell proliferation,
and cellular cytotoxicity (52); synthesis of PGE2 and leukotriene C
by macrophages (53); detoxification of xenobiotics through glutathione S-transferase reactions (9); inhibition of apoptosis in monocytes, lymphocytes, and neutrophils (49 –51); and replication of
HIV (54). It seems reasonable to speculate that the location and
3339
3340
INCREASED GSH UPTAKE IN MONOCYTE RESPIRATORY BURST
42. Sundaresan, M., Z. X. Yu, V. J. Ferrans, K. Irani, and T. Finkel. 1995. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science 270:296.
43. Lowe, G. M., C. E. Hulley, E. S. Rhodes, A. J. Young, and R. F. Bilton. 1998.
Free radical stimulation of tyrosine kinase and phosphatase activity in human
peripheral blood mononuclear cells. Biochem. Biophys. Res. Commun. 245:17.
44. Konishi, H., M. Tanaka, Y. Takemura, H. Matsuzaki, Y. Ono, U. Kikkawa, and
Y. Nishizuka. 1997. Activation of protein kinase C by tyrosine phosphorylation
in response to H2O2. Proc. Natl. Acad. Sci. USA 94:11233.
45. Roebuck, K. A., L. R. Carpenter, V. Lakshminarayanan, S. M. Page, J. N. Moy,
and L. L. Thomas. 1999. Stimulus-specific regulation of chemokine expression
involves differential activation of the redox-responsive transcription factors AP-1
and NF-␬B. J. Leukocyte Biol. 65:291.
46. Zheng, M., F. Aslund, and G. Storz. 1998. Activation of the OxyR transcription
factor by reversible disulfide bond formation. Science 279:1718.
47. Bize, I., and P. B. Dunham. 1995. H2O2 activates red blood cell K-Cl cotransport
via stimulation of a phosphatase. Am. J. Physiol. 269:C849.
48. Morales, A., M. Miranda, A. Sanchez-Reyes, A. Biete, and J. C. Fernandez-Checa.
1998. Oxidative damage of mitochondrial and nuclear DNA induced by ionizing
radiation in human hepatoblastoma cells. Int. J. Radiat. Oncol. Biol. Phys. 42:
191.
49. Um, H.-D., J. M. Orenstein, and S. M. Wahl. 1996. Fas mediates apoptosis in
human monocytes by a reactive oxygen intermediate dependent pathway. J. Immunol. 156:3469.
50. Hyde, H., N. J. Borthwick, G. Janossy, M. Salmon, and A. N. Akbar. 1997.
Upregulation of intracellular glutathione by fibroblast-derived factor(s): enhanced
survival of activated T cells in the presence of low Bcl-2. Blood 89:2453.
51. Watson, R. W. G., O. D. Rotstein, M. Jimenez, J. Parodo, and J. C. Marshall.
1997. Augmented intracellular glutathione inhibits Fas-triggered apoptosis of activated human neutrophils. Blood 89:4175.
52. Droge, W., K. Schulze-Osthoff, S. Mihm, D. Galter, H. Schenk, H.-P. Eck,
S. Roth, and H. Gmunder. 1994. Functions of glutathione and glutathione disulfide in immunology and immunopathology. FASEB J. 8:1131.
53. Rouzer, C. A., W. A. Scott, O. W. Griffith, A. L. Hamill, and Z. A. Cohn. 1982.
Arachidonic acid metabolism in glutathione-deficient macrophages. Proc. Natl.
Acad. Sci. USA 79:1621.
54. Garaci, E., A. T. Palamara, M. R. Ciriolo, C. D’Agostini, M. S. Abdel-Latif,
S. Aquaro, E. Lafavia, and G. Rotilio. 1997. Intracellular GSH content and HIV
replication in human macrophages. J. Leukocyte Biol. 62:54.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
28. Meister, A., and M. E. Anderson. 1983. Glutathione. Annu. Rev. Biochem. 52:
711.
29. Ishii, T., Y. Sugita, and S. Bannai. 1987. Regulation of glutathione levels in
mouse spleen lymphocytes by transport of cysteine. J. Cell. Physiol. 133:330.
30. Watanabe, H., and S. Bannai. 1987. Induction of cystine transport activity in
mouse peritoneal macrophages. J. Exp. Med. 165:628.
31. Rimaniol, A.-C., S. Haik, M. Martin, R. Le Grand, F. D. Boussin,
N. Dereuddre-Bosquet, G. Gras, and D. Dormont. 2000. Na⫹-dependent highaffinity glutamate transport in macrophages. J. Immunol. 164:5430.
32. Kaplowitz, N., J. C. Fernandez-Checa, R. Kannan, C. Garcia-Ruiz, M. Ookhtens,
and J.-R. Yi. 1996. GSH transporters: molecular characterization and role in GSH
homeostasis. Biol. Chem. Hoppe-Seyler 377:267.
33. Garcia-Ruiz, C., J. C. Fernandez-Checa, and N. Kaplowitz. 1992. Bidirectional
mechanism of plasma membrane transport of reduced glutathione in intact rat
hepatocytes and membrane vesicles. J. Biol. Chem. 267:22256.
34. Sexton, D. J., and B. Mutus. 1995. Platelet glutathione transport: characteristics
and evidence for regulation by intraplatelet thiol status. Biochem. Cell Biol. 73:
155.
35. Lu, S. C., W.-M. Sun, J. Yi, M. Ookhtens, G. Sze, and N. Kaplowitz. 1996. Role
of two recently cloned rat liver GSH transporters in the ubiquitous transport of
GSH in mammalian cells. J. Clin. Invest. 97:1488.
36. Aw, T. Y., M. Ookhtens, and N. Kaplowitz. 1986. Mechanism of inhibition of
glutathione efflux by methionine from isolated rat hepatocytes. Am. J. Physiol.
251:G354.
37. Ohno, Y., and J. I. Gallin. 1985. Diffusion of extracellular hydrogen peroxide into
intracellular compartments of human neutrophils: studies utilizing the inactivation of myeloperoxidase by hydrogen peroxide and azide. J. Biol. Chem. 260:
8438.
38. Fialkow, L., C. K. Chan, S. Grinstein, and G. P. Downey. 1993. Regulation of
tyrosine phosphorylation in neutrophils by the NADPH oxidase: role of reactive
oxygen intermediates. J. Biol. Chem. 268:17131.
39. Finkel, T. 1999. Signal transduction by reactive oxygen species in non-phagocytic cells. J. Leukocyte Biol. 65:337.
40. Hecht, D., and Y. Zick. 1992. Selective inhibition of protein tyrosine phosphatase
activities by H2O2 and vanadate in vitro. Biochem. Biophys. Res. Commun. 188:
773.
41. Denu, J. M., and K. G. Tanner. 1998. Specific and reversible inactivation of
protein tyrosine phosphatases by hydrogen peroxide: evidence for a sulfenic acid
intermediate and implications for redox regulation. Biochemistry 37:5633.