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Department of Immunochemistry, German Cancer Research Center (DKFZ), Heidelberg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation was enhanced through pathways involving Rac, Vav1,
PKC
, p56lck, p59fyn, and IB kinases. In a
cell-free system, the autophosphorylation of rFyn was stimulated by GSH
disulfide but not by HP. These findings suggest that the oxidation of
the cellular thiol pool may play a role as an amplifying mechanism for
TCR/CD3 signals in immune responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A large body of evidence indicates that the activation of certain signal cascades and transcription factors can be induced or enhanced by reactive oxygen species or other oxidants (for examples, see Refs. 3, 4, 5, 6, 7, 8, 9, 10, 11). This phenomenon was mostly interpreted as an oxidative stress response that protects the cells against potentially lethal stress. Various types of oxidative stress were found to induce in certain bacteria and mammalian cells the expression of proteins with cytoprotective activity against oxidative stress (3, 4, 10, 12, 13). When the oxidative stress was induced with unphysiologically high millimolar concentrations of hydrogen peroxide (HP), it was found that the activity of several protein tyrosine kinases (PTKs) such as Lck (14, 15, 16), ZAP 70 (17), and Syk (18) was strongly enhanced. This enhancement may result either from direct oxidative activation or indirectly from the oxidative inhibition of a tyrosine phosphatase that normally down-regulates these PTKs (19, 20). Millimolar concentrations of HP were found to cause also the activation of the mitogen-activated protein kinases (MAPKs) extracellular signal-related kinase (ERK) 1 and ERK2 in Jurkat T cells and in cardiac myocytes (21, 22). In view of the unphysiologically high concentrations of HP that have been used in these earlier studies, it was not clear, however, whether and how these redox effects may contribute to the regulation of the immune system under physiological conditions.
The physiological relevance of redox effects in the immune response
against environmental pathogens in vivo was suggested by the markedly
increased susceptibility to Listeria infection of mice
lacking the gp91 protein of the NADPH oxidase (23, 24). In
the physiological microenvironment of T cells, HP is produced by
activated macrophages at an estimated rate of 2–6 x
10-14 µmol/h per cell and may reach 10–100
µM in the vicinity of these cells (25, 26, 27).
Physiologically relevant concentrations of HP were previously shown to
augment IL-2 production by mitogenically stimulated T cells in
different experimental systems (28, 29). In one of these
studies, 200 µM HP was shown to cause an increase in IL-2 gene
expression, in the AP-1 transcription factor activity and in the
expression of c-Jun but not c-Fos mRNA
(29). Treatment of Jurkat cells with 200 µM HP in the
absence of a mitogenic stimulus was found to stimulate the expression
of c-Jun and the transient expression of c-Fos but not the production
of IL-2 (30). Concentrations of 30–100 µM HP were also
shown to induce NF-B transcription factor activity in one subline of
Jurkat cells but not in others (7).
IL-2 production by ex vivo derived mitogenically stimulated murine
lymphocytes was shown to be enhanced also by high but physiologically
relevant concentrations of L-lactate (LAC; 10–30 mM),
i.e., another metabolite from activated macrophages (31, 32). Because these concentrations of LAC were found to cause
also a decrease in the intracellular GSH level, and the enhancement of
IL-2 production was reversed by the addition of exogenous GSH, it was
suggested that the IL-2 production is modulated by the intracellular
GSH level or by the GSH/GSH disulfide (GSSG) ratio (32).
Taken together, these studies suggested the possibility that even
moderate changes in the GSH status of T cells may play an
important immunopotentiating role in the immune system. Therefore, the
studies in this report have been focused mainly on the regulatory role
of the intracellular thiol status. To investigate the
effect of a mild oxidation of the intracellular GSH pool on the signal
cascades in T cells in more detail, we used mainly the GSH reductase
inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU; Refs. 33, 34) as an alternative to HP and LAC because it provided a
more selective means to modulate the intracellular thiol status.
Previous studies have shown that treatment of T cells with BCNU causes
a dose-dependent decrease in the intracellular GSH level and a
corresponding increase in GSSG, a profound activation of the
transcription factor AP-1, and a moderate activation of the
transcription factor NF-B (35).
The two signaling pathways derived from the TCR and CD28 receptor were
shown to involve a complex scenario of intracellular biochemical events
that finally result in multiple cellular responses. The two signaling
pathways eventually merge and synergistically stimulate the activity of
different members of the MAPK family, i.e., Jun-N-terminal kinase (JNK)
and p38 MAPK, and induce the transcription factor NF-B and the
expression of IL-2 by a combination of transcriptional activation and
mRNA stabilization (36, 37). The activation of Src family
PTKs such as Lck and Fyn is one of the earliest events in TCR signal
transduction. Lck and Fyn knockout mice display severe immunological
defects (38). Once activated, Lck and/or Fyn induce signal
cascades that include among other components PLC
(39),
PKC family members (40, 41) such as PKC
(42), Vav1 (39), Rac (43, 44),
and finally JNK and c-Jun (45), i.e., a common component
of the transcription factors AP-1 and NF-AT (46). Because
stimulation at the TCR in the presence of the costimulatory CD28 signal
leads to T cell activation and IL-2 production, whereas repeated TCR
stimulation in the absence of the costimulatory signal leads to anergy
and peripheral tolerance (47, 48), we addressed in a first
set of experiments the question whether exposure of T cells to
physiologically relevant concentrations of HP or LAC may modulate
selectively TCR/CD3 signaling, CD28 signaling, or both.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BCNU was obtained from Bristol Arzneimittel (Munich, Germany).
Wortmannin, Trolox, cyclosporin A, MKK886, N-acetylcysteine
(NAC), and pyrrolidine dithiocarbamate (PDTC) (Sigma, St. Louis, MO),
and glycerol trinitrate (GTN) (Merck, Darmstadt, Germany) were
purchased from the indicated suppliers. All other reagents were
obtained either from Sigma or from Roche Molecular Biochemicals
(Mannheim, Germany). The Abs were obtained from the indicated sources:
anti-phospho-p38 and anti-phospho-p42/44, New England Biolabs
(Beverly, MA); anti-p38, anti-Fyn (FYN3), and anti-Lck
(2102), Santa Cruz Biotechnology (Santa Cruz, CA); anti-IB
kinases (IKK)
and anti-JNK, PharMingen (San Diego, CA);
anti-Flag (M2), Sigma; anti-hemagglutinin (HA) (12CA5), Roche
Molecular Biochemicals; and anti-Myc (9E10), Upstate Biotechnology
(Lake Placid, NY). Anti-TCR(CD3) (OKT3) and anti-CD28 Abs were
derived from hybridomas and purified. Rac cDNAs provided by Dr. S.
Gutkind (National Institutes of Health, Bethesda, MD) (49)
were Myc tagged and inserted into pEF-BOS-derived vectors. The
expression vectors encoding Vav
319–356 (50), MEKK1
DN (51), MLK3 KR (52), PKC- KR
(53), HA-JNK (54), and MKK7 DN
(55) were previously described. The reporter plasmids
(B)3-luc (56), 4XRE/AP-luc
(57), and the IL2 promoter luciferase construct
(58) have been described previously. The IL-2 luciferase
reporter construct contains the human IL-2 promoter (residues 577 to
+53) inserted into MluI and HindIII cloning sites
of pGL2-Basic (Promega, Madison, WI).
Cell culture, transfections, and stimulations
Jurkat T leukemia cells were grown at 37°C in RPMI 1640 medium containing 10% (v/v) heat-inactivated FCS, 10 mM HEPES, 1% (v/v) penicillin/streptomycin, and 2 mM glutamine (all obtained from Life Technologies, Gaithersburg, MD). Jurkat cells were grown in an incubator at 37°C and 5% CO2 and transfected by electroporation using a gene pulser (Bio-Rad, Richmond, CA) at 250V/950 µF. Stimulation of Jurkat cells was performed in a final volume of 500 µl by adding anti-CD3 (final concentration 10 µg/ml, clone OKT3) and/or anti-CD28 (final concentration 10 µg/ml, clone 9.3) Abs.
Isolation of primary human peripheral T lymphocytes and determination of IL-2 production
PBLs (95% pure) were prepared from heparinized blood of healthy donors by density centrifugation on Ficoll gradients (Lymphoprep Nycomed Pharma, Oslo, Norway) as described (59). After lysis of B cells with sheep erythrocytes, the cells were stimulated with anti-CD3 plus anti-CD28 Abs and/or BCNU (20 µM) for the indicated periods of time. Secreted IL-2 was determined in the supernatant by a kit from BioSource (Nivelles, Belgium) according to the instructions of the manufacturer.
Western blotting
Proteins were extracted from cells in Nonidet P-40 lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM PMSF, 10 mM NaF, 0.5 mM sodium vanadate, leupeptin (10 µg/ml), 1% (v/v) Nonidet P-40, and 10% (v/v) glycerol). Equal amounts of protein were separated by SDS-PAGE before blotting to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membrane was then incubated in a small volume of TBST containing various dilutions of the primary Abs, followed by the detection of the respective proteins with an appropriate secondary Ab coupled to HRP. Secondary Abs were visualized by enhanced chemiluminescence according to the instructions of the manufacturer (Amersham Lifescience, Freiburg, Germany).
Measurement of intracellular GSH
Intracellular GSH and GSSG levels were determined as described (35).
In vitro kinase assays
Cells were lysed in Nonidet P-40 lysis buffer and the IKK, JNK,
p56Lck, and p59Fyn proteins contained in the cell lysate were
immunoprecipitated. The precipitate was washed three times in lysis
buffer. IKK and JNK precipitates were washed two times in kinase buffer
(20 mM HEPES/KOH pH 7.4, 25 mM ß-glycerophosphate, 2 mM DTT, and 20
mM MgCl2). The kinase assay was performed in a
final volume of 20 µl kinase buffer containing 2 µg of bacterially
expressed GST-c-Jun 5–89(5–89) or GST-IB- 1–54(1–54) protein, 5 µCi
[-32P]ATP, and 20 µM ATP for 20 min at 30°C.
Immunoprecipitated p56Lck and p59Fyn were washed two times with kinase
buffer (20 mM PIPES, 10 mM MnCl2) and incubated
in the presence of different concentrations of GSH or GSSG. After
treatment, immune complexes were washed once with kinase buffer and
suspended in a total reaction volume of 20 µl kinase buffer
containing 20 mM PIPES pH 7.2, 10 mM Cl2, 5 µCi
[-32P]ATP, and 20 µM ATP for 20 min at 30°C. The
reaction was stopped by the addition of 5 x SDS sample buffer.
The reaction products were separated by SDS-PAGE, autoradiographed, and
quantified using a phosphorimager.
Luciferase assays
Reporter assays were performed essentially as described (60). Briefly, cells were washed with isotonic buffer and lysed in 100 µl of lysis buffer (Promega, Mannheim, Germany). The luciferase assays were performed according to the manufacturer’s instructions (Promega) and quantified in a Duo Lumat LB 9507 (Berthold, Wildbad, Germany). The results were normalized to the activity of ß-galactosidase expressed by a cotransfected lacZ gene under the control of a constitutive Rous sarcoma virus promoter.
Preparation of recombinant Fyn
The recombinant Fyn protein was isolated from baculovirus-infected Sf9 (Spodoptera frugiperda) insect cells, and purified by affinity chromatography on a phosphotyrosine column plus anion exchange chromatography (61).
EMSA
Jurkat cells (5 x 106) were treated as indicated, and nuclear extracts were prepared essentially as described (62). Briefly, cells were washed twice with TBS buffer (25 mM Tris-HCl pH 7.4, 137 mM NaCl, 5 mM KCl, 0.7 mM CaCl2, 0.1 mM MgCl2). Cells were centrifuged and the pellet was resuspended in 200 µl cold buffer A (10 mM HEPES/KOH pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF) by gentle pipetting. After incubation for 10 min on ice, 5 µl of 10% Nonidet P-40 was added, and cells were lysed by vortexing. The homogenate was centrifuged for 30 s in a microfuge, and the pellet containing the cell nuclei was dissolved in 30 µl buffer C (20 mM HEPES/KOH pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, and 1% (v/v) aprotinin). The extract was centrifuged for 5 min in a microfuge at 4°C, and 5 µg of proteins contained in the supernatant was used for band shift assays.
Binding of NF-B to its cognate DNA (60) and of
CD28RE/AP-binding proteins (57) were measured
as described. The sense sequences of the oligonucleotides were 1)
NF-B: 5'-AGTTGAGGGGACTTTCCCAGGC-3'; and 2) CD28RE/AP:
5'-TCTGGTTTAAAGAAATTCCAAAGAGTCATCAG-3'.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the effects of the macrophage products LAC and HP on
TCR-mediated signals and costimulatory signals, we transiently
transfected Jurkat T cells with a luciferase reporter gene construct
fused to the human IL-2 promoter. The transfected cells were incubated
for 18 h in medium and then stimulated for 8 h with
anti-CD3, anti-CD28 Abs, or the combination of both. If
indicated, the cultures were also treated with HP at a final
concentration of 50 µM for 1 h or LAC at a final concentration
of 10–30 mM for 8 h before Ab stimulation. The results showed
that treatment with HP or LAC had relatively little effect on the
transcriptional activity after stimulation with anti-CD3 Abs in the
absence of anti-CD28 Abs but caused a substantial enhancement in
the presence of anti-CD28 Abs alone or in combination with
anti-CD3 Abs (Fig. 1
). Considering
that anti-CD3 Abs caused in the presence of anti-CD28 an
3-fold increase in transcription, the data indicated that exposure
to HP or LAC can substitute at least partly for the TCR-mediated signal
but not for the costimulatory signal. Therefore, we stimulated the
Jurkat cells in all subsequent experiments with anti-CD28 Abs in
combination with anti-CD3 Abs or PMA.
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B and CD28RE/AP
elements from the IL-2 promoter
To determine the influence of the thiol redox state on the
transcriptional activity under the control of the IL-2 promoter, we
measured the transcription from the reporter gene construct after
treatment of the transfected Jurkat cells with various anti-CD28
Abs in combination with anti-CD3 Abs or PMA in the presence or
absence of BCNU. In a first set of experiments we showed that BCNU
causes a dose- and time-dependent decrease in the intracellular
GSH/GSSG ratio of the Jurkat cells (Fig. 2A) and aggravates the
decrease in the GSH/GSSG level that is seen after treatment with
anti-CD3/CD28 Abs alone (Fig. 2B). These changes were
similar to the changes seen with the physiologically relevant
concentration of 50 µM HP and with 100 µM of the NO donor glycerol
trinitrate (GTN) (Fig. 2B). BCNU caused only a moderate
enhancement of transcription from the IL-2 promoter in the absence of
the stimulatory agents, but a substantial enhancement in the presence
of these agents (Fig. 3A). The
2- to 3-fold enhancement after BCNU treatment was again in the same
order of magnitude as the relative enhancement that was achieved in
anti-CD28-stimulated cells by anti-CD3 Abs (Fig. 1
).
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B). Analogous
experiments with a reporter gene under the control of NF-B binding
sites gave similar results (data not shown). The transcription of a
reporter gene under control of NF-AT binding sites, in contrast, was
not markedly affected by BCNU treatment irrespective of whether
transcription was induced by costimulation (data not shown). To
investigate the effect of BCNU on the binding of nuclear proteins to
the CD28RE/AP element in unstimulated and stimulated Jurkat cells, we
analyzed also the DNA/protein complexes by EMSA. The results showed
that the stimulatory agents induced the binding of proteins in complex
I and enhanced the binding of complexes II and III proteins (Fig. 3C). In the absence of the stimulating agents, BCNU had no
effect on complex I, but enhanced the binding of proteins in complex II
and III. If applied in combination with anti-CD3/CD28 Abs, BCNU did
not further enhance the binding of complex III proteins, but strongly
augmented the binding of proteins in complexes I and II. Supershift
experiments using subunit-specific Abs revealed the occurrence of the
NF-B p65 protein in complexes I and II and the predominant
localization of c-Rel within complex I (data not shown). Enhancement of the CD3/CD28-induced activation of IKK by BCNU treatment
Because the CD28RE/AP element is contacted also by proteins of the
NF-B/Rel family (63), and because NF-B activity was
previously shown to be enhanced by BCNU (35), we examined
the effect of BCNU on the activation of NF-B in more detail. In a
first set of experiments, we determined whether BCNU may induce or
enhance the NF-B DNA-binding activity whether administered alone or
in combination with PMA, anti-CD3, and anti-CD28 Abs. The
DNA-binding activity of NF-B was determined by EMSA. BCNU was found
to enhance the induction of NF-B DNA-binding by the stimulating
agents but had little effect by itself (Fig. 4A). To test whether the
enhanced induction of the NF-B DNA-binding activity may result from
an increased activation of IKKs or from IKK-independent mechanisms as
reported for the UV-induced NF-B activation (64), the
IKK complex was isolated from extracts of BCNU-treated or untreated
Jurkat cells by immunoprecipitation with monoclonal anti-IKK
Abs. The subsequent analysis by immunocomplex kinase assays with
recombinant GST-IB as substrate revealed that the
CD3/CD28-induced IKK activity was strongly enhanced by BCNU (Fig. 4B). This was associated with an increased phosphorylation
of IKK itself, indicating that the BCNU treatment acted upon a
target further upstream of the IKKs.
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The three known families of MAPKs, i.e., ERK, p38 MAPK, and JNK
(65), play a key role in immune responses by inducing the
transactivation potential of several immunologically important
transcription factors (66). Their activity is controlled
by another set of protein kinases (MAPK kinases), which are activated
in turn by various signals including cytokines (66). To
analyze the effect of BCNU on the activation of the three MAPK
families, we treated Jurkat T cells with graded concentrations of BCNU
and determined in different aliquots of the cell extracts the JNK
activity, the phosphorylation of p38 MAPK, and the phosphorylation of
ERK1/2 using phospho-specific Abs. The results (Fig. 5A) showed that the kinase
activity of endogenous JNK, as measured by the immunocomplex kinase
assay, is strongly augmented by 10–100 µM BCNU but inhibited at
higher concentrations. The analysis of p38 MAPK and ERK1/2
phosphorylation by immunoblotting with phospho-specific Abs revealed
that the phosphorylation of p38 MAPK was similarly enhanced by 10–100
µM BCNU and inhibited at higher concentrations, whereas p42/44
(i.e., ERK1/2) was not detectably phosphorylated in the presence of
BCNU (Fig. 5A). Concentrations of 10–100 µM BCNU
decreased the intracellular GSH/GSSG ratio of costimulated Jurkat cells
typically to values <10 (see Fig. 1).
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). The results showed that both agents enhanced the activation of
JNK and the phosphorylation of p38 MAPK in
anti-CD3/anti-CD28-stimulated Jurkat cells to a degree that was similar
to that seen with BCNU (Fig. 5B). In contrast, higher
concentrations of GTN (
0.5 mM) or HP (0.5 mM) inhibited the
activity of JNK and the phosphorylation of p38 MAPK (data not shown),
indicating that these signal pathways are enhanced by moderately
oxidative conditions but inhibited by strongly oxidative
conditions. BCNU treatment enhances MAPK activation and IL-2 production in primary human T cells
To ensure that the regulatory redox effect operates also in
primary T cells, we stimulated human peripheral T lymphocytes with
anti-CD3 plus anti-CD28 Abs together with or without 20 µM
BCNU. The results (Fig. 6) showed that
the kinase activity of the endogenous JNK as measured by c-Jun
phosphorylation as well as the phosphorylation of p38 MAPK were
markedly enhanced by BCNU treatment (Fig. 6A). In addition,
BCNU treatment was found to cause a substantial increase in IL-2
production (Fig. 6B).
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To characterize the regulatory redox effects in more detail, we
treated Jurkat T cells with BCNU in the absence or presence of the
antioxidants NAC or PDTC, the 5-lipoxygenase inhibitor MK886, the
phosphatidylinositol 3-kinase antagonist wortmannin, the calcineurin
inhibitor cyclosporin A, the PTK inhibitor herbimycin A, and the
vitamin E-analogous antioxidant trolox that prevents peroxidation of
membrane lipids. These experiments revealed that the BCNU-induced JNK
activation and p38 MAPK phosphorylation were not affected by trolox,
MK886, wortmannin, or cyclosporin A, but were profoundly inhibited by
NAC, PDTC, or herbimycin A (Fig. 7A), indicating that one or
more tyrosine kinase species may be involved in the activation cascade.
The inhibitory effect of the structurally unrelated antioxidants NAC
and PDTC provided additional support for the interpretation that the
GSH system modulates the signaling cascade by redox regulation.
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, and MKK7. These experiments showed that the activation of JNK
by BCNU was markedly inhibited by all dominant negative proteins under
test (Fig. 7B). Because Vav, Rac, and PKC are activated
early during T cell activation (67), it is suggested that
the redox-sensitive components are located upstream of these components
at a very early point in the signal cascade. This conclusion was
further supported by the fact that the activation of JNK by ectopic
expression of Rac was not blocked by NAC (data not shown). Activation of Lck and Fyn
Because the previous experiments suggested
collectively that the redox-sensitive target may be a relatively early
component of the signal cascade, we studied the effect of
BCNU on PTKs and on the tyrosine phosphorylation of signal proteins.
Because the two PTKs Lck and Fyn were previously shown to be activated
in T cells with a sulfhydryl-reactive reagent or high (i.e.,
physiologically not relevant) concentrations of HP (14, 15, 16, 68), we treated Jurkat cells again for various time periods with
BCNU and determined at first the kinase activity of the endogenous Lck
by immunoprecipitation and in vitro kinase assay. These experiments
showed that BCNU stimulated (within 5 min) the autophosphorylation of
Lck (Fig. 8A) and the
phosphorylation of its substrate protein enolase (data not shown).
Similarly, BCNU stimulated also the autophosphorylation (Fig. 8B) and kinase activity (data not shown) of Fyn. When cell
extracts from BCNU-treated Jurkat T cells were analyzed by Western
blotting with anti-phosphotyrosine Abs, another protein of 74 kDa was
also found to be phosphorylated within 5 min after BCNU treatment
followed by two proteins of 95 and 56 kDa (Fig. 8C). The
protein of 95 kDa was identified by immunoblotting and
immunoprecipitation as the Vav protein (data not shown), which is known
to be inducibly phosphorylated by Lck (43, 44) and Fyn
(69). To determine whether GSH may interact directly with
the Fyn kinase protein, we studied the effect of GSSG on rFyn in a
cell-free system. The results showed that GSSG enhanced strongly the
autophosphorylation of rFyn in a dose-dependent fashion (Fig. 8D), whereas HP (0.5–2 mM), thioredoxin (10–150 µg/ml),
or butylated hydroxyanisol (100–500 µM) had no conspicuous effect
(data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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With these experimental conditions, we have then shown 1) that the
transcription from the IL-2 promoter can be enhanced by moderate
oxidative changes in the intracellular thiol pool of the T cells, and
2) that such moderately oxidative conditions are associated with the
stimulation of a signal pathway that leads to the selective activation
of the MAPKs JNK and p38 MAPK but not ERK/1 and ERK/2. The
redox-sensitive components of this signal cascade were shown to be
located at a very early point in the cascade and tentatively identified
as p56 Lck and p59 Fyn. Most of these experiments were performed with a
Jurkat T cell line, but similar results were also obtained with primary
human T cells (Fig. 6). The TCR expression was not markedly altered in
Jurkat or primary human T cells by BCNU at the relevant concentrations
(25–100 µM) and after various time intervals (6–36 h) as tested
cytofluorometrically with anti-CD3 Abs (data not shown).
The changes that were seen under these moderately oxidative conditions resembled only partly the effects of the strongly oxidative conditions that have been reported previously. In line with our results in this report, millimolar concentrations of HP were previously shown to enhance the activity of Lck (16). However, at the relatively high concentration used by these authors and especially if applied in combination with vanadate, HP may enhance indirectly the activated and autophosphorylated state of Lck and other PTKs of the Src family by inhibiting corresponding protein tyrosine phosphatases (19). Beiqing et al. (30) reported that HP caused a substantial increase in the expression of c-Jun and AP-1 DNA-binding activity in Jurkat cells but failed to activate the transcription factor NF-AT. HP even inhibited the transcription under control of NF-AT or the IL-2 promoter in cells that had been stimulated with PMA plus PHA. Because NF-AT activity requires the phosphorylation of c-Jun, this inhibitory phenomenon may be related to our observation that the activation of JNK and the phosphorylation of p38 MAPK were inhibited by higher (i.e., 0.5 mM) concentrations of GTN or HP (data not shown). Others have found that millimolar concentrations of HP cause also the activation of ERK1 and ERK2 in Jurkat T cells and cardiac myocytes (21, 22). These MAPKs were not detectably activated under our conditions. Activation of JNK and p38 MAPK in the absence of ERK1/2 activation was previously observed also in human 293 cells after treatment with the S-alkylating agent methyl methanesulfonate, and there was suggestive evidence for a determining role of the intracellular GSH pool also in those experiments (70). Taken together, these earlier studies and our experiments with 50 µM BCNU lead to the conclusion that the moderate oxidation or depletion of intracellular thiols has relatively selective effects on cellular signal cascades. BCNU inhibits the GSH reductase enzyme by interacting with a functionally important sulfhydryl group, but is not a nonspecific sulfhydryl reactive reagent (34). Because an experimentally induced decrease in the GSH:GSSG ratio by three different agents, i.e., by BCNU, HP, or the NO donor GTN, had essentially the same effect on the activation of c-Jun and p38 MAPK, it is suggested that the redox modulation of T cell activation involves a shift of the GSH redox state and/or the oxidative modification of certain thiol groups in signal proteins. Activation of JNK without concomitant activation of ERK2 has also been observed in T cells stimulated with natural CD28 ligands (71); a similar activation pattern was found in human skin fibroblasts after irradiation with UV-A (72), suggesting that UV-A may cause a similar type of oxidative stress with similar effects on redox-sensitive signal proteins.
Furthermore, our analysis of the signaling events in BCNU-treated cells
indicated that the oxidative modification of certain thiol groups in
one or a few early components of the signal cascade leads to the
activation of Vav, NF-B, JNK, and p38 MAPK. This has the interesting
implication that many Vav/Rac-regulated processes, including the
organization of cytoskeletal proteins (73), may be subject
to regulation by redox changes in the cellular thiol pool. Our
experiments with pathway-specific inhibitors excluded the possibility
that these redox-mediated effects involve lipid peroxidation,
5-lipoxygenase, phosphatidylinositol 3-kinase, or
Ca2+-dependent signaling processes. The
redox-mediated activation of JNK and p38 MAPK appears to involve PTKs
because the activation was completely inhibited by herbimycin A and by
dominant negative versions of Vav, PKC, and Rac, i.e., signaling
components that are located downstream on PTKs.
Our experiments on the oxidative activation of rFyn by GSSG in a
cell-free system (Fig. 8) suggest that Fyn is (one of) the
redox-sensitive targets responsible for the redox modulation of T cell
signaling. P59fyn and
p56lck are PTKs of the Src family that are
predominantly expressed in lymphoid cells and involved in Ag receptor
signaling (74, 75, 76). It has been shown in various cells
that oxidative stress causes formation of mixed disulfides
between low m.w. thiols and sulfhydryl groups of certain
cytosolic proteins (S-thiolation). Mixed disulfide formation
with GSH (i.e., S-glutathiolation) was found to
stimulate certain enzymes and to inhibit others (77, 78, 79, 80).
The details of the chemical modification p59fyn
and GSSG remain to be analyzed but may involve the motif CPxxxxxxMxxCW
(see Ref. 81) that is shared by the PTKs,
p59fyn, p56lck, and
p60c-src. Similar motifs occur also in
p69ltk (82) and
p72syk (83).
The mild oxidative changes in the intracellular thiol pool occur not
only in pathological conditions of oxidative stress but are likely to
play an important role in the normal immune response under
physiological conditions. In the immediate vicinity of activated
macrophages, T cells are exposed to 10-4 M HP
(25, 26, 27), i.e., a concentration that was found to cause a
similar change in the GSH/GSSG ratio (Fig. 2B) and a similar
increase in IL-2 promoter activity (Fig. 1A) as the
treatment with BCNU (Figs. 2 and 3). Moreover, several types of
leukocytes including activated macrophages and granulocytes perform
glycolysis and release LAC into the extracellular space even under
aerobic conditions (84, 85). The interstitial fluid in the
immediate vicinity of activated macrophages is likely to contain 20–40
mM LAC (31). LAC concentrations may even systemically
reach concentrations of 20–30 mM in conditions of lactic acidosis
(86). Exposure of T cells to 10–30 mM
LAC was previously shown to decrease the
intracellular GSH level, enhance the production of IL-2 (31, 32), and enhance also the activation of CTL (87).
By amplifying the TCR-mediated signal transduction, the exposure of T
cells to HP and LAC from activated macrophages may enable the infected
host to start the immune response against an invading pathogen long
before optimal doses of Ag accumulate. The decrease in the signal
threshold may be critically important for the immune system to win the
race with rapidly multiplying pathogens. Decreasing the threshold may
also increase the risk of autoimmune processes. T cells isolated from
the synovial fluid of patients with rheumatoid arthritis were recently
found to have predominantly a decreased intracellular GSH level and the
"primed" CD45RO phenotype (88).
Taken together, our experiments indicate that moderate changes in the GSH status as they are typically induced by several different macrophage products of small m.w. may amplify the TCR-mediated signal pathway at a very early point and, thereby, play an important auxiliary role in the immune system of higher organisms. The contribution of these oxidative changes to the functional changes of T cells in autoimmune processes and other pathological states remains to be investigated.
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2 Current address: McKinsey & Company, 50672 Cologne, Germany.
3 Current address: Faculty of Health and Science, Ben-Gurion University, Beersheba, Israel.
4 Current address: Department of Immunology and Cell Biology, University of Münster, Röntgenstr. 21, 48149 Münster, Germany.
5 Address correspondence and reprint requests to Dr. M. Lienhard Schmitz or Dr. Wulf Dröge, Department of Immunochemistry, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
6 Abbreviations used in this paper: GSH, glutathione; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; GSSG, GSH disulfide; GTN, glycerol trinitrate; HA, hemagglutinin; HP, hydrogen peroxide; JNK, Jun-N-terminal kinase; LAC, L-lactate; MAPK, mitogen-activated protein kinase; NAC, N-acetylcysteine; PDTC, pyrrolidine dithiocarbamate; PTKs, protein tyrosine kinases; ERK, extracellular signal-related kinase; IKK, IB kinases.
Received for publication May 10, 2000. Accepted for publication July 26, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and transcription of human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 87:9943.B activity by cysteine and cysteine derivatives. AIDS 5:497.[Medline]
B transcription factor and HIV-1. EMBO J. 10:2247.[Medline]
B and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO J. 12:2005.[Medline]
B and AP-1. Proc. Natl. Acad. Sci. USA 91:1672. B in a tyrosine kinase-dependent mechanism and in combination with vanadate activate the p56lck and p59fyn tyrosine kinases in human lymphocytes. Blood 82:1212.B activation through CD28 requires reactive oxygen production by 5-lipoxygenase. EMBO J. 14:3731.[Medline]
B and the activator protein-1. Eur. J. Biochem. 221:639.[Medline]
ß T cell development is abolished in mice lacking both Lck and Fyn protein tyrosine kinases. Immunity 5:429.[Medline]
to activate JNK and IL-2 promoter in T lymphocytes. EMBO J. 17:3101.[Medline]
B kinase complex by MEKK1, a kinase of the JNK pathway. Cell 88:213.[Medline]
, a selective upstream regulator of JNK/SAPK and IL-2 promoter activation in Jurkat T cells. Eur. J. Immunol. 29:132.[Medline]
and cellular stresses. EMBO J. 16:7045.[Medline]
B dependent transcription involves direct interference with transactivation. Proc. Natl. Acad. Sci. USA 94:13504. B by preventing the degradation of I B- and I B-ß. J. Biol. Chem. 273:1288.B activation is antagonized by E1B 19 K. Mol. Cell. Biol. 16:4052.[Abstract]
B by UV. EMBO J. 17:5170.[Medline]
regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70:389.[Medline]
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,
Indicates the location of the DNA-NF-
indicates the
position of a constitutively DNA-binding protein. B,
Jurkat cells were stimulated for 3 h with anti-CD3/anti-CD28 Abs
and BCNU (10 µM) as shown. Endogenous IKK
