and glutathione disulfide in immunology and immunopathology. WULF. DR#{212}GE,'KLAUS SCHULZE-OSTHOFF,. SABINE MIHM, DAGMAR. GALTER, HEIKE.
REVIEWS
Functions
of glutathione
immunology
and
Department
of Immunochemistry,
Even
supply
a moderate
elevates
the
Deutsches
increase
in the
intracellular
Krebsforschungszentrum,
cellular
glutathione
(GSH) and glutathione disulfide (GSSG) levels and potentiates immunological functions of lymphocytes in vitro. At low GSSG levels, T cells cannot optimally activate the immunologically important transcription factor NFxB, whereas high GSSG levels inhibit the DNA binding activity of NFxB. The effects of GSSG are antagonized by reduced thioredoxin (TRX). As the protein tyrosine kinase activities p56lck and p59fY’ are activated in intact cells by hydrogen peroxide, they are likely targets for
GSSG action. These redox-regulated nal cascades for NFxB activation
enzymes
trigger
sig-
from the molecules,
and transduce signals T cell antigen receptor, from CD4 and CD8 and from the IL-2 receptor 13-chain. The effec-
tor phase
of cytotoxic
T cell responses
and
IL-2-depen-
dent functions are inhibited even by a partial depletion of the intracellular GSH pool. As signal transduction is facilitated by prooxidant conditions, we propose that the well-known immunological consequences of GSH depletion ultimately may be results of the accompanying GSSG deficiency. As HIV-infected patients and SlY-infected rhesus macaques have, on the average, significantly decreased plasma cyst(e)ine and intracellular GSH levels, we also hypothesize that AIDS may be the consequence of a GSSG deficiency as well.-Droge, W., Schulze-Osthoff, K., Mihm, S., Galter, D., Schenk, H., Eck, H. -P., Roth, S., Gm#{252}nder,H. Functions of glutathione and glutathione disulfide in immunology and immunopathology. FASEBJ. 8, 1131-1138 (1994) Key tion
Words:
transcription
factors
redox regulation
GSSG regula-
NFxB
AGAINST environmental hazin bacteria, as exemplified by the adaptive response against oxidative stress (1). Reactive oxygen intermediates (ROI)2 such as superoxide anions or hydrogen peroxide are produced during various electrontransfer reactions (2). If exposed to hydrogen peroxide or superoxide anions, certain bacteria respond with the synthesis of protective enzymes. The response involves activation of a
ADAPTIVE
DEFENSE
ards
already
have
MECHANISMS
been
set of redox reactive
found
transcription
factors (Oxy R or Sox R,
Sox 5, respectively) that up-regulate inducible protective genes (3, 4). The immune system of higher organisms has evolved as an even more sophisticated adaptive defense mechanism. Recent evidence suggests that this system also uses the ancient principle of redox regulation. Substantial quantities of hydrogen peroxide are produced by macrophages and neutrophils in the inflammatory environment, and similar concentrations of hydrogen peroxide were found to stimulate the and
0892-6638/94/0008-1131/$01.50.© FASEB
in
immunopathology
SCHULZE-OSTHOFF, SABINE MIHM, DAGMAR ECK, STEFFEN ROTH, AND HELMUT GMUNDER
HANS-PETER
cysteine
disulfide
DR#{212}GE,’KLAUS
WULF
ABSTRACT
and glutathione
D-69120
GALTER,
Heidelberg,
HEIKE
SCHENK,
Germany
activation of immunologically and immunological functions. tive
substances
disulfide play
and role
in this
such
(GSSG),
an
relevant transcription factors Not unexpectedly, redox reacas glutathione (GSH), glutathione the thioredoxin (TRX) redox couple
important
context.
This
review
summa-
the available evidence for the redox regulation munologically relevant genes and for the eminent GSH and GSSG in immunological responses.
of imrole of
rizes
REDOX
REGULATION
IMMUNOLOGICALLY
TRANSCRIPTION
OF NFxB
AND
OTHER
RELEVANT
FACTORS
The nuclear transcription factor xB (NFxB) is involved in the inducible transcription of several immunologically important genes including those of the interleukin-2 (IL-2) receptor a-chain, tumor necrosis factor-a (TNF-a), major histocompatibility complex antigens, and c-fos (reviewed in refs 5, 6). NFxB protein has been found in many different cell types including B and T lymphocytes, macrophages, and monocytes (6). Transcriptional activation of NFxB-dependent genes can be induced by dissociation of a preexisting inactive complex composed of the NFxB p5O/p6S heterodimer and an inhibitory protein IxB, as illustrated in Fig. 1. Dissociation of this complex permits the p50/p65 heterodimer to be translocated into the nucleus and to exert its transactivating function (7). As the activation of NFxB is accompanied by degradation of IxB (8), there is a possibility that this process is actually triggered by the activation of a specific protease or by modification of IxB. Redox processes are involved further upstream in the signaling cascade (Fig. 1). Hydrogen peroxide is reportedly produced by activated macrophages at a rate of 2-6 x 10 mol/h per cell (9, 10). The inhibitory effect of activated macrophages on mitogenic responses of lymphocytes was shown to be mediated at least partly by hydrogen peroxide (11, 12) and the inhibition of the mitogenic response requires
‘To
whom
correspondence
and
reprint
requests
should
be
addressed.
2Abbreviations: butylhydroxyanisol;
BCNU, l,3-bis(2-chloroethyl)-l-nitnzourea; BSO, L-buthionine-(S,R)-sulfoximine; GSSG, glutathione disulfide; IxB, inhibitor
glutathione; interleukin-l; ethanol; NAC, p50/p65,
IL-2R,
interleukin-2
N-acetyl-cysteine;
NFxB
heterodimer;
receptor; NDGA,
PDTC,
2-ME,
nordihydroguaiaretic pyrolidine
BHA, GSH, of ,cB; IL-l, 2-mercaptoacid;
dithiocarbamate;
TCR, T cell receptor
for antigen; TNFa, tumor necrosis factor-cs; TPA, tetradecanoylphorbolacetate; TRXr,,d, reduced thioredoxin; AP-l, activator protein-l; APC, antigen-presenting cells; ROl, reactive
oxygen
intermediates;
chloram-phenicolacetyltransferase; cells.
1ICF1a,
T
cell-specific
LAK,
factor-la;
lymphokine-activated
CAT, killer
1131
Ill_V
ulWVJ
NFxB
ACTIVATION
BY CYTOKINES
RECEPTOR-MEDIATED
WA ILl TNF. “C.
Figure
1.
Redox
regulation
of
NFxB
by
glutathione
disulfide
(GSSG). BCNU, 1,3-bis(2-chloroethyl)-i-nitrosourea; BSO, Lbuthionine-(S,R)-sulfoximine; GSSG, glutathione disulfide; IxB, inhibitor of xB; IL-i, interleukin-i; IL-2R, interleukin-2 receptor; 2-ME, 2-mercaptoethanol; p5O/p65, NFxB heterodimer; PDTC, pyrolidine dithiocarbamate; TCR, T cell receptor for antigen; TNFo#{231} tumor necrosis factor-a; TPA, tetradecanoylphorbolacetate; TRXre,i, reduced thioredoxin.
about 10 M hydrogen peroxide (13), sO it is reasonable to assume that concentrations of this order of magnitude do in fact occur at the surface of the activated macrophages. Physiologically relevant concentrations of hydrogen peroxide rapidly induce NFB-speciflc DNA binding and transactivating activity in a human T cell line (Jurkat R) and in cells of the cervix carcinoma line HeLa (14-16). The general importance of this effect has occasionally been questioned in view of the fact that some other T lineage cells such as the human Molt-4 line fail to activate NFxB upon exposure to hydrogen peroxide under standard cell culture conditions (Fig. 2). However, NFxB can be induced by hydrogen peroxide in Molt-4 cells if cultured in a modified NCTC 135 culture medium (described in ref 17) that contains more physiological amino acid concentrations, including cystine but no cysteine, nucleotides, or nucleosides (Fig. 2). If cultured in
standard
RPMI
higher
GSSG
medium, levels
AND
OTHER
SIGNALS
NFxB activity can be induced in T lineage cells not only by hydrogen peroxide but also by other agents such as TNF-a, IL-I, or tetradecanoylphorbolacetate (TPA). Recent evidence indicates that these agents use also redox processes for signal transduction (14). TNF-a, for example, was shown to induce the production of reactive oxygen intermediates in several cell types (20, 21). Experiments with antioxidants provided further support for the conclusion that these oxidative processes are part of the signal cascade to NFxB activation. Cysteine, N-acetyl-cysteine (NAC), 2-mercaptoethanol (2-ME), butylhydroxyanisol (BHA), nordihydroguaiaretic acid (NDGA), and vitamin E analogs are potent inhibitors of NFxB activation (14, 15, 22-26). Cysteine and the xenobiotic thiol pyrolidine dithiocarbamate (PDTC) appear to inhibit NFxB activation by several different mechanisms depending on their concentration (15, 27). REDOX-SENSITIVE CASCADE
PROTEINS
IN THE
SIGNAL
Several studies suggest that the activation of NFxB is triggered through a cascade of membrane-bound and cytoplasmic protein kinases including tyrosine kinases of the src family (28-30). At least two members of the src family, p56 and p59f’Y’, were found to be activated by hydrogen peroxide and by the thiol oxidizing agent diamide (29-31). The protein tyrosine kinase p56 is associated with CD4
MOLT-4
JURKAT
RPMI H202’
-
TPA-
NCTC RPMI ______________________
NCTC
+
-
+11 -
-
++-
+
-
-
++
JR
+1 I
NFKB-
-
+
-
-
-
++
ll
#{247} +1
-
-
++
-
Molt-4 cells show a priori markedly
that
are
barely
increased
by hydrogen
peroxide, suggesting that the activation of NFxB may be induced by changes of GSSG levels rather than by high GSSG levels per se. Further studies will have to show whether the
1
2
3
4
5
6 -
7
.
8
-S
-
9
10111213141516
-,
oxidative burst in the inflammatory environment also stimulates NFxB activation in vivo, and whether oxidative conditions are also required for the classical antigen-triggered activation of NFxB-dependent genes or whether they trigger an independent or auxillary signal pathway. There is a strong indication that the T cell-specific factor-la (TCF1a), i.e., another immunologically relevant transcription factor, is also subject to redox control. Like NFxB, TCF1a activity of fetal thymocytes in submersion cultures is potentiated under conditions of elevated oxygen concentrations (18). The transcription factor activator protein-i (AP-i) is a heterodimer of Fos and Jun proteins that controls the expres-
Figure 2. Different patterns of NFxB activation by hydrogen peroxide in Molt-4 and Jurkat JR cells. Cells of the human T cell lines Molt-4 (20) or Jurkat JR (8) were incubated overnight at a density of 10 cells/20 ml in standard RPMI medium or modified NCTC
sion of another set of genes including that of the T cell growth factor IL-2 (19). In HeLa cells, AP-i activity is
or hydrogen
peroxide
stimulated
vested
nuclear
to
some
extent
by
hydrogen peroxide (19). show only marginal AP-l to hydrogen peroxide (16) (Fig. 2).
Human
T Image cells, in contrast,
activity
after
1132
Vol. 8
exposure
November
1994
The
FASEB
Wie
L1
135
culture
and
medium
mosphere (20). NFxB mined by electrophoretic
Journal
(11) and (30
LM).
extracts
then stimulated with TPA (20 ng/ml) Two hours later, the cells were harwere
and AP-l mobility
prepared
under
nitrogen
DNA binding activity shift assays as described
at-
was deterin ref 20.
DROGE
ET AL.
REVIEWS and
CD8
molecules
and
is associated
p59fYu1
with
(32-34). Both tyrosine of the corresponding likely
IL-2 the
receptor
/3-chains
T cell receptor/CD3
kinases surface
whereas complex
are activated by cross-linking receptor molecules and are
to
play an important role as signal transducing in T cell-mediated immune responses. Proteintyrosine-phosphatases, conversely, have a reactive cysteine residue in their active site, which must be in the reduced form for catalytic activity (35). It remains to be determined
molecules
whether
elevated
GSSG
tyrosine
residues
by
levels stabilize inhibiting
the
and GSSG/GSH ratios by inhibiting glutathione reductase (27). Pretreatment with BSO also inhibits this effect (27). The effects of these agents and the putative role of GSSG in the activation of NFxB are schematically illustrated in Fig. 1. The simplest interpretation of the available data is that hydrogen peroxide elevates intracellular GSSG levels and thereby acts indirectly on the signal cascade of NFxB activation.
the phosphorylated corresponding
phos-
phatases.
REACTIVE CYSTEINE RESIDUES TRANSDUCING PROTEINS AND
IN SIGNAL TRANSCRIPTION
FACTORS ROLE OF GSSG ACTIVATION
IN THE
INDUCTION
OF
NFxB
A direct interaction between hydrogen peroxide and any protein in the signal cascade has not yet been demonstrated. It is well established, however, that hydrogen peroxide converts
reduced
GSH
into GSSG
by the glutathione
peroxidase
re-
action. signal NFxB
An important role of GSSG in the NFxB activating cascade is suggested by the facts that 1) the potent inducer TPA increases intracellular GSSG levels and GSSG/GSH ratios in T lineage cells (36), and 2) the TPAinduced activation of NFxB is inhibited by L-buthionine(S,R)-sulfoximine (BSO) (S. Mihm and W. DrOge, unpub-
lished observation), synthesis (37) that
a specific inhibitor of glutathione depletes both intracellular GSH
bioand
GSSG levels. Experiments with human T lineage cells (Molt-4) in DTT-supplemented cultures suggest that the hydrogen peroxide-induced activation of NFxB may also involve GSSG (27). Treatment with BSO inhibits under these conditions the activation of NFxB by hydrogen peroxide alone or in combination with TPA. Moreover, the TPAinduced activation of NFxB in DTTsupplemented cultures is markedly enhanced by i,3-bis(2-chloroethyl)-l-nitrosourea (BCNU), an agent that elevates intracellular GSSG levels
TABLE
1. Examples of proven or putative
Cysteine
clusters
in the vicinity
Proto-oncogene
tyrosine
redox reactive cysteine
of positively
charged
As GSSG plays an important role in protein disulfide formation and protein folding, it is suggested that the NFxB activating signal cascade may require disulfide formation at a critical cysteine residue of one of the regulatory proteins in the cascade. A widely used procedure to identify potentially
redox
reactive
proteins
the use of thiol
reactive
residues in proteins
amino
acids
kinase
p59b
xRxxRxPCPxxCPxxxHxxxxHCxKKxPxxRPx
p5fik
xRxxRxxxPxxCPxxxxxxxRxCxKxxPxx
RPx
xxxxxxxxCxPCKxxKxx
Thioredoxin Phospholipase
C-’12
PLC--y2
(cys 348/353/355/360)
PLC-’12
(cys
1082/1090)
PLC-72
(cys
1184/1199)
Single cysteine
residues
Phospholipase
xRCxRxxCRCxxxxCxxxPxxKP xHxPKxxRxxxCPxxxxxxC xCxxRxxxxxxxxxxxx#{231}RxxRRRx
in the vicinity
of multiple
positively
charged
amino
acids
C-y2
PLC-y2
(cys
RxxRxxHCRxRx RxRxKxKHCRxxRxxRHx
586)
PLC-72 (cys 693) PLC-y2
(cys
xCKPxxKxKx
937)
Transcription factors NFxB and Rel Proteins ‘IC,
involves
agents such as diamide or N-ethylmaleimide (for example, see ref 38). This method must be used, however, with the reservation that the functional inactivation of a given protein by a highly reactive and unphysiological agent does not prove that the thiol groups being tested are also subject to redox regulation in intact cells. Mammalian cells contain approximately 10 mM GSH and approximately 0.5 mM GSSG in the cytosole and mitochondrial matrix (39, 40). Although GSSG/GSH ratios may vary among different cell types, it is clear that most mammalian cells have strongly reducing intracellular conditions. Therefore it is generally assumed that the cysteine residues of cytoplasmic and nuclear proteins are largely in the reduced state. However, at least one cysteine residue in each of the proto-oncogene tyrosine kinases 59fn and 5filck is located next to a proline and close to other positively charged amino acids and an additional cysteine (Table 1). A cationic environment stabilizes the deprotonated S group and thereby renders the thiol group
cysteine;
RxxRxRxxC
H, histidine;
K, lysine;
P, proline;
R, arginine;
x, one of the other amino
acids;
C, cystelne
adjacent
to a positively
charged
amino
acid.
GLUTATHIONE
FUNCTION
IN IMMUNOLOGY
AND
IMMUNOPATHOLOGY
1133
Il_V
Il_VVJ
highly reactive and susceptible to oxidation. A second cysteine in the vicinity can lead to formation of a cyclic disulfide
and thereby may cause an even stronger decrease of the redox potential. This is typically exemplified by the conserved sequence motif CxPCK of the well-known oxidoreductase TRX (Table 1). Whereas the oxidation of free cysteine molecules to cystine or GSH to GSSG is associated with the loss of one degree of freedom, the formation of the intramolecular disulfide bridge requires only little change of
Like TRX,
entropy.
p59’”
and p56
can also be expected
to undergo oxidation easily even within the relatively ing intracellular environment. Similar clusters with cationic amino acids have been found in phospholipase
another
in signal transduction (Table I). NFxB and related factors of the v-Rel and c-Rel oncoprotein family share a characteristic seThe
quence
protein
reducseveral C-
involved
transcription motif
with
factors
a cysteine
and
three
arginine
residues
in
the DNA binding region (41-43) (Table 1). It is, therefore, not surprising that NFxB is easily inhibited in vitro by oxidation and reactivated by thiols such as 2-mercaptoethanol or dithiotreitol (38). Studies with several physiologically relevant oxidizing and reducing agents revealed that NFxB DNA binding is inhibited by physiologically relevant concentrations of GSSG even in the presence of a several-fold excess of thiols, and that it is reactivated by physiologically relevant concentrations of reduced TRX (27).
SECOND LEVEL OF REDOX REGULATION GSSG: INHIBITION OF NFxB DNA BINDING ACTIVITY IN INTACT CELLS
BY
Several experimental approaches have been used that the redox modulation of NFxB DNA binding
to show activity
also operates
in vivo and modulates
tivity in intact cells (27). Up until identified as the only physiologically
this regulatory
the transcriptional acnow, GSSG has been relevant
mediator
effect (see Fig. 1). The DNA binding
of NFxB
is inhibited
relevant
concentrations
in cell-free
of GSSG
systems
of
even in the presence
of a
NFxB-dependent genes is expected to require a wellbalanced level of GSSG. At low GSSG levels, NFxB is not optimally activated whereas high levels of GSSG convert the heterodimer into a reversibly oxidized form with little or no
of
f
oxidafive inhlbition ot DNA binding actlvlty
p
optimal
r
GSSG
3. Illustration of the requirement for optimal NFxB activation.
1134
Vol. 8
Figure
November 1994
intracellular GSSG level (arbitrary units)
for intermediate
binding activity (Fig. 1). As GSSG levels are deterlargely by the availability of cysteine (44), it makes sense that T lineage cells generally have a tightly controlled cysteine supply that is limited by an extremely weak membrane transport activity for cystine. In T cells, this transport activity is more than 10-fold lower than the transport activity for cysteine, alanine, or arginine (45-47). Cystine is the quantitatively most important cysteine source in blood plasma and standard cell culture media, and thus the most limiting precursor for the biosynthesis of GSH and GSSG. As NFxB activation and DNA binding are sequential events (Fig. 1), cells can potentially enhance the effective NFxB activity by changing redox states. The inflammatory environment with its oxidative conditions may be the optimal site to induce the nuclear translocation of NFxB, whereas the subsequent migration of the lymphocyte to a less oxidative environment may provide more favorable conditions for NFxB DNA binding. Exposure to lactateproducing cells may provide an additional mechanism to facilitate the shift to lower GSSG levels as discussed later. A strong potentiation of NFxB activity has been demonstrated in human T lineage cells (Molt-4) that were shifted to reducing conditions 1 h after TPA stimulation (27). In contrast to NFxB, AP-i is almost exclusively localized in the nucleus, which contains considerably lower concentrations of GSSG. AP-I is also slightly less sensitive than NFxB to oxidative inhibition by GSSG in vitro, and instead more sensitive to oxidized TRX (27). It is therefore not surprising that elevated GSSG levels in intact cells inhibit selectively the DNA binding activity of NFxB and not that of AP-i. BCNU treatment of T lineage cells (Molt-4) even potentiates expression of the chloramphenicolacetyltransferase (CAT) reporter
gene under
control
EFFECTS
OF
FACTORS
NFxB
of AP-i
TRX
ON
AND
sites (27).
THE
TRANSCRIPTION
AP-1
activity
by physiologically
large excess of thiols, and three independent procedures that elevate GSSG levels in intact cells were found to inhibit the DNA binding activity and transactivating activity in intact cells (ref 27 and unpublished observations by S. Mihm and W. Droge). GSSG thus appears to have two mutually antagonistic effects as illustrated in Fig. 3, and the optimal activation of
activation NF-KB
DNA mined
levels of
The
FASEB
Whereas NFxB DNA binding activity is inhibited by GSSG, it is enhanced by thiols including dithiotreitol, cysteine, glutathione, and reduced TRX (27, 48, 49). The latter is by far the most effective and apparently the only physiologically relevant thiol compound with this function. TRX was found to restore the DNA binding activity of oxidized NFxB in vitro and to augment gene expression from HIV-LTR in intact cells as shown by CAT assays (48, 49). The induction of NFxB activation and nuclear translocation, in contrast, is strongly inhibited by TRX (16, 50), indicating that TRX antagonizes the effects of GSSG at both levels of redox regulation (Fig. 1). This leads to the prediction that the intracellular GSSG levels have to be adequately balanced against the corresponding TRX levels in order to support the activation and nuclear translocation of NFxB without excessive inhibition of its DNA binding activity (27). In agreement with this prediction, TRX high producer cell lines were found to require indeed higher concentrations of the GSSG precursor cystine in the culture medium than low producer lines (51). As discussed previously, an increased cellular cysteine supply causes not only an increase in intracellular GSH levels but also an (even stronger) increase in intracellular GSSG (44).
EFFECT OF HYDROGEN PEROXIDE IMMUNOLOGICAL FUNCTIONS The activation gen peroxide
Journal
ON
and potentiation of NFXB activity by hydrosuggested the possibility that this agent may
DROGE
ET AL.
REVIEWS have immunopotentiating effects. This has not been generally confirmed. However, studies of mixed lymphocytes cultures revealed that several classes of agents that prevent the formation or the effects of oxygen radicals also inhibit the stimulation of lymphocytes proliferation (52). Studies of the murine T cell clone Di0.G4.l revealed that 10 LM hydrogen peroxide causes a marked augmentation of the rate of DNA synthesis in the absence but not in the presence of concanavalin A (53), suggesting that a strong mitogenic stimulus may either bypass the requirement for reactive oxygen intermediates or may by itself induce the generation of reactive oxygen intermediates within the cell. A study of mitogenically stimulated accessory cell-depleted splenic T cell preparations from mice revealed that the production of IL-2 is
strongly increased by hydrogen peroxide or superoxide anion (O2) if the cultures are supplemented with lactate (13). In view effect
of the may
strong well
be
T cell stimulation.
glycolytic
activity
physiologically
As lactate
of macrophages, this in the course of
relevant
can be converted
intracellu-
larly into pyruvate, and pyruvate reacts with hydrogen peroxide (54) and competes with the glutathione peroxidase reaction (53), it is conceivable that lactate facilitates the shift from initially high to low GSSG levels. Proliferative and cytotoxic T cell responses in mixed lymphocyte cultures are
only marginally
increased
by low concentrations
of hydrogen
peroxide and strongly inhibited by higher concentrations even in the presence of lactate (13). It remains to be determined whether a brief exposure of lymphoid cells to a combination of hydrogen peroxide and lactate may also augment the proliferative and cytotoxic T cell response.
ROLE OF THIOL COMPOUNDS IN THE REGULATION OF IMMUNOLOGICAL FUNCTIONS: IS GSSG
INVOLVED
AGAIN?
Studies of lymphocyte functions in vitro have been greatly facilitated by the empirical finding that these functions are
strongly
potentiated
by thiols (13, 37, 45). This is exemplified
by the fact (among others) that immunologists are routinely adding 2-ME to the culture medium when studying immunological responses of murine lymphocytes in vitro (55). The main function of 2-ME is to enhance the cysteine supply to the lymphoid cells (45). Under standard cell culture conditions, 2-ME is rapidly oxidized and generates a cysteine containing mixed disulfide in the culture medium, which is transported into lymphocytes more effectively than cystine itself (45) (see Fig. 1). The intracellular reduction of the mixed disulfide yields cysteine, which serves as a limiting substrate for the biosynthesis of glutathione (37, 56). The intracellular glutathione level, in turn, determines the level of GSSG. Experiments with T lineage cells (Molt-4) have shown that an increasing cellular supply of cysteine in the
physiologically
relevant
of intracellular tional increase
GSH levels but also to a more than proporof intracellular GSSG (44). In view of the
range
leads not only to an increase
redox-sensitive
tyrosine
kinase activities
of p561
and
p59fYf
(Fig. 1), there is a possibility that the mixed disulfide of 2-ME and cysteine (45) may also act directly on these signal transducing proteins, thereby substituting for GSSG rather than serving as its precursor. Glutathione is a limiting factor for the immune system to the extent that certain T cell functions can be potentiated in vivo by administration of glutathione (57). Even a partial depletion of the intracellular glutathione pool by treatment with BSO has dramatic consequences for a variety of lymphocyte functions (58-69). Collectively, these studies mdi-
GLUTATHIONE
FUNCTION
IN
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
cated that IL-2-dependent functions including T cell proliferation, the development of large CD8 T cell blasts, cytotoxic T cell activity, generation of lymphokine-activated killer cells (LAK), and the proliferation of human CDI6 NK cells are particularly sensitive against GSH depletion (6369), whereas the production of the lymphokines IL-2 and
interferon-y,
and the expression
of their
respective
mRNA
species, are not markedly affected (66, 67). Essentially similar results were obtained in cultures without BSO when a glutathione deficiency was induced simply by limiting the extracellular cysteine supply (17, 46, 47, 64, 66, 69). The effect of glutathione on the IL-2-dependent proliferation and cytotoxic T cell activation is not understood in detail. Although glutathione-depleted cells were reported to ex-
press a normal
IL-2 receptor
density,
IL-2 receptor
synthesis
and turnover may be decreased (68). As the IL-2 receptor achain is transcriptionally controlled by NFxB (5), it is possible that this effect is in essence the consequence of a GSSG deficiency. Moreover, signal transduction by the IL-2 receptor /3-chain involves the redox-regulated protein tyrosine kinase p56lck (33), which is activated by hydrogen peroxide and diamide. The IL-2 receptor-mediated signal transduction, thus, may also depend on GSSG (Fig. I). The cytotoxic T lymphocyte effector function is triggered by interaction of its antigen receptors and CD8 molecules with the target cell. This process thus involves p59’#{176} and ps6lck tyrosine kinase activities (Fig. I), both of which were reported to be redox sensitive and enhanced by oxidative conditions (29). In spite of the relatively strong effects of glutathione depletion on the generation of large CD8 T cell blasts and cytotoxic T cell activity (64, 65), it was noted that perform and protease (BLT-esterase) activities, i.e., two typical functions of cytotoxic T lymphocytes, are not markedly affected by glutathione depletion (64, 67). This is compatible with the interpretation that triggering of the CTL effector phase via the T cell receptor/CD3/CD8 complex is inhibited as a result of the GSSG deficiency, as illustrated in Fig. 1. We therefore have to face the previously unexpected possibility that all the reported immunological consequences of the glutathione deficiency may in essence be consequences of a GSSG deficiency. The striking resistance of IL-2 production even against severe GSH depletion (66) can be interpreted by the assumption that this lymphocyte function may require 59fYn and p65lck tyrosine kinase activities only during a brief moment of antigenic or mitogenic stimulation. IL-2 production does not require an IL-2 receptor-mediated signal transduction pathway nor a continued expression of the antigen receptor-mediated signal pathway. In contrast to the IL-2-dependent functions, IL-2 production and IL-2 mRNA expression were found to be inhibited by glutathione and potentiated by lactate (70). As lactate raises
intracellular
a potent
pyruvate
scavenger
concentrations,
of hydrogen
peroxide
and
pyruvate
is
(54) and thereby
capable of inhibiting the glutathione peroxidase reaction (53), it is possible that the inhibition of IL-2 mRNA expression may also be mediated by GSSG rather than by GSH directly.
EVIDENCE FOR A LIMITING AND GLUTATHIONE IN VIVO
PHYSIOLOGICAL CONDITIONS:
OF A GSSG Recent cysteine
AND IS AIDS
ROLE OF UNDER
CYSTEINE
PATHOLOGICAL THE
CONSEQUENCE
DEFICIENCY?
studies of healthy human subjects indicated that the supply and the intracellular glutathione levels also
1135
REVIEWS have a strong influence with
intracellular
on the T cell system in vivo. Persons
glutathione
tein had significantly
levels
higher
of 20-30
numbers
nmol/mg
of CD4
T cells than
persons with either lower or higher glutathione levels (71). This may be explained by the mutually antagonistic effects of GSSG at the two levels of redox regulation of NFxB (Fig. 1). Persons who moved during a 4-week observation period from the optimal to the suboptimal range (10-20 nmol/mg protein) experienced, on average, a 30% decrease in number of CD4 T cells. This decrease was prevented by
treatment with NAC (71). Moreover, markedly decreased mean plasma cystine and cysteine concentrations and decreased intracellular glutathione levels have been found in HIV-infected persons at all stages of the disease and in rhesus macaques 2 weeks after SIV infection (17, 72-74). We therefore considered NAC for the treatment of these patients (75, 76). Because of technical difficulties, intracellular GSSG levels have not been determined but are also expected to be markedly decreased in HIV patients. In contrast, significant changes of mean plasma amino acid levels have not been observed in HIV-infected chimpanzees or in SIVinfected African green monkeys, i.e., in two species that do not show AIDS-like symptoms after retroviral infection (77).
MODULATION OF IMMUNE RESPONSES IN VIVO BY CYSTEINE OR CYSTEINE DERIVATIVES: BASIS FOR VACCINATION PROGRAMS AND FOR THE IMMUNOTHERAPY “Professional”
OF
CANCER
antigen-presenting
cells (APC)
such as macro-
phages present not only antigen to the specific T cell receptors but also deliver certain “costimulatory signals” and release substantial amounts of cysteine that raise the intracellular glutathione levels of activated T cells in their vicinity (64). This cysteine delivering function appears to be physiologically important for the T cell response because the early phase of blast transformation increases the demand for cys-
teine (53, 78). Earlier
studies
have shown
that the optimal
activation of cytotoxic T cells requires in addition to antigen and IL-2, certain metabolic activities of the stimulator cell (79). More recent studies of cytotoxic and protective responses against a larger panel of tumor cells and tumor cell variants in vivo indicated that the capacity to release cysteine into the extracellular space is one of the limiting factors that may determine the immunogenic properties of any stimulator cell if administered in vivo in relatively large numbers
(47). Cells that cannot
release
substantial
teine can, nevertheless, be immunogenic and in vitro if administered in relatively
Exogenous
glutathione
amounts
of cys-
for T cells in vivo small numbers (78).
may even inhibit
T cell responses
under these conditions. If administered at high numbers, however, such “nonprofessional” stimulator cells induce an almost complete inhibition that is reversed by exogenous glutathione (78). Exogenous glutathione thus potentiates the response against high doses of nonprofessional stimulator cells, suggesting that a glutathione deficiency may indeed be responsible for the induction of immunological nonresponsiveness under conditions of excessive antigenic stimulation by nonprofessional stimulator cells. The induction of a glutathione deficiency by excessive antigenic stimulation is in line with the observation that T cells have a particularly strong demand for cysteine during the rapid increase of cell volume (blast transformation) after antigenic stimulation (78).
1136
Vol. 8
November 1994
CONCLUDING
REMARKS
proFor many lymphocyte
years, thiols were little more than functions in vitro. More recently,
broadened
a tool to study the interest has
to include:
#{149} the role of cysteine, GSH, and GSSG in the regulation of the immune system; #{149} the role of cysteine and glutathione deficiencies in pathological conditions; #{149} therapeutic effects of cysteine derivatives such as NAC and 2-oxo-4-thiozolidine-carboxylate (OTC, procysteine); #{149} negative consequences of superoptimal levels of cysteine,
GSH,
or GSSG;
#{149} the modulation of immune responses in vivo by cysteine or cysteine derivatives as a basis for vaccination programs, immunotherapy of cancer, etc. This brief review is inevitably incomplete, but it may provide guidelines for further studies. It is clear that GSH is one of the limiting factors that determine the magnitude of immunological functions in vitro and in vivo; a successful clinical application of GSH or its precursor cysteine will require a more detailed understanding of their positive and negative effects under various conditions. Moreover, we must be prepared to reevaluate one of the central dogmas concerning the role of GSH. GSH was viewed mostly as an important antioxidant that protects cells against oxidative stress. However, GSH is also the substrate for GSSG synthesis. The intracellular GSSG level is strongly dependent on the GSH level, and GSSG is one of nature’s mildest oxidants. Studies of the redox regulation of transcription factors suggest that all reports published about the immunological consequences of a GSH deficiency may possibly reflect the consequences of a GSSG deficiency. Finally, as HIV-infected patients and Slyinfected rhesus macaques have, on average, significantly decreased plasma cyst(e)ine and intracellular GSH levels, we propose that AIDS may also be the consequence of a GSSG deficiency. The
assistance
manuscript
of Mrs.
is gratefully
I. Fryson
in the preparation
of this
acknowledged.
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