priming of IL-12 production
* First Department of Internal Medicine, Faculty of Medicine, School of Medicine, and
Faculty of Health Sciences, Gunma University, Maebashi, Japan; and
Basic Research Laboratories, Ajinomoto Co., Kawasaki, Japan
Correspondence: Kunio Dobashi, M.D., Ph.D., First Department of Internal Medicine, Gunma University Faculty of Medicine, School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma, 371-8511, Japan. E-mail: dobashik{at}med.gunma-u.ac.jp
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priming
increased the GSH/GSSG ratio and enhanced IL-12 production through p38,
and DEM negated the priming effect of IFN- on p38 activation and
IL-12 production as well as on the GSH/GSSG ratio. These findings
reveal that glutathione redox regulates LPS-induced IL-12 production
from monocytes through p38 MAP kinase activation and that the priming
effect of IFN- on IL-12 production is partly a result of the
glutathione redox balance.
Key Words: immune response • antigen-presenting cell • signal transduction
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and IL-2 production, and has an important role in maintaining
the in vivo balance between Th1 and Th2 responses [1
].
Several investigators have shown that the expression of p40 and p35 in
human monocytes is highly regulated and that priming by IFN-
augments p40 and p35 mRNA expression in response to LPS
[6
, 7
]. Glutathione is the most abundant nonproteinous tripeptide containing a sulfhydryl group in virtually all cells, and it plays a significant role in many biological processes. It also constitutes the first line of the cellular defense mechanism against oxidative injury and is the major intracellular redox buffer in ubiquitous cell types [8 ]. Evidence suggests that the intracellular redox status regulates various aspects of cellular function [9 ]. Although studies have demonstrated that glutathione plays an important role in the initiation and maintenance of T-cell-dependent immune responses [10 11 12 ], little is understood about the relationship between glutathione and immune responses in antigen-presenting cells (APC) such as monocytes, macrophages, and dendritic cells. A recent study demonstrated that the glutathione level in murine APC determines which of the Th1 or Th2 response predominates [13 ]. We also showed that IL-12 production is regulated by the glutathione redox of murine macrophages [14 ]. In human APC, however, the redox regulation of IL-12 production has not been determined.
Many extracellular stimuli elicit specific biological responses through the activation of mitogen-activated protein (MAP) kinase cascades. The MAP kinases constitute an important group of serine/threonine signaling kinases that by modulating the phosphorylation, and hence the activation status of transcription factors, link transmembrane signaling with gene induction events in the nucleus. Three major subgroups of MAP kinases have been characterized in mammalian cells: extracellular signal-regulated kinase (Erk), c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase [15 ]. Although LPS activates all three MAP kinases in monocytes [16 17 18 ], the relationship between the activation of these kinases and the induced cytokine expression remains obscure. Recent studies suggest that LPS activates p38 MAP kinase, which subsequently promotes the induction of IL-12 production in murine APC [19 , 20 ]. Conversely, a redox regulation of signal transduction that may involve glutathione has been suggested [21 ]. In fact, reduced glutathione (GSH) and oxidized glutathione (GSSG) by thiol/disulfide-exchange reactions influence the redox status and activity of redox-sensitive enzymes, including protein kinase and phosphatases [22 ]. However, the relationship between glutathione redox and MAP kinase activity in human monocytes is not still clear.
The present study shows that the intracellular redox balance of the
GSH/GSSG ratio regulates LPS-induced IL-12 production through p38 MAP
kinase activation in human monocytes. Furthermore, we demonstrate that
the enhancement of LPS-induced IL-12 production by IFN- priming of
human monocytes is, at least in part, a result of the increased
intracellular GSH/GSSG ratio leading to the enhanced p38 MAP kinase
activation.
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was obtained from Pepro Tech (Rocky Hill,
NJ). Nicotinamide adenine dinucleotide phosphate reduced form
(ß-NADPH), 5-5'-dithiobis-2-nitrobenzoic acid (DTNB), and glutathione
reductase were obtained from Wako Pure Chemical Industries (Osaka,
Japan). To specifically inhibit p38 MAP kinase activity
[23
], SB203580 and SKF86002 were obtained from
Calbiochem-Novabiochem Corp. (La Jolla, CA) and dissolved in
dimethylsulfoxide (DMSO; Sigma Chemical Co.).
Cell culture/human monocytes
Peripheral blood mononuclear cells (PBMC) were separated from
leukocyte concentrates obtained from healthy volunteers by density
gradient centrifugation in lymphocyte separation medium (ICN
Biomedicals, Aurora, OH). To separate monocytes, PBMC were resuspended
in fresh RPMI 1640 medium (Gibco BRL, Life Technologies, Rockville, MD)
containing 10% fetal bovine serum (FBS; Equitech-Bio, Ingram, TX;
complete medium) and seeded in flat-bottom polystyrene dishes. The
cells were incubated for 1 h at 37°C to allow for attachment.
Adherent cells consisted of more than 90% of monocytes, as assessed by
differential counting on May-Giemsa-stained smears. In some
experiments, monocytes were prepared by magnetic cell sorting and
biomolecular separation (MACS; monocyte isolation kit; Miltenyi Biotec,
Bergisch Gladbach, Germany). No difference was observed between MACS
and adherent-purified monocytes with regard to the responses analyzed
in this study. Monocytes were cultured in complete medium and
maintained in humidified 5% CO2/95% air. For cytokine
production, monocytes (2x105/500 µl) were incubated with
or without 1000 U/ml IFN- for 24 h. The cells were then washed
and incubated with or without glutathione modulators in fresh RPMI 1640
medium containing 1% FBS. The cells were washed once again and
stimulated with 1 µg/ml LPS in complete medium for 24 h.
Thereafter, the cells were sedimented by centrifugation, and the
supernatants were stored at -20°C before cytokine quantitation.
THP-1 cells
Human monocytic THP-1 cells (American Type Culture Collection,
Rockville, MD) were cultured in RPMI 1640 medium with 4.5 g/l glucose,
10 mM HEPES, 1 mM sodium pyruvate, and 50 µM 2-mercaptoethanol
supplemented with 10% FBS and maintained in humidified 5%
CO2/95% air. Subconfluent cells were washed and
resuspended with in fresh RPMI-1% FBS. THP-1 cells
(1x106/500 µl) were incubated with 1.2% DMSO for
24 h, because DMSO enhances the ability of human myeloid cell
lines to produce IL-12 [24
]. Glutathione modulators or
IFN- were added, and then the cells were washed and stimulated as
described above.
Cell exposure to glutathione modulators
To increase cellular GSH, monocytes or THP-1 cells were
incubated with GSH-OEt for 4 h at various concentrations (0.05,
0.5, and 5 mM). To deplete cellular GSH, the cells were incubated with
DEM (0.6, 6, and 60 µM) for 4 h or BSO (5, 50, and 500 µM) for
24 h [25
]. Glutathione modulators were added in the
presence of 1% FBS to minimize the influence of FBS on cellular GSH.
Cell viability determined by trypan blue dye exclusion was always over
90%.
Measurements of intracellular GSH and GSSG concentrations
After incubation with glutathione modulators or IFN-,
monocytes or THP-1 cells were washed three times with cold washing
buffer [0.1 M sodium phosphate, 5 mM ethylenediaminetetraacetate
(EDTA), pH 7.5] and immediately thawed in 100 µl lysis buffer (0.1%
Triton-X, 0.1 M sodium phosphate, 5 mM EDTA, pH 7.5). Five minutes
later, the lysates were acidified with 15 µl of 0.1 N HCl, and
protein was precipitated with 15 µl of 50% sulfosalicylic acid.
After centrifugation, supernatants were collected for GSH and GSSG
assays. The total cellular glutathione concentration was assayed by the
GSSG-reductase-DTNB recycling procedure according to Tietze
[26
] as modified by Buchmuller-Rouiller and coworkers
[25
]. GSH was oxidized sequentially by DTNB and reduced
by ß-NADPH in the presence of glutathione reductase. Formation of
2-nitro-5-thiobenzoic acid was monitored by comparing the absorbance at
405 nm with that of standard samples of GSH in the lysis buffer. GSSG
was assayed according to Griffith [27
]. Briefly,
standard solutions of GSSG or aliquots of samples were supplemented
with 2 µl 2-vinylpyridine per 100 µl sample volume. All solutions
were adjusted to pH 7.5 with triethanolamine. After a 60-min incubation
at room temperature, the assay proceeded as described for total
glutathione.
Quantitation of IL-12 (p70), IL-1ß, and IL-8
The concentrations of the heterodimeric form (p70) of IL-12,
IL-1ß, and IL-8 in resulting supernatants were measured using
commercially available enzyme-linked immnunosorbent assay (ELISA) kits
(Quantikine TM, R&D Systems, Minneapolis, MN), according to the
manufacturer’s instructions. The assay detected >5 pg/ml IL-12 p70,
>1 pg/ml IL-1ß, and >3 pg/ml IL-8.
Preparation of complementary RNA (cRNA) probes
A human IL-12 p40 cDNA fragment containing residues 625–1022
(Gen Bank accession no. M65290) [28
] and a p35 cDNA
fragment containing residues 583–908 (Gen Bank accession no. M65291)
[28
] were amplified by polymerase chain reaction (PCR).
The synthesized sense and antisense PCR primers for IL-12 p40 were
5'-GAGTCTGCCCATTGAGGTCAT-3' and 5'-AATTTTCATCCTGGATCAGAACC-3'; those
for p35 were 5'-TTTATGAAGACTTGAAGATGTACCAG-3' and
5'-TCAAAGTTTTATAAAAATGACAACGG-3' (Kurabo, Osaka, Japan)
[29
]. The PCR products were fractionated on agarose gels
and then cloned into the pGEM-t Easy vector (Promega Corp., Madison,
WI). Sequencing analysis (Perkin Elmer Corp. PE Applied Biosystems,
Foster City, CA) confirmed the identity of the amplified DNA. Human
glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA was cloned as
described above, except that a fragment of it containing residues
71–1053 was amplified by PCR using a synthesized primer
(5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' sense; 5'-CATGTGGGCCATGAGGTCCACCAC-3'
antisense) [30
]. The cRNA probes for human IL-12 p40 and
p35, human G3PDH were synthesized using [
-32P] UTP
(ICN Biomedicals) and T7 RNA polymerase (Promega Corp.).
Northern blot hybridization
Total RNA was extracted from monocytes or THP-1 cells using the
TRIzol reagent (Gibco BRL, Life Technologies) in a modification of the
acid guanidinium thiocyanate-phenol-chloroform method of Chomczynski
and Sacchi [31
]. Total RNA from 2 x
106 monocytes or 15 µg total RNA from THP-1 cells per
lane were size-fractionated by electrophoresis through 1.4% agarose
gels containing 0.66 M formaldehyde and transferred overnight in 20x
SSC (1x SSC=150 mM sodium chloride and 15 mM trisodium citrate) to
Hybond-N membranes (Amersham Pharmacia Biotech, Tokyo, Japan). The RNA
was then immobilized by UV irradiation on a UV Stratalinker
(Stratagene, La Jolla, CA). After prehybridization, the membrane was
hybridized at 60°C overnight in hybridization buffer [50%
formamide, 0.2% sodium dodecyl sulfate (SDS), 5% dextran sulfate, 50
mM HEPES, 5x SSC, 5x Denhardt’s solution, and 100 µg/ml denatured
salmon sperm DNA] containing a human IL-12 p40 or p35 cRNA probe. The
membrane was washed and exposed to X-ray film (Hyperfilm; Amersham
Pharmacia Biotech) at -70°C. After detecting IL-12 mRNA, the probes
was stripped, and the blots were rehybridized with a control human
G3PDH cRNA probe. The mRNA level was quantified by densitometry using
NIH Image Version 1.62, and the optical density of the IL-12 p40 and
p35 bands was corrected by comparison with G3PDH mRNA in the same blot.
Measurement of p38 MAP kinase and Erk activity
The activity of p38 MAP kinase was measured using commercially
available kits (p38 MAP kinase assay kit, New England Biolabs, Beverly,
MA), according to the manufacturer’s instructions. In brief, activated
p38 MAP kinase was immunoprecipitated from equal amounts of cytoplasmic
protein using antiphospho-p38 MAP kinase (Thr 180/Tyr 182) antibody.
The in vitro kinase reaction was performed using the immunoprecipitated
p38 MAP kinase and activating transcription factor 2 (ATF-2) as the
substrate. ATF-2 phosphorylation was measured by Western blotting using
antiphospho-ATF-2 (Thr 71) antibody. Erk activity was measured by
analyzing Elk-1 phosphorylation as substrate using antiphospho-Elk-1
(Ser 383) antibody by Western blotting according to the manufacturer’s
instructions (p44/42 MAP kinase assay kit, New England Biolabs). We
normalized cytoplasmic protein contents using the bicinchoninic acid
(BCA) protein assay (Pierce, Rockford, IL) and detected the contents of
ß-actin in the blots using anti-ß-actin antibody.
Statistical analysis
All values are expressed as mean ± SE. The
nonparametric analysis of variance (Kruskal-Wallis method) determined
significance among groups. We used the Mann-Whitney U test to analyze
significant differences between individual groups, and a value of
P < 0.05 was considered significant.
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View this table: [in a new window] |
Table 1. Effects of Glutathione Modulators on Intracellular Contents of GSH and
GSSG in Human Monocytes
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Figure 1. Effects of glutathione modulators on LPS-induced IL-12 production in
human monocytes. Cells were incubated with GSH-OEt and/or DEM for
4 h and then stimulated with LPS (1 µg/ml) for 24 h. IL-12
p70 (A), IL-1ß (B), and IL-8 (C) proteins in culture supernatants
were evaluated by ELISA. Values represent mean ± SE
of five experiments. *, P < 0.05 compared with
LPS-stimulated monocytes.
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Figure 2. Northern blots of the effects of glutathione modulators on LPS-induced
IL-12 mRNA expression. (A) Human monocytes were incubated with GSH-OEt
(5 mM) and/or DEM (60 µM) for 4 h and stimulated with LPS (1
µg/ml) for 6 h, and then total RNA was extracted and hybridized
with human p40 and p35 cRNA probes. The blots were stripped, and then
human G3PDH probe was used as a loading control. (B) The mRNA level was
quantified by densitometry. The optical density of the p40 (open bars)
or p35 (solid bars) mRNA band was corrected for G3PDH, and the results
were expressed as a percentage of the value obtained for control
(LPS-stimulated monocytes).
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-glutamylcysteine synthetase [32
]. A
24-h incubation with BSO promoted a dose-dependent decrease in the
GSH/GSSG ratio (P<0.05; Fig. 3C
).
 View larger version (27K): [in a new window] |
Figure 3. Effects of glutathione modulators and IFN- on glutathione
redox and LPS-induced IL-12 mRNA expression in THP-1 cells. After
incubating DMSO-treated THP-1 cells with (A) IFN- for 24 h, (B)
GSH-OEt for 4 h, (C) BSO for 24 h, and (D) DEM for 4 h,
the ratios of GSH/GSSG were determined by enzyme assay. Values
represent the mean ± SE of five experiments. Amounts
of GSH and GSSG did not differ among control cells in each group. *,
P < 0.05 compared with control. (E) Cells were
incubated with IFN- (1000 U/ml), GSH-OEt (5 mM), BSO (500 µM), and
DEM (60 µM) and stimulated with LPS (1 µg/ml) for 6 h, and
then total RNA was extracted. The expression of IL-12 p40 mRNA was
evaluated by Northern blotting as described in the legend to Figure 2
.
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Effect of IFN- on glutathione redox and LPS-induced IL-12
production in THP-1 cells
Intracellular concentrations of GSH were increased in
DMSO-treated THP-1 cells incubated for 24 h with IFN-, and GSSG
levels were decreased, resulting in a dose-dependently increased ratio
of GSH/GSSG (P<0.05; Fig. 3A
). Although IL-12 p70 protein
was undetectable in DMSO-treated THP-1 cells stimulated by LPS alone,
exposure to 1000 U/ml IFN- increased LPS-induced IL-12 p70 protein
production to a detectable level (10.90±2.29 pg/ml, n=6).
In addition, IFN- augmented IL-12 p40 mRNA expression (340%; Fig. 3E
) in DMSO-treated THP-1 cells stimulated by LPS.
Effects of IFN- in human monocytes and glutathione modulators in
IFN--primed monocytes on glutathione redox and LPS-induced IL-12
production
In human monocytes, as in THP-1 cells, IFN- (1000 U/ml, 24 h) increased intracellular GSH levels and decreased those of GSSG,
resulting in a significantly increased ratio of GSH/GSSG
(P<0.05; Table 2 ). Moreover, in IFN--primed monocytes (1000 U/ml, 24 h),
GSH-OEt (5 mM, 4 h) increased GSH levels and the GSH/GSSG ratio
further (Table 2)
. Conversely, incubating IFN--primed monocytes with
DEM (60 µM, 4 h) promoted a significant decrease in GSH levels
and the GSH/GSSG ratio (P<0.05; Table 2
) to those in the
absence of IFN-.
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View this table: [in a new window] |
Table 2. Effects of Glutathione Modulators on Intracellular Contents of GSH and
GSSG in IFN--Primed Monocytes
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significantly enhanced LPS-induced IL-12 p70
protein production (P<0.05; Fig. 4A
) as well as the expression of p40 and p35 mRNA (355% and 443%,
respectively; Fig. 4B
). Furthermore, 5 mM GSH-OEt increased LPS-induced
IL-12 protein production and mRNA expression in IFN--primed
monocytes (Fig. 4A
and 4B)
. Conversely, DEM significantly and
dose-dependently inhibited LPS-induced IL-12 p70 protein production
(P<0.05; Fig. 4A
) and considerably suppressed LPS-induced
p40 and p35 mRNA expression (by 69% and 62%, respectively; Fig. 4B
)
in IFN--primed monocytes. DEM negated the priming effect of
IFN- on LPS-induced IL-12 protein production and mRNA expression.
Similarly, LPS-induced IL-1ß production was also enhanced
significantly by priming with IFN- (P<0.05; Fig. 4C
).
Besides, in IFN--primed monocytes, GSH-OEt increased, and DEM
(P<0.05) inhibited LPS-induced IL-1ß production
significantly (Fig. 4C)
and negated the priming effect of IFN- on
LPS-induced IL-1ß production.
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Figure 4. Effects of IFN- in human monocytes and glutathione modulators in
IFN--primed monocytes on LPS-induced IL-12 production. Human
monocytes were primed with or without IFN- (1000 U/ml) for 24 h, incubated with GSH-OEt and/or DEM for 4 h, and then stimulated
with LPS (1 µg/ml). (A and C) After 24 h, IL-12 p70 (A) and
IL-1ß (C) proteins in culture supernatants were evaluated by ELISA.
Values represent mean ± SE of five experiments. *,
P < 0.05 compared with LPS-stimulated monocytes.  ,
P < 0.05 compared with IFN--primed monocytes
stimulated with LPS. (B) After 6 h, total RNA was extracted, and
expression of IL-12 p40 and p35 mRNA was evaluated by Northern blotting
as described in the legend to Figure 2
.
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on LPS-induced p38
MAP kinase and Erk activation in human monocytes affect
LPS-induced p38 MAP kinase in human monocytes. Little or no p38 MAP
kinase was activated in unstimulated monocytes (Fig. 5
). LPS significantly activated p38 MAP kinase, which was maximal at
10 min and sustained for up to 80 min (Fig. 5A)
. The activation of
LPS-induced p38 MAP kinase was enhanced by GSH-OEt and suppressed by
DEM (Fig. 5B) . Furthermore, IFN-, which increases the GSH/GSSG
ratio, enhanced LPS-induced p38 MAP kinase activation, and DEM
negated this effect of IFN- (Fig. 5C)
. GSH-OEt, DEM, or IFN-
alone failed to stimulate p38 MAP kinase activation (Fig. 5B
and 5C) .
In contrast to p38 MAP kinase, LPS-induced Erk activation was not
modulated by GSH-OEt or DEM (Fig. 5D)
.
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Figure 5. Effects of glutathione modulators and IFN- on LPS-induced p38 MAP
kinase activation. P38 MAP kinase activity was measured in an in vitro
kinase reaction using ATF-2 as substrate and probing for
antiphospho-ATF-2 (Thr 71) in Western blots. (A) Human monocytes were
stimulated with LPS (1 µg/ml) for the indicated periods. (B) Human
monocytes were incubated with GSH-OEt (5 mM; lanes 3 and 4) or DEM (60
µM; lanes 5 and 6) for 4 h and then stimulated with or without
LPS for 10 min. (C) Human monocytes were primed with IFN- (1000
U/ml; lanes 3–6) for 24 h, further incubated with DEM (60 µM;
lanes 5 and 6) for 4 h, and then stimulated with or without LPS
for 10 min. (D) Effects of glutathione modulators on LPS-induced Erk
activation. Erk activity was measured in an in vitro kinase reaction
using Elk-1 as substrate and probing for antiphospho-Elk-1 (Ser 383) in
Western blots. Human monocytes were incubated with GSH-OEt (5 mM; lanes
3 and 4) or DEM (60 µM; lanes 5 and 6) for 4 h and then
stimulated with or without LPS for 10 min. The corresponding bottom
panels are Western blots using anti-ß-actin antibody to demonstrate
the equality of cytoplasmic protein contents.
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(P<0.01;
Fig. 6A
). Similarly, SKF86002 significantly attenuated LPS-induced
IL-12 production enhanced by GSH-OEt or IFN- (P<0.01;
Fig. 6B
).
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Figure 6. Effects of SB203580 and SKF86002 on LPS-induced IL-12 production in
human monocytes. Human monocytes were cultured with 0.1% DMSO (control
vehicle) or 10 µM SB203580 (A) and 0.1% DMSO (control vehicle) or 10
µM SKF86002 (B) for 1 h before stimulation with LPS (1 µg/ml).
After 24 h, IL-12 p70 protein in culture supernatants was
evaluated by ELISA. In addition, cells were incubated with GSH-OEt (5
mM) for 4 h (lanes 3 and 4) or IFN- (1000 U/ml) for 24 h
(lanes 5 and 6) before stimulation. Values represent mean ±
SE of five experiments. *, P < 0.01
compared with LPS-stimulated monocytes. , P < 0.01
compared with GSH-OEt-treated monocytes with LPS. #, P < 0.01 compared with IFN--primed monocytes stimulated with LPS.
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affected
the glutathione redox balance and increased the intracellular GSH/GSSG
ratio, which was likely to enhance LPS-induced p38 MAP kinase
activation and IL-12 production. This is the first study of the redox
regulation of IL-12 production and signal transduction in human
monocytes. The modulation of IL-12 production has an important role in controlling the balance between Th1 and Th2 responses [33 ]. A recent study by Peterson et al. [13 ] showed that glutathione depletion in murine APC decreases the production of IL-12 and leads to polarization from the typical Th1 cytokine profile toward Th2 response patterns, suggesting that glutathione levels in APC play a central role in determining which of the Th1 and Th2 cytokine responses predominate during an immune state. We have also shown that IL-12 production was positively or negatively polarized by augmenting or depleting the intracellular contents of GSH in murine peritoneal macrophages by GSH-OEt or DEM, respectively [14 ]. To study the importance of glutathione redox in cellular processes, many investigators have tested the effects of glutathione modulators that increase or deplete intracellular glutathione [13 , 14 , 25 , 34 , 35 ]. In the present study, we used three glutathione modulators, GSH-OEt, DEM, and BSO, and confirmed that GSH-OEt actually increased intracellular GSH and the ratio of GSH/GSSG in human monocytes, and DEM decreased them (Table 1) . Consistent with our previous observation in murine macrophages [14 ], we observed that LPS-induced IL-12 production was controlled arbitrarily by these agents in human monocytes. GSH-OEt enhanced, whereas DEM suppressed, LPS-induced IL-12 p70 protein production as well as the expression of p40 and p35 subunit mRNA (Figs. 1A and 2) . Conversely, LPS-induced IL-8 production was not modulated by GSH-OEt or DEM (Fig. 1C) . In addition, we considered that the inhibitory effect of DEM on LPS-induced IL-12 production was not attributed to DEM toxicity because coexistence of GSH-OEt and DEM negated the effect of DEM on IL-12 production consistent with the glutathione redox balance. Therefore, we suggest that the glutathione redox balance regulates LPS-induced IL-12 production via the expression of p40 and p35 subunit mRNA in human monocytes and that this balance is affected by the intracellular GSH/GSSG ratio. The latter theory is supported by the present study. GSH-OEt increased, and BSO decreased the GSH/GSSG ratio and LPS-induced IL-12 mRNA expression in THP-1 cells, respectively (Fig. 3B 3C and 3E) , but DEM did not alter the GSH/GSSG ratio or influence IL-12 mRNA expression (Fig. 3D and 3E) .
A recent study shows that LPS activates p38 MAP kinase, which promotes IL-12 production in murine macrophages [19 ]. In addition, another study has demonstrated that mice deficient in MKK3, the specific upstream MAP kinase kinase for p38 MAP kinase, have defects in p38 MAP kinase activation and IL-12 production [20 ]. The present study found that LPS is a powerful stimulator of p38 MAP kinase activation (Fig. 5A) and that the selective blockade of this kinase activation inhibited LPS-induced IL-12 production (Fig. 6) , suggesting that p38 MAP kinase activation positively regulates LPS-induced IL-12 production in human monocytes. Furthermore, we demonstrated that GSH-OEt augmented, and DEM suppressed LPS-induced p38 MAP kinase activity (Fig. 5B) , and the selective blockade of this kinase activation markedly abrogated the augmenting effect of GSH-OEt on LPS-induced IL-12 production (Fig. 6) . Conversely, the ERK pathway has also demonstrated to play a role in the control of LPS-induced IL-12 production [19 ]. However, we showed in this study that LPS-induced ERK activation was not modulated by GSH-OEt or DEM (Fig. 5D) . These findings suggest the pivotal role of intracellular glutathione redox on p38 MAP kinase activation in LPS-induced IL-12 production in human monocytes.
Many authors [6
, 7
, 36
37
38
]
have demonstrated that IFN- priming of human monocytes increased
their ability to produce IL-12. In particular, the priming effect of
IFN- on p40 gene promotor region has been analyzed in detail
[36
37
38
]. However, there are no studies that glutathione
redox is related to the up-regulation of LPS-induced IL-12 production
by IFN-. In this study, IFN- increased intracellular GSH levels
and the ratio of GSH/GSSG in monocytes and THP-1 cells (Table 2
and
Fig. 3A ), and IFN- priming of these cells augmented LPS-induced
IL-12 production (Figs. 3E
and 4
, A and B) in agreement with
demonstrated findings [6
, 7
] . Moreover,
IFN- enhanced LPS-induced p38 MAP kinase activity (Fig. 5C)
, and the
selective blockade of this kinase activation largely inhibited the
LPS-induced IL-12 production enhanced by IFN- (Fig. 6)
. These
results agree with the finding that the increased GSH/GSSG ratio
enhanced LPS-induced IL-12 production through p38 MAP kinase activation
mentioned above. Furthermore, in IFN--primed monocytes, DEM negated
the priming effect of IFN- on the LPS-induced IL-12 production (Fig. 4A and 4B)
and p38 MAP kinase activation (Fig. 5C)
, as well as on the
GSH/GSSG ratio (Table 2)
. In addition, priming with IFN- enhanced
LPS-induced IL-1ß production as previously shown [39
,
40
], and this priming effect was enhanced by GSH-OEt and
abolished by DEM in accord with the change of the GSH/GSSG ratio (Fig. 4C
and Table 2
). Therefore, we propose that the enhancement of
LPS-induced IL-12 production from monocytes by IFN- priming is a
result of a change in glutathione redox: The increased GSH/GSSG ratio
activated p38 MAP kinase and up-regulated IL-12 production. This
regulation can be observed in other cells. Recently, we have
demonstrated that IFN- and IL-4 affect glutathione redox balance,
and this balance regulates LPS-induced IL-12 production from human
alveolar macrophages [41
]. However, IFN- priming
effect on IL-12 production cannot be explained only by the contribution
of glutathione redox, because IFN- priming dramatically increases
IL-12 production despite a mild increase in the GSH/GSSG ratio, and
multiple regulatory elements such as nuclear factor-
B (NF-B)
[37
] and F1 complex including Ets-2, IFN-regulatory
factor-1 (IRF-1), c-Rel, and Ets-related factors [36
,
38
] have been implicated in IFN- priming on the p40
gene promoter region.
Then the question would arise how the intracellular glutathione redox
regulates p38 MAP kinase activation. Recently, Hashimoto et al.
[42
] proposed that tumor necrosis factor
(TNF-)-induced p38 MAP kinase activation is inversely regulated by
intracellular GSH levels in human pulmonary vascular endothelial cells.
The mechanism of this proposal is supported by their findings that
reactive oxygen species (ROS) including H2O2,
which induced by TNF-, stimulate p38 MAP kinase activation and that
scavenging of ROS by the increase in intracellular GSH attenuates
TNF--induced p38 MAP kinase activation. Conversely, in this paper,
we have already demonstrated that intracellular GSH positively
regulates p38 MAP kinase activation. Therefore, we consider other
mechanisms of glutathione redox regulation of this kinase activation.
Some enzymes that bear an accessible thiol essential for activity can form protein-mixed and intramolecular disulfides by reacting with small disulfide moieties, including those of glutathione, namely GSSG [22 ]. Conversely, GSH, which can reduce a wide variety of disulfides by transhydrogenation, is a major reductant of cellular protein disulfides [22 ]. These enzyme activities depend on protein S-thiolation/dethiolation, i.e., the oxidation of protein sulfhydryls to mixed disulfides and their reduction back to sulfhydryls [43 ]. Therefore, the balance of the reaction from cellular thiol to disulfide, including that of glutathione redox, must be able to regulate the activity of these enzymes [22 ]. Park et al. [44 ] examined whether the MAP kinase superfamily is regulated by the thiol redox mechanism. They showed that selenite, which can oxidize sulfhydryl groups, inhibits the p38 MAP kinase signaling pathways in human embryonic kidney cells. This observation is similar to our finding that the loss of the thiol/disulfide ratio of intracellular glutathione caused by DEM suppresses p38 MAP kinase activation. Hence, we postulate that glutathione redox regulates p38 MAP kinase activation through protein S-thiolation/dethiolation in the p38 MAP kinase signaling pathway, and further research is required to clarify this issue.
The present study suggests that LPS-induced IL-12 production from human
monocytes is regulated by the glutathione redox, specifically the
intracellular GSH/GSSG ratio, during the mediation of p38 MAP kinase
activation and that IFN- priming of monocytes leads to an increase
in intracellular GSH/GSSG, which probably enhances LPS-induced p38 MAP
kinase activation and IL-12 production. IL-12 is a key cytokine that
differentiates Th0 from Th1. Therefore, the notion that the glutathione
redox balance of APC, e.g., monocytes, macrophages, and dendritic
cells, regulates the balance between Th1 and Th2 responses through
IL-12 production may not only help explain the differences in "Th1"
and "Th2" diseases but also provide a therapeutic option for
altering the Th1-Th2 balance in allergic and autoimmune diseases, as
well as in other conditions.
Received February 22, 2001; revised June 4, 2001; accepted September 28, 2001.
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of lipopolysaccharide-inducible p35 and p40 genes Blood 86,646-650, and IL-1ß production in human myeloid leukemia cell lines and peripheral blood mononuclear cells Blood 83,1847-1855This article has been cited by other articles:
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