From the
Our previous studies in human monocytes have demonstrated that
interleukin (IL)-10 inhibits lipopolysaccharide (LPS)-stimulated
production of inflammatory cytokines, IL-1
Interleukin-10 (IL-10),
NF
When incubated with
We have previously shown in human monocytes that IL-10
inhibits cytokine mRNA accumulation by acting at the level of gene
transcription
(6) , indicating a possible mechanism of IL-10
action through transcription factors. Several approaches using mutation
or deletion of
Although treatment of cells with TPCK and PDTC may elicit broader
effects than inhibition of NF
Our data, however, do not rule out the possibility that
IL-10 exerts its inhibitory action on cytokine gene transcription
through other transcription factors. While transcription factors such
as AP-1, NF-IL-6, Oct-1, Sp-1, and CREB are not affected by IL-10,
IL-10 does cause a rapid nuclear localization of the transcription
factor, p91, a component of the protein complex which binds to the
interferon-
The fact that IL-4 inhibits cytokine synthesis and cytokine mRNA
accumulation by enhancing cytokine mRNA degradation suggests the
existence in human monocytes of a transcription factor-independent
mechanism. This mechanism is not stimulated by IL-10 in human
monocytes, as suggested by our observation that IL-10 has little or no
effect on cytokine mRNA stability. Interestingly, in murine peritoneal
(35) and porcine alveolar
(36) macrophages, IL-4
inhibits cytokine gene transcription with little or no effect on mRNA
stability, whereas, in these same systems, IL-10 appears to act by
enhancing mRNA degradation
(4) . These observations suggest that
IL-4 and IL-10 generate distinct signals affecting distinct steps in
cytokine mRNA metabolism and that the mechanisms employed by these two
inhibitory cytokines vary depending on the animal species.
IL-10
action on NF
The protease
activities involved in the processing of p105 and I
In conclusion, the present study
shows that the cytokine synthesis inhibitory activity of IL-10 may be
mediated by inhibition of NF
, IL-6, IL-8, and tumor
necrosis factor (TNF)-
by blocking gene transcription. Using
electrophoretic mobility shift assays (EMSA), we now show that, in
monocytes stimulated with LPS or TNF
, IL-10 inhibits nuclear
localization of nuclear factor
B (NF
B), a transcription
factor involved in the expression of inflammatory cytokine genes.
Several other transcription factors including NF-IL-6, AP-1, AP-2, GR,
CREB, Oct-1, and Sp-1 are not affected by IL-10. This selective
inhibition by IL-10 of NF
B activation occurs rapidly and in a
dose-dependent manner and correlates well with IL-10's cytokine
synthesis inhibitory activity in terms of both kinetics and dose
responsiveness. Furthermore, compounds such as tosylphenylalanyl
chloromethyl ketone and pyrrolidinedithiocarbamate that are known to
selectively inhibit NF
B activation block cytokine gene
transcription in LPS-stimulated monocytes. Taken together, these
results suggest that inhibition of NF
B activation may be an
important mechanism for IL-10 supression of cytokine gene transcription
in human monocytes. IL-4, another cytokine that inhibits cytokine mRNA
accumulation in monocytes, shows little inhibitory effect on
LPS-induced NF
B activation. Further examination reveals that,
unlike IL-10, IL-4 enhances mRNA degradation and does not suppress
cytokine gene transcription. These data indicate that IL-10 and IL-4
inhibit cytokine production by different mechanisms.
(
)
originally
identified as a cytokine synthesis inhibitory factor, inhibits
production of a wide range of cytokines in various cell types (for
review, see Ref. 1). For instance, IL-10 suppresses synthesis of IL-1,
IL-6, IL-8, and tumor necrosis factor (TNF)-
in
monocytes/macrophages
(2, 3, 4, 5, 6) , of IL-1, IL-8,
and TNF
in neutrophils
(5) , and of IL-2, TNF
,
interferon-
, granulocyte/macrophage colony-stimulating factor, and
others in T cells
(7) . This inhibition of cytokine production
is accompanied by reduced accumulation of mRNAs
(2, 3, 4, 5, 6) . Nuclear run-on
experiments in human monocytes have demonstrated that IL-10 inhibits
lipopolysaccharide (LPS)-induced synthesis of IL-1
, IL-6, IL-8,
and TNF
by acting mainly at the level of gene transcription
(5, 6) , raising the possibility of IL-10 affecting the
activities of transcription factors involved in cytokine gene
expression. Gene promoters for IL-1
, IL-6, IL-8, and TNF
contain multiple cis-acting motifs including those that bind
such transcription factors as nuclear factor
B (NF
B),
NF-IL-6, AP-1, AP-2, CREB, GR, Oct-1, and Sp-1
(8, 9, 10, 11) . Motifs for NF
B,
NF-IL-6, AP-1, AP-2, and CREB are common to all four cytokine gene
promoters, and recent data have clearly indicated that NF
B,
NF-IL-6, and AP-1 are critical for the induction of these cytokine
genes
(8, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) .
B was originally identified as a heterodimeric complex
consisting of two subunits, p65 and p50 (reviewed in Ref. 22). In most
cells including monocytes, NF
B is constitutively present in the
cytoplasm as an inactive p65-p50-I
B ternary complex. Various
stimuli including LPS induce rapid dissociation of the inhibitory
subunit I
B. The resultant heterodimer of p50 and p65 translocates
into the nucleus and initiates transcription of appropriate genes in
synergy with other transcription factors such as NF-IL-6. Like
NF
B, NF-IL-6 exists in the cytosol and responds to stimulation by
rapidly translocating into the nucleus
(23) . By contrast, c-Fos
and c-Jun, the two components of AP-1, are synthesized de novo during stimulation
(24) . Our present data suggest that
IL-10 causes selective inhibition of NF
B activation in
LPS-stimulated human monocytes, and that this NF
B inhibition may
be a mechanism by which IL-10 causes the suppression of the cytokine
production. Our data further demonstrate that IL-4 inhibits cytokine
production by a mechanism that does not involve NF
B.
Cell Preparation and Incubation
Human peripheral
blood mononuclear cells (PBMC) were prepared as described
(5) .
For some experiments, purified monocytes were prepared from PBMC by
elutriation
(25) . The elutriated monocyte fraction consisted of
>95% monocytes as determined by histologic staining and by
immunofluorescence assay for CD14 antigen. Cell incubation was
performed as described previously
(5) .
Assays for Cytokine Proteins
Cytokines released
into cell culture media were determined by using commercially available
specific enzyme-linked immunosorbent assay kits (IL-6 and TNF kits
from Biosource, Camarillo, CA; IL-1
and IL-8 kits from R&
Systems, Minneapolis, MN). The assays were performed in duplicate. The
data presented are from representative experiments. Data points are the
means of two determinations, which were within 10%, mostly 5%, of the
mean.
Northern Blot Analysis
Cytokine mRNA levels in
cells after various treatments were determined by Northern blotting as
described previously
(6) . Cytokine cDNA probes were from
R& Systems.
Nuclear Extract Preparation and Electrophoretic Mobility
Shift Assay (EMSA)
At the end of the specified incubation time,
cells (15-20 10
per sample) were collected by
centrifuging at 350
g for 5 min at 4 °C. After
washing once with phosphate-buffered saline, the cell pellet was
suspended in 0.5 ml of buffer A (10 m
M Hepes-NaOH, pH 7.8, 15
m
M KCl, 2 m
M MgCl
, 0.1 m
M EDTA,
1 m
M dithiothreitol, and 1 m
M phenylmethylsulfonyl
fluoride). The suspension was transferred to a microcentrifuge tube
then centrifuged at 750
g for 5 min. The supernatant
was removed by aspiration, and the pellet was resuspended in 0.2 ml of
buffer A. After 10 min on ice, Nonidet P-40 was added to 0.5% and the
suspension was centrifuged at 1330
g for 15 min. The
resultant nuclear pellet was suspended in 15 µl of buffer B (20
m
M Hepes-NaOH, pH 7.9, 1.5 m
M MgCl
, 0.5
m
M dithiothreitol, 0.42
M NaCl, 0.2 m
M EDTA,
25% glycerol, and 0.5 m
M phenylmethylsulfonyl fluoride). After
15 min on ice with vigorous stirring, the suspension was centrifuged at
16,300
g for 10 min. Fifteen µl of the resultant
supernatant was diluted with 75 µl of buffer C (20 m
M
Hepes-NaOH, pH 7.9, 50 m
M KCl, 0.2 m
M EDTA, 0.5
m
M dithiothreitol, and 0.5 m
M phenylmethylsulfonyl
fluoride) to obtain the final nuclear extract preparation. EMSAs for
NF
B and other transcription factors in nuclei were performed by
using gel shift assay system (Promega), except that 8% (instead of 4%)
gels were used. The reaction mixture contained 2 µl of 5
gel shift binding buffer, 1 µl of
P-labeled consensus
oligonucleotide (oligo) probe of appropriate specificity, 2 µl of
water, and 5 µl of nuclear extract (1 µg of protein).
Nuclear Run-on Assay
Cytokine gene transcription
rates were measured by nuclear run-on assays as described previously
(6) .
mRNA Stability Analysis
Cytokine mRNA stability
was analyzed as described previously
(6) .
Other Reagents
Human recombinant IL-10 and IL-4
were from Schering-Plough. Human recombinant TNF was obtained from
R& Systems. LPS, TPCK, TLCK, and PDTC were purchased from Sigma.
Consensus oligonucleotide probes for various transcription factors,
except for NF-IL-6 were from Promega. The probe for NF-IL-6 was
synthesized as reported
(26) . Antibodies against p65 and p50
subunits of NF
B were from Santa Cruz Biotechnology.
Phosphorothioate analogs of NF
B oligonucleotides, both sense and
antisense, were prepared as reported
(13) .
IL-10 Selectively Inhibits NF
In order to determine the effects of IL-10 treatment
on transcription factors involved in the synthesis of proinflammatory
cytokines, nuclear extracts were prepared from appropriately treated
PBMC, and EMSA was performed using specific B
Activation
P-labeled
cis-acting DNA elements. Thus, when nuclear extracts from
LPS-treated cells were incubated with a
P-labeled NF
B
oligonucleotide probe, a
P-labeled protein-oligonucleotide
complex was formed (Fig. 1 A). This complex formation was
inhibited in the presence of an excess of unlabeled NF
B
oligonucleotide, but not in the presence of an excess of unlabeled
Oct-1 oligonucleotide, showing that the NF
B oligonucleotide
specifically competes with
P-labeled probe. Furthermore,
specific antibodies to the NF
B components p50 and p65 caused a
supershift of the
P-labeled complex, confirming the
identity of the protein in the complex as NF
B (Fig. 1).
Hence, the
P-labeled oligonucleotide probe used in these
experiments complexed with cellular NF
B and LPS stimulated
NF
B translocation into the nucleus. The data that anti-p50
antibody caused the total shift of the
P-oligonucleotide-NF
B complex while anti-p65 antibody
caused only a partial shift show that LPS-stimulated nuclear NF
B
exists as both p50-p50 homodimer and p50-p65 heterodimer, with the
homodimer predominating.
Figure 1:
Effect of IL-10 on LPS- and
TNF-induced NF
B activation in human PBMC. A, the
nuclear protein sample from LPS-stimulated cells (see below) was used
to examine the specificity of the complex observed by EMSA using a
P-labeled NF
B oligonucleotide probe as described
under ``Materials and Methods.'' Competition experiments were
performed using unlabeled NF
B and Oct-1 oligonucleotide probes at
100
molar excess. In ``supershift'' experiments,
antibodies were used at a concentration of 1 µg per assay.
B, cells were stimulated with 80 ng/ml LPS or 20 ng/ml
TNF
in the absence or presence of 10 ng/ml IL-10 for 1.5 h before
the extraction of nuclear protein for EMSA.
Incubation of PBMC with IL-10 alone for up
to 1.5 h had no effect on nuclear NFB. However, when IL-10 was
added 5 min before LPS, NF
B translocation induced by LPS was
completely inhibited (Fig. 1 B). Under the same conditions,
production of IL-6 and IL-8 was also inhibited (data not shown).
Stimulation of PBMC with a different stimulus, TNF
, caused nuclear
localization of NF
B, but to a smaller degree than LPS.
TNF
-induced NF
B translocation (and cytokine synthesis under
the same experimental conditions) was inhibited by IL-10 treatment.
Thus, IL-10 inhibits NF
B activation in PBMC in response to two
distinct stimulating agents.
P-labeled NF-IL-6 cis-acting element, nuclear
extracts from LPS-stimulated PBMC generated a
P-labeled
protein-oligonucleotide complex, which was competed away with unlabeled
NF-IL-6 oligonucleotide but not with unlabeled Oct-1 or NF
B
oligonucleotide. This complex formation was not affected by IL-10
treatment. LPS-stimulated localization of AP-1 into the nucleus, when
assessed under similar conditions, was also not inhibited by IL-10.
When incubated with the
P-labeled cis-acting
elements for AP-2, CREB, GR, Oct-1, and Sp-1, nuclear extracts from
resting cells produced the appropriate
P-labeled
protein-oligonucleotide complexes. None of these complexes was affected
by either LPS or IL-10 (data not shown). These results suggest that
IL-10 causes selective inhibition of NF
B activation in
LPS-stimulated PBMC.
Characteristics of IL-10 Inhibition of NF
The inhibitory effects of IL-10 on NFB
Activation
B
activation and cytokine synthesis were dependent on IL-10 concentration
(Fig. 2). Nearly complete inhibition of both NF
B
translocation and cytokine synthesis occurred at 1-10 ng/ml
IL-10. However, substantial NF
B activity was still detected at 0.1
ng/ml IL-10, a concentration that inhibited TNF
and IL-1
synthesis (60-75% inhibition) more effectively than it did IL-6
and IL-8 synthesis (20-35% inhibition) (Fig. 2). This
differential inhibition pattern suggests that the threshold levels of
NF
B necessary for the initiation of cytokine gene expression vary
among different cytokines and confirms our previous conclusion that
cytokine gene expression is differentially regulated in LPS-stimulated
monocytes
(6) . This experiment was performed using a
PBMC-derived preparation that contained >95% monocytes. The
lymphocyte fraction obtained from the same PBMC preparation produced
neither cytokines nor NF
B translocation. Thus, cytokine production
and NF
B activation and their inhibition by IL-10 in LPS-stimulated
PBMC were due primarily to monocytes and not to lymphocytes, although
the lymphocytes accounted for 80% of the PBMC population.
Figure 2:
Effects of IL-10 concentrations on
LPS-induced NFB activation and cytokine release in human
monocytes. A, various concentrations of IL-10 were added 5 min
before the addition of 80 ng/ml LPS. After 1.5 h, nuclear protein was
extracted and EMSA was performed as described under ``Materials
and Methods.'' B, an aliquot of cell suspension from each
of the culture samples used for EMSA was further incubated for a total
period of 4 h. Cytokines released to the supernatants were measured by
enzyme-linked immunosorbent assay. The amounts of IL-1
, IL-6,
IL-8, and TNF
produced by LPS stimulation in the absence of IL-10
were 2.4, 17.0, 120.6, and 3.1 ng/ml,
respectively.
In
experiments discussed above where a complete inhibition of NFB
activation was observed, IL-10 was added 5 min before LPS. The same
complete inhibition was noted when IL-10 was added simultaneously with
or 2 h before LPS, suggesting that IL-10 acts rapidly to inhibit
NF
B activity and that the signal(s) generated by IL-10 lasts for
at least 2 h (Fig. 3). Similar rapid inhibitions have also been
observed with both cytokine protein synthesis and cytokine gene
expression
(6) , demonstrating a strong correlation between
cytokine gene expression and NF
B activation. In addition, the
nuclear appearance of NF
B in response to LPS preceded cytokine
mRNA accumulation, and nuclear NF
B activity reached a maximum
within 30 min of LPS addition and remained virtually unchanged over the
next 2.5 h (data not shown). When IL-10 was added 0.5 h or 1 h after
LPS during an LPS stimulation period of 1.5 h, substantial inhibition
of NF
B activity was still observed and was more pronounced at 0.5
h than at 1 h (Fig. 3). This observation supports the view that
nuclear NF
B undergoes rapid turnover during LPS stimulation
(27) .
Figure 3:
Effect of IL-10 addition time on
inhibition of LPS-induced NFB activation in human PBMC. IL-10 (10
ng/ml) was added 2 h prior to, 0, 0.5, or 1 h after LPS (80 ng/ml).
Cells were exposed to LPS for a total period of 1.5 h. For all other
details, see ``Materials and
Methods.''
Inhibition of NF
To determine whether
inhibition of NFB May Be Sufficient for Suppressed
Synthesis of Inflammatory Cytokines
B activation is sufficient for suppression of
cytokine production, two non-peptide inhibitors of NF
B activation
were investigated for their ability to inhibit cytokine mRNA synthesis.
TPCK, a chymotrypsin-like serine protease inhibitor, is a potent
inhibitor of NF
B activation
(28) . In our system, TPCK
inhibited LPS-induced NF
B activation as well as the accumulation
of IL-1
, IL-6, IL-8, and TNF
mRNAs in a dose-dependent
manner, and, at 20 µ
M, both responses were completely
inhibited (Fig. 4). TLCK, an analog of TPCK with inhibitory
activity against trypsin-like serine proteases
(28) , inhibited
neither NF
B activation (data not shown) nor cytokine mRNA
accumulation in LPS-stimulated PBMC (Fig. 4). In LPS-stimulated
human monocytes, it has been published that the antioxidant, PDTC,
inhibits NF
B activation
(29, 30) and TNF
synthesis
(30) in a dose-dependent manner. In our experiments,
PDTC by itself caused slight accumulation of cytokine mRNAs, but a
dose-dependent inhibition of LPS-induced accumulation of various
cytokine mRNAs was still observed (Fig. 4). Given the relative
specificity with which these two compounds (TPCK in particular) inhibit
NF
B among various transcription factors tested
(28, 29) , our present data are consistent with NF
B
inhibition being responsible for the inhibition of cytokine mRNA
accumulation.
Figure 4:
Effects of TPCK, TLCK, and PDTC on
LPS-induced NFB activation and cytokine mRNA accumulation in human
PBMC. A, cells were treated with TPCK at the specified
concentrations for 30 min prior to stimulation with 80 ng/ml LPS for an
additional 30 min. Nuclear protein was extracted and EMSA was performed
as described under ``Materials and Methods.'' B,
various inhibitors as indicated were added to the cell suspension for
30 min before the addition of LPS. Cells were cultured for an
additional 2.5 h before extracting total RNA for Northern blot
analysis. For details, see ``Materials and
Methods.''
An antisense oligonucleotide to the p65 subunit of
NFB inhibits IL-8 production in Jurkat T lymphocytes
(13) .
We have also examined the effect of this antisense oligonucleotide on
the production of IL-1
, IL-6, and TNF
by LPS-stimulated PBMC.
Preincubation of cells with the antisense oligonucleotide for 4-8
h resulted in an inhibition of cytokine synthesis by 50% to 70%,
probably due to insufficient uptake of the oligonucleotide. Upon longer
preincubations, the oligonucleotide itself induced cytokine synthesis.
Taken together, these data suggest that inhibition of NF
B
activation may be sufficient for suppression of the synthesis of
various inflammatory cytokines.
IL-4 Does Not Inhibit NF
The
effect of IL-10 on LPS-induced NFB Activation
B activation was compared with
that of IL-4, because IL-4 is a potent inhibitor of cytokine synthesis
in human monocytes
(5) . Under the present experimental
conditions, maximal IL-4 inhibition of LPS-induced synthesis of the
cytokines was 80% (IL-1
, IL-6, and IL-8) or 70% (TNF
). The
steady state levels of cytokine mRNAs, as determined by Northern blot
analysis, were also similarly inhibited. However, at 10 ng/ml, that was
optimal for its inhibitory effects on cytokine synthesis, IL-4 caused
no significant suppression of the NF
B activation (Fig. 5),
suggesting that the inhibition of cytokine synthesis by IL-4 does not
involve NF
B.
Figure 5:
Effect of IL-4 on LPS-induced NFB
activation in human PBMC. Cells were stimulated with 80 ng/ml LPS in
the presence of 10 ng/ml each of IL-10 or IL-4 for 1.5 h. Nuclear
protein was extracted and EMSA was performed as described under
``Materials and Methods.''
Nuclear run-on experiments revealed no inhibition
by IL-4 of transcription of cytokine genes in LPS-stimulated monocytes
(Fig. 6), while mRNA stability analysis showed that IL-4 treatment did
enhance the degradation of various cytokine mRNAs (Fig. 7). These
results are consistent with IL-4 not inhibiting NFB activation and
indicate that IL-4 and IL-10 inhibit cytokine synthesis in
LPS-stimulated human monocytes through distinct mechanisms.
B sequence, overexpression of NF
B proteins,
antisense oligonucleotides to NF
B, and the NF
B DNA binding
sequence have provided data to strongly implicate NF
B in the
expression of cytokine genes in monocytes
(8, 12, 13, 14, 31, 32) .
Additional studies have further suggested that NF
B acts
synergistically with NF-IL-6 and AP-1 to effect cytokine gene
expression
(17, 19) . In the present study, we
demonstrate that IL-10 inhibits NF
B activation in stimulated human
monocytes without affecting AP-1, NF-IL-6, or other transcription
factors that are believed to be involved in cytokine gene
transcription. This selective inhibition of NF
B activation occurs
rapidly and in a dose-dependent manner and correlates well with the
inhibition of cytokine gene transcription in terms of both time course
and dose-response, indicating that IL-10 inhibition of cytokine gene
expression may be mediated by the blockade of NF
B activation.
B only, it is noteworthy that these
compounds do not affect other transcription factors such as AP-1,
Oct-1, CREB, and Sp-1
(28, 29) and that a structural
analog of TPCK is without effect on NF
B activation or on cytokine
mRNA accumulation (Fig. 4). This selectivity with regard to
NF
B inhibition and the inhibition of cytokine mRNA accumulation by
TPCK and PDTC in conjunction with the observations that antisense
oligonucleotides and the NF
B binding sequence inhibit cytokine
mRNA synthesis would indicate that the blockade of NF
B activation
might indeed be sufficient for the inhibition of cytokine gene
transcription, thus supporting the view that NF
B is a potentially
important target for IL-10's inhibitory action in human
monocytes.
response region
(33) . However, the importance
of this complex in cytokine gene transcription has yet to be
determined. Recently, it has been reported that in LPS-stimulated
macrophages from mice lacking the p50 subunit of NF
B, TNF
and
IL-1
release are normal
(34) , suggesting
NF
B-independent regulation of the cytokine genes. However, it
remains unclear whether the in vivo role of p50 in cytokine
gene regulation can be compensated for by other members of the NF
B
family ( e.g. p65). A definitive assessment of the role of
NF
B in gene expression of inflammatory cytokines must await
evaluation of animals with targeted disruption of both p50 and p65.
B most likely involves the blockade of a reaction
required for release of I
B from the NF
B complex in intact
cells. Experiments using cell-free preparations have suggested that
protein kinases such as protein kinase C phosphorylate I
B causing
its release and ensuing activation of NF
B
(37) . Although
phorbol myristate acetate-induced activation of NF-
B appears to
involve protein kinase C, other NF
B activating agents such as
TNF
and IL-1 appear not to require protein kinase C
(38, 39) . A recent study has demonstrated that several
NF
B activators including LPS, IL-1, TNF
and phorbol ester
activate a chymotrypsin-like serine protease that rapidly degrades the
inhibitory subunit I
B
(28) . Additionally, the p50
component of the NF
B complex is derived from the inactive
cytosolic precursor protein p105 through a proteolytic cleavage
(27) catalyzed presumably by an ATP-dependent serine protease
activity
(40) , and this proteolytic cleavage occurs within 15
min after LPS addition
(27) . That this latter process might
also be a target for IL-10 action is suggested by the fact that the p50
homodimer predominates in the nucleus from LPS-stimulated monocytes and
that this translocation is inhibited by IL-10.
B are likely
regulated by reactive oxygen intermediates such as hydroxyl radical,
based on the observation that various NF
B inducers produce
reactive oxygen intermediates and that antioxidants and radical
scavengers inhibit NF
B activation
(29) . Because IL-10
blocks NF
B activation in response to distinct NF
B inducers
such as LPS and TNF
, IL-10-induced signals seem to interfere with
the activity of a common messenger produced by different signaling
pathways, and this messenger might well be a reactive oxygen
intermediate. Therefore, it will be interesting to determine whether
IL-10 inhibits metabolism of precursor proteins and formation of
reactive oxygen intermediates.
B activation in activated human
monocytes, and that the major mechanism by which IL-4 inhibits cytokine
synthesis in human monocytes is, distinctively, the enhancement of
cytokine mRNA degradation.
- p-tosyl-
L-lysine
chloromethyl ketone; PDTC, pyrrolidinedithiocarbamate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.